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THE NUTRIENT AND PHYTONUTRIENT COMPOSITION OF ONTARIO GROWBEANS AND =IR EFFECT ON AZOXYMETHANE INDUCED COLONTC PRENEOPLASIA IN RATS
Heten Anita MiUie Samek
A thesis submitted in confonnity with the requirements for the degree of Masters of Science Graduate Department of Nutritional Sciences University of Toronto
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The author retains ownership of the L'auîeur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenivise de ceile-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. TEE NUTRIENT AND PaYTONUTRIENT COMPOSITION OF ONTARIO GROWN BEANS AND THEIR EFFECT ON AZOXYMETHANE INDUCED COLONIC PRENEOPLASIA IN RATS
Master of Science, 200 1 Helen A. Samek Graduate Department of Nutritional Sciences University of Toronto
Beans have long been recognized for their nutritionai quality. Epidemiological studies have shown that populations consurning substantial amounts of beans in their daily diets
are at low risk of developing many chronic diseases including colon cancer. The ht aim of this thesis was to Uivestigate both the nutritional and phytonuûient composition of raw and processeci Navy, Kidney and Fava beans. The nutrient and phytonutrient analysis of raw was in good agreement with published literature. The nutrient and phytonutrient content of canned beans, homogenized with the liquid content found in the can, appeared to be slightly higher than those beans that were not homogenized with the liquid contents, suggesting leaching. The second airn was to investigate the effects of canneci navy bean consumption on the development of preneoplastic lesions in the colons of azoxyrnethane-treated F344 Fisher rats. The incidence of aberrant crypt foci
(ACF) and aberrant crypts (AC), induced by azoxymethane in rats fed csuzned navy beans showed signifiant reductiom in the number and sïze compareci to rats consuming a bean-fke diet This study demonstrates that Navy, Kidney and Fava beans contain anticarcinogenic phytonutnent compounds capable of inhibithg chemically-induced preneoplastic lesions in the rat colon. 1 would like to extend my deepest appreciation and gratitude to my supervisor Dr. A. Venketeshwer Rao, whose endless support and supervision guided me through this thesis. Thdyou for your continued patience, encouragement and for giving me the opportunity to see myself grow as a strong, capable individual.
1 will forever be indebted to Dr. David Yeung for his expertise, his continued belief in my capabilities, his desire to see me succeed and above al1 for being such a good fiiend. Your have instillecl the desire in me to reach for the stars.
I am sincerely grateful to Dr. David Jenkins for his insight and guidance. Your helpfül suggestions and encouragement were very much appreciated.
A big thank you to the H.J. Heinz Company of Canada and The Ontario Bean Growers Association for generously helping to fùnd this research project. 1 am also grateful to the University of Guelph for assisting me with some of my analysis.
I would Iike to thank my fellow graduate students Sujatha Chakravarthi and Linda Kim for their support when the bill seemed almost too big to climb, and to Debbie Gurfinkle whose insight always helped me see the brighter side of things. 1would also like to thank al1 of the mernbers of Dr. Rao's Lab; Dr. Sanjiv Agarwd, Anita Agarwal, Nalini Shiwnarain, Judlyn Fernandes and Honglei Chen whose fiiendship was often at times a driving force.
1 would also like to extend my appreciation to Margaret Hardy, Janet Campbell, Emalia D'Souza and Voula Philips for their patience and helpfùl hand.
To Rose Ricupero, Nicole Dollo and Anna-lisa Masci. You have al1 been an endless pillar of strength. Your love and support continues to redefme the tme meaning of fnendship.
1 want to thank my father whose zest for knowledge has always inspired me to be the best 1 can be. 1 have looked up to you Erom day one and you have continued to support dl my endeavors with much support and understanding. You will always be a part of al1 my successes. 1 would also like to thank my mother and rny brothers for their encouragement and support. Without your love, littie would be possible.
To John Emanoilidis. You have been there through it ALL. Your belief that 1 cm accomplish al1 that 1 desire has allowed me to continue through the toughest of times. I could not have done this without your everlasting encouragement, understanding and patience, your unwavering support, and above dl, your love. TABLE OF CONTENTS
Page List of Tables
List of Figwes
Publications Ansing fiom this Thesis Research xiii
CHAPTER 1 : UWRODUCTION 1
1.1 introduction 2
CHAPTER 2: HYPOTHESIS AND OBJECTIVE 7
2.1 Rationaie 8
2.2 Hypothesis
2.3 Objectives
2.3.1 Overall Objective
2.3.2 Specific Objectives
CHAPTER 3 : LITERATURE REVIEW
3. f History
3.2 Patterns of Bean Conswnption
3.3 Nutrient Profile
3.3.1 Mamnutnent Composition
3 -3.1.1 Protein
3.3.1.2 Carbohydrate
3.3.1.3 Fat
3.3.2 Micronutrient Composition 3.3.2.1 Vitamins
3.3.2.2 Minerais
3.4 HdthBenefits of Beans
3 -4.1 Coronary Heart Disease
3 A2 Diabetes Mellitus
3.4.3 Obesity
3.4.4 Cancer
3.5 Health Benefits of Phytonutrients
3 .S. 1 Phytic Acid
3.5.2 Saponins
3.5.3 Oligosaccharides
3 SA Trypsin Inhibitors
3.5.5 Lectins
3.6 Summary
CHAPTER 4: PROXIMATE ANALYSIS
4.1 introduction
4.2 Objective
4.3 Materials and Methods
4.3.1 DryBeanSamples
4.3.2 Dry Bean Preparation
4.3.3 Nutrient Determination
4.4 Results 4.5 Discussion 65
4.6 Summary 71
CHAPTER 5: PHYTONUTRIENT COMPOSïîION 77
5.1 Introduction 78
5.2 Materials and Methds 79
5.2.1 Dry Bean Samples and Dry Bean Preparation 79
5.2.2 Phytonutrient Determination 79
5.3 Results 84
5 -4 Discussion 95
5.5 Summary 99
CHAPTER 6: THE EFFECT OF NAVY BEAN CONSUMPTION ON
AZOXYMETHANE-INDUCED COLONIC PRENEOPLASIA 1O 1
6.1 Introduction 102
6.2 Objective 103
6.3 Materials and Methods 103
6.4 Results 1OS
6.5 Discussion 118
CHAPTER 7: GENERAL DISCUSSION 124
7.1 General Discussion 125
CHAPTER 8: SWMMARY 136
8.1 Swnmary 137
CHAPTER 9: CONCLUSION 140
9.1 Conclusion 141 CHAPTER 10: FUTURE DIRECTIONS
10.1 Future Directions
CHAPTER 1 1 : REFERENCES
1 1.1 References
viii LIST OF TABLES
Page
Table I .1 Legume Classification and their Biotanical and Cornmon Name
Table 3.3.1 Nutrient Content of Selected Cooked Beans
Table 3.3.2.1 Vitamin Content of Selected Cooked Beans
Table 3.3.2.2 Mineral Content of Selected Cooked Beans
Table 4.4.1 Proximate Composition of Raw and Canned Navy Beaas
Table 4.4.2 Proximate Composition of Raw and Canned Kidney Beans
Table 4.4.3 Proximate Composition ofRaw and Canned Fava Beans
Table 4.4.4 Proximate Composition of Tirne-Treated Cooked Navy Beans
Table 4.4.5 Total Dietary Fiber Content in Raw and Camed Navy,
Kidney and Fava Beans
Table 4.4.6 Total Dietary Fiber Content in Raw and Canned Navy,
Kidney and Fava Beans as reported by USDA Handbook No.8
Table 4.4.7 TotaI Folate Content in Raw and Canned Navy,
Kidney and Fava Beans
Table 4.4.8 Total Folate Content of The-Treated Cooked Navy Beans
Table 5.3.1 Phytonutrient Composition of Raw and Canned Navy Beans
Table 5.3.2 Phytonutrient Composition of Raw and Canned Kidney Beans
Table 5.3.3 Phytonutrient Composition of Raw and Canned Fava Beans
Table 5.3.4 Phytonutrient Composition of The-Treated Cooked Navy Beans
Table 6.3.1 Mamonutrient Composition of Experimental Diets
Table 6.3.2 Micronutrient Composition of Experimental Diets Table 6.4.1 Effects of Dry Bean Consumption on Body Weight,
Food lntake, Protein Efficiency and Food Efficiency
Table 6.4.2 Mean Daily Food and Protein Intake
Table 6.4.3 Effect of Dry Bean Consumption on AOM-Induced
ACF in F344 Fisher Rat Colon NOTE TO USER
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This is reproduction is the best copy available LIST OF FIGURE3
Page
Figure 3 -3.1 Cornparison of Macronutrient Content of Oilseed Legumes
(Soy Bean) and Grain Legumes (Kidney Beans) 17
Figure 4.4.1 Percentage Decrease of Proximate Content fiom Raw Navy Bean
Seeds at Various Cooking Intervais 59
Figure 4.6.1 Pacentage of Recommended Daily IntaLe of Mamnutrients, Fiber
and Folate fÎom One-Cup Serving of Canned Navy Beans 73
Figure 4.6.2 Percentage of Recommended Daily Intake of Macronutrients, Fiber
and Folate fiom One-Cup Seniing of Canned Kidney Beans 74
Figure 4.6.3 Percentage of Recommended Daiiy Intake of Macronutrients, Fiber
and Folate fiom One-Cup Serving of Cmed Fava Beans 75
Figure 5.3.1 Percentage Reduction of Phytonutrient Content of Canned Navy Beans in
Tomato Sauce and Without Tomato Sauce From Raw Bean Seed 9 1
Figure 5.3.2 Percentage Reduction of Phytonutrient Content of Canned Kidney Beans
in Water and Without Water From Raw Bean Seed. 92
Figure 5.3.3 Percentage Reduction of Phytonutrient Content of Canned Fava Beans
in Water and Without Water From Raw Bean Seed. 93
Figure 5.3.4 Percentage Reduction of Phytonutrient Content of Tirne-Treated
Cooked Navy Beans From Raw Bean Seed. 94
Figure 6.4.1 Changes in Fecal Output Behueen Ban-fed Diets and
Control-fed Diets Over The
Figure 6.4.2 Percent ACF Decrease in Bean-fed Animals fiom Controls
xii PUBLICATIONS ARISING FROM TECESIS RESEARCH
Samek HA, Rao, AV. The effect of navy bean coasumption on azoxymethane-induced colonic preneoplasia in rats. Nutr and Cancer, 200 1 (in press).
Samek HA, Rao, AV. The nutrient and phytonutrient composition of Ontario grown beans. Nutr Res, 200 1 (in press).
Samek HA,Rao, AV. The eflect of navy bean consumption on azoxymethane-induced colonic preneoplasia in rats. FASEB Conference, Ottawa, 2001.
... Xlll CHAPTER 1
- INTRODUCTION - 1.1 INTRODUCTION
Beans, which are categorized as legumes, have been cultivateci for thousands of years and
have played an important role in traditional diets throughout the world (Messina, 1999;
Deshpande et al., 1984). Bean crops have been especially important as a staple in
developing areas like Afiica, Latin America and Asia, pviding an eoonomical source of energy and high quality protein (Gd and Anderson, 1994; Gupta, 1983). However, in societies where the consumption of meat is quite high, such as those found in Western civilizations, bean consumption is infiequent and in small amounts. Its position as a less significant component in the Canadian diet may be a result which label bans as the
"poor man's rneat", suggesting an inverse relation between bean intake and incorne
@dessina, 199 1). Other reasons for a lower co~lsumptionof beans may be related to long cooking tirnes and social inconveniences. Unfortunately, the lack of consumer knowledge with regard to the nutritional content in beans, may also be responsible for its low intake.
Despite their rninor dietary role in Canada, beans offer nutritional value matched by few other foods (Geil and Anderson, 1994). Recent dietary guidelines suggest that excessive fat intake, cholesterol, and sodium should be avoided. On a 100 kcal basis, beans (except oilseeds, i.e. soybeans and peamts) contain 80% less total fat than lem ground beef and are low in sodium. They contain no cholesterol and are excellent sources of wrnplex carbohydrates and fiber. Beans also provide a good source of vegetable protein, and vitamins and minerais. in addition, beans are less expensive than animal food products and have a considerably longer shelf life than severai animal, nuit and vegetable products when stored under appropriate conditions (Haytowitz et al., 198 1 ;Sgarbiere, 1989). A number of epidemiologicai studies have shown that populations coIlSwning substantial arnounts of beans in their daily diets face a lower risk of developing many chronic diseases including cancer (Correa, 198 1 ;Jain et al., 1999; Key et al., 1997; Mills et al., 1989).
More recently beans have been shown to contain several biologically active compounds, known as phytonutrients, which include trypsin inhibitors, phytohaemagglutenins, phytic acid, saponins, oligosaccharides and others (Anderson et al., 1979; Messina, 1999;
Valdemiro et al., 1982). Although traditionally these compounds have been termed
'antinutrients' due to their effects on the absorption and utilization of nutrients, recent research indicates that these cornpounds may be responsible for producing some of the health benefits in beans which may reduce the nsk of various chronic diseases faced by western civilizations (Kennedy, 1995; Messina, 199 1; Thompson, 1993; Wang and
Wixoa, 1999).
Of al1 the legumes, the soybean is the one that has received the greatest amount of attention. The phytonutrients in soybean have been studied extensively, and provide much supporting evidence for the role of soybean phytonutrients in tbe prevention of cancer, heart disease, and otha degrnerative diseases (Anderson et al., 1999; Kushi et al.,
1999; Second International Symposium on the role of Soy in Preventing and Treating
Chronic Eisease, 1996). However, the legume family consists of many other varieties of beans, and recent studies ïndicate that grain legumes also contain severai of the same phytonutrients that have been identified in soybean for thev potential health benefits.
Legumes are divided into two separate and distinct categories; the oilseeds such as soybeans, primady grown for their protein and oil content; and the grain legumes such as lentils or kidney beans which are grown for their protein contri'bution to the diet (Geil and
Anderson, 1999; Sgarbieri, 1989). Table 1.1 shows the various types of beans and their bo tani cal names.
Within Canada, Ontario leads in the production of beans. Many factors including soi1 characteristics, climatic conditions, agridtural practices, bean varinia and methods of processing can dl influence the nutritional and phytochemicai composition of these beans
(Deshpande, 1984; Meiners et al., 1976). However, little information is available with regard to the nutritional and phytochemical contents in the bans grown in Ontario, and their effects on chronic disease.
Cancer is one of the leading causes of mortdity in North America with colon cancer being the second most common cause of death (World Cancer Research Fund, 1997).
The incidence and mortdity rate due to colorectal cancer is rising with more than
870,000 new cases diagnosed woddwide since 1996 (World Cancer Research Fund,
1997). As a result, the primary goal in dealing with this epidernic has focused on dietary prevention strategies (Potter, 1996). The presence of typsin inhibitors, lectins, phytic acid, oligosaccharides and saponins in soybeans have shown to be a contributor to Table 1.1 Legume Classification and their Botanical and Cornmon Name.
Legume Classification Botanical Name Common Name Oilseeds Glycine max Soybeans Peanuts
Grain Legumes Phaseofus vulgaris Pinto beans Navy beans Red kidney beans Black beans
Lima beans
Kgna unguiculata Black-eyed bean Viciafaba Broad beans (fava beans) 1: Daîa obtained fiorn USDA Nutrient Database for Standard Refcrence ( Hancibook No. 8). 2: Geil and Anderson, 1994. reduced cancer nsk in those individuals who consume than (Messina, 1999; Messina and
Bames, 199 1). Based on the publishai literature relating to the physiological role of
soybeans, there is convincing evidence to suggest that beans can play an important role in
the prevention of cancer. Therefore it is hypothesized that inaeased knowledge
regarding the nutritional and phytochemïcal composition of Ontario grown beans and the
role of beans in cancer prevention will stimulate increased consumption and generate the development of new and innovative ninctionai foods for markets by the food industry. CHAPTER 2
- mTHESIsANDOBJECTlVE- A number of epidemiologicai snidies have shown that populations consuming substantial
amounts of beans in their daily diets are at a lower risk of many chronic diseases that are
characteristic of Western countries (Jain et al., 1999; Mills et al., 1989; World Cancer
Research Fund, 1997). Biologically-active compowids known as phytonutrients present
in soybeans, have been extensively studied for their role in preventing and treating
various chronic diseases (Messina, 1999; Second International Symposium on the role of
Soy in Prevenîing and Treathg Chronic Disease, 1996). Studies have shown that
cornmon beans such as Kidney beans, Fava beans and Navy beans, among other varieties,
also wntain these phytonutrient that were identified in soy beans for their potential
clinical application (Second International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, 1996).
