ESTIMATION OF OLIGOSACCHARIDE CONTENT

IN LOCALLY AVAILABLE CEREALS AND PULSES

SAMPATH SUNKOJI.
B.Tech (Dairy Technology)

THESIS SUBMITTED TO
ACHARYA N.G.RANGA AGRICULTURAL UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE
IN
FOOD SCIENCE AND TECHNOLOGY

FACULTY OF P.G. STUDIES

POST GRADUATE AND RESEARCH CENTRE

ACHARYA N.G.RANGA AGRICULTURAL UNIVERSITY
RAJENDRANAGAR, HYDERABAD - 500 030.

2005
 CERTIFICATE

Mr. SAMPATH SUNKOJI. has satisfactorily prosecuted the course of research and
that the thesis entitled "ESTIMATION OF OLIGOSACCHARIDE
CONTENT IN LOCALLY AVAILABLE CEREALS AND PULSES
submitted, is the result of original research work and is of sufficiently high standard to
warrant its presentation to the examination. I also certify that the thesis or part thereof
has not been previously submitted by him for a degree of any University.

Date : ( Dr. T. MADHAVA RAO)

Place : Hyderabad Major Advisor
 CERTIFICATE

This is to certify that the thesis entitled " ESTIMATION OF

OLIGOSACCHARIDE CONTENT IN LOCALLY AVAILABLE CEREALS
AND PULSES submitted in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE IN FOOD SCIENCE AND TECHNOLOGY for
Acharya N. G. Ranga Agricultural University, Hyderabad is a record of the bonafide
research work carried out by Mr. SAMPATH SUNKOJI. under my guidance and
supervision. The subject of the thesis has been approved by the students advisory
committee.

No part of the thesis has been submitted for any other degree or diploma.
The published part has been fully acknowledged. All the assistance and help received
during the course of investigation have been duly acknowledged by the author of the
thesis.

(Dr. T. MADHAVA RAO)

Chairman of the Advisory committee

Thesis approved by the student Advisory Committee

Chairman : (Dr. T. MADHAVA RAO) _______________
Assoc. Professor of LPT & Dy. Director
Administrative office,
A.N.G.R. Agricultural University
Rajendranagar, Hyderabad - 30.

Member : (Dr. K. KONDAL REDDY _______________

Associate Professor
Department of LPT
College of Veterinary Science
Rajendranagar, Hyderabad -30.

Member : (Dr. N. LAKSHMI DEVI) _______________

Associate Professor
Department of Foods & Nutrition
Post Graduate and Research Centre
Rajendranagar, Hyderabad -30.
 CONTENTS

Chapter No. Title Page No.

I INTRODUCTION

II REVIEW OF LITERATURE

III MATERIALS AND METHODS

IV RESULTS AND DISCUSSION

V SUMMARY AND CONCLUSION

LITERATURE CITED
 LIST OF TABLES

Table Title Page No.

No.
1 Sources available for dietary oligosaccharides

2 Novel microbial enzymes uses for oligosaccharide production

3 Reaction conditions of hydrolysis of polysaccharides to

oligosaccharides

4 Applications of oligosaccharides in different food industries

5 Moisture content in raw cereals and pulses

6 Moisture content in germinated cereals and pulses

7 Observed concentration of raffinose by HPLC analysis

8 Observed concentration of stachyose by HPLC analysis

9 Observed concentration of maltotriose by HPLC analysis

10 Observed concentration of maltotriose by HPLC analysis

11 Observed concentration of maltopentaose by HPLC analysis

12 Observed concentration of maltohexaose by HPLC analysis

13 Observed concentration of maltoheptaose by HPLC analysis

14 Oligosaccharide content in raw cereals and pulses

15 Oligosaccharide content in germinated cereals and pulses.

 LIST OF FIGURES

Figures Title Page No.

No.
1 Results chromatogram of raffinose (0.5 per cent) standard

2 Results chromatogram of stachyose (0.5 per cent) standard

3 Results chromatogram of maltotriose (0.5 per cent) standard

4 Results chromatogram of maltotetraose (0.5 per cent) standard

5 Results chromatogram of maltopentaose (0.5 per cent) standard

6 Results chromatogram of maltohexaose (0.5 per cent) standard

7 Results chromatogram of maltoheptaose (0.5 per cent) standard

 ACKNOWLEDGEMENTS

In all reverence, I pay my profound and sincere obeisance to the Almighty

for leading in the right direction, in my quest for knowledge.

I express my sincere and wholehearted thanks to my Major Advisor and

Chairman of the advisory committee Dr. T. Madhava Rao, Associate Professor and
Deputy Director (Vety), Administrative office, Rajendranagar, Hyderabad, for
reposing confidence in me and accepting me as his student. I further extend my
sincere gratitude for his timely guidance, diligence, studiousness and constant
encouragement given to me throughout the course of the study and in preparation of
this thesis, without whose help this thesis would not have seen the light of the day.
It gives me immense pleasure in extending my sincere gratitude to
Dr. K. Kondal Reddy, Associate Professor, Department of Livestock Products
Technology, College of Veterinary Science, Rajendranagar, Hyderabad, for acting as
a member of the advisory committee and for his invaluable guidance, timely
suggestions during the research work and in completion of this thesis.
I extend my wholehearted gratitude to Dr. N. Lakshmi Devi, Associate
Professor, Department of Foods and Nutrition, Post Graduate and Research Centre,
Rajendranagar for being, member of the advisory committee, for her right guidance
for her timely support during my research work.
I take pleasure in thanking with profound sense of reverence to

Dr. B.V.R. Rao, Professor and University Head, Department of LPT, College of

Veterinary Science, Rajendranagar, Hyderabad for his guidance besides providing

valuable suggestions and support during the conduct of this study.

I feel it appropriate to express my sincere thanks to Dr. Y. N. Reddy,

Programme Director, Food Science and Technology Programme Post Graduate and

Research Centre, Rajendranagar, Hyderabad. I also feel it equally important to thank

Dr. P. Rajyalakshmi, Dr. N. S. Sumathi, Dr. K. Umamaheswari, Dr. M. Sudhakar

Reddy, Dr. Veeranna Goud, Dr. J. Dilip Babu, Dr. Kanwaljith Kaur, Dr. B.

Srinivasan and all the faculty members for their kind cooperation during my study.

I take it as a privilege to receive the blessings of my beloved father

Shri. S. Neelakantam, who was the main source of inspiration for taking up this

course and the study. He has been the guiding spirit in all my endeavours. I also take

it as equally important to owe my sincere regards and gratitude to my mother Smt. S.

Kousalya who wished success in all my ventures and provided constant

encouragement. I am greatly indebted to my affectionate sister Smt. S. Saritha and

my brother-in-law Shri. Narendra Chari whose help and humour brought my study

twinkle.

I take pleasure in expressing my sincere thanks to all my friends Balesham,

Pradeep, Siddeshwar, Naveen, Rajashekar, Bhaskar, Raghavender, Shilpa,

Vandana, Sulaxana, Sameeraja, and Anuradha who supported me in all my efforts

and extended their timely assistance in preparation of this thesis.

My greatful thanks to my friends Mallikarjun, Arun, Srikanth, Shashi,

Narender Raju, Vijaya Bhasker, Vijay for their cooperation and encouragement.

My very special thanks to Baburao, Venkataiah, Yedukondalu, Ravi, and

Umesh Reddy for their help and coordination.

I remember the savy camaraderie that my friends, classmates, senior and

juniors has extended and whose friendship I always cherish.

Finally, I thank all the people who directly or indirectly helped me to complete
my thesis successfully

(SAMPATH SUNKOJI)
 DECLARATION

I, Mr. SAMPATH SUNKOJI here by declare that the thesis entitled

ESTIMATION OF OLIGOSACCHARIDE CONTENT IN LOCALLY
AVAILABLE CEREALS AND PULSES submitted to Acharya N.G. Ranga
Agricultural University for the Degree of MASTER OF SCIENCE in FOOD
SCIENCE AND TECHNOLOGY is a result of original research work done by me.
It is further declared that the thesis or any part there of has not been published earlier
in any manner.

Date : (SAMPATH SUNKOJI)

Place : Hyderabad
Author : SAMPATH SUNKOJI
I.D. NO. : FST-2003-008
Title of the thesis : ESTIMATION OF OLIGOSACCHARIDE
CONTENT IN LOCALLY AVAILABLE
CEREALS AND PULSES.
Degree to which it is : MASTER OF SCIENCE
submitted
Faculty : FACULTY OF P.G. STUDIES

Major field : FOODS SCIENCE & TECHNOLOGY

Major advisor : Dr. T. MADHAVA RAO

University : ACHARYA N.G.RANGA AGRICULTURAL

UNIVERSITY
Year of submission : 2005

ABSTRACT

An attempt was made to estimate the oligosaccharide content in cereals and pulses

and to study the effect of germination on different oligosaccharides ( stachyose, raffinose,

maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose) in cereals and

pulses, considering their importance as prebiotics. Quantification, involved hot water

extraction (98C/ 2 hrs) of oligosaccharides, pre-filtration (soy cloth), filtration

(membrane- 0.25) followed by high performance liquid chromatography (HPLC)

analysis, with a refractive index (RI) detector and using oligosaccharide standards as

reference. Repetitive injection of different concentrations of standard sugars was done to

check the detector response.

 The seeds of 32 different pulses and cereals were analyzed. Mainly stachyose

content was observed (in mg/ gm of dry matter) in green gram (1.24), sorghum (traces),

barley (traces), Wheat (traces) and black gram (8.302).

The raffinose content was observed (in mg/ gm of dry matter) in barley (traces),

wheat (5.2), cow pea (4.261), green peas (2.04), black gram (traces), pigeon pea (1.28)

and soya (3.04).

The maltotriose content was observed (in mg/ gm of dry matter) in sorghum

(0.533), chick pea (0.39), maize (0.52), rajma (0.486) and Lathyrus sativus (0.58).

In few samples, more than one oligosaccharide were observed like in green gram

(stachyose, maltohexaose), sorghum (stachyose, maltotriose), barley (stachyose,

raffinose), wheat (stachyose, raffinose) and black gram (stachyose, raffinose). In few

samples like ragi, bajra and rice maltooligosaccharide content studied was absent.

The germination of seeds for 48 hours resulted complete disappearance of

stachyose, raffinose in cereals and pulses. The maltotriose content in pulses completely

disappeared, but in cereals, 45.1 %, 57.3 % loss was observed in sorghum and maize

respectively, whereas complete loss in remaining cereals was observed.

The results suggested that the oligosaccharides present in dormant seeds were

hydrolyzed during germination; germination reduces the availability of prebiotic

oligosaccharides.

Identified rich sources of oligosaccharides are black gram, soy, cowpea and

wheat, which may be used for extraction in their natural form by membrane separation.

These extracted oligosaccharides can be used as prebiotics in development of functional

foods.
 CHAPTER-I:

INTRODUCTION

Diet plays a major role in not only providing the essential nutrients but also in

giving a feeling of satisfaction and well being. For the maintenance of good health and

reducing the susceptibility to discases, diet modulates different body mechanisms.

Viewing such an important link of health with diet, the concept of functional foodswas

introduced (Vijendra et al., 2001).

In the area of functional foods the probiotic and prebiotic foods have tremendous
market. A prebiotic is a food ingredient that is only digested in the large intestine by
good bacteria, where it selectively stimulates the growth and / or activity of good bacteria
to benefit the health of the host (Gibson and Roberfroid, 1995). Since they are not
digested or absorbed in the upper gastrointestinal tract, they are used selectively by
beneficial bacteria. Regular consumption of foods containing prebiotics is a great way of
promoting a healthier digestive system. Both probiotics and prebiotics containing
product can be called as symbiotic product.

The ingestion of carbohydrates from a variety of food sources is essential for

maintaining health. In the gastrointestinal tract (small & larger intestines) carbohydrates

undergo digestion and respectively fermentation by enzymes produced in the intestines

and by bacterial enzymes.

Dietary carbohydrates represent a complex group of components, which have

physiological and nutritional properties. Within the dietary carbohydrates, it has become
clear over the last decade, that the group of non-digestible oligosaccharides (NDO) play,

or likely to play an important nutritional role (Cummings et al., 1997).

Non-digestible oligosaccharides can be defined as a non-digestible food

ingredient that beneficially affects the host by selectively stimulating the growth and/or

the activity of one or a limited number of bacteria in colon and thus improve the host

health (Gibson et al., 1995).

Various types of oligosaccharides have been found as natural components in

many common foods including fruits, vegetables, seeds, onion, garlic, milk, honey, and

in Japanese traditional foods such as sake and sweet sake used as seasonings.

Non-digestible oligosaccharides posses excellent functional properties (like

solubility, gelation, viscosity, cations binding etc.,) which effects the final quality of the

food. The type of chemical structure leads to physical properties such as swelling, extent

of insolubility in water (ex: inulin) resistance to hydration etc and the research on their

characteristics found that these oligosaccharides had excellent physiological properties

(like anti-carcinogenic effect, anti-diabetic, anti-cardiovascular, higher rate of mineral

absorption etc.) that are both scientifically interesting and beneficial to human health

(Masao, 2002).

Cereals and pulses are important component of both human and livestock diets.

The oligosaccharides are major components in many food cereals and pulses and the
nutritional activity of grains is frequently associated with the presence of these

oligosaccharides.

Recently Japanese researchers reported that oligosaccharide content in

yakoon, soy can separate by using membrane processing (Kamada et al., 2002).

No data is available on the in individual oligosaccharides content in cereals and

pulses. However, some reports [Pazur et al., (1962); Kuo et al., (1988)] suggested that

germination reduces certain oligosaccharide content in pulses, which in turn reduces the

flatulence. Therefore, an attempt is made to study the levels of oligosaccharides content

in Indian cereals and pulses and the effect of germination on deterioration of

oligosaccharides. The present study was carried out with following objectives:

To estimate the Fructo-oligosaccharides (FOS) content in raw and germinated

cereals and pulses.

To identify the levels of different types of FOS present in cereals and pulses.
 CHAPTER II
REVIEW OF LITERATURE
2.1 Introduction:
In the area of functional foods the probiotic and prebiotic foods have tremendous
market. The concept of probiotics has existed for many years and a number of food
products that contain one or more of these bacterial strains are commercially available.
The probiotic approach adds live bacteria to food in sufficient numbers to survive
passage through the stomach and reach the intestine, where they exert their positive
function. Mainly, bifidus and lactobacillus bacteria have a positive influence on the
human system. By adding prebiotic substances to food we can encourage the growth of
probiotics in the gut.
A prebiotic is a food ingredient that is only digested in the large intestine by
good bacteria, where it selectively stimulates the growth and / or activity of good bacteria
to benefit the health of the host (Gibson and Roberfroid, 1995). Since they are not
digested or absorbed in the upper gastrointestinal tract, they are used selectively by
beneficial bacteria. Regular consumption of foods containing prebiotics is a great way of
promoting a healthier digestive system. Both probiotics and prebiotics containing
product can be called as symbiotic product.

In the carbohydrates, non-digestible oligosaccharides are having similar

properties of prebiotics as mentioned above; hence they can be called as prebiotic
nondigestible oligosaccharides. The oligosaccharides are carbohydrates with low degree
of polymerization and consequently low molecular weight. They have been defined as
carbohydrate having 2 to 20 monosaccharide units.
The concept of nondigestible oligosaccharides originated from the observation
that the anomeric carbon atom of the monosaccharide unit of some dietary
oligosaccharides has configuration that makes their osidic bonds nondigestible by
hydrolytic activity of the human digestive enzymes.
The main categories of nondigestible oligosaccharides presently available or in
development as food ingredients include carbohydrates in which the monosaccharide unit
is fructose, galactose, glucose, and/or xylose (Cummings and Roberfroid, 1996; Delzenne
and Roberfroid, 1994). According to Crittenden & Playne (1996), the prebiotic market
is expanding rapidly and the demand for novel compounds may not be limited to
oligosaccharides although these are current market leaders.

2.2 OCCURENCE OF OLIGOSACCARIDES

The occurrence of nondigestible oligosaccharides from the natural sources like
fruits, vegetables, seeds, roots, and milk etc oligosaccharides can be manufactured in the
process of enzymatic synthesis from simple sugars or enzymatic hydrolysis from more
complex carbohydrates (Murphy, 2001). Sources available for dietary oligosaccharides
are presented in Table 1.

Table 1: Sources available for dietary oligosaccharide (Murphy, 2001)

Type of Natural occurrence Industrial production
oligosaccharides process
Fructo-oligosaccharides Fruits and vegetables, onions, Synthesis from saccharose
banana, garlic, etc.
Galacto- Human milk Enzymatic synthesis from
oligosaccharides lactose
Lactulose - Synthesis from lactose
Lactosucrose, - Synthesis from saccharose
glycosylsucrose and/or lactose
(Iso)malto- - Hydrolysis or glycosyl
oligosaccharides transfer from starch
Xylo-oligosaccharides - Hydrolysis from polyxylans
Stacchyose, raffinose Soya bean
Palatinose- - Synthesis from starch
oligosaccharides
Gentio-oligosaccharides - Enzymatic synthesis from
glucose
Cyclodextrins - Synthesis from starch
2.3 PREPARATION OF NONDIGESTIBLE OLIGOSACCHARIDES:
Three methods are used to prepare the nondigestible oligosaccharides. They are,
Hot water extraction from roots, fruits, vegetables, cereals and pulses like inulin,
soybean oligosaccharides.
Alcoholic extraction of oligosaccharides by using ethanol or alcohol in a
combination with or without water.
Partial enzymatic hydrolysis of oligosaccharides (like oligofructose, the
hydrolysates of inulin) or polysaccharides (like xylooligosaccharides on xylan
polysaccharide or pectin hydrolysates)
Enzymatic syntheses from one or a mixture of disaccharides using osyl-transferase
like fructooligosaccharides from lactose or lactosucrose from a mixture of sucrose
and lactose.

2.3.1 Hot Water Extraction:

In this process, the extraction of naturally occurring oligosaccharides from
different sources, in a manner very similar to the extraction of sucrose from sugar beets
(diffusion in hot water), followed by refining using technologies similar to the sugar and
starch industries (membrane processing/ ion exchangers), and then evaporation and
spray-drying.

The source material is first ground to fine powder / paste and then subjected to
dilution to avoid gelatinization while heating. Total water and sample subjected to hot
water at 90oC for about 2 hours. And then the crude extract subjected to the pre-filtration
with soy cloth so as to avoid the blocking of the membranes used for filtration. The
membrane processing is used to separate the proteins and lipids from the oligosaccharide
solution.
Urano et al., (1997) reported membrane separation of oligosaccharides using
standard saccharides and nanofiltration (NF).
After membrane separation process the oligosaccharide solution may be subjected
to drying by using either spray drying or freeze-drying.
2.3.2 Alcoholic extraction
One of the most commonly used method of extractions of low molecular weight
carbohydrates from food is to boil a defatted sample with 80% alcohol solution.
The monosaccharides and oligosaccharides are soluble in alcoholic solutions
whereas proteins, polysaccharides and dietary fibre are insoluble. By filtering the
boiled solution, the soluble components are collected.
The molecules other than oligosaccharides that are soluble can be
separated by using clarifying agents or by passing through ion exchange resins.
Prior to analysis, the alcohol can be removed from the solution by
evaporation under vacuum, so that an aqueous solution of sugar remains.
Hymowitz et al.,(1972) used ethanol-water (80:20 v/ v) and satisfactory
results were obtained from oligosaccharide extraction in samples.
Sosulski et al., (1982) also used 80% methanol (v/ v) for oligosaccharide
estimation.
Kuo et al., (1988) used 80% ethanol for analysis of oligosaccharide
content in different cereals.

2.3.3 Hydrolysis from complex carbohydrates (Polymers):

The oligosaccharides have been obtained from biological polymers by hydrolysis

under conditions that leave the desired oligosaccharides intact for subsequent isolation.
The enzymes and acids are used as hydrolytic agents and often hydrolysis yield a
different series of oligosaccharides from the same polymer. The sources of specific
oligosaccharides include polysaccharides, other oligosaccharides and glycosides, with the
polysaccharides as the most common source.
2.3.3.1 Hydrolysis by enzymes:
The use of enzymatic procedure for hydrolyzing polymers is dependent on the
availability of suitable enzyme preparations. These enzymes are divided into two groups;
the exo-hydrolases and the endo-hydrolases.
The exo-hydrolases cleave polysaccharides by successive removal of residues
from one end of the polymeric chain. If the polymer is of uniform structure, a high
percentage of the polymer is converted into a specific oligosaccharide. For example exo-
hydrolases used to prepare isomaltose from dextrans (Jeanese et al., 1953).
The enzymes of endo-hydrolase type are more common in biological materials
and many types of endo-hydrolases have been used for preparing oligosaccharides.
These hydrolysates effect random fragmentation of polysaccharides to give a homologous
series of oligosaccharides, as for example, malto-oligosaccharides from amylase (Dube
and Nordin, 1962), and xylo-oligosaccharides from xylan (Whistler and Masak, 1955).
Accordingly, a vide array of oligosaccharides can be obtained from these polysaccharides
depending on the specificity of the enzyme employed. Source of oligosaccharides by
enzymatic hydrolysis are given in Table 2.
Table 2: Source of oligosaccharides by enzymatic hydrolysis
Source Enzyme Product
Starch MTSase Trehalose
(glycosyltransferase)
MTHase (-amylase) Trehalose
-Glucosidase Nigero-oligosaccharide
Maltotrihydrolase -Maltrotriose
Maltotetraohydrolase -Maltotetraose
Maltopentaose from -Maltopentaose
amylose
Maltohexaohydrolase -Maltohexaose

2.3.3.2 Hydrolysis by acids:

The controlled acid hydrolysis of polysaccharides, oligosaccharides and
glycosides result in reaction mixtures from which numerous oligosaccharides have been
obtained. If the hydrolysis constants are known, these can be utilized in selecting the
proper conditions for hydrolyzing the polymer.
The procedures that have been employed can be grouped into the following types:
autohydrolysis, hydrolysis in dilute acids, in concentrated acids, acetolysis, and
hydrolysis by acidic resins.
 The autohydrolysis procedure is applicable to polysaccharides composed with
acidic groups. These polysaccharides ionized in solutions for auto hydrolysis to occur,
when a solution of the polymer is heated for a sufficient period of time. According to
Charlson et al., (1955) uronic acid containing oligosaccharides are prepared by this
method.
Hydrolysis with diluted acids is generally achieved with hydrochloric or sulphuric
acid in the concentration range from 0.01N to 2.0 N. Often hydrolysis is affected at high
temperatures, but in some cases it may happen at room temperature. If prolonged periods
of hydrolysis are employed, dehydration of the oligosaccharide may occur (Sweeley and
Walker, 1964). Such procedures yield the methyl glycosides, which may then be
converted in to the free oligosaccharides by enzyme or controlled acid hydrolysis. The
random hydrolysis leads to a homologous series of oligosaccharides. The malto-
oligosaccharides from amylase (Whelan et al., 1953; Pazur and Budovich, 1956) and
isomalto-oligosaccharides from dextrans are examples of this type (Turvey and Whelan,
1957). Reaction conditions of acid hydrolysis to produce oligosaccharides are given in
Table 3.
Table 3: Reaction conditions for acid hydrolysis of polysaccharides to form
oligosaccharides. (Pigman and Horton, 1970)

Polymer Type of Acid concentration Temperature (oC)

oligosaccharide and Time (hr)
Amylose Malto 0.33 N H2SO4 100, 2
Malto 0.1 N HCl 100, 4
Dextran Isomalto 0.33 N 100, 10
Nigeran Nigero 1 N H2SO4 85, 3
Pustulan Gentio 1 N H2SO4 100, 2
Glucomannan Gluco-manno 30% HCOOH 100, 4
Aspen Xylo 1 N H2SO4 90, 8
hemicellulose
Inulin Inulo 0.01 N HCl 70, 0.5
Anogeissus Galacto 0.01 N H2SO4 100, 1
schimperi gum

When the glycosidic linkages of a polymer are resistant to hydrolysis in dilute

acids, hydrolysis can be effected in concentrated acids at refluxing temperatures for
prolonged period of time. The oligosaccharides so prepared include oligosaccharides like
chito-oligosaccharides etc. (Barker et al., 1958).
Acetolysis , involving simultaneous use of sulfuric acid and acetic anhydride, and
another method for hydrolyzing such resistant polymers. Initially, Wolform and Dacons
(1952) prepared oligosaccharides from cellulose by acetolysis. .
Painter (1960) also prepared oligosaccharides by using water soluble
polystyrenesulfonic acid resin. This method was used by Painter et al., (1962) to obtain
oligosaccharides from glycoproteins with yields several times greater than conventional
hydrolysis with dilute acids.

2.4 FUNCTIONAL PROPERTIES

Oligosaccharides isolated from the native plants provide many functional
properties that affect the technological function of foods. These functional properties also
influence the food products properties during its processing and final product quality and
characteristics. The primary properties of isolated oligosaccharides used for food
development are related to their solubility, viscosity and gelation- forming ability, water-
binding capacity, oil-binding capacity and mineral and organic molecule-binding
capacity.

2.4.1 SOLUBILITY:
The oligosaccharides are as a rule are very soluble in water, but solubility will
vary with the oligosaccharide with respect to its source and structure (chain length). As
the chain length increases the solubility will decrease. And the small chain length
oligosaccharides are easily soluble in water. The temperature and concentration of the
oligosaccharide will also have affect on the solubility. Small chain oligosaccharides are
readily soluble at room temperature (ex: oligofructose) and as the chain length increases
the solubility at room temperature decreases (ex: inulin). The solubility of
oligosaccharides is proportional to the temperature. Most of the oligosaccharides are
completely soluble at 85-90oC.
This solubility behavior of oligosaccharides has been utilized to effect a
separation of the oligosaccharide from inorganic salts and is of particular importance in
chromatographic methods based on partition coefficients (Pigman and Horton, 1970)
Because of easy dispersion of inulin in water, inulin has potential as fiber source in
beverages (Silva, 1996; Nelson, 2001). But, Frank (2002) observed that inulin is
moderately soluble in water (maximum 10% at room temperature)
Kaur and Guptha (2002) observed that oligofructose provide about 30-50% of the
sweetness of the table sugar and also more soluble than sucrose about 80% in water at
room temperature (Frank, 2002).

2.4.2 VISCOSITY:
Viscosity is another property of oligosaccharides that provides rheological change
in food systems. Generally, as the molecular weight or chain length of the
oligosaccharides increases, the viscosity of the solution with oligosaccharides increases.
This property depends on parameters like concentration of oligosaccharides in solution,
solution temperature, pH, shear conditions of processing and ionic strength. Primarily,
long chain polymers such as inulin, bind significant water and exhibit high solution
viscosity.
However, in general, highly soluble oligosaccharides, those that are lower
branched or are relatively short chain oligosaccharides have low viscosities. These low
viscosity oligosaccharides (ex: oligofructose) are generally used to modify texture or
rheology, manage water migration, influence the colligative properties of the food system
and improve the marketability of the food product as a health-promoting or functional
food product.
Wendy et al., (2005) indicated that thickened beverages made with inulin are
acceptable and enhanced fiber content in the juice with little effect on sensory
characteristics.
2.4.3 GELATION:
Gelation is an important attribute of some oligosaccharide ingredients which add
form or structure to various food products. Gelation represents the association of polymer
units to form a network of junction zones. The gel formed by this process encapsulates
water and other components in solution to form a firm 3-dimensional structure. Gel
formation depends on the type of oligosaccharide, its concentration, temperature,
presence of ions (for example calcium), pH and the presence of other rheology modifiers
in the food system. Particle gel forming polymers such as inulin and oligofructose are
typically used at much higher concentrations than the molecular gel forming gums, and
are used with the gums in systems to influence system rheology and overall texture.
Oligosaccharides in higher concentrations in liquids will give gel formation.
According to Frank (1993), when inulin mixed with aqueous liquids, it forms
gels with short spreadable texture, which can be easily be incorporated in to foods to
replace fat up to 100 per cent.
Spiegel et al.,(1994) identified inulin and stated that it is responsible for as being
creamy appearance in yoghurt having a less chalky and more creamy texture and was
sweeter with less sour / fermented taste and after taste.

2.4.4 WATER BINDING / HOLDING CAPACITY:

The oligosaccharides interact with water differently, dictating how the
oligosaccharides are used and how they function in a food system. This interaction is
generally described as water uptake, hydration, adsorption, absorption, binding or holding
with two most common being water-binding capacity (WBC) and water-holding capacity
(WHC). WHC refers to the amount of water, the gel system retains within its structure
without pressure or stress, while WBC refers to the amount of water the gel system
retains after it is stressed, as following centrifugation. The WBC of oligosaccharides
likely has greater practicality, because food manufacturing / processing typically uses
some form of physical stress (for example, extrusion, mixing or kneading,
homogenization, and so on). The oligosaccharide source does not directly influence the
WBC, but rather the source determines the physicochemical properties of the
oligosaccharide, such as chain length, particle size, and porosity. These properties in turn
influence the WBC and its use and the conditions of its use in food development.
However, other factors in the food system can also influence WBC, such as pH, ionic
strength, concentration of the fiber component and interaction with other water binding
ingredients (that is, sugar, starches, and so on).
Schaller and Smith (1999) reported that ice crystals formation was reduced
when 50% of the corn syrup was incorporated with inulin in ice cream during thermally
abusive storage conditions by binding the water molecule.
Terry et al.,(1999), observed that yoghurt made with 10 percent inulin with a
degree of polymerization of 12-16 was found to increase firmness and decrease syneresis
compared to yoghurt made with controls (no inulin).

2.4.5 EMULSIFICATION:
Many dietary oligosaccharides are fat and / or oil dispersible and some also bind
oil. The oil binding capacity is in part related to its chemical composition but is more
largely a function of the porosity of the oligosaccharide structure rather than the affinity
of the molecule for oil. By hydrating oligosaccharides with water, the water occupies the
pores, significantly reducing oil binding. This technique is used successfully with batters
and spreadings and film coatings to reduce the oil uptake during frying operations, and
reduces the total fat content of the final food product, enhancing crispiness.
Frank, (2002) used inulin in preparation of table spreads for the purpose of fat
emulsion at about 2-10 per cent level.

2.4.6 CATION-EXCHANGE CAPACITY:

Some oligosaccharides from fruit and vegetable have cation exchange capacity
(CEC) from unmethylated galacturonic acid residues and phytic acid from cereal food is
also able to bind cations such as calcium, cadmium, zinc and copper (with this property
cations will be transported from small intestines to large intestines). The type of
oligosaccharide, the system pH, ionic strength and the chemical nature of the cation
influence the CEC (Thibault et al., 1992). However the effect of pH dependent. These
primary functional properties of several isolated oligosaccharide sources provide the
means to make high-fiber foods with high eating quality.

2.5 HEALTH BENEFITS OF OLIGOSACCHARIDES:

A wide variety of oligosaccharide components can provide beneficial
physiological influences and have several health-promoting implications
2.5.1 Effect on Cancer:
In a review of possible mechanistic effects for colon cancer inhibition Reddy
(1999) has emphasized the importance of examining both probiotic and prebiotic activity
and the possible synergistic effect when both are used together.
There is evidence that increasing the number of bifidobacteria in the colon by
introducing prebiotics and reducing intestinal pH can have direct impact on carcinogenic
substances in the large intestines (Goldin and Gorbach, 1980).

Prebiotics will encourage the bacteria (lactic acid bacteria) multiplication. In

addition to lactic acid production the bacteria also produce short chain fatty acids (acetic,
palmetic and butyric acid) which prevent growth of putrefactive bacteria (Rasic, 1983).
Butyric acid has been shown to increase apoptosis in human colonic tumor cell
lines (Hague et al., 1993).
Buddington et al., (1996) further observed that significant reduction in
nitroreductase (carcinogenic substance) activity when used 4 gm/d dietary supplement of
fructo-oligosaccharides.

Rowland et al., (1998) reported that feeding of inulin and oligofructose reduced
the activity of enzymes implicated in carcinogenesis and decreased the incidence of
tumors after exposure to known carcinogens (Kato, 2000).

The synthetic oligosaccharides decreased colonic tumors in transgenic mice prone

to develop aberrant crypt foci, a phenomenon associated with an increased activity of gut
and lymphoid tissue (Pool et al., 1993).
 Delzenne, (2003) observed an increase in apoptotic index and a decrease in
aberrant crypt foci in colon of rats receiving 25-100g inulin type
fructooligosaccharides/kg diet.

2.5.2 Effect on Triglyceride Metabolism

Kaur et al., (1988), observed in rats that there was significant reduction in
triglyceride content of blood and liver, when chicory roots (inulin) was included in the
diet.
The activity of lipogenic enzymes were found to be lower in livers of rats, fed
with Oligofructose when compared with control, suggesting that oligofructose feeding
could decrease lipogenic flux and thus liver very-low-density lipoprotein (VLDL)
secretion capacity (Gibson 1990; and Arbeeny et al., 1992).

The oligofructose, a mixture of nondigestible and fermentable fructans when fed

to rats at 10 per cent level triacylglycerol content in VLDL decreased (Fiordaliso et al.,
1995).

The triacylglycerol lowering action of oligofructose was reported to be due to a

reduction in the novo fatty acid synthesis by the liver through inhibition of lipogenic
enzymes (namely acetyl-CoA carboxylase, fatty acid synthatase, malic enzyme, ATP
citrate lyase, and glucose-6-phosphate dehydrogenase) (Kok et al., 1996) regulated only
through modification of gene expression.

Kok et al., (1998) stated that when rats fed with lipid rich diet containing
100g fructo-oligosaccharides / Kg, a decrease in triacylglycerolaemia occurred without
any protective effect on hepatic triglycerol accumulation and lipogenesis.

Jackson et al., (1999) reported that daily addition of 10g inulin in the diet
significantly reduced the fasting insulin concentrations in 54 healthy middle aged men
and women during the 8 week test period.
 Several non-digestible but fermentable dietary oligosaccharides are able to
regulate lipemia and triglyceridemia in both humans and animals (Delzenne and Nadine,
2001).
In non-obese rats and hamsters, Delzenne and Williams (2002) observed a
decrease in hepatic and serum triglycerol, when inulin type fructans were added to high
carbonate diet at concentrations of 25-100g/kg for several weeks.

2.5.3 Effect on Mineral metabolism

Non digestible oligosaccharides have been found to stimulate absorption of
several minerals and to improve mineralization of bone.
Ohta et al., (1993) reported that the absorption effect increased with an increase
of fructooligosaccharides dosage and at the 15 per cent fructooligosaccharides level
showed highest absorption rate was shown in rats.
Chronic supplementation of inulin or oligofructose in diets decreased or prevented
the loss of calcium and phosphorous from the bones of gastrectomized rats (Ohta et al.,
1994) and the loss of bone mineral density by overectomized rats (Taguchi et al., 1994).

Coudray et al., (1997) studied the effect of inulin (40g/d) on absorption of body
calcium, magnesium, iron, and zinc in healthy adults and found that inulin increased
calcium absorption and had no effect on other minerals metabolism. Vanden et al.,
(1999) also reported an increased absorption of calcium in adolescents on oligofructose
ingestion:

In a study in post-menopausal women, it was shown that short chain fructo-

oligosaccharides given at the dose of 10g/day for 5 weeks increased magnesium
absorption (+12.5%) and the plasma Mg level (Tahiri et al., 2001) . where as in girls, an
increase in Ca absorption was observed in girls at menarche who had received 5 g fructo-
oligosaccharides /day (Griffin et al., 2002) .

Intake of prebiotics acidifies the intestinal contents (by producing short chain
fatty acids) which solubilizes minerals (mainly Ca and Mg salts) (Coudray et al., 2003).
 The increased presence of butyrate, which is a selective source of energy for the
intestinal epithelial cells, improves the absorptive capacity of the mucosa. Both the
phenomena are thought to be at the basis of the repeatedly observed increase in mineral
absorption from food in human volunteers (Coudray et al., 1997; Griffin et al., 2002).

2.5.4 Effect on Immunity

Causey et al., (1998) provided evidence that long chain inulin stimulated the
human immune system by binding to specific lectin-like receptors on leukocytes.
Kelly et al., (1998) studied the impact of inulin on immune system by using
B6C3F1 mice to examine the immunomodulating properties of a longer-chain inulin with
an average degree of polymerization of 22 units and reported an increase in the speed of
macrophage response.

Intestinal immunity was enhanced by supplementing dietary oligosaccharides.

Synthetic fructans (oligosaccharides) at a dose of 50-80 gm/kg diet increased peyers
patches in mice and at a dose of 10mg/kg, promoted caecal and colonic macrophages
level in rats (Schley & Field, 2002).

Due to prebiotic effects of oligosaccharides, bifidobacteria are able to stimulate

production of cytokine (tumor necrosis factor and interleukin 6) and reactive
molecules (NO, H2O2) by macrophages in vitro. Overall improvement in immunity has
also been reported in humans with a diet enriched in short chain fructo-oligosaccharides
(Saavedra et al., 2002).

2.5.5 Effect on Constipation

Constipation is an ailment encountered in elderly people. Many factors contribute
to the development of constipation with aging such as changes in diet and fluid intake,
intake of drugs, decrease in the intestinal motility physical inactivity etc.
 Kameoka et al., (1986) reported that when patients fed 6 or 12g of FOS daily, the
average number of days between stools gradually reduced to a 1-day interval of normal
value.
Hidaka et al., (1991) observed that administration of oligofructose relieved
constipation.
Several studies in humans suggested that fermentation of oligosaccharides
stimulate colonic motility (Roberfroid 1993). Lactulose is used predominantly as a
pharmaceutical in the treatment of constipation and hepatic encephalopathy (Clausen and
Mortensen, 1997).
Hond et al., 2000 observed a significant increase in stool frequency in healthy
volunteers having one stool every 2-3 days by including inulin with degree of
polymerization of more than 25 in the diet. Den et al., (2000), observed increased stool
frequency and faecal bulk when subjects were fed with 15 g/d inulin and a placebo o
15g/day sucrose.

2.5.6 Effect on Diabetic

Benefits of oligosaccharides in diet may go beyond a direct effect on serum
glucose on insulin levels. Oligosaccharides as dietary fiber improves glucose tolerance
and beneficial in the treatment of both type II diabetics and I. Fiber property has been
shown to slow down the digestion of oligosaccharides in the intestines and in turn
decrease the post parental glucose uptake.
Oku et al., (1984) reported that in streptozotocin treated rats (diabetic), ingestion
of a diet containing 20 per cent oligofructose for 2 months has decreased postprandial
glycemia.

In diabetic subjects, taking 8 g of oligosaccharides /day for 14 days led to a

decrease in fasting blood glucose (Yamashita et al., 1984).

Inulin containing food products may be beneficial as it influences by reducing

glucose uptake and there by reducing post prandial hyperglycemia (Kim et al., 1998).
 Feeding fructo-oligosaccharides at 100g/kg diet to male wistar rats for 30 days
reduces postprandial glycemia and partially restores insulin secretion. This effect is due
to the higher secretion of glucagone like peptide in portal vein due to increase in
production of this peptide in colon (Kok et al., 1998).

Oligofructose given at the dose of 10% in the diet of rats for a period of 30 days,
reduced postprandial glycemia by 17% and insulinemia by 26% respectively (Kaur and
Gupta, 2002).

2.5.7 Effect on Cardiovascular Diseases:

By feeding the oligosaccharides, the process of fermentation is increased in the
gastrointestinal tract there by increase in production of short chain fatty acids. The high
proportion of propionate produced in the caecum (which reaches the liver through the
portal vein) is a key factor in reduction of hepatic triglycerol synthesis.

Fiordaliso et al., (1995) and Gupta et al., (1996) reported that in short term (3
weeks) and long term (16 weeks) administration of fructan type oligosaccharides to rats
decreased total cholesterol level in blood serum.
Davidson et al.,(1998) reported lowering in both total and LDL serum cholesterol
after feeding inulin (18g/day for 3 weeks) to slightly hypercholesterolemia human
volunteers.

According to Demigne et al., (1999), short chain fatty acids (SCFA) are known to
modulate hepatocyte metabolism; acetate may be considered to be both a lipogenic and
cholestrogenic substrate whereas propionate acts as an inhibitor of hepatic lipid synthesis.

Delzenne et al., (2002) reported modest reduction in total cholesterol and LDL-
cholesterol by exhibiting prebiotic properties, when diet was supplemented with fructo-
oligosaccharides (dose 8-20g/day).
 According to Delzenne (2003), oligosaccharides have been shown to modulate
hepatic lipid metabolism in rats and hamsters with an effect on triglycerol accumulation
and/ or serum lipids.

2.5.8 Effect on Uremia and Nitrogen/ Urea Disposal

Feeding rats, a diet supplemented with inulin and oligofructose (10%) for a few
weeks, decreased uremia in both normal and nepherectomized rats (Delzenne et al., 1995
and Younese et al., 1997).

Dietary inulin effectively enhanced faecal nitrogen excretion and reduced renal
excretion of nitrogen in rats (Younese et al., 1995).

Inulin and oligofructose serve as an energy source for intestinal bacteria. When
the fermentable carbohydrate intake is high the amount of ammonia required to sustain
maximal bacterial growth may become insufficient and blood urea is then required as a
ready source for bacterial protein synthesis in the caecum (Younese et al 1995 and Tetens
et al., 1996).

The consumption of non-digestible carbohydrates had resulted in a higher fecal

excretion of nitrogen (Mortensen, 1992). In addition to increasing total nitrogen transfer
to colon, it is important to limit the formation of ammonia and various end products of
protein catabolism which have been proposed as causative risk factors for colonic
carcinogenesis in the distal part of the large bowel (Lupton and Marchand, 1989).

Chicory root extract has also been reported to inhibit xanthine oxidase, an enzyme
involved in the synthesis of uric acid from purines and thus can be useful in relief from
gout.

2.5.9 Suppression of Pathogens / Gastrointestinal Health

 The prebiotics are thought to decrease incidence of intestinal disease in humans.

Oligosaccharides in the gastrointestinal tract can promote the specific organisms growth

and suppress the non-indigenous organisms.

This mechanism may include competition between the indigenous flora and non-

indigenous organisms for nutrients present in limited quantities. Indigenous flora

produces metabolites (SCFA), which inhibit multiplication of non-indigenous organisms

also create adverse conditions to non-indigenous organisms. Any one or all of these

mechanisms may be responsible for suppression of pathogens in gut (Fuller, 1992).

Masai et al., (1987) observed significant increase in number of bifidobacteria and

a decrease in number of clostridia when rats supplemented with soybean oligosaccharides

at a dose of 10g given twice daily for 3 weeks.

Prebiotics may suppress the growth of pathogens for overall beneficial health of

host (Roberfroid, 2001).

For the galacto-oligosaccharides, Tanaka et al,. (1983) and Ito et al,. (1993) have

reported evidence for an increase in both bifidobacteria and lactobacilli and decrease in

the pathogenic bacterial count for doses ranging from 3 to 10 g/day.

2.6 APPLICATION OF OLIGOSACCHARIDES IN FOOD INDUSTRY

The functional foods, in addition to their basic nutritive value being will contain
the proper balance of ingredients which will help us to function better and more
effectively in many aspects of our lives including helping us directly in the prevention
and treatment of illness and disease (Goldberg, 1994). The human nutrition has moved
from a focus on the prevention of nutrient deficiencies to an emphasis on health-
maintenance and reduces risk of chronic diseases. A variety of foods and their
components are emerging as factors capable of modifying growth, development,
performance and disease resistance.
The increasing awareness of nutrition, health and quality food consciousness of
consumers and the keen competition in the market, compel the food industry to search for
those ingredients which impart specific functionalities to food products while reserving
or enhancing the nutritional quality of food stuff in order to sell their products profitably.
Different functional attributes of oligosaccharides are due to the difference in
their chain length, their functional properties they can be used in different food industries.
Application of oligosaccharides in different food industries given in Table 3.

Table 4: Applications of oligosaccharides in different food industries

Application Functionality
Dairy Products Sugar and fat replacement, synergy with
sweeteners, Body and mouthfeel, foam
stability, fiber and prebiotic.
Frozen desserts Sugar and fat replacement, texture and
melting, synergy with sweeteners, fibre and
prebiotic.
Table spreads Fat replacement, texture and spread ability,
emulsion stability, fibre and prebiotic.
Baked goods and breads Fibre and prebiotic, moisture retention and
sugar replacement.
Breakfast cereals Fibre and prebiotic, crispness and
expansion.
Fillings Sugar and fat replacement, Texture
improvement
Fruit Preparations Sugar replacement, synergy with
sweeteners, body and mouthfeel, fibre and
prebiotic.
Salad dressings Fat replacement, body and mouthfeel
Meat products Fat replacement, texture and stability, and
 fiber
Diabetic products and meal replacers Sugar and fat replacement, synergy with
sweeteners, low caloric value, body and
mouthfeel, fibre and prebiotic.
Chocolate Sugar replacement, fiber, and heat
resistance
Tablets Sugar replacement, fibre and prebiotic

With increasing consumer health consciousness and also increasing awareness of

physiologically functional foods, the future for products containing oligosaccharides
seems to be greatly promising. These functional oligosaccharides may play an important
role especially for the reduction of lifestyle-related diseases in the near future as well as
the improvement of human health.
 CHAPTER - III

MATERIALS AND METHODS

The work was carried out in Department of Livestock Products Technology, College

of Veterinary Science, Rajendranagar and Post Graduate Research Centre of Home Science

College, Hyderabad.

3.1 Procurement of raw materials

The following cereals and pulses were procured from the local markets of Hyderabad.

Cereals Pulses
Sorghum Green gram
Rice Soya bean
Barley French beans
Ragi Lathyrus sativus
Wheat Cowpea
Bajra Pea
Maize Black gram
Pigeon
Chick Pea

3.1.2. Chemicals

The oligosaccharides standards were procured from Sigma Aldrich Chemicals

(U.S.A)

Standard oligosaccharides
Raffinose
Stachyose
Maltotriose
Maltotetriose
Maltopentaose
Maltohexaose
Maltoheptaose

HPLC grade Acetonitrile was procured from the Qualigens Fine Chemicals,

Mumbai.
 HPLC grade water was prepared at the Department of Livestock Products

Technology by triple glass distillation unit.

3.1.3 Apparatus used in the experiments

Mortar-pestle.

Weighing balance (Adairdutt, Model- 120).

Syringe filter.

Syringe filter membranes (cellulose acetate) (0.25).

HPLC Waters model No.2695.

Detector (Refractive Index detector of Waters No.2414).

