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This is to certify that the

thesis entitled

Oligosaccharides of cowpeas

presented by

Maurice A. Akpapunam

has been accepted towards fulfillment
of the requirements for

M.S. Food Science

degree in

 

 

guLKQ/y //”Z!e do; 7255

Major professor

 

Date February 21, 1978

 

0-7639

 

OLIGOSACCHARIDES OF COWPEAS (VIGNA SINENSIS)

 

By

Maurice Abiamefune Akpapunam

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science
and Human Nutrition

1978

ABSTRACT

OLIGOSACCHARIDES OF COWPEAS (VICNA SINENSIS)

 

By

Maurice Abiamefune Akpapunam

Thirteen cultivars of cowpeas (yign§_sinensis) grown in the
Southern United States were first subjected to proximate analysis
and then to the identification and quantitative determination of
their oligosaccharides.

Paper chromatography supplemented by acid hydrolysis and
enzyme hydrolysis showed the presence of sucrose, raffinose,
stachyose, and verbascose. Quantitative analysis resulted in the
following concentration estimates of these sugars in cowpeas on
a dry basis: sucrose 2.2%, raffinose 1.2%, stachyose 3.4%, and
verbascose 0.9%.

Sucrose varied considerably between varieties, raffinose
was fairly constant, while stachyose and verbascose showed little
variation.

The non-digestible oligosaccharides, raffinose, stachyose,
and verbascose represented approximately 9% of all sugars present

in cowpeas.

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to
Professor Pericles Markakis, his major professor, for his encouragement
and able guidance throughout the course of this work and during the
preparation of this manuscript.

Special thanks and appreciation are also expressed to
Professor LeRoy Dugan, Jr., Professor Donald H. Dewey, and Dr. Jerry N.
Cash for serving in the guidance committee and for reviewing the
manuscript and giving helpful suggestions. Dr. Jerry N. Cash was
particularly helpful in preparing the slides from which the pictures
in this manuscript were made and the author is very grateful to him.

He also wishes to thank Professor Milo B. Tesar of Crop Science
Department, Michigan State University, and Professor D. L. Chambliss of
Auburn University, Auburn, Alabama, for providing the samples of cowpeas
used for this study.

The author is most grateful to the Federal Government of

Nigeria for sponsoring his studies.

11

TABLE OF CONTENTS

Page

LI S T OF TABLE S O O O O O 0 O O O O O O O O O O O O O O O O O O O 0 iv

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . v
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . 6
Methods Used in Separation and Analysis of Sugars . . . . . 6
Oligosaccharides of Legumes . . . . . . . . . . . . . . . . 15

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . .V. 19

Preparation of Samples . . . . . . . . . . . . . . . . . . . l9
Separation and Identification of the Oligosaccharides . . . l9
Characterization of the Oligosaccharides . . . . . . . . . . 21
Quantitative Analysis . . . . . . . . . . . . . . . . . . 23
Determination of "Total Carbohydrate" . . . . . . . . . . . 26

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 27
CONCLUSIONS 0 O O O O O O O O O O O O O O O O O O O O O O O O 0 O 43

BIBLIWWHY O O O O O O O O O O O O O O O O O O O O O O O O O O O 44

iii

Table

LIST OF TABLES

Page
The Botanical Varieties of the Cultivated Cowpea . . . . . 4
Proximate Composition of the Cowpea Cultivars . . . . . . 29
RG and Rf Values of Authentic Sugars and Cowpea Sugars . . 31

Oligosaccharides of 13 Cowpea Cultivars . . . . . . . . . 4O

Carbohydrates, Oligosaccharides, and Non-Digestible
Oligosaccharides, Average and Range of the 13 Cultivars . 42

iv

Figure

LIST OF FIGURES

Thirteen cultivars of cowpeas (Vigna sinensis) used
for this study . . . . . . . . . . . . . . . . . . . .

Descending paper chromatography of standards and
cowpea Oligosaccharides . . . . . . . . . . . . .

Descending paper chromatography of standards and
cowpea Oligosaccharides (variety, Giant Blackeye) . .

Descending paper chromatography of standards and
cowpea Oligosaccharides (variety, Giant Blackeye) . .

Descending paper chromatography of the product of
invertase action on sugars of cowpeas . . . . . . . .

Descending paper chromatography of acid hydrolysates
of cowpea Oligosaccharides (variety, California No. 5)

Plot of Log [R /(1-R )] against number of hexose units
f f
in cowpea Oligosaccharides . . . . . . . . . . . . . .

Plot of Log [R /(1-R )] against number of hexose units
f f
in COWpea Oligosaccharides . . . . . . . . . . . . . .

Page

28

30

32

33

35

36

38

39

INTRODUCTION

Matured dried beans are staple foods in many countries. In
these countries they serve as major sources of protein in the diet.
As a result much research had been conducted on the protein quality
and quantity of these leguminous seeds. In recent years, the role
of the raffinose group of Oligosaccharides (raffinose, stachyose,
and verbascose) in causing flatulence in man and other animals
consuming leguminous seeds has opened up another field of research
related to these seeds.

The flatus effect and the discomfort experienced after
eating beans and other leguminous seeds are said to be due to lack
of a-galactosidase activity in the human digestive tract. The
Oligosaccharides thus pass into the large intestine where they
are fermented by bacteria with the formation of gas and generation
of flatus. Relationships between molecular weight of the oligo-
saccharides and their levels to flatus formation has been reported
by Cristofaro et al. (1972). Stachyose and verbascose are reportedly
more involved in flafus formation than raffinose.

In nature these Oligosaccharides occur freely as soluble
reserves in plant tissues or in plant storage organs.

Raffinose is a trisaccharide, consisting of one molecule

each of D-galactose, D-glucose and D-fructose. It has been shown

that the fructose and glucose components are joined as in sucrose.
The galactose molecule is attached to the glucose with an a (1-6)
bond. Partial hydrolysis could yield either melibiose (galactose,

glucose) and fructose or sucrose (glucose, fructose) and galactose.

 

      

Sucrose
M
CH2 H HOCH2 O H
H
H
OH H
HO 0 H0 H 2OH
OH
W
Melibiose

Structure of Raffinose

Raffinose occurs widely distributed in plants, and next to
sucrose, it is probably the most abundant oligosaccharide of the plant
kingdom. It occurs in sugar beets and is concentrated in sugar beet
molasses. Cotton seed contains a good proportion and is generally
the material used for its preparation.

Stachyose is the best known tetrasaccharide. It is composed
of two molecules of D-galactose and one each of D-glucose and
D-fructose. It is also known as O-a-galactopyranosyl (1 + 6) -
O-a—D-galactopyranosyl - (1 + 6) — O-a-D-glucopyranosyl - (1 + 2) -

B—D-fructofuranoside.

 

Structure of Stachyose

On partial hydrolysis with dilute acid, stachyose yields
D-fructose, and the trisaccharide, manninotriose. Complete hydrolysis
yields D-fructose, D-glucose and two molecules of D-galactose. The

tubers of Stachys tuberifera are the classical source of stachyose and

 

may contain up to 75% (dry weight) of stachyose (Bailey, 1965).
Verbascose, like the others is a non-reducing heterogeneous

oligosaccharide. It is composed of D-galactose, D-glucose and
D—fructose. It is also referred to as O-a-D—galactopyranosly -

(1 +6) - O—a-D-galactopyransyl - (l + 6) - O-a-D-galactopyranosyl -
(l + 6) - a-D-glucopyranosyl - (1 + 2) - B-D-fructofuranoside. Upon
hydrolysis, with dilute acid, D-fructose and a tetrasaccharide,
verbascotetraose are formed. This upon further hydrolysis yields

three molecules of D-galactose and one of D-glucose.

 

Structure of Verbasocse

Verbascose occurs in many plants that synthesize sucrose,
raffinose, and stachyose, but the most important source is the roots
of mullein Verbascum thapsus.

Several workers including Nigam and Giri (1961), Korytnyk

and Metzler (1962), Gould and Greenshields (1964), Kim et a1. (1973),

Cerning et a1. (1975), Tanaka et a1. (1975) and others have attempted
to separate and quantitate this group of oligosaccharides in various
legume seeds. Their findings have so far indicated the presence of
these oligosaccharides in relatively large amounts.

This present study was set to investigate the presence of
these oligosaccharides in the cowpea. Cowpea or blackeye peas is a
tropical legume with its origin in the continent of Africa from where
it was introduced to the West Indies, North America, and other tropical
and subtropical countries. It belongs to the genus Vigna. There are

three well cultivated species of this genus: Vigna sinensis (common

 

cowpea), Vigna unguiculata (catjang), and Vigna sesquipedalis

(yardlong bean). Some of the differences between these distinct

botanical varieties are given in the table below.

Table 1. The Botanical Varieties of the Cultivated Cowpeaa

 

 

 

 

 

Seed Pod

Botanical Variety Length Shape Length Shape

Var. sinensis 6-9 mm Sub reinform 200-300 mm Not flabby

(common cowpea) to sub- pendent when

;g}obose mature

Var. unquiculata 5-6 mm Oblong or 80-130 mm Not flabby

(catjang) cylindric or inflated,
erect or
ascending
when mature

Var. sesquipedalis 8-12 mm Elongated 300-10001mn Fleshy and

(yardlong bean) kidney inflated when
green, shrink
on drying

 

 

 

 

 

aTable taken from "Tropical Pulses," by J. Smartt (1976).

The seed of this leguminous crop is very nutritious and it
is widely consumed in many countries as a pulse and as a vegetable.
Because of its high protein content (Table 2), the cowpea has attracted
attention as a possible source of protein for the diet of many people
in different parts of the world. Several high yielding varieties of
this crop have been introduced. Unfortunately this crop like the other
members of the legume family contains relatively large amounts of the
raffinose group of oligosaccharides.

There have been some attempts to determine the quantity of
this group of oligosaccharides in this bean (Nigam and Giri, 1961;
Lee et a1., 1972). There is, however, no general agreement as to the
quantity of these components, in particular, verbascose, in the beans.
The discrepancies arose possibly because these components vary with the
stage of maturity of the bean seeds (Korytnyk and Metzler, 1962) and
perhaps with the variety of the beans. Thus, apart from determining
the quantity of these oligosaccharides in the matured, dried, bean
seeds, an attempt has been made, in this study, to investigate possible
varietal differences among these groups of oligosaccharides in thirteen

cultivars of the beans.

LITERATURE REVIEW

Methods Used in Separation and
Analysis of Sugars

 

 

Recent developments in the chromatographic method of analysis
have provided investigators in many fields with an exceedingly powerful
and useful tool for separation and identification of sugars in foods
and other substances. All the different methods ranging from paper
chromatography, paper electrophoresis, thin-layer and column chroma-
tography have been used at one time or another depending on the
availability of equipment, time, the extent of investigation and
perhaps the amount of material to be analyzed. Paper chromatography
is used in identification work. Paper electrophoresis likewise
separates sugars for possible qualitative identification or for
confirmation. Column chromatography provides a means of obtaining

sufficient material for confirmatory chemical studies.

Paper Chromatography

 

The classical work of Partridge (55, 56), was the first attempt
at separation of sugars on paper. Flood, Hirst and Jones (20) placed
the paper chromatography of reducing sugars on a quantitative basis
using the Somogyi micro-copper reagent. Using hypoiodite oxidation,
Hawthorne (28) also determined quantitatively the sugars separated

on paper.

Application of paper chromatography for separation of
oligosaccharides is similar to that for monosaccharides. Excellent
reviews on these determinations are given by Bailey and Pridham (5),
Hough and Jones (31), Heftmann (29), and Albon and Cross (1).

Korytnyk and Metzler in 1962 studied the formation of raffinose
and stachyose in lima beans. They utilized paper chromatography for
the separation and identification of the sugars. Nigam and Giri (49)
employed paper chromatography for the separation and quantitation of
oligosaccharides present in eight pulses.

Bailey and Pridham (6) and Bailey (4) have outlined a number
of procedures for the structural identification of oligosaccharides.

In recent years colorimetric methods have achieved popularity
over the classical analytical methods which utilize the reducing
properties of sugars to determine their quantities gravimetrically or
titrimetrically. This is because they are simple and are applicable
to very small amounts of materials such as are separated by chroma-
tographic procedures. Volumetric procedures involving the use of
potassium ferricyanide, ceric sulfate, copper sulfate, and sodium
hypoiodite are applicable to the determination of small amounts of
reducing sugars after separation by partition chromatography. However,
experience shows that these methods require considerable skill, they
are time consuming and sensitive to slight variations in the conditions.

The anthrone and 1-naphtholsu1fate colorimetric reagents are
excellent for standard sugar solutions, but when applied to the

analysis of sugars separated by partition chromatography, the presence

of only traces of residual solvent developer have been found to render
them useless. Residual phenol, for instance, holds tenaciously to the
filter paper, interfering with the green color produced by the anthrone
reagent. Another disadvantage of the anthrone reagent is the high cost
and the fact that its solution in sulfuric acid is not stable. The
method also suffers from the disadvantage that, while it is satis-
factory for free sugars and their glycosides, it is of limited use

for methylated sugars and the pentosans. Residual propionic acid is
also known to interfere with the l-naphthol sulfate reagent. This
limits its use for determination of disaccharides for which butanol-
propionic acid-water is an excellent solvent for separation.

Aniline phthalate and aniline trichloroacetate have been
utilized for the colorimetric determination of sugars and their
derivatives; the reagents have, however, proved unsatisfactory for
ketoses.

The phenol-sulfuric acid method of Dubois et al. (7) has proved
to be very useful for the quantitative determination of simple sugars,
oligosaccharides, polysaccharides, and their derivatives. The method
is particularly useful for the determination of small quantities of
sugars separated by paper partition chromatography with the phenol-
water solvent and also for the sugars separated with solvents which
are volatile; for example, butanol-ethanol-water, ethylacetate-acetic
acid-water, or methylethyl-ketone-water. The reagents are stable,
also the color produced is stable and insensitive to changes in

conditions.

\o

A number of reagents have been utilized for detection of sugar
spots. Among these is ammoniacal silver nitrate (55) which is said to
be universal in application. It, however, has some disadvantages; for
example, it is known to react with a very wide range of reducing sub-
stances other than the sugars. Also sugar spots may migrate from the
wet spot to the dry area of the filter paper. Another disadvantage of
the method is the need for controlled heating. To overcome some of
these disadvantages, Trevelyan et a1. (74) introduced a method which
eliminates the heating step. Also in this method, reagents are applied
in organic solvents, thus reducing the danger of migrating spots.

Triphenyltetrazolium chloride (47) can be used as a spray
reagent for sugars. It is, however, time consuming and it is not good
for non-reducing sugars. A solution of benzidine in glacial acetic
acid has been used by Horrocks (30) as a chromatographic spray reagent
for detection of sugar spots. A.modification of this method involving
the use of a mixture of benzidine in glacial acetic acid and trichloro-
acetic acid in water (67) is very suitable for both reducing and non-
reducing sugars.

Other spray reagents include aniline hydrogen phthalate (57),
a-naphthol-phosphate (1), naphthoresorcinol-trichloroacetic acid (56),
and dinitrosalicylate reagent (78). While most of these reagents
have been used for monosaccharides, only very few are applicable

to oligosaccharides and polysaccharides.

10

Paper Electrophoresis

 

This technique is used for separating charged materials using
a support, buffers, and an electric current. The manipulations are
in many ways similar to paper chromatography except for the actual
development of the chromatograms in the migration chamber. The tech-
nique can be used to provide useful confirmatory evidences regarding
the structure of an oligosaccharide. In fact, it is suggested that
wherever possible, electrophoresis should be used in conjunction with
paper chromatography and the identity of an oligosaccharide checked by
comparing its electrophoretic rate of movement with that of an authentic
specimen.

Several papers and reviews have been presented on paper
electrophoresis; details can be obtained from Bailey and Pridham (5),
Foster (18, 19), and Block et a1. (9). Most observations on the elec-
trophoretic behavior of oligosaccharides have been carried out using an
alkaline borate buffer. Foster (19) recorded the rates of movement,
relative to D—glucose (MG values), of various glucopyranosyl disac-
charides, and Bailey and Pridham (5) have examined the mobilities of
the raffinose family of oligosaccharides and a series of l—4—B-linked
D-mannose oligosaccharides (D.P. 2-5). In the case of the raffinose
series, the behavior is highly characteristic of the group, the
mobilities increasing in the order; raffinose<=stachyose<=verbascose<
tetragalactosyl—sucrose. (This is the reverse order of that observed
in paper chromatography.) With mannose oligosaccharides, however, the
more usual behavior of decreasing rate of movement with increasing

molecular weight was observed.

11

Some other electrolyte systems which have been used for this
technique include sodium arsenite, basic lead acetate, sodium hydroxide,
sodium tungstate, sodium-metavanadate, and ammonium or sodium molybdate.
With the exception of ammonium molybdate all the other electrolytes

were applied mainly to monosaccharides.

Gas-Liquid Partition Chromatography

 

Separation of carbohydrate derivatives by gas-chromatography is
quite recent. The delay probably might be due to some restrictions of
the method. For example, the material subjected to gas-chromatography
must be volatile, stable to the operating conditions of the instrument,
and not irreversibly adsorbed on the chromatographic column. Methyl
glycosides of monosaccharides fulfilled these requirements and were
the first group of carbohydrate derivatives investigated. Using
programmed temperatures for the separation, oligosaccharide esters
have also been separated and analyzed by this technique.

The gas-chromatographic technique has the advantage of speed,
and the fact that the fractions can be analyzed quantitatively and
collected for identification is another added advantage. The
equipment is, however, very expensive. Detailed reviews on its

use for carbohydrates are given by Kircher (41) and Bishop (8).

Thin-Layer Chromatography

 

The first attempts to separate sugar mixtures by thin-layer
chromatography were made in 1961 (70, 69, 54). Since then many papers

concerning this problem have continued to appear. Poor separation of

12

some of the more common sugars and the low capacity of the
chromatoplates are two disadvantages of this technique.

In most earlier reports, silica gel G, kieselguhr, gypsum,
cellulose powder, as well as silica gel and/or kieselguhr impregnated
with boric acid, sodium tetraborate, bisulfulite and acetate have been
used on the chromoplates. Recently thin-layer chromatography on silica
gel impregnated with sodium hydrogen phosphate or sodium monohydrogen
phosphate in phosphoric acid was successfully employed by Ovodov et a1.
(52). They claimed the method is quite suitable for mono, oligo, and
polysaccharides.

Tanaka et a1. (72) developed a procedure using thin-layer
chromatography and thiobarbituric acid reagent for the quantitative
determination of oligosaccharides in legume seeds. They reported
excellent recovery.

Cerning et al. in 1975, also utilized thin—layer chromatography
in their studies of the carbohydrate composition of horse beans.

Excellent results were reported.

Column Chromatography

 

Column chromatography is fast becoming an important tool for
separation of carbohydrates. A good account of this technique is
given by Binkley and Wolfrom (7). Many materials have been used as
the stationary phase for columns. Among these are charcoal, cellulose
powder, starch, celite, clay, silica gels, sephadex, and ion-exchange

materials.

l3

Separation of oligosaccharides on column was reviewed by Bailey
and Pridham (5). According to them, the separation can be effected by
three main methods:

1. Partition chromatography on cellulose or celite,
2. Adsorption chromatography on charcoal, and
3. Ion-exchange resin chromatography.

Newly developed column packings for high performance liquid
chromatography has greatly simplified the separation of sugars in food
substances. Palmer in 1975 described a simple, rapid and versatile
method for the separation and determination of sugars via high pressure
liquid chromatography. He claimed that virtually any combination of
monosaccharides, disaccharides and/or higher oligomers can be separated
by this method. Havel et a1. (27) determined the oligosaccharides
released during hydration of textured soy with the aid of high
performance liquid chromatography.

Floridi in 1974 applied automatic ion-exchange chromatography

for simultaneous determination of sugars and sugar alcohols in foods.

Specific Enzyme Methods
A number of methods have been described which utilize enzymes
for the determination of carbohydrates in biological fluids, such as,
blood and urine (15, 16, 62). Many of these methods are presently
employed for the determination of carbohydrates in foods (32, 76, 39).
D-glucose, for example, can be determined in a mixture of

sugars with a coupled enzyme system containing glucose oxidase or

14

more properly, B—D-glucose: oxygen oxido-reductase. The principle

behind the reaction is summarized below:

lucose
D glucose oxidase D—gluconic acid lactone + hydrogen peroxide
Hydrogen peroxide catalase water + oxygen

Oxygen + reduced leuko-chromogen'--+ oxidized colored chromogen

A test kit, glucostat, is commercially available, for
quantitative colorimetric analysis. The kit contains buffer, reduced
chromogen, catalase, and glucose oxidase.

D-fructose has been determined in mixtures containing only
glucose or sucrose by differences in reducing power before and after
oxidation of the glucose with glucose oxidase (79).

D-galactose may be determined quantitatively in mixtures of
monosaccharides by a coupled enzyme system containing galactose oxidase
in a way similar to that used for D-glucose. The enzyme is known to be
active on free D—galactose as well as D-galactopyranosyl residues of
01130 and polysaccharides. In fact, Avigad et al. (2) found that the
tetrasaccharide, stachyose, was oxidized six times faster than
D-galactose. It is evident that this enzyme is useful for the
quantitative analysis of oligosaccharides.

Rot et a1. (62) defined certain conditions which will permit
adaptation of the galactose oxidase reaction to a generally useful
method for the determination of galactose in a variety of situations.

These include, that the sample should be at approximately neutral pH,

15

and free of the interfering substances enumerated below: protein
precipitants and other substances capable of injuring enzymes in gen-
eral (e.g., large amounts of ethanol) should be obviously eliminated.
One further condition which has been found to affect the galactose oxi-
dase reaction adversely appears to be high ionic strength or osmolarity.
Thus, high concentrations (exceeding approximately 0.5 M) of simple
salts: chlorides, sulfates, and phosphates of sodium or potassium
and organic compounds (glucose, glycine, tris, succinate, sucrose)

have been found to inhibit the enzyme.

Oligosaccharides of Legumes

 

Legume seeds are particularly rich in oligosaccharides of
the galactosyl sucrose type (raffinose, stachyose, and verbascose).

Nigam and Giri (49) examined the seeds of some pulses in-
cluding cowpea (Vigga sinensis) for their oligosaccharide composition.
They found the chromatographic pattern of all the pulses examined to
be the same. The carbohydrates were non-reducing and yielded glucose,
galactose, and fructose on hydrolysis. The oligosaccharides were con-
firmed as raffinose, stachyose, and verbascose. The determination of
each of these component sugars in the various pulses showed uniformity.
Each one contained a large amount of verbascose followed by stachyose
and then sucrose, with relatively small amounts of raffinose. They
stated that this raffinose family of oligosaccharides seemed quanti—

tatively more important than the polysaccharides which occur in the

pulses in a much lower amount.

16

Nigam and Giri also observed that soaking the leguminous seeds
in water brought about a reduction in the amount of these oligosac-
charides in the seeds. After about 48 hours soaking, the raffinose
and stachyose content completely disappeared. Sucrose was reported
constant during the soaking and the germination that followed. Fructose
was observed to increase with time. They could not strike any balance
between the disappearance of the ologosaccharides and the formation of
sucrose and the monosaccharides. They regarded the phenomenon as a
result of a—galactosidase activity of the germinating seeds which
hydrolyzes the oligosaccharides to monosaccharides and sucrose.
Glucose, fructose, and to a large extent galactose play an important
function in plant growth and the synthesis of cellulose and stem
protein.

Gomyo and Nakamura (24) confirmed the presence of raffinose
in legumes, stating that it could be synthesized from uridine
diphosphate, galactose, and sucrose. They also observed that in seeds
raffinose appears during maturation and disappears during germination,
a view also maintained by Gould and Greenshields (25), and Nigam and
Giri (49).

Cerning et a1. (12) reported varying amounts of verbascose,
stachyose, raffinose, sucrose, and in some cases, traces of glucose
and fructose in horse beans. They obtained an average value of 5.7%
for these ethanol soluble sugars in these beans.

Korytnyk and Metzler (42) studied the formation of raffinose

and stachyose in lima beans. They observed that a decrease in stachyose

17

was accompanied by a corresponding increase in fructose, whereas the
relative amount of sucrose and raffinose remained fairly constant.
They suggested a stepwise formation of the sugars. Sucrose may be
formed from fructose and uridine diphosphate glucose, whereas, at a
later stage of development, raffinose is formed from sucrose and a
corresponding galactose intermediate. Similarly, stachyose could

be formed from raffinose by a transgalactosylation reaction.

Pritchard et a1. (58) indicated that the composition of the
ethanol soluble fractions of winter field beans varied considerably
between varieties. They maintained that the main components were
oligosaccharides and sucrose or its hydrolysis products.

Tanaka et a1. (72) determined the oligosaccharides of soybeans,
mungbeans, adazuki beans, and white beans. They did not report the
presence of verbascose, but their value for raffinose, stachyose, and
sucrose agreed fairly well with those of other workers.

Soybeans seem to have a higher percentage of sucrose than most
other legumes. The values obtained by Kawamura (37), showed sucrose
highest followed by stachyose and then raffinose. This sequence agrees
with the findings of Tanaka et al. (72) and the data of Hardinge et a1.
(26).

Studies on soybeans have shown that the raffinose group of
oligosaccharides tend to disappear after germination and soaking or
autoclaving (40, 38, 80). Germination and irradiation of navy beans
also reduce the oligosaccharides content (68). One may, therefore,

consider that soaking and/or blanching as well as germination and

18

irradiation of leguminous seeds might well serve as a method for the
removal of oligosaccharides from these seeds.

The above reviews have all indicated the presence, in beans,
of the raffinose group of oligosaccharides. There is, however, no
general agreement about the quantities of each sugar in the various
legumes. The variations in the reported values could be as a result
of the different methods of analysis. It could also have been due to
the stage of maturity of these beans as well as the fact that these

sugars vary with the variety of the beans.

MATERIALS AND METHODS

Preparation of Samples

 

Thirteen different varieties of dried, matured cowpeas (Vigga
sinensis) grown in the Southern United States were used for this
study.

A 10 gram sample of the well-ground seeds from each of the
varieties was covered with 70% cold ethyl alcohol and extracted with
constant agitation for 12 hours. The resulting mixture was then fil—
tered through a Buchner funnel. The residue was washed three to four
times with small portions of cold 70% alcohol. The combined extracts
were then concentrated under vacuum at about 40°C. The concentrated
syrup was clear and needed no further clarification. It was made up
to 25 ml with distilled water and stored in the refrigerator (0-5°C).

Attempts at extraction with 80% hot ethanol produced a syrup
containing oil and some slimy materials which could not be removed

by centrifugation.

Separation and Identification of
the Oligosaccharides

 

 

The sugars were separated by paper chromatography using
Whatman No. 1 filter paper (46 x 57 cm) with serrated lower edge

to prevent irregular solvent flow.

19

20

Known volumes of the sample extracts were spotted on a line
7.5 cm from the top edge of the filter paper. Standard sugars:
sucrose, glucose, fructose, stachyose, raffinose, galactose, and
melibiose were also applied at 2.5 cm intervals along the same line.
The standards had a concentration of 1% (w/v) in 10% (v/v) l-propyl
alcohol. To avoid obtaining wide spots and consequent spreading, each
spot was dried by a current of hot air from an electric hair dryer after
the application of about 1 A (1 ul, 0.001 ml) from a microsyringe. The
paper was then left to air dry for about 30 minutes. Next, the paper
was placed in a chamber which had been saturated with the developing
solvent. After about an hour, the developing solvent was added through
a small hole on top of the chamber cover onto a trough holding the paper.

The overrun development method was employed because of the very
low Rf values of the oligosaccharides. The development lasted for
48 hours.

The following solvent systems were tried:

A. n-butanol, ethanol, water (5:3:2 by volume);

B. n-butanol, ethanol, water (10:1:2 by volume);

C. 1-butanol, pyridine, water (6:4:3 by volume); and

D. 1-butanol, acetic acid, water (4:1:5 by volume).
Of these, the first and third solvents (A and C) produced good
separation. Solvent A, however, was preferred to Solvent C because
of the irritating odor of the latter. Both are monophasic mixtures
and therefore easy to work with, since one does not have to worry

about their eventual separation.

21

At the end of the development, the paper was brought out and
dried in air for about an hour. It was then sprayed with benzidine
reagent to detect the sugar positions. The paper was allowed to dry
in air and then heated in an oven at 100°C to 105°C for 5-10 minutes.

The benzidine spray reagent was prepared from two solutions:

0 1 g benzidine in 40 ml glacial acetic acid--Solution A; and

0 30 g of trichloroacetic acid in 40 ml of water--Solution B.
These solutions were mixed before use and diluted with about 10 ml of
acetone. The reagent reacts with almost all sugars producing intense
dark brown coloration with most of them and yellow with fructose.
Other reagents tried include a-naphthol-phosphoric acid for detecting

ketoses and triphenyl-tetrazolium chloride for reducing sugars.
Characterization of the Oligpsaccharides

Approximately 100-200 ul of sugar extracts were applied to the
filter paper by transverse streaking. Several sheets of paper were
streaked with the extract in order to obtain sufficient quantities of
individual sugars after elution. The portion occupied by the sugar
was eluted from the papers following the procedure described below
for quantitative analysis. The eluates of each individual sugar were
combined, concentrated, and subjected to the following tests.

1. Test for Reducing Sugars. Ten ml of the eluted sugar was

 

made alkaline with 1N NaOH solution in a test tube. The tube was then
placed in a water bath at 25°C. After about 2 minutes, 2 m1 of 0.5%

triphenyltetrazolium chloride was added to the tube. The mixture,

22

properly shaken, was left in the bath for exactly 30 minutes. Formation
of a red precipitate after that time indicates presence of reducing
sugars.

2. Test for Presence of Fructose in the Sugar Structure. The

 

separated sugar spots on the paper were sprayed with a-naphthol-
phosphate reagent (1). Purple coloration of the spots indicates
the presence of fructose in the structure of the sugars.

3. Raybin's Test (for Sucrose and Sucrose Containing Compounds).

 

Five m1 of the eluted sugar in 5 m1 of N/20 NaOH (at 10°C) was shaken in
a closed test tube with 7-10 mg of diazouracil until the latter dis-
solved. A blue-green color developed within a few minutes. The color
faded, turning brownish on standing at room temperature. The blue-green
color was given by all the cowpea sugars.

4. Hydrolysis of the Oligosaccharides. Some quantities of the

 

eluted sugar solution were hydrolyzed with 1N H2804, for 2 hours at
100°C. The resulting hydrolysates were then rechromatographed using
n-butanol, ethanol, water (5:3:2 by volume) on Whatman No. 1 filter
paper. Enzymic hydrolysis with o—galactosidase and invertase were
also attempted.

5. Correlation of Structure with Papergram Mobility. This
test was performed with the original sample extract. Five to ten
microliters of the extract together with standard sugars (sucrose,
raffinose, and stachyose) were spotted on two filter papers. The
papers were developed along one direction (unidimensional) with

n-butanol, ethanol, water (5:3:2 v/v) and l-butanol, pyridine, water

23

(6:4:3 v/v), respectively. The sugar spots were identified with the
benzidine spray reagent. The Rf values of the individual sugar
components from the two chromatograms were determined and from this
the partition function, a, (log a = Log [Rf/(l-Rf)] was calculated.
Log 0 was then plotted against the number of hexose units (molecular
size) in each component.

The above procedure was repeated for the glucose terminal
series of sugars obtained by invertase hydrolysis of the oligosac-

charides of the cowpeas. The separation on the filter paper was with

n-butanol, ethanol, water (5:3:2 v/v) solvent system.

Quantitative Analysis

 

Two methods were used: the phenol-sulfuric acid colorimetric
method (17) and the modified resorcinol-hydrochloric acid method (61).
The former was applied to the determination of sucrose, raffinose, and
stachyose and the latter to the determination of verbascose.

The sugars were first separated on Whatman No. 1 filter paper
and then eluted for the analysis. As shown on the following sketch,
at a distance of 7.5 cm from the straight edge of a 46 x 57 cm Whatman
No. 1 paper one linear portion (5 cm) was reserved as a blank space, a
second portion (31 cm) was streaked with the cowpea extract and a third
portion (10 cm) was spotted with authentic sucrose, raffinose, and
stachyose solutions. The streaking of the extract was done in
duplicate. Each portion, 15 cm long, contained 50 ul of the extract.
The paper was then developed as in the qualitative analysis using

Solvent A.

24

 

7'5 cm { Blank Extract Suc Raf St Suc = Sucrose
————-: i 3

 

Raf = Raffinose

St = Stachyose

 

 

WWW/WWW

Sketch of filter paper set up.

After drying, the strip containing the standard sugars was cut
and sprayed with the a-naphthol-phosphoric acid reagent, to locate the
sugar positions. The strip was then matched against the main paper and
portions corresponding to each sugar was cut out from the main paper.
Each section containing the sugar was further cut into smaller pieces
and placed in a beaker containing 10 ml of deionized water. The beaker
was then left covered for 30 minutes, with occasional shaking. A por-
tion was cut from the blank space corresponding to the sugar portions

and treated as above. The extracted sugars were then filtered through

25

sintered glass wool and collected in test tubes with covers. One ml
of each of these was then pipetted into other test tubes. To this

was added 1 m1 of 5% phenol reagent. After mixing, 5 m1 of concen-
trated sulfuric acid was added quickly to each of the tubes. The
mixture was shaken to mix properly and then cooled to room temperature,
using running water from the tap. The absorbance of the resulting
yellow-orange color was determined at 490 nm with a Beckman model

DU Spectrophotometer.

Standard curves were prepared for each sugar using concen-
trations in the range of 10-70 pg per ml. The blank absorbance value
was subtracted from the absorbance of each of the sample sugars before
reading off the concentration from the standard curve.

The above method could not be applied for the determination of
verbascose, because no authentic verbascose was available to prepare
a standard curve. Instead the eluted sugar was partially hydrolyzed
with yeast invertase (also with 0.05 N HCl) to liberate fructose which
was determined with Roe's reagent (61). To 1 or 2 ml of the hydrolysate
was added 2 ml of 0.1% alcohol resorcinol followed by 6 ml of 30% HCl.
The tube was heated in a water bath at 80°C for eight minutes. The
purple color obtained was measured with a spectrophotometer at 520 nm.

A standard curve was prepared using fructose ranging from
0.025 to 0.1 mg. The obtained fructose values were converted to

verbascose by multiplying by a factor of 4.6.

26

Determination of "Total Carbohydrate"

 

The percentage of total carbohydrate content of the cowpeas was
obtained by subtracting from 100 the percentage of contents in moisture,
ash, protein (N x 6.25), fiber, and lipid. All the components with the
exception of fiber were determined by the methods outlined in the AOAC
(50) methods of analysis. The fiber was determined by the modified

trichloroacetic acid digestion reagent method of Whitehouse et a1. (77).

RESULTS AND DISCUSSION

Figure l is a display of the thirteen cowpea cultivars used

for this study. They all belong to Vigna sinensis. The color and size

 

of the seeds varied among cultivars. The color ranged from creamy-white
to dark brown. The pinkish color of SA Dandy variety is a result of
pretreatment against infestation. The true color is creamy-white.

The composition of the cowpeas is given in Table 2. It shows
that the seeds are high in protein and carbohydrate, low in oil and
fairly high in crude fiber and ash. The protein, oil, ash, and carbo-
hydrate showed some uniformity in all the varieties while the crude
fiber varied to some extent. All the values are the means of two
determinations, which varied by less than 4%.

Separation and Identification of the
Oligosaccharides

 

 

Figure 2 shows the separation of the oligosaccharides of some
of the cowpea varieties on paper. The chromatographic pattern of all
the varieties examined were found to be the same. The chromatogram
showed that there are four types of oligosaccharides in these cowpea
varieties. The RC and Rf values of authentic sugars and of the sugars
of cowpeas with two different solvents are shown in Table 3. The

solvents were: n-butanol, ethanol, water, 5:3:2 by volume, and

l-butanol, pyridine, water, 6:4:3 by volume. These data indicate

27

28

 

Figure 1. Thirteen cultivars of cowpeas (Vigna sinensis) used for
this study. (Sa Dandy cowpeas true color is creamy-white;
pink color is the result of pretreatment.)

Note: A = California No. 5 Blackeye; B = Mississippi
Silver Skin; C = Knuckle Purple Hull.

29

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a.mm s.hm m.mm m.sm s.on A.mm m.wm A.mm s.om o.em o.mm n.5m m.mm Amoemumecae any
mmumuvmnoaumo
m.H o.~ m.H w.H w.H m.H m.H A.H ~.H m.H w.a m.H m.H mesa“;
s.m m.m m.m s.m H.m ~.m m.m m.m m.m m.m ~.m s.m o.m gm<
a.m a.~ a.m H.s o.m m.m H.m ~.m s.s s.m s.s s.~ ~.~ amass
m.s~ h.- m.m~ m.s~ m.s~ m.- m.- o.H~ e.m~ m.s~ ~.s~ m.o~ m.o~ Am~.o x zv
afiououm
a.m A.HH H.0H m.m o.oH a.oa o.a a.a H.oH H.OH s.m H.m a.~ «unease:
nun" 3.1 “Job “Jan .d“x ,5 ,0 .u "Sun 1a.d [Luv nuns nuns
mm mm m. mm Mm mm mm um um mm we we we
a u M u nu a a. . a. a u A s .d n. can a. I.
N514 D... 39 as In. Com 8% ”.0 3T? T53 J 39 Cal;
3 a an 90 3T.— _ 3T. IS 3‘ u 900 O
.A 1 my D. ,A a T.e nus .A I. s a a m
a t. A Wu P T... as “M... Ma r.
.4 I C4 Trd I E
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mum>HuH=U
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mAvo>wooou mm camamm w ooa\wv mum>wuaao momsou ma mo cowuwmoqaoo oumauxoum

30

 

Figure 2. Descending paper chromatography of standards and cowpea
oligosaccharides.

Solvent: n-butanol, ethanol, water, 5:3:2 by volume.
Standards: 1, melibiose; ii, stachyose; iii, raffinose;
iv, glucose; v, fructose; vi, galactose; vii, sucrose.
Varieties: Ba Sa Dandy; CeMississippi Silver Skin;

D -California No. 5; E- Alabama 963.8 GI—S; F- Giant
Blackeye; G=Pinkeye Purple Hull; A and HaAlabama

562 3-1-2.

31

Table 3. R and R Values of Authentic Sugars and Cowpea Sugars

 

 

 

 

G f
RG Values Rf Values
(B-E-W) a (B-E-W) a (B-P-W) b
Authentic Cowpea Authentic Cowpea Authentic Cowpea
Sugars Sugars Sugars Sugars Sugars Sugars
Sucrose .69 .68 .24 .24 .23 .24
Raffinose .30 .28 .11 .11 .ll .11
Stachyose .13 .12 .05 .04 .04 .04
Verbascose -— .06 -- .02 .02 .02

 

 

 

 

 

 

 

aB-E-W:

bB-P-W:

n-butanol, ethanol, water, 5:3:2 by volume.

1-butanol, pyridine, water, 6:4:3 by volume.

the presence of sucrose, raffinose, stachyose and probably a pentasac-
charide in cowpeas. Figures 3 and 4 are sketches of the separation of
these sugars on paper with the two solvent systems.

Increasing the separation time from 48 hours to 60 hours did not
show any spot between origin and the pentasaccharide. Increasing the
spotted quantity of extract did not result in the appearance of more
spots on the chromagtogram.

When the separation was first carried out with the fresh
extracts there were no spots indicating the presence of glucose or
fructose (Figure 2). When the extracts were kept in the refrigerator
(0-5 C) and then analyzed after five months in a way identical to that

for the fresh extracts, two more spots appeared on the chromatogram.

32

 

Start Line A Stac. Raf. Suc.
F—fi——"""—‘——-‘

87 o

I Q
..

 

 

Solvent Front

 

 

 

Figure 3. Descending paper chromatography of standards and cowpea
oligosaccharides.

Solvent: n-butanol, ethanol, water (5:3:2 v/v).
Standards: Stac., Stachyose; Raf., Raffinose;
Suc., Sucrose; A a Giant Blackeye Cowpea.

33

 

Start Line Stac Ref Sue

A
vr— I;

 

a

' <7
0 D
O

 

Solvent Front

 

 

 

Figure 4. Descending paper chromatography of standards and cowpea
oligosaccharides.

Solvent: l-butanol, pyridine, water (6:4:3 v/v).
Standards: Stac., Stachyose; Raf., Raffinose;
Suc., Sucrose; A = Giant Blackeye Cowpea.

34

Cerning et a1. (12) reported similar findings with horse beans.
The monosaccharides were probably formed by hydrolysis of the

oligosaccharides through glycosidases extracted along with the sugars.

Further Identification and Confirmation
of Individual Sugars

1. Test for Reducing Segar. The sugars did not give any color
when treated with triphenyltetrazolium chloride reagent. This is an
indication that they are non-reducing (49, 47).

2. Test for Presence of Fructose and Sucrose in the Sugars.
When the sugars were treated with a-naphtylamine-phosphate (l), a
purple color was produced. This is a positive test confirming the
presence of fructose in the sugars. Further structural evidence was
given by the diazouracil test (60). The blue-green color given by all
the sugars was a positive indication of the presence of sucrose in the
structures of these sugars.

3. Hydrolysis of the Segars. Partial hydrolysis, with dilute
mineral acid and yeast invertase gave fructose, glucose, melibiose, a
trisaccharide, and a tetrasaccharide (Figure 5). Complete hydrolysis
with stronger mineral acids gave fructose, glucose, and galactose only
(Figure 6). All these findings are consistent with the structure of
the oligosaccharides, sucrose, raffinose, stachyose, and verbascose.

4. Correlation of Structure with Papergram Mobility. In a
homologous series of sugars a linear relationship exists between Log

[Rf/(l-Rf)] (partition function), and the number of hexose units in

35

 

Start Line D

0")
1C2!

‘—

8, Mel Sue Glu Fr

 

v

C]

8
0

mo!

 

 

Solvent Front

 

 

Figure 5.

Descending paper chromatography of the product of invertase
action on sugars of cowpeas.

Solvent: n-butanol, ethanol, water (5:3:2 by volume).

Me1., Melibiose, Suc., Sucrose; G1u., Glucose; Fr., Fructose;
Fr., Fructose; A = Sucrose hydrolysate; B = Raffinose hydrol-
ysate; C = Stachyose hydrolysate; D = Verbascose hydrolysate.

36

 

Start Line
Mel Sec 2 3 g l, Gal Glue F;

 

no 0

00 o

 

Solvent Front

 

 

 

Figure 6. Descending paper chromatography of acid hydrolysates of
cowpea oligosaccharides.

Variety: California No. 5 Cowpea.

Solvent: n-butanol, ethanol, water (5:3:2 by volume).
Fr., Fructose; Suc., Sucrose; G1uc., Glucose;

Gal., Galactose; Me1., Melibiose.

1 = Disaccharide; 2 8 Trisaccharide; 3 = Tetrasaccharide;
4 = Pentasaccharide.

37

oligosaccharides (22, 23). In general, increasing the size of an
oligosaccharide by one monosaccharide unit is found to decrease the
logarithm of the partition function by an amount which depends on the
type of hexose unit being added and its mode of attachment. Based on
this principle, French and Wild in 1953 obtained linear relationships
for sucrose, raffinose, stachyose, and verbascose, and for their
hydrolysis products: D—glucose, melibiose, manninotriose, and
verbascotetraose.

Similar relationships were obtained for the oligosaccharides
separated from the cowpeas, Figures 7 and 8. This further supports the
contention that the sugar occurring behind stachyose on the filter paper
is a pentasaccharide and belongs to the same series of sugars as the
confirmed ones: sucrose, raffinose, and stachyose; it is probably

verbascose.

Ollgosaccharide Content of the Cowpea
Varieties

 

Table 4 shows the oligosaccharide content of the 13 cultivars
analyzed. The sucrose, raffinose, and stachyose contents were deter-
mined by the phenol-sulfuric acid method (17). Recovery tests with
this method gave sucrose 102.5%, raffinose 90.0%, and stachyose 97.5%.
The resorcinol-HCl method (61) by which the verbascose content was
determined was equally very reliable with 91.0% recovery.

0f the four oligosaccharides separated from the cowpeas,
sucrose was the most variable in amount. It ranged from a low value

of 1.8% to a high value of 3.1% with an average of 2.2% on a dry basis.

38

 

 

 

ASH
ad
I
3 0.1
\
EH
5..
00
O
._'l
0.01 1 ‘ ‘ 4
1 2 3 4 5

Number of Hexose Units

Figure 7. Plot of Log [Rf/(l-Rf)] against number of hexose units in
cowpea oligosaccharides.

Solvent: n-butanol, ethanol, water, 5:3:2 by volume.

A: Sucrose terminal series, sucrose, raffinose, stachyose,
and verbascose from cowpeas.

B: Glucose terminal series, glucose, melibiose, mannino-
triose, and verbascotetraose, obtained by invertase
hydrolysis of sucrose, raffinose, stachyose and verbascose
extracted from cowpeas.

39

1.0F'

Log [Rf/(l - Rf)]
c>
L.
l

 

 

0.01 1 l A j
1 2 3 4 5 6

Number of Hexose Units

 

Figure 8. Plot of Log [R /(l-Rf)] against number of hexose units in
cowpea oligosaccharides.

Solvent: 1-butanol, pyridine, water, 6:4:3 by volume.
A: Sucrose terminal series, sucrose, raffinose, stachyose,
and verbascose from papergram separation of cowpea extract.

40

 

 

Table 4. Oligosaccharides of 13 Cowpea Cultivars (g/100 g dry basis)
Varieties Sucrose Raffinose Stachyose Verbascose
1 California a
No. 5 2.4 i0.09 1.11 0.08 3.6i 0.18 0.91:0.05
2 Cabbage
Cowpea 2 1: 0.04 1.21:0.03 3.41:0.11 0.91 0.06
3 Aub. 70.3 1.8: 0.03 1.1i0.05 3.110.06 0.910.01
4 Pinkeye
Purple Hull 2.1:0.01 1.310.01 3.6: 0.03 0.9: 0.06
5 Mississippi
Silver Skin 1.9:0.05 1.1t0.01 2 910.09 1.03:0.05
6 Magnolia
Blackeye 2.4: 0.02 1.2: 0.06 3 4:0.01 1.010.01
7 Alabama
963.8 Gl-5 1.81:0.07 1.11:0.05 4 li20.14 0.06:0.01
8 Alabama 562
3-1-2 1.92:0.04 1.3i0.04 3 5i0.09 0.610.03
9 Knuckle
Purple Hull 1.8: 0.04 1.1i0.08 3 4: 0.13 1.010.03
10 Big Boy
Cowpea 3.0i0.06 1.1: 0.06 3 610.01 1.0: 0.06
11 Sa Dandy
Cowpea 2.0i 0.14 1.2i0.02 3 3:0.01 0.9:0.06
12 Tenn. White-
crowder 3.1:0.05 1.1: 0.12 2.9:0.21 0.8:0.04
13 Giant
Blackeye 2.6iO.10 1.2i0.19 3.8i 0.03 0.9i0.02

 

 

 

 

 

8Mean of four determinations.

bOne standard deviation.

41

Raffinose was fairly constant at about 1.2%. Stachyose was the most
predominant sugar in almost all the varieties. The exception was in
Tenn. Whitecrowder variety, where it fell slightly below sucrose. The
mean value for stachyose was 3.4%. Two varieties, Mississippi Silver
Skin and Tenn. Whitecrowder had the lowest stachyose value of 2.9%
while the Alabama 963.8 Gl-S variety had the highest value of 4.1%.
The rest were uniform at about 3.5%. Verbascose varied very little
between varieties. The two Alabama varieties (Alabama 963.8 01-5 and
Alabama 562 3-1-2) were particularly low in verbascose with an average
of 0.6% as compared to the others ranging from 0.8% to 1.0%.

The sucrose values compared fairly well with the values
obtained by Nigam and Giri (49), and Lee et a1. (43). The values for
raffinose, stachyose, and verbascose did not agree with those obtained
by either of these groups of workers. Nigam and Giri (49) reported the
following values: sucrose 1.5%, raffinose 0.4%, stachyose 2.0%, and
verbascose 3.1%. Lee et a1. (43) obtained 1.86% for sucrose, 0.1% for
raffinose, 1.66% for stachyose; they reported trace amount of verbascose.

The total oligosaccharide content of the cowpea varieties was
fairly constant, Table 5. It ranged from 6.9% to 8.6% with an average
of 7.7% on dry basis. Table 5 also shows the average and range of the
non-digestible oligosaccharides (stachyose, raffinose, and verbascose)
in the cowpeas. It shows that about 9% of the sugars of the cowpea are
not digestible and hence contribute to the flatus problem that might be

experienced by consuming the beans.

Table 5. Carbohydrates, Oligosaccharides, and Non-Digestible

42

Oligosaccharides, Average and Range of the 13 Cultivars

(g/100 g, dry basis)

 

 

Average Range
Total carbohydrates (excluding
fiber) 63.6 61.3-65.7
Total oligosaccharides 7.7 6.9-8.6
Non-digestible oligosaccharides
(stachyose, raffinose,
verbascose) 5.5 4.8-5.9

 

 

 

CONCLUSIONS

The cowpeas contained a substantial amount of the raffinose
group of oligosaccharides (raffinose, stachyose, and verbascose)
together with sucrose. Sucrose varied considerably between varieties.
Raffinose was fairly uniform, while stachyose and verbascose showed
little variation.

Cowpea is also rich in protein and other important nutrients.
In order, therefore, to utilize the full benefits of this crop, efforts
should be made to evolve an efficient and a more economical way to
eliminate the raffinose family of oligosaccharides from the beans

and its by-products.

43

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10.

11.

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