In Canada, Ontario is one of the major producen and processors of a wide varïety of beans, yet the nutritional and phytonutnent composition have not been studied well. A number of factors such as soi1 characteristics, clirnatic conditions, agricultural practices, and processing methods can influence the nutritional and phytonutrient composition of these beans (Deshpande et al, 1984; Meiners, 1976; Meiners et al., 1976). In spite of the health benefits of common beans, very linle information is available with regards to the composition of dry and processed Ontario grown beans. Once their nutrient and phytonutrient compositions are identified and their levels established, effective strategies can be dweloped to inaease their coosumption in Canada and provide a means for the use of these beans in clinical application
Colon cancer is a prevalent disease worldwide with rising incidence and mortality rates
(World Cancer Research Fund, 1997). Among ail caacers, it is one that is highiy correlateci with a diet typical of Western countries, i-e. high in saîurated fat, and low in dietary fiber and plant foods (Potter, 1996; Correa, 198 1). Therefore, dietary intervention may be beneficial. Based on the published literatwe reiating to the physiological role of soybeans in cancer prevention, there is convincing evidence to suggest that cornmon beans can play a role in the prevention of cancer development as well. Therefore, bean constituents such as fiber, folate or its phytochemical composition may provide protective dietary elements within the colon.
Raw and canned Ontario grown beans contain substantial amounts of macronutrients, micronutrients and phytonutrients that be affected through processing and can also have an effect on colonic preneoplasia in rats. 23 OBJECTIVES
23.1 Overail Objective
To investigate the macronutrients, micronutrients and phytonutrient composition of
Ontario grown beans, and their role in the prevention of colon cancer in a rat model.
23.2 Specific Objectives
a) To detemllne the macronutnent and various micronutrient composition of raw Ontario grown Kidney beans, Fava beans and Navy beans. b) To detemine the macronutrient and various micronutrient composition of commerciaily
processed canned Ontario grown Kidney beans, Fava beans and Navy beans. c) To determine the phytonutrient composition of raw Ontario grown Kidney beans, Fava
beans and Navy beans. d) To determine the phytonutrient composition of commercially processed canned Ontario
grown Kidney beans, Broad beans and Navy beans. e) To investigate the effect of cooking times on the macronutrienî, micronutrient and
phytonutrient composition of Ontario pwnbeans. f) To determine if feeding commercially processed canned Navy beans prevents colonic
preneoplasia in rats. CHAPTER 3
- LITERATURE REVIEW - 3.1 HISTORY
The health benefits of beans have been known for centuries. Primitive people grew and
consurned beans as a dietary staple long before modem nutrition researchers endorsed
their significant health virtues (Messina, 1999). Dry or common beans are seeds that are
categorized as legumes which corne hma large family of plants that are disthguished
by their seed-bearing pods (Sgarbieri, 1989). Of the 13,OOO species of legumes, on1 y
about 20 are commonly consumeci by humans (Sathe, 1984; Sgarbieri, 1989). Legumes
can be separated into two classes: oil seeds such as soy bans and peanuts, which are
grown for their protein and oil content; and grain legumes, such as common or dry beans
(i.e. kidney beans), lentils, lima beans, cowpeas, Fava beans, chickpeas and common
peas, which are grown primarily for their protein source (Gd and Anderson, 1994).
Beans, peas and lentils are know as 'pulses' fkom the Latin word "puise", an Ment bean
porridge.
Dry bans are among the oldest cultivated foods. It appears that the first cultivation of
legumes occdin Southeast Asia approximately 9750 B.C. (Sgarbieri, 1982). The
next oldest sites of agriculture, which date about 8000 B.C., are those in the Middle East,
in a region commoniy known as the Fertile Cresent. It is suspectai that this is the place of ongin of chiclpeas, Fava beans and lentils. The dependence of China and India on vegetarian diets, led to the development of soybean-based imitations of dairy products by the Chinese, and the utilization of legurnes for food in India (Sgarbiere, 1989). nie first cultivations of legumes in the New World occurred around 4000 B.C. in both
Pen, and Mexico, from which the pdcegradually spread to other parts ofNorth
America (Lucier, 2000). Today the world production of major legumes is estimated to be
186,503,ûûO metrk tons annuaiiy (United Nations; Food - Domestic Utilization, 1998).
In Canada, Ontario is one of the major producers and processors of a wide variety of beans.
3.2 PATTERNS OF BEAN CONSUMPTION
Beans are a dietary staple in some parts of the world, providing a significant portion of total protein intake. Pulses, together with nuts and seeds, have been estimated to provide about 5.6% total energy in economically developing codesand 2.4% in developed corntries (Deshpande et al., 1984; Sgarbieri, 1989). Beans make the greatest contribution to dietary energy supply in some areas of sub-Saharan Africa, where 1 1- 17% of total energy is derived from them, followed by the Middle East, Asia, and North Afnca
(Deshpande et al., 1984; Sgarbien, 1989). In some areas of China, they provide as much as 10% totai energy. In Central Arnerica, they are usually eaten with nce and provide 5% of total energy. Consurnption appears to be the Least in Europe, Australia, New Zealand and North America (United Nations, Food - Domestic UtiIization Data, 1998; Lucier et al, 2000).
In Canada, the consumption of beans bas been declinhg for years. Acwrding to the
United Nations, Food - Domestic UtilUation Data for 1998, Canadian wnsumption of beans is 1.1 kg / person annuaîiy. This equates to only 0.5% of total energy intake per
pason. This is less than half the amount consumed by American families yearly.
It is quite possible that bean coasumption is low primarily because of its label as the
'*or man's meat", a metaphor which is consistent with the inverse relation between
intake and incorne (Messina, 1999). Beans contain a number of components responsible
for flatulence, causing social inconveniences. One likely reason for lower consumption
in Western civiiizations is probably related to their prolonged cooking and preparation
tune (Lucier,et al, 20). Western counnies are fast paced, and prefer easy-to-prepare
and convenience foods (Lucier,et al, 2000). However, it is likely that the primary reason
for low consumption is due to the lack of consumer knowledge regardhg their nutritional
content and potential contribution to a healthy diet. It should therefore be the goal of the
Ontario Ministry of Agiculture, Canadian food companies and health professionals to
increase the profile of beans resulting in a higher mnsumption of this 'health benefiting'
food in Canada.
3.3 hWTRIENT PROFILE
Recent dietary guidelines published by Health and Welfar~Canada suggest that excessive intake of total fat, saturated fat, cholesterol, sugar, and sodium should be avoided and reconmiend an increased wnsumption of complex carbohydrate and fiber (Health
Canada, 1997).. The nutrient composition of beans malces them ideally suited to meet these dietary recommendations for good health as they are low in fat and sodium, and are cholestero1 fiee (Gdand Anderson, 1994). provide an excellent source of complex carbohydrate and fiber (Anderson et al., 1984. Dry beans supply a dense source of plant protein and contain substantial sources of various vitamias and rninerals. In light of their nutritional value, beans make a valuable contn'bution to a normal healthy diet.
33.1. MACRONUTRIENT COMPOSITION
The macronutrient composition of sorne commonly consumeci beans is shown in Table
3.3.1.
Beans generally provide a good source of protein, carbohydrate, fiber and vitamïns and minerals (Sathe et al, 1984). Legumes are divided into two separate and distinct categories; the oilseeds such as soybeans and the grain legumes such as Kidney bans
(Geil and Anderson, 1994; Sgarbieri, 1989). The nutrient contribution fiom each of these categones is sornewhat different. The oilseed beans contain higher protein md fat, whereas the beans definecl as grain legumes are higher in carbohydrate and fiber. Both categories also provide a number of vitamins and minerals, however the vitamin and minera1 contribution fiom each category also has their differences as a result of their legume category. Figure 3.3.1 displays the macronutrient differences between the
Kidney bean (grain legume) and the Soy bean (oil seed legume). Table 33.1 Nutrient content of selected cooked bans w100g)
Bean Wate Energy Protein Fat Carb Ash Dieâary r (k-0 (8) (g) hydrate 0 Fiber
Broad Beans 83.70 62.00 4.80 0.50 10.10 0.90 3 -60
, (Faba beans) ' Garbanzo 60.21 164.00 8.86 2.59 27.41 0.92 7.60 Beans (chickpeas) a KidneyBeans 66.94 127.00 8.67 0.50 22.18 1.O9 7.40 .(red) a
, Lentils " 69.64 1 16.00 9.02 0.38 20.14 0.83 7.90 LimaBeansa 69.79 115.00 7.80 0.38 20.89 1.15 7 -00 NavyBeansa 63.18 142.00 8.70 0.57 26.31 1.23 6.40 Peanuts, 1.55 585.00 23.68 49.66 21.51 3.60 8 .O0 roasted (whole parts) ' Pinto Beans a 93.39 22.00 1.86 0.32 4.10 0.34 NIA Soy Beans ' 62.55 173.00 16-64 8.97 9.92 1.91 6.00 a: Data obtained hmUSDA Nutrient Database for Standard Reference ( Handbook No. 8). b:. N/A: data not listed Figure 3.3.1 Cornparison of Macronutrient Content of Oilseed Legumes (Soy Bean) and Grain Legumes (Kidney Bean) 3.3.1.1 PROTEIN
Bean protein can make a valuable addition to the diet. The protein content of dry beans provides between 20% to 30% of energy, with the exception of soy beans which provides
34% (Sathe et al., 1984; Sgarbieri, 1982). A serving of cooked beans (approximately L O0 g or !A cup) will provide an average of 7 to 9 grams of protein (Messina, 1999; Sathe et al., 1984; Sgarbieri, 1982). This is considerably more protein than in most other vegetables, and double the amount of protein found in 1 cup of 2% low-fat milk (Geil and
Anderson, 1994).
Cuxrently there is much interest in bean protein worldwide as health concerns and cost of animal protein sources increase (Gd and Anderson, 1994; Sanvar et al, 1989; Sgarbieri,
1982). Even though beans have been recognized for their high protein content, the protein quality of beans have been questioned. The legume protein is relatively nch in essential amino acids such as lysine. When compared with a reference protein such as eggs, they are moderately deficient in the sulfùr-containing amino acids methionine (Geil and Anderson, 1994; Messina, 1999). The significance of this problem has been exaggerated. This is because tests, such as the protein-efficiency ratio, which measures protein quality, are most oflen conducted with rats, which require approximately 50% more methionine for growth than humans (Messina, 1999). Consequentiy, because bean proteins are low in sulnir-containiig amino acids, the protein-efficiency ratio of bans tend to be quite low. A mixture of legumes and a complementary source of sulfur- containing amino acids, such as those found in cereal grains, will greatly improve its protein efficiency. Some examples of food complementary combinations are baked
beans on toast and corn tortillas with refned beans.
Beans provide an excellent source of complex carbohyârate. The carbohydrate content of
cooked beans ranges fiom 5 to 23 g per 100 g serving (Gupta, 1983; Sgarbiere, 1989).
The total carbohydrate component of Qy beans makes up 50 - 70% by weight (Sgarbiere,
198 1). The main carbohydrate component is starch ranging fkom 38 - 45%, with small
amounts of sugars such as finoseand strarchyose which are considered indigestible
and cause flatulence, and hcto-oligosaccharides (Gd and Anderson, 1994).
Fructo-oligofnictosaccharides(FOS), have recently attracted considerable attention as nonabsorbable dohydrates with prebiotic properties (Jenkins et al., 1999). The addition ofFOS to the diet has shown to significantly increased the nwnber of colonic bifidobacteria, which are regardd as beneficial strains in the colon. Reddy et al. (1997), found that the addition of 10% FOS to the diet significantly inhibited the formation of aberrant crypt foci formation and crypt multiplicity in the colons of azoxymethane- induced rats. Jenkins et al. (1999), suggests that the addition of FOS to the diet may play a role in reducing blood choiesterol, stimulate immune bction and enhance vitamin synthesis. Legumes seeds also contain large amounts of resistant starch (RS) which resists digestion
in the srnail intestine and then passe into the large intestine where it is fennented by
bacteria to produce short-chah fatty a&, with a high proportion of butyrate (Cummings
and MacFarlane, 1992; Englyst and Cummings, 1987; Scheppach et al., 1988). Studies
show foods that promote the production of butyrate are associated with a lower risk of
bowel cancer (MacIntyre et al., 1993). Resistant starch may be quantitatively more
important than dietary fiber as a substrate for fermentation (Ahmed et al., 2000). Starchy
foods nch in RS have shown to inauence the regulation of glucose metabolism by
lowerhg postprandiai hyperinsulinernia/hypergiycemia(Lehrer-Metzer et ai., 1996;
Raben et al., 1994).
It has also been found that RS may lower the level of plasma cholesterol (behall et al.,
1989), which might be a consequeme of hepatic metabolism regulation (Sacquet et al.,
i 983). RS also increases fecal bullc (Jenkins et al., 1998) and lowers fecal pH (Philipps
et al., 1995), factors that are usually considered as markers of healthy colonic mucosa
(Cummings et al., 1992).
In addition to FOS and RS, beans aIso contain a significant amount of dietary fiber.
Legumes provide berneen 1 and 7 g of fiber per 100 g of cooked serving. Bean fiber is primarily compriseci of soluble fiber, which contributes to 213 of its composition, and
insoluble fiber, providing only a third (Hughes and Swanson, 1989). Fiber comprises a
heterogeneous group of nonstarch polysaccharides such as cellulose, hemicellulose and
pectins and non-carbohydrate substances such as phytic acid (depending on the nature and source of fiber in the diet) (Reddy, 1999). Epidemiological and laboratory animal studies have suggested an inverse relationship between colon cancer and dietary intake of fiber-rich foods (Howe et ai., 1992; Reddy, 1995; Steinrnetz and Potter, 1991).
3.3.13 FAT
Most beans are low in fat and contain approximately 0.5 g per 100 g cooked se-g. The fat content of dry beaas range hm0.8 - 1.5% with the exception being soy beans and peanuts which contain 19% and 46% of fat (Geil and Anderson, 1994). This relates to
7.3 g of fat per lûûg seniing of oooked soy beans and 32 g of fat in H cup of roasted peanuts (Gd and Anderson, 1994). The fatty acid composition of dry beam is primarily unsaturateci fatty-aciâs with a-linolenic acid present in the greatest amount (Sgarbieri,
1989). However, because the total fat composition of grain legumes is quite low, the dietary contribution of a-linolenic acid by beans is generdly minor. In the case of soybeans which are high in fat, they contribute significantly to a-linolenic acid intake.
Because beans are plant foods, they are cholesterol fkee. The low fat content of dry beans is important in light of dietary recornrnendations, which suggest lower fat and cholesterol intakes to reduce the risk of developing chronic diseases (Health Canada, 1997). 333 MICRONUTRIENT COMPOSITION
The micronutrient cmposition of cofll~fionlyconsumed beans is shown in table 2.3.2.1.
Dry beans provide sources of water-soluble vitamim. These include thiamin, riboflavin, niacin, and folacin, otherwise knowu as folate. Legumes are thought to be some of the best sources of folate (Meiners et al., 1976). The folic acid in human health has been idaitified in recent studies. There is convincing evidence to suggest that adequate intake of this nutrient may help prevent a number of health problems at ali stages of life. This vitamin has been shown to prevent neural tube defects in the fetus (Oladapo, 2000) and lower the risk of ischemic heart disease and strokes by lowering plasma homocysteine levels in older persons (Brattstrom and Wilchen, 2000; Law, 2000).
There is also evidence to suggest that folic acid treatment reduces the incidence of cancer
(Freudenheim et al., 199 1; Jacob, 2000; Law, 2000; Thurmon, 200 1; Willet, 2000).
Folate plays a vital role in normal ce11 division and the repair of DNA damage (Willet,
1994). Low folate in patients with many types of cancers has been observed. nie
Nurses' Health Study showed that women taking folic acid-containing multivitamins for at least 15 years were 75% Iess likely to develop colon cancer than women who did not.
It was also found that patients with ulcerative colitis who took 1000 mcg/day of folk acid supplements had a lowereà risk of developing colon cancer (Harvard Women's Health
Watch, 2001). Song et al. (2000), investigated the effects of O, 2, 8, or 20 mg folatekg on the Table 33.2.1 Vitamin content of selected cooked beans.
(mg) (mg) Brod 0.09 .O8 Beans (faba 1 / Garbanzo 1 0.12 / 0.06 Beans
Kidney Beans (red)
- Lima 0.16 0.05 Beans NaV 0.20 0.06 Beans %' Peanuts, 0.44 0.09 roasted (whole parts)' Pinto 0.67 0.05 Beans Soy Beans 1 0.15 1 0.28 ac a: Data obtained fiom USDA Nub .nt Database for Standard Reference ( ~LdbookNo. 8). b: N/A:data not Iisted. c: Values expressed per 100 g of cooked, boiled, senring. development of intestinal polyps in mice. Their labontory fouad increasing dietary
folate levels significantly decreased the number of colonic ACF in a dose-dependant
manner compared to the folate-deficient diet, after three months. The number of ACF
was also inversely melateci with serum folate concentrations.
Recent surveys have indicated that folic acid intakes in North Arnerica are below the RN1
/ DRI. The average intake of folic acid bmfoods have been ody about half of the
current nutrition recommendations of 4OO mcg/day. One serving of cooked beans can
provide more than half the current recommendations for folate intake (Heaith Canada,
1997). Alaiough some of these water-soluble vitamins are heat labile, ordinary cooking
of dry beans (those cooked at home), appears to pose little problems as far as nutnent
retention is concemed (Deshpande et al., 1984).
