Blueberry husks, rye bran and multi-strain probiotics affect the severity of colitis induced by dextran sulphate sodium

ORIGINAL ARTICLE

Blueberry husks, rye bran and multi-strain probiotics affect theseverity of colitis induced by dextran sulphate sodium

ASA HAKANSSON1, CAMILLA BRANNING2, DIYA ADAWI3, GORAN MOLIN1,

MARGARETA NYMAN2, BENGT JEPPSSON3 & SIV AHRNE1

1Food Hygiene, 2Division of Applied Nutrition and Food Chemistry, Lund University, Lund, Sweden, and 3Department of

Surgery, Lund University, Malmo University Hospital, Malmo, Sweden

AbstractObjective. The enteric microbiota is a pivotal factor in the development of intestinal inflammation in humans butprobiotics, dietary fibres and phytochemicals can have anti-inflammatory effects. The aim of this study was to evaluate thetherapeutic effect of multi-strain probiotics and two conceivable prebiotics in an experimental colitis model. Material andmethods. Sprague-Dawley rats were fed a fibre-free diet alone or in combination with Lactobacillus crispatus DSM 16743,L. gasseri DSM 16737 and Bifidobacterium infantis DSM 15158 and/or rye bran and blueberry husks. Colitis was induced by5% dextran sulphate sodium (DSS) given by oro-gastric tube. Colitis severity, inflammatory markers, gut-load of lactobacilliand Enterobacteriaceae, bacterial translocation and formation of carboxylic acids (CAs) were analysed. Results. The diseaseactivity index (DAI) was lower in all treatment groups. Viable counts of Enterobacteriaceae were reduced and correlatedpositively with colitis severity, while DAI was negatively correlated with several CAs, e.g. butyric acid. The addition ofprobiotics to blueberry husks lowered the level of caecal acetic acid and increased that of propionic acid, while rye bran incombination with probiotics increased caecal CA levels and decreased distal colonic levels. Blueberry husks with probioticsreduced the incidence of bacterial translocation to the liver, colonic levels of myeloperoxidase, malondialdehyde and seruminterleukin-12. Acetic and butyric acids in colonic content correlated negatively to malondialdehyde. Conclusions. Acombination of probiotics and blueberry husks or rye bran enhanced the anti-inflammatory effects compared with probioticsor dietary fibres alone. These combinations can be used as a preventive or therapeutic approach to dietary amelioration ofintestinal inflammation.

Key Words: Blueberry husks, dietary fibre, experimental colitis, multi-strain probiotics, rye bran

Introduction

Inflammatory bowel disease (IBD) is believed to be a

result of an abnormal gastrointestinal (GI) immune

response leading to inflammation, triggered by one

or more environmental risk factors in genetically

predisposed individuals. The enteric microbiota is

implicated increasingly as a pivotal factor in the

development of intestinal inflammation in humans

and experimental animals [1,2], but which bacterial

characteristics drive the inflammatory reaction re-

mains to be elucidated. Bacteria inhabiting the

interface between the lumen and the mucosa may

play a role in the inflammatory process, since

dysfunctions in innate immunity are related to

intestinal inflammation [2,3].

A hallmark of IBD is the infiltration of large

numbers of phagocytic leucocytes into the mucosal

interstitium [4] and a majority of the cells in the active

lesions of ulcerative colitis (UC) are activated neu-

trophils and macrophages [5,6], causing production

and release of reactive oxygen species (ROS). This is

associated with an increased lipid peroxidation ob-

served in the mucosa of UC and in experimental

colitis models [7,8]. Overproduction of ROS has

been demonstrated in colonic tissue from

IBD patients, suggesting that colonic inflammation

Correspondence: Siv Ahrne, PhD, Division of Applied Nutrition and Food Chemistry, Chemical Center, P.O. Box 124, SE 221 00 Lund, Sweden. Tel: �46 46

2228 327. Fax: �46 46 2224 532. E-mail: [email protected]

Scandinavian Journal of Gastroenterology, 2009; 44: 1213�1225

(Received 25 March 2009; accepted 8 July 2009)

ISSN 0036-5521 print/ISSN 1502-7708 online # 2009 Informa UK Ltd.

DOI: 10.1080/00365520903171268

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produces high levels of oxidants that probably exceed

the low antioxidant capacity in IBD patients [9]. By

secretion of pro-inflammatory products, polymor-

phonuclear leucocytes can injure epithelial cells [10]

and transepithelial migration can affect the epithelial

permeability. This leads to deterioration of the

epithelial barrier function and exposure of the under-

lying tissue and bloodstream to harmful luminal

components, e.g. bacteria and endotoxins [11]. As a

consequence, direct stimulation of epithelial cytokine

production can occur [12]. Myeloperoxidase (MPO),

a constituent of the primary granules of neutrophils,

has been widely used as a marker for the presence and

activation of neutrophil granulocytes [13].

Alterations of the composition of the GI micro-

biota may alter host immunity and the course of

inflammation [14]. Administration of probiotics

and/or fermentable dietary fibres, i.e. potential

prebiotics, could be strategies to affect the balance

of the microbiota in a positive direction. Certain

strains of Bifidobacterium or Lactobacillus can be

given orally as probiotics and may alleviate intestinal

inflammatory response and halt the vicious circle of

inflammation [15]. The human diet consists of a

large variety of fruits, vegetables and cereals that

contain dietary fibres and phytochemicals, with the

potential to reach the colon and be metabolized by

the microbiota [16,17]. Carbohydrates are the most

important substrate for the colonic microbiota and

are degraded mainly to carboxylic acids (CAs).

