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Accepted Manuscript
Stability of Vitamin B12 with the Protection of Whey Proteins and Their Effectson the Gut Microbiome
HuanhuanWang Yikai Shou Xuan Zhu Yuanyuan Xu Lihua Shi ShashaXiang Xiao Feng Jianzhong Han
PII S0308-8146(18)31798-9DOI httpsdoiorg101016jfoodchem201810033Reference FOCH 23694
To appear in Food Chemistry
Received Date 5 October 2017Revised Date 28 September 2018Accepted Date 6 October 2018
Please cite this article as HuanhuanWang Shou Y Zhu X Xu Y Shi L Xiang S Feng X Han J Stabilityof Vitamin B12 with the Protection of Whey Proteins and Their Effects on the Gut Microbiome Food Chemistry(2018) doi httpsdoiorg101016jfoodchem201810033
This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customerswe are providing this early version of the manuscript The manuscript will undergo copyediting typesetting andreview of the resulting proof before it is published in its final form Please note that during the production processerrors may be discovered which could affect the content and all legal disclaimers that apply to the journal pertain
1
Stability of Vitamin B12 with the Protection of Whey Proteins and Their Effects on the
Gut Microbiome
Running title Stability and Effect of Vitamin B12Whey Complexes
HuanhuanWangadagger huanvalhznueducn
Yikai Shouadagger 403278497qqcom
Xuan Zhub zhuxuanmailzjgsueducn
Yuanyuan Xub 362120957qqcom
Lihua Shib 624872970qqcom
Shasha Xiangb xsscherishqqcom
Xiao Fengb 249259608qqcom
Jianzhong Hanb jzhan99zjgsueducn
aSchool of Medicine Hangzhou Normal University Hangzhou 310018 China
bSchool of Food Science and Bioengineering Zhejiang Gongshang University Hangzhou
310018China
daggerThese authors contributed equally to this work
Corresponding author Dr Xuan Zhu Email zhuxuanmailzjgsueducn Phone
+8657128008902 School of Food Science and Bioengineering Zhejiang Gongshang
University Xuezheng Str 18 Hangzhou 310018 China
Abstract
Cobalamin degrades in the presence of light and heat which causes spectral changes and loss
of coenzyme activity In the presence of beta-lactoglobulin or alpha-lactalbumin the thermal-
and photostabilities of adenosylcobalamin (ADCBL) and cyanocobalamin (CNCBL) are
increased by 10-30 Similarly the stabilities of ADCBL and CNCBL are increased in the
presence of whey proteins by 197 and 22 respectively when tested in gastric juice for 2
h Due to the limited absorption of cobalamin during digestion excess cobalamin can enter
the colon and modulate the gut microbiome In a colonic model in vitro supplementation with
cobalamin and whey enhanced the proportions of Firmicutes and Bacteroidetes spp and
reduced those of Proteobacteria spp which includes pathogens such as Escherichia and
Shigella spp and Pseudomonas spp Thus while complex formation could improve the
stability and bioavailability of cobalamin these complexes might also mediate gut
microecology to influence human nutrition and health
2
Keywords cobalamin whey protein gut microbiome stability
3
1 Introduction
Cobalamin (vitamin B12) is a naturally occurring organometallic compound containing
cobalt that serves as an important water-soluble vitamin for human health The recommended
daily intake for cobalamin is 24 microg (Rucker Suttie McCormick amp Machilin 2001) This
vitamin functions as a cofactor for two classes of human enzymes namely isomerases and
methyltransferases Consequently cobalamin deficiency can cause disturbances in cell
division leading to neuropathy nervous system disease and pernicious anemia (Allen 2010)
Cobalamin has four bioactive forms and many analogues which different in their upper
andor lower functional groups For example the adenosyl can be replaced by a methyl
hydroxyl or cyano group to form methyl- hydroxo- or cyano-cobalamin respectively
Cyanocobalamin (CNCBL) is not found in nature but is used as a supplement for humans and
animals Cobalamin is not stable during food processing or storage as it is photo- and
heat-labile (Ahmad Hussain amp Fareedi 1992) Moreover the aerobic photodecomposition of
cobalamin processes more rapidly than the anaerobic photodecomposition (Demerre amp
Wilson 1956) meaning common food processing can enhance degradation All forms of
cobalamin are irreversibly inactivated under irradiation However some enzymes requiring
adenosylcobalamin (ADCBL) and methylcobalamin can also protect these compounds from
decomposition (Demerre amp Wilson 1956)
Whey protein typically consists of beta-lactoglobulin alpha-lactalbumin bovine serum
albumin and immunoglobulins The main protein (65) in bovine whey is beta-lactoglobulin
(18 kDa) a globular protein with 162 amino acid residues Beta-lactoglobulin belongs to the
lipocalin family and is able to bind small hydrophobic molecules in the internal cavity of its
β-barrel Because of multiple binding sites beta-lactoglobulin can bind a variety of ligands
including fatty acids polyphenols and vitamins such as cobalamin (Sawyer Brownlow
Polikarpov amp Wu 1998) Alpha-lactalbumin (14 kDa) the second most prevalent protein is a
small globular protein containing 123 amino acid residues with a large alpha-helical domain
and a small beta-sheet domain (Cawthern Narayan Chaudhuri Permyakov amp Berliner 1997)
The two domains are separated by a deep cleft and linked by a calcium binding loop It has
been reported that alpha-lactalbumin may function as a carrier of hydrophobic lipids vitamins
and metabolites
Ligand-binding proteins such as lactoglobulins and lactalbumins have been used to bind
low-molecular weight molecules for their protection and delivery (de Wolf amp Brett 2000)
The maximum cobalamin binding capacities of beta-lactoglobulin alpha-lactalbumin casein
blood serum album and proteose-peptone were determined to be 850 690 98 80 and 370
μgg respectively (Gizis Kim Brunner amp Schweigert 1965) Beta-lactoglobulin and
alpha-lactalbumin have also been reported to bind vitamin B12 and protect it from
4
decomposition (Gizis Kim Brunner amp Schweigert 1965 Johns et al 2015) Some
researchers have suggested that lactoferrin can dramatically increase the photostability and
solubility of cobalamin (US Pat 6500472B2 2002)
Due to limited absorption of cobalamin during digestion excess cobalamin can enter the
colon and modulate the gut microbiome Cobalamin is the only vitamin produced exclusively
by bacteria and archaea but it is used by all domains of life Synthesis of cobalamin is
energetically costly requiring nearly 30 different enzymes (Martens Barg Warren amp Jahn
2002) and only 20 of prokaryotes have the capacity to produce cobalamin (Degnan Barry
Mok Taga amp Goodman 2014) Cobalamin is a precious commodity and has been suggested
to be important not only as a nutrient but also as a signaling molecule for the spatial and
functional organization of gut microecology
In this study we examined whether beta-lactoglobulin or alpha-lactalbumin could
enhance the stability of cobalamin during food processing and positively affect the
composition of a model human gut microbiome The results obtained from this research
provide insights into possible applications of ligand-binding proteins as carriers of cobalamin
in the development of functional foods and pharmaceuticals
2 Method and Materials
21 Chemical reagents
Beta-lactoglobulin (purityge90) and alpha-lactalbumin (purityge85) were purchased
from Beingmate Company (Hangzhou China) and used without further purification ADCBL
and CNCBL were purchased from Sigma-Aldrich Chemical Company (St Louis US) and
used without further purification
22 Sample preparation
Stock solutions of beta-lactoglobulin alpha-lactalbumin ADCBL and CNCBL were
prepared at a final concentration of 10 μM in 1 mM phosphate buffer at pH 60
Cobalamin-whey protein complexes were prepared by mixing different concentrations of
protein (025 05 075 μM) with the ligand (025 μM) in phosphate buffer All samples were
prepared and incubated for approximately 1 h at room temperature in 200 mL flasks
(DURAN no 2121654 Schott GmbH Mainz Germany) while shaking (50 rpm) in the dark
23 Photodecomposition and heat treatment procedures
Briefly 200 mL flasks containing the cobalamin-whey protein complexes were placed
under a UV LED bulb (9 W) (Leiman Co Ltd Shenzhen China) with a light intensity of ca
100 μmol m-2 s-1 Each flask was placed 10 cm from the light source Samples were analyzed
5
every 15 min for up to 120 min Similarly the 200 mL flasks containing cobalamin-whey
protein complexes were placed into a water bath at 80 degC (Sonorex TK 52 ultrasonic water
bath Bandelin Electronics Berlin Germany) Samples were analyzed every 15 min for up to
120 min in the dark to avoid light degradation All experiments were performed in triplicate
24 In vitro stomach digestion
The stability of cobalamin in gastric fluid (SGF) was determined by subjecting samples
to simulated digestion in the presence of pepsin (pH 13 37 degC 2 h) SGF was prepared as
described previously (Tokle Lesmes Decker amp McClements 2012) with slight modifications
Briefly SGF was prepared by dissolving 1 (wv) pepsin 3 g of sodium chloride and 9 ml
of HCl in 1 L of water the pH was adjusted to 13 using 1 M HCl Cobalamin-whey protein
complexes (01 g) were incubated in 100 mL of SGF with shaking (100 strokesmin) in a
water bath at 37 degC for 2 h to simulate stomach conditions
25 Cobalamin determination
To extract cobalamin from 10 mL samples of B12-whey complexes 50 mL of sodium
acetate buffer (pH 60) was added in the presence of sodium cyanide (1) and the solution
was heated in a water bath (Type 1004 water bath GFL Gesellschaft fuumlr Labortechnik
Burgwedel Germany) for 40 min at 90 degC The pH was adjusted to 7 and 10 mL hexane was
added before the mixture was centrifuged for 15 min at 4010 g (Varifuge 30 Heraeus
centrifuge Heraeus Instruments Hanau Germany) The aqueous layer was collected and
passed through a solid phase extraction column (SPE) (CEC181M6 United Chemical
Technologies Bristol PA USA) which had been prewashed with 3 mL of methanol and 3
mL of Milli-Q water with the help of a pump (AL 15 Knf Neuberger Hamburg Germany)
to control the flow (1 drop per second) The column was washed three times with Milli-Q
water and 3 mL of methanol was used to elute cobalamin The solvent was evaporated to
dryness and the residue was subsequently dissolved in 1 mL of Milli-Q water before being
passed through a membrane filter (02 μm) (Macherey-Nagel Duumlren Germany) and analyzed
by HPLC using a RP-18 column (2504 mm ID 5 microm Merck Darmstadt Germany)
Cobalamin was detected using a modified HPLC method that was previously reported
(Gu Zhang Song Li amp Zhu 2015) All chromatographic separations were performed at
room temperature The mobile phases consisted of a mixture of methanol with 01 formic
acid (A) (Merck Darmstadt Germany) and ultra-purified water with 01 formic acid (B)
which was degassed by an ultrasonic water bath the flow rate was 05 mL per min The
gradient elution was programmed as follows 0-2 min 20 A 2-3 min 20-25 A 3-11 min
25-35 A 11-19 min 35-20 A 20-22 min 100-100 A 22-26 min 100-20 A and
6