Due to the low fat content of beans they are coincidentally poor sources of fat-soluble
vitamins A, and E.
Typical mineral contribution of commonly consumed bans is shown in Table 3.3.2.2.
Beans are primarily considered a rich of essential minerais. These include calcium, iron, magnesium, phosphorus, potassium, zinc and copper (USDA Handbook No. 8, 1972).
Although there is considerable variation among beans, they can provide approximately 50 mg of calcium per 90- 100 g of cooked sening. This equates to Table 33.2.2 Mineral content of selected cooked
Bem Calcium Ibn Magnesium Phosphorus Potassium Zinc Copper Manganese Sodium (mg) (mg (mg) (mg) (mg) (mg) (mg) (mg) (mg) 1 Broad Beans 36.0 1.5 43 .O (Faba beans) ac
Gar banzo 49.0 2.8 48.0 Beans 9 (chickpeas) ac Kidney 28.0 2.9 45.0 Bems (=a 4 qc
Lentils 9C 19.00 33. 36.0 3 Lima Beans 17.0 2.3 43 .O *c 9 Navy Beans 70.0 2.4 59.0 ac Peanuîs, 54.0 2.2 176.0 roasîed 6 (whole parts) Pinto Beans 15.00 0.6 18.00 ac 6 Soy Beans 102.00 5.1 86.0 4 a: Data obtained hmUSDA Nutrient Database for Standard Reference ( Handbook No. 8). b: N/A: data not listed. c: Values expressed per 100 g of cooked, boiled, serving. appoximately 6% of the recommmded daily intake of calcium for adults. One cup of cooked beans can also pmvide 20 - 25% of the requirements for phosphorus, magnesium and manganese, about 20% of potassium and copper, and 10% of zinc (Geil and
Anderson, 1999; Nutrition Recommendations, Health Canada, 1997). The iron content in cooked beans is appmximately 1.5 - 2.0 mg per 100 grams. This compares favorably with iron recommendations of 8 - 13 mg/day for adults. However, it is important to note that the high mineral content of cooked beans rnay not necessarily equate to high bioavailability of some of these minerals. Minerals hmplant sources are generally less bioavailabIe than minerals derived fiom animal sources (Geil and Anderson, 1999). Dry beans contain a nurnber of components, including fiber, phenolic compounds and phytic acid which may react with these minerals to decrease th& bioavailability (Thompson,
1993). Dry beans are naturally low in sodium (except for peanuts), making them nutntionally advantageous to people who are on low sodium diets.
3.4 HEALTH BENEFITS OF BEANS
Beans can be an important cornponent of any healthy diet. The preseace of their fùnctional components enabled them to provide health benefits beyond basic nutrition
(Kushi, 1999). However, fuhue research should be directed at the protective and therapeutic deof beans in combating conditions such as coronary heart disease, diabetes mellitus, obesity, and cancer, al1 of which are faced by western civilizations. 3.4.1. CORONARY HEART DISEASE
Coronary heart disease is a major medical concem in most Western cornries (Anderson et al., 1999). It accounts for more thaa half the deaths in North America, and it is also a significant concern in developing counhies around the world (Cam1 and Kurowska,
1995). Epidemiological data suggest an inverse relationship between the intake of complex carbohydrates and dietary fiber, and coronary heart disease (Anderson et al.,
1984; Anderson et al., 1990). Die- modification is necessary to lower risk factors, such as elevated semm cholesterol, in combating heart disease (Anderson et al., 1990). It has been documentd that certain high fiber fdscan produce significant hypocholesterolemic effects (Lukm et al., 1962). Significant reduction in total cholesterol is important, as a 1% decrease in semcholesterol rdtsin a 2% demease in the risk of coronary heart disease (Gdand Anderson, 1994). Although the mechanisms responsible for the hypocholesteroIemic effects observed in various studies on fibre are not certain, it is hypothesized that férmentation of dietary fiber in the colon generates short-chah fatty acids, which may inhibit hepatic cholesterol synthesis (Anderson et al.,
1999).
In a study conducted by Groen et al., (1962), Trappist monks wnsuming lactovegetarian diets, which included 100 - 150 glday of dned beans, were shown to have serum cholesterol levels that were significantly lower than Benedictine monks who were consuming typical Western diets. Luyken et al., (1962), obsdsirnilar results using
100g/day of dned brown beans. Jenkins et al., (1983), showed a significant decrease in the total serum cholesterol levels of male hypercholesterolemic pateints, consuming 140 g/day of a variety dried beans when compareci to other cabhydrate sources. There have been a number of human studies which have investigsted the cholesterol-lowexing effects of a variety of dried beans in amounts ranging hm75 - 200 glday (Anderson, 1984;
Jenkins et al., 1985; Shutler et al., 1989). All of these studies indicated significantiy lowered cholesterol levels ranging hm-5.2% to -18.7%. However, it shodd be noted that the hypocholesterolemic effeds produced by the consumption of dried beans were most noticeable in subjects whose initial semcholesterol levels were the highest.
Canned beans account for the majority of total bean consumption (Haytowitz, 198 1).
Several studies have indicated that canned bans might also play a dein the management of hypercholesterolania Shutla et al. (1 989), and Anderson et al. (1984), both demonstrated a cholesterol-lowering effect, following the consumption of canneci beans in amounts ranging fiom 69 - 150 g/day.
3.4.2. DIABETES MELLITUS
Diet remains an important factor in controllhg diabetes mellitus. Recent work on the dietary management of diabetes has focused on two factors: dietary fiber content and the nature of the carûohydrate eaten (determined by their glycemic index) (Crapo et al.,
1977; Jenkins et al., 1978). A number of studies have implicated dietary fiber as a major component in certain foods, which may provide important health benefits to diabetics
(Anderson and Akanji, 1991; Jenkins et al., 1978; 1983; 1987). Beans are among these foods as they provide significant sources of dietary nber (Gd and Anderson, 1994;
Marlett, 1992). Other studies have shown that the glycemic index of foods is also an
important factor in managing diabetes (JenLins et al., 1981; 1985; 1987; 1988; Wolever
et al., 1990; 1991).
The glycemic index (GI)is a system of classification in which the glucose response to
foods are indexed against a standard (white bread) (Wolever, 1990). Beans have been
identified as a potentially clinically usehl food pducing relatively flat giycernic
responses. Specific incorporation of beans into the diet have been associated with reduced blood glucose and insulin Ievels. Jenkins et al- (198 l), investigated the effect of
62 different foods on blood glucose levels of healthy fasting volunteers. Blwd glucose levels were measured over 2 hours and expressed as a percentage of the area under the glucose response curve. Out of the 62 commonly eaten foods (vegetables, breakfsst cereals, cereals and biscuits, Mts, dairy products and beans), beans showcd the Iowest rise in blood glucose.
It should be noted that not al1 dohydrate sources raise blood glucose to the same extent when fed equivaIent arnounts. in a study that investigated the effects of 50 g of 8 varieties of legurnes and 24 cornmon foods (grains, breads and pasta, breakfast cereals, biscuits and tuberous vegetables) found both the mean peak rise in blood glucose concentration and mean area under the cuwe of the subjects who ate beans were at least
45% lower than those subjects who ate the other fmds (Jenkins et al, 1980). JenLins et ai. (1984), aiso investigated the effkct of varying the amount (25 g or 50g) and type (bread or beans) of carbohydrate in test meals on the bldglucose response. The blood giucose ara after a hdf bread portion was 48% that of the fbll bread meal. Fifty gram of white pea beans gave a blood glucose response 4 1% that of 50 g of bread, and a combineci meal of bread (25 g) and beans (25 g) gave a blood glucose response of 60% of the fidl bread med.
Incorporation of low O1 foods (such as beans) into the diet have also been associated with reductions in low-density lipoprotein cholesterol and triglyceride levels in hyperlipidemic patients. The substitution of 140 g of dried beans for other sources of starch in the diets of 7 male hyperhpidanic patients showed reductions in mean fasting senmi triglyceride levels by 25% and a reduction in total serum cholesterol levels by 7% (Jenkins et al,
1983). Jeakins et al. (1988), also examined the effect of low GI traditional foods consumed by hyperlipidemic and non-hyperlipidemic patients for 3 months. Only subjects with raised triglyceride levels showed significant reductions in total cholesterol,
LDL cholesterol and serum triglyceride with no change in HDL cholesterol.
These studies indicate that the reintroduction of low GI legumes into a Western diet not only benefits individuals who have developed diabetes, but those whose lipid levels are high and are at risk for disease Obesity is a wmplex disease with sexious health consequences. Dietary intervention for
the puIpose of weight loss is a challenge faced by dietitians and the medical community.
Beans intaLe reduces the risk of developing obesity much in the same way it protects
against diabetes and other chronic diseases because they are low in fat and high in fiber
(Anderson et al., 1999; Geil and Andason, 1994). Foods with a high fiber content may produce a feeling of satiety (Anderson et al, 1999). as they provide slower passage through the alimentary canal prolonging gastnc emptying, increase insulin sensitivity, and dampen postprandial glycernia (Geil and Andetson, 1994). Therefore, high fiber, low fat foods, such as beans, can be viewed as an important component in weight- reducing diets (Rolls, 1995).
Leathwood et al. (1988), investigated the effects of bean puree versus potato puree on plasma glucose and hunger, and fond beans to produce a slower, sustained rise in plasma glucose than potatoes. Through the use of a hunger rating questionnaire, the study also found delayed hunger sensations and prolonged feelings of satiety in those subjects consuming bean puree compared to those who had potato puree.
Studies also suggest that protein may be more satiating than carbohydrate or fat (Rolls,
1995). The observations of enhanceci postmeai satiety associated with protein compareci with other ma-nutrients may suggest that an inwease in legume protein intake may provide weight-1oss benefits. There is aiso a potential nsk of pater rend stress b y indganimal protein intake (Anderson, 1999). Therefore it would be unwise for
obese individuals to consume di& with a high animai protein content shce these people
are more likely to be at a higher risk for developing diabetes, glucose intolerance or
hypertension. Similar to soy protein which appears to lessen the workload of the kidneys
when compareci to animal protein (Kontessis et al., 1990), beau protein may also have
advantages in treating obese individuais- They are Iow in fat, contain high fiber, and
provide an adequate protein source, al1 of which serve as important factors for the
management of weight loss and weight maintenance (Health Canada, 1992).
3.4.4 CANCER
It is estimateci that up to 70% of al1 cancers are related to diet (Doll and Petro, 198 1). A
major protective dietary factor against cancer appears to be a diet high in fiber and low in
fat, with an increased consumption of fiuits and vegetables (Potter, 1996; World Cancer
Research Fund, 1997). The Iow fat, high fiber composition of beans makes thern ideally
suited for this purpose (Geil and Anderson, 1994).
The role of beans in cancer prevention is unclear. Most reviews discussing epidemiological studies related to this topic found that the number of studies suggesting an inverse association are aimost matched by those which found a positive association between legume intake and cancer risk (Steinmetz and Potter, 199 1). ui a recent report set forth by the World Cancer Research Fund, it was noted that 58 epidemiological studies had examined the association between bmconsumption and cancer risk. The result of this investigation showed that of 58 studies, 29 (50%) reported a decreased nsk with higher bean intake, 22 (38%) reported an increased risk, and 7 studies (12%) indicated no association between bean intake and cancer. In an epiderniological study conducted by Jah et al. (1999), which was relead following the above investigation reported a statisticaily significant decreased association between increased intakes of beans, lentils, and nuts and prostate cancer risk. This study is of partidar interest, as it was conducted in three major geographicai areas within Canada: Metropolitan Toronto,
Montreai, and Vancouver. The study indicates that beans grown in Canada contain sorne beneficiai properties. This is important as envionmental conditions and processing techniques Vary form country to country, which could have an effect on the nutritional and hctional quality of the beans.
Recent interest on the health benefits of beans bas focused aromd a nurnber of biologically active components referred to as phytochemicals (Anderson, 1979; Kemedy,
1995; Messina, 1999; Messina and Messina, 199 1; Second International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, 1996; Thompson, 1993;).
Although they were once refmed to as 'antinutrients' because of their potential to reduce the availability and utilization of nutrients, and cause growth inhibition (Limer, 1994;
Messina and Messina, 1991 ;Thompson, 1993; Wang and Wixon; 1999), they are now being looked upon as important compounds for the prevention of various chronic diseases
(Second International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, 1996). Among these chernicals are phytic acid, lectins, saponinî, trypsin
inhiiitors and oligosaccharides.
The clinical applications of phytonutrients have received a great deal of interest in recent
years. In particular, the phytonutrients present in soybeans have been studied extensively, with a number of reviews discussing the hedth significance of these hdings
(Anderson et al., 1995; Kennedy, 1995; Kushi et al., 1999; Limer, 1993; Messina, 1999;
Messina and Bames, 199 1; "ïilompson, 1993; Wang and Wixon, 1999 ). At a recently held international conférence on the role of soy in preventing and treating chmnic diseases, several papers were presented providing supporting evidence for the deof soy bean phytonutrients in the prevention of cancer, heart disease and other degenerative diseases (Second International Soy Symposium on the Role of Soy in Preventing and
Treating Chronic Disease). Beans may aIso contain several of the same phytonutrients that were identified in soybeans. However, to date analysis of these phytonutrients in the beans grown in Ontario have not been prefod.
Phytic acid is a storage fom of phophorus in plant seeds (Wixon and Wang, 1999). Its concentration in legumes account for approximately 1 - 3% on a dry weight basis
(Deshpande et al., 1984; Reddy et al., 1982). It has been looked upon as an anthutrient because of its ability to bind to protein, starch and various minerais, thus reducing and slowing th& digesti'bility (Thompson, 1993; Reddy et ai., 1982; Limer, 1993).
However, these properties could be considered as desirabIe in the prevention of diabetes, cardiovascular disease and cancer (Wang and Wixon, 1999).
Phytic acid has a clear benefit for people with diabetes meîlitüs by delaying glucose release hmstarch. Long-terni exposure to phytic acid appears to be beneficial in prevention of diabetes by delaying the nse of glucose in the blood and preventing the development of tissue resistance to insulin (Wang and Wixon, 1999). Jenkins et al
(1NO), found legumes to have the lowest starch digestibility and blood glucose response compareci to 0thcarbohydrate foods. A number of other studies have also found benefits in improving diabetes control with the addition of phytic acid (Simpson et al.,
198 1; Thompson et al., 1987; Yoon et al., 1983).
The potential preventative effect of phytic acid against cardiovascular disease is Wrely due to its hypolipidemic and hypocholesterolemic properties (Wang and Wixon, 1999).
The addition of 0.2 - 9% phytic acid to the diets of rats significantly reduced the plasma cholesterol and triglyceride levels (KIevay, 1977; Shma, 1980, 1984; Jariwalla et al.,
1990). Because phytic acid can bind to zinc, it may lower the senun zinc to çopper ratio, which tends to predispose humans to cardiovascular disease (Klevay, 1977).
A number of in vitro and ahalstudies have demonstrated potential preventative effects of phytic acid against cancers in the colon, mamary glands and other tissues (Wang and
Wixon, 1999). When phytic acid (1%) was added to the drinking water of rats one to two weeks prior to the administration of azoxymethane (AOM), a colon specific carcinogen, and up to 5 months afterwards, significant decreases in tumor size and volume in the colons of rats were observed (Shamsuddin et al., 1988; 1989). Risk of mammary cancer risk was also reduced with the addition of phytic acid to the diet of mice, as indicated by decreased cell proliferation, nuclear aberration and intraductai proliferation (Thompson and Zhang, 1991).
The mechanisms by which phytic acid may reduce the risk of cancer have been suggested in various studies (Shamsuddin et al., 1988, 1989; Shamsuddin and Ullah, 1989;
Thompson, 1989; Graf and Eaton, 1985,1990; niompson and Zhang, 199 1).
A) Phytic acid rnay bind with pmxidant ~e=+and reduce DNA damage by fiee radical formation during lipid oxidation (Graf and Eaton, 1990). B) A reduction in available zinc will indirectiy reduce the amount of DNA needed for ce11 prolifdon (Thompson,
1993). The ability of phytic acid to bind with zinc may also be anticarchogenic, as zinc is needed during DNA synthesis. C) The slowing down of starch digestion by phytic acid may cause some of the starch to escape to the colon. Fermentation of unabsorbed starch produces short-chah fatty acids (SCFA). The presence of SCFA would lower the pH environment in the colon, which in turn would provide protection against colon cancer by reducing the solubility of bile acids and neutalizing ammonia which may act as tumor promoters (Newrnark and Lupton, 1990). Saponins are naturally occuring amphiphilic heat-stable glycosidic compounds present in
many plants (Price et ai, 1987). The major source of saponins in the human diet are
beans (OakenfÛiI, 198 1). They coosist of a steroid or triterpene group linked to one or
more sugar residues of differait types. The aiterpene portion is hydrophobic and the sugar portion is hydrophilic. These Wace characteristics are responsible for saponins biological activities, inciuding hypocholesterolemic (Oakdland Sidhu, 1990; Carroll and Kurowska, 1995), immunostirnulatory (Wu et al., 1990), and anticarcinogenic
(Kilnichi et ai., 199 1) properties.
