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This is to certify that the

thesis entitled

DETERMINATION OF DIETARY FIBER, AND TOTAL AND
INDIGESTIBLE STARCH AND PROTEIN IN A SELECTED
SAMPLE OF DRY BEAN (Phaseolus vulgaris L.)

GENOTYPES

presented by

Maria Teresa Ospina

has been accepted towards fulfillment
of the requirements for

MQS her degree in Plant Breeding
and Genetics

   
 

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Date 04-07-00

 

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mm mm."

DETERMINATION OF DIETARY FIBER, AND TOTAL AND INDIGESTIBLE
STARCH AND PROTEIN IN A SELECTED SAMPLE OF DRY BEAN
(Phaseolus vulgaris L.) GENOTYPES

 

By

Maria Teresa Ospina

AN ABSTRACT OF A DISSERTATION

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

MASTER OF SCIENCE

Plant Breeding and Genetics Program
Departments of Crop and Soil Science

2000

Professor George L. Hosfield

ABSTRACT

DETERMINATION OF DIETARY FIBER, AND TOTAL AND INDIGESTIBLE
STARCH AND PROTEIN IN A SELECTED SAMPLE OF DRY BEAN
(Phaseolus vulgaris L.) GENOTYPES
By

Maria Teresa Ospina

Dry seeds of common bean (Phaseolus vulgaris L.) are considered hard to digest

 

and often cause gastrointestinal discomfort. Carbohydrates and protein contribute to this
distress. Dry bean also has low starch digestibility and its incomplete digestion and
absorption in the small intestine leads to fermentation after it enters the colon. In the bean
sample preparation the particle size of cook and raw beans was comparable by grinding
the freeze-dried samples so they would pass through a 40 and 60-mesh screen,
respectively. In order to establish variability among different bean genotypes,
determination of indigestible starch was performed on forty-one different bean genotypes,
and two processing technologies — cooked and raw beans. In the determination of total
dietary fiber, an indigestible residue is obtained. Statistics were used to declare
significance for the variables indigestible residue, and indigestible starch. Cooked beans
had higher amounts of indigestible residue and indigestible starch than raw beans.
Determinations were also made for indigestible protein. In most of the genotypes, higher
mean values of indigestible protein were obtained in cooked bean samples than raw bean
samples. Results indicated that cooking beans increases the amount of indigestible starch

and probably indigestible protein when compared with raw bean seeds.

ACKNOWLEDGEMENTS

I would like to express my gratitude to my committee members, Dr. George L.
Hosfield, Dr. Maurice Bennink, Dr. James Kelly, and Dr. David Douches for their
assistance in the design and execution of my research project. A special thanks goes to
Dr. Bennink for his invaluable help in my lab work. Gratitude is expressed to the
Agricultural Research Service, USDA, Michigan State University, and the Bean/Cowpea

Collaborative Support Program (CRSP) for financial support during this study.

TABLE OF CONTENTS

 

Page
LIST OF TABLES ........................................................................... vii
LIST OF FIGURES .......................................................................... ix
APPENDIX: LIST OF TABLES .......................................................... x
GENERAL INTRODUCTION ............................................................. 1
GENERAL LITERATURE REVIEW
Nutritional Composition of Dry Bean ............................................ 5
Carbohydrates .............................................................. 5
Starch ........................................................................ 5
Dietary Fiber ................................................................ 5
Proteins ..................................................................... 6
Lipids ........................................................................ 7
Minerals ...................................................................... 8
Vitamins ..................................................................... 8
Dry Bean Digestibility ............................................................. 9
Sample Preparation ........................................................ 9
Starch Digestibility ........................................................ 10
Indigestible Starch ......................................................... 11
Breeding for Digestibility in Dry Beans ................................ 12
STUDY 1. METHODOLOGY TO DETERMINE INDIGESTIBLE STARCH
IN DRY BEANS (Phaseolus vulgaris L.)
INTRODUCTION ........................................................................... 1 3
MATERIALS AND METHODS
Plant Materials ............................................................. 17
Sample Preparation ........................................................ 17
Particle Size Distribution ................................................. 18
Total Dietary Fiber (TDF) Assay ....................................... 19
Determination of Indigestible Starch ................................... 21
Glucose Assay ............................................................. 21
Calculations ................................................................ 22
RESULTS AND DISCUSSION
Particle Size Determinations ............................................. 24

V

STUDY II. DETERMINATION OF INDIGESTIBLE STARCH IN A SELECT
SAMPLE OF DRY BEAN (Phaseolus vulgaris L) GENOTYPES

 

Page

INTRODUCTION ........................................................................... 30
MATERIALS AND METHODS

Plant Materials ....................................................................... 33

Field Plot Procedures ............................................................... 33

Indigestible Starch Assay .......................................................... 35

Indigestible Protein Determination ............................................... 36

Statistical Analysis .................................................................. 37
RESULTS AND DISCUSSIONS ......................................................... 39

Breeding Implications .............................................................. 53
GENERAL CONCLUSIONS .............................................................. 58
LIST OF REFERENCES ..................................................................... 60

vi

STUDY I

Table 1.

STUDY II

Table 1.

Table 2.

Table 3.
Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

LIST OF TABLES

Residue obtained from the Total Dietary Fiber (TDF)
Assay and indigestible starch (IS) calculated in bean
Sample treatments cooked and raw, ground with 40 and
60-mesh screens ..................................................

Identification, commercial class, seed coat color, seed
weight, and yield of41 dry bean genotypes. . .

Forms of the analysis of variance for several nutritional
quality factors of 41 dry bean genotypes and two
preparation methods in a split-plot arrangement of
treatments .........................................................

Analysis of variance for the total dietary fiber from raw
and cooked bean samples of 41 dry bean genotypes .......

Analysis of variance for the indigestible starch from raw
and cooked bean samples of 41 dry bean genotypes .......

Means and low, medium and high groupings for the total
dietary fiber of 41 dry bean genotypes prepared by two
methods, raw and cooked .......................................

Means and low, medium and high groupings for the
indigestible starch on a total flour basis of 41 dry bean
genotypes prepared by two methods, raw and
cooked .............................................................

Means and low, medium and high groupings for the
indigestible starch on a mg basis (resistant starch) of 41
dry bean genotypes prepared by two methods, raw and
cooked .............................................................

Means and low, medium and high groupings for the
indigestible protein as a percent of the total protein of 41
dry bean genotypes prepared by two methods, raw and
cooked .............................................................

vii

Page

29

34

38

41

42

43

44

45

47

Table 9. Means squares for the variables total dietary fiber and
indigestible starch of the treatment effect in each
genotype ........................................................... 52

viii

LIST OF FIGURES

STUDY I Page

Figure 1. Methodology to determine indigestible starch in dry
beans .............................................................. 23

Figure 2. Particle Size Distribution in Cooked, Raw, Raw +
Soaked, and Raw + Humidified Bean Samples Ground
Through a 40-Mesh Screen .................................... 27

Figure 3. Particle Size Distribution in Raw and Raw + Soaked
Bean Samples Ground Through a 60-Mesh Screen 28

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

APPENDIX: LIST OF TABLES

Page

Means for total starch and total protein on a total flour
basis for 41 dry bean genotypes by two methods, raw and
cooked ............................................................. 67
Indigestible protein in raw beans in duplicate ‘A” ......... 68
Indigestible protein in raw beans in duplicate ‘B’ .......... 69
Indigestible protein in cooked beans in duplicate ‘A’ 70
Indigestible protein in cooked beans in duplicate ‘B’ 71
Mean squares for the variable indigestible protein (g) for

41 Dry bean genotypes prepared by two methods, raw
and cooked ........................................................ 72
Ranking of each raw bean genotypes for indigestible
starch on a per flour and a per starch basis .................. 73
Ranking of each cooked bean genotypes for indigestible
starch on a per starch basis ..................................... 74

Spearman’s rank correlation coefficient (rs statistic)
between the ranks of raw and cooked beans of 41dry bean
genotypes for indigestible starch on a per g of flour basis
and on a per 100 mg starch basis. ............................. 75

GENERAL INTRODUCTION

Dry seeds of common bean (Phaseolus vulgaris L.) have contributed essential

 

protein, carbohydrates, vitamins, and minerals to human diets for 8 millennia. In general,
bean seeds contain about twice the amount of protein found in cereals. Bean seed protein
is rich in the amino acid lysine but deficient in sulfur-containing amino acids. Cereal
grain proteins, on the other hand, are rich in sulfur-containing amino acids but deficient in
lysine. Thus, bean protein and cereal protein complement one another’s amino acid
deficiency; and, thus, can provide a more utilizable protein to human diets than if each
were consumed individually (Bressani, 1973).

In the new world there is botanical, archeological, and biochemical evidence that
beans were domesticated in the Americas (Debouck et al. , 1988) about 7,000 years ago
(Kaplan, 1965). Wild bean accessions were found widely distributed, from Chihuahua in
Mexico [reported by Nabhan (1985)], to San Luis in Argentina [reported by Burkart and
Brucher (1953), Debouck, et al. , 1988]. Archeological evidence demonstrates that the
oldest bean plants were found in Jujuy, Argentina, Ancash, Peru, and Tehuacan and
Puebla, Mexico. The wide distribution of beans in the American hemisphere shows that
the bean plant can readily adapt to different environments.

Phaseolin, the most important storage protein in P. m, was found to be a
useful marker for evolutionary studies (Gepts, 1988). This usefulness is the result of

phaseolin’s molecular complexity. which can be detected by its migration through an

electric field. The process by which charged particles move through a stationary liquid
under the influence of an electric field is known as electrophoresis. Because of its
complexity each phaseolin type is most likely unique and can, therefore, be used to trace
the evolutionary origin of common bean genotypes (Gepts, 1984 and 1988). Considering
their electrophoretic profiles, different phaseolin types have been identified. The type of
phaseolin found in wild bean populations clearly showed the pattern of bean distribution
in the Americas. Beans with the M and S type phaseolin were found in Mexico,
Guatemala, and Costa Rica. Colombia was considered a transition zone for phaseolin type
because CH and B type phaseolins were found. Peru, Bolivia, and Argentina had I, T, C,
K, H, and J types of phaseolin.

Years after the discovery of the New World by Columbus in 1492, beans were
widely cultivated in Western Europe. Common bean reached continental Europe between
the early sixteenth and seventeenth centuries and England by 1594 [Kueneman et al.,
1978 (cited by Evans, 1976)]. From Western Europe, beans were distributed to the
Southeast of Europe, Iran, India, and some places in Asia and Africa. Most of the bean
varieties consumed in Europe were introduced there from the southern Andean Region.
Beans were also brought back to the Americas, especially to Argentina and USA by
Spanish explorers (Gepts et al., 1988). Seeds of P. m were transported from Brazil
to Africa by sailors engaged in the slave trade.

On a weight basis dry beans contain about 63% total carbohydrate, 22% crude
protein, 9% water, 4% ash, and 2% fat (Watt and Merril , 1963). The carbohydrate
fraction of bean seeds is made up of dietary fiber which is about 17%, starch about 40%,

2

and sugars about 6%. Sugars found in beans are sucrose (2.8%), raffinose (0.4%),
stachyose (2.9%), and about 1% of other hexoses and pentoses (Tomkinson, 1986). Crude
fiber is considered an old term and its use is being discontinued because the method of
analysis that includes extraction of the plant material first with acid and then with alkali,
recovers only variable portions of cellulose, hemicelluloses, and lignin present in dietary
fiber (Spiller, 1986). The definition of dietary fiber includes cellulose, hemicellulose,
pectin, lignin, and intracellular polysaccharides such as gums, mucilages, and starch that
is not digested by mammalian digestive enzymes (Trowel et al., 1976).

Dry beans are considered hard to digest and often cause gastrointestinal
discomfort. Many people experience flatulence, cramps, and diarrhea after eating beans
(Fleming, 1981). Both, carbohydrates and protein contribute to gastrointestinal
discomfort. Nutritionists commonly accept the fact that beans have low protein
digestibility, because high levels of nitrogen are lost when beans are consumed. Beans
also have low starch digestibility with both intrinsic factors and cooking method
contributing to this phenomenon (Hellendom, 1969). Englyst et a1. (1985) and Cummings
(1983) have shown that incomplete digestion and absorption of starch in the small
intestine lead to fermentation of the undigested starch after it enters the colon.

Since the 19605, research has shown that the nature of starch (amylose and
amylopectin) is the cause of its low digestibility in a wide range of foods (Borchers,
1961; Sandstedt et al. , 1962). Thome et al. , (1983) showed that amylose and amylopectin
differed in biochemical reaction to enzyme digestion. Recently, Chung (1996) suggested
that starch indigestibility in P. m was mainly due to the crystallization of cell walls

3

in the cotyledon. The crystallization apparently encapsulates the starch granules; thus,
rendering them inaccessible to attack by digestive enzymes (Chung, 1996).

Starch indigestibility in dry bean may be under genetic control. Murphy (1973)
suggested genetic selection for improved starch digestibility might lead to beans with
reduced flatulence. The bean variety ‘Pike’s Jacob’s Cattle’ was reported to be a gasless
variety. One of its parents was ‘Jacob’s Cattle,’ a reported gasless variety, and the other
parent was a Black Mexican variety. Evaluation of human intestinal gas formation after
consumption of ‘Pike’s Jacob’s Cattle’ showed that the amount of intestinal gas evolved
was less than what one would expect among cultivars of P. m. Murphy (1973)
concluded that flatulence appeared to be under genetic control and can be reduced by
genetic selection. From the foregoing, one can formulate several questions regarding
indigestibility and flatulence from eating beans. Is it possible that some bean varieties are
more digestible than others because of differences in starch and protein quantity and
physical-chemical characteristics? Since the majority of the macromolecular content of a
bean seed is starch, how digestible is starch? Is starch digestibility under genetic control?
Are varietal differences in starch digestibility large enough to be altered through
selection? Does cooking make starch more indigestible than the starch in raw beans?

The answers to the proposed questions formulated the basis for the present study.
The research objectives were to: 1.) Develop suitable methodology for measuring
indigestible components in bean seeds, 2.) Determine if there is variability of indigestible
components, especially starches in bean seeds, and 3.) Measure the range of variability
for indigestible components in bean seeds among a diverse group of genetic stocks.

4

GENERAL LITERATURE REVIEW

Nutrient Composition of Dry Beans

Carbohydrates

 

The carbohydrate content in dry bean seeds is about 60% on a dry basis (Kay,
1979; Price et al., 1988). Monosaccharides, oligosaccharides, polysaccharides comprise
the carbohydrates in beans. The largest portion of the bean carbohydrate fraction is starch,
a storage polysaccharide. In the literature reported values for starch content in dry beans
vary from 24 to 56% due to differences among cultivars and analytical procedures
(Cerning etal., 1975; Lai etal., 1979; Reddy et al., 1984).

M.

Starch is composed of two different glucose polymers — amylose and
amylopectin, and in dry bean seeds, starch is organized into semicrystalline granules.
Amylose is a long chain of glucose units linked by 1,4-oc-D-glucopyranosidic bonds.
Amylopectin is a branched structure of glucose molecules with interconnecting
1,6-oc-D-glucopyranosidic bonds (Dreher et al., 1984; Whistler et al. , 1984). The
molecular weight of amylopectin is higher than that of amylose. Unlike amylopectin,
amylose has glucose chains with strong (1-4) bonds, which make it difficult for
enzymatic digestion (Thome et al., 1983).

