Lecturer in Food Metabonomics Department of Food Biosciences Tuohy - Presentation.pdf · Lecturer...

Speaker 16

Dr Kieran Tuohy

Lecturer in Food MetabonomicsDepartment of Food Biosciences

University of Reading

k.m.tuohy@reading.ac.uk

• Introduction

• The human gut microflora

• Functional foods – PREBIOTICS

• Prebiotic discovery and efficacy in vitro and in vivo

• Case study – galactooligosaccharides from lactose;pectic-oligosaccharides from citrus fruit waste

• Concluding remarks

Outline

• Re-design food production process to reduce waste─ efficacy, impact on product, cost

• Stabilise, reduce, recover value (material or energy)─ economic relevance, technological issues

• Fractionate to added value products─ identify suitable technologies, high value products, lucrative markets─FUNCTIONAL FOODS

Approaches to food waste

• In our bodies there are 20 x more bacteria than human cells

• Human genome – 30,000 genes• Bacteria – 1,600 genes• More than 1,000 bacterial species within the gut• Redundancy but also genetic, evolutionary and

ecological plasticity• Microbial mega-genome with metabolic potential

many fold greater than human

The Human Gut Microflora

Gut Physiology

Diet: infant and adultGut

Microflora

Microbial Interactions: Successional Development

Homeostasis

Immune System

1. Must be non-digestible

2. Selective fermentation in the colon

3. Must improve health

PREBIOTICS‘Non digestible food ingredients that selectively stimulate a limited number of bacteria in the colon, to improve host health’

(after Gibson and Roberfroid, 1995)

Health benefits of prebiotics

Microflora

modification

Laxation Lipid metabolism

Diarrhoea/IBS

Colon cancer

Blood glucose

Mineral absorption

IBD

Immune func in children

PREBIOTICS

Prebiotic Health Benefits

Modified from Crittenden 2006

prebiotics

stimulategrowth

colonocytes

Bifidobacteria

Pathogens

faecal bulking improved bowel habit

colonisationresistance

block adhesion

reduce exogenousand endogenousintestinal infection

eliminate pro-IBDantigens

dendritic cell

M cell

Th1

Tr

Th2IFNg

IL-10TGF-b

immunomodulationanti-inflammatory

suppress IBD inflammation

allergy prevention

lamina propria

Selective proliferation of beneficial bacteria

Prebiotic Health BenefitsStimulation of beneficial microbial activities

SCFA

peristalsis

prebiotics

selectivefermentation

improvedbowel habit

reduced cancer risk and IBDinflammation

colonocytes

increasedmineralabsorption

antagonism ofpathogens andputrefactive bacteria

fewer toxicbacterialmetabolites

reduced pH

Ca++ Mg++

reduced cancer risk

trophic and anti -neoplasticeffects

induce

de novolipogenesis

controlled serum lipids and cholesterol

Modified from Crittenden 2006

• Retail prices of functional foods are typically 30 to 500 times higher than comparable conventional foods

• Depending on the definition of functional foods, estimated market size is between $30 and $60 billion (Japan, US, EU)

• Over the past ten years the market has grown at about 10% per annum (World Bank)

• Prebiotics European market alone is currently worth £59 million and is expected to increase to £121 million in 2010 (Frost & Sullivan)

• Leading prebiotics – fructans (inulin, oligofructose, galactooligosaccharides, lactulose)

Functional Foods market

Determining prebotic potential in vitro and in vivo

Caecum

Ascendingcolon

Transverse colon

Descendingcolon

Rectum

• In vitro gut models (batch, complex)

• Faecal microbiology (16S rRNA)

• Faecal metabolites (GC, NMR)

• Biomarkers of disease ─ blood lipids─ faecal water genotoxicity─ immunology

• Metabolite profiles – blood, urine, faecesTuohy et al., Curr.Pharma Design (2005)

31 Volunteers fed Prebiotic Biscuits for 21 days

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

Log 1

0 cel

ls/g

faec

es.

Total bacteria

Bacteroides spp.

Bifidobacterium spp.

Clostridium spp.

Lactobacillus spp.

Active biscuits ( ) Placebo biscuits ( )Tuohy et al., Brit. J. Nutr. (2001)

Receptor activity

Enhanced persistence

Novel structural diversity

Microbial polysaccharides

Algal polysaccharides

BacterialEPS

Monosaccharidesoligosaccharides

Con

trol

led

hydr

olys

isPh

age

hydr

olys

is

Equi

libriu

m

synt

hesi

s

Species specificity Oligosaccharides

Gly

cosy

l tr

ansf

er

Raw material Target prebiotic property

Raw material

Manufacturing technologies

Plant polysaccharides

HYDROLYSIS SYNTHESIS

Synthesis

Enzyme reactorUltrafiltration

Retentate recycle

Permeate

Lactose

GOS

Drying

Sucrose

IMO

β-Galactosidase

Dextransucrase+ dextranase

Alternansucrase

Acceptors

Prebiotics from lactose

Enzyme reactor

Lactose

GOS

Dryingβ-galactosidase

• Lactose in whey is a major problem for the dairy industry• Cost associated with disposal• Can convert to high value prebiotics

Manufactured by Friesland Foods (EU)Clasado (UK) Yakult (Japan)

Tzortzis et al., Appl. Microbiol. Biotech. (2005)

Galacto-oligosaccharidesGOS

Oligosaccharide % (dry weight)Disaccharides 33Trisaccharides 39Tetrasaccharides 18Pentasaccharides 7

Higher oligosaccharides 30 4 8 12 16 20 24 28 32 36 40

Time (min)

Galb1,4Glc

Galb1,6Galb1,4GlcGalb1,3Galb1,4GlcGalb1,4Galb1,4Glc

Galb1,3Galb1,6Galb1,4GlcGalb1,6Galb1,6Galb1,4GlcGalb1,3Galb1,3Galb1,4GlcGalb1,6Galb1,3Galb1,4GlcGalb1,3Galb1,4Galb1,4Glc Galb1,6Galb1,4Galb1,4Glc

+

b-Galactosidase Galb1,4Glc

Glc

+ …

-0.8-0.6-0.4-0.2

00.20.40.60.8

1

V1 V2 V3

Total bacteria Bifidobacterium spp.

Lactobacillus spp. Bacteroides spp.

Clostridium histolyticum group

Novel GOS

Bi2muno® GOS/B. bifidum3-stage gut model

counting by FISH with group-specific 16S rDNA probes

D Log 10bacteria

ml-1

Tzortzis et al., J. Nutr. (2005)

-0.2

-0.1

0

0.1

0.2

0.3

0.4

3.6g B2M/d 7g B2M/d 7g VGOS/d 7.6g Sucrose/d

Log1

0 (c

ell/g

)

Total Bacteria Bifidobacterium spp.

Lactobacillus spp Bacteroides spp

Clostridium histolyticum group

Novel GOS in vivo

Bi2muno® GOS/B. bifidumHuman volunteer trial, 2-weeks feeding

counting by FISH with group-specific 16S rDNA probes

Enzymereactor

U/F

Retentate recycle

Permeate

50g dextran dm-3

100120140160180200220240260

20 25 30 35 40 45 50 55 60Elution Time (min)

Ref

ract

ive

Inde

x (m

V) 19.5 kDa9.5 kDa

1.15 kDa0.18 kDa6250 U enzyme dm-3, 60 min

residence time19800 U enzyme dm-3, 90 min residence time62500 U enzyme dm-3, 60 min residence time

Hydrolysis

Pectin composition

Pectic oligosaccharides

Dead-endUF cell10 000

NMWCO

High methoxy citrus pectinLow methoxy apple pectin

Pectic oligosaccharides

Amano endo-pectinase

0 10 20 30 40 50 60Time (min)

PGA Hydrolysate

POS1(HM citrus pectin)

POS2(LM apple pectin)

46 8 10

High performance anion exchange chromatography

66.5

77.5

88.5

99.510

TotalBifid

obacter

iaBac

teroides

Clostridia

Lactobac

illiEubac

teria

051024

TotalBifid

obacter

iaBac

teroides

Clostridia

Lactobac

illiEubac

teria

0

5

10

24

Log

10ce

lls/g

Pectic oligosaccharides

Batch culture, 1% CHO, pH 6.8, bacteriology by FISH

FOS POS

* *

Manderson et al., Appl. Environ. Microbiol (2005)

Toxin

“Decoy”oligosaccharides

Antiadhesive saccharides

Bacteria

Anti-adhesive activity

0102030405060708090

100

E. coli N

CTC 129

00

E. coli N

CTC 1312

7

E. coli N

CTC 1312

8

E. coli O

111:H

27

E. coli O

119:H

4

E. coli O

128:H

12

Dsv. d

esulfu

rican

s

L. acid

ophilus

L. gas

seri

**

* * * **

*

Adh

esio

n re

lativ

e to

con

trol

(%)

0

20

40

60

80

100

120

140

No toxin 0.01 0.1 1 10 100 Toxin OnlyConcentration of POS mg ml-1

% C

ell s

urvi

vabi

lity

*

***** * *

VT1

VT2

Inhibition of verocytotoxin

Flash-extracted POS

Olana-Martin et al., FEMS Microbiol. Letts (2003)

Prebiotic potential ofBergamot POS

-10

-5

0

5

10

15

20

25

Bifidobacteria Bacteroides Lactobacilli Clostridia Eubacteria

Diff

eren

ces

in b

acte

rial p

opul

atio

n si

ze (%

)

FOSBergamot

Mandalari et al., Appl Microbiol Biotech (2007)

Biorefining of functional food ingredients

Antioxidants

Prebiotics

Glucose

Prebiotics

Antiadhesives

Pretreatment

Pectinase

Hemicellulase

Cellulase

Fractionation

Fractionation

Sugar beetpulp

Citruswaste

Distillers’grains

• Prebiotics appear to be a viable, high value option for food waste management

• Success dependant on the waste, waste transfer process and cost

• Cost:benefit ratio set to improve with increasing market growth

• Market growth dependant upon establishment of prebiotic health benefits

Concluding remarks

Thank you!• LTN

• Profs Glenn Gibson, Bob Rastall (UR)

• Dr George Tzortzis (Clasado)

• Dr Giusy Mandalari, Dr Arjan Narbad (IFR)

Speaker 17

Tom Berry

Principal Sustainability AdvisorBusiness Programme

Forum for the Future

t.berry@forumforthefuture.org.uk