MOLECULAR STRUCTURE AND PHYSICOCHEMICAL PROPERTIES OF STARCH
ISOLATED FROM PHASEOLUS VULGARIS
By
Sharon Hooper

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Food Science-Doctor of Philosophy
2014

ABSTRACT
MOLECULAR STRUCTURE AND PHYSICOCHEMICAL PROPERTIES OF STARCH
ISOLATED FROM PHASEOLUS VULGARIS
By
Sharon Hooper
The health benefits of legumes have been known for many years. They are
considered to be a food staple in the diet of humans. Dry beans have been implicated in the
prevention and management of type II diabetes mellitus and in the reduction of risks associated
with colon cancer. Dry beans are good sources of complex carbohydrates, protein, dietary fiber,
vitamins and minerals. However, use of the starch fraction of dry beans is limited due to the lack
of molecular structure-functionality relationships. The specific objectives of this study were: (1)
to determine and compare the physicochemical properties of starch isolated from six varieties of
Phaseolus vulgaris, namely black bean, dark red kidney bean, light red kidney bean, navy bean,
pinto bean and small red bean, (2) to determine and compare the digestibility, estimated
glycemic index, and molecular weight distributions of isolated raw, canned and stovetop-cooked
bean starches, (3) to investigate the effects of canning and a traditional cooking method (boiling)
on the digestibility and molecular weight distributions of starch isolated from dried bean.
The physicochemical characteristics of starches isolated from six varieties of Phaseolus
vulgaris grown in Michigan were determined. Starch yield varied from 22.8% to 34.1% on a
whole seed basis (~ 10% moisture content). The raw isolated bean starches exhibited high
resistant starch contents ranging from 41.9% (light red kidney bean) to 55.7% (pinto bean),
whilst total amylose content varied from 28.0% (pinto bean) to 29.8% (dark red kidney bean).
Significant differences were observed for the gelatinization transition temperatures, pasting
parameters, and resistant starch contents of the isolated dark red kidney and light red kidney bean

starches when compared to isolated starch from black, navy, pinto and, small red beans. All
isolated bean starches displayed the characteristic C-type X-ray diffraction pattern of legumes.
The crystallinity and B-type starch polymorph contents ranged from 36.1% to 24.9% and 19.6%
to 15.6% respectively. This study demonstrated that the chemical compositions of beans are
different for different varieties, and thus the varieties exhibit different physicochemical
properties.
The weight average molecular weight distribution (Mw) of starches isolated from native,
canned, and stovetop-cooked beans were analyzed using high-performance size exclusion
chromatography with multi-angle laser light scattering and refractive index detectors (HPSECMALLS-RI). Results revealed that amylose of isolated native dark red kidney bean starch had
the smallest Mw (1.0 x 106 g/mol), whereas isolated native pinto bean starch had the largest
value of Mw (1.8 x 106 g/mol). The Mw values of amylopectin for isolated native bean starches
ranged from 2.4 x 107 g/mol to 3.9 x 107 g/mol. Isolated canned and stovetop-cooked bean
starches displayed a mono-modal Mw distribution, with a reduction in high molecular weight
fractions, whereas isolated native bean starch Mw distribution was bi-modal. Results of in vitro
α-amylase starch hydrolysis showed ranges of rapidly digestible starch (RDS) (1.95-2.71%),
slowly digestible starch (SDS) (14.36-17.39%), and resistant starch (RS) (78.95-83.7%) among
the tested isolated native Phaseolus vulgaris starches. However, RDS, SDS, and RS fractions in
isolated canned bean starches ranged from 7.58-13.21%, 20.75-24.90%, and 63.17- 68.54%,
respectively. Isolated stovetop-cooked bean starches yielded similar results: RDS, 7.37-10.61%;
SDS, 22.55-26.84%; and RS, 62.55-68.59%). The hydrolysis indices (HI) and glycemic indices
(GI) were marginally greater for isolated canned bean starches than for stovetop-cooked starches.

Copyright by
SHARON HOOPER
2014

iv

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. M. Bennink, former advisor, for his wisdom, guidance,
dedication, and kindness, and sincerely thank my committee, Drs. L. Bourquin, K. Cichy,
P.K.W. Ng (chair), and G. Strasburg for their invaluable contributions, patience, encouragement,
and support, all of which made this dissertation possible.
I wish to express my gratitude to the Department of Food Science and Human Nutrition for
offering me a fellowship and several teaching assistantships that supported my doctoral research
without financial burden.
I would like to express my gratitude to Dr. YuLai Jin of Kellogg Company who helped
me tremendously with my molecular weight determinations. To all my friends who have given
me suggestions, shown support during my pursuit of the Ph.D. degree at Michigan State
University, I sincerely say “Thank you”
I am extremely grateful to my mom, brothers and sisters for their sacrifice, shoulders of
support and love during the course of my Ph.D. studies.
Almighty God, my Rock, in whom I live and move and have my being, none of this
would have been possible without you. Thank you for always being with me.

v

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................................ix
LIST OF FIGURES .....................................................................................................................x
CHAPTER 1 ................................................................................................................................1
INTRODUCTION .......................................................................................................................1
LITERATURE CITED ................................................................................................................6
CHAPTER 2 ................................................................................................................................9
LITERATURE REVIEW ............................................................................................................9
2.1. Legumes ................................................................................................................................10
2.2. Production and Utilization ....................................................................................................10
2.3. Starch ....................................................................................................................................12
2.4. Legume Starch ......................................................................................................................14
2.5. Crystallinity...........................................................................................................................15
2.6. Gelatinization ........................................................................................................................19
2.7. Pasting ...................................................................................................................................19
2.8. Retrogradation.......................................................................................................................22
2.9. Resistant Starch .....................................................................................................................22
2.10. Starch Hydrolysis and Glycemic Index ..............................................................................23
2.11. Molecular Weight ...............................................................................................................26
2.11.1. Starch Solubilization ........................................................................................................26
2.11.2. Size Exclusion-Chromatography .....................................................................................27
2.11.3. Light Scattering ................................................................................................................28
LITERATURE CITED ................................................................................................................30
CHAPTER 3 ................................................................................................................................38
PHYSICOCHEMICAL PROPERTIES OF STARCH ISOLATED FROM SIX VARIETIES OF
PHASEOLUS VULGARIS GROWN IN MICHIGAN ..............................................................38
3.1. ABSTRACT ..........................................................................................................................39
3.2. INTRODUCTION ................................................................................................................40
3.3. MATERIALS AND METHODS ..........................................................................................42
3.3.1. Materials ............................................................................................................................42
3.3.2. Starch Isolation ..................................................................................................................42
3.3.3. Proximate Analysis of Starch.............................................................................................44
3.3.4. Nitrogen Determination .....................................................................................................44
3.3.5. Trace Mineral Analysis ......................................................................................................44
3.3.6. Physicochemical Properties of Bean Starch ......................................................................45
3.3.6.1. Total starch content .........................................................................................................45
3.3.6.2. Starch Damage ................................................................................................................46
vi

3.3.6.3. Pasting Properties............................................................................................................46
3.3.6.4. Gelatinization and Retrogradation ..................................................................................47
3.3.7. Resistant Starch ..................................................................................................................47
3.3.8. Amylose and Amylopectin Contents .................................................................................48
3.3.9. X-ray Diffraction ...............................................................................................................49
3.3.9.1. Crystallinity.....................................................................................................................49
3.3.9.2. A-type and B-type Polymorphic Composition ...............................................................49
3.3.10. Statistical Analysis ...........................................................................................................50
3.4. RESULTS AND DISCUSSION ...........................................................................................51
3.4.1 Proximate Analysis .............................................................................................................51
3.4.2. Total Starch, Starch Damage, Amylose Content and Resistant Starch Contents ..............52
3.4.3. Pasting Characteristics .......................................................................................................55
3.4.4. Gelatinization and Retrogradation Properties ....................................................................58
3.4.5. Crystallinity........................................................................................................................62
3.5. CONCLUSIONS...................................................................................................................64
APPENDICES .............................................................................................................................66
APPENDIX A ..............................................................................................................................67
X-RAY DIFFRACTION STANDARD CURVE USED IN THE DETERMINATION OF THE
POLYMORPHIC COMPOSITION OF ISOLATED NATIVE STARCH .................................67
APPENDIX B ..............................................................................................................................71
PASTING PROPERTIES OF ISOLATED NATIVE BEAN STARCHES ................................71
LITERATURE CITED ................................................................................................................72
CHAPTER 4 ................................................................................................................................78
EFFECTS OF DIFFERENT COOKING METHODS ON THE DIGESTIBILITY AND
MOLECULAR WEIGHT DISTRIBUTION OF ISOLATED BEAN (PHASEOLUS VULGARIS)
STARCHES .................................................................................................................................78
4.1. ABSTRACT ..........................................................................................................................79
4.2. INTRODUCTION ................................................................................................................80
4.3. MATERIALS AND METHODS ..........................................................................................84
4.3.1. Materials ............................................................................................................................84
4.3.2. Stovetop-Cooking of Dry Beans ........................................................................................84
4.3.3. Canning of Dried Beans .....................................................................................................84
4.3.4. Starch Extraction ................................................................................................................85
4.3.5. Moisture Content ...............................................................................................................85
4.3.6. Starch Digestibilty .............................................................................................................85
4.3.6.1 Hydrolysis Index (HI) and Glycemic Index (GI) .............................................................86
4.3.7. Preparation of Starch Dispersions for the HPSEC-MALLS-RI System ............................87
4.3.8. Molecular Weight Determination ......................................................................................88
4.3.9. Statistical Analysis .............................................................................................................89
4.4. RESULTS AND DISCUSSION ...........................................................................................90
4.4.1. In vitro Digestibility ...........................................................................................................90
4.4.2. Molecular Weight Determination ......................................................................................101
4.5. CONCLUSIONS...................................................................................................................106
APPENDICES .............................................................................................................................107
APPENDIX C ..............................................................................................................................108
vii

ENZYMATIC HYDROLYSIS OF ISOLATED NATIVE, CANNED AND STOVETOPCOOKED BEAN STARCHES ...................................................................................................108
APPENDIX D ..............................................................................................................................111
WEIGHT AVERAGE MOLECULAR WEIGHT DETERMINATIONS ...................................111
LITERATURE CITED ................................................................................................................114
CHAPTER 5 ................................................................................................................................120
GENERAL CONCLUSIONS ......................................................................................................120
CHAPTER 6 ................................................................................................................................126
FUTURE RECOMMENDATIONS ............................................................................................126

viii

LIST OF TABLES

Table 3.1. Chemical Composition of Starches from Phaseolus vulgaris (g/100g) .....................52
Table 3.2. Physicochemical Properties of Bean Starch of Six
Phaseolus vulgaris Varieties (g/100g) .........................................................................................55
Table 3.3. Pasting Properties of Isolated Bean Starch from Six
Phaseoluss vulgaris Varieties ......................................................................................................58
Table 3.4. Gelatinization and Retrogradation Parameters for Isolated Bean Starches ................61
Table 3.5. Crystallinity and B-type Polymorph Composition of Starch Isolated
from Dry Beans ............................................................................................................................64
Table 1-B. Pasting Properties of Isolated Native Bean Starches .................................................71
Table 4.1. Nutrient Label for Sunbeam Giant Enriched White Bread .........................................87
Table 4.2. Rapidly Digestible Starch, Slowly Digestible Starch and Resistant Starch Contents of
Isolated Starch from Raw, Stovetop-Cooked, and Canned Beans ...............................................96
Table 4.3. Hydrolysis Index and Glycemic Index of Starch Isolated from
Six Bean Varieties........................................................................................................................100
Table 4.4. Weight Average Molecular Weight (Mw) of Amylose and Amylopectin in Isolated
Native Bean Starch ......................................................................................................................103
Table 1-C. Absorbance of Glucose Released During Enzymatic Hydrolysis of Isolated Native
Bean Starches ...............................................................................................................................108
Table 2-C. Absorbance of Glucose Released During Enzymatic Hydrolysis of Isolated Canned
Bean Starches ...............................................................................................................................109
Table 3-C. Absorbance of Glucose Released During Enzymatic Hydrolysis of Isolated
Stovetop-cooked Bean Starches ...................................................................................................110
Table 1-D. Light Scattering Weight Average Molecular Weights of Isolated Native, Canned and
Stovetop-cooked Bean Starches ...................................................................................................113

ix

LIST OF FIGURES

Figure 2.1. Chemical structure of amylose and amylopectin.......................................................13
Figure 2.2. Unit cell geometry of hexagonal and monoclinic unit cells. .....................................17
Figure 2.3. Double helices arrangement of A-type and B-type crystallites in starch granules....18
Figure 2.4. Typical RVA curve, showing the main parameters used to describe pasting ...........21
Figure 3.1. Wet isolation of starch from dry beans......................................................................43
Figure 3.2. Pasting (Rapid Visco Analyzer) profiles of starches isolated from black (BB), dark
red kidney (DR), light red kidney (LR), navy (NB), pinto (PB) and
small red (SR) beans ....................................................................................................................57
Figure 1-A. X-ray diffractograms of B-type starch (0–100% potato, P) and pure A-type (100–0%
waxy corn, W) ..............................................................................................................................68
Figure 4.1. Starch hydrolysis curves for isolated native bean starches of six
Phaseolus vulgaris varieties ........................................................................................................93
Figure 4.2. Starch hydrolysis curves for isolated stovetop-cooked bean starches of six
Phaseolus vulgaris varieties ........................................................................................................94
Figure 4.3. Starch hydrolysis curves for isolated canned bean starches of six
Phaseolus vulgaris varieties ........................................................................................................95
Figure 4.4. Relationship of RDS with GI of raw (A), canned (B) and stovetop-cooked (C) bean
starch isolated from six Phaseolus vulgaris varieties ..................................................................99
Figure 4.5. Molecular weight distributions of isolated native starch from six
Phaseolus vulgaris varieties ........................................................................................................102
Figure 4.6. Molecular weight distributions of starch isolated from stovetop cooked beans of six
Phaseolus vulgaris varieties ........................................................................................................104
Figure 4.7. Molecular weight distributions of starch isolated from canned beans of six
Phaseolus vulgaris varieties ........................................................................................................105
Figure 1-D. Isolated native black bean starch dn/dc ....................................................................112
Figure 2-D. Isolated native navy bean dn/dc ...............................................................................112

x

CHAPTER 1
INTRODUCTION

1

In many countries the incidence of diabetes, obesity and cardiovascular diseases is
increasing rapidly. Foods are now consumed for both nutrition and health benefits; their
components may potentially exert a positive impact in the prevention and treatment of chronic
diseases. The health benefits of legumes have been known for many years. They are considered
to be a food staple in the diet of humans (Leterme, 2002). Dry beans have been implicated in the
prevention and management of type II diabetes mellitus and in the reduction of risks associated
with colon cancer. Several studies have demonstrated that the consumption of dry beans or
legumes, low glycemic index foods, may be protective against developing type II diabetes
(Broughton, Hernandez, & Blair, 2003; Duranti, 2006; Geil & Anderson, 1994; Guillon &
Champ, 2002; Hangen & Bennink, 2002). The glycemic index (GI) of a food is related to the
rate at which glucose is absorbed from the small intestine (Jenkins et al., 1981; Jenkins, 1982;
Wolever, Jenkins, Thompson, Wong, & Josse, 1987). Low GI foods elicit a more sustained but
lower level or degree of increase in post-prandial blood glucose and insulin, and reduce gastric
emptying of the stomach (Esfahani et al., 2009; Jenkins, Wolever, Taylor, Barker, & Fielden,
1980; Thorne, Thompson, & Jenkins, 1983). Dry beans give a low glycemic response relative to
other high-carbohydrate-containing foods; this may be due to a combination of several factors
such as the structure-derived higher resistance to digestion of bean starches, the presence of fiber
and/or the partial or incomplete gelatinization of starch during cooking. The physicochemical
properties and molecular structures of starch that affect digestion are poorly understood.
Apart from being eaten as a vegetable, grain legumes can be considered as a source for
raw material (starch) for the processing industry. For example, isolated starch from peas has
been used in extruded bakery products, dressings, instant soups and puddings (Guillon & Champ,
2002). Starch is the most abundant carbohydrate in the legume seed (22-45%, w/w) and can
2

provide up to 70-80% of the calories consumed by humans worldwide (Mikulíková, Masár, &
Kraic, 2008). Starch is a polymeric mixture of essentially linear amylose and branched
amylopectin α-D-glucan molecules (Bird, Lopez-Rubio, Shrestha, & Gidley, 2009; Fox & Robyt,
1992; Kim & Huber, 2010). The functionality of starch is dependent on amylose and
amylopectin, as well as on the physical organization of these macromolecules into the granular
structure (Cheetham & Tao, 1998; Zobel, 1988)
The commercial production of legume starches is still small compared to the overall
production of starch (Guillon & Champ, 2002). Bean starch is under-utilized in the food
industry due to the high cost of isolation, lack of knowledge of its physical and molecular
structures, and limited understanding of how these structures affect its functionality and end-use
(Hoover, Hughes, Chung, & Liu, 2010). Starches isolated from different botanical sources (e.g.,
corn, wheat and potato) differ in chemical structure, morphology and functionality.
Digestibility of native and cooked starches from grain legumes is known to be relatively
low when compared to that of most cereals and tubers. The difference in digestibility may be
due to legume starches having higher amylose content, higher gelatinization temperature, and
higher capacity to retrograde (Björck, Granfeldt, Liljeberg, Tovar, & Asp, 1994; Tovar,
Granfeldt, & Bjorck, 1992; Tovar & Melito, 1996). The digestion of starch, which affects the
rate of glucose release and absorption in the body, plays an important role in human health and
nutrition. Starch can be classified into three main groups based on its rate of digestion, namely
rapidly digestible starch (RDS) which provides a quick source of energy, slowly digestible starch
(SDS), and resistant starch (RS). Resistant starch is not digested in the small intestine and passes
into the colon where it is fermented by natural microflora to produce short chain fatty acids (e.g.
butyrate), that retard or reduce colon carcinogenesis (Bennink, Rondini, & Barrett, 2012; Noah et

3

al., 1998). Determining the physicochemical properties and structural features of starch that are
associated with these three starch fractions will provide information to plant breeders to aid in
the development of bean varieties with specific functional properties as well as enable food
scientists to create bean foods that are high in SDS and RS to prevent or reduce the impact of
chronic diseases such as diabetes. In general, results from this study will provide invaluable
information to the growing knowledge base on food legumes.

The specific objectives of this study were:
1. To determine and compare the physicochemical properties of starch isolated from six
varieties of Phaseolus vulgaris, namely, black bean, dark red kidney bean, light red
kidney bean, navy bean, pinto bean and small red bean.
2. To determine and compare the digestibility, estimated glycemic index, and molecular
weight distributions of isolated raw, canned and stovetop-cooked bean starches.
3. To investigate the effects of canning and a traditional cooking method (boiling) on the
digestibility and molecular weight distributions of starch isolated from of dried bean.

The following dissertation is divided into: (1) Literature review, (2) Physicochemical
properties of starch isolated from six varieties of Phaseolus vulgaris grown in Michigan, (3)
Effects of different cooking methods on the digestibility and molecular weight distibutions on
isolated bean (Phaseolus vulgaris) starch, (4) General conclusions, and (5) Recommendations for
future studies. The chapters of this dissertation were written according to the Food Chemistry

4

journal article format and thus some information such as introduction and laboratory procedures
are very similar for certain chapters.

5

LITERATURE CITED

6

LITERATURE CITED

Bennink, M., Rondini, E., & Barrett, K. (2012). Nutrition and human health benefits of dry beans
and pulses. In M. Siddiq & M. Uebersax (Eds.), Dry beans and pulses: production,
processing and nutrition (pp. 335–358). Oxford, UK.: Blackwell Publishing Ltd.
Bird, A. R., Lopez-Rubio, A., Shrestha, A. K., & Gidley, M. J. (2009). Modern Biopolymer
Science. Modern Biopolymer Science (pp. 449–510).
Björck, I., Granfeldt, Y., Liljeberg, H., Tovar, J., & Asp, N. (1994). Food properties affecting the
digestion and absorption of carbohydrates. American Journal of Clinical Nutrion, 59, 699S–
705S.
Broughton, W., Hernandez, G., & Blair, M. (2003). Beans (Phaseolus spp.)–model food
legumes. Plant and Soil, 252, 55–128.
Cheetham, N. W. H., & Tao, L. (1998). Variation in crystalline type with amylose content in
maize starch granules : an X-ray powder diffraction study. Carbohydrate Polymers, 36,
277–284.
Duranti, M. (2006). Grain legume proteins and nutraceutical properties. Fitoterapia, 77(2), 67–
82.
Esfahani, A., Wong, J. M. W., Mirrahimi, A., Srichaikul, K., Jenkins, D. J. A., & Kendall, C. W.
C. (2009). The glycemic index: physiological significance. Journal of the American College
of Nutrition, 28 Suppl, 439S–445S.
Fox, J. D., & Robyt, J. F. (1992). Modification of starch granules by hydrolysis with
hydrochloric acid in various alcohols, and the formation of new kinds of limit dextrins.
Carbohydrate Research, 227, 163–170.
Geil, P. B., & Anderson, J. W. (1994). Nutrition and health implications of dry beans: a review.
Journal of the American College of Nutrition, 13(6), 549–58.
Guillon, F., & Champ, M. M.-J. (2002). Carbohydrate fractions of legumes: uses in human
nutrition and potential for health. The British Journal of Nutrition, 88 Suppl 3, S293–306.
Hangen, L., & Bennink, M. R. (2002). Consumption of black beans and navy beans (Phaseolus
vulgaris) reduced azoxymethane-induced colon cancer in rats. Nutrition and Cancer, 44(1),
60–5.
Hoover, R., Hughes, T., Chung, H. J., & Liu, Q. (2010). Composition, molecular structure,
properties, and modification of pulse starches: A review. Food Research International,
43(2), 399–413.

7

Jenkins, D. J. (1982). Lente carbohydrate: a newer approach to the dietary management of
diabetes. Diabetes Care, 5(6), 634–641.
Jenkins, D. J., Wolever, T. M., Taylor, R. H., Barker, H., Fielden, H., Baldwin, J. M., Goff, D.
V. (1981). Glycemic index of foods: a physiological basis for carbohydrate exchange. The
American Journal of Clinical Nutrition, 34(3), 362–6.
Jenkins, D. J., Wolever, T. M., Taylor, R. H., Barker, H. M., & Fielden, H. (1980). Exceptionally
low blood glucose response to dried beans: comparison with other carbohydrate foods.
British Medical Journal, 281(6240), 578–80.
Kim, H.-S., & Huber, K. C. (2010). Physicochemical properties and amylopectin fine structures
of A- and B-type granules of waxy and normal soft wheat starch. Journal of Cereal Science,
51(3), 256–264.
Leterme, P. (2002). Recommendations by health organizations for pulse consumption. The
British Journal of Nutrition, 88 Suppl 3, S239–42.
Mikulíková, D., Masár, S., & Kraic, J. (2008). Biodiversity of Legume Health-promoting
Starch. Starch - Stärke, 60(8), 426–432.
Noah, L., Guillon, F., Bouchet, B., Buléon, A., Molis, C., Gratas, M., & Champ, M. (1998).
Digestion of carbohydrate from white beans (Phaseolus vulgaris L.) in healthy humans. The
Journal of Nutrition, 128(6), 977–85.
Thorne, M. J., Thompson, L. U., & Jenkins, D. J. (1983). Factors affecting starch digestibility
and the glycemic response with special reference to legumes. The American Journal of
Clinical Nutrition, 38(3), 481–8.
Tovar, J., Granfeldt, Y., & Bjorck, I. M. (1992). Effect of Processing on Blood Glucose and
Insulin Responses to Starch in Legumes. Journal of Agricultural and Food Chemistry, 40,
1848–1851.
Tovar, J., & Melito, C. (1996). Steam-Cooking and Dry Heating Produce Resistant Starch in.
Journal of Agricultural and Food Chemistry, 44, 2642–2645.
Wolever, T. M., Jenkins, D. J., Thompson, L. U., Wong, G. S., & Josse, R. G. (1987). Effect of
canning on the blood glucose response to beans in patients with type 2 diabetes. Human
Nutrition. Clinical Nutrition, 41(2), 135–40.
Zobel, H. F. (1988). Molecules to Granules: A Comprehensive Starch Review. Starch - Stärke,
40(2), 44–50.

8

CHAPTER 2
LITERATURE REVIEW

9

2.1. Legumes
Dry beans are a part of the plant Leguminosae family. The family is generally
characterized by edible seeds borne in pods that often open along two seams, by butterfly-like
flowers (the flower is irregular and made up of five petals) and by compound stipulate leaves.
Both the edible pods and the seeds are called beans or pulses. There are over 18,000 species of
legumes though only about 20 are normally consumed by humans (de Almeida Costa, da Silva
Queiroz-Monici, Pissini Machado Reis, & de Oliveira, 2006; Leterme, 2002). Most widely used
of these include Phaseolus vulgaris (common bean), Vigna unguiculata (cowpeas), Pisum
sativum (common pea), Lens culinaris (lentil), Arachis hypogaea (peanut) and Glycine max
(soya bean). Legumes are second only to the grasses (cereals) in providing food crops for the
world. They are among the most nourishing vegetables eaten by mankind (de Almeida Costa et
al., 2006; Sathe & Salunkhe, 1981; Venter & Eyssen, 2001). Grain legumes occupy an important
place in human nutrition, especially in the dietary patterns of low income groups in developing
countries where animal protein is expensive (Broughton et al., 2003). In general, legumes are
good sources of complex carbohydrates, protein, dietary fiber, vitamins and minerals. In
addition, the fat content of the majority of legumes falls between one and two percent, except for
soybeans, which have a higher fat content. The predominant fatty acid found in legumes is
linoleic acid (de Almeida Costa et al., 2006; Tharanathan & Mahadevamma, 2003).

2.2. Production and Utilization
Leguminosae are second to cereal crops in agricultural importance based on area
harvested and total production. According to the Food and Agriculture Organization of the
10

United Nations (FAO), in 2012 more than 24 million metric tons of dry beans were produced
worldwide. Phaseolus vulgaris, generally known as the common bean, is produced in a range of
crop systems and environments in regions as diverse as Latin America, Africa, China, Europe,
the United States and Canada (FAO, 2014). North Dakota and Minnesota produce
approximately 50% of the total bean crop each year in the United States and the top five beans
grown include pinto, navy, black, red kidney (light and dark), and great northern beans (Miller,
2014). The common bean is the most widely cultivated of all beans.
Traditionally, grain legumes are processed and consumed as human food in a variety of
ways. Processing improves palatability of beans and also increases the bioavailability of
nutrients. Fresh or canned unripe pods or seeds are eaten as vegetables. Pounding, grinding and
milling of dry grain is commonly practiced to obtain split seeds (dhals) or cotyledons with or
without the seed coat. The most common method of cooking is to boil the entire seed or the
dehusked cotyledons in water with salt. The dry beans are usually first soaked overnight
(approximately 16 hours) and then cooked in boiling water. The process of soaking softens the
seed to reduce cooking time and may also remove anti-nutritional factors such as tannins.
Commercial canning has increased the convenience of using dry beans (Anderson, Smith, &
Washnock, 1999; Tharanathan & Mahadevamma, 2003). Bean flour is used in food products
prepared by baking, steaming or deep frying in oil. Processing methods have been developed
using flour or isolated protein concentrates to make instant foods and meat analogs, especially
from soybeans (Salunkhe & Kadam, 1983). The use of dry beans for food is dependent upon the
seed size, shape, color and flavor characteristics and is often associated with particular social or
ethnic groups (Ensminger & Ensminger, 1993). Taste and variety preference vary significantly

11

from one country to another. Popular uses include soups, mixed-bean salads, rice and peas,
beans boiled with meat or other vegetables, or cereals and baked beans (Geil & Anderson, 1994).

2.3. Starch
Starch is the major carbohydrate storage material in many higher plants. Starches
isolated from different botanical sources show diverse granule morphology and the granules vary
in shape (spherical, oval, polygonal, disk, elongated and kidney shapes), and in size (1 µm-100
µm in diameter). The granule size and shape influence the physicochemical characteristics of
starch, as well as its end use (Chibbar, Ambigaipalan, & Hoover, 2010; Hoover, Hughes, Chung,
& Liu, 2010). Starch is a polymer in which the monomeric unit is the six-carbon sugar Dglucose. The polymerization of glucose into starch produces two types of polymers, amylose
and amylopectin, which represent approximately 98-99% of the dry weight of starch. These
polymers have different structures and properties. Amylose is an essentially linear polymer with
α–1,4 linkages, whereas amylopectin is a highly branched polymer with short α–1,4 linked
chains and 5-6% non-randomly distributed α–1,6 linkages (Figure 2.1.). Amylose has an average
molecular weight of approximately 1 x 105 to 1 x 106 g/mol, with a degree of polymerization by
number (DPn) of 324-4920. Conversely, amylopectin is a much larger molecule than amylose
with a molecular weight of 1 x 107 to 1 x 109 g/mol and DPn of 9600-15900 (Sajilata, Singhal, &
Kulkarni, 2006; Tester, Karkalas, & Qi, 2004; Wang, Bogracheva, Hedley, Centre, & Asp,
1998). The unit chains present in amylopectins (18-25 units) are shorter than those found in
amylose molecules (Tester et al., 2004). Amylose and amylopectin are deposited into starch
granules to produce semi-crystalline starch granules that vary in shape, size, and composition

12

Figure 2.1. Chemical structure of amylose and amylopectin. Source Tester, Karkalas, and Qi
(2004).

13

depending on botanical source and growing environment. The amylose and amylopectin
contents of normal starches are 20-30% and 70-80%, respectively. Mutant lines of maize
starches have been obtained with amylose contents in the range of 0% (waxy maize) to 70%
(Hylon VII) (Jane, 1995; Pérez, Baldwin, Gallant, & Jane, 2009; Tester et al., 2004). Starch is
semi-crystalline in nature with approximately 70% of the starch granule being amorphous and
about 30% crystalline, as shown in diffraction experiments. The main component of the
amorphous regions is amylose, while the crystalline region is primarily occupied by amylopectin
(Sajilata et al., 2006). Variations in the structure of amylose and amylopectin, such as average
molecular size, average degree of polymerization, and chain length distribution can effect
differences in the physicochemical properties of starch granules (Thitipraphunkul, Uttapap,
Piyachomkwan, & Takeda, 2003)

2.4. Legume Starch
The starch contents of beans and other legumes range from 13 to 49% of their dry weight.
Extensive research has been conducted on cereal, potato, sweet potato and cassava starches due
to their availability and wide usage in food and non-food applications. However, there is a lack
of information on structure-property relationships among bean starches. This is in part due to the
difficulty encountered in isolating pure starches from legumes because of their high protein
content, as well the effect of fine fiber co-settling with the starch and the high cost of isolation
(Chibbar et al., 2010; Hoover et al., 2010; Huang et al., 2007).
Legume starch granules vary in shape: round, oval, spherical, elliptical and irregular; and
in size: width (5-55 µm) and length (5-70 µm). Most legume starches are comprised of simple

14

granules (granules are that formed singly). However, a mixture of simple and compound
granules (granules that form in aggregates) was reported for wrinkled peas and smooth peas.
The amylose contents of legume starches range from 24-48%. Still, it is difficult to compare the
amylose contents among and between legumes due to differences in where they were grown,
cultivar, physiological state of the seed, and the methodology used for determination (Chibbar et
al., 2010; Hoover et al., 2010; Shimelis, Meaza & Rakshit, 2006).

2.5. Crystallinity
Much of the information about starch granule crystalline properties has been acquired
from X-ray powder diffraction studies. Starch granules from various botanical origins generally
show one of three types of X-ray diffraction patterns: A-type (cereal starches); B-type (tuber,
root, high amylose and retrograded cereal, and legume starches) and C-type (legume starches).
The C-type pattern is a mixture of both A- and B-types (Cheetham & Tao, 1998). There is a Vtype conformation, which results from amylose being complexed with substances such as
aliphatic fatty acids, emulsifiers, butanol and iodine to form a double helix (Buléon, Gérard,
Riekel, Vuong, & Chanzy, 1998). The main difference between A- and B-types of starches is in
their crystalline unit cells. A unit cell is a small repeating entity in a crystal structure. Each type
unit cell differs in the three unit cell edge lengths and the three internal angles (Figure 2.2.)
(Massa, 2004). The A-type starch adopts a close-packed monoclinic array with eight water
molecules present per unit cell, while the B-type is a more open hydrated structure, consisting of
double helices packed in a hexagonal array. For the B-type thirty-six water molecules per unit
cell fill the large central channel formed by the hexagonically packed double helices;

15

approximately half of the water molecules are tightly bound to the chains, and the other half are
connected only to other water molecules (Figure 2.3.) (Bello-Pérez, Rodriguez-Ambriz, SanchezRivera, & Agama-Acevedo, 2009; Chibbar et al., 2010; Gernat, Radosta, Damaschun, &
Schierbaum, 1990; Imberty, Chanzy, & Perez, 1988; Pérez, Baldwin, & Gallant, 2009).
Legume starches upon X-ray diffraction are characterized by strong intensity peaks
corresponding to approximately 2θ=15°, 17° and 23°. Also, some legume starches have shown a
small peak at 2θ =5.6°, which is characteristic of B-type starches; however, the intensity of peak
was very low (Singh, Nakaura, Inouchi, & Nishinari, 2008). In an experiment using microfocus
synchrotron wide-angle diffraction mapping on a series of C-type starch granules from smooth
peas, it was found that C-type starch granules globally contained 60% of A-type polymorph and
40% of the B- type polymorph; additionally, the two polymorphs were present in each granule.
The A-type polymorph was located essentially in the outer part of the granules whereas the Btype polymorph was found mostly in their centers (Buléon et al., 1998). These types of starch
polymorphs depend partly on the chain length making up the amylopectin lattice, the density of
packing within the granules, and the presence of water (Sajilata et al., 2006). The relative
proportions of A- and B- in the C-type polymorphs and how they relate to functional properties
(such as gelatinization, pasting and swelling) and digestibility is unknown. Ambigaipalan et al.
(2011) studied the structure of faba bean, black bean and pinto bean cultivar starches at different
levels of granule organization and their physicochemical properties and found that the B
polymorphic content followed the order faba bean >black bean >pinto bean, ranging from 25.0%
to 7.9%, while crystallinity ranged from 20.3% to 23.1% for these beans. In general, the
crystallinity for legume starches ranges between 17 and 34%, which could be influenced by
moisture content, size and number of crystallites arranged in a crystalline array and polymorphic

16

content (Hoover et al., 2010). Singh et al. (2008) postulated that the proportion of longer
amylopectin chains may be responsible for the differences in crystallinity observed among
starches from different legumes. High performance liquid chromatography (HPLC) analyses
conducted on the distribution of amylopectin chain lengths in starches from 20 species
established that there was a close relationship between the weight-average chain lengths of the
amylopectins and the crystal type of starch granules. Short amylopectin chain lengths favored
A-type crystallinity, while long chain lengths showed B-type crystallinity and intermediate chain
lengths were associated with C-type crystallinity (Hizukuri, Kaneko, & Takeda, 1983; Hizukuri,
1985).

Figure 2.2. Unit cell geometry of hexagonal and monoclinic unit cells. Source: Massa (2004).

17

Figure 2.3. Double helices arrangement of A-type and B-type crystallites in starch granules.
Source: Sarko and Wu (1978).

18

2.6. Gelatinization
Gelatinization has been defined as the phase transition of starch granules from an ordered
state to a disordered state, which takes place during heating in excess water (Hermansson &
Svegmark, 1996). When starch granules are heated in excess water, the hydrogen bonds between
glucose residues weaken, granules take up water resulting in swelling, and granular structure
and crystallinity are lost. The starch is dispersed in the heated water and is susceptible to
hydrolysis by α-amylase (Bertolini, 2009; Jenkins & Donald, 1998).
The gelatinization process is usually studied by differential scanning calorimetry (DSC).
A starch sample is heated at a defined rate and the changes in heat capacity are measured as a
function of temperature. Differential scanning calorimetry (DSC) measures the gelatinization
transition temperatures (To, onset; Tp, peak; Tc, conclusion) and enthalpy of gelatinization (ΔHgel).
These transition temperatures are said to be affected by the molecular design of the crystalline
region, which is related to the distribution of amylopectin short chains and not the amylose to
amylopectin ratio (Jane et al., 1999; Singh, 2011). In general, higher gelatinization temperatures
and enthalpies of gelatinization have been observed in starches comprising amylopectin with
longer branch chains because longer chains more easily form stable double helical crystallites
than shorter chains (Jane et al., 1999).

2.7. Pasting

Pasting is the phenomenon following gelatinization of a starch and involves granular
swelling, exudation of amylose and amylopectin, and total disruption of the starch granule.
Pasting viscosity profiles are commonly analyzed using a Rapid Visco Analyzer (RVA). A
19

typical profile is presented in Figure 2.4. Pasting temperature is the point at which the
temperature rises above the gelatinization temperature. The peak viscosity indicates the
maximum viscosity reached during the heating and holding cycle and is indicative of the water
holding capacity of a starch; peak temperature occurs at peak viscosity. The breakdown
viscosity is normally regarded as a measure of the disintegration of the starch granules as they
are heated due to the rupture of granules and the release of soluble amylose (Abdel-Aal, Hucl,
Chibbar, Han, & Demeke, 2002; Gupta, Bawa, & Semwal, 2009; Karim, Norziah, & Seow,
2000). As the starch-water mixture is cooled, re-association between starch molecules,
especially of amylose, results in the formation of a gel and the subsequent increase in viscosity
(i.e., total setback increases). Total setback involves retrogradation, or re-ordering, of the starch
molecules (Kim, Lee, Baik, Joo, & Yoo, 2007; Rupollo et al., 2011).

Pasting properties of starch are affected by amylose and lipid contents and by branch
chain-length distribution of amylopectin (Gupta et al., 2009; Jane et al., 1999). Starches with
greater amylose, lipid and phospholipid contents have higher pasting temperatures, lower peak
viscosities and shear-thinning (breakdown viscosity), and higher setback viscosities (Du, Jiang,
Ai, & Jane, 2014; Jane et al., 1999; Zhang, Ao, & Hamaker, 2008; Zhang & Hamaker, 2003).
Waxy wheat flour starch, conversely, has been shown to have significantly lower peak and
pasting temperatures, higher peak viscosities and lower setback viscosity than non-waxy or
normal wheat flour (Abdel-Aal et al., 2002; Gupta et al., 2009). This is because waxy wheat
flour starch is essentially made up of 100% amylopectin molecules. Amylopectin with longer
branch chains display larger peak viscosity and lower pasting temperatures than their shorter
chain counterparts (Rosin, Lajolo, & Menezes, 2002).

20

Figure 2.4. Typical RVA curve, showing the main parameters used to describe pasting. Source:
Bertolini (2009).

21

2.8. Retrogradation
With continued heating after gelatinization, swollen granules will undergo pasting.
When cooled, the starch chains are able to re-associate with each other through hydrogen
bonding, forming an ordered structure that is more resistant to digestion. This process is known
as retrogradation (Englyst, Kingman, & Cummings, 1992; Englyst & Hudson, 1996; Miles,
Morris, Orford, & Ring, 1985; Miles, Morris, & Ring, 1985). Both amylose and amylopectin
will re-associate during this process with amylose retrogradation occurring at a faster rate than
that of amylopectin. Amylopectin is limited in its retrograding capabilities by its branched
structure, and the polymers that do re-associate are less firmly bound than those of retrograded
amylose (Copeland, Blazek, Salman, & Tang, 2009; Englyst et al., 1992). Therefore, recrystallized (retrograded) amylopectin requires a lower temperature (70ºC) to reverse
retrogradation upon reheating whereas amylose requires a temperature of 160ºC (Miles, Morris,
Orford, et al., 1985; Miles, Morris, & Ring, 1985). Retrogradation can result in effects such as
precipitation, gelation, changes in consistency and opacity, and decreased storage stability, all of
which are unwanted side effects for industrial applications (Hermansson & Svegmark, 1996).
The rate of starch retrogradation is dependent on many factors such as botanical source, ratio of
amylose to amylopectin, the structure of both amylose and amylopectin, temperature as well as
the presence of other compounds such as lipids and proteins (Thitipraphunkul et al., 2003).

2.9. Resistant Starch
Resistant starch has been defined as the fraction of starch which escapes digestion in the
small intestine, and may be fermented in the large intestine (Englyst et al., 1992). The resistance
of starch to digestion can be attributed to several factors, which have led to the emergence of
22

four categories of starch. They are as follows. RS1: starch that is physically inaccessible to
digestion by entrapment in a non-digestible matrix (e.g., seeds, grains and tubers with intact cell
walls); RS2: native or ungelatinized starch granules protected from digestion due to the
conformation of the granule as in raw bananas and potatoes; RS3: retrograded starch produced
via heating and subsequent cooling (e.g., corn flakes); and RS4: chemically modified starch,
including starches which have been esterified or cross-bonded) (Englyst et al., 1992; Nugent,
2005; Topping et al., 2003). The formation and levels of resistant starch present in foods are
greatly influenced by processing techniques, in particular those that use heat and moisture, for
example, canning, extrusion, and microwave heating. Dehulling legumes by steam treatment and
cooking for instance resulted in higher levels of resistant starch (Tovar et al., 2002). The
resistant starch most frequently found in processed foods is mainly retrograded starch.
Research has shown that resistant starch imparts biological benefits, of which some
benefits are like those of traditional fiber, and others are unique to resistant starch (Haralampu,
2000). Resistant starch that reaches the large intestine can act as a substrate for microbial
fermentation, the end-products being hydrogen, carbon dioxide, methane and short chain fatty
acids (SCFA). The short chain fatty acids are thought to be protective against colorectal cancer (
(Englyst & Hudson, 1996; Nugent, 2005)

2.10. Starch Hydrolysis and Glycemic Index
In human nutrition, starch plays a major role in supplying the metabolic energy that
enables the body to perform energy-requiring functions. It is broken down to glucose by
amylase enzymes in the human digestive system, and glucose is absorbed from the small
intestine into the bloodstream and used as an energy source. The rate at which glucose is

23

absorbed from the intestinal tract appears to be an important parameter in controlling the
homeostasis of blood glucose. Glycemic index and glycemic load have been used to assess the
relative risks of foods that have been implicated in the prevention of diabetes, coronary heart
disease and obesity. The glycemic index (GI) is a measure of the extent to which a test food
increases blood glucose levels during the 2 hours after consumption, compared to eating an
equivalent amount of reference carbohydrate either glucose or white bread (Jenkins et al., 1981;
Ludwig et al., 1999; Wolever, Jenkins, Wóng, Josse, & Thompson, 1987). Foods with a high GI
cause a more rapid rise in blood glucose and insulin levels compared to foods with a low GI.
Dry beans have a relatively low glycemic index, varying from 27-42% relative to glucose and
40-59% that of white bread when compared to other carbohydrate sources (Bennink et al., 2012;
Foster-Powell, Holt, & Brand-Miller, 2002). This low GI property can be attributed to the slow
rate of bean starch digestion.
Thus, in the context of digestibility, starch is classified into three fractions, rapidly
digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) (Berry, 1986;
Englyst et al., 1992). According to Englyst et al. (1992) RDS is the amount of starch digested in
the first 20 min of a standard in vitro digestion reaction mixture. RDS causes a rapid increase in
blood glucose concentration after ingestion of carbohydrate foods. SDS is the fraction of starch
digested slowly but completely in the human small intestine and is defined as the starch that is
digested after the RDS fraction, between 20 min and 120 min of a standard in vitro digestion
reaction. RS is the fraction that escapes digestion in the small intestine, and is analytically
defined as the starch that is not digested within the first 120 min of the standard digestion
reaction.

24

The major enzymes involved in starch hydrolysis are amylases and amyloglucosidases.
Alpha-amylases are the main endo-splitting enzymes involved in the hydrolysis of α-1,4 bonds
of starch (amylose and amylopectin). The nature and the distribution of the hydrolysis products
(maltose, glucose, and dextrins of high molecular weights) depend on the source of the amylases
and the substrate. Amyloglucosidase is an exo-acting enzyme that hydrolyzes the α-1,6 linkages
in starch and produces glucose from the non-reducing end of the starch molecule (Annison &
Topping, 1994; Colonna, Leloup, & Buléon, 1992).
Several factors have been proposed to limit starch hydrolysis, including particle size of
substrate, amylose content, crystallinity, degree of gelatinization, extent of retrogradation, and
the presence of other components interacting with starch (amylose-lipid complexes) (Colonna et
al., 1992; Du et al., 2014; Jane et al., 1999). Hydrolysis of native and cooked starches from grain
legumes is known to be relatively slower when compared to most cereal starches (A-type
crystalline pattern). This characteristic can be attributed to legume starches having larger
amylose content, higher crystallinity (when raw), and higher capacity to retrograde (Björck et al.,
1994; Noah et al., 1998; Tovar et al., 1992; Tovar & Melito, 1996)
Zhou et al. (2010) conducted a study to determine the changes in the molecular weight
distribution of starch fractions following digestion of foods in the human small intestine.
Volunteers with ileostomies consumed six selected foods: breakfast cereal (muesli), white bread,
oven-baked French fries, canned mixed beans and a custard containing either a low-amylose
maize starch (LAMS) or a high-amylose maize starch (HAMS). The digesta total resistant starch
contents (expressed as a fraction of ingested starch) were: muesli, 8.9%; bread, 4.8%; fries,
4.2%; bean mix, 35.9%; LAMS custard, 4.0%; HAMS custard, 29.1%. Chromatographic
analysis showed that the starches were fractionated into high molecular weight (43,500 kDa),

25

medium molecular weight (420 kDa) and low molecular weight (8.5 kDa) fractions. It was
found that a low molecular weight fraction (8.5 kDa) was predominant in the undigested residue.
Canned beans and HAMS custard contained starches with lesser digestibility and consisted
mostly of low molecular and medium molecular weight fractions and had very little high
molecular weight fractions. These results suggested that not only were the rate and extent of
starch digestion, and the SDS and RS contents of a food, affected by the amylose content of
starch, but also by the molecular weights of the amylose and amylopectin present in that starch.

2.11. Molecular Weight
Several analytical techniques have been used for the separation and structural
characterization of starch including ultracentrifugation, size exclusion chromatography and light
scattering (Bello-Pérez et al., 2009; Ratnayake, Hoover, & Warkentin, 2002; Wyatt, 1993; Yoo
& Jane, 2002). However, many problems have been linked with the separation and
characterization of the two polymer fractions of starch. These include solubilization, degradation
aggregation and retrogradation (Chiaramonte, Rhazi, Aussenac, & White, 2012).

2.11.1. Starch Solubilization
In order for starch to be accurately characterized the macromolecule needs to be
completely dissolved in the solvent of choice. Solubilization should be under moderate
conditions which ensure that the starch structure remains intact. Starch is insoluble in cold
water, but becomes soluble in excess water under certain heating conditions; however, depending
26

on the temperature and length of heating, depolymerization can occur (Bello-Pérez et al., 2009).
Starch dissolution is usually accomplished with dimethyl sulfoxide (DMSO) or aqueous DMSO
solutions, sometimes with the addition of salts such as LiBr and LiNO3.(Cave, Seabrook, Gidley,
& Gilbert, 2009; Othman, Al-Assaf, & Hassan, 2010; Yokoyama, Renner-Nantz, & Shoemaker,
1998). Zhong, et al. (2006) determined the molecular weights of rice starch amylose and
amylopectin using DMSO, with either 50 mM LiBr or 10% water or both for batch mode multiangle laser light scattering. They found that DMSO/50 mM LiBr was a better solvent for
dissolving and reducing starch aggregates than DMSO/10% water.

