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This is to certify that the

dissertation entitled

PROCESSING STRATEGIES TO IMPROVE DRY BEAN (PhaseoTus vulgaris)
DIGESTIBILITY AND FOOD UTILIZATION

presented by

Liiiian Geraldine Occefia

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in TFOOd SCIENCE

 

 

Major professor

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Date ApriT 29, 1994

 

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

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PROCESSING STRATEGIES TO IMPROVE DRY BEAN (Phaseolus vulgaris)
DIGESTIBILITY AND FOOD UTILIZATION

By

Lillian Geraldine Oooefia

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition
1994

ABSTRACT

PROCESSING STRATEGIES TO IMPROVE DRY BEAN (Phaseolus vulgaris)
DIGESTIBILITY AND FOOD UTILIZATION

By

LillianGeraldineOccefia

Dry edible beans serve as a major source of protein in many developing countries.
However, many constraints, including antinutritional and flatulence factors which interfere
with protein digestibility, limit wider utilization of dry edible beans. Processing strategies
designed to eliminate these factors and provide a precooked ingredient suitable for food
formulations are warranted.

Whole and split dry beans (Phaseolus vulgaris) soaked and hydrated for 16 hrs.
(25°C) were subjected to hot water (60°C/60 mins.), steam (100°C/15 mins.) and
differential enzymatic pretreatments. Cooked CONTROL beans contained all original soak
and rinse waters, cook water or condensate (formulation water) prior to milling to a
homogeneous slurry whereas leachate of EXTRACT and cooked beans was replaced with
fresh formulation water. Bean slurries pre-heated to 95°C were dried on a double drum-
dryer. Enzymatic pretreatment involved the use of both indigenous ("sprout slurry") and
commercial enzymes (Viscozyme, Alcalase, Neutrase and Celluclast).

The drum-dried bean meals were analyzed for proximate composition (moisture,
5%; protein, 23%; fat, 2%; and ash, 4%), total soluble sugars (stachyose, 0.3% to
0.8%), dietary fiber (soluble, 5% and insoluble, 15-21%), total mineral content (plasma
emission spectrometry) and in-vitro starch and protein digestibilities. Functional properties
of the drum-dried bean meals, including color, water absorption index, water solubility
index and degree brix of the soluble fraction were also evaluated.

The resultant bean meals were differentiated by soluble losses and quality
characteristics. Hot water extraction provided an approximately 8% increase in protein
digestibility (in-vitro). Soluble dietary fiber content was approximately 5%, whereas
insoluble dietary fiber averaged 18%. Hot water extraction resulted in significant reduction
of minerals (K, Ca, Al, Cu, Fe, Zn and Mg) and soluble sugars (stachyose, 0.3%-0.8% ).

The processed product had neutral color and did not possess the frequently
objectionable "beany" flavor. The DDBMs were incorporated in a pumpkin quick bread
formulation (0%, 20%, 40%, and 60% levels of substitution) and a weaning beverage
(5%, 8% and 10%). Acceptable quality breads were produced at the 20% and 40% levels
of substitution. Enzyme pre-digested DDBMs possess great potential in weaning food
formulations, especially at the 8% concentration level. The use of indigenous enzymes
(i.e., sprouting ) to modify the functional properties of DDBM used in weaning beverage
formulations warrants additional research.

Microwave preconditioning of dry beans demonstrated the potential of microwave
energy to improve whole bean seed cooking, ensure rapid processing of beans by
disrupting starch granules and facilitating gelatinization. The physical and functional
properties (swelling power, solubility, degree of syneresis, appparent viscosity and
elasticity) of pure bean starch were significantly affected by microwave heating.

To my parents, who have provided me a strong foundation
to go through life's challenges

iv.

ACKNOWLEDGMENTS

My sincere appreciation goes to my Graduate Committee members: Dr. Maurice
Bennink, Dr. George Hosfield, Dr. Mary E. Zabik and Dr. Wanda Chenoweth, for their
valuable advice and guidance throughout my Ph.d Graduate program:

I am most grateful to my Advisor, Dr. Mark A. Uebersax, who has become a
mentor, colleague, teacher and friend; thank you for providing me the support and
opportunities for my professional growth.

I am fortunate to have wonderful friends in:

Ahmad Shirazi, who was always encouraging and available to lend a
helping hand and listening ear

Sharon Hart, Miriam Nettles, Virginia Vega, Nirmal Sinha, Danny
Campos, Leticia Carpio, Cheribeth Tan, who always cheered
me up when the going got rough

Danny, Letlet and Muhammad Siddiq, for helping and staying up with me
during those last nights before the defense.

My brother, Luis, who is always around when I need help.

I am forever grateful to my family for their love, support and encouragement.

Sincere gratitude is extended to Gerber Products Company in Fremont, M1 for the
use of their facilities; thank you Jerry Stroven and Duane Dougan, for all your assistance.

I also thank the Bean-CowpealCRSP for partial support of this project

To the Aggie family of Xavier University, especially Fr. Ledesma & the Food Tech
faculty and staff, for holding the fort while I was on study leave

Above all,IamgratefultotheAlmightyforkeepingmein thepalmofI-Iishand.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................... xi
LIST OF FIGURES ............................................................................. xiii
LIST OF PLATES ............................................................................... xvii
INTRODUCTION ............................................................................... 1
REVIEW OF LITERATURE ................................................................. 4
Chemical Composition of Dry Beans (Phaseolus vulgaris) .................................... 4
Carbohydrates ....................................................................... 4

Proteins ............................................................................... 11

Minerals and Vitamins .............................................................. 12

Lipids ................................................................................. 13

Tannins and Polyphenols ........................................................... 14

Bean Flavor Constituents ........................................................... 14

Nutritional Digestibility and Physiology of Dry Edible Beans ........................ 16
Factors Affecting Digestibility of Dry Legume Starch and Protein ............ 16
Digestibility of Legume Protein .................................................... 22
Digestibility of Legume Starch ..................................................... 25

Flatulence Associated with Legume Consumption ............................... 27

Raffinose, Stachyose and Verbascose ...................................... 28

Dietary Fiber ................................................................... 31

Resistant or Indigestible Starch 33
Hypocholesterolemic and Hypoglycemic Responses of Bean Dietary Fiber. 39
Processing and Utilization of Dry Edible Beans as Specialized Foods .............. 44

Bean Sprouts, Curds and Ferments ............................................... 46

vi

Bean Meals, Flours and Powders ................................................ . 47

Functional Characteristics and Applications ...................................... 48
Drum-Drying of Legumes ................................................... 51
Description of Equipment and Process System .................... 51

Control of Drying Process ............................................ 53

Thermal Extrusion ................................................................... 54
Microwave Heating of Legumes ................................................... 56
Mechanism of Microwave Heating ........................................ 56
Microwave Effects on Food Composition ................................. 57

Potential for Dry Edible Beans as Weaning Foods .............................. 61
Potential for Soybean as a High -Protein Weaning Beverage .................. 66
MATERIALS AND METHODS ............................................................. 72
Hot Water/Steam Extractions of Whole Navy Beans ................................... 72
Enzymatic Pre-digestion of Whole Navy Beans ....................................... 75
Aqueous Extractions and Enzymatic Pre-digestion of Split Navy Beans ............ 76
Drum—Drying of Bean Meals .............................................................. 78
Chemical and Biological Analyses ....................................................... 79
Proximate Composition ............................................................. 79
Moisture ........................................................................ 79

Protein .......................................................................... 80

Fat ............................................................................... 80

Ash .............................................................................. 80

Soluble Sugars by HPLC ........................................................... 80
Extraction of Soluble Sugars ................................................ 80
Separation and Quantitation .................................................. 82

Total Mineral Content ............................................................... 83
Insoluble and Soluble Dietary Fiber ............................................... 83

vii

In-vitro Starch Digestibility ......................................................... 86
Determination of Total Starch 86

Determination of Available Starch 88

In-vitro Protein Digestibility ........................................................ 9O
Methionine ............................................................................ 92
Amino Acid Analysis ................................................................ 92
SDS-PAGE Electrophoresis ........................................................ 93

Assessment of Antinutritional Factors 94

Screening for Heat-Stable Trypsin Inhibitory Activity .................. 94
Screening for Phytohemagglutinins ........................................ 96

Phytic Acid .................................................................... 96

Physical and Functional Properties of Drum-Dried Bean Flours ..................... 98
Color .................................................................................. 98
Cellular Structure and Starch Granule Appearance .............................. 98
Solubility ............................................................................. 99
Water Absorption Index ...................................................... 99

Water Solubility Index ........................................................ 99

Water Holding Capacity ............................................................ 99
Bulk Density ......................................................................... 100
Nitrogen Solubility Index (NSI) ................................................... 100
Incorporation of Drum-Dried Bean Flour in Food Product ..................... 100
Quick Bread .......................................................................... 100
Weaning Food Beverage ............................................................ 102
Objective Quality Evaluation of Bean Products ......................................... 103
Viscosity ............................................................................. 103

pH .................................................................................... 103
Texture ....... ~ ......................................................................... 103

viii

Volume of the bread ................................................................. 103

Density ................................................................................ 105
Moisture .............................................................................. 105
Microstructure ....................................................................... 105
Weaning Food/Beverage .................................................................. 105
Suspension Stability ................................................................ 105
Visual Stability ................................................................ 105

Micro Quantitative Method ................................................... 105
Consistency .......................................................................... 107
Apparent Viscosity ........................................................... 107

Relative Viscosity ............................................................. 107
Physico-Chemical Tests ........................................................... . 107
Color ........................................................................... 107

pH .............................................................................. 107

Soluble solids ................................................................. 107

Total Solids .................................................................... 108
Microwave Preconditioning of Whole Dry Navy Beans .............................. 108
Texture ................................................................................ 108
Color .................................................................................. 108
Cellular Structure and Starch Granule Appearance .............................. 110
Microwave Heating of Bean Starch ...................................................... 110
Bean Starch Isolation ............................................................... 110
Bean Starch Chemical Composition ............................................... 111
Heating of Bean Starch ............................................................ . 111
Microwave Heating ........................................................... 111
Conventional Heating ........................................................ 112

Bean Starch Physical Properties ................................................. . 112

ix

Starch Swelling Power and Solubility ..................................... 112

Degree of Syneresis .......................................................... 112
Gel Rheological Properties .................................................. 112
Scanning Electron Microscopy (SEM) ..................................... 112
Statistical Analysis ......................................................................... 113

CHAPTER I. EXTRACTIVE PRETREATMENTS TO IMPROVE DRY

BEAN DIGESTIBILITY ...................................................................... 114
Introduction ................................................................................. 114
Materials and Methods ..................................................................... 115
Results and Discussion .................................................................... 117
Conclusion .................................................................................. 159

CHAPTER II. FUNCTIONAL CHARACTERISTICS OF DRUM-DRIED
BEAN MEALS AND THEIR INCORPORATION IN SELECTED FOOD

SYSTEMS .......................................................................................... 162
Introduction ................................................................................. 162
Materials and Methods .................................................................. . 163
Results and Discussion .................................................................... 166
Conclusion .................................................................................. 189

CHAPTER III. MICROWAVE HEATING CHARACTERISTICS OF

WHOLE BEANS AND BEAN STARCH ................................................. 191
Introduction ................................................................................. 191
Materials and Methods ..................................................................... 192
Results and Discussion .................................................................... 195
Conclusion .................................................................................. 209

SUMMARY AND RECOMMENDATIONS .............................................. 213

LIST OF REFERENCES ...................................................................... 216

Table 1.

Table 2.
Table 3.
Table 4.
Table 5.

Table 6.
Table 6.
Table 7.

Table 7.

Table 8.

Table 9.

Table 10.

Table 11.
Table 12.
Table 13.

Table 14.
Table 15.
Table 16.

LIST OF TABLES

Nutritional composition of selected raw dry edible beans (Phaseolus

vulgaris) ............................................................................... 8
Constraints limiting utilization of dry beans ........................................ l7
Pumpkin bread formula ............................................................... 102
Proximate chemical composition (% db) of split bean drum-dried meals ....... 120
Proximate chemical composition of non-extracted (CONTROL) with

subsequent enzyme-predigested treated whole drum-dried bean meals ....... 121
Total mineral content (ppm) split bean drum-dried meals ......................... 125
(Cont.'d) Total mineral content (ppm) split bean drum-dried meals ............. 126

Total mineral content (ppm) of non—extracted (CONTROL) with subsequent
enzyme pre-digested treated whole drum-dried bean meals.................... 127

(Cont.'d) Total mineral content (ppm) of non-extracted (CONTROL) with

subsequent enzyme pre-digested treated whole drum-dried bean meals ...... 128
Effect of extractive pre-treatments on the total starch content of bean drum-

dried meals ........................................................................... 132
Effect of extractive pre-treatments on the in-vitro digestibility of starch in raw

split navy beans and split bean drum-dried meals 135
Effect of enzymatic pre-digestion on in-vitro digestibility of starch in raw

whole Navy beans and whole bean drum-dried meals .......................... 136
Amino acid content (p moles) of split drum-dried bean meals .................... 156
Methionine content of drum-dried bean meals ...................................... 156

Heat stable Trypsin Inhibitor Activity (TIA) inositol phosphates (%) and
phytate content (%) in split bean drum-dried meals ............................. 158

Visual reaction for screening of dry bean lectin (phytohemagglutinin) .......... 160
Physical and functional properties of split bean drum-dried bean meals ........ 169

Physical characteristics of pumpkin breads substituted with 0-60% drum-
dried bean meal ..................................................................... . 172

xi

Table 17.

Table 18.

Table 19.
Table 20.

Multiple Blade Shear Compression Mode of pumpkin breads with 0 to 60%
drum-dried bean meal substitution ................................................. 175

Mean values of physical Texture Profile Analysis (T PA) of drum-dried bean
meal pumpkin breads ................................................................ 176

Physical characteristics of weaning bean beverages ............................... 180

Microwave pre-conditioning and re-heating times (min.) using a Nordicware
microwave pressure cooker ....................................................... . 201

xii

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

Figure 7.
Figure 8.

Figure 9.

Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.

Figure 15.

Figure 16.
Figure 17.

Figure 18.
Figure 19.
Figure 20.

LIST OF FIGURES

Proximate composition of selected raw legumes (Phaseolus vulgaris) ......... 5
Macro-mineral content of selected raw legumes (Phaseolus vulgaris) .......... 6
Micro—mineral content of selected raw legumes (Phaseolus vulgaris) .......... 6
Vitamin content of selected raw legumes (Phaseolus vulgaris) .................. 7
Energy values of selected raw legumes (Phaseolus vulgaris)................... 7

Outline summarizing factors influencing digestibility and bioavailability of
nutrients in dry beans ............................................................. . 18

Nitrogen digestion, absorption and retention ..................................... 19

Proposed mechanism of gas production involving oligosaccharides and

resistant starch ..................................................................... . 29
Outline illustrating processing strategies and edible by-products of common
dry beans in LDC and industralized countries ................................... 45
Schematic components of a double surface drum-drier ......................... . 52
World production of major food legumes .......................................... 67
Illinois method for processing soymilk from whole soybeans .................. 69
Flowchart for hot water extraction of dry navy beans ............................ 73
Designation of treatment tenninology and extraction treatment differentiation
used during cooked bean preparation............................................ 74
Flowchart for enzyme pre-digestion of whole and split Navy beans prior to
drum-drying ......................................................................... 77

Flowchart for the extraction of soluble sugars from drum-dried bean meals. 81

Flowchart of enzymatic method to determine the insoluble and soluble dietary

fiber of drum-dried bean meals .................................................. . 84
Flow diagram for determination of total starch .................................... 87
Flowchart for the determination of available starch ............................... 89
Flowchart for in-vitro protein digestibility ......................................... 91

xiii

Figure 21.
Figure 22.

Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.

Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Figure 34.

Figure 35.

Figure 36.

Figure 37.
Figure 38.
Figure 39.

Figure 40.

Figure 41.

Figure 42.

Figure 43.

Flowchart for the Trypsin Inhibitor Assay (TIA) ................................. 95
Flowchart for the separation and quantitative determination of phytic acid as
inositol phosphate ................................................................. . 97
Flowchart of utilization of drum-dried bean flour in pumpkin bread ........... 101
Flowchart of the objective evaluation of drum-dried bean meal products ...... 104

Experimental flowchart for the preparation of the weaning bean beverage... 106
Experimental design for the microwave pre-conditioning of dry navy beans. 109

The experimental flow chart for study [-1. Extractive pre-treatment to

improve dry bean digestibility ..................................................... 116
Moisture-solids balance for drum-dried bean meals .............................. 118
Soluble sugars of split drum—dried bean meals .................................... 123

Soluble sugar content of enzyme pre-digested whole drum-dried bean meals 123

Insoluble and soluble dietary fibers of split bean drum-dried meals ............ 130
Insoluble and soluble dietary fibers of whole bean drum-dried meals ......... 130
Total starch content of raw and split bean drum-dried meals .................... 133

Total starch content of raw and enzyme pre-digested whole bean drum-

dried meals ......................................................................... . 133
Hydrolysis of starch in raw and split bean drum-dried meals by pancreatic
alpha—amylase ....................................................................... 137
Hydrolysis of starch in raw and whole bean drum-dried meals by in-vitro
pancreatic alpha-amylase ........................................................... 138
In-vitro protein digestibility of split bean drum-dried meals ..................... 151
In-vitro protein digestibility of whole bean drum-dried meals ................... 151
Graphical representation of the SDS-gel electrophoretic pattern of enzyme
pre-digested whole drum-dried bean meals .................................... . 153
The experimental flowchart for Study H: Functional characteristics of
drum-dried bean meals and their incorporation in selected food systems... 164
Flowchart of the experimental design for Study H-2: Incorporation of
drum-dried bean flour in pumpkin bread ....................................... . 165
A generalized Texture Profile Analysis (TPA) curve and a typical pumpkin
bread TPA curve .................................................................... 177

Comparative visual stability of bean beverage DDBMs and soy formula ...... 182

xiv

Figure 44.

Figure 45.

Figure 46.

Figure 47.
Figure 48.

Figure 49.

Figure 50.

Figure 51.

Figure 52.

Figure 53.

Figure 54.

Figure 55.

Figure 56.

Figure 57.

Figure 58.

Figure 59.
Figure 60.

Figure 61.

Absorbance vs concentration for 5% EXTRACT and 5% Viscozyme bean

beverages ............................................................................ 184
Absorbance vs Concentration of 8% EXTRACT and 8% Viscozyme bean

beverages ............................................................................ 184
Absorbance vs Concentration of 10% EXTRACT and 10% Viscozyme

bean beverages ...................................................................... 184
Comparative absorbance of 5% bean beverages ................................. . 186

Comparative absorbance of 8% EXTRACT and enzyme pre-digest
(Viscozyme) beverages ............................................................ 186

Comparative absorbance of 10% EXTRACT and enzyme pre-digest bean
beverages ............................................................................ 186

Comparative transmission (%) of bean beverage separated supemate
from DDBM EXTRACT and soy formula ....................................... 187

Comparative visual stability of bean beverage from DDBM and soy

formula .............................................................................. 187
Viscosity of bean beverages from drum-dried bean meals at different
concentration levels ................................................................. 188
Time for the bean beverage (5%) to flow through the two marks of an
Oswald pipette ...................................................................... 188
Apparent viscosities of 5% EXTRACT bean beverages with different levels
of emulsifiers ........................................................................ 190
Time for 5% EXTRACT and 5% Viscozyme bean beverages with
caregeenan (0.02 & 0.03%) to flow through the two marks of an Oswald
pipette ................................................................................ 190
Comparative transmission (%) of 5% EXTRACT with and without
carageenan ........................................................................... 190
Comparative transmission (%) of 5% Viscozyme bean beverage with and
without carageenan ................................................................. 190
The experimental flowchart for Chapter III: Microwave pre-conditioning of
dry beans (Study 1) and microwave heating of bean starch (Study H) ....... 193

Moisture content of dry navy beans soaked at 25°C for various time periods. 196

Maximum force required to shear Navy beans pre-conditioned by
microwavesin a Nordicware pressure cooker ................................... 197

Maximum force required to shear pre—conditioned Navy beans after re—
heating (0.5 min., 740 W) in a Nordicware pressure cooker ................. 197

XV

Figure 62.

Figure 63.

Figure 64.

Figure 65.

Figure 66.

Figure 67.
Figure 68.
Figure 69.

Figure 70.

Figure 71.

Total increase (% above soak weight) in weight of pre-conditioned Navy
beans after re-heating ( 0.5 min., Nordicware pressure cooker)......... 198

Maximum force required to shear Navy beans cooked in a conventional
retort (45 min., 116°C) or subjected microwave energy levels in
Nordicware pressure cooker ..................................................... . 198
Color of final product as affected by post-soaking moisture content and
microwave energy levels used for pre-conditioning (Nordicware pressure
cooker) ............................................................................... 199

Swelling power pattern of bean starch with conventional and microwave
he at in g ............................................................................... 204

Swelling power pattern of corn starch with conventional and microwave
he at i n g ............................................................................... 204

Solubility Index (SI) of corn and bean starches during microwave heating... 205
Solubility Index (SI) of corn and bean starches during conventional heating. 205

Degree of syneresis (%) of 8% (w/w) starch gels stored at room
temperature for 1 week ............................................................. 207

Apparent viscosity (poise) of 8% (w/w) starch gels stored at room
temperature for 7 days ............................................................. 208

Apparent Elasticity of 8% (w/w) starch gels stored at room temperature
for 7 days ............................................................................ 208

xvi

Plate 1.

Plate 2.

Plate 3.

Plate 4.

Plate 5.

Plate 6.
Plate 7.

Plate 8.

Plate 9.

Plate 10.

Plate 1 1.

Plate 12.

Plate 13.

Plate 14.

LIST OF PLATES

Raw split meal (a-b) under LSM microscopy and (c-d) under polarizing
light microscopy ...................................................................... 142

EXTRACT DDBM (a—b) under LSM microscopy and (c-d) under polarizing
light microscopy ...................................................................... 143

LSM micrographs of non-extracted (CONTROL) (a-b) and enzyme
pre- digested whole bean drum- dried meals. Viscozyme (c- d), Sprout (e- f),
Alcalase (g- -,h) Neutrase (i-j) and Celluclast (k- l) ............................ 144-145

LSM micrograph of canned and subsequently freeze-dried whole navy bean... 146

LSM micrographs of (a) soft canned bean and (b) firm canned bean
(92 RNK 8P) .......................................................................... 147

General appearance of a) raw navy bean splits and b) EXTRACT DDBM...... 167

Crystalline structure of drum-dried bean meal under a stereomicroscope
(50x): a) EXTRACT, b) VISCOZYME ............................................ 168

Appearance of pumpkin quick breads prepared with different levels (20%,
40% and 60%) of pre-treated DDBMs (CONTROL, EXTRACT,
VISCOZYME and SPROUT) ....................................................... 171

General appearance of pumpkin quick breads prepared with CONTROL
DDBM substituted at 20% to 60% reference sample in 100% wheat flour
formulation ............................................................................ 173

LSM micrographs of pumpkin bread cellular microstructure: a) Reference
(100% all-pu urpose flour), b) CONTROL DDBM, c)" EXTRACT DDBM,
d) VISCOZYME DDBM, e) SPROUT DDBM... ... 179

Appearance of weaning beverage prepared with different levels (5%, 8%,
10%) of pretreated (a) EXTRACT and Viscozyme DDBMs and
.(b) CONTROL and Sprout DDBMs ................................................ 183

Structural components of dry (a) and soaked (b) (25°C, 16 hours) navy
beans. Pictures were taken from the cross-section of the seeds using a
LSM .................................................................................... 202
Cellular structure of navy beans pre—conditioned (a) at 590 watts for 5 min.
and subsequently re-heated (b) at 740 watts for 3.5 min. from frozen state.
Pictures were taken from a cross-section of the whole beans using LSM ..... 203

8% starch gels stored at room temperature for 7 days .............................. 207

xvii

Plate 15. Scanning Electron Micrographs of (a) Bean and (b) Corn Starch granules
microwave-heated at 70°C for 2 minutes .......................................... 210

Plate 16. Scanning Electron Micrographs of (a) Bean and (b) Corn Starch granules with
conventional heating at 70°C for 2 minutes ....................................... 211

xviii

INTRODUCTION

Seeds of the annual legume (Phaseolus vulgaris L.), commonly termed dry edible
beans, serve as the major alternative source of protein and calories in lesser developing
countries (LDC's) of Latin America and Africa, and are also frequently considered for
applications in world food relief programs in regions experiencing sustained crop failure.
Although per capita bean consumption is significantly much less in developed countries
compared to LDCs, it is projected to increase due to focused dietary recommendations
as well as diet and health positioning currently presented by health authorities. Dry bean
fiber is now being promoted as the "Fiber of the 90's" due to potential
hypocholesterolemic and hypoglycemic effects .

Major constraints still limit wider dry bean utilization. Biological or nutritional
concerns include antinutritional and flatulence factors which interfere with protein
digestibility and bioavailability of minerals. The preference for convenient and fast foods
in industrialized economies also presents a major drawback to consumer utilization of
beans due to the excessive time and expense involved in their preparation. Thus, the
technological challenge is to develop highly digestible, preprocessed, prolonged shelf-life
food products 'and ingredients from dry edible beans with minimum process inputs and
appropriate technologies. The World Health Organization (WHO) and the United Nations
International Children and Educational Fund (UNICEF) which have been promoting and
supporting appropriate "complementary" or "weaning" feeding practices in LDCs, have
encouraged the use of indigenous food resources such as dry beans. Dry edible beans

therefore possess potential in the development of improved, low-cost weaning food

2
formulations, which could alleviate protein and energy malnutrition in young children.

This dissertation was conducted in conjunction with the Bean/Cowpea
Collaborative Research Support Program (cusp) of coordinated projects in Africa and
Latin America focusing on removal of constraints to the production and utilization of beans
and cowpeas. The program is funded by a Title XII grant from the United States Agency
for International Development (U SAID)/BIFAD with the ultimate goal of reducing hunger
and malnutrition in developing countries. In many of the countries where beans and
cowpeas are staple foods, responsibility of production and preparation for family
consumption rests with women and children. It is envisioned that village-level food
centers, possibly managed by cooperatives, could centrally process locally produced raw
materials such as dry edible beans into nutritious foods which can be distributed through
day-care centers, school lunch programs and nutrition intervention clinics. Such a
strategy will not only benefit infants, young children, pregnant and lactating women, but
will also serve as an activity which will directly involve women in management and
employment enterprises.

The purpose of this research was to investigate possible processing strategies
which will reduce antinutritional and digestibility factors, as well as decrease the time
involved in the preparation of dry beans. Individual studies were conducted to address
specific hypotheses associated with the objectives, and are discussed in three Chapters.
Chapter I (Studies [-1 and I-2) involved the development of shelf-stable drum-dried bean
meals which possess potential in preparation of improved weaning food formulations. The
effects of different extractive pretreatrnents (hot water, steam and enzymatic ) on drum-
dried bean meal composition and digestibility were evaluated in-vitro. ther, the
feasibility of the use of germinated sprouted dry beans to serve as an indigenous source of
enzymes was investigated. Chapter 11 (Studies II-l, II-2 and II-3) was undertaken to
characterize the functional properties of the drum.dried bean meals and to evaluate their
performance in selected food systems (quick bread and weaning food beverage). Chapter

3

III (Studies III-1 and 111-2) was conducted to assess the feasibility of using microwave
energy as a preconditioning treatment to reduce cooking time of dry beans. The physical
properties of isolated bean starch during microwave heating and conventional cooking

were further compared in this study.

REVIEW OF LITERATURE

CHEMICAL COMPOSITION OF DRY EDIBLE BEANS (Phaseolus
vulgaris)

Although dry edible beans clearly differ in size and seedcoat color, there is a very
basic commonality among commercial classes (navy, pinto, dark and light red kidney,
Great Northern, pink, cranberry and black turtle soup beans). The compositional
components of dry edible beans including protein, starch, fiber and minerals are generally
very similar among these classes of dry beans (Figures 1, 2, 3 and 4). Further, the total
dietary caloric energy derived from different bean classes is identical (Figure 5). Dry
beans are of lower caloric value than soybeans (Glycine max L. Merr.) and peanuts
(Arachis hypgea L.), both of which contain significantly higher amounts of lipids. These
data (Table 1) are compiled from a major USDA funded analytical screening project and
represent numerous sampling points of raw edible beans. It is most evident that only minor
variations in each component exists among the diverse bean types presented. Thus, the
interchange or substitution of different bean types within a major seedcoat class in recipes
which require milling, mashing or mixing can be readily accomplished without dramatic
functional or nutritional consequences (Uebersax and Occefia, 1991).

An outlined overview of the major constituents is provided to specifically delineate
and to further demonstrate the similarity in composition among different commercial

classes of dry beans.

Carbohydrates
The total carbohydrates of dry legumes range from 24.0% in winged beans to
68.0% in cowpeas (Reddy et al., 1984). Starch constitutes the most abundant legume

 

 

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I G. Northern

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Protein

Moisture

Figure l. Proximate composition of selected raw legumes (Phaseolus vulgaris)

Source: Uebersax and Ocean. 1991

 

 

 

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Black Crberry G.North. Kidney Navy Pink Pinto

Figure 2. Macro-mineral content of selected

 

raw legumes (Phaseolus vulgaris)
(SamUebaxumemlWl)
20
D Sodium
I Zinc
Cl Manger“
H Copper
I Iron
8 10"

 

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Figure 3. mac-mineral cmtentof selected
raw legumes (Phaseolus vulgm's)

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legumes (Phaseolus vulgaris)

 

82

Figure 4. Vitamin content of selected raw
Energy
Figure 5. Energy Values of Selected Raw

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9

carbohydrate ranging between 24.0% to 56.6%. Total sugars represent only a small
portion of the total carbohydrate content.

Sugars. Among the sugars, oligosaccharides of the raffinose family (raffinose,
Stachyose, verbascose, and ajugose) predominate in most legumes and account for 31.1%
to 76.0% of the total sugars (Akpapunam and Markakis, 1979; Kon, 1979; Rockland et al.,
1979; Ekpenjong and Borchers, 1980; Reddy and Salunkhe, 1980; Fleming, 1981 and
Sathe and Salunkhe, 1981). Walker and Hymowitz (1972) found that the sugar content
ranged from 4.4% to 9.2% in 28 varieties of Phaseolus vulgaris. Sucrose accounted for
46.4% of total sugars present, while rafftnose and Stachyose made up 10.4% and 43%,
respectively. The predominance of a particular oligosaccharide depends on the type of
legume. For example, Stachyose represents the major oligosaccharide in most varieties of
Phaseolus vulgaris while verbascose is the major oligosaccharide in black gram, red
gram, mung bean and broad beans (faba beans); raffinose is present in moderate to low
amounts.

Starch. Variations in reported starch contents (24-56%) are partially due to
differences in cultivars and analytical procedures. The physimhemical properties and
internal molecular structures of bean starches differ depending on the original source,
maturation and environmental factors. Most bean starch granules have wide variability in
size (1-80 u) and shape which could be due to genetic control and seed maturity. For dry
beans, a wide variability in shape (oval, oblong, round) is found in starch granules from
the same source (Reddy et al., 1984). Biliaderis et a1. (1979) reported that the major
portion of legume starches have molecular weights higher than 2 x 106 kd , and over 90%
of them have molecular weights above 4 x 104 ltd.

Amylose contents ranging from 10% to 66%, may influence starch solubility, lipid
binding, and other functional properties. Biliaderis et al. (1981) fractionated legume
starches and reported that the variability between amyloses from different legumes may be

due to: l) maturity of seed, 2) genetic control of amylose synthesis, 3) cultivar

l O

differences, and/or 4) seed storage history. A high degree of amylose polymerization may
confer structural stability to the granule but may also be partially responsible for its
resistance toward in-vitro alpha-amylolysis. Most legume starches have gelatinization
temperatures of 60°C to 90°C and are characterized by no distinctive pasting peak, but
rather, a very high viscosity which remains constant or increases during cooling. The
crystalline nature of the starch granule manifests birefringence (exhibited by the appearance
of a "Maltese cross" under polarizing light) which is lost when starch is completely
gelatinized. Indigestible or resistant bean starch and its possible role in flatulence is an
area of interest in bean fiber research (Thompson et al., 1987 ).

Lai and Varriano-Marston (1979) indicated that bean starch had lower swelling
power than wheat starch within the temperature range 60°C to 74°C; however, above
75°C, bean starch surpassed wheat starch in swelling power. The restricted swelling,
plus the single-stage swelling pattern, have been interpreted to explain the crystalline
(ordered) and amorphous (unordered) regions of starch granules and the presence of strong
binding forces which relax within a temperature range. The restricted swelling
characteristics are similar to those shown by chemically cross-linked starches (Schoch and
Maywald, 1968; Lineback and Ke, 1975; Vose, 1977; Lai and Varriano-Marston, 1979;
Naivikul and D'Appplonia, 1979; Li and Chang, 1981; Sathe and Salunkhe, 1981).
According to Schoch and Maywald (1968), these restricted swelling pastes are classified as
Type C starches which show no pasting peak, but rather possess a very high viscosity
which remains constant or increases during cooling and have relatively constant cold-paste
viscosity during a holding period at 50°C.

An important feature of dry beans is their relatively high content of non-starch
polysaccharides (N SP), and their reported hypocholesterolemic and hypoglycemic efi‘ects.
Although in general beans contain slightly more insoluble than soluble fiber, they are rich
sources of soluble NSP, particularly in comparison with cereals grains. Cellulose is the
major component of fiber in smooth and wrinkled peas, red kidney and navy beans, while

11

in other legumes (lupins, lentils and broad beans), hemicellulose is the major component.
Cotyledon cell walls contain higher levels of pectin than cellulose. The seed coats are
primarily composed of cellulose (29 - 41%) with small amounts of lignin (1.2 — 1.7%).
Total dietary fiber is higher in cooked beans than in raw beans. Water-soluble fractions can
be lost with cooking water even though the beans are intact, while long boiling may

cause modifications in certain constituents of other fiber fractions.