Dietary Fiber.

 

Dietary fiber is composed of those portions of plant cells that are not digested by

human enzymes, such as polysaccharides (cellulose, hemicellulose, oligosaccharides,
pectins, gums, waxes) and lignin (Trowell et al. , 1976). Starch was also found not to be
totally digested by alimentary enzymes. This starch is called resistant starch
(Englyst et al. , 1987). Two types of dietary fiber are distinguished -- soluble and
insoluble. The content of dietary fiber in P. m ranges from 17.5 to 20%; thus, dry
beans are considered a good source of dietary fiber (Prosky et a1. , 1985). The type and
amount of fiber consumed determines the variation of function of dietary fiber in the
gastrointestinal tract and utilization of nutrients. Lignin, cellulose, and hemicellulose are
considered insoluble forms of dietary fiber. Pectin, gums, mucilages, and starch constitute
the soluble dietary fiber. Water-insoluble dietary fiber affects the passage of materials in
the gastrointestinal tract and reduces the absorption of nutrients. Water-soluble dietary
fiber affects the metabolism of carbohydrates and lipids (Munoz, 1986). The plant
species, its age, and growth conditions determine properties and metabolic effects of
dietary fiber. This may explain some of the variation in experimental results obtained by
different researchers, even when using the same technique. Chemical methodologies
allow estimation of total, insoluble and soluble dietary fiber (Lee et al., 1992).
EM

Protein content in legumes is among the highest found in the plant kingdom and is
considerably greater than in the cereals. Bean protein ranges from 19 to 30% with an
average of 25% (Bressani, 1973; Tomkinsom, 1986). Cereals contain about 9 to 18%

protein. Oat (Avena sativa L.) is the cereal with the highest protein content, about 18%.

 

True protein digestibility of legumes varies from 59.5% in Cajanus to 93.9% in Pisum

6

sativum, and evaluations in P. vulgaris showed variations among 76.8 to 84.1% (Jaffe,
1973; Bressani, 1973).
Variability for protein content in P. vulgaris, as well as other nutrients and amino

acids, is affected by variety and location (Bressani, 1973). Bean proteins have higher
amounts of lysine and threonine than methionine and cystine. Salt-soluble globulin —a

storage protein of B. v_ulgg's_— can be resistant to enzymes that may account for the low
digestibility of protein (Seidl et al. , 1969).
M

Most legumes are low in total lipid content. Lipids in legumes are composed of
neutral lipids (e.g., triacylglycerol), phospholipids (e.g., phosphatidycholine), and
glycolipids (esterified steryl glucoside). Neutral lipids are the predominant class of lipids
that vary from 32 to 51%, of the total lipid fraction. Phospholipids constitute 23 to 38%,
and glycolipids 8 to 12% of total lipids (Salunkhe et al. , 1989). The content of lipids
increases 20% during seed maturation. Neutral lipid content increases faster than
phospholipids or glycolipids during this stage of the plant growth cycle. Phospholipids
and glycolipids are part of the seed membrane (Salunkhe et al. , 1989). Components of the
fatty acids such as palmitic, oleic, linoleic, and linolenic acids were found in neutral
lipids, phospholipids, and glycolipids of legumes (Mahadevappa et al., 1978). The
content of lipids in beans is generally less than 2.0% (Watt et al. , 1963; Tomkinson,
1986). Drumm et al., (1989) showed a 1.8 to 2.6 g per 100 g dry weight lipid content

among four bean market classes (navy, dark red kidney, pinto, and black turtle soup).

Drumm et al., (1989) also found that linolenic acid, an unsaturated fatty acid and palmitic
and stearic acids; both saturated fatty acids, were the major fatty acids in these beans.
Significant differences were found among the four bean classes for each lipid component:
neutral lipids, phospholipids, and glycolipids (Drumm et al., (1989).
M

The mineral content of a substance is found by heating at 500°C until all that
remains is ash. Legumes are a good source of minerals like calcium, iron, magnesium,
copper, zinc, and potassium (Salunkhe et al. , 1985). The amount of calcium found in
legumes is higher than that found in cereals (Douty et al. , 1982). The content of ash
(minerals) in P. M varies from 3.5 to 4.1% (Kay, 1979). Minerals and their
association with other components can be altered with processing of food. Mineral
availability can be inhibited by dietary components such as proteins and complex
carbohydrates.
Vitamins

Legumes are sources of thiamin, niacin, riboflavin, and folic acid (Salunkhe,
1989). Kasper (1986) stated that there are two possibilities of how dietary fiber
substances effect vitamin metabolism. First, dietary fiber can effect ingested vitamins, by
reducing their absorption from the intestine. Studies with humans and animals have
demonstrated that the absorption of vitamin A, carotene, and vitamins B,, B6, 8,, is
influenced when lignin, pectin, and cellulose are added to the diet. Second, intestinal

vitamin synthesis is effected by the interaction between dietary fiber and vitamin

metabolism. Experiments with animals showed that carbohydrates, which are difficult to
digest, such as raw potato starch, pectin, and cellulose stimulate the synthesis of B

vitamins and vitamin K in the intestine (Kasper, 1986).

Dry Bean Digestibility

Sample Preparation

 

Drying of samples before grinding is important to assure consistency in results.
Particle size may also affect the results of digestibility evaluations (Horvath et al., 1986).
When drying bean flour above 60°C or in a microwave oven, in vitro values of dietary
fiber and lignin increased (Heller et al. , 1977); in the same way, when particle size
decreased, dietary fiber decreased as well as other fractions varying non-uniformly
(Heller et al. , 1977). Heller et a1. (1977) concluded that the optimum way to dry samples
of food for grinding was by freeze-drying.

There are different methods in sample preparation for studies of indigestible
components of food that mimic human digestion. Food can be chewed before enzyme
digestions (Muir et al. , 1993) or it can be pulverized through a mincer with 0.9 cm
diameter holes (Englyst et al. , 1992). The mincing methodology to prepare food samples
for digestibility studies gave more consistent data than when the samples were chewed
(Englyst et al. , 1992). Although chewing of samples has its limitations in determining
indigestible residue in food, this method may be more appropriate than milling or

homogenizing samples (Muir et al., 1994).

Starch Digestibility

 

Starch digestion occurs through the action of salivary and pancreatic a-amylases,

which produce maltose and a-dextrins. Maltose in turn is catabolized by intestinal
glucosidases that produce glucose (Manners, 1978). Digestibility in dry bean depends on
the condition of the seed and on cooking time. Hellendoorn (1973) stated that starch
digestibility in raw bean increases 20% after soaking, and can increase rapidly up to 95%
after one hour of cooking under pressure. Chung (1996) found that when beans were
cooked for a long time, the cell walls of cotyledons become rigid, encapsulating the
granules; thus, producing a barrier to enzymatic hydrolysis. The rate of digestion of
starch and sugars in beans is determined by the physical form of the food and not by the
large complex polymers of starches (Wahlquist et al. , 1978; Jenkins et al., 1987).
Availability of glucosidic bonds of starch to enzyme attack can determine the starch
digestion rate. Starch is incompletely digested in the small intestine (Englyst et al. , 1992).
The amount of starch that escapes digestion in the small intestine varies depending on the
food preparation, the starch form, and the method of analysis. Undigested starch can vary
from less than one percent to more than 20% (Schweizer, et al. , 1990). Starch that
escapes digestion within the small intestine, enters the colon where it serves as an energy
source for colonic bacteria (Cummings, 1983). Digestion of starch can be altered by
dietary fiber components, such as cellulose, hemicellulose, and lignin. The protease and
amylase inhibitors in P. Maris can reduce starch digestion, as well as the lectin residues

present after the cooking process. On the other hand, starch digestion may interfere with

10

the availability of proteins, lipids, minerals, and vitamins.

Indigestible components, such as dietary fiber, effect the functioning of the
digestive processes in humans by decreasing the availability of nutrients. Nutrient
assimilation requires movement of food through the digestive tract, enzymatic hydrolysis
of complex nutrients, and absorption of smaller compounds in the small intestine.
Schneeman et al., (1986) concluded from in vitro experiments that dietary fiber restrains
digestive enzyme activity and may slow starch or carbohydrate hydrolysis. Any
undigested dietary constituent that is not digested in the small intestine can act as a
substrate for fermentation in the large intestine (Fleming, 1986), and dietary fiber is
considered a fermentable substrate. Beans are a high fiber food that often cause quantities
of intestinal gas to be produced (Calloway et al., 1971).

Indigestible Starch

 

Several techniques reported by Johansson etal., (1984); Holm et a1. , (1986);
Tovar et al. , (1990); Englyst et al. , (1992), and Muir et al., (1993) for determination of
indigestible starch utilized the same general principle, which is the digestion of starch by
the enzyme a-amylase producing dextrins and maltose. These components are removed
by filtration or centrifugation. The remaining starch (resistant starch) is determined by
solubilizing the starch with 2 M KOH, and digesting the resistant starch to form glucose
with amyloglucosidase. Glucose is quantified to estimate the equivalent amount of

resistant starch.

ll

Breeding for Digestibility in Dry Beans

 

Since the 19605, the improvement of food quality in dry bean has been an
important plant-breeding objective. Breeders have used various approaches to raise the
protein content, remove anti-nutritional compounds, improve the amino acid balance and
digestibility, and lower flatulence in beans. However little breeding work has been done
to improve digestibility of carbohydrates including starch. Murphy (1973) mentioned that
genetic selection might be feasible in dry bean to lower flatulence of which undigested
starch is a large contributor (Tomkinson, 1986). There is a paucity of information on the
genetic variability of indigestible components in beans including protein and starch to

provide guidance for the breeder in plant improvement programs.

12

STUDY I

METHODOLOGY TO DETERMINE INDIGESTIBLE STARCH IN DRY BEANS
(Phaseolus vulgaris L.)

INTRODUCTION

In dry bean (Phaseolus vulgaris L.) the improvement of digestibility and food

 

quality of cooked seeds is an important research objective for food technologists,
nutritionists, and plant breeders. The incomplete digestion and absorption of food in the
small intestine lead to the fermentation of the undigested chemical constituents after they
enter the colon. Seed proteins and carbohydrates are the main indigestible components in
dry bean. Of the carbohydrate fraction, starch can be a significant source for fermentation
in the large intestine (Tovar et al. , 1990). Indigestible starch in dry beans causes
gastrointestinal discomfort including flatulence, cramps, and diarrhea (Fleming, 1981).
Persons in the food industry and consumers generally agree that dry bean
consumption would increase if the digestibility of this food crop could be improved and
the associated gastrointestinal difficulties ameliorated. There is anecdotal (‘Jacob’s
Cattle’ bean) and experimental evidence (Murphy, 1973) that genetic variability exists in

dry beans for flatulence and related gastrointestinal discomfort. However, the flatulence

described by Murphy (1973) was probably mostly due to oc-oligosacharrides.
Indigestible starch can be determined in vitro in a step-wise fashion that includes

the digestion of bean flour with enzymes, solubilization of resistant starch, and the

13

enzymatic degradation of resistant starch to glucose. Enzymatic digestions can be

preformed using the total dietary fiber (TDF) assay suggested by Lee et al. (1992). In this

assay, three sequential enzymatic digestions are carried out by a-amylase, protease, and
amyloglucosidase, where each enzyme has a specific function in the process of digestion;
the resulting TDF is also called indigestible residue (IR). The indigestible starch
remaining in TDF is determined by solubilizing the indigestible starch with a base (e.g.,
2N NaOH), and digesting the starch with amyloglucosidase to produce glucose. Finally,
the amount of glucose is determined and equated to the quantity of indigestible starch in
the sample.

Nutrient determinations in most foods are determined on the raw product.
However, dry beans are generally soaked and must be cooked to render the seeds
palatable, inactivate heat labile anti-nutrients, and permit the digestion and assimilation of
protein and starch (Deshpande et al. , 1983). Wassimi et al. , (1988) found a significant
amount of protein loss in soaked and cooked beans. Moreover, the variation in protein
loss found in the study by Wassimi et al. (1988), demonstrated that consideration other
than the nutrient content of raw beans is important. Because of the possibility of nutrient
loss in beans during their preparation for eating, it would be logical to determine
indigestible starch on both raw and cooked beans. Moreover, Chung (1996) found that
when beans are cooked, the cell walls of cotyledons become rigid and encapsulate starch
granules. Chung’s (1996) research provides evidence that cooked bean starch may be less

digestible than raw bean starch.

14

For analysis of food components, samples should be homogeneous before
subsampling. A lack of homogeneity between subsamples may lead to a large sampling
error in the experiment. Freeze-drying before comminution is recommended for sample
preparation (Heller et al. , 1977) because freeze-drying reduces undesirable changes in the
sample. The comminution of a food may be accomplished either by chewing or milling.
The chewing technique is a natural method of food comminution because it gives a better
approximation of the physiological digestive process than does the milling method.
Although chewing the sample may be more appropriate from a physiological stand point,
milling and homogenizing are preferred to obtain consistent results. Milling and
homogenizing a sample may underestimate the true resistant starch; however, the data
would be consistent and reproducible from laboratory to laboratory.

In many cases the particle size of the food sample should be reduced for analysis
(Horwitz, 1988) and for homogenization. Mills are used to reduce food into small
particles, the size of which simulates food in the intestinal tract. Mills are fitted with
screens that control the size of particles. For dried foods, particles of 20-mesh screen size
should be used for determining moisture, total protein, or mineral content (Proctor et al. ,
1998). Particles for lipids and carbohydrate analysis should be of a size to pass through a
40-mesh screen (Proctor et al., 1998). In preparing bean samples to evaluate digestibility,
one must ensure that the particle sizes between raw and cooked samples are comparable.
The importance of particle size in digestion studies was reported by Cummings (1986),
who found that when the same source of fiber was fed at two different particle sizes,
greater stool output occurred with the larger particle size. The result of Cumming’s work

15

(1986) suggests that larger particles are more slowly digested than smaller ones. The
hypothesis that particle sizes between raw and cooked beans are different is tenable.
Soaking and cooking cause structural changes in cells that most likely effect the size of
particles resulting after beans are ground.

Because of the importance of indigestible starch in human nutrition and consumer
acceptance of beans as a food, this study was undertaken. The specific objective was to
develop an appropriate methodology to determine indigestible starch in dry bean that was

accurate, repeatable, and useful to plant breeders.

16

MATERIALS AND METHODS

Plant Materials
‘Montcalm’ dark red kidney bean was the experimental material on which particle

size distribution and indigestible starch determinations were made. ‘Montcalm’ has a

typical kidney shaped seed that weighs approximately 65 g - 100 seed". ‘Montcalm’ was
grown in the nursery during the summer of 1996 on a M°Bride Sandy Loam (Coarse -—
loamy, mixed, fi'igid Alfic Fragiothods) soil at the Montcalm Research Farm near

Stanton, MI. Seeds were harvested from mature plants, cleaned of plant and soil debris

and stored in Kraft® paper bags at 16°C until laboratory evaluations were made.
Sample Preparation

Four treatments were imposed on ‘Montcalm’ dark red kidney bean to ascertain
the effects of sample preparation and particle size on the amounts of TDF and indigestible
starch.

Treatment I- cooked beans. One hundred-gram samples of beans were soaked in
distilled water in a beaker for 12 hours. The beans were drained and placed into a 1.0-L
beaker to which 500-g of distilled water was added. The beakers were placed on a hot
plate and brought to a slow boil. Beans were cooked until eating soft which took
approximately one hour. Eating soft was determined tactilely by placing individual beans
between the thumb and index finger and lightly squeezing the beans. Beans that ruptured

readily upon the light compression force and had a paste-like consistency were

17

considered eating soft. After the beans were determined eating soft, the water remaining
in the beaker was drained, and the beans cooled and lyophilized in a Virtis-Genesis 12EL
freeze dryer.

Treatment 2- raw beans. One hundred-gram samples of raw beans were used
without soaking and lyophilizing.

Treatment 3- soaked raw beans. One hundred-gram samples of beans were
soaked for 12 hours in a beaker containing 500-g of distilled water. After the beans were
soaked, the beans were drained, dried, and lyophilized in a Virtis-Genesis 12EL freeze
dryer.

Treatment 4- raw and humidified beans. One hundred gram samples of beans
were placed in a humidity chamber and held at 40°C until beans gained about 50% of
their initial weight. The beans were lyophilized in a Virtis-Genesis 12EL freeze dryer.
Particle Size Distribution

Bean samples from each treatment, i.e., cooked, raw, raw + soaked, and raw +
humidified were ground to a flour using a Wiley mill fitted with a 40-mesh screen. After
the samples were ground, the particle size distribution of the flour was determined on an
instrument containing screens that were layered from top to bottom and with the
following mesh sizes: 26, 40, 48, 54, 74, 94, and 120. The ground bean flour from each
sample from each treatment was weighed and placed on top of the 26-mesh screen (top
most screen). The instrument was shaken for 10 minutes to allow different sized particles

to percolate through the various screens. The residues were then collected from each

18

screen and weighed. The flour that passed through the l20-mesh screen was collected in
the pan at the bottom of the screens. The amount of a bean flour sample collected in each
of the mesh screens from each of the treatments was reported as a percentage of the initial
weight of bean flour in grams placed in the 26-mesh (top) screen.

Bean flour samples from the raw and raw + soaked treatments were ground with a
Wiley mill fitted with a 60-mesh screen, and the particle size distribution determined in
the same way as for the samples ground through the 40-mesh screen described above.
Total Dietary Fiber (TDF) Assay

The determination of indigestible starch in beans includes digestions with
enzymes, solubilization of starch, and enzymatic degradation to glucose. Enzymatic
digestions were performed using the Total Dietary Fiber (TDF) assay (Fig. 1) suggested
by Lee et al., (1992). In this assay, three sequential enzymatic digestions were carried out
by a-amylase, protease, and amyloglucosidase, where each enzyme had a specific
function in the process of digestion. The indigestible starch remaining in TDF was
solubilized with 2 N NaOH, digested with amyloglucosidease, and the glucose that
resulted from the enzyme digestion was determined spectrophotometrically.

Total dietary fiber (TDF) was determined on the cooked, raw, and raw + soaked
treatments (Fig. 1). The raw + humidified treatment was eliminated from further
consideration because the particle size distribution profiles were greatly discordant from

the raw and cooked particle size distributions. The amount of bean sample used for each

assay was 1.000 :t 0.005 g. Duplicate 1.000 i 0.005 g samples of each of the three

19

preparation treatments were placed into a 400-ml beaker to which 40 ml of a
2-(N-Morfolino) ethanesulfonic acid/Tris (hydroxymethyl) aminomethane (MES/TRIS)

buffer pH 8.2 solution was added and stirred at low speed until all clumps disappeared.
Heat-stable a-amylase solution (50 ul) was added to the solution while stirring. Beakers

were covered with aluminum foil and placed into a water bath at 95°C with constant
shaking for fifteen minutes. The beakers were then uncovered, placed in a water bath at
60°C. Distilled water (10 ml) was added to each beaker to rinse the beaker walls. A
protease solution (100 pl) was added to each beaker and the samples incubated at 60°C

with constant agitation for 30 minutes. A 5 ml solution 0.561N hydrochloric acid (HCL)

was added to each beaker and pH was adjusted to a range of 4.0 - 4.7 using either 1N
HCL or 1N sodium hydroxide (NaOH). Amyloglucosidase solution (300 pl) was added
to each beaker while stirring. Beakers were covered with aluminum foil and placed in a
water bath to incubate at 60°C for 30 minutes. A 225 ml of a 95% ethyl alcohol solution,
which was brought to 60°C, was added to each digested sample. Samples were left to
stand for one hour at room temperature to precipitate. The samples were filtered under
vacuum through crucibles with sintered glass. The residue remaining in each crucible was
washed twice, first with 78% alcohol, then 95% alcohol, and finally acetone (15 ml each)
during the filtering process. The residue remaining in the crucibles was considered the
dietary fiber (indigestible residue). This residue was left in the crucibles, which were
dried in an oven at 105°C overnight. After the crucibles were removed from the oven they

were cooled in dessicators and weighed to the nearest 0.1 mg. The total dietary fiber

20

residue from each sample was used to determine the amount of indigestible starch.
Determination of Indigestible Starch

The residue of each sample obtained from the TDF assay was gently scraped from
the crucible and transferred into beakers and weighed. A 2N NaOH solution (IO-ml) was
added to each beaker while stirring to solubilize the starch (Fig. 1). When the residue in
the beakers was completely mixed, beakers were allowed to stand at room temperature
for one hour. Ten ml of a 1M (pH 4.5) acetate buffer was added to each beaker and the
pH of the solution was adjusted to 4.5 using 6N HCL. Water was added to each beaker to
bring the net weight to 100 g, and 400 pl of the solution was pipetted into each of four
small screw-capped tubes (four tubes per beaker). Three units of amyloglucosidase (from
Aspergillus niger, Boehringer Mannheim) in 100 ul of 0.1 M acetate buffer (pH 4.5) was
added to each screw-capped tube. The tubes were incubated at 55°C overnight. Glucose
was determined from 40 pl of the solution from each screw-capped tube.
Glucose Assay

A working glucose standard was prepared in duplicate by pipetting into test tubes
10, 20, 30, and 40 pl of a standard solution containing 100 mg/dL of glucose. Distilled

water was added to each test tube to bring each sample to a total volume of 40-pl.

Duplicate reagent blanks of 40-pl of distilled water were used. One ml of the combined
enzyme color reagent (SIGMA Kit No. 510A) containing Glucose Peroxidase (PGO) and
o-Dianisidine dihydrochloride, was added to each test tube, mixed and incubated for

45 minutes in the dark at room temperature (Fig. 1). Samples were analyzed by

21

transferring 40 pl of the amyloglucosidase digest solution into test tubes containing 1 ml
of the combined-enzyme color reagent. The tubes were mixed and incubated in the same
way as the standards. Absorbance was read with a spectrophotometer at 450 nm using
micro plastic cuvettes. Distilled water was used to zero the spectrophotometer. The four
readings from each solution were averaged. Absorbance versus glucose concentration was
used to calculate the slope and intercept of the glucose standard curve.
Calculations

A constant multiplication factor (mf) from dilutions in each of the three assays
was calculated (Fig. 1, Footnote m). The factor mfl represents the amount of residue used
for the determination of resistant starch taken from the total residue weight (indigestible
residue) during the total dietary fiber assay. Factor mf2 (Fig. 1, Footnote ‘2’) is for the 0.4
ml that was removed from beakers containing 100 ml of the solution during the assay of
resistant starch determination. The factor mf3 (Fig. 1, Footnote ‘3’) represents the 40 pl
taken from 500 pl of the solution during the amyloglucosidase hydrolysis of resistant
starch. The 0.9 value is the equivalent to the weight of glucose as starch is being formed,

since 0.1 is liberated as water when glucose molecules are added to the polymer.

The product between the amount of glucose (pg) found in 40 pl of the solution
and the factors mfl , mf2, mf3, and 0.9, was taken as the amount of indigestible starch

present in a bean flour sample.

22

BEAN SAMPLE PREPARATION

U
TOTAL DIETARY FIBER

 

 

(Removal of sugars and digestible starch and protein)

1.0 g bean sample
U
Heat-Stable (Jr-Amylase
(Gelatinization and starch hydrolysis)

U
Protease
(Protein hydrolysis)

U
Amyloglucosidase
(Starch and dextrin hydrolysis)
U

Ethanol Precipitation, Filtration, Washes

U
Dry residue (total fiber)

U

INDIGESTIBLE STARCH DETERMINATION

Solubilization (2 N NaOH)

U
Brought to pH 4.5 and diluted to 100 ml

U
400 pl taken to be hydrolyzed ' " ‘2’

*(l)

 

Amyloglucosidase (100 pl)
(Digestion of resistant starch)

Incubation
U

40 pl taken for glucose analysis

U

GLUCOSE ANALYSIS

nitric.)

 

(Sample digestion)

U

Combine enzyme-color reagent
U

Determination of absorbance with Spectrophotometer

" ”’ Step that corresponds to mfl; "‘ " ‘2’ Step that corresponds to mt2; " " ' ‘3’ Step that corresponds to mB

Figure 1. Methodology to determine indigestible starch in dry beans.

23

RESULTS AND DISCUSSION

Particle size determinations

The flour from the cooked, raw, raw + soaked, and raw + humidified bean
treatments had different particle sizes when they were ground with a Wiley mill fitted
with a 40-mesh screen (Fig. 2). For ground beans that were cooked, about 90% of the
particles were extremely fine and passed through the 120-mesh sieve. The remaining 10%
of the sample was collected from the 74, 94, and 120 mesh-screens (Fig. 2A). Particles of
the raw and raw + soaked bean treatments were larger than for the cooked bean treatment,
and less than 1% of the particles passed through the 120—mesh screen (Figs. ZB and 2C).
For these two-sample preparation methods, the majority of the flour particles were
collected from the 74, and 94-mesh screens. About 6, 15, 33, 14, and 24% of the flour
from the raw + humidified treatment were collected from the 48, 54, 74, 94, and 120-
mesh screen, respectively (Fig. 2D).

Because the particle sizes of the raw and raw + soaked bean treatments were not
comparable to the cooked bean samples, raw and raw + soaked beans were ground in a
mill fitted with a 60-mesh screen. When the particle size distribution was determined on
the raw bean treatment, only about 1% of the flour sample passed through the 94-mesh
screen (Fig. 3A). When raw beans were soaked for 12 hours and freeze-dried before
grinding (raw + soaked treatment), about 9% of the sample passed through the 94-mesh
screen. About 52% of the flour from the raw + soaked treatment passed through the 120-
mesh screen (Fig. 3B). Examination of data indicated that the cooked beans ground in a

24

mill fitted with a 40-mesh screen and raw + soaked beans ground in a mill fitted with a
60-mesh screen were more similar in particle size distribution (Figs. 2A and 3B) than the
other profiles. The distribution of particle sizes from the humidified bean treatment did
not follow the pattern of the other samples (Fig. 2D). For this reason this treatment was
not used for the indigestible starch determinations. When raw beans (not soaked or
humidified) were ground in mills with either a 40 or 60-mesh screen, frequent screen
breakage occurred. However, raw beans that were soaked for 12-hours or humidified and
freeze-dried rarely caused screens to break when the beans were ground. Soaking and
freeze-drying of raw + soaked beans appeared to facilitate an easy grinding of the
samples and essentially eliminated screen breakage. Although hardness tests were not
conducted, the particles from the freeze-dried raw + soaked beans may have been more
friable than particles from raw beans not soaked and freeze-dried. Alternatively, the
smaller and more uniform particle sizes associated with ground raw beans that were
soaked and freeze-dried before grinding may have exerted less force against the screen
and, thus, caused less screen damage than the larger and less uniform particles that
resulted when raw beans without soaking or freeze drying were ground.

Once a satisfactory method to ensure comparable particle sizes of raw and cooked
beans was achieved, an analytical procedure to determine the amount of indigestible
starch in a sample could be developed. After many preliminary experiments, the
analytical procedure finally chosen (Fig. 1) enabled the direct and quantitative
determination of TDF and indigestible starch on the same sample.

The amount of indigestible starch detected was 22.7% in the cooked bean sample

25

and 1.5% in the raw + soaked freeze-dried bean sample, when both samples were ground
using a 60-mesh screen (Table 1).

Grinding beans and sieving the resultant flour through the appropriate screen to
give raw and cooked beans similar particle sizes eliminated the problem of
overestimating the amount of indigestible starch in raw beans compared to cooked beans.
The importance of particle size in digestion studies was reported by Cummings (1986),
who found that when the same source of fiber was fed at two different particle sizes,
greater stool output occurred with the larger particle size. The result of Cumming’s work
(1986) suggests that larger particles are more slowly digested than smaller ones. When
raw beans were ground and sieved through a 40-mesh screen the larger particles
compared to the particles from cooked beans ground and sieved through the same
40-mesh screen were more slowly digested leading to an inflated value of indigestible
starch. The accurate estimation of indigestible starch in dry bean should lessen errors in
data and provide plant breeders with a protocol to screen breeding lines for starch

digestibility with a high degree of precision.

26

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26

A. Raw

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Figure 3. Particle Size Distribution in Raw and Raw + Soaked Bean Samples
Ground Through a 60-Mesh Screen.

28

 

Table 1. Residue obtained from the Total Dietary Fiber (TDF) assay and
indigestible starch (IS) calculated in bean sample treatments, cooked and raw, ground

with 40 and 60-mesh screens.

 

40-mesh screen

60-mesh screen

 

 

Total dietary Indigestible Total dietary Indigestible
Fiber Starch fiber Starch -
Treatment (mg) (mg/ g of flour) (mg) (mg/g of flour)
Cooked 393.4 22.7 - -
Raw“ 330.8 13.6 238.7 2.7
Raw + soaked 251.9 2.0 195.6 1.5
Humidified 317.6 10.8 - -

 

* Grinding raw beans through a 60-mesh screen was difficult because frequent

screen breakage occurred.

29

STUDY II
DETERMINATION OF INDIGESTIBLE STARCH IN A SELECT SAMPLE OF

DRY BEAN (Phaseolus vulgaris L.) GENOTYPES
INTRODUCTION

During most of the 20‘h century, crops used for human consumption have been
improved through plant breeding for numerous characteristics of which those of high
economic importance receiving the most attention. Yield and pest resistance are generally
the major goals of breeding programs. Not with standing the importance of yield,
breeders do not neglect food-quality improvements. Food quality includes characteristics
that have a direct impact on human nutrition and those related to preparation for eating
(culinary).

Breeding for a new trait of interest includes basic steps. First, the breeder must
decide on a screening procedure. Second, germplasm is screened, and then useful genetic
variability is determined. Finally, parents are selected for inheritance studies. The
screening technique used must be accurate, reproducible, capable of dealing with small
samples, and cost effective. In the evaluation of germplasm, adapted genotypes, elite

lines, exotic lines, and introductions can be included.

 

Dry edible bean (Phaseolus vulgaris, L.) is an important food globally. Many
developing nations depend on beans for nutrition, because often protein from animal
sources is scarce and/or expensive. In addition to high protein, beans are a source of

30

vitamin B, iron, complex carbohydrates, and soluble fiber. In developed countries such as
the US, United Kingdom, and Canada, replacing high meat and high fat diets with beans
may lower cholesterol, and may be associated with a lower incidence of stroke, heart
disease, and colon cancer. With changes in nutritional guidelines and culinary habits one
may anticipate an increase in the domestic consumption of dry beans. Annual
consumption of dry beans in the US. has increased from 5.4 to 9 lbs. since 1980 (Lucier,
1999). This increase in consumption may be the result of the recognition that including
beans in the diet may confer important health benefits.

Despite wide acknowledgement of the crop’s nutritional superiority compared to
cereals, root crops, and other vegetables, beans are underutilized partly because they can
cause gastrointestinal discomfort after they are ingested. The major discomforts
associated with eating beans are cramps, flatulence and diarrhea (Tovar et al, 1990).
There is anecdotal evidence that some beans are easier to digest than others and do not
cause gastrointestinal problems.

The problems of low digestibility of dry bean and its physiological effects are of
interest to nutritionists and plant breeders. Indigestible starch in beans may be the greatest
contributor to flatulence and related gastrointestinal discomfort among the seed
constituents. Chung (1996), stated that crystallization of the cell walls in the cotyledon
acts as a physical barrier to enzyme hydrolysis and swelling of the starch granules in
cooked beans. Chung (1996), found that the longer it took beans to cook, the greater the
crystallization of the cell walls. In addition, there was variation of cell wall rigidity
between navy and pinto beans.

31

The possibility for genetically improving bean digestibility has been suggested
by Murphy (1973). Variability of flatulence response was observed in humans after
subjects ate beans from a cross between a reportedly gasless bean variety with a ‘normal’
bean genotype (Murphy, 1973).

Because of the interest by researchers in improving digestibility in beans and the
inclusion of a digestibility improvement component into a breeding program, this study
to ascertain variation in starch digestibility was undertaken. The main objective of the
research was to measure in vitro starch digestibility in a selected sample of 41 dry bean
genetic stocks. A second objective was to ascertain the potential of specific genetic stocks

as parents in a breeding program to improve starch digestibility and reduce flatulence.

32

MATERIALS AND METHODS

Plant Materials

Forty-one genetic stocks (entries) of dry bean (Table 1) from various US. and
international breeding programs formed the experimental material for this study. The
forty-one entries (Table 1) differed from one another for seed color, shape, and size, the
commercial class, and yield. The entries had been previously evaluated for adaptation to
Michigan bean production areas and several were evaluated for thermal processing traits.
Field Plot Procedure

The forty-one entries were planted in a 6 x 7 rectangular lattice with 3 replications
in a Misteguay silty clay soil [fine, ellitic (calcareous), frigid typic Haplaquolls] at the
Saginaw Valley Bean and Sugarbeet Research Farm, Saginaw, MI in 1996. Seeds of each
entry were precision-drilled with a tractor mounted air planter into 4 row plots that were 5
m long and spaced 0.5 m apart. Within-row spacing was 0.12 m. At the time of planting,
a mixed fertilizer consisting of 33.0 kg N- ha'1 and 11 kg P- ha'l to which 10 and 2.5% Mn

and Zn, respectively, were banded in rows. A pre-plant incorporated application of

4.7 L -ha’l Eptam (S-ethyl dipropylthiocarbamate) + 2.3 L - ha‘I Dual (S-metolachlor) was
applied to control weeds. Subsequent hand weeding was practiced as needed. Mature

plants of the forty-one entries were harvested and threshed from 8 m of row of each plot.

Seeds were cleaned and sized using slotted metal sieves and stored at 23°C until samples

were prepared for laboratory analyses.

33

Table 1. Identification, commercial class, seed coat color, seed weight, and yield of 41
dry bean genotypes.

 

 

 

Seed Weight Yield
Identification Commercial Class Seed Coat Color (g - 100 seeds ‘1) (Kg - ha‘ 1)"
N80242 Small white White 18 2700
Black Turtle Soup Tropical black Black 20 2429
Seafarer Navy White 1 7 2328
Domino Tropical black Black 21 3335
Sanilac Navy White 21 2739
Tuscola Tropical black Black 22 2282
8217-111-24 Undefined White 19 1648
Jalpataqua Tropical black Black 23 3151
Nep-2 Navy White 18 2200
San Fernando Tropical black Black 19 3341
Bunsi Navy White 18 2277
ICA Pijao Tropical black Black 20 2906
Aurora Small white White 14 2322
P766 Undefined Black 20 271 l
Jamapa Tropical black Black 20 2953
Protop—Pl Pinto Brown/mottle 34 1239
FF4-13-MMMM Tropical black Black 24 2707
C-20 Navy White 16 2163
F leetwood Navy White 1 8 2593
Mexico 12-1 Undefined Brown 21 2697
Carioca Pinto Brown-beige/ mottle 22 3003
Laker Navy White 19 2365
Black Magic Tropical black Black 19 2956
Swan Valley Navy White 18 2741
Midnight Tropical black Black 20 2814
BAT 41 Small dark red Red(opaque) 20 2181
15-R-148 Small dark red Red (shiny) 20 1470
BAT 1507 Small dark red Red (shiny) 21 2672
BAC 95 Small dark red Red (shiny) 21 2807
Harblack (0) Tropical black Black (opaque) 19 2786
Harblack (Sh) Tropical black Black (shiny) 19 3335
Cumulus Navy White 22 2521
N84004 Navy White 17 2582
C-20 Mutant Navy White 19 2118
Jacob’s Cattle Heirloom (specialty) Red/white spotted 50 1310
Huron Navy White 2 1 2523
Mayflower Navy White 19 2809
Albion Navy White 19 2555
N87602 Navy White 17 2720
N78042 Navy White 2 l 2699
Huetar Small dark red Red (opaque) 21 1733
Mean 2522
LSD (P = 0.05) 754
LSD (P = 0.01) 1009
CV (%) 18.1

 

*Data on seed class and seed color provided by USDA-ARS Bean Food Quality Genetics program in the
Department of Crop and Soil Sciences at Michigan State University.

34

Indigestible Starch Assay

Indigestible starch was determined on both raw and cooked beans. Raw beans
were soaked in water for 12 hours, drained, and lyophilized in a Virtis-Genesis 12EL
freeze dryer. Beans were cooked in tin cans following the procedure of Hosfield and
Uebersax (1980) and Hosfield et al.,(1984). These cooking procedures involved placing

beans with a fresh weight equivalent of 100 g total solids into nylon mesh bags and

soaking them for 30 min at 25°C followed by a 30 min soak at 91°C. All soaking was
done in distilled water to which 0.28 g CaCl2 was added per liter of water to produce a
solution containing 100 ppm Ca ion. Soaked beans were cooled and drained for 5 min
and placed in number 303 (100 x 75 mm) tin cans. Cans were sealed and processed in a
retort without agitation for 45 min at 116°C. When the cans were removed from the
retort, they were cooled and stored inverted for a minimum of 2 weeks at room
temperature. Cooked beans were removed from the cans, drained and lyophilized in a
Virtis-Genesis 12EL freeze dryer.

The freeze-dried raw and cooked bean samples were ground in a mill fitted with a
40-mesh screen for cooked beans and 60-mesh screen for raw beans. Cooked beans
ground and sieved through a 40-mesh screen, and raw beans ground and sieved through a
60-mesh screen had comparable particle sizes wich were representative of physiological
values for particle size when beans are chewed and swallowed by humans (Heller et al. ,
1977). After the beans were ground, the milled flour was partitioned into three parts for

the following analyses: (1) total protein, (2) total starch, and (3) indigestible starch. The

35

partitioned flour was placed into capped vials, and then refrigerated until evaluated in the
three respective assays. All analyses were performed in duplicate.

Indigestible starch was determined on duplicate raw and cooked samples, using
the total dietary fiber (TDF) assay described by Lee et al. , (1992) in which digestible
macromolecules are removed, followed by the resistant starch determination and the
glucose analysis (Johansson et al. , 1984).

Indigestible Protein Determination

Kjeldahl nitrogen methodology (AACC method 46-12, 1983) was used to
determine the content of protein in each raw and cooked bean sample from the TDF
assay. Evaluations were performed on one-half of the TDF assay residue remaining after
enzymatic digestions of bean samples from each duplicate. A catalyst tablet and 5 ml of
sulfuric acid were added to each digestion tube containing the preweighed TDF assay
residue. Tubes were place in a digestion block and heated slowly to 400°C for five hours.
When digestion was completed, tubes containing the samples were allowed to cool before
the distillation and titration procedures were performed. To calculate the percentage of
protein in each bean sample, the percentage of nitrogen was multiplied by 6.25, and the

following equation was used:

(ml of HCl titrated) (Normality of HCl) (1.4007) (6.25)

 

% protein in the sample =
TDF A residue weight (g)

36

Statistical Analysis

All data collected were subjected to an analysis of variance (ANOVA) appropriate
to a randomized complete-block design with a split-plot arrangement of treatments.
Genotypes were the whole-plot and the cooked and raw treatments were sub-plots. The
model used for split-plot experiments showing the separate experimental error variances
for the whole-plot and subplot is:

Y.-,-,.=# + at + p.+ (1.1+ fli+ (61.6).) + cm.

i =1,..a, k=1,..r, j= 1,..b

Where a is the general mean, a, is the effect of the ith level of factor A (genotypes), ,0, is
the effect of the kth block (replications), d”, is the whole-plot random error, ,6,- is the effect
of the jth level of factor B (treatments, cooked and raw), (046),, is the interaction effect
between factors A and B, and 9.7;. is the subplot random error. The ANOVA for the whole-
plot error and the sub-plot error from the previous model with fixed effects for factors A
and B is presented in Table 2.

Statistical analyses were executed by SAS (SAS Institute, 1988). Statistical
analyses were performed on the cooked and raw bean treatments separately. The variables
— indigestible residue, indigestible starch, and resistant starch were evaluated. Mean
separations were performed using the LSD test criterion with the mean square error term

set at the 95% confidence level.

37

Table 2. Form for the analyses of variance of several nutritional quality factors of forty-one dry
bean genotypes and two preparation methods in a split-plot arrangement of treatments.

 

 

 

Degrees of
Source of variation freedom Sum of Squares Mean square
expectations
Replicates r-l ab 2 (Yk — Y. ..)Z = SS(Rep)
Whole-plot (A) a-I n z (Y,.. — Y...)2 = SS(A) 02. + be:d + rb9’A
Error (Whole-plot) (r-1)(a-1) b 2 (Yik — Y... — Y..k +Y...)2 cl, + boi,
SS(EA)
Sub-plot (B) b-1 ra 2 (Y, — Y...)2 = 88(8) 02. + “19,8
lnteraction(A*B) (a-l)(b-l) r 2 (YU _ Y... _ Y.) .+Y...)2 02c ”Veg/{B
SS(AB)
Error (Sub-plot) a(r-l)(b-l) Z (Yijk— Y”, _ Y.jk +Y...)2 0'2c
SS(EB)
Total rab-l

 

where 03, =1/(a-DZa’1. 933= Van-0213’]: 93AB=I/(a-l)(b-1)Z(GB):U

38

RESULTS AND DISCUSSION

Total starch and protein were determined on each of the 41 cooked bean samples.

The total starch of cooked beans ranged from 31.6 g-100 g”l (15-R-148) to 49.8 g-100 g'l
(N 80242) (Appendix, Table 1). Total protein content of cooked beans ranged from 17.2
g - 100 g" (N87602) to 27.8 g - 100 g" (15-R-148) (Appendix, Table 1). A paired
comparison t-test (P=0.10, data not given) indicated that the total starch of raw beans was
not significantly different than the cooked beans (mean 41.5 cooked beans vs 40.2 raw
beans) (Appendix, Table l). The raw beans ranged in total starch from a low of 3 l .1 g-
100 g " for ‘Jalpataqua’ to a high of 46.8 g- 100 g " for Jacob’s Cattle (Appendix, Table
1). When the 41 entries of raw beans were taken as a whole, raw bean protein was 2%
higher than the cooked beans (24.3 vs 22.4, respectively) (Appendix, Table 1). This
difference was highly significant based on a paired comparison t-test (P=0.0001, data not
given). Wassimi et al., (1988) reported a 2% loss in protein, on average, when beans were
cooked compared to the raw bean percentages. Raw bean protein ranged from 18.5 for
N87602 to 29.2 for 8217-111-24 (Appendix, Table 1). There was significant variability
between duplicate laboratory samples for indigestible protein (Appendix, Tables 2, 3, 4,
and 5).

Indigestible protein was determined on duplicate samples of each genotype for
raw (Appendix, Tables 2 and 3) and cooked (Appendix, Tables 4 and 5) beans. There
were large differences between field replications for the indigestible starch of raw and

cooked beans. This variability is obviated by the large coefficients of variation (CV)

39

noted (Appendix, Table 6). The analysis of variance for total dietary fiber in bean flour
(Table 3) indicated significant differences between genotypes (whole-plots) and the raw
and cooked treatments (sub-plots). There were no significant differences among
genotypes for indigestible starch (Table 4), but the differences among treatments, i.e., raw
and cooked beans, when the data were averaged over all the genotypes, were highly

significant (Table 4). There was an increased quantity of total dietary fiber for cooked
beans (22.1 mg - 100 mg flour ") compared to raw beans (19.9 mg - 100 mg flour ") on an

experiment mean basis (Table 5). This 2.2 mg - 100 mg flour " increase in the dietary
fiber of cooked beans compared to raw beans was not significant (data not shown).

The mean indigestible starch on a per gram flour basis was 12.4 g for raw beans
and 33.9 g for cooked beans (Table 6). When the indigestible starch was considered, on a

per 100 mg starch basis, the raw beans averaged 3.1 mg - 100 mg starch" over the 41

genotypes and 8.2 mg - 100 mg starch" averaged over the 41 cooked bean genotypes
(Table 7). The finding that cooked beans had increased indigestible starch compared to
raw beans agrees with observations made by Chung (1996) who found that cooking
beans, even for 10 minutes, increased the amount of indigestible starch compared to raw
beans. Considering this data, Chung (1996) concluded that the increase in indigestibility
that he observed in cooked versus raw beans was due to polymerization of the cell walls
in cotyledons. When cell walls polymerize or become crystalline in structure they may
encapsulate the starch granules, thus, rendering the starch granules inaccessible to

enzymatic digestion. The observation in the present study where cooked beans had more

40

Table 3. Analysis of variance for the total dietary fiber from raw and cooked bean
samples of 41 dry bean genotypes.

 

 

 

Source Variation DF SS MS F Value Pr>F
Replicates 1 0.8 0.8 0.77 0.3865
Genotypes

(Whole plot, Factor A) 40 500 13 6.57 0.0001
Replicates x Genotype

(Whole plot error) 40 76 2.0 1.88 0.0234
*Treatment

(Sub-plot, Factor B) l 198 198 196 0.0001
Genotype x Treatment

(Interaction AxB) 40 59 1.5 1.46 0.1160
Error

(Sub-plot error) 41 4 l 1
Total 163 875
CV (%) 4.8

 

*Treatments were raw and cooked beans

41

Table 4. Analysis of variance for the indigestible starch from raw and cooked bean
samples of 41 dry bean genotypes.

 

 

 

Source Variation DF SS MS F Value Pr>F
Replicates 1 0.000874 0.00874 52 0.0001
Genotypes

(Whole plot, Factor A) 40 0.001305 0.000033 1.4 0.1365
Replicates x Genotype

(Whole plot error) 40 0.000920 0.000023 1.4 0.1654
*Treatment

(Sub-plot, Factor B) 1 0.19059 0.019059 1 127 0.0001
Genotype x Treatment

(Interaction ArB) 40 0.001126 0.000028 1.7 0.0540
Error

(Sub-plot error) 41 0.000693 0.000017
Total 163 0.023978
CV (%) 17.7

 

‘Treatments were raw and cooked beans.

42

Table 5. Means and low, medium, and high groupings for the total dietary fiber of 41dry
bean genotypes prepared by two methods, raw and cooked.

 

 

 

 

Raw Cooked

Total Dietary Fiber Total Dietary Fiber
Genotype (mg - 100mg flour '1) Group" Genotype (mg - 100mg flour '1) Group"
N78042 15.1 1 N78042 15.7 1
N80242 17.3 1 Nep-2 17.1 1
Mayflower 17.8 1 N80242 19.1 1
Nep-2 18.0 1 Midnight 19.7 1
Midnight 18.1 1 Mayflower 19.9 1
C-20 Mutant 18.3 11 Swan Valley 20.1 11
Jacob’s Cattle 18.4 11 Tuscola 20.4 11
Harblack (O) 18.5 11 C-20 Mutant 20.6 [1
Swan Valley 18.7 11 BAT 1507 20.8 11
N84004 18.9 11 N87602 21.4 11
Huron 18.9 11 N84004 21.4 11
Harblack (Sh) 18.9 11 Albion 21.4 11
N87602 19.0 11 Harblack (O) 21.7 11
Albion 19.0 11 Laker 21.7 11
Bunsi 19.4 11 C-20 Mutant 21.9 11
Laker 19.5 11 Harblack (Sh) 21.9 11
C-20 Mutant 19.5 11 Cumulus 21.9 11
Domino 19.7 11 Huetar 21.9 11
Huetar 19.7 11 Mexico 12-1 21.9 11
Seafarer 19.7 11 San Fernando 22.0 11
Cumulus 19.8 11 BAC 95 22.3 11
BAT 41 19.8 11 Seafarer 22.4 11
8217-111-24 20.1 11 Jacob’s Cattle 22.4 11
Tuscola 20.2 11 BAT 41 22.4 11
San Fernando 20.4 11 8217-111-24 22.5 11
FF4-13-MMMM 20.6 11 Huron 22.5 11
BAC 95 20.8 11 Domino 22.5 11
Fleetwood 20.9 11 Bunsi 22.5 11
Mexico 12-1 21.0 11 Carioca 22.8 11
Carioca 21.0 11 P766 22.9 11
Black Turtle Soup 21.0 11 Aurora 22.9 11
Aurora 21.1 11 Black Turtle Soup 23.2 11
BAT 1507 21.3 11 Jalpataqua 23.4 11
ICA Pijao 21.3 11 ICA Pijao 23.6 11
Sanilac 21.6 11 Sanilac 23 .9 11
Jalpataqua 21.7 11 lS-R- 148 24.2 111
Black Magic 21.8 11 Black Magic 24.6 111
Protop-Pl 22.2 111 FF4-13-MMMM 25.3 111
Jamapa 22.3 111 Fleetwood 25.4 111
P766 22.6 111 Protop-Pl 26.1 111
15-R-l48 23.3 111 Jamapa 26.3 111
Mean 19.9 Mean 22.1
CV (%) 2.8 CV (%) 3.4
LSD 1.5 LSD 1.9

 

*Group; 1 = low, 11 = intermediate, 111 = high.

43

Table 6. Means and low, medium, and high groupings for the indigestible starch on a

total flour basis of 41 dry bean genotypes prepared by two methods, raw and cooked.

 

 

 

 

Raw Cooked

Indigestible Starch Indigestible Starch
Genotype (mg - g flour '1) Group" Genotype (mg - g flour '1) Group"
Protop-Pl 7. 1 I Mayflower 27.0 I
Harblack (Sh) 8.0 l 15-R-148 28.0 1
Midnight 8.1 1 FF4-13-MMMM 28.4 11
C-20 Mutant 8.1 1 N87602 29.4 11
N87602 8.5 1 Jamapa 29.6 11
Cumulus 8.5 1 Jalpataqua 30.2 11
Jacob’s Cattle 8.8 l Harblack (Sh) 30.3 11
N78042 9.5 11 Swan Valley 30.8 11
P766 9.7 11 San Fernando 30.8 11
Swan Valley 9.9 11 Black Magic 31.1 11
Seafarer 10.0 11 Aurora 3 1 .3 11
N80242 10.0 11 Tuscola 31 .4 11
F leetwood 10.2 11 Harblack (O) 31.6 11
Jalpataqua 10.2 11 N78042 31.7 11
FF4-13-MMMM 10.5 I 1 Fleetwood 31.7 11
Harblack (O) 1 1.2 11 Mexico 12-1 32.4 11
Carioca 1 1.2 11 Protop-Pl 32.4 11
Nep—2 11.3 11 P766 32.8 11
Jamapa 12.2 11 Nep-2 33.0 11
C-20 12.4 11 8217—111-24 33.1 11
Black Magic 12.4 11 BAT 1507 33.3 11
San Fernando 12.8 11 C-20 33.4 11
N84004 12.9 11 Huetar 33.6 11
Aurora 13.1 11 Domino 33.9 11
Black Turtle Soup 13.1 11 ICA Pijao 33.9 11
ICA Pijao 13.1 11 Jacob’s Cattle 34.0 11
Huetar 13.1 11 Midnight 34.2 11
Mayflower 13.3 11 BAC 95 34.3 11
Tuscola 13 .3 11 Seafarer 34.5 11
Domino 13 .6 11 Huron 34.6 11
15-R-148 13.7 11 Bunsi 34.9 11
Albion 13.9 11 Black Turtle Soup 35.3 11
Sanilac 14.0 11 Laker 36.4 11
Laker 14.2 11 Carioca 36.6 11
BAC 95 14.3 11 C-20 Mutant 37.1 11
Bunsi 15.7 11 Albion 37.1 11
Mexico 12-1 16.0 11 N84004 39.5 11
BAT 1507 16.4 111 BAT 41 41.1 11
Huron 19.8 111 Sanilac 41.9 111
BAT 41 20.9 111 N80242 44.9 111
8217-111-24 21.5 111 Cumulus 48.7 111
Mean 12.4 Mean 33.9
CV (%) 28.7 CV (%) 15.5
LSD 0.0 LSD 0.0

 

*Group; 1 = low, 11 = intermediate, 111 = high.

44

Table 7. Means and low, medium, and high groupings for the indigestible starch on a
starch basis of 41 dry bean genotypes prepared by two methods, raw and cooked.

 

 

 

 

Raw Cooked

Indigestible Starch Indigestible Starch
Genotype (mg - 100mg starch'l) Group" Genotype (mg - 100mg starch‘1) Group"
Midnight 1.8 1 Mayflower 6.3 1
Harblack (Sh) 1.8 1 Tuscola 6.7 l
N87602 1.9 l FF4-13-MMMM 6.7 l
Protop-Pl 1.9 1 Mexico 12-1 7.0 l
Jacob’s Cattle 1.9 11 Swan Valley 7.2 1
Swan Valley 2.2 II N78042 7.2 1
Cumulus 2.2 11 Jamapa 7.2 1
N78042 2.3 11 Black Magic 7.3 l
C-20 Mutant 2.3 11 San Fernando 7.3 1
P766 2.5 11 N87602 7.3 l
Seafarer 2.5 11 Jalpataqua 7.4 l
FF4-13-MMMM 2.6 11 Harblack (Sh) 7.5 1
N80242 2.6 II Jacob’s Cattle 7.9 II
Nep-2 2.6 11 Fleetwood 7.9 11
Fleetwood 2.6 11 Nep-2 7.9 11
Carioca 2.9 11 C-20 7.9 11
Jamapa 3.0 11 Harblack (O) 7.9 11
San Fernando 3.0 11 ICA Pijao 7.9 11
Tuscola 3.1 11 Midnight 8.0 11
Albion 3.1 11 Carioca 8.1 11
N84004 3. 1 11 Protop-Pl 8.2 11
C-20 3.1 11 Albion 8.3 11
Black Turtle Soup 3.2 11 Laker 8.3 11
ICA Pijao 3.3 11 BAT 1507 8.3 11
Jalpataqua 3.3 11 Huetar 8.3 11
Harblack (O) 3.3 11 Seafarer 8.4 11
Huetar 3.4 11 Aurora 8.4 11
BAC 95 3.4 II Bunsi 8.4 11
Aurora 3.4 11 Domino 8.4 11
Domino 3.4 11 BAC 45 8.5 11
Sanilac 3.5 11 Sanilac 8.5 11
Mayflower 3 .5 ll 8217-111-24 8.7 11
15-R-148 3.5 11 C-20 Mutant 8.7 11
Black Magic 3.5 11 lS-R-148 8.9 11
Laker 3.7 11 Huron 8.9 11
Bunsi 3.7 11 N80242 9.0 11
Mexico 12-1 3.9 11 Black Turtle Soup 9.4 11
Huron 4.4 11 P766 9.8 11
BAT 1507 5.2 111 BAT 41 10.1 111
BAT 41 5.4 111 N84004 10.3 111
8217-111-24 5.6 111 Cumulus 11.9 111
Mean 3.1 Mean 8.2
CV (%) 28.6 CV (%) 15.0
LSD 1.8 LSD 2.5

 

*Group; 1 = low, 11 = intermediate, 111 = high.

45

indigestible starch than raw beans may be due to the cell wall polymerization
phenomenon described by Chung (1996). Cooking beans also increased the percentage of
indigestible protein compared to that found in raw beans (Table 8).

There was considerable variation associated with the assays for indigestible starch
and indigestible protein (Tables 6, 7, and 8). The coefficients of variability for
indigestible starch of the 41 genotypes were 28.7 % (Table 6) and 28.6% (Table 7) for
raw beans and 15.5% (Table 6) and 15.0% (Table 7) for cooked beans on a per mg of
flour and per 100 mg of starch basis, respectively. There was large variability for
indigestible protein of raw and cooked beans denoted by the coefficients of variability.
The coefficients of variability were 34.9% and 27.5% for the raw and cooked samples,
respectively (Table 8).

The variability noted among data for indigestible starch (Tables 6, 7), and protein
(Table 8) was most likely due to analytical error. The methodology to determine
indigestible components in raw and cooked bean flour involved (1) grinding beans to a
small and homogeneous particle size, (2) determining the indigestible residue, and (3)
determining the undigested starch and protein that is in the indigestible residue. Since the
determination of indigestible starch and protein required scraping small samples of
residue from the sintered glass crucibles, pipetting small quantities of solution, and
reading glucose formation from 40 m1 of solution; small analytical errors could be
magnified by the various multiplication factors used in calculating data, thus, leading to
large errors in the final values calculated.

The four genotypes with the highest quantities of total dietary fiber in the raw

46

Table 8. Means and low, medium, and high groupings for the indigestible protein as a
percent of the total protein of 41 dry bean genotypes prepared by two methods, raw and cooked.

 

Indigestible Protein (as a percentage of the total seed protein)

 

 

 

Raw Cooked
Genotype (%) Group“ Genotype (%) Group“
Laker 6.4 1 Swan Valley 10.2 1
Sanilac 9. 1 1 Laker 12.0 1
8217-111-24 9.2 l N78042 12.2 1
Albion 9.9 1 F leetwood 12.6 1
Swan Valley 10.1 1 Huron 12.9 I
020 Mutant 10.5 1 Sanilac 12.9 I
Tuscola 10.7 1 Tuscola 13 .5 1
N84004 1 1.0 11 N84004 14.0 1
N87602 l 1.1 11 Jacob’s Cattle 14.1 I
N80242 1 1.1 11 N80242 14.1 I
N78042 l 1.8 11 BAC 95 14.4 1
Huron 12.1 11 C—20 15.8 1
Domino 12.1 11 Carioca 16.3 11
Seafarer 12.5 11 Bunsi 17.3 11
San Fernando 12.6 11 Aurora 17.5 11
Harblack (O) 12.6 11 Albion 17.6 11
FF4-13-MMMM 12.8 11 8217-111-24 17.8 11
Jamapa 13.0 11 Mayflower 18.0 11
Bunsi 13.2 11 15-R-148 18.3 11
Mayflower 13.8 11 BAT 41 18.4 11
Jalpataqua 13.9 11 San Fernando 18.6 11
Black Turtle Soup 14.0 11 Mexico 12-1 18.7 11
BAC 95 14.0 11 ICA Pijao 18.8 11
Protop-P 1 14. 1 ll Nep-2 19.4 11
Fleetwood 14.4 11 Black Magic 19.8 11
C-20 14.6 11 Black Turtle Soup 19.9 11
BAT 41 14.8 11 Midnight 19.9 11
Mexico 12-1 14.9 11 Jalpataqua 20.5 11
Aurora 15.0 111 Cumulus 20.7 11
Carioca 15.4 111 FF4-13-MMMM 20.7 11
P766 15.5 111 Harblack (O) 20.9 11
ICA Pijao 16.5 111 020 Mutant 21.8 11
Nep-2 16.6 111 N87602 21.8 11
Harblack (Sh) 17.0 111 Protop-Pl 22.6 111
Huetar 17.5 111 Huetar 22.6 111
BAT 1507 18.0 111 Domino 23.1 111
15-R-148 18.5 111 Harblack (Sh) 23.3 111
Cumulus 18.9 111 BAT 1507 23.3 111
Black Magic 19.3 111 P766 25.5 111
Jacob’s Cattle 20.0 111 Seafarer 25.6 111
Midnight 20.8 111 Jamapa 26.6 111
Mean 13 .7 Mean 17.9
CV (%) 34.9 CV (%) 27.5
LSD 1.8 LSD 2.5

 

*Group; 1 = low, 11 = intermediate, 111 = high.

47

bean treatment were Protop-Pl, ‘Jamapa,’ P766, and 15-R-148 (Table 5). These four lines

made up the high group of lines for total dietary fiber and ranged from 22.2 to 23.3 mg-
100 mg flour "(Table 5). The raw bean entries, N78042, N80242, ‘Mayflower,’ Nep-2,

and ‘Midnight’ formed the group with the lowest values for dietary fiber (Group I) (Table
5). The dietary fiber values for this group ranged from 15.1 to 18.1 mg-100 mg'1 of flour.
The remaining 32 genotypes exhibited intermediate dietary fiber values and ranged from
18.3 to 21.8 mg of dietary fiber - 100 mg"l of flour (Table 5).

Although the ranges of dietary fiber values for cooked beans (Table 5) was higher
than the raw beans—15.7 to 26.3 (cooked beans) versus 15.1 to 23.3 (raw beans) mg of

dietary fiber - 100 mg'l of flour, respectively— many of the same genotypes fell into the
same respective groupings noted for the raw beans. For example, 15-R-148, Protop-Pl ,
and ‘Jamapa’ were in the high group (Group III) for total dietary fiber of cooked beans
and also in the high group for the total dietary fiber of raw beans (Table 5). The five
genotypes that made-up the low group (Group I) of entries for total dietary fiber were the
same for both raw and cooked beans.

The genotypes Nep-2 and ‘San Fernando’ were expected to be close in mean
values for total dietary fiber but fell into different groups —low (Nep-2) and intermediate

(San Fernando), respectively— in the cooked treatment (Table 3). Nep-2, a white seeded
bean, is a EMS mutant of ‘San F emando,' a black seeded bean (Mob, 1971), and
presumably differs from San Fernando only by the P allele the, “ground gene” of

Kooiman, (1931), which is necessary for the plant to produce color in the seed coat. The

48

difference in dietary fiber between ‘San F ernando’ and Nep-2 may be due to flavonoid
compounds present in black beans (Beninger et al., 2000) but not in white beans that can
complex with seed components, namely starch and protein, thus rendering these
macromolecules indigestible. An increased indigestible starch and/or protein in the seed
would be reflected in a higher quantity of total dietary fiber.

‘Jacob’s Cattle,’ that was evaluated by Murphy, (1973), and reported as a probable
gasless variety, had a higher amount of dietary fiber when seeds were cooked than in the
raw seeds. Other genotypes had lower values for dietary fiber of raw and cooked beans
than ‘Jacob’s Cattle’ (Table 5).

Mean values of indigestible starch on a per gram flour basis for the individual
genotypes for raw beans ranged from 7.1 to 21.5 mg (Table 6) and 1.8 to 5.6 mg on a
100 mg of starch basis (Table 7). For raw bean flour, the genotype 8217-III-24 had the
highest amount of indigestible starch on both a per gram flour and per 100 mg of starch
basis (Tables 6 and 7, respectively). Other genotypes with high quantities of indigestible
starch on a per gram flour basis in the raw bean were BAT 41 and ‘Huron’ with 20.9 and
19.8 mg ~ g'l flour, respectively (Table 6). In the raw bean, Protop-Pl had the lowest
amount of indigestible starch on a per gram flour basis (Table 6), but ‘Midnight’ had the
least amount of indigestible starch on a per 100 mg of starch basis (Table 7). In the raw
bean, low quantities of indigestible starch on a per gram of flour (Table 6) were also
associated with the genotypes ‘Harblack (Sh)’, ‘Midnight,’ C-20 Mutant, N87602,

‘Cumulus’, and ‘Jacob’s Cattle.’ The range of values for the genotypes in the low

49

indigestible starch on a per gram flour basis was 7.1 to 8.8 mg - g" of flour (Table 6).

For the cooked beans, means of the individual genotypes for indigestible starch
ranged from 27.0 mg to 48.7 mg on a per g of flour basis (Table 6) and 6.3 to 11.9 mg on
a per 100 mg of starch basis (Table 7). ‘Cumulus’ had the highest level of indigestible
starch on both a per g of flour and 100 mg starch basis (Tables 6 and 7). In the cooked
bean group, ‘Mayflower’ had the lowest amount of indigestible starch on both a per g of
flour and 100 mg of starch basis. Spearman’s rank correlation (rS statistic) (Appendix,
Tables 7, 8 and 9) computed between the ranks of individual genotypes for indigestible
starch on a per g of flour basis and on a per mg starch basis were high value and highly
significant for both raw (rs=0.9) and cooked (rs=0.7) beans, indicating that the genotypes
ranked similarly for indigestible starch on a per g of flour basis and per 100 mg starch
basis.

Comparison of the rankings of means for total dietary fiber (Table 5) and
indigestible starch on a per g flour basis when beans were cooked (Table 6), showed that

lS-R-148 had a high quantity of total dietary fiber (24.2 mg - 100 mg flour ") but a low
quantity of indigestible starch on a per g flour basis (28.0 mg - g flour "). Although the
indigestible starch quantity of 15-R-148 was comparatively low on a per g flour basis, the
indigestible starch of this entry on a per 100 mg starch basis tended to be high

(8.9 mg - 100 mg") (Table 7) compared to the other entries. 15-R-l48 had the lowest
amount of total starch 31.6 g - 100 mg" of the 41 entries evaluated (Appendix, Table 1).

Also among the cooked bean samples, ‘Sanilac’ had the highest amount of total dietary

50

fiber of the intermediate ranked group for this trait (Table 5), but was in the group that
ranked high for the amount of indigestible starch on a per g flour basis (Table 6). There
does not appear to be a strong association between the quantity of total dietary fiber and
indigestible starch when all 41 entries are considered together. Single degree of freedom
analyses for total dietary fiber and indigestible starch on a per g flour basis showed that
12 of the 41 entries evaluated had non-significant mean squares for the total dietary fiber
trait but all the entries had significant mean square for the indigestible starch on a per mg
flour basis (Table 9).

‘Mayflower’ contrasted with 15-R-148 for quantities of total dietary fiber and
indigestible starch on both a per g flour and 100 mg starch basis of cooked beans.
‘Mayflower’ had a low quantity of indigestible starch on a per g flour basis (27.0 mg - g")
(Table 6) and per mg starch basis (6.3 mg - 100 mg") (Table 7). The total starch content

of cooked ‘Mayflower’ bean was comparatively high (42.9 g - 100 g") among the 41

entries and exceeded the mean of the entries by 1.4 g ' 100 g" (Appendix, Table 1). Other
entries such as ‘Tuscola,’ ‘Swan Valley,’ and N78042 fell into the low or low side of the
intermediate group for total dietary fiber (Table 5) and had low amounts of indigestible
starch on a per 100 mg starch basis (Table 7), but their quantities of indigestible starch on
a per g flour basis (Table 6) were intermediate between the high and low groupings.
Means for indigestible protein of cooked beans (Table 8) ranged from 10.2 %
(‘Swan Valley’) to 26.6% (‘Jamapa’) and 6.4% (‘Laker’) to 20.8% (‘Midnight’) for raw

beans (Table 8). Cooked bean genotypes, ‘Seafarer’ (25.6%), P766 (25.5%), ‘Domino’

51

Table 9. Mean squares for the variables total dietary fiber and indigestible starch of the
treatment effect in each genotype.

 

 

Total Dietary Fiber Indigestible Starch
Identification df (mg/ 100 mg flour) (mg/g flour)
N80242 1 1.8162“ 0.001224“ “
Black Turtle Soup 1 2.2943“ 0.000494"
Seafarer 1 3.4172“ 0.000601 ““
Domino 1 4.03 84*“ 0.000409"
Sanilac 1 2.4315“ 0.000776“ “
Tuscola 1 0.0132 NS 0.000326“
8217-111-24 1 2.7114“ 0.000135"
Jalpataqua 1 1.3268 NS 0.000398“
Nep—2 1 0.4628 NS 0.000472"
San Fernando 1 1.3719 NS 0.000328"
Bunsi 1 5.071 1““ 0.000369"
ICA Pijao 1 2.5106“ 0.000435"
Aurora 1 1.7715“ 0.000331“ “
P766 1 0.0344 NS 0.000534"
Jamapa 1 7.2983" 0.000304"
Protop-Pl 1 6.8787“ “ 0.000641“ “
FF4-13-MMMM 1 10.5034" 0.000319"
C-20 1 2.7620“ 0.000442"
Fleetwood 1 9.1 150““ 0.000465M
Mexico 12-1 1 0.5120 NS 0.000271"
Carioca 1 1 .5640“ 0.000646“ “
Laker 1 2.4747“ 0.000490"
Black Magic 1 3.5054" 0.000349"
Swan Valley 1 1.0686 NS 0.000439"
Midnight 1 1.4702 NS 0.000684”
BAT 41 1 3.4966“ 0.000407"
15-R-148 1 0.4173 NS 0.000204M
BAT 1507 1 0.0936 NS 0.000287"
BAC 95 1 1.0468 NS 0.000397"
Harblack (O) 1 4.9608" 0.000419"
Harblack (Sh) 1 4.3942" 0.000499"
Cumulus 1 2.3328“ 0.001614”
N84004 1 3.2146“ 0.000712”
C-20 Mutant 1 3.2146“ 0.000712"
Jacob’s Cattle 1 8.2723" 0.000635”
Huron 1 6.6973M 0.000218"
Mayflower 1 2.3709“ 0.000188“ “
Albion 1 2.9088“ 0.000540"
N87602 1 2.8614“ 0.000438“
N78042 1 0.2601 NS 0.000492"
Huetar 1 2.3973“ 0.000418”

 

“, *“ Significant at the 0.05 and 0.01 levels of probability, respectively; NS = Non significant

52

(23.1%), BAT 1507 (23.3%), and ‘Harblack (Sh)’ (23.3%) had a high quantity of
indigestible protein. ‘Swan Valley’ (10.2%), ‘Laker’ (12.0%), and N78042 (12.2%) had
low quantities of indigestible protein (Table 8). In the raw bean, genotypes ‘Midnight’
(20.8%), ‘Jacob’s Cattle’ (20.0%), and 15-R-148 (18.5%) showed high quantities of
indigestible protein, and ‘Laker’ (6.4%), ‘Mayflower’ (13.8%), and N87602 (11.1%) had
a low percentage of indigestible protein (Table 8). Samples of cooked beans of ‘J amapa’
and N78042 showed high and low mean values, respectively for both total dietary fiber
(Table 5) and indigestible protein (Table 8). The raw samples of genotypes 15-R-l48 and
‘Mayflower’ had high and low or intermediate contents, respectively of total dietary fiber
(Table 5) and indigestible protein (Table 8).

The mean square for duplicates for indigestible protein was significant (0t=0.05)
indicating a lack of consistency among the two laboratory analyses for each genotype.
The lack of consistency between duplicate samples probably contributed to the large

coefficient of variability (Table 8).

Breeding implications.

 

A loss of nutrients occurs when a food component is not digested and absorbed in
the small intestine. Moreover, undigested food provides a substrate for microbial
digestion in the colon with accompanying gastrointestinal discomfort including flatus
production. F latulence occurring after consuming a meal of beans is considered by many

as the most important factor limiting the consumption of dry bean. Hence, the

53

improvement of digestibility in dry bean would not only improve the bioavailability of
starch and protein but also reduce flatulence from eating beans and enhance consumer
acceptance of this important crop.

Significant variability among genetic stocks for indigestible components of the

seed —total dietary fiber, starch, and protein— found in this study indicate that these traits
can be altered by selection. Improved digestibility of macromolecules and a reduction in
flatus potential could probably be achieved by selecting in breeding populations those
recombinants with low quantities of indigestible components. Generally, the breeding
objectives of a program dictate which traits are selected and procedures used. In this
regard, the bean breeder interested in improving the food value of the crop is faced with
several challenges. The questions proposed for the present study include: should the
breeder concentrate selection efforts to reduce flatulence, reduce indigestible starch,
—thus improving the bioavailability of this nutrient — or improve the total dietary fiber
component? Total dietary fiber consists of the indigestible residue after bean flour is
treated with digestive enzymes and is composed mainly of cell wall components
(cellulose and hemicellulose), lignin, undigested starch, protein, sugars, and ash. By
reducing the indigestible residue through selection, the breeder is effectively reducing
total dietary fiber. Since dietary fiber is an important component in health improvement
strategies to reduce serum cholesterol and the incidence of some types of cancer,
selection for a reduced content of indigestible residue (total dietary fiber) in bean seeds

may have a negative impact on improving dry bean for some factors contributing to

54

human health. However, selection for a low dietary fiber content would be in agreement
with a breeder’s desire to improve digestibility and lower flatulence. In other words, one
of the characteristics of beans that promote their utility as a healthy food — a good source
of fiber — is a characteristic that limits bean consumption because of the potential for
gastrointestinal stress including flatulence. Faced with this dilemma, the breeder might
proceed with a strategy that permits one to increase the quantity of total dietary fiber
(indigestible residue) and lower the quantity of indigestible starch. Although the
correlation between the total dietary fiber and indigestible starch was not determined in
this study, there does not appear to be a strong association between these two traits
(Tables 5, 6, and 7). For example, ‘Jamapa’ had the highest quantity of total dietary fiber
in cooked beans (26.3%) (Table 5) but had a relatively low quantity (Group II) of
indigestible starch on a per g flour basis (Table 6) compared to the other genotypes and
ranked 37‘h highest for this trait. Other examples of the lack of a strong association
between a line’s total dietary fiber and indigestible starch are provided in Tables 5 and 6.
The development of a selection index along with selection either upward or downward to
some ideal value for each of the traits may be a method to increase total dietary fiber and
decrease indigestible starch in beans through selection and breeding.

Seed characteristics that could be involved in breeding for improved carbohydrate
digestibility in beans are total starch, indigestible residue (or dietary fiber), and
indigestible starch. Digestibility determinations can be made on a per gram of flour used
for laboratory evaluations or on a per 100 mg of starch determined from total starch
values of beans. After the evaluation of these parameters, the breeder would decide which

55

one to use in the selection program. A useful parameter for the breeder to consider to
improve the digestibility of beans through selection is the indigestible starch on a per 100
mg of starch basis. A bean genotype with low indigestible starch on a per 100 mg of
starch, although containing high indigestible starch on a per g of flour, would be a good
parent to include in a plant breeding program. Determination of total dietary fiber could
be considered the most cost efficient evaluation since it is obtained directly from the
enzyme digestions during the assay. The total dietary fiber is the indigestible residue
remaining after the flour is digested with enzymes and, thus, contains the undigested
macromolecules present in the flour sample.

The results of this study point out the need to determine both the total seed starch
(e. g. appendix Table 1) and the indigestible starch on a per g of flour basis (e.g. Table 6).
In a breeding program the selection of a segregant on the basis of high total starch and
low indigestible starch (per g of flour) would be equivalent to selecting a segregant with

low total starch and high indigestible starch (per g of flour).

In Phaseolus vulgaris evidence indicates two major centers of domestication —in

 

Mesoamerica and in the Andes— which led to two groups of cultivars with contrasting
agronomic characteristics (Gepts and Debouck, 1991). These two gene pools are
distinguished by their unique electrophoretic pattern of phaseolin —a reserved seed
protein (Gepts and Bliss, 1986; Gepts et al., 1986; and Gepts, 1988). Cultivars with “S”
type phaseolin generally have comparatively small seeds and are classified in the

Mesoamerican center of domestication; cultivars with “T” type phaseolin generally have

56

large seeds (e. g. Kidney beans) and are classified in the Andean center of domestication.
In this study all the bean genetic stocks used except Jacob’s Cattle represented the
Mesoamerican center of domestication. Jacob’s Cattle total dietary fiber and contents of

indigestible starch were unremarkable for cooked beans; however, in a separate study
(Ospina, unpublished, 1999) ‘Montcalm’ dark red kidney bean — a genotype
representing the Andean center of domestication — had an indigestible starch value for

cooked beans of 22.7 mg ~ g flour " (data not shown). This value was 4 mg lower than
‘Mayflower’ a Mesoamerican cultivar evaluated in the current study with the lowest
value among genetic stocks for cooked bean indigestible starch (on a per g flour basis)
(Table 6). The low content of indigestible starch in ‘Montcalm’ suggests that there could
be a possible difference between the Andean and Mesoamerican bean genotypes for total
dietary fiber and starch digestibility. Future research could determine if there is a
relationship between genetic origin of a bean cultivar and its content of indigestible
starch. Although there is no published data on starch indigestibility in dry beans and the
minimum and maximum values for this trait are currently unknown, results from the
present study are useful as a point of reference for bean breeders. The indigestible starch
content of ‘Montcalm’ indicates that it would be wise to evaluate germplasm with more

genetic diversity.

57

GENERAL CONCLUSIONS

In the determination of indigestible starch in dry beans, comparable particle size
of the cooked and raw bean flour is important to obtain accurate results. To obtain
comminuted cooked and raw bean flour with similar particle sizes, cooked beans should
be freeze dried and ground through a 40-mesh screen, and raw beans should be soaked,
freeze dried, and ground through a 60-mesh screen. The grinding of raw beans without
the soaking process caused frequent screen breakage when ground through a 60-mesh
screen, thus, rendering flour from raw, non-soaked beans unacceptable because of wide
variability of particle size. Humidification of bean seed was time consuming and made
this process impractical. The measurement of the particle size distribution of bean flour
by using several different sizes of mesh screens was a useful way to compare the particle
size of bean flour regardless of treatment. Differences among genotypes for indigestible
starch in raw and cooked beans could be detected using enzymatic methodologies once
raw and cooked bean particle sizes were comparable.

Significant differences were detected between the experiment means for
indigestible starch on both a per gram flour and per 100 mg starch basis of raw and
cooked seeds of the 41 genotypes evaluated in the present study. However, no differences
were detected between the experiment means for raw and cooked beans for total dietary
fiber. Several genotypes had similar values for dietary fiber regardless of whether the
flour samples came from cooked or raw beans. This finding suggests that cooking of
beans does not always increase the amount of indigestible residue in beans.

58

Also, it could be inferred that bean genotypes with high dietary fiber content and
low indigestible starch have high amounts of indigestible protein and ash. Evaluation of
indigestible protein in the 41 bean genotypes showed that cooked samples had higher
values than raw samples, except for Jacob’s Cattle. Beans are known to lose protein
during cooking and the amount lost may be as high as 2%.

Results obtained in this study suggest that there could be a possible difference in
content of indigestible starch between Andean and Mesoamerican bean genotypes.
Further research is necessary to determine if there is a relationship between genetic origin

of the bean genotype and its content of indigestible starch.

59

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66

APPENDIX

A.__—_—a_’l'

._.—._ .._..____il-

Table 1. Means for total starch and total starch and total protein on a total flour basis for
41 dry bean genotypes prepared by two methods, raw and cooked.

 

 

 

 

 

Total Starch (g‘100g’1) Total Protein (g- IOOgfl

Genotype Cooked Raw Cooked Raw
N80242 49.8 38.6 20.1 24.6
Black Turtle Soup 37.6 41.2 20.8 22.5
Seafarer 41.2 40.1 20.8 22.1
Domino 40.3 39.9 24.2 26.1
Sanilac 49.3 40.6 21.0 23.7
Tuscola 46.5 43.5 24.4 25.4
8217-111-24 38.1 38.3 22.4 29.2
Jalpataqua 40.7 31.1 22.9 25.5
Nep-2 41.7 43.3 23.4 25.1
San Fernando 42.4 41.9 23.7 25.7
Bunsi 41.6 42.3 23.0 27.3
ICA Pijao 42.8 40.4 22.7 24.9
Aurora 37.3 38.3 24.0 26.4
P766 33.5 39.0 22.6 27.7
Jamapa 41.1 41.3 20.6 24.8
Protop-Pl 39.5 37.8 26.0 29.1
FF4-13-MMMM 42.0 40.9 21.2 22.4
C-20 42.2 39.4 21.8 25.0
Fleetwood 40.3 38.4 20.9 23.4
Mexico 12-1 46.0 41.1 22.0 20.5
Carioca 45.2 38.8 21.8 22.2
Laker 43.8 39.0 22.9 23.8
Black Magic 42.8 35.4 21.8 24.4
Swan Valley 43.0 44.7 24.0 24.9
Midnight 43.0 46.2 21.7 23.3
BAT 41 40.9 38.8 24.6 25.1
15-R-148 31.6 39.2 27.8 28.5
BAT 1507 40.1 31.3 23.4 25.1
BAC 95 40.4 42.2 23.0 22.0
Harblack (O) 39.9 33.6 20.4 22.8
Harblack (Sh) 40.4 44.1 21.1 21.8
Cumulus 38.5 41.3 22.1 24.4
N84004 38.5 41.3 24.5 24.8
020 Mutant 42.4 35.0 20.1 22.6
Jacob’s Cattle 43.3 46.8 21.7 23.3
Huron 38.8 44.8 24.2 21.8
Mayflower 42.9 38.3 23.7 25.3
Albion 44.9 44.8 19.8 21.1
N87602 40.0 45.5 17.2 18.5
N78042 44.0 41.3 21.1 22.9
Huetar 40.3 39.1 21.7 25.1
Experiment mean 41.5 40.2 22.4 24.3
CV (%) 8.2 8.9 8.4 9.3

 

67

Table 2. Indigestible protein in raw beans in duplicate ‘A’.

 

 

Indigestible Total Initial wt Total
Protein Residue protein in protein sample for protein Indigestible
Identification in residue from TDF residue flour TDF sample protein
(a) (g) (g) (g) (g) (g) (g)
N80242 0.316 0.1518 0.02064 0.24545 1.0008 0.224565 0.0919
Black Turtle Soup 0.173 0.1968 0.03405 0.22521 1.0010 0.225435 0.1510
Seafarer 0.152 0.1955 0.02972 0.02972 1.0012 0.215809 0.1377
Domino 0.198 0.2057 0.04073 0.26106 1.0013 0.261399 0.1558
Sanilac 0.129 0.21 1 1 0.02723 0.23729 1.0007 0.237456 0.1 147
Tuscola 0.157 0.1997 0.03135 0.25376 1.0001 0.253785 0.1235
8217-111-24 0.185 0.1926 0.03563 0.03563 1.0016 0.292047 0.1220
Jalpataqua 0.162 0.2180 0.03532 0.25476 1.0007 0.254938 0.1385
Nep-Z 0.245 0.1859 0.04555 0.25111 1.0013 0.251436 0.1811
San Fernando 0.141 0.1926 0.02716 0.25635 1.0006 0.256504 0.1059
Bunsi 0.210 0.1886 0.03961 0.27237 1.0004 0.272479 0.1454
ICA Pijao 0.205 0.1935 0.03967 0.24896 1.0009 0.249184 0.1592
Aurora 0.255 0.2084 0.05314 0.26427 1.0015 0.264666 0.2008
P766 0.192 0.2396 0.46000 0.27695 1.0017 0.277421 0.1658
Jamapa 0.148 0.2219 0.24834 0.24834 1.0008 0.248539 0.1321
Protop-Pl 0.197 0.2245 0.04423 0.29085 1.0016 0.291315 0.1518
FF4- l 3-MMMM 0.137 0.1972 0.02642 0.22355 1.0004 0.223639 0.1181
020 0.175 0.1970 0.03448 0.24967 1.0018 0.2501 19 0.1379
Fleetwood 0.197 0.2089 0.041 15 0.23403 1.001 1 0.234287 0.1756
Mexico 12-1 0.181 0.2067 0.03742 0.20458 1.0015 0.204887 0.1826
Carioca 0.182 0.2201 0.04006 0.22144 1.001 1 0.221684 0.1807
Laker 0.069 0.21 1 1 0.01457 0.23813 1.0012 0.238416 0.061 1
Black Magic 0.292 0.2236 0.06529 0.24420 1.0002 0.244249 0.2673
Swan Valley 0.126 0.2048 0.02580 0.24944 1.0016 0.249839 0.1033
Midnight 0.322 0.2082 0.06704 0.23259 1.0018 0.233009 0.2877
BAT 41 0.159 0.2117 0.03366 0.25071 1.0013 0.251036 0.1341
15-R-148 0.207 0.2593 0.05368 0.28461 1.0013 0.284980 0.1884
BAT 1507 0.212 0.2071 0.04391 0.25082 1.0011 0.251096 0.1749
BAC 95 0.103 0.2096 0.02159 0.21960 1.0009 0.219798 0.0982
Harblack (O) 0.132 0.1868 0.02466 0.22753 1.0006 0.227667 0.1083
Harblack (Sh) 0.200 0.1931 0.03862 0.21794 1.0003 0.218005 0.1772
Cumulus 0.270 0.2006 0.05416 0.24412 1.0014 0.244462 0.2215
N84004 0.1 10 0.1799 0.01979 0.24805 1.0013 0.248372 0.0797
020 Mutant 0.130 0.1807 0.02349 0.22591 1.0011 0.226159 0.1039
Jacob’s Cattle 0.288 0.2049 0.05901 0.23296 1.0004 0.233053 0.2532
Huron 0.120 0.201 1 0.02413 0.21803 1.0009 0.218226 0.1 106
Mayflower 0.190 0.1799 0.03418 0.25212 1.0019 0.252599 0.1353
Albion 0.082 0.1956 0.01604 0.21124 1.0013 0.21 1515 0.0758
N87602 0.106 0.1933 0.02049 0.18507 1.0007 0.185200 0.1106
N78042 0.197 0.1549 0.03052 0.2291 1 1.0018 0.229522 0.1330
Huetar 0.303 0.1914 0.05799 0.25025 1.0010 0.250500 0.2315

 

68

Table 3. Indigestible protein in raw beans in duplicate ‘B’.

 

 

Indigestible Total Initial wt Total
Protein Residue protein in protein sample for protein Indigestible
Identification in residue from TDF residue flour TDF sample protein
(g) (g) (g) (g) (g) (g) (g)
N80242 0.185 0.1716 0.031746 0.24545 1.0014 0.245794 0.1292
Black Turtle Soup 0.145 0.1995 0.028928 0.22521 1.0014 0.225525 0.1283
Seafarer 0.126 0.1926 0.024268 0.21555 1.0004 0.215636 0.1 125
Domino 0.1 13 0.1978 0.022351 0.26106 1.0017 0.261504 0.0855
Sanilac 0.074 0.2163 0.016006 0.23729 1.0012 0.237575 0.0674
Tuscola 0.1 15 0.1997 0.022966 0.25376 1.0001 0.253785 0.0905
8217-111-24 0.093 0.1961 0.018238 0.29158 1.0003 0.291667 0.0625
Jalpataqua 0.161 0.21 14 0.035645 0.25476 1.0014 0.255117 0.1397
Nep—2 0.245 0.1541 0.037755 0.25111 1.0016 0.251512 0.1501
San Fernando 0.214 0.1757 0.037599 0.25635 1.0020 0.256863 0.1464
Bunsi 0.168 0.1933 0.032474 0.27237 1.0007 0.272561 0.1 191
ICA Pijao 0.203 0.2110 0.042833 0.24896 1.0018 0.249408 0.1717
Aurora 0.128 0.2028 0.025958 0.26427 1.0005 0.264402 0.0982
P766 0.179 0.2251 0.040293 0.27695 1.001 1 0.277255 0.1453
Jamapa 0.148 0.2160 0.031968 0.24834 1.0005 0.248464 0.1287
Protop-Pl 0.197 0.1935 0.038119 0.29085 1.0012 0.291 199 0.1309
FF4-13-MMMM 0.149 0.2072 0.030873 0.22355 1.0011 0.223796 0.1380
C-20 0.201 0.1923 0.03 8652 0.24967 1.0018 0.2501 19 0.1545
Fleetwood 0.127 0.2086 0.026492 0.23403 1.0009 0.23424] 0.1 131
Mexico 12-1 0.121 0.1952 0.023619 0.20458 1.0018 0.204948 0.1152
Carioca 0.138 0.2049 0.028276 0.22144 1.0010 0.221661 0.1276
Laker 0.082 0.1961 0.016080 0.23813 1.0013 0.238440 0.0674
Black Magic 0.144 0.2008 0.028915 0.24420 1.0017 0.244615 0.1182
Swan Valley 0.127 0.1939 0.024625 0.24944 1.0017 0.249864 0.0986
Midnight 0.183 0.1641 0.030030 0.23259 1.0007 0.232753 0.1290
BAT 41 0.217 0.1875 0.040688 0.25071 1.0017 0.251136 0.1620
15-R-148 0.220 0.2356 0.051832 0.28461 1.0013 0.284979 0.1819
BAT 1507 0.230 0.2012 0.046276 0.25082 1.0019 0.251297 0.1841
BAC 95 0.198 0.2024 0.040075 0.21960 1.0007 0.219754 0.1824
Harblack (O) 0.223 0.1790 0.039917 0.22753 1.0015 0.227891 0.1431
Harblack (Sh) 0.200 0.1780 0.035600 0.21794 1.0009 0.218136 0.1632
Cumulus 0.191 0.2004 0.038276 0.24412 1.0020 0.244608 0.1565
N84004 0.180 0.1897 0.034146 0.24805 1.0006 0.248199 0.1376
C-20 Mutant 0.130 0.1862 0.024206 0.22591 1.0010 0.226136 0.1070
Jacob’s Cattle 0.183 0.1872 0.034258 0.23296 1.0019 0.233403 0.1468
Huron 0.154 0.1857 0.028598 0.21803 1.0016 0.218379 0.1310
Mayflower 0.190 0.1876 0.035644 0.25212 1.0013 0.252448 0.1412
Albion 0.137 0.1881 0.025770 0.22124 1.0013 0.211515 0.1218
N87602 0.106 0.1946 0.020628 0.18507 1.0015 0.185348 0.1113
N78042 0.170 0.1399 0.023783 0.2291 1 1.0014 0.229431 0.1037
Huetar 0.156 0.1897 0.029593 0.25025 1.0011 0.250525 0.1181

 

69

Table 4. Indigestible protein in cooked beans in duplicate ‘A’.

 

 

Indigestible Total Initial wt Total
Protein Residue protein in protein sample for protein Indigestible
Identification in residue from TDF residue flour TDF sample protein
(a) (g) (g) (g) (g) (g) (g)

N80242 0.175 0.1842 0.03224 0.20148 1.0005 0.201581 0.1599
Black Turtle Soup 0.195 0.2338 0.04559 0.20758 1.0010 0.207788 0.2194
Seafarer 0.217 0.2302 0.04995 0.20803 1.0001 0.208051 0.2401
Domino 0.228 0.2358 0.05376 0.24164 1.0012 0.241930 0.2222
Sanilac 0.148 0.2412 0.03570 0.21039 1.0013 0.210664 0.1694
Tuscola 0.165 0.2341 0.03863 0.24442 1.001 1 0.244669 0.1579
8217-111-24 0.176 0.2204 0.03879 0.22445 1.0018 0.224854 0.1725
Jalpataqua 0.172 0.2393 0.041 16 0.22891 1.0012 0.229185 0.1796
Nep-2 0.35 0.1579 0.05527 0.23408 1.0010 0.234314 0.2359
San Fernando 0.182 0.2347 0.04272 0.23699 1.0015 0.237345 0.1799
Bunsi 0.192 0.2270 0.04358 0.23019 1.0007 0.230351 0.1892
ICA Pijao 0.134 0.2301 0.03083 0.22689 1.0009 0.227094 0.1357
Aurora 0.191 0.2158 0.04122 0.23961 1.0010 0.239850 0.1718
P766 0.319 0.2556 0.08154 0.22626 1.0014 0.226577 0.3599
Jamapa 0.249 0.2797 0.06965 0.20661 1.0019 0.207003 0.3365
Protop-Pl 0.282 0.2781 0.07842 0.25969 1.0003 0.259768 0.3019
FF4-13-MMMM 0.171 0.2688 0.04596 0.21 153 1.0010 0.21 1742 0.2171
C-20 0.185 0.2225 0.041 16 0.21750 1.0013 0.217783 0.189

F leetwood 0.1 10 0.2735 0.03009 0.20859 1.0008 0.208757 0.1441
Mexico 12-1 0.201 0.2349 0.04721 0.22015 1.0007 0.220304 0.2143
Carioca 0.172 0.2455 0.04223 0.21837 1.0015 0.218698 0.1931
Laker 0.142 0.2316 0.03289 0.22907 1.0009 0.229276 0.1434
Black Magic 0.177 0.2576 0.04560 0.21808 1.0016 0.218429 0.2087
Swan Valley 0.103 0.2233 0.02299 0.24027 1.0009 0.240486 0.0956
Midnight 0.297 0.2003 0.05949 0.21735 1.0007 0.217502 0.2735
BAT 41 0.213 0.2373 0.05054 0.24602 1.0017 0.246438 0.2051
15-R-148 0.207 0.2593 0.05368 0.28461 1.0013 0.284980 0.1884
BAT 1507 0.271 0.2511 0.06805 0.23377 1.0001 0.233793 0.2911
BAC 95 0.106 0.2196 0.02299 0.23032 1.0011 0.230573 0.0997
Harblack (O) 0.160 0.2379 0.03806 0.20361 1.0008 0.203773 0.1868
Harblack (Sh) 0.252 0.2230 0.05620 0.21118 1.0019 0.211581 0.2656
Cumulus 0.229 0.2039 0.04669 0.22077 1.0013 0.221057 0.21 12
N84004 0.103 0.211 1 0.02174 0.24489 1.0016 0.245282 0.0886
C-20 Mutant 0.245 0.2090 0.05121 0.20676 1.0013 0.207029 0.2473
Jacob’s Cattle 0.120 0.2325 0.02790 0.21700 1.0013 0.217282 0.1284
Huron 0.107 0.2314 0.02776 0.24234 1.0004 0.242437 0.1021
Mayflower 0.258 0.2040 0.05263 0.23675 1.0005 0.236868 0.2222
Albion 0.159 0.2229 0.03544 0.19849 1.0008 0.198649 0.1784
N87602 0.235 0.2073 0.04872 0.17161 1.0005 0.171696 0.2837
N78042 0.169 0.1598 0.02701 0.21062 1.0015 0.210936 0.1280
Huetar 0.233 0.2104 0.04902 0.2171 1 1.0016 0.217457 0.2254

 

70

Table 5. Indigestible protein in cooked beans in duplicate ‘B’.

 

 

Indigestible Total Initial wt Total
Protein Residue protein in protein sample for protein Indigestible
Identification in residue from TDF residue flour TDF sample protein
(g) (g) (g) (g) (g) (g) (g)
N80242 0.147 0.1674 0.02461 0.20148 1.0011 0.201702 0.1220
Black Turtle Soup 0.184 0.2039 0.03752 0.20758 1.0013 0.207850 0.1805
Seafarer 0.264 0.2145 0.05663 0.20803 1.0003 0.208300 0.2719
Domino 0.267 0.2170 0.05794 0.24164 1.0017 0.242075 0.2393
Sanilac 0.089 0.2080 0.01851 0.21039 1.0013 0.210664 0.0879
Tuscola 0.143 0.191 1 0.02733 0.24442 1.0007 0.244836 0.1 1 16
8217-111-24 0.189 0.2172 0.04105 0.22445 1.0001 0.224472 0.1829
Jalpataqua 0.244 0.2172 0.05299 0.22891 1.001 1 0.229162 0.2312
Nep-2 0.234 0.1517 0.03550 0.23408 1.0005 0.234197 0.1516
San Fernando 0.218 0.2091 0.04558 0.23699 1.0012 0.237274 0.1921
Bunsi 0.167 0.2155 0.03599 0.23019 1.0010 0.230420 0.1562
ICA Pijao 0.237 0.2296 0.05442 0.22689 1.0011 0.227140 0.2396
Aurora 0.191 0.2236 0.04271 0.23961 1.0019 0.240065 0.1779
P766 0.161 0.2126 0.03423 0.22626 1.0016 0.226622 0.1510
Jamapa 0.164 0.2473 0.04056 0.20661 1.0016 0.206941 0.1960
Protop-Pl 0.160 0.2442 0.03907 0.25969 1.0013 0.260028 0.1503
FF4-13-MMMM 0.178 0.2350 0.04183 0.21153 1.0010 0.211742 0.1976
C-20 0.141 0.1955 0.02757 0.21750 1.0006 0.217631 0.1267
Fleetwood 0.093 0.2440 0.02269 0.20859 1.0015 0.208903 0.1086
Mexico 12-1 0.165 0.2140 0.03531 0.22015 1.0013 0.220436 0.1602
Carioca 0.131 0.2219 0.02907 0.21837 1.0007 0.218523 0.1330
Laker 0.104 0.2147 0.02233 0.22907 1.0006 0.229207 0.0974
Black Magic 0.171 0.2402 0.04107 0.21808 1.0019 0.218494 0.1880
Swan Valley 0.107 0.2441 0.02612 0.24027 1.0014 0.240606 0.1086
Midnight 0.158 0.1702 0.02689 0.21735 1.0007 0.217502 0.1236
BAT 41 0.177 0.2269 0.04016 0.24602 1.0004 0.2461 18 0.1632
15-R-148 0.188 0.23 79 0.04473 0.27813 1.0008 0.278353 0.1607
BAT 1507 0.205 0.1997 0.04094 0.23377 1.0009 0.233980 0.1750
BAC 95 0.202 0.2149 0.04341 0.23032 1.0013 0.230619 0.1882
Harblack (O) 0.212 0.2243 0.04755 0.20361 1.0017 0.203956 0.2331
Harblack (Sh) 0.192 0.2213 0.04249 0.21118 1.0006 0.211307 0.2011
Cumulus 0.188 0.2378 0.04471 0.22077 1.0007 0.220925 0.2024
N84004 0.204 0.2291 0.04674 0.24489 1.0005 0.245012 0.1908
C-20 Mutant 0.185 0.21 10 0.03904 0.20676 1.0008 0.206925 0.1887
Jacob’s Cattle 0.148 0.2256 0.03339 0.21700 1.0018 0.217391 0.1536
Huron 0.171 0.221 1 0.03781 0.24234 1.0014 0.242679 0.1558
Mayflower 0.164 0.1981 0.03249 0.23675 1.0015 0.237105 0.1370
Albion 0.159 0.2179 0.03465 0.19849 1.0008 0.198649 0.1744
N87602 0.131 0.1994 0.02612 0.17161 1.0015 0.171867 0.1520
N78042 0.154 0.1578 0.02430 0.21062 1.0014 0.210915 0.1152
Huetar 0.193 0.2559 0.04939 0.2171 1 1.0007 0.217262 0.2273

 

71

Table 6. Mean squares for the variable indigestible protein (g) for 41 dry bean
genotypes prepared by two methods, raw and cooked.

 

 

 

Source of Variation df Indigestible Protein (g) Pr>F
Duplication (D) 1 0.0177 0.0044“
Genotype (G) 40 0.0033 0.0394“
Duplication x Genotype 40 0.0739 0.6483 NS
Error 82

Corrected Total 163

CV (%) 28.9

 

D = replications; G = genotypes

*, ** Significant at the 0.05 and 0.01 levels of probability, respectively; NS = Non-

significant

72

Table 7. Ranking of each raw bean genotype for indigestible starch on a per flour and a

per starch basis.

 

Indigestible starch on a per flour basis

Indigestible starch on a per starch basis

 

 

Mean Mean Difference
Genotype (mg/g flour) Rank Genotype (mg/ 100 mg starch) Rank (di) (di)2
8217-111-24 21.5 1 8217-111-24 5.6 l 0 0
BAT 41 20.9 2 BAT 41 5.4 2 0 0
Huron 19.8 3 BAT 1507 5.2 4 -1 1
BAT 1507 16.4 4 Huron 4.4 3 1 1
Mexico 12-1 16.0 5 Mexico 12-1 3.9 5 0 0
Bunsi 15.7 6 Bunsi 3.7 6 0 0
BAC 95 14.3 7 Laker 3.7 8 -1 1
Laker 14.2 8 Black Magic 3.5 21 -13 169
Sanilac 14.0 9 15-R-148 3.5 11 -2 4
Albion 13.9 10 Mayflower 3.5 14 -4 16
lS-R-l48 13.7 11 Sanilac 3.5 9 2 4
Domino 13 .6 12 Domino 3 .4 12 0 0
Tuscola 13 .3 13 Aurora 3 .4 18 -5 25
Mayflower 13.3 14 BAC 95 3.4 7 7 49
Huetar 13.1 15 Huetar 3 .4 15 0 0
ICA Pijao 13.1 16 Harblack (O) 3.3 26 -10 100
Black Turtle Soup 13.1 17 Jalpataqua 3.3 28 -1 1 121
Aurora 13.1 18 ICA Pijao 3.3 16 2 4
N84004 12.9 19 Black Turtle Soup 3.2 17 2 4
San Fernando 12.8 20 C-20 3.1 22 -2 4
Black Magic 12.4 21 N84004 3.1 19 2 4
C-20 12.4 22 Albion 3.1 10 10 144
Jamapa 12.2 23 Tuscola 3.1 13 10 100
Nep-2 1 1.3 24 San Fernando 3.0 20 4 16
Carioca 1 1.2 25 Jamapa 3.0 23 2 4
Harblack (O) 11.2 26 Carioca 2.9 25 1 1
FF4-13-MMMM 10.5 27 Fleetwood 2.6 29 -2 4
Jalpataqua 10.2 28 Nep-2 2.6 24 4 16
Fleetwood 10.2 29 N80242 2.6 30 -1 1
N80242 10.0 30 FF4-13-MMMM 2.6 27 3 9
Seafarer 10.0 3 1 Seafarer 2.5 3 1 0 0
Swan Valley 9.0 32 P766 2.5 33 -6 1
P766 9.7 33 C-20 Mutant 2.3 38 -5 25
N78042 9.5 34 N78042 2.3 34 0 0
Jacob’s Cattle 8.8 35 Cumulus 2.2 36 -1 1
Cumulus 8.5 36 Swan Valley 2.2 32 4 16
N87602 8.5 37 Jacob’s Cattle 1.9 35 2 4
C-20 Mutant 8.1 38 Protop-Pl 1.9 41 -3 9
Midnight 8.1 39 N87602 1.9 37 2 4
Harblack (Sh) 8.0 40 Harblack (Sh) 1.8 40 0 0
Protop-Pl 7.1 41 Midnight 1.8 39 2 4

 

73

Table 8. Ranking of each cooked bean genotype for indigestible starch on a per flour and
a per starch basis.

 

 

 

Indigestible starch on a per flour basis Indigestible starch on a per starch basis

Mean Mean Difference
Genotype (mg/g flour) Rank Genotype (mg/ 100 mg starch) Rank (di) (di)2
Cumulus 48.7 1 Cumulus 11.9 1
0 0
N80242 44.9 2 N84004 10.3 5 3 9
Sanilac 41.9 3 BAT 41 10.1 4 -1 1
BAT 41 41.1 4 P766 9.8 24 -20 400
N84004 39.5 5 Black Turtle Soup 9.4 10 -5 25
Albion 37.1 6 N80242 9.0 2 4 16
C-20 Mutant 37.1 7 Huron 8.9 12 -5 25
Carioca 36.6 8 15-R-148 8.9 40 -32 1024
Laker 36.4 9 C-20 Mutant 8.7 7 2 4
Black Turtle Soup 35.3 10 82l7-III-24 8.7 22 -12 144
Bunsi 34.9 1 1 Sanilac 8.5 3 8 64
Huron 34.6 12 BAC 95 8.5 14 -2 4
Seafarer 34.5 13 Domino 8.4 18 -5 25
BAC 95 34.3 14 Bunsi 8.4 1 1 3 9
Midnight 34.2 15 Aurora 8.4 31 -16 256
Jacob’s Cattle 34.1 16 Seafarer 8.4 13 3 9
ICA Pijao 33.9 17 Huetar 8.3 19 -2 4
Domino 33.9 18 BAT 1507 8.3 21 -3 9
Huetar 33 .6 l9 Laker 8.3 9 10 100
C-20 33.4 20 Albion 8.3 6 14 196
BAT 1507 33.3 21 Protop-Pl 8.2 25 -4 16
8217-111-24 33.1 22 Carioca 8.1 8 14 196
Nep-2 33.0 23 Midnight 8.1 15 8 64
P766 32.8 24 ICA Pijao 7.9 17 7 49
Protop-Pl 32.4 25 Harblack (O) 7.9 29 -4 16
Mexico 12-1 32.4 26 C-20 7.9 20 -6 36
Fleetwood 3 1 .7 27 Nep—2 7.9 23 4 16
N78042 31.7 28 Fleetwood 7.9 27 1 1
Harblack (O) 31.6 29 Jacob’s Cattle 7.9 16 13 169
Tuscola 31.4 30 Harblack (Sh) 7.5 35 -5 25
Aurora 31.3 3 l Jalpataqua 7.4 36 -5 25
Black Magic 31.1 32 N87602 7.3 38 -6 36
San Fernando 30.8 33 San Fernando 7.3 33 0 0
Swan Valley 30.8 34 Black Magic 7.3 32 2 4
Harblack (Sh) 30.3 35 Jamapa 7.2 37 -2 4
Jalpataqua 30.2 36 N78042 7.2 28 -8 64
Jamapa 29.6 37 Swan Valley 7.2 34 3 9
N87602 29.4 38 Mexico 12-1 7.0 26 12 144
FF4-13-MMMM 28.4 39 FF4-13-MMMM 6.7 39 0 0
15-R-148 28.0 40 Tuscola 6.7 30 10 100
Mayflower 27.0 41 Mayflower 6.3 41 0 0

 

74

Table 9. Spearman’s rank correlation coefficient (r, statistic) between the ranks of
raw and cooked beans of 41 dry bean genotypes for indigestible starch on a per g of flour
basis and on a per 100 mg starch basis.

 

Spearman’s rank correlation
coefficient (r,)

 

Raw Cooked

 

Indigestible starch on a per g of flour basis
vs. 0.9 0.7
Indigestible starch on a per 100 mg of starch basis

 

 

75