2.11.2. Size Exclusion-Chromatography
Size-exclusion chromatography (SEC) is a well established-method for the analysis of
polymers and macromolecules such as starches and proteins. In this type of chromatography,
separation is based on molecular size rather than chemical properties. Technically, molecules
are separated on the basis of hydrodynamic size and not weight-averaged molecular weight
(MW). The stationary phase in SEC consists of a column packing material (usually microbeads)
with pores comparable in size to the molecules to be fractionated. The porous beads of a
polymeric gel or silica beads will allow small molecules into their pores but not large ones.
When a solute is injected into such a column, the smaller molecules are distributed through a
larger volume of solvent than is available to the large molecules. As a result, smaller dissolved
molecules flow more slowly through the column because they penetrate deep into the pores,
whereas large dissolved molecules flow quickly through the column because they do not enter
the pores. Therefore, larger molecules are eluted from the column sooner than smaller

27

molecules, which enables the separation of molecules by size (Othman et al., 2010; Podzimek,
2014; Thrathnigg, 2000). The elution volume of a molecule is related to its hydrodynamic
volume (Cave et al., 2009).
In order to determine the specific molecular weight (Mw) of a molecule, column
calibration is required. Standards of known molecular weights are used to convert the elution or
retention volume to a molecular weight for a given column set. In SEC analysis of starch,
dextrans and pullulans are the standard polymers commonly used (Cave et al., 2009; Huang et
al., 2007; Xie et al., 2012). Dextran is branched glucan composed of chains of varying lengths,
with molecular weights ranging from 1 x 103 kDa to 410 x 103 kDa. The straight chain consists
of α-1,6 glycosidic bonds between glucose molecules, while branches begin from α-1,3 linkages.
Pullulans, on the other hand are linear polysaccharides made of repeated units of maltotriose
with molecular weights ranging from 3 x 103 kDa to 1600 x 103 kDa (Cave et al., 2009).

2.11.3. Light Scattering
The interaction of light with matter can yield important information about the structure of
starch and its polymers. The phenomenon of light scattering occurs when electromagnetic
radiation hitting a molecule is partly scattered. Charge separation occurs by the interaction of the
electrons within the molecule with the oscillating electric field component of light. An
oscillating dipole is created and the molecule emits scattered light. Almost all of the scattered
light has the same wavelength as the incident radiation and comes from elastic scattering, known
as Rayleigh scattering (Wyatt, 1993). In multi-angle laser light scattering (MALLS) the
scattering intensity is measured at several different angles. The two major principles that are
used in light scattering to determine the molar mass are:

28

1. The intensity of light scattered (LS) is proportional to the product of the polymer (Mw)
and the polymer concentration (C).
LS α Mw x C (dn/dc)2
Where (dn/dc) is the refractive index increment, which expresses the variation of the refractive
index of a solution with solute concentration.
2. The angular variation of the scattering is directly related to the radius of the polymer
(Wyatt, 1993)
Recently, high performance SEC equipped with both multi-angle light scattering
(MALLS) and differential refractometer (RI) detectors has been used routinely to determine
absolute molecular weights of starches (Zhong et al., 2006; Yokoyama, Renner-Nantz, &
Shoemaker, 1998; Othman, Al-Assaf, & Hassan, 2010; Cave, Seabrook, Gidley, & Gilbert,
2009). HPSEC-MALLS-RI is a rapid method to separate and monitor starch components and
debranched fragments. Molecular weight measurements with light scattering is an absolute
method and is independent from column calibration (Podzimek, 2014; Wyatt, 1993). The
average weight molecular weights of bean, rice, canna, barley, wheat, banana and potato
amylopectins determined by this method were found to be in the range of 1.3-56.8 x 108 g/mol
(Yoo & Jane, 2002).

29

LITERATURE CITED

30

LITERATURE CITED

Abdel-Aal, E. S. M., Hucl, P., Chibbar, R., Han, H. L., & Demeke, T. (2002). Physicochemical
and Structural Characteristics of Flours and Starches from Waxy and Nonwaxy Wheats.
Cereal Chemistry, 79(3), 458–464.
Ambigaipalan, P., Hoover, R., Donner, E., Liu, Q., Jaiswal, S., Chibbar, R., Seetharaman, K.
(2011). Structure of faba bean, black bean and pinto bean starches at different levels of
granule organization and their physicochemical properties. Food Research International,
44(9), 2962–2974.
Anderson, J. W., Smith, B. M., & Washnock, C. S. (1999). Cardiovascular and renal benefits of
dry bean and soybean intake. The American Journal of Clinical Nutrition, 70(3 Suppl),
464S–474S.
Annison, G., & Topping, D. L. (1994). Nutritional role of resistant starch : Chemical Structure vs
Physiological Function Primary Structure of Starch Components, (35), 297–320.
Bello-Pérez, L. A., Rodriguez-Ambriz, S., Sanchez-Rivera, M., & Agama-Acevedo, E. (2009).
Starch Molecular Structure. In A. Bertolini (Ed.), Starches: Characterization, Properties,
and Applications (1st ed., pp. 33–57). CRC Press.
Bennink, M., Rondini, E., & Barrett, K. (2012). Nutrition and human health benefits of dry beans
and pulses. In M. Siddiq & M. Uebersax (Eds.), Dry beans and pulses: production,
processing and nutrition (pp. 335–358). Oxford, UK.: Blackwell Publishing Ltd.
Berry, C. S. (1986). Resistant starch: Formation and measurement of starch that survives
exhaustive digestion with amylolytic enzymes during the determination of dietary fibre.
Journal of Cereal Science, 4(4), 301–314.
Bertolini, A. C. (Ed.). (2009). Starches: Characterization, Properties, and Applications (1st ed.,
p. 288 pages). CRC Press.
Björck, I., Granfeldt, Y., Liljeberg, H., Tovar, J., & Asp, N. (1994). Food properties affecting the
digestion and absorption of carbohydrates. American Journal of Clinical Nutrion, 59, 699S–
705S.
Broughton, W., Hernandez, G., & Blair, M. (2003). Beans (Phaseolus spp.)–model food
legumes. Plant and Soil, 252, 55–128.
Buléon, a., Gérard, C., Riekel, C., Vuong, R., & Chanzy, H. (1998). Details of the Crystalline
Ultrastructure of C-Starch Granules Revealed by Synchrotron Microfocus Mapping.
Macromolecules, 31(19), 6605–6610.

31

Cave, R. a., Seabrook, S. a., Gidley, M. J., & Gilbert, R. G. (2009). Characterization of starch by
size-exclusion chromatography: The limitations imposed by shear scission.
Biomacromolecules, 10(8), 2245–2253.
Cheetham, N. W. H., & Tao, L. (1998). Variation in crystalline type with amylose content in
maize starch granules : an X-ray powder diffraction study. Carbohydrate Polymers, 36,
277–284.
Chiaramonte, E., Rhazi, L., Aussenac, T., & White, D. R. (2012). Amylose and amylopectin in
starch by asymmetric flow field-flow fractionation with multi-angle light scattering and
refractive index detection (AF4–MALS–RI). Journal of Cereal Science, 56(2), 457–463.
Chibbar, R. N., Ambigaipalan, P., & Hoover, R. (2010). REVIEW: Molecular Diversity in Pulse
Seed Starch and Complex Carbohydrates and Its Role in Human Nutrition and Health.
Cereal Chemistry, 87(4), 342–352.
Colonna, P., Leloup, V., & Buléon, A. (1992). Limiting factors of starch hydrolysis. European
Journal of Clinical Nutrition, 46 Suppl 2, S17–32.
Copeland, L., Blazek, J., Salman, H., & Tang, M. C. (2009). Form and functionality of starch.
Food Hydrocolloids, 23(6), 1527–1534.
De Almeida Costa, G. E., da Silva Queiroz-Monici, K., Pissini Machado Reis, S. M., & de
Oliveira, A. C. (2006). Chemical composition, dietary fibre and resistant starch contents of
raw and cooked pea, common bean, chickpea and lentil legumes. Food Chemistry, 94(3),
327–330.
Du, S.-K., Jiang, H., Ai, Y., & Jane, J.-L. (2014). Physicochemical properties and digestibility of
common bean (Phaseolus vulgaris L.) starches. Carbohydrate Polymers, 108, 200–5.
Englyst, H. N., & Hudson, G. J. (1996). The classification and measurement of dietary
carbohydrates. Food Chemistry, 57(1), 15–21.
Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of
nutritionally important starch fractions. European Journal of Clinical Nutrition, 46(2), S33–
50.
Ensminger, M. E., & Ensminger, A. H. (1993). Foods & Nutrition Encyclopedia, Two Volume
Set (2nd ed., Vol. 9, pp. 169–170). Boca Raton, Florida: Taylor & Francis.
Foster-Powell, K., Holt, S. H., & Brand-Miller, J. C. (2002). International table of glycemic
index and glycemic load values: 2002. Am J Clin Nutr, 76(1), 5–56.
Geil, P. B., & Anderson, J. W. (1994). Nutrition and health implications of dry beans: a review.
Journal of the American College of Nutrition, 13(6), 549–58.

32

Gernat, C., Radosta, S., Damaschun, G., & Schierbaum, F. (1990). Supramolecular structure of
legume starches revealed by X-ray scattering. Starch - Stärke, 42(5), 175–178.
Gupta, M., Bawa, A. S., & Semwal, A. D. (2009). Morphological, Thermal, Pasting, and
Rheological Properties of Barley Starch and Their Blends. International Journal of Food
Properties, 12(3), 587–604.
Haralampu, S. G. (2000). Resistant starch — a review of the physical properties and biological
impact of RS. Carbohydrate Polymers, 41, 285–292.
Hermansson, A., & Svegmark, K. (1996). Developments in the understanding of starch
functionality. Trends in Food Science and Technology, 71, 345–353.
Hizukuri, S. (1985). Relationship between the distribution of the chain length of amylopectin and
the crystalline structure of starch granules. Carbohydrate Research, 141, 295–306.
Hizukuri, S., Kaneko, T., & Takeda, Y. (1983). Measurement of the chain length of amylopectin
and relevance to the origin of crystalline polymorphism of starch granules. Biochimica e
Biophysica, 760, 188–191.
Hoover, R., Hughes, T., Chung, H. J., & Liu, Q. (2010). Composition, molecular structure,
properties, and modification of pulse starches: A review. Food Research International,
43(2), 399–413.
Huang, J., Schols, H. a., van Soest, J. J. G., Jin, Z., Sulmann, E., & Voragen, A. G. J. (2007).
Physicochemical properties and amylopectin chain profiles of cowpea, chickpea and yellow
pea starches. Food Chemistry, 101(4), 1338–1345.
Imberty, A., Chanzy, H., & Perez, S. (1988). The Double-helical Ndure of the Crystalline Part of
A-starch. Journal of Molecular Biology, 201, 365–378.
Jane, J. (1995). Starch Properties, Modifications, and Applications. Journal of Macromolecular
Science, Part A, 32(4), 751–757.
Jane, J., Chen, Y., Lee, L., McPherson, E., Wong, K., Radosavlijevic, M., & Kasemsuwan, T.
(1999). Effects of amylopectin branch chain length and amylose content on the
gelatinization and pasting properties of starch. Cereal Chemistry, 76, 629–637.
Jenkins, D. J., Wolever, T. M., Taylor, R. H., Barker, H., Fielden, H., Baldwin, J. M., … Goff,
D. V. (1981). Glycemic index of foods: a physiological basis for carbohydrate exchange.
The American Journal of Clinical Nutrition, 34(3), 362–6.
Jenkins, P. J., & Donald, A. M. (1998). Gelatinisation of Starch: A combined
SAXS/WAXS/DSC and SANS Study. Carbohydrate Research, 308, 133–147.

33

Karim, A. A., Norziah, M. H., & Seow, C. C. (2000). Methods for the study of starch
retrogradation. Food Chemistry, 71, 9-36
Kim, S.-H., Lee, B.-H., Baik, M.-Y., Joo, M.-H., & Yoo, S.-H. (2007). Chemical structure and
physical properties of mung bean starches isolated from 5 domestic cultivars. Journal of
Food Science, 72(9), C471–7.
Leterme, P. (2002). Recommendations by health organizations for pulse consumption. The
British Journal of Nutrition, 88 Suppl 3, S239–42.
Ludwig, D. S., Majzoub, J. A., Al-Zahrani, A., Dallal, G. E., Blanco, I., & Roberts, S. B. (1999).
High Glycemic Index Foods, Overeating, and Obesity. PEDIATRICS, 103(3), e26–e26.
Massa, W. (2004). Crystal Structure Determination (Vol. 7, p. 210). Springer Science &
Business Media.
Miles, M. J., Morris, V. J., Orford, P. D., & Ring, S. G. (1985). The roles of amylose and
amylopectin in the gelation and retrogradation of starch. Carbohydrate Research, 135(2),
271–281.
Miles, M. J., Morris, V. J., & Ring, S. G. (1985). Gelation of amylose. Carbohydrate Research,
135(2), 257–269.
Noah, L., Guillon, F., Bouchet, B., Buléon, A., Molis, C., Gratas, M., & Champ, M. (1998).
Digestion of carbohydrate from white beans (Phaseolus vulgaris L.) in healthy humans. The
Journal of Nutrition, 128(6), 977–85.
Nugent,A. P. (2005). Health properties of resistant starch. Nutrition Bulletin, 30(1), 27–54.
Othman, Z., Al-Assaf, S., & Hassan, O. (2010). Molecular Characterisation of Sago Starch Using
Gel Permeation Chromatography Multi-Angle Laser Light Scattering. Sains Malaysiana,
39(6), 969–973.
Pérez, S., Baldwin, P. M., Gallant, D. J., & Jane, J. (2009). Starch. (Third Edit., pp. 193–236).
Elsevier.
Podzimek, S. (2014). Truths and myths about the determination of molar mass distribution of
synthetic and natural polymers by size exclusion chromatography. Journal of Applied
Polymer Science, 131(7), 40111-40120.
Ratnayake, W. S., Hoover, R., & Warkentin, T. (2002). Pea Starch: Composition, Structure and
Properties — A Review. Starch - Stärke, 54(6), 217–234.
Rosin, P. M., Lajolo, F. M., & Menezes, E. W. (2002). Measurement and Characterization of
Dietary Starches. Journal of Food Composition and Analysis, 15(4), 367–377.

34

Rupollo, G., Vanier, N. L., da Rosa Zavareze, E., de Oliveira, M., Pereira, J. M., Paraginski, R.
T., Elias, M. C. (2011). Pasting, morphological, thermal and crystallinity properties of
starch isolated from beans stored under different atmospheric conditions. Carbohydrate
Polymers, 86(3), 1403–1409.
Sajilata, M. G., Singhal, R. S., & Kulkarni, P. R. (2006). Resistant Starch?A Review.
Comprehensive Reviews in Food Science and Food Safety, 5(1), 1–17.
Salunkhe, D. K., & Kadam, S. S. (1983). Handbook of World Food Legumes, Volume I:
Nutritional Chemistry, Processing Technology and Utilization: Vol 1 (1st ed., pp. 1–4).
Boca Raton, Florida: CRC Press.
Sarko, A, & Wu, H.-C. H. (1978). The crystal structures of A-, B- and C-polymorphs of amylose
and starch. Starch - Stärke, 30(3), 73–78.
Sathe, S., & Salunkhe, D. (1981). Isolation, partial characterization and modification of the great
Northern bean (Phaseolus vulgaris L.) starch. Journal of Food Science, 46, 417-421.
Shimelis, E; Meaza, M; Rakshit, S. (2006). Physico-chemical properties, pasting behavior and
functional characteristics of flours and starches from improved bean (Phaseoulus vulgaris
L.) varieties grown in East Africa. Agricultural Engineering International CIGR Journal,
VIII, 1–19.
Singh, N. (2011). Functional and Physicochemical Properties of Pulse Starch. In B. K. Tiwari, A.
Gowen, & B. Mckenna (Eds.), Pulse Foods: Processing,Quality and Nutraceutical
Applications (1st ed., pp. 92–119). Elsevier Inc.
Singh, N., Nakaura, Y., Inouchi, N., & Nishinari, K. (2008). Structure and viscoelastic properties
of starches separated from different legumes. Starch/Staerke, 60(7), 349–357.
Tester, R. F., Karkalas, J., & Qi, X. (2004). Starch—composition, fine structure and architecture.
Journal of Cereal Science, 39(2), 151–165.
Tharanathan, R. ., & Mahadevamma, S. (2003). Grain legumes—a boon to human nutrition.
Trends in Food Science & Technology, 14(12), 507–518.
Thitipraphunkul, K., Uttapap, D., Piyachomkwan, K., & Takeda, Y. (2003). A comparative study
of edible canna (Canna edulis) starch from different cultivars. Part II. Molecular structure of
amylose and amylopectin. Carbohydrate Polymers, 54(4), 489–498.
Thrathnigg, B. (2000). Size-exclusion Chromatography of Polymers. In R. . Meyers (Ed.),
Encyclopedia of Analytical Chemistry (pp. 8008–8034). Chichester: John Wiley & Sons
Ltd.

35

Topping, D. L., Morell, M. K., King, R. a., Li, Z., Bird, A. R., & Noakes, M. (2003). Resistant
Starch and Health—Himalaya 292, a Novel Barley Cultivar to Deliver Benefits to
Consumers. Starch - Stärke, 55(12), 539–545.
Tovar, J., Granfeldt, Y., & Bjorck, I. M. (1992). Effect of Processing on Blood Glucose and
Insulin Responses to Starch in Legumes. Journal of Agricultural and Food Chemistry, 40,
1848–1851.
Tovar, J., & Melito, C. (1996). Steam-Cooking and Dry Heating Produce Resistant Starch in.
Journal of Agricultural and Food Chemistry, 44, 2642–2645.
Tovar, J., Melito, C., Herrera, E., Rascón, A., Pérez, E., & Pe, E. (2002). Resistant starch
formation does not parallel syneresis tendency in different starch gels. Food Chemistry,
76(4), 455–459.
Venter, C. S., & Eyssen, E. Van. (2001). More legumes for better overall health. South African
Journal of Clinical Nutrition, 14(3), 32–38.
Wang, T. L., Bogracheva, T. Y., Hedley, C. L., Centre, J. I., & Nr, N. (1998). Starch : as simple
as A , B , C ?, 49(320), 481–502.
Wolever, T. M. S., Jenkins, D. J. A., Wóng, G. S., Josse, R. G., & Thompson, L. U. (1987).
Effect of Canning on the Glycaemic Response to Beans in Patients with Type 2 Diabetes.
Canadian Institute of Food Science and Technology Journal, 20(5), 320-325
Wyatt, P. J. (1993a). Light scattering and the absolute characterization of macromolecules.
Analytica Chimica Acta, 272(1), 1–40.
Xie, J., Zhao, J., Hu, D.-J., Duan, J.-A., Tang, Y.-P., & Li, S.-P. (2012). Comparison of
Polysaccharides from Two Species of Ganoderma. Molecules, 17(1), 740–752.
Yokoyama, W., Renner-Nantz, J., & Shoemaker, C. F. (1998). Starch Molecular Mass and Size
by Size-Exclusion Chromatography in DMSO-LiBr Coupled with Multiple Angle Laser
Light Scattering. Cereal Chemistry, 75(4), 530–535.
Yoo, S. H., & Jane, J. L. (2002). Molecular weights and gyration radii of amylopectins
determined by high-performance size-exclusion chromatography equipped with multi-angle
laser-light scattering and refractive index detectors. Carbohydrate Polymers, 49(3), 307–
314.
Zhang, G., Ao, Z., & Hamaker, B. R. (2008). Nutritional property of endosperm starches from
maize mutants: a parabolic relationship between slowly digestible starch and amylopectin
fine structure. Journal of Agricultural and Food Chemistry, 56(12), 4686–94.

36

Zhang, G., & Hamaker, B. R. (2003). A three component interaction among starch, protein, and
free fatty acids revealed by pasting profiles. Journal of Agricultural and Food Chemistry,
51(9), 2797–800.
Zhong, F., Yokoyama, W., Wang, Q., & Shoemaker, C. F. (2006). Rice starch, amylopectin, and
amylose: Molecular weight and solubility in dimethyl sulfoxide-based solvents. Journal of
Agricultural and Food Chemistry, 54(6), 2320–2326.
Zhou, X., Baik, B.-K., Wang, R., & Lim, S.-T. (2010). Retrogradation of waxy and normal corn
starch gels by temperature cycling. Journal of Cereal Science, 51, 57–65.

37

CHAPTER 3
PHYSICOCHEMICAL PROPERTIES OF STARCH ISOLATED FROM SIX
VARIETIES OF PHASEOLUS VULGARIS GROWN IN MICHIGAN

38

3.1. ABSTRACT
The physicochemical characteristics of starches isolated from six varieties of Phaseolus
vulgaris grown in Michigan were determined. Starch yield varied from 22.8% to 34.1% on a
whole seed basis (~ 10% moisture content). The raw isolated bean starches exhibited high
resistant starch contents ranging from 41.9% (light red kidney bean) to 55.7% (pinto bean),
whilst total amylose content varied from 28.0% (pinto bean) to 29.8% (dark red kidney bean).
Significant differences were observed for the gelatinization transition temperatures, pasting
parameters, and resistant starch contents of the isolated dark red kidney and light red kidney bean
starches when compared to isolated starch from black, navy, pinto and, small red beans. All
isolated bean starches displayed the characteristic C-type X-ray diffraction pattern of legumes.
The crystallinity and B-type starch polymorph contents ranged from 36.1% to 24.9% and 19.6%
to 15.6% respectively. This study demonstrated that the chemical compositions of beans are
different for different varieties, and thus the varieties exhibit different physicochemical
properties.

39

3.2. INTRODUCTION
The common bean (Phaseolus vulgaris) is one of the most important group of legumes
consumed by humans globally. The genus encompasses more than 50 species and the annual
production of dry beans is approximately 24 million metric tons worldwide (Gepts, 2001). Dry
beans play an important role in the diets of many people in developing regions, by providing an
affordable source of essential nutrients, such as protein (22% of seed weight), vitamins, minerals,
and calories, in developing regions (Duranti & Gius, 1997; Geil & Anderson, 1994; Guillon &
Champ, 2002; A. Kaur et al., 2013) . Dry beans are considered healthy for all societies because
they are high in complex carbohydrates, proteins, dietary fiber, and folate while being low in fat,
and sodium (Anderson et al., 1999; Broughton et al., 2003). The inclusion of legumes in the diet
have been reported to yield other health benefits including anti-diabetic, hypocholesteremic,
antioxidative and anticancer activities (Anderson et al., 1999; Bennink, Rondini &Barrett, 2012;
(Englyst, Vinoy, Englyst, & Lang, 2003; Tharanathan & Mahadevamma, 2003).
Starch is arranged in semi-crystalline granules that vary in size, shape and molecular
structure among different and within the same plant species. Starch is comprised primarily of
two polymers, amylose (linear) and amylopectin (highly branched) (Chibbar, Ambigaipalan, &
Hoover, 2010; Hoover, Hughes, Chung, & Liu, 2010; Hughes et al., 2009; Jane, Wong, &
McPherson, 1997). The functional properties of starch depend on many factors, including but
not limited to the ratio between its two polymers, degree of crystallinity and botanical source.
Legume starches provide distinct and unique functional properties to food systems due to their
higher gelatinization temperature, greater tendency to retrograde, and greater resistant starch
content when compared to that of cereal starches (Mikulíková et al., 2008). Dry bean starch is
digested slower, has a lower glycemic index and produces more short chain fatty acids in the
40

large intestine than cereal or tuber starches (Englyst, Kingman, & Cummings, 1992; Fleming &
Vose, 1979; Shekib, 1994). These are important attributes particularly for developed countries,
where there is a high prevalence of diabetes and obesity. Due to the importance of legume
starches in the human diet, knowledge of their structure-function relationships would be of
interest in expanding their efficacy and utilization as new ingredients in the food industry (de
Almeida Costa, da Silva Queiroz-Monici, Pissini Machado Reis, & de Oliveira, 2006;
Ambigaipalan et al., 2011). Additionally, variation in starch properties among varieties of
Phaseolus vulgaris could provide valuable information for plant breeders. The objective of this
study was to determine the composition and physicochemical properties of starch isolated from
six varieties of Phaseolus vulgaris.