The effects of different saponins on lipid metabolisrn in animals and humans were summarized by Oakenfùll and Sighu (1990). in the majority of these cases, substantial reduction in plasma cholesterol consentrations was reported. This was also associateû with increases in the output of feces and bile acids and neutral staol output. Of the twenty experirnents conducted, only six did not show any significant reductions in plasma cholesterol concentrations. In animal studies the evidence for hypocholesterolemic effect of saponins is strong particularly when fed in the presence of cholesterol. In human studies, controlling for other dietary substitutions, feeâing saponins resulted in a significant blood cholesterol lowering effect. The mechanism by which saponins Iowa cholesteml may be explaineci by its ability to
bind with dietary cholesterol, thus preventing its absorption (Reshef et al., 1976).
Saponins can also bind to bile acids, which may reduce their resorption (Oakdl and
Sidhu, 1986) and therefore more cholesterol is needed to compensate for the bile acid
losses in feces, resulting in lower concentrations of serum cholesterol.
The biological characteristics of saponins suggest that they may also have some
anticarcinogenic properties. In vitro studies by Rao and Sung (1995), showed soya
saponins to have a dose-dependant growth inhiiitory effect against human colon
carcinoma cells. Koratkor and Rao (1997), investigated the effects of soya saponins on
colon cancer in the mouse. They found that when they provided 3% soya saponins, by weight, to the diets of rnice that were administered azoxymethane, significant reductions in the incidence of aberrant crypt foci occurred compared to those mice that were not receiving soya saponins.
Saponins appear to function as anticarchogens by their ability to bind to primary bile acids. This in tum prevents their bacterial conversion to secondary bile acids which consequently reduces their ability to promote turnourigenesis (Oakenfull and Sidhu,
1983). In addition to the bile acids, saponins also have the ability to bind to cholesterol
(Reshef et al., 1976) and prevent cholesterol oxidation in the colon, which is also known to be responsible for colon cancer promotion (Kendall et al., 1992). Saponins may also prevent carcinogenesis by hctioning directly as cytotoxins to cancer cells, and or fiuictioning as fiee-radical scavengers (Wang and Wixon, 1999). A major factor which appears to limit the consumption of beans is theV ability to produce
gas in the gastroiatestinal tract. One of the first studies to be published which showed
diets containhg beans markedly increased flatuience was published in 1966 (Steggerda
and Dimmick, 1966). The primary reason for the gas production is due to the presence of
fructooligosaccharides (FOS) present in the beans. Fianilence will occur with bean consumption busethe human intestjnal mucosa lacks the enzyme q-galactosidase necessary to cleave the l,6-galactosidic linlrage. These FOS pass into the large intestine where they undergo fermentation by anaerobic bacteria, forming large amounts of carbon dioxide, hydrogen and methane (Messina, 1999; Rackis et al., 1970). Because of the discodort and the social embarrassrnent associated with flatulence, many people avoid eating beans al1 together.
Some reports indicate that it is possible to remove substantid amounts of FOS through various processing methods @eshpande et al., 1984). Cooking has proven to be effective in causing some reduction in the gas promoting activity of various beans (Deshpande et al., 1984). It is possible to also remove considerable amounts of FOS and reduce flatulence by changing the water in which beans are boiled one or more times (Anderson et al., 1979). There are however benefits associated with FOS consumption (Messina,
1999)-
Research indicates that FOS are important nonabsorbable carbohydrates with prebiotic properties. Studies indicate that they are able to favorably stimulate the growth of bifidobacteria, which are regarded as benefkiai strains in the colon, thus promothg
health and longevity of the colon (Anderson et al, 1999; Reddy a al., 1997). In diet
intervention studies, Gibson et al. (1995). demonstrateci that dietary administration of
FOS significantly increaseù fecal bifidobacteria, whereas bacteriodes, clostridia and
hobacteria and or gram-positive cocci were decreased on total fecal bacterial count.
These bifidobacteria, colonizing at the expense of enteropathogens, may bind the
ultimate carcinogen by physically removing it via feces (Reddy, 1999).
In addition to selective modulation of bifidobacteria, FOS increase the production of
short-chain fatty acids (SCFA), especially butyrate, in the colon by microbial
fmentation (Reddy, 1999). Foods that promote the production of butyrate are
associateci with a lower risk of bowel cancer (MacIntyre et al, 1993).
Recent studies have examined the effect of feeding FOS and their ability to inhibit
makers of colon cancer. Koo and Rao, (1 99 1), reported that administration of both
bifidobacteria and 5% FOS to fernale mice given DMH resulted in approximately 50% as many aberrant crypts (AC) as in the wntrol animals. They also observed a decrease in the number of aberrant crypt foci (ACF) compared to the control animals. Reddy et al.
(1997), also found a significant inhibition of ACF formation after feeding FOS to F344
AOM-induced rats. A number of other studies that have investigated the role of FOS consurnption on the incidence of colonic preneoplastic markers in animals, are surnmarized in a recent revkw by Brady et al, (2000). Human studies have also been summarized here, however they are more indirect and provide more cirCUmSfStI1tial evidence than that offiby the animal studies.
Because of tbeir gtowth-promoting effect on bifidobacteria and their ability to be fermentecl into SCFA, it has been hypothesized that FOS may promote health of the colon, increase longevity and decrease the risk of colon cancer (Benno et al., 1989; Koo and Rao, 1991; Mitsuoka, 1982).
3.5.4 TRYPSIN INHIBITORS
Protease inhibitors are abundant in raw cereals and legumes, particularly soybeans. They are protein molecules that bind with trypsins and interfère with protein hydrolysis during digestion (Wang and Wixon, 1999). The two major types of trypsin inhibitors in soybeans are the Kunitz and Bowman-Birk inhibitor (BBI). The Kunitz inhibitor is themally unstable and is destroyed during most processing procedures, while the
Bowman-Bisk inhibitor is relatively heat stable and can survive normal heat treatment, with approximately 5 - 20% of the activity still rernaining (Messina, 1999; Rackis and
Gumbman, 198 1; Hathcock, 1991; Wang and Wixon, 1999).
Although protease inhibitors have been associated with pancreatic cancer in animal studies, recent research indicates they may ais0 act as anticarcinogenk agents. This is suggested by animal studies, in vitro ce11 culture work and epidemiological data, which showed low cancer mortality rate in human populations with high intaLe of protease
inhibitors (Troll and Kennedy, 1989; Messina and Barnes, 199 1; Messina and Messina,
199 1; Second International Symposium; The Role of Soy in Preventing and Treating
Chronic Disease).
In vitro, pmtease inhibitors have been shown to suppress the malignant transformation of cells induced by different types of carchogens, e.g. radiation, W light, chernical carcinogens and steroid homiones (Kennedy, 1984; Kennedy and Billings, 1987).
The BBI fkom soy beans has show the greatest suppression of carcinogenesis assays in animals. These assays have indicated the BBI to be able to inhibit or prevent the development of chemically-induced cancer in the colon by 100% (St. Clair et al., 1WO), suppress carcinogenesis in the liver by 7 1% (St. Clair et ai., 1990), in the lung by 48%
(Witschi and Kennedy, 1989) and the oral epithelium by 86% (Kennedy et al., 1993).
There appears to be multiple mechanisms by which protease inhibitors interfere with the development of cancer. The inhibitor may reduce the digestion of proteins, which in tum will decrease available amho acids needed to feed the growing cancer cells (Troll, 1989).
Deprivation of amino acids such as leucine, phenylalinine and tyrosine have shown to prevent the growth of mouse hepatoma and mammary adenocarcinorna (Troll et al.,
1987). These inhibitors may also play a significant role in stopping onwgme expression begun by carcinogen exposure. Protease inhibitors were shown to be able to suppress the formation of superoxide anion radicals (.02-)and hydrogen peroxide (H 2O 2 ) formation induced by tumor promoters, (e.g TPA), in phagocytic neutrophils. These oxygen reactive species can damage and or modify ceiiular DNA thus promoting oncogme expression (Kennedy and Billings, 1987; Troll, 1987, 1989). The inhibitors may also act as an anticarcinogenic agent by inhiiiting the induction of the DNA polymerase a, an enzyme responsible for the amplification of virus-modified DNA (Heilbronn et ai., 1985).
3.5.5 LECTINS
Lectins are proteins which have the unique ability to bind to the carbohydrates found on the surface of many mamrnalian cells (Wright, 1975). Also refend to as glycoproteins, they are of non-immune origin (Rhodes, 1996), which appear to have fiinctions which vary Erom one lectin to another (Liener, 1983; Sgmieri and Whitaker, 1982). Their biological actions include agglutinating erythrocytes, agglutinating bacteria, inducing lyrnphcyte transformation, stimulating mass ce11 degranulation and agglutinating and damaging neoplastic cells in prefaence to the normal adult equivalents (Lis and Sharon,
1973). This latter property appears to provide some lectins with a protective effect against experimental carcinogenesis in laboratory anirnals (Inbar et al., 1972, Wright,
1975).
Lectins are ubiquitous in nature and are present in many of the food items commonly consumeci in the human diet (Nachbar and Oppenheim, 1980). They are particularly plentifùl in nuts and more so in seeds (Nachbar and ûppenheim, 1980; Rhodes, 1996).
They are typically giobular proteins which are highly resistant to digestion by mammalian enzymes and sunive passage through the digestive tract (Jae and Vega
Lette, 1968; Brady et al., 1978; Nachbar et al., 1980).
Much of the existing literature on lectins have discdtheir toxicity. Many lectins are
toxic or aahirally bound to toxins (Limer, 1983; Wright, 1975). The toxicity of lectins is
characterized by growth inhibition in experimental animals and diarrhea, nausea, bloating
and vomiting in humans (Liener, 1989). The toxic effects of lectins relate to their
binding to specific receptor sites on the epithelial ceii of the intestinal mucosa. This can
cause lesions, dimption and abnormal development of the microviliae (Liener, 1989) thus affècting the absorption of nutrients. However, a common observation is that a high intake of vegetables (including legumes) and therefore lectins, Uicreases intestinal motility as well as mucus and gas production, which decreases the likelihwd of coloncancer (Jordinson et al., 1999; Wright, 1975). In this way, the body is protected fiom their toxicity (Wright, 1975).
From the before mentioned mechanism, a number of researchers have inferrecl that lectins in foods may play a role in protecting against intestinai cancer. A number of studies have found some dietary lectins (fkom legumes and mushrooms) to inhibit proliferation of colorectal carcinoma ce11 lines (Koniala< et al., 1992; Yu et ai., 1993) and legume lectins to stimulate differentiation of undifferentiated colon cancer cells (Jordinson et al., 1999).
Severai mechanisms for the body's response to lectins as a protector against intestinal cancer have been postulateci (Etzler and Branstrator, L 974; Wright, 1975): 1) mast-ce11 degranulation causes inflamn~ationand increases mucus production, thus increasing
faecd bulk which may sweep away malignant cells and carcinogens; 2) lectins bind to the
mucosal cells, induces mitosis and increases epitheliai hanover, providing les of an
opportunity for neoplastic cells to take; 3) neoplastic cells may be susceptible to damage
by direct contact with lectins.
Because lectins occur in great amounts in plant foods, especially in legumes, theh
quantity in legumes and possible role in the prevention of colon carcinogenesis should be
investigated.
Although only a fwr major phytonutnents present in beans are discussed, other
biologically active phytonutrients are also present in beans. Several recent reviews have
addressed this topic (Messina and Bennink, 1998; Sgarbieri and Whitaker, 1982;
Thompson, 1993).
There is considerable interest in the role that legumes may play in the prevention of chronic diseases. Much of this interest has focused recently on the role of soybeans.
However, there have been relatively few studies that have focused on elucidating the effects of beam on disease nsk. The epidemiological studies that have addressed this question have focused on the roles of 0th- foods or nutrients and have collected limited information on legume intake and its relation to disease prevention. nierefore it is the aim of this thesis to investigate the presence of vatious fhctional components that may exist in legumes, and fiirther understand the role of legumes as a food item that may potentially bring health benefïts to the diet beyond basic nutrition. CHAPTER4
- PROXIMATE ANALYSIS - 4.1 INTRODUCTION
Dietary guidelines for good health published by He& and Welfare Canada suggest
avoiding excessive intalces of total fat, saturated fat and cholesterol, as well as sugar
and sodium. On a 100 lccal basis, legumes (with the exception of oilseeds) contain
80% less total fat and only 25% of the sodium content of Lean ground beef (Haytowitz
et al, 198 1). They contain no cholesterol and provide good sources of polyunsaturated
fatty acids (Sathe et al, 1984).
Dry beans also provide a good source of complex carbohydrates (Sathe et al, 1984;
Geil et al, 1994) quality vegetable protein, which is of special interest worldwide due to increased health concems regarding high fat animal protein sources (Gd et al,
1994).
Beans are rich in dietary fiber, which has shown to lower cholesterol, blood glucose and more recenly, have a protective effect against colon cancer. Dry beans are also a good source of water soluble vitamins such as folate. Epidemiological studies show that folate appears to have an inverse association to cancer risk and other chronic diseases (Rodgers, 1990; Rodgers et al, 1993; Steinmetz et al, 1993).
ln spite of the recognized nutritional contribution of legumes to the diet, the annual per capita consurnption of legumes in Canada is low (1.1 kg) (United Nations - Food
Domestic Utilization Data, 1998). This is a resuit of the popularity of animal products combined with long cooking times requirsd to prepare beans for consumption To compensate for low bean intakes due to excessive peparation tirne, food companies provide canned legumes for consumption. Lack of knowledge regarding the nutritionai and health benefits of beans may also contn'bute to low consumption.
Although food legumes represent a large numba species, only the dry beans of
PhtzseoIm wlgaris have attained popularity in North Amerka. Navy beans are used commercially in preparîng canned pork with beans, baked beans and bans in tomato sauce, while kidney beans are popular in chili and sirnilar products. Broad beans of the Vicia faba species appear to be more popular with Middle Eastern dishes such as
Foule.
The most popular processing methods employed for dry beans in western counines include cooking and canning. These processing methods however, influence the nutritive quality of the bans consumed. Therefore it is important to obtain data on the nutritional contribution of raw, cooked and commercially processed legumes that are of nutritional and economical importance in Canada.
4.2 OBJECTIVE
To investigate the macronutrient, folate and total fibre compsition of raw and canned
(with and without liquid contents in can) Navy, Kidney and Fava beans. 43 MATERIALS AND METHODS
43.1 Dry Beur Samples
In view of their economical and nutritionai importance, only Broad beans, Red Kidney beans and Navy beans and their processed equivalents wae used for nutrient estimation. Dry bans seeds were obtained hmthe Ontario Bean Growers
Association and processeci samples were obtained hmthe H.J. Heinz Company of
Canada. Three lots fkom three different kinds of legumes were used. One analysis was conducted on each lot.
43.2 Dry Bean Preparation
Ruw Bean Sampks: Raw bean samples were received and ground dominto a fine powder. A Wyllie Mill with a mesh size of 40 was used to achieve the unifonn particle size for al1 samples.
Canned Bean Sampfes
Each canned bean sample were either homogenized using the liquid contents in the can or homogenized after by rinsing the bans and draining the liquid content. With regard to the canned Kidney beans and Fava beans, the liquid content in the can was water. The liquid content in the canned Navy bans was tomato sauce. Each bean sample was then placed in flask cylinders for fieeze drying. Following freeze drying, each sample was placeâ into the WyUie Mill, using a mesh size of 40 to obtain uniform particle size, and ground into a fme powder. Cookd / Time-Treaîeà Navy &an Samples
Tripkate lots of raw Navy bean seeds were obtained hmthe Ontario Bean Growers
Association. The beam were soaked for three ho-, blanched for 6 minutes at a temperature of 76"C,followed by cooking under pressure at 120°C for 25,50 and 75 minutes. Most processors follow this samc procedure for preparing canned beans.
The beans were homogenized without the water contents, fkze dried and ground into a fine powder using a Wyllie Mill (mesk size 40) in order to achieve uniform size.
The proximate composition and phytonuîrient composition was obtained for the triplicate bean lots at each cooking tirne interval.
Storuge
Al1 raw and processed (fieeze dried) beans were stored in piastic bags, in a cold room, at a tempurature of 4°C.
4.3.3 Nutrient Determination of Ontario Grown Beans
Proximafe Anaiysis
AOAC Official Methods of Analysis were used to detennine proximate analysis
(percent moisture, total proteins, total iipids, and ash) of raw and processed beans.
Total carbohydrate content of the products were determined by the difference.
Total dietary fiber content of three selected beans was measured by the method described by Englyst et al, 1982. Foiate Determination
Samples were analysised for folate within the Laboratory Services Division at the
University of Guelph, Ontario. The analysis was conducteci using current standard published procedures for folate determination. Proximate andysis of raw and cooked Navy beam, Kimbans and Fava beaus are shown in Tables 4.4.1. to 4.4.3.
The data fkom the present andysis was found to be in close agreement with other available data with regard to the raw legumes. Moisture values for uncooked legumes were generally in good agreement with data from the USDA Handbook No. 8, except for the Fava beans, which showed slightly higher values than the published data
(Meiners, 1976; Watt and Merxil, 1972). The protein, fat and ash values determineci for the samples in the current analgrsis were also in good agreement with the data tabulateci in Handbook No. 8.
With regard to the processed samples, data for moishue, protein, fat, ash and carôohydrate for Fava and Kidney bans fiornogenized with liquid contents in the cm) are generally in good agreement with data for these components as given in
Handbook No. 8 (Appendix A). With regard to the samples which were rinsed and drained fiom the liquid contents in the can, the proxirnate values for protein, fat, carbohydrate and ash (with the exception of moishire) appear to be greater than that of the beans anaiyzed with the liquid contents in the can. Tabk 4.4.1 Prosimite Composition of Raw and Canneci Navy Beans
CHO Protein Fat Ash Mois ture
Raw Bean 61.13 21.93 126 3.31 12.37 * 0.08 * 0.08 * 0.07 * 0.24 & 0.08
Processed Navy 23.06 6.19 0.41 2.02 6938 Beans r 0.51 h 0.09 0.29 r 0.06 * 0.25 (Baked Berras, homogenized witb tomato sauce) frocessed Navy 22.73 6.08 0,40 2.01 68.20 Beans 0,60 * 0.13 * 0.03 r 0.05 h 0.82 (Baked Beans, homogenized with out tomato sauce) a: each value represents the mean standard error of the mean for three analysis from each of three purchase lots (N=3). Table 4-4.2 ProUmrite Composition of Raw and Cameci Kidney Beans
CHO Protein Fat Ash Moisture
Raw Bean 62-45 2 1.97 1.05 3.05 11.47 * 0.76 0.72 * 0.011 * 0.13 f 0.21
Processed 14.07 4.46 0.36 1.66 78.62 Kidney Beans I. 0.38 * 0.12 k 0.02 k 0.06 * 0.51 homogenized with water contents in cm
Processed 18.77 5.87 037 2.07 72.49 Kidney Beans h 036 k 0.13 h 0.02 * 0.04 0.52 (Beans homogenized with out water contents in con) a: each value represents the mean * standard error of the mean for three analysis from each of three purchase lots (N=3). Table 4.4.3 Proximate Composition of Raw and Canned Fava &ans
CHO Protein Fat Ash Moisture
Raw Bean 59.92 25.03 1.42 2-45 11.18 * 0.13 *OS% & 0.01 * 0.12 =t 0.36
Processed Fava 12.67 9.82 0.26 1.12 75.42 Beans a0.27 h 0.18 * 0.02 * 0.06 * 0.59 (Beans homogenlzed with water contents in can)
Processed Fava 23.82 9.13 0.58 2.82 63.18 Beans * 1.04 * 0.53 * 0.02 aO.11 * 1.66 (Beans homogenized with out water contents in can) a: each value represents the mean + standard error of the mean for three anaïysis from each of three purchase lots (Ns3)- The proximate analysîs of Navy beam cooked at various time intervals to detennine the effeçts of cooking on nutrient retention are provided in Table 4.4.4.
These results indicate that as the cooking continued, the buuis took on more water. As they took on more water, their macronuûient composition deciined. Figure 4.4.1 illustrates the changes in percent composition of Navy beans as they continue to be cooked. Table 4.4.4. Proximate Composition of Thne-Treated Cooked Navy Beans
CHO Protein Fat Ash Moisture
Blanched for 29.01 9.61 0.52 1.02 60.88 6 minutes r 0.83 r 020 =t0.004 r 0.03 0.68 (75.9Oc)
Pressure 2 1.22 8.07 0.43 1.12 7033 Cooked for 25 * 0.43 r 0.12 r 0.02 r 0.06 r 0.47 minutes (1 19.9OC)
Pressure 21.08 6.99 0.40 0.84 70.13 Cooked for 50 * 0.54 k 0.21 r 0.02 r 0.09 r 1.12 minutes (1 l9.9OC)
Pressure 19.06 636 038 0.85 73.06 Cooked for 75 r 0.18 r 0.05 r 0.01 0.06 r 0.18 minutes (1 19.9OC) a: each value represents the mean * standard error of the mean for three annlysis from each of three purchase lob (N=3). b: samples are drained from water after cooking. Figure 4.4.1. % Decrease of Proximate Content From Raw Navy Bean Seeds at Various Cooking Intervals
CHO PRO FAT ASH
Proximate Content NOTE TO USER
Page(s) not included in the original manuscript are unavailable from the author or university. The manuscript was microfilmed as received.
This is reproduction is the best copy available The total fiber content of raw and cooked Navy beans, Kidney beans and Fava beans are show in Table 4.4.5. Vdues reported for fiber content in Handbook No. 8 are provided in Table 4.4.6.
Values found in the current analysis for raw bean total dietary fiber were much less than those values reported in Handbook No. 8. Considerable differences were noted in the raw Navy and Fava beans. The raw Kidney beans value reporteci here was in closer proximiîy to the value reported in Handbook No. 8.
With regard to the canned beans, the values are generally in good agreement to those reported in Handbook No.8. Table 4.4.5. Total Dietary Fiber Content In Raw and Canned Navy, Kidney and Fava Beas.
p: / 1oog Raw Beans Camed beans NaW 14.50 f 0.8 3.55 f O.lO(witb tomrrto sauce) 2.83 * 0.60 (without Tomato sauce)
3.60 IO.10 (without water from can)
14.92 f 0.6 5-67 k 0.40 (without water from cm) a: each value rcpresenb the mean *standard error of the mean for üuee andysis fmm each of three purchme lots (N=3). b: sarnples are drairied from water after cooking.
Table 4.4.6. Total Dietary Fiber Content in Raw and Canned Navy, Kidney and Fava Beans as Reported By USDA Handbook No. 8.
g / 100g Raw Bems Cirnned beruis Nav~ 24-40 5.10
a: Data obtained from USDA Huidbook No. 8. The folate content of raw and cooked Navy, Kidney and Fava beans are shown in
Table 4.4.7 and 4.4.8.
Folate values for raw and processed Navy, Kidney and Fava beans were found to be considerably low in this study compared to those values provided in Handbook No. 8.
The percentage of vitamin retention foliowing processing was ver-low. With the exception of the Fava bean, virtually 100% of folate was lost after processing of canned beans. The Fava beans showed a 77% reduction of the folate content in those beans not homogenized with the liquid content in the can, and only a 6 1% reduction in folate of those beans that were homogenized with the liquid content in the can.
With regard to the time / treated cooked navy beans, a 94%, 96%, 97.6% and 99% reduction in folate composition was seen in the 6 minute blanched, 25,50 and 75 minute pressure cooked beans respectively. Table 4.4.7. Folate Content of Raw and Canned Navy, Kidney and Fiva &ans
Navy Bean Kidney Bean Fava Bean
Raw Bean 99.54 f 4.08 103.93 +, 19.63 85.44 I26.25
Canned kans 0.26 f 0.014 0.18 f 0.02 32.93 f 7.16 (homogenized with iiquid content In cm)
Canned Beans 0.01 .O02 0.09 t 0.003 19.48 f 6.78 ( bomogenlzed with out liquid content in can) a: eacb value represents the mean r standard error of the mean for three anaiysis from each of three purchase lob (n=3).
Table 4.3.8. Folate Content of TbTreated Cooked Navy Beans
mcg / 100 g
Raw Bean Blanched for Pressure Pressure Pressure 6 minutes Cooked for 25 Cooked for 50 Cooked for 75 (75.9OC) minutes minutes minutes (1 19.9OC) (1 19.PC) (1 19.9OC)
a: each vaiue represents the mean standard error of the mean for three anaiysis from each of three purcbasc lots (n=3). b: samples are drained from water after cooking. 4.5 DISCUSSION
Proximate Composition of Raw Beans
Data on proximate composition (moisture, protein, fat, carbohydrate and ash) values are provided for raw, canneci and cooked legumes in Tables 4.4.1 through 4.4.3. With regard to the raw legumes, the data hmthe present analysis was found to be in close agreement with other available data. Moisture values for uncodred legumes were generally in good agreement with data tabuiated in the USDA Handbook No. 8, except for the Fava beans, which showed slightly higher values than the published data (Meiners, 1976; Watt and
Merril, 1972). Meiners (1 W6), explained that the moisture content of dried legumes can be greatly affecteci by relative humidity of the surrounding atmosphere at harvest and during storage. Any differences in moisture content between the current anaiysis and data fiom other published sources may be due to past environments and sources of the samples (Meiner et al., 1976).
The protein, fat and ash values detennined for the samples in the curent analysis were also in good agreement with the data tabulateci in Handbook No. 8. Variations among lots of raw legurnes are shown by the standard enors reported in Tables 4.4.1 - 4.4.3 for each of the proximate components which were detennined by laboratory analysis.
Results of triplkate analysis for the different market lots of the legumes analysed were in remarkably good agreement. The slight variation displayed by the SE rnay be primarily caused by differences among bean samples. These differences, while not great (- i 1%) were seen in the carbohydrate, protein and ash of the Fava beans; carbohydrate and protein in Kichey and Navy beans. As previously mentioned, these differeaces may be caused by past environments, Le., varied climatic conditions.
Proximate Composition of Canned Beans
One of the most popular methods for preparing besns in western countries is canning.
Canned beans contain water, which may or rnay not be used in preparing beans dishes dependhg on cultural or personal preferences. Some cooking practices prefer to drain the water fiom the canned beans, whiie others choose to include it in their fmished product.
Meiners et al. (1976), examined the cooking water of legumes to determine the proximate composition remaining when different varieties of legwnes were cooked. Their study found that the water in which the beans were cooked contained some residues. The cooking water contained about 1 to 3% protein, a srnall amount of ash and between 4 to
10% other materials, of which was suspected to be primarily carbohydrate. Therefore, in the current study, the analysis included examining the bean samples homogenized with the liquid contents in the can and also the bean samples ~sedand drained fiom the liquid content in the can.
Data for proximate composition of canned legumes on Navy, Kidney and Fava are provided in tables 4.4.1 - 4.4.3. Data for moishue, protein, fat, ash and carbohydrate for Fava and Kidney bans (homogenized with liquid contents in the can) are generally in
good agreement with data for these compments as given in Handbook No. 8 (page 14).
Dismepancies in data of the curent analysis and that of Handbook No. 8 are easity
distinguishable with regard to the canned navy beans (homogenized with tomato sauce).
It is important to point out that the canned navy beans in the current snidy containeci
tomato sauce, whereas those reported by Handbook No. 8 are prepared with water.
However, the moisture content of the canneci navy beans is in close proximity to that of
Handbook No. 8 which may suggest that the tomato sauce present in the current analysis
may compensate for the water content found in other canned navy beans. The greater differences are seen in the protein, carbohydrate and ash content, al1 of which rnay be explained by the presence of tomato sauce.
With regard to the samples which were rinsed and drained fiom the liquid contents in the cm, the proximate values for protein, fat, carbohydrate and ash (with the exception of moishue) appear to be greater than that of the beans analyzed with the liquid contents in the can. On a g/100g basis, the lack of water content in the can would make the serving of beans more concentrateci with macronutrients. This could explain why the proximate values for the beans homogenized without water are slightly higher than those with water.
Cooking such as boiling, is also a popular method of preparing beans. The length of time required to cook beans may b~gout several changes in physical and nutritional qualities. in addition, prolonged cooking may even reàuce the nutritive quality of bans (Deshpmde et al., 1984). Sevdworkers have studied the changes in cooking methoâs
on proximate composition of dry beans (Carpenta, 198 1; Deshpande, 1984; Halaby et
ai., 198 1 ;Meiners et al., 1976; Sgarbieri, 1986; Sathe et al, 1984'; Sathe et ai, 1984~1,but
none have examineci the nuaients lost during the heking procedure at various tirne
intemals.
With the need for accurate tables of food composition, most attention has focused on the
effects of cooking on nutrient content and nutrient retention. in the current study,
proximate analysis was conducted to obtain the nutrient composition of Navy Beans at various cooking time intewals to determine the effects of cooking on nutrient retention.
The redts of this experiment are show in Table 4.3.4.
Table 4.4.4 indicates that as cooking continues, the beans take on more water. As they take on more water, their mamonutrient composition declines. Figure 4.4.1 illustrates the changes in percent composition of navy beans as they continue to be cooked.
Fiber Composition of Raw, Cunned und Cooked Beum
Values for total dietary fiber content in selected beans are reported in Table 4.4.5. The values for total fiber content reported in the USDA Handbook No. 8 are provided in
Table 4.4.6. In contrest to the gendygood agreement between data nOm this research and that which is provided in Handbook No. 8 for protein, fat and ash contents, values reporteci here for total dietary fiber for raw beaas were much less than total dietary fiber values reported in Handbook No. 8. Considerable differences were noted in the raw Navy and
Fava bans. The raw Kidney beans value reported here was in closer proximity to the value reported in Handbook No. 8. With regard to the canned beans, the values are generally in good agreement to those reporteci in Handbook No.8.
The methods used for measuring die- fiber are intended to rnimic the enzymatic and chernical reactions of food in the mouth, stomach and small intestine. Discrepancies in published values for some raw and canned foods wuld be explained by the characteristics of the enzymes used and, to some extent, by the principle of the method used (Baker and
Holden, 198 1; Englyst, 1982; Mongeau and Brassard, 1982; Paul and Southgate, 1978).
The value for cannecl baked beans in tomato sauce reported by Englyst et al. (1982), is
3.87g/100g. This is in good agreement with the current reported value of Navy bans in tomato sauce, which also used the Englyst method for fiber detennination.
Folate Composition of Raw, Cunned and Cooked Beans
Augustine et ai (198 1), reported the minerai and vitamin composition of raw and cooked samples of nine classes of Paseolus wuIgaris species. Among those finding were the results of the folate content within the legumes. On a 1OOg dry weight basis, the mean folate valws for raw beans was 0.30 mg. Mer cooking, the value fell to 0.22 mg, indicating that 73% of the vitamin was retained In the curent study however, the percentage of vitamin retention following processing was much lower. With the exception of the Fava bean, virtually 100%of folate was lost after processing of canneci beans. The Fava beans showed a 77% reduction of the folate content in those beans not homogenized with the liquid content in the cm, and only a 6 1% reduction in folate in those beans that were homogenized with the liquid content in the can.
Although folate values provideci by Handbook No. 8 are much higher than those found in this study, it is possible that commercial processing techniques have a significant effect on folate retention. Legumes are generally thought to be good sources of folate, however, it appears that not dl 1egumes will retain nutrients to the same extent under commercial processing techniques.
With regard to the time / treated cooked navy beans, a 94%, 96%, 97.6% and 99% reduction in folate composition was seen in the 6 minute blanched, 25,50 and 75 minute pressure cooked beans respectively.
Aithough the values reported for folate in this study is much lower than those reported in other published works, the folate value for canned navy beans and the final the/ treated value (which is intended to mimic the coolring method of pmcessed canned beans) are in good agreement, indicating almost a 100% reduction in folate content. It is difficult to speculate why the reporteci values in this shuiy are so different than those
in pubiished work (Augustine et al, 198 1; USDA Handbook No. 8). It may be a result of
different geographical growing location, growing seasons, processing techniques and
analyticai techniques.
The nutrient contents of selected beans, fiom the current study, suggest they are ideally
suited to meet major dietary recommendations for good heaith. The fat content found in
canned Navy beans (in tomato sauce), Kidney and Fava beans (wmbined with the water
content from the cm) in the current study is very low in fat. On a 2000 kcal per day diet,
a one-cup serving (250 ml) of Navy, Kidney or Fava beans will contribute ody 2%, 1.8% and 1.3% calories from fat. This is approximately 80% las total fat than that provided by lean ground beef and becawe beans contain a much higher ratio of unsaturateci to saturated fats, a serving of beans will provide a higher quality fat.
Beans are generally thought to provide a high quality plant protein. The protein contribution by the Navy bans (with tomato sauce), Kidney and Fava beans (with water content nom the can) in this study (based on a 20kcal per day diet) dlprovide 23%,
16% and 20% of the recommended nutrient intake, respectively. The protein content of legumes is of special interest worldwide due to high cost and the increased health con- regardhg hi& fat animal protein sources. Hence, bean protein is a good alternative source.
Dietary guidelines reçommend coosuming increased amounts of starches and complex carbohydrates, which also makes beans ideally suited for Canada's Guide to Healthy
Eatîng. A one-cup serving (baseci on a 2000 kcal diet) of camed Navy beans (in tomato sauce), Kidney and Fava beans (with the water contents fkom the can) provide 2 1%, 13% and 1 1.5% of the recommended intake of total carbohydrates.
Based on the current analysis, a one-cup shgof canned Navy beans (in tomato sauce)
Kidney and Fava beans (with out the water content fiom the cm) will pmvide between
28-39%, 17924% and 27-38% (respectively), of the current recommendation of 25 -
35g/day of fiber. A number of articles have implicated dietary fiber as a major component in certain foods which may provide important health benefits in diabetes, hypercholesterolemia and cancer (Anderson and Akanji, 199 1; Jaikins et al, 1980;
Saperstein et al, 1978) making beans fhctionally beneficial in the diet.
The following figwes illustrate the percentage of the recommended nutrient intake obtaineà hmconsuming a one-cup serving of canned Navy, Kidney and Fava beans, based on a 2000 kcal diet. Figue 4*6.1 Percentage of Recommendd Daüy Intake of Macronutrients, Fiber and Folate From One-Cup Sewing of Canned
N~VYBeans
Protein CHO Fat
8 W / TS: represents canned Navy beans in tornato sauce 8 WO 1 TS: represenh canned Navy beans with tomato sauce removed Figure 4.6.2 Percentage of Recommended Daily Intake of Macronutrients, Fiber and Folate From One-Cup Serving of
Canned Kidney &ans
em W / H20: represents cannd Kidney beans with water content in can 9' WO / H20: represents canned Kidney beans without water content in can Fipn4.6.3 Percentage of Recommended Daily lntake of MacronuMonts, Fiber and Folate From One-Cup Servhg of
Canned Fava Beans
Proteln CHO Fat FIbr Folde
W / H20: represents canned Fava beans with water content in can WO / H20: represents canned Fava &ans withoui water content in cari Figures 4.6.1 to 4.6.3 indicate that a srnall portion of the macronutnents, folate and fiber are leached out into the liquid content of the cm. Preferences to consume beans with or without the liquid content found in camed beans appear to alter the percentage of macronutrients, folate and fiber wntriiuted to the diet per sening. Codgthe canned beans with the liquid portion of the can wiIi provide a slightly higher nutrient intake. CHAPTER 5
- PHYTONUTRIENT COMPOSITION - 5.1 INTRODUCTION
Recent studies have suggested that dry legumes may contain a nurnber of biologically
active compounds refdto as phytonutrients. These phytonutrients which include
trypsin inhibitors, phytates, haemagglutinins, saponins and oligosaccharides, have
traditionaily been labeled "anti-nutrients" because of th& ability to reduce the
availability and utilization of nutrients (Thompson, 1993). They are predominantly
present in raw legumes seeds. Cooking and or processing may alter the levels of these
kt-liable phytonutrients (Deshpande et al., 1984). Studies now indicate that as a result
of the compositional changes that occur during processing, these phytonutrients rnay
occur at levels that may exert significant beneficial properties (Anderson and Wolf, 1995;
Deshpande et al., 1984; Liener, 1993; Thompson, 1993; Wang and Wixon, 1999).
Much research has been conducted on the phytonutrients found in soybeans. These
studies have shown their beneficial effects of include lowering serum cholesterol,
decreasing the riskof heart disease, maintaining bone density, and lowering the frepuency
of breast, colon and prostate cancer (Kennedy, 1995; Messina and Barnes, 199 1 ; Second
International Symposium on the Role of Soy in Preventing and Treating Chronic Disease,
1996; Thompson, 1993; Wang and Wixon, 1999).
Although soybeans have been studied extmsively with regard to its phytochemical composition and their role in disease prevention, legumes being hmthe sarne family may also contain a similar profile of the beneficial phytonutrients. Very little information about the phytonutrient composition of ôeans is available in the literatlue. Therefore this study was undertaken with the objective of analyzing the phytonuûient composition of commonly w13sumed bans grown in Ontario.
5.2 MATERIALS AND METHODS
52.1 Dry Bern Samples and Preparation
See Chapter 4 (page 46).
5.2.2. Phytonutnent Determination
Fat fiee samples were used for the estimation of trypsin inhibitor activity, saponins, isoflavones, phytic acid and lectins.
Preparation of Fat Free Dry Samples
The dry powder afier priliminary processing was weighed accurately into a thimble and plugged with a rnetal cap and filter paper. The magnetic thimble was placed into a
Soxtec Fat Analyzer and fat was extracted using petroleum ether for 20 minutes. The fat fiee residue was collecteci in glas cups, cooled and weighed.
Tsr>sin Inhibitors Detennination
Trypsin ïnhibitory Activity was detdned by the method of Roy Rao (197 1). The activity of the enzyme, trypsin, was assayeà using casein as substrate and inhibition of this activity was measured in the bean extracts. 500 mg of defaîted, pulverized bean material was treated with 20 ml of O. 1M sodium phosphate buffer (pH 7.5). The samples were shaken for 1 hour and centrifûged at 220 rpm for 30 min at lS°C. The supernatants were diluted in such a way that i~hbitionwas observai at 60,80, and 100% of control enzyme activity.
The incubation mixture consistai of O. lm1 of trypsin (0.05 mghl in 0.00 1 M HCL), 0.2 ml 2% casein solution in phosphate buffer, 0.06ml of bean extract and 0.04ml of sodium phosphate buffer (pH 7.5). incubation was carried out at 37C for 20 min. The treatment was stopped by the addition of O.6%mlS% TCA. Corresponding blanks were run concurrently and absorbance measured at 280 nm in a spectrophotometer.
One trypsin unit (TU) is arbitrarily defïned as an hcrease of 0.01 absorbance units at
280nm under the conditions describeci. Trypsin inhibitor activity is defined as the number of trypsin units inhibited (TUI).
Phytic Acid Determination
Phytic acid was estimated by the method of Davies and Reid (1979).
One gram of ground, fat free sample was extracted with 25ml of O.SN HN03 for 3 hours by continuous shaking, followed by cenûifûging at 12000 rpm for 20 min. The supernatant was collected and made up to suitable volume. 0.28ml of the filtrate was combineci with 02ml of fdcammonium sulphate solution (2 1mg in l OOml of H20) and
placed in a boiling water bath for 20 min.
The contents were cooled and Idof isoamyl alcohol was added and rnixed. 0.02ml of
10Y0 ammonium thiocyanate solution was added and shbthoroughly, and centrifûged
at 3000 rpm for 10 min. The alcohol layer was then separated and the color intensity was
read at 465nm against isoamyl alcohol blank after 15 min. Sodium phytate standard (4 -
40 ug) was nui simultaneously with the samples and results were expressed as mg phytic
acid / 100g.d.wt.
Saponin Determination
Saponin estimation was detennined using method of unpublished work by Gurnnkle and
Rao, 200 1.
Finely ground, defatted beans material was extracted in methanol(70ml) for 4hr at 60C, with gentle shaking. The saponin containing methanol extract was filtered and the bean residue washed with additional methanol bringing the total volume of methanol extract to
100 ml. A clean-up procedure was then applied to separate the saponin nom non-saponin constituents of the methanol extract. 5 mi aliquot was mixed in a 1: L ratio with 0.4 M ammonium sulfate and left ovefnight at room temperature. This resulted in the formation of non-saponin precipitate. The precipitate was removed by centrifugation at 2000g for 30 mh. îne precipitaie was then washed thtee times with 1: 1 mixture of methanol and
0.4 M ammonium dphate to release my entrapped saponias. The saponin-containing
cenhifùgate was then slowly passed through an Oasis HLB extraction cartridge (Waters
Corporation; 500 mg adsobent, 12-ml barrel). The saponins were retained by the
adsorbent The cartridge was washed with 10 ml of water and the saponins were
extractecl hmthe cartridge with 6 ml of methanol. 5 ul were applied to a TLC plate for
saponin detemination by direct densitometry.
Huemaggiutin~tionActivity Determination (ZecfinAc fivity)
Haemagglutinin activity (HU) was estimated by the method describeci by Weder et al.,
1997. One gram of finely ground beau sample was combined with Sm1 of PBS (pH 7.2).
Extraction took place by continuous shaking for 24 hours at 4C. the mixture was
centrifiiged at 16000 rpm fio 20 0 at OC. The supernatant was collecteci and diluted
serially with PBS for a total of 6 dilutions (6,25ûug/ml to 200,000ug~ml).0.025ml of each dilution was lightly shaken with 0.025ml of 2% erythrocyte suspention (0.4ml of erythrocyte pellets with 19.6rnl of PBS). The mixture was left for 1 hour and shook slightly to detect agglutination activity.
One unit of haemagglutination activity (HU) is defined as the amount of material per ml in the last dilution giving visible haemagglutination. Samples wae analysised for fiuctoi>ligosaccharide content at the ORAFTl Company in
Belgium. The analysis was conducteci using the method described by Prosky and
Hoebregs (1 999). 53 RESULTS
The selected phytonutrient content in Navy, Kidney and Fava beans are shown in Tables
5.3.1. to 5.3.4.
The literature suggests that cooking generaily inactivates the heat sensitive factors such
as trypsin inhibitors, lectins, phytates and oligosaccharides, while the heat stable factors
such as saponins, may ody be slightly reduced during heat processing (Deshpande et al,
1984; Reddy et al, 1982). In the current study, lectin activity (HA) is stilî present following processing (Tables 5.3.1 to 5.3.3). The HA in the current study suggests a reduction by 96% in canned kidney beans with water and 97.4% reduction in caaned
Kidney bans without water. It also indicates a 77.4% decrease in Fava beans with water, and 84.87% without water. The Navy beans in tomato sauce showed a 98.8% reduction in HA and a 98.9% reduction when the tomato sauce was rernoved (Figure 5.3.1). The
HA ofthe time / treated Navy beans appear to be increasingly inactivated as processing continued (Table 5.3.4.). This is to be expected as lectins are a heat sensitive factor. The appreciable decrease in HA fiom the raw Navy bean seed at 6,25,50 and 75 minutes were 57.8%, 96.5%, 98% and 98.9%, respectively.
The trypsin inhibitory activity (TIA), in the cumnt study, appears to be somewhat affected by commercial heat processing. The loss of TIA in Navy beans cooked at different theintervais (Figure 5.3.4.) also suggests the presence of heat labile and non- labile inhibitory components in the bean seed (Singh & iambunathm, 198 1). in the current study, only 69.2 1%, 55.85% and 62.56% of TM of Kidney beans with water, Tibk 53.1. Phytonutrient Composition of Raw and Canned Navy Beans
TN Saponin Pytic Acid Fructo- EiIJ (xid) Oligo- YO YO Saccharide
Piocessed Navy 2137.57 0.27 1.32 136 1633.06 Beans f 93.9 f 0.03 f 0.03 f 13.7 (B8kcd Beans, homogenized with tonuto sauce)
Processed Navy 1063.43 0.26 1.19 0.7 1573.69 Beans f 42.2 f 0.07 f 0.03 140.1 (Baked Beans, homogenfied with out tomato sauce) a: each vdue represents the mean mtandard error of the mean for three maiysis, one from each of three purchue lots (N=3). Table 53.2. Pbytonutrient Composition of Raw and Canncd Kidney Beans
TIU Saponln Pytic Acid Fructo- HU (XIO~) 01ig0- YO YO Saccharide
Raw Bern
Proc& Kiàney 7749.28 0.25 131 N/A 223339 Beans f 819.2 f 0.11 f 56.6 ma= homogenized with water contents in can)
Pr- Kidney ci397S9 0.24 1.20 055 181838 Beans f 564.7 f 0.05 f 0.01 f 34.1 (&ans homogenized with out water contents in can) a: each value represents the merin * standard error of the mean for theanaiysis, one from each of three purchase lots (N=3). Table 533 Phytonutrient Composition of Raw and Canned Fava Beans
TIU Saponin Pytic Acid Fructo- EiIJ Oligo- (XI 03) YO YO Saccharide
Processeâ Fava 1543.4 1 0.00 1254 N/A 2035.49 Beans f 813 f 0.04 f: 49.8 (Be-s homogeirizeà with water contents in can)
Processed Fava 702S8 0.00 1.45 0.55 1361.88 Seans t 58.6 f 0.85 f 0.08 f 63.9 (Beans homogenized witb out water contents in cm) a: each value represents the mean *standard trror of the mean for üuee anaiysb, one from each of three purchase lots (N=3). Table 53.4. Phytonutrient Composition of Tirne-Treated Cooked Navy Beans
TIU Saponin Pytic Acid Fructo- HU (xi@) Oiigo- VO ./O Saccharide
Bhnched for 3739.82 033 1.59 N/A 61717.42 6 minutes (75.90C) I733 f0.09 I1697.2
Pressure Cooked 299736 032 1.24 NIA 5114-96 for 25 minutes f 98.7 f 0.02 f 88.6 (119.9OC)
Pressure Cooked 2551.72 0.28 1.16 N/A 2860.69 for 50 minutes f68.8 f 0.01 f 84.9 (1 l9.9OC)
Pressure Cooked 2366.77 0.27 1.14 NIA 1561.21 for 75 minutes + 33.77 + 0.02 f 535 (1 19.9OC) a: each value represenb the mean h standard error of the mean for three anrilysis, one from each of tbree purchrise lots (N=3). Navy beans with tomato sauce and Fava bans with wata (respectively), still ranained dercommercial processing. When TIA was measured for the same beans without the liquid contents hmthe can, 56.33%, 27.78% and 28.48% TIA remained (respectively).
The commerciaily mimicked the/ treated navy bean samples indicate an appreciable loss of TIA also. As cookuig progressai under the various thne intervals, TM decreases by 2.28%, 2 1.68%, 33.32% and 38.15%, in the bans cooked for 6,25,50 and 75 minutes
(respectively), hmthe raw bean. The find theinterval indicates 6 1.85% stïll remaining after pressure cooking.
The range of phytate reduction fiom the raw seeâ in Navy, Kidney and Fava beans drained hmthe liquid contents in the cmwas 50.62%, 48.49% and 26.02%, respectively. When the liquid contents of the canned beans was not removed, the phytic acid reduction was only 45.22%, 43.77% and 2 1.42% respectively (Figure 5.3.1. - 5.3.3).
With regard to the time / treated cooked Navy beans, phytate content appreciably decreased by 34.02%, 48.54%, 5 1.86% and 52.6996, as cooking continued.
Oligosaccharide content for the 3 canned samples without the liquid contents f?om the cmshowed a 79.47%, 83.18% and 82.75% deaease respectively. Although oligosaccharide content for cmed Kidney and Fava beans with water contents are not reported, the Navy bean sarnple with tomato sauce shows only a 60.1 1% reduction.
With regard to the saponin analysis, the current study showed a 28.94% and 3 1.57% reduction in saponin content of Navy beans with and without tomato sauce (respectively). Canned Kidney beans showed a higher level of saponin retention with the water content
(13.7996) and without the water content (17.24%) hmthe can compareci to the canneci
Navy beaos in tomato sauce. When viewing the effect of cooking Navy beans at different
time intervals, saponin content decreased gradually. The reduction ranged hm13.15%
to 28.94%, which is in good agreement with previously mentioned, published work.
Figures 5.3.1 - 5.3.4. iIlustrate the phytonutrient content remaining in canned beans with or without the liquid contents combined with the beans. These figures appear to suggest that the removal of the heat stable factors in canned beans is primarily due to leaching losses (when the water or tomato sauce in the can is removed). When the liquid contents hmthe can is not removed, a lower loss of heat stable phytonutrïents are observecl. Figure 5.3.1 % Reduction of Phytonutrient Content of Canned Navy Beans in Tomato Sauce and Witbout Tomato Sauce From Raw Navy Bean Seed
TIA Saponln Phytirtû F-Oligo HU Figure 53.2 % Reduction of Phytonutrient Content of Canned Kidney Beans in Water and Without Water From Raw Kidney Beans Seed
-- --. .-*- Raw WM20 O WOM20
-CI -- 1.- -- -Y Saponin Phytaîe Figure 5.33. % Reduction of Phytonutrient Content of Canned Fava Beans With Water and Without Water From Raw Fava Bean Seed Figure 53.4. ./o Reduction of Phytonutrient Content of Time Treated Cooked Navy Beans From Raw Navy Bean Seed
Raw 6 min 25 min 50 min 75 min 5.4 DISCUSSION
Dry beaos contain a numba of components once refdto as antinuîrients, including
trypsin inhibitors, saponhs, hearnagglutinins (lectins), Phytates, and oligosaccarides,
among others. Processing and heat treatment are required to prepare grain legume seeds
for food use. Cooking or processing will aita the anthutrient levels in beans and their biological properties, permitting more efficient digestion and assimilation of protein and starch.
Cooking generally inactivates the heat sensitive factors such as trypsin inhibitors, lectins, phytates and oligosaccharides, while the heat stable factors such as saponins, may only be slightly reduced during heat processing (Deshpande et al, 1984; Reddy et al, 1982).
Complete inactivation ofthe heat sensitive factors cannot be achieved. Thompson et al
(1983), found that heat processing or cooking can only reduce the levels of lectins.
De Mulelnaere (1964), found lectins to also be resistant to inactivation by dry heat.
Jaffe (1980), reported that lectins in bans were heat resistant and were not completely destroyed.
In the current study, lectin activity, othenuise known as heamagglutiation activity (HA), is still present following processing (Tables 5.3.1 to 5.3.3). A number of studies have reported almost complete inactivation of HA in Phuseolus wlgaris stain of legumes
(Liener, 1977; 1979). There is generally good agreement with HA of processeci Kidney beans and Navy beans in the current study. However, the Fava bean retains 15 - 20% of HU after processing. This coud be explained by the different variety hmwhich the
Fava bean is derived (Vicia faba) and the presence of more heat stable isomeric forms in bis bean.
The HA of the time / treated Navy bans appear to be increasingly inactivated as pnxessing continues (Table 5.3.4.). This is to be expected as lectins are a heat sensitive factor. The appreciable decrease in HA hmthe raw Navy bean seeâ at 6,25,50 and 75 minutes were 57.8%, 96.5%, 98% and 98.9%, respectively.
To date no studies have indicated at what level lectins will exibit their potential benefïcial effects. Howwer, Noah et al (1980), have suggested that through adequate cooking, resuiting in almost complete inactivation of HA, will not bring about symptoms of lectin toxicity reported in humans.
Trypsin inhibitors, being protein are subject to denaturation and inactivation by heat. In the cument study, trypsin inhibitory activity (TIA) appears to be reasonably destroyed by commercial heat processing. The loss of TIA in Navy beans cooked at different time intervals (Figure 5.3.4.) also suggests the presence of heat labile and non-labile inhibitory components in the bean seed (Singh & Iambunathan, 198 1). The resistance to complete destruction of TIA in legurnes by heat is reported by a number of researchers (Anderson et al, 1995; Ellenrieder et al, 1980; Kennedy, 1995). In the mentstudy, TIA in Kidney aud Fava beans with water and Navy beans with tomato sauce stül remainexi afier commercial processing. Whea TIA was measured for the same beans without the liquid contents, lasTIA ranained in al1 three samples. This suggests that a substantial amount of TM is leached out into the water. Depending on individual cooking practices and preferences (cooking and mnsuming canned beans with or without the liquid contents in the am), this could have a significant impact on the amount of trypsin inhibitor units one consumes, when eating cannexi legumes.
The commercially rnirnicked time / treatd navy bean samples indicate an appreciable
Ioss of TIA also. As cooking progressed under the various time intervals, TIA decreased gradually bmthe raw bean. The final time interval of the pressure cooked samples indicated a somewhat higher TIA than the canneci Navy bans with or without tomato sauce. This suggests that TM is destroyed more readily thn,ugh commetcial processing rather than pressure cooking, as done within the home.
In general, two types of TI are recognized: the heat labile Kuntz Inhibitor (KI) and the heat stable Bowman Birk inhibitor (BBI). Levels of TI in these beans and in processed beans may be a reflection of relative amounts of these inhibitors presence.
Reduction in phytates and oligosaccharides have been observed in Navy, Kidney and
Pinto beans after cooking for 90 min at lûû°C (Tyer et al, 1980). Other researchers have also reported similar fîndings @ahpande et al, 1984; Reddy et al, 1982; Tinsley et al, 1985). The range of phytate reduction fimm the raw seed in Navy, Kidney and Fava
beans appeared to follow the same pattern as that suggested in the literature.
With regard to the time I treated cooked Navy beans, phytate content appeared to be more
readily destmyed within the initial oooking pied and then uitimately reached some sort
of threshold point during prolonged cooking (Figure 5.3.4.). The results therefore
indicated that even after prolonged cooking times, appmximately 50% of the phytic acid
content will be retained in the bean.
Oligosaccharide content for the 3 bean samples without the liquid contents fiom the can
showed a decrease. Although oligosaccharide content for canned Kidney and Fava beans
with water contents are not reporteci, the Navy bean sample with tomato sauce shows only a 60.1 1% reduction. This showed a considerably different phytic acid content remaining compareci to Navy beans without tomato sauce, suggesting a substantial amount of leaching into the tomato sauce.
Saponins are heat stable factors and may not be reduced significantly during heat processing. Although there was no saponin detection in the raw or processed Fava bean, the analysis indicated their presence in both the navy and kidney beans.
There have been very few studies on the saponin content of cooked or processed beans.
However, Price et al (1 987), suggest that cooking and canning have Little effect on the saponin content of various beans. The cwent study showed a 28.94% and 3 1.57% reduction in saponin content of Navy beans with and without tomato sauce (respectively). Cameci Kidney beans showed a higtier level of saponin retention with the water content
(13.79%) and without the water content (1 7.24%) firom the can compareci to the canned
Navy bans in tomato sauce. When viewing the effect ofcooking Navy beans at different time intervals, saponin content decxeased gradually. The range of saponin losses were in good agreement with previously memtioned, published work.
The current study indicates that certain phytonutrients are present in Ontario grown beans. Traditionally, these same phytonutrients were considered toxic. However, recent studies now suggest that they may be responsible for exerting beneficial effects against various chronic diseases. In fact, these same studies have indicated that in many cases, the same interactions that make them antinutritive are also responsible for their beneficial effects. However, these health benefits are only possible at certain levels of phytonutrient intake.
To date, much of the clinical applications with regard to phytonutnents in beans have focused on those found in soy beans, with a number of reviews discussing the health significance of these findings (Anderson et al., 1995; Kennedy, 1995; Kushi et al., 1 999;
Liener, 1993; Messina, 1999; Messina and Barnes, 199 1; Thompson, 1993; Wang and
Wixon, 1999 ). Dry- beans also contain several of the sarne phytonutrients identified in soybeans. Whether they exert the same protection as what was seen with soybean consumption, is stiil to be deteRllitled.
Observations hmthe cwent study showed that the phytonutrient levels fiom processed bans where generaily lower than soy beans. The reason for this is not clear, however the fact that soy- beans are oilseed, as opposed to grain legumes, may explain the differences.
It is also possible that through commercial processing and canning beans with water, grain legwnes undergo a higher level of leaching than oilseeds and are therefore unable to retain their phytonutrients to the same extent as soybeans.
Attention should focus on finding ways to preserve phytonutrients in canried grain legumes. Additionally, Merstudies should also investigate whether the phytonuûients in grain legumes can exert the same physiological benefits as those observed fkom soy beans. CHAPTER 6
- THE EFFECT OF NAVY BEAN CONSUMPTION ON AZOXYMETHANE-INDUCED COLONIC PRENEOPLASIA - 6.1 INTRODUCTION
Colon cancer is one of the leading causes of cancer relateddeaths in the world (World
Cancer Research Fund, 1997). in the rnid 19903, more than 870,000 new cases where
diagnosed worldwide (WHO, 1997), which accounted for almost IO% of al1 new cancers
(World Cancer Research Fund, 1997). Colon cancer is second oniy to lung cancer in
North America, and its incidence and mortaiity rates are rising (Pappalardo et al, 1996).
Epidemiological data suggests that colon cancer is the foxm of cancer most associated
with diet (Potter, 1996) accounting for up to 90% of geographical differences in
wlorectal cancer incidence (Pappalardo et al, 1996; World Cancer Research Fund, 1997).
This has caused speculation that signifiant decreases in colon cancer incidence could be
achieved through dietary modification (Do11 and Peto, 198 1). It is suggested that a major
protector against cancer appears to be a diet high in fiber, low in fat, with generous
intakes of plant fwds that are nch in nuûients and phytochemicals (Anderson et al,
1999). Foods that posses these characteristics are nuits, vegetables and legumes. From
the legume family, beans (Phoseolus vulguris), are low in fat, contain a good source of dietary fiber and a wide range of phytochemicals that have been linked to the reduction in colon cancer (Hughes et al, 1997). However, bean consumption has received little attention with regard to their potential role in reducing the incidence of colon cancer.
To date, among the legurne fdy,soy beans have received the most amount of attention for their role in the prevention of cancer. Phytochemical contents of soy beans have been suggested as playing an important role in disease prevention (Messina, 199 1; 1999). Many researchers have used animal models to evaluate the capacity of phytochemicals to
reduce the omet and progression of colon carcinogaiesis. Inositol hexaphosphate (Ullah
and Shamsuddin, 1990), saponins (Koratkar and Rao, 1997), polyphenols (Kim et al,
1994), protease inhibitors (Kennedy, 1994; St. Clair et al, 1990) and oligosaccharides
(Koo and Rao, 199 1) are among phytochemicals that have show to protect against or
prevent carcinogaiesis in animals.
Recent studies reveal that dry beans also contain several of the same wmponents found
in soy beans suggested to have anticarcinogenic effects, yet very little information exists
on dry bean consumption and the incidence of colon cancer.
6.2 OBJECTIVE
In a rat model, the objective of this study, therefore, was to investigate the role of bean
consumption on chemically-induced preneoplastic lesions in the colon of rats.
6.3 MATERIALS AND METHODS
Animals
A total of 48 male Fisher rats (F344) at 3 weeks of age weighing approximately 58 grams fiom Charles River, Inc. Montreal, Canada, were used in this study. The animals were individually housed in plastic cages with comcob bedding under a 12: 12-hour light-dark cycle and controlled temperature (22OC) and humidity (50°C) conditions. Food and water were provided ad libitum. The care and housing for the rats cornplieci with the guidelines unda the Canadian Council on Anunal Care. The protocol was approved by the Ethics
Cornmittee for the use of animais, University of Toronto.
Preparation und Composition of Diers
The macro- and micro- nutnent composition of dl four experimentd diets fed to the rats are outlined in Table 6.3.1 and 6.3.2.
Al1 diets prepared for this study were imcalorîc. The bean flour was quantitatively used to replace the AIN-93M diet. Rats in the control group consumeci 100% bean- fiee, AIN-
93M diet with casein (Dyets Inc., Bethelhem, PA) while al1 other rats consurned one of thtee prepared mixtures of AIN-93M diet and canned navy beans without tomato sauce.
The three diet mixes were as follows:
a) Test group 1 : 10% of the diet supplied by navy beans and 90% AIN93M diet.
b) Test group 2: 25% of the diet supplied by navy beans and 75% AIN93M diet.
c) Test group 3: 50% of the diet supplied by navy beans and 50% AlN93M diet.
Navy beans in tomato sauce were obtained fiom the H.J. Heinz Company of Canada, Ltd.
The beans were drained, rinsed and fieeze dried. The dry beans were then ground into a fine flou to achieve the same consistency as the AM-93M diet. The diets were mixed and prepared according to the composition percentage of the bean diet. Fresh diets were prepared every month, and al1 diets were refiigerated to prevent spoilage. Table 63.1 Composition of Esperimentd Diets Fed to Rats (%loo@
Beans O 10 25 50 CHO 72.07 72-11 72.16 72.27 Protein 14 14-42 15.04 16.08 Fat 4 3.73 3.32 2.63 Fiber 5 5.39 5.98 6.95 Methionine 0.32 0.28 0.24 0.16 0% rat diet consists of 100% AIN-93M diet mix 10% bean diet consists of 10% canned navy beans and 90% AIN-93M diet mix 25% bean diet consists of 25% canned navy beans and 75% AIN-93M diet mix 50% bean diet consists of 50% canneci navy beans and 50% AIN-93M diet mix Canned Navy beans supplied by the H.J. Heinz Company of Canada AIN-93M Diet mix supplied by Dyets Inc., Bethelhem, Pennsylvania Table 63.2 Contribution of AIN 93M Diet to the Vitamin and Minerai Content of the Rat Diet (35gkg)
Ingrdient 0% Beans Diet 10% Bean Diet 25% Bean Diet 50% Bean Diet
Hturnin Mk fm&) Niacin Thiamine Riboflavin Folic Acid (mcg) Pantothenic acid Vitamin B6 Vi tamin B 12 Vitamin A Vitamin E Vitamin D
Miireral Muc (ml0 Calcium Potassium Sodium Magnesium Iron Zinc Manganese Selenium a: 0% rat diet consists of 100% AIN-93M diet mix b: 10%bean diet consists of 10% canned navy beans and 90% AIN-93M diet mix c: 25% bean diet wnsists of 25% canned navy beans and 75% AIN-93M diet rnix d: 50% bean diet consists of 50% canned navy beans and 50% AM-93M diet mix e: Canned Navy beans supplied by the H.J. Heïnz Company of Canada f: AIN-93M Diet mix supplied by Dyets Inc., Betheihern, Pennsylvania Studj Design
Animais were acclimatization for a modof one weelr, where they consumd labonitory
Purina rat chow. The animais were then randomly assigned to one of the four groups,
which wntained 12 rats in each treatment. Following acclimatization, rats were placed
on their respective diets for 4 weeks to determine the protein efficiency ratio of the dry
beans used in this study. Ahthe rats had consumeci their respective diets for 4 weeks,
treatment with carcinogen begaa. Rats hmal1 groups received 3 intraperitoneal
injections of azoxymethane (AOM) (Sigma-Aldrich Canada LTD., Ontario, Canada) each
consisting of 5 mg/kg of body wt, over the course of one week, totaling 15 mgkg body wt for each rat. AOM was dissolve-in 0.9% saline to a concentration of 5 mghi a day prior to injections. Body weights and food intake were monitored every week for the duration of the experiment. Fecal weights were obtained the week following the 1st
AOM injection and the final week of the experiment. Upon completion of the experiment (100 days after AOM injections) rats were euthanized by cardiac puncture and cervical dislocation under CO2.
Coion Prepara tion
Colons were immediately removed £kom the rats, washed fiee of fecal material with 0.9% saline, cut longitudinally and spread out on filter paper. The colons were then fixed flat in 10% buffered Formalin (VWR Canlab hc., Ontario, Canada) between two filter papers for one week before ACF detennination. ACFEnurnerution
The method usPd for ACF enurneration was followed by that outlined by Bruce et al
(1993) and Mclellan (1990). Fixed colons were stained with 0.2% meîhylene blue for 15-
20 min in a petn dish. The colons were then scored for Abernuit Crypt Foci (ACF) and
Aberrant Crypts (AC) on the mucosal surface of the colon with a Light microscope under a mapification of x4O. ACF and AC were disthguished hmnormal crypts by their darker staining, enlarged and slightly elongated size, thick epithelial lining, siightly elongated cryptal opening, and increased pericryptal zone. The total number of ACF and
AC were recordeci for al1 colons. Total ACF were divided into 3 separate groups to reflect foci of different aggressive states. ACF which consistecl of 1 - 3 AC's were classifiecl as small, 4 - 6 AC's; medium and ACF that consistai of 7 AC's or greater were regarded as Iarge.