Among the CAs, butyrate most effectively protects

the intestinal mucosa against injury and promotes

mucosal healing [18]. Blueberry and rye bran are

especially rich sources of dietary fibres and phyto-

chemicals. Diet can also affect the composition of

the GI microbiota [19]. Specific intestinal bacteria

can metabolize phenolics to varying degrees, produ-

cing a variety of aromatic metabolites which can

either be retained by the bacterial cell or released in

the lumen [20]. Antimicrobial effects of polyphe-

nols, as well as antioxidant activities, have been

demonstrated [21].

In view of the experimental limitations imposed by

studies in humans, animal models are appealing, and

in a model reproducing some of the UC character-

istics, dextran sulphate sodium (DSS) is used to

induce colitis. The DSS model has become a

frequently applied research tool for the pathogenesis

of UC and the development of therapeutic agents

[22]. In the present study the DSS model was

applied in rat, the aim being to clarify the therapeutic

effect of a multi-strain probiotics alone or together

with blueberry husks and rye bran (two conceivable

prebiotics). Colitis severity, inflammatory markers,

the gut-load of lactobacilli and Enterobaceriaceae,

bacterial translocation and formation of CAs along

the hindgut have all been investigated. The idea

behind the treatment regimens was that both pro-

biotics and dietary fibres can exercise positive effects

on the intestinal environment and mitigate inflam-

mation, and that they can possibly complement each

other.

Material and methods

Animals

Female Sprague-Dawley rats (n�36), initial weight

of 21392 g, were purchased from Mollegard (Viby,

Denmark). The animals were housed individually at

a constant room temperature and humidity in a 12 h

light/dark cycle and acclimatized for 1 week before

commencement of the trial. They were given free

access to water, while the feed intake was restricted

to 23 g (dwb, dry weight basis) per day. Manipula-

tion and experimental procedures followed general

guidelines in the care and use of laboratory animals,

and the protocol was approved by the Animal Ethics

Committee at Lund University.

Blueberry husks, rye bran and probiotics

Husks from wild-growing, low-bush, blueberries of

Vaccinium myrtillus L were derived from pressed

berries and freeze dried (Probi AB, Lund, Sweden),

and the bran derived from commercial Swedish rye

was supplied by Lantmannen (Jarna, Sweden).

A mixture of three bacterial strains representing

two Lactobacillus species and one Bificobacterium

species was used: The strains L. crispatus DSM

16743 and L. gasseri DSM 16737 (Probi AB)

originate from the healthy vaginas of fertile women.

Bifidobacterium infantis DSM 15158 has been iso-

lated from the stool of a healthy, breast-fed infant

(Probi AB).

The bacterial preparations of lactobacilli were

made from fresh cultures in LCM (Lactobacillus-

carrying medium) [23]. The cultures were incubated

anaerobically at 378C for 48 h, harvested by cen-

trifugation and resuspended in bacterial suspension

medium (sodium chloride, 8.5 g/l; bacteriological

peptone (Oxoid, Unipath Ltd., Basingstoke, Hamp-

shire, England), 1 g/l; Tween 80, 1 g/l; L-cysteine

hydrochloride monohydrate (Merck, Darmstadt,

Germany) 0.2 g/l), before being given to the rats.

The frozen bacterial culture of B. infantis (Probi

AB) was allowed to thaw, the cells were harvested by

centrifugation and resuspended in bacterial suspen-

sion medium. The amounts of bacteria in the multi-

strain probiotics given daily to the rats were 2�1010

CFU (colony-forming units) for B. infantis DSM

15158 and 5�1010�1�1011 CFU for L. gasseri

DSM 16737 and L. crispatus DSM 16743.

1214 A. Hakansson et al.

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Treatment groups and study design

Five test diets and one control diet were prepared as

described in Table I. The rats were randomly

allocated to one of the six treatment groups, each

group comprising 6 animals: colitis control (C

group), probiotics (P group), rye bran (R group),

rye bran and probiotics (RP group), blueberry husks

(B group) and blueberry husks and probiotics (BP

group).

Rye bran and blueberry husks were included at a

level of 80 g dietary fibre/kg in the diets (dwb). The

dry matter content of the diets was adjusted with

wheat starch. The wheat starch has been shown to be

completely digested and hence does not contribute

to any CA formation [24]. The content of dietary

fibre in rye bran was 48.0 g/100 g (dwb), while the

content of dietary fibre in blueberry husks was

40.8 g/100 g (dwb). Of the dietary fibre in rye

bran, 5.1 g/100 g (dwb) could be considered as

Klason lignin, i.e. components not soluble in 12 M

H2SO4, whereas the amount of Klason lignin in

blueberry husks was 14.1 g/100 g (dwb). The non-

starch polysaccharides in rye bran mainly consisted

of xylose (39%), glucose (30%) and arabinose

(21%), and in blueberry husks the non-starch

polysaccharides mostly consisted of glucose (39%),

uronic acids (25%) and xylose (20%).

Probiotics were mixed daily with the feed at

feeding time. The rats were allowed to adapt to the

diets for 7 days after which a 7-day experimental

period followed. Daily food intake was measured for

all animals. Colitis was induced by 5% (w/w) DSS

(MW�36,000�50,000; ICN Biomedicals Inc., Aur-

ora, Ohio, USA) dissolved in water and given once a

day by oro-gastric tube. The mean starting weight of

the rats was 22393 g. On day 7, post-induction of

colitis, the animals were anaesthetized by subcuta-

neous injection of a mixture (1:1:2) of Hypnorm

(Division of Janssen-Cilag Ltd., Janssen Pharmaceu-

tica, Beerse, Belgium), Dormicum (F. Hoffmann-La

Roche AG, Basel, Switzerland) and water at a dose

of 0.15 ml/100 g. Under an aseptic technique, a

laparotomy was performed through a midline inci-

sion, arterial blood was withdrawn for cytokine

assay, samples from the caudate lobe of the liver

were taken for bacteriological analysis and the

caecum and colon were dissected. The luminal

content was gently removed, the tissues were rinsed

with isotonic saline and samples were collected for

bacteriological analysis, as well as for CAs (caecal,

proximal and distal colon; stored at �408C) and

assay activity of MPO and malondialdehyd (MDA).

pH was measured in the caecal content before

storage at �408C. Samples for bacteriological eva-

luation were placed in sterile tubes containing

freezing media, and all samples were immediately

frozen at �708C for later determination.

Dietary fibres and carboxylic acids

A gravimetrically method by Asp et al. [25] was used

to determine soluble and insoluble dietary fibre in

the raw materials. The composition of the insoluble

and soluble fibre residues was analysed by gas-liquid

chromatography (GLC) for the neutral sugars as

their alditol acetates and spectrophotometrically for

the uronic acids [26].

The amount of CAs (acetic, propionic, isobutyric,

butyric, isovaleric, valeric, caproic, heptanoic,

succinic and lactic acid) was analysed by a GLC

method [27]. The caecal and colonic contents were

Table I. Composition of the test diets g/kg dry feed.

Component C group P group R group RP group B group BP group

Rye bran1 0 0 166.7% 166.7% 0 0

Blueberry husks2 0 0 0 0 196.1% 196.1%Casein 120 120 120 120 120 120

DL-methionine 1.2 1.2 1.2 1.2 1.2 1.2

Maize oil 50 50 50 50 50 50

Mineral mixture3 48 48 48 48 48 48

Vitamin mixture4 8 8 8 8 8 8

Choline chloride 2 2 2 2 2 2

Sucrose 100 100 100 100 100 100

Wheat starch5 670.8 670.8 504.1 504.1 474.7 474.7

Abbreviations: C group�control; P group�supplement of probiotics; R�supplement of rye bran; RP�supplement of rye bran and

probiotics; B�supplement of blueberry husks; BP�supplement of blueberry husks and probiotics.

%Corresponding to 80 g dietary fibres/kg diet (dw); 1rye bran (Lantmannen); 2blueberry husks (Probi AB); 3containing (g kg�1):

0.37 CuSO4 �5H2O, 1.4 ZnSO4 �7H2O, 332.1 KH2PO4, 171.8 NaH2PO4 �2H2O, 324.4 CaCO3, 0.068 KI, 57.2 MgSO4, 7.7 FeSO4 �7H2O,

3.4 MnSO4 �H2O, 0.02 CoCl �6H2O, 101.7 NaCl (Apoteket, Malmo, Sweden); 4containing (g kg�1): 0.62 menadione, 2.5 thiamin

hydrochloride, 2.5 riboflavin, 1.25 pyridoxine hydrochloride, 6.25 calcium pantothenate, 6.25 nicotinic acid, 0.25 folic acid, 12.5 inositol,

1.25 p-aminobenzoic acid, 0.05 biotin, 0.00375 cyanocobalamin, 0.187 retinol palmitate, 0.00613 calciferol, 25 d-a-tocopheryl acetate,

941.25 maize starch (Apoteket); 5wheat starch (Cerestar, Krefeld, Germany).

Effects of synbiotics on experimental colitis 1215

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homogenized with an Ultra Turrax† T25 basic

(IKA†-Werke, Staufen, Germany) after adding in-

ternal standard (2-ethylbutyric acid; Sigma Chemi-

cal Company, St Louis, Mo., USA).

After complete derivatisation, the samples were

injected onto an HP-5 column (Hewlett Packard,

GLC, HP 6890, Wilmington, Del., USA) and Chem

Station software (Hewlett Packard) was used for the

analysis.

Assessment of the severity of colitis

The severity of colitis was assessed daily from day 0

to day 7 using a clinical Disease Activity Index (DAI)

that has been validated [28] and shown to correlate

histologically with pathological findings [29]. DAI

values ranging from 0 to 4 were calculated using:

stool consistency (normal, loose, diarrhoea), pre-

sence or absence of faecal blood (test slides with

Hemoccult II (SmithKline Diagnostic, USA) and

macroscopic evaluation of the anus) and weight loss.

The arithmetical mean of the three scores deter-

mines the DAI value. Weight changes were based on

the starting weight of each rat at initiation of

treatment. Weight-loss scores were determined as

0�no weight loss; 1��0�4.99% weight loss; 2�5.00�9.99% weight loss; 3�10.00�19.99% weight

loss; 4��20.00% weight loss. Stool scores were

determined as 0�normal stool, well-formed pellets;

2�loose stool, pasty stool that does not stick to the

anus; 4�diarrhoea, liquid stool that sticks to the

anus. Bleeding scores were determined as 0�no

bleeding; 2�positive Hemoccult test, 4�gross

bleeding.

Myeloperoxidase and malondialdehyde activity

MPO was estimated in the whole colonic tissue [30],

which contains mucosa and muscle layers. Colonic

tissues were collected, weighed prior to storage at

�708C until time of assay [31].

Colonic content of malondialdehyde (MDA) was

determined as an index of lipid peroxidation, using

MDA 586 (Oxis International Inc., Portland, Oreg.,

USA), a colorimetric assay designed to quantify

MDA [32]. Whole colonic tissues were collected,

rinsed in ice cold Dulbecco’s phosphate buffered

saline (PBS), weighed and then frozen immediately

at �708C for later evaluation.

Interleukin-12 (Rt IL-12)

Serum IL-12 production was assayed by solid-phase

sandwich ELISA. The commercially available IL-12

ELISA kit was purchased from BioSource Interna-

tional, Inc., USA, and the assay recognized both

natural and recombinant Rt IL-12 (p70), as well as

the free p40 subunit. The analysis of cytokine

protein expression was done according to the pro-

cedures described in the protocol and all samples

were analysed in duplicate.

Viable count

Liver samples (0.36190.14 g) of the caudate lobe

were placed in room temperature for 5 days to

stimulate multiplication of possible existing bacteria

translocated from the intestinal tract. Before cultur-

ing, the liver samples were placed in an ultrasonic

bath for 5 min and swirled for 2 min on Chiltern

(Therma-Glas, Gothenburg, Sweden). Viable

counts were obtained from violet-red bile glucose

(VRBG) agar (Oxoid), incubated aerobically at 378Cfor 24 h (Enterobacteriaceae count), from Rogosa agar

(Oxoid), incubated anaerobically (Gas Pack System,

Gas Pack; Becton-Dickinson Microbiology Systems,

Cockeynsville, Md., USA) at 378C for 72 h (lacto-

bacilli count), and from brain heart infusion (BHI)

agar (Difco, Detroit, Mich., USA), incubated both

aerobically and anaerobically, as described above,

at 378C for 72 h (aerobic and anaerobic count,

respectively).

Caecal tissues were placed in an ultrasonic bath

for 5 min and swirled for 1 min on Chiltern. Viable

counts were obtained from Rogosa agar (Oxoid) that

was incubated anaerobically, as described above, at

378C for 72 h (lactobacilli count) and from VRBG

(Oxoid) incubated aerobically at 378C for 24 h

(Enterobacteriaceae count). The number of colonies

formed on each plate was counted and corrected for

the weight of the original tissue.

Randomly amplified polymorphic DNA (RAPD)

Quantitatively predominant colonies were randomly

picked, and re-cultured on Rogosa agar. Crude

cell extracts were made from pure colonies, and

one microlitre was used in the polymerase chain

reaction (PCR) [33]. Agarose gel electrophoresis

was run; the gels were stained with ethidium

bromide and photographed under ultraviolet (UV)

illumination.

16S rRNA gene sequencing

Amplification of the 16S rRNA genes was carried

out using the universal primers ENV1 (5?-AGA

GTT TGA TII TGG CTC AG-3?, Escherichia coli

numbering 8 to 27) and ENV2 (5?-CGG ITA CCT

TGT TAC GAC TT-3?, E. coli numbering 1511 to

1492) [34]. The PCR mixture contained 0.2 mM of

each primer, 2 ml template DNA, 5 ml 10�PCR

buffer (100 mM Tris-HCL, 500 mM KCl, pH 8.3),

200 mM of each deoxyribonucleotide triphosphate,


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2.5 mM MgCl2 and 2.5 U of Taq DNA polymerase

(Roche Diagnostics, Mannheim, Germany) in a final

volume of 50 ml. PCR was performed in a PCR

Mastercycle 5333 (Eppendorf) with the following

profile: 1 cycle at 948C for 3 min, followed by

30 cycles of 968C for 15 s, 508C for 30 s, and 728Cfor 90 s, with an additional extension at 728C for

10 min. The amplification products were checked by

running the products on 1.5% (w/v) agarose gel

(Type III, High EEO; Sigma) after ethidium bro-

mide staining and were visualized under UV light.

Amplicons were single-strand sequenced by MWG

(Biotech, Ebersberg, Germany) and the 16S rDNA

sequences (mostly around 500 bp) generated from

the mucosal samples were subjected to BLAST

search against GenBank [35] for approximate phy-

logenetic affiliation and aligned to 16S rDNA

encoding sequences of selected Lactobacillus strains

(type strains) retrieved from the Ribosomal Data

Base (RDP-II) [36].

Statistics

DAI scores, MPO activity, lipid peroxidation, Rt

IL-12 and lactobacilli and Enterobacteriaceae counts

(Figures 1�6 and Table IV) were presented as

medians with 25 and 75 percentiles. The statistics

were conducted in SigmaStat† version 3.0 (SPSS

Inc., Chicago, Ill., USA). Differences between

all groups were evaluated by Kruskal-Wallis

test one-way ANOVA on ranks followed by all

pairwise multiple comparison procedures (Student-

Newman-Keuls method), if appropriate. When com-

paring only two groups, the Mann-Whitney rank

sum test was used. The correlation between expecta-

tions of benefit was ascertained using Pearson’s

correlation coefficient. Calculation of the incidence

of translocation was conducted in QuickStat version

2.6 and was evaluated by the Fisher exact test.

Caecal pools of CAs were calculated as the

concentrations of each acid (mmol/g) multiplied by

the caecal amount. The values were extrapolated to a

complete intake of dietary fibre during the experi-

mental period. The proportion of individual CAs

was calculated as a percentage of total CAs for each

rat before statistical evaluation. Feed intake, body-

weight change, caecal content, caecal tissue weight,

caecal pH and CAs (Tables II and III) were

presented as means9SEM (standard error of

the mean). Data from rats given diets containing

probiotics were compared with the control or the

corresponding fibre diets. Rats given rye bran or

blueberry husks, without any probiotics, were com-

pared with those given the control diet. For statis-

tical evaluation of the differences between samples,

two-way ANOVA was used to determine the effects

of dietary fibre (Fibre), probiotics (Pro) and their

interactions (Fibre�Pro). The probiotic and the

dietary fibre effects were then evaluated by using

one-way ANOVA followed by Tukey’s procedure.

The Minitab statistical software (Release 14) was

used to make these evaluations. P-values of less than

0.05 were considered significant.

Results

Colitis severity

The products were well tolerated by all animals, with

no adverse side effects during the period of intake.

There was no mortality among the experimental

groups.

DAI score increased with each treatment day and

differences between groups reached statistical sig-

nificance on day 3 of the DSS regimen ( pB0.05)

(Figure 1). By days 6 and 7, the DAI score was

significantly lower in all treatment groups compared

with the colitis control group (C group) ( pB0.01).

On the seventh day, rats in the C group had

diarrhoea, weight loss, gross rectal bleeding, erosion

and/or superficial ulcer of the colonic mucosa in the

distal part.

The rats consumed approximately half of the feed

provided during the experimental period (72�105 g)

(Table II) and the body-weights were corrected for

the feed intake. Compared with the C group, the

body-weight change was positive in all treatment

groups ( pB0.05), with the exception of the R

group.

The caecal tissue weights were significantly higher

in the C group than in the R and B groups ( p�0.01

and p�0.03, respectively; Table II).

Carboxyolic acids in the hindgut

The pH of the caecal content was significantly

lower in groups R and B compared with the C group

( pB0.001; Table II).

The caecal pools and levels of CAs were sig-

nificantly higher in the B group compared with the

C group ( p�0.002 and p�0.03, respectively),

and also for the pools compared with the R group

( p�0.013; Table III). The higher caecal amount of

CAs was due to acetic acid and butyric acid. The R

group exhibited significantly higher CA levels than

the C group in the distal part of colon ( p�0.04;

Table III).

In general, the proportion of acetic acid was

significantly higher throughout the hindgut in the

B group than in the R group ( p�0.026). In

the caecum, the proportion of butyric acid was

significantly higher in the R group than in the C

group, while the proportion of propionic acid and


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minor acids was significantly lower in the B group

compared with the C group ( p�0.02 and p�0.005,

respectively). In the distal part of colon, the propor-

tion of lactic acid was significantly lower in the R

group and B group compared with the C group ( p�0.027 and p�0.026, respectively; Table III).

Concerning the effects of probiotics, the caecal

pool of CAs was significantly higher in the RP group

than in the R group ( p�0.002), whereas the level in

the distal part of colon was lower ( pB0.05; Table

III). The caecal pool of CAs was significantly lower

in the P group than in the C group ( pB0.05),

Figure 1. Effects of 5% dextran sulphate sodium (DSS) on the time-course of changes in the disease activity index (DAI) over the 7-day

experimental period. DAI scores are expressed as medians (25 and 75 percentiles). The asterisk (*) denotes pB0.05 and asterisks (**)

denote pB0.01 compared with colitis control. Day 3: *Control (C) versus treatment with rye bran (R), blueberry husks (B) or blueberry

husks and bacteria mixture (BP). Day 4: *Control versus bacteria mixture (P), **control versus rye bran, rye bran and bacteria mixture

(RP), blueberry or blueberry husks and bacteria mixture. Days 5 and 6: The same as day 4. Day 7: **Control versus all treatment groups.

Figure 2. Effects of dextran sulphate sodium on colon mucosal

myeloperoxidase activity. Data are expressed as medians (25 and

75 percentiles). The asterisk (*) denotes pB0.05 compared with

all groups; the hash (#) denotes pB0.01 between the rye bran

group (R) and the rye bran and bacteria mixture group (RP).

Figure 3. Effects on colon mucosal lipid peroxidation by induc-

tion of colitis by dextran sulphate sodium. Data are expressed as

medians (25 and 75 percentiles). The asterisk (*) denotes pB0.05

and asterisks (**) denote pB0.01 compared with the colitis

control group (C).


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whereas the level in the distal part of colon was

higher, resulting in a significantly increased propor-

tion of butyric acid ( pB0.05; Table III).

Inflammatory markers

As assessed by elevated MPO activity in the colonic

tissue, administration of DSS for 7 days caused

significantly lower levels of neutrophil infiltration

in the BP group compared with all other groups

( pB0.05), while MPO in the RP group was lower

than that in the R group ( pB0.05; Figure 2).

Compared with the C group, MDA levels were

significantly lower in all treatment groups ( pB0.01),

with the exception of the P group (Figure 3).

Serum IL-12 was significantly lower in the B

group, and the BP group, as compared with the C

group ( pB0.05; Figure 4).

Translocation to liver

With the exception of the R group, the incidence of

translocation to the caudate lobe of the liver,

measured as incidence of positive plate counts,

decreased significantly in all treatment groups com-

pared with the C group ( pB0.01; Table IV).

Lactobacilli and Enterobacteriaceae counts in caecal

tissue

The viable count of lactobacilli was significantly

higher in the R group, compared with the C group

( pB0.05; Figure 5). When comparing the groups

receiving the probiotic mixture, the viable count was

significantly higher in the RP group than in the P

group and the BP group, and the count of the BP

group was significantly higher than the B group

( pB0.05; Figure 5). The caecal counts of Enter-

obacteriaceae significantly decreased in all treatment

groups compared with the C group ( pB0.05; Figure

6). A significant decrease was also found between the

R group and RP group, and between the R group

and B group. The Enterobacteriaceae count of the BP

Figure 4. Interleukin-12 concentrations in serum are expressed as

medians (25 and 75 percentiles). Asterisks (*) denote pB0.05

compared with the colitis control group (C) and with the groups

treated with bacteria mixture (P), rye bran (R), and rye bran and

bacteria mixture in combination (RP).

Figure 5. Total lactobacilli counts are expressed as medians (25

and 75 percentiles). The hash (#) denotes pB0.05 between the

rye bran group (R) and the control (C) and blueberry husk group

(B), and also the rye bran and bacteria group (RP) compared with

the bacteria group (P) and the blueberry husks and bacteria group

(BP). The asterisk (*) denotes pB0.05 between the blueberry

husk group and the blueberry husks and bacteria group.

Figure 6. Total Enterobacteriaceae counts are expressed as medians

(25 and 75 percentiles). The asterisk (*) denotes pB0.05 between

the control group (C) and all the other test groups. The hash

(#) denotes pB0.05 when comparing the rye bran group (R)

versus rye bran and bacteria group (RP), the rye bran

group versus blueberry husks group (B) and the blueberry husks

group versus blueberry husks and bacteria mixture group (BP).


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Table II. Body-weight change, caecal content and caecal pH in rats fed a fibre-free diet (C group) supplemented with probiotics (P group),

rye bran (R group), rye bran and probiotics (RP group), blueberry husks (B group) or blueberry husks and probiotics (BP group)1,2.

p-value

C group P group R group RP group B group BP group Fibre Pro Fibre�Pro

Feed intake, g/d 10.391.1a 12.991.0 13.491.1a,b 13.090.9 15.090.4b 13.590.5* 0.016 NS NS

Body-weight

change, g/g

feed3

�0.2890.15a 0.0390.05* �0.0090.04a 0.1390.03* 0.1190.02b 0.0490.02 0.003 0.014 0.005

Caecal content, g 1.690.5 1.190.3 1.690.2 2.190.4 2.690.3 2.290.4 0.028 NS NS

Caecal tissue

weight, g

1.490.1a 1.290.1 1.090.1b 1.290.1 1.090.1b 1.190.1 0.018 NS NS

Caecal pH 7.290.1a 7.390.1 6.690.1b 6.590.2 6.590.1b 6.590.1 B0.001 NS NS

1Values are expressed as means9SEM, n�6. Asterisks indicate statistically significant difference from rats fed the same diet but without

bacteria: *pB0.05, **pB0.01, ***pB0.001; 2between control, rye bran and blueberry husks, i.e. those without any added probiotics,

without a common letter differ, pB0.05; 3body-weight change during the experimental period divided by the total feed intake during the

experimental period.

Table III. Carboxylic acids in the hindgut of rats fed a fibre-free diet (C group) supplemented with probiotics (P group), rye bran (R group),

rye bran and probiotics (RP group), blueberry husks (B group) or blueberry husks and probiotics (BP group)1,2.

p-value

C group P group R group RP group B group BP group Fibre Pro Fibre�Pro

Caecum3

Acetic 5193a 5892 6091b 5991 6892c 6192* B0.001 NS 0.004

Propionic 1491a 1291 1191a 1290 1091b 1391* NS NS 0.026

Butyric 791a 991 1191b 1292 1091a,b 1191 0.031 NS NS

Lactic 1094 992 891 691 591 591 NS NS NS

Succinic 694 290 391 391 391 391 NS NS NS

Minor4 1291a 1091 992a 891 591b 790 B0.001 NS NS

Total

mmol/g 4498a 5993 6896a,b 7994 7597b 8898 B0.001 0.015 NS

mmol 10299a 59910* 135931a 344936** 223924b 299971 B0.001 NS B0.001

Proximal5

Acetic 3695a 5392** 5394a 5592 6693b 6392 B0.001 B0.05 B0.01

Propionic 591 991* 691 892 692 891 NS B0.05 NS

Butyric 391 791** 692 791 591 791 NS B0.05 NS

Lactic 3494a 1693** 2197a 1692 1191b 1092 B0.001 B0.01 NS

Succinic 591 891** 894 591 391 591 NS NS NS

Minor4 1794 891* 891 991 991 491* B0.01 B0.05 NS

Total

mmol/g 7492a 5194 5796b 6696 4294b 4694 B0.001 NS B0.01

Distal6

Acetic 4795a 5092 5693a,b 5193 6292b 5993 0.002 NS NS

Propionic 991 891 1091 991 791 791 NS NS NS

Butyric 591 890* 891 991 792 691 NS NS NS

Lactic 1895a 1692 992b 1292 891b 1093 0.004 NS NS

Succinic 893 692 1294 1192 491 793 NS NS NS

Minor4 1393a 1294 391b 991** 1291a 593* 0.027 NS 0.043

Total

mmol/g 6296a 102911** 98913b 5895* 6596a,b 7897 NS NS B0.001

1Values are expressed as the means of levels (mmol/g) or pools (mmol/caecum) and standard errors or percentages of the total CA, n�6.

Asterisks indicate statistically significant difference from rats fed diets without probiotics: *pB0.05, **pB0.01, ***pB0.001; 2values of

control, rye bran and blueberry husks, i.e. those without any added probiotics, without a common letter are statistically different, pB0.05;3rats fed the fibre-free diet, rye bran diet, blueberry husks diet or the blueberry husks and probiotics diet; n�5; 4formic, isobutyric,

isovaleric, valeric, caproic and heptanoic acid; 5rats fed the rye bran diet; n�3; 6rats fed the rye bran and probiotics diet; n�5.


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group was significantly higher than that of the B

group ( pB0.05).

Identification of caecal lactobacilli

A total of 108 isolates from caecum tissue, picked

from countable Rogosa agar plates, and representing

18 isolates per group, were subjected to RAPD

typing, and the RAPD types dominating each group,

based on recurrent band pattern, were identified to

species level. L. reuteri represented the majority of

isolates from the C group, and in 4 out of 6 groups

L. reuteri and L. animalis constitute a dominant part

of the lactobacilli flora of the caecum tissue (group

C, 72% L. reuteri and 22% L. animalis; group R,

67% L. reuteri and 17% L. animalis; group RP, 67%

L. reuteri and 22% L. animalis; group BP, 69%

L. reuteri and 23% L. animalis). The composition of

the lactobacilli flora was different in group P where

28% of the isolates were L. gasseri (67% L. reuteri), and

in group B it was dominated by L. animalis (91%).

Correlations

A linear relationship was observed between the DAI

at day 7 and Enterobacteriaceae counts (r�0.54;

pB0.001). There was also a positive correlation

between the DAI score and MPO activity (r�0.39;

pB0.05) and between body-weight change during

the last 7 days and the total level of CAs in the

caecum (r�0.61; pB0.001). The DAI at day 7

correlated negatively to the total caecal concentra-

tion of CAs (r��0.55; p�0.001), acetic acid (r��0.63; pB0.001) and butyric acid (r��0.54; p�0.001). A negative correlation was found between

the MDA and the acetic acid (r��0.40, p�0.02)

and the butyric acid (r��0.41, p�0.01) concen-

trations in the distal part of the colon.

Discussion

In the present study, we have shown that probiotics,

rye bran and blueberry husks together or alone were

able to attenuate the inflammatory response of DSS.

During the course of the experiment, treated rats

had an attenuated inflammatory response, evidenced

by lower DAI values from day 3 to 7. The original

scoring of DAI by Cooper et al. [29] was used, but

the limits of body-weight change have been more

precisely defined in the present study. With the

modified scoring limits the judgement between

scores 1 and 2, and 2 and 3, may be objectively

facilitated.

Body-weight loss is clinically relevant and is

included in the DAI. Patients with IBD are exposed

to nutritional risk since malnutrition and loss of

body-weight are predominant clinical features [37],

and except for poor nutritional intake and malab-

sorption, the inflammatory reactions affect the

catabolism [38]. By supplementation of probiotics,

rye bran or blueberry husks, it was feasible to

prevent the body-weight loss of the animals during

progression of colitis.

Viable count of the caecal tissue verified an

increase in lactobacilli in the R, RP and BP groups.

Lactobacilli are regarded as beneficial and a decrease

in numbers has been found in the mucosa of patients

with UC [39]. It has been suggested that the

differential physiology of the intestinal mucosa

caused by inflammation may influence the composi-

tion of lactobacilli [40]. In contrast, all treatments

decreased the Enterobacteriaceae counts as compared

with the C group, although the B group showed the

strongest reduction. Blueberry husks have previously

been shown to have an antimicrobial effect, sug-

gested to be mediated by the polyphenols [41], and a

positive correlation was observed in the present

study between the Enterobacteriaceae count and

DAI. The Gram-negative, sometimes pathogenic or

opportunistically pathogenic Enterobacteriaceae are

increasingly linked with IBD [42] and experimental

colitis [43]. This family includes species such as

E. coli and Klebsiella pneumonia, but all members of

the Enterobacteriaceae family have lipopolysaccharides

(LPS) associated with the cell wall. E. coli strains

associated with IBD and with colitis in dogs [44]

share the ability to replicate in cultured epithelial cells

and macrophages and belong to a group of adherent

and invasive E. coli. Cats with signs of intestinal

inflammation had an increased mucosal Enterobacter-

iaceae load [45]. It has been shown that a subset

of bacteria comprising E. coli, other species of

Enterobacteriaceae and Clostridium correlated with

abnormalities in mucosal architecture, up-regulation

Table IV. Incidence of translocation to the caudate lobe of the

liver.

VRBG BHI anaerobic BHI aerobic Rogosa

C group 5/6 5/6 5/6 2/6

P group 0/6** 0/6** 0/6** 0/6**

R group 2/6 2/6 2/6 2/6

RP group 0/6** 0/6** 0/6** 0/6**

B group 0/6** 0/6** 0/6** 0/6**

BP group 0/6** 0/6** 0/6** 0/6**

Abbreviations: C group�control; P group�supplement of pro-

biotics; R�supplement of rye bran; RP�supplement of rye bran

and probiotics; B�supplement of blueberry husks; BP�supple-

ment of blueberry husks and probiotics; VRBG�violet-red bile

glucose; BHI�brain heart infusion.

**Denotes pB0.01 compared with the control group.


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of pro-inflammatory cytokines (IL-12, IL-8 and IL-

1) and the clinical disease activity [45].

In the present study, IL-12 in plasma was mea-

sured as an inflammatory mediator with systemic

effects, and a decrease in IL-12 was found in the

groups given blueberry husks in the diet. The

antimicrobial effects from polyphenols in blueberry

may suppress pro-inflammatory components of the

intestinal microbiota, and hence reduce the stimula-

tion of macrophages, resulting in a subsequent less-

pronounced induction of IL-12. The role of different

cytokines in the hierarchy of inflammatory mediators

orchestrating the inflammatory process in IBD is not

fully elucidated. In contrast, the importance of IL-12

in active IBD has been shown experimentally by

demonstrating that the IL-12 cytokine profile in

colonic tissue is up-regulated due to DSS treatment

[46] and that antibodies to IL-12 can abrogate

experimental colitis [47]. Further, clinical data

show that gene transcription of IL-12 is up-regulated

in active UC [48]. No significant effects of probiotics

on the IL-12 levels could be recorded in the present

study, even if Lactobacillus, in a strain-specific

manner, can modulate a host immune response by

altering the profiles of cytokines released in the gut

by epithelial and immune cells [49], and Lactobacillus

has been shown to modify and potentiate the

macrophage activity and response to stimulants

[50]. A proposed mechanism of DSS action includes

increased macrophage activation [51]. It is impor-

tant to point out that as well as the possible effects of

the Lactobacillus strains in the probiotic mixture, the

resident Lactobacillus flora might exercise effects.

L. murinus and L. reuteri usually comprise a con-

siderable part of the intestinal Lactobacillus popula-

tion of rats [52,53]. With the exception of two of the

treatment groups, this is consistent with results from

the present study. However, in the P group, the

dominating species were L. gasseri and L. reuteri, and

L. animalis was the major component of the dom-

inating lactobacilli of the B group. Bacteria with

tannase activity from faeces of mice have been

identified as L. animalis or L. murinus (i.e. L.

animalis is relatively closely related to L. murinus)

[54]. This might suggest that the fibre-free diet did

not provide optimal survival and persistence condi-

tions for L. animalis, while blueberry husks improved

growth performance and competing ability.

In the present study bacterial translocation to the

caudate lobe of the liver was significantly inhibited in

all groups receiving probiotics and also blueberry

husks alone. It has previously been shown that

Gram-negative bacteria can be found in portal blood

and liver biopsies from patients with UC undergoing

colectomy [55], and circulating and agglutinating

antibodies against various enteric bacteria have been

identified in active UC [56]. The indigenous flora

and an intact mucosa are vital components of body

defences against luminal pathogenic bacteria, and a

part of the therapeutic strategy for UC aims at

restoring the intestinal integrity and function, as

increased intestinal permeability might facilitate the

translocation of bacteria from the gut and give rise to

potential infection and inflammation [57].

A positive correlation between DAI values and

MPO was found in the present study and the

mucosal activity of MPO was significantly decreased

in the BP group. Up-regulation of MPO has been

described in DSS-induced colitis [58] and DAI has

been shown to be correlated with MPO [29]. MPO

is a potent pro-oxidative enzyme, and is one of the

chief mediators of neutrophil-dependent tissue da-

mage [59]. There is a direct relationship between

MPO activity and the number of neutrophils in

tissue [60]. A positive correlation between the

amounts of macrophages and neutrophils in colonic

tissue of patients suffering from UC has been found,

suggesting an active role of macrophages in neutro-

phil recruitment in UC [5].

MDA, which is an indicator of the degree of lipid

peroxidation induced by reactive oxygen species [61],

was decreased in the colonic tissue of groups given

blueberry husks or rye bran. Oxidants produced by

activated neutrophils are involved in the initiation and

perpetuation of the inflammatory process by increas-

ing the number of neutrophils and macrophages that

induce a self-sustaining loop [62].

Lipid peroxidation is mediated by oxygen free

radicals and is believed to be an important factor for

the damage of cell membranes [63]. The protective

effect of the dietary supplements may be due to the

scavenging effects of the polyphenols, in rye bran

and blueberry husks, to the oxidative damage caused

by ROS at the induction of colitis. Serum MDA

levels increase in patients with UC, which suggests

that lipid peroxidation could have an important role

in the pathogenesis of UC [64].

In the present study, the distal colonic concentra-

tion of acetic and butyric acids correlated negatively

to MDA in colonic tissue, and rye bran gave

comparatively higher levels of CAs in the distal

colon than the control diet. Since UC appears

distally, dietary fibres that release their fermentation

products in this part of the colon may offer an

advantage. A slow fermentation of rye has been

obtained by others [65] and may be caused by

arabinoxylan, the main fibre component in rye. On

the one hand, in the present study the caecal levels of

CAs increased, whereas the level in the distal colon

decreased with the addition of probiotics to the rye

bran diet. On the other hand, the damage induced

by DSS has been reported to affect the distal colon


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and caecum preferentially [66], with less damage

evident in the proximal colon [66] and in the ileum

[67]. Therefore, an increased production in the

caecum would be favourable in this model. With

this in mind, it is interesting to observe that the RP

group yielded the highest amounts of butyric acid.

The B group had a higher proportion of acetic

acid in the caecum compared with the C group or

the R group, which can be explained by the

comparatively high content of uronic acids in blue-

berry husks. Interestingly, the proportion of acetic

acid decreased, whereas that of propionic acid

increased when probiotics were added. This may

be a consequence of a change in the composition of

the microbiota, in this case indicated by the higher

count of lactobacilli in the caecum in the groups

supplemented with probiotics. The phenomenon

can possibly be of some clinical value since propionic

acid is decreased in patients with IBD [68], and both

propionate and butyrate have been shown to de-

crease cytokine release from human neutrophils to a

higher extent than acetate [69]. Indeed, a negative

correlation was found between DAI and several

specific CAs, including butyric acid. This is in line

with the observation that severe colitis is character-

ized by low CA levels and impaired utilization of

butyric acid [70,71].

Several studies have proposed that dietary manip-

ulations can be beneficial for the management of

intestinal inflammatory conditions like UC. This is

interesting in view of the fact that most pharmaceu-

ticals currently available, although effective, are not

devoid of potentially serious side effects, limiting

their chronic use [72]. The present study demon-

strates that administration of especially blueberry

husks supplemented with a mixture of probiotic

strains attenuates DSS-induced colonic injury and

inflammation in rats.

Acknowledgements

This study was funded by the Functional Food

Science Centre (FFSC) at Lund University. We

thank Lantmannen (Jarna, Sweden) and Probi AB

(Lund, Sweden) for kindly supplying the rye bran,

the freeze-dried blueberry husks and probiotics.

None of the authors has any conflicts of interest to

declare, but Siv Ahrne, Goran Molin and Bengt

Jeppsson are minority stockholders in Probi AB. All

of the co-writers, took part in the design of the study,

the evaluation of the results and the writing of the

manuscript. Asa Hakansson and Camilla Branning

contributed equally to the work, performed on the

animal experiments and the experimental studies.

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Documents

Blueberry husks, rye bran and multi-strain probiotics affect the severity of colitis induced by dextran sulphate sodium