Proteins

Dry bean (Phaseolus vulgaris) protein content ranges from 18.8 to 29.3% (Meiners
et al., 1976; Varriano-Marston and De Omana, 1979; Hosfield and Uebersax, 1980 and
Chang and Satterlw, 1982). Koehler et a1. (1987) investigated thirty-six varieties of eight
types of dry beans for protein content (kjeldahl nitrogen) and protein quality (digestibility)
as determined by Tetrahymena pynformis on the raw bean flour. There was a great deal of
variability among different commercial classes of Phaseolus vulgaris and also among
cultivars within a class. The amount of protein ranged from 17.5% for the Pinto (cultivar
NW-590) to 28.7% for the red kidney (cultivar Royal Red). Except for kidney beans,
there was little varietal difference in protein content. Pinto beans had the highest protein
quality with six varieties having values of 90 % or greater and the remaining having values
of 80 % or greater. Red kidney beans had the lowest protein quality with a mean value of
59%. The proteins in beans can be classified into two categories: metabolic and storage
proteins. The storage proteins tend to be found in the globulin fractions (salt soluble Gl )
while the metabolic (enzymatic or non-storage) proteins are primarily found in the (water
soluble GZ) albumin fraction (Deshpande and Nielsen, 1987; Nielsen, 1991). The
storage proteins have high affinity for water and contribute dramatically to the nutritional
and functional properties of the cooked seed (Boulter, 1981). They also serve as a source
of nitrogen and carbon compounds for the germinated seedling.

12

Total biological utilization of the legume protein is relatively low. Protein
digestibility may be impaired possibly by the presence of numerous anti-nutritional
compounds which must be removed or destroyed during processing (Bressani et al., 1982
and Aw and Swanson, 1985). Raw legumes are poorly digested, but adequate heat
treatment improves the digestibility significantly. However, in many parts of the world.
the thermal treatment provided for bean preparation in the home setting is not sufficient to
inactivate toxic phytohcmagglutinins (lectins), but is often just sufficient to heat and hydrate
the beans (Coffey et al., 1985). Gomez-Brenes et a1. (1975) reported that peak digestibility
and Protein Efficiency Ratios (PER) of dry P. vulgaris were obtained after soaking for 8
or 16 hours and cooking at 121°C for 10 to 30 minutes. Heating for longer than this
resulted in lowered protein quality and decreased available lysine. Bean proteins have been
reported to be resistant to in-vitro enzymatic digestion (Satire etal., 1981; Bressani, 1975);
however, heating improved in-vitro susceptibility to enzymatic hydrolysis (Sathe et al.,
1982).

Bean proteins are relatively rich in essential amino acids, particularly lysine,
threonine, isoleucine, leucine, phenylalanine and valine (Sathe et a1. 1981; Bressani,
1975). However, they are deficient in sulfur-containing amino acids, particularly
methionine and cystine. This explains their complementation with cereal grains (deficient
in lysine) in plant-based diets. Besides their low content, the bioavailability of the sulfur-
containing amino acids is also quite low even after cooking (Liener and Thompson, 1980).

Minerals and Vitamins

The total ash content of P. vulgaris ranges from 3.5% to 4.1% (db) (Table 1).
Beans are generally considered to be substantial sources of calcium and iron. They also
contain significant amounts of phosphorus, potassium, zinc and magnesium (Augustin et
al., 1981, Koehler et al., 1987, Meiners et al., 1976, Watt and Merrill, 1963 and Sgarbieri
et al., 1978). Differences among bean classes (Figures 2 and 3) and environmental factors

l3

influence variability (Augustin et al., 1981). Ash content decreases after cooking due to
leaching, with losses ranging from 10 to 70%. The wide range of reported losses could be
due to different soaking and cooking methods. Although dry beans are relatively rich
sources of some minerals, high mineral values cannot be equated with high bioavailability
of some of these minerals because of the presence of significant amounts of phytic acid
(myoinositol hexaphosphoric acid) which impedes the absorption of multivalent cations,
e. g. calcium, magnesium, zince and iron (Augustin and Klein, 1989).

Dry edible beans provide several water soluble vitamins (thiamine, riboflavin,
niacin and folic acid), but very little ascorbic acid. However, variability of vitamin content
is high. Both genetic and environmental factors, e.g., storage time and conditions as well
as differences in analytical methodologies could attribute to the variability among these
reported values (Augustin and Klein, 1989). Commercial methods of preparation of
canned beans cause a significant loss of water soluble vitamins.

The mineral and vitamin composition of dry beans are summarized in Figures 2,3
and 4 and Table 1. These data further demonstrate the compositional similarity among

various dry bean classes.

Lipids

Dry beans possess relatively low total fat content, generally 1-2% (Korytnyk and
Metzler, 1963 ; Koehler and Burke, 1981; Sathe and Salunkhe, 1981; Drum et al.,
1990). Neutral lipids are the predominant class and account for 60% of the total lipid
content. Phospholipids make up 24-35%, while glycolipids account for up to 10% of the
total lipid content of legume seeds. The fatty acid composition of legumes shows a
signifith amount of variability. However, legume lipids are generally highly unsaturated
(1% to 2%), with Phaseolus vulgaris containing 63.3% (Watt and Merrill,l963;

Takayama et al., 1965), hence the storage of legumes can result in a loss of quality,

14

nutritional value and functionality. Unsaturated linolenic acid ispresent in the highest
concentration, while palmitic acid is the predominant saturated fatty acid.
Tannins and Polyphenols

The procyanidin and condensed tannin content of dry beans ranges from 0.4 to
1.0% (Sgarbieri and Garruti, 1986). These compounds are localized in the bean seed coat
with low or negligible amounts present in the cotyledon (Ma and Bliss, 1978). The
hydroxyl groups of the phenol ring enable the tannins to form crosslinks with proteins
which may be implicated in post-harvest seed hardening or decreased digestibility (Ma and
Bliss, 1978). During cooking, polyphenolic compounds are partially leached into the
cooking medium and may interact with cotyledonary constituents, yet complete destruction
of the tannins does not occur during heat processing (Elias etal., 1979; Bressani and Elias,
1980; Bressani et al., 1982). According to Leakey (1988), some colored seeded beans also
produce particular flavonoids, proanthocyanidins, which polymerize to form phlobaphenes
implicated in the afterdarkening process with seed storage (aging). Afterdarkened beans
resist water inhibition, become hard-to—cook and are less digestible than freshly harvested
seeds.

Bean Flavor Constituents

Native Flavor Precursors. The development of the characteristic flavors of
dry beans accompanies heat treatment, with non-enzymatic browning (Maillard) reactions
contributing significantly to the formation of numerous flavor compounds. The formation
of Maillard reaction products has been shown to be influenced by the temperature of the
reaction and the amine and carbonyl source (Hodge, LE. and Hofreiter, B.T., 1972).

Oxidative degradation, catalyzed by heat, enzymes, light or metals, leads to the
formation of hydroperoxides which decompose to react with other components in the

system to produce off-flavors.

15

Ofi-flavor. Phenolic acids are localized in the cotyledon of the bean. These
compounds are characterized by sour, bitter, phenol-like, and astringent flavor notes, and
contribute to the formation of adverse flavors and colors, and changes in nutritional quality
during processing (Sosulski, 1979).

Saponins are present in dry beans and are present following processing. These
compounds are suspected of causing bitter off-flavors and may be associated with frost
damaged seed. Further off-flavor development during post-harvest storage has been
associated with mold growth (Uebersax, M.A. and Bedford, 1980; Maddex, 1978).

All dry beans posess remarkably similar compositional and nutritional
characteristics and serve well to supply energy and nutrients in diverse dietary forms. The
associated common traits of dry bean have practical applications commercially for

cormninuted or formulated foods, and in developmental world food relief programs.

NUTRITIONAL DIGESTIBILITY AND PHYSIOLOGY OF DRY EDIBLE
BEANS

Dry legumes are important dietary sources of protein, complex carbohydrates,
essential nutrients and energy. However, legume protein is generally less digestible than
protein in grains and animal products (Tobin and Carpenter, 1978; Wolzak, Elias and
Bressani, 1981; Wolzak, Bressani and Gomez Brenes, 1981). Table 2 and Figure 6
summarize the major constraints to utilization of dry edible beans (Uebersax et al., 1991).
The relatively poor digestion of legume protein limits the biological value of the protein and
reduces the overall contribution of legumes towards providing a nutritionally well balanced
diet. Bean starch, the main component of dry legumes, is digested at a slower rate than
starch from grains and root crops (Thompson et al., 1987).

Protein digestibility is expressed in terms of calculated responses related to body
nitrogen balance. "True" digestibility gives consideration to the component of the fecal
nitrogen originating from the body (mucosal cells of the gastrointestinal tract and digestive
enzymes) or endogenous N, while "apparent" digestibility considers only the relationship
between total fecal N and total dietary N. The relationships of absorption, retention and
digestibility are shown diagrarmnatically in Figure 7.

Factors Affecting Digestibility of Dry Legume Starch and Protein.

A factor that affects digestion of legume protein and starch is the physical form of
the protein and starch when it enters the stomach and small intestine. Grains and root
vegetables are usually ground and/or cooked prior to consumption. Grinding and cooking
serve to break the cell structures and to release the protein and starch so that proteolysis and
amylolysis can occur. Legumes are generally consumed as whole seeds that have been
cooked. Cellwallstructuresincookedbeansareintactandsunoundtheproteinbodiesand

starch granules. Chewing rrracerates sorrre of the cells, but most of the cells remain whole

16

l7
Tablez- CominljminngUfinnonofDryBe-n

 

Constraints
PHYSICAL

Reference

0 Preparation (time, convenience and expense) Usher-uni

. Post Harvest Stora e Changa
(seed hardening)g

BIOLOGICAL
. Anti-nutrients
- pretense inhibitors end lectin:

- enrylnee inhibitors

- procyanidin lnlencdone

- Digestibility
4am.- acid

- protein

- elerch

. Flatulence Factors
- oligosaccharides

- protein:

- retrograded starch

.Mr

W 0989)

Sofa-Dada: a II. (1979);
Vern-rem

nd Jar-hat (1981);
Stanley and

Ann- (1985):
Shauna n M1989)

Lin-u I. 8.0978);
'lbanpeon n al.(1983.
1986) June at al.( 19»)
Cdey a a1. 0985)

laflen £11973);
Pom. J. R. and
Whitaker. LR.( 1977):
Pick. LE. and
Weber, G. (1979)

Bressani ad. (1982)

Map (1982):
Your 6 IL (1983)

Wolzak a a1. (1981);
Chat, LC. and L
D. Sundae (1981);
Emmi a al. (1982)
Cdfey a :1. (1985)
“had. (1937)

How, I. and F.

Saulaki (1985);

Harpoon a :1.

GM); Bellini all

Srieune (1989);Tover a aL(l990);
Englyn nd Cunning

(1990)

WY¢¢ (1972);
Plain (1981ng Olsen
net. (1981)

m (1971)

Brian, RN. and
LH. My (1990)

Helm (1978);

Planing (198le Reddy
n nL. 1934

 

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ALIMENTARY CHANNEL
FOOD N (I)
Body Tissue Body Tissue
/ ENDOGENOUS N
\K I /
ABSORBED yk METABOLISM
RETAINED <, pg» RETENTION
3 DIGESTION
Urine N V Urine N
(U) (U)
EXCRETED FECAL N EXCEED
(F)
W DEW W
True Apparent
Digestibility (D) N Intake I_- (EI- B) II;E
r-rE-r-rr-az-rm 1-5-3;
Biologist! V-lue (BV) Wad“ r . (r ~ Fk) I . r
143.33-31-33) IE 3.
Net Protein Utilization (NPU) W I I
N Intake

where I,FandU areirrtake, fecal andurinarynitrogen valuee.respeetively.onthe rest diet,
andfiandUkarethecorrespondingvaluesonanon-proteindiet. NPUcanalsobedefmed
inter'rnsofbodyN.

Figure 7. Nitrogen digestion. absorption and retention (Pellett. 1978)

20

and enter the digestive tract intact. Thus, the cell structures render legume protein and
starch less available to the digestive enzymes except for the small amounts of starch that
leaches from the cells (Wm'sch et al.,l986; Golay et al., 1986).

Another factor affecting digestion of both protein and starch is the amount of total
dietary fiber in the diet. Phaseolus vulgaris beans typically contain 17.5 - 20% dietary fiber
and are regarded as an excellent source of dietary fiber (Prosky et al., 1985; Anderson and
Bridges, 1988). As an individual increases dietary fiber intake, nutrient digestibility
and/or absorption tends to decrease (Kies, 1981). When dietary fiber was increased from
16.4 g/day to 37.4 g/day, protein digestion decreased 6%, fat digestion decreased 2% and
total energy digestibility dmsed by 1.8% Decreased digestibility of protein and starch is
presumably due to physical entrapment and/or premature release of food particles from the
stomach, although direct inhibition of digestive enzymes by fiber cannot be excluded
(Schneeman, 1982). Dietary fiber can encase nutrients in a fibrous matrix which impedes
digestive enzymes from gaining access to protein and starch or fiber can reduce absorption
of digested protein and starch by slowing or preventing diffusion of the digestive products
to the mucosal cells (Johnson and Gee, 1981; Elsenhaus et al., 1981). Food is normally
held in the stomach until it is reduced to particles less than 1 mm, but fiber can cause the
premature release of large particles from the stomach. Large particles represent another
type of physical entrapment which limits digestion.

A potential factor which could also limit protein and starch digestion is the presence
of naturally occurring protease and amylase inhibitors. During processing and cooking,
nrost of these inhibitors are inactivated; however, residual levels of active inhibitors may
remain, particularly in improperly cooked beans. Rayas-Duarte et al.(1992) reported that
trypsin inhibitory activity (TIA) in boiled (30 mins.) Great Northern beans exhibited
minimum heat stability (2.5-5% TIA retention) in the whole intact bean matrix, medium
heat stability in the dry bean flour, and maximum heat stability (97-100% TIA retention )
in the extracted bean albumins compared to unheated samples. Individuals accustomed to

21

consuming beans with residual levels of inhibitors possess an enlarged pancreas which
secretes more digestive enzymes to compensate for the enzymes which have been
inactivated by the inhibitors (Schneeman, 1982; Madar et al., 1976 ). As a result of
pancreatic hyperplasia and hypertrophy, adequate amounts of digestive enzymes are
secreted and digestion is not limited. The best evidence to support this conclusion is based
on the experience with exogenous amylase inhibitors ( "starch blockers"). It has been
demonstrated that chronic consumption of amylase inhibitors was ineffective in reducing
starch digestion and caloric utilization. Essentially the same results are found with
continued consumption of protease inhibitors (Madar et al., 1976 ).

Legumes also contain lectins which are toxic factors that interact with glycoprotein
on the surface of red blood cells and cause them to agglutinate (Singh, 1988). Einhoff et
a1. (1985) have shown that Leguminosa lectins will bind both the storage protein and the
glycosidase enzymes and that both the lectins and the lectin-bound proteins are found in the
protein bodies. They suggested that lectins act during maturation of the plant to contribute
to an orderly arrangement of the storage proteins in the protein bodies. Phytohemagglutinin
(PHA), the lectin of dry beans, is a 648 protein with two subunits of molecular weight
34 kd and 36 kd. Estimates of PHA's molecular weight range from a low of 115 kd to a
high of 150 kd (Coffey, 1985). Cooking beans will inactivate much of the lectin activity
in raw beans; however, fully cooked beans can still contain a significant amount of the
original lectin activity (Thompson et al., 1983; Rea et al., 1985). Reaidi et al.(1981)
reported one cultivar of P. vulgaris contained appreciable amounts even after 20 min.
boiling. For complete elimination of the toxic effects, pre-soaked (minimum 4-5 h) kidney
beans should be heated for either 4 hr at 90°C, 90 min. at 95°C or 10 min at 100°C
(Annual Report, 1982, Rowett Research Institute). It is difficult to measure the effect of
residual lectins on digestion and absorption. In rats, plasma protein losses into the
intestine is one of the main signs of lectin toxicity. Ingested kidney bean lectins bind to

membrane-glycocalyx receptors on microvilli in the proximal part of the small intestine, and

22

is associated with considerable disruption of intestinal microvilli (Annual Report, 1982,
Rowett Research Institute). Data provided by Donatucci et al. (1987) strongly suggest that
small quantities of lectins are sufficient to reduce absorption of glucose and presumably
amino acids and small peptides.

Phytic acid, another antinutritional factor, has been reported to reduce the biological
availability of minerals. The study of Tecklenbm'g et al. (1984) indicated phytic acid to be
partitioned with the protein fraction during air classification processing. The high degree of
correlation between protein content and Zn, Fe, K and Mg, and between phytic acid and
these minerals suggested that these elements are present as metallic phytates. Sandberg et
a1. (1989) reported that addition of 10 umol inositol hexa- and penta-phosphate reduced
iron solubility from the reference 39% to 0.2% and 6%, respectively, while the same
amount of inositol tetra- and tri-phosphate slightly increased iron solubility. This indicates
that degradation of inositol hexa- and pentaphosphates seemed to significantly reduce the
inhibiting effect on iron bioavailability.

Huisman et al. (1990) made a comparison of the effects of antinutritional factors
present in Phaseolus vulgaris on piglets, rats and chickens. They found that live-weight
gain was markedly reduced during feeding 200 g raw beans in piglets, but this response
was not shown in the rats and chickens. When the supply of dietary protein is adequate,
there is no reduction in pancreas weight, but weight of the intestine was increased in all
tlnee species due to feeding raw beans.

Digestibility of Legume protein.

There have been two prevalent concepts to explain poor digestion of legume
protein: (a) legumes contain protease inhibitors and lectins which reduce legume protein
digestion (Jaffe and Vega Lette, 1968; Liener, 1983) and (b) the inherent structure of
legume protein is such that it resists proteolysis. Deshpande and Nielsen ( 1987) provided
evidence that heated legume protein is not resistant to trypsin proteolysis in-vitro; however,

23

their research does not preclude the possibility that a portion of the trypsin digestion
products are resistant to other proteolytic enzymes which are necessary to hydrolyze the
protein to absorbable end products (free amino acids, di- and tripeptides).

Globulin G1 is the major protein fraction of Phaseolus beans (50 to 75% of the
total bean protein). Marquez and Lajolo (1981) found that the globan GII fraction
(phytohemagglutinin or lectin) was 60% digestible, whereas the glutelin fraction was only
40% digestible. Trypsin inhibitor is also poorly digested, only 38% digestible ( Boulter,
1984). Heated albumin was only 53% digestible, but unheated albumin was 100%
digestible. It was noted that the heated albumin formed large aggregates which were
probably inaccessible to proteolytic enzymes (Marquez and Lajolo, 1981). If the
albumins were in the bean rather than isolated, it is likely that its digestibility would be
approximately 100%. Nonextractable proteins are probably located in the cell walls and
hulls and these nonextractable proteins comprise about 10 - 12% of the total protein. The
digestibility of the nonextractable proteins in Phaseolus beans is not known; but, based on
the digestibility of proteins in brans and hulls of other plant products, one would expect
relatively poor digestion of the nonextractable proteins. Additional factors such as tannins
(Elias et al., 1979; Bressani et al., 1982; Aw and Swanson, 1985 ), dietary fiber, residual
lectin activity and protein encasement within cell walls will decrease total protein
digestibility to the relatively low levels commonly reported for Phaseolus vulgaris. Several
hypotheses have been presented to explain decreased protein digestibility of the various
fractions. Steric hinderance of proteolysis by the carbohydrate moieties of the
glycoproteins (globulin G11 and protease inhibitor fractions) is one possibility for the
measured digestibility of these two fractions ( Chang and Satterlee, 1981 ). They have
hypothesized that poor legume protein digestibility is due to the compact, dense nature of
bean protein which prevents proteolytic enzymes from reaching the internal catalytic sites.
Dissociation of protein fractions by urea and/or sulflrydryl agents resulted in a significant
improvement in protein digestibility in soy protein (Rothenbuhler and Kinsella, 1986) and

24

sorghum protein (Hamaker, 1987). The presence of carbohydrate-protein bonds and
proteimprotein bonds have been proposed to reduce protein digestibility in legumes.
Carbohydrate-protein and protein-protein bonds are not susceptible to mammalian proteases
and reduce overall protein digestibilities. Formation of carbohydrate-protein bonds is
thought to occur via oxidative coupling of two activated benzene rings, one from cell wall
phenolic compounds and the other from tyrosine. Similarly, protein crosslinking is
thought to occur via peroxidase-catalyzed coupling of two tyrosine residues. If the
nonextractable protein is in cell walls and hulls and if it is poorly digested, at least part of
the poor digestibility would likely be due to crosslinking of protein with carbohydrate or
other protein.

The high level of bean protein (about 25% db) is about double many corrunon cereal
grains; however, this factor of quantity cannot exclude the concept of protein quality which
is most important to nutritional comparisons among protein sources. The nutritional value
of a protein is a direct consequence of the qualitative and quantitative distribution of amino
acids comprising the primary structure of the protein. Overall quality of the protein is
determined by the essential arrrino acids. Methionine, a sulfur containing amino acid, has
consistently been shown to be fust linriting in dry bean. Cereal grains (wheat, corn) are
limiting in lysine. Thus, these two protein sources are "complementary” and when blended
in a diet provide for each other's limitations. Batterham et a1. (1990) determined the
utilization of ileal digestible lysine in soya-bean meal by pigs. They reported that values for
the ileal digestibility of lysine in protein concentrate are unsuitable in dietary formulations
as the assay does not reflect the proportion of lysine that can utilized by the pig. Their study
indicated that with heat-processed meals, a considerable proportion of the lysine is
absorbed in a form(s) that is(are) inefficently utilized.

A comprehensive review focusing on molecular strategies to improve protein
quality has been conducted by de Lumen (1992). Techniques used to increase the
methionine content of tobacco seeds using heterologous seed protein genes from peas,

2 5
common bean and soybean have long term potential utility in modifying lysine, tryptophan
and threonine contents in legumes.

Digestibility of Legume Starch.

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

The "glycemic index" has been developed to aid dietary treatment of diabetics
(Jenkins et al., 1982) and is defined as :

x 100

glucose response curve for the

equivalent amount of glucose
Canned beans have been reported to have a low glycemic index indicating that bean starch
is digested more slowly than starch from most other foods. Elevated blood glucose
promotes triglyceride and cholesterol synthesis by the liver which results in more very low
density lipoproteins (triglycerides and cholesterol) being secreted into the blood (Anderson
et al., 1984a & b; Jenkins et al., 1984; Thompson et al., 1984). Furthermore, elevated
blood glucose promotes glycosylation of proteins in the vascular system (Jenkins et al.,
1988). The net effect of elevated blood lipids and unwanted protein glycosylation is an
increased risk of cardiovascular disease. Thus, consumption of legume starch reduces the
risk of premature cardiovascular disease compared to consuming starch with a high
glycemic index. While slow starch digestion is desirable, it is undesirable for the starch to

be digested too slowly since there will be incomplete digestion and absorption in the small

26

intestine. Undigested starch will pass to the colon where rapid fermentation will take place
and flatulence will result.

Two major factors affect the rate and perhaps the extent of starch digestion: l)
gastric emptying time and 2) accessibility of the glycosidic bonds in starch to pancreatic
amylase and other digestive enzymes. If the rate of starch digestion is constant, starch
which is released from the stomach slowly and over an extended time period will be
digested and absorbed more slowly than when gastric emptying is rapid and over a short
time period. There is a strong negative correlation between postprandial changes in blood
glucose and gastric emptying (Mourot et al., 1988 ). Likewise, if gastric emptying is
constant, starch digestion rate determines how high the blood glucose rises in response to
starch ingestion. The digestion rate of starch strongly influences the glycemic response to
different foods (Thompson, 1988; Jenkins et al., 1982). The glycemic index of a food is
obviously a function of both gastric emptying and starch digestion rate.

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

A significant but less recognized factor which limits starch digestibility is the
tendency for some starches to retrograde. Retrogradation is a phenomenon initiated when
gelatinized starch cools slowly, hydrogen bonds within or between starch chains reform,
and water between the starch chains is squeezed out resulting in a compacted dense
molecular structure with differentiated functional properties. Amyloses with degree of
polymerization (DP) between 200 and 1200, have a high tendency to retrograde to
crystalline ”beta sheet” type configurations. The average DP for legume amylose is DP
1000 - 1400 (Biliaderis et al., 1981), which is highly conducive to formation of

27

retrograded crystalline starch structures. Retrograded amylose is indigestible within the
small intestine (Englyst and Cummings, 1987). Legume starch has a higher percentage of
amylose than most starches which increases the potential for formation of indigestible
retrograded starch. During cooking some of the amylose leaches from the starch granule.
Since the cell structure in cooked beans remain intact, much of the leached amylose remains
within the cell and fills spaces between protein bodies and starch granules. If amylose
retrogradation occurs, another indigestible matrix (similar to fiber in cell walls) in and

around the protein and starch granules is formed (Bennink and Srisuma, 1989).

Flatulence Associated with Legume Consumption

Flatulence refers to the passage of gas through the rectum. Any material that is not
digested and absorbed in the small intestine is a susceptible substrate for microbial
catabolism within the colon with the subsequent production of variable levels of flatus.

Suspected Causes of Flatulence. Establishing a cause and effect relationship
between specific factors and flatulence has been a long and active area of research. Alvarez
(1942) suggested that swallowed air or the diffusion of gasses from the blood into the
intestinal lumen were responsible. Danhof et al. (1953) proposed that because of the high
carbon dioxide found in flatus, secretions in the intestine (especially the pancreatic
secretion) may be the causal factor. As early as 1929, Kantor ( 1929) linked bean diets and
gas production to bacterial fermentation within the lumen to the intestine. An early
suggestion that carbohydrates in dry beans may in part be responsible was made by
Anderson (1924). He was able to demonstrate that the primary gases evolved in anaerobic
cultivation of various carbohydrate media were high percentages of carbon dioxide and
hydrogen and low percentages of nitrogen and oxygen. Olson et al. (1975) reported that
protein rich fractions from beans and soybeans did not significantly contribute to flatulence
in rats and humans. Tomlin et al. (1991) reported that fermentation gases make the highest
contribution to normal flatus volume, and that a ”fiber free" diet eliminates these without

28

changing residual gas release of around 200 ml/24 h. Figure 8 illustrates the proposed
mechanism for gas production involving oligosaccharides, indigestible starch and dietary
fiber.

Rafflnose, Stachyose and Verbascose. The oligosaccharides of the
raffinose family have been implicated in flatus formation (Fleming, 1981a): raffinose (a
trisaccharide containing galactose, glucose and fructose, with the galactose moiety having a
1-6 bond); Stachyose (a tetrasaccharide nrade up of two adjacent galactose units, glucose
and fructose with the 1-6 bond) and verbascose (a pentasaccharide made up of three
adjacent galactose units, glucose and fnlctose with the 1-6 bond). These sugars cannot be
digested by humans because the human intestinal mucosa and pancreatic secretions do not
contain the enzyme (a -1, 6 galactosidase) necessary to split these oligosaccharides into
simple sugars. Figure 8 illustrates the proposed mechanism for gas production involving
oligosaccharides, indigestible starch and indigestible starch. Ruttloff et a1. (1967) found no
enzymatic hydrolysis of raffinose in the intestinal mucosa of rats, pigs, and humans. Other
studies on the absorption and degradation of oligosaccharides containing B-galactosyl
groups show that less than 1% of the administered dose was able to pass through the
intestinal wall of man and animals (Taeufel et al., 1960). The indigestible oligosaccharides
pass through the small bowel and reach the colon where bacterial fermentation produces
large amounts of methane (Olson et al., 1981). Oral alpha-galactosidase (Novozyme)
therapy was found feasible to promote oligosaccharide hydrolysis and reduce intolerance
symptoms in healthy adult subjects after ingestion of refried black beans (Solomons et al.,
1991). This in situ, intraintestinal assisted digestion had been previously proven
succcessful for lactose and sucrose intolerance.

Steggerda et a1. (1966) reported that low molecular weight carbohydrate fractions
were especially potent in gas production compared with fat, protein, and complex
polysaccharide fractions when human subjects consumed various fractions of soybean

meal. Steggerda and Dimmick (1966) showed that differences in human flatus production

29

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and gas composition occurred with different kinds and quantities of bean diets. They
observed the average concentration of carbon dioxide in the collected flatus changed from
11% on a controlled non-gas producing diet to 51% when large quantities of pork and
beans were consumed. Calloway (1966) subjected pure carbohydrates to fermentation with
colonic bacteria and found they had the capability to hydrolyze the a-1,6 galactoside of
Stachyose. Up to this time no a-(l,6) galactosidase could be found in mammals
(Gitzelmann & Ajurricchio, 1965) leading to speculation that sugars of raffinose family
may cause much of the intolerance associated with beans.

I Cristofaro et al., (1973) found that diets containing Stachyose and verbacose
exhibited the highest flatus activity. Rackis et a1. (1970), using an in-vr'tro assay, showed
that toasted, dehulled, and defatted soybean meal had gas producing factors of sucrose,
raffinose and Stachyose and gas-inhibiting factors of phenolic (syrigic and ferrulic) acids.
The lipids, proteins and water-insoluble polysaccharides of soybean meal had no gas
producing activity.

Murphy et al. (1972) using human subjects and the California Small White bean
(CSW), showed that the flatulent activity was associated in the 60% and 85% aqueous
extracted ethanol phase which contained raffinose and Stachyose. But purified raffinose
and Stachyose fed alone at levels found in CSW did not increase the carbon dioxide level of
the flatus. The sugars, however, made a major contribution to the hydrogen component of
the breath and flatus during the period of high flatus volume; but the major factors
responsible for the increase in carbon dioxide volume remained unidentified.

Raffinose and Stachyose were reduced by high temperature extrusion of pinto bean
high starch fractions (Borejszo and Khan, 1992). Loss of these sugars could be possibly
due to Maillard reaction between charged protein groups and reducing sugars.

Fleming (1981), using rats, showed a significant positive correlation between
hydrogen production and raffinose plus stachyose, and glucans and pentosans
hydrolyzable in dilute acid. Studies by Olson et al. (1975;1982) revealed that flatus-

3 l
producing capacity was not totally eliminated by removal of oligosaccharides, suggesting
that there are other substances that contribute to flatulence. The role of oligosaccharides in
plant biology can be elucidated through molecular techniques, and knowledge of
biosynthetic pathways for raffin'ose oligosaccharides enables control and modification of
these sugars to alleviate the problem of flatulence associated with bean consumption (de
Lumen, 1992).

Dietary fiber. Studies indicated that even after the removal of oligosaccharides,
dry beans could still induce appreciable flatus (Olson et al., 1975; Fleming, 1981 & 1982;
Hellendoom, 1976, 1978; Kamat & Kulkarni, 1981). Wagner et a1. (1977) then compared
a cooked California Small White (CSW) bean which contained 4% oligosaccharides, to
oligosaccharide free CSW solids (residue fiom hexane and 70% ethanol extraction of
CSW). The oligosaccharides served as a source of hydrogen when ingested by rats in life
support systems. His results indicated that if the oligosaccharide content was the only
hydrogen source in CSW, it would have to be 25 times more potent; the Stachyose would
have to be seven times as potent as free the CSW solids. Thus, CSW must contain at least
one 70% alcohol-insoluble substance which, in addition to the oligosaccharides, was
essential to bring about the observed quantitative physiological response in rats to whole
beans.

Dietary fiber, being the indigestible component for man, has been shown to be
fermented by microorganisms in the colon. Reddy et a1. (1984) mentioned that fiber may
be involved in the fermentation by microorganisms with subsequent flatulence production.
Analysis of feces for the presence of fiber showed that the greater part of cellulose and
lignin can be recovered in the stool. Calloway et al. (1980, 1983) measured the
digestibility of pectin and hemicellulose in humans on an input/output basis. Of the
subjects fed pectin diets, 15.3% of the pectin was digested by women ileostomy subjects,
46.5% was digested by men ileostomy subjects, respectively, while 96.5% and 95% of
the pectin was digested by men and women subjects with intact colons. When subjects

32

were fed hemicellulose diets, 65% of the hemicelluloses were digested by women
ileostomy subjects and 63% by male ileostomy subjects. However, 95 and 97% of the
hemicelluloses were digested by men and women subjects with intact colons.

Apparent digestibility of total fiber ranged from 47 to 82% during the experimental
period using human subjects fed a strictly controlled diet of a fiber supplement. Prynne
and Southgate (1979) reported values of 70% to 80% for the apparent digestibility of the
fiber in a control segment during the experimental period.

Farrell et a1. (1978) fed human subjects neutral detergent fiber and observed a
complete disappearance of hemicellulose from diets with decreased transit time, while 80%
and 55% of the neutral detergent fiber in the diet disappeared from low and high fiber diets,
respectively.

Fleming (1981) showed a significant positive correlation between hydrogen
production and glucans and pentosans hydrolyzable in dilute acid. Significant negative
correlations were found between hydrogen production and starch or lignin contents.
Marthinsen and Fleming (1982) measured the breath and flatus gasses of humans
consuming high-fiber diets and found xylan and pectin diets resulted in a production of
high flatus volume, hydrogen and carbon dioxide excretion. Cellulose and corn bran
generally produced breath and flatus gas excretion at levels equivalent to a fiber-free diet.

Hellendoom (1978) found that while most of the hemicelluloses and soluble pectins
were fermented in the large intestine, some could be recovered from the stool. Tadesse and
Eastwood (197 8) reported a hemicellulose preparation increased hydrogen production in
man, but cellulose, lignin, and pectin did not. Fleming (1981), using the smooth seeded
field pea, found hydrogen production in the rat to be closely associated with the quantity of
oligosaccharides remaining in the seed meal. However, the flatulent activity of the whole
seed appeared to be due in equal parts to the indigestible oligosaccharides and components
of the cell-wall fiber.

33

Fleming (1983) compared the effects of selected purified fibers to those derived
from cereals or legume seeds. Rats were fed diets containing 10% dietary fiber from
selected sources and 10% protein. Pectin reduced rat weight, while Feed Efficiency Ratio
(FER), Protein Efficiency Ratio (PER) and apparent protein digestibility values compared
to a fiber-free diet. Cellulose, xylan and raffinose had no influence on feed intake, weight
gains or FER. Cellulose and xylan increased PER values and the rates of food passage
through the gastrointestinal tract, but decreased the apparent protein digestibility values.
Finally, the cell-wall-fiber fraction of beans had little effect on feed consumption, growth,
FER and PER. The cell-wall- fiber fraction reduced apparent protein digestibility and the
hull fraction accelerated food passage relative to the fiber-free diet.