3.2 Sample preparation

The sample preparation sequence is outlined in the following flow diagram

(3.2.1 and 3.2.2). The prepared samples was used for analysis by HPLC with 70 per

cent acetonitrile as eluent as described by Kamada et al., (2002) and the standards of

oligosaccharides (Sigma Chemicals, USA) were used for quantification of different

fructo-oligosaccharides in the samples of cereals and pulses. Empower software was

used to integrate the peaks for the purpose calculation of peak areas.
3.2.1 Fresh sample

Collection of commonly available cereals and pulses (as listed under 3.1)

Grinding sample into fine uniform powder

Addition of water [1:40 (w/v)]

Heat treatment (water bath at 980C / 2 hrs)

Cooling to 45 0C

Initial filtration (soy cloth)

Secondary filtration (ordinary filter paper)

Tertiary filtration (what man No.42 paper)

Final filtration through Millipore (syringe) membrane (0.25)

Analysis for concentration of oligosaccharides by HPLC

3.2.2 Germinated sample

Collection of commonly available cereals and pulses (as listed at 3.1)

Steeping and germination for 48 hours

Pulverizing

Addition of water [1:40 (w/v)]

Heat treatment (water bath at 980C / 2 hrs)

Cooling to 45 0C

Initial filtration (soy cloth)

Secondary filtration (ordinary filter paper)

Tertiary filtration (what man No.42 paper)

Final filtration through Millipore (syringe) membrane (0.25)

Analysis for concentration of oligosaccharides by HPLC

3.3 Analysis

3.3.1 Moisture estimation

The moisture content of the samples was determined as per the method

described by AOAC (1984).

Procedure:

Approximately 10 gm of sample was taken into a petri dish with lid and the

extract weight was noted down (w1).

The sample was dried in a hot air oven at 100 to 1100C till constant weight

was obtained.

The sample was cooled in a dessicator and the final weight was taken (w2).

Calculation:

Initial weight (w1) Final weight (w2)

Percent moisture = ---------------------------------------------------- X 100
Initial weight (w1)
3.3.2 HPLC analysis

HPLC Parameters:

(1) Pump: Isocratic pump No.2695 separation module.

(2) Detector: Refractive Index detector of waters No.2414, internal

temperature of 40 0C.

(3) Injection:

Injection volume 10L

Auto sampling for 100 vials

(4) Mobile phase:

Acetonitrile and water [70:30 (v/v)]. Both were of HPLC grade.

(5) Column:

Waters Spherisorb (5m), ammonia column.

Dimensions: 4.6 mm x 250 mm.

Temperature: 650C

Flow rate: 1.6 ml / min.

(6) Run time: 30 min.

(7) Software: Empower

 3.3.2 Calculation of concentration of oligosaccharides

Oligosaccharide concentration =

Sample peak area Standard weight

= ------------------------ X ---------------------- X Purity of standard (100%)
Standard peak area Sample weight

3.4 Statistical analysis

Statistical analysis was carried out by the procedures laid down by Snedecor and

Cochran (1989). The data was tabulated and subjected to statistical analysis at the end of

the study for Mean, Standard Deviation (SD). For standardizing HPLC analysis of

oligosaccharides five replications were taken and from which mean and SD were

calculated. For executing sample analysis, Mean of two replications was presented in

results.
 CHAPTER IV

RESULTS AND DISCUSSION

Oligosaccharides are the functional food ingredients used in different products as

prebiotics to promote the health of host. The probiotic market is expanding rapidly and the

demand for novel compounds may not be limited to oligosaccharides alone but expandable

to different other functional food ingredients.

In the present study, oligosaccharides levels were estimated in cereals and pulses.

The effect of germination on oligosaccharides content in cereals and pulses was also

estimated. The results are presented under the following sections.

4.1 Estimation of moisture content and dry matter in cereals and pulses.

4.1.1 Estimation of moisture content in raw cereals and pulses

4.1.2 Estimation of moisture content in germinated cereals and pulses.

4.2 Standardizing HPLC analysis of oligosaccharides.

4.3 Estimation of oligosaccharides in samples

4.3.1 In fresh samples (raw)

4.3.2 In germinated samples

4.1 Estimation of Moisture content

The moisture content in raw and germinated cereals and pulses was estimated

according to AOAC procedure as mentioned at 3.3.1

4.1.1 Estimation of Moisture content in raw cereals and pulses

Moisture content is an important parameter, which effects the final calculation of

the oligosaccharide content in dried cereals and pulses. In each product the moisture
content variation is expected. Therefore, the expression of oligosaccharide content on total

weight of the seed is not appropriate when comparing data with other sources of

oligosaccharides. Since the oligosaccharides are present as minor fractions of total seed

contents, the moisture content will have major impact on the oligosaccharide calculations.

The results of moisture content of cereals and pulses obtained are presented in Table 5.

Table 5: Moisture content in raw cereals and pulses

Sl. No Name of sample Moisture content Dry matter

% %
1. Green gram 10.45 89.55
2. Sorghum 10.90 89.10
3. Chick Pea 9.71 90.29
4. Maize 14.90 85.10
5. Barley 9.71 90.29
6. French bean 12.40 87.60
7. Ragi 13.12 86.88
8. Lathyrus sativus 10.60 89.40
9. Wheat 12.89 87.11
10. Cowpea 12.50 87.50
11. Pisum Sativum (pea) 16.05 93.95
12. Black gram 09.60 90.40
13. Pigeon pea 13.55 86.45
14. Bajra 11.90 88.10
15. Soya 8.19 91.81
16. Rice 13.76 86.24

The observed results are in confirmation with the published results Aykroyd (1966)

. The moisture content varied from 8.19% (in soya) to 16.05% (in green pea).
4.1.2 Moisture content in germinated cereals and pulses

Usually during germination, seeds absorb water by a process called imbibition.

After water entering into seed coat, seed swelling starts and initiates germination.

During germination, water intake of each seed varies; the dry matter content

reduces as moisture content increases. So moisture content in germinated seeds was carried

and presented data in Table-6. It may be observed that the moisture content varied among

different germinated seeds and moisture content in germinated pulses was higher than the

cereals. It may due to higher hydration capacity of proteins presented in higher proportion

in pulses.

The germinated whole seeds were subjected to fine grinding to get uniform

sampling and AOAC procedure was followed as mentioned in para 3.3.1

Table 6: Moisture content in germinated cereals and pulses.

Sl. No Name of sample Moisture content Dry matter

% %
1. Green gram 64.12 35.88
2. Sorghum 34.29 65.71
3. Chick Pea 57.46 42.54
4. Maize 33.26 66.74
5. Barley 32.18 67.82
6. French bean 57.14 42.86
7. Ragi 36.45 63.55
8. Lathyrus sativus 56.39 43.61
9. Wheat 43.51 56.49
10. Cowpea 58.47 41.53
11. Pisum sativum (pea) 58.27 41.73
 12. Black gram 54.63 45.37
13. Pigeon pea 56.67 43.33
14. Bajra 34.95 65.05
15. Soya 62.10 37.90
16. Rice 33.17 66.83

4.2 Standardizing HPLC analysis.

Generally, analysis of sugars by HPLC gives poor quantification. The peak areas of

repetitive injections of either samples or standard sugars differ to some extent. The errors

associated with repetitive injection are: (a) the precise injection of small quantities of

samples is very difficult and (b) detector responses fluctuate with time. In the following

experiment an auto sampler was used to avoid first error and an experiment with different

levels of standards was carried out to check the second factor. Precisely 10.0l of water

standard solutions containing different concentrations (1.2, 1.0, 0.7, 0.5, 0.4 % (w/v)) were

injected five times into HPLC with parameters given at 3.3.2

The data obtained is presented below with respect to standard:

4.2.1. Raffinose

The standard solution of raffinose at different concentrations was injected for five

times. Raffinose was eluted at the retention time as shown in the chromatogram - 1. The

Mean, Standard Deviation and coefficient of variation of the observed concentration are

presented in Table 7. Coefficient of variation for 1.2% concentration was less and

considered as standard for further calculations.

Table 7: Observed concentration of raffinose by HPLC analysis

Sl. No. Known Observed concentration

Concentration (%) Mean* Standard Co-eff.
Deviation Variation
 1 1.2 1.196 0.009 0.752
2 1.0 0.992 0.008 0.806
3 0.7 0.693 0.015 2.164
4 0.5 0.492 0.013 2.642
5 0.4 0.394 0.011 2.791
* - Mean of five observations. Hello

4.2.2. Stachyose

The standard solution of stachyose at different concentrations was injected for five

times. Stachyose was eluted at the following retention time as shown in the chromatogram -

2. The Mean, Standard Deviation and coefficient of variation of the observed concentration

are presented in Table -8. Coefficient of variation for 1.2% concentration was less and

considered as standard for further calculations.

Table 8: Observed concentration of stachyose by HPLC analysis

Sl. No. Known Observed concentration

Concentration (%) Mean* Standard Co-eff.
Deviation Variation
1 1.2 1.186 0.009 0.758
2 1.0 0.989 0.008 0.808
3 0.7 0.693 0.012 1.731
4 0.5 0.492 0.007 1.422
5 0.4 0.394 0.006 1.522
* - Mean of five observations.

4.2.3. Maltotriose

The standard solution of maltotriose at different concentrations was injected for five

times. Maltotriose was eluted at retention time as shown in the chromatogram - 3. The

Mean, Standard Deviation and coefficient of variation of the observed concentration are

presented in Table 9. Coefficient of variation for 1.2% concentration was less and

considered as standard for further calculations.

 Table 9: Observed concentration of maltotriose by HPLC analysis.

Sl. No. Known Observed concentration

Concentration (%) Mean* Standard Co-eff.
Deviation Variation
1 1.2 1.194 0.006 0.502
2 1.0 0.991 0.007 0.706
3 0.7 0.694 0.010 1.440
4 0.5 0.492 0.007 1.422
5 0.4 0.397 0.006 1.511
* - Mean of five observations.

4.2.4. Maltotetraose

The standard solution of maltotetraose at different concentrations was injected for

five times. Maltotetraose was eluted at the retention time as shown in the standard

chromatogram - 4. The Mean, Standard Deviation and coefficient of variation of the

observed concentration are presented in Table -10. Coefficient of variation for 1.2%

concentration was less and considered as standard for further calculations.

Table 10: Observed concentration of maltotetraose by HPLC analysis

Sl. No. Known Observed concentration

Concentration (%) Mean* Standard Co-eff.
Deviation Variation.
1 1.2 1.193 0.006 0.502
2 1.0 0.992 0.009 0.907
3 0.7 0.693 0.007 1.010
4 0.5 0.494 0.009 1.821
5 0.4 0.396 0.007 1.767
* - Mean of five observations.
4.2.5. Maltopentaose

The standard solution of maltopentaose at different concentrations was injected for

five times. Maltopentaose was eluted at the retention time as shown in the standard

chromatogram-5. The Mean, Standard Deviation and coefficient of variation of the

observed concentration are presented in Table 11. Coefficient of variation for 1.2%

concentration was less and considered as standard for further calculations.

Table 11: Observed concentration of Maltopentaose by HPLC analysis

Sl. No. Known Observed concentration

Concentration (%) Mean* Standard Co-eff.
Deviation Variation
1 1.2 1.196 0.007 0.585
2 1.0 0.984 0.005 0.711
3 0.7 0.697 0.008 1.147
4 0.5 0.495 0.009 1.818
5 0.4 0.398 0.008 2.010
* - Mean of five observations.

4.2.6. Maltohexaose

The standard solution of maltohexaose at different concentrations was injected for

five times. Maltohexaose was eluted at the following retention time as shown in the

standard chromatogram 6. The Mean, Standard Deviation and coefficient of variation of

the observed concentration are presented in Table 12. Coefficient of variation for 1.2%

concentration was less and considered as standard for further calculations.

Table 12: Observed concentration of maltohexaose by HPLC analysis

Sl. No. Known Observed concentration

Concentration (%) Mean* Standard Co-eff.
Deviation Variation.
1 1.2 1.192 0.009 0.755
 2 1.0 0.995 0.007 0.904
3 0.7 0.690 0.10 1.449
4 0.5 0.494 0.008 1.619
5 0.4 0.396 0.006 1.515
* - Mean of five observations.

4.2.7. Maltoheptaose

The standard solution of maltoheptaose at different concentrations was injected for

five times. Maltoheptaose was eluted at the following retention time as shown in the

standard chromatogram 7. The Mean, Standard Deviation and coefficient of variation of

the observed concentration are presented in Table 13. Coefficient of variation for 1.2%

concentration was less and considered as standard for further calculations.

Table 13: Observed concentration of maltoheptaose by HPLC analysis

Sl. No. Known Observed concentration

Concentration (%) Mean* Standard Co-effe.
Deviation Variation.
1 1.2 1.198 0.008 0.667
2 1.0 0.997 0.008 0.802
3 0.7 0.696 0.008 1.149
4 0.5 0.494 0.007 1.417
5 0.4 0.395 0.007 1.772
* - Mean of five observations.

The results obtained by repetitive injections of all standard sugars at different

concentrations has shown that there is no variation in the observed concentration indicating

that detector response is valid at various concentrations of sugars. Such validation of

detector response is essential to extrapolate the results in samples with different levels of

sugars.
4.3 Estimation Oligosaccharides in Samples

After standardizing the quantification of standard sugars by HPLC, the samples

extracted from cereals and pulses were injected. The obtained peak areas for different

sugars were integrated and concentration was calculated on dry matter basis taking into

account the moisture content of cereals and pulses estimated (Table 5).

The results of oligosaccharide content in fresh cereals and pulses on dry matter

basis are presented in Table -14. Very insignificant variation was observed in

oligosaccharide content between the two replications.

Table 14: Oligosaccharides content in fresh/ raw cereals and pulses

S.No. Sample Oligosaccharides mg/gm of dry matter *

Sta Raf M.tri M.tet M.pen M.hex M.hep
1 Green gm 1.2493 - - - - 3.9781 -
2 Sorghum Tr - 0.5333 - - - -
3 Chick pea - - 0.3908 - - - -
4 Maize - - 0.5267 - - - -
5 Barley Tr Tr - - - - -
6 Rajma - - 0.486 - - - -
7 Ragi - - - - - - -
8 Lathyrus - - 0.5882 - - - -
9 Wheat Tr 5.2 - - - - -
10 Cow pea - 4.261 - - - - -
11 Pisum - 2.046 - - - - -
12 Black gm 8.1024 Tr - - - - -
13 Pigeon - 1.2801 - - - - -
14 Bajra - - - - - - -
15 Soya - 3.0401 - - - - -
16 Rice - - - - - - -
* - the values presented are means of two replications.
Tr- Traces, Sta Stachyose, Raf- Raffinose, M. tri- Maltotriose, M.tet- Maltotetraose,
M.pen- Maltopentaose, M.hex- Maltohexaose, M.hep- Maltoheptaose.

Earlier research workers who estimated sugars, reported mostly on the

concentration of raffinose and stachyose in cereals and pulses. The oligosaccharides in

pulses determined to asses the degree of flatulence that they may cause after ingestion due

to their non-digestibility.

In a number of mature leguminous seeds, the verbascose contents were higher than

that of raffinose ( Schweizer et al.,1978; Quemener and Brillouet, 1983).

The concentrations of Stachyose and galactinol and the measured equilibrium

constant for the galactosylation of raffinose would indicate that stachyose needs to be

spatially separated within seed tissues for its synthesis to continue (Kandler and Hopf,

1980). The synthesis of Stachyose appeared to be limited by the availability of raffinose.

Green gram

In the present study, stachyose (1.24mg/gm) and maltohexaose (3.97mg/gm)

content were observed in green gram as presented in Table -14. In a similar study, Kuo

et.al, (1988) reported 16.7mg of stachyose / gram of defatted dry matter, where he used

80% ethanol for extracting oligosaccharides. His study did not indicate presence of

maltohexaose. It appears that the method of ethanol and water extraction may have some

influence in the determination of concentration of stachyose due to its solubility.

Maltohexaose was identified only in green gram. It can be used as a marker to identify

adulteration of green gram flour in other flours of pulses.

Sorghum

In the present study, the traces of stachyose and maltotriose were observed (Table -

14). Stachyose was observed in traces and 0.533 mg of maltotriose was observed per 1 gm

of dry matter. The above results are coinciding with the results of Kuo et.al, (1988) with

regard to stachyose only; where they reported that sorghum contains traces of stachyose

and raffinose. It appears that they did not attempt to detect the level of maltotriose and its

level was not reported.

Chick pea

In chick pea, maltotriose content was observed in higher proportion (0.390 mg/ gm)

and stachyose, raffinose peaks were slightly recognizable but are not considerable, when

compared with the other peaks. The above results were in contrast with the results reported

by Sosulki et al., (1982), where he observed raffinose content of 0.45 per cent and

stachyose content of 1.72 per cent. He used gas chromatography for analysis and diluted

samples by 20 times with 80 per cent ethanol. It appears that the method of analysis used to

quantify sugars or the variety of chick pea may have influenced the observed concentration

of sugars. Further studies may be required to confirm the inconsistency in reported results.

Maize

In maize, maltotriose (0.526mg/ gm), negligible peaks of raffinose and absence of

stachyose were observed as shown in Table-14. The above results were similar to the

results reported by Kuo et al., (1988), with regard to stachyose only, where they reported

the absence of stachyose, but detected raffinose at a level of 2.1mg/ gram in maize defatted

meal.
Barley

In barley, the traces of stachyose and raffinose were observed as shown in Table

14. Kuo et al., (1988) observed traces of stachyose but raffinose was about 6.3mg/ gm of

defatted meal. These results are contrary to the present study with regard to raffinose. It

may due to the difference in sample extraction methods used for quantification and also

due to using of defatted sample by the earlier researchers. It was reported by Mayer and

Poljkoff (1966) that raffinose is a precursor for stachyose. In the present study, stachyose

content was also not in higher proportion assuming that raffinose may have converted into

stachyose.

Rajma

In Rajma only maltotriose was observed at a concentration of 0.486mg/gm of dry

matter (Table -14). No published literature on oligosaccharide content of rajma (French

bean) is available for comparison. No other oligosaccharide was noticed in Rajma samples.

Ragi and Bajra

In ragi and bajra no oligosaccharide content was observed. Published reports on

oligosaccharide content of Ragi and Bajra are also not available for comparison. Millets

reported to contain less than 1% unavailable carbohydrates (cellulose, hemicelluloses,

lignin, oligosaccharides and non cellulose.) matter (Kent, 1983). Therefore it may be

presumed that oligosaccharide content may be negligible in smaller millets like ragi and

bajra.

Lathyrus sativus (Kesari Dhal)

 It is cultivated in limited scale due to its toxic principles present in dhal. In

Lathyrus sativus, maltotriose content was 0.588 mg/ gm of dry matter. The sugar

composition of Lathyrus sativus was not reported for comparison. Since it contains, toxic

principle (amino propionic acid and amino propiono nitrite) responsible for lathyrism,

probably no research was done to analyse the oligosaccharide composition.

Wheat:

In wheat, stachyose was found in traces and raffinose content was 5.2 mg/ gm of

dry matter (Table -14). The results are slightly different compared to the data reported by

Kuo et al., (1988). They reported traces of stachyose and 7.0 mg/ gm of raffinose in

defatted meal of wheat. The slight difference may be attributed to defatting of the samples

used for analysis by the reference authors. Abou and Appolonia (1972) also observed

raffinose in wheat bran, embryo scutellum and aleurone and they stated that raffinose

appeared late in the development of wheat grain, but they did not mention the content in

their samples.

Cow pea:

In cow pea, raffinose content was 4.26 mg/ gm on dry matter basis (Table -14). In a

similar work Kuo et al., (1988) reported that cow pea contains raffinose of 3.7 mg/gm in

defatted meal. This slight deviation in the content of raffinose may be due to the difference

in regional and climatic conditions where crop was raised or may be due to the genetic

difference in the crop.

Pisum sativum (Green Pea):

 In green pea, the raffinose content observed was 2.046 mg/ gm on dry matter basis

(Table 14). This is in total contrast to the results reported by Kuo et al., (1988). The

authors observed stachyose content of 32.3 mg/ gm and raffinose content of 11.6 mg/ gm of

defatted meal. It may be mentioned that they used 80 % ethanol for dilution and sample

extraction.

It appears that the methods of ethanol and water extraction may have some

influence in the determination of concentration of sugar due to their solubility, especially

when the content of raffinose and stachyose are in large proportion. Pigman and Horton,

(1970) reported that solubility of raffinose and stachyose is optimum in water, if the

concentration is low. However, if the concentration is higher, the alcohol appears to be an

ideal solvent for extraction and proper quantification of raffinose and stachyose. Probably

alcohol may be a better solvent to break the complexes of protein and sugars and to release

the sugar for estimation.

Black gram:

In black gram, the raffinose was found in traces and stachyose content was 8.102

mg/ gm on dry matter basis (Table- 14). Nearly similar results were presented by Reddy

and Salunkhe (1980). In their experimental analysis, the stachyose content was 8.90.3

mg/ gm dry matter and raffinose was present in traces in defatted meal.

Pigeon Pea

In pigeon pea, only raffinose content was observed at concentration of 1.280 mg/

gm of dry matter. This result is close to the values reported by Iyengar and Kulkarni

(1977). They observed raffinose content of 1.05 per cent on total weight of sample. This

slight deviation may be attributed to the genetic variation of the plants.

Rice

In this study, no oligosaccharide was observed in rice sample as given n Table -14.

Similar results were reported by Kuo et al., (1988). They observed absence of stachyose

and raffinose in rice. The absence of oligosaccharides in rice may be due to high content of

starch.

Soya bean

In the present study, the presence of raffinose and absence of other oligosaccharides

was observed as given in Table - 14. Raffinose content of 3.04 mg/ gm on dry matter was

observed but these results were in contrast with results of Kuo et al., (1988). They reported

raffinose content of 12.6 mg/ gm in defatted dry matter. This deviation may be due to the

fat content of soy (nearly 21%), and extraction method in sample preparation, which effects

solubility of oligosaccharides.

4.3.2 Effect of germination on oligosaccharide content

In seeds, many metabolic changes take place during germination. There is a scope

for larger molecules to undergo breakdown reactions first. Therefore, to study the effect of

germination on oligosaccharide content of cereals and pulses, analysis was done after 48

hrs after soaking and germination.

The results of oligosaccharide content in germinated cereals and pulses on dry

matter basis are presented in Table -15.

Table 15: Oligosaccharide content in germinated cereals and pulses

Sl.No. Sample Oligosaccharide mg/ gm of dry matter*
Sta Raf. M.tri M.tet M.pen M.hex. M.hep
1 Green gram - - - - - - -
2 Sorghum - - 0.2928 - - - -
3 Chick pea - - - - - - -
4 Maize - - 0.2306 - - - -
5 Barley - - - - - - -
6 Rajma - - - - - - -
7 Ragi - - - - - - -
8 Kesari Dhal - - - - - - -
9 Wheat - - - - - - -
10 Cow pea - - - - - - -
11 Green peas - - - - - - -
12 Black gram - - - - - - -
13 Pigeon pea - - - - - - -
14 Bajra - - - - - - -
15 Soya - - - - - - -
16 Rice - - - - - - -
* the values presented are means of two replications.

From the data presented in Table -15, it can observed that there was total loss of

oligosaccharides during germination in almost all cereals and pulses except sorghum and

maize. Maltotriose present in chick pea, rajma, and lathyrus sativus completely

disappeared after 48 hours of germination. Complete loss of maltotriose may be due to

higher activity of enzymes breakdown in to simple sugars, whereas in sorghum and maize,

maltotriose decreased to 45.1% and 57.3% after 48 hours of germination respectively.

This may be due to the tough seed coat which may not allow more water and takes more

time for germination.

 Similar results were reported by Adjei et al., (1976) during germination, who stated

that seeds undergo marked metabolic changes and the reserve carbohydrates including the

oligosaccharides are hydrolyzed.

The content of oligosaccharides i.e. malto-oligosaccharides, stachyose, raffinose

might have decreased drastically due to their conversion in to simple sugars.

In the present study, the complete loss of raffinose in cowpea, Pisum sativum, black

gram, pigeon pea and soy was observed in germinated beans. This may be because of

hydrolysis of oligosaccharides by -galactosidase enzyme.

Supporting to observations, Reddy and Salunkhe (1980) indicate high activity of -

galactosidase in resting seeds and increase during the first two days of germination. Similar

results were reported by Silva and Luh (1979), where they observed complete

disappearance of raffinose and stachyose sugars in black eye and pink beans after

germination for four days.

East et al., (1972) and Hsu et al., (1973) also reported that raffinose and stachyose

in soybeans disappeared as a result of germination.

In a similar study by Kim et al., (1973), it was reported that about 70 per cent of the

raffinose and stachyose were removed from soy beans by combination of various

treatments, involving pH adjustments, soaking and germination.

Identified rich sources of oligosaccharides in black gram, soy, cowpea and

wheat may be used for extraction in their natural form. These extracted oligosaccharides

can be used as prebiotics in functional foods development.

 Overall, the present results indicate that certain pulses are good sources of

oligosaccharides such as green gram (stachyose and maltohexaose), cowpea (raffinose),

black gram (stachyose), soya (raffinose), green pea (raffinose), rajma (maltotriose) and

Lathyrus sativus (maltotriose) similarly certain cereals such as sorghum (maltotriose),

maize (maltotriose) wheat (raffinose) are also contain good quantities of oligosaccharides.

The above sources may be used to extract the oligosaccharides in their natural state by

membrane separation process as prebiotics similar to work reported in chicory root and

yakoon (Kamada et al., 2002).

 CHAPTER - V

SUMMARY AND CONCLUSION

The present study was carried out to estimate the oligosaccharide content in

dormant and germinated cereals and pulses and to study the effect of germination on

different oligosaccharides (stachyose, raffinose, maltotriose, maltotetraose,

maltopentaose, maltohexaose, maltoheptaose) in cereals and pulses. All the cereals and

pulses were collected from local super markets and standard sugars were procured from

Sigma Aldrich Chemicals (USA). Estimation, involved hot water extraction (98 oC/ 2

hrs) of oligosaccharides, pre-filtration (soy cloth), filtration ( membrane- 0.25) followed

by high performance liquid chromatography (HPLC) analysis, HPLC Waters model

No. 2695 with a refractive index (RI) detector (Waters No. 2414) and using

oligosaccharide standards as reference. Experiments were carried out in the laboratory of

Department of Livestock Products Technology, College of Veterinary Science and Post

Graduate Research Center, Acharya N.G. Ranga Agricultural University, Rajendranagar,

Hyderabad. The results drawn from this study are summarized here under.

Initially powdered samples of dormant and germinated cereals and pulses were

tested for moisture content. The moisture content varied from 1.45 % (in maize)

to 7.45% (in green pea).

Sample solutions were prepared by diluting sample 40 times with water.

Repetitive injection of different concentrations of standard sugars was done, to

check the detector response and observed that there was no variation in the

observed concentrations and expected concentration.

 Stachyose content was observed (mg/ gm of dry matter) in green gram (1.24),

sorghum (Traces), barley (Traces), Wheat (Traces) and black gram (8.302).

Raffinose content was observed (mg/ gm of dry matter) in barley (traces), wheat

(5.2), cow pea (4.261), green peas (2.04), black gram (traces), pigeon pea (1.28)

and soya (3.04).

Maltotriose content was observed (mg/ gm of dry matter) in sorghum (0.533),

chickpea (0.39), maize (0.52), Rajma (0.486) and

Kesari dhal (0.58).

Maltotetraose, maltopentaose, and maltoheptaose were not found in cereals and

pulses.

Cereals and pulses were subjected to germination for 48 hours and analyzed for

oligosaccharide content and found complete disappearance of stachyose, raffinose

in cereals and pulses. Maltotriose in pulses completely disappeared, but in cereals

45.1 %, 57.3 % loss was observed in sorghum and maize respectively, whereas

complete loss in remaining cereals was observed.

The present study conducted is a preliminary step towards the screening of cereals

and pulses to identify rich sources of oligosaccharides. The results showed slight

variations with literature cited, may be due to variation in sample preparation methods

and analytical techniques. The identified rich sources of oligosaccharides in black gram,

soy, cowpea and wheat may be useful for extraction in their natural form by membrane

separation process. These extracted oligosaccharides can be used as prebiotics in

development of functional foods.