Dr.

Ashish Kumar Singh

Senior Scientist
Dairy Technology Division
NDRI, Karnal
 Introduction
 Composition of diet - influence intestinal
physiology, metabolism of microflora
 Dietary carbohydrate improve intestinal
physiology
 Carbohydrate - divergent in molecular
size, monosaccharide unit, linkage between
units
 Certain Dietary carbohydrate escape
digestion
 Reach lower part of intestine- perform
functions known as Non Digestable
carbohydrate
Dietary carbohydrates classified - molecular size or DP
Monosaccharide
Oligosacharide - DP 2-10
Polysaccharide - DP > 10 (Sako et al.,1999)
Oligo and polysaccharide introduced as functional food
ingredient
 Galacto, fruto, malto, isomalto, genito, xylooligosaccharide,
lactulose and lactosucrose
One important OS-- galactooligosaccharide -- being
used in infant formulae, bread, beverage for curing
constipation (Playne,1997)
Oligosaccharides
 Complex carbohydrates made up of 2-10
or more monomer units
 Naturally occurring structural
components of plants
 Presents in mammary secretions too
 Some formed during normal processing
and chemical reactions.
 Various Kind of Oligosaccharides
(OS)
Raw Material Products
Starch Malto-OS, Isomalto-OS,
Cyclodextrins, maltitol, gentio-
OS, Trehalose,
Sucrose Glycosylsucrose, FOS,
Lactosucrose, raffinose
isomaltulose, xylosucrose,
stachyose
Lactose Galcto-OS, Lactulose, Lactitol

Xylan, Agar, Mannan, Xylo-OS, Manno-OS, agaro-OS,

Chitin, Chitin/chitosan-OS
 Properties of OS
Physico-chemical Sweetness, Bitterness,
Properties Hygroscopic, Stability,
Freezing point depression

Functional Properties Colour formation, Water

binding ability, gel formation,
emulsification, Stabilization of
active substances

Biological Properties Digestibility, nondigestibility,

noncariogenicity,
bacteriostatic action
Novel Physiological Functions of
OSs
 As prebiotic
 As immunomodulatory agent
 Improved mineral absorption
 Anti-Carcinogenic effect
 Anti-arteriosclerosis effect
Prebiotics

 Prebitocs are dietary  OSs are not hydrolyzed

food ingredients, by gastric acids or
usually complex intestinal enzymatic
carbohydrates, not secretions, passed to
large intestine and
digested by host selectively as sole
enzymes and source of carbon by
selectively utilized by Bifidobacteria and
beneficial bacteria for other LABs. Hence
their growth. they are called as
bifidogenic factors.
Bifidobacteria

 One of a few
predominant bacteria in
the colonic microflora
 Bifidobacteria
throughout the life.
produces ß-
 Exert antimicrobial galactosidase enzyme
effect,immunomodulation that digest OSs and
properties, reduction of produce short chain
risk of cancer, improve fatty acids (SCFAs).
GI health.
 Predominant products of carbohydrate metabolism
in the human colon
End Bacterial group involved Metabolic fate
product
Acetate Bacteroides, Bifidobacteria, Metabolized in muscle,
Eubacteria,Lactobacilli, Clostridia, kidney, heart and brain
Ruminococci, Peptococci, Veillonella,
Peptostreptococci, Propionibacteria,
Fusobacteria, Butyrivibrio
Propionate Bacteroides, Propionibacteria, Cleared by the liver,
Veillonella possible glucogenic
precursor,suppresses
cholesterol synthesis
Butyrate Clostridia, Fusobacteria, Metabolised by the
Butyrivibrio, Eubacteria, colonic epithelium,
Peptostreptococci regulator of cell growth
and differentiation
 End product Bacterial group involved Metabolic fate

Ethanol,succinate, Bacteroides, Bifidobacteria, Absorbed,further

lactate, pyruvate Lactobacilli, Eubacteria, fermented to short-
Peptostreptococci, Clostridia, chain fatty acids
Ruminococci,
Actinomycetes,
Enterococci, Fusobacteria,
Hydrogen Clostridia, Ruminococci, Partially excreted in
Fusobacteria breath,
metabolizedby
hydrogenotrophic
bacteria

Gibson, 1999
OSs and Mineral Absorption
 SCFAs production- lowering of pH and
increased solubilization of minerals
 Higher calcium and magnesium
absorption after ingestion of
commercially available OSs preparations
 FOS have been shown to improve iron
bioavailability
 However, no consistency regarding
mineral metabolism and OSs intake
OSs and Cancer Prevention
 Colorectal cancer- carcinogen production
as as result of bacterial activity
 OSs help in suppressing the activity of
enzymes that convert procarcinogens to
carcinogens
 Help in proliferation of Bifidobacterium,
that activate cell wall and extracellular
immune system components
 Lactulose protect against DNA damage
Effect of SCFA on colonic epithelial cells at
different stages of the adenoma-carinoma sequence

Normal Mucosa Stimulation of proliferation in the basal

crypt (acetate, propionate, butyrate)

Hyper-proliferation Inhibition of proliferation in the upper

crypt ( butyrate)
Early Adenoma

Induction of apoptosis (butyrate)

Intermediate Adenoma
Inhibition of proliferation ( butyrate,
propionate)
Late Adenoma
Stimulation of differentiation (butyrate)
Different effects on DNA (butyrate)
Carcinoma
Regression of carcinomatosis
Metastases (interlukin-2 + butyrate)
-----------------------------OSs and Cancer Prevention

 4 g FOS/day decreased - glucuronidase

and glycocholic acid hydroxylase
activities, but had no effect on
nitroreductase
 Certain other experiments showed no
significant change in the activity of
enzymes
 In-vitro studies are better to control the
other factors and easy to carry out.
Role of OSs in Immunomodulation

 Proven anti-microbial properties through

competitive inhibition, lowering of pH,
prevent adhesion to intestinal cell wall.
 Lactulose- showed promising results in
treatment of shigella carrier
 Reduction in incidence of atopic aczema
(Allergy) in infants most probably by
minimizing the exposure of allergen and
enhancing the growth of bifidobacteria
 Non-adhesive Adhesive

MMicroorganisms
= M cells of intestinal epithelium
Immune Response L = Lymphocytes
APC = Antigen presenting cells
Intestinal Epithelium
M Th = T-helper cells
IL = Interleukines
L
TGF = Tumour growth factor
L L
IFN = Interferon
APC
L TNF = Tumour necrosis factor
Ig = Immunoglobulin

TH
IgG ↑

Cell TGF-β↓ Antibody B IgM ↑

IL-2 ↑ IL-4 ↓ mediated
mediated
IFN- γ ↑
+ IgE ↓
response IL-10 ↓ response

B
TH2
TH1
IgA
IL-2 ↑
IFN-γ ↑
TNF-α ↑
IFN-α ↑

Viruses Tumors
Natural killer cells ↑
Macrophages ↑
Cytotoxic T-lymphocytes ↑
Reduction in cholesterol &
Triglyceride level

 Similar mechanisms as postulated for

dietary fibers
 Lesser absorption
 Reduction in hepatic synthesis- Low serum
triglyceride levels in rats
 Production of SCFAs and precipitation of bile
salts due to deconjugation and acidification
 Human clinical trials are inconclusive
 Colonic Bacteria & SCFA Related
Effects

Act as immunomodulators i.e.

absorb procarcinogens, promote
Inhibit growth attack on malignant cells
Lower blood
of many cholesterol &
harmful triglycerides
microbes Colonic Reduce food
Improve Bacteria intolerances
mineral food allergies
absorption SCFA
Restore normal
Reduce liver intestinal
toxins i.e.blood microflora
amines & Produce nutrients e.g. B-group
ammonia vitamins, Folic acid, digestive
enzymes
 Reduced Controlled
cancer risk serum lipids
Improved De novo lipid
and IBD & cholesterol
bowl habit genesis
inflammation

Colonocyte

Induce
peristalsis
Trophic & Ca++ Mg++
neoplastic Increased
effect mineral
absorption
SCFA Reduced pH

Selective Fewer toxic

Antagonism of
fermentation bacterial
pathogens &
putrefactive metabolites
bacteria
Oligosaccharides
Reduced cancer
risk

Health Effects of Non Digestible Oligosaccharides

 Prebiotics Dietary Polysaccharides

Bifidobacteria Butyrate, Unfermented Fiber

Propionate, Acetate

Energy supply
Lower pH Stool
Higher
water Bulking
content

Lower putrefactive Lower

Lower secondary
enzyme activity
putrefactive bile acids
substances
Improvement
of bowl Lipid
Mineral absorption Cancer Prevention movement metabolism
Local Effects Systemic Effects
Fecal bulking ( ) Cholesterol
Bacteria TG ( insulin, blood glucose)
Selective ^ bacteria Blood ammonia levels
SCFA production Urea
Selective in SCFA B-vitamins
Mineral absorption Immune function
B-vitamin synthesis glutamine?
Abbreviations: SCFA, short chain fatty acids; TG:
triglyceride = increase; = decrease
Potential effects of non absorbable carbohydrates
physiological effects Jenkins et al., 1999
The most common prebiotic.
A chain of about 20 fructose molecules with
one terminal glucose. Also called Fructans.
Found in roots of chicory, Jerusalem
artichokes, dahlias, dandelions and related
species.
Humans need to eat 2-4 grams/day to
stimulate probiotics in the gut.
SUCROSE INULIN OLIGOFRUCTOSE
Sources for inulin production
Jerusalem
artichoke

Dahlia roots

Asparagus root

Ginger

Chicory roots
Percent inulin content (on fresh weight basis)
from some important sources
Source Inulin (%)
Garlic 15–20
Asparagus root 10–15
Salisfy 15–20
Jerusalem artichoke 15–20
Dahlia tubers 15–20
Chicory root 15–20

Gupta and Kaur,1997

Both inulin and oligofructose are used
worldwide to add fiber to food products
without any off flavour or increase in
viscosity.

These properties allow the formulation of high

fiber foods that look and taste like standard

food formulations.
Both inulin and oligofructose are used
worldwide to add fiber to food products
without any off flavour or increase in
viscosity.

These properties allow the formulation of high

fiber foods that look and taste like standard

food formulations.
Inulin
 Inulin is a plant OS found naturally in more
than 36,000 types of plants.
 Fermented by a limited number of colonic
bacteria

Inulin
Fermented 10% Gases
by
50% VFA
Colonic
Microflora 40% Bacterial Biomass
Inulin
n or m equal the number of fructose units
Inulin intake in the U.S. = from 1 to 4 grams daily.
Inulin Production Processing
Chicory Root
Decolorization

Washing, Slicing Followed Purified Inulin

by Extraction Juice

Raw Juice Evaporate/Concentrate

Dimeralization
Spray Dry

Ultrafiltration Dry Inulin Powder

Colon Health Benefits of Inulin

 15 grams of inulin a day for fifteen days

feeding resulted in
 Beneficial bacteria.by about 10%
 Reduction in Gram+ve bacteria
 Production of SCFAs
 energy production
 butyric acid has cancer-preventing
properties within the intestine
 Recent animal research also shows that
inulin prevents precancerous changes in the
colon
---------------------------------------------------------Colon Health Benefits of Inulin

 Antitumor - due to action of butyrate

 butyrate may induce growth arrest and
cell differentiation and unregulated
apoptosis,
 Hypolipidemic
 may lower serum triglycerides in some
humans via decreased triglyceride
synthesis in the liver
 may lower cholesterol levels in some
type 2 diabetics. Propionate, a product of
inulin fermentation may inhibit HMG-CoA
reductase, the rate-limiting step in
cholesterol biosynthesis.
Fructo-oligosaccharides (FOS)
 Naturally occurring carbohydrate in almost 2000
plant foods
 Short or medium chain of ß–D-Fructose linked
through ß(2 1) linkage
 Combination of three sugars 1-Ketose (GF2),
Nystose (GF3) and 1F-ß- fructofuranosylnystose
(GF4)
 Produced by the action of a fructofuranosyl
furanosidase on sucrose or partial hydrolysis of
fructose polymers
 Higher sucrose concentration (60-85%) –
Desirable
FOS

 Stable at neutral pH and high temp.,

boiling in acidic conditions degrade it
 0.4-0.6 times as sweet as sucrose
 Low calorific value (1.5 Kcal/g)
 Promote growth of intestinal beneficial
bacteria
 Exert secondary health promoting effects
in in-vitro, in-vivo and human
experimentations
Lactulose
 Chemically known as 4-o- β- D-
galactopyranosyl fructose
 Formed during heating of milk
 Alkali isomerisation process is used for
commercial production
 White crystalline powder, readily soluble
in water
 Degree of sweetness – 0.48 - 0.62
 Lactulose as bifidus factor
 Utilized by bifidobacteria with concomitant
production of lactic acid
 An important ingredient for infant formulae
 0.5lactulose incorporation in infant formula –
stimulate the bifidobacteria without laxative
effect
(Nagendra et al.,1994)
 1.2 –1.5% lactulose in diet increased
bifidobacteria, lowered down pH and inhibited
putrefactive gm – ve bacteria
Other application of lactulose

 Control of chronic constipation

 Prevention of portal systematic
encephalopathy
 Controlling salmonella colonization
 Inhibit dehydroxylation of primary bile
acids in experimental animals
 Exert anti- toxin effect
 Anti- carcinogenic effect
Galactooligosaccharides
 Naturally occurring OS in human milk
 Galactose molecules linked through β(1-6)
linkage with terminal glucopyranosyl unit
through α(1-4)bond
 Commercially produced by the
transglucosyl activity of β– galactosidase
 Colorless, relative sweetness 0.4
 Slightly viscous solution
 Stable at – 100- 160 C
- 2-3 pH
 Nature of Oligosaccharide
 Lactose- β-D-Gal-(1-4)-D-Glu
 Allolactose - β-D-Gal-(1-2)-D-Glu
β-D-Gal-(1-3)-D-Glu
β-D-Gal-(1-6)-D-Glu
 Transgalactosylation- β-D-Gal-(1-3)-D-Gal
β-D-Gal-(1-6)-D-Gal
 6` Galatosyl lactose
3` Galactosyl Lactose
 Tetra, penta and hexasaccharides
(Prenosil et al.,1987;Smart, 1993)
 Galactooligosaccharide manufacturers
• Yakult Honsha Co. ltd
• Nissin Sugar Mfg. Co. Ltd
• Snow Brand Milk Products in Japan
• Borculo Whey Products in Netherlands
(Playne et al.,1997)
One GOS product on Japanese market
(Oligomate 55) contains atleast 55% 4`GOS in
solid material
Oligomate solution – slightly more viscous than
HFCS, relative sweetness is about 35 of sucrose
(Sako et al., 1999)
 Enzymatic hydrolysis
 Enzyme β-D-Galactosidase used for
hydrolysis
 Main products - D-glucose, D-galactose
 Transferase reaction involves lactose, its
hydrolytic products
 Particularly galactose to form trans- GOS

(Smart, 1993)
 GOS - DP 2 to 6, depends upon source of
enzyme, consist of - β1-3, β1-4, β1-6 bonds
(Gibson, 2004)
β-D-Galactosidase
 Widely distributed in nature
 β-D-galactosidase is commonly known as
lactase (Gekas et al.,1985)
 Sources-Plants, bacteria, yeast, fungi,
animal organs
 All lactases are not safe
 Few are safe, already being used in
hydrolysis
 Yeast and bacterial originated lactases are
generally used in milk (optimum pH 6 -7)
 Fungal lactases for acid whey hydrolysis
(optimum pH 2.5 - 4.5) (Gekas, 1985)
Mechanism

 E+ Lac Enz - Lactose

 E- Lac Galactosyl-enzyme + glu
 Galactosyl enzyme + acceptor
Galactosyl acceptor + E
Acceptor water - free galactose
 Acceptor sugar - galactooligosaccharide
formed
 Reaction called as trans- Galactosylation
(Zarate,1990; Mahoney,
1998)
(Prenosil et al.,1987;Zarate et al.,1990)
Oligosaccharide formation : effect
of different factors

 Source of enzyme
 Nature and concentration of
substrate
 Type of process (immobilized/free)
 Process condition and medium
composition
 Degree of lactose conversion
 Source of Enzyme
 Most important factor to determine no. and
type of oligosaccharide (Smart, 1993)
 Most of studies based on E. coli (Mahoney,
1998)
 Lactase from Kluveromuces fragilis
reported highest OS concentration
 At initial lactose conc. of 35%, 44.6% of OS
produced at 93% conversion of lactose both
have neutral pH optimum
(Zarate, 1990)
 Galactosidase from K. fragilis and K. lactis
are generally very similar
(Mahoney, 1992)
 Maxilact 2000 (K.lactis galactosidase)
found to produce a maximum amount of
trisaccharides (8%)
(Zarate et al.,
1990)
 Lactase from A. niger reported low
formation of OS (1-2%)
(Mahoney,
1998)
Concentration & Nature of
substrate
 Reaction leads to shift towards OS formation –
when lactose conc. higher

(Ekharte, 1997)
 Initial conc.4.4% lactose- only 10% of total
sugar OS
 If initial lactose concentration 13.3% - 26% of
total sugar OS
(Lopez leiva, 1990)
 Lactose in buffer solution give larger amount
of OS than milk or its products
 In first case OS – 25 to 44.6%
 In second case OS – 1 to 25% (Mahoney, 1992)
Type of Process

 Includes immobilized or free enzyme

 Immobilized galactosidase produces more
di-, tri- or tetra oligosaccaharide

(Ekharte, 1997)
 Immobilized lactase with higher initial
lactose conc., showed lower production of
OS than free enzyme
(Prenosil, 1987)
Process conditions & Medium
composition
 In specific cases higher temp, pH, salt conc -
produce high level of oligosaccharides

 But only for E. coli, B. circulans (Mahoney, 1992)

 In a study, increasing enzyme conc of Maxilact

2000 from .05-.1% didn’t have a significant effect
on OS production when initial lactose conc was
23%
(Rustom et al.,1998)
 Degree of Lactose Conversion
 Most favourable end product depend upon water
activity present in mixture (Ekharte,1997)

 Highest level of OS – at highest lactose starting

levels of 15 – 50% (Mahoney, 1998)

 At long incubation times – all products were

rehydrolyzed

 After 4 h of incubation all OS rehydrolyzed (Zarate et

al.,1990)
Commercial Production of GOS
Lactose solution

Enzymatic reaction

Decolourization

Demineralization

Filtration

Concentration Drying

GOS syrup GOS powder

Mechanism of GOS Formation

Complex
E Lac
Complex
Glu

E
Acceptor GOS
Physiological Functions of GOS

 Well established “Bifidogenic” role

 Check constipation
 Reduction in blood ammonia level
 Improved mineral absorption
 Prevention of colon cancer
 Check enzymatic activity
 6’ GOS feeding decreased the conversion of
procarsinogens into carcinogenic form
Functional Properties

Flavour
Colour Emulsification

Water Formulated Humectants

Binding Foods

Encapsulation Stabilization
Gelation
Application in Food Products

 As sweetener
 As fat mimetic
 In infant formulae
 Synbiotic products
 As humectant
 As bulking agent
 OS BASED PRODUCTS
AVAILABLE IN MARKET
TYPE OF PRODUCT COMPANY DESCRIPTION
PRODUCT NAME
Prebiotic InufloraTM Naturally Tablet or powder form
Vitamins

Prebiotic Inu-Lean Naturally Chocolate mint wafers — contain

Vitamins inulin and 500 mg calcium

Prebiotic ProSure Abbott Nutrition and energy beverage,

Laboratories contains 2.4 g FOS/8 oz

Synbiotic Blended Stonyfield Flavored yogurt containing six

Yogurt live active cultures and inulin

Synbiotic Synbiotic UAS Supplement with probiotic

Sachets Laboratories cultures and FOS

Brannon, 2003
Toxicity Studies With OSs
Generally recognized as safe (GRAS) status
No mutagenic effect, only diarrhea or
intestinal disturbances
Oligosaccharides Acute Chronic
Toxicity Toxicity
GOS 15g/kg 1.5g/kg
FOS 10g/kg 2.17g/kg
Inulin 25g/day