26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored
by a Diode Array Detector (Waters US) at 361 nm
26 In vitro intestinal digestion
Colonic fermentation of the cobalamin-whey complex was conducted according to
Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child
who had not received antibiotic treatment in the previous three months were collected and
maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as
previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was
immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)
xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred
immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive
medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone
(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl
45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O
0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride
400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150
p-aminobenzoic acid 20 folate 10) and cysteine (08)
Six parallel reactors inoculated with immobilized gut microbiota were operated for 10
days The reactors were continuously fed nutritive media differing only in their
supplementation with cobalamin-whey complexes or cobalamin only The fermentation was
performed under typical conditions of the proximal colon according to previously described
procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix
2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium
was replaced every 12 h During the simulated fermentation the pH was maintained at 60
throughout the experiment with the addition of 2 M NaOH and the temperature was
maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2
into all reactors and medium vessels
The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of
fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05
μM) were added to the nutritive media for 10 days during fermentation All six reactors were
sampled daily and samples were frozen at -80 degC for pyrosequencing
27 DNA isolation PCR 16S rDNA analysis
DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01
Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
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solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
1
Stability of Vitamin B12 with the Protection of Whey Proteins and Their Effects on the
Gut Microbiome
Running title Stability and Effect of Vitamin B12Whey Complexes
HuanhuanWangadagger huanvalhznueducn
Yikai Shouadagger 403278497qqcom
Xuan Zhub zhuxuanmailzjgsueducn
Yuanyuan Xub 362120957qqcom
Lihua Shib 624872970qqcom
Shasha Xiangb xsscherishqqcom
Xiao Fengb 249259608qqcom
Jianzhong Hanb jzhan99zjgsueducn
aSchool of Medicine Hangzhou Normal University Hangzhou 310018 China
bSchool of Food Science and Bioengineering Zhejiang Gongshang University Hangzhou
310018China
daggerThese authors contributed equally to this work
Corresponding author Dr Xuan Zhu Email zhuxuanmailzjgsueducn Phone
+8657128008902 School of Food Science and Bioengineering Zhejiang Gongshang
University Xuezheng Str 18 Hangzhou 310018 China
Abstract
Cobalamin degrades in the presence of light and heat which causes spectral changes and loss
of coenzyme activity In the presence of beta-lactoglobulin or alpha-lactalbumin the thermal-
and photostabilities of adenosylcobalamin (ADCBL) and cyanocobalamin (CNCBL) are
increased by 10-30 Similarly the stabilities of ADCBL and CNCBL are increased in the
presence of whey proteins by 197 and 22 respectively when tested in gastric juice for 2
h Due to the limited absorption of cobalamin during digestion excess cobalamin can enter
the colon and modulate the gut microbiome In a colonic model in vitro supplementation with
cobalamin and whey enhanced the proportions of Firmicutes and Bacteroidetes spp and
reduced those of Proteobacteria spp which includes pathogens such as Escherichia and
Shigella spp and Pseudomonas spp Thus while complex formation could improve the
stability and bioavailability of cobalamin these complexes might also mediate gut
microecology to influence human nutrition and health
2
Keywords cobalamin whey protein gut microbiome stability
3
1 Introduction
Cobalamin (vitamin B12) is a naturally occurring organometallic compound containing
cobalt that serves as an important water-soluble vitamin for human health The recommended
daily intake for cobalamin is 24 microg (Rucker Suttie McCormick amp Machilin 2001) This
vitamin functions as a cofactor for two classes of human enzymes namely isomerases and
methyltransferases Consequently cobalamin deficiency can cause disturbances in cell
division leading to neuropathy nervous system disease and pernicious anemia (Allen 2010)
Cobalamin has four bioactive forms and many analogues which different in their upper
andor lower functional groups For example the adenosyl can be replaced by a methyl
hydroxyl or cyano group to form methyl- hydroxo- or cyano-cobalamin respectively
Cyanocobalamin (CNCBL) is not found in nature but is used as a supplement for humans and
animals Cobalamin is not stable during food processing or storage as it is photo- and
heat-labile (Ahmad Hussain amp Fareedi 1992) Moreover the aerobic photodecomposition of
cobalamin processes more rapidly than the anaerobic photodecomposition (Demerre amp
Wilson 1956) meaning common food processing can enhance degradation All forms of
cobalamin are irreversibly inactivated under irradiation However some enzymes requiring
adenosylcobalamin (ADCBL) and methylcobalamin can also protect these compounds from
decomposition (Demerre amp Wilson 1956)
Whey protein typically consists of beta-lactoglobulin alpha-lactalbumin bovine serum
albumin and immunoglobulins The main protein (65) in bovine whey is beta-lactoglobulin
(18 kDa) a globular protein with 162 amino acid residues Beta-lactoglobulin belongs to the
lipocalin family and is able to bind small hydrophobic molecules in the internal cavity of its
β-barrel Because of multiple binding sites beta-lactoglobulin can bind a variety of ligands
including fatty acids polyphenols and vitamins such as cobalamin (Sawyer Brownlow
Polikarpov amp Wu 1998) Alpha-lactalbumin (14 kDa) the second most prevalent protein is a
small globular protein containing 123 amino acid residues with a large alpha-helical domain
and a small beta-sheet domain (Cawthern Narayan Chaudhuri Permyakov amp Berliner 1997)
The two domains are separated by a deep cleft and linked by a calcium binding loop It has
been reported that alpha-lactalbumin may function as a carrier of hydrophobic lipids vitamins
and metabolites
Ligand-binding proteins such as lactoglobulins and lactalbumins have been used to bind
low-molecular weight molecules for their protection and delivery (de Wolf amp Brett 2000)
The maximum cobalamin binding capacities of beta-lactoglobulin alpha-lactalbumin casein
blood serum album and proteose-peptone were determined to be 850 690 98 80 and 370
μgg respectively (Gizis Kim Brunner amp Schweigert 1965) Beta-lactoglobulin and
alpha-lactalbumin have also been reported to bind vitamin B12 and protect it from
4
decomposition (Gizis Kim Brunner amp Schweigert 1965 Johns et al 2015) Some
researchers have suggested that lactoferrin can dramatically increase the photostability and
solubility of cobalamin (US Pat 6500472B2 2002)
Due to limited absorption of cobalamin during digestion excess cobalamin can enter the
colon and modulate the gut microbiome Cobalamin is the only vitamin produced exclusively
by bacteria and archaea but it is used by all domains of life Synthesis of cobalamin is
energetically costly requiring nearly 30 different enzymes (Martens Barg Warren amp Jahn
2002) and only 20 of prokaryotes have the capacity to produce cobalamin (Degnan Barry
Mok Taga amp Goodman 2014) Cobalamin is a precious commodity and has been suggested
to be important not only as a nutrient but also as a signaling molecule for the spatial and
functional organization of gut microecology
In this study we examined whether beta-lactoglobulin or alpha-lactalbumin could
enhance the stability of cobalamin during food processing and positively affect the
composition of a model human gut microbiome The results obtained from this research
provide insights into possible applications of ligand-binding proteins as carriers of cobalamin
in the development of functional foods and pharmaceuticals
2 Method and Materials
21 Chemical reagents
Beta-lactoglobulin (purityge90) and alpha-lactalbumin (purityge85) were purchased
from Beingmate Company (Hangzhou China) and used without further purification ADCBL
and CNCBL were purchased from Sigma-Aldrich Chemical Company (St Louis US) and
used without further purification
22 Sample preparation
Stock solutions of beta-lactoglobulin alpha-lactalbumin ADCBL and CNCBL were
prepared at a final concentration of 10 μM in 1 mM phosphate buffer at pH 60
Cobalamin-whey protein complexes were prepared by mixing different concentrations of
protein (025 05 075 μM) with the ligand (025 μM) in phosphate buffer All samples were
prepared and incubated for approximately 1 h at room temperature in 200 mL flasks
(DURAN no 2121654 Schott GmbH Mainz Germany) while shaking (50 rpm) in the dark
23 Photodecomposition and heat treatment procedures
Briefly 200 mL flasks containing the cobalamin-whey protein complexes were placed
under a UV LED bulb (9 W) (Leiman Co Ltd Shenzhen China) with a light intensity of ca
100 μmol m-2 s-1 Each flask was placed 10 cm from the light source Samples were analyzed
5
every 15 min for up to 120 min Similarly the 200 mL flasks containing cobalamin-whey
protein complexes were placed into a water bath at 80 degC (Sonorex TK 52 ultrasonic water
bath Bandelin Electronics Berlin Germany) Samples were analyzed every 15 min for up to
120 min in the dark to avoid light degradation All experiments were performed in triplicate
24 In vitro stomach digestion
The stability of cobalamin in gastric fluid (SGF) was determined by subjecting samples
to simulated digestion in the presence of pepsin (pH 13 37 degC 2 h) SGF was prepared as
described previously (Tokle Lesmes Decker amp McClements 2012) with slight modifications
Briefly SGF was prepared by dissolving 1 (wv) pepsin 3 g of sodium chloride and 9 ml
of HCl in 1 L of water the pH was adjusted to 13 using 1 M HCl Cobalamin-whey protein
complexes (01 g) were incubated in 100 mL of SGF with shaking (100 strokesmin) in a
water bath at 37 degC for 2 h to simulate stomach conditions
25 Cobalamin determination
To extract cobalamin from 10 mL samples of B12-whey complexes 50 mL of sodium
acetate buffer (pH 60) was added in the presence of sodium cyanide (1) and the solution
was heated in a water bath (Type 1004 water bath GFL Gesellschaft fuumlr Labortechnik
Burgwedel Germany) for 40 min at 90 degC The pH was adjusted to 7 and 10 mL hexane was
added before the mixture was centrifuged for 15 min at 4010 g (Varifuge 30 Heraeus
centrifuge Heraeus Instruments Hanau Germany) The aqueous layer was collected and
passed through a solid phase extraction column (SPE) (CEC181M6 United Chemical
Technologies Bristol PA USA) which had been prewashed with 3 mL of methanol and 3
mL of Milli-Q water with the help of a pump (AL 15 Knf Neuberger Hamburg Germany)
to control the flow (1 drop per second) The column was washed three times with Milli-Q
water and 3 mL of methanol was used to elute cobalamin The solvent was evaporated to
dryness and the residue was subsequently dissolved in 1 mL of Milli-Q water before being
passed through a membrane filter (02 μm) (Macherey-Nagel Duumlren Germany) and analyzed
by HPLC using a RP-18 column (2504 mm ID 5 microm Merck Darmstadt Germany)
Cobalamin was detected using a modified HPLC method that was previously reported
(Gu Zhang Song Li amp Zhu 2015) All chromatographic separations were performed at
room temperature The mobile phases consisted of a mixture of methanol with 01 formic
acid (A) (Merck Darmstadt Germany) and ultra-purified water with 01 formic acid (B)
which was degassed by an ultrasonic water bath the flow rate was 05 mL per min The
gradient elution was programmed as follows 0-2 min 20 A 2-3 min 20-25 A 3-11 min
25-35 A 11-19 min 35-20 A 20-22 min 100-100 A 22-26 min 100-20 A and
6
26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored
by a Diode Array Detector (Waters US) at 361 nm
26 In vitro intestinal digestion
Colonic fermentation of the cobalamin-whey complex was conducted according to
Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child
who had not received antibiotic treatment in the previous three months were collected and
maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as
previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was
immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)
xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred
immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive
medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone
(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl
45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O
0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride
400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150
p-aminobenzoic acid 20 folate 10) and cysteine (08)
Six parallel reactors inoculated with immobilized gut microbiota were operated for 10
days The reactors were continuously fed nutritive media differing only in their
supplementation with cobalamin-whey complexes or cobalamin only The fermentation was
performed under typical conditions of the proximal colon according to previously described
procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix
2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium
was replaced every 12 h During the simulated fermentation the pH was maintained at 60
throughout the experiment with the addition of 2 M NaOH and the temperature was
maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2
into all reactors and medium vessels
The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of
fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05
μM) were added to the nutritive media for 10 days during fermentation All six reactors were
sampled daily and samples were frozen at -80 degC for pyrosequencing
27 DNA isolation PCR 16S rDNA analysis
DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01
Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
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solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
2
Keywords cobalamin whey protein gut microbiome stability
3
1 Introduction
Cobalamin (vitamin B12) is a naturally occurring organometallic compound containing
cobalt that serves as an important water-soluble vitamin for human health The recommended
daily intake for cobalamin is 24 microg (Rucker Suttie McCormick amp Machilin 2001) This
vitamin functions as a cofactor for two classes of human enzymes namely isomerases and
methyltransferases Consequently cobalamin deficiency can cause disturbances in cell
division leading to neuropathy nervous system disease and pernicious anemia (Allen 2010)
Cobalamin has four bioactive forms and many analogues which different in their upper
andor lower functional groups For example the adenosyl can be replaced by a methyl
hydroxyl or cyano group to form methyl- hydroxo- or cyano-cobalamin respectively
Cyanocobalamin (CNCBL) is not found in nature but is used as a supplement for humans and
animals Cobalamin is not stable during food processing or storage as it is photo- and
heat-labile (Ahmad Hussain amp Fareedi 1992) Moreover the aerobic photodecomposition of
cobalamin processes more rapidly than the anaerobic photodecomposition (Demerre amp
Wilson 1956) meaning common food processing can enhance degradation All forms of
cobalamin are irreversibly inactivated under irradiation However some enzymes requiring
adenosylcobalamin (ADCBL) and methylcobalamin can also protect these compounds from
decomposition (Demerre amp Wilson 1956)
Whey protein typically consists of beta-lactoglobulin alpha-lactalbumin bovine serum
albumin and immunoglobulins The main protein (65) in bovine whey is beta-lactoglobulin
(18 kDa) a globular protein with 162 amino acid residues Beta-lactoglobulin belongs to the
lipocalin family and is able to bind small hydrophobic molecules in the internal cavity of its
β-barrel Because of multiple binding sites beta-lactoglobulin can bind a variety of ligands
including fatty acids polyphenols and vitamins such as cobalamin (Sawyer Brownlow
Polikarpov amp Wu 1998) Alpha-lactalbumin (14 kDa) the second most prevalent protein is a
small globular protein containing 123 amino acid residues with a large alpha-helical domain
and a small beta-sheet domain (Cawthern Narayan Chaudhuri Permyakov amp Berliner 1997)
The two domains are separated by a deep cleft and linked by a calcium binding loop It has
been reported that alpha-lactalbumin may function as a carrier of hydrophobic lipids vitamins
and metabolites
Ligand-binding proteins such as lactoglobulins and lactalbumins have been used to bind
low-molecular weight molecules for their protection and delivery (de Wolf amp Brett 2000)
The maximum cobalamin binding capacities of beta-lactoglobulin alpha-lactalbumin casein
blood serum album and proteose-peptone were determined to be 850 690 98 80 and 370
μgg respectively (Gizis Kim Brunner amp Schweigert 1965) Beta-lactoglobulin and
alpha-lactalbumin have also been reported to bind vitamin B12 and protect it from
4
decomposition (Gizis Kim Brunner amp Schweigert 1965 Johns et al 2015) Some
researchers have suggested that lactoferrin can dramatically increase the photostability and
solubility of cobalamin (US Pat 6500472B2 2002)
Due to limited absorption of cobalamin during digestion excess cobalamin can enter the
colon and modulate the gut microbiome Cobalamin is the only vitamin produced exclusively
by bacteria and archaea but it is used by all domains of life Synthesis of cobalamin is
energetically costly requiring nearly 30 different enzymes (Martens Barg Warren amp Jahn
2002) and only 20 of prokaryotes have the capacity to produce cobalamin (Degnan Barry
Mok Taga amp Goodman 2014) Cobalamin is a precious commodity and has been suggested
to be important not only as a nutrient but also as a signaling molecule for the spatial and
functional organization of gut microecology
In this study we examined whether beta-lactoglobulin or alpha-lactalbumin could
enhance the stability of cobalamin during food processing and positively affect the
composition of a model human gut microbiome The results obtained from this research
provide insights into possible applications of ligand-binding proteins as carriers of cobalamin
in the development of functional foods and pharmaceuticals
2 Method and Materials
21 Chemical reagents
Beta-lactoglobulin (purityge90) and alpha-lactalbumin (purityge85) were purchased
from Beingmate Company (Hangzhou China) and used without further purification ADCBL
and CNCBL were purchased from Sigma-Aldrich Chemical Company (St Louis US) and
used without further purification
22 Sample preparation
Stock solutions of beta-lactoglobulin alpha-lactalbumin ADCBL and CNCBL were
prepared at a final concentration of 10 μM in 1 mM phosphate buffer at pH 60
Cobalamin-whey protein complexes were prepared by mixing different concentrations of
protein (025 05 075 μM) with the ligand (025 μM) in phosphate buffer All samples were
prepared and incubated for approximately 1 h at room temperature in 200 mL flasks
(DURAN no 2121654 Schott GmbH Mainz Germany) while shaking (50 rpm) in the dark
23 Photodecomposition and heat treatment procedures
Briefly 200 mL flasks containing the cobalamin-whey protein complexes were placed
under a UV LED bulb (9 W) (Leiman Co Ltd Shenzhen China) with a light intensity of ca
100 μmol m-2 s-1 Each flask was placed 10 cm from the light source Samples were analyzed
5
every 15 min for up to 120 min Similarly the 200 mL flasks containing cobalamin-whey
protein complexes were placed into a water bath at 80 degC (Sonorex TK 52 ultrasonic water
bath Bandelin Electronics Berlin Germany) Samples were analyzed every 15 min for up to
120 min in the dark to avoid light degradation All experiments were performed in triplicate
24 In vitro stomach digestion
The stability of cobalamin in gastric fluid (SGF) was determined by subjecting samples
to simulated digestion in the presence of pepsin (pH 13 37 degC 2 h) SGF was prepared as
described previously (Tokle Lesmes Decker amp McClements 2012) with slight modifications
Briefly SGF was prepared by dissolving 1 (wv) pepsin 3 g of sodium chloride and 9 ml
of HCl in 1 L of water the pH was adjusted to 13 using 1 M HCl Cobalamin-whey protein
complexes (01 g) were incubated in 100 mL of SGF with shaking (100 strokesmin) in a
water bath at 37 degC for 2 h to simulate stomach conditions
25 Cobalamin determination
To extract cobalamin from 10 mL samples of B12-whey complexes 50 mL of sodium
acetate buffer (pH 60) was added in the presence of sodium cyanide (1) and the solution
was heated in a water bath (Type 1004 water bath GFL Gesellschaft fuumlr Labortechnik
Burgwedel Germany) for 40 min at 90 degC The pH was adjusted to 7 and 10 mL hexane was
added before the mixture was centrifuged for 15 min at 4010 g (Varifuge 30 Heraeus
centrifuge Heraeus Instruments Hanau Germany) The aqueous layer was collected and
passed through a solid phase extraction column (SPE) (CEC181M6 United Chemical
Technologies Bristol PA USA) which had been prewashed with 3 mL of methanol and 3
mL of Milli-Q water with the help of a pump (AL 15 Knf Neuberger Hamburg Germany)
to control the flow (1 drop per second) The column was washed three times with Milli-Q
water and 3 mL of methanol was used to elute cobalamin The solvent was evaporated to
dryness and the residue was subsequently dissolved in 1 mL of Milli-Q water before being
passed through a membrane filter (02 μm) (Macherey-Nagel Duumlren Germany) and analyzed
by HPLC using a RP-18 column (2504 mm ID 5 microm Merck Darmstadt Germany)
Cobalamin was detected using a modified HPLC method that was previously reported
(Gu Zhang Song Li amp Zhu 2015) All chromatographic separations were performed at
room temperature The mobile phases consisted of a mixture of methanol with 01 formic
acid (A) (Merck Darmstadt Germany) and ultra-purified water with 01 formic acid (B)
which was degassed by an ultrasonic water bath the flow rate was 05 mL per min The
gradient elution was programmed as follows 0-2 min 20 A 2-3 min 20-25 A 3-11 min
25-35 A 11-19 min 35-20 A 20-22 min 100-100 A 22-26 min 100-20 A and
6
26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored
by a Diode Array Detector (Waters US) at 361 nm
26 In vitro intestinal digestion
Colonic fermentation of the cobalamin-whey complex was conducted according to
Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child
who had not received antibiotic treatment in the previous three months were collected and
maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as
previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was
immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)
xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred
immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive
medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone
(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl
45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O
0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride
400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150
p-aminobenzoic acid 20 folate 10) and cysteine (08)
Six parallel reactors inoculated with immobilized gut microbiota were operated for 10
days The reactors were continuously fed nutritive media differing only in their
supplementation with cobalamin-whey complexes or cobalamin only The fermentation was
performed under typical conditions of the proximal colon according to previously described
procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix
2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium
was replaced every 12 h During the simulated fermentation the pH was maintained at 60
throughout the experiment with the addition of 2 M NaOH and the temperature was
maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2
into all reactors and medium vessels
The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of
fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05
μM) were added to the nutritive media for 10 days during fermentation All six reactors were
sampled daily and samples were frozen at -80 degC for pyrosequencing
27 DNA isolation PCR 16S rDNA analysis
DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01
Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
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Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
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Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
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Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
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Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
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Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
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(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
3
1 Introduction
Cobalamin (vitamin B12) is a naturally occurring organometallic compound containing
cobalt that serves as an important water-soluble vitamin for human health The recommended
daily intake for cobalamin is 24 microg (Rucker Suttie McCormick amp Machilin 2001) This
vitamin functions as a cofactor for two classes of human enzymes namely isomerases and
methyltransferases Consequently cobalamin deficiency can cause disturbances in cell
division leading to neuropathy nervous system disease and pernicious anemia (Allen 2010)
Cobalamin has four bioactive forms and many analogues which different in their upper
andor lower functional groups For example the adenosyl can be replaced by a methyl
hydroxyl or cyano group to form methyl- hydroxo- or cyano-cobalamin respectively
Cyanocobalamin (CNCBL) is not found in nature but is used as a supplement for humans and
animals Cobalamin is not stable during food processing or storage as it is photo- and
heat-labile (Ahmad Hussain amp Fareedi 1992) Moreover the aerobic photodecomposition of
cobalamin processes more rapidly than the anaerobic photodecomposition (Demerre amp
Wilson 1956) meaning common food processing can enhance degradation All forms of
cobalamin are irreversibly inactivated under irradiation However some enzymes requiring
adenosylcobalamin (ADCBL) and methylcobalamin can also protect these compounds from
decomposition (Demerre amp Wilson 1956)
Whey protein typically consists of beta-lactoglobulin alpha-lactalbumin bovine serum
albumin and immunoglobulins The main protein (65) in bovine whey is beta-lactoglobulin
(18 kDa) a globular protein with 162 amino acid residues Beta-lactoglobulin belongs to the
lipocalin family and is able to bind small hydrophobic molecules in the internal cavity of its
β-barrel Because of multiple binding sites beta-lactoglobulin can bind a variety of ligands
including fatty acids polyphenols and vitamins such as cobalamin (Sawyer Brownlow
Polikarpov amp Wu 1998) Alpha-lactalbumin (14 kDa) the second most prevalent protein is a
small globular protein containing 123 amino acid residues with a large alpha-helical domain
and a small beta-sheet domain (Cawthern Narayan Chaudhuri Permyakov amp Berliner 1997)
The two domains are separated by a deep cleft and linked by a calcium binding loop It has
been reported that alpha-lactalbumin may function as a carrier of hydrophobic lipids vitamins
and metabolites
Ligand-binding proteins such as lactoglobulins and lactalbumins have been used to bind
low-molecular weight molecules for their protection and delivery (de Wolf amp Brett 2000)
The maximum cobalamin binding capacities of beta-lactoglobulin alpha-lactalbumin casein
blood serum album and proteose-peptone were determined to be 850 690 98 80 and 370
μgg respectively (Gizis Kim Brunner amp Schweigert 1965) Beta-lactoglobulin and
alpha-lactalbumin have also been reported to bind vitamin B12 and protect it from
4
decomposition (Gizis Kim Brunner amp Schweigert 1965 Johns et al 2015) Some
researchers have suggested that lactoferrin can dramatically increase the photostability and
solubility of cobalamin (US Pat 6500472B2 2002)
Due to limited absorption of cobalamin during digestion excess cobalamin can enter the
colon and modulate the gut microbiome Cobalamin is the only vitamin produced exclusively
by bacteria and archaea but it is used by all domains of life Synthesis of cobalamin is
energetically costly requiring nearly 30 different enzymes (Martens Barg Warren amp Jahn
2002) and only 20 of prokaryotes have the capacity to produce cobalamin (Degnan Barry
Mok Taga amp Goodman 2014) Cobalamin is a precious commodity and has been suggested
to be important not only as a nutrient but also as a signaling molecule for the spatial and
functional organization of gut microecology
In this study we examined whether beta-lactoglobulin or alpha-lactalbumin could
enhance the stability of cobalamin during food processing and positively affect the
composition of a model human gut microbiome The results obtained from this research
provide insights into possible applications of ligand-binding proteins as carriers of cobalamin
in the development of functional foods and pharmaceuticals
2 Method and Materials
21 Chemical reagents
Beta-lactoglobulin (purityge90) and alpha-lactalbumin (purityge85) were purchased
from Beingmate Company (Hangzhou China) and used without further purification ADCBL
and CNCBL were purchased from Sigma-Aldrich Chemical Company (St Louis US) and
used without further purification
22 Sample preparation
Stock solutions of beta-lactoglobulin alpha-lactalbumin ADCBL and CNCBL were
prepared at a final concentration of 10 μM in 1 mM phosphate buffer at pH 60
Cobalamin-whey protein complexes were prepared by mixing different concentrations of
protein (025 05 075 μM) with the ligand (025 μM) in phosphate buffer All samples were
prepared and incubated for approximately 1 h at room temperature in 200 mL flasks
(DURAN no 2121654 Schott GmbH Mainz Germany) while shaking (50 rpm) in the dark
23 Photodecomposition and heat treatment procedures
Briefly 200 mL flasks containing the cobalamin-whey protein complexes were placed
under a UV LED bulb (9 W) (Leiman Co Ltd Shenzhen China) with a light intensity of ca
100 μmol m-2 s-1 Each flask was placed 10 cm from the light source Samples were analyzed
5
every 15 min for up to 120 min Similarly the 200 mL flasks containing cobalamin-whey
protein complexes were placed into a water bath at 80 degC (Sonorex TK 52 ultrasonic water
bath Bandelin Electronics Berlin Germany) Samples were analyzed every 15 min for up to
120 min in the dark to avoid light degradation All experiments were performed in triplicate
24 In vitro stomach digestion
The stability of cobalamin in gastric fluid (SGF) was determined by subjecting samples
to simulated digestion in the presence of pepsin (pH 13 37 degC 2 h) SGF was prepared as
described previously (Tokle Lesmes Decker amp McClements 2012) with slight modifications
Briefly SGF was prepared by dissolving 1 (wv) pepsin 3 g of sodium chloride and 9 ml
of HCl in 1 L of water the pH was adjusted to 13 using 1 M HCl Cobalamin-whey protein
complexes (01 g) were incubated in 100 mL of SGF with shaking (100 strokesmin) in a
water bath at 37 degC for 2 h to simulate stomach conditions
25 Cobalamin determination
To extract cobalamin from 10 mL samples of B12-whey complexes 50 mL of sodium
acetate buffer (pH 60) was added in the presence of sodium cyanide (1) and the solution
was heated in a water bath (Type 1004 water bath GFL Gesellschaft fuumlr Labortechnik
Burgwedel Germany) for 40 min at 90 degC The pH was adjusted to 7 and 10 mL hexane was
added before the mixture was centrifuged for 15 min at 4010 g (Varifuge 30 Heraeus
centrifuge Heraeus Instruments Hanau Germany) The aqueous layer was collected and
passed through a solid phase extraction column (SPE) (CEC181M6 United Chemical
Technologies Bristol PA USA) which had been prewashed with 3 mL of methanol and 3
mL of Milli-Q water with the help of a pump (AL 15 Knf Neuberger Hamburg Germany)
to control the flow (1 drop per second) The column was washed three times with Milli-Q
water and 3 mL of methanol was used to elute cobalamin The solvent was evaporated to
dryness and the residue was subsequently dissolved in 1 mL of Milli-Q water before being
passed through a membrane filter (02 μm) (Macherey-Nagel Duumlren Germany) and analyzed
by HPLC using a RP-18 column (2504 mm ID 5 microm Merck Darmstadt Germany)
Cobalamin was detected using a modified HPLC method that was previously reported
(Gu Zhang Song Li amp Zhu 2015) All chromatographic separations were performed at
room temperature The mobile phases consisted of a mixture of methanol with 01 formic
acid (A) (Merck Darmstadt Germany) and ultra-purified water with 01 formic acid (B)
which was degassed by an ultrasonic water bath the flow rate was 05 mL per min The
gradient elution was programmed as follows 0-2 min 20 A 2-3 min 20-25 A 3-11 min
25-35 A 11-19 min 35-20 A 20-22 min 100-100 A 22-26 min 100-20 A and
6
26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored
by a Diode Array Detector (Waters US) at 361 nm
26 In vitro intestinal digestion
Colonic fermentation of the cobalamin-whey complex was conducted according to
Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child
who had not received antibiotic treatment in the previous three months were collected and
maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as
previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was
immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)
xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred
immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive
medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone
(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl
45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O
0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride
400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150
p-aminobenzoic acid 20 folate 10) and cysteine (08)
Six parallel reactors inoculated with immobilized gut microbiota were operated for 10
days The reactors were continuously fed nutritive media differing only in their
supplementation with cobalamin-whey complexes or cobalamin only The fermentation was
performed under typical conditions of the proximal colon according to previously described
procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix
2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium
was replaced every 12 h During the simulated fermentation the pH was maintained at 60
throughout the experiment with the addition of 2 M NaOH and the temperature was
maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2
into all reactors and medium vessels
The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of
fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05
μM) were added to the nutritive media for 10 days during fermentation All six reactors were
sampled daily and samples were frozen at -80 degC for pyrosequencing
27 DNA isolation PCR 16S rDNA analysis
DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01
Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
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solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
4
decomposition (Gizis Kim Brunner amp Schweigert 1965 Johns et al 2015) Some
researchers have suggested that lactoferrin can dramatically increase the photostability and
solubility of cobalamin (US Pat 6500472B2 2002)
Due to limited absorption of cobalamin during digestion excess cobalamin can enter the
colon and modulate the gut microbiome Cobalamin is the only vitamin produced exclusively
by bacteria and archaea but it is used by all domains of life Synthesis of cobalamin is
energetically costly requiring nearly 30 different enzymes (Martens Barg Warren amp Jahn
2002) and only 20 of prokaryotes have the capacity to produce cobalamin (Degnan Barry
Mok Taga amp Goodman 2014) Cobalamin is a precious commodity and has been suggested
to be important not only as a nutrient but also as a signaling molecule for the spatial and
functional organization of gut microecology
In this study we examined whether beta-lactoglobulin or alpha-lactalbumin could
enhance the stability of cobalamin during food processing and positively affect the
composition of a model human gut microbiome The results obtained from this research
provide insights into possible applications of ligand-binding proteins as carriers of cobalamin
in the development of functional foods and pharmaceuticals
2 Method and Materials
21 Chemical reagents
Beta-lactoglobulin (purityge90) and alpha-lactalbumin (purityge85) were purchased
from Beingmate Company (Hangzhou China) and used without further purification ADCBL
and CNCBL were purchased from Sigma-Aldrich Chemical Company (St Louis US) and
used without further purification
22 Sample preparation
Stock solutions of beta-lactoglobulin alpha-lactalbumin ADCBL and CNCBL were
prepared at a final concentration of 10 μM in 1 mM phosphate buffer at pH 60
Cobalamin-whey protein complexes were prepared by mixing different concentrations of
protein (025 05 075 μM) with the ligand (025 μM) in phosphate buffer All samples were
prepared and incubated for approximately 1 h at room temperature in 200 mL flasks
(DURAN no 2121654 Schott GmbH Mainz Germany) while shaking (50 rpm) in the dark
23 Photodecomposition and heat treatment procedures
Briefly 200 mL flasks containing the cobalamin-whey protein complexes were placed
under a UV LED bulb (9 W) (Leiman Co Ltd Shenzhen China) with a light intensity of ca
100 μmol m-2 s-1 Each flask was placed 10 cm from the light source Samples were analyzed
5
every 15 min for up to 120 min Similarly the 200 mL flasks containing cobalamin-whey
protein complexes were placed into a water bath at 80 degC (Sonorex TK 52 ultrasonic water
bath Bandelin Electronics Berlin Germany) Samples were analyzed every 15 min for up to
120 min in the dark to avoid light degradation All experiments were performed in triplicate
24 In vitro stomach digestion
The stability of cobalamin in gastric fluid (SGF) was determined by subjecting samples
to simulated digestion in the presence of pepsin (pH 13 37 degC 2 h) SGF was prepared as
described previously (Tokle Lesmes Decker amp McClements 2012) with slight modifications
Briefly SGF was prepared by dissolving 1 (wv) pepsin 3 g of sodium chloride and 9 ml
of HCl in 1 L of water the pH was adjusted to 13 using 1 M HCl Cobalamin-whey protein
complexes (01 g) were incubated in 100 mL of SGF with shaking (100 strokesmin) in a
water bath at 37 degC for 2 h to simulate stomach conditions
25 Cobalamin determination
To extract cobalamin from 10 mL samples of B12-whey complexes 50 mL of sodium
acetate buffer (pH 60) was added in the presence of sodium cyanide (1) and the solution
was heated in a water bath (Type 1004 water bath GFL Gesellschaft fuumlr Labortechnik
Burgwedel Germany) for 40 min at 90 degC The pH was adjusted to 7 and 10 mL hexane was
added before the mixture was centrifuged for 15 min at 4010 g (Varifuge 30 Heraeus
centrifuge Heraeus Instruments Hanau Germany) The aqueous layer was collected and
passed through a solid phase extraction column (SPE) (CEC181M6 United Chemical
Technologies Bristol PA USA) which had been prewashed with 3 mL of methanol and 3
mL of Milli-Q water with the help of a pump (AL 15 Knf Neuberger Hamburg Germany)
to control the flow (1 drop per second) The column was washed three times with Milli-Q
water and 3 mL of methanol was used to elute cobalamin The solvent was evaporated to
dryness and the residue was subsequently dissolved in 1 mL of Milli-Q water before being
passed through a membrane filter (02 μm) (Macherey-Nagel Duumlren Germany) and analyzed
by HPLC using a RP-18 column (2504 mm ID 5 microm Merck Darmstadt Germany)
Cobalamin was detected using a modified HPLC method that was previously reported
(Gu Zhang Song Li amp Zhu 2015) All chromatographic separations were performed at
room temperature The mobile phases consisted of a mixture of methanol with 01 formic
acid (A) (Merck Darmstadt Germany) and ultra-purified water with 01 formic acid (B)
which was degassed by an ultrasonic water bath the flow rate was 05 mL per min The
gradient elution was programmed as follows 0-2 min 20 A 2-3 min 20-25 A 3-11 min
25-35 A 11-19 min 35-20 A 20-22 min 100-100 A 22-26 min 100-20 A and
6
26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored
by a Diode Array Detector (Waters US) at 361 nm
26 In vitro intestinal digestion
Colonic fermentation of the cobalamin-whey complex was conducted according to
Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child
who had not received antibiotic treatment in the previous three months were collected and
maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as
previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was
immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)
xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred
immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive
medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone
(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl
45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O
0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride
400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150
p-aminobenzoic acid 20 folate 10) and cysteine (08)
Six parallel reactors inoculated with immobilized gut microbiota were operated for 10
days The reactors were continuously fed nutritive media differing only in their
supplementation with cobalamin-whey complexes or cobalamin only The fermentation was
performed under typical conditions of the proximal colon according to previously described
procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix
2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium
was replaced every 12 h During the simulated fermentation the pH was maintained at 60
throughout the experiment with the addition of 2 M NaOH and the temperature was
maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2
into all reactors and medium vessels
The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of
fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05
μM) were added to the nutritive media for 10 days during fermentation All six reactors were
sampled daily and samples were frozen at -80 degC for pyrosequencing
27 DNA isolation PCR 16S rDNA analysis
DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01
Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
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solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
5
every 15 min for up to 120 min Similarly the 200 mL flasks containing cobalamin-whey
protein complexes were placed into a water bath at 80 degC (Sonorex TK 52 ultrasonic water
bath Bandelin Electronics Berlin Germany) Samples were analyzed every 15 min for up to
120 min in the dark to avoid light degradation All experiments were performed in triplicate
24 In vitro stomach digestion
The stability of cobalamin in gastric fluid (SGF) was determined by subjecting samples
to simulated digestion in the presence of pepsin (pH 13 37 degC 2 h) SGF was prepared as
described previously (Tokle Lesmes Decker amp McClements 2012) with slight modifications
Briefly SGF was prepared by dissolving 1 (wv) pepsin 3 g of sodium chloride and 9 ml
of HCl in 1 L of water the pH was adjusted to 13 using 1 M HCl Cobalamin-whey protein
complexes (01 g) were incubated in 100 mL of SGF with shaking (100 strokesmin) in a
water bath at 37 degC for 2 h to simulate stomach conditions
25 Cobalamin determination
To extract cobalamin from 10 mL samples of B12-whey complexes 50 mL of sodium
acetate buffer (pH 60) was added in the presence of sodium cyanide (1) and the solution
was heated in a water bath (Type 1004 water bath GFL Gesellschaft fuumlr Labortechnik
Burgwedel Germany) for 40 min at 90 degC The pH was adjusted to 7 and 10 mL hexane was
added before the mixture was centrifuged for 15 min at 4010 g (Varifuge 30 Heraeus
centrifuge Heraeus Instruments Hanau Germany) The aqueous layer was collected and
passed through a solid phase extraction column (SPE) (CEC181M6 United Chemical
Technologies Bristol PA USA) which had been prewashed with 3 mL of methanol and 3
mL of Milli-Q water with the help of a pump (AL 15 Knf Neuberger Hamburg Germany)
to control the flow (1 drop per second) The column was washed three times with Milli-Q
water and 3 mL of methanol was used to elute cobalamin The solvent was evaporated to
dryness and the residue was subsequently dissolved in 1 mL of Milli-Q water before being
passed through a membrane filter (02 μm) (Macherey-Nagel Duumlren Germany) and analyzed
by HPLC using a RP-18 column (2504 mm ID 5 microm Merck Darmstadt Germany)
Cobalamin was detected using a modified HPLC method that was previously reported
(Gu Zhang Song Li amp Zhu 2015) All chromatographic separations were performed at
room temperature The mobile phases consisted of a mixture of methanol with 01 formic
acid (A) (Merck Darmstadt Germany) and ultra-purified water with 01 formic acid (B)
which was degassed by an ultrasonic water bath the flow rate was 05 mL per min The
gradient elution was programmed as follows 0-2 min 20 A 2-3 min 20-25 A 3-11 min
25-35 A 11-19 min 35-20 A 20-22 min 100-100 A 22-26 min 100-20 A and
6
26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored
by a Diode Array Detector (Waters US) at 361 nm
26 In vitro intestinal digestion
Colonic fermentation of the cobalamin-whey complex was conducted according to
Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child
who had not received antibiotic treatment in the previous three months were collected and
maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as
previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was
immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)
xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred
immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive
medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone
(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl
45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O
0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride
400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150
p-aminobenzoic acid 20 folate 10) and cysteine (08)
Six parallel reactors inoculated with immobilized gut microbiota were operated for 10
days The reactors were continuously fed nutritive media differing only in their
supplementation with cobalamin-whey complexes or cobalamin only The fermentation was
performed under typical conditions of the proximal colon according to previously described
procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix
2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium
was replaced every 12 h During the simulated fermentation the pH was maintained at 60
throughout the experiment with the addition of 2 M NaOH and the temperature was
maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2
into all reactors and medium vessels
The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of
fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05
μM) were added to the nutritive media for 10 days during fermentation All six reactors were
sampled daily and samples were frozen at -80 degC for pyrosequencing
27 DNA isolation PCR 16S rDNA analysis
DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01
Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
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solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
6
26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored
by a Diode Array Detector (Waters US) at 361 nm
26 In vitro intestinal digestion
Colonic fermentation of the cobalamin-whey complex was conducted according to
Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child
who had not received antibiotic treatment in the previous three months were collected and
maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as
previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was
immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)
xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred
immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive
medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone
(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl
45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O
0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride
400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150
p-aminobenzoic acid 20 folate 10) and cysteine (08)
Six parallel reactors inoculated with immobilized gut microbiota were operated for 10
days The reactors were continuously fed nutritive media differing only in their
supplementation with cobalamin-whey complexes or cobalamin only The fermentation was
performed under typical conditions of the proximal colon according to previously described
procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix
2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium
was replaced every 12 h During the simulated fermentation the pH was maintained at 60
throughout the experiment with the addition of 2 M NaOH and the temperature was
maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2
into all reactors and medium vessels
The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of
fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05
μM) were added to the nutritive media for 10 days during fermentation All six reactors were
sampled daily and samples were frozen at -80 degC for pyrosequencing
27 DNA isolation PCR 16S rDNA analysis
DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01
Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
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solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
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180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
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Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
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Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
7
recover DNA from trace amounts of sample has been shown to be effective for most bacteria
Blank samples consisted of unused swabs processed via DNA extraction protocols and tested
Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the
manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology
Co Ltd Hang Zhou Zhejiang Province China)
We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of
samples from in vitro colonic stimulation as a template and the primers 319F
5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo
All reactions were carried out in a 25 μL total volume containing approximately 25 ng of
genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water
to adjust the volume PCRs were performed on a Master cycler gradient thermocycler
(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC
for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC
for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for
10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the
DNA extraction process ultrapure water was used as a negative control to exclude
false-positive results PCR products were normalized using AxyPrep TM Mag PCR
Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the
quantification step regardless of the PCR volume submitted for sequencing The amplicon
pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics
Danvers MA USA) and the size and quantity of the amplicon library were assessed using
the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for
Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)
(Illumina) was combined with the amplicon library (expected at 30) and clustered to a
density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs
except for one library that was sequenced with both protocols using the standard Illumina
sequencing primers which eliminated the need for a third (or fourth) index read
Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial
Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was
used to select operational taxonomic units (OTUs) by making an OTU table Sequences were
assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU
and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal
Database Project) To estimate alpha diversity the OTU table was rarified and four metrics
were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique
OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
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solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
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Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
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Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
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1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
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Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
8
used to generate principal component analysis (PCA) The heatmap of important gut
microbiome families was constructed using Mev 4-9-0
28 Statistics
All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos
test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant
3 Results and Discussion
31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin
Whey proteins were associated with slow light-induced ligand decomposition In the
absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and
ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After
irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and
075 μM alpha-lactalbumin were extended significantly compared to those in the absence of
alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL
and ADCBL were lower than that of cobalamin alone The extent of decomposition in the
presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached
until 120 min after UV irradiation Compared with others CNCBL with 05 μM
alpha-lactalbumin had the greatest stability following irradiation In contrast to other
alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the
slowest decomposition rate
In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and
ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly
enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and
ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of
decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up
to 120 min after UV irradiation Compared with others CNCBL plus 05 μM
beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to
other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the
highest rapid decomposition rate
CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate
than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation
indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to
CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin
Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus
beta-lactoglobulin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
9
UV-light easily induces the decomposition of cobalamin in multiple steps starting with
cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on
the pH These intermediates decompose further to deoxyadensoine to form cobamides neither
of which are bioactive in humans (Schneider amp Stroinski 1987)
Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin
with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp
Schweigert 1965) the differences in the protective effects of the various proteins can be
attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp
Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures
of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin
as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the
carbonyl content was increased due to oxidation of tryptophan histidine and methionine in
all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin
were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior
of both molecules Thus whey proteins can offer protection to cobalamin against
photodecomposition However a strange phenomenon was observed alpha-lactalbumin
which contains one methionine residue provided better protection to ADCBL during the
irradiation than beta-lactoglobulin which contains four methionine residues This
phenomenon is most likely because exposed methionine residues and thiol groups of proteins
have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation
(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine
(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein
increases the decomposition rate of CNCBL also increases Additionally during irradiation
treatment higher concentrations of whey protein can lead to more substances with thiol
groups By way of an explanation a recent study stated that cysteine and other reducing
substances such as methional and dimethyl disulfide from methionine can rapidly destroy
CNCBL under irradiation (Mukherjee amp Sen 1957)
32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of
cobalamin
Thermal treatment is an essential element in food processing and the stability of
nutrients during thermal treatment is directly related to food quality Cobalamin is not only
photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was
reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced
significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared
with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
10
CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC
Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was
significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein
enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low
concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL
complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the
presence of whey proteins Similarly there were no differences in the CNCBL decomposition
rates in the first 45 min suggesting that even low concentrations of whey protein afford
sufficient protection Similar results were observed for ADCBL-whey protein complexes in
the first 30 min These results indicate that low concentrations of whey protein are an efficient
protective agent for cobalamin during food processing
In solution all forms of cobalamin are liable in the presence of vitamin C thiamine
nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40
of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al
1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting
from hydrolysis of propionamide side chains in the corrin ring to form deamidated
cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also
stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat
sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed
during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis
Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin
and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat
treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower
than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin
dimers and trimers via disulfide bond formation Meanwhile the thiol group in
beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers
of beta-lactoglobulin some of which are hydrophobically associated products are also
formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable
improvement of cobalamin is due to an increase in the number of exposed hydrophilic
binding sites and fewer unexposed thiol groups during heating unlike the changes caused by
irradiation treatment
Many studies demonstrated that more attention has been paid to the stability of
cobalamin under different storage conditions than to the light and thermal sensitivities of
cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate
vitamin B12 from substances by encapsulation lyophilization and addition of iron salts
EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
11
to its degradation However all of the aforementioned methods were associated with an
enhanced stability of cobalamin in pharmaceutics products all of which contain a high
amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that
lactoferrin provided adequate protection to cobalamin against light-induced decomposition
during food processing By contrast in this study the addition of beta-lactoglobulin or
alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low
concentrations of cobalamin due to light and heat treatments during food processing
33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro
stomach digestion
Due to the low pH ADCBL was reduced by approximately 25 after 120 min of
treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was
reduced by only 10 after 120 min treatment in IVSDS With supplementation of
alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were
significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the
decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS
almost 95 of CNCBL and ADCBL were retained with the protection provided by
beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL
(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin
following the same trend as that observed in heat treatment
The bioavailability of vitamin B12 in humans and animals is low as a substantial
amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte
Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be
easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4
vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz
Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of
dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in
the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some
studies have reported that cobalamin from milk is more efficiently absorbed than a single
supplementation Researchers also reported that the stability of cobalamin at pH 3 was
significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In
the in vitro stomach digestion model used in this study both beta-lactoglobulin and
alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment
Particularly with the protection conferred by whey protein the stability of ADCBL was
significantly improved in contrast to that of single ADCBL The addition of
beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
12
indicates that this method can certainly increase cobalamin bioactivity for food industrial
applications
34 Effects of cobalaminwhey protein complexes on microecology
Most microbiome studies on the human intestine have relied on data obtained from stool
samples (Clarke et al 2014) and some studies were unable to find significant influences of
prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case
because some influences of those substance may function in the upper portion of the intestinal
tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that
has escaped absorption can mostly influence the microbiome of the proximal colon and we
herein simulated the microbial community using an in vitro intestinal digestion model to
investigate the effects of cobalamin
After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a
mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity
analysis demonstrated a significant contrast among various supplementation combinations of
cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between
alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes
of the other combinations were similar However a gradual increase in the alpha diversities of
beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was
observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for
recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and
CNCBL due to an increase in the alpha diversity of the gut microbiome
The bacterial community composition was different between single and combination
supplementation of cobalamin These differences were shown by PCA plots which explained
79 of the total variation in the three primary principal axes and showed clear grouping
based on single or combination supplementation (Fig 5A) The microbial ecology with
different supplementation was diverse at the phylum level However the most abundant
populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a
majority of the communities in all samples (Fig 6) The combination of CNCBL and whey
protein significantly improved the relative abundance of Verrucomicrobia by 34 After
adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating
that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved
the growth of Firmicutes (Fig 4B)
Among various supplementations significant differences in heatmaps were obvious at
the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared
between all samples by the mean relative abundances to identify the bacterial composition
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
13
influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of
microbiota profiles represents eight cobalamin supplementation diets The samples taken from
whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were
distinguished from the others Differences in the stabilities of the microbiota profiles under
cobalamin and combination supplementation did not reach significance which was in contrast
to the variation in whey protein supplementation alone The CNCBL supplementation
stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese
Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig
5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased
Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae
Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover
alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae
Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae
Combined supplementation increased the proportions of Firmicutes and Bacteroidetes
and reduced the proportions of Proteobacteria which includes several genera of pathogens
such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that
Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic
Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al
2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region
leading to conformational changes in the secondary structure of mRNA which masks the
ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak
Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66
bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader
sequence of a corresponding mRNA that regulates translation initiation and gene expression
by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin
biosynthetic cob operon and the transporter btuB gene was repressed by the addition of
vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the
bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp
Goodman 2014) vitamin B12 and complexes could mediate the microecological structure
especially promoting Firmicutes abundance An increase in the ratios of
ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL
supplementation which was partially related to IBD These results also indicate that excess
CNCBL would likely lead to an inflammatory environment
The effect of a 10-day high-dose whey protein and cobalamin supplementation on the
microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts
have also been observed between carnivores and vegans who normally have deficient vitamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
14
B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during
cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)
Those changes included a reduction in potential pathogens and an increase in bacterial
diversity richness Thus far no systematic research on the effect of cobalamin on the
microbiome has been reported although many people exhibit excess vitamin B12 upon
supplementation Recently researchers have paid a substantial amount of attention on the
effect of cobalamin on the microbial community However most research has focused on the
inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)
Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in
human gut microbes to distinguish differences in their interactions with cobalamin Our
results demonstrate that supplementation of cobalamin and whey can enhance the proportions
of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria
4 Conclusion
Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and
beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during
food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the
thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20
Meanwhile the pH stability of the cobalamin-complex is of paramount importance for
bioavailability during stomach digestion Under the protection of whey proteins the stabilities
of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22
respectively compared with those of the others Following absorption of cobalamin in the
small intestine the residual cobalamin or complexes worked as a modular unit on the gut
microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the
ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum
Proteobacteria which led to a better and healthier colonic environment Despite the relatively
small sample size we observed a significant modulatory effect of vitamin B12 on the gut
microbiome Whey protein-ligand complexes with cobalamin significantly improved the
stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing
human nutrition
Acknowledgements
This work was supported by National Natural Science Foundation of China (No
31501452 and 81302781) the Scientific Research Foundation of Education Department of
Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the
most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
15
international communication for construction of the first ranked discipline of Zhejiang
Gongshang Univerisity (No 2017SICR106)
Conflict of interest statement
The authors declare that they have no conflict of interest
References
Ahmad I Hussain W Fareedi A A (1992) Photolysis of cyanocobalamin in aqueous
solution Journal of Pharmaceutical Analysis 10 9-15
Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and
Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and
cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)
1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short
communication Casein hydrolysate and whey proteins as excipients for
cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132
Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R
(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary
between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148
Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin
B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653
Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current
Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)
Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of
Biological Chemistry 272(49) 30812-30816
Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology
Ecology 57(2) 324336
Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole
PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated
dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920
Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides
thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic
Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of
milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55
10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application
in systems for controlled delivery and uptake of ligands Pharmacological Reviews
52(2) 207-236
Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in
the gut Cell Host and Microbe 15(1) 47-57
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
16
Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American
Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and
binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352
Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in
soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59
Heep I amp Odenthal H (2015) US Pat 9089582B2
Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal
12(4) 345-359
Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk
International Journal of Food Science and Technology 50 421-430
Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding
proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761
Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage
compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)
180-187
Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285
Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of
benzimidazole bases into cobamides Journal of Bacteriology 195(9) 1902-1911
Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and
Pharmacology 11(1) 26-31
Nahvi A Sudarsan N Ebert M S Zou X Brown K L amp Breaker R R (2002) Genetic control by a metabolite binding mRNA Chemistry and Biology 9(9)
1043-1049
Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of
Vitamin B 12 Gene Regulation by Cobalamin-Based Photoreceptors Annual Review of Biochemistry 86 485-514
Rajkumar H Mahmood N Kumar M Varikuti S R Challa H R amp Myakala S P
(2014) Effect of probiotic (VSL3) and omega-3 on lipid profile insulin sensitivity inflammatory markers and gut colonization in overweight adults A randomized
controlled trial Mediators of Inflammation 2014 348959-348960
Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc
Sawyer L Brownlow S Polikarpov I amp Wu S Y (1998) β-lactoglobulin Structural
studies biological clues International Dairy Journal 8(2) 65-72
Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter
Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber
coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions Food and Function 3(1) 58-66
Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2
Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural
element RNA 9 1084-1097
Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of
Microwave Heating on the Loss of Vitamin B(12) in Foods Journal of Agricultural and Food Chemistry 46(1) 206-210
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
17
Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by
Riboswitches Annual Review of Microbiology 59 487-517
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
18
Figure legends
Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the
presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The
triangle block diamond and round markers represent ADCBL (025 μM) in the presence of
075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and
round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM
beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and
round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG
and alone respectively
Fig 2 Stability of cobalamin after heat treatment in the presence or absence of
alpha-lactalbumin or beta-lactoglobulin
A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence
of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and
gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and
alone respectively C) The black white dot pattern and gray bars represent CNCBL (025
μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black
white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05
025 μM beta-LG and alone respectively
Fig 3 Stability of cobalamin during stomach digestion in the presence of
alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone
(gray bar)
A) ADCBL B) CNCBL
Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota
from an in vitro intestinal simulator
A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c
d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison
of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each
letter indicates that a significant difference (plt005) exists
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
19
Fig 5 Data analysis of microbiota from an in vitro intestinal simulator
A) Principal component analysis plots of microbiota from in vitro intestinal simulations by
complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial
abundance from in vitro intestinal simulations Each row represents family-like phylogenetic
groups of bacteria whose mean abundance differed significantly between complexes
Hierarchical clustering of the bacterial fingerprints and various supplementations collected
from samples of in vitro intestinal simulations
Fig 6 Stacked bar plots showing the average percentage of bacterial populations from
an in vitro intestinal simulator in the presence of cobalamin and complexes
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
20
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
21
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
22
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
23
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
24
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin
25
26
Highlights
Whey protein enhanced thermalphoto stability of cobalamin during food processing and
storage
Whey protein improved the stability and bioavailability of cobalamin during digestion
Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin