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CHANGES IN CELL WALL STRUCTURE AND STARCH DIGESTIBILIT Y
DURING COOKING OF DRY BEAN (Phaseolus vulgaris L.)

By

Yongsoo Chung

A DISSERTATION

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

Department of Food Science and Human Nutrition

1996

ABSTRACT
CHANGES IN CELL WALL STRUCTURE AND STARCH DIGESTIBILITY
DURING COOKDIG OF DRY BEAN (Phaseolus vulgaris L.)
BY

Yongsoo Chung

The indigestible starch fractions of dry bean have been studied by examining the
raw and cooked seeds. The effects of selected heat treatments on cell wall structural
appearance and starch bioavailability were enzymatically and microscopically examined.
An enzymatic procedure using B-amylase and pullulanase was used to selectively digest
starch fractions. The starchy plant tissue evaluated included: navy beans, pinto bean,
sweet potato and wheat.

The degree of gelatinization measured by the enzymatic method showed lower
scores than that obtained by the microscopic birefringent method on differentially cooked
beans. The lower values measured by the enzymatic method resulted from inaccessibility
of the enzyme within gelatinized starch. Cooking whole beans for 10 min or longer
caused crystallization within cell wall. The crystalline structure was observed under
polarized microscope appearing a white band around cell walls. The crystallized cell walls
of cooked bean was mechanically resistant and acted as a physical barrier not only to
enzyme hydrolysis but also to swelling of starch granules leading to a residual of
ungelatinized starch during cooking. The rigidity of the crystallized cell wall that changed

during cooking was investigated by measuring available starch after reheating of freeze-

dried and milled flours. The longer the cooking time, the greater the rigidity of the
crystallized cell wall of the cooked beans. The cell wall rigidity varied between bean types
and among the process conditions studied. DSC therrnography demonstrated that the
crystalline structure formed during cooking was more stable than starch that demonstrated
to be associated with resistant starch fraction. Starches of cooked sweet potato and wheat
were readily available to undergo enzymatic degradation since no crystallization occurred
within cell walls to limit enzyme accessibility.

The attributes of physical appearance, palatability and nutritional bioavailability are
essential components associated with food quality of cooked dry beans. Dry bean cell
walls undergo structural changes during cooking that limit starch digestibility.
Crystallized cell walls encapsulating starch granules were observed in cooked bean
exudate (brine). The amount of starch in brine was not correlated with brine viscosity
however total protein and insoluble dietary fiber in the brine were positively correlated

with increased brine viscosity.

Dedicated to my wife, J unghee, and parents
for their love, patience and moral support

iv

ACKNOWLEDGMENTS

I give thanks God, Jesus Christ, for being my Lord, providing me this opportunity
to study at Michigan State University, guiding and leading me until now and forever. My
heartfelt gratitude goes to my advisor, Dr. Mark A. Uebersax, for the constant guidance,
encouragement and valuable advice during my graduate study. He has become my
mentor, teacher and wonderful friend. Genuine appreciation is also extended to members
of my graduate committee, Dr. Maurice. R. Bennink, Dr. George. L. Hosfield and Dr.
Perry. K. W. Ng for their guidance, comments and suggestions.

I express the special appreciation to my wife, Junghee, for her sacrificial support
and endless love. My sweet sons, Myunghoon and Myungjoon, have became a source of
energy and joy to accomplish the study. Special gratitude and love go to my wonderful
parents and family for their love and encouragement.

Thanks are extended to many fellow students at Michigan State University,
especially Jai Neung Kim, for their assistance and friendships throughout this study.
Special appreciation goes to Dr. Joanne Whallon for her assistance in microscope study.
Special recognition is expressed to the research committee of the Bean/Cowpea
Collaborative Research Support Program (CRSP) and USDA-ARS for providing funding

during graduate study.

TABLE OF CONTENTS

Page
LIST OF TABLES ................................................ ix
LIST OF FIGURES ............................................... xi
LIST OF PLATES ................................................ xii
INTRODUCTION ................................................ 1
REVIEW OF LITERATURE ........................................ 3
Physical and Chemical Properties of Bean Starch .................... 3
Granule Composition and Structure ......................... 3
Granule size, shape, and microscopic appearance ......... 4
Molecular structure ............................... 6
Granule Crystallinity .................................... 7
Swelling Power and Solubility ............................. 8
Gelatinization and Pasting ................................ 9
Starch gelatinization and melting concept ............... 11
Method for determining starch gelatinization ............ 14
Birefringence end point method ................ 14
Viscosity method .......................... l4
X-ray diffraction method ..................... 15
Amylose / Iodine blue value method ............. 1?
Differential Scanning Calorimetry (DSC) method . . . 18
Laser light scattering method .................. 19
Other method .............................. 19
Factors Affecting Gelatinization Process within Food Systems
Time and temperature ........................ 19
Moisture ................................. 22
Ingredients ............................... 23

Processing procedures ....................... 28

Retrogradation ........................................ 30
Nutritional Attributes of Dry Bean Starch ......................... 33
Factors Affecting Digestion of Starch ....................... 33
Digestibility of Legume Starch ............................ 36
Indigestible Starch ..................................... 39

Cell Wall Components—and Chemistry in Plant Seeds ................. 46
Definition of Dietary Fiber ............................... 46
Components of Dietary Fiber ............................. 46
Organization of Cell Wall Structure ........................ 48
Dicotyledonous Plants ............................ 48
Monocotyledonous Plants ......................... 49

Constituents of Cell Walls ............................... 50
Fibrillar Polysaccharides ........................... 50

Matrix Polysaccharides ............................ 51

‘ Pectic substances ........................... 51

Hemicelluloses ............................. 52

CHAPTER I. INDIGESTIBLE STARCH COMPONENTS IN DRY BEANS

Introduction ........................................................ 55
Materials and Methods .............................................. 59
Results and Discussion .............................................. 65
Conclusions ........................................................ 78

vii

CHAPTER II. HEAT-INDUCED CHANGES IN CELL WALL STRUCTURES
REDUCE BIOAVAILABILIT Y OF BEAN STARCH

Introduction ........................................................

Materials and Methods ..............................................

Results and Discussion ..............................................

Conclusions ........................................................

CHAPTER III. CHARACTERIZATION OF BRINE OBTAINED FROM CANNED

BEAN POSSESSING DIFFERENTIAL QUALITY

Introduction .......................................................
Materials and Methods ..............................................

Results and Discussion ..............................................

LIST OF TABLES

Page
Table 1. Granule dimensions and shapes of legume starches ................. 5
Table 2. Gelatinization temperature ranges reported for legume starches ....... 10
Table 3. Effect of sucrose concentrations on the gelatinization endotherrn of wheat
starch ............................................................ 25
Table 4. Effect of salt concentrations on the gelatinization endotherm of wheat
starch ............................................................ 27
Table 5. Components of dietary fiber ........................................ 47
Table 6. Total solids and carbohydrate contents of whole beans and brine after
cooking .......................................................... 66
Table 7. Degree of gelatinization of beans held in different conditions measured by
the enzymatic method ............................................. 67
Table 8. Starch available by B-amylase and pullulanase after different cooking and
reheating treatment ................................................ 69
Table 9. Available starch of flour samples prepared differentially on reheating . . . 89
Table 10. Visual characteristics of canned navy beans and brine ............... 106
Table 11. Canning characteristics of eight selected navy beans ............... 113
Table 12. Average value of canning characteristics of eight selected navy
beans grouped by visual appearance ........................... 114
Table 13. Percentage of proximate composition in brine .................... 117
Table 14. Average percentage of brine composition of eight selected navy beans
grouped by visual appearance ..................................... 118
Table 15. Correlation matrix indicating relationships among visual appearance,
canning quality and brine composition ......................... 120

Table 16. T-test for variables grouped by brine characteristics based on total solids
and alcohol insoluble solids .......................................

Table 17. T-test for variables grouped by bean integrity based on total solids and
alcohol insoluble solids ...........................................

LIST OF FIGURES

Page
Figure 1. Schematic representation of X-ray diffraction patterns of various
starches use for their classification into the A-, B-, C-, and V-type
starches .......................................................... 17

Figure 2. Experimental protocol used to assess the degree of starch gelatinization in
differentially cooked and prepared navy beans ..................... 58

Figure 3. Flow sequence to determine degree of gelatinization ................ 62

Figure 4. Experimental protocol used to assess starch availability and cellular
appearance of foods prepared under selected preparation procedures . . . 83

Figure 5. Available starches of navy bean (experimental line, N84004) prepared with
different cooking times ............................................ 91

Figure 6. Available starches of pinto bean (var. Carioca) prepared with different
cooking times .................................................... 93

Figure 7. Available starches of sweet potato prepared with different cooking
times ............................................................ 94

Figure 8. Available starches of wheat prepared with different cooking times ..... 96

Figure 9. DSC thermograms of pinto bean flour prepared after differential
cooking treatments ............................................... 101

Figure 10. Flowchart of the enzymatic method to determine the insoluble and
soluble dietary fiber of alcoholic precipitated flour ................ 102

LIST OF PLATES
Page

Plate 1. Light microscopy images of soaked beans presented as representative
fields
a, c) transmitted and b, d) polarized ................................... 74

Plate 2. Polarized light microscopy images of the cross sections for differentially
prepared beans
a) soaked b) cooked for 40 min ..................................... 75

Plate 3. Polarized light microscopy images of the fresh beans cooked for different
times
a) 10 min, b) 20 min, c) 30 min and d) 40 min ...................... 76

Plate 4. Light microscopy images of intact cooked beans heated for 2 hr at 98°C . . 77

Plate 5. Polarized light microscopy image of reheated (10 min) bean flour
prepared after differential cooking treatments.
a) soaked, b) 10 min cooked and c) 40 min cooked .................. 97

Plate 6. Light microscopy images illustrating cell wall structure for differentially

cooked navy beans

a) 10 min cooked b) 40 min cooked ................................. 98
Plate 7. Polarized light microscopy images of bean and sweet potato tissue cooked

for 40 min

a) whole bean b) whole sweet potato ............................... 100

INTRODUCTION

Legume consumption has increased in the western world due in-part to the
improved recognition of beans as a good source of dietary fiber and protein. In addition,
the high quality of carbohydrate associated with controlled digestion and absorption is
increasingly being recognized. Legume starch is well-known as a low glycemic index
food. It has been shown that the low glycemic index diets improve metabolic variables not
only in diabetes (Brand, et al., 1991) and hyperlipidemia (Jenkins, et al., 1987a) but also in
healthy subjects (Jenkins, et al., 1987b). The resistance of legume starches towards
hydrolytic enzymes have been of great interest to nutritionists, since they have been found
to exhibit a lower glycemic index than the cereals (Jenkins, et al., 1980). However,
legume starch granules are contained within thick cell walls, which is a critical factor
determining the rate of digestion. The starch granules contained within intact cell walls
pass the stomach and enter the small intestine. The indigestible starch also reaches colon
and provide substrates for bacteria to ferment which result in flatulence.

The degree of starch gelatinization in cooked beans is of importance to their
complete digestibility. Seeds of food legumes are known to contain starch fractions that
do not gelatinize and therefore are resistant to digestion. In vitro enzymatic methods have
been used for estimation of degree of starch gelatinization, these methods provide results
which are closely related with true in viva digestibility if appropriate enzymes and

conditions are maintained. Heating of beans during processing is required for palatability.

However, the integrity of cell wall polymerized during cooking is a concern to maximum
digestability.

The research reported in this dissertation was directed and conducted toward the
development and assessment of accurate methodology for starch gelatinization of cooked
whole beans and to attain a greater understanding nature of changes within cell wall
during cooking. Both approaches are necessary for implementing technological strategies
for improvement of dry bean utilization.

This research was conducted and reported in three independent Chapters. Chapter

titles and their respective Null hypotheses (Ho) are presented:

Chapter 1: Indigestible starch components in dry beans

Ho: The presence of whole cells with crystallization in the cell wall do not
prevent starch gelatinization and lower starch digestibility.

Chapter 2: Heat-induced changes in cell wall structures reduce bioavailability of bean
starch

Ho: The crystallization of cell wall in cooked beans is not a limiting factor
associated with starch gelatinization and bioavailability.

Chapter 3: Characterization of brine obtained from canned beans possessing
differential quality

Ho: The compositional components of brine and physical bean quality after
processing are not controlled by genetic background.

REVIEW OF LITERATURE
Physical and Chemical Properties of Bean Starch

legumes are the dicotyledonous seeds of plants that belong to the family
Leguminoase, which contains about 600 genera with 13,000 species. The major
carbohydrate of the legume seed is starch. Within each parenchyma cell, starch granules
are embedded within a protein matrix (Powrie et al., 1960; Sefa-Dedeh and Staley, 1979
and McEwen et al., 1974). Legumes contain 24% (winged beans) to 68% (cowpeas) total
carbohydrate on a dry basis of which starch makes up a larger portion ranging from 24 to
56% (Reddy et al., 1984). These variations in starch contents are due to different
cultivars and analytical procedures (Pritchard et al., 1973 and Ceming-Beroard et al.,
1975). Starches contain two types of glucose polymers: amylose and amylopectin. The
arnylopectin molecules are highly branched and in general, larger than amylose molecules.
The starch found in legumes has oblong granules which vary in size by species. Dry bean
starch granule is resistant to swelling and rupture and generally contains high amylose
content (30 - 37%) (Hoover and Sosulski, 1985). Starch granules can greatly influence
the cooking characteristics of legumes. Gelatinization temperatures ranging from 60°C to
over 75°C are relatively high compared to cereals and may contribute to processing
variability (Hahn et al., 1977).

Granule Composition and Structure

The major energy reserve of most plant tissue is starch, which is most abundant in

seeds, roots and tubers. Following the biosynthesis, starch is stored in the plant as

compact micron-sized granules that are partly crystalline. This relatively inert and water-
insoluble fraction provides energy reserves which facilitate starch isolation and analytical
handling (Zobel, 1984).

Granule size, shape, and microscopic appearance

The size and shape of legume starch granules are shown in Table 1. The granule
size is quite variable and ranges from 4 to 85 pm depending on the starch source. Most
bean starch granules are oval (greater length titan width), although spherical, round,
elliptical, and irregular granules are also found. This wide variation in granule size and
shape is generally attributed to genetic control and seed maturity. For dry beans,
however, a wide variability in shape is found in starch granules from the same source
(Reddy et al., 1984).

Light microscopic studies of legumes starches clearly reveal two distinct starch
granule characteristics: the presence of a hilum and the presence of lamellae. The hila
have been described as furrows, grooves, cracks and stria (Naivikul and D'Appolonia,
1979), cracking dark bands (Hall and Sayre, 1971), and microfibrils (Donovan, 1979).

The hilum and lamellae observed under the light microscope are not seen under a
scanning electron microscope. Instead, their surfaces appear to be smooth with some
occasional "scar-like" features. These latter structures could arise from adhering cell wall
materials or proteins, to both.

Under the polarized light microscope, intact granules exhibit a well-defined

birefringence pattern with a dark cross. This is indicative of the highly organized nature of

 

 

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the granule. Amylopectin may be responsible for the crytallinity since waxy starches with
essentially no amylose are very birefringent (Hood, 1982).
Molecular structure

Generally, starch is defined as a polymer of D-glucose units. It is composed of
two different polymers, a linear compound, amylose, and a branched component,
amylopectin. Traditionally, amylose was thought to be a linear glucose polymer in which
the individual monomers were connected solely by 0t-1,4 glucosidic linkages. It is now
recognized that it has some elements of nonlinearity (Hood, 1982; Whistler and Daniel,
1984). The molecular structure is not well defined. It has a molecular weight of 150,000
- 1,000,000 depending on its biological origin. Greenwood and Thomson (1962)
indicated that structural differences exist along the amylose molecule and that it is not
completely linear. Amylopectin is a ramified structure containing 94-96% (at-1,4 and 4-6%
(at-1,6 linkages. The average chain length is 20-26 glucose units. Amylopectin does not
give a native starch-iodine (Amylose-iodine) blue color but rather a purple and sometimes
reddish brown color depending upon its source. The molecular weight of amylopectin is
on the order of 107- 108. Its molecular structure has been studied by many investigators
(Haworth et al., 1937; Meyer, 1952; Wheland, 1971; French, 1972; Robin et al., 1974;
Yamaguchi et al., 1979; and Manners and Matheson, 1981). Several structural models
have been proposed for amylopectin molecule to account for its physicochemical
properties. In 1937, Haworth et al., proposed the "laminated" structure, i.e., the ratio of
A and B chains is 1:(n-2), where A chains are attached to the macromolecule by a single

linkage from the potential reducing-group, and B chains are linked to two or more other

chains, where n is the total number of chains. In the same year, Staudinger and Husemann
suggested the "comb" structure. All of the chains except one were designated A chains.
Meyer (1952) originated the "tree type" structure in which branching from a centering
region is predominant. Wheland (1971) later revised the Meyer structure such that the
A:B ratio was approximately 1:1. A cluster—type structure was originally proposed by
Nikuni (1969) for starch molecules; then independently, for the amylopectin component by
French (1972), and later by Robin et al. (1974). More recently, an extended cluster model
has been proposed by Yamaguchi et a1. (1979). Manners and Matheson (1981) supported
the structure of the cluster-type because it was more in accord with the physical
properties, the observed structural features, and mode of biosynthesis within the starch
granule than were other hypothesized models. It is becoming increasingly apparent that,
except for high-amylose starch, the molecular structure of amylopectin is similar among
cultivars (Hood, 1982).

The molecular structure of starch components and their arrangement are related to
the crystalline properties of the granule. According to the amylopectin structure proposed
by Robin et a1. (1974), the Ot-l,6 linkage rich regions represent the amorphous area within
the granule, whereas the outer short chains (DPSIS) form the highly organized helical
structures contribute to the crystalline properties of the granules.

Granule Crystallinity

Starch granules contain both crystalline (ordered) and amorphous (unordered)
regions. This crystallinity gives rise to the birefringent property of starch granules (Elbert,

1965). X-ray diffractometry has been used to reveal the presence and characteristics of

crystalline structures of starch granules (Katz and Van Itallie, 1930; Sarko and Wu, 1978).
Legume starches have been shown to exhibit a "C" type X-ray diffraction pattern (Hoover
and Sosulski, 1985; Kawamura, 1969; Sarko and Wu, 1978; Colonna et al., 1980; Lai and
Varriano-Marston, 1979). This is intermediate between the A (cereal) and the B type
(tuber). The reasons for these differences are not properly understood. Hizukuri (1985)
has suggested that slight differences in the chain length and chain profile of the constituent
amylopectin molecules may be responsible for these differences in X-ray patterns. Hoover
and Sosulski (1985) have shown that most legume starches are characterized by two very
pronounced lines centered at 17.2 and 181° 26 angles, which correspond, respectively, to
interplanar spacing of 5.15 and 4.98 A (l A = 0.1 nm).

Swelling Power and Solubility

Legume starches usually have restricted swelling behavior. Leach et a1. (1959)
indicated that legume starch swelling power is lower than that exhibited by wheat starch.
Many workers have studied the swelling pattern of various legume starches; Lineback and
Ke (1975), chick pea and horse bean; Lai and Varriano—Marston (1979), black bean,
yellow pea, and navy bean; Lii and Chang (1981), red bean. They have observed the same
single-stage pattern. Lai and Varriano-Marston (1979) indicated that bean starch had
lower swelling power than wheat starch within the temperature range 60°C to 74°C; above
7 5°C, however, bean starch surpassed wheat starch in swelling power. The restricted
swelling, plus the single-stage swelling pattern, have been interpreted to explain the
crystalline and amorphous regions of starch granules and the presence of strong binding

forces which relax at one temperature range. Reddy et a1. ( 1984) suggested that the

swelling ability and solubility depend on starch source, temperature and pH. Lai and
Varriano-Marston (1979), Comer and Fry ( 1978), and Sathe et al. (1981) reported that
solubility of legume starches is less than 30%.

Gelatinization and Pasting

Starch, when heated in the presence of excess water, undergoes an order-disorder
phase transition called gelatinization over a temperature range characteristic of the starch
source (Donovan, 1979; Biliaderis et al., 1980). The above phase transition is associated
with the diffusion of water into the granule and subsequent hydration and swelling of the
starch granule. This endothermic reaction results in loss of crystallinity, decreased
relaxation time of water molecules, and amylose leaching (Stevens and Elton, 1971;
Lelievre and Mitchell, 1975; Donovan, 1979; Hoover and Hadziyev, 1981).
Most legume starches have gelatinization temperatures of 60°C to 90°C, with the
exception of wrinkled peas which ranges over 99°C (Reddy et al., 1984). The
gelatinization temperature ranges of various legume starches are presented in Table 2.
The Brabender viscosity pattern of bean starches showed restricted swelling characteristics
similar to those shown by chemically cross-linked starches (Schoch and Maywald, 1968;
Lineback and Ke, 1975; Vose, 1977; Lai and Varriano-Marston, 1979; Naivikul and
D'Appplonia, 1979; Lii and Chang, 1981; Sathe and Salunkhe, 1981). According to
Schoch and Maywald (1968), these restricted swelling pastes are classified as Type C
starches which show no pasting peak, but rather a very high viscosity which remains
constant or increases during cooling and have relatively constant cold-paste viscosities

during a holding period at 50°C. Kawamura and Fukaba (1957) classified legume starches

10

Table 2. Gelatinization temperature ranges reported for legume starches

 

 

Gelatinization
Starch Source Temperatures Range (°C) Reference
Lima bean 70 to 85 Schoch & Maywald (1968)
Lentil 64 to 74 Schoch & Maywald (1968)
58 to 61 Biliaderis et al. (1979)
Yellow pea 63 to 73.5 Schoch & Maywald (1968)
Navy bean 66 to 77 Schoch & Maywald (1968)
68 to 74 Biliaderis et a1. (1979)
Garbanzo bean 62.5 to 72 Schoch & Maywald (1968)
65 to 71 Biliaderis et a1. (1979)
Mung bean 60 to 78 Schoch & Maywald (1968)
63 to 69 Biliaderis et al. (1979)
Wrinkled pea 69 to 83 Schoch & Maywald (1968)
> 99 Biliaderis et a1. ( 1979)
Black gram 71.5 to 74 Sathe et a1. (1982)
Black bean 63.8 to 76 Lai & Varriano-Marston (1979)
Smooth pea 65 to 69 Biliaderis et al. (1979)
Red kidney bean 64 to 68 Biliaderis et al. (197 9)
Faba bean 61 to 66 Biliaderis et al. (1979)
61 to 69 Lorenz (1979)
Soybean (Amsoy 71) 73 to 81 Wilson et al- (1978)
Pea 54 to 66 Comer & Fry (197 8)
Red bean 63 to 70 Lii & Chang (1981)
Adzuki bean 83 to 89 Biliaderis et a1. (1979)

 

11

into two categories based on hot paste characteristics: 1) those which do not have a
substantial rise in viscosity during heating (25°C to 925°C) and cooling (925°C to 25°C)
cycles and 2) those which show a distinct rise in viscosity during heating and cooling
cycles. Lai and Varriano—Marston (1979) studied the gelatinization temperature range of
black bean, and reported temperature ranges of 638°C to 76°C, which is considerably
higher than that of wheat starch, 556°C to 63°C. These results are similar to those
reported for navy and mung bean starches according to Schoch and Maywald (1968).

Rosenthal et al. (1970) studied the pasting characteristics of jack bean and guandu
bean starches and reported that there was no peak at low concentrations (up to 6%). At
these low concentrations, no sensible retrogradation was observed and their viscosity
curves were similar to cross-bonded starch. Vose (1977) studied the effects of acidic and
basic conditions during pasting of double-milled air-classified pea flour. He found that at
acidic pH values up to pH 4, there was a steady increase in viscosity during the pasting
process, including a resistance to shear breakdown when 8% slurries were maintained at
96°C for 15 min; however, at pH 3 a pasting peak occurred, which was followed by a
decrease in viscosity and then a set-back during cooling. Lii and Chang (1981) also
observed that the different steeping solutions used during starch isolation did not affect the
viscosity pattern. Sathe and Salunkhe (1981) indicated that the gelation of purified bean
starch could yield a stable gel at concentrations of 7% or above (w/v).
Starch gelatinization and melting concept

Starch granules are not soluble in cold water, but can reversibly imbibe water and

swell slightly. However, as the temperature is increased, the starch molecules vibrate

12

more vigorously, breaking the weaker intermolecular bonds (firstly in the amorphous area)
and allowing their hydrogen bonding sites to engage more water molecules. This
penetration of water, and the increased separation of more and longer segments of starch
chains, increases randomness in the general structure and decreases the number and size of
crystalline regions. Continued heating in the presence of abundant water results in a
complete loss of crystallinity as judged by loss of birefringence. This process is called
"Gelatinization" and the temperature at which gelatinization occurs is called the
"Gelatinization Temperature". For a population of granules, the range for a gelatinization
temperature is usually between 5-10°C, indicating that fractions of granules exhibit
different gelatinization temperatures.

Gelatinization process also can be described as the melting of starch crystallites
which should be amenable to thermodynamic analysis (Lelievre, 1973). Marchant and
Blanshard (197 8) postulated three constituent processes for starch gelatinization based on
nomquilibrium thermodynamics: (1) diffusion of water into starch granules, (2) a
hydration—facilitated helix-coil transition which is a melting process, and (3) swelling as a
result of crystallite disintegration (melting). Using a light-scattering method to study
melting and swelling processes during gelatinization, Blanshard (1979) reported that
gelatinization may be described as a "Semicooperative process". Each granule has its own
degree of crystallinity with its unique energy characteristics. The imposition of a 2°C
temperature rise may result in certain granules being totally gelatinized but, in others, only
some of the crystallites will have their gelatinization threshold exceeded. If the

temperature increase is applied continuously, eventually the whole population of granules

13

will be gelatinized. Since the amorphous region hydrate initially, French (1984) has
proposed that swelling of amorphous phase which occurs when starch is heated in excess
water contributes to the disruption of the crystallite regions by tearing molecules from the
crystallites.

Aside from swelling during gelatinization, the viscosity of the medium also
increases. When the granule swells to the point that internal bonding can no longer
maintain its integrity, it ruptures and the viscosity declines. During heating, amylose
leaches from the granule and forms an extragranular network (Allen et al., 1977; Miller et
al., 1973). Viscosity changes during the early heating stages appear to be due largely to
this extragranular material. During the later stages when maximum viscosity is
approached or attained the swollen granules and the extragranular material both contribute
to apparent viscosity. Both the molecular and granular structures contribute to the
increase in the viscosity. Based on the changes in the viscosity of starch in excess water
system during and after heating, Olkku and Rha (1978) summarized the step of
gelatinization as follows: (1) granules hydrate and swell to several times of their original
size, (2) granules loss their birefringence, (3) clarity of the mixture increases, (4) marked,
rapid increase in consistency occurs and reaches a maximum, (5) linear molecules dissolve
and diffuse from ruptured granules, (6) upon cooling, uniformly dispersed matrix forms a
gel or paste-like mass.

Method for determining starch gelatinization

Degree of gelatinization can be determined qualitatively and /or quantitatively by

physical, chemical, and biochemical methods (Watson, 1964; Wong and Lelievre, 1982).

14

Birefringence End Point Method Starch granules show some degree of
birefringence owing to a highly oriented nature of D-glucosyl units in amorphous and
crystalline regions. Loss of birefringence is a characteristic of starch that undergoes
thermal gelatinization. The measurement requires an optical microscope with crossed
polarizing filters and a heating stage (Schoch and Maywald, 1956; Watson, 1964).
Watson (1964) determined the degree of gelatinization by measuring the percent loss of
birefrigence (2, 10, 25, 50, 75, 90, and 98%) of the granules in the field during uniform
rates of heating. The 98% point is taken as the gelatinization temperature end-point. In
practice, usually only initial gelatinization and 98% loss temperatures are recorded.
Watson's method was modified by Berry and White (1966) to follow the progress of the
gelatinization by recording the light output on a photocell as a function of hot stage
temperature. This method is often used to determine the degree of gelatinization, because
of its simplicity in equipment and application. It is limited, however, to dilute granule
suspensions (0.1 - 0.2%).

Viscosity Method In common practice, the most widely used method for
determining the degree of gelatinization is based on changes in viscosity during
gelatinization. A Brabender Viscolamylo/Graph, the most commonly used instrument,
provides information not only on gelatinization but also on the properties of the paste
during cooling. This instrument records the torque required to balance the viscosity that
develops when the starch slurry is subjected to programmed heating and cooling cycles.
The temperature at which the first major rise in viscosity occurs is highly dependent on

starch concentration and is generally higher than gelatinization temperatures obtained by

15

loss of birefringence. Viscosity is measured in arbitrary units that reflect paste
consistency; accordingly, Brabender Viscoamylograph results are described in term of
paste properties and pasting temperatures at given time. This methodology is highly
empirical and thus, reported data obtained by this technique should include instrument
model, torsion spring used, bowl speed, volume of slurry, slurry concentration (including
basis), and start, hold, and final temperatures.

Freeman and Verr (1972) observed the gelatinization process and paste
development of starch slurry with the simple rotational viscometer, Brookfield Synchro
Lectric viscometer. However, this technique is quite tedious compared to Brabender
Viscoamylograph because it lacked a temperature control system. A more sophisticated
rotational viscometer with programmable temperature control system, Haake Rotovisco is
becoming increasingly used for studying gelatinization process, paste development and
rheology of the paste (Voisey and deMan, 1976; Lee et al., 1995).

X-Ray Diffraction Method In addition to providing information about the
crystal structure of the crystallites of starch granules, x-ray diffraction can give
information about the relative amounts of crystalline and amorphous phases. Therefore, it
can be used as a tool to measure the extent of gelatinization (Lugay and Juliano, 1965;
Varriano - Marston et al., 1980; Owus-Ansah et al., 1982).

X-rays are a form of electromagnetic radiation with a wavelength typically
between 0.1 and 1.0 nm, which is comparable to the molecular spacing in a crystal. When
an x-ray beam impinges on a crystal which is held in a special mount that allows the crystal

to be rotated with respect to the incident beam, diffraction occurs. Diffraction is the

phenomenon that occurs whenever a wave motion interacts with an obstacles. The
diffracted beams are recorded to obtain information on the structure of the crystal and the
molecules within the crystals. In x-ray analysis of starch granules, satisfactory results are
obtained if the specimens are properly prepared and mounted. Coarse powder gives less
intense pattern. Therefore, the samples have to be less than 200 mesh inside and packed
as densely as possible into a sample holder. The finished surface must be smooth and flush
with the face of the sample holder. Upon recording the diffracted beams, the patterns are
analyzed based on their interplanar spacings and relative intensities of the diffraction lines.
Native starches are classified according to their wide-angel X-ray diffraction
patterns, as shown in Figure 1, as A-, B-, or C-type starches (Zobel, 1988; Biliaderis,

1991a). An additional diffraction pattern known as V-type crystallinity corresponds to

 

@0
Ce)

 

 

 

Figure 1. Schematic representation of X-ray diffraction patterns of various starches use
for their classification into the A-, B-, C-, and V-type starches (Zobel, 1988)

l7

structures of helical inclusion complexes of amylose, which is typical of amylose-lipid
complexes (Biliaderis, 1991a). The A—type pattern is exhibited by cereal starches (rice,
wheat, and com); the B-type pattern is shown by tuber, fruit and high-amylose corn
starches and by retrograded starch. C-type pattern starch is an intermediate between A-
and B- type starches and it is typical of legume seed starches (e. g., pea and bean starch).
The V-type structure has not been found in native cereal starches, but it may form due to
heating of lipid-containing starches (Biliaderis, 1991b).

Amylase / Iodine Blue Value Method Amylose complexes with iodine to yield
brilliant blue color and this characteristic has been used as an analytical tool to measure
amylose content. A quantitative method to determine the amount of amylose present in
solution base has been developed by McCready and Hassid (1963) and modified by Gilbert
and Spragg (1964). The interaction of amylose with iodine generates a helical inclusion
complexes in which the iodine molecules occupy the central cavity of the helical
polysaccharide. The helix consisted of six glucose residues per turn, with a pitch of 8A.
The absorbance of the blue color is measured with a spectrophotometer at 600 nm.
Application of amylose / iodine blue complex method to determine the degree of
gelatinization has been used in many food products. Roberts et a1. (1954) utilized this
method to determine an index of parboiling of rice. Birch and Priestly (1973) modified
their method for parboiled rice by treating with alkali solution to distinctively differentiate
degree of gelatinization. Wootton et a1. (1971) also used amylose / iodine method to

determine degree of gelatinization in biscuits.

18

Differential Scanning Calorimetry (DSC) Method The term "Differential
Scanning Calorimetry" was initially a source of some confusion in thermal analysis. The
parent technique to DSC is "Differential Thermal Analysis (DTA)". The purpose of
differential thermal systems is to record the difference between an enthalpy change which
occurs in a sample and that in some inert reference material when both are heated.

The important difference between DTA and DSC is that in DSC the sample and
reference are each provided with individual heaters which allows the determination to be
conducted with no temperature difference between sample and reference. Running in this
mode allows two important simplifications compared to DTA. First, because the sample
and reference pans are maintained at the same temperature, the calibration constant for the
instrument is independent of temperature. Obviously, this greatly simplifies the
experimental technique since the calibration constant need only be determined for one
standard material. Second, since the sample and reference pans have independent heater,
the difference in heat flow to the sample and reference in order to maintain equal
temperature can be measured directly. Thus, data are obtained in the form of differential
heat input (dH/dt) vs temperature (or time, since constant heating rate are used). These
data are readily used to obtain temperature and an enthalpy of transitions or reactions.

For optimum peak sharpness and resolution, the contact surface between pan and
sample should be maximized, which is usually accomplished by having the sample as thin
discs, or film, or fine granules. Frequently, in applications in foods, the sample will be
dispersed or soluble in water, which obviates any problem with contact surface.

Calibration of the instrument is generally canied out with a high purity metal with

19

accurately known enthalpy of fusion and melting point. The most commonly used
calibrant is indium (AH = 6.8 cal/gm, mp = 156.4°C).

Stevens and Elton (1971) applied DSC to measure heat of gelatinization of
starches, and since then many groups have applied DSC in the study of gelatinization
(Longston and LeGrys, 1981; Ghiasi et al., 1982; Raemy and Loliger, 1982). Banks and
Greenwood (1975) suggested this method was valid by considering gelatinization to be the
analogue of a melting process for a crystal.

Laser Light Scattering Method The use of polarize laser-light scattering is a
relatively new technique for following changes in the internal structure of starch granule
during gelatinization. Since the anisotropic scattering indicates a spherulic organization
(French, 1984). Many investigators (Marchant et al., 1977; Marchant and Blanshard,
1978, 1980a, 1980b) have successfully utilized this technique to observe the changes of
starch granule during gelatinization.

Other Methods Other methods in addition to those previously described are
available, e.g., enzymatic digestibility, nuclear magnetic resonance, light-extinction,
solubility or sedimentation of swollen granules, and absorption of con go red measurement.
However, these methods are less popular due to the difficulties in their procedures or
degree of accuracy in measuring the degree of gelatinization.

Factors affecting gelatinization process within food systems

Time and Temperature Many investigators have studied the influence of time

and temperature on the extent of starch gelatinization in order to design the efficient starch

cooking systems and / or to optimize both process and product quality.

20

Most kinetic studies on starch gelatinization were performed under isothermal
conditions. Since the temperature can then be considered as an independent variable, and
the time dependence can be determined. In practice, particularly in processing of food
constituents. processes to promote desirable reactions are not accomplished isothermally,
but the product undergoes a real temperature change (thermally dynamic) and there is a
significant lag time within the product. Lund (1984) proposed in his work and cooperated
with Wirakartakusumah ( 1981) on the isothermal and nonisotherrnal kinetics of starch
gelatinization:

water + ungelatinized starch —> gelatinized starch
heat

The reaction is reversible, and under isothermal conditions the form of the rate
expression is:
d[UG]

r = - —- = k [HzO]"°[UG]n
(it

where m, n = order of reaction for water and UG, respectively
k = the reaction rate constant
UG = ungelatinized starch.

If excess water is present, the reaction rate constant k and [H20]'° can be
combined into an apparent or empirical pseudo rate constant Ka.
d[UG]
= - Ka [UG]"
dt
For n = 1, a first order reaction, the integrated form is
[UGJ

1n = Ka t
[UGr]

 

where UG, = initial amount of ungelatinized starch at time t
UG, = final amount of ungelatinized starch at time t

The expression [UGJ / [UG,] is unity when gelatinization reaches 100 %
completion provided the time is sufficient at any given temperature. However, if the
extent of starch gelatinization is temperature dependent (i.e., the starch is not complete
gelatinized at a given temperature even if held an infinite time), the argument [UG,] /
[UG,] must be modified to describe the extent of reaction.

[UG, - UG,]

1n = Ka t
[UGi - UGu]

 

where UGu = ultimate amount of ungelatinized starch at the given temperature

Then two procedures were used to determine the reaction rate constants. The
isothermal method uses data generated by measuring the extent of reaction as a function
of time at a given constant temperature. The second approach is based on the heat
evolution or temperature scanning method using DSC data. The results indicate that the
ultimate extent of gelatinization is directly dependent on heating temperature.
Furthermore, that a significant degree of gelatinization has been accomplished at the end
of lag time. However, this methodology cannot describe the rate of gelatinization over the
entire range of no gelatinization to maximum extent of gelatinization. This would suggest
that the process of gelatinization is complex and not readily described by pseudo first
order kinetics. This observation is consistant since there is a diverse population of starch

granules, each with its unique degree of crystallinity. The process can be empirically

IQ
I‘J

modeled but interpretation of mechanism from the model should be done with great care.
Pravisani et al. (1985) examined the kinetics of starch gelatinization in potato and
confirmed the previous studies that at temperatures below certain thresholds,
gelatinization was not completed even for long reaction time.

Moisture At temperature above that for complete gelatinization, several studies
have shown that there is a minimum water to starch ratio (W/S) in order to achieve
complete gelatinization (W ootton and Bamunuarachchi, 1979; Eliasson, 1980; Marchant
and Blanshard, 1980).

Wirakartakusumah (1981) studied the influence of moisture on starch
gelatinization using DSC and followed the changes in enthalpy of various W/S of rice
starch suspension at temperature above the gelatinization temperature (150°C, at 10°C /
min). At WIS ratio 2 2:1, only two endotherrns are obtained. The first endotherm is
observed at an onset temperature (To) of about 71°C. This single endotherm is
characterized as the gelatinization phase change. In addition to the first endotherm, the
second endotherm is obtained in the temperature range between 90 - 110°C. This higher
temperature endotherm is associated with melting of an amylose-lipid complex. A similar
type of endotherm which occurred at 100°C was also reported by Kuimiya et a1. (1980)
from maize and wheat starches. As the W/S ratio decrease to less than 1.5:1, the
gelatinization endotherm develop a trailing shoulder with a marked decrease in the area
(enthalpy). This shouldering may be due to the destabilizing effect of water and heat

treatments on the amorphous and crystalline region of starch granules. The onset

23

temperature (To) and temperature range of gelatinization shift to higher temperature as
the water content decreases.

Wirakartakusumah (1981) also reported that more than 3 moles of water / hexose
unit are required to start gelatinization of rice starch and, about 13.5 moles of water /
hexose unit are required to complete gelatinization. These values are in good agreement
with the data reported by Donovan (1979) on potato starch. Therefore, it is concluded
that a critical amount of water is necessary to initiate and to complete gelatinization.

Ingredients

mm It is well established that physical characteristics of starch
pastes may be radically altered by the presence of polar substances containing large
hydrophobic groups. Practical instances of utilizing these effects include: (1) addition of
sulfonated oils to textile starch sizes as “softeners” and antiskining agents, (2) use of soaps
to modify viscosity of starch sizing in paper coatings, (3) the former application of
polyoxyethylene monostearate as a bread softener, (4) use of monoglyceride to alter the
paste consistency of dehydrated potato flakes, and (5) use of glycerol monostearate and
other mono- and di-glycerides to prevent stickiness in cooked rice kernels. These effects
are due primarily to the formation of a complex between the fatty adjunct and amylose, the
linear fraction of starch polymers (Larsson, 1980: Marringat and Juliano, 1980; Ohashi et
al., 1980; Eliasson et al., l981a,b; Hoover and Hadziyev, 1981; Ghiasi et al., 1982). It is
postulated that the use of pork back fat pieces in “pork and beans” has a direct impact on

the starch characteristics of navy beans during canning.

Gray and Schoch (1962) performed a definitive study on the effect of nine fatty
adjuncts on swelling and solubilization characteristics of starches from various sources.
As a broad generalization, lower polar adjuncts containing four or five carbon atoms (e. g.,
butyl and emyl alcohols) markedly assist the hydration of corn starch when cooked in
water, causing a substantial reduction in the gelatinization temperature and an increase in
the extent of solubilization. Fatty adjuncts containing a hydrocarbon residue of about 12
carbon atoms tend to complex with the amylose below 85°C, consequently repressing
swelling and solubilization. This complex breaks down at higher temperatures, and the
surfactant may thereafter assist swelling. Compounds containing a hydrocarbon residue of
18 carbon atoms (stearate) generally appear to form complexes which are stable at least
up to 95°C, and hence repress swelling and solubilization over the entire gelatinization
temperature ranges.

The time of addition and time for diffusion of adjunct into the granule are also very
important and can cause substantial differences in properties of the cooked starch or cereal
grains. If the adjunct is allowed to diffuse into granule significantly, swelling and
solubilization may be greatly reduced. Whereas, if the adjunct is added immediately before
cooking, the amylose-lipid complex will largely form extragranularly and the adjunct will
have little effect on swelling and solubilization.

MW Ionic and nonionic soluble constituents affect
gelatinization characteristics of starches. Wootton and Bamunuarachchi (1980) presented
a study on the effect of sucrose and NaCl on wheat starch gelatinization. However, the

situation regarding the influence of sugar and salt on starch gelatinization is not quite

25

clear. Much of the previous research dealt with low starch concentration and, hence,
makes correlation between the earlier studies difficult and some of these techniques are
more suited to qualitative, rather than quantitative, interpretation.

Table 3 demonstrates the results of Wootton and Bamunuarachchi (1980) on AHG
(enthalpy of gelatinization), the extent of gelatinization and temperatures at which the
endotherms began (T 0) reached their peak (Tp), and concluded (Tc), for wheat starch /
water systems containing various levels of sucrose in the aqueous phase. This can be seen
that To and Tc were not affected by increased levels of sucrose while Tp was increased.
These results agreed with the findings of earlier workers, who indicated that increasing
sucrose concentration caused increases in pasting temperatures corresponding with the
change in Tp. From the lack of change in To and Tc, it appears that while the peak
temperature of the gelatinization endotherm rises with increased sucrose concentration,
the range over with the process extends is not affected by the concentration of the solutes.

Sucrose has a restrictive effect on the heat of gelatinization. This effect is widely

known and several explanations for this phenomenon have been proposed including

Table 3. Effect of sucrose concentrations on the gelatinization endotherm of

 

 

 

wheat starch
Sucrose cone. in Extent of Endotherrn
aqueous phase A He Gelatinization temp. (°C)
(%) (cal Lg) (%) To Tp Tc
0 4.7 100 50 68 86
15 3.2 68 50 70 86
30 2.8 60 50 73 86
45 2.3 49 50 75 86

 

From Wootton and Bamunuarachchi (1980)

competition between starch and sucrose for available water, sucrose inhibition of granular
hydration, and sucrose-starch interactions. From the review paper of Lund (1984), he
suggested that the actual mechanism by which sucrose restricts the gelatinization of starch
is not simply lowering available water in proportion to the level of sucrose present in the
system. If it is, then a linear relationship between a AHG and the sucrose level in the
aqueous phase may be expected however the relationship between these two parameters is
not linear (Table 3).

Since the rising cost of sucrose and the increased availability of corn syrup and
high fructose syrup, many investigators have studied the effects of different sugars on
starch gelatinization (Koepsel, 1977; Hoseney et al., 1978; Savage and Osman, 1978).
They concluded that monosaccharides delay gelatinization less than disaccharides, except
maltose, which acted like a monosaccharide. By using DSC, Spies and Hoseney (1982)
reported that the gelatinization-delaying characteristics of a sugar solution were related to
the water activity of the solution and to the molecular size of the sugar. As the water
activity of a sugar solution decreases, gelatinization temperature of starch in that solution
increases. But at equal water activities, sugars with longer chain length delay
gelatinization more than do shorter chain sugars.

Evans and Haisrnan (1982) examined starch gelatinization in the present of
nonionic constituents and found that the effect of temperature and extent of gelatinization
could be explained through solute effect on available water and melting point. The
influence of NaCl on starch gelatinization was also studied by Wootton and

Bamunuarachchi (1980). As shown in Table 4, salt has a different and more complex

27

effect on starch gelatinization than does sucrose. Changes in salt concentration affected
all three temperatures associated with the gelatinization of the endotherm. A variation in
the temperature range over which the gelatinization endotherm extends also occurs with
added salt. Evans and Haisman (1982) reported that other salts such as sodium sulfate
and sodium hydrogen phosphate (pH 7.0) increase gelatinization temperature, whereas
CaClz decreased and then increased gelatinization temperature with increased
concentration. Oosten (1982) proposed a hypothesis to explain this phenomenon. His
hypothesis is that starch acts as a weak acid ion exchanger and that cations tend to protect
and to stabilize the granule structure while anions function as gelatinizing agents by

rupturing hydrogen bonds.

Table 4. Effect of salt concentrations on the gelatinization endotherm of wheat starch

 

 

 

Salt cone. in Extent of Endotherrn
aqueous phase A HG Gelatinization temp. (°C)

(%) (call g) (%) To Tp Tc

0 4.7 100 50 68 86

3 2.7 57 58 71 88

6 2.5 53 64 75 88

9 2.6 55 68 78 88

12 2.7 57 65 77 . 88

15 2.7 57 65 77 88

21 2.8 60 61 80 90

30 3.3 70 59 79 91

 

From Wootton and Bamunuarachchi (1980)

W Hydrocolloids have been used extensively in food products to
modify texture, improve moisture retention, control water mobility, and maintain overall

product quality during storage. Because starch is a basic ingredient of so many foods, the

28

study of changes within Starch granules and pasting properties in the presence of gums
were needed to extend knowledge of the function of starch in starch/gum based products.

Christianson et a1. (1981) studied the gelatinization of wheat starch as modified by
Xanthan gum, Guar gum and Cellulose gum. The results indicated that the gum hasten the
onset of initial paste viscosity and substantially increase final peak of viscosity of wheat
starch. Further investigation in hydrocolloid interaction with starches, Christianson (1984)
proposed the two possible reasons why gums enhance the effect of gelatinization. First,
there may be interaction between exudate from the granule (solubilize amylose and low
molecular weight amylopectin) and the gums. Second, the addition of thickening gums
would mean that the forces being exerted on the granules in the shear field are much larger
than those encountered in starch-water suspensions of equal starch concentration. These
increased forces should significantly affect granule breakdown and the amount of material
exudate into medium.

Praeessing procedures Treatment of starch and cereal grains prior to cooking
can significantly alter cooking and / or gelatinization characteristics (Gough and Pybus,
1971; Donovan et al., 1983; Lorenz and Kulp, 1979, 1981, 1982; Lund, 1984).

In processing of cereal grains, perhaps the most widely known pretreatment is
parboiling of rice. Generally, parboiling consists of two processing operations: soaking
and / steaming. During soaking, rice is hydrated so that sufficient water is present to
allow subsequent gelatinization. During steaming, gelatinization and solubilization of
starch occur which contributes to the final quality of milled rice. The gelatinization

characteristics of the remaining ungelatinized starches in the intact rice kernels are altered

29

as a result of the parboiling process. The parboiled rice tends to gelatinize over a
temperature range that is both broader and lower than the untreated rice. This indicates
that the starch granules are damaged and destabilized in the previous parboiling treatment,
but the crystalline structure have not been disordered completely (Lund, 1984).

Soaking and/ or steeping are the indispensable steps to provide sufficient moisture
in the grain for gelatinization or facilitating further processing steps. Usually moderately
high water temperature (SO-60°C) is used to hasten the hydration process. Gough and
Pybus (1971) observed an interesting characteristic of gelatinization for wheat starch when
it was steeped for 72 hr at 50°C. The treatment changed and narrowed the temperature
range for gelatinization from 52 to 61°C before steeping to 65.4 to 658°C after steeping.
However, there was no change in the x-ray pattern between the raw and treated starches.
Similar studies conducted by Lorenz and Kulp (1979) for wheat starch, Wirakartakusumah
(1981) for rice starch steeped at various times and temperatures confirmed the previously
reported data.

The occurrence of increasing and narrowing the range of temperature for
gelatinization during steeping has been thought to be similar to the annealing process of
semicrystalline polymers. In synthetic polymers, exposure to an appropriate temperature
and solvent environment causes a spontaneous ordering of the polymer molecules. The
ordering process is known as annealing and generally occurs at a temperature near the
melting point of the polymer. Since starch is a semicrystalline material consisting of
amorphous and crystalline regions, holding a suspension of granules just below

gelatinization temperature would be expected to give rise to more perfectly ordered

30

crystals with higher melting points. The increase in crystallinity level was continued by x-
ray examination, according to Ahmed and Lelievre (1978). Another processing procedure
which is known as "Heat-Moisture Treatment" also causes changes in gelatinization
properties, physicochemical and functional properties of starch. This treatment consists of
starch granules with limited moisture content exposed to excessive high temperature for a
period of time. Lorenz and Kulp (1981) reported that the "heat-moisture treatment"
causes an increase in the bread-baking potential of potato starches. Further, Lorenz and
Kulp (1982) studied the effect of the "heat-moisture treatment" on tuber starches
(arrowroot and cassava). They reported that the physical properties of treated tuber
starches approached those of wheat starch. X-ray diffraction studies also show the change
of B-pattem of tuber starches toward an A-pattem of cereal starch. Donovan et al.(1983)
reported that the heat-moisture treated potato and wheat starches showed a boardening of
the gelatinization temperature range and a shifting of the endotherrnal transition toward
higher temperatures, compared to untreated starches. The interpretation of these results
was also given to the effects of recrystallization and the perfection of the small crystalline
regions of the granule. Changes in gelatinization properties by either annealing or heat-
moisture treatments could have significant effect on cooking characteristics of starch.
This area warrants further investigation.

Retrogradation

Starch granules, when heated in excess water above their gelatinization
temperature, undergo irreversible swelling, resulting in amylose leaching into solution. In

the presence of a high starch concentration, this suspension will form an elastic gel on

31

cooling. The molecular interactions that occur after cooling have been called
retrogradation. These interactions are found to be both time and temperature dependent.
Miles et al. (1985b) showed, with the aid of physical techniques, that amylose gelation
occurs as a result of a phase separation, which produces regions that are rich and deficient
in polymer and that, if the amylose concentration is sufficiently high, the region rich in
polymer forms a three dimensional network

Amylose crystallization was found to be a secondary process, occurring in the
region rich in polymer. Miles et al. (1985b) and Ring et a1. (1987) attributed the initial gel
fumness during gelation to the formation of an amylose matrix gel and the subsequent
slow increase in gel fumness to amylopectin crystallization. Heterogeneous acid
hydrolysis of waxy maize amylopectin gels, followed by gel permeation chromatography
studies on the residue, revealed that amylopectin crystallization occurs by association of
amylopectin molecules with DP 5 15 (Ring et al., 1987). The crystallization of
amylopectin was shown to be reversible at temperatures below 100°C, whereas the initial
gelation and crystallization of amylose was irreversible even at 100°C (Miles et al., 1985b;
Ring et al., 1987). This indicates a greater degree of molecular interaction in the latter.
X-ray diffraction and shear modules studied on various starches showed that the initial
rates of development and stiffness of gels were sequenced arnon g starch sources as
follows: smooth pea > maize > wheat > potato; while the long-term increase followed the
order: smooth pea > potato > maize > wheat (Orford et al., 1987). The latter process was
found to be more important at high starch concentrations. Differences in the fine structure

of amylopectins, and also in the extent of association between starch components, may

32

account for the above observed retrogradation tendencies. The freeze-thaw stability of
starch gels is an important characteristic of food starches. This stability is determined by
gravirnetric measurement of the water exuded (syneresis) from a gel after it has been
frozen and thawed. This exudation occurs owing to the reassociation of linear starch
molecules (retrogradation). This high retrogradation tendencies of legume starches was
also shown by Hoover and Sosulski (1985) and Tjahjadi and Breene (1984) by
measurement of the degree of syneresis during low temperature storage. The high degree
of syneresis makes native legume starches unsuitable for use in foods requiring low
temperature storage. However, modification by acetylation and hydroxy-propylation was
found to significantly decrease the extent of syneresis to levels that may be acceptable to

food processors (Hoover and Sosulski 1986; Hoover et al. 1988).

33

Nutritional Attributes of Dry Bean Starch

Dry legumes are dietary staples in many parts of the world and are important
dietary sources of protein, complex carbohydrates (starch and dietary fiber), other
essential nutrients, and energy. The digestion of starch in dry legumes is lower than in
cereal grains and many other foodstuffs. Factors contributing to decreased digestibility
include: 1) increased dietary fiber intake; 2) intact cell walls which hinder the digestive
enzymes from gaining access to the starch and protein; 3) residual lectin activity; 4) limited
proteolysis of certain protein sub-fractions; 5)incomplete hydration of the starch granule;
and 6) amylose retrogradation. Slow digestion of starch is beneficial since it reduces
hyperglycemia and hypertriglyceridemia which are risk factors in the development of
cardiovascular disease. However, it is likely that slow digestion of starch and retrograded
amylose are major contributors to the flatulent problem frequently associated with legume
consumption. The challenge to the food processor and plant geneticist is to produce
legumes that continue to provide a low glycemic index and a good source of fiber with
improved protein digestibility and decreased flatulence. However, producing a low
glycemic, fiber rich food with minimal flatulence may be mutually exclusive.

Factors Aflecting Digestion of Starch

A factor that affects digestion of both legume proteins and starches is the physical
form of the protein and starch when it enters the stomach and small intestine. Grains and
root vegetables are usually ground and/or cooked prior to consumption. Grinding and
cooking serve to break the cellular structures and to release the protein and starch so that

proteolysis and amylolysis can occur. Legumes are generally consumed as whole seeds

34

that have been cooked. Cell wall structures in cooked beans are intact and surround the
protein bodies and starch granules. Chewing macerates some of the cells, but most of the
cells remain whole and enter the digestive tract intact. Thus, the cellular structures render
legume protein and starch less available to the digestive enzymes except for the small
amounts of starch that leaches from the cells (W firsch, et al., 1986; Golay, et al., 1986).
Another factor affecting digestion of both protein and starch is the amount of total
dietary fiber in the diet. Phaseolus vulgaris beans typically contain 17.5 - 20% dietary
fiber and are regarded as an excellent source of dietary fiber (Prosky, et al., 1985;
Cummings, et al., 1985; Anderson and Bridges, 1988). As an individual increases dietary
fiber intake, nutrient digestibility and/or absorption tends to decrease (Kies, 1981; Miles,
et al., 1988). Decreased digestibility of protein and starch is presumably due to physical
entrapment and/or premature release of food particles from the stomach, although direct
inhibition of digestive enzymes by fiber cannot be excluded (Schneeman, 1982). Dietary
fiber can encase nutrients in a fibrous matrix which impedes digestive enzymes from
gaining access to protein and starch and fiber can reduce absorption of digested protein
and starch by slowing or preventing diffusion of the digestive products to the mucosal
cells (Southgate, 1973; Johnson and Gee, 1981; Flourie, et al., 1984; Elsenhaus, et al.,
1981; Vahouny, et al., 1981). Food is normally held in the stomach until it is reduced to
particles less than 1 mm (Meyer, et al., 1979; Meyer, et al., 1981); but fiber can cause the
premature release of large particles from the stomach (Meyer and Doty, 1988). Large
particles represent another type of physical entrapment which limits digestion (Williams, et

al., 1984; Doty and Meyer, 1988). The many possible effects of fiber on nutrient

35

bioavailability have been thoroughly reviewed (Vahouny and Kritchevsky, 1982; Trowell,
et al., 1985; Vahouny and Kritchevsky, 1986).

A potential factor which could limit protein and starch digestion is the presence of
naturally occurring protease and amylase inhibitors. During processing and cooking, most
of these inhibitors are inactivated; however, residual levels of active inhibitors may
remain, particularly in improperly cooked beans. It is reported that for individuals
accustomed to consuming beans with residual levels of inhibitors, the pancreas enlarges
and secretes more digestive enzymes to compensate for the enzymes which have been
inactivated by the inhibitors (Schneeman and Lyman, 1975; Madar, et al., 1976). As a
result of pancreatic hyperplasia and hypertrophy, adequate amounts of digestive enzymes
are secreted and digestion is not limited. The best evidence to support this conclusion is
based on the experience with exogenous amylase inhibitors ("starch blockers"). Several
groups demonstrated that chronic consumption of amylase inhibitors was ineffective in
reducing starch digestion and caloric utilization (Savaiano, et al., 1977; Granum and
Eskeland, 1981; Bo-Linn, et al., 1982; Carlson, et al., 1983; Garrow, et al.,1983).
Essentially the same results are found with continued consumption of protease inhibitors
(Madar, et al., 1976).

Legumes contain lectins which can potentially reduce nutrient digestion and
absorption. Cooking beans will inactivate much of the lectin activity in raw beans;
however, fully cooked beans can still contain a significant amount of the original lectin
activity (Thompson, et al., 1983; Rea, et al., 1985). The deleterious effects of raw beans

or comparable levels of isolated lectins are well documented (S garbieri and Whitaker,

36

1982; Liener, 1986). But, it is difficult to measure the effect of residual lectins on
digestion and absorption. Data provided by Donatucci et a1. (1987) strongly suggest that
small quantities of lectins are sufficient to reduce absorption of glucose and presumably
amino acids and small peptides. There maybe sufficient residual lectin activity in cooked
beans to cause at least a small decrease in protein and starch digestibility.

Digestibility of Legume Starch

Legume starch is digested more slowly than starch from most other sources. The
rate of in viva starch digestion is illustrated by the rise in blood glucose and insan
following the consumption of starch. The "glycemic index" has been developed to aid

dietary treatment of diabetics (Jenkins, et al., 1982) and is defined as:

glucose response curve for a food

 

glucose response curve for the equivalent amount of glucose X 100
Canned beans have a low glycemic index compared to other food groups. Thus, bean
starch is digested more slowly than starch from most other foods. The importance of the
glycemic index is that elevated blood glucose promotes triglyceride and cholesterol
synthesis by the liver which results in more very low density lipoproteins (triglycerides and
cholesterol) being secreted into the blood (Anderson, et al., 1984; Jenkins, et al., 1985;
Thompson, 1988). Also, elevated blood glucose promotes glycosylation of proteins in the
vascular system (Jenkins, et al., 1988). The net effect of elevated blood lipids and
unwanted protein glycosylation is an increased risk of cardiovascular disease. Thus,

consumption of legume starch reduces the risk of premature cardiovascular disease

37

compared to consuming starch with a high glycemic index. While slow starch digestion (a
low glycemic index) is desirable, it is undesirable for the starch to be digested too slowly
since there will be incomplete digestion in the small intestine. Undigested starch will pass
to the colon where rapid fermentation will take place and flatulence will result.

Two major factors affect the rate and perhaps the extent of starch digestion. The
first factor is gastric emptying time and the second factor is accessibility of the glycosidic
bonds in starch to pancreatic amylase and other digestive enzymes. If the rate of starch
digestion is constant, starch which is released from the stomach slowly and over an
extended time period will be digested and absorbed more slowly than when gastric
emptying is rapid and over a short time period. There is a strong negative correlation
between postprandial changes in blood glucose and gastric emptying (Mourot, et al.,
1988). Likewise, if gastric emptying is constant, starch digestion rate determines how
high the blood glucose rises in response to starch ingestion. The digestion rate of starch
strongly influences the glycemic response to different foods (Thompson, 1988; Jenkins, et
al., 1982). The glycemic index obviously reflects both gastric emptying and starch
digestion rate.

Legume starch digestion rate is affected by conditions which slow or prevent
digestive enzymes from gaining access to the glycosidic bonds of starch. Much of the
legume starch reaches the stomach and small intestine within intact cell walls. Thus, the
fiber matrix of cell walls is the first parameter to hinder starch digestion since amylase
must penetrate the cell wall before amylolysis can proceed. Another parameter affecting

digestion rate is the hydration state of the starch; starch must be hydrated to be digested.

38

Many types of starch, including legume starch, must be gelatinized to hydrate the
starch and the extent of gelatinization often determines the rate of starch digestion (Holm.
et al., 1988; Daniel and Whistler, 1985). When beans are cooked, the starch is not fully
hydrated (Golay, et al., 1986) and there are data which suggest that some of the starch still
retains its bifringence (Lai and Varriano-Marston, 1979; Hahn, et al., 1977; Varriano-
Marston and DeOmana, 1979). It appears that more energy is required to break the bonds
between starch chains in legume starch than in most other types of starch (Biliaderis, et al.,
1981; Hoover and Sosulski, 1985a, 1985b).

Another factor which limits starch digestibility is the tendency for some starches to
retrograde. When gelatinized starch cools slowly, some of the water between the starch
chains is squeezed out and hydrogen bonds within or between starch chains reform.
Amylose molecules with a degree of polymerization (DP) between 200 and 1200 glucose
units, have a high tendency to retrograde to crystalline "beta sheet" type configurations.
The average DP for legume amylose is DP 1000 - 1400 (Biliaderis, et al., l98la), which is
highly conducive to formation of retrograded crystalline starch structures. Retrograded
amylose is indigestible within the small intestine (Englyst and Cummings, 1987). Legume
starch has a higher percentage of amylose than most starches which increases the potential
for formation of indigestible retrograded starch. During cooking some of the amylose
leaches from the starch granule. Since the cell structure in cooked beans remain intact,
much of the leached amylose remains within the cell and fills spaces between protein

bodies and starch granules. If amylose retro gradation occurs, another indigestible matrix

39

(similar to fiber in cell walls) in and around the protein and starch granules is formed. This
retrograded amylose matrix is another factor which will slow starch digestion.

Indigestible Starch

It is feasible that normally digestible carbohydrates (such as starch) may not be
completely digested if the organism lacks sufficient digestive capacity relative to the
amount or type of carbohydrate ingested. This may be due to consumption of
carbohydrates which are less accessible or resistant to hydrolysis, a deficiency in the
hydrolyzing capacity of the individual (inherited or temporary), the result of insufficient
reaction time to digest and absorb the carbohydrate properly, or the result of a too rapid
food transit (Hellendoorn, 1978).

Stephen (1983), using starch mixtures from navy beans, rice and potatoes, directly
measured the passage of carbohydrates by inserting an aspirator at the human ileocecal
junction. It was observed that after serving meals of different proportions to subjects, that
the percent starch could be recovered at ranged from 2.3 to 20.1% (mean of 9.3%) for
smaller meals, and 2.2 to 10.9% (mean of 6.0%) for the larger meals. The conclusion
drawn from this work was that 2 to 20% of the dietary starch escaped absorption in the
small intestine.

Starch digestibility may also be dependent on cooking time, preparation of dry
beans or the type of starch itself. Accordingly, 5 to 15% of bean starch can remain
indigestible even after prolonged cooking (Hellendoom, 1969). Faki and Bhavanishangar
(1983), using in vitro and in vivo studies, demonstrated that apart from oligosaccharides,

the starch and hemicelluloses of chickpea, cowpea and horse gram contributed

40

substantially to the total flatulent effect. Roasting or boiling were ineffective in reducing
flatulence, but removal of oligosaccharides and hemicelluloses by preliminary water
soaking and sieving followed by precipitation of protein resulted in a product significantly
non-flatulent as well as non-nutritive.

All starch resisting digestion in the small intestine is subjected to bacterial
fermentation in the large intestine with the subsequent production of volatile fatty acids.
Most of the starch which reaches the colon is not totally resistant to pancreatic amylase,
but its hydrolysis is retarded so that it is not completely digested during its passage
through the small intestine . The reasons for this incomplete digestion are separated into
intrinsic (physical inaccessibility, resistant starch granules and retrograded starch) and
extrinsic factors.

Physical inaccessibility occurs when starch is contained within undisrupted plant
structures such as whole or partly milled grains and seeds. The cell walls may entrap
starch and prevent its complete swelling and dispersion(Wiirsch, et al., 1986) thus
delaying or preventing its hydrolysis with pancreatic amylase in the small intestine. It has
also been observed that after a meal of sweetcom, peas and beans, up to 20% of fecal
solids may be starch contained in recognizable, undigested food (Englyst, 1985).

The actual crystalline structure of the starch granule is suggested to depend on the
chain length of amylopectin. A-type starch is the normal pattern for cereal starch
granules, B-type is typical of potato, arnylomaize and retrograded, and C-type
(combination of A and B patterns) is characteristic of certain pea and bean starches. In

general, starch granules showing X-ray diffraction patterns B or C tend to be the most

41

resistant to pancreatic amylase, though the degree of resistance is dependent on the plant
source (Fuwa et al., 1980; Englyst and Cummings, 1990). Cooking disrupts the granules
and facilitates the hydrolysis of the starch contained within them. Crystallization at higher
temperature and lower water content will favor the formation of A pattern, and lower
temperature and high water content, the B pattern. On cooling, gelatinized starchy foods
will retrograde, solubility of the starch molecule decreases and so does its susceptibility to
hydrolysis by acid and enzymes.

The extent of crystalline bonding in amylopectin is limited by the branch length.
Therefore amylopectin retrogrades to a lesser extent than amylose, the retrograded
amylopectin being not so firmly bound as retrograded amylose. Pure amylose crystallized
with the B pattern can be solubilized only by autoclaving. The resistance to dissolution is
due to the extensive network of intra- and interhelical hydrogen bonds that stabilize the
double helical structure of crystalline amylose. Retro graded starch may be separated into
that redispersed at 100°C (mainly retrograded amylopectin) and that resistant to dispersion
in boiling water (mainly retrograded amylose). Human studies suggest that the mainly
retrograded amylose fraction resists virtually complete digestion in the small intestine
(Englyst & Cummings, 1985; 1987).

Socorro et a1. (1989) reported that dietary fiber decreased digestibility of black
bean, corn, rice and wheat starches by porcine enzyme, especially when whole grain fiber
was used. However, black bean and rice starch digestibility by human pancreatic or-
arnylase was not affected by fiber, while corn and wheat starch was slightly inhibited. The

use of the human enzyme is therefore recommended for in vitro amylolysis assays,

42

although the difficulties of extrapolating results obtained with animal enzymes to humans
should be carefully considered .

Studies by Fernandez and Berry (1989) reported that germination sharply
increased the susceptibility of chickpea starch to digestibility by or-amylase, but no change
in the appearance of SEM could be attributed to germination. In viva and in vitra
methods with experimental rats and commercial digestive enzymes, respectively, were
used by Nnanna and Phillips (1990) in assessing the protein and starch digestibility and
flatulence potential of germinated cowpeas. Germination reduced the flatulence potential
of seeds . In viva digestibility of starch and protein was also significantly increased by
germination. Germination did not affect in vitra protein digestibility, but reduced in vitra
digestibility of freeze-dried and 70°C-dried starch. Cooking in boiling water significantly
increased in viva protein and starch digestibility of both ungerminated and germinated
seed.

Tovar et. al. (1990a) found the starch content of a raw red kidney bean (Phasealus
vulgaris) flour (RBE) was higher than that of a cooked and blended (CBB) and of a
cooked, freeze-dried, and milled (CBF) preparation. However, wet homogenization as
well as pepsin pretreatment of CBF increased the starch yield, indicating that starch in the
cooked samples is not completely available to enzymatic degradation unless cell wall
entrapped granules are released by mechanical or enzymatic disruption of the fibrous
walls. This could partially explain a number of inconsistencies in digestibility values
reported in the literature (Fleming and Vose, 1979; Jenkins et. al., 1982; Wllrsch et al.,

1986; Socorro et. al., 1989 and O'Dea and Wong, 1983). Influence of encapsulated and

43

resistant starch fractions on dietary fiber values was also noticed. CBF showed
remarkably low values of in vitra amylolysis rate and starch digestibility index in a
digestion/dialysis system, features that seemed to depend also on the integrity of cell walls.

Large differences exist in the degree to which different starch containing foods
affect the blood glucose levels of both normal volunteers and diabetics. These differences
appear to relate to the digestibility of the starch and the factors determining this, including:
the interaction of starch with fiber, antinutrients (e. g., phytate) and protein in the food,
together with the nature of the starch itself and its physical form (e.g., raw or cooked,
ground or whole). In this respect legumes exemplify a class of foods, high in fiber, protein
and antinutrients, with a starch which is digested slowly in vitro. They also produce
relatively small blood glucose rises after consumption by both normals and diabetics and in
the long term result in improved diabetic control. Identification of foods which yield the
response and further understanding of factors determining starch digestibility will allow
greater therapeutic use of diet in the management of diabetics and disorders of
carbohydrate metabolism.

Thompson, et al. (1987) determined digestion of wheat starch (W S) and red
kidney bean (RKB) starch by pancreatic (PA) and salivary (SA) amylose in the presence or
absence of lectins. Compared with WS, digestion of RKB starch by PA and SA was
70.0% and 66.6% lower, respectively. RKB lectin added to WS at the hemagglutinin
activity level in RKB starch resulted in significantly decreased digestion with PA (63.9%)
and SA (43.8%) as did heated RKB lectin with insignificant hemagglutinin activity (41.1%

with PA, 35.8% with SA). Jack bean lectin (concanavalin A) also resulted in reduced rate

of starch digestibility. Kinetic analyses revealed noncompetitive inhibition by RKB lectins
on both arnylases. Results confirmed the role of lectins in reducing the rate of starch
digestion and its possible health benefit

Wiirsch, et al. (1986) studied the factors responsible for the slow digestibility of
starch in leguminous seeds by examining microscopically the cooked seeds after various
treatments and by measuring starch digestion in vitro. Starch in leguminous seeds is
entrapped in parenchyma cells and swells only partially during cooking. The cr-amylase
cannot easily penetrate within the gelatinized starch granules due to steric hindrance and
the physical nature of the leguminous starch. Disruption of the cells, especially before
cooking increases the susceptibility of starch to a-amylase digestion.

The objective of a comprehensive study recently conducted was to determine
whether legumes in a physical form which is rapidly digested in vitro give rise to
proportionately greater metabolic responses in viva than legumes which are slowly
digested in vitro (O'Dea and Wong, 1983). Samples of cooked whole and ground lentils
were incubated in vitro with pancreatic amylase for 30 min and the percentage starch
hydrolysis determined. Grinding the lentils before cooking resulted in a 5-fold increase in
the rate of starch hydrolysis (whole lentils 12.1%, ground lentils 60.9%). For the in viva
studies six healthy, young lean subjects consumed two test meals containing 50 g starch:
whole lentils and lentils that had been ground finely before cooking. Postprandial glucose
and insulin responses were measured over 4 hrs. Peak glucose and insan responses
occurred 60 min postprandially for the whole lentils and 30 min postprandially for ground

lentils. Although the increase in plasma glucose after ground lentils (1.6mM) was

45

significantly higher than that after whole lentils (0.09mM), there was no difference in the
magnitude of the insulin responses. These results indicate that, unlike for cereals, the rate
of intestinal starch hydrolysis is not the major factor determining the metabolic responses
to legumes. By virtue of their low post prandial glucose and insulin responses,
irrespective of their physical form and digestibility, legumes would appear to be ideal for

inclusion in the diet of diabetics.

46

Cell Wall Components and Chemistry in Plant Seeds

Definition of Dietary Fiber

Dietary Fiber (DF) was defined by Trowell (1974) as that part of plant material in
our diet which is resistant to digestion by secretions of the human digestive tract. As this
definition did not include polysaccharides present in some food additives (such as plant
gums, algal polysaccharides, pectins, modified celluloses, and modified starches) Trowell
(1976) extended the definition to include all the polysaccharides and lignin that are not
digested by endogenous secretions of the human digestive tract. Accordingly, the term
DF now refers mainly to nonstarchy polysaccharides and lignin in the diet (Southgate,
1976).

Plant cell walls are the main source of DF, and most of our DF intake comes from
the cell walls in foods such as fruits, vegetables, and cereal products. The principal
components of DF are complex polysaccharides and some of which are associated with
lignin and protein.

Components of Dietary Fiber

The main components which make up DF are summarized in Table 5. The
parenchymatous tissues are particularly important in connection with DF, because the
walls of these tissues comprise the bulk of the DF from fruits and vegetables, and the
endosperm of cereals. The parenchymatous tissues have mainly thin primary cell walls,
whereas the lignified tissues have cell walls that have ceased to grow and have undergone
secondary thickening. The lignified tissues are of greater importance with some cereal

product, e. g., wheat bran and bran based product.

47

 

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The cellulose microfibrils, the main structural elements, contain highly ordered
crystalline regions, in which linear chains of cellulose molecules are highly packed, and
less ordered amorphous regions in which the cellulose chains are less closely packed and
in which other polysaccharides may be found. The microfibn’ls are closely associated with
hemicelluloses, pectic substances, and glycoproteins deposited as an amorphous matrix of
macromolecules.

Organization of Cell Wall Structure
Dicotyledonous plants

The seeds of dicotyledonous plants can be classified into those which are free of an
endosperm, referred to as nonendospermic (e. g.. bean, pea, etc.) and those which have an
endosperm, referred to as endospermic (as in certain leguminous species, e.g., guar, locust
bean, etc.). The nonendospermic seeds usually have starch as the main storage
polysaccharide, and their cell wall material is derived mainly from the tissues of the
cotyledons and there is some contribution from the testa (which are usually lignified). The
cell wall polysaccharides of the cotyledons are similar to those of parenchymatous tissues.
The main cell wall polymers of parenchymatous tissues of dicotyledons are pectic
substances, hemicelluloses (e. g., xyloglucans), and cellulose, whereas those of lignified
tissues are lignin, hemicelluloses (glucuronoxylans), and cellulose; usually different types
of hemicellulosic polysaccharides occur in the cell walls of the two types of tissues.

By contrast to nonendospermic seeds, all the endospermic leguminous seeds
contain galactomannans, which are located in the endosperm cell walls (Meier and Reid,

1982). The galactomannans are essentially linear molecules but are highly substituted on

49

C-6 of the B-(l-4)-linked-D-Man p residues with single Gal p residues, which confers on
them properties which are quite different from those of unbranched, cellulose-like, water-
insoluble mannans and glucomannans. The galactomannans are hydrophilic and are
usually obtained from the crushed seeds (or endosperms) by hot water extraction. Based
on the interactions of galactomannan with water and other polysaccharides, uses of
galactomannan form guar and locust bean seeds appears to slow glucose absorption in the
small intestine by interacting with intestinal mucosa (Johnson and Gee, 1981).
Monocotyledonous plants

The cell walls of certain organs of monocotyledonous plants, particularly those of
cereal grains, are an important source of DF. All cereals have endosperrnous seeds; the
endosperm of wheat, for example, represents about 80 to 85% of the grain, and is the
source of white flour. The cell wall polysaccharides of parenchymatous tissues of cereals
are mainly arabinoxylans and B-D-glucans, but they all contain small but significant
amounts of cellulose, usually associated with glucomannan.

The most notable differences between the cell walls of the endosperm (and
aleurone layer) of cereals and those of parenchymatous tissues of dicotyledons are that
unlike the latter, the former are virtually free of pectins and pectic substances, and the
amount of cellulose is very low (Mares and Stone, 1973; Fincher, 1975; Bacic and Stone,
1981). Despite the relatively low levels of cellulose, the endosperm cell walls consist of a
microfibrillar phase embedded in an amorphous matrix (Mares and Stone, 1973); in this
respect they are similar to most primary plant cell walls. The primary cell walls of most

cereal grains have cellulose microfibrils, which are closely associated with glucomannan.

50

and these fibrillar structures are embedded in an amorphous matrix of hemicelluloses,
which consist mainly of arabinoxylans and/or B-D-glucans, some of which are cross-linked
by phenolic esters and /or proteins.

Constituents of Cell Walls

All the wall layers consisted of two phases, a microfibrillar phase and a matrix
phase. The microfibrillar phase is distinguishable from the matrix phase by its high degree
of crystallinity and its relatively homogeneous chemical composition.

Fibrillar polysaccharides

The fiber fractions termed fibrillar polysaccharides include the basic structural units
of microfibn'ls which have extremely long, thin structures and are made up mainly of
cellulose molecules, which are aligned parallel to the long axis of the microfibril. Cellulose
is an unbranched B 1,4-glucan. The cellulose chains are held in a crystalline lattice within
the microfibril. The lattice is stabilized by both intramolecular and intermolecular
hydrogen bonds.

It is clear from X-ray diffraction and chemical studies that the bulk of the
microfibril is made up of crystalline B 1,4-glucan. However, there is evidence that some
degree of structural heterogeneity may exist within the microfibril. First, or-cellulose
fraction of cell walls almost always contains a small amount of some sugars other than
glucose, usually mannose and xylose. Secondly, there is less crystalline region examined
in the electron microscope with negative stains, in which the non- glucose residue would be

expected to be found in the less crystalline region of the microfibril.

51

Matrix polysaccharides

Matrix polysaccharides are made up of linearly orientated polymers, which are
present at all stages of the development of the wall, and also of highly branched
polysaccharides that are deposited at particular stages of growth. These polysaccharides
may, at the surface of the microfibril, be incorporated into its structure. There are two
major fractions in the matrix polysaccharides.

Pectic substances

By definition, pectic substances are polysaccharides that are solubilized from the
cell wall by aqueous solutions of chelating agents such as ethylenediaminetetra-acetate or
ammonium oxalate. The solvent action of the chelating agents depends on their ability to
combine with Ca” and Mg“. The soluble pectic substances comprise the methyl ester,
pectin, the deesterifred pectic acid and its salts, pectates and certain neutral
polysaccharides. The pectic polysaccharides are made up of a group of a polysaccharides
rich in galacturonic acid, rharnnose, arabinose and galactose. They are characteristic of
the middle lamella and primary wall of dicotyledonous plants, and to a lesser extent of
monocotyledonous plants. They may also be linked covalently to phenols, cellulose and
protein. The most abundant component of the pectin polysaccharides are polyuronic
acids. The polyuronic acids composed mainly of two different polysaccharide:
rhamnogalacturonan and homogalacturonan (Albersheim, 1974).

WE) This polysaccharide is a major component of the
middle lamella and primary cell wall of dicotyledonous plants, with the greatest

concentration in the middle lamella. It contains a backbone of a1,4—linked galacturonic

52

acid and all-linked rhamnose. Many of the galacturonic acid residues of R6 are methyl
esterifred, and some may contain acetyl group esterifred to their hydroxyl groups. The
backbone is long and the degree of polymerization is around 2000. RC contains a number
of different side-chains, attached to the C4-position of rhamnose. These side-chains are
composed principally of arabinose and galactose.

W Homogalacturonans are made up of orl,4-linked chains of
galacturonic acid, which may be partly methyl esterified. Other sugar residues are absent
or present only in low quantities. They are found in considerable quantities in some fruits,
and are also present in the primary walls of suspension-cultured dicotyledonous cells.
Homogalacturonon with a low degree of methyl esterification may be referred to as ‘pectic
acid’, while more highly methylated molecules may be referred to as ‘pectinic acid’, or
simply as ‘pectin’. This terminology can also apply to molecules containing covalently-
linked homogalacturonan and rhamnogalacturonan.

W This is a highly-branched molecule containing a backbone of orl,5-
linked arabinose and side-chains of single arabinose residues linked by or(1-2) or or(1-3)
bonds to the main chain.

Liam Galactans are largely unbranched chains of BlAlinked D-
galactopyranose residues, with little or no additional sugar material present in the
molecule. In some cases a few of the galactose residues appear to be 1,6-linked.

Hemicelluloses

Hemicelluloses are the polysaccharides solubilized by alkali from the depectinated

(and delignified) cell walls. Selvendran and O’Neill (1982) suggested that some of the

53

alkali-soluble polymers are polysaccharide—protein-polyphenol complexes. In addition to
polysaccharides, small amount of glycoproteins, both hydroxyproline-rich and
hydroxyproline-poor, are also present, and the associated sugars are mainly arabinose and
galactose. The hernicellulose is connected with cellulose microfibrils by strong hydrogen
bonding. In contrast to the pectins, the hemicelluloses vary greatly in different cell types
and in different species.

Kym These polysaccharides have a backbone of BIA—linked xylose residues.
The backbone is substituted by or-linked ¢o—methylglucuronic acid on C2 of some xylose
residues, by or-linked arabinose on C2 or C3 and by acetyl esters on C2 or C3. The
primary cell wall of monocotyledonous plants include, as a major hemicellulose, an
arabinoxylan in which arabinose is the dominant side-chain. Secondary walls of
monocotyledonous plants contain an arabinoxylan with rather more glucuronic acid. The
primary walls of dicotyledonous plants have small amounts of glucuronoarabinoxylan,
containing both glucuronic acid and arabinose side-chains. The secondary walls of
dicotyledonous plants contain glucuronoxylan, with only a low proportion of arabinose, as
their major hemicellulose.

The xylans are capable of crystallization under certain conditions, though they are
not thought to be crystalline in the cell wall. They may crystallize with either a two-fold
or a three-fold screw axis, depending on the degree of acetylation.

WWW Mannans and galactomannans have been found in
the cell walls of some seed endosperrns, where they function as a food reserve. In some

cases, they also have a role in imbibing and retaining water. They contain a BIA-linked

mannose chain, and where galactose is present it is linked by an or(l-6) bond to mannose.
The mannans are able to form very hard, crystalline structures.

Qalactans The galactan subgroup of the hemicelluloses is mainly composed of
arabinogalactans. Their molecules consist of a main chain of B-galactopyranose units
linked to each other by [31,3-linkages.

W Xyloglucan is the principal hemicellulose of the primary walls of
dicotyledonous plants. ' It consists of a backbone of B(1-4)-linked glucose residues, to the
majority of which xylose residues are attached by or(1—6) bonds. Xyloglucans are also
found as storage polysaccharides in some seed endosperm cell walls. Primary walls of
monocotyledonous plants contain small amounts of xyloglucan, with a lower xylose:

glucose ratio than in dicot primary walls.

CHAPTER 1 INDIGESTIBLE STARCH COMPONENTS IN DRY BEANS

Introduction

Procedures to determine the degree of gelatinization of starch within a food matrix
or purified starch have been developed by several researchers. It has been recognized that
selective use of appropriate enzymes and proper handling are important to consistently
determine the degree of gelatinization of bean (Phaseolus vulgaris L.) starch in-situ. The
enzymatic methods are based on the hydrolysis of gelatinized starch by glucoarnylase to
form glucose (Chiang and Johnson, 1977). These methods are useful to differentiate
between native (raw) starch and gelatinized (cooked) starch. However, these methods
are not suitable to distinguish between gelatinized starch and retrograded starch due to the
limited selectivity of glucoamylase. According to Kainuma et al. (1981), the combination
of B-amylase (E.C.3.2.1.2) and pullulanase (E.C.3.2.l.41) (BAP) provides a highly
sensitive approach for the detection of structural changes in raw, gelatinized and
retrograded starch. flamylase is unable to act on raw starch and pullulanase (debranching
enzyme) requires thermal disruption of the three dimensional structure of amylopectin
molecules for active hydrolysis. For instance, corn starch paste induced to the retrograded
state through frozen storage (13 days at -20°C), resulted in 21% of the retrograded starch
being over estimated by glucoarnylase compared with data obtained using the BAP.
Another example is that 35% of the rice starch paste hydrolyzed by glucoarnylase was not
detected by the B-amylase and pullulanase enzyme complex. It has been recognized that
prOper sample handling is necessary for accurate assessment of total starch content and

degree of gelatinization of raw and processed bean starch, because the starch is

55

56

encapsulated within cell walls. This structural partitioning and the appearance of resistant
starch are among the unique characteristics of processed beans compared to cereal grain
(Tovar et al., 1990a).

The degree of starch gelatinization in cooked beans is of importance to their
complete digestibility. Most studies associated with indigestible starch fractions of
leguminous seeds have used the terminology “resistant starch” (RS), which is defined as
dietary starch that does not digest in the small intestine (Englyst and Cummings, 1985).
It has been recognized that the portion of R5 in cooked beans consists of not only
resistant starch granules but also ungelatinized starch. Previous research has shown that
some portion of the bean starch retains its crystalline structure after prolonged cooking
(Lai, and Varriano-Marston, 1979; Varrano-Marston and DeOmana, 1979). Loss of
birefringence under polarized light microscopy is commonly used as an indicator of starch
gelatinization (Schoch and Maywald, 1956; Watson, 1964). The appearance of
birefringency due to crystalline structures may be a valuable phenomenon to observe
crystallization within cell walls. It has been suggested that a rigid cell wall is a physical
barrier to digestive enzymes and is an important determinant limiting the relatively low
bioavailability of starch in cooked beans (Wtirsch et al., 1986; Flemming et al., 1988).
The cell walls in cooked beans may also inhibit starch gelatinization due to limited swelling
potential of the starch granule and restricted or limited water availability necessary for
complete gelatinization ('Ihorne et al., 1983; Wilrsch et al., 1986).

The purpose of this study was to investigate the degree of starch gelatinization

during cooking of whole beans using enzymatic and microscopic techniques. Enzymatic

57

digestion of the starch fraction of cooked beans was conducted with B-amylase and

pullulanase to measure the degree of starch gelatinization during differential heating times.

Experimental Plan
Whole dry navy beans (variety Seafarer) were soaked in water for 12 hrs and thermally
processed with differential time at 115.5°C. Samples are prepared as fresh cooked and
lyophilized. Degree of gelatinization was measured by an enzymatic method and a visual
microscopic method. Reheating was conducted on lyophilized sample to investigate the
rigidity of crystallized cell walls.
The null hypothesis tested in this research is stated as follows:

Ho: The presence of whole cells with crystallization in the cell wall do not prevent
starch gelatinization and lower starch digestibility.

The experimental protocol used in this study is outlined in Figure 2.

58

WHOLE DRY NAVY BEANS

12 HR. SOAK (at 24°C)

1

THERMAL PROCESSING
(10, 20, 30 and 40 min at 115.5°C)

I FRESH COOKED I FRESH COOKED-
‘ - LYOPHILIZED

 

 

 

 

 

 

l

DEGREE OF GELATINIZATION w

 

 

 

I MILLING I

 

‘ I DEGREE OF GELATINIZATION v15

 

 

 

RBI-[EATING
(10, 20, 30 and 40 min at 94°C)

* l

Available Starch

 

 

 

Figure 2. Experimental protocol used to assess the degree of starch gelatinization in
differentially cooked and prepared navy beans. (Degree of gelatinization BAP:
B-amylase and pullulanase enzymatic method; VIS: microscopic method)

59

Materials and Methods

Materials

Navy beans (Phaseolus vulgaris, var. Seafarar) were obtained from the Michigan
Foundation Seed Association, Okemos, Michigan and stored at 4°C until used. The seed
was produced in the 1993 growing season. Bean samples were randomly selected and
used directly as whole bean seeds for cooking and preparation procedures.

Thermal Processing

Dry bean samples were soaked in prepared soaked water (distilled water
containing 100 ppm Ca”) for 12 hrs at room temperature prior to processing. The soaked
beans were thermally processed in 4 oz cans (204x214) at 115.5°C in a still retort. Beans
were cooked for 10, 20, 30 and 40 min. The experiment was replicated twice for each
cooking time. Distilled water containing 100 ppm Ca” was used as brine. The hermetic
seal of all cans were punctured immediately after the cooling cycle due to inadequate
heating to achieve commercial sterility.

Sample Preparation
Cooked (fresh) sample

Cooked whole beans (canned) were referred to as "fresh cooked". Seed coats
were removed from the fresh cooked sample and the cotyledons were macerated using a
mortar and pestle for immediate microscopic observation and enzymatic assay.
Cooked-Lyophilized sample

Cooked samples including brine were frozen, freeze—dried and milled by passing

through a Udy Cyclone Mill (U dy Co., Fort Collins, CO) equipped with a 20 mesh screen.

60

All samples were held in tightly capped glass bottles in a desiccator to minimize physical
and chemical changes prior to analyses.
Cooked-Lyophilized + Reheated sample

Additional heating of the cooked-lyophilized sample was conducted by suspending
80 mg meal in distilled water (8 ml) in a covered culture tube (50 ml) and reheating for 10,
20, 30, and 40 min at 94°C in a water bath with occasional shaking.

Light Microscopy

The samples for microscopic examination were prepared by macerating cotyledons
from fresh cooked or lyophilized beans with a mortar and pestle. A portion of the
resultant bean paste was suspended in distilled water and mounted on a glass slide and
observed under light microscopy. Fully cooked samples were prepared by hand cutting
the whole beans with a razor blade and then suspending and mounting cross sections on a
glass slide. A laser scanning confocal microscope (Zeiss 210, West Germany) was used
with a red laser (Helium-neon emitting at 633 nm) and a blue laser (Argon-ion emitting at
488 nm). Photomicrographs of representative specimens were obtained after viewing
numerous fields and used as the basis to draw conclusions.

Determination of Starch Gelatinization
Enzymatic hydrolysis

The degree of gelatinization (DG) of bean starch was estimated by the method of
Kainuma et al.(1981) using a combination of B—amylase and pullulanase (BAP). One ml of
the enzyme solution contained 3.4 IU of pullulanase (EC. 3.2.1.41, Sigma No. P5420)

and 0.8 IU of B-amylase (EC. 3.2.1.2, Boehringer Mannheim No. 102822).

61

Samples (80 mg, db) were placed in a glass homogenizer (30 mL) with 8 mL of
distilled water and dispersed with up and down movement of a polyester piston (10-20
times) by hand. Two samples of 2.0 mL were transferred to 25—mL volumetric flasks.
One flask was filled with acetate buffer (0.8M, pH 6.0) and used for the determination of
DC of the sample (DISPERSED SAMPLE). To the other flask, 0.2 mL of 10M sodium
hydroxide solution was added. The flask was then heated 3-5 min at 50°C to gelatinize
the starch completely; 1.0 mL of 2M acetic acid is then added to neutralize to pH 6.0.
This flask is then filled with 0.8M acetate buffer, pH 6.0. It is used as the reference of
100% degree of gelatinization (ALKALINE GELATINIZED SAMPLE). Each sample
solution (4.0 mL) from both dispersed and alkaline gelatinized sample was mixed with
enzyme solution (1.0 mL) and the mixture was incubated 30 min at 40°C with shaking.
After the reaction, the enzyme was inactivated by boiling for 5 min. Then 1.0 mL of the
reaction mixture was diluted five times and 1.0 mL and 0.5 mL of the diluted reaction
mixture were analyzed by the Somogyi—Nelson procedure (Robyt and Whelan, 1968) for
reducing sugar and the phenol-sulfuric acid assay (Dubois et al., 1956) for total
carbohydrate, respectively. The procedure and flow diagram for the BAP method are
shown in Figure 3.

Degree of gelatinization (D6) of a starch sample was expressed as the percentage
of the degree of hydrolysis compared to the completely gelatinized starch sample under
alkaline conditions.

A / B

Degree of gelatinizationup (%) = — x 100
A'IB'

62

Glass Homogenizer

4— add 8 m1 of distilled water
4— add 80 mg (db) sample
o— gentle dispersion
(up and down 10-20 times)
.__ take two 2 ml of sample

 

 

DISPERSED SAMPLE GELATINIZED SAMPLE
25 ml v lum ' k 25 ml volumetric flask

add 0.2 ml 10M NaOH soln _fi
heat 3-5 min at 50° C
add 1.0 ml 2M acetic acid ——’

 

fill to 25 ml with
0.8M acetate buffer

 

 

 

Sample solution 1 Sample solution 2
+— take 4 ml sample solution
We

add 1 ml enzyme solution ——9
incubate at 40°C for 30min ——-v

lml wi

 

 

 

inactivate enzyme
by boiling for 5min '
take lml of reaction mixture __,,
1:31.932: 125111111:
I‘_ add 4 ml distilled water ‘_“|
| l |
1 ml for 0.5 ml for 1 ml for 0.5 ml for
Somogyi- phenol- Somogyi- phenol-
Nelson sulfuric acid Nelson sulfuric acid
assay assay assay assay
A B A’ 8’

Figure 3. Flow sequence to determine degree of gelatinization (DG): DG (%) =
[ (A / B) + (A’ / B’) ] x 100; (Modified: BAP method of Kainuma et al., 1981)

63

where, A = amount of reducing sugar in dispersed sample
B = amount of total carbohydrate in dispersed sample
A' = amount of reducing sugar in alkaline gelatinized sample
B' = amount of total carbohydrate in alkaline gelatinized sample
Direct polarized microscopy
The degree of gelatinization was quantitated by counting the number of cells which
consisted of gelatinized, partially gelatinized, and ungelatinized starch granules.
Classification was made based on visual rating of cellular brightness using a 5 point scale:
1 = ungelatinized, bright-white / numerous occlusions; 3 = partially gelatinized, light-
white / few occlusions; 5 = gelatinized, clear / no occlusions. Three representative
photomicrographs under polarized light were taken on fresh cooked samples and the
degree of gelatinization estimated by visual microscopy method is defined as “degree of
gelatinizationvxs” and calculated as follows:
X + 3Y + 52

Degree of gelatinizationws (%) = x 100
5 (X + Y +Z)

 

where, X = number of ungelatinized cells
Y = number of partially gelatinized cells
Z = number of gelatinized cells
Statistical Analysis
Degree of gelatinization measured by enzymatic methods was performed in
duplicate and each sample was analyzed in triplicate. All other determinations were made

in triplicate. The mean, standard errors, mean square errors, one factor ANOVA were

conducted using Super ANOVA software (version 1.11, 1991, Abacus Concepts, Inc.,

64

Berkeley, CA). Mean separations were performed using LSD with the mean square error

term at the 95 % confidence level.

65

Results and Discussion

General

Table 6 shows total soluble solids and carbohydrate relationships during cooking
of beans in brine. The total solids of beans decreased with increased cooking time while
brine solids increased. These results demonstrated that the cooking process caused the
leaching of solids into the brine in response to bean tissue breakdown. The amount of
total carbohydrate leached into the brine after 10 min cooking was 18.5 % and was
significantly lower than that of extended cooking times. The prolonged cooking over 10
min did not affect the amount of total carbohydrate leached into brine. The amount of
available starch in brine, which is measured by B-amylase and pullulanase, increased with
cooking time up to 30 min and dropped at 40 min of cooking time.

Table 7 shows the degree of gelatinization of beans for the different preparation
conditions used and measured by the BAP method. The degree of gelatinization increased
linearly up to 15% after 30 min cooking of the fresh cooked sample. However, further
cooking resulted in a reduced hydrolytic response. It was observed that the pattern of
enzyme hydrolysis on starch in brine (Table 6) was similar to that of the degree of
gelatinization in freshly cooked beans (Table 7). This relationship may be attributed to
release of encapsulated starches from the bean cotyledon tissues during cooking. A
previous study by Tovar (1995) supports this explanation. He presented his work on
hydrolysis of cooked brown beans and cooking water. Beans were boiled in water for 2
hrs and beans and the cooking water were freeze-dried separately, powdered and fractions

observed. Both fractions contained cell wall-surrounded starch in an encapsulated form.

la

C0
lir.

Table 6. Total solids and carbohydrate contents of whole beans and brine after cooking1

 

 

 

 

Cooking Total Solids (%) % of Total Solids in Brine
Time (min)
Bean Brine Total CHO Available Starchz
10 36.2 21: 0.8a 2.3 i 0.6a 18.5 i 1.2a 0.1 i 0.1a
20 33.4 :l: 1.13 2.3 i 0.6a 27.2 i 2.0b 1.6 i 0.2b
30 33.0 :t 3.2a 2.5 :l: 0.4a 28.5 i 0.6b 2.2 i 0.4c
40 31.6 i 2.2a 2.7 i 0.3a 29.0 i 1.8b 0.4 i 0.1a

 

1 Values are means of duplicate. Means in a column with different letters were
significantly different (p < 0.05).
2 Available by the BAP enzyme method (Ii-amylase and pullulanase)

67

Table 7. Degree of gelatinization of beans held in different conditions measured by
the enzymatic method1

 

 

Conditions Degree of GelatinizationBAp (%)
Soaked 5.3 i 0.6ab
Cooked (Fresh)

10 min 6.2 i 0.4ab

20 min 10.1 i 1.1b

30 min 15.4 :t 3.3c

40 min 7.2 i 0.8ab
Cooked - Lyophilized

10 min 2.1 i 0.7a

20 min 2.5 i 1.3a

30 min 5.4 i 4.6ab

40 min - 2.4 21: 2.0a
Soaked - Lyophilized + Reheated

(30 min / 94°C; Homogenate) 116.7 :1: 12.3d
Corn Starch (30 min / 94°C) 100.4 :l: 5.2d

 

1 Treatments were done in duplicate and each sample was analyzed in triplicate.
Means in a column with different letters were significantly different (p < 0.05).

68

He noticed that the cooking process caused a breakdown of cotyledons and starches
released from the seeds were encapsulated within intact cell walls. This similar pattern
suggests that susceptibility of the enzymes to starch has been changed during cooking due
to conformational change of starch by gelatinization and the integrity of cell wall due to
crystallization.

Heating generally ruptures cell walls and frees starch granules from their cellular
compartrnentalization. It also has been observed that some carbohydrates leach from
intact beans into the brine during cooking. However, this study showed that
crystallization within the cell walls of beans during heating results in individual cells which
resist rupture and starch release. The heating of beans caused cell walls to crystallize
which prevented the release of starch, even though prolonged heat caused more
gelatinization of starch within cells as shown by microscopic observations.

The degree of gelatinization of the lyophilized samples throughout different
cooking times was significantly less than that of the fresh samples. Retrogradation of
starch occurred during preparation by freeze-drying. Over 50% of gelatinized starch in
the fresh cooked samples was retrograded during the freeze-drying process. Bean
starches from the soaked, lyophilized and reheated homogenate treatments were
completely hydrolyzed by the enzyme, B—amylase and pullulanase, indicating that the cell
wall was readily ruptured and starches were available for digestion.

The extent of starches hydrolyzed by B—amylase and pullulanase upon different
cooking and reheating treatments is shown in Table 8. Freeze-dried flours prepared with
different cooking time without subsequent reheating showed no significant differences on

starch bioavailablity as measured by the enzyme digestion. It is proposed that low

69

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digestibility values obtained for the soaked and uncooked sample was due to ungelatinized
starch granules while those obtained for any cooked samples was caused by crystallization
of cell walls. Many studies have revealed that starch granules of the cooked bean are
encapsulated within thick-walled cell (Wfirsch et al., 1986; Tovar et al., 1991). Tovar
reported that the thick and mechanically resistant cell walls acted as a physical barrier to
swelling and enzymatic breakdown of starch granules in vitro. In this study, we found that
the rigidity of the cell walls crystallized during the cooking process varied with cooking
time (Table 8). Reheating of the 10 min-cooking and freeze-dried flour showed higher
values for available starch than those cooked for 20, 30, and 40 min. Approximately 32%
of the total starch measured as free starches of soaked and freeze-dried flour, was
liberated by the reheating process in the 10 min cooked sample while only 10% of the total
starch was available in the 40 min-cooking and freeze-dried sample. The amount of starch
released after reheating showed no significant difference among 20 min, 30 min, and 40
min cooked samples. Tovar et al. (1990b) observed that milling of the freeze-dried bean
samples cooked in boiling water for 60—70 min resulted in a flour that retained cell
structure. We confirmed their observations and additionally demonstrated that the
crystallized cell walls induced by initial heating (just 10 min) was resistant to enzymatic
attack and milling. However, the milling process of the soaked and freeze-dried sample
liberated starch granules and rendered them available through heat induced gelatinization.
There were no significant differences among reheated samples of the soaked-freeze-dried

and milled bean flour heated for differential times (Table 8). These data confirm previous

71

studies that milling of the samples before cooking increased starch availability (Golay et
al., 1986; Fleming et al., 1988).

A mechanism of crystallization of a thick cell wall during cooking based on the
above observations could be proposed as follows. The cell wall of raw bean was readily
broken by milling. However, once heat was applied to the soaked bean, crystallization of
cell walls inhibited the mechanical breakdown and release of starch granules. Thus, most
starch granules are entrapped within thick cell walls after cooking. Crystallized cell walls
become barriers to enzyme hydrolysis and continuation of starch granule swelling. During
the soaking process, most starch granules absorb water and swell which increases
potential to be gelatinized during heating. However, some starch granules remain intact
after soaking and are not able to be gelatinized even with prolonged heating because the
crystallized cell walls inhibit expansion of starch granules after cooking. These data are
conclusive that during cooking of whole beans some portion of starch granules remain
intact. The structural characteristics of these intact starch granules result in reduced
bioavailability.

Microscopy

Plate 1 shows the appearance of aqueous suspensions of cold soaked (12 hrs) and
lyophilized bean meal viewed under transmitted (a,c) and polarized light (b,d). These
duplicate fields demonstrate the diversity and typical appearance of cellular structures
within the sample and illustrate the free starch granules (a,b) and starch granules bound
within the cell (c,d). These samples received no heat treatment and the granules are

clearly ungelatinized. These liberated starch granules (Plate lb) showed characteristic and

72

clear birefringence light scattering patterns while starch granules packed within
parenchyma cells (Plate 1d) appeared white. The appearance of the cell wall of the soaked
beans viewed under the polarized microscope did not show any evidence of crystalline
structure. One of the typical characteristics associated with gelatinized starch is a loss of
birefringence patterns within the starch granule when viewed under polarized light. Cross
sections of soaked bean and cooked bean, which is viewed under polarized microscope,
were shown in plate 2.

Two distinctive features were observed in cooked whole beans (Plate 3a-d): 1) a
decreased percentage of ocular illumination (used as an indicator of the degree of
gelatinization) resulted with increased cooking times (10 min, 33.9%; 20 min, 38.6%; 30
min, 39.6% and 40 min, 43.5%) and 2) crystallization of the cell wall (as evidenced by a
white band) resulted during the initial heating period of 10 minutes. Cell walls had a
highly dense crystalline appearance after this minimal heat treatment The white ring was
produced by crystallization within the cell wall. There was no crystallization in cell walls
of soaked beans (Plate 1d). However, cooking for 10 min or longer caused crystallization
within cell wall. The energy provided during this 10 minute cock was not sufficient to
soften cotyledonary tissue and render beans palatable. However, it did transform the cell
walls into firm and stable structures which resisted breakdown during subsequent cooking
of up to 40 minutes duration (Plate 3b-c-d).

Plate 4 shows that ungelatinized starch can occur in beans cooked for 2 hrs.
Prolonged cooking in an open kettle causes disruption of the testa (seed coat / hull) and

liberation of most of the solids into brine. The samples in Plate 4 were taken from whole

73

beans which remained intact after cooking for 2 hrs. The observation of prolonged
cooking without the expected complete gelatinization of starch suggests that there is a
physical barrier which limited the hydration or granular swelling typically observed for
gelatinized starch. It is suggested that the heat-induced crystalline structure observed in

the cell wall limits complete gelatinization of intact starch granules.

74

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Plate 1. Light microscopy images of soaked beans presented as representative fields
a, c) transmitted and b, d) polarized

75

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Plate 2. Polarized light microscopy images of the cross sections for differentially
prepared beans
a) soaked b) cooked for 40 min

76

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a) 10 min, b) 20 min, c) 30 min and d) 40 min

   

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Plate 4. Light microscopy images of intact cooked beans heated for 2 hr at 98°C
a) transmitted b) polarized

78

Conclusions

Heating of whole beans in a liquid medium (brine) caused crystallization of
constituents within cell walls which prevented cellular breakdown and release of starch to
the extra-cellular milieu. These starches can not be readily digested and, thus, are not
biologically available as nutrients. The heat induced crystallization observed in cell walls
after heating were resistant to disruption using mechanical homogenization. The degree of
gelatinization of the fresh cooked beans measured enzymatically was lower (about 60%
when compared at 30 min cook) than that measured microscopically due to limited
enzymatic access to the crystallized cell wall. The rigidity of cell walls cooked for 10 min
was sufficiently firm to hinder enzyme access yet sufficiently weak to be broken by
reheating. The prolonged cooking (over 10 min) increased the rigidity of crystallized cell
wall resulting in firmer structure that limited starch availability on reheating of the freeze-
dried flour. The freeze-drying process caused retrogradation of the gelatinized starches in
fresh cooked beans and it lowered available starches over 50% as determined by BAP
method.

The results obtained from this study demonstrate “ungelatinizable starch” in fully
cooked edible beans. These data have direct implications on the maximum nutritional
bioavailability and digestibility of bean products. Thus, there is a negative nutritional
impact using beans as a dietary staple within subsistent populations dependent upon beans
as an energy and protein resource and an economic impact depressing total bean
consumption within cultures sensitive to intestinal gas formation and flatulence.

Improving the gelatinization potential of bean starch granules could increase bean

79

hydration capacity (cooked bean yield), increase available caloric value, reduce flatulence

and perhaps decrease cooking time required to render a palatable product.

 

Ho: The presence of whole cells with crystallization in the cell walls do not prevent
starch gelatinization and lower starch digestibility.

Reject the HO as stated and conclude that the presence of whole cells with
crystallization in cell walls prevent starch gelatinization which lowers starch
digestibility.

 

CHAPTER 2 HEAT-INDUCED CHANGES IN CELL WALL STRUCTURES
REDUCE BIOAVAILABILITY OF BEAN STARCH

Introduction

Legume consumption has increased in the western world due to their recognition
as a good source of dietary fiber and protein. In addition, the high quality of carbohydrate
related with digestion and absorption is increasingly being recognized. Legume starch is
well-known as a low glycemic index food. Low glycemic index diets have been shown to
improve metabolic variables not only in diabetics (Brand, et al., 1991) and hyperlipidemia
(Jenkins, et al., 1987a) but also in healthy subjects (Jenkins, et al., 1987b). The resistance
of legume starches towards hydrolytic enzymes has been of great interest to nutritionists,
since they have been found to exhibit a lower glycemic index than the cereals (Jenkins, et
al., 1980).

Recently, there has been much interest in characterizing resistant starch (RS), since
it functions as a component of dietary fiber, in escaping digestion in the small intestine, but
being readily fermented in the colon by microorganisms (Asp et al., 1986; ijrck et al.,
1986, 1987; Englyst and Cummings, 1985; Englyst and MacFarlane, 1986; Siljestrom and
Asp, 1985; Wyatt and Horn, 1988). Most of the starch which reaches the colon is not
totally resistant to pancreatic amylase, however its hydrolysis was retarded so that it is not
completely digested during its passage through the small intestine. Many factors are
involved in formation of resistant starch in leguminous seeds during cooking. One of the
important and perhaps unique characteristics is thermally induced crystallization of the cell

wall. The crystallized cell walls entrap starch granules and prevent their complete swelling

8O

81

and dispersion. This results in granules being physically inaccessible for its hydrolysis with
pancreatic amylase (W firsch et al., 1986). As a result of encapsulation, some of the bean
starch remain intact and ungelatinized (retains crystalline structure) during even a
prolonged cooking. In my previous work, it has been found that a minimal heating (10
min at 115.5°C) caused crystallization of cell wall which resisted breakage from milling
and homogenization processes.

The purpose of this study is to investigate the changes in cell wall structure of dry
beans, sweet potato, and wheat, all starchy foods during cooking and the effect of
reheating on the integrity of crystallized cell walls. Starch bioavailability was examined in

vitro using an enzyme complex, B-amylase and pullulanase.

Experimental Plan

Previous research conducted by USDA (G.L. Hosfield, Research Genetist),
Michigan State University (JD. Kelly, Professor) and The Michigan Dry Edible Bean
Research Advisory Board agronomist (Gregory V. Vamer, Director) has shown
significant variability among bean cultivars and breeding lines for canning quality. One
navy bean (experimental line, N 84004) and one pinto bean (variety, Carioca) were
selected to compare starch bioavailability with other sources of starchy foods including: 1)
sweet potato and 2) wheat. The experimental line, N 84004, has been consistently
characterized as possessing excessively soft texture (extensive tissue breakdown and

cellular disruption) upon canning and is thus unacceptable for commercial use. Carioca

82

has previously been shown to possess low starch digestibility among a screening selection
of pinto beans (Mridvika et al., 1994).
The null hypothesis tested in this research is stated as follows:

Ho: The crystallization of cell wall in cooked beans is not a limiting factor
associated with starch gelatinization and bioavailability.

The experimental protocol is outlined in Figure 4.

83

Navy bean (N84004), Pinto bean (Carioca)
Sweet Potato, Wheat (Chelsea)

12 HR. SOAK (at 24°C)

THERMAL PROCESSING
(10 and 40 min at 115.5°C)

l

Cooked Whole Samples

Polarized Light Microscopy FREEZE-DRYING
(with brine)

l

MILLING

l

REHEATING
(10, 20, and 30 min at 94°C)

1

STARCH BIOAVAILABILITY
(by BAP method)

Figure 4. Experimental protocol used to assess starch availability and cellular appearance
of foods prepared under selected preparation procedures

C0<
The
WOU

SW6:

84

Materials and Methods

Materials

Navy beans (experiment line, N 84004) were obtained from the Cooperative
Elevator Company during 1992 crop year. Pinto beans (cultivar, Carioca) were obtained
from the USDA, ARS: Sugar Beet and Bean Research Center in East Lansing, MI. Bean
samples were field-dried, harvested, sorted, packaged, and stored in a cooler maintained at
4°C prior to processing. Sweet potatoes (Ipomoea batatas Lam.) were obtained from
local market and wheat (Triticum aestivum L., cultivar, Chelsea) samples were obtained
from Department of Crop and Soil Science at Michigan State University, East Lansing.

Thermal Processing

Each sample was soaked for 12 hrs at room temperature in distilled water with 100
ppm Ca++ prior to processing. A simmering brine containing only 100 ppm Ca” was
added before sealing. The soaked samples were thermally processed in 4 oz cans (204 x
214) at 115.5°C for 10 and 40 min. The experiment was replicated twice for each cooking
time. All cans were punctured to disrupt hermetic seal immediately after the cooling cycle
due to inadequate heating to assure commercial sterility.

Sample preparation

Seed coats of cooked whole beans were removed for microscopic observations.
Cooked whole seeds including brine were frozen at -20°C and freeze-dried for two days.
The lyophilized samples were ground with a Udy Cyclone Mill to a particle size that
would pass through a 20 mesh screen to produce a milled flour suitable for analyses.

Sweet potatoes were cut into cubes [maximum V2 inch (12.7mm), minimum V4

ll}

en

pn

0n]

85

inch(6.35mm)] prior to soaking and the same procedure used for beans was applied.
Chelsea wheat was cooked and the cooked whole wheat including kernels was freeze-
dried and milled to have wheat flour. All samples were held in tightly capped glass bottles
in a desiccator to minimize physical and chemical changes prior to analyses. Additional
heating treatment to flour samples was conducted by suspending 80 mg meal in distilled
water (8 ml) in a covered culture tube (50 ml) and reheating for 10, 20 and 30 min at 94°C
in a water bath with occasional shaking.