Statistical Analysis
Al1 statistical analysis was done using unpaired two-tailed students t-test in the Excel5.0 program (Microsoft Corp., Redmond, WA). P values of 6.4 RESULTS Al1 aniinals remained healthy throughout the experimental period. Body weights, food intake' food efficiency ratio (FER)and protein efficiency ratio (PER)of rats in various groups are shown in Table 6.4.1 and 6.4.2. Table 6.4.1 Effects of Dry Bean Consumpth on Body Weigbt, Food Intake, Protein Efficacy and Food Enicacy in Rats. Week Groups Body Weight Food Intake FER PER (glweek) (s) (g) Week 1 Control 91.34S.17 74.65f 1.87 0.4410.0 1 3.1 53.O3 10% ban-fed 93.55+2.96 76,5821.35 0.46M.O 1 3.1 M.02 25%bean"fed 93.50f2.83 77.35i1.25 0.4Sf0.0 1 3.08M.02 WM~an-fed 90.77I2.79 75.95I1.42 0.4&û.O 1 2.72&&.03* Week6 Control 208.23S.3 1 109.48*1.87 0.27*0.01 1.9M.07 (F0110"ng 10% bean-fed 2 10.1 1*4.02 1 10.01*1.35 O.17Hl.O 1 1.2ZM.09 AOM injati-1 25% be811dfed 206.1 W.16 1 11 .OoIl.ZS O. 17M.O 1 1.1 8M.05* 50% bean-fed l91.87*4.67* 1 10.4W1.42 0.16&0.01* 1.12M.20* Week20 Control 345.73k4.73 119.38*1.87 0.05M.O 1 0.35M.05 (Prior ta 10% ban-fed 348.1 S4.85 123.8B1.27 0.O4M.O 1 0.3M.03 euthanesia) 25% bean-fed 339.4w4.70 13 l.OS*l.63* O.06kO.O 1* 0.34M.04 50% bean-fed 320.725.75* 128.4&1.28* 0.07*0.0 1* 0.34M.05 a: FER, food efficiency ratio (body weight gain (g) /food intake Wday); PER, protein efficiency ratio (body weight gain (g) /protein intake (*y). b: Values are Means *SEM. c: * Significantly different nom control PcO.05 (n=12 for each group). Table 6.4.2 Mean Daiiy Food and Protein Intake. Week Groups Mean Daily Food Mean Daily Protein Intake Intake (g) (d Week 1 Control 10.66M.25 1 -49I0.03 10% bean-fed 1 0.94I0.27 1 S7I0.07 25% bean-fed 1 1.05I0.23 1.66I0.05 50% bean-fed 10.7 1B.25 1.72f0.03 Week 6 Control 2.18I0.02 (Following AOM 10% ban-fed injections) 25% ban-fed 50% bean-fed 15.77H.29 2.5 1I0.0 1 * Week 20 Control 17.04M.33 2.3910.060 (Prier to euthanasia) 10% bean-fed 17.68I0.25 2.55M.05 25% bean-fed 1 8.72I0.23* 2.82îû.03 * 50% bean-fed 18.34,+0.36* 2.95M.02* a: Values are Means * SEM. b: * Significantly different fiom control (P~0.05). c: n=12 for each group. Bo& Weights Body weights did not significantly (P<0.05) diffa between the mntrol group and those rats consuming the 10% and 25% bean diathroughout the stdy @od. However, there was a significant (P Food Intuke Mean weekly and daily food intalces did not significantly (P FER FER was calculated by deteniining the body weight gain (g/week) of the rats divided by their food intake (g/week). There was a significant decrease (P<0.05) in the FER in rats consuming the 50% bean diet, folIowing AOM, compared to the cuntrol diet. There was however a significant increase in the FER in the 25% and 50% bean groups compared to control within the latter period of the study. This could be explained by their significantly higher food intakes during the same tirne. PER PER was calculateci by determining the body weight gain (g/week) of the rats divideci by their protein intake (g/week). The dtsindicate a significantly lower (PQ).05) PER value in the 50% bean-fed rats at the end of week one. This is surprishg since there was no significant difference (P4.05) in the mean daily protein intake dwing the same time period. Week six aiso showed significantly (W0.05)Lowa PER values for the 25 and 50% bean-fed groups compared to controis. However, the daily protein intake for the same thefiame indicates significantly (P<0.05)higher levels ofprotein for these two groups. This may suggest that the quaiity of protein in these diets may be questionable. Week twenty shows significantly (P This is of great interest as food intakes were significdy (P<0.05)higher in these groups compared to the control group. Furthemore, the macronutrient composition of the diets (Table 6.3.1 ) indicate higher protein contribution to the diet hmthe 25 and 50% bean diets. Increased food intake coupied with a higher protein diet should ideally produce significant increases in PER values. However, this is not the case, which Mersupports the possibility that the quality of bean protein in this diet is poor. Fecal Output Fecal output was measured at the time following AOM injections and once again prior to euthanasia. There were significant (R0.05)increases in fecal output in the 25% and 50% bean-fed groups compared to the control group following the AOM injections. Significant (PI0.05) inmases in fecal output were aiso obswed in aIl three beau-fcd groups compareci to the control gmup at the point prior to euthanasia. Al1 three bean-fed groups also showed significant (P a: $ Significant difference from control following AOM injections. b: t Significant difference hmcontrol prior to euthanasia. c: + Significant difference fiom time AOM injections were given to time at which animals were sacrificed. Aberrant Cgpt Foci and Aberrant CwtDatelopment The dose response effect of bean co~sumptionon AOM-induced ACF incidence is shown in Table 3. Although there were no statistid differences in the total number of ACF between the control group and the bean-fed groups, some protective trends were noticed. A reduction of 14.14% (65.83I6.26), 19.03% (62.08I8.73) and 5.11% (72.756.59) in ACF âevelopment was observed in rats consuming 10,25, and 50% bean diets respective1y, over the control group (76.67I9.55). There were significant (R0.05) decreases in the total number of AC development in the 10% and 25% bean- fed groups compared to control animals (1 75* 15.6 1, 170.17I25.37 and 254.83*33.69, respectively). A 17.8% reduction in the total number of AC was observed in the 50% bean-fed group, but this difference was not significant. When ACF were classified according to their size (1-3 AC's or small, 4-6 AC'Sor medium and 7 or pater AC's or large) no significant differences were seen among al1 bean-fed groups and the control group with regard to small size ACF. The 10% bean-fed group showed significant (RO.05) decreases in medium size ACF wmpared to control, and although no significant differences were observed in the 25% and 50% bean-fed groups with regard to medium size ACF, there was a 38.9% and 2 1.47% respective reduction in medium size ACF over the control group. Ail bean-fed animals showed significant (Pe0.05) decreases in large size ACF over the control. Table 6.4.3 Eflect of Dry &an Consumption on AOM-Incidence ACF in F344 Fisher Rat Colon. Group Total ACF Total AC Small ACF Medium ACF Large ACF Control 76.67k9.55 254.83G3.69 46.925.33 24.83*4.03 4.92M.85 (6 1 %) (32%) (6.40%) 10% 65.836.26 175.oî 15.6 1 * 50.W5.50 15.17* 1.15' 0.67*0.30* Bean-Fed (76%) (23%) (1 -00%) 25% 62.08I8.73 170.17*25.37* 46.25*6.32 1 5. 18k2.80 0.58*0.24* Bean-Fed (75%) (24%) (0.93% 50% 72.75*6.59 209.4222.55 52.33*4.83 19.5W2.35 1 .25I0.52* Bean-Fed (72%) (27%) (1 -72%) a: ACF, aberrant crypt foci; AC, Aberrant crypt. b: Size of ACF: small; 1-3 AC/foci, medium; 4-6 AC/foci, large; 7 or greater AC/foci . c: Values are means * SEM. d: * Significant difference fiom control (Pe0.05). e: (%) Percentage of total ACF Figure 6.4.2. Percent ACF Decrease in Bean-fed Animah from Controb. *Percent ACF decrease in bean-fed animals from controls. ACF were classified according to their size as small(1-3 ACIfoci), medium (4-6 ACIfoci) and large (>7 ACtfwi). n= 12. 6.5 DISCUSSION Dry beans supply a good source of protein, fiber, essential vitamins and minerais, are low in fat and are regarded as a healthy food item (Gdand Anderson, 1994). Dry bean intake has been shown to have protective and therapeutic effects against conditions such as coronary heart disease, diabetes mellitus and obesity (Gdand Anderson, 1994), however, to date few data exist on the intake of dry beans and the incidence of colon cancer. Although there is hited epidemiologid data which has investigated the consumption of beans and the incidence of cancer, some researchers have reportai a decrease in colon cancer incidence in areas of the world where dry bean intake is high, i.e. Latin America and Mexico, and a higher incidence in wlon cancer where dry bems consumption is low such as the U.S. and Canada (Correa, 198 1; Lucier et al, 2000). The results of this snidy show that canned Navy bauis without tomato sauce can significantly inhibit the incidence of AOM-induced colonic preneoplastic lesions in rats. AOM-induced colon cancer in male Fisher rats at a dose of 1Smg/kg of body weight is a comrnonly used mode1 for evaluating cancer promoting or inhibithg capacity of dietary components. (Holt et al, 1996). Colon cancer begins with the appearance of ACF, which sequentiaily develop into polyps and adenornas and ultimately carcinomas. Therefore ACF are reliable endpoint biornarkers of colon cancer for short-term animal studies (Bruce et al, 1993; Bird, 1995). In this study, protective trends were noticed in those animals that consumed the various percentages of a bean diet. Although oniy the 10% and 25% bean-fed groups showed a significanî decrease in total AC, all ban-fed groups showed reductions in the incidence of total ACF and AC over the control-fed animals. The results suggest a stronger pmtective effect against colon cancer in those gcoups consuming 10% and 25% of their diet as beans followed by the 50% bean-fed diet. With regd to the size of the ACF, no significant reductions were made in those ACF which were classified as small. The 10% bean-fed group showed a significant decrease in the medium size ACF, and although not significant, the 25% bean-fed group showed the next greatest protective effect, followed by the 50% bean-fed group against the control. Al1 three bean-fed groups showed a significant decrease in the large size ACF, with the greatest reduction occuning in the 109'0 and 25% bean-fed groups, followed by the 50% bean-fed group. This is of great importance because large foci are suggested to have more growth potential over smaller foci, hence they are more aggressive (Bird, 1995). Aggressive foci respond differently to growth factors or promoters than less aggressive foci ailowing thern to get promoted more rapidly to the next step in carcinogenesis. While the decrease in total ACF in the bean-fed groups is not significant, the pronound reductive effect seen in ACF of increasing size (Fig. 6.4.2) suggests that beans may slow the growth of ACF detainhg them hmgrowing into bigger foci. The results of the data appear to suggest that a greater protective effect against preneoplastic biomarkers of colon cancer occurred within the 10% and 25% bean-fed group followed by the 50% bean-fed group. This is surprishg since (Hughes et al 1997), found that rats fed a 59% bean (pinto) diet had significantly fewer colon adenocarcinornas and tumors than rats that were fed a casein diet. A number of components within beans have been suggested for playing a role in the prevention of cancer, whidi may also provide some explanation for the protective trend that was observed. Dry beans are generally considered to be a good source of vegetable protein, however legume proteins have some limitations. They are in general deficient in the sulfur- containhg amino acid methionine (Sgarbieri, 1989 and Gupta, 1983). in this study, the group that showed the least protective effect compareci to the control was the 50% bean- fed group. The mean body weight of this group was dso significantly lower than that of the control group, following the AOM injections, wwhich continued until the end of the study. Since bean Bour quantitatively replaceci AW3Mdiet, it is possible these rats suffered hma methionine deficiency. Methionine deficiency may produce the aiteration in DNA methylation, which is characteristic of tumors and premalignant tissue (World Cancer Research Fund, 1997). It has been reporteci that rats actually have a methionine requkment that is -50% higher than humans (Sarwar et al, 1989). Therefore the combination of lower methionine intake coupled with higher requirement for methionine could explain why the least protective effects where seen in the 50% bean-fed SOUP. Hughes study supplemented the 59% pinto bean diet with methionine and saw no significant differences in body weight between the bean group and control, and also saw a signincant decrease in the development of colonic adenocarcinornas and tumors in rats codgthe bean diet compareci to the control. Despite the decrease in body weight in the 50% bean-fed group, the 10% and 25% bean- fed groups indicated no significant differences in body weight throughout the study. This is important to note because a 10% and 25% inclusion of bans into the daily diet is more realistic for human consumption and easier to achieve that 50% of the daily diet supplernented by beans. Dry bans also contain some of the richest sources of total die- fiber (Hughes et al, 1997; Hughes and Swanson, 1989), and have been among the more fiequently investigated dietary factors in studies of the etiology of colon cancer (Kushi et ai, 1999). Hughes et al (1 997) suggests that the dietary fiber in dry beans is a good candidate for the anticarcinogenic properties of dry beans. This is because dry bean fiber contains considerable amounts of insoluble fiber known for diluting carcinogens or bile acid promoters (Jenkins et al, 1987). Insoluble fiber also results in increases in fecal output. In this study, there was a significant increase in fecal output in al1 bean-fed groups at the end of the study compared to the control-fed group. It is suggested that smd fecal masses may be associated with bowel disease which cm weaken the intestinal wall, and allow components such as mutigens or carcinogens to becorne more highly concentrated in the luminal contents (Burkitt, 1973). Insoluble fibers cause inmeases in fecal output and reduce the length of time toxic components remain in the colon (Fleming et al, 1985). Dry beaus also contain soluble fiber which have shown to be fermented into SCFA such as acetate, butynite and propnonate, thus lowering colonic pH and Uiauencing bacterial metabolism (Fleming et al, 1985). In addition to a good source of dietary mer, dry beans also wntain a number of biologically-active compounds refmed to as phytochemicals. Phytochemicals such as saponins, phytic acid, protease inhibitors, polyphenols, lectins and oligosaccharides al1 have demonstrated to have anticarcinogenic properties (Thompson, 1993). To date much of the research that has investigated the anticarcinogenic effects of legurne phytochernicals has focused on those found in soy beans (Messina, 1999). Vey little is known about the phytochemicals in dry beans, however these phytochemicals have been investigated for their anticarcinogenic properties using various other sources. Koratkar and Rao (1 997), found soy bean saponins to play a role in inhibithg the incidence of ACF in the colons of mice. Sirnilarly, Pretlow et al (1992), a found protective effect in rat colon carcinogenesis nom phytate obtained fiom corn or nce. Kennedy et al (1993) and St. Claire et al (1990), demonstrated the efffetiveness of tyrpsin inhibitors on reducing the incidence of cancer, and Kim et al (1994) showed the ability of phenolics to inhibit colon cancer in Fisher rats. Future investigations should examine the individual effectiveness of dry bean phytochemicals on the incidence of colon cancer, since very little is know with regard to their capacity to prevent carcinogenesis. In conclusion, the results of this stuây suggest that dry beans (navy beans) have a beneficial effed on AOM-induad colonic preneoplastic markets of colon cancer. However, dry beau acceptance in Western diets still remains low. Further clinical trials are required not only to revive dry beans conswllption as a healthy part of the daily diet, but to also encourage its inclusion into the diet as a healthful food known for its protective effects against disease. CHAPTER 7 - GENERAL DISCUSSION - 7.1 DISCUSSION Among al1 cancers, colon cancer is the one that is highly influenced by the diet and has great potential to be prevented (Potter, L 996). A diet which includes nuits, vegetables and whole grains, includuig legumes appear to play a significant role in the prevention of this disease. Insufficient data hmhuman and animal sîudies exists for the purpose of determining the effect of relatively high consumption on cancer risk. The only other evidence with regard to bean consumption and cancer nsk, cornes mdyfiom experiments that have investigated individual constituents of pulses that have biological properties and may prevent cancer in experimentally induced animals. No studies have investigated the cumulative effect of macronutrients, micronutrients, fiber and certain phytonutrients on the incidence of preneoplasia in rats. Therefore the overail objective of this study wes to investigate the effect of consuming cornmercially processed Navy beans on chemically-induced colonic preneoplasia in rats. To accomplish this objective an investigation of the nutnent and phytonutrient content of beans was undertaken. Although this information is already available in published literature, environmental conditions and industrial processing techniques Vary, which may result in different nutrient and phytonutrient values. This was observeci as a result of the current analysis. The first phase of this analysis showed Kidney beans, Fava beans and Navy beans, all grown and processed in Ontario, contain the nutritional and phytonutrient components found to be beneficial in preventing cbronic diseases of the Westem countrîes. Cooking or processing is required for legume consumption. It is necessary not only to tenderize the seed, devdop aroma, acceptable flavour and texture, but to also to improve the bioavailability and utilization of nutrïents and phytonutrients. However, as a result of the cooking process, compositional changes occur within the legwne nutrient and phytonutrient content. Data for moisture, protein, fat, ash and carbohydrate for canned Fava and Kidney beans (homogenized with liquid contents in the can) were generaiîy in good agreement with data provided in Handbook No. 8 (Appendix). There were discrepancies noted hm cment analysis and that of Handbook No. 8 with regard to the canned Navy beans (homogenized with tomato sauce). These discrepancies may be a factor of the beans being cooked in tomato sauce instead of water. These beans may also require slightly different processing conditions than beans processed and canned in water, which too may alter the proximate values for moisture, fat, ash and carbohydrates. For the samples that were nnsed and drained fiom the liquid contents in the cm, the proximate values for protein, fat, carbohydrate and ash (with the exception of moisture) appear to be slightly greater than that of the beans analyzed with the liquid contents fiom the cm. This suggests a portion of the macronutrients were leached out into the liquid content of the cm. As a remit, it would appear that the utilization of the liquid content within a can of beans would provide more nutrients to the diet- In contrast to the generaI1y good agreement between data fiom this research and that which is provided in Handbook No. 8 for protein, fat and ash contents, values reporteci here for total dietary fiber for raw beans were much less than total dietary fiber values reported in Handbook No. 8. Considerable differences were noted in the raw Navy and Fava beans. The raw Kidney beans value reported here was in closer pmximity to the value reported in Handbook No. 8. The methods used for meamring dietary fiber are intended to mimic the enzyxnatic and chernical reactions of food in the mouth, stomach and smaii intestine. Discrepmcies in published values for some raw and canned foods could be explained by the characteristics of the enzymes used and, to some extent, by the pruiciples of the various methods available for use (Baker ami Holden, 198 1 ;Englyst, 1982; Mongeau and Brassard, 1982; Paul and Southgate, 1978). The literature suggests that approxmiately 73% of folate is retained after cooking (Augustine et al, 198 1). In the cument study however, the percentage of vitamin retention following processing was much lower. With the exception of the Fava bean, virtually 100% of folate was lost afier processing of the canned Navy and Kidney beans. Although folate values provided by Handbook No. 8 are much higher than those found in this study, it is possible that commercial processing techniques have a significant effect on folate retention. Legumes are generally thought to be good sources of folate, however, it appears that not al1 legumes will retain nutrients to the same extent under commercial processing techniques. It may also be a resuit of diffmnt geographical pwing location, growing seasons, processing techniques and analytical techniques. Further investