Production of prebiotics from hemicellulose - LTH X 4 X 5 Before growth ... 2P-B 8 25 60 0.30 3P-A 11 26 48 0.30 3P-B 8 21 58 0390.39 Falck et al (2014) ... Eva Nordberg Karlsson Funding

Production of prebiotics from hemicellulosehemicellulosePATRICK ADLERCREUTZ, DIV. OF BIOTECHNOLOGY

Gut microbiota

• Gut microbiota – microorganisms in our gastrointestinal tract

• The gut microbiota has a large influence on our health and well-being

• How can we influence our gut microbiota?

Probiotics and prebiotics

• Probiotics – beneficial microorganisms in the gut microbiota

• Prebiotics – substrates promoting beneficial microorganisms

Arabinoxylan, AXOS and XOS

• Arabinoxylan (AX), and oligosaccharides derived from AX (AXOS and XOS) are prebiotics

• XOS and AXOS have positive effects on glucose metabolism, lipid metabolism and metabolic , pdisorders

• The effects depend on the molecular size and• The effects depend on the molecular size and structure

B k t t l (2011) C it R F d S i N t 51 178Broekaert et al. (2011) Crit. Rev. Food Sci. Nutr. 51, 178

Selective enzymatic hydrolysis ofSelective enzymatic hydrolysis of arabinoxylan

24)- β-D-Xylp-(1 4)-β-D-Xylp-(1 4)-β-D-Xylp-(1 4)-β-D-Xylp-(1

3

α-L-araf1

α-L-araf1

ff

α-L-arabinofuranosidaseα-L-arabinofuranosidase

endo- β-1,4-xylanase

Xylanase hydrolysis of rye flour arabinoxylanXylanase hydrolysis of rye flour arabinoxylan

450

X2

X3

X

AXOS

HPAEC-PAD300

6 h

X

X4

X5

HPAEC PAD

100

2002 hnC

100

0 h

-100 2 4 6 8 10 12 14 16 18 20 22 25

10

Min

Falck et al (2013) ( )J. Agric. Food Chem. 61, 7333

Processing of cereal residues

Products

Lund UniversityANTIDIABETIC FOOD CENTREANTIDIABETIC FOOD CENTRE

A Centre of Excellence in Research and Innovation

Duration 2007 2017 Duration 2007-2017 Budget 34 000 000 USD Approx. 50 senior researchers from 4 faculties Approx. 50 senior researchers from 4 faculties Mission: Preventing type 2 diabetes with food.

Effects on mice

HFD Oat0 Oat1 Rye0 Rye1 Rye2 Guar LFD

Glucose (mM) 6.5 ± 0.3 6.1 ± 0.2 6.1 ± 0.3 7.2 ± 0.2 6.6 ± 0.3 6.0 ± 0.2 6.0 ± 0.3 5.4 ± 0.3

Fructosamine (mM) 505 ± 17 550 ± 9 515 ± 25 470 ± 16 523 ± 25 435 ± 18 497 ± 40 535 ± 14

ALT (U/L) 4.4 ± 0.4 4.3 ± 0.4 5.8 ± 0.9 3.1±0.4 3.4 ± 0.5 1.4 ± 0.2 2.3 ± 0.4 2.9 ± 0.2

K Berger, P Falck, C Linninge, U Nilsson, U Axling, C Grey, H Stålbrand, E Nordberg Karlsson, M Nyman, C Holm, P Adlercreutz (2014) J. Agric. Food Chem. 62, 8169

PCA plot The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

Body weightLiver weight

ALT

b ill

Barley0

1

Insulin

Fructosamine

LactobacillusC. leptum group

Acetic acidValeric acid HFD

Oat0 Oat1

orr)

[2]

0.5

Fat (%)

Glucose

HDLAkkermansia muciniphila

Propionic acid

Iso-butyric acid Butyric acid

Iso-valeric acid

Valeric acidTot SCFA

Barley1

Rye0

Rye1Guar

p(co

rr)[2

],t(

co

0

Cholesterol

LDL

Bifidobacterium

Rye2-0.5

-1

Berger et al (2014) J. Agric. Food Chem. 62,

-1.5-1.5 -1 -0.5 0 0.5 1

p(corr)[1], t(corr)[1]

Agric. Food Chem. 62, 8169

Oat products stimulate Lactobacilli

Berger et al (2014) J. Agric. Food Chem. 62, 8169

Rye and guar products stimulate BifidobacteriaRye and guar products stimulate Bifidobacteria

Berger et al (2014) J. Agric. Food Chem. 62, 8169

Why are bifidobacteria stimulated by the oligosaccharide-rich product?

Bifidobacterium adolescentis consumes AXOS from rye flourAXOS from rye flour

80.0

60.0

70.0

X3HPAEC-PAD

40.0

50.0

nC

X2

AXOS

HPAEC PAD

20.0

30.0 X4 X5 Before growth

4.4 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0-5.0

10.0

After growth

Min

Falck et al (2013) J. Agric. Food Chem. 61, 7333

Growth of Weissella strains* on hydrolysed birchwood xylan *isolated from Indian food (by 16S rDNA either W. cibaria or W. confusa)

HPAEC-PAD

a: Standards

c-f: After growthXOS

b: Before growth

XOS consuming strains!