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

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

Starch digestibility may also be dependent on the type of native starch, cooking
time, and procedure used for preparation of dry beans. Accordingly, 5 to 15% of bean
starch can remain indigestible even after prolonged cooking (Hellendoorn, 1969). Paid
and Bhavanishangar (1983), using in-vitro and in-vr’vo studies, showed that apart from

34

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

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

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

The actual crystalline structure of the starch granule is suggested to depend on the
chain length of amylopectin and has been classified using x-ray diffraction techniques into
groups termed A, B and C. A-type starch is the normal pattern for cereal starch granules,
B-type is typical of potato, amylomaize and retrograded starch, and C—type (combination of
A and B patterns) is characteristic of certain pea and bean starches. In general, starch
granules showing X-ray diffraction patterns B or C tend to be the most resistant to
pancreatic amylase, though the degree of resistance is dependent on the plant source
(Fuwa,Takay and Sugimoto,l989, as cited by Englyst and Cummings, 1990). Cooking

_35

disrupts the granules and facilitates the hydrolysis of the starch contained within them.
Crystallization at higher temperature and lower water content will favor the A pattern, and
lower temperature and high water content, the B pattern. On cooling, gelatinized starchy
foods will retrograde, solubility of the starch molecule decreases and so does its
susceptibility to hydrolysis by acid and enzymes.

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

Socorro et al (1989) reported that dietary fiber decreased digestibility of black
bean, corn, rice and wheat starches by pancreatic porcine enzyme, especially when whole
grain fiber was used. However, black bean and rice starch digestibility by human
pancreatic a-amylase was not affected by fiber, while corn and wheat starch was slightly
inhibited. The use of the human enzyme is therefore recommended for amylolysis assays,
although the difficulties of extrapolating results obtained with animal enzymes to humans
should be carefully considered.

Studies by Fernandez and Berry (1989) reported that germination sharply increased
the susceptibility of chickpea starch to digestibility by a-amylase, but no change in the
appearance of SEM could be attributed to germination. In-vr’vo and in-vr'tro methods with
experimental rats and commercial digestive enzymes, respectively, were used by Nnanna
and Phillips (1990) in assessing the protein and starch digestibility and flatulence potential

36

of germinated cowpeas. Germination reduced the flatulence potential of seeds. In-vr‘vo
digestibility of starch and protein was. also significantly increased by germination.
Germination did not affect in-vitro protein digestibility, but reduced in vitro digestibility of
freeze-dried and 70°C-dried starch. Cooking in boiling water significantly increased in-
vivo protein and starch digestibility of both ungerminated and germinated seed.

Tovar et. a1. (1990) found the starch content of a raw red kidney bean (Phaseolus
vulgaris) flour (RBE) was higher than that of a cooked and blended (CBB) and of a
cooked, freeze-dried, and milled (CBF) preparation. However, wet homogenization as well
as pepsin pretreatment of CBF increased the starch yield, indicating that starch in the
cooked samples is not completely available to enzymic degradation unless cell wall
entrapped granules are released by mechanical or enzymatic disruption of the fibrous walls.
This could partly explain a number of inconsistencies reported in literature (Fleming and
Vose, 1979; Jenkins et. al., 1982a and b; Wursh et. al., 1988; Socorro et. al., 1989 and
O'Dea and Wong, 1983). Influence of encapsulated and resistant starch fractions on
dietary fiber values was also observed. CBF showed remarkable low values of in-vr’tro
amylolysis rate and starch digestibility index in a digestion/dialysis system, features that
seemed to depend also on the integrity of cell walls. In a related study, Tovar et a1. (1991)
suggested the persistence of starch granules (resistant starch i.e., retrograded amylose, 3-
9% dwb) enclosed in cotyledon cells as the primary reason for the limited enzymatic
availability of starch in preeooked flours (PCF) as evidenced by SEM of PCFs of milled
boiled and freeze-dried red kidney beans, white beans and lentils demonstrating relatively
large particles which contained cell structures filled with starch. The susceptibility to
enzymatic hydrolysis of PCFs was enhanced with pepsin preincubation or with additional
boiling, resulting in an apparently thinner surface. Homogenization of PCF's resulted in
the largest increase in in-virro a-amyloysis rate, with almost complete disruption of the
cotyledon cell walls. When these PCFs were subjected to in - viva feeding studies with
rats, between 8% (beans) and 11% (lentils) of the total starch ingested appeared in the

37

feces, indicating a relatively low starch digestibility; 60% of the fecal starch in the bean-fed
animals and 70% in the lentil-fed group was retrograded amylose (T ovar et al., 1992).

The rate of wheat starch digestibility in the presence or absence of polyphenols
(catechin or tannic acid) and/or phytic acid at concentrations found in legumes was
determined in an in-vitro dialysis system. Addition of tannic acid and phytic acid reduced
the starch digestibility 13 and 60% respectively, at 5 hr. Combined tannic and phytic acid
reduced the digestibility at a level (63%) which did not differ significantly from that with
only phytic acid. These results agree with those observed in an earlier study by Thompson
et. a1. (1983). There was no additive or synergistic effect between these two antinutrients.
Catechin had no significant effect on rate of starch digestibility, or it may have been
obscurred by the large reduction by phytic acid. A future dose-response study of these
antinutrients and starch digestibility may help understand the interaction between phytate
and polyphenols.

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

Thompson et. al. (1987) determined digestion of wheat starch (WS) and red
kidney bean (RKB) starch by pancreatic (PA) and salivary (SA) amylose in the presence

38

or absence of lectins . Compared with WS, digestion of RKB starch by PA and SA was
70.0% and 66.6% lower, respectively. RKB lectin added to WS at the hemagglutinin
activity level in RKB starch resulted in significantly decreased digestion with PA (63.9%)
and SA (43.8%) as did heated RKB lectin with insignificant hemagglutinin activity (41.1%
with PA, 35.8% with SA). Jack bean lectin (concanavalin A) also resulted in reduced rate
of starch digestibility. Kinetic analyses revealed noncompetitive inhibition by RKB lectins
on both amylases. Results confirmed the role of lectins in reducing the rate of starch
digestion and its possible health benefit.

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

The objective of a comprehensive study recently conducted was to determine
whether legumes in a physical form which is rapidly digested in-vitro gave rise to
proportionately greater metabolic responses in-vr’vo than legumes which are slowly
digested in-vr'tro (O'Dea and Wong, 1983). Samples of cooked whole and ground lentils
were incubated in vitro with pancreatic amylase for 30 min and the percentage starch
hydrolysis determined. Grinding the lentils before cooking resulted in a 5-fold increase in
the rate of starch hydrolysis (whole lentils 12.1%, ground lentils 60.9%). For the in-vivo
studies six healthy, young lean subjects consumed two test meals containing 50 g starch:
whole lentils and lentils that had been ground finely before cooking. Postprandial glucose
and insulin responses were measured over 4 h. Peak glucose and insan responses
occm'red 60 min postprandially for the whole lentils and 30 min postprandially for ground
lentils. Although the increase in plasma glucose after ground lentils (1.6 mM ) was

39

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

Hypocholesterolemic and Hypoglycemic Responses of Bean Dietary Fiber

Hypocholesterolemic Effect. There is considerable literature evidence that
foods which contain high levels of water-soluble dietary fiber such as oats or bean
products, and purified forms of water-soluble dietary fiber reduce blood cholesterol
(Kritchevsky, 1987 as cited by Gatenby, 1990; Anderson, 1985; Anderson et al., 1984 ;
Kirby et al., 1984; Storch et al., 1984). Soluble fiber may bind bile acids and cholesterol
in the intestine, decreasing their absorption. Bacteria in the colon convert primary bile
acids into secondary bile acids, which are less well absorbed. Thus, less bile acid is
returned to the enterohepatic circulation which drives de novo hepatic synthesis of
cholesterol to serve as a precursor for bile acid synthesis, with resultant decreases in the
amount of cholesterol available for lipoprotein synthesis (Anderson and Tietyen - Clark,
1986; Kirby et al., 1981; Kay, 1982). However, not all sources of soluble fibers increase
fecal bile acid excretion, and the magnitude of increased excretion may be small (Anderson,
et al, 1984; Chen and Anderson, 1986).

Short-chain fatty acids (SCFA) may also mediate the hypocholesterolemic effects of
soluble fiber. Soluble fibers are almost completely fermented in the colon to SCFA,
primarily acetate, propionate and butyrate. These fatty acids are absorbed into the portal
vein and appear to inhibit hepatic cholesterol synthesis. The increased fecal loss of bile
acids coupled with reduced hepatic cholesterol synthesis may decrease hepatic secretion of
very-low-density lipoprotein (VLDL) cholesterol, a. precursor to LDL cholesterol (Chen, et

40

al., 1981). SCFA may also inhibit cholesterol synthesis in peripheral tissues, resulting in
an increase in peripheral LDL receptors and an increase in LDL clearance (Chen and
Anderson, 1986; Kirby et al., 1981).

Effects of oat-bran or bean supplemented diets were assessed for 10
hypocholesterolemic men who were observed for 3 weeks on a metabolic ward. Oat-bran
or bean (100 g db) supplements decreased serum cholesterol and LDL - cholesterol
concentrations by 23% below initial values without alterations in cholesterol or fat intakes
(Anderson et al., 1984). However, in an earlier study, Jenkins et al., (1983) reported that
low- and- high density lipoprotein cholesterol levels remained unaltered when dried beans
where fed to 7 male hyperlipidemic patients to substitute for other som'ces of starch. Mean
fasting serum triglyceride levels were reduced by 25% while total serum cholesterol levels
were 75% lower than the non-bean supplemented control group.

Anderson and Chen (1982) found bean supplemented diets (115g dried beans/day;
50 g total and 20 g soluble fiber) to selectively lower atherogenic LDL cholesterol fractions
while preserving anti-atherogenic HDL cholesterol levels. This effect probably relates to
the high soluble storage polysaccharide content of legumes. Short chain fatty acid
fermentation products of soluble fibers may attenuate hepatic cholesterol synthesis.

The hypolipidemic effects of canned beans were also studied by Anderson (1985)
in 10 hypercholesterolemic men. After 3 weeks on the canned-bean diet (122g daily; 24 g
total and 7 g soluble fiber), serum cholesterol values averaged 13% lower and triglycerides
values 12% lower than control values.

Daily consumption of 100—135g dried beans (dry measure) reduces serum
cholesterol levels ~ 20% short-term, hypothetically reducing risk for CHD by 40%
(Anderson et al., 1984 ). Smaller portions of 100-200g cooked or canned beans lower
serum cholesterol ~12% short-term and 20-25% long-term (Anderson et al., 1984b;

Anderson, 1985). Nutritional therapy combining "High Carbohydrate Fiber” (HCF) diets

41

with bean supplementation is well tolerated and associated with no major side effects,
except for reported increased flatulence and eructations.

Swain et a1. (1990) concluded that oat-bran had little cholesterol-lowering effect
and that high-fiber and low-fiber dietary grain supplements about equally reduce serum
cholesterol levels probably because they replace dietary fats. This confirms earlier studies
by Anderson et al.( 1984) where serum cholesterol levels decreased by 19% and low-
density lipoprotein levels decreased by 23-24% with both the oat-bran and bean diets.
Since there was no randomized low-fiber control group, this suggests only that oat bran
and beans are equivalent, not that either one have direct - cholesterol - lowering properties.

Hypoglycemic Effect. Jenkins et al.(1985) indicated that selection of low
glycemic index carbohydrate foods may be a useful adjunct to the management of
hyperlipidimia. They found that reduction in the mean glycemic index (GI) of diets of 12
hyperlipidemic patients resulted in a significant reduction in total and LDL serum
cholesterol and serum triglyceride by comparison with the mean lipid values for the
preceding and subsequent months on the control,diets. Low glycemic responses have been
observed for legumes compared with other starchy foods such as bread, potato and certain
breakfast cereals (Potter et al., 1981; Jenkins et al., 1981, 1982b; Walker and Walker,
1984; Tappy et al., 1986).

Jenkins et al., (1980) incubated carbohydrate portions of lentils, soya beans and
whole meal bread with human digestive juices. Results suggested that the rate of digestion
might be an important factor determining the rise in blood glucose concentration after a
meal and that supplementing chemical analysis with iii-vitro and in-vivo food testing might
permit identification of useful foods for diabetics.

To test the effect of processing on digestibility and the glycemic response to a
leguminous seed, a group of eight healthy volunteers took a series of breakfast test meals
containing either lentils which had been processed in four different fashions or the same
amount of carbohydrate as white bread. Processing variables included : 1) boiling 20

42

minutes, consumed whole; 2) boiled 20 minutes blended to paste; 3) Boiled 60 minutes and
4) Boiled and drying 250°F/ 12 hours. Lentils boiled for 20 min. resulted in a flattened
blood glucose response in comparison to bread. This was unaltered by blending the lentils
to a paste or boiling them for an additional 40 minutes. However the blood glucose
response was significantly enhanced by drying the boiled blended lentils for 12 h at 250°F.
In vitro digestion with human saliva showed the rate of sugars released from the food
related positively to the blood glucose rise. Breath hydrogen studies indicated that
carbohydrate malabsorption was too small to account for differences in the blood glucose
response. These results emphasize the importance of processing in determining
digestibility and hence the glycemic response to a food (Jenkins et al.,l982).

Wolever et a1. (1986) fed 50 g carbohydrate portions of five varieties of beans, both
cooked and canned, to groups of diabetic patients, and found their glycemic indices to be
lower than that of white bread ascribed a GI of 100. They concluded that the glycemic
effect of dried legumes was increased by the canning process. This observation may be of
significant dietary influence because canned beans are also more convenient to use then
dried beans.

The effect of a 50 g starch meal prepared with pre-cooked instant bean flakes on
glucose and insulin plasma levels and on glucose oxidation rate as measured by continuous
indirect calorimetry was assessed in six healthy human volunteers during 4 h following the
meal. Comparison was done with the same load of starch as potato flakes to which the
fiber and protein content of the bean meal had been added. Thirty minutes after ingestion,
plasma glucose and insulin rise was less after beans than after potatoes (0.4 vs 2.0 mol -1
and 8 vs 38 U-ml'l, respectively). The elevation of glucose oxidation was markedly less
during the 2 h following the ingestion of beans when compared to potatoes. The bean flake
meal gave rise to only moderate elevation of plasma glucose and insulin levels and of

glucose oxidation rate when given to four non-insulindependent-diabetics mellitus

43

patients. The slow carbohydrate property of legumes was related to the histological
structme of the seed rather than due to their fiber content (Tappy et. al., 1986).

Jenkins et al. (1980) conducted a study where normal volunteers took 50-g
carbohydrate portions of eight types of dried legumes and 24 common foods drawn fiom
among grains, cereals and pasta, breakfast cereals, biscuits, and tuberous vegetables. Both
themeanpeakrisein blood glucoseconcentrationandmeanareaundertheglucosecurveof
the subjects who ate beans were at least 45% lower than those of subjects who ate the other
foods. These results suggest a potentially valuable role for dried leguminous seeds in
carbohydrate exchanges for individuals with impaired carbohydrate tolerance.

Leathwood and Pollet (1988) reported the effects of slow release carbohydrates in
the form of bean flakes on plasma glucose and satiety in man. After a meal containing
potato, plasma glucose levels rose sharply, but fell below initial levels 2 to 3 hours later.
In contrast, there was a low, sustained increase in blood glucose after consumption of bean
puree. In a related study, consumption of bean puree delayed the return of hunger
(increased satiety) and decreased ratings for propensity to eat a tasty snack between meals.
Jenkins et al., (1982) also found slow release dietary carbohydrate to improve second meal
tolerance. A breakfast of lentils produced a significant reduction (71%) in the blood
glucose area and flattened the plasma insulin and gastric inhibitory polypeptide responses
in comparison to whole meal bread of identical carbohydrate content. The lentil breakfast
was followed by a significantly flatter blood glucose response to the standard bread lunch
which followed 4 hours later. These results, together with breath hydrogen studies,

indicate that the flattened response to lentils is not due to carbohydrate malabsorption.

PROCESSING AND UTILIZATION OF DRY EDIBLE BEANS AS
SPECIALIZED FOODS

Conventional processing techniques for dry edible beans include simmering or
"crock-pot” low temperature cooking, commercial canning, roasting and germination or
sprouting. Figure 9 summarizes in a flowchart processing strategies and edible by-
products of common dry beans. Dry beans are handled, stored and processed in diverse
manners in Lesser Developed Countries (LDC) and in industrialized systems appropriate
for developed economies.

Greater utilization of prepared dry edible beans has been advocated by
governmental nutrition programs for the prevention and treatment of malnutrition in young
children and nutritional maintenance of youth and adult populations in LDCs. Dry edible
beans are also frequently considered for applications in world food relief programs in
LDCs and regions experiencing sustained crop failure.

In many parts of the world, the primary responsibility for bean and cowpea
production rests with women and children. While women's role in agriculture vary by
country and region, it is not unusual for them to play a major role in seed selection,
planting, cultivation, harvesting, storing, and processing and preparation. Women play
multiple roles of providing for their family's sustenance, clothing, overseeing the health
and nutrition of the children, and frequently supplementing family income. In African and
Latin American countries where beans are staple foods, women carry on their shoulders
and/or head water vessels and wood for cooking fuel, long distances, frequently in arid or
mountainous terrain to their village or anal home sites. Traditional cooking of dry edible
beans in these countries involve excessive expenditure of time and fuel. The development
of appropriate preparation technologies for use at the household and village-level would
facilitate processing and dietary availability of beans and other legumes. Valuable time

44

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could thus be devoted to more effective child care or additional income-generating
activities. Drum-dried bean meals possess potential for pre-cooked, prolonged shelf-life
weaning food formulations which can provide both protein and energy to the infants as
well as offer preparation convenience for mothers.

The demand for convenient food items has resulted in specialized pre-processed
food products and ingredients from dry edible beans, possessing diversified functional
properties which may be used directly, or utilized as a component of formulated foods
(Uebersax et al., 1991). The technological challenge presented is to achieve these
improvements within the scope of minimum process inputs and appropriate technologies.
Potential increased consumption of novel bean products is dependent on the individual
processing economics associated with the specific product.

Bean Sprouts, Curtis and Ferments

Utilization of legumes in LDC‘s still entail long and tedious preparation
procedures. However, traditional methods of legume preparation, such as germination
and fermentation, have found wide acceptability in Afiican and Asian countries for their
potential in improving digestibility and reducing antinutritional factors. Germination and
fermentation have been reported to reduce phytic acid which interferes with protein
digestibility and mineral bioavailability (Rao and Belavady, 1978; Silva and Luh, 1979;
Sathe and Salunkhe,]982) and oligosaccharides which have been associated with
flatulence (Rackis et al., 1970; Calloway, et al., 1971; Murphy et al., 1972; Rao and
Belavady, 1978 and Fleming, 1981). Enzymatic treatment of beans have also been
reported to remove much of the oligosaccharides (Calloway et al., 1971).

Germination results in at least a ten-fold increase in phytase activity and a
simultaneousdecreaseinphytate,andifdonecarefully,thebean seedcanstillbeusedfor
cooking in the customary manner (Reddy et al., 1978). Sprouting was found to increase
the digestibility coefficient of red kidney beans from 29.5% in the raw to 66.4% in the

47

cooked kidney bean. Nielsen and Liener (1984) reported a thiol protease with an acid pH
optimum to be responsible for the disappearance of the major storage protein, 01, during
germination. During the process of sprouting, enzymes released from the embryo
hydrolyze portions of the linear and branched starch molecules into short-chained dextri-
maltose which does not swell when cooked into a gruel (Ebrahim, 1983). Sprouted grains
may also be consumed fresh or when followed by seed coat removal, can be roasted or
ground for use in soups or side dishes.

Soybeans are fermented to produce a variety of nutritious Oriental food products
including tofu (calcium precipitated curd), natto, misc, tempeh, shoyu and tamari. Indian
fermented delicacies like idly and dosa can be prepared from black gram (Phaseolus
mango); dhokla and khaman utilize Bengal gram (Cicer arr'etr'nwn).

'Dhal' is prepared by cooking beans until soft, mashing, mixing with water, then
boiling to yield a homogeneous gruel. They can also be cooked with vegetables, spices
and condiments. Roasting and puffing of dhal and whole seeds to improve flavor and
maintain maximum protein quality involve the application of dry heat (100°C to 200°C)
for a short period (1-5 min). Dry beans and other legumes could be prepared by methods
which include protein and production or fermentation to improve quality and digestibility.
Swanson and Raysid (1984) found that there was a significant increase in protein
digestibility and PER in tempeh made from red beans (P. Vulgaris L.) and corn. Further
research is needed to assess the potential of Phaseolus vulgaris in fermented foods.

Bean Meals, Flours and Powders

Beans may be consumed as whole cooked seeds where seed distinctives are readily
perceived by the consumer, or as cornminuted meals, pastes or curds where seed integrity
is lost and the functional properties are a direct function of the seed components and their
interaction with waterandheat. Inproductsutilizingbeansin milledormashedforms,the
consumer may not readily discern the bean type (Uebersax and Occeiia, 1991). High

48

protein food ingredients can be produced as either flour processed by pin milling & air
classification, or as concentrates and isolates processed by alkali, salt and acid extraction
with subsequent use of isoelectric precipitation or ultrafiltration (Uebersax et al., 1991).
Chang and Satterlee (1979) produced bean protein concentrates containing 72% and 81%
protein by wet processing using water extraction techniques.

Dry beans have been milled into whole flour and air-classified into high starch and
high protein fractions (Kon,l979; Ekpenyong and Borchers, 1980; Reddy and
Salunkhe,1980; Aguilera et al., 1982 a and b). High protein flour fractions generally
contain twice the original whole bean seed protein content. Bean bulls are readily separated
by cracking rolls and air aspiration and then milled into a high fiber fraction. Air
classification of roasted beans produced high fiber, starch and protein fractions, each
suitable as food ingredients in a variety of food products such as cookies, doughnuts,
quick breads and leavened doughs (Defouw, et al., 1982; Dryer et al., 1982; Zabik et
al., 1983; Uebersax and Zabik, 1986). Instant precooked bean powders have also been
prepared by soaking, cooking, slurrying and drum or spray drying of edible beans
(Bakker, 1973).

Functional Characteristics and Applications. Much research has been
conducted to investigate the functionality of high protein bean products to broaden their
use in various foods. Air-classified high protein bean flour contains a residual starch which
offers some particularly desirable functional applications. Lee et al. (1983) reported that
wheat flour substituted with 10% navy bean fractions (whole, hull and high protein and
starch ) resulted in increased water absorption and decreased mixing performance. Whole
bean flour and high starch flours produced viscous solutions with controlled swelling at
high temperature. Bean slurries remained stable during high temperature holding, and upon
cooling, showed moderate increase in viscosity, indicating a tendency toward association

of starch molecules. Zabik et al. (1983) demonstrated that dry roasted air-classified navy

4 9

bean protein flours substituted for 10% bread flour exhibited increased water absorption
but decreased arrival and peak times of the flours processed with increased roasting
temperature. Mixing stability of the flours showed a decreasing trend as the bean/bead ratio
and roasting time increased. Lorirner et al. (1991) studied the effect ofprime and cull navy
bean high-protein flours on rheological parameters and microstructure of composite
doughs. Composite flours with increased protein exhibited the expected increase in
absorption, delay in arrival and peak time and reduction in stability. The microstructure of
composites with cotyledon flour from culled beans showed slightly greater gluten
disruption than was evident in the doughs prepared from the wheat-prime cotyledon
composites.

Kohnhorst et al. (1990) reported that navy and kidney bean flours formed
significantly higher volume foams but had lower foam stability compared to soy products
at pH 6.5, possibly indicating compositional or structural differences between the
proteins. The emulsion capacity, gel strength and foaming properties of navy and kidney
bean flours were similar and were significantly higher than that of soy flours.

Navy bean flours substituted for wheat flour in baked products such as pumpkin
and banana breads and fiied "doughnuts holes" increased protein quality and quantity
since they are relatively high in lysine. Dry roasted navy bean flour incorporated into
pumpkin bread increased tenderness because of the reduced gluten formation and of the
diluted nature of the gluten. Volume decreased with increasing levels of bean flour
substitution because of decreased gluten formation. The pumpkin bread with bean flour
was darker than the control made from wheat flour because of increased reducing sugars in
the navy bean flour, promoting Maillard browning. Pumpkin bread containing 35% navy
bean protein substitution appeared to be optimum and contained about 25% more protein
than the control (Dryer et al., 1982). Extruded navy bean flour incorporated at either 20 or
35% level of substitution produced acceptable quick breads (Kane et al., 1991).
Doughnuts with 13% bean substitution had less fat, were softer and showed less firming

50

after 6 days storage compared to control doughnuts and other bean substitutions (Uebersax
et al., 1981 and 1982). Raised doughnut holes prepared at a 25% navy bean protein
florn' substitution level exhibited greater tenderness than did the wheat flour control (Zabik
et al., 1983).

Volume and crumb color of bread were not adversely affected at a 10% bean
protein substitution, and raised the protein level by 20% (Zabik et al., 1983). Silaula et
al. (1989) reported that dry-roasted navy and pinto bean high -protein fractions blended
with bread or whole wheat flour increased tenderness and reduced lightness of breads.
Substitution of legume flour for bread or wheat flour dilutes the gluten and interferes with
the formation of a well-defined protein-starch complex. Substitution of 5 and 10% high-
protein flour for bread flour increased absorption and lengthened arrival and peak times in
farinograph studies but decreased dough stability, causing a faster rate and greater
magnitude of breakdown ( Lorimer et al. 1991). Navy bean flour incorporated into
Chinese steam bread decreased volume but increased tenderness with increased bean
substitution (Uebersax and Zabik , 1983 ).

DeFouw et al. (1982) reported that navy bean hulls incorporated into sugar-snap
cookies resulted in less acceptable interior color as the level of hull substitution increased.
Texture was not significantly affected by less than 30% substitution with unroasted or
moderately roasted hulls. Flavor, top grain and spread were adversely affected as the level
of substitution increased. Navy bean protein substitution for wheat flour increased water
holding capacity and cookie tenderness, but decreased cookie spread slightly (Zabik et al.,
1983).

Breads prepared with wheat flom' blend (10% pinto flour) had a slight decrease in
volume, while those with bread and pinto flour blend had fewer differences in volume and
tenderness (Uebersax and Zabik, 1986a and b). Noodles produced with pinto bean flour at
% wheat substitution were more tender and slightly darker, with decreased flavor and
acceptability as substitution level increased (U ebersax and Zabik, 1986).

51

Numerous studies assessing the funtional characteristics of other legume flours
have been conducted. For instance, heat processing of winged bean flour have been
observed to lower nitrogen solubility, emulsification and foam capacity, but increased
water and fat absorption capacity (Narayana and Rao, 1982). Sathe and Salunkhe (1981)
reported that Great Northern bean flour had lower oil and water absorption capacities
compared to those of soybean and sunflower flours. Emulsion capacity and stability of the
flour was the lowest compared to those of the protein concentrates and isolates and

albumin and globulin proteins.

Drurrr - Drying of Legumes. Drum drying of dry beans into precooked flakes
and powders could be used to produce ingredients suitable for convenience foods or
replacements for cooked bean purees or refried beans. Drum-driers have high drying rates
and high energy efficiencies, and are suitable for slurries in which the particles are too
large or thick for spray drying. Bakker et al. (1967 and 1969) produced instant pea bean,
pea and lentil powders using a modified single-drum—drier. An acceptable drum-dried
infant cereal using banana (40% dw), rice (10.5% dw) and soybean (16.5% dw)was
developed on a commercial scale to supply the needs of the Costa Rican Program for
Food and Nutrition aimed at children under age 2. The process consisted of the following
steps: 1) cooking the soybeans at 121°C for 30 minutes; 2) boiling the rice for 5 minutes to
gelatinize the rice starch; 3) inactivation of the banana enzymes by boiling with the rice and
soybeans for 3 minutes, and 4) drum-drying to 5% moisture (Lastreto et al., 1986).

W A basic drum dn'cr. as
illustrated in Figure 10, is comprised of one or more hollow steel drums (rollers) which
slowly rotate and are heated internally by pressurized steam to 120-1700C. Drum-driers
use conduction heat transfer; condensation of the steam inside the drum provides energy

52

 

 

 

 

 

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Figure 10, Schematic Components of a Double Surface Drum-Drier: A - Steam-
Heated Drums; 3, C - Slurry “Feed and Puddle”; D - “Nip"; E -
Dehydration Surface; F - “Doctor” Blade (knife); G - Dehydrated
Sheet; H - Conveyor; l - Vapor Hood.

53

for vaporization of water at the external drying surface ( Batty and Folkman, 1983). The
drum are mounted to rotate about the symmetrical axis. A feeding device is used to apply
a thin, uniform layer of food material to be dried on the hot cuter sm'face. There are a
variety of methods for distributing the stock feed over the surface, including a) dipping,
b) spraying and c) spreading or use of auxiliary feed rollers. Before the drum completes
one revolution (within 20 secs to 30 mins.), the feed material applied on the periphery is
dried as the heated drum rotates toward the 'doctcr' blade (knife ) which contacts the drum
surface uniformly along its length, and scrapes the thin layer of dry material from the drum
surface.

Drum driers are classified as single-drum, double-drum, or twin—drum systems. A
single-drum drier has only one roll while a double-drum drier has two rolls which rotate
toward each other at the top. The two parallel drums of a twin-drier may either rotate
toward each other or away from each other at the top, and are not spaced close together
(Harper, 1979). The single drum is widely used as it has greater flexibility, a larger
proportion of the drum area is available for drying, easier access for maintenance and no
risk of damage caused by metal objects falling between the drums (Fellows, 1988).

Was, Therearefourbasicdrumdriercontrols inan
atmospheric double drum drier; these include : a) puddle level, b) drum speed (rpm),
c) steam pressure and d) drum clearance ("nip"). The speed of the rotation is adjusted so
that the desired moisture is obtained when the product is scraped off the drum (Batty and
Folkman, 1983). Other control points of the drum—drying process include initial solids
content, feed rate and drum-drier temperature. A typical commercial single drum dryer has
the following dimensions: 1.65 m in diameter and 4 m long. The drums are run at about 8
to 9 atm. steam pressure (115-135 psig) with surface temperature of about 240°C. The
rotation speed of the drums is 3.5 to 4 rpm and the effective drying surface 31 m2. .
Pretreated slurry is pumped through a tangential steam heater where starch is fully

gelatinized. The hot slurry enters the flash tank which serves as a surge reservoir for

5 4
uniformly supplying slurry to the drum drier. Following application and dehydration, the
thin continuous sheets of dried beans falling from the drier knives is broken by a screw
conveyor and the resultant flakes collected are passed through a Fitzpatrick comminuting
mill to provide uniform particle size.

Developments in drum design to improve the sensory and nutritional qualities of
dried food include the use of auxiliary rolls to remove and reapply food during drying, the
use of high-velocity air to increase the drying rate or the use ofchilled air to cool down the
product. Drums may be enclosed in a vacuum system to dry food at lower temperatures
which is especially suitable for high-value heat-sensitive food products (Fellows, 1988).

Thermal Extrusion

High starch navy, pinto and black bean flour fractions yield good expansion
properties during high pressure and high temperature extrusion (Zabik et al., 1983b;
Aguilera et al., 1984). When the cooking and extrusion processes are combined, the
increased pressure differential will facilitate expansion and a final dry formed 'bean'
particle which readily hydrates and softens when reconstituted is achieved. Die
configuration can be designed to produce a variety of intricate product shapes which can be
further Optimized to produce simulated beans. Appropriate binders and coating agents can
be added to improve appearance of pre-ccoked or quick cooking bean products resulting in
promising prototypes of extrusion cooked - formed 'beans' (Uebersax et al., 1991).

Legume flours can also find application in extruded snack items, breakfast cereals,
pasta products and infant foods. Extrusion of millet flour to prepare instant infant foods
yielded a product that was much less dispersible ( d0% for millet flour at 85°C compared
to >70% for corn starch at 35°C). Increased lipid and fiber contents of millet flour
probably contributed to these observations. However, a similar decrease in soluble starch
was observed in both materials when the extrusion moisture content decreased from 17%
to 12%. More amylopectin was dispersed from the millet extrudate when the extrusion

55

moisture content was 13.6% and 15.0% (Waniska, RD. and Gomez, M.H., 1992). The
formation of amylose-lipid complexes reduces the digestibility and water solubility of
cooked starches (Galloway et al., 1989). Amylose in starch will complex with lipids
during extrusion cooking. As starch in the presence of lipids is exposed to increasing
amounts of heat and pressure, the amount of amylose-lipid complexing increases (Huber,
1991). Extruded navy bean and lentil flours substituted (20 and 35%) for all-purpose
flour in pumpkin bread demonstrated the feasibility of extruded starchy legumes as food
ingredients (Hartet al., 1991).

Giese (1992) provided a review of new types of pasta developed from
unconventional raw materials such as legumes. High temperature extrusion cooking allows
added proteins such as egg albumen to form a coagulated network to improve pasta
quality. Thin-walled macaroni formulations, incorporating durum wheat semolina with
15% and 25% drum-dried navy bean flour or 15% whole raw navy bean meal, have been
successfully developed at Michigan State University (MSU) using a high temperature
twin-screw extrusion cooker (Lin et. al., 1993).

Borejszo and Khan (1992) reported lower levels (3.69%) of flatulence causing
sugars (raffinose and Stachyose) in extruded pinto bean high starch fractions compared to
non-extruded samples (5.29%) with higher reduction occurring at higher process
temperatme (163°C). According to Chauchan et al. (1988), protein efficiency ratio values
of extruded rice-legume blends (rice-soybean, 2.25; rice-bengal gram, 2.30 and rice-
black gram, 2.28) were improved compared to the respective raw blends (2.10, 1.89 and
1.98 respectively). They also demonstrated that extrusion processing reduced the phytates
in the products to the extent of 20.3% to 26.8%. Ummadi et a1. (1993) determined the
effect of low and high impact extrusion processing on iron dialyzability, forms of iron,
tannin content and degradation of phytic acid in navy beans, chickpeas, cowpeas and
lentils. Dialyzable iron increased following low impact extrusion in all the legume

products, but only in navy beans with high impact extrusion. Low impact extrusion

56

resulted in increased total soluble and ionic iron. However, high impact extrusion did not
result in any change in total soluble iron but increased ionic iron in cowpeas and lentils.
Degraded phytate products increased to 51-71% in extruded legumes. Tannin content
decreased with low and high impact extrusion.

Microwave Heating of Legumes

Microwave heating has a direct potential for use as a preconditioning treatment for
common dry beans to reduce processing time, and to inactivate antinutritional factors
(Uebersax et al., 1991).

Mechanism of Microwave Heating. Microwaves are generated by a
magnetron (a device that converts electrical energy at low frequencies) into an
electromagnetic field with centers of positive and negative charges that change direction at a
frequency of 109/sec. As microwaves enter the product, they interact with regions of
positive and negative charges on water molecules, (electrical dipoles) that rotate the
molecules in the electrical field by forces of attraction and repulsion between oppositely
charged regions of the field and the dipoles. This results in the disruption of hydrogen
bonds between adjacent water molecules and generates heat by "molecular friction"
(Decareau, 1988; Mudgett, 1989; Giese, 1992). The molecules in the microwave paths
oscillate around their axes in response to reversal of the electric field that occurs 915 or
2450 million times per second, creating intermolecular friction that results in the generation
of heat (Cross and Fung, 1989). Food cooked by microwave involves heat generated from
within the food through a series of molecular vibrations. In contrast, conventional heating
methods transfer thermal energy from product surfaces toward their center 10 - 20 times
slower than microwave heating (Mudgett, 1989). In developing products for microwave
processing, it is important to recognize that microwaves are a form of energy, not a form of
heat, and are only manifested as heat upon interaction with an aqueous material as a result

of one or more energy-transfer mechanisms.

57

Microwave Effects on Food Composition

Magnum. Greater losses in moisture in microwave-cooked compared to
conventionally heated products could be due to a greater rise in post-oven temperature,
causing more dehydration through evaporation and increased shrinking. Very few studies
provided data on water content of foods prior to and following microwave cooking
( Schiffmann, 1988).

Emma, Hafez et al. (1985) conducted experiments to determine in vivo protein
digestibility and metabolizable nitrogen using male Sprague-Dewley rats. They observed
that proper microwave treatment increased the digestibility of soybean. Optimum
microwave heating time was around 9-12 minutes for 1 kg whole soybeans to improve
weight gain, digestibility and intestinal proteolytic activity under experimental conditions
maintained during the study. They also did not observe alteration of fatty acid composition
of soy oil. 7

A study by Yoshida and Kajimoto (1988) showed that microwave heating might be
effective in the inactivation of the trypsin inhibitor in whole soybeans. Microwave
treatment of whole soybeans for 8-10 minutes after soaking to approximately 25% moisture
would be optimal to prepare soyflour or soy grits without a burnt odor, and completely
inhibiting trypsin activity. The loss of molecular species of soy triglycerides containing
more than four double bonds in a triglyceride was less than that obtained from native
soybeans prior to soaking (8.6% moisture). In an earlier study, Yoshida and Kajimoto
(1986), found that microwave heating for about 5 minutes did not cause a loss of
unsaturated fatty acids, nor did it change molecular species of soybean triacylglycerols.

Susceptibility to heat processing has been observed with protein containing
significant amounts of carbohydrates. Interactions between functional groups with the
protein chain or between the protein chain and other food constituents during heating lead
to cross-linkages which are resistant to normal digestive processes.

58

Overall, nutritional effects of microwaves on animal proteins appear minor,
however, many studies have demonstrated that microwave heating may aid retention of
total protein in foods (Decareau, 1986). For example, microwave treatment of potatoes
favored protein retention over other preparation methods (boiling or oven-baked)

investigated (Mudgett, 1982).

W Overall, much work needs to be done on the effects of
microwave heating on the carbohydrate fraction of foods. Aref et al. (1972) observed the
inactivation of alpha-amylase in wheat flour after 60-second exposures to microwave
energy without pronounced deleterious effects on the principal characteristics of the flour or
the subsequently prepared doughs . The exposme also drastically reduced the number of
viable organisms in the flow, but seemed to cause a relatively high loss in moisture.

Microwave baking is usually done at 896 to 2450 MHz, the range also used in
normal household ovens. The lower frequency is to be recommended in some cases, as this
enables both higher penetration depth to be obtained and thus avoids the risk of the core not
being sufficiently baked.

The main problem of baking with microwaves is the lack of both crust formation
and surface browning. This may be achieved by short-time conventional re-baking for
about 4-5 min at a temperature of ZOO-300°C.

Flour with a high alpha—arnylase concentration and low protein content have been
used for dough preparation. In microwave baking, the entire loaf is heated rapidly, and the
development of 002 and steam in the dough is accelerated, thus resulting in breads with a
relatively high specific volume despite the low protein content The temperature range
essential for alpha-amylase is reached rapidly, so that the amount of undesired
decomposition products remain low.

Microwaves are currently used for commercial leavening and proofing, thus

enabling the process of gas development and adhesive formation to be completed within a

5 9
period of only 2-4 min. Microwave treatment might require changes in the dough recipe,
leading to new final products. A lack of crust formation and differences in the color were
observed in whole-grain baked products, and the microwave breads were tough and
rubber-like (Schiffmann, 1988). The taste was less significant than that of conventionally
prepared bread because the microwave products lacked both caramelization and
development of aroma components. Loaf volume and crumb condition were not of the
quality obtained in conventional bread production. The following are advantages of
microwave baking: 1) provides a final product with a higher nutritive value than
conventional baking, provided the same ingredients are used; 2) baking times are reduced
to a few minutes - the entire baking process of a 1 -kg loaf only takes 7-8 minutes and 3)
microwave equipment requires less space.

Microwavable pasta and pasta made from a primary material other than wheat have
been developed Strategies for improving pasta performance during microwave heating
include modifying the wall thickness and adding selected ingredients such as egg albumen
to assist shape retention. The increased protein content allows for the formation of
polypeptide chains which interact to form a fibrillar network which subsequently entraps
starch granules and slows gelatinization (Giese, 1992). Cabello et al. (1992) reported a
highly acceptable precooked and dehydrated pasta suitable for microwave heating using
15% substituted navy bean cotyledon flour. The navy bean flour pasta had a richer yellow
color which was favored over 100% semolina pasta. Microwave cooked legume pasta
made from different levels of extruded drum-dried bean flour demonstrated higher cooked
weight and was less firm than 100% semolina control pasta. Microwave preparation (740

watts, 5 mins.) also resulted in lower cooking loss than conventional cooking (100°C, 2
mins.) (unpublished data).

 

60

WE“ No general statement can be made flomreported
literature studies regarding total fat in microwave versus conventionally prepared food

products due to the differing procedlnes, conditions, and products used (Decareau, 1982).
Industrial investigations of the function of fats, oils and emulsifiers in microwave
food formulations reported fats/oils and emulsifiers to function optimally, and not to be
chemically altered by microwaves (Durkee Foods Corporation, 1989). Emulsifiers
interface with water, within the food matrix, due to the polar hydroxyl end of the
emulsifier. This interaction may maintain moisture within microwave prepared foods.
They also interact with proteins, starches, and gases such as air, further stabilizing and
improving texture (Connerton and Shuleva, 1989). Fats/oils can function
thermodynamically as a "heat source" to absorb energy, generate heat, and release heat to
adjacent non-fat molecules of lower polarity. Lipids remain totally unchanged by the

microwave energy, thereby aiding moisture retention within a product.

Mina“ Investigation of some vegetables cooked by oven heat and in a
microwave oven revealed negligible loss of water and minerals regardless of cooking
method, although microwave heating was suggested to be superior to conventional heating
since less nutrients were lost to the aqueous cooking medium. More recent studies showed
that nutrient retention in his and vegetables was greater with microwave heating than
with conventional heating due to less water-to-product ratio and consequent reduced
leaching in foods cooked with microwave energy, therefore the microwave heating
increased retention of nutrients. Specific studies like those of four potato varieties prepared
by various home cooking methods and microwave cooking suggested that the latter
enhances macro-nutrient retention.

Mineral determinations for meat drippings showed significant differences of
selected minerals (Na, Cl, P, Fe) from oven-roasted pork, beef and lamb drippings. It

was suggested that the greater concentration of minerals in the drippings from

61

conventionally cooked roasts could be related to the lower moisture content of the

drippings. Therefore comparisons of absolute mineral losses remain unclear.

Wm, Studies have indicated that destruction of thiamine in food systems is
primarily related to heat level rather than exposure to microwaves. Studies with pork, beef
and lamb suggested that vitamin retention was not related solely to length of exposure to
high temperatures. The contribution of vegetables to the riboflavin and niacin content in the
diet is minimal, so few studies have investigated microwave effects on these components.

Thereviewofmany selected studiesindicatedthat noappreciablelossesinascorbic
acid were attributed to microwave heating compared to conventional heating methods.
Any increased retention of ascorbic acid in microwave-cooked foods cannot be attributed
entirely to the dielectric heating. A combination of factors including ratio of water-to-
vegetable, cooking length, and variability of cooking loads may account for the variations
in reported data (Harrison, 1980).

Except for significant losses of Vitamin A in microwave-cooked meat, limited data

on fat-soluble vitamins are available.
Potential for Dry Edible Beans as Weaning Foods

The Codex Alimentarius Commission (Food and Agriculture Organization/World
Health Organization, 1976) prepared the official guidelines ”Recommendations for
International Standards of Infant Foods." Dried products included in this section are based
on cereals (wheat, rice, barley, oatmeal, rye, millet, corn, sorghum) and legumes (ex.
soybeans) of low water content processed under conditions that allow their reconstitution
withmilkorwater. Iftheproduct istobemixedwithwater,itisrequiredthattheprotein
content be at least 15% (db) and protein quality at least 70% of casein value. The addition

of protein concentrates, isolates and essential amino acids is allowed only in concentrations

62

needed to meet these criteria. Supplemental sugar and fruit addition is also allowed under
these guidelines.

The Thirty-fourth World Health Assembly of the World Health Organization
(1981) adopted the "International Code of Marketing of Breast-milk substitutes” in the
form of a recommendation which aims to protect and promote breast-feeding and ensure the
proper use of breast-milk substitutes when these are necessary to ensure adequate nutrition.
The Code defines "Infant formula" as a breast-milk substitute formulated industrially in
accordance with applicable Codex Alimentarius standards, to satisfy the normal nutritional
requirements of infants between four and six months of age, and adapted to their
physiological characteristics. Infant formula may also be prepared at home, in which case it
is described as "home-prepared" formula. "Weaning food" or "complementary food" are
defined as any food, whether manufactured or locally prepared, suitable as a complement
to breast-milk or to infant formula, when either becomes insufficient to satisfy the
nutritional requirements of the infant. Such food is also commonly called as "breast-milk
supplement" (WHO, 1981). WHO and United Nations International Children and
Educational Fund (UNICEF) promote and support appropriate and timely complementary
(weaning) feeding practices, usually when the infant reaches four to six months of age, and
further encourage the use of locally available food resources.

In LDCs, research efforts on weaning food formulations have focused on the
utilization of indigenous or locally-available protein-rich foods such as legumes and oil
seeds. Digestibility and flatulence-producing components are important factors to consider
when feeding legumes to children. Processing techniques including dehulling, fine
grinding, roasting or any form of prolonged cooking and sometimes germination and
fermentation, have been found to reduce flatus from beans.

A challenge limiting starch-based gruels such as rice and corn as primary

supplement stocks are their characteristically high ingestion volumes required to achieve

6 3

adequate nutrient delivery (low nutrient density) and the high viscosity which might cause
choking in young children. The ability of starch to bind more water leads to dilution which
increases bulk which subsequently limits the amount of nutrients that could be derived
per serving of gruel. Industrial methods which have been reported to modify starch
structure to lower its water-binding capacity have included enzyme pre-treatment
(amylase), precookin g or extrusion and drum»drying, and germination or sprouting
(Lastreto et al., 1982 and Marero et al., 1988).

Enzymes released from the embryo of sprouted grains hydrolyze portions of the
starch molecules into short-chained dextri-maltose which does not swell when cooked into
a gruel (Ebrahim, 1983) and therefore possess great potential in the preparation of flour
from sprouted grains for use in weaning foods. Flour from sprouted grains can be used in
greater amounts to give the same viscosity as flour from unsprouted grain, thereby
improving nutrient and energy density (Mosha and Svanberg, 1983; Desikachar, 1981 and
Brandtzaeg et al., 1981). Weaning foods suitable for use in the Philippines were
developed by Marero et al.(1988) and prepared from flours obtained from germinated rice-
mungbean, germinated rice-cowpea, germinated corn-mungbean and germinated com-
cowpea. The low viscosity and high caloric density formulations were found stable for 6
months, microbially safe and well-tolerated by infants. The blends had amino acid values
which approximated the FAO reference pattern, except for the S-containing amino acids.
The rice-based formulations were demonstrated to be superior to the corn-based gruels
based on chemical score for cystine/methionine, net dietary protein energy and PER. The
use of germinated legumes was also reported to increase the micronutrient contents of the
weaning food formulations (with the exception of calcium ), compared to control
formulations without the incorporation of germinated legumes. Kusin et al. (1984)
reported that in rural Indonesia, growth faltering from the third month and the high

percentage of wasting at age 0-3 years in preschool children were mainly due to the

6 4
inadequacy ofweaning foods andthelowintakesofvitaminsandmineralsresultingfiom
low consumption of pulses and leafy vegetables .

In South and CentralAmericaarldpartsofcentral andeastem Africa, drybeansare
commonly served without dehulling, a form unsuitable for children. Thus, young children
are merely given the broth or soup in which the beans have been cooked, resulting in poor
protein digestibility, diarrhea, and other gastrointestinal distresses. Protein digestibility of
the soup was lowered by the tannins present in the seed-coats of colored beans and are
partially leached into the cookin g medium. The hydroxyl groups of the phenol ring enable
the tannins to form crosslinks with proteins which may be implicated with decreased
digestibility (Ma and Bliss, 1978 and Haslam 1979; Elias et al., 1979; Aykroyd and
Doughty, 1982; Bressani and Elias, 1980; Bressani et al., 1983 and 1988). The Institute
of Nutrition of Central America and Panama (INCAP) introduced in the late 1950's
"Incaparina", a mixture of predominantly vegetable protein origin having a nutritional value
similar to that of milk and suitable for the mixed feeding of infants and young children.
The first one tested clinically was "Mixture 8" made with entirely indigenous materials
having the following formulation : lime-treated corn flour flour (50%), sesame meal (35%),
cottonseed meal (9%), torula yeast (3%) and powdered kikuyu leaf (3%), and was
supplemented with vitamin A. "Mixture 9" was the first mixture recommmended for
commercial production , and was made of lime-treated (CaCO;;; 1%) corn flour (29%),
sorghum (29%), cottonseed flour (38%), torula yeast (3%), and supplemented with 4,500
ID. of vitamin A per 100 grams of the dry product. Alternate formulas were subsequently
developed with soy, peanut and sesame as the alternate vegetable protein sources and with
rice as the cereal component (Scrimshaw, 1980). These modifications were recommended
due to agricultural availability and least cost formulation considerations. Other
recommendations to improve the product included the addition of amylase to make a
thinner gruel, and marketing it as a precooked product. However, these were not endorsed
protocols due to inherent additional costs, departure from the traditional practice of

plottil

COHCCI

feedln
BaI-A.
in Ind
witho
lochnl
ftrmc
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65

preparing thick corn gruels or the potential encouragement of feeding by bottle which
would have introduced problems of contamination and displacement of breast milk. Other
efforts which have have been made to develop low-cost nutritive infant foods included : 1)
Del Valle et al.'s (1981) "Soyaven," an infant formula already in commercial production;
2) "Nutrisoy", a blend of corn and soybeans (Easterbrook, Gregg, 1986; malted and roller
dried sorghum and cowpea weaning food formulations (Malleshi et al., 1989); 3) sesame
protein-based weaning food (Moharram et al., 1989); 4) ultrafiltered chickpea protein
concentrate in infant formula (Ulloa et al., 1988; Reddy et al., 1990).

Traditional germinated legume and cereal seeds have also been used in mixtures for
feeding young children in India. An Incaparina-type of precooked weaning food, named
Bat-Altar ("nutritious child"), played an important role in government nutrition programs
in India (Scrimshaw, 1980 and Reddy et al., 1990). It was provided to the consumer
without cost and largely displaced imported weaning foods. Traditional processing
techniques which have been utilized include roasting, malting (germination), puffing and
fermentation (Reddy et al., 1990). Legumes which have been successfully utilized in
commercial preparations include soybeans, green and black gram, chickpeas, pigeon peas,
cowpeas and groundnuts. Multimixes consisting of local staples (ex. cassava and
plantain), legumes and vegetables (ex. dark-leafy greens), or sometimes even meat, raised
not only the protein but also the vitamins, minerals and energy content as well. The
addition of fat or legume oil is recommended when the legume or pulse component is
intrinsically low in fat. Gupta and Sehgal (1991) reported lower levels of phytic acid,
polyphenols and saponins and higher in-vitro digestibility of low-cost weaning mixtures
prepared by mixing malted pearl millet, roasted amaranth, roasted green gram, jaggery (a
low-grade molasses residual fraction) and malted barley. Olaofe (1988) compared the
mineral contents of imported baby foods with those of selected Nigerian grains (maize,
sorgum, cowpea and soya beans) suitable for baby foods and found the iron contents to

be in the same range but calcium to be much lower in the Nigerian grains. Corn-flour

66

infant feeds consumed in Nigeria were reported to have considerably lower amounts of
mineral elements than popular brands of infant formula (Fatoki, 1990). Moharram et al.
(1989) also reported that weaning foods based on sesame protein in combination with dry
skim milk and either corn or rice starch were successfully prepared and found to be feasible
for production in developing countries .

Potential for Soybeans as a High-Protein Weaning Beverage

The soybean and suitable processing methodology have been extensively studied
for use in weaning foods. This review is presented to provide a basis for use of these
technologies in preparation of dry bean (Phaseolus vulgaris) based products.

W Soybeans (Glycine max (L.) Merr.) are the most
widely grown of the grain legumes. It is essentially a temperate crop but can be grown up
to an elevation of 5000 meters depending upon the location. Production of soybeans is
dominant in both developed countries and lesser developed countries (Figure 11). Dry
beans, peas and peanuts are of similar total production scale, with noted differentiation
among predominant sectors (Uebersax and Occer‘la, 1991). Soybeans serve as the major
source ofprotein to supplement a variety of grains in the diet in LDCs and are utilized to
help alleviate malnutrition, especially in the Asian region. The approximate composition of
soybeans is as follows: protein, 40%; lipid, 20%; cellulose and hemicellulose, 17%;
sugars, 7%; crude fiber, 5% and ash, 6% (Erickson et al., 1980). Isolated soy proteins
are now used commercially in many food applications such as infant formulas, emulsified
meats, imitation cheese, bakery products, high-protein diet products and formulated
soymilk (Haytowitz and Matthews, 1989).

 

The preparation of
soymilk requires five basic steps: cleaning the soybeans soaking, grinding, filtering the
soy residue and cooking the soymilk. Commercial production of soymilk include

Total Production (1000 MT)

1 20000

67

 

100000 .

80000-

60000-

40000 -

20000

       
   
   

 

 

Soybean Grantham DryPesr Dryberns Olickpees Broadbuns

Source of production

Lesser Developed Countries
I Developed Countries

\\\\\\\\\

\\\\\\

 

 

\ \ \ \
I I I I I
A A \ \ \ \ \ \ \ H
1 l . .
r ‘I

Lentils

 

 

 

 

 

 

Legume

Figure l 1. World production of major food legumes (Scmce: Data
adaped frcrn FAO, 1989).

68

additional steps of dehullin g, formulating, homogenization, sterilization and packaging.
The flowsheet for commercial soymilk production using the Illinois Method is outlined in
Figure 12. The advantages of this method include: very high yield ( 89% solids, 95%
protein); removal of oligosaccharides and beany flavor. However, the use of an expensive,
powerful hornogenizer to produce a smooth, stable emulsion is a major disadvantage.

Cleaning reduces or eliminates damaged soybeans in which activated lipoxygenase
has catalyzed oxidation of the unsatm'ated fatty acids producing compounds with the
characteristic beany flavor. Good quality soymilk can be made without dehulling of the
soybeans, but dehulling offers the following advantages: improved protein recovery,
improved soymilk flavor and slightly whiter color; improved digestibility; reduced
oligosaccharides, soaking time, grinding energy and bacterial count.

Soybeans are generally soaked in about three times their weight of water. Cold
soaking requires a longer time ( 8 to 10 hours at 20°C or 14 to 20 hours at 10°C) for
hydration of soybeans compared to hot soaking. Longer soaking periods result in greater
loss of soluble components, particularly the water-soluble carbohydrates. Soaking reduces
energy input required for grinding, enables better dispersion and suspension of the solids
during extraction and increases yield and decreases cooking time. Soaking in alkaline
solution containing sodium bicarbonate or sodium citrate are reported to decrease the
beany soymilk flavor, tenderize the soybeans leading to reduced cooking time and
enhanced homogenization, reduce oligosaccharides and accelerate the inactivation of the
soybean trypsin inhibitor.

Blanching or steaming in a solution of sodium bicarbonate initiates inactivation of
the lipoxygenase causing the beany flavor, washes out water-soluble oligosaccharides and
inactivates trypsin inhibitors. Hot water grinding to achieve a 150 mesh colloidal
suspension further inactivates the lipoxygenase. Filtering the insoluble soy residues fiom
the slurry is done using a decanter centrifuge and results in improved flavor, mouthfeel
and facilitates removal of oligosaccharides.

69

SOYMILK PROCESSING PROCEDURE

 

Whole Soybeans

i

Soaking in 0.5% NaHCO3 solution for 6 to 12 hours
(Bean : Water 8 1:3)
(Water hardness 84 ppm on CaCO3 basis)

 

 

 

 

i

[ Blanching by boiling for 10-20 min

 

 

 

Draining and rinsing

 

Grinding with a Fitzpatrick mill
(Fust 0.25 inch screen and second. 0.073 inch screen)

 

 

i

Heating slurry to 82 C
Homogenizing at 3500 psi and 500 psi
Adding water to give 12% solid

 

 

i

Neutralizing to pH 6.8 to 7.2 with 6N HCl

Formulating by adding water. sugar. flavoring
(0.02% salt. 0.2% vertillirl, calcium 25 mM, earcWater=1:10)

 

i

| Heating to 82 c
l Homogenizing at 3500 psi and 500 psi

i

Battling & sterilizing at 121 C: 15 min.
Cooling

i

| Shelf-Stable Ready-to-Use Beverage

_—

 

 

Figure 12. Illinois Method for processing soymilk from whole soybeans (Nelson et al., 1975)

7 0

Cooking or heating the soymilk destroys the spoilage microorganisms, inactivates
trypsin inhibitors and also reduces viscosity to facilitate extraction and give higher yields of
protein and solids. Heating soymilk to 100°C for 14 to 30 minutes or at 110°C for 8 to
22 minutes was reported by Van Buren et al. (1964) to inactivate 80 to 90% of the native
trypsin inhibitors.

Appropriate formulation of soymilk using sweetening and flavoring agents
increases the acceptability of soymilk. Flavored soyrnilks already currently commercially
produced in Asian nations include vanilla, milk, egg, strawberry, apple, peanut, chocolate,
coffee, almond, orange (Taiwan), malt (Hong Kong), pandan (Singapore and Malaysia),
and fruit, vegetable, beef, yakult, lactic acid, honey and sesame (Japan). The additon of

soy oil, lecithin or an emulsifier improves the mouthfeel of soymilk.

WWW There are three
major problems to be overcome to produce good quality soymilk : 1) elimination of the
beany/bitter flavor, 2) inactivation of trypsin inhibitors and 3) removal of flatulence-
causing oligosaccharides.

Wilkens et al.(1967) reported that lipoxygenase (or lipoxydasc) present in the
soybean is responsible for the undesirable flavors and odors upon grinding soybeans and
is the cause of the beany flavor in soymilk. Soybean lipoxygenase catalyzes the oxidation
of cis,cis 1,4 pentadiene containing fatty acids to form 1,3, cis, trans hydroperoxides.
These hydroperoxides decompose to form greenish beany, painty flavors. Several
methods have been developed to eliminate off-flavors in soymilk Lipoxygenase is often
inactivated by hot water blanching of soybeans in boiling water prior to cracking or hot
water grinding with boiling water to produce a slurry having a temperature of 80°C or
above and holding at this temperature for 10 minutes. Removal of the soy oil to prevent it
from reacting with the lipoxygenase has also been suggested to yield a bland soymilk.
Steinkraus (1973) patented a method for producing defatted soy flour which involves

71

extracting full-fat soy flour with 95% ethanol, followed by a mixture of equal volume of
95% ethanol and n-hexane. This procedure extracts remaining phospholipids which tend
to form off flavors. The beany flavor in soymilk was reported to be reduced by
fermentation with Lactobacillus acidophilus (Wang et al., 1974) orAspergillus oryzae and
Rhizopus oligosporus (Ebine, 1976). Nelson et al.(1976) reported that soaking and/or
blanching soybeans in 0.5% alkaline solution (NaHCO3) improves soymilk flavor,
removes oligosaccharides and decreases cooking time. BadenhOp (1969) reported that
alkaline soaking reduces a beany flavor compound, 1-ccten-3-ol. Acidic grinding (below
pH 3 ) will inactivate lipoxygenase, but is not utilized commercially. Flavor of soymilk has
also been improved by slurryin g soy flour in hot water or in-line dispersement of soyflour
which minimizes water contacting time followed by rapid hydrothermal cooking (154°C
for 30—40 sec) (Johnson et al., 1981), or by ultra-high temperature (UHT) sterilization of
soymilk by steam injection at 140°C for 4 sec.

Two of the five or more trypsin inhibitors have been purified and studied : the
Kunitz (1.4%) and the Bowman Birk (0.4%) inhibitors. Van Buren et al. (1964) indicated
that 90% of trypsin inhibitors in soymilk could be inactivated at 100°C for 14 to 30
minutes or at 110°C for 8 to 22 minutes.

Soybeans contain the oligosaccharides raffinose (1.1%) and Stachyose (3.8%).
Oligosaccharides can be removed by alkaline soaking of soybeans (Nelson et al., 1976),
heat treatment and removal of soy fiber residue. Sugimotc and Van Buren (1970) also
used an enzyme system of alpha galactosidase and invertase to remove oligosaccharides.
These enzymes are a commercial product extracted from Aspergillus saitoi free from
protease activity. The soymilk-enzyme mixture was incubated for 3 hours at 55°C, then
boiled for 10 minutes to stop the enzyme reaction. The enzyme treated soymilk contained
no oligosaccharides because they had been hydrolyzed by enzymes to their constituent

monosaccharides.

MATERIALS AND METHODS
Sample Preparation

Commercially produced whole and split dry navy beans grown in the 1991 crop
year were obtained from the Cooperative Elevator Company , Pigeon, MI. The beans were
field-dried, harvested, sorted, packaged and transported to Michigan State University
where they were stored in a walk-in cooler maintained at 4°C (39°F) prior to use.

Extractive Pretreatments

Hot Water/Steam Extractions of Whole Navy Beans
Whole dry navy beans were soaked for 16 hours at 25°C (77°F) in stainless steel
tanks and kettles. The hydrated beans were subjected to either hot water (60°C/60 mins)
(140°F/60 mins) or steam extraction (100°C/15 mins) (212°F/15 mins) procedures as
outlined in Figure 13, respectively. "CONTROL" meal contained cooked beans and all
original soak and rinse waters, cook water or condensate. Leachate of "EXTRACT" beans
was discarded and replaced with fresh formulation water (Figure 14). Cooking involved
holding the beans at the designated temperatures and times in covered stainless steel tanks
and kettles (Groen Model N60 Sp, Illinois) equipped with either a vortex mixer or rotating
paddles to provide gentle agitation which would facilitate extraction and avoid thermal
gradients. Extraction prowdures were as follows:
a) Aqueous extraction. Hydrated beans were submerged in water at the ratio of a
3:1 water to bean. The mixture was continuously agitated and held at
60°C (140°F) for 30 minutes for split beans, and for 60 minutes for the
whole beans. The cooking water was drained and mixed with the soak and
rinse waters. The accumulated formulation water was added to the

"CONTROL" beanspriortomillingina 1:1 watertobeanratio

72

73

HOT WATER EXTRACTION OF DRY NAVY BEANS

Was

Soak 820 ‘—.l

 

DRY NAVY BEANS
(hi/hole or Split)
L

l-I20:8aluu(‘3:l)

 

 

 

 

 

 

COLD-WATER SOAK
(25 Cl 16 Hrs )

 

 

 

 

 

 

-——--.- Saki-{20*

——> RinseI-UO—DI

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

, INDIRECT STEAM HEAT
‘- cw! “20 <— (Ccoking/Extraction) —>Cooluing mo ——
Whole : 60 C / 60 min.
split: 60C/30mirr.
W
am, Wm COOKED BEANS “if; Wm ..—
l=l° mam mm“ 2 1°“) 1:1' moan...
COOKIE!) BEANS COOKED BEANS
(CONTROL) (EXTRACT)
CONTROL EXTRA
Homogeneous Slurry Homogeneouscglurry
S ion Suspension
Fitzpatrickmill 0.03" Fitzpatrickmill 0.03”
-l-{E -
Direct: Steam mica Direci1 Steamed
(Tangential Heating) (Tangential Heating)
95 C 95 C
DOUBLE DRUM- DRY DOUBLE DRUM- DRY
(Dammit/soybean oil Wuhan oil
added as process'm and added as processirt aid
SIZE REDUCTION SIZE REDUCTION
(FLAKES)
FINTSI-IER (1/8") FINISHER (1/8")
CONTROL EXTRACT
[ Drum-dried Meal Drum-dried Meal 1

 

 

 

 

Figure 13. Flowchart for hot water extracrion of dry navy beans

74

 

W

HOT WATER or STEAM
(60 mm C) (15 urinal 100 C)

 

 

 

 

 

BEAN DILUTION DIFFERENTIATION

 

 

 

 

 

[

 

 

 

Accumulated th
(mmmweofin mm, mm m
(131:1-I20:Beans) 8 (111; H20: Beans)

 

 

 

 

 

l ? |

CONTROL II._’IREATMENT1ERMINOLOGL_. EXTRACT

 

 

 

 

Figure 14. Designation of treatment terminology and extraction
treatment differentiation used during cooked bean

preparation.

E M)

Slum
both
with

Bioi

75

while an equivalent amount of fresh formulation water was added to the
"EXTRACT" beans.

b) Steam extraction. Hydrated beans were held in a stainless steel mesh basket and
placed into a covered steam kettle. Live steam was injected into the base of
the covered kettle and continuously refluxed through the beans during the
60 minute extraction pa'iod. Condensate accumulated at the base of the
kettle below the bean surface and was drained and added to the accumulated
formulation water. After heating, the beans and formulation water (1:1)
were milled to a homogeneous slurry with a Frtzpatrickmill (Model D
Comminuting Machine, Fitzpatrick 00., Chicago, IL) using a 0.04 inch

mesh screen.

Enzymatic I’m-digestion of Whole Navy Beans

Whole dry navy beans soaked for 16 hours at 25°C (77°F) were milled to a
slurry and subjected to enzymatic pretreatment. Enzymatic pie-digestion involved the use of
both commercial and indigenous enzyme preparations. Commercial enzyme preparations
with selective substrate specificity and activity were obtained from Novo Nordisk

Bioindustrials Inc. (Danbury, CT).

1. Commercial enzyme preparations were as follows:

a) Viscozyme120L (a multienzyme complex of the carbohydrases
cellulase, B-glucanase, hemicellulase, arabanase and
xylanase; activity of 120 FBG/ml);

b) Celluclast 1.5L (cellulase from fungus Tn'choderma reeset' ; activity of
1500 NCU/g)

c) Neutrase (metallo—bacterial protease produced by a selected strain of
neutral Bacillus subtilis; activity of 0.5 AU/g) and

90m

76

d) Alcalase Food Grade (an endoproteinase prepared from Bacillus
lichemfonnis; activity of 2.5 AU/g ).
II. Indigenous preparation was obtained as follows :
Seed germination was conducted in the seed research facilities of the
Michigan Department of Agriculture, East Lansing, MI, using the
the standard procedure for dry beans of the Association of Official
Seed Analysts (1991). Hundred dry seed were uniformly positioned
on 2 cm centers using a manual seed depositor on clean, saturated
fiber padding. The whole navy bean seeds were allowed to
germinate in chambers for three days under controlled temperature
and dual light cycle (20°C at night and 30°C during daylight), and
humidity. The sprouted beans were pureed (referred to as ”sprout
slurry") and held frozen ( 4°C] 24°F) until used to serve as an
intrinsic source of catabolic enzymes with substrate diversity.
Small batches (approximately 5 kg) of the "CONTROL”

(soak, rinse and heating water added back) and hot water-extracted
whole bean bean slurries were individually incubated and
predigested for 16 hours (overnight ) at 4°C (39°F) with each of
the selected commercial enzymes ( 0.1 % w/w ) and "sprout slurry"
(5% w/w) prior to drum-drying (Figure 15). A batch with no
added enzyme served as a comparative control for the experiment.

Aqueous Extraction and Enzymatic Pie-digestion of Split Navy Beans
Commercial scale-up of the drum-drying process utilizing split navy beans was
conducted following the aqueous extraction (Figure 13) and enzyme pie-digestion

77

      
      

DRY NAVY BEANS
(Whole or Split)

“Tn-nn-n‘ (3:1)

COLD-WATER SOAK
(25C [16 Hrs. )

   

 

. H20 INDIRECT STEAM HEAT
‘_ Coolant 6‘ (Cooking/Extraction)
Whole: 60 C / 60 min.
Split: 60 C / 30 min.

1:1:H2028eam

NE) , L . f L I prout

Fem) like amj | WI tear ”Earl its?“ flat"
I

INCUBATION
Wholerl6HrsJ4C
S lit : 2Hrs./20C ‘

DOUBLE DRUM-DRY
moi!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 15. Flowchart for enzyme predigestion oof whole and
split navy beans prior to drum-dryin g
‘Commereial- ~scalenmsusing splitbeansalsopreparedforthese
designated treatments

78

(Figure 15) protocols. The cooking time was reduced to 30 minutes holding at 60°C
(140°F) to minimize excessive disintegration of the split beans. Two separate batches of
the "CONTROL" split bean slurries were incubated (55°C / 2 hours) (131°F12 hours) and
pre—digested with either the Viscozyme (0.1% w/v) or "sprout slurry” (3% w/v) prior to
drum-drying.
Drum-Drying of Bean Meals

Various drum-drying systems were utilized during selected phases of this research.
Each system was steam heated and operated with batch sizes appropriate to maintain full

operating capacity. Nomenclature and description of these drum-drying systems are as

follows:

a) W. A 6” x 8" (38 cm x 52 cm) fabricated double-drum-drier with a
heating surface area of 1134 cm2 was utilized to produce the enzyme pre-digested whole
bean drum-dried meals.

”211262315. Aqueous and steam extracted whole bean drum-dried meals were
produced using a pilot-scale American drum-drier (Overton Machine Co., Dowagiac, MI)
with drum dimensions of 12" x 19 1/8" (77 x 123 cm) and (total surface area of 10.24
n2 ) (9509 cm2)with drying surface area nip to doctor blade equal in 5.12 n2 (4755 em2).
It was set up to simulate a single drum operation by running one drum as a cold applicator
(Bakker-Arkema et al., 1966).

c) W. A 5' 4" x 2' (413 cm x 155 cm) atmospheric double
drum—drier (Overton Machine Co., Dowagiac, MI) with a heating surface area of 62.83
{:2 (58357 cm2)was used to produce split bean drum—dried meals. Drying conditions of
4.5 rpm and 75 psig per drum were maintained.

After holding the slurries at the desired temperatures and times, total solids content
was checked using a Computrac Moisture Analyzer Model MA5A (Quintel Corp.) to

control slurry viscosity and ensure a uniform film formed on the dryer surface. The

79

temperature of the slurry was then brought up to 90-950C (203°F) by steam injection,
prior to pumping the slurry into a stainless steel flash tank (Perma-San Model 75 OVC,
Process Equipment Corp., MI) serving as a reservoir for continuously supplying slurry to
the drum-drier. The slurry was pumped through a tangential steam heater which
gelatinized the starch and reduced microbial load of the slurry. A 0.1% mixture (1:1 ) of
lecithin and soya oil was sprayed on the surface of the drum-drier to prevent the sheets
from sticking to the drum-drier surface. The drum-dried bean sheets obtained from the
drum heating smface were scraped by the drier knives (doctor blades) and collected using
a continuous belt conveyor, weighed and flaked through a finisher (Reeves Pulley Co.,
Indiana) with a 1/4" (1.61 cm) stainless steel mesh screen. Drum-dried bean flakes were
thoroughly mixed to obtain a homogeneous product and stored in sealed polyethylene
bags which were held in fiber drums at 4°C (39°F) . The drum-dried bean flakes were
further milled into flour using a Udy Cyclone Mill with a 20 mesh screen (Udy Co., Fort
Collins, CO), prior to analyses.

Chemical and Biological Analyses
All the analyses were conducted using the described methodologies and were
replicated three times, unless otherwise stated.

Proximate Composition

Moisture. Approximately 2.0 grams of the raw bean or drum-dried bean meal
sample was weighed into previously weighed aluminum dishes and dried to a constant
weight at 80°C (176°F) (ca 7 hrs.) in partial vacuum having pressure equivalent to 25 mm
Hg. Percentage moisttu'e was calculated from the weight loss on a fresh weight basis
(AACC Method 4440):

(Eq. 1) % Moisture = loss in moisture (g)! initial weight of sample (g) x 100

80

Protein. Protein content was determined by the classical Kjeldahl nitrogen
analysis using a modification of the AOAC (1984 ) method. Five millimeters of
concentrated sulfuric acid and one catalyst (1(2804 and Selenium) tablet ( (Tecator Co.,
Sweden) were added into each of the digestion tubes containing the pre-weighed samples
(ca 150 mg). The tubes were slowly heated until digestion was complete. The protein
content was calculated on a dry weight basis using a nitrogen conversion factor of 6.25.

(Eq. 2) % Protein = % Total "Kjeldahl" Nitrogen x 6.25

Fat. Approximately 3.0 grams of the raw bean or drum-dried bean meal sample
was refluxed for 4 hours with 60.0 ml petroleum ether using a Goldfish Extractor, or for
25 mins with 40.0 ml petroleum ether using the Soxtec System HT6 (Tecator, England).
Solvent was evaporated to yield a fat soluble residue. Percent crude fat was calculated on a
dry weight basis (AACC Method 30-25).

(Eq. 3) % Crude Fat = wt. of fat (g)/ dry weight of sample (g) x 100

Ash. Approximately 2.0 grams raw bean or drum-dried bean meal sample was
weighed into pro-weighed crucible amd incinerated at 525°C for 24 hours. The ash was
cooled in a dessicator and weighed at room temperature. Percent ash was reported on a dry
weight basis (AACC Method 08—01).

(Eq. 4) % Ash = weight of incinerated residue (g)/ dry weight of sample (g) x100
Soluble Sugars Quantitated by High Performance Liquid Chromatography
(HPLC)

The extraction of the soluble sugars for the drumcdried bean meals was determined
using a modified HPLC procedure developed by Agbo (1982) as outlined in Figure 16.

Extraction of Soluble Sugars. Approximately 2.0 g representative raw or

drum-dried bean meal sample was placed in a 50-ml polyethylene centrifuge tube. Twenty-
five ml of 80% ethanol was added and the mixture was then vortexed. The tube was

(1)

F

81

 

 

2.0 g Sample

 

 

 

*— Addwrnisonernrnoi

Shakaiamhathataoc
forlSmin

 

I Sample Mixture

 

 

 

L...

 

 

 

Residue a

 

 

 

‘_ AddSmIMemanol

 

l

 

 

Residue b

 

 

Maroinimmnnoi
tproceedaain (l)

 

l

I

 

 

 

Cenu'ifugeumrpmforiim'm
1

Supernatanta
+b +c

 

 

 

   
  

 

 

 

 

 

 

 

 

 

 

 

 

Residue c I
scard

 

Supernatant c I

 

 

 

 

 

 

 

 

 

I Pre-filter I

 

 

a lasin (1) ‘_ Add2ml10‘h
] leadacetab
Supernatantb I Mixture
I cram eSm'InZMrpm
I l
Supernatant2a Residue2
Di
AdlemllO‘h
oxalicacid
[ I
Supernatant 2b Residue I
Bringlovohna
manners-not
'mZSleolmithk

 

HPLC ANALYSIS

 

 

 

Figure 16. Flowchart for the extraction of soluble sugars from
drum-dned bean meals (adapted from Agbo, 1982)

82

placed in a shaking water bath (Model 1024 Tecator, Hoganas, Sweden) and held at
80°C (176°F) for 15 minutes. The sample was then cooled to room temperature and
centrifuged using a Sorvall RC-SB refrigerated centrifuge (Du Pont Inc., IL) at 2000 rpm
(755 x g) for 3 minutes. One ml of 10% lead acetate (w/v) solution was added to the
supernatant to precipitate ethanol soluble proamines, then the tube was again centrifuged
for another 3 minutes at 2000 rpm (755 x g). One ml of 10% oxalic acid (w/v) solution
was added to the filtrate to precipitate excess lead acetate. The sample was again
centrifuged at 2000 rpm (755 x g) for another 3 minutes. The final supernatant was
evaporated down to 3 ml and made-up to a final volume of 5-ml using 80% ethanol.
Extracts were stored at -4°C (25°F) prior to analysis.

Separation and Quantitation. The extracted sample was filtered through a
0.45am millipore filter (Waters Associates, Milford, MA). The filtered sample was
injected into a 125 A, 10 um, 3.9 x 300 mm ID. Carbohydrate Analysis column (Waters
Associates, Milford, MA). The elution solvent was acetonitrilezwater (65:35, v/v) at a
flowrate of 2.0 ml/min. A 50 ul sample was injected using a 100 ul pressure-lock
Hamilton microsyringe.

The HPLC consisted of a Solvent Delivery System 6000A, a Rheodyne Injector
equipped with a 50-ul sample loop and a Differential refractometer R401 (Waters
Associates, Inc.). The refractometer was set at 4x to resolve the soluble sugars as discrete
peaks.

A mixed standard solution was prepared containing a known amount of standard
glucose, sucrose, raffinose and Stachyose sugars (Sigma Chemical Co., St. Louis, MO).
Quantitation and identification of the sugars were done using the external standard method
programmed in the Data Module Model 730 (Waters Associates, Inc.) to calculate the
absolute amounts of each component in the injected sample. These data were used to
express the percent sugar content based on a dry bean flour weight.

83

Total Mineral Content

Mineral content of raw beans, drum-dried bean meals and water used during
processing was determined using an inductively coupled argon plasma (ICP) emission
spectrometer (Janell-Ash Model 955 Atomcornp) equipped for simultaneous analysis of 19
elements (Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, P, Pb, Se, T1 and
Zn) and maintained by the Department of Pharmacology and Toxicology at Michigan State
University. Triplicate samples were combined with 2 ml of concentrated Baker Instra-
analyzed nitric acid in 15 ml screw-capped teflon vials (Tuff-Tainer, Pierce Chem. Co.,
Rockford, 11). The samples were incubated overnight at 70-75°C (167°F), cooled and
quantitatively transferred to 10 ml Class A volumetric flasks containing 1.0 ml of 100 ppm
Yttrium (internal standard). They were diluted to final volume with water purified by a
Millipore water purification system Class 1 (Millipore Corp, Bedford, MA). Along with
the bean samples, blanks and standard materials were prepared to serve as controls.

Mineral content was determined and values reported in ppm on a dry weight basis.

Insoluble and Soluble Dietary Fiber

The enzymatic method of Prosky et al. (1988) for determining insoluble and
soluble dietary fiber is outlined in Figure 17. Four replicate 1.0 g drum-dried bean meal
samples were weighed into 400 ml polyethylene beakers to which was added 50 ml of pH
6.0 phosphate buffer. A 0.1 ml heat-stable bacterial a-amylase (Sigma Chemical Co., St.
Louis, MO) was added, the beaker covered with aluminum foil and the mixture was
incubated in a shaking boiling water bath for 15 minutes. The mixture was then cooled to
room temperature and pH adjusted to pH 7.5102 using a 0.275N NaOH solution. Five
milligrams of protease was next added and the mixture was incubated for 30 minutes in a
1024 Tecator (Hoganas, Sweden) shaking water bath maintained at 60°C (140°F). The
mixture was cooled and pH was adjusted to pH 4.0-4.6 with 0.325M HCI. A 0.3 ml
amyloglucosidase solution (Sigma Chemical Co., St. Louis, MO) was added and the

84

Drum-dried Bean Meal Sample (1.0 g)
m butter (50 ml: pH at»
a . mylaan (0.1 ml)

Incubate
(100 015 mins.)

Adjust pH 7.5:;02

I..— MWEdmg)

Incubate
(60 C00 mins.)

 

Adjust pH 4 .0-4. 6

ME— Amylogbeoaidam, $32.13 (03 ml)

(60Cf3'0mins.)

 

lOmldaiaIind
WIDOCZX)

 

Residue

WIN-e- (2:):

lOml95§etbel .
lOmlaauma w

 

 

 

 

Oven-Dry 0! -Dry
105 C 105 C

i
nrzr’l‘riill-‘éhi arm nréfli'k‘ll’l'fim (snr)

Figure 17. Flowchart of the enzymatic method to determine the insoluble and
soluble dietary fiber of drum-dried bean meals (Prosky et al.,l988)

85

mixture incubated for another 30 minutes at 60°C (140°F). Insoluble and soluble fiber
fractions were determined sequentially on this digest.

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

(Eq. 5) IDF(%)= mg insoluble residue- .

 

sample weight. (mg)

Soluble fiber. The weight of combined filtrate and water washings was
adjusted to 100 g with water. Four 100 ml portions of 95% ethanol preheated to 60°C
(140°F) was added and allowed to precipitate at room temperature for at least an hour. The
enzyme digest was filtered through previously ashed and weighed fritted crucibles
containing Celite. The residue was sequentially washed with three 20 ml portions of 78%
ethanol, two 10 ml portions of 95% ethanol and finally two 10 ml portions of acetone. The
residues were dried and analyzed for protein and ash following the same protocol for the
insoluble fiber. Percent Soluble Dietary Fiber (SDF) was calculated as follows:

(Eq. 6) SDF(%)= -mg soluble residue - .

 

‘ sample weight. (mg)

86

In-ritro Starch Digestibility

Total and available starch content of the drum-dried bean meal samples were
determined using a modification of the enzymatic methods of Holm et al.(1986 and 1985).
The released glucose was measured by the colorimetric Glucose oxidase/peroxidase Assay
using a glucose diagnostic kit (#510-A, Sigma Chemical Co., St. Louis, MO). The
combined enzyme-color reagent contains the two enzymes, glucose oxidase and
horseradish peroxidase, and the color reagent, o—dianisidine. The procedure is based upon
the following coupled enzymatic reactions:

 

 

Glucose Oxidase
Glucose + 2H20 + 02 > Gluconic Acid 4» 211202
Peroxidase
H202 + o-Dianisidine > Oxidized o-Dianisidine
(Colorless) (Brown)

The intensity of the brown color measured at 450 nm is proportional to the original
glucose concentration. The total amount of glucose in samples was calculated from a
standard curve prepared from known concentrations of glucose. The total starch content in
each sample was obtained by multiplying the mg of glucose in each sample by a factor of

0.9 to account for the weight of the water gained during the hydrolysis of starch to glucose.

Total Starch. The modified enzymatic/colorimetric methods of Holm et al.
(1986) and Tovar et al. (1992) were followed to determine total starch content of the drum-
dried bean meals (Figure 18). A 500 mg drum-dried bean sample was weighed into a
beaker which was tared containing a magnetic stir bar. The sample was dispersed in 10.0
ml distilled water and 10.0 ml 4N NaOH, and allowed to stand at room temperature for
1.0 hour. Twenty milliliters of 0.08 M phosphate buffer was added and pH was adjusted to
6.0 with 5N HCl. The mixture was incubated with 50 ul of thermostable a-amylase Type

87

 

 

 

 

 

 

500 m sample
Distilled H20 (10 ml)
4N NaOH (10 ml)
Stand in Room Temperature
(1 hour) 0.03 M Phosphate Buffer(20 ml)
5N HCl
Adj u5t to
pH = 6.0
a-amylase (T ermamyl)
(50 ul)
Incubate (90-100 C;
1.0 hour)
Cool; Mak up to 50 g
50 ul amyloglucosidase/buffer solution
(450 ul)
Incubate
(55 - 60 C; overnight)
1
40 ul Enzyme-color Reagent (5.0 ml)

Incubate
(45 mins; oom Temp)

Absorbance (450 nm)

Figure 18. Flow diagram for determination of tOtal starch.

[611

the

37‘

101

St.
2.C

inc

“'21

31¢

ca;

88

XII-A (Bacillus lichenifomu's, Sigma Chemical Co., St. Louis, M0) at 90-100°C(203-
212°F) for 1.0 hour. The mixture was cooled to room temperattue and distilled water
added so that the total aqueous weight is 50.0 g. A 50.0 ul sample aliquot was pipetted
into a screw—capped tube and incubated with 450 ul of amyloglucosidase enzyme solution
(Aspergillus niger (Boehringer Mannheim) 10 mg of lyophilizate/ml of 3.2 M (NH4)2804;
)lbuffer solution (dilute enzyme stock solution 1:10 with 0.1M sodium acetate, pH 4.5)
and incubated overnight at 55-60°C(140°F). The mixture was cooled to room temperature
and 40 ul sample aliquot used for the glucose assay.

Available Starch. Figure 19 outlines the protocol for determining in-vitro
digestibility of starch. A 500 mg sample was weighed into a 100 ml beaker tared with a
magnetic stir bar and mixed with 10.0 ml of deionized—distilled water (For raw bean
samples and reference cornstarch and all-purpose flour, samples were gelatinized with 10.0
ml distilled water in a 90-100°C(212°F) water bath for 20 mins. and then cooled to room
temperature before proceeding to the next steps). BC] (200 ul , 5N)was added to bring
the pH to approximately 1.5. Pepsin (100 mg) was added and the mixture incubated at
37°C (99°F) for 1.0 hour. Twenty ml of 0.1M phosphate buffer (pH 6.0 - 7.0, and
containing 0.1% Thimersal)® and 215 ul of 4N NaOH were added and pH was adjusted
to 6.0. Deionized distilled water was added so that the total aqueous weight was 50 g. The
mixture was incubated with 15.3 ul of porcine pancreatic a-amylase (Sigma Chemical Co.,
St. Louis) at 37°C(99°F). The rate of starch hydroysis was monitored by removing a
2.0-ml sample of the digest at appropriate time intervals (15 and 60 min., and 24 hours of
incubation). This aliquot was immediately added to 8.0 ml of 95% ethanol. Precipitation
was allowed to occur for 1.0 hour at room temperature and then centrifuged at 2000 g
(3000 rpm) for 10 mins. (For zero time sampling, a 2.0 ml sample was taken prior to
adding the porcine a-amylase solution). A 50.0ul supernatant was pipetted into a screw-
capped tube and incubated with 450 ul of amyloglucosidase (Aspergillus niger, 14 enzyme

89

500 m mple

 

Heat
(90-100 C; 20 mins)
5N HQ (200 uL)
squirt. EC 3.4. 23.1 (100 mg)
Incubate
( 37 C: 60 mins)
0.1M Phosphate buffa'whnl
*— 4N NaOH 215 111) )
Distilled water (20 ml)
Porcine amylase solution, EC 3.2.1.1 (15.3 141)
Adjust to pH 6
Incubate
(37 C)

Remove 2.0 m1 sample
(0.15. 60 & 380 min)

Centrifuge
(2000xg; 10 mins)

Amyloglucosidase. EC 3.2.1.3
solution ( 450 1:1)

Pipette ul supematant
Incubate
(24 hrs.: f5 - 60 C)

GLUCOSE ASSAY
(Glucose oxidase/Peroxidase reagent)

Figure 19. Flowchart for the determination of available starch ( Holm. 1984)

9 0
units/mg protein, 10 mg protein/ml; Boehringer Mannheim) enzyme solution overnight at
55-60°C (131°F). The mixture was cooled to room temperature and 100 ul was used for
the glucose assay.

The release of free glucose was measured with a glucose oxidase/peroxidase
reagent and the starch content was calculated as glucose x 0.9. Reagent blanks with the
starch degrading enzymes were run in each case. Corn, potato and wheat starch
suspensions and all-purpose (wheat) flour were assayed as reference samples.

(Eq. 7) % Total or Available Starch = WW2. x 100

sample weight (mg)

In-vitro Protein Digestibility

The sequential multi-enzyme system (AOAC, 1990) for determining in-vitro
protein digestibility is outlined in Figure 20. Drum-dried bean meal samples and control
casein (ANRC Na caseinate) were weighed to contain approximately 10 mg nitrogen.
Nitrogen content of the drum-dried bean meal samples was determined by Kjeldahl method
(Eq. 2). Ten ml of deionized distilled water was added to the casein control (Sigma
Chemical Co., St. Louis, MO ) or to the sample in a test tube containing a stirring bar.
Protein suspensions were held in a water bath maintained at 37°C (99°F)for at least an
hour. One ml of "Enzyme A" solution (porcine pancreatic trypsin, Type IX; bovine
pancreatic achymotrypsin, Type II and porcine intestinal peptidase, Grade 1; Sigma
Chemical Co., St. Louis, MO) which had previously been equilibrated to pH 8.01-0.03,
was added to the protein suspension after it had also been equilibrated to pH 8.01003.
After incubation with Enzyme A for 10 minutes at 37°C (99°F), 1.0 ml of "Enzyme B"
solution (bacterial protease, Pronase E; Sigma Chemical Co., St. Louis, MO) was added
to the protein suspension which was further incubated for 9 minutes in a water bath
maintained at 55°C(l31°F). At precisely 20 minutes from the addition of "Enzyme A"
solution, pH was read at 37°C(99°F). The extent to which pH of the protein suspension

91

Sampl (10 mg N)

deionized distilled H2000 ml)
Soaék
(1 hr: 7 C)
Equilibrate to pH 8.03003
Enzyme A (Tm. EC 3.21.4 &
a-Chymotrypsin, EC 3.21.1)
In b
(10 mic“; 3‘7 C)
Enzyme B (Protease, EC 3 l4 16)
In b
(9.0 “sags C)
Read pH (X)
(at precisely 20 min. after addition
of Enzyme A; 37 C)
Calculate % Ligestibility =
23484-2256 (X)

Figure 20. Flow chart for in-vitro protein digestibility (AOAC, 1990)

9 2
dropped was used as a measure of protein digestibility. Percent digestibility was calculated
as follows:
(Eq. 8) % Protein Digestibility = 234.84 - 22.56 (X)
whereX=pHat20min.

Methionine Content

The improved gas chromatography procedure of MacKenzie (1977) for the
analysis of methionine in plant materials was followed. A 200 ul of the cyanogen bromide
reagent (5% in 50% formic acid) was added to 20 mg of the drum-dried bean meal in a
microcentrifuge tube. After 2 hours at room temperature, the tube was centrifuged at 2000
g (1300 rpm) for 15 min. and 2 ul aliquots of the supernatant were injected directly on the
chromatographic column using a Hamilton syringe (No.7001).

All analyses were performed using a SRI 8610 gas chromatograph (SRI
Instruments, California) equipped with both flame ionization and flame photometric
detectors. The column ( 4 ft x 1/8" SS ) was packed with Porapak QS, 80-100 mesh
(Applied Science Labs., State College, Pa, USA.) and conditioned at 160°F (110°C) for
2 hr. with a hydrogen carrier gas. The GC conditions used were as follows: column
temperature, thermal gradient 145 to 160°C (293 to 320°F) at 5°C] min., and
l60°(320°F) for 3 min. Quantitation and data reduction were performed by a laboratory
data system using an internal standard program.

(139- 9) nmolcsMcthioninc= MW x 100
(g) protein

Amino Acid Analysis

Oxidation, acid hydrolysis and amino acid anlysis of the oxidized samples were
performed at the Macromolecular Structure Facility maintained in the Biochemistry
Department at Michigan State University. Constant boiling HCl (200141 (Pierce Chemical
Co., Rockford, IL) was added to the bottom of the vacuum vial. Three alternate vacuum

93

nitrogen flushing steps were required to ensure the oxygen-flee atmosphere. Vapor phase
hydrolysis was conducted at 112-116°C for 24 hours. Following hydrolysis, the samples
were re-dried with ethanolzwaterztriethylamine (2:2:1, v/v), and derivatized. The
derivatization reagent consisted of a 7:1:l:1 solution of ethanol, triethylamine, water and
phenyllisothiocyanate (PITC). After 10 min. derivatization, the samples were vacuum-dried
and redissolved in 500 111 of 5 mM sodium phosphate, pH 7.8. A Waters 600 HPLC
system (Waters Associates, Milford, MA) was used for the analysis of the derivatized
amino acids. The two component (A and B) gradient mobile phase system consisted of the
following : A, 15 mM sodium acetate buffer, pH 5.9, and 0.05% triethylamine and B,
acetonitrile:H20 (60:40). An injection volume of 40 it! and ultraviolet absorbance at 254
nm were used for detection of the Pl'I‘C-labelled amino acids.

SDS-PAGE Electrophoresis

Crude extracts of the raw and drum-dried bean meals were subjected to
discontinuous SDS-PAGE using an electrode buffer at pH 8.3 following the procedure of
Hames (1990). A 7.5% to 24% acrylamide gradient with a 3.75% acrylamide stacking gel
was employed (180 x 160 x 1.5 mm slab, Protean 16 cm electrophoresis unit, Bio—Rad
Laboratories, Richmond, CA). The sample and standards were loaded onto the gel and the
gel was run at 20 mA constant current. The SDS-PAGE gels were stained with silver
nitrate to resolve bands, and destained with a 5% MeOH, 7.5% HOAc solution. Standard
proteins used for molecular weight estimation were from an electrophoresis calibration kit
for molecular weight determination of low molecular weight proteins (Electrophoresis
Calibration Kit, Pharmacia-LKB, Piscataway, NJ). The kit consisted of phosphorylase b
(94 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD),
soybean trypsin inhibitor (20.1 kD), and a - lactalbumin (14.4 kD).

94

Assessment of Antinutritional Factors
Screening for Heat-Stable Trypsin Inhibitor Activity (TIA)

A modification of the spectrophotometric assay of Kakade et al. (1974) by
Rayas-Duarte et al. (1992) to determine trypsin inhibitor activity using the substrate
BAPNA (N -benzoyl-DL-arginine-p—nitroanilide),is summarized in Figure 21.
Approximately 5.0 gram drum-dried bean meal samples were homogenized with
100 ml deionized-distilled water using a high-shear Polytron (Brinkman
Instruments Co, Westbury, NY), Model [’1‘ 10/35, at medium speed (setting=5).
'Ihe homogenized bean solutions were centrifuged at 15000 g (10000 rpm)
for 30 minutes at 20°C(68°F) . The supernatants were used to assay for . TIA.
The total sample volume was adjusted to yield a final volume of 400 ul. Control
tubes contained 400 ul of deionized water. The reagent blank tubes contained 400
ulofdistilledwater+200ulof30%aceticacid,whilethe sampleblanktubes
contained400ulofsample+21Dul of30%aceticacid.Samplealiquotswere
selected to yield a 40-60% inhibition of trypsin activity compared to a control
containing no sample.

A 400 ul sample aliquot of trypsin (EC 3.4.21.4 bovine pancreas Type III-
8, Sigma Chemical Co, St. Louis, MO) was added to all tubes at 10 second
intervals toensure thatallofthetubeswereincubatedforthesametimeperiod.
The tubes were then incubated at 37°C (99°F) for a total of 10 minutes before
addition of 1.0 ml BAPNA solution (Sigma Chemical Co., St. Louis, MO).
The tubes were incubated for another 10 minutes before addition of 200 ul of 30%
acetic acid to all tubes except the reagent and sample blank tubes. The tubes were
vortexed before reading absorbance using a Spectronic-70 spectrophotometer at
410 nm.

One unit oftrypsin activity (TA) was defined as an increase of0.001
absorbance unit at 410 nm of the reaction mixture under assay conditions. One unit

95

 

I 5.0 g Sample I

 

 

I . Hogenize I

(3.0 mm; Polytrtn, Medium Speed (05))
Cenuifuge

(15000 xG/1000 rpm: 30 min: 25 C)

Residue Supernate
f (Din-d) (Stock)

 

 

 

 

 

 

 

 

 

Aliquot
(20 it!)

380 ul deionized-distilled n20
Trypsin. EC 1421.4 (0.4 ml)

I VortexI

 

 

 

 

Incubate
(37 C shakrn' g waterbath: 10min.)

 

_BAPNA‘(1.0 ml):
@ 15 sec. intervals

Incubate
(37 C shaking water built; 10 min.)

0.2ml30%Aceticacid

 

 

 

 

Vortex I

l

AbsorbanceJ

 

(410nm)

 

Figure 21. Flowchart for the trypsin inhibitor assay (TIA)
(Kakade etal., 1974)

96
oftrypsininhibitoryactivityCTIA)wasdefinedat asa10%decreaseinthenumber

of units of TA under assay conditions. Thus, a 50% decrease in units of TA (i.e.,

50% inhibition of TA) would equal 5 units of TIA (Rayas-Duarte, etal., 1992).

Screening for Phytohemagglutinin

Phytohemagglutinin (PHA) was determined using Sigma Kit No. P-0526
(Sigma Chemical Co., St. Louis, MO) containing purified Phasolus vulgaris
erythroagglutinin (kidney bean lectin) as immunogen. A 0.01 g/ml slurry of raw
bean or drum-dried bean meal in deionized distilled water was prepared. Different
aliquots (10 to 40 ul) ofthe raw and drum-dried bean meal slurries were
pipetted into round bottom microtiter wells and reacted with 10 ul of
antibody. The reaction of the antibody with purified Phaseolus vulgaris lectin
(Sigma Chemical Co., St. Louis, MO) was used as a reference. For comparative
control so as to distinguish sample settling fiorn agglutination, the same quantifies
of raw and drum-dried bean meal slurries were pipetted into adjacent rows of
wells, but no antibody was added to it. The formation of a lectin-antibody

precipitate was monitored every 30 minutes.

Phytic Acid

Purified inositol phosphate fractions were prepared by hydrolysis of sodium
phytate using a modification of the procedure of Graf and Dintzis (1982) as
summarized in Figure 22. A 0.5 g drum-dried bean sample was extracted with
20 ml of 0.5M HCl for 2 hr. at 20°C under vigorous mechanical agitation. The
extract was centrifuged and the supernatant decanted, frozen overnight and filtered
using No. 1 Whatman filter paper. The inositol phosphates were separated from the
filtrate by ion-exchange using plastic columns with glass wool filter and containing

0.65 ml resin (AG 1-X8, ZOO-400 mesh). The filtrate was passed through the

97

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.5 g Sample
a 20 ml 0.5M HCl
Extract
(Mechanical agitation; 2 hr.: 20 C)
Centrifuge
(15 min.)
I
I
Supernatant Residue
(Discard) ,
Freeze
(Overnight)
Filter
(Whatrnan No. l)

 

 

I

Make-up to Volume
(1:1; 1120: Sample)

1

 

 

 

 

 

 

 

 

 

 

 

 

I Collect eluent I

 

 

 

[Evaporate to dryness I

I‘

ma 1

 

 

 

 

 

Pass through column
(AG 1-X8, 200400 mesh; 0.65 ml resin)
10ml 0.02m HCI
‘ , 5.rnl 0.025M HCI
fig
‘ .‘ 1.0ml 2M HCl (10x)

1.0mlHPLCgradewater

Figure 22. Flowchart for the separation and quantitative determination of phytic

acid as inositol phosphates

98

column (ca 0.4 ml/min) followed by 10 and 5 ml rinsings of 0.025M HCI. The
inositol phosphates were eluted from the resin with ten l-ml portions of 2M HCl,
and the eluent evaporated to dryness and diluted with 1.0 ml of HPLC Baker
Analyzed reagent water. Inositol phosphates were then identified and

quantified by HPLC using the method of Sandberg et al. (1989).

Physical and Functional Properties of Drum-Dried Bean Meals
Color

Drum-dried bean meal surface color was evaluated using a Hunter Lab Model D25-
PC2 system (Hunter Associates Laboratory, Inc., Reston, Virginia) which includes an

optical sensor and a sensor interface unit (SIU) interfaced to a Zenith computer. The
calorimeter measures reflectance on three coordinates labeled L, aL, and bL. The L value

measures darkness (0) to lightness (100); aL ( green to red) and bL (blue to yellow)

values were expressed as hue angle by calculating the corresponding x, y and 2 values. A
white tile (L: 95.35, aL = -O.6, bL=+0.4) was used to standardize the system.

Cellular Structure and Starch Granule Appearance

The microstructure of the drum-dried bean meals and starch granule appearance
were observed under Laser Scanning Confocal Microscope (LSM) (Carle Electron Optics
Limited, Tokyo, Japan). A polarizing light microscope (Carl Zeiss, West Germany) was
also utilized to determine the effect of the extraction pretreatrnents and the drum-drying
process on the gelatinization and birefringence of bean starch. Visual examination and

selected representative photomicrographs were prepared as the basis to draw conclusions

Solubility

Drum-dried bean meal solubility characteristics were determined by dissolving
approximately 0.5 g sample in 50.0 ml distilled water. The mixture was slowly stirred on
a magnetic stir plate at room temperature for one hour, after which it was filtered through a

99

Whatrnan No. 1 filter paper. The filtrate was weighed and dried in a vacuum-oven. The
following solubility indices were determined:

Water Absorption Index (WAI) was determined by weighing the wet
residue.

(Eq- 1 1) WAI = missus)
sample dry weight (g)

Water Solubility Index (WSI) was determined by weighing the dry
soluble solids.

(EQ- 10) WSI= 331W
sample dry weight/50 ml water

Soluble Solids (SS, °B) of the filtrate from the above preparation was
determined using an Abbe refractometer (Bausch and Lomb)

and recorded as degrees Brix (°B).
Water Holding Capacity (WHC)

WHC, frequently referred to as hydration capacity (HC), was determined using
AACC (1984) Method 56-20. Triplicate 2.0 g samples were weighed into centrifuge
tubes. Forty ml distilled water was added and the tubes vortexed to suspend the sample.
The suspension was allowed to stand for 10 min., inverting the tubes three times at the end
of the 5-min and 10-min periods. The tubes were then centrifuged for 15 minutes at 1000
g (and the supernatant decanted. The tubes were then inverted and allowed to drain for 5
minutes. The wet samples were weighed and hydration capacity calculated using the
formula:

(Eq. 11) WHC = 4» ° -
sample wt (dry basis)

100

Bulk density

Bulk density was determined as the weight of drum-dried bean meal sample
tightly packed into a 11X) ml graduated cylinder and recorded as g/lm ml.

Nitrogen Solubility Index (NS!)

NSI of the drum-dried bean meals was determined using AACC Method 46-23. A
5.0 g sample was weighed into a 400 ml beaker and mixed with 200 ml distilled water.
The mixture was stirred with a mechanical stirrer at low speed for 120 min at
30°C(86°F). The mixture was centrifuged at 15000 rpm (for 10 min and the supemate
was filtered through a funnel plugged with glass wool to obtain a clear liquid. Percent
water-soluble nitrogen was determined from the liquid using the Kjeldahl method. Nitrogen
solubility index, expressing water soluble nitrogen as a percentage of the total nitrogen was
calculated as follows:

(Eq. 12) Nitrogen Solubility Index (NSI) = W x100
% total nitrogen

Incorporation of Drum-Dried Bean Flour in Food Products

Quick Bread

The procedure for baking the pumpkin bread is outlined in Figure 23. The pumpkin
bread formula (Rombauer and Becker, 197 8) is presented in Table 3. The composite flours
consisted of all-purpose and drum-dried navy bean flour blended in 80:20, 60:40 and
40:60 ratios, respectively. Batter samples of 325 grams were poured into 3" x 5" (7.62 cm
x 12.7 cm) greased baking pans and baked at 177i2°C (350°F) for 45 min. in a National
Reel Type Test Baking Oven. Bread loaves were removed from the pan 30 min after
baking, for 15 min of additional cooling and weighed to determine the percentage weight
loss during baking.

101

 

WEIGH INGREDIENTS

1

PREPARATION J

 

 

 

(Ingedient Blending & Mixin

 

 

 

BAKE
(177L045 mins)

 

 

 

(30 mins.)

I

Lwratctt j

I

I EVALUATE I

I coon I

 

 

 

 

Color IMoistureW Density 7 Texture
(CC) I a. ab) I V (96) ) cm)

Figure 23. Flowchart of utilization of drum-dried bean flour in pumpkin bread

102

Table 3. Pumpkin Bread Formula1

 

 

Ingredient Weight (g)
All purpose or composite flour2 100.65
Baking powder, SAS 0.45
Baking soda 2.00
Salt 3.00
Cinnamon, ground 0.50
Cloves, ground 0.25
Brown sugar, dark 133.35
Vegetable shortening 31.35
Egg, fresh whole 50.00
Pumpkin, Canned 123.00
Milk, whole fluid 60.50
Vanilla 1.23

 

I Rombauer and Becker, 1978 as cited by Alani et al., 1989

2 Composite flours contained all-purpose/drum-dried bean flow in 80:20. 60:40, 40:60 rados

Weaning Food

The drum—dried bean meals were utilized to formulate a bean-based weaning
food/beverage similar to soymilk. Different concentrations ( 5%, 8% and 10%) of the
”EXTRACT"(aqueous), and "ENZYME-DIGEST" (Viscozyme) drum-dried bean meals
were prepared by homogenizing ('I‘ekmar Tissumizer) weighed quantities of the sample
with 200 ml distilled water for 2 min. Objective evaluations (Figure 24) were conducted
to assess the quality of the bean weaning food/beverage.

Selected emulsifiers (carageenan products and xanthan gums) at differential levels
were combined with the 5% EXTRACT and ENZYME-DIGEST drum-dried bean meal
samples to enhance stability of the beverage.

103

Objective Quality Evaluation of the Bean Products

Quick Bread

Viscosity. Batter viscosity was determined using a Brookfield viscometer (model
DV-II) equipped with a no. 7 spindle and rotating at 20 to 100 rpm, depending on the
sample consistency. Results were recorded as centipoise (cps) at 23i3°C (73°F).

pH. A portable pH meter (VWR Scientific Inc.,San Francisco, CA) was used to
measure the pH of the pumpkin batter.

Volume. Bread volume was measured by rapeseed displacement and results
were recorded as cubic centimeters (cm3).

Texture

Tenderness. The standard shear compression cell of the Kramer Shear
Press (Model TMS-90, Food Technology Corp., Maryland) equipped with a 300-lb
transducer was used and texture reported as Newtons per gram. Bread slices 0.5 cm thick
were used for evaluation.

Texture Profile Analysis (TPA). A Texture Press TMS-90 was used,
equipped with a parallel plate compression cell TPA-l (Food Technology Corp.,
Maryland). The TPA test is a two-bite compression mode programmed to a specified
percentage of deformation. A 1.0 cm thick pumpkin bread sample sliced from the middle
portion of the loaf was trimmed using a 0.5 cm x 0.5 cm square-shaped dough cutter. The
bread slice was placed at the center of the lower compression plate. The upper
compression plate of the transducer was initially lowered down to about 1 cm above the
sample. By pressing the "RUN” command, the bread slice was compressed twice with a
designated 50% compression distance. After each two-stage compression sequence, the
TPA force-distance curve and complete TPA analysis data was plotted directly using the
TMS-90 Control Panel.

104

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Lam beam

=8: «...—:33 23.5 eta—E...

 

 

 

ZO—ths—Srm >.—.—r_<DO ”Er—8356

 

105

Density. The trimmed pumpkin bread slice was weighed and its volume
calculated by measuring the lenght x width x height to determine density as follows :

(Eq. 13) Density = Wane:
Volume of Bread slice

Moisture. AACC Method 4440 (1983) was used to determine the moisture of
the pumpkin bread. Approximately 20.0 g pumpkin bread sample was dried in a vacuum
oven at 90°C (194°F) overnight.

Microstructure. Cellular structure of the pumpkin bread was observed under
Laser Scanning Confocal Microscope (LSM) (Carle Zeiss, Inc., West Germany) and
observations and conclusions drawn after visual viewing of multiple samples and fields.
Representative fields were prepared as photomicrographs.

Weaning Food/ Beverage

Suspension Stability

The different concentrations (5%, 8% and 10%) of drum-dried bean

slurries were prepared by adding deionized distilled water to the drum-dried bean meals
(Figrn'e 25) and thoroughly mixing using a Tekrnar tissue homogenizer prior to conducting
the following indices of stability:

Visual S tability

The visual stability of the weaning food/beverage was evaluated by placing the
homogeneous bean beverage in a 50-ml conical graduated centrifuge tube and the settled
solids measured at 15 minute interval periods. The amount of the separated supernatant
wasplottedagainsttimeandwasrecorded.

Micro-quantitative method

a. Absorbance/% Transmission of the weaning food/ beverage was measured
using a Spectronic 70 spectrophotometer (Bausch and Lomb, 1].). Differential quantities
(10, 20, 40 and 60 ul) of the homogeneous suspended weaning food/beverage slurry

106

PREPARATION OF THE WEANING BEAN BEVERAGE

DRUM—DRIED BEAN MEAL
( EXTRACT, ENZYME PRE-DIGEST)

I I

5% 8.0% 10.0%

I I

HOMOGENIZE
( 2 min)

 

 

 

 

OBJECTIVE EVALUATION

 

 

 

Figure 25. Experimental flowchart for the preparation of the weaning bean beverage

107

were mixed with a constant quantity (3.0 ml) of distilled water and absorbance read, prior
to settling, at 565 nm. This procedure provided a design to quantitate the influence of
concentration on stability.

b. For each of the suspended beverage, a 100 rd aliquot of the separated supemate
was taken at 15 minute intervals during static holding at room temperature. The
transmission (%) data was used to calculate an index of stability/settling of solids with
time.

Consistency

Apparent Viscosity

Apparent viscosity of the bean beverage was evaluated using a Bookefield
viscometer (Model DV-II) with a no. 2 spindle and rotating at 100 rpm for 5 min. Data
was recorded as centipose (cps).

Relative viscosity

Relative viscosity of the bean beverage was determined by recording the time
required, at room temperature, for the bean beverage sample to flow through the fixed
distance between the two marks of an Oswald pipette. Results were reported as time of

flow in seconds.

Physico-Chemical Tests

Color of the different concentrations of weaning food/ beverage was
evaluated using the Hunter Lab Model D25-PC2 system standardized against the white tile.

pH of the beverage was measured using a Fischer Accumet (Fisher
Scienctific, Pittsburgh, PA) pH meter standardized against a standard buffer solution
(pH=7.0).

Soluble Solids of the beverage was measured using an Abbe refractometer
(Bausch and Lomb, Il).

108

Total Solids (%) of the different concentrations of bean beverage were
detemined by weighing 50.0 g samples in tared aluminum pans and allowed to dry to a
constant weight in an oven at 87°C(l89°F), cooled and reweighed to determine weight

loss.

Microwave Preconditioning of Whole Dry Navy Beans

The procedure described by Shirazi et al. (1992) was followed in this study
(Figure 26). Whole dry navy beans were ambient soaked at different time periods and
allowed to equilibrate at 4°C for 2 days. The beans were then rehydrated to different
moisture contents (ranging from 47% to 54%) and subjected to microwave heating at
three microwave energy levels (440, 590 and 740 watts) for 2 minutes in a Radarange
microwave oven (Model-RS458P, Amana Refrigeration, Inc.). A microwavable pressure
cooker, Nordicware's Tender Cooker (Northland Aluminum Products, Inc., Minneapolis,
MN), was used to contain the beans and reduce dehydration during heating. The heated
beans were stored at -18°C(0°F). The frozen preconditioned beans were subsequently
microwave heated for 30 seconds.

Microwave heating time was established after a come-up time ranging from 3 to 7
minutes. Heat distribution patterns were monitored using MIW-02-10740 fiber optic
probes of a Luxtron 755 Multichannel Fluoroptic Thermometer. Control beans were boiled
in an open kettle for 15, 30 and 45 mins.

Texture of both the microwave and conventionally cooked beans were evaluated
using a Kramer Texture Test System ( Model TMS-90) equipped with the FI‘ 3000
transducer and the No. C-15 standard multiple blade shear compression cell. Texture was
expressed as Newtons] 50 g bean sample.

Color of 50.0 g beans was obtained using a Hunter Lab Model D25-PC2 (Hunter
Associates Laboratory, Inc., Reston, Va.) system standardized against a white tile.

109

 

 

 

Equilibration
(4 C; 48 hrs.)

 

 

 

Microwave Preconditioning
mins.)

I I
new
I I

Freeze
(-18 C)

V
Microwave Reheating
(740 w; 30 secs.)

Figure 26. Experimental design for the microwave preconditioning of dry navy beans

 

 

 

110

Cellular Structure and Starch Granule Appearance of microwave pre-
conditioned beans was observed under both Scanning Electron Microscope (SEM) (Model
I SM 35CF, Japan Electron Optics Limited, Tokyo, Japan) and Laser Scanning Confocal
Microscope ( LSM ) (Carle Zeiss, Inc., West Germany). Microwave heated bean samples
were submerged in liquid nitrogen bath, split into halves and freeze-dried overnight. The
dried samples were mounted on circular aluminum stubs with adhesive mounting tab and
coated with 20 nm of gold by "Film Vac”, sputter-coated and examined under SEM. The

same sample preparation was carried out for LSM examination.

Microwave Heating of Bean Starch
Navy beans (Fleetwood cultivar [firm texture) grown during the 1987 crop year in
research plots in Saginaw, Michigan, were used in this study.

Bean Starch Isolation

Bean starch was isolated using the method described by Naivikul and D'Appolonia
(1979) as modified by Srisuma, N. (1989). Whole bean flour (500 g) was extracted with
1.5 liter of 0.016N NaOH by mixing in a Waring blendor for 2 min. The water-soluble
material was removed by centrifugation (2000 g for 20 min) (1300 rpm). The precipitate
formed was sieved through 60 mesh screen retaining the non-starch components, yielding
crude starch. The crude starch was washed with deionized-distilled water (3x), 80%
ethanol (2x) and deionized-distilled water (2x). The supernatant and floating sludge were
removed and discarded after each washing. The primary starch was air dried at 40°C
(104°F) for two days and sieved through a 60-mesh screen to obtain the final bean starch
sample. Commercial corn starch (Argo R Pure Corn Starch, CPC International Inc.,
Englewood Cliffs, NJ) was used as a comparative control.

l l 1
Bean Starch Chemical Composition

Proximate Analyses. Moisture, ash and protein contents were determined using the
standard methods (AACC, 1983 and AOAC, 1984, respectively).

Amylase Content. The amylose content of the isolated bean starch was determined
by a modification of the improved colorimetric procedure of Morrison and Laignelet
(1983). Approximately 80.0 mg of the bean starch was weighed into a screw-capped test
tube to which 10.0 ml of urea-dimethylsulphoxide (UDMSO) was added. After mixing,
the tube was placed in a boiling water bath for 90 min. with intermittent mixing until the
bean starch-UDMSO solution became transparent and homogeneous. The solution was
cooled to room temperature, and a 1.0 ml aliquot of the solution was measured into a 100

ml volumetric flask. Ninety-five ml of deionized—distilled water was added with 2.0 ml of
Iz-KI solution (2 mg 12, 20 mg Kl/ml). Final sample volume was adjusted to 100 ml. The

absorbance of the sample solution was measured at 635 nm, 15 min. after the Iz-KI
addition, using UDMSO-Iz-KI solution as a blank. The blue value is defined as the
absorbance/cm at 635 nm of 10 mg anhydrous starch in 100 ml dilute Iz-Kl solution at
20°C (Srisuma, 1989). Blue value was converted into amylose content using the
following equation :

(Eq. 14) Amylose (%) = (28.414 x Blue Value) - 6.218 .

Heating of Bean Starch

Microwave Heating. Starch slurries were heated in screw-capped test tubes using a
Radarange microwave oven (Model - RS458P; Amana Refrigeration, Inc. ) with a 700 watt
rated power. Desired temperatures were pre-set on the microwave and actual temperatures
of the heated bean slurries were monitored using a Luxtron 755 unit with MIW—02-10740
fiber optic probes.

112

Conventional Heaa'ng. Starch slurries were heated in screw-cap test tubes ( 14 mm
ID.) and plawd in a thermostat-controlled water bath. Temperatures were monitored
using heating probes inserted inside the tubes and connected to a temperature measuring
device (Model T-l99, Omega Engineering Inc., Stramford CT).

Bean Starch Physical Properties

Starch Swelling Power and Solubility. Swelling power and solubility of bean
starch were determined for the temperature range from 70°C(158°F) to 90°C (194°F),
using a modified version of the method of Leach et al. (1959).

Degree of Syneresis. Ten grams of 8% (w/w) starch slurry was prepared in a
culture test tube (14 mm ID.). The slurry was mixed well, heated in the microwave oven
or in the water bath, and intermittently mixed (15 secs interval, 3x) using a vortex mixer.
After holding the temperature at 70°C for two minutes, the sample was cooled in a
20°C(68°F) water bath for 2 minutes. The starch gel was then incubated at room
temperature for 7 days prior to further analyses. Degree of syneresis (D8) of bean starch
was determined by measuring the liquid exuded after the storage period. DS was

expressed as the percentage weight loss of the gel after discarding the exudate.

Gel Rheological Properties. After weighing the samples prepared above, the gel
rheological properties were evaluated using back-extrusion (Hickson et al., 1982). A
cylindrical plunger (8.51 mm diameter) attached to the load cell of the Instr-on Universal
Testing Machine (Model 1122) was set to travel through the gel at a constant speed of 50
mm/min. The force required to drive the plunger was registered by a load cell attached to a
Hewlett Packard data acquisition microcomputer.

Scanning Electron Microscopy. Cellular structure and starch granule appearance of

microwave and conventionally heated starch samples were compared using Scanning

113

Electron Microscopy (SEM). Starch slurry (10%, wlw) in a culture test tube was heated in
the microwave oven and held at 70°C (158°F) for 2 minutes. Same procedure was
followed for conventional heating of samples in a water bath maintained at 70°C (158°F).
After heating, the starch paste was prepared for SEM examination using a method modified
from Pearse (1960 a & b), Bancroft (1975 a, b &c) and Varriano-Marston et a1. (1985).
This involved dropping the starch paste in a - 70°C(-94°F) n-propanol bath (temperature
maintained by solidified acetone and liquid N2), transferring the frozen starch droplet into
liquid N2 bath and freeze~drying the starch droplet. The dried samples were mounted on
circular aluminum stubs with adhesive mounting tab and then coated with 20 nm of gold by
"Film Vac,” Sputter-coated and examined under a Scanning Electron Microscope (JEOL
Model JSM 35CF, Center for Electron Optics, Michigan State University).

Statistical Analysis

The StatView 11 (1987) computer program for the Macintosh II was used for data
calculations and statistical analyses. All mean values are reported plus or minus one
standard deviation.

One-way and two-way analyses of variance were determined using the ANOVA
program. Mean squares were reported after rounding to two decimal places. Comparisons
between treatment means were provided by Fisher's PLSD (Protected Least Significant
Difference) test, such that different letters among treatments denote significant differences
(p < 0.05). Significant F ratios were indicated by asterisks: p<0.05 (*). Regression
analysis was also determined for the'measurement of suspension stability of the bean
weaning food/beverage.

CHAPTER I EXTRACT IVE PRETREATMENTS TO IMPROVE DRY
BEAN DIGESTIBILITY

Introduction

Processing strategies designed to eliminate antinutritional and flatulence factors in
dry edible beans include soaking, extractive pretreatments. germination, dehulling and
various cooking and drying methods (Uebersax et al., 1991) Soaking of legume seeds
reduces the toxicant content, soluble sugars and minerals due to leaching losses.
Enzymatic treatment of beans have been reported to remove much of the flatulence-causing
oligosaccharides (Calloway ct al., 1971). Germination and fermentation have also been
reported to reduce phytic acid which interferes with protein digestibility and mineral
bioavailability, and oligosaccharides which have been associated with flatulence. Cooking
of dry edible beans is essential to render palatability and inactivate toxic substances. Steam
blanching inactivates enzyme systems (Liener, 1962; 1978), enhances digestibility by
inactivating biologically active inhibitors, and stabilizes flavor by controlling the oxidation
of chemical constituents (Drumm et al., 1990).

Dry bean utilization could be substantially enhanced if they are conveniently
available as shelf-stable flours. Drum-drying of bean meals presents the following
advantages: 1) allows for utilization of split and culled beans which reduces costs and
eliminates the need for maintaining the integrity of beans during digestion and extraction
procedures, 2) enables use of selective extracts, and 3) products are pre-cooked and
readily hydratable as a nutritious product with controlled product consistency.

The Null Hypothesis tested in this study is stated as follows :

Ho: the pretreatment of dry beans by physical and biological methods will
not enhance their digestibility through removal or inactivation of interfering
components prior to drum-drying.

114

115

The objectives of this research were : l) to develop hot water/steam extraction and
enzymatic pretreatments procedures 2) produce drum-dried bean meals (DDBMs) and
3) establish chemical and biological compositional differentiation among the pretreated
drum-dried bean meals. Two separate studies are discussed in this Chapter : Study I-l
involved the "commercial-scale" development of DDBMs prepared from split dry navy
beans and the evaluation of the effects of hot water and enzymatic pretreatments on
DDBM composition. Study [-2 was the "bench-scale" development of DDBMs from
whole dry navy beans and the characterization of the effects of differential enzymatic
pretreatments on resultant DDBMs. Whenever appropriate, comparisons between split

and whole DDBMs were discussed.

Materials and Methods

Whole or split dry navy beans were soaked for 16 hours at 25°C in stainless steel
tanks and kettles. The different extractive pretreatments were carried out after static
hydration of the beans. Weights of materials were recorded during the semicontinuous
process to establish a mass balance. The experimental flowchart for this study is
presented in Figure 27.

The chemical and biological analyses conducted to evaluate the effectiveness of the
different pretreatments for enhancing digestibility of the drum-dried bean meals have been
described in detail. Proximate composition was determined using standard methodologies
for Moisture (AACC Method 44-40), Protein (AOAC, 1984), Fat (AACC Method 30-25)
and Ash (AACC Method 08-01). Soluble sugars of raw beans and drum-dried bean meals
were determined by HPLC following a modification of the extraction procedure
developed in the MSU Legume Laboratory (Agbo, 1982). Mineral content of raw beans,
drum-dried bean meals and water used during processing was determined using an
inductively coupled argon plasma (ICP) emission spectrometer (Jartell-Ash Model 955
Atomcomp) equipped for the simultaneous analysis of 19 elements and maintained by the

116

 

 

IIHOLE NAVY BEANS SPLIT NAVY BEANS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Hot Water
and Steam Enzymatic Hot Water
Extractions Predlgadons Extraction
DRUM-DRIED BEAN MEALS I
l 7
Iowa-fl 3.....1...

 

 

 

 

Figure 27. The experimental flowchart for Study I-l: Extractive
pretreatments to improve dry bean digestibility.

117

Department of Pharmacology and Toxicology at Michigan State University. The
enzymatic method of Prosky et al. (1988), with modifications from Sigma Chemical Co.
(St. Louis, MO), was used to determine the insoluble and soluble dietary fiber content of
the drum-dried bean meal samples. A modification of the enzymic/colorimetric method of
Holm et al. (1986) was followed to determine starch content of the drum-dried bean meals.
The rate of starch hydroysis was evaluated as described by Holm et al. (1985). ln-vitro
protein'digestibility was determined using a sequential multi-enzyme system (AOAC,
1990). The procedure of MacKenzie (1977) for the analysis of methionine in plant
materials using improved gas chromatography was followed. A modification of the ion-
exchange method of Graf and Dintzis (1982) was used to extract the phytates and the
HPLC method of Sandberg and Ahderinne (1986) was followed to separate and
quantitatively determined the inositol tri-, tetra-, penta- and hexaphosphates of the DDBMs.
The method of Kakade et al.(1974) was used to assay for trypsin inhibitory activity.
Phytohemagglutinin was determined using Sigma Kit No. P-0526 (Sigma Chemical Co.,
St. Louis, MO) containing purified Phaseolus vulgaris erythroagglutinin as immunogen.

Results and Discussion

Steam extraction (100°C/ 15 mins) of whole dry navy beans was conducted in
preliminary runs and compared with the aqueous pretreatment. Hot water extraction
resulted in a substantial reduction in crude ash, soluble sugars and total mineral content
due to leaching of soluble components. Based on the results obtained, the hot water
pretreatment was recommended as the method of extraction to use prior to drum-drying
because of greater potential to leach out undesirable soluble components and the relative
adaptability for appropriate application in village-level situations with LDC‘s. Data
obtained for steam extracted DDBMs are summarized in Appendix A and will be referred

to whenever discussion is deemed appropriate.

118

Yield

Figure 28 illustrates the moisture-solids balance for the drum-drying of bean meals
collected during commercial-scale runs. Soaked and hydrated navy bean splits were
subjected to hot water extraction prior to drum-drying of pre-heated bean slurries. The
mass balance was prepared to provide an overall yield estimate. Material balances are
useful in comparing actual operations with theoretical values for determining nutrient loss
as well as waste generation. An initial 400 lbs of dry navy beans with @13% moisture
was hydrated and hot water extracted. The beans were divided into two lots: 1) Half of the
cooked beans (200 lbs.) served as CONTROL with all the accumulated formulation water
added back to yield a final 118 lbs. of drum-dried bean flakes (63% F.W.; 68% solids).
2) The other half of the bean lot (200 lbs.) was the EXTRACT with leachate discarded and
fresh formulation water added back to obtain a final yield of 143 lbs. drum-dried bean
flakes (72% F.W.; 77% solids).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

comm. Dry Beam EXTRACT Dry Beam
200 lbs Fresh Wt. @ rsxmc 200 lbs Fresh Wt. @ 13%MC
174 lbs. Solids 174 lbs. Solids
DDBM comet. DDBM mcr
1261baFreshWt.@6%MC 1431thm§hWL@6%MC
ll8lbs. Solids 1341bt5011dl
YIELD YIELD
aw. : 63% aw. : 72%
Solid: = 689‘» Solids: 77%

OverallProces Yleld Estllrnte: KW. : 67%; Sollfi :73%

Figure 28: Moisture-solids balance for drum-dried bean meals

1 l9
Moisture loss brought about by the extreme high temperature during the drum—drying
process (... 240°C) contributed to the losses in DDBM yield. Additional losses were
attributed to the physical loss of slurry during milling and pumping and due to losses of
flaking sheets at the drum-dryer. These data were obtained during the experimental process
sequences and represent two independent batches. Therefore this mass balance is useful to
describe the general process yield. However, no inferences may be drawn regarding the

extraction treatments
Proximate Composition

Tables 4 and 5 summarize the proximate composition of split and whole
DDBMs, respectively. Moisture contents of split DDBMs were significantly different
(p < 0.05) (ranging from 5.1% to 12.6% ). Enzyme pre-digestion with Viscozyme
resulted in significantly higher (p < 0.05) protein (26%) and carbohydrate contents
(74%) than raw split beans and the other split DDBMs. Crude fat content was also
significantly different (p < 0.05) between the split DDBMs, although it was generally low
for both the raw beans and the DDBMs. Processing had a significant influence
(p < 0.05) on ash content of the DDBMs.

Crude protein content of whole DDBMs was not significantly affected by the type
of enzyme utilized for pre-digestion. Pre-digestion with the proteases (Alcalase and
Neutrase) both resulted in slightly lower fat values of the whole DDBMs. Ash content of
whole beans increased with processing and enzyme pretreatment, except for the Celluclast
pre-digested DDBM which was lower than the raw whole bean.

The lower carbohydrate content of the EXTRACT DDBM could have resulted from
loss of soluble components upon removal of the leachate. Sprouting has been reported to
result in an increase in ash content apparently caused by loss of starch (Chavan and

120

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Kadam, 1989), but this effect was not observed for the Sprout DDBMs. Ash content has
been reported to decrease after cooking due to leaching, but this was not the case for
DDBMs, which were significantly higher in ash content than the raw bean. However, the
values were still within the reported range of values for dry beans (3.5% to 4.1%).

Soluble Sugars

Soluble sugars (particularly the oligosaccharides raffinose and Stachyose) were
present in lower amounts following hot water extraction compared to steam extraction
(Appendix A). Figure 29 illustrates that sugar content of split bean CONTROL and
EXTRACT DDBMs are not significantly different (p < 0.05). However, the differential
enzymatic pretreatments employed had an influence on the soluble sugar content of whole
DDBMs, especially the simple sugars (glucose and sucrose) which increased with
processing (Figure 30). The aqueous extraction in this study resulted in a more significant
reduction of the soluble sugars compared to the steam pretreatment, indicating leaching of
soluble components (Appendix A). However, there was no significant difference (p <
0.05) in theoligosaccharide content between CONTROL DDBM and EXTRACT DDBM,
although the latter did possess slightly less Stachyose. This suggests that the hot water
extraction conditions (60°C/30 mins.) may not have sufficiently leached out the soluble
components. A temperature of 60°C was deemed sufficient to initiate bean starch
gelatinization, mobilize soluble constituents and yet not result in a mushy product which
woulddecrease yieldandeaseofbean separation The enzymatic pretreatments (Viscozyme
and Sprout) lowered the Stachyose content of split DDBMs, particularly in the case of the
Sprout split DDBM. According to Chavan and Kadam (1989), germination or sprouting of
grains causes a significant reduction in raffinose saccharides. Viscozyme exhibited a
darker surface color (brown hue), indicating occurrence of Maillard reaction as a result of
increased glucose content. Borejszo and Khan (1992) reported reduction of raffinose and
Stachyose by high temperature extrusion of pinto bean high starch fractions, possibly due

123

 

 

 

 

 

 

 

l
.0

Aiv 38.-o.—

 

Figure 29. Soluble sugars of split drum-dried bean meals ( p < 0.05)

 

 

 

 

 

 

 

 

iv 38.!—

Figure 30. Soluble sugar content of enzyme predigested
whole drum-dried bean meals (p < 0 05)

124

to Maillard reaction between charged protein groups and reducing sugars. The enzyme
predigested whole DDBMs also exhibited lower stachyose compared to the CONTROL
DDBM, clearly indicating the potential of selective enzyme pretreatments to reduce
oligosaccharides. Processing increased glucose content of the DDBMs as a result of
starch gelatinization. The CONTROL DDBM exhibited the highest glucose content,
brought about by the retention of the formulation waters; glucose content of the EXTRACT
DDBM was lower as a result of the removal of the leachate. The Sprout split DDBM
demonstrated almost two times the sucrose content of the CONTROL DDBM. According
to Chavan and Kadam, (1989), during germination of wheat, starch content is decreased
whereas reducing sugars are increased The sucrose peak may actually be a composite of
sucrose and some other disaccharide (such as maltose) having almost the same retention

time as sucrose.
Total Mineral Content

Tables 6 and 7 summarize the total mineral content of split and whole DDBMs,
respectively. Calcium and iron concentrations of split beans increased with processing.
Sprout split DDBM demonstrated a significantly higher (p < 0.05) calcium value than the
other split DDBMs while CONTROL DDBM exhibited a higher iron content compared to
the other DDBMs. Processing had no significant influence on the zinc, phosphorus,
magnesium, manganese and copper contents of split DDBMs.

Calcium content of Alcalase pretreated DDBM is significantly lower (p < 0.05)
compared to that of the other enzyme pretreated whole DDBMs. Sprout pretreated DDBM
demonstrated a significantly higher (p < 0.05) iron value than the other pretreated
DDBMs. The differential enzymatic pretreatments likewise had no significant effect on the
zinc, manganese, copper and potassium values of the whole DDBMs. However, iron,
phosphorus, aluminum and sodium concentration increased with processing.

125

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High mineral values of dry beans cannot be equated with high bioavailability of
some minerals because of the presence of significant amounts of phytic acid which impedes
the absorption of multivalent cations like calcium, magnesium, zinc and iron. Lola and
Marltakis (1975) reported that majority of the total phosphorus content of dry beans is
present as phytic acid or its calcium, magnesium and potassium salts. According to
Tecklenburg et al.(1984), total phosphorus content correlated well with phytic acid content
and protein content. Results of this study demonstrated a slight increase in phosphorus and
a significant increase in calcium for the Sprout split DDBMs, contrary to the observation
of (Chavan and Kadam, 1989) that calcium, total phophorus and phytate-P decreased
during sprouting of millets.

Dietary Fiber

The insoluble dietary fiber comprised approximately 73% to 80% of Total Dietary
Fiber ('I'DF), with the soluble fiber making up approximately 25% of TDF. CONTROL
and EXTRACT split DDBMs were not significantly different (p < 0.05) in their soluble and
insoluble fiber components (Figure 31). Dietary fiber content of whole DDBMs was
influenced by the enzyme used for predigestion (Figure 32). According to Hughes (1991),
dietary fiber extracts or isolates derived from whole foods possess a ratio of soluble to
insoluble dietary fiber similar to that of food from which they are derived, but are higher in
total dietary fiber. Dry beans contain approximately 7% soluble dietary fiber and 13%
insoluble dietary fiber. Dry bean dietary fiber is primarily structural (includes lignin,
cellulose and hemicellulose) in nature, and not easily isolated or extracted because it is
intimately involved in maintaining cellular structure and cannot be easily separated from the
other cellular components. Nonstructural dietary fibers include the soluble pectin, gums
and mucilages. Srisuma et al. (1991) reported that the cell wall strcuture of bean cotyledon
possesses a high content of matrix polysaccharides (Hot water soluble polymer, 25.7% -

Total Dietary Flber (S)

130

 

     

  
  
    

   
 

 

 

  

 

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Raw 0mm EXTRACT Viscozyme Sprout
Figure 31. Imoluble and soluble dletary fibers
of split been drum-dried meals

 

 

 

  
 
 
      
 
 
 
 

   
 

 

  
  
  

Total Dletary fiber (%)

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ll],
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Figure 32. Insoluble and soluble dietary fiber: of
whole drum-dried been meals

131

322.5% and Hemicellulose B, 14.6% to 19.2%) with less cellulose, Hemicellulose A and
lignin.

Results of this study demonstrated a higher content of insoluble dietary fiber for
the DDBMs (16 - 22.1% IDF) compared to the figure (13%) reported by Hughes (1991)
for dry beans, although the soluble fiber approximated the reported value of 7%. The
higher TDF content obtained in this study is attributed to the loss of moisture with
processing, thereby increasing the concentration of TDF. The IDF and SDF contents of
DDBMs subjected to enzymatic pretreatments more closely approximate the figures
reported by Hughes (1991), and indicate the potential of enzymatic predigestion as a
processing pretreatment to influence product dietary fiber content. The higher TDF value
obtained for the raw bean meal also suggest the inherent presence of indigestible or
resistant starch which was made more accessible to enzymatic digestion during drum-
drying and enzymatic pretreatments. Dietary fiber has been implicated in the flatulence
problem associated with consumption of dry beans, and has been attributed to the soluble

component pectin.

Effect of Extractive Pretreatments on the Irv-vitro Digestibility and
Microstructure of Starch in Drum-dried Bean (Phaseolus vulgaris) Meals

Starch Content

Table 8 and figures 33 and 34 summarize the effects of various extractive
pretreatments on the apparent starch content of drum-dried split and whole bean meals,
respectively. All the raw and drum-dried bean meal (DDBM) samples were significantly
lower (p < 0.05) in total starch content than the reference samples, cornstarch (95%) and
all-purpose flour (wheat) (75%). Starch content of raw whole and split navy bean meals
were estimated to be 48 and 60%, respectively. Split CONTROL (54%), Viscozyme
(61%) and Sprout (57%) DDBMs had slightly higher starch contents than their

132

Table 8. Effect of enzymatic pretreatments on the total starch content of

bean drum-driedmeals1

 

 

'I'reatrrrentz,3 Starch content
(% dbL
A. Split Drum-Dried Bean Meals
Raw 68.02 1 2.71b
CONTROL 57.29 1 2.2111
EXTRACI‘ 60.16 1 0.04a
Viscozyme 64.58 1 3.0%
Sprout 60.35 1 1.00ab
Reference Samples
C 99.76 1 2.81d
All-Purpose Flour 75.04 1 1.22c
LSD(0_05) 5.07
B. Whole Drum-Dried Bean Meals
Raw 55.77 1 1.24ac
CONTROL 56.08 1 7.31a
Sprout 56.07 1 0.25ab
Viscozyme 61.92 1 3.16b
Alcalase 49.77 1 7.86a
Neutrase 59.74 1 2.3bc
Celluclast 60.16 1 3.39bc
Reference Samples
C 99.76 1 2.81e
All-Purpose Flour 75.04 1 1.22d
LS13(0.05) 9.33

 

1 n=2; Values are the means of two assays

Means within a treatment group followed by different letters are significantly different (p < 0.05)

2 Hot Water Extraction (60°C/60 minutes)

3 Raw.the been meal; CONTROL. non-extracted; Sprout. 1% germinaed bean slurry;
Connnereial Enzyme preparations (V iseozyrne. Alcalase. Neutrase and Celluclast)

133

 

 

3:22.30 5:8

Cornstarch All-purpose

Sprout

Flgure33. Total starch content ofraw and splltbean drum-dried meals

 

100

/./
/

 

w w m 0

3:22.30 5:8

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. .
_
m
u

hmm

Treatment
Figure 34. Total starch content of raw and enzyme pro-digested

whole bean drum-dried meals

134

corresponding whole drume bean meals (52%, 59% and 51%, respectively) due to
hull loss in split beans. The starch content of the split CONTROL DDBM ( 54%) was
not significantly different (p < 0.05) floor the EXTRACT DDBM samples (53%), but
significantly lower (p < 0.05) than the enzyme predigested samples, Viscozyme (61%) and
Sprout (57%). The enzyme pre-digested whole DDBMs yielded significantly higher
(p < 0.05) apparent starch contents (55% to 59%) than the CONTROL DDBM (52%),
except for the Alcalase (44%) and Sprout (51%) treated DDBMs which were not
significantly different (p < 0.05).
In-vitro Starch Availability

Tables 9 and 10 summarize the digestibility of starch (%) in variously treated split
and whole DDBMs held under different incubation periods (0, 15, 60 and approx. 380
mins.). Figures 35 and 36 depict the a-amylolysis curves of split and whole
DDBMs, respectively. All the split and whole DDBMs were hydrolyzed at a significantly
lower (p < 0.05 ) rate than the boiled (20 min. at 100°C) cornstarch used as a reference
sample. The other reference, boiled all-purpose flour, exhibited a lower degree of
hydrolysis than cornstarch, but was not significantly different (p < 0.05) from the
EXTRACT and enzyme predigested split (V iscozyme and Sprout) DDBMs. The boiled
raw bean flours (split and whole) were not significantly different (p < 0.05) from the
CONTROL DDBMs and exhibited the lowest degree of hydrolysis. The various extraction
pretreatments employed prior to drum-drying all resulted in an increased rate of
amylolysis, with the enzyme pre-digested DDBMs demonstrating the largest increment in
starch availability. The degree of hydrolysis increased in the following order : a) for split
bean, raw = CONTROL < EXTRACT = Sprout < Viscozyme; whereas b) for whole
beans, raw = CONTROL = Sprout <Viscozyme < Neutrase = Alcalase = Celluclast
(Figures 36 and 37).

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Degree 0! hydrolysis (%)

 

 

 

 

 

 

 

 

 

 

0r . r T r - r r f I - - —O— EXTRACT
0 20 40 60 80
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0 100 200 300 400

Incubation the(nh)

Figure 35. Hydrolysis of starch in raw and split bean drum-dried meals
by pancreatic a-amylase

138

 

 

 

 

 

 

 

 

 

 

 

so
1
t!
V
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D
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Incubation time (min)

Figure 36. Hydrolysis of starch in raw and whole bean drum-dried meals
by in-vitro pancreatic a-amylase

139

Degree of hydrolysis for whole beans which were preheated with hot water versus
steam cooking increased as follows: raw < HWC < SC < SE < HWB (Appendix C). An
additional gelatinization step (boiling for 20 min.) prior to the starch assay, of previously
pre-gelatinized split bean DDBMs further increased the rate of amylolysis (Appendix C).

Total and available starch contents of the drumodried bean meals were determined
by following the method of Holm et al. (1986 and 1985, respectively). The following
steps were incorporated prior to enzyme digestion : a) solubilization with 4N NaOH at
room temperature for 1 hour to measure total starch content and b) pepsin incubation at
37°C for 1 hr. at pH 1.5 for the determination of "potentially available" starch. According
to Tovar et al. (1990b), these pretreatments enhanced accessibility of starch to amylases
and destroy the integrity of remaining intact cell walls. The alkaline pretreatment used
provides for the dispersion of crystalline raw starch and retrograded amylase fractions,
resulting in higher starch yields (Englyst and Cummings, 1990). Underestimation of
starch content as a consequence of encapsulated starch have been previously reported for
precooked legume flours (PCFs). The NaOH or KOH treatment thus provides a way to
measure "total starch content" by solubilizing so-called "resistant starch". A preincubation
with pepsin has a thinning effect on the cell surface with accompanying alteration of
fiber/protein associations occurring in the cell walls (Tovar et al., 1990a).

Total starch yields of the CONTROL and EXTRACT split DDBMs were not
significantly different (p < 0.05), indicating that the aqueous extractive pretreatment
employed did not have a significant effect on starch content (p < 0.05), as it did for
relatively more soluble simple sugars. Total starch yields of the CONTROL and
EXTRACT DDBMs were significantly lower (p < 0.05) compared to that of the raw bean
meal. Tovar et al. (1990b) reported that differences in susceptibility to enzymatic attack in
PCFs and raw flours are mainly due to the release of starch granules during milling of
uncooked seeds, whereas starch-containing cotyledon cells remain in the PCFs.

140

The enzyme pro-digested split bean DDBMs, Viscozyme (commercial), possessed
significantly higher (p < 0.05) starch contents than the CONTROL and EXTRACT
DDBMs. The Sprout DDBM was not significantly different from the CONTROL and
EXTRACT DDBMs. Both Viscozyme and Sprout contain cell wall degrading enzymes
which could hydrolyze cell wall or starch with a corresponding increase in released
glucose. The reference samples, cornstarch and all-purpose flour, possessed
significantly higher (p < 0.05) starch contents than the boiled raw seed and DDBM flours.

The enzyme pre-digested whole bean DDBM flours did not differ significantly
(p < 0.05) in their apparent starch contents. However, the cell-wall degrading enzymes,
Viscozyme and Celluclast. resulted in the highest starch yields, followed by the protease,
Neutrase. These results again indicate the need for the disruption of cotyledon cell wall in
order for the starch to be exposed to the amylases. The Sprout DDBM possessed similar
starch content as the unextracted sample, CONTROL, indicating that the indigenous
amylases present during germination did not sufficiently hydrolyze the starch.

The "potentially available" starch represents the amount of starch which can be
hydrolyzed by amylolytic enzymes, and is starch measrn'ed after pepsin or homogenization
pretreatments of cooked beans (Tovar et al., 1990a; Tovar, 1992). According to Holm et
al. (1985), preincubation with pepsin increased the starch availability of raw and boiled
wheat to a-amylase, indicating a large fraction of the starch was encapsulated in a protein
matrix. The disintegration of protein aggregates released highly available gelatinized starch
that was readily hydrolyzed by a subsequent incubation with a -amy1ase. The release of
starch from the protein matrix indicates that gelatinization alone is not the only factor

involved in increasing the starch susceptibility to enzyme action.

141

Laser Scanning and Polarizing Light Microscopy

Abundant free starch granules with adhering protein bodies were observed in the
navy bean flour from milled raw seeds (Plate 1a). The raw bean starch granules exhibited
birefringence, as evidenced by the presence of the characteristic "Maltese cross" pattern
when viewed under polarizing light microscopy (Plate 1d). The differential extractive
pretreatments employed prior to drum-drying resulted in microstructural changes as
demonstrated by extensive disruption of the cotyledon cells and exposure of the starch
granules (Plates 2a and b, and 3a to e). No evidence of defined cellular structures nor
intact starch granules were identified in bean meals subjected to enzymatic pre-digestion of
the slurries prior to drum-drying, particularly those meals pre-digested with the commercial
enzymes Neutrase and Celluclast (Plates 3i to j) which appeared amorphous. For
comparative purposes, LSM micrographs of flour obtained from canned and subsequently
freeze-dried whole navy beans are shown in Plate 4. Abundant agglomerates of intact
cellular structures entrapping starch granules were evident in these hydrated and thermally
processed canned samples. Similar intact cellular structures were observed under LSM of
canned whole navy beans (Plate 5). Further, when viewed under polarizing light
microscopy, the "Maltese crosses" were still present with relatively greater frequency
"crosses" observed in the "firm" (92 RNK 8P) breeding line compared to the "soft" (92
RNK 17E) breeding line, indicating incomplete and differential gelatinization of starch in
these thermally processed samples (data not shown).

Plate 1a illustrates the free starch granules released by the breakdown of cell
walls of raw navy bean seeds during milling. A high proportion of the starch granules
iembedded in a protein matrix (Plate 1d), as evidenced by the preponderance of fluorescent
protein bodies. The "Maltese cross" observed under polarizing light microscopy (Plate 1d)
is a manifestation of birefringence exhibited by ungelatinized crystalline starch granules.
The short boiling step (20 mins. at 100°C) employed prior to the enzyme digestion of the

 

Plate '- Raw Split mcal (a—h) undcr LSM microscopy and (c-d) under polarizing
light microscopy

143

'0 Ltll

 

Plate 2. EXTRACT DDBM (ll-bl UlldCl' LSM microscopy and (c-d) under polarizing
light microscopy

144

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Plate 3. LSM micrographs of non-extracted (CONTROL) (a-b) and enzyme pre-
digested whole bean drum-dried meals: Viscozyme (c-d). Sprout (c-D.
Alcalase (g-h). Neutrase (i-j) and Celluclast (k-l)

145

 

 

 

lflvn ‘
...——

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146

e ZOOM-20 T-Zs CI368 It“ F0 L488

0 ZOOHIZO TIZr CISlO 3'405 F0 L488

 

Plate 4. LSM micrograph of canned and subsequently freeze-dried
whole navy bean

147

 

Plate 5. LSM micrographs of (a) soft canned and (b) firm canned bean (92 RNK 8P)

148

raw bean may have resulted in partial swelling of the bean starch granules. The a-amylases
cannot easily penetrate through the protein matrix encapsulating starch (Holm et al., 1986)
or within incompletely gelatinized granules due to steric hindrance (Wursch, 1986). These
factors could explain the lowest degree of hydrolysis (Figure 5) demonstrated by boiled
raw bean seeds. I

Birefringence was not exhibited by pregelatinized EXTRACT DDBM under
polarizing light microscopy (Plate 2), indicating complete gelatinization during the drum-
drying process which involved exposure to temperatures as high as l49°C(300°F).
Wursch et al. (1986) observed that a-amylase digested starch granules progressively from
outside of the cell walls. Thus, the absence of the intact cotyledon cells in DDBMs which
may be attributed to wet homogenization (Fitzmill) of heated beans prior to drum-drying
has resulted to a greater surface area being available to the amylases, rendering the DDBMs
susceptible to amylolytic attack with resultant increased digestion rates.

According to Tovar et al. (1992), acidic preincubation of PCFs with pepsin,
simulating the gastric phase of digestion, resulted in a greater rate of hydrolysis. The
resulting thinned or even missing cell surface and higher amylolytic rate observed in PCFs
may be regarded as an enzymatic rather than merely a pH dependent effect. Wursch et al.
(1986) also reported a minor change in digestion of leguminous starch during incubation
under acidic conditions in the absence of pepsin. Results of LSM in this study
demonstrated that enzymatic pre-digestion of bean slurries prior to drum-drying affected the
integrity of the cell walls and facilitated the amylolytic process by exposure of starch (Plate
3a-f). DDBMs from whole beans incubated (prior to drum-drying) with the proteases
Alcalase (food grade) and Neutrase, exhibited a higher degree of hydrolysis than those
pre-digested by Viscozyme and Sprout, indicating potential denaturation of the pretein
mauix and increased accessibility of a-amylase to the substrate. The LSM micrographs
(Plate 3d and e) illustrate the disruption of the starch granules increased the total granule

149

surface area exposed to enzyme action. These results further confirm the observation of
Wong et al., (1985) and Tovar et al., (1989) of a possible occurrence of protein/starch
interactions which decrease carbohydrate digestibility.

The presence of intact cellular structures encapsulated by a protein matrix
(fluorescing) in whole beans canned and subsequently freeze-dried (Plate 4) confirm the
observations of Tovar et al. (1992) that freeze-dried PCFs contained more intact cells than
vacuum-dried flour. Plate 5 also demonstrates the same observation with canned whole
beans ("frrm" versus "soft" cultivars) hydrated and thermally processed, clearly
demonstrating that the high retort temperature 115°C (240°F) was not sufficient to disrupt
cell walls, or to completely gelatinize legume starch, and that gelatinization alone is not the
only factor involved in increasing starch susceptibility to amylolytic activity. These
results are contrary to the observation of Hughes and Swanson (1989) that flours prepared
after autoclaving and warm-air drying of common beans do not contain intact cells.
Commercially canned beans have been previously reported to contain resistant starch
(Siljestrom and Bjorck, 1990). During cooking, some of the amylose leaches out from the
starch granule, remaining within the intact cell and filling spaces between protein bodies
and starch granules. If amylose retrogradation occurs, another indigestible matrix is formed
in and around the protein and starch granules, thereby contributing to slow protein and
starch digestion (Bennink and Srisuma, 1989). During methodology development and
screening, it was observed that canned and freeze-dried samples (as in Plate 4 ) had to
undergo a gelatinization step (boiling for 20 mins.) prior to the starch availability assay to
enable normal digestion rates. Lower rates of digestibility were observed for samples
tested without this additional heat treatment(data not shown). Tovar et al.(1990b) reported
that a second boiling treatment promotes a further rise in the enzymatic susceptibility of
starch. This could also account for the increased degree of hydrolysis observed with

150

pregelatinized split DDBMs subjected to a boiling step (20 mins.) prior to the starch assay
(data not shown).

The reference cornstarch exhibited the highest degree of hydrolysis, while the all-
purpose (wheat) flour was not significantly different (p < 0.05) from the enzyme pre-
digested DDBMs. Holm et al. (1985) reported that pure starch was more available to
salivary a-amylase in-vitro than starch present in flour. The cohesive properties of wheat
proteins result in the formation of glutinous lumps when these samples are being dispersed,
making it difficult for the enzymes to degrade the starch completely to glucose.

lit-vitro Protein Digestibility

In-vitro protein digestibility of raw split navy beans improved significantly
(p < 0.05) with processing ( Figure 37). The effect of the differential aqueous extractive
pretreatments employed prior to drum-drying on in-vitro protein digestibility of the split
DDBMs were not significantly different (p < 0.05).

The differential enzymatic pretreatments (Figure 38) did not significantly enhance
(p < 0.05) in-vitro protein digestibility of whole raw bean meals, with the exception of the
Celluclast predigested DDBM which did increase. The CONTROL DDBM (no enzyme
predigestion) demonstrated a significantly higher (p < 0.05) protein digestibility value
than the enzyme pre-digested DDBMs. Heat treatment greatly improves the digestibility of
most dry bean protein fractions. The improved performance upon heating is partially
attributed to inactivation of antinutritional factors such as protease inhibitors and lectins
(Nielsen, 1991) and opening of the protein structure through denaturation (Hsu et al.,
1977). However, processing can also cause a decrease in protein digestibility via the
nonenzymatic browning reaction and thermally induced cross-linking reactions. Hence, a
sensitive in-vitro protein digestibility method should detect digestibility changes due to
protease inhibitors, substrate-bound inhibitors and processing effects. The multienzyme
technique utilized in this study for estimating in-vitro apparent protein digestibility can

5 hotel- Dlluflblllty

5 Protein Dlguflblmy

151
IN-VITRO PROTEIN DIGESTIBILITY 0F DRUM-BRIE) BEAN MEALS

W

 

 

 

 

 

 

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drum-driedbeanmeals

152

detect the effects of trypsin inhibitor and heat treatment on protein digestibility (Hsu et al.,
1977). Results of this study demonstrated the improvement in protein digestibility of the
DDBMs with heat processing. Although the effects of the aqueous extractive
pretreatments were not significantly different, the EXTRACT split DDBMs exhibited a
slightly higher (%) protein digestibility over the CONTROL DDBM which indicated the
importance of discarding leacheate, thereby removing soluble antinutritional factors. The
high temperature involved in drum-drying gelatinized starch, opening the carbohydrate-
protein structure and facilitating hydrolysis of proteins by the proteolytic enzymes. This
same phenomenon was reported for thermoplastic extrusion involving high temperature,
short time heat processing, thereby increasing digestibility of puffed corn grits (Hsu et al.,
1977). The slightly higher (%) protein digestibility exhibited by the Split DDBMs over the
whole DDBMs demonstrated the significance of the removal of bean hulls (containing less
digestible proteins) to improving bean digestibility. Results also indicated that the
enzymatic pretreatments with Viscozyme and Sprout (both containing amylases which
increased reducing sugars), could have resulted in Maillard reaction as evidenced by the
resultant browning reaction in the DDBMs, thereby lowering digestibility of whole
DDBMs compared to CONTROL DDBM. According to Hsu et al. (1977), digestibility of
bread was decreased due to the Maillard reaction between the reducing sugars which was
created from the hydrolysis of starch by the enzymes in the barley malt, and the proteins.
The effect of enzymatic predigestions on improved protein digestibility has potential,
although it is also clearly demonstrated that the nature of the specific enzyme involved is

critical.
Sodium Dodeeyl Sulfate (SDS) Gel Electrophoresis

Figure 39 is a graphical representation of the electrophoresis gel which was run
for the whole DDBMs subjected to enzymatic predigestion. Gel Lane (A) represents the

153

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154

protein components present in uncooked or untreated whole (raw) dry bean. CONTROL
DDBM (B) represents the processed sample with the retained formulation water. General
banding patterns between raw (A) and cooked (B) bean flour are quite similar with little
indication of major band position shifts. However, the cooked sample bands were
generally less intense than those obtained for raw beans. Gel lanes (C - F) present the
CONTROL DDBM (B) upon exposure to selective enzyme pretreatments of the slurry.

It was noted that each enzyme pretreatment resulted in a new band (Rm 60) not
evident in the raw or untreated DDBM. Sprout DDBM (C) demonstrated an increased
number of protein bands and intensity compared to CONTROL DDBM. It is speculated
that these effects (C) have resulted from protein synthesis during the germination or
sprouting process when enzymes are generated and overall enzymatic activity increased.
However, no dramatic increase in total crude protein was noted for the Sprout-treated
sample (Table 5). Whole DDBM subjected to Viscozyme (D), a multienzyme complex
made up of cell-wall degrading enzymes, also demonstrated increased numbers of protein
bands and a general increase in intensity of handing patterns, which could be a result of
the release of the matrix proteins associated with the cell wall. The banding pattern
obtained for Celluclast (E), a cellulase which is also a cell-wall degrading enzyme, could
also represent release of cell wall associated proteins. Alcalase (F) and Neutrase (G) are
both proteases and clearly demonstrate increased in hydrolytic degradation of large
molecular weight proteins and partitioning of peptides. Neutrase (G) exhibited increased
evidence of hydrolysis and extensive degradation through the formation of lower molecular
weight peptides following enzymatic predigestion. These data demonstrate no major
changes in protein patterns as a result of cooking; however, selective hydrolysis of the
native proteins was evident upon enzymatic digestion and may be associated with

differential ill-vitro protein digestibility.

155

Amino Acid Composition

Table 11 summarizes the amino acid composition of split raw beans and DDBMs.
Methionine content of split beans increased with processing; CONTROL DDBM was
almost two times the amount of the EXTRACT and Viscozyme DDBMs. Cystine was not
present in any of the DDBMs. Lysine in EXTRACT DDBM was two times that found in
raw split beans; lysine in CONTROL DDBM was similar to that in raw beans, while a
decrease was observed in the enzyme pre-digested Viscozyme. Isoleucine, valine and
phenylalanine concentration increased with processing of DDBMs, with the EXTRACT
DDBM containing the highest concentration followed by the CONTROL and either a
decrease or a similar level observed in Viscozyme compared to the raw bean meal. The
sulfur amino acids cystine/cysteine (Cys) and methionine are of primary concern in protein
utilization because they are first-limiting in dry beans. Cys has been found to be more heat-
sensitive than methionine. Measuring Cys of beans receiving heat treatment is important in
estimating the deterioration of nutritive value due to heating. In navy beans, Cys
destruction was 30% at 100°C for 10 mins. but increased with increased cooking time
(Dhurandhar and Chang, 1990). In this study, cysteine content for both the raw beans and
DDBMs was 0.00 prnole, which could be a result of an analytical problem. Lysine content
for the Viscozyme-treated DDBM was lower than that of the other DDBMs. This could be
attributed to the non-enzymatic browning (Maillard) reaction, demonstrated by the brown
hue of the flakes. Lysine losses resulting from Maillard reaction involve the reaction of the
E-amino group on the lysine residue which becomes blocked by a sugar, yielding a
biologically unavailable lysine residue. Heating of dry bean protein improves the rate of
release of methionine by in-vitro treatment with enzymes (Rayas-Duarte et al., 1988).
Methionine increased with processing, and was highest in the CONTROL DDBM

indicating retention of the amino acid with the original formulation water. This

156

Table ll. Amino acid content (packs) of split drum-dried bean meals.

 

 

 

 

Amino acid RAW CONTROL EXTRACT VISCOZYME
Asparfic 1337.63 3030.13 4264.42 2047.05
Giuramie 2952.91 5052.50 7155.03 3374.45
Hydroxyproline 43.58 0.00 97.94 43.23
Serine 1390.13 2360.30 3536.96 1706.30
Giyeine 1609.90 2343.92 4553.57 2007.64
Histidine 430.43 322.74 1337.53 566.54
Arginine 557.13 920.70 1393.47 631.51
Threonine 1111.19 2027.04 3349.60 1431.46
Alanine 1349.96 2406.43 3463.09 1609.24
Praline 1352.50 2433.36 3314.26 1652.53
Tyrosine 120.37 193.79 653.42 145.10
Valine 979.03 1367.45 2931.52 1133.35
Methionine 122.34 290.43 142.60 145.94
Cysteine 0.00 0.00 0.00 0.00
lsoleucine 677.03 1224.71 200757 305.93
Leucine 1256.67 2191.53 3659.46 1479.15
Phenylalanine 601.14 1009.36 1796.19 675.71
Lvsine 310.94 396.81 670.65 250.51

Table 17.. Methionine contentofsplit beandrurn-dried menial

Truman Methionine
' (uncles Met/Mdp'ocin)

 

A. Split Benn Drum-Dried Meals

Raw 19126; 5.253
comor. 203.21;29.ma
met 196.99; 153a
Viscozyrrz 201.95;13.70a
Sprout 207.96; 7.49:1

B. Whole Bean Drum-Dried Meals
Raw 192.7o;3.39a
CONTROL 220.67;0.67e
Vmoayme 22617;6.36d
Sprout 205.09:2.41b
m 21550;6.36c
Nennaae 210.66;2.36e
Ccmndm 210.66;2.33e

 

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157

observation confirms previous reports that methionine was not decreased by conventional
cooking or alkaline treatment (Table 12).

Trypsin Inhibitory Activity (TIA)

Table 13 summarizes the units of TIA present in split raw and DDBMs.
Processing significantly reduced (p < 0.05) TIA from 18,703 units of TIA per gram of
raw split beans to a range of 1000 to 3300 units of TIA per gram of split DDBM. The
EXTRACT DDBM was significantly lower in residual TIA compared to the other split
DDBMs.

The antinutritional factor trypsin inhibitor (TI) has been reported to be heat
resistant, and 50-80% of the original trypsin inhibitor activity is retained after various heat
treatments. TIs are high in cystine. Trypsin activity of navy beans (1.9 to 2.7%) was
retained even after cooking for 40 min at 100°C. Heat inactivation of TI activity in soaked
whole navy beans was more effective compared to dry roasting (Dhurandar and Chang,
1990). The significant reduction (p < 0.05) of trypsin activity in the split DDBMs
indicated that they have received adequate heat processing. The drum-drying process called
for an extremely high temperature of approximately 240°C; in addition, pretreatments
including a 16-hr soaking period and hot water extraction (60°C/30 min) facilitated
effective reduction of TI.

Phytic Acid

Table 13 summarizes the results of the HPLC analyses of split bean DDBMs for
separation and quantitative determination of inositol tri-, tetra, penta and hexaphosphates
(1P3 - 1P6). Fractional data are expressed as a percentage of the sum of inositol
phosphates. Soaking has been reported to decrease the content of phytate in cereals and
legumes. This is readily apparent in the degradation of phytate (1P6) in the EXTRACT

DDBM (leachate discarded), with a corresponding increase in the formation of lower

158

Table 13. Heat Stable Trypsin Inhibitor Acrivity (mum. inositol phosphates (%) and phytate
content (%) in split been drum-dried meals

 

..lmsitalflasnhatafla). 1‘0le
Treatment3'4 units of'l'IA lg DDBM 193 194 195 195

 

Phytate
(%)
Raw 18.7031318d 6.72 6.33 16.21 70.74 100
CONTROL 3.292130c ND ND 25.39 74.61 100
EXTRACT 1.0881113 16.27 9.34 20.71 53.68 100
Viscozyme 2.5061-41b 4.78 1.21 15.75 78.26 100
Sprout 3.221112% ND 4.95 18.22 76.83 100

 

1 n=3, Means within a column followed by different letters are significantly different (p < 0.01)
2 Rayas-Duarte et al., 1991

3HotWarerExtraction

4 Raw,Whole Bean Meal; comm. Non-extracted; EXTRACT, Fresh formulation;
Viscozyme, Commercial preparation; Sprout, 1% germinated slurry

5 Total Phytate 8 Sum of 1P3, TP4, [PS and 1P6 (Inositol tri-, tetra-, penta- and hexaphosphates)

159

inositol phytates. No difference in phytate content was exhibited by the raw, CONTROL
and enzyme pre-digested DDBMs. These data demonstrate the potential of discarding the
soak water in reducing phytate content in processed bean products. The heat treatment
employed in drum-drying did not effectively reduce the phytate content of DDBMs.

Lectin (Phytohemagglutinin)

Table 14 summarizes results of a diagnostic assay for screening
phytohemagglutinin activity. Only the raw bean split demonstrated the presence of
agglutination within half an hour when reacted with the antibody (erythroagglutinin) for
lectin. The EXTRACT DDBM showed no visible precipitation after reacting with the
antibody, after a holding period of one hour at room temperature.

Phytohemagglutinin is also heat stable, but the absence of agglutination when reacted with
an antibody again indicated sufficient heat treatment of the DDBMs to inactivate the bean
lectin. Lectin activity in raw navy beans was half as much in raw red kidney beans, and
cooking for 10 min at 100°C inactivated all lectin activity in navy beans. Wet heat was
more effective in inactivating lectins than dry heat (Dhurandar and Chang, 1990). Coffey
et al. (1992) reported that purified phytohemagglutinin is sensitive to enzymatic treatments,
particularly proteolytic digestion, hence the hemagglutinating activity and enzyme pre-
digested DDBMs warrant investigation. Further, erythroagglutination by lectin is affected
by the molecular properties of the lectin, cell surface properties, metabolic state of cells and
conditions of assay such as temperature, cell concentration and mixing, hence the lectin

assay method to use should take these factors into consideration.
Conclusion

The composition of the drum-dried bean meals varied with the process
pretreatments utilized. CONTROL and EXTRACT DDBMs did not differ significantly in

their oligosaccharide content, but were significantly different in their in-vitro starch

160

Table 14. Visual reaction for screening of dry bean lecrin ( phyooheningglnnnin)1

 

 

Sample Sample Volume Visual Reaction
(ul)
Andbody (Ab) 10 +
Raw Navy Bean Splits O
1 -
20 -
30 -
40 -
Raw Beans with
Ab (ul)
10 +-
20 +4-
30 +++
4O +—+-++
INDBhiEXHILACT‘
10 -
20 -
3o -
4o -
INDBthDGniACHT
with Ab (10 til)
10 -
20 -
30 -
4o -
EkkmmdeWuur 10 -

 

1 n=3

2 + indicates presence of a precipitate; number of + indicates the degree of precipitation
- indicates absence of a precipitate

161

digestibilities. Protein digestibility of the DDBM was significantly enhanced by the drum—
drying process with increases approximating 9 % over the unprocessed bean.

The enzymatic pretreatments of DDBMs prior to drum-drying significantly
increased starch availability by disrupting intact cellular structures and exposing starch. It
was proposed that the denaturation of the protein matrix increased the accessibility of the
a-amylases to starch molecules. Sprouting of beans demonstrated potential as an extractive
pretreatment to improve dry bean digestibility and warrants further investigation for
applications in appropriate village-level situations in LDCs.

Results of this study suggest that dry bean digestibility has been substantially
improved by the extractive pretreatments employed. Oligosaccharide content was reduced
by the aqueous and enzymatic pretreatments. Trypsin activity has been significantly
reduced and phytohemagglutinin was inactivated by the heat treatments utilized. Results
indicated that indigestible starch may be the main factor affecting dry bean digestibility,
which could also be implicated with the flatulence problem associated with dry bean

consumption.

 

Ho: the pretreatment of dry beans by physical and biological methods will
not enhance their digestibility through removal or inactivation of interfering
components prior to drum-drying.

Reject the Ho as stated and conclude that pretreatment and processing
influences bean digestibility.

CHAPTER II FUNCTIONAL CHARACTERISTICS OF DRUM-DRIED
BEAN MEALS AND THEIR INCORPORATION IN
SELECTED FOOD SYSTEMS

Introduction

Decline in overall bean consumption in the United States and potential dietary
benefits of beans have prompted investigations into the use of legume flours in food
systems (Zabik et al., 1983a and b). Legumes are successful in increasing the quality of
protein through amino acid complementation and through increasing the total quantity of
protein in formulated food products. The utilization of bean flour from dry roasted split
navy beans has been investigated for bread making (D'Appolonia, 1978). The effect of
the addition of navy bean flour and concentrate on the rheological properties of dough and
baking quality of bread were studied by Sathe et al. (1981) and Lorimer et al. (1991). Lee
et al. (1983) reported on the physicochernical characteristics of dry-roasted navy bean flour
fractions and demonstrated differential functional performance of fractions based on
processing conditions. The substitution of navy bean flour for wheat flour in pumpkin and
banana breads and doughnut holes produced acceptable and nutritious products (Uebersax
et al., 1982; Uebersax and Zabik, 1986).

Drum drying of dry beans into precooked flakes, meals or powders could be used
to produce versatile ingredients in baked products, convenience food formulations or
replacements for cooked bean purees or refried beans. There is also potential in the
development of improved weaning food formulations especially suitable for the LDCs.

The Null Hypothesis tested in the studies is stated as follows:

HozDrum-driednavybeanmealsdonotpossesspotential foruseinfoodsystems.

The purpose of this research was to characterize the functional properties of

preheated drum—dried navy bean meals produced using selective predigestion or extraction

162

1 6 3
treatments and to evaluate the suitability of these bean meals in model formulated food

systems (quick bread and weaning beverage).

This chapter is comprised of three studies as outlined in Figure 40. Study II-l
involved the characterization of the physical and functional properties of the drum-dried
bean meals (DDBM); Study II-2 focused on the utilization of these various drum-dried
bean meals in a quick bread (pumpkin) 1311111101. and Study II-3 involved the incorporation
of DDBM in a weaning beverage formulation. Drum-dried meal prepared by aqueous
exu'action of split navy beans termed "EXTRACT" (soak, rinse and cook waters discarded
and replaced with fresh formulation water) and enzyme predigested (Viscozyme) split
bean drum-dried meal were utilized in Study II-3.

Materials and Methods

Split dry navy (Phaseolus vulgan's) beans grown in the 1991 crop year were
obtained from the Cooperative Elevator Company, Pigeon, MI. The beans soaked and
hydrated for 16 hours. (25°C) were subjected to hot water extraction (60°C/30 mins.)
prior to milling to a homogeneous slurry. Heated bean slurries were dried using a double
drum-dryer.

The physical and functional properties of the drum-dried bean meals, including
water holding capacity (WHC) and nitrogen solubility (N SI) were evaluated using
standard AACC (1984) methods (Figure 40). The microstructure of the DDBMs was
observed under Laser Scanning Confocal Microscopy (LSM).

The recipe of Rombauer and Becker (197 8) was followed in making the pumpkin
quick bread. The composite flours consisted of wheat flour and drum-dried bean meals
blended in 80:20, 60:40 and 40:60 ratios, respectively (Figure 41). The drumodried bean
meals were ground to a flour using the Fitzrnill with a 0.04 mesh screen. All formulation
ingredients were weighed to the nearest 0.01 g, sealed in clear polyethylene bags,
randomly coded and held at —23.9°C until blended, mixed to a batter and baked. Three

164

FUNCTIONAL CHARACTERISTICS OF DRUM-DRIED BEAN MEALS
AND THEIR INCORPORATION IN SELECTED FOOD SYSTEMS

SPLIT NAVY BEANS

3511-:an PRETREATMENTS
1

| | l | l
CONTROL EXTRACT VISCOZYME SPROUT

DRUM-DRIED BEAN MEALS

 

 

 

 

 

 

 

 

 

 

Functional Properties Incorporation in Food Sysrems
Color Solubility WSI WAl WHC Bulk NSI Quick Bread Beverage
density (20%. 40%. 60%) (5%. 8%. 10%)

 

1

OBJECTIVE QUALITY EVALUATION

O . I O O m.
gure . Th perrmental flowchart for Study II . Functronal CharaCterlstrcs of Dru
H 40 dr'feccleean Meals and their Incorporation in Selected Food Systems

165

SPLIT BEAN DRUM-DRIED BEAN FLOUR

l
I | I
CONTROL EXTRACT VISCOZYME SPROUT

l l

 

Pumpkin Bread

SUBSTITUTION LEVELS

 

 

0% 20% 40% 60%

BAKING
(3 X)

 

OBJECTIVE QUALITY EVALUATION

Figure 41. Flowchart of the experimental desi for Stud II-2' Incorpo '
drum-dried bean flour in pumpkin Buread y . ranon Of

166

replications of each variable were baked in a rotary oven at 177°C (350°F) for 45 mins.
Objective quality evaluations were conducted on both the batter and baked products
(Figure 41).

Different concentrations ( 5%, 8% and 10%) of the weaning food beverage were
prepared by homogenizing DDBM slurries with 200 - ml deionized distilled water in a 400-
ml polyethylene beaker for 2 minutes. A Tissumizer (T ekrnar Co., Cincinnati, OH) with
the large probe size (# 180) was used. Figure 24 summarized theevaluation ofthe
physical quality characteristics of the fully prepared bean beverage.

Results and Discussion

Physical and Functional Properties of Drum-Dried Bean Meals

Plate 6 illustrates the general appearance of the raw split beans and split bean
DDBM, as well as representative crystalline structures of the DDBM observed under a
stereomicroscope (Wild Type 256530, Heerbrugg, Switzerland). Plate 7 shows the fine
crystals of the EXTRACT DDBM compared to the coarse crystals of Viscozyme DDBM.
The microstructure of the DDBMs and starch granule appearance were also observed under
LSM (Plate 2) and discussed in detail in the study on starch digestibility (Chapter I).

The physical and functional properties of the split DDBMs are summarized in Table
15. The DDBMs exhibited a neutral color, and although CONTROL samples appeared
slightly darker than the EXTRACT samples due to the retention of the original formulation
water, they were not significantly different (p < 0.05). The DDBMs possessed a bland
flavor, and did not exhibit the distinct and frequently objectionable "beany" flavor.

The different extractive pretreatments did not have a significant effect (p < 0.05) on
the water holding capacity (WHC) and soluble solids content (°Bx) of the DDBMs, but
had a significant influence (p < 0.05) on solubility indices (WSI and WAI) and bulk
density (g/100 ml) . WAI and W8] give indications of likely cold-paste viscosity and
gelatinization, respectively (Holm et al., 1985). The enzymatic predigestion treatments

167

\ \\ \ HI: \\ SI’IIIS

(‘()\'I'R()l

G \\ \'l‘lxR l' \ IR \( "|‘I()\

Plate (1. General appearance of (a) raw navy bcan splits and
(h) [iX'l‘RAC'l' DDBM

 

Plate 7. Crystalline structure ot‘drum-dried bean meal under a
stereomicroscope (50x): (a) EXTRACT (b) VISCOZYME

 

169

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170

(Viscozyme and Sprout) significantly increased (p < 0.05) the water absorption index
(WAI) of the DDBMs. WAI serves as an index of product functionality and is therefore
important to obtain a maximum value. Avin et al., (1992) reported an increase in WAI of
red kidney beans with increased extrusion temperature, but no significant influence on

density and water solubility index (W SI).
Incorporation of Drum-Dried Bean Meals in Food Products

Quick Bread

A quick bread (pumpkin) formulation was utilized in this study because it
minimizes the problems previously encountered with incorporation of legume rich breads
and bread products (decreased flavor, volume and color) by using spices, ensuring
consistent specific volume through the method of preparation and decreased color change.
Quick breads are baked as breads, yet they lack a strong gluten development and contain a
high ratio of sugar to flour, as in a cake system; hence, incorporation of protein substitutes
is feasible (Dryer et al., 1982).

There were no significant differences (p < 0.05) in the color of the pumpkin breads
with increasing levels of drum-dried bean meal substitution (Plate 8 and Table 16). Since
these products are characteristically dark, no objective difference was observed in the color
of the products due to the promotion of Maillard browning reaction generally associated
with increased levels of reducing sugars inherent in the bean flour.

Bread loaf volume decreased with increasing levels of drum-dried bean meal
substitution, with differences most significantly apparent (p < 0.05) between the Reference
(all-purpose flour, 100% level) and breads prepared with 60% drum-dried bean meal
(particularly, CONTROL and EXTRACT) (Plate 9). Bread volume (cm3) decreased with
a corresponding increase in crumb density (Table 16) when levels of DDBM substitution
were increased, because of increased gluten formation and its inherently more compact
product structm'e. A preliminary and essential phase prior to the development of the gluten

171

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174

is the hydration of the protein and starch of the flour. With higher levels of substitution
(60%), it was possible that the DDBMs were not fully hydrated by the available fluid.

Shear force values increased (i.e., decreased tenderness) with increased level of
bean meal substitution; however, no statistical differences were detected among the various
substituted-pumpkin breads (Table 17). It is likely that increased shear force values are a
result of the decreased gluten formation and the resulting decrease in size and number of the
air cells present within the crumb structure. With higher levels of DDBM substitution
(60%), bread density is greater as a result of decreased air cells needed to develop and
expand gluten, which in turn leads to increased shear resistance force.

Table 18 summarizes the textural data obtained from the Texture Profile Analysis
(TPA) of the pumpkin breads. The TPA is a two bite method of texture analysis in which
the food is compressed twice and a complete texture profile of the sample is calculated from
the data recorded. A generalized TPA curve, a typical pumpkin bread (CONTROL DDBM)
curve and the definition and calculations of textural parameters are illustrated in figure 42.
Hardness is the area under the primary force deformation curve. Adhesiveness refers to
the area under the third curve measured during the second bite in the downstroke mode.
Cohesiveness is obtained from the ratio of area 2 (adhesive force) to area 1(hardness) in the
upstroke mode during the first bite. Stringiness is measured as the distance of area 3
(adhesiveness) from area 1 (hardness) measured in the upstroke mode, while springiness
is measured as the distance ofarea 2.

A typical pumpkin bread curve consists of only two peaks with areas corresponding
to the parameters hardness and adhesiveness. Hardness values increased with increasing
levels of drum-dried bean meal substitution, for all the different pretreated DDBMs.
Increased adhesiveness. gumminess (hardness x cohesiveness) and chewiness (gumminess
x springiness) were also observed with increased DDBM substitution levels. Conversely,
springiness and cohesiveness of the pumpkin breads decreased as substitution with drum-
dried bean meals was increased. These TPA results confirm observations that as DDBM

175

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FORCE

177

 

 

 

 

 

 

 

 

A Hardness 1
Fracturability
Point
Hardness 2
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Downstroke Upstroke Downstroke Upstroke
or HI

Typical Product Curve
(control pumpkin bread)

Figure 42. A generalized Texture Profile Anal

0

 

 

 

 

First Bite

 

 

Second Bite

 

 

 

 

PARAMETER FORMULA

V

Hardness Value of Hardness 1

Cohesiveness Area 2/Area 1
Adhesiveness Area 3

Stringiness Distance of Area 3
Gumminess Hardness x Cohesiveness
Chewiness Gumminess x Springiness
Springiness Distance of Area 2
Fracturability User Defined

pumpkin bread TPA curve.

 

TIME or DISTANCE

UNITS

Lbs. or Newtons
Dimensionless (ratio)
ln.-Lbs. or Newton-Cm
In. or Cm
Dimensionless (ratio)
Dimensionless (ratio)
In. or Cm

Lbs. or Newtons

ysis (TPA) curve and a typical

178

substitution levels increased, pumpkin bread volume decreased, with a corresponding
increase in crumb density and shear force.

Plate 10 demonstrates the cellular microstructures of the various pumpkin breads
substituted with 20% DDBM. The pumpkin bread with all-purpose flour (100%)
demonstrated a thick protein matrix enveloping starch granules. The microstructure of the
DDBM-substituted pumpkin breads were characterized by a thinner, discontinuous gluten
matrix over the flow starch granules. The protein matrix is indicated by the fluorescent
bodies. The flour and bean starch granules can not be easily distinguised, but widt the low
level of substitution, flour starch granules predominate. Microstructure of pumpkin breads
with the enzyme pre-digested DDBMs (Viscozyme and Sprout) (Plate 10d and e)
demonstrated a more extensive gluten matrix compared to those substituted with
CONTROL or EXTRACT DDBMs (Plate 10b and c), and could partly account for the
slightly taller height of resultant pumpkin breads.

Weaning Food/Be verage

The beverage concentrations utilized in this study were 5%, 8% and 10% drum-
dn'ed bean meal (DDBM) suspensions. Preliminary trials utilizing lower concentrations
(2.5% and 4%) of the beverages (Appendix D) demonstrated very limited stability,
whereas concentrations higher than 10% exhibited too viscous a consistency for a weaning
beverage.

Table 19 summarizes the physical characteristics of the bean beverages. The
soybean control and the DDBM beverages all exhibited a neutral color. The extractive
pretreatments employed did not have a significant effect (p < 0.05) on the color of the
weaning beverage. Total solids increased with the increased beverage concentrations, and
the enzyme predigested beverage exhibited a higher soluble solids content compared to the
product with EXTRACT DDBM.

179

 

Plate 10. LSM micrographs of pumpkin bread
cellular microstructure: a) Reference
(100% all-purpose flour)
b) CONTROL DDBM c) EXTRACT
DDBM d) VISCOZYME DDBM
e) SPROUT DDBM

 

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181

Visual stability of the weaning bean beverages was expressed as the amount of
clear, separated supernatant (ml) plotted against time (min), within a 2—hour observation
period A commercially prepared soybean based weaning formula (Isornil Soy Protein
Formula, Ross Laboratories, Columbus, OH )(15%) was utilized as a comparative control.
Frgure 43 demonstrates that for both the EXTRACT and enzyme predigested (Viscozyme)
bean beverages, suspension stability increased with increased beverage concentration.

The 15% soybean formula was completely stable with time and did not exhibit
any settling of solids. With very low concentrations of the beverage (2.5%, Appendix D),
settling of solids with a corresponding increase in separated supernatant, occurred almost
immediately after homogenization. The 10% Viscozyme sample closely approximated the
stability of the soybean control; however, the consistency was highly viscous. In general,
the Viscozyme DDBM beverages were relatively more stable than the EXTRACT DDBM
suspensions. Plate 11 demonstrates the effect of extractive pretreatments as well as
concentration on the relative stabilities of the weaning beverages. After a one hour static
holding period, the 8% and 10% concentrations were still stable, particularly the enzyme-
predigested (V iscozyme) sample. However, the 10% level might be too viscous a product
for a weaning food. A recommended weaning product is one which will produce a low
paste viscosity or high calorie density to prevent choking of infants, hence the 8% level
was judged to be a more appropriate level.

A micro-quantitative method designed to evaluate beverage stability involved
measuring spectrophotometric Absorbance (565 nm) (or % Transmission, Appendix D) of
dilute suspensions prepared using differential quantities (ranging from 2.5 to 40 ul) of the
weaning beverages prior to the settling of the solids immediately after the homogenization
step. Figures 44 to 46 illustrate the effects of the different concentrations (5%, 8% and
10%) of EXTRACT and Viscozyme DDBMs on the stability of the bean beverages. The
regression equations derived demonstrated good correlation between the absorbance

readings and concentration, and provided a tool to quantitate the influence of DDBM

182

 

 

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200

100

‘l'lmo (min)
Figure 43. Comparative visual stability of bean beverage

from DDBMs and soy formula

183

VISCOZYME

Wing, r

G ‘ _ ' “"“""cr-m.,m....

Plate 1 1. Appearance of weaning beverage prepared with different
levels (5% 8%, l()‘7c)of pretreated (a) EXTRACT and
Viscozyme DDBMs and (b) CONTROL and Sprout
DDBMs

 

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185

concentration on stability of the beverage suspensions. As sample concentration was
increased, absorbance reading increased (decreased % transmission) which indicated a
decrease in settling of solids or a corresponding increase in suspension stability. Figures
47 to 49 demonstrates that at higher concentrations (8% and 10%), the Viscozyme
DDBM beverage exhibited higher absorbance readings, indicating a relatively more stable
suspension than the EXTRACT DDBM beverage.

Figures 50 and 51 illustrate the comparative transmission (%) of the DDBM
beverages over time, another index of stability/settling of solids with time. The higher
beverage concentration (10%) exhibited lower transmission (%), indicating decreased
settling of solids or increased suspension stability compared to the lower concentration
(5%). The 15% soybean control exhibited consistent transmission (%) with time,
indicating no separation of solids and therefore complete stability during the period studied.

Figure 52 summarizes the apparent viscosities of the various weaning bean
beverages measured using a Brookefield viscometer. The 8% and 10% concentrations of
the enzyme prc-digest (V iscozyme) beverage were significantly (p < 0.05) higher than the
corresponding concentrations of the EXTRACT beverage. The 5% beverages did not
differ significantly (p < 0.05). Relative viscosities of the 5% beverages are illustrated in
Figure 53. Viscozyme was significantly (p < 0.05) higher than the 5% EXTRACT
beverage, confirming the results for apparent viscosity . The 8% and 10% concentrations
produced extremely viscous suspensions which were too thick to pass the Oswald pipet;
hence relative viscosities were not measured. Figures 54 and 55 illustrate the apparent
and relative viscosities of the bean beverages with the addition of commercial emulsifiers
and stabilizers (Methylcellulose, xanthan gum, Carageenan systems - Seakem®, Lactarin
MU®, Viscarin GP and SD®; details in Appendix D) obtained from FMC Corporation
(Marine Colloids Division, Philadelphia, PA) . Results demonstrate the dramatic increase
in apparent viscosity with the addition of 1% levels of carageenan systems (LactarinO,

Viscarin® GP and SD and Seakem®). The emulsified beverages demonstrated greater

Absorbance (0“ an)

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from DDBM and soy formula

188

 

 

   

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Figure 52. Viscosity of bean beverages from
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Figure 53. Time for the bean beverage (5 %) to flow through
the two marks of an Oswald pipette

Viscozyme

Extract

l 8 9
stability (Figures 56 and 57) by exhibiting less settling of solids over a given period
compared to those bean beverages without added emulsifier. The levels of emulsifiers
utilized in this study were based on the recommendations of the manufacturer according to
the dispersion media (water or milk) and functional categories or applications (gelation,

viscosity and cold solubility).

Conclusion

Results demonstrated the feasibility of incorporating drum-dried navy bean meals in
pumpkin quick breads and weaning food/beverages. Acceptable quality breads were
produced at the 20% and 40% levels of substitution. Enzyme predigested DDBMs possess
great potential in weaning food formulations, especially at the 8% concentration level. The
use of indigenous enzymes ( i.e., sprouting ) to modify the functional properties of DDBM
used in weaning beverage formulations warrants additional research.

Potential optimization of pretreatments exists to obtain specific functional properties
of the meals. High quality products were obtained by the extractive pretreatments and
drum-drying process. The functional properties demonstrated through incorporation of
DDBM in a quick bread indicated potential use in a broader spectrum of baked products.
Potential of the drum.dried bean meals in weaning food formulations to enhance the

nutrient density of infant diets also warrants further research.

 

Ho: Drum-dried navy bean meals do not possess potential for use in food systems.

Reject the Ho and conclude that drum-dried bean meals possess functional
properties suitable for incorporation into nutritional food products.

 

190

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CHAPTER III MICROWAVE HEATING CHARACTERISTICS OF WHOLE
BEANS and BEAN STARCH

Introduction

A major deterrent to the utilization of dry beans is the long cooking times involved
in their preparation. Consumers favor convenience foods which provide consistently high
quality with minimum delay. Commodities receiving some pre-preparation which reduce
cooking requirements enhancing the potential for microwaveable reheating are sought.
Although numerous specialized applications of microwave energy for processing or
cooking food products have been developed, little attention has been directed towards its
utilization in legumes. Microwave heating has a direct potential for use as a
preconditioning treatment for process time reduction of dry beans.

Starch granules undergo physico-chemical changes during thermal processing. Dry
bean starch granules are resistant to swelling and rupture; their gelatinization temperatures
range from 60°C to over 75°C, relatively high compared to many standard cereal grains
(Hahn et al., 1977). Bean starch has a higher percentage of amylose ( generally 30-37%)
than most plant starches, which increases the potential for formation of indigestible
retrograded starch (Hoover and Sosulski, 1985). During cooking, some of the amylose
leaches from the starch granule. Since the cell structure in cooked beans remain intact,
much of the leached amylose remains within the cell and fills spaces between protein bodies
and starch granules. The unique properties of bean starch can have practical applications in
food systems, particularly in modifying product textural quality.

The Null Hypothesis of this study is stated as

Ho: Microwave energy will not adequately disrupt starch granules and facilitate

gelatinization in rim to irrrprove whole seed cooking, functional properties
and digestibility.

191

192

The objectives of the studies were : 1) to evaluate microwave heating as a
pretreatment to dry beans prior to processing, storage and final preparation and 2) to
compare the effects of microwave and conventional heating on the physical and functional
properties of navy bean starch.

This chapter is comprised of two parts : Study I - the microwave preconditioning
of whole dry navy beans and evaluation of resultant bean characteristics; and Study 11 -
determination and comparison of the effects of conventional and microwave heating on

the physical and functional properties of isolated navy bean starch were compared.
Materials and Methods

Microwave Preconditioning of Whole Dry Navy Beans

The experimental plan for this Chapter is presented in Figure 58. Standard (C-20)
whole dry navy beans were ambient soaked for different time periods and allowed to
equilibrate at 4°C for 2 days. The beans were thus rehydrated to different moisture
contents ( range 47% to 54% ) and subjected to microwave heating at three microwave
energy levels (440, 590 and 740 watts) for 2 minutes in a Radarange microwave oven
(Model - RS458P, Amana Refrigeration, Inc.). A microwavable pressure cooker
(Nordicware) was used to contain the beans and reduce dessication during heating. The
heated beans were subsequently packaged in polyethylene ziplock bags and stored at
(-)18°C.

The frozen preconditioned beans were heated in the microwave oven for 30
seconds. Microwave heating time was established after a come-up time ranging from 3 to 7
minutes. Control beans were conventionally cooked in an open kettle for 15, 30 and 45
minutes.

Texture of microwave and conventionally cooked beans were evaluated using a

Kramer Texture Test System ( Model TMS-90) equipped with the FT 3000 transducer and

MICROWAVE HEATING CHARACTERISTICS OF WHOLE BEANS and BEAN ST ARCH

193

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DRY NAVY BEANS
PRODUCT APPUCATION] CHARACTBUZATION OF
TRIAL HEATED ST ARCH
WHOLE BEANS Mm
' ‘Wb'fj" BEAN STARCH
W7 . [4.8 % Mm
. 34.9 % Amylase
'MI' ca' _owxva r'iiacoanmomo -02¢ a. Man
. 47445 Noam
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. Room Town
. PM Levels: MICRO“! AVE
440 W. 590 W, 740W TING
. Tune - 2 sin. “EA
. 70. an. 90 C , 100 c
. 2 min.
FROZB‘J STORAGE
- 18 C I
FUNCTIONAL PROPERTIES
CHARACIERIZATION
MICROWAVE HEATING] - Nu”! ram
, . Sobrbrhrylnda
I . Degree (JIM
. W Viscosity & elasticity
EVALUATION OF
PRODUCT
. Tantra
. Color
. Mia-cam

 

 

 

Figure 58. The experimental flowchart for Chapter III: Microwave preconditioning of dry

 

 

 

 

 

 

 

 

 

 

 

CONVHITIONAL

 

 

 

 

 

beans (Study 1) and microwave heating of bean starch (Study 11)

 

194

the No. 015 standard multiple blade shear compression cell. Color of beans was
obtained using a Hunter Lab Model D25-PC2 (Hunter Associates Laboratory, Inc.,
Reston, Va.).

Cellular structure and starch granule appearance of microwave pre-conditioned
beans was observed under SEM (Model JSM 35CF, Japan Electron Optics Limited,
Tokyo, Japan) and LSM (Carle Zeiss, Inc., West Germany).

Microwave Heating of Bean Starch

Navy beans (Fleetwood cultivar [firm texture) grown during the 1987 crop year in
research plots in Saginaw, Michigan, were used in this study. Bean starch was isolated
using the method described by Naivikul and D'Appolonia (1979) with slight modifications.
Commercial corn starch (Argo R Pure Corn Starch, CPC International Inc., Englewood
Cliffs, NJ) was used as a comparative control. Proximate Analyses were conducted using
the standard methods (AACC, 1983 and AOAC, 1984). The amylose content was
determined by a modification of the colorimetric procedure of Morrison and Laignelet
(1983).

Starch slurries were heated in screw-cap culttue tubes ( 14 mm ID.) using a
Radarange microwave oven (Model - RS458P; Amana Refrigeration, Inc.) with a 700 watt
rated power. Temperatures were monitored using a Luxtron 755 unit with MIW-02-10740
fiber optic probes. For conventional heating, starch slurries were heated in screw-cap test
tubes (14 mm ID.) placed in a thermostat-controlled water bath.

Bean starch physical properties including swelling power, solubility, degree of
syneresis, gel rheological properties and examination of cellular structure and starch

granule appearance using SEM were evaluated.

195

Results and Discussion

Microwave Preconditioning of Whole Dry Navy Beans

Figure 59 illustates the moisture gain in dry beans during soaking, yielding a
typical water uptake response which plateaued after 5 hours of soaking at room
temperature. Moisture content of the soaked beans ranged from 50 to 55% during an
overnight soak period (16 hours). These results are consistent with numerous previous
reports (Uebersax, M., 1985; Tittiranonda, A., 1984; Burr, et. al., 1968).

Significant differences in bean texture (p < 0.05) were observed after microwave
preconditioning (Figure 60). Preconditioned samples with higher initial moisture content
(54%) possessed a softer texture (required a lower shear force ) compared to samples with
lower initial moisture content (47%). Figure 61 demonstrates that texture of the frozen
preconditioned samples was significantly softer (p < 0.05) after the 30-second cook.
Lower microwave energy level (440W) was associated with longer come-up times which
resulted in significantly higher weight gains (Figure 62) (p < 0.05) during cooking due
partly to longer sample/water contact. Figure 63 illustrates that as weight gain (% above
soak weight) increased, the texture of microwave cooked bean samples approached that of
the conventionally retorted 115°C (240°F) canned bean samples, regardless of the
preconditioning microwave power levels used. Initial bean hydration level and the
maintenance of moisture during microwave heating are critical factors which affect final
product texture. Sufficient thermal energy and adequate moisture are needed to produce a
desirable and palatable texture. A desirable texture of cooked beans was associated with a
weight gain of approximately 30% above the initial bean soak weight. Figure 64
demonstrates that color of the final product (expressed as lightness, L) was a function of
both initial moisture content of the bean and microwave processing power level. In

general, beans of softer texture, darker color and higher yield were produced using the

Content (96, w.b.)

Holatura

196

 

 

 

 

 

 

60

1 + fl
50 -'
40 d
30 -
20 -

I

10 r r

0 1 O 2 O

Soak Tlme (mln.)

Figure 59. Moisture content of dry navy beans soaked at 25 C for

various time periods

Shear Force (N)

Shear Force (N)

197

 

4000
c 1%“!qu
WWW) I 440W
‘ Z 590w
:
D 740W
3000‘

 

 

 

 

L

 

 

50 54
Been Mdature Quint (5, rub.)

Figure 60. Maximum force required to shear navy beans preconditioned
by microwaves In a Nordicware Presure Cooker

 

zooo
WWau-p I 440W
Minn-”much
I 590w
. b I 740W

 

 

 

47 50 54
Been MdstnreCartcat (%, sub.)

Figure 61. Maximum force required to shear preconditioned navy bears

after reheating (0.5 min., 740 W) in a Nordicware
premre cooker

198

 

MW 440W
‘ (Mbowavel’owulaveh)

ISOOW

Weight gain (5 above soaked wt.)

l

 

 

 

 

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limilillliili!

 

 

50 54
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Figure 62. Total incense (‘5 above soak weight) in weight of preconditioned

Cooked Product Texture
(Max Shear Force, N)

navy beans after retreating (0.5 min., Nordicware pressure cooker)

 

1000
1400 - '
y - 1034.5 ° 10%-Leeann) Fl‘z - 0.4e2
1
1200 -

 

 

 

 

 

we. Cab
(95 Above Seded Weight)

Flgure63. Maximum forcerequlredtoshearnavybeanscookedlna
conventional retort (45 minutes, 116 C) or subjected to
microwave energy levels in a Nordicware Pressure Cooker

Lightness (L) value

199

 

 

 

80
J
60‘ .—
E El 440w
40.. E 590W
E E 740W
20‘ 'E
O :—

 

 

 

 

 

SO

Moisture Content (96, Mb.)

Figure 64. Color of final product as affected by post-soaking moisture content
and microwave energy levels used for preconditioning (Nordicware
pressure cooker)

200

pressure cooker at lower microwave energy levels with subsequent longer total cooking
times (Table 20).

Plate 12 demonstrates that the average diameter of starch granules in soaked beans
was approximately twice as large as that in dry seeds as a result of water uptake during the
16 hours of ambient soaking. Microstructm'al changes including cellular disruption and
separation were observed as a result of microwave preconditioning and reheating (Plate

13).

Microwave Heating of Bean Starch

The chemical composition of corn and isolated navy bean starch is presented in
Table 21. The moisture and protein contents of the two starches are significantly different
(p < 0.05). Corn starch has a much lower (p < 0.05) amylose content than navy bean
starch.

There was a significant difference (p < 0.05) in the swelling power between
microwave and conventionally heated corn and bean starches. Both corn and bean
starches microwave heated at 70°C exhibited a higher swelling power compared to those
conventionally heated (Figures 65 and 66). With microwave heating, the characteristic 2-
stage pattern of swelling of corn starch was not observed. For both methods heat
treatments, corn starch granules exhibited a much higher degree of swelling compared to
bean starch. Bean starch demonstrated the characteristic restricted swelling pattern under
microwave heating. Swelling power of conventionally heated bean starch more than
doubled when heated within the range of 70-80°C. Microwave heating did not have a
significant effect (p < 0.05) on swelling power at the same temperatme range. Microwave
heating at 70°C had a significant effect (p < 0.05) on swelling potential of bean starch
which was observed to be more than double that of starch heated using the conventional

heating method

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Swelling Power

Swelling Power

 

204

 

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70 90

00
Temperature (degree C)

Figure 65. Swelling power pattern of bean
starch with conventional and
microwave heating

 

 

 

 

7 0 80
Temperature (degree C)

Figure 66. Swelling power pattern of corn starch
with conventional and microwave heating

205

 

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Figure 68. Solubility Index (81) corn and bean starches
during conventional hen g

206

Solubility of both starches was low with microwave treatment, indicating some
leaching of amylose molecules out of the starch granules (Figures 67 and 68). Corn starch
solubility was higher than bean starches throughout the temperature range 70—90°C for
microwave heating. Increased solubility of starches with increased temperature was
observed for conventional heating. The restricted swelling power and solubility patterns of
the bean starch indicates the existence of strong bonding forces within the granules,
allowing the starch to gradually relax when heated ova the temperature range 70-900C.

Conventional heating resulted in a significantly higher degree of syneresis for both
corn and bean starches (p < 0.05) than observed for microwave heating, as demonstrated
by a greater amount of exudate. Corn starch gels have a well recognized ability to hold
liquid, and are expected to have only slight exudate, as illustrated by the microwave heated
sample (Figure 69). However, the conventionally heated corn starch sample exhibited a
very low liquid holding capacity, as demonstrated by the greater gel shrinkage and higher
percentage of exudate. The conventionally heated bean starch sample exhibited a
significantly higher (p < 0.05) degree of syneresis and percentage of exudate compared to
microwave-heated bean starch, as shown in Plate 14 This observation was consistent with
results which demonstrated relatively low swelling power at 70°C (Figure 65).

According to Ring (1985), starch gel rigidity is affected by granule deformability
and the strength and stability of matrix amylose gel. Results demonstrated conventional
heating had a significant increase (p < 0.05) on the rigidity of heated corn starch gels
compared to microwave heating, while bean starch gels exhibited a slightly softer gel when
microwaved (Figures “IO-71). Corn starch has been previously reported to have a low
amylose content and weak bonding strength within its starch granules, which could partly
account for its ease of deformation. Bean starch possesses a higher amylose content

(p < 0.05) and a higher tendency for retrogradation than corn starch.

207

 

Degree of Syneresls (%)

 

 

 

ShrehMeat Treatment
Figure 69. Degree of syneresis(%) of 8% (w/w)

starch gels stored at room
temperature for 1 week

x!“ mm \\ l~ lll‘..\'l'|.\(;. 700C

 

Plate 14. 8% starch gels stored at room temperature for 7 days

Apparent Vlacoalty (polaa)

Apparent Eaatlelty (NlernZ)

208

 

 

 

 

 

Starch/Heat Treat-eat

Figure 70. Apparent viscosity (poise) of 8%(w/w) starch gels
atoredatroomtemperaturefor‘ldays

1.0

 

 

 

 

4 "f: 55] //
o.o '

 

 

Strch [Red Meat

Figure‘ll. ApparentElasticityof 8% (wlw)starchgelsstoredatroorn
tem‘ldaperaturefor

209

The Scanning Electron Micrographs (SEM) of corn and bean starches microwave
and conventionally heated at 70°C for 2 minutes are shown in Plates 15 and 16,
respectively. Corn starch granules appeared to have collapsed and melted under both
methods of heat treatment, possibly as a result of solubilization of the amylose. However,
for the microwave heated corn starch, disintegration of the starch granule was more
pronounced with matrix formation of the leached materials. These data for corn starch
coincide with the very high swelling power, without the characteristic 2-stage pattern,
observed at 70°C .

The more resistant bean starch granules still appeared swollen under both methods
of heating. Fibrous-like exudates, generally recognized to be amylose, leached out of the
granules. However, the microwave-heated starch appeared to have a greater quantity of
fibrous-like exudate leaching , with some evidence of matrix formation. The bean starch
granules also exhibited shrinkage and deformation within the central region, indicating a
weak bonding region.

Conclusion

Microwave preconditioning of dry beans demonstrated the potential of microwave
energy to improve whole bean seed cooking, ensure rapid processing of beans by
disrupting starch granules and facilitating gelatinization. A number of factors must be
controlled in microwave processing of beans to assure appropriate quality. These factors
include: a) bean initial moisture, b) the water to bean (liquid/solids) ratio, c) microwave
power level (wattage) and d) heating period. Palatable products may be obtained upon
application of a suitable combination of these factors.

The physical and functional properties of pure bean starch were significantly
affected by microwave heating. Microwave heated starch gels exhibited a significantly
lower degree of syneresis and a slightly softer texture than did conventional heated gels.
Amylose molecules leached out from the bean starch granules when microwave heated at

210

 

Plate 15. Scanning Electron Micrographs of (a) Bean and
(b) Corn starch granules microwave—heated at
700C for 2 minutes

211

  

a ISKU X466 6683 19.6U eaten

SKU H488 8886 19.5D £5099

 

Plate 16. Scanning Electron Micrographs of (a) Bean and
(b) Corn starch granules with conventional

heating at 700C for 2 minutes

212

70°C for 2 minutes, compared to conventionally heated bean starch. Microwave heating
of isolated bean starch slurries confirmed the potential for the use of microwave
preconditioning of dry beans in rim to facilitate subsequent cooking. Further work to
provide continuous in-line process treatments which could facilitate improved ease of

cookability in bean is warranted

 

Ho: Microwave energy will not adequately disrupt starch granules and facilitate
gelatinization in rim to improve whole seed cooking and functional
properties.

Reject the Ho as stated and conclude that microwave energy
preueatments dramatically influence cooking properties of dry beans.

 

SUMMARY and RECOMMENDATIONS

The research conducted in the course of this dissertation has been directed towards
the improved utilization of dry edible beans. The hypothesis and objectives have been
focused on the development of processing methodologies which may be adaptable to
centralized small and intermediate-scale processing operations and provide products of
utility for feeding of young children. All of the studies incorporated practical applications
of fundamental technologies and have provided results which indicate that improved
functional and nutritional properties may be obtained through process manipulation.

The process pretreatments employed in this research influenced the composition of
the drum-dried bean meals. CONTROL and EXTRACT DDBMs significantly differed in
their in-vitro starch digestibilities. Oligosaccharide content was reduced by the aqueous
and enzymatic pretreatments. The use of enzymatic predigestions was conducted with
commercially available enzyme preparations to provide fundamental information regarding
the changes in intrinsic properties of bean meals. The use of germinated seed as a resource
for development of indigenous enzymes was provided to assure linkage to appropriate
village-based technology . The enzymatic pretreatments of DDBMs significantly increased
starch digestibility by disruption of intact cellular structures and denaturation of protein
matrix, thereby increasing accessibility of the amylases to starch. The results reported
indicated that the use of enzymatic digests derived from indigenous enzyme sources has
potential for immediate adoption. Further research is warranted in the development of
selected enzymes from native plant materials.

The dmmdrying technology utilized in this research has direct application for
village-level and cooperative centralized processing. Such a facility would enable the
Pmduction of nutritious food ingredients derived from dry edible beans for use in a variety
0f products. Protein digestibility of DDBMs was significantly improved by the drum-
drying process. Trypsin activiity was significantly reduced, and phytohemagglutinin was
inaCtivated by the high heat treatment utilized during the drum-drying process.

213

2 l 4

The feasibility of incorporating DDBMs in pumpkin quick breads and weaning
food/beverages was demonstrated. Acceptable breads were produced at the 20% and 40%
levels of substitution. Enzyme pre-digested DDBMs possess great potential in weaning
food formulations, particularly at the 8% concentration level. The research conducted on
the functional properties of prepared bean meals has demonstrated that these products are
functionally suitable for use in standardized foods. Further research is warranted to
develop specific recipes for use in regionalized traditional foods.

The potential for development of weaning beverages with reduced viscosity and
improved nutritional value has a significant potential for improving the nutritional status of
young children. Ingredients for these foods have been prepared and provide a readily
accessible option. The bean ingredient could be blended with other appropriate ingredients
mixed with water and available without additional cooking. Work is warranted to develop
additional native products suitable for blending with the DDBM to enhance and optimize the
formulation.

The use of microwave energy although appearing as an advanced technology, was
demonstrated to have potential as a pretreatment for enhancing the cookability of dry beans,
and for modifiying physical and functional propterties of bean starch for applications in
food systems. Such a protocol could be established in a centralized and public sector
framework to facilitate pre-processing of dry beans. Whole dry beans could be prepared
and pretreated with microwave energy to enable enhanced cookability and storage of dry,
shelf-stable bean products with improved cooking potential. This methodology could be
established as a continuous operation and provide sound whole seed for consumer use.
Such atechnology wouldreducetherequirements forlongtermcookingandthe associated
requirements for fuel gathering and use.

The development and use of technologies to enhance the use of dry beans must be

Pursued within the context which is sensitive to the endproduct outcome. The products

derived under a village-level organizational scheme could benefit families, especially those

21 5
with young children, and the overall economic development within the region through the
utilizationn of women in the development and processing of value-added nutritious food
products. Additional processing methodologies could be incorporated into this central pre-
processing regime. These enterprises would enable women to serve in managerial,
supervisory and labor roles.

The research conducted in this dissertation has provided technological
methodologies to improve the utilization of dry edible beans. This work provides a
framework for further research in this area. It is essential that additional research be
focused on village-level adaptation of these technologies in the framework of the Bean
Cowpea-CRSP to assure the sociological integration of technologies designed to enhance
the use of dry beans throughout the world.

 

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