41

3.3. MATERIALS AND METHODS
3.3.1. Materials

Six varieties of the species Phaseolus vulgaris, black bean (Zorro), dark red kidney
(Redhawk), light red kidney bean (CELRK), small red bean (Merlot), pinto bean (P07863) and,
navy bean (Medalist) were obtained from USDA-ARS located in East Lansing, MI, USA. The
beans were harvested in 2011.

3.3.2. Starch Isolation
Starch was extracted from six varieties of Phaseolus vulgaris according to the procedure
of Hoover and Sosulski (1985) with some modifications (Figure 3.1.). Beans (450 g) were
steeped in distilled water (500 mL, 0.01% sodium metabisulfite) for 24 h at room temperature.
The swollen seeds were rinsed with distilled water and homogenized in a Waring blender for 4
min on a low setting. The homogenates were sequentially passed through 250 μm, 180 μm and
75 μm sieves. The filtrate was allowed to settle at room temperature for 18h and the supernatant
discarded. The sediment was suspended in excess 0.2% sodium hydroxide, then allowed to stand
for 4h, after which the supernatant was removed. This procedure was repeated ten times. The
final sediment was suspended in distilled water, passed through a 75 μm sieve, neutralized to pH
7.0 with hydrochloric acid (1 M), filtered and washed with distilled water. The residue was air
dried and stored at room temperature in a desiccator for further analyses. Extraction was done in
duplicate.

42

Dry beans (450 g)
Steep in H2O-0.01% Na2S2O3 (100 mL)
Rinse with H2O
Grind in blender for 4 min at low speed
Filter through 250, 180 and 75 µm sieves sequentially
Let settle at room temperature for 18 h
Discard supernatant
Suspend sediment and let settle for 4 hr in excess 0.2% NaOH
Discard supernatant
Repeat process ten times (0.2% NaOH wash)
Suspend sediment in H2O
Filter via 75 µm sieve
Neutralize with 1 M HCl
Filter and wash residue
Dry residue at room temperature
Starch powder

Figure 3.1. Wet isolation of starch from dry beans.

43

3.3.3. Proximate Analysis of Starch
Quantitative estimations of moisture, fat, and ash of isolated dry bean starches were
performed using standard AOAC Methods [925.09, 920.39, 923.03 (2000)] respectively.
Analyses were performed in duplicate, and mean values were reported.

3.3.4. Nitrogen Determination
Total nitrogen content of isolated dry bean starch was determined by the Dumas
combustion method at A & L Great Lakes Laboratories (Fort Wayne, IN) in accordance with
AOAC Method 968.06 (2000). Each of the isolated starch samples per variety was analyzed in
duplicate.

3.3.5. Trace Mineral Analysis
For each of the six varieties, isolated bean starch sample (500 mg dry weight) was placed
in a test tube and ultra-pure nitric acid (3 mL) added. The mixtures were shaken overnight in a
water bath (Dubnoff Metabolic Shaker Incubator, Precision Scientific Co., USA) for 16 h at
25°C. Following extraction, samples were placed in a digestion block (Martin Machine,
Ivesdale, IL) and incubated for 4 h at 125°C with refluxing. Following digestion, samples were
cooled for 5 min before adding hydrogen peroxide (2 mL) and incubated at 125°C for an
additional hour. This step was repeated before raising the digestion block temperature to 200°C
and this temperature was maintained until each sample was completely dry. Cooled samples
were then resuspended in 2% ultra-pure nitric acid (3 mL) and incubated overnight prior to
analysis using ICP-OES (inductively coupled plasma-optical emission spectroscopy; CIROS ICP
Model FCE12, Spectro, Kleve, Germany). To ensure batch-to-batch accuracy, all samples were

44

digested and measured alongside tomato leaf standard purchased from the National Institute of
Standards and Technology (SRM 1573A; Gaithersburg, MD). Each of the isolated starch
samples per variety was analyzed in duplicate, and mean values were reported.

3.3.6. Physicochemical Properties of Bean Starch
3.3.6.1. Total starch content
Total starch content in isolated bean starch was determined in duplicate according to the
procedure in the Total Starch Assay Kit (Megazyme International Ireland Ltd. Co., Wicklow,
Ireland). Briefly, isolated bean starch (100 mg) was placed in a glass test tube (16 mL) followed
by the addition of 0.2 mL of 80% (v/v) ethanol to aid in dispersing the sample. Thermostable αamylase (3 mL of 3,000 U/mL of Ceralpha reagent) diluted (1:30) in sodium acetate buffer (100
mM, pH 5.0) was added to the sample and the tube was incubated in a boiling water bath for 6
min with vortexing at 2, 4, and 6 min. The tube was then placed in a water bath at 50ºC and 0.1
mL of amyloglucosidase (3300 U/mL of soluble starch) was added to the tube. The isolated bean
starch sample was incubated for 30 min. Following incubation, the contents of the tube were
transferred to a volumetric flask (100 mL) and the volume adjusted with distilled water to 100
mL. The contents of the volumetric flask were then transferred to a beaker (250 mL) and 3 mL
of the contents were placed in centrifuge tube and a centrifuged at 1500 x g for 10 min at 25ºC.
An aliquot (0.1 mL) of the supernatant was pipetted to the bottom of a test tube (15 mL),
followed by addition of glucose oxidase-peroxidase-aminoantipyrine (GOPOD) reagent (3 mL)
and was then incubated in the water bath at 50oC for 20 min. A spectrophotometer (GENESYS
10S UV-Vis, Thermo Fischer Scientific Inc., USA) was used to measure the absorbance for the

45

isolated starch sample at 510 nm against the reagent blank (0.1 mL of distilled water and 3 mL of
GOPOD reagent). Total percent starch on a dry weight basis was calculated based on formulas
outlined in the Total Starch Kit. Each of the isolated starch samples per variety was analyzed in
duplicate.

3.3.6.2. Starch Damage
Megazyme Starch Damage Assay Kit (Megazyme International Ireland Ltd. Co.,
Wicklow, Ireland) based on the thermostable α-amylase and amyloglucosidase procedure was
used according to manufacturer’s instructions to determine the starch damage content for all
starch samples. During the assay damaged starch granules were first hydrated and then
hydrolyzed to maltosaccharides and α-limit dextrins by purified fungal α-amylase. The reaction
was terminated with dilute sulfuric acid and aliquots were treated with amyloglucosidase to
hydrolyze dextrins to glucose which was measured by glucose oxidase/peroxidase reagent
mixture (GOPOD). Each of the isolated starch samples per variety was analyzed in duplicate.

3.3.6.3. Pasting Properties
The pasting properties of the bean starches were determined using a Rapid Visco
Analyzer RVA-4 (Newport Scientific Pty Ltd, Warriewood, Australia). Aqueous starch slurries
(3 g starch, 25 mL distilled water) were equilibrated at 50 °C for 1 min, heated at a rate of 6
°C/min to 95°C, held at 95°C for 5 min, cooled at a rate of 6°C/min to 50°C, and held at 50°C
for 2 min. The spindle speed was 960 rpm for the first 10 s (to disperse the sample) and then 160
46

rpm for the remaining 23 min. Each of the isolated starch samples per variety was analyzed in
duplicate.

3.3.6.4. Gelatinization and Retrogradation
Gelatinization temperatures were measured and recorded on a differential scanning
calorimeter (TA Instruments, DSC Q100, DE, USA) equipped with a thermal analysis data
station. Isolated starch (5 mg, dry weight) was weighed in a hermetic aluminum DSC pan and
distilled water (20 µL) was added with a microsyringe to the DSC pan (TA Instruments,
Newcastle, DE, USA). The pan was sealed, reweighed, and allowed to stand at room
temperature for 24 h. The scanning temperature range and the heating rate were 20–120°C and
10 °C min-1, respectively. The heated pans were cooled at room temperature for 3 h and kept at
4 °C ± 2°C for 7 days. After the period of storage, samples were scanned under the same
conditions as mentioned above. The thermograms were recorded using an empty pan as
reference. The transition temperatures reported were the onset (To), peak (Tp) and conclusion
(Tc) temperatures of the gelatinization endotherm. The enthalpy of gelatinization (ΔH G) and
retrogradation (ΔH R) were determined by integrating the area between the thermogram and a
base line under the peak, and was expressed in terms of Joules per gram (J/g) of dry starch. Each
of the isolated starch samples per variety was analyzed in duplicate.

3.3.7. Resistant Starch
The resistant starch contents of duplicate isolated bean starch samples were measured
using the Resistant Starch Assay kit by Megazyme (Megazyme International Ireland Ltd. Co.,
Wicklow, Ireland) according to manufacturer’s instructions. Starch samples were incubated in a
47

shaking water bath with pancreatic α-amylase and amyloglucosidase (AMG) for 16 h at 37°C;
non-resistant starch was solubilized and hydrolyzed to D-glucose by the combined effect of both
enzymes. The reaction was terminated by the addition of an equal volume of ethanol and the
resistant starch was recovered as a pellet upon centrifugation and washed twice by suspension in
aqueous ethanol (50% v/v) followed by centrifugation and decantation of the liquid. The
resistant starch in the pellet was dissolved in KOH (2 M) by vigorously stirring in an ice-water
bath with a magnetic stirrer and neutralized with acetate buffer. The starch was quantitatively
hydrolyzed to glucose with AMG. D-glucose was measured using the GOPOD reagent, thus
giving a measure of the resistant starch content of the bean starch samples. The non-resistant
starch was determined by pooling the original supernatant and the washings, adjusting the
volume to 100 mL and measuring D-glucose content with the GOPOD reagent.

3.3.8. Amylose and Amylopectin Contents
Megazyme Amylose/Amylopectin Assay Kit (Megazyme International Ireland Ltd. Co.,
Wicklow, Ireland) based on the thermostable α-amylase and amyloglucosidase procedure was
used according to manufacturer’s instructions to determine the amylose and amylopectin content
for all starch samples. Starch samples were dispersed by heating in DMSO and starch was
precipitated in ethanol. The starch precipitate was dissolved in an acetate/salt solution;
amylopectin was specifically precipitated by the addition of the lectin concavalin A and removed
by centrifugation. The amylose in the supernatant was enzymatically hydrolyzed to D-glucose
which was measured with the GOPOD reagent. The total starch in a separate aliquot of the
acetate/salt solution was similarly hydrolyzed to D-glucose and measured using the GOPOD
reagent. The concentration of amylose in each starch sample was estimated as the ratio of
48

GOPOD absorbance at 510 nm of the supernatant of Con A precipitated sample, to that of total
starch sample. Each of the isolated starch samples per variety was analyzed in duplicate.

3.3.9. X-ray Diffraction
3.3.9.1. Crystallinity
X-ray diffractograms were obtained using an X-ray diffractometer (XDS 2000, Scintag
Inc, CA,USA) with copper K-alpha emission radiation and operating conditions of target voltage
35kV; current 35 mA; scanning range 3-35°; scan speed 0.20°/min; step time 6 s; divergence slit
width 2.00 mm; scatter slit width 4.00 mm and receiving slit width 0.200 mm. The moisture
contents of all starch samples were adjusted to approximately 11.3% (moisture contents of the
isolated starches were in this range, Table 3.1) by storage at room temperature in a dessicator
over saturated LiCl for 14 days. Crystallinity of the starches was quantitatively estimated
following the method of Nara & Komiya, (1983). The crystallinity (%) was calculated by the
equation:
Crystallinity (%) = 100 × Ac/ (Ac + Aa)
where Ac is the crystalline area on the X-ray diffractogram and Aa is the amorphous area.

3.3.9.2. A-type and B-type Polymorphic Composition
Polymorphic composition (proportion of A-type and B-type) of starch samples were
calculated using the method outlined by Davydova et al. (1995) and modified by Zhou et al.
(2004). The ‘B’ polymorph content was calculated by determining the ratio of the area under the
diffraction peak centered at 15.2º 2θ to the total crystalline area (as described above). Peaks in
diffractograms were de-convoluted with eXPFIT (Nix RM, 2010). A calibration curve was
49

prepared by mixing pure B-type starch (0–100% potato) and pure A-type (100–0% waxy corn)
starch. The area of the peak occurring at 15.2º 2θ was directly proportional to the %A
polymorph in the calibration mixtures (r = 0.988).

3.3.10. Statistical Analysis
All statistical analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC,
USA). Analyses of variance (ANOVA) were performed and the mean separations were evaluated

by Tukeys HSD test (p ˂ 0.05).

50

3.4. RESULTS AND DISCUSSION

3.4.1. Proximate Analysis
The chemical characteristics of the starches isolated from six varieties of Phaseolus
vulgaris beans are summarized in Table 3.1. The yields of starches recovered from the dry beans
varied from 22.8% (pinto bean) to 34.1% (dark red kidney bean). Isolation of pure starches from
legumes is difficult due to the presence of protein and fiber co-settling with the starch during the
extraction process (Huang et al., 2007). The disparity in yield may be due to beans before
isolation having initially having different starch contents, as well as slight variations in the
isolation process. The lipid contents of all the starch samples were less than 1% , which is not
surprising since dry beans have a low lipid content. The ash contents ranged from 0.04% to
0.08%. The low ash content suggests that the starches are relatively free from contamination by
fine fiber. The nitrogen contents (0.09–0.10%; Table 3.1) were low in all the dry bean starches.
Both dark red kidney and light red kidney bean starches yielded the highest level of phosphorus
(0.013%), while the other starches contained approximately 0.0030%. These results were all
similar to those obtained by other investigators (Ambigaipalan et al., 2011; Chung, Liu, Hoover,
Warkentin, & Vandenberg, 2008; Chung, Liu, Pauls, Fan, & Yada, 2008; Hoover & Ratnayake,
2002; Hoover & Sosulski, 1985; Wani, Sogi, Wani, Gill, & Shivhare, 2010; Zhou, Hoover, &
Liu, 2004).

51

Table 3.1. Chemical Composition of Starches from Phaseolus vulgaris (g/100g)a
Starch Source

Yield

Lipid

Ash

Moisture

Nitrogen

Phosphorus

(%)b

(%)

(%)

(%)

(%)

(%)

Black bean

26.7 ± 0.7

0.035 ± 0.0

0.064 ± 0.0

11.45 ±0.5

0.097 ± 0.00

0.0020 ± 0.00

Dark red kidney

34.1 ± 5.7

0.015 ± 0.0

0.068 ± 0.0

14.34 ± 0.2

0.101 ± 0.00

0.0130 ± 0.00

31.1 ± 1.1

0.055 ± 0.0

0.078 ± 0.0

12.37± 0.9

0.097 ± 0.00

0.0130 ± 0.00

Navy bean

28.9 ± 0.1

0.055 ± 0.0

0.051 ± 0.0

11.73 ±0.1

0.094 ± 0.00

0.0030 ± 0.00

Pinto bean

22.8 ± 2.3

0.074 ± 0.0

0.035 ± 0.0

12.47 ±0.2

0.089 ± 0.00

0.0020 ± 0.00

Small red bean

25.0 ± 4.4

0.094 ± 0.0

0.075 ± 0.0

11.52 ±0.5

0.092 ± 0.00

0.0020 ± 0.00

bean
Light red kidney
bean

a

Data represent means and standard deviations of duplicate analyses

b

Yield = (dry mass of isolated starch/ mass of beans used for isolation) x 100%

3.4.2. Total Starch, Starch Damage, Amylose Content and Resistant Starch Contents
All the isolated starch preparations had total starch contents greater than 90% (Table 3.2),
with light red kidney bean starch being the highest (96.2%). The variation in total starch results
for the isolated starches can be due to fiber (which co-settles with starch during extraction)
adhering to some bean starch samples more than others and artificially raising the total starch
measured.
The percent starch damage ranged from 0.16% to 0.29%. Starch damage affects the
susceptibility of the starch granules to α- amylolysis as well as to hydration and water binding.
As starch damage increases, the starch granules become more susceptible to digestive enzymes
52

(Chung, Lim, & Lim, 2006). In this study the values for starch damage was very low and
therefore would not have significant effect on the studied starch characteristics. Based on the
data in Tables 3.1.and 3.2., the starches isolated were relatively free from contaminants, such as
protein and fat. Legume starches have a high amylose content (24-65%) compared to that of
cereal starches (Ambigaipalan et al., 2011; Hoover & Sosulski, 1985; Singh, Singh Sandhu, &
Kaur, 2004). The amounts of amylose in the isolated bean starch samples were all around 28%
and not significantly different among the varieties studied (Table 3.2.). Others have reported
similar values for the common bean (Ambigaipalan et al., 2011; Hoover & Ratnayake, 2002).
Many methods have been developed for estimating the amylose content of various
materials. The most common methodologies utilize the iodine binding capacity of amylose.
However, amylopectin and other starch contaminants (e.g., lipids) are able to complex with
iodine causing an overestimation of the spectrophotometric measurement of amylose (Mestres,
Matencio, & Pons, 1996). The method utilized in this study was enzymatic and removed
amylopectin before measurement by complexation with concavalin A. Thus, comparison of
amylose contents reported in literature among and between legumes is difficult due to
differences in the methodologies used to determine amylose and factors affecting amylose
content (Chibbar et al., 2010).
Resistant starch (RS) refers to a portion of starch that is resistant to enzymatic hydrolysis
in the small intestine and passes into the colon to be fermented by gut microflora (Englyst et al.,
1992). In the present study pinto beans contained the greatest amount of resistant starch
(55.70%), whilst light red kidney bean had the lowest (41.92%). The resistant starch contents of
starches isolated from dark red kidney and light red kidney beans were significantly lower than
those of the other bean starches. According to Annison & Topping (1994) amylose content has

53

been known to be a factor in the formation of resistant starch. In general, starches with high
amylose contents are more resistant to enzyme hydrolysis (Li, Jiang, Campbell, Blanco, & Jane,
2008). However, in the current study amylose content was not able to account for the differences
observed in resistant starch contents of the isolated starches from the bean varieties. Differences
in amylopectin chain lengths may explain the variation in resistant starch contents. Amylopectin
with longer chain lengths are less susceptible enzyme hydrolysis due to their ability to form
double helices (Li et al., 2008).
The type of resistant starch measured in the raw bean starches was RS2, which refers to native,
ungelatinized starch granules. The RS2 content of the native bean starch is reduced once
processing conditions cause gelatinization (Bravo, Siddhuraju, & Saura-Calixto, 1998;
Ekanayake, Nair, Asp, & Jansz, 2006).

54

Table 3.2. Physicochemical Properties of Bean Starch of Six Phaseolus vulgaris Varieties
(g/100g)a,b
Starch

Total Starch

Starch Damage

Amylose

Resistant Starch

(%)

(%)

(%)

(%)

Black bean

95.96 ± 2.30 a

0.16 ± 0.01 d

28.50 ± 2.19 a

52.89 ± 0.40 b

Dark red kidney
bean

90.87 ± 1.92 a

0.25 ± 0.01 b

28.50± 1.44 a

45.20 ± 0.80 c

Light red kidney
bean

96.28 ± 1.80 a

0.29 ± 0.01 a

28.63 ± 0.87 a

41.92 ± 0.62 d

Navy bean

91.69 ± 4.51 a

0.16 ± 0.01 d

28.05 ± 1.91 a

51.93 ± 0.77 b

Pinto bean

91.99 ± 5.32 a

0.24 ± 0.01 b

28.01 ± 1.71 a

55.70 ± 0.50 a

Small red bean

92.74 ± 4.22 a

0.21 ± 0.01 c

28.26 ± 1.83 a

51.10 ± 0.80 b

a

Data represent means and standard deviations (n = 4)

Values within the same column followed by the same letter are not significantly different (P ˂ 0.05)
from each other by Tukey's HSD (honest significant difference) test
b

3.4.3. Pasting Characteristics
Pasting properties provide a picture of the functional behavior of a starch in water
undergoing heating and cooling periods (Bello-Pérez et al., 2009). Pasting temperature is a
measure of the temperature at which a starch suspension starts to thicken (Huang et al., 2007). It
also gives an indication of the minimum temperature required for cooking a specific or particular
starch. The peak viscosity indicates the water holding capacity of the starch while final viscosity
relates to the ability of the starch to form a paste or gel upon cooling (Shimelis, Meaza, Rakshit,
2006).
55

The RVA profiles of the studied bean starches are presented in Figure 3.1. and the
quantitative data are shown in Table 3.3. Bean starches from dark red kidney and light red
kidney beans had lower peak viscosities compared to starch samples from navy and pinto beans.
Pinto bean had the highest pasting temperature (79.9°C). With respect to the peak viscosity,
dark red and light red kidney bean starches had lower peak viscosities than that of navy bean
starch. Black bean starch exhibited the highest break down viscosity (1102.5 cP). High
breakdown viscosity reflects granular swelling that makes the starch granules more susceptible to
shear (Hughes et al., 2009). This indicates that dark red kidney and light red kidney bean
starches have more resistance to shear. Setback viscosity values for all bean starches ranged
between 2265.5 cP and 4349.5 cP. The faster retrogradation tendency of dark red kidney bean
and light red kidney bean starches, indicated by their high setback values, implies that they
would be favorable in food products such as gluten-free noodles (Otto, Baik, & Czuchajowska,
1997). The lower pasting temperatures, lower peak and breakdown viscosities, and high setback
viscosity observed for light red kidney and dark red kidney bean starches when compared to the
other bean starches are indicative of starches with lower amylose content (Abdel-Aal, Hucl,
Chibbar, Han, & Demeke, 2002; Chung, Liu, Peter Pauls, et al., 2008; Gupta, Bawa, & Semwal,
2009; Jane et al., 1999). However, in this present study the amount of amylose cannot account
for the differences observed since there were no significant differences in amylose content
among the starches. Generally, pasting properties of starches have been shown to be influenced
by molecular structure (such as molecular weight of amylose and amylopectin, amylopectin
chain branch length,and crystallinity) and composition (amylose/ amylopectin ratio and lipid complexed amylose) (Hughes et al., 2009; Jane et al., 1999). The high weight average molecular

56

weights of amylopectins (Table 4.3.) for dark red kidney and light red kidney bean starches may
contribute to their pasting differences when compared to those of the other bean starches.

10000

120

9000

Temperature Profile

Viscosity (cP)

7000

80

6000
5000

60

4000
40

3000

Temperature (°C)

100

8000

BB
DR
LR
NB
PB
SR

2000

20

1000
0

0
0

2

4

6

8

10

12

14

Time (min)

Figure 3.2. Pasting (Rapid Visco Analyzer) profiles of starches isolated from black (BB), dark
red kidney (DR), light red kidney (LR), navy (NB), pinto (PB) and small red (SR) beans.

57

Table 3.3. Pasting Properties of Isolated Bean Starch from Six Phaseolus vulgaris
Varietiesa,b
Starch Source

Pasting
Temperature
(ºC)

Peak Viscosity
(cP)

Breakdown
Viscosity
(cP)

Setback Viscosity

Final Viscosity

(cP)

(cP)

Black bean
76.9 ± 0.3 b

4388.7± 81.0 a

1102.5± 41.7 b

3598.2± 38.5 a,b

6884.5± 84.1 a

74.9± 0.3 c

2812.3± 213.9 b

118.3± 43.5 d

4349.5± 436.3 a

7043.5± 674.6 a

74.4± 0.6 c

2935.8± 199.8 b

162.2± 16.6 d

4109.5± 197.3 a

6883.0± 413.7 a

78.4± 0.0 b

4793.5± 51.6 a

1631.0± 79.2 a

3273.5± 101.1 a, b

6436.0± 73.5 a

79.9± 0.3 a

3245.0± 7.8 b

364.0± 38.9 c

2265.5± 33.9 b

5146.5± 13.4 a

77.9± 0.1 b
4234.3± 110.0 a
950.0± 47.4 b
Data represent means and standard deviations (n = 4)

3604.3± 100. a, b

6888.5± 162.6 a

Dark red
kidney bean
Light red
kidney bean
Navy bean
Pinto bean
Small red
bean
a

Values within the same column followed by the same letter are not significantly different (P ˂ 0.05)
from each other by Tukey's HSD (honest significant difference)test
b

3.4.4. Gelatinization and Retrogradation Properties
Differential scanning calorimetry was used to study the thermal properties of the isolated
bean starches. The gelatinization transition temperatures [To (onset), Tp (peak) and Tc
(conclusion)] and the enthalpy of gelatinization (ΔH) are presented in Table 3.4. There were
significant differences (P<0.05) in To, Tp and Tc among the studied varieties of Phaseolus
vulgaris starches. The onset gelatinization temperature (To) varied from 65.5ºC (light red kidney
bean) to 68.8ºC (small red bean). The peak transition temperatures of light red kidney and dark
red kidney bean starches were significantly different from those of the other bean starch samples.

58

A similar trend was also observed for the conclusion transition temperature as well. The smaller
transition temperatures may be a result of the low resistant starch contents of light red and dark
red kidney bean starches. Conversely, isolated pinto bean starch had the largest resistant starch
content and the highest transition temperatures when compared to those of the other starches
(Table 3.2.). Black bean starch showed the largest enthalpy of gelatinization (14.5 J/g). Higher
gelatinization temperature was an indication of more perfect crystals (Huang et al., 2007). Noda
et al. (1998) suggested that the gelatinization transition temperatures are influenced by the
molecular organization of the crystalline region, which is related to the distribution of
amylopectin short chains, and not by the proportions of crystalline region, which correspond to
the amylose/amylopectin ratio. The enthalpy of gelatinization is thought to reflect the total
crystallinity of the amylopection polymer (Tester et al., 2004; Tester & Morrison, 1990).
Conversely, Cooke & Gidley (1992), proposed that the enthalpy of gelatinization does not show
the loss of crystallinity but indicates the loss of the double helical order. Isolated pinto bean
starch had the lowest degree of crystallinity (Table 3.5.). The retrogradation properties of bean
starches are summarized in Table 3 4. The To, Tp and Tc values of retrograded starches were
lower than those obtained for the initial gelatinization. Light red kidney and pinto bean starches
presented the lowest To values and the bean starch isolated from black bean showed the highest
Tp. Transition temperatures for all the isolated bean starches were not affected by the amount of
amylose present (Table 3.2.).
For retrograded starch, the enthalpy value provides a measure of the energy
transformation taking place during the melting of the recrystallized amylopectin chains. Storing
gels at lower temperatures results in the formation of starch crystals that melt at lower
temperatures. Due to the limited dimensions of the amylopectin chains, the stability of
59

amylopectin crystallites is lower than that of amylose. Recrystallized amylopectin melts in the
range 40-100ºC while amylose is much higher (120-170ºC) (Liu, 2005). Starch samples having
amylopectin with longer chains tend to retrograde faster than those with shorter chains (Jane et
al., 1999).

60

Table 3.4. Gelatinization and Retrogradation Parameters for Isolated Bean Starches a
Starch Source

To d

Tp

Tc

ΔH (J/g)

Raw b
Black bean

67.0 ± 0.1 a,b

74.8 ± 0.0 a

87.2 ± 0.1 a

14.5 ± 0.3 a

Dark red kidney
bean

66.4 ± 0.3 b,c

72.3 ± 0.4 b

83.6 ± 0.3 c

13.6 ± 0.3 a,b

Light red kidney
bean

65.5 ± 0.0 c

71.7 ± 0.1 b

83.0 ± 0.4 c

12.4 ± 0.2 d,c

Navy bean

67.6 ± 0.3 a,b

75.7 ± 0.1 a

86.5 ± 0.6 a, b

11.9 ± 0.0 d

Pinto bean

67.4 ± 0.1 a,b

75.3 ± 0.2 a

87.2 ± 0.3 a

13.1± 0.0 b,c

Small red bean

68.0 ± 0.1 a

75.4± 0.1a

86.2 ± 0.8 b

13.3 ± 0.1 b,c

Black bean

47.7 ± 0.4 a

61.3 ± 0.4 a

74.5 ± 0.1 a

6.1 ± 0.2 a

Dark red kidney
bean

47.3 ± 0.1 a

59.4 ± 0.3 b

72.3 ± 0.4 b,c

5.1 ± 0.1 b

Light red kidney
bean

46.2 ± 0.4 b

57.6 ± 0.2 c

71.4 ± 0.3 c

6.0 ± 0.6 a

Navy bean

47.4± 0.1 a

58.6 ± 0.0 b

72.5 ± 0.4 b,c

5.2 ± 0.0 b

Pinto bean

46.9 ± 0.7 a,b

58.6 ± 0.3 b

73.6 ± 0.0 a,b

6.2 ± 0.1 a

Small red bean

47.3 ± 0.1 a

58.6 ± 0.4 b

72.3 ± 0.3 b,c

5.9 ± 0.3 a

Retrograded c

a

Data represent means and standard deviations (n = 2).

b

Values within the same column for raw samples followed by the same letter are not significantly
different (P ˂ 0.05) from each other by Tukey's HSD (honest significant difference) test.
c

Values within the same column for retrograded samples followed by the same letter are not significantly
different (P ˂ 0.05) from each other by Tukey's HSD test.
d

To: onset temperature, Tp: peak temperature, Tc: conclusion temperature, ΔH: enthalpy change.

61

3.4.5. Crystallinity
The proportion of starch present in the crystalline form was the greatest for navy bean
starch (36.06%), whereas, pinto bean starch had the lowest value (24.86%) (Table 3.5.). Similar
values of crystallinity have been reported for legume starches (black bean starch, 32.1% and
pinto bean starch 33% at a moisture content of 19%) (Zhou et al., 2004). Ambigaipalan et
al.(2011) studied the structure of faba bean, black bean and pinto bean cultivar starches and
crystallinity ranged from 20.3% to 23.1%. Isolated pinto bean starch with its low crystallinity
had the largest resistant starch content (55.86%) (Table 3.2.). However, it should be noted that
the degree of crystallinity is not always linked with the resistance of starch has to the activity of
amylases (Jane et al., 1999). For example, native potato starch has a high degree of crystallinity
and is very resistant to amylases whereas, cereal starches are characterized by a high degree of
crystallinity but are more susceptible than potato starch to enzymatic activity (Englyst, 2004;
Jane et al., 1999).
Crystallinity differences among legume starches is attributed to or influenced by: (1)
crystallite size, (2) number of crystallites that are arranged in a crystal array (3) moisture content
and (4) proportions of A-type and B-type polymorphs. Crystallites are microscopic crystalline
regions found within a solid polymer below the crystalline melting temperature (Hoover et al.,
2010a). Some investigators calculate the B-type polymorph based on an X-ray diffraction peak
centered at 5.2° 2θ (Davydova et al., 1995). This peak was minimal or non-existent in the X-ray
diffractograms of the starches in this study. This could be attributed to the moisture content
(11.3%) of the starch samples. Zimeri and Kokin (2002) found that pre-solubilized inulin's
crystallinity depended on moisture content and that crystallinity was low at low moisture
contents. The A-type polymorph contents of the starches used in this study was determined from
62

a calibration curve derived from mixtures of pure B-type (0–100% potato) and pure A-type
(100–0% waxy corn). An X-ray diffraction peak centered at 15.2º 2θ was directly proportional
to the amount of A-type polymorph in the sample (r = 0.988). The amount of B-type polymorph
ranged from 15.22 % to 19.59% (Table 3.5). Ambigaipalan et al. (2011) reported that the B-type
polymorph contents for faba bean starch (20.6-25.0%), pinto bean starch (21.3-23.1%) and black
bean starch (15.4-17.9%). These B-type polymorph values are comparable to the values
determined in this study. All dry bean starches show a characteristic C-type X-ray diffraction
pattern which is a mixture of A and B-type crystalline structures (Chibbar et al., 2010;
Davydova, Leont, Genin, Sasovc, & Bograchevap, 1995; Hoover & Ratnayake, 2002; Hoover &
Sosulski, 1985a; Pérez, Baldwin, & Gallant, 2009; Zhou et al., 2004). X-ray diffraction pattern
may depend on starch origin as well as environmental growth conditions (Hoover et al., 2010).

63

Table 3.5. Crystallinity and B-type Polymorph Composition of Starch Isolated from Dry
Beansa
Starch Source

Crystallinity (%)

B-type Polymorph (%)

Black bean

26.79 ± 3.5 b,c

15.22 ± 0.16 b

Dark red kidney bean

25.35 ± 0.030 b,c

17.36 ± 0.050 a,b

Light red kidney bean

27.94 ± 1.4 b,c

17.04 ± 2.6 a,b

Navy bean

36.06 ± 3.6 a

19.09 ± 0.60 a

Pinto bean

24.86 ± 0.50 c

17.25 ± 1.4 a,b

Small red bean

31.17 ± 1.8 a,b

19.59 ± 0.22 a

Values followed by the same letter within a column are not significantly different (P ˂ 0.05) from each
other by Tukey's HSD (honest significant difference) test.
a

3.5. CONCLUSIONS
The findings of the current study showed that the physicochemical properties of starches
isolated from dark red kidney and light red kidney beans differed from those of the other isolated
bean starches. Consequently, dark red kidney and light red kidney bean starches may have
different end-use applications than the other common bean starches. Dark red kidney and light
red kidney bean starches were found to have lower resistant starch contents, lower onset, peak,
and conclusion gelatinization temperatures, lower pasting temperatures, lower peak viscosities,
lower breakdown viscosities and higher setback viscosities, when compared to starches isolated
from black, navy, pinto and small red beans. C-type X-ray diffraction patterns were found for
the isolated bean starches of all six studied varieties. Starch from pinto beans displayed the
highest resistant starch value (55.7%) and had the lowest degree of crystallinity (24.9%). No
64

significant differences were observed in amylose contents among the bean starches. Therefore,
amylose content did not contribute to the differences observed in the physicochemical properties
of the isolated starch samples studied.

65

APPENDICES

66

APPENDIX A
X-RAY DIFFRACTION STANDARD CURVE USED IN THE DETERMINATION OF THE
POLYMORPHIC COMPOSITION OF ISOLATED NATIVE STARCH

A calibration curve was prepared by mixing pure B-type starch (0–100% potato)
and pure A-type (100–0% waxy corn). X-ray diffractograms were obtained using an X-ray
diffractometer (XDS 2000, Scintag Inc, CA,USA) with copper K-alpha emission radiation and
operating conditions of target voltage 35kV; current 35 mA; scanning range 3-35°; scan speed
0.20°/min; step time 6 s; divergence slit width 2.00 mm; scatter slit width 4.00 mm and receiving
slit width 0.200 mm. The moisture contents of all starch samples were adjusted to approximately
11.3% (moisture contents of the isolated starches were in this range, Table 3.1.) by storage at
room temperature in a desiccator over saturated LiCl for 14 days. X-ray diffractograms used to
generate the calibration curve are displayed in Figure 1-A.

67

700

0%W-100%P

600
Intensity

500
400
300
200
100
0
0

10

20

30

40

Intensity

Diffraction angle (2θ)

10%W-90%P

1200
1000
800
600
400
200
0
0

10

20

30

40

Intensity

Diffraction angle (2θ)

800
700
600
500
400
300
200
100
0

25%W-75%P

0

10

20

30

40

Diffraction angle (2θ)

Figure 1-A. X-ray diffractograms of B-type starch (0–100% potato, P) and pure A-type
(100–0% waxy corn, W)

68

Figure 1-A. (cont'd).
1000

Intensity

800

50%W-50%P

600
400
200
0
0

10

20

30

40

Diffraction angle (2θ)

1000

75%W-25%P

Intensity

800
600
400
200
0
0

10

20

30

40

Diffraction angle (2θ)

1200

Intensity

1000

90%W-10%P

800
600
400
200
0
0

10

20

30

Diffraction angle (2θ)

69

40

Figure 1-A. (cont'd).
1200

100%W-0%P

Intensity

1000
800
600
400
200
0
0

10

20

30

Diffraction angle (2θ)

70

40

APPENDIX B

PASTING PROPERTIES OF ISOLATED NATIVE BEAN STARCHES

Table 1-B. Pasting Properties of Isolated Native Bean Starchesa,b
Names

BB1
BB1
BB2
BB 2
DR1
DR1
DR2
DR2
LR1
LR1
LR2
LR2
NB1
NB1
NB2
NB2
PB1
PB1
PB2
PB2
SR1
SR1
SR2
SR2

Peak
Viscosity
(RVU)
369.0833
378.8333
362.1667
352.8333
242.0833
280.4167
213.5000
201.4167
318.6667
194.1667
180.7500
285.0000
396.4167
402.5000
529.0000
455.5833
285.5833
285.0000
254.9167
256.1667
347.9167
362.2500
356.4167
344.8333

Trough
Viscosity
(RVU)
269.5833
288.0833
274.0833
263.6667
231.1667
277.5000
199.5833
189.7500
306.3333
181.4167
167.5833
269.1667
265.1667
261.9167
357.2500
310.0833
252.6667
245.2500
231.7500
230.6667
265.1667
273.3333
281.4167
274.8333

Breakdown
Viscosity
(RVU)
99.5000
90.7500
88.0833
89.1667
10.9167
2.9167
13.9167
11.6667
12.3333
12.7500
13.1667
15.8333
131.2500
140.5833
171.7500
145.5000
32.9167
39.7500
23.1667
25.5000
82.7500
88.9167
75.0000
70.0000

Final
Viscosity
(RVU)
585.0000
596.4167
560.9167
552.5000
636.3333
764.3333
489.0833
458.0833
762.0000
433.9167
412.6667
685.7500
532.0000
540.6667
715.6667
612.5833
457.2500
456.5000
402.0833
399.6667
574.6667
604.9167
562.3333
554.2500

a

Setback
Viscosity
(RVU)
315.4167
308.3333
286.8333
288.8333
405.1667
486.8333
289.5000
268.3333
455.6667
252.5000
245.0833
416.5833
266.8333
278.7500
358.4167
302.5000
204.5833
211.2500
170.3333
169.0000
309.5000
331.5833
280.9167
279.4167

Peak
Time
(min)
4.5996
4.5996
4.5996
4.5330
5.4663
6.9996
4.8663
4.7330
4.5996
6.2663
5.5996
4.4663
4.6663
4.5996
4.3330
4.3996
5.1996
5.0663
5.3330
5.3996
4.5996
4.5996
4.7330
4.7996

Pasting
Temperature
(°C)
76.7500
76.7000
76.7500
77.5500
75.1500
75.1000
75.1000
74.3500
73.5000
75.2000
74.3500
74.4000
78.3500
78.4000
77.5500
77.5500
79.2500
80.0500
80.0500
80.1000
77.5500
77.4500
78.4000
78.3500

RVU: rapid visco units
BB: black bean, DR: dark red kidney bean; LR: light red kidney bean; NB: navy bean; PB: pinto bean;
SR: small red bean
b

71

LITERATURE CITED

72

LITERATURE CITED

Abdel-Aal, E. S. M., Hucl, P., Chibbar, R., Han, H. L., & Demeke, T. (2002). Physicochemical
and Structural Characteristics of Flours and Starches from Waxy and Nonwaxy Wheats.
Cereal Chemistry, 79(3), 458–464.
Ambigaipalan, P., Hoover, R., Donner, E., Liu, Q., Jaiswal, S., Chibbar, R., … Seetharaman, K.
(2011a). Structure of faba bean, black bean and pinto bean starches at different levels of
granule organization and their physicochemical properties. Food Research International,
44(9), 2962–2974.
Ambigaipalan, P., Hoover, R., Donner, E., Liu, Q., Jaiswal, S., Chibbar, R., Seetharaman, K.
(2011b). Structure of faba bean, black bean and pinto bean starches at different levels of
granule organization and their physicochemical properties. Food Research International,
44(9), 2962–2974.
Anderson, J. W., Smith, B. M., & Washnock, C. S. (1999). Cardiovascular and renal benefits of
dry bean and soybean intake. The American Journal of Clinical Nutrition, 70(3 Suppl),
464S–474S.
Annison, G., & Topping, D. L. (1994). Nutritional role of resistant starch : Chemical structure vs
physiological function primary structure of starch components. Annual Review of Nutrition,
(35), 297–320.
AOAC. (2000). Official Methods of Analysis. (A. Horwitz, Ed.) (17th ed.). Gaithersburg, MD:
Association of Official Analytical Communities International.
Bello-Pérez, L. A., Rodriguez-Ambriz, S., Sanchez-Rivera, M., & Agama-Acevedo, E. (2009).
Starch Molecular Structure. In A. Bertolini (Ed.), Starches: Characterization, Properties,
and Applications (1st ed., pp. 33–57). CRC Press.
Bennink, M., Rondini, E., & Barrett, K. (2012). Nutrition and human health benefits of dry beans
and pulses. In M. Siddiq & M. Uebersax (Eds.), Dry beans and pulses: production,
processing and nutrition (pp. 335–358). Oxford, UK.: Blackwell Publishing Ltd.
Bravo, L., Siddhuraju, P., & Saura-Calixto, F. (1998). Effect of Various Processing Methods on
the in Vitro Starch Digestibility and Resistant Starch Content of Indian Pulses. Journal of
Agricultural and Food Chemistry, 46(11), 4667–4674. Broughton, W., Hernandez, G., &
Blair, M. (2003). Beans (Phaseolus spp.)–model food legumes. Plant and Soil, 252, 55–128.
Chibbar, R. N., Ambigaipalan, P., & Hoover, R. (2010). Review: Molecular Diversity in Pulse
Seed Starch and Complex Carbohydrates and Its Role in Human Nutrition and Health.
Cereal Chemistry, 87(4), 342–352.

73

Chung, H.-J., Lim, H. S., & Lim, S.-T. (2006). Effect of partial gelatinization and retrogradation
on the enzymatic digestion of waxy rice starch. Journal of Cereal Science, 43(3), 353–359.
Chung, H.-J., Liu, Q., Hoover, R., Warkentin, T. D., & Vandenberg, B. (2008). In vitro starch
digestibility, expected glycemic index, and thermal and pasting properties of flours from
pea, lentil and chickpea cultivars. Food Chemistry, 111(2), 316–321.
Chung, H.-J., Liu, Q., Peter, P, K., Fan, M. Z., & Yada, R. (2008). In vitro starch digestibility,
expected glycemic index and some physicochemical properties of starch and flour from
common bean (Phaseolus vulgaris L.) varieties grown in Canada. Food Research
International, 41, 869-875.
Cooke, D., & Gidley, M. J. (1992). Loss of crystalline and molecular order during starch
gelatinisation: origin of the enthalpic transition. Carbohydrate Research, 227, 103–112.
Davydova, N.-, Leont, S. P., Genin, Y. V, Sasovc, A. Y., & Bograchevap, T. Y. (1995a). Some
physico-chemical properties of smooth pea starches. Carbohydrate Polymers, 27, 109–115.
De Almeida Costa, G. E., da Silva Queiroz-Monici, K., Pissini Machado Reis, S. M., & de
Oliveira, A. C. (2006). Chemical composition, dietary fibre and resistant starch contents of
raw and cooked pea, common bean, chickpea and lentil legumes. Food Chemistry, 94(3),
327–330.
Duranti, M., & Gius, C. (1997). Legume seeds: protein content and nutritional value. Field
Crops Research, 53(1-3), 31–45.
Ekanayake, S., Nair, B. M., Asp, N.-G., & Jansz, E. R. (2006). Effect of Processing of Sword
Beans (Canavalia gladiata) on Physicochemical Properties of Starch. Starch - Stärke, 58(5),
215–222.
Englyst, E. (2004). Resistant starch- Nutritional and biological activity. Polish Journal of Food
and Nutrition Sciences, 13, 51–64.
Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of
nutritionally important starch fractions. European Journal of Clinical Nutrition, 46(2), S33–
50.
Englyst, K. N., Vinoy, S., Englyst, H. N., & Lang, V. (2003). Glycaemic index of cereal products
explained by their content of rapidly and slowly available glucose. The British Journal of
Nutrition, 89(3), 329–40.
Fleming, S. E., & Vose, J. R. (1979). Digestibility of raw and cooked starches from legume seeds
using the laboratory rat. The Journal of Nutrition, 109(12), 2067–75.
Geil, P. B., & Anderson, J. W. (1994). Nutrition and health implications of dry beans: a review.
Journal of the American College of Nutrition, 13(6), 549–58.
74

Gepts, P. (2001). Phaseolus vulgaris. In Encyclopedia of genetics (pp. 1444–1445). Academic
Press.
Guillon, F., & Champ, M. M.-J. (2002). Carbohydrate fractions of legumes: uses in human
nutrition and potential for health. The British Journal of Nutrition, 88 Suppl 3, S293–306.
Gupta, M., Bawa, A. S., & Semwal, A. D. (2009). Morphological, Thermal, Pasting, and
Rheological Properties of Barley Starch and Their Blends. International Journal of Food
Properties, 12(3), 587–604.
Hoover, R., Hughes, T., Chung, H. J., & Liu, Q. (2010a). Composition, molecular structure,
properties, and modification of pulse starches: A review. Food Research International,
43(2), 399–413.
Hoover, R., Hughes, T., Chung, H. J., & Liu, Q. (2010b). Composition, molecular structure,
properties, and modification of pulse starches: A review. Food Research International,
43(2), 399–413.
Hoover, R., & Ratnayake, W. S. (2002a). Starch characteristics of black bean, chick pea, lentil,
navy bean and pinto bean cultivars grown in Canada. Food Chemistry, 78(4), 489–498.
Hoover, R., & Sosulski, F. (1985a). Studies on the Functional Characteristics and Digestibility of
Starches from Phaseolus vulgaris Biotypes. Starch - Stärke, 37(6), 181–191.
Huang, J., Schols, H. a., van Soest, J. J. G., Jin, Z., Sulmann, E., & Voragen, A. G. J. (2007).
Physicochemical properties and amylopectin chain profiles of cowpea, chickpea and yellow
pea starches. Food Chemistry, 101(4), 1338–1345.
Hughes, T., Hoover, R., Liu, Q., Donner, E., Chibbar, R., & Jaiswal, S. (2009). Composition,
morphology, molecular structure, and physicochemical properties of starches from newly
released chickpea (Cicer arietinum L.) cultivars grown in Canada. Food Research
International, 42(5-6), 627–635.
Jane, J., Chen, Y., Lee, L., McPherson, E., Wong, K., Radosavlijevic, M., & Kasemsuwan, T.
(1999). Effects of amylopectin branch chain length and amylose content on the
gelatinization and pasting properties of starch. Cereal Chemistry, 76, 629–637.
Jane, J. L., Wong, K. S., & McPherson, A. E. (1997). Branch-structure difference in starches of
A and B-type x-ray patterns revealed by their naegeli dextrins. Carbohydrate Research,
300(3), 219–227.
Kaur, A., Kaur, P., Singh, N., Virdi, A. S., Singh, P., & Rana, J. C. (2013). Grains, starch and
protein characteristics of rice bean (Vigna umbellata) grown in Indian Himalaya regions.
Food Research International, 54(1), 102–110.

75

Liu, Q. (2005). Understanding Starches and their Role in Foods. In S. W. Cui (Ed.), Food
Carbohydrates: Chemistry, Physical Properties and Applications (1st ed., pp. 310–355).
Taylor and Francis Group, LLC.
Mestres, C., Matencio, F., & Pons, B. (1996). A Rapid Method for the Determination of
Amylose Content by Using Differential Scanning Calorimetry. Starch - Stärke, 48, 2–6.
Mikulíková, D., Masár, S., & Kraic, J. (2008). Biodiversity of Legume Health-promoting Starch.
Starch - Stärke, 60(8), 426–432.
Nara, S., & Komiya, T. (1983). Studies on the Relationship Between Water-satured State and
Crystallinity by the Diffraction Method for Moistened Potato Starch. Starch - Stärke,
35(12), 407–410.
Noda, T., Takahata, Y., Sato, T., Suda, I., Morishita, T., Ishiguro, K., & Yamakawa, O. (1998).
Relationships between chain length distribution of amylopectin and gelatinization properties
within the same botanical origin for sweet potato and buckwheat. Carbohydrate Polymers,
37(2), 153–158.
Otto, T., Baik, B.-K., & Czuchajowska, Z. (1997). Wet Fractionation of Garbanzo Bean and Pea
Flours. Cereal Chemistry, 74(2), 141–146.
Pérez, S., Baldwin, P. M., & Gallant, D. J. (2009). Starch. Starch (Third Edit., pp. 149–192).
Elsevier.
Shekib, L. A. (1994). In-vitro digestibility and microscopic appearance of germinated legume
starches and their effect on dietary protein utilization. Food Chemistry, 50(1), 59–63.
Shimelis, E; Meaza, M; Rakshit, S. (2006). Physico-chemical properties, pasting behavior and
functional characteristics of flours and starches from improved bean (Phaseoulus vulgaris
L.) varieties grown in East Africa. Agricultural Engineering International CIGR Journal,
VIII, 1–19.
Singh, N., Singh Sandhu, K., & Kaur, M. (2004). Characterization of starches separated from
Indian chickpea (Cicer arietinum L.) cultivars. Journal of Food Engineering, 63(4), 441–
449.
Tester, R., Karkalas, J., & Qi, X. (2004). Starch—composition, fine structure and architecture.
Journal of Cereal Science, 39,151-165.
Tester, R., & Morrison, W. (1990). Swelling and gelatinization of cereal starches. I. Effects of
amylopectin, amylose, and lipids. Cereal Chemistry, 67,558-563.
Tharanathan, R. ., & Mahadevamma, S. (2003). Grain legumes—a boon to human nutrition.
Trends in Food Science & Technology, 14(12), 507–518.

76

Wani, I., Sogi, D., Wani, A., Gill, B., & Shivhare, U. (2010). Physico-chemical properties of
starches from Indian kidney bean (Phaseolus vulgaris) cultivars. International Journal of
Food Science and Technology, 45, 2176–2185.
Zhou, Y., Hoover, R., & Liu, Q. (2004). Relationship between ?-amylase degradation and the
structure and physicochemical properties of legume starches. Carbohydrate Polymers,
57(3), 299–317.
Zimeri, J. (2002). The effect of moisture content on the crystallinity and glass transition
temperature of inulin. Carbohydrate Polymers, 48(3), 299–304.

77

CHAPTER 4
EFFECTS OF DIFFERENT COOKING METHODS ON THE DIGESTIBILITY AND
MOLECULAR WEIGHT DISTRIBUTION OF ISOLATED BEAN (PHASEOLUS
VULGARIS) STARCHES

78

4.1. ABSTRACT
The weight average molecular weight distribution (Mw) of starches isolated from native,
canned, and stovetop-cooked beans were analyzed using high-performance size exclusion
chromatography with multi-angle laser light scattering and refractive index detectors (HPSECMALLS-RI). Results revealed that amylose of isolated native dark red kidney bean starch had
the smallest Mw (1.0 x 106 g/mol), whereas isolated native pinto bean starch had the largest
value of Mw (1.8 x 106 g/mol). The Mw values of amylopectin for isolated native bean starch
ranged from 2.4 x 107 g/mol to 3.9 x 107 g/mol. Isolated canned and stovetop-cooked bean
starches displayed a mono-modal Mw distribution, with a reduction in high molecular weight
fractions, whereas isolated native bean starch Mw distribution was bi-modal. Results of in vitro
α-amylase starch hydrolysis showed ranges of rapidly digestible starch (RDS) (1.95-2.71%),
slowly digestible starch (SDS) (14.36-17.39%), and resistant starch (RS) (78.95-83.7%) among
the tested isolated native Phaseolus vulgaris starches. However, RDS, SDS, and RS fractions in
isolated canned bean starches ranged from 7.58-13.21%, 20.75-24.90%, and 63.17- 68.54%,
respectively. Isolated stovetop-cooked bean starches yielded similar results: RDS, 7.37-10.61%;
SDS, 22.55-26.84%; and RS, 62.55-68.59%). The hydrolysis indices (HI) and estimated
glycemic indices (GI) were marginally greater for isolated canned bean starches than for
stovetop-cooked starches.

79

4.2. INTRODUCTION
The common bean (Phaseolus vulgaris) is one of the main leguminous plants consumed
by humans. Dry beans are rich in proteins, carbohydrates, minerals and dietary fiber. The
consumption of dry beans has been linked to reduced risk of diabetes, obesity and heart disease
(Anderson et al., 1999). The major carbohydrate found in beans is starch.
Starch is composed of two distinct types of macromolecules: amylose and amylopectin.
Amylose is essentially linear with α–1,4 glycosidic linkages, whereas amylopectin is highly
branched with short α–1,4 linked glucose chains and 5-6% non-randomly distributed α–1,6
glycosidic linkages. Amylose has an average molecular weight of approximately 1 x 105 to 1 x
106 g/mol, with a degree of polymerization by number (DPn) in the range of 324-4920.
Conversely, amylopectin is a much larger molecule than amylose with a molecular weight of 1 x
107 to 1 x 109 g/mol and DPn of 9600-15900 (Sajilata, Singhal, & Kulkarni, 2006; Tester,
Karkalas, & Qi, 2004; Wang, Bogracheva & Hedley, 1998). The unit chains present in
amylopectins (18-25 units) are shorter than those found in amylose molecules (Tester et al.,
2004). The amylose and amylopectin contents of normal starches are within the ranges of 2030% and 70-80%, respectively (Gidley et al., 2010; Han & Lim, 2004; Hasjim, Lavau, Gidley, &
Gilbert, 2010a; Yoo & Jane, 2002). The functionality of starch depends to a great extent on the
molecular structure, size and weight of these components (Othman et al., 2010). Starch
molecular weight is often influenced by botanical source, starch isolation and dissolution
procedures, as well as the technique used to determine its molecular weight.
In order for starch to be accurately characterized, the macromolecule needs to be
completely dissolved in the solvent of choice. Dissolution is usually accomplished with
dimethyl sulfoxide (DMSO) or aqueous DMSO solutions, sometimes with the addition of salts
80

such as LiBr and NaNO3 (Yokoyama, Renner-Nantz, & Shoemaker, 1998; Othman, Al-Assaf, &
Hassan, 2010; Cave, Seabrook, Gidley, & Gilbert, 2009). Several techniques have been used for
the separation and structural characterization of starch including ultracentrifugation, sizeexclusion chromatography and light scattering (Wyatt, 1993; Bello-Pérez, Rodriguez-Ambriz,
Sanchez-Rivera, & Agama-Acevedo, 2009; Ratnayake, Hoover, & Warkentin, 2002; Yoo &
Jane, 2002). Size-exclusion chromatography (SEC), which utilizes calibration standards is
commonly used to determine the weight average molecular weight (Mw) of starch. Recently,
high-performance-size-exclusion chromatography (HPSEC) equipped with multi-angle laser
light scattering (MALLS) and refractive index (RI) detectors has been used to determine Mw of
polymers. This method provides absolute molecular weight without the need for calibration
standards (Podzimek, 2014; Syahariza, Sar, Hasjim, Tizzotti, & Gilbert, 2013; Witt, Gidley, &
Gilbert, 2010; Yoo & Jane, 2002).
Starch can be classified into rapidly digestible starch (RDS), slowly digestible starch
(SDS), and resistant starch (RS) according to the rate of glucose release through enzymatic
action and its absorption in the gastrointestinal tract (Englyst, Kingman, & Cummings, 1992).
RDS is the starch fraction that is digested within the first 20 min of incubation with α-amylase
and amyloglucosidase. It is also the portion of starch that causes a sudden increase in blood
glucose level after ingestion. SDS is the starch fraction that is digested completely, but slowly,
in the small intestine and is designated as the portion of starch digested from 20 to 120 min of
incubation, RS is the fraction of starch remaining after 120 min of in vitro enzymatic digestion
and is fermented in vivo by bacteria in the large intestine (Englyst, 2004; Englyst et al., 1992;
Englyst & Englyst, 2005).

81

Dietary starch varies greatly in digestibility and studies have shown that legume starches
are more resistant to digestion than cereal starches (Englyst, Kingman, & Cummings, 1992).
The type and extent of processing treatment can mainly affect the digestibility of starch by
influencing its gelatinization and retrogradation (Annison & Topping, 1994; Sajilata et al., 2006).
When starch is fully gelatinized and dispersed, the starch becomes easily digestible. However as
the gel cools and ages, the polymers once more form a partially crystalline structure which is
able to resist digestion (Muir & O’Dea, 1992). The structure formed is known as retrograded
resistant starch (Roger & Colonna, 1992). Amylose content, degree of crystallinity, and the
presence of other components interacting with starch (amylose-lipid complexes) are also able to
influence the digestibility of starch. High amylose starches (containing 70% amylose) and
starches with a high degree of crystallinity limit enzymatic starch hydrolysis. (Colonna et al.,
1992; Du et al., 2014; Jane et al., 1999).
Beans must be processed (such as by roasting, boiling and canning) prior to consumption
in order to improve their palatability. These different heat treatments can cause alterations in the
bean structure and also influence its nutritional characteristics (Kaur & Sandhu, 2010; Rehman &
Shah, 2005). The heating of starch granules, or dry beans in moderate or excess water, such as
during normal home cooking or canning, causes starch to gelatinize. This results in the rupture
and disintegration of the semi-crystalline granular structure of starch via the disruptions of interand intra-molecular hydrogen bonds of starch molecules (Tester & Morrison, 1990a, 1990b).
Gelatinization increases the susceptibility of starch to digestive enzymes, as the swelling of
starch granules increases the accessibility of enzymes to penetrate into the granules. Although
cooking or another heat treatment gelatinizes granular starch, it is noted that starch granules in
cooked bean seeds may be trapped in the protein and cell-wall matrices, inhibiting both the

82

swelling of starch granules and the solubility of starch molecules during cooking. When the rate
of starch digestion is decreased in the human digestive tract, postprandial glucose and insulin
responses are reduced and/or delayed (Thorne et al., 1983). Determination of starch digestibility
and resistant starch, content of food ingredients and processed foods will provide nutritional
information to consumers and others (Perera, Meda, & Tyler, 2010; Roger & Colonna, 1992).
The objective of this study is to determine the effect of canning and traditional cooking on the in
vitro enzymatic digestibility and molecular weight distributions of isolated bean starches.

83

4.3. MATERIALS AND METHODS
4.3.1. Materials

Six varieties of the species Phaseolus vulgaris, black bean (Zorro), dark red kidney
(Redhawk), light red kidney bean (CELRK), small red bean (Merlot), pinto bean (P07863), and
navy bean (Medalist) were obtained from the USDA-ADRS located in East Lansing, MI, USA.
The beans grown in Michigan were harvested in two growing seasons, 2011 and 2012 for each
bean variety.

4.3.2. Stovetop-Cooking of Dry Beans
The cooking of the beans was carried out in a traditional manner done in many
households. Briefly, dried beans (1.5 kg) was soaked in distilled water (3000 mL) overnight at
room temperature. The water was discarded and the soaked beans were rinsed with distilled
water. The beans were then placed in a 5-quart stainless steel pot, distilled water was added
(3000 mL) and the beans were brought to a boil (100°C)for 15 min on a Kenmore electric stove.
The heat was reduced and the beans were allowed to simmer gently (using the simmer option on
the stove) for 1h. The cooked beans were allowed to cool to room temperature (approximately
25°C). The cooked beans were stored covered in their cooking water at room temperature
(25°C). After two days, starch was extracted from the beans.

4.3.3. Canning of Dried Beans
Dried beans (3 kg) were soaked in distilled water (6000 mL) for 14h. The water was
discarded and the beans were rinsed with distilled water. Approximately 450 g amounts of
soaked beans were placed in cans (No. 3), hot distilled water (90°C) was added to just cover the
84

top of the beans, lids were placed on top and the cans were sealed with a Dixie Double Seamer
(Athens, Georgia, USA). The sealed cans were then placed in a basket in an FMC retort (FMC
Corporation, Food Processing Systems Division, Madera, CA) that heated the cans to 125°C for
15 min and subsequently cooled them to 38°C for over 35 min. The canned beans were stored at
room temperature (25°C) for two days before starch extraction. For each variety, starch was
extracted from the combined contents of all the cans of that variety.

4.3.4. Starch Extraction
Starch was isolated from native, canned, and stovetop-cooked beans according to the
method of Hoover & Sosulski (1985). Air-dried isolated starch samples were stored in a
desiccator at room temperature for future analyses.

4.3.5. Moisture Content
Quantitative estimations of moisture of isolated native, canned and stovetop-cooked bean
starches were performed using standard[ AOAC Method 925.09 (2000)]. Analyses were
performed in duplicate, and mean values were reported

4.3.6. Starch Digestibilty
Starch digestibility was analyzed according to the method described by Englyst et al.
(1992) with modifications. For each of the six varieties, isolated bean starch (0.5 g, w/w) and 0.1
M sodium acetate buffer (pH 5.2, 20 mL) were added to a 50-mL screw-capped polypropylene
centrifuge tube and mixed by vortexing for 5 min. After mixing, guar gum (50 mg) and 6 glass

85

beads were added to each tube. The tubes were then incubated with pancreatin (P-7545, 8× USP
Specifications, Sigma-Aldrich Co., St. Louis, MO), invertase (I-4504, Sigma-Aldrich Co., St.
Louis, MO) and amyloglucosidase (from A. niger, 3300 U/ml of soluble starch, Megazyme Ltd,
Co. Wicklow, Ireland) mixture (5 mL) at 37°C. After 20 min (G20) and 120 min (G120) of
incubation of a 0.5 mL aliquot of hydrolysate from the tube were removed and added to absolute
ethanol (5 mL) in another tube to stop the enzymatic reaction. The glucose released in each
measurement was determined using a glucose oxidase peroxidase diagnostic kit (Megazyme Ltd,
Co.Wicklow, Ireland). The remaining contents of each 50 mL tube was heated in a boiling water
bath, treated with 7 M KOH (10 mL), and hydrolyzed further with 0.15 mL of amyloglucosidase
(50 U/mL) to determine total glucose (TG) after 180 min. Glucose content was also measured
using the glucose oxidase peroxidase diagnostic kit. The RDS, SDS, and RS percentages of the
total starch were calculated from the values of G20, G120 and TG as follows:
RDS = G20 × 0.9
SDS= (G120-G20) × 0.9
TS = TG × 0.9
RS = TS- (RDS + SDS)

4.3.6.1. Hydrolysis Index (HI) and Glycemic Index (GI)
The hydrolysis curve used to calculate the hydrolysis indices of samples was obtained
utilizing the procedure as described in Section 4.3.6, except that hydrolysates were removed and
analyzed at 20, 40, 60, 90, 120, and 150 min time points. Percent glucose and starch were
calculated for each time point (Englyst et al., 1992) and plotted against time (x-axis) to obtain the
hydrolysis curve for each bean variety. The hydrolysis index (HI) was obtained by dividing the

86

area under the hydrolysis curve (0-180 min) of the isolated bean variety starch sample by the
area obtained for the hydrolysis curve of a standard material (white bread, Table 4.1.). The area
under the curve was calculated using the trapezoidal method (Microsoft Excel, 2007).
HI ={[Area under curve of isolated bean starch (dry weight)]/ [Area under curve of
white bread (dry weight)]} x 100
The estimated glycemic index (GI) was predicted with the formula of Goñi, GarciaAlonso, & Saura-Calixto (1997):
GI = 39.71 + (0.549 x HI).

Table 4.1. Nutrient Label for Sunbeam Giant Enriched White Breada
Ingredients

Mass per Serving Size (g)b

Total carbohydrate

28

Dietary fiber

1

Sugars

3

Protein

4

Total fat

1.5

Trans. fat

0

a

White bread used in the digestibility study was commercially produced and was purchased from a local
supermarket located in East Lansing, MI
b
Serving size: 57 g

4.3.7. Preparation of Starch Dispersions for the HPSEC-MALLS-RI System
Starch solutions (10 mg/mL) were prepared for HPSEC analyses. Briefly, isolated starch
sample (50 mg) was weighed into a screw-capped glass culture tube. 50 mM LiBr in 90%
87

DMSO-10% de-ionized water solution (5 mL) was added. The tightly capped tube was vortexed
for 10 s to disperse the starch. The tubes were placed in a water bath at 80ºC and individual
tubes were vortexed every 15min. After two hours, the tubes were removed and shaken on an
orbital shaker (Cole-Parmer, IL, USA) at 200 rpm overnight at room temperature. The starch
dispersions were centrifuged (Sorvall RT6000B centrifuge, Thermo Scientific Inc., USA) at
4600 x g for 10 min at 20ºC and the supernatants were analyzed with the HPSEC-MALLS-RI
system. Analyses for bean starch samples were done in duplicate.

4.3.8. Molecular Weight Determination
The weight-average molecular weights of isolated bean starches were determined using
high performance size-exclusion chromatography (HPSEC, Agilent 1200, Agilent Technologies,
Santa Clara, CA, USA). HPSEC analyses were conducted using PSS columns: precolumn,
Gram10000 and Gram1000 (Polymer Standards Services, Mainz, Germany) with packing
materials having pore diameters of 10 μm. A Wyatt miniDawn TREOS multi-angle laser light
scattering detector (Wyatt Tech. Corp., Santa Barbara, CA, USA) and a Wyatt Optilab refractive
index detector (Wyatt Tech. Corp., Santa Barbara, CA, USA) were used to measure light
scattering and refractive index values, respectively. The laser sources of these detectors were
operated at 658 nm. The HPSEC analyses of the starches isolated from bean samples were
performed at 70ºC for 120 min, with sample concentrations of 10 mg/mL, injection volume of
100 μL and eluent flow rates of 0.2 mL/ min. 50 mM LiBr in 90% DMSO-10% de-ionized water
was used as the solvent. The data processing was performed using the ASTRA 5.3.4.18 software
package (Wyatt Tech. Corp., Santa Barbara, CA, USA). Pullulan standards: (342 g/mol, 57191;

88

6 x 103 g/mol, 18179; 110 x 103 g/mol, 47053; 800 x 103 g/mol, 18789; 1600 x 103 g/mol,
61943) and dextran standards: 1 x 103 g/mol, 00268; 5 x 103 g/mol, 00269; 50 x 103 g/mol,
00891; 410 x 103 g/mol, 00895) (Polymer Standards Services, Mainz, Germany) were used to
verify the light scattering method. Four runs were done for each variety and sample treatment.

4.3.9. Statistical Analysis
All statistical analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC,
USA). Analyses of variance (ANOVA) were performed and the mean separations were evaluated

by Tukeys HSD test (p ˂ 0.05).

89

4.4. RESULTS AND DISCUSSION
4.4.1. In vitro Digestibility
The rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch
(RS) contents of the isolated native, canned and stovetop-cooked bean starch samples are
presented in Table 4.2. All isolated native bean starch samples were lower in RDS (1.95-2.71%)
and higher in RS (78.95-83.71%) when compared to canned bean starch samples and stovetopcooked bean starch samples. The RDS contents of isolated stovetop cooked and canned bean
starches ranged between 7.58-13.21% and 7.37-10.61% respectively. RDS is rapidly and
completely digested in the small intestine and is associated with more rapid elevation of
postprandial plasma glucose. The RDS and RS values for native isolated bean starches were
comparable to the respective RDS and RS contents of raw potato starch determined by Englyst et
al., (1992). Native starches are very resistant to hydrolysis by pancreatic amylase (Chung, Liu,
& Hoover, 2009). Ancona, Campos, Guerrero, & Ortiz ( 2011) postulated that the crystalline
structure of native starch protects their glycosidic bonds and limits enzymatic hydrolysis. The
RS levels of isolated native starches determined by the Megazyme method (Table 3.2.) were
considerably lower than those obtained using Englyst et al. (1992) protocol. The disparity may
be due to a significant reduction in the duration of enyme digestion (2 h) used in Englyst et al.
(1992) protocol as opposed to the 16 h of enzyme digestion employed in the Megazyme protocol.
Additionally, instead of directly measuring glucose originating from RS as in the Megazyme
protocol, Englyst et al. (1992) method indirectly estimates total RS. This indirect measurement
of RS includes any RS2 and RS3 in the sample (Perera, Meda, & Tyler, 2010).
In starches that have undergone heat-moisture treatments, gelatinization has taken place,
which occurs with a loss in crystallinity. During gelatinization the starch granules are swollen
90

and ruptured and are more susceptible to hydrolysis by α-amylase (Bertolini, 2009; Jenkins &
Donald, 1998). It can be postulated that a greater degree of crystallinity in starch reduces
enzymatic starch hydrolysis (Hoover & Zhou, 2003). This phenomenon was observed for
isolated native navy bean starch which had the highest level of crystallinity (36.1% , Table 3.5.).
However, in this present study, the reverse also occurred where isolated native pinto bean starch
had the lowest degree of crystallinity (24.9%, Table 3.5.) also had the lowest level of RDS and
the highest level of RS (Table 4.2.). It is possible that enzymatic hydrolysis is not only
dependent on overall crystallinity but on an interplay of many factors such as amylose content
and amylopectin branch chain lengths. Amylose contents of the isolated native bean starch
samples did not affect the enzymatic digestibility in this study since no significant differences
were observed in the amylose contents of the six bean varieties. Slowly digestible starch is a
desirable form of dietary starch. Isolated native light red kidney bean starch had the largest
amount of SDS (17.39%) and the smallest amount of RS (78.95%). Only marginal differences in
the RDS, SDS and RS contents were observed among isolated canned and stovetop-cooked
starches. In general, isolated starches from canned and cooked beans contained greater amounts
of RDS, SDS and RS when compared to their native starch counterpart samples.
The extent of hydrolysis (after 180 min) among the native starches ranged from 24.9%
for black beans to 27.3% for light red kidney beans (Figure 4.1.). All isolated native bean
starches showed similar hydrolysis rates during the initial 40 min with small differences in the
latter stages (Figure 4.1.). Hoover and Sosulski (1985) showed that after 6h digestion with
porcine pancreatic α-amylase, four dry bean starches had hydrolysis rate values ranging from
26% for isolated native navy bean starch to 35% for isolated native black bean starch. In this
current study isolated native black bean starch was enzymatically hydrolyzed to a lesser extent

91

(7.7%) than in the study conducted by Hoover and Sosulski (1985). Variations in the extent of
starch hydrolysis may have been due to different enzyme and substrate concentrations in this
study compared to the study conducted by Hoover and Sosulski (1985). The higher degree of
hydrolysis for light red kidney bean starch appears to be associated with its higher RDS content.
A similar phenomenon was also observed with isolated native small red bean and dark red
kidney bean starches. The enzyme starch hydrolysis profiles for isolated stovetop-cooked and
canned bean starches are illustrated in Figures 4.2. and 4.3. Isolated starches from stovetopcooked and canned beans displayed a greater extent of starch hydrolysis (39.8-8.4%) than the
starches that were isolated from raw whole beans (24.9-27.3%) (Figure 4.1.). Although, the
extent of starch hydrolysis was found to be higher in the processed starch samples than the native
starches; however, when compared with the degree of starch hydrolysis for corn starch (74%)
(Hoover & Sosulski, 1985a), the processed dry bean starches were more resistant to α-amylase
hydrolysis. This may be due to bean starch not being fully gelatinized when processed and
therefore the physical nature of the starch granule limits starch hydrolysis and/or leads to the
formation of retrograded resistant starch (RS3) (Björck et al., 1994; Tovar et al., 1992; Tovar &
Melito, 1996). The resistant starch content of the processed isolated bean starches ranged from
62.5% to 68.6%.

92

Starch Hydrolysis (%)

30
25
BB

20

NB

15

PB

10

DR

5

LR
SR

0
0

20

40

60

80

100

120

140

160

180

200

Time (min)

Figure 4.1. Starch hydrolysis curves for isolated native bean starches of six Phaseolus vulgaris
varieties.a
a

BB: isolated native black bean starch; NB: isolated native navy bean starch; PB: isolated native pinto

bean starch; DR: isolated native dark red kidney bean starch; LR: isolated native light red kidney bean
starch; SR: isolated native small red bean starch

93

50

Starch Hydrolysis (%)

45
40
35

BBS

30

NBS

25

PBS

20

DRS

15
10

LRS

5

SRS

0
0

50

100

150

200

Time (min)

Figure 4.2. Starch hydrolysis curves for isolated stovetop-cooked bean starches of six Phaseolus
vulgaris varieties.a
a

BBS: isolated stovetop-cooked black bean starch; NBS: isolated stovetop-cooked navy bean starch; PBS:

isolated stovetop-cooked pinto bean starch; DRS: isolated stovetop-cooked dark red kidney bean starch;
LRS: isolated stovetop-cooked light red kidney bean starch; SRS: isolated stovetop-cooked small red
bean starch.

94

60

Starch Hydrolysis (%)

50
BBC

40

NBC

30

PBC

20

DRC
LRC

10

SRC

0
0

50

100

150

200

Time (min)

Figure 4.3. Starch hydrolysis curves for isolated canned bean starches of six Phaseolus vulgaris
varieties. a
a

BBC: isolated canned black bean starch; NBC: isolated canned navy bean starch; PBC: isolated canned

pinto bean starch; DRC: isolated canned dark red kidney bean starch; LRC: isolated stovetop cooked light
red kidney bean starch , SRC: isolated canned small red bean starch.

95

Table 4. 2. Rapidly Digestible Starch, Slowly Digestible Starch and Resistant Starch
Contents of Isolated Starch from Raw, Stovetop-Cooked, and Canned Beans a,b
Starch Source
Ra w c
Black bean
Dark red kidney
bean
Light red kidney
bean
Navy bean
Pinto bean
Small red bean
Canned d
Black bean
Dark red kidney
bean
Light red kidney
bean
Navy bean
Pinto bean
Small red bean
Stovetop-Cookede
Black bean
Dark red kidney
bean
Light red kidney
bean
Navy bean
Pinto bean
Small red bean

RDS (%)

SDS (%)

RS (%)

1.95 ± 0.02 b,d c
2.23± 0.01 a,b,c

14.69 ± 0.05 a,b
14.96 ± 0.41 a,b

83.34 ± 0.03 a,b
82.50 ± 0.42a,b

2.71 ±0.12 a

17.39 ±1.45 a

78.95±1.34 c

1.61 ± 0.16 d,c
1.55 ± 0.25 d
2.41 ±0.24 a,b

14.44 ±0.51 b
14.36 ± 0.47 b
16.64 ±0.25a,b

83.70 ±0.35a
83.56 ±0.71 a
80.95 ±0.01b,c

7.58 ±0.66 c
8.82 ±0.78 b,c

24.90 ±0.82 a
22.64 ±0.56 a

67.52 ±1.50 a
68.54 ±0.22 a

9.96 ±1.08 a,b,c

24.05 ±1.58 a

65.99 ±0.49 a

13.21 ±1.93 a
11.96 ±0.67 a,b
12.89 ±0.24 a,b

20.75 ±0.73 a
24.87 ±3.99 a
22.75 ±1.49 a

66.03 ±2.66 a
63.17 ±3.32 a
64.37 ±1.73 a

9.72 ±1.90 a
7.37 ±0.49 a

24.51 ±1.67a,b
26.32 ±0.31 a,b

65.77 ±0.23 a,b
66.32 ±0.80 a,b

8.87 ±1.03 a

22.55 ±1.58 a

68.59 ±0.54 b,

10.61 ±1.53 a
8.58 ±1.65 a
8.87 ±1.03 a

26.84 ±2.33 a,b
24.58 ±0.49 a,b
22.55 ±1.58 b

62.55 ±0.80 b
66.85 ±2.14 b
68.59 ±0.54 a

a

RDS: rapidly digestible starch; SDS: slowly digestible starch; RS: resistant starch, all calculated as
percentage of total starch based on Englyst et al., (1992).
b

Data represent means and standard deviations of duplicate measurements.

c

Values within the same column for raw samples followed by the same letter are not significantly
different (P ˂ 0.05) from each other by Tukey's HSD (honest significant difference) test
d

Values within the same column for canned samples followed by the same letter are not significantly
different (P ˂ 0.05) from each other by Tukey's HSD test
c

Values within the same column for stovetop-cooked samples followed by the same letter are not
significantly different (P ˂ 0.05) from each other by Tukey's HSD test

96

The calculated hydrolysis index (HI) and glycemic index (GI) of isolated native, canned
and stovetop-cooked bean starches are reported in Table 4.3. The HI expresses the digestibility
of starch in foods in relation to the digestibility of starch in white bread (Thorne et al., 1983).
Values obtained for HI for isolated raw bean starches are in the range of 17.9 to 21.5 with raw
light red kidney bean starch having the lowest hydrolysis index. For isolated stovetop- cooked
and canned bean starches the hydrolysis index values were in the range of 35.2 to 42.1.
The glycemic index (GI) concept is a tool for ranking foods with respect to their blood
glucose raising potential (Thorne et al., 1983). The estimated glycemic index (GI) is derived
from in vitro digestion kinetics (Goñi et al., 1997). The calculated GI values for isolated native
bean starches were higher in the present study than those reported by Ambigaipalan et al. (2011)
for isolated native starch from different cultivars of black bean (49.6 vs ~39%) and pinto bean
(49.7 vs ~36%). The substantial difference could be ascribed to the methods used for analysis.
The Englyst (1992) method was used in this study because it mimicked in vivo digestion,
whereas Ambigaipalan et al. (2011) used an American Association of Cereal Chemists approved
method (AACC, 32-40) and also used the following equation to predict glycemic index:
GI = 8.198 + 0.862HI. The derivation of this equation is based on the regression analysis of HI
and GI under the conditions of that particular experiment (Granfeldt, Björck, Drews, & Tovar,
1992).
The main determinant of the glycemic index of foods has been found to be RDS (Englyst
& Hudson, 1996; Englyst, Vinoy, Englyst, & Lang, 2003). Regression analyses of results from
the three processing treatments (native, canned, and stovtop-cooked beans) of the six bean
varieties in the present study (Figure 4.4.) confirmed that RDS (r2 = 0.83, 0.76, 0.49) is strongly
correlated to GI. Glycemic indices have been positively correlated to RDS and SDS contents of

97

starches (Madhusudhan & Tharanathan, 1995, 1996). Dried beans are low glycemic index foods,
which makes them beneficial in the management of diabetes (Thorne et al., 1983).

98

A

y = 0.5491x - 25.457
R² = 0.8526

RDS (%)

3
2
1
0
49

49.5

50

50.5

51

51.5

52

GI

RDS (%)

B

y = 1.3646x - 72.724
R² = 0.7594

15
10
5
0
58

59

60

61

62

63

GI

C
y = 0.5878x - 26.863
R² = 0.4924

RDS (%)

15
10
5
0
58

59

60

61

62

63

GI

Figure 4.4. Relationship of RDS with GI of raw (A), canned (B) and stovetop-cooked (C) bean
starch isolated from six Phaseolus vulgaris varieties.

99

Table 4.3. Hydrolysis Index and Glycemic Index of Starch Isolated from Six Bean
Varietiesa
Starch Source
Rawd
Black bean
Dark red kidney
bean
Light red kidney
bean
Navy bean
Pinto bean
Small red bean
Cannede
Black bean
Dark red kidney
bean
Light red kidney
bean
Navy bean
Pinto bean
Small red bean
Stovetop-Cookedf
Black bean
Dark red kidney
bean
Light red kidney
bean
Navy bean
Pinto bean
Small red bean

HI b

GI c

18.29 ± 0.25 c,d
18.91± 0.12 b,c

49.64 ± 0.14 c,d
50.08± 0.07 b,c

21.52 ±0.48 a

51.51 ±0.26 a

17.87 ± 0.52 d
18.02 ± 0.20 c,d
19.69 ±0.17 b

49.49 ± 0.03 d
49.67 ± 0.11 c,d
50.51 ±0.09 b

35.2 ±1.98 a
36.84 ±0.67 a,b

59.03 ±1.09 a
59.93 ±0.37 a

39.63 ±0.39 a,b

61.45 ±0.21 a

42.09 ±2.53 a
41.61 ±0.69 a
39.17 ±1.21 a, b

62.81 ±1.39 a
62.54 ±0.38 a
61.20 ±0.66 a

40.02 ±1.69 a,b
39.00 ±0.14 a,b,c

61.67 ±0.92 a,b
61.11 ±0.07 a,b,c

39.88 ±0.027 a,b

61.59 ±0.015 a,b

42.05 ±0.18 a
37.21 ±1.48 b,c
35.26 ±0.34c

62.79 ±0.096 a
60.13 ±0.81 c
59.06 ±0.18 c

a

Data represent means and standard deviations of duplicate
HI: hydrolysis index =[ area under hydrolysis curve (white bread)/ area under hydrolysis curve (starch)]
x 100
b

c

GI: glycemic index = 39.71 + 0.549 x HI

d

Values within the same column for raw samples followed by the same letter are not significantly
different (P ˂ 0.05) from each other by Tukey's HSD (honest significant difference) test
e

Values within the same column for canned samples followed by the same letter are not significantly
different (P ˂ 0.05) from each other by Tukey's HSD test
f

Values within the same column for stovetop-cooked samples followed by the same letter are not
significantly different (P ˂ 0.05) from each other by Tukey's HSD test

100

4.4.2. Molecular Weight Determination
Starch is composed of two macro-molecules, amylose and amylopection (Hoover &
Sosulski, 1985; Hughes et al., 2009). When starch is fractionated by size-exclusion
chromatography and viewed on a chromatogram, two peaks are observed. The first peak
represents the branched and higher molecular weight amylopectin, while the second peak
corresponds to the mainly linear and lower molecular weight fraction of amylose. The weight
average molecular weights (Mw) of amylose and amylopectin of isolated native bean starches
are shown in Table 4.4. and the Mw distribution profile is displayed in (Figure 4.5.). Amylose
fraction of isolated native dark red kidney bean starch had the smallest Mw (1.0 x 106 g/mol),
whereas native pinto bean starch had the largest Mw (1.8 x 106 g/mol). The weight average
molecular weights of amylopectin for isolated native bean starch in the present study ranged
from 2.4 x 107 g/mol to 3.9 x 107 g/mol. The above Mw values for amylopectin were lower than
those reported by Du et al. (2014) for native pinto bean starch (5.3 x 108 g/mol), native red
kidney bean starch (8.3 x 108 g/mol) , native black bean starch (3.1 x 108 g/mol) and native navy
bean starch (3.27 x 108 g/mol). Du et al. (2014) used aqueous starch dispersions for HPSECMALLS-RI to determine the Mw of the starches. Heating starch in water can cause degradation
as well as the formation of aggregates which result in starch polymers having higher measured
Mw (Cave et al., 2009). The lower Mw values for amylopectin measured in this study may be
attributed to the method used to dissolve starch samples as well as shear scission and degradation
during SEC separation (Cave et al., 2009). Regression analyses of results from the six bean
varieties in the present study showed that Mw for amylose was strongly correlated to GI (r2 =
0.55), RDS (r2 = 0.50), SDS(r2 = 0.49), RS (r2 = 0.42). Mw for amylopectin was not correlated
with GI, SDS, RS and RDS values for isolated native starches (data not shown).
101

Starch Concentration (mg/mL)

0.16
0.14
0.12
BB

0.1

DR
0.08

LR

0.06

NB

0.04

PB
SR

0.02
0
1.00E+00

1.00E+02

1.00E+04

1.00E+06

1.00E+08

1.00E+10

Molecular weight (g/mol)

Figure 4.5. Molecular weight distributions of isolated native starch from six Phaseolus vulgaris
varieties.a
a

For abbreviations, see Figure 4.1.

102

Table 4. 4. Weight Average Molecular Weight (Mw) of Amylose and Amylopectin in
Isolated Native Bean Starch a
Starch Source
Weight Average Molecular Weight (x 106 g/mol)
Amylose

Amylopectin

Black bean

1.2

24.0

Dark red kidney bean

1.4

31.0

Light red kidney bean

1.8

34.0

Navy bean

1.0

35.0

Pinto bean

1.6

29.0

Small red bean

1.5

26.0

a

Results of 4 injections. A calibration curve was used to calculate the weight average molecular weights.

The weight average molecular weight distribution profiles for isolated stovetop-cooked and
canned bean starches are displayed in Figures 4.6. and 4.7., respectively. Almost all samples of
the isolated canned bean starches exhibited a monomodal distribution, that is, where only one
peak was evident; indicating that starch degradation had occurred. Seemingly, the high pressures
and temperatures during the canning process (Bravo et al., 1998) were able to modify the starch
structure of both amylose and amylopectin, which led to reduction in the high molecular weight
starch fractions when compared to those isolated native bean starches (Figure 4.5.). It is possible
that the molecular degradation affected amylopectin more than amylose (compare Figures 4.5.,
4.6. and 4.7.). The weight average molecular weight distribution of isolated stovetop cooked

103

bean starches showed some remnants of a high molecular weight fraction (Figure 4.7.). The
exact mechanisms of the molecular weight reduction of starch are not completely clear. In
general, some amount of polymer degradation takes place with a subsequent reduction in high
molecular weight fractions. This reduction in molecular weight appears to occur through the
breaking of covalent bonds by the heating process (Einde, Goot, & Boom, 2003).

0.16
Concentration (mg/mL)

0.14

BBST

0.12

DRST

0.1

LRST

0.08

NBST

0.06

PBST

0.04

SRST

0.02
0
1.00E+00

1.00E+02

1.00E+04 1.00E+06 1.00E+08
Molecular weight (g/mol)

1.00E+10

Figure 4.6. Molecular weight distributions of starch isolated from stovetop cooked beans of six
Phaseolus vulgaris varieties.a
a

For abbreviations, see Figure 4.1.

104

0.16

Concentration (mg/mL)

0.14
0.12
BBC

0.1

DRC

0.08

LRC

0.06

NBC

0.04

PBC
SRC

0.02
0
1.00E+00

1.00E+02

1.00E+04

1.00E+06

1.00E+08

1.00E+10

Molecular weight (g/mol)

Figure 4.7. Molecular weight distributions of starch isolated from canned beans of six Phaseolus
vulgaris varieties.a
a

For abbreviations, see Figure 4.3.

105

4.5. CONCLUSIONS
Information regarding starch digestibility, such as digestion rate and enzyme resistance,
are of importance for diet-related disorders including obesity and diabetes (Hasjim, Lavau,
Gidley, & Gilbert, 2010). All isolated native bean starches were low in rapidly digestible starch
(1.95-2.71%) and high in resistant starch (78.95.0-83.71%). In vitro digestibility showed that
light red kidney bean starch, dark red kidney bean starch and small red bean starch were more
susceptible to hydrolysis by pancreatic α-amylase than the other three studied varieties. The
main determinant of the glycemic indices of the isolated native bean starches, isolated canned
bean starches and isolated stovetop-cooked bean starches was found to be the proportion of
rapidly digestible starch. The weight average molecular weights of amylose for the starches
were comparable to isolated starches reported in the literature. However, amylopectin Mw
results were lower and this could be due to shear scission during SEC analysis or the method
used for dissolution. The weight average weight molecular distribution for amylose was found
to be strongly correlated to glycemic index. The correlation between amylopectin Mw and GI
was weak. In general, isolated canned and stovetop-cooked starches had higher amounts of
RDS, lower amounts of SDS and RS, and larger glycemic indices than those of native isolated
bean starch. This indicates that the hydrothermal treatments of stovetop-cooking and canning
significantly alter the digestibility and molecular weight of the isolated starch studied.

106

APPENDICES

107

APPENDIX C
ENZYMATIC HYDROLYSIS OF ISOLATED NATIVE, CANNED AND STOVETOP-COOKED
BEAN STARCHES

Table 1-C. Absorbance of Glucose Released During Enzymatic Hydrolysis of Isolated Native
Bean Starches
ABS
Time
(min) BB1 BB1
20 0.123 0.123
40 0.200 0.210
60 0.314 0.321
90 0.467 0.479
120 0.591 0.582
150 0.739 0.741
180 0.742 0.789

BB2 BB2 NB1 NB1 NB2 NB2
0.122 0.122 0.109 0.111 0.118 0.117
0.192 0.196 0.191 0.196 0.206 0.209
0.304 0.303 0.307 0.304 0.303 0.317
0.467 0.479 0.453 0.457 0.474 0.487
0.588 0.588 0.599 0.592 0.579 0.581
0.715 0.706 0.782
0.78 0.766 0.772
0.759 0.784 0.793 0.846 0.771 0.852

PB1
0.115
0.188
0.301
0.475
0.571
0.766
0.772

PB1
PB2
PB2
0.116 0.102 0.107
0.183 0.188 0.190
0.307 0.296 0.301
0.479 0.469 0.482
0.59 0.544 0.553
0.743
0.74 0.779
0.788 0.759 0.809

DR1 DR1 DR2 DR2 LR1 LR1 LR2 LR2 SR1
SR1
SR2
SR2
20 0.148 0.15 0.142 0.155 0.158
0.15 0.157 0.163 0.134 0.137 0.145 0.148
40 0.260 0.269 0.265 0.272 0.283 0.294 0.241 0.249 0.219 0.219 0.232 0.235
60 0.388 0.388 0.389 0.392 0.421 0.458 0.405 0.405 0.333 0.340 0.356 0.366
90 0.605 0.620 0.624 0.642 0.586
0.6
0.59 0.642 0.515 0.539 0.537 0.542
120 0.740 0.756 0.719 0.731 0.809 0.803 0.731
0.75 0.678 0.710 0.686 0.701
150 0.842 0.877 0.868 0.892 0.948 0.919 0.901 0.965 0.804 0.794 0.763 0.771
180 0.972 1.023 0.979 1.042 0.905 0.944 0.897 0.978 0.782 0.914 0.861 0.893
a
ABS: absorbance
b
BB: black bean, DR: dark red kidney bean; LR: light red kidney bean; NB: navy bean; PB: pinto bean;
SR: small red bean

108

Table 2-C. Absorbance of Glucose Released During Enzymatic Hydrolysis of Isolated Canned
Bean Starches
ABS
Time
(min) BB1
BB1
20 0.2169 0.267
40
0.429 0.431
60
0.498 0.489
90
0.684 0.687
120
0.868 0.852
150
0.91 0.917
180
0.951 0.978

BB2 BB2
0.257 0.275
0.449 0.465
0.582 0.591
0.787 0.785
0.907 0.923
0.929
0.94
0.978 0.979

NB1 NB1 NB2 NB2 PB1
PB1
PB2
PB2
0.385 0.378 0.447 0.464 0.373 0.378 0.355 0.347
0.643 0.637 0.661 0.647 0.523 0.518 0.461 0.459
0.738
0.76 0.777 0.781 0.746 0.738 0.689 0.699
0.864 0.876 0.956 1.004 0.754 0.739 0.869 0.869
0.932 0.926 1.007 1.055 0.931 0.935 1.054 1.046
1.047 1.063 1.189 1.208
1.01 1.019 1.061 1.082
1.134 1.145 1.036 1.054 1.234 1.239 0.996 1.043

DR1
DR1 DR2 DR2 LR1 LR1 LR2 LR2 SR1
SR1
SR2
SR2
20
0.28 0.277 0.307 0.309 0.274 0.278 0.313 0.311 0.397 0.393 0.385 0.387
40
0.531 0.533 0.52
0.52 0.399 0.404 0.434 0.433 0.516 0.506 0.477 0.466
60
0.646 0.656 0.648 0.649 0.577 0.576 0.555 0.561 0.558 0.565 0.584 0.614
90
0.755
0.83 0.731 0.749
0.75 0.771 0.788
0.78 0.748 0.757 0.738
0.75
120
0.881 0.887 0.893 0.895 0.854 0.875 0.847
0.85 1.009 0.999 0.924 0.957
150
1.002 0.995 0.941 0.935 0.986 0.991 0.983 0.978 0.999 1.014 0.973 0.991
180
1.061 1.071 1.047 1.087 1.001 1.002 1.043 1.054 1.245 1.267 1.094 1.091
a
ABS: absorbance
b
BB: black bean, DR: dark red kidney bean; LR: light red kidney bean; NB: navy bean; PB: pinto bean;
SR: small red bean

109

Table 3-C. Absorbance of Glucose Released During Enzymatic Hydrolysis of Isolated
Stovetop-cooked Bean Starches
ABS
Time
(min) BB1 BB1
20 0.318 0.332
40
0.42 0.427
60 0.689 0.701
90 0.818 0.821
120 0.881 0.87
150 0.975 0.984
180 1.026 1.056

BB2 BB2
0.274
0.249
0.412
0.434
0.628
0.638
0.789
0.816
0.857
0.878
0.891
0.896
0.995 1.0008

NB1 NB1 NB2 NB2 PB1
PB1
PB2
PB2
0.331 0.337 0.283 0.285 0.236 0.244 0.257 0.334
0.446 0.428 0.434 0.449 0.368
0.35 0.347 0.382
0.733 0.743 0.712 0.717 0.619 0.618 0.624 0.627
0.81 0.787 0.805
0.81 0.685 0.723
0.75 0.733
0.924 0.907 0.933
0.95
0.82 0.811 0.878 0.897
0.935 0.957 0.962 0.937 0.909 0.896 0.919 0.922
0.972 0.982 0.968 0.939 0.989 0.931 1.014 1.011

DR1 DR1 DR2 DR2
LR1 LR1 LR2 LR2 SR1
SR1
SR2
SR2
20 0.207 0.238 0.233
0.243 0.239 0.269 0.262 0.282 0.256 0.232 0.277 0.276
40 0.321 0.348 0.405
0.408 0.286 0.318 0.311 0.307 0.295 0.309 0.245 0.253
60 0.582 0.661 0.635
0.632 0.606 0.612 0.623
0.62 0.563 0.559
0.57 0.569
90 0.726 0.719 0.689
0.685 0.703 0.711 0.703 0.702
0.65 0.667
0.62 0.629
120 0.809 0.815 0.831
0.842 0.908 0.882 0.909 0.867 0.723 0.812 0.768 0.734
150 0.973 0.994 0.963
0.944 0.907 0.919 0.911 0.915 0.789 0.825 0.812 0.832
180 0.991 0.963 0.956
0.941 1.008 0.988 0.972 0.991 0.906
0.92 0.941 0.892
a
ABS: absorbance
b
BB: black bean, DR: dark red kidney bean; LR: light red kidney bean; NB: navy bean; PB: pinto bean;
SR: small red bean

110

APPENDIX D

WEIGHT AVERAGE MOLECULAR WEIGHT DETERMINATIONS

One of the two major principles that are used in light scattering to determine the molar
mass is:
1. The intensity of light scattered (LS) is proportional to the product of the polymer (Mw)
and the polymer concentration (C).
LS α Mw x C (dn/dc)2
Where (dn/dc) is the refractive index increment, which expresses the variation of the refractive
index of a solution with solute concentration (Wyatt, 1993).

Differential Index of Refraction (dn/dc) Determination
Isolated starch samples were vacuum oven dried at 70°C for 18 h. Starch samples
(25mg) were placed in a screw capped glass tube (14mL) and DMSO-50 mM LiBr-H2O (90%
DMSO-10% H2O) (5mL) was added. The tubes were placed in a water bath held at 80°C for 2 h.
The tubes were votexed every 5 min to mix for the first 30min, then vortexed every 10min. The
tubes were then taken and were allowed to shake at 200rpm overnight (~16hrs) at room
temperature (25°C). The tubes were centrifuged (4000 rpm) at 20°C for 10 min (Eppendorf
model 5810R). The supernatants were decanted into scintillation vials and serial dilutions of 0
(blank), 1, 2, 3, 4 and 5mg/mL were made using DMSO-50 mM LiBr-H2O (90% DMSO-10%
H2O)as the solvent. Mixtures were vortexed for 10 s and injected (400 µL) using Agilent
G1329A autosampler to an Agilent 1200 system (G1311A Quaternary Pump with a G1322A
111

degasser) directly connected from pump to the dRI detector (Wyatt Corp., Optilab rEX,
wavelength at 658nm). The samples were kept at room temperature (~25C). The HPLC flow
rate was at 0.1mL/min (25°C), while the dRI detector temperature was set at 40°C. Data
collection and dn/dc calculation was done by using ASTRA software.

Figure 1-D. Isolated native black bean starch dn/dc.

BB starch dn/dc: 0.0438±0.0010 mL/g r²: 0.997

Figure 2-D. Isolated native navy bean dn/dc.
112

NB starch dn/dc: 0.0454±0.0011 mL/g r²: 0.997

Table 1-D. Light Scattering Weight Average Molecular Weights of Isolated Native, Canned and
Stovetop-cooked Bean Starches
Starch Sourcea

Molecular
Weight (g/mol)
Native
Canned
Stovetop-cooked
BB1
2.34E+08
9.89E+06
6.26E+06
BB1
2.55E+08
6.98E+06
9.23E+06
BB2
1.89E+08
1.50E+07
1.09E+07
BB2
6.46E+07
1.15E+07
1.06E+07
DR1
4.83E+07
8.38E+06
8.29E+06
DR1
4.73E+07
7.69E+06
9.45E+06
DR2
2.54E+07
1.70E+07
1.66E+07
DR2
9.96E+07
1.63E+07
5.45E+06
LR1
5.67E+07
1.26E+07
1.07E+07
LR1
5.29E+07
9.89E+06
6.92E+06
LR2
1.11E+08
1.06E+07
7.50E+06
LR2
7.25E+07
1.07E+07
6.88E+06
NB1
3.13E+07
7.85E+06
1.56E+07
NB1
3.07E+07
8.36E+06
5.80E+06
NB2
3.54E+07
1.56E+07
1.65E+07
NB2
1.92E+07
1.06E+07
9.33E+06
PB1
8.68E+07
5.52E+06
1.16E+07
PB1
5.20E+08
9.51E+06
4.67E+07
PB2
1.00E+08
6.60E+06
1.09E+07
PB2
1.97E+07
1.60E+07
1.11E+07
SR1
2.25E+07
8.52E+06
8.83E+06
SR1
2.49E+07
7.27E+06
1.30E+07
SR2
2.20E+08
1.13E+07
8.38E+06
SR2
2.48E+07
1.11E+07
1.12E+07
a
BB: black bean; DR: dark red kidney bean; LR: light red kidney bean; NB: navy bean; PB:
pinto bean; SR: small red bean

113

LITERATURE CITED

114

LITERATURE CITED

Ambigaipalan, P., Hoover, R., Donner, E., Liu, Q., Jaiswal, S., Chibbar, R., Seetharaman, K.
(2011). Structure of faba bean, black bean and pinto bean starches at different levels of
granule organization and their physicochemical properties. Food Research International,
44(9), 2962–2974.
Seetharaman, K. (2011). Structure of faba bean, black bean and pinto bean starches at different
levels of granule organization and their physicochemical properties. Food Research
International, 44(9), 2962–2974.
Ancona, D. B., Campos, M. R. S., Guerrero, L. a C., & Ortiz, G. D. (2011). Structural and some
nutritional characteristics of Velvet bean (Mucuna pruriens) and Lima bean (Phaseolus
lunatus) starches. Starch/Staerke, 63(8), 475–484. 9
Anderson, J. W., Smith, B. M., & Washnock, C. S. (1999). Cardiovascular and renal benefits of
dry bean and soybean intake. The American Journal of Clinical Nutrition, 70(3 Suppl),
464S–474S.
Annison, G., & Topping, D. L. (1994). Nutritional role of resistant starch: Chemical Structure vs
Physiological Function Primary Structure of Starch Components. Annual Review of
Nutrition, (35), 297–320.
AOAC. (2000). Official Methods of Analysis. (A. Horwitz, Ed.) (17th ed.). Gaithersburg, MD:
Association of Official Analytical Communities International.
Bello-Pérez, L. A., Rodriguez-Ambriz, S., Sanchez-Rivera, M., & Agama-Acevedo, E. (2009).
Starch Molecular Structure. In A. Bertolini (Ed.), Starches: Characterization, Properties,
and Applications (1st ed., pp. 33–57). CRC Press.
Bertolini, A. C. (Ed.). (2009). Starches: Characterization, Properties, and Applications (1st ed.,
p. 288 pages). CRC Press.
Björck, I., Granfeldt, Y., Liljeberg, H., Tovar, J., & Asp, N. (1994). Food properties affecting the
digestion and absorption of carbohydrates. American Journal of Clinical Nutrion, 59, 699S–
705S.
Bravo, L., Siddhuraju, P., & Saura-Calixto, F. (1998). Effect of Various Processing Methods on
the in Vitro Starch Digestibility and Resistant Starch Content of Indian Pulses. Journal of
Agricultural and Food Chemistry, 46(11), 4667–4674.
Cave, R. a., Seabrook, S. a., Gidley, M. J., & Gilbert, R. G. (2009). Characterization of starch by
size-exclusion chromatography: The limitations imposed by shear scission.
Biomacromolecules, 10(8), 2245–2253.

115

Chung, H.-J., Liu, Q., & Hoover, R. (2009). Impact of annealing and heat-moisture treatment on
rapidly digestible, slowly digestible and resistant starch levels in native and gelatinized
corn, pea and lentil starches. Carbohydrate Polymers, 75(3), 436–447.
Colonna, P., Leloup, V., & Buléon, A. (1992). Limiting factors of starch hydrolysis. European
Journal of Clinical Nutrition, 46 Suppl 2, S17–32.
Du, S.-K., Jiang, H., Ai, Y., & Jane, J.-L. (2014). Physicochemical properties and digestibility of
common bean (Phaseolus vulgaris L.) starches. Carbohydrate Polymers, 108, 200–5.
Einde, R. M., Goot, A. J., & Boom, R. M. (2003). Understanding Molecular Weight Reduction
of Starch During Heating-shearing Processes. Journal of Food Science, 68(8), 2396–2404.
Englyst, E. (2004). Resistant starch – Nutritional and Biological Activity. Polish Journal of Food
Nutrition Sciences, 13(54), 51–64.
Englyst, H. N., & Hudson, G. J. (1996). The classification and measurement of dietary
carbohydrates. Food Chemistry, 57(1), 15–21.
Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of
nutritionally important starch fractions. European Journal of Clinical Nutrition, 46(2), S33–
50.
Englyst, K. N., & Englyst, H. N. (2005). Carbohydrate Bioavailability. British Journal of
Nutrition, 94, 1–11.
Englyst, K. N., Vinoy, S., Englyst, H. N., & Lang, V. (2003). Glycaemic index of cereal products
explained by their content of rapidly and slowly available glucose. The British Journal of
Nutrition, 89(3), 329–40.
Gidley, M. J., Hanashiro, I., Hani, N. M., Hill, S. E., Huber, A., Jane, J. L., Gilbert, R. G.
(2010). Reliable measurements of the size distributions of starch molecules in solution:
Current dilemmas and recommendations. Carbohydrate Polymers, 79(2), 255–261.
Goñi, I., Garcia-Alonso, A., & Saura-Calixto, F. (1997). A starch hydrolysis procedure to
estimate glycemic index. Nutrition Research, 17(3), 427–437.
Han, J. a., & Lim, S. T. (2004). Structural changes of corn starches by heating and stirring in
DMSO measured by SEC-MALLS-RI system. Carbohydrate Polymers, 55(3), 265–272.
Hasjim, J., Lavau, G. C., Gidley, M. J., & Gilbert, R. G. (2010a). In vivo and in vitro starch
digestion: are current in vitro techniques adequate? Biomacromolecules, 11(12), 3600–8.
Hasjim, J., Lavau, G. C., Gidley, M. J., & Gilbert, R. G. (2010b). In vivo and in vitro starch
digestion: Are current in vitro techniques adequate? Biomacromolecules, 11(12), 3600–
3608.
116

Hoover, R., & Sosulski, F. (1985a). Studies on the functional characteristics and Digestibility of
starches from Phaseolus vulgaris biotypes. Starch - Stärke, 37(6), 181–191.
Hoover, R., & Sosulski, F. (1985b). Studies on the Functional Characteristics and Digestibility of
Starches from Phaseolus vulgaris Biotypes. Starch - Stärke, 37(6), 181–191.
Hoover, R., & Zhou, Y. (2003). In vitro and in vivo hydrolysis of legume starches by α-amylase
and resistant starch formation in legumes—a review. Carbohydrate Polymers, 54(4), 401–
417.
Hughes, T., Hoover, R., Liu, Q., Donner, E., Chibbar, R., & Jaiswal, S. (2009). Composition,
morphology, molecular structure, and physicochemical properties of starches from newly
released chickpea (Cicer arietinum L.) cultivars grown in Canada. Food Research
International, 42(5-6), 627–635.
Jane, J., Chen, Y., Lee, L., McPherson, E., Wong, K., Radosavlijevic, M., & Kasemsuwan, T.
(1999). Effects of amylopectin branch chain length and amylose content on the
gelatinization and pasting properties of starch. Cereal Chemistry, 76, 629–637.
Jenkins, P. J., & Donald, A. M. (1998). Gelatinisation of Starch: A combined
SAXS/WAXS/DSC and SANS Study. Carbohydrate Research, 308, 133–147.
Kaur, M., & Sandhu, K. S. (2010, January). In vitro digestibility, structural and functional
properties of starch from pigeon pea (Cajanus cajan) cultivars grown in India. Food
Research International, 29. 410-415.
Madhusudhan, B., & Tharanathan, R. N. (1995). Legume and cereal starches—why differences
in digestibility? — Part II. Isolation and characterization of starches from rice (O. sativa)
and ragi (finger millet, E. coracana). Carbohydrate Polymers, 28(2), 153–158.
Madhusudhan, B., & Tharanathan, R. N. (1996, April). Structural studies of linear and branched
fractions of chickpea and finger millet starches. Carbohydrate Research, 71, 123-130.
Muir, J. G., & O’Dea, K. (1992). Measurement of resistant starch: factors affecting the amount
of starch escaping digestion in vitro. The American Journal of Clinical Nutrition, 56(1),
123–7.
Othman, Z., Al-Assaf, S., & Hassan, O. (2010). Molecular Characterisation of Sago Starch Using
Gel Permeation Chromatography Multi-Angle Laser Light Scattering. Sains Malaysiana,
39(6), 969–973.
Perera, a., Meda, V., & Tyler, R. T. (2010). Resistant starch: A review of analytical protocols for
determining resistant starch and of factors affecting the resistant starch content of foods.
Food Research International, 43(8), 1959–1974.

117

Podzimek, S. (2014). Truths and myths about the determination of molar mass distribution of
synthetic and natural polymers by size exclusion chromatography. Journal of Applied
Polymer Science, 131(7), 40111-40121.
Ratnayake, W. S., Hoover, R., & Warkentin, T. (2002). Pea Starch: Composition, Structure and
Properties — A Review. Starch - Stärke, 54(6), 217–234.
Rehman, Z., & Shah, W. H. H. (2005). Thermal heat processing effects on antinutrients, protein
and starch digestibility of food legumes. Food Chemistry, 91(2), 327–331.
Roger, P., & Colonna, P. (1992). The influence of chain length on the hydrodynamic behaviour
of amylose. Carbohydrate Research, 227, 73–83. doi:10.1016/0008-6215(92)85061-4
Sajilata, M. G., Singhal, R. S., & Kulkarni, P. R. (2006). Resistant Starch?A Review.
Comprehensive Reviews in Food Science and Food Safety, 5(1), 1–17.
Syahariza, Z. a, Sar, S., Hasjim, J., Tizzotti, M. J., & Gilbert, R. G. (2013). The importance of
amylose and amylopectin fine structures for starch digestibility in cooked rice grains. Food
Chemistry, 136(2), 742–9.
Tester, R. F., Karkalas, J., & Qi, X. (2004). Starch—composition, fine structure and architecture.
Journal of Cereal Science, 39(2), 151–165.
Tester, R., & Morrison, W. (1990). Swelling and gelatinization of cereal starches. I. Effects of
amylopectin, amylose, and lipids. Cereal Chemestry,67,551-557. Retrieved from h
Thorne, M. J., Thompson, L. U., & Jenkins, D. J. (1983). Factors affecting starch digestibility
and the glycemic response with special reference to legumes. The American Journal of
Clinical Nutrition, 38(3), 481–8.
Tovar, J., Granfeldt, Y., & Bjorck, I. M. (1992). Effect of Processing on Blood Glucose and
Insulin Responses to Starch in Legumes. Journal of Agricultural and Food Chemistry, 40,
1848–1851.
Tovar, J., & Melito, C. (1996). Steam-Cooking and Dry Heating Produce Resistant Starch in.
Journal of Agricultural and Food Chemistry, 44, 2642–2645.
Wang, T. L., Bogracheva, T. Y., & Hedley, C. L. (1998). Starch : as simple as A , B , C ?,
Carbohydrate Polymers, 49(320), 481–502.
Witt, T., Gidley, M. J., & Gilbert, R. G. (2010). Starch digestion mechanistic information from
the time evolution of molecular Size distributions. Journal of Agricultural and Food
Chemistry, 58(14), 8444–8452.
Wyatt, P. J. (1993). Light scattering and the absolute characterization of macromolecules.
Analytica Chimica Acta, 272(1), 1–40.
118

Yokoyama, W., Renner-Nantz, J., & Shoemaker, C. F. (1998). Starch Molecular Mass and Size
by Size-Exclusion Chromatography in DMSO-LiBr Coupled with Multiple Angle Laser
Light Scattering. Cereal Chemistry, 75(4), 530–535.
Yoo, S. H., & Jane, J. L. (2002). Molecular weights and gyration radii of amylopectins
determined by high-performance size-exclusion chromatography equipped with multi-angle
laser-light scattering and refractive index detectors. Carbohydrate Polymers, 49(3), 307–
314.

119

CHAPTER 5
GENERAL CONCLUSIONS

120

In the present study, starches were isolated from native, canned and stovetop-cooked
dried beans and their physicochemical properties, digestibility and molecular structures, and how
these features are interrelated were determined. The general findings include:
All isolated native starch samples had total starch contents greater than 90%, with light
red kidney beans having the highest total starch (96.2%). This indicated that the major
component of the starch isolates was in fact starch. The disparity in the total starch contents for
the native isolated bean starches could be due to fiber co-settling with the starch during
extraction and adhering to some bean starch samples more than others both of which could
decrease the total starch content. The amounts of amylose in the isolated native bean starches
varied from 28.01 to 28.63% and were not significantly different from each other, thus it is
unlikely the amylose content had a significant influence on any differences noticed in the studied
starch characteristics. Resistance starch determined by the Megazyme assay revealed that
isolated native pinto bean starch contained the greatest amount of resistant starch (55.70%),
whilst light red kidney bean had the lowest (41.92%). The resistant starch contents of starches
isolated from dark red kidney bean and light red kidney were significantly lower than those from
the other isolated native dry bean starches. Therefore, these two isolated native bean starches
would have a different nutritional impact when compared to the other four isolated native bean
starches from Phaseolus vulgaris.
Significant differences (P<0.05) were observed in gelatinization onset (transition)
temperature (To), peak temperature (Tp) and conclusion temperature (Tc) among the studied
isolated native bean starches. The onset gelatinization temperature (To) varied from 65.5ºC
(isolated native light red kidney bean starch) to 68.8ºC (isolated native small red bean starch).
The peak transition temperatures of isolated native light red kidney and dark red kidney bean
121

starches were significantly different from those of the other bean starch samples. A similar trend
was also observed for the conclusion transition temperatures as well. The ranges of To (46.247.7°C), Tp (58.6-61.3°C) and Tc (71.4-74.5°C) values of retrograded isolated native starches
were lower than those obtained for the initial gelatinization. Both amylopectin and amylose
were involved in the retrogradation process, since isolated native bean starches were allowed to
retrograde for 7 days at 4°C.
Results revealed that isolated native bean starches from dark red kidney and light red

kidney beans had lower peak and break down viscosities than native starch isolated from navy
and pinto beans. This indicated that dark red kidney and light red kidney bean starches would
swell less and have more resistance to shear. Therefore they would be suitable in foods that
require excessive stirring or need to be pumped through a pipe. The setback viscosity values for
the isolated native bean starches ranged between 2265.5 cP and 4349.5 cP. The smaller
resistance starch contents of isolated native light red kidney and dark red kidney bean starches
when compared to the other isolated native bean starches, may account for their lower pasting
temperatures. All isolated native bean starches exhibit a C-type X-ray diffraction pattern.
Crystallinity was the greatest for navy bean starch (36.06%), whereas, pinto bean starch had the
lowest value (24.86%). In the present study no relationship was observed between crystallinity
and the other starch characteristics studied.
The findings of the current study showed that the physicochemical properties of native
starches isolated from dark red kidney and light red kidney beans differed from those of the other
isolated bean starches. Consequently, native dark red kidney and light red kidney bean starches
may have different end-use applications than the other common bean starches.

122

The digestibility of the isolated native, canned and stovetop-cooked bean starches were
investigated using the in vitro Englyst method. The rapidly digestible starch (RDS), slowly
digestible starch (SDS) and resistant starch (RS) contents, and the glycemic indices of the
isolated native, canned and stovetop-cooked bean starches were determined. All isolated native
bean starch samples were lower in RDS (1.55-2.71%) and higher in RS (80.95-83.70%) when
compared to isolated canned bean starch samples and isolated stovetop-cooked bean starch
samples. The RDS contents of isolated stovetop-cooked and canned bean starches ranged
between 7.3-10.61% and 7.58-13.21%, respectively. Native starches are in a relatively noswollen form, without having undergone heating, and thus is less susceptible to enzymatic
hydrolysis whereas isolated canned and stovetop-cooked starches have undergone gelatinization
and have lost some degree of crystallinity. Isolated native light red kidney bean starch had the
largest amount of SDS (17.39%) and the smallest amount of RS (78.95%). Only marginal
differences in the RDS, SDS and RS contents were observed among isolated canned and
stovetop-cooked starches. In general, isolated starches from canned and stovetop-cooked beans
contained greater amounts of RDS and SDS and lower amounts of RS when compared to their
isolated native starch counterpart samples.
The extent of hydrolysis after 180 min among the native starch samples ranged from
24.9% for black beans to 27.3% for light red kidney beans. All isolated native bean starches
showed similar hydrolysis rates during the initial 40 min with small differences in the latter
stages.. Isolated starches from stovetop-cooked and canned beans displayed a greater extent of
starch hydrolysis (39.8-48.4%) than the starches that were isolated from raw whole beans (24.927.3%). The glycemic index (GI) values for isolated native bean starches ranged from 49.41 to
51.5l. Isolated native light kidney bean starch had the highest GI value. The glycemic indices

123

for isolated canned and stovetop-cooked starches from all six bean varieties ranged from 59.03 to
62.81. There were only marginal differences in the GI values among the isolated canned and
stovetop-cooked bean starches. Strong relationships were found between glycemic index and
RDS content for all the bean starches studied. Results, indicate that dry beans are low glycemic
index foods, which make them beneficial in the management of diabetes.
The weight average molecular weights (Mw) of amylose and amylopectin of isolated
native bean starches were determined. Amylose of isolated native dark red kidney bean starch
had the smallest Mw (1.0 x 106 g/mol), whereas amylose of native pinto bean starch had the
largest Mw (1.8 x 106 g/mol). The Mw of amylopectin for isolated native bean starch samples in
the present study ranged from 2.4 x 107 g/mol to 3.9 x 107 g/mol. The above amylopectin Mw
values were lower than those reported in literature (5 x 108-8 x 108). The lower Mw values for
amylopectin may be attributed to the method used to dissolve starch samples as well as to shear
scission and degradation during size-exclusion chromatography (SEC) separation. Findings
from this present study showed the Mw for amylose was postively correlated to GI (r2 = 0.55),
RDS (r2 = 0.50), SDS(r2 = 0.49), and RS (r2 = 0.42) for isolated native bean starches. Mw for
amylopectin was weakly correlated with GI, SDS, RS and RDS values for isolated native bean
starches which suggests that amylopectin Mw is unable to predict the variability observed in GI,
SDS, RS and RDS values obtained.
Isolated canned bean and stovetop-cooked bean starches generally exhibited a
monomodal distribution in their Mw distribution, which indicated that the hydrothermal
treatments degraded starch, which is bimodal in nature upon evaluation with size-exclusion
chromatography.

124

Beneficial properties of dry bean starch confirmed in this research indicate that dry bean
starch can be utilized as a new starch source in the food industry, with unique functional
properties. Findings from this study will not only add to the growing knowledge base of dry
bean starches, but provide information regarding bean starch digestibility, such as digestion rate
RDS, SDS and RS contents, which are of importance for diet-related disorders including obesity
and diabetes.

125

CHAPTER 6
FUTURE RECOMMENDATIONS

126

1. `Findings from this study showed that isolated bean starches have low glycemic indices
(GI). In vitro methods to measure GI may prove helpful for the initial screening of
Phaseolus vulgaris varieties; however, in accordance with the definition, the GI must be
confirmed in vivo by clinical trials. Therefore, studying the oral glucose tolerance and
insulin response (more sensitive) of cooked and canned bean starches in healthy
individuals is recommended.
2. In order to fully link the molecular structure of bean starch to functional properties (eg.,
digestibility) and to make meaningful comparisons with the structures of cereal starches,
it is recommended for future studies that the fine structures of amylose and amylopectin
be characterized. This should include, but not be limited to, average chain length,
average chain length distribution, degree of polymerization and level of branching.
3. The present study examined the molecular weight distribution and physicochemical
properties of isolated starch from single lines of six varieties of Phaseolus vulgaris.
Investigating these same parameters using several lines for each bean variety as well
different genus (e.g., Vigna unguiculata) may yield results that truly represent the species
in general.
4. The weight average weight molecular distribution is affected by the method used to
dissolve starch. Investigating the effects of various methods for starch dissolution is
recommended to determine an optimum method which reduces starch degradation.

127