Patel et al (2013) FEMS Mi bi l L tt 346 20FEMS Microbiol. Lett. 346, 20

GH43 β-xylosidase from Weissella sp. strain 92

P Falck, J Linares-Pastén, P Adlercreutz, E Nordberg Karlsson (2015) Glycobiology

GH43 β-xylosidase from Weissella sp. strain 92


Hydrolysis of XOS by Weissella β-xylosidase

1000

600

800

200

400X2X3

0 5 10 15 20 250

200 X3X4

mM


Kinetics of hydrolysis by Weissella β-xylosidase

Substrate kcat (s-1) KM (mM) kcat/KM(s-1mM-1)

Nit h l β D l id 258 ± 11 7 4 ± 1 1 34 9 ± 5 4p-Nitrophenyl-β-D-xylopyranoside 258 ± 11 7.4 ± 1.1 34.9 ± 5.4(14)-β-D-xylobiose (X2) 961 ± 25 7.2 ± 0.5 134 ± 10(14)-β-D-xylotriose (X3) 900 ± 13 6.5 ± 0.3 138 ± 7(14) β D xylotriose (X3) 900 ± 13 6.5 ± 0.3 138 ± 7(14)-β-D-xylotetraose (X4) 770 ± 7 17 ±0.3 54.3 ± 0.9


Weissella β-xylosidase. Activity on arabinose substrates

• Low activity on p-nitrophenyl-α-L-arabinofuranoside• No activity on AXOSNo activity on AXOS• The bacteria do not grow on AXOS


Further development of (A)XOS production processes

Process steps.pFrom rye bran to prebiotic products

• Heat pretreatment• Starch degradation (amylase, amyloglucosidase)• Protein degradation (protease)Protein degradation (protease)• Separation steps to remove small molecules

(ethanol precipitation)(ethanol precipitation)• Xylan hydrolysis (xylanase)

Process schemesProcess schemes

F l k t l (2014) BiFalck et al (2014) BioresourceTechnol. 174, 118

ProductsProducts

M AXMass AX

Yield, % (w/w) Yield, % (w/w)

Content, % (w/w) A/X

Products based on supernatants isolated before heat pretreatmentp p2S-A 22 12 11 0.622S-B 20 10 11 0.473S-A 23 10 9 0.633S B 23 11 10 0 523S-B 23 11 10 0.52Products based on supernatants isolated after heat pretreatment1-A 17 33 41 0.381-B 13 33 53 0.342P-A 11 23 45 0.382P-B 8 25 60 0.303P-A 11 26 48 0.303P-B 8 21 58 0 393P B 8 21 58 0.39

Falck et al (2014) Bioresource Technol. 174, 118Falck et al (2014) Bioresource Technol. 174, 118

Product composition

Falck et al (2014) Bioresource Technol. 174, 118

Process steps.pFrom rye bran to prebiotic products

• Heat pretreatment• Starch degradation (amylase, amyloglucosidase)• Protein degradation (protease)Protein degradation (protease)• Separation steps to remove small molecules

(ethanol precipitation)(ethanol precipitation)• Xylan hydrolysis (xylanase)

Hydrolysis by R marinus xylanase (GH10)Hydrolysis by R. marinus xylanase (GH10)

HPAEC-PAD

After removal of singleAra substituents

Aft l f d blAfter removal of doubleAra substituents

Original sample


Hydrolysis by Pentopan Mono BG (GH11)Hydrolysis by Pentopan Mono BG (GH11)

HPAEC-PAD

After removal of singleAra substituents

After removal of doubleAra substituents

Original sample


Time course of xylanase catalysed hydrolysisTime course of xylanase catalysed hydrolysis


Conclusions

• Xylanases are useful for production of prebioticoligosaccharides from arabinoxylan

• The products have positive health effects in mice on a high fat diet

• Different xylanases produce different AXOS• Selective stimulation of beneficial gut bacteria (such as g (

Bifidobacteria) is possible• Weissella strains proven to use XOS. Putative probiotics.

AcknowledgementsAcknowledgements

Div. of BiotechnologyPeter FalckPatrick AdlercreutzEva Nordberg Karlsson

Funding

Antidiabetic Food CentreEva Nordberg KarlssonCarl GreyJavier Linares-PasténAnna Aronsson

Antidiabetic Food Centreat Lund University

Div. of BiochemistryHenrik Stålbrand

C ll b t ithi AFC

The Swedish Research Council (VR)

Collaborators within AFCKarin BergerCecilia HolmCaroline Linninge

Vinnovag

Ulf NilssonUlrika AxlingMargareta Nyman

Documents

Production of prebiotics from hemicellulose - LTH X 4 X 5 Before growth ... 2P-B 8 25 60 0.30 3P-A 11 26 48 0.30 3P-B 8 21 58 0390.39 Falck et al (2014) ... Eva Nordberg Karlsson Funding