Light Microscopy

The samples for microscopic examination were prepared by macerating cotyledons
from fresh cooked samples with a mortar and pestle. A portion of the resultant sample
paste was suspended in distilled water and mounted on a glass slide and observed under a
light microscope. Additionally, some samples were prepared by hand cutting the whole
samples with a razor blade and then suspending and mounting cross sections on a glass
slide. A laser scanning confocal microscope (Zeiss 210, West Germany) was used with a
red laser (Helium-neon emitting at 633 nm) and a blue laser (Argon-ion emitting at 488
nm). Photomicrographs of representative specimens were obtained after viewing
numerous fields and used to draw conclusions.

Available Starch

Available starch of each treatment was measured with some modification of
enzymatic method using B—amylase and pullulanase (Kainuma et al., 1981). The
procedure of estimating degree of gelatinization was applied to measure available starch

only on the fraction termed DISPERSED SAMPLE. Reaction mixture hydrolyzed by the

86

enzymes are analyzed by the Somogyi-Nelson procedure (Robyt and Whelan, 1968) for
reducing sugar and maltose was used to make standard solutions. The available starch
content was obtained by multiplying the ttg of maltose in each sample by a factor of 0.95
to account for the weight of the water gained during the hydrolysis of starch to maltose
and by dividing a dilution factor of 128.

maltose (pg / ml of reaction mixture) x 0.95

g starch/ 100 g sample (db) = x 100
128

 

Differential Scanning Calorimetry (DSC)

DSC measurements were performed with a Du Pont Model 2920 Modulated DSC
instrument equipped with a 2200 Thermal Analysis data station. Samples of
approximately 10 mg were weighed accurately into aluminum sample pans and the pans
were hermetically sealed. An empty sealed pan was used as a reference. The DSC was
run from 20 to 250°C at the 10°C/min heating rate and the analysis was performed in a
nitrogen atmosphere (50cc/min). Transition enthalpy (AH) computed as call g were
calculated from the area under the curve described by recorded trace and a straight
baseline joining T, (initial transition temperature) and Tc (completion transition
temperature).

Statistical Analysis

Available starch measured by enzymatic methods was performed in duplicate and
each sample was analyzed in triplicate. The mean, standard errors, mean square errors,

one factor ANOVA were done using Super ANOVA software (version 1.11, 1991,

87

Abacus Concepts, Inc, Bekeley, CA). Mean separations were performed using LSD with

the mean square error term at the 95% confidence level.

at-

3~f

88

Results and Discussion

Starch Availability

Available starch obtained for all starchy food differentially prepared in this study is
shown in Table 9. For navy beans (experimental line, N 84004), reheating of the soaked,
freeze-dried and milled flour (SFM) caused release of starch granules from cells and thus,
was available to the hydrolytic enzymes. However, not all cells were broken down by the
milling process. The evidence presented in the microscopic image (Plate 5a) of the SFM
revealed that intact cells remained. The BAP assays indicated that entrapped starch
granules did not undergo hydrolytic degradation with the enzymes. An average of 8.5g of
starch per 100g navy bean (dry basis) was available upon reheating of the SFMs. The
available starch of cooked (10 min and 40 min), freeze-dried and milled flour (CFM) was
significantly lower than that of SFM even when additional heat treatment was applied.
This observation confirms previous work by Tovar et al. (1990b) that the difference in
susceptibility to enzymatic attack observed between precooked flours and the
corresponding raw flours were mainly due to the release of starch granules during milling
of uncooked seeds, whereas starch-containing cotyledon cells remained in the precooked
red kidney bean flours.

After reheating the CFMs, there was an increase in the amount of available starch.
A 10 min additional heating of the cooked flour (CFMlo) showed a 7-fold increase in
available starch while the treatment of 40 min cooked flour (CFMio) sample increased only

3-fold when compared to the cooked flour which had no reheating treatment. Starch

Table 9. Available starch of flour samples prepared differentially on reheatingl

 

Available starch (g starch / 100 g sample)

 

 

 

Soaked Cooked
Reheating (min) 10 min 40 min
Navy bean (N84004)
0 0.30 i 0.143 0.95 :l: 0.35a 1.10 i 0.14a
10 8.95 i 0.21e 6.60 i 1.98c 3.55 i 0.07b
20 8.15 i 0.2lde 6.80 :l: 0.57c 2.85 i 0.5%
30 8.75 :l: 0.50de 7.70 i 0.14cd 3.45 i 0.07b
Pinto bean (Carioca)
0 0.35 i 0.07a 1.10 i 0.42a 0.70 :1: 0.713
10 11.80 i 2.55d 12.85 i 0.78de 7.00 i 0.28bc
20 13.30 i 4.12de 12.45 i 1.63d 5.20 :l: 2.55b
30 16.60 i 1.98e 11.10 i 3.54cd 7.40 i 0.28bc
Sweet Potato
0 27.15 :I: 1.49a 57.95 :t 3.75b 58.75 :1: 0.07b
10 64.00 :1: 3.68b 60.10 :t 4.10b 59.85 :1: 2.33b
20 63.20 :l: 1.98b 58.55 i 6.2% 61.15 :l: 5.5%
30 62.50 :1: 0.71b 61.40 :t 2.26b 59.75 :1: 0.35b
Wheat

0 0.20 :t 0.013 18.25 :l: 64% 27.40 i: 0.40bc
10 33.95 :1: 2.33c 26.50 i 7.07bc 30.00 :l: 1.56bc
20 29.70 :1: 4.53bc 27.60 :l: 7.21bc 36.10 i 4.53c
30 32.35 :1: 3.18c 29.70 i1.13bc 36.10 :1: 3.110

 

1 Means within a specific product (columns and rows) followed by different letters are

significantly different (P<0.05), n = 2

90

leaching as a consequence of thermal cell wall disruption was observed with polarized
light microscope (Plate 5b, c). Tovar (1991) showed a starch release and a melted
appearance of particulate structures by scanning electron microscope (SEM) with
additional heat treatment. However, some encapsulated starch granules remained intact
within a crystallized cell wall. In our study, the rigidity of the cell wall was varied with
cooking time. There was a significant difference in available starch between CFM“) and
CFMio navy bean samples after additional heat treatment (Table 9). Relative starch
hydrolysis of navy bean (experimental line N84004) prepared on different cooking time
after reheating treatment was shown Figure 5. Reheating of CFMlo enabled 80% of starch
to be hydrolyzed by the enzymes while reheating of CFMio allowed only 40%. The
rigidity of crystallized cell walls in beans cooked for 10 min was significantly less than that
of 40 min cooked samples.

Starch availability by reheating of SFM and CFM of pinto beans showed similar
trends observed with navy bean flours. Again, the cell wall of cooked pinto beans
encapsulating starch granules is a factor lowering the enzymatic availability of starch.
However, starches available to the enzymes after reheating of SFM in pinto beans were
relatively higher than that of navy beans. Available starch on pinto bean CFMio and
CFM“ with an additional heating was 11-fold and 6.5-fold increased, respectively when
compared to the flour without an additional heating. The incremental increase in available
starch when an additional heat treatment was applied to pinto beans (cultivar Carioca) was
twice that obtained for navy beans (experimental line, N84004). The results from cooked

beans indicate that the longer the cooking time, the harder the rigidity of cell wall of the

91

 

120-

 

100. c

 

804

 

 

El Soaked
10 min cooked
I40 min cooked

 

 

 

Relative hydrolysis (%)

 

 

 

 

 

 

 

 

 

 

20
Reheating (min)

   

Figure 5. Available starches of navy bean (experimental line, N84004) prepared with
different cooking times

 

92

cooked beans. Figure 6 demonstrated that relative starch hydrolysis upon reheating.
Reheating of CFM“) enabled 90% of starch available by the enzymes while reheating of
CFMao allowed only about 40%. It is concluded that the rigidity of crystallized cell wall
after cooking varies among varieties and process conditions. The factors affecting the
rigidity of crystallized cell walls could be due not only to cooking time but also other
factors such as age of seeds, storage conditions, cell wall components and the genotype.

Results also demonstrated available starch of SFM and CFM of sweet potato with
reheating. About 40% of starch in soaked sweet potato was hydrolyzed by the enzymes
even without gelatinization. Reheating of SFM enabled most starches to be available for
enzymes due to complete disruption of cellular structure or complete gelatinization.
Reheating of CFM showed no significant difference in available starch of both sweet
potatoes cooked for 10 min and 40 min. There were no significant differences in the
amount of starch hydrolysis, which is maximum, among all flour samples reheated for
different times (Figure 7). Cooking caused sweet potato starches to gelatinize and to be
retained inside the cells. In contrast to bean flours, starch granules were entrapped within
the cells, however, there was no evidence of cell wall crystallization in cooked sweet
potato. Most starches were readily available to the hydrolytic enzymes (Plate 7)

Cooking whole wheat increased available starch gradually and dramatically with
increased cooking time (Table 9). Sixty percent and eighty-six percent of wheat starch
were hydrolyzed by the cooking for 10 min and 40 min, respectively. In raw wheat, the
starch is embedded in a protein matrix and this restricts the availability of starch for

amylolysis (Holm et al., 1985). The disruption of the protein matrix during cooking

93

 

 

 

 

 

Relative hydrolysis (%)
$

 

 

 

 

 

........

...........
.....
...........

1O

 

Reheating (min)

 

 

 

......

 

 

 

El Soaked
10 min cooked
I40 min cooked

Figure 6. Available starches of pinto bean (var. Carioca) prepared with different

cooking times

 

94

 

 

   
 

  

 

 

 

 

 

 

 

 

  

 

 

 

 

 

120-
b
100- b b b b
’e‘ _
E 80‘/
.9
(I)
3‘ ._
e 60/
g a DSoaked
.5 / D 10 min cooked
.g 40 I40 min cooked:
(6
g 20_/
O 10 20
Reheating (min)

Figure 7. Available starches of sweet potato prepared with different cooking
times

95

(reheating) contributed to the increased starch hydrolysis. No hydrolysis was detected in
raw ground wheat. However, the release of starch granules from the wheat endosperm by
grinding soaked and freeze-dried sample made almost all starch available to the enzymatic
hydrolysis (Figure 8). Reheating of the SFM wheat flour led to the gelatinization of all the
starches and thus approached the maximum attainable availability. Holm and Bjorck
(1988) reported that the availability to or-amylase of starch in the drum-dried flours was
restricted substantially by the protein structure. In our study the reheating of the cooked-
milled wheat samples increased the availability of starch by eliminating the obstacle to the
enzymatic hydrolysis. Reheating the wheat flour showed the same level of starch
availability regardless of reheating time. Once the highly organized structures within
starch granules are destroyed, the availability is not increased further by disintegration of
the granule.

Polarized light microscopy

Plate 5 shows polarized light microscopy image of reheated (10 min) bean flour.
All free starches were gelatinized but the cells unbroken by milling retained adhesion in the
soaked, freeze-dried and milled flour(SFM) sample. Most cell walls were broken by
reheating but some encapsulated starch granules remained intact in the CFM“, sample
while more intact cells remained after reheating in the CFM“, sample due to rigidity of the
cell walls. A view of cooked navy beans under increased magnification focused directly
on the cell wall is shown in Plate 6. A thick cell wall was evident (likely due to
crystallization) on both cooked beans (10 min and 40 min) however there was no apparent

difference on thickness due to the heating period. Polarized light microscopy image of

96

 

 

Relative hydrolysis (%)
$_
V

 

 

 

 

 

0 10 20
Reheating (min)

 

 

 

30

 

 

 

a 40 min cooked

Figure 8. Available starches of wheat prepared with different cooking times

 

97

200l-40 TIZI CI795 IIlSI F. L00!

’OOIIZO I'll

\ 5"
\

ll
ZOOM-2| I-ZI

 

Plate 5. Polarized light microscopy image of reheated (10 min) bean flour prepared after
differential cooking treatments
a) soaked, b) 10 min cooked and c) 40 min cooked

98

CIBEB 8-281

 

b

Plate 6. Light microscopy images illustrating cell wall structure for
differentially cooked navy beans
a) 10 min cooked b) 40 min cooked

99

cooked (40 min) bean and sweet potato were shown in Plate 7. It is quite clear that starch
granules were entrapped within cell walls in both cooked samples. Crystallization
occurred within cell walls of beans and ungelatinized starch remained within the cells
(Plate 7a). However, no evidence of crystallization occurred within cell walls of cooked
sweet potato and it is clear that most starches were gelatinized since birefringence is lost
within the cells (Plate 7b).

Differential scanning calorimetry (DSC)

DSC was used to characterize the endothermic heat flow of flour samples. DSC
thermograms obtained are illustrated in Figure 9. The DSC scans constitute
measurements of pinto bean flours, soaked flour (SFM), 10 min and 40 min cooked flour
(CFMm and CM). All flour samples exhibited two endothermic transitions. One is
over a temperature range 95 to 180°C, which may be due to dissociation of a crystalline
structure of bean flour. The broad range of the first peak obtained from bean flours (solid
state and mixture of food components) includes the melting temperature of retrograded
starch and resistant starch, which was estimated at ~ 160°C. These results are consistent
with those reported by Sievert and Pomeranz (1990 and 1989) and Ring et al. (1987).
The other endothermic transition occurred in the range from 190 to 230°C. The peak
temperature was ~210°C. Transition enthalpies AH (cal/g) measured on SFM, CFMio and
CFM“, of pinto bean were 0.59, 1.51 and 2.09, respectively. The findings from
thermoanalytic measurements indicated that a crystalline structure which is more stable
than resistant starch was formed during cooking processes. Further research is needed on

separation and characterization of the crystallized cell wall.

8'288

. 33°"\

ZOOM-48 T-2s 8'95 F8 L488

 

Plate 7. Polarized light microscopy images of bean and sweet potato tissue cooked for
40 min
a) whole bean b) whole sweet potato

1m

10min

Cooked

40min

 

 

Heat Flow (meal / sec)

 

50 100 150 200 250
Temperature (°C)

I=igure 9. DSC thermograms of pinto bean flour prepared after differential
cooking treatments

102

Conclusions

The rigidity of cell wall crystallized during cooking beans varied with cooking
time. The longer the cooking time, the greater the rigidity of cell wall of the cooked
beans. The cell wall integrity varied between bean type and among process conditions.
Starch granules of cooked beans encapsulated within crystallized cell wall are restricted
from enzymatic hydrolysis while starches of cooked sweet potato are readily available
since no crystallization occurs within cell wall. Based on DSC thermograms, the
crystalline structure formed during cooking processes is more stable than resistant starch.
These findings are consistent with physiological responses (low glycemic index,

flatulence), commonly observed with bean consumption.

 

H.: The crystallization of cell wall in cooked beans is not a limiting factor
associated with starch gelatinization and bioavailability.

Reject the [-10 as stated and conclude that the crystallization of cell wall is a critical
limiting factor for starch gelatinization and bioavailability.

 

CHAPTER 3 CHARACTERIZATION OF BRINE OBTAINED FROM CANNED
BEANS POSSESSING DIFFERENTIAL QUALITY
Introduction

During thermal processing of the beans differential amounts of nutrients are
leached into the cooking brine which lowers consumer acceptability because of the
formation of cloudy, viscous, or grainy brine. Moreover, the nutritional value of beans is
lowered if the brine is discarded. Much research has been conducted on nutrient loss and
on approaches to minimize nutrient loss through processing as well as genetic
improvement (Augustin and Klein, 1989). A better understanding of the relationship
between canned bean quality and brine composition would provide insight to appropriate
processing and selection of breeding lines. The purpose of this study is to investigate plant
materials selected for their differential canned bean and brine characteristics and to

characterize the leached components present in the brine of these materials.

Experimental Plan

Much effort has been conducted to improve canning quality of dry edible bean
genetically and technologically by USDA (G.L. Hosfield, Research Genetist) and
Michigan State University (M.A. Uebersax and JD. Kelly, Professors). Navy beans
(Phaseolus vulgaris) grown at Michigan State University Agricultural Experimental
Station in 1993 crop year have shown diversities on characteristics of brine and integrity
of cooked bean. Eight cultivars (and/or experimental lines) of navy beans were selected
based on visual canning quality of beans and brines. The quality characteristics were

categorized into two intersecting groups: 1) bean integrity (intact or split), 2) brine

103

104

viscosity (thick / viscous / cloudy or thin / fluid / clear). Relationships of the categorized
bean and brine with physical characteristics of canned beans and chemical composition of
brine were investigated.

The null hypothesis tested in this research is stated as follows:

11,: the compositional components of brine and physical bean quality after
processing are not controlled by genetic background.

Materials and Methods

Thermal Processing Procedure

Moisture content of dry bean samples was determined using a Burrows Digital
Moisture computer 700 (Burrows equipment Co., Evanston, IL). Dry bean samples
(equivalent to 100 g dry solids) were placed in nylon mesh bags and soaked in water for
30 minutes at room temperature (21°C). Immediately after this soaking, beans were
transferred to a 88°C water bath for an additional 30 min. All soaking was performed in
water containing 100 ppm calcium ion prepared using distilled water and reagent grade
CaClz. After hot soaking, beans were momentarily cooled under cold tap water,
completely drained and weighed. After weighing, beans were filled into 303x406 cans and
covered with boiling brine (1.249% NaCl, 1.561% sucrose and 100 ppm calcium). Cans
were sealed and processed in a still retort for 45 minutes at 116°C. After thermal
processing, cans were uniformly cooled to 38°C under cold tap water and stored for two
weeks at room temperature before quality evaluation. The storage period after processing

permits canned beans to completely equilibrate with the canning medium.

Canning Quality Evaluation

After the cans were opened, the free flow brine was decanted and retained for
observation and analyses. The washed-drained weight of processed beans was determined
by decanting the can contents on a number 8 mesh sieve, rinsing them in cold tap water to
remove adhering brine, and draining for 2 rrrin on the sieve positioned at a 15° angle. One
hundred grams of washed processed beans was weighed and transferred to a sample dish
for color measurement. Texture was determined by using a Texture Test System (Model
TSM-90) equipped with the FT 3000 transducer and a Standard Multiblade Shear
Compression Cell (Model CS-l, Food Technology Corp., Reston, VA). The water
content of canned beans (final moisture percentage) was determined from the 100g texture
samples. These were oven dried at 80°C until the weight remained constant. The
identification and descriptions of navy bean samples selected for use in this study are

shown in Table 10.

Brine Characteristics

Representative samples of brines were obtained for chemical analysis through
stirring and pipeting the brine suspensions. Caution was exercised to assure a
homogeneous distribution.
Total solid content

Approximately 3 g of brine sample were weighed in a pre-tarred aluminum tray
and dried in a vacuum oven at 75°C with 27 "Hg overnight.

% total solid = dried weight of sample (g) / initial weight of sample (g) x 100

106

Table 10. Visual characteristics of canned navy beans and brine

 

 

 

Quality Class Description

Cultivar/ Breeding line Bean integrity Brine appearance
Fleetwood Intact Thick / Viscous / Cloudy
Crestwood Intact Thick / Viscous / Cloudy
Midland Split Thick / Viscous / Cloudy
I 92912 Split Thick / Viscous / Cloudy
Mayflower Split Thin / Fluid / Clear
Seafarer Split Thin / Fluid I Clear

N 90563 Intact Thin / Fluid / Clear

N90598 Split Thin/Fluid/Clear

 

107

Alcoholic insoluble solids
About 3 g of brine sample were taken in a pre-tarred aluminum tray and four
volumes of 85% ethanol were added. They were stirred with a glass rod and incubated for
an hour. The alcohol supematants were decanted and precipitates were dried at 40°C in
convection oven overnight.
dried weight of alcohol precipitates (g)

% alcoholic insoluble solids = x 100
initial weight of samples (g)

 

Protein content (Kjeldahl nitrogen analysis)

The protein content of the brine samples was determined using AACC method 46-
12, 1983. Five milliliters of concentrated sulfuric acid and one catalyst tablet were added
into each digestion tube containing pre-weighed sample (ca 5 g of brine). This tube was
then slowly heated to 400°C until the digestion was completed (approximawa 5 hr). The
protein content was determined on a dry basis using a nitrogen conversion factor of 6.25.

The following equation was used to calculate the % protein of sample.

(ml of HCI titrated) (Nomality of HCI) (1.4007) (6.25)

 

% protein of sample =
Sample weight (g)

Total starch (Kainuma et al., 1981)
Dried powders prepared from alcohol precipitation were used to measure the
starch content. The starch content was quantitatively determined using an enzymatic

method. This method is based on enzymatic hydrolyses using B—amylase and pullulanase

108

and thus termed “BAP” method. The enzymes, B-amylase and pullulanase, were used to
hydrolyze the alkali gelatinized starch sample and Phenol-sulfuric acid assay was used to
measure maltose units in the digested sample. The total starch content in each sample was
obtained by multiplying the mg of maltose in each sample by a factor of 0.95 to account
for the weight of the water gained during the hydrolysis of starch to maltose.

Dietary fiber content (Lee et al., 1992)

Sample Preparation Canned bean brine contains relatively high amounts of
sucrose (1.56% by weight, formula preparation). The desugaring procedure by extracting
with 85% EtOH was employed prior to determining dietary fiber. Five hundred ml of the
85% ethanol were added into the centrifuge tube containing about 50g of homogenized
brine sample. After stining and incubation for an hour, the samples were centrifuged at
4000 rpm for 10 min, the supernatant decanted and the residues dried overnight at 40°C.
The dried sample was used to measure dietary fiber, pectin content, and total starch. The
enzymatic method of Lee et al. (1992) for determining insoluble and soluble dietary fiber is
outlined in Figure 10.

Digestion Approximately 1 g of dried alcohol precipitate powder was accurately
weighed into 600mL beaker and 40mL of MES/TRIS buffer (0.05M, pH 8.2) was added.
After stining on magnetic stirrer until sample is completely dispersed, 50ttL of heat stable
or—amylase solution (No.A-0164) was added and incubated for 35 min at 95°C with
continuous agitation. After this starch hydrolysis, the treated solution was cooled to 60°C
and any precipitate resuspended by dispersion with a spatula and rinsed with 10 mL H20.

Protein hydrolysis was canied out by adding 100uL protease (No.P-3910) solution and

109

Alcohol precipitate powder (1.0g)
in 600 ml beaker

MES /T'RIS buffer (40 ml: 0.05M, pH 8.2)
(ll-amylase (50 #1)
Incubate
(95°C / 35 min)

Cool to 60°C

I‘— Protease (100 pl)

Incubate
(60°C / 30 min)

Adjust pH 4.04.7

Amyloglucosidase (300 pl)
Incubate
(60°C II30 min)

Cool

I
Filter
(Fritted aucible porosity No.2)

 

Resgue Filirate
10 ml deionized-
distilled water (2x)

If I
Residue Washings ———-."— 100 ml 95% alcohol (60°C)
. (4X)

 

 

Prec' irate
Washings:
10 ml 95% alcohol (2x) ._p Discard Filter
10 ml acetone (2x) (Potted aucible porosity No.2)

1
h l

Residue Filtrate
Washings:
20 ml 78% ethanol (3x)
Discard 10 ml 95% ethanol (2x)
10 ml acetone (2x)
Oven dry (105°C) Oven dry (105°C)

1 l

INSOLUBLE SOLUBLE
DIETARY FIBER (IDF) DIETARY FIBER (SDF)

 

Figure 10. Flowchart of the enzymatic method to determine the insoluble and soluble dietary fiber of
alcoholic precipitated flour (Lee et al., 1992)

110

incubation at 60°C for 30 rrrin with continuous agitation. Following protein hydrolysis,
5mL of 0.561N HCI was dispensed into beaker and pH was adjusted to 4.0-4.7 at 60°C
using 1N HCI or 1N NaOH solution. Starch hydrolysis was catalyzed using 300ttL of
arnyloglucosidase (No. A-9913) solution maintained at 60°C for 30 min with constant
agitation.

Insoluble Dietary Fiber The solution was cooled and filtered through a
previously ashed and weighed fritted glass crucible (No.2) containing celite, using the
Fibertec System E 023 Filtration Module (Tecator, England). The residue was washed
with two 10 ml portions of deionized-distilled water. The filtrate and water washings
were set aside for the determination of soluble fiber. The residue was washed with two 10
ml portions of 95% ethanol and then with two 10 m1 portions of acetone. The crucible
containing the residue was dried overnight in a 105°C (221°F) air oven. Residue from one
set of duplicates was analyzed for protein, while the duplicate was incinerated overnight at
525°C (977°F) to analyze for ash content. Blank determinations were maintained
throughout the procedure. Percent Insoluble Dietary Fiber (lDF) was calculated using the
following formula:

IDF (%) = mg insoluble residue - . .

r 1 r + n r '
sample weight (mg)

Soluble Dietary Fiber The weight of combined filtrate and water washings was
adjusted to 100 g with water. Four 100 ml portions of 95% ethanol preheated to 60°C
(140°F) was added and allowed to precipitate at room temperature for at least an hour.

The enzyme digest was filtered through previously ashed and weighed fritted crucibles

111

containing Celite. The residue was sequentially washed with three 20 ml portions of 78%
ethanol, two 10 ml portions of 95% ethanol and finally two 10 ml portions of acetone.
The residues were dried and analyzed for protein and ash following the same protocol for

the insoluble fiber. Percent Soluble Dietary Fiber (SDF) was calculated as follows:

SDF(%) = mg soluble residue -
| 1% pretein in residee + 20 ash in residue) x mg residue | x 1!!!
sample weight (mg)
Uronic acid

Estimates of pectin content were made based on total uronic acid. Preparation and
digestion steps were the same as for the dietary fiber. Zero point three (0.3) mL of the
hydrolysate were mixed with 0.3mL of a solution containing 2g of sodium chloride and 3g
of boric acid / 100m] in a 50-mL tube. Five mL of concentrated sulfuric acid was added
and vortex-mixed. The tube was placed in heating block at 70°C for 40 min and cooled to
room temperature.

When cool, 0.2 mL of dimethylphenol solution (0.19 in 100 mL of glacial acetic
acid) was added and vortex-mixed immediately. Between 10 and 15 rrrin later, the
absorbance at 400nm and 450nm were read against a water reference. The difference
obtained by subtracting the reading at 400nm from that at 450nm was used to calculate
sample concentration with glucuronic acid standards over the range 0.025 to 0.125

mg/mL.

Results and Discussion

The description of visual characteristics of the canned beans and brine used in this
study are presented in Table 10. The integrity of cooked beans was divided into two
groups: intact and split beans. Brine characteristics was grouped by viscosity and
appearance: thick/ viscous / cloudy and thin / fluid / clear. Brine characteristics was not
related with integrity of cooked beans. Cooked beans whose brine appearance was
viscous and cloudy showed either intact or split. Similar observation was made with the
cooked beans in which brine appearance was fluid and clear.

Under identical process conditions, the eight navy bean cultivars selected for study
exhibited canning quality differences (Table 11). Soaking dry beans before canning is
considered as a necessary step to decrease cooking time, increase drained weight and
ensure uniform bean expansion in the can during processing (Nordstonn and Sistrunk,
1977; Quast and da Silva, 1977). Soaked weight indicates the seed hydration ability
during soaking treatment. The hydration ability during soaking did not influence the
potential for bean breakage, i.e., there is no apparent relationship between bean breakage
and soaked weight. However, soaked weights of beans in which brine appeared thick and
viscous was slightly less than that obtained for beans in which brine appeared thin and
fluid. Seed coat characteristics were suggested to be major factors in controlling seed
hydration during soaking (Smith and Nash, 1961; Quast and da Silva, 1977) and may be
associated with soluble solid leaching.

The processed yield of canned beans was determined by its washed drained weight.

A high water holding capacity of beans with an intact seed coat is one of the most desired

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overall product quality characteristics in canned bean products. Differences in drained
weight occurred among the cultivars studied (Table 11). The Fleetwood cultivar
possessed highest drained weight (301.0 g) with intact bean appearance. Drained weight
of cooked beans in which brine appearance was thick and viscous was significantly higher
than that obtained for cooked beans in which brine appearance was thin and fluid (Table
12).

Bean texture is also a primary canning quality character because texture affects the
perceived stimuli for chewing and , hence, influences to a large degree a consumer’s
acceptance of a food product. Beans may be unacceptable if they are perceived as either
“too firm” or “too soft” after cooking. The experimental line N 90563, in which the bean
is intact and brine is fluid and thin, showed highest texture value (954.5 N). The alternate
experimental line N 90598 showed lowest texture value (633.7 N), in which the bean
appearance was split and brine character was fluid and thin (Table 11). Bean integrity
appeared to have an associated relationship with texture value. Hosfield and Uebersax
(1985) presented results showing that a firming effect of associated with addition of
CaClz during soaking and processing was helpful in decreasing seed coat splitting during
canning. In this study however, Midland, in which bean appearance is split showed
relatively high texture value (906.4 N) while Fleetwood, in which bean appearance is
intact showed relatively low texture value (657.7 N). Further research designed to elicit
control factors determining texture value of cooked beans and cultivar quality is

warranted.

116

The amount of bean solids (%, w/w) is another quality term used for determining
water holding capacity of canned beans. Theoretically, percent (%) bean solids and
drained weight of canned beans demonstrate an inverse relationship. However, Fleetwood
showed lowest total solids with highest drained weight and N 90563 possessed lowest
water holding capacity (highest total solids) resulting in lowest drained weight (Table 11).
Average value of total solids in each group showed no significant differences among
treatments (Table 12).

Table 13 presents proximate composition of the brine of the canned navy beans.
Average percentage of brine composition grouped by visual appearance is shown in Table
14. Solids in viscous brine were higher than those in fluid brine. The amount of protein in
viscous brine was usually higher than that in thin brine. Most of the solids consisted of
soluble dietary fiber (SDF) and starch ranging from 61.2% to 41.6%. From Tables 13 &
14, it is evident that starch leakage from the bean was not related with bean breakage.
Fleetwood was mostly intact but had higher starch content in the brine whereas N 90598
had severely split bean but 'had less starch in brine. In general, viscous brine contained
more SDF and protein titan did the fluid brine. The amount of insoluble dietary fiber (IDF)
appeared to have some effect on viscosity of the brine. In summary, there was no one
specific component identified which adequately served as a single determinant of bean
brine viscosity. Combinations of the components such as protein, soluble dietary fiber and
starch and their interactions are suspected to be the determining factors. Further studies
on brine composition are required to assess the effects of each component for their

individual contributions toward viscosity changes in brine.

 

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Statistical analysis (Correlation matrix)

Several studies have examined the relationships among and within physico-
chemical characteristics and canning quality of beans (Uebersax, 1985;; Srisuma, 1989;
Ruengsakulrach, 1990; Hsia, 1994). In this study, an effort was made to investigate
possible correlations among the various parameters.

The relationships among visual appearance of cooked bean and brine, bean canning
quality and brine composition are shown in a correlation matrices presented in Table 15.
Lower left side of the matrices was obtained from the relationships with brine composition
based on total solids while upper right side of the matrices was the relationship based on
alcohol insoluble solids. The correlation coefficients between visual appearance and bean
integrity (canning quality) in lower left side of the matrices demonstrate similar levels of
significance to those in upper right side because the values are not dependent on either
total solids or alcohol insoluble solids of the brine.

Relationships between bean integrity and canning quality character-i566

Correlation analysis was conducted by grouping and designating the bean integrity
as “intact = l” and “split = 2”. No correlation was found between cooked bean integrity
and texture (r = - 0.022). As described above, texture of cooked bean could not be
estimated only by degree of visual bean integrity, either intact or split, among bean
samples selected in this study. The correlation between bean integrity and drained weight

is also very low (r = - 0.149). Total solids of cooked beans exhibited a negative

120

 

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121

correlation with bean integrity (r = - 0.410). Intact beans showed high value of total
solids while split beans possessed low value of total solids.
Relationships between brine characteristics and canning quality characteristics

Correlation analysis was conducted by grouping and designating the brine
characteristics as “thin / clear = 1” and “thick / cloudy = 2”. Drained weight and the
thickness of brine showed fairly strong correlation (r = 0.726), which is statistically
significant at 95 % confidence level (P<0.05). As the viscosity in brine increases, the
drained weight of cooked beans increased. High viscous brine was associated with low
soaked weight of beans (r = - 0.629). Texture of cooked bean showed no correlation with
brine viscosity and clarity (r = - 0.085). A non-significant negative correlation was
obtained between total solids and brine appearance.
Relationships between bean integrity and brine composition

The amount of starch in brine showed close relationship with visual integrity of
cooked bean (r = 0.609, AIS; r = 0.534, TS). Thermal processing broke down cellular
structure and released cellular components into the brine matrix. The greater the percent
breakdown of bean resulted increased amount of starch in the brine. However, negative
correlations were obtained between bean integrity for either the amount of total protein or
percent total solids in brine.
Relationships between brine characteristim and brine composition

As can be seen, strong positive correlations were obtained between brine
characteristics and percent total solids in the brine. Thick and cloudy brines contain high

percent solids estimated by both total solids and alcohol soluble solids. The amount of

protein in brine is strongly and positively correlated with brine viscosity and clarity (r =
0.791, TS; r = 0.511, A18). The more protein present in the brine, the greater the brine
viscosity. Another factor affecting brine viscosity is the amount of insoluble dietary fiber
(IDF). An important physical property of IDF is its capacity to bind water. A high
correlation between amount of the insoluble dietary fiber in the brine and brine viscosity
was observed (r = 0.612). No significant correlations were found between the amount of
starch in brine and brine viscosity.

Statistical analysis (T-test)

Two tailed T-test was conducted for variables grouped both by brine
characteristics and bean integrity based on total solids and alcohol insoluble solids (Table
16 and Table 17). The statistical analysis demonstrated the same trend as shown on the

correlation study.

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Conclusions

Brine characteristics (thick / viscous / cloudy and thin / fluid / clear) were not
related with integrity (intact and split) of cooked beans. The viscous brine contained more
SDF and protein than did the fluid brine. There was no one specific component identified
which adequately served as a single determinant of bean brine viscosity. No strong
correlation was found between cooked bean integrity and canning quality characteristics.
Relationships between brine characteristics and canning quality characteristics
demonstrated that drained weight was positively correlated with brine thickness. Soaked
weight of beans was negatively correlated with brine viscosity. The amount of protein and

IDF in brine was strongly correlated with increased brine viscosity.

 

Ho: the compositional components of brine and physical bean quality after
processing are not controlled by genetic background.

Reject the Ho as stated and conclude that the compositional components of brine
and physical quality after processing are controlled by genetic background.

 

SUMMARY AND CONCLUSIONS

This research was conducted to provide a greater understanding of structural
changes in components within seeds during thermal processing to improve starch
digestibility of dry bean.

The degree of starch gelatinization in cooked beans is of importance to their
complete digestibility. An enzymatic procedure using B—amylase and pullulanase was used
to selectively digest the gelatinized starch fractions. The effects of selected heat
treatments on cell wall structural appearance and starch bioavailability were enzymatically
and microscopically examined.

The degree of gelatinization measured by the enzymatic method showed lower
scores than that obtained by the microscopic birefringent method on differentially cooked
beans. The lower values measured by the enzymatic method resulted from inaccessibility
of the enzyme within gelatinized starch. Cooking whole beans for 10 min or longer
caused crystallization within cell wall. The crystalline structure was observed under
polarized microscope and appeared as a white band around cell walls. The crystallized cell
walls of cooked bean was mechanically resistant and acted as a physical barrier not only to
enzymatic hydrolysis but also to swelling of starch granules leading to a residual
ungelatinized starch during cooking. The rigidity of the crystallized cell wall that changed
during cooking was investigated by measuring available starch after reheating of cooked,
freeze-dried and milled (CFM) flours. The longer the cooking time, the greater the

rigidity of the crystallized cell wall of the cooked beans. Reheating of CFM“, enabled

126

80% of starch to be hydrolyzed by the enzymes while reheating of CFM40 allowed only
40% starch hydrolysis for the navy bean (experimental line, N 84004). The cell wall
rigidity varied between bean types and among the process conditions studied. Differential
scanning calorimetry (DSC) thermography demonstrated that the crystalline structure
formed during cooking was more stable than starch that is commonly referred to as
“resistant starch fraction”. Starches of cooked sweet potato and wheat were readily
available to undergo enzymatic degradation since no crystallization occurred within cell
walls to limit enzyme accessibility.

The attributes of physical appearance, palatability and nutritional bioavailability are
essential components associated with food quality of cooked dry beans. Dry bean cell
walls undergo structural changes during cooking that limit starch digestibility. The
crystallized cells encapsulating starch granules were observed in cooked bean exudate
(brine). The amount of starch in brine was not correlated with brine viscosity however
total protein and insoluble dietary fiber in brine were strongly correlated with increased
brine viscosity.

These studies have provided evidence for mechanisms limiting overall potential for
maximum nutritional bioavailability and digestibility of cooked bean products. The
development and implementation of appropriate process conditions for preparing dry bean
could increase available caloric value for populations who consume beans as a dietary
staple, reduce flatulence and perhaps decrease cooking time required to render a palatable
product. To enhance understanding of the mechanisms associated with cell wall

complexes, further research is required to separate and characterize the crystallized cell

128

walls and to assess the effect of salts in soaking solution on the rigidity of the crystallized

cell wall structures which were demonstrated through these studies.

BIBLIOGRAPHY

BIBLIOGRAPHY

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Albersheirn P. 1976. The primary cell wall. In Plant biochemistry. J. Bonner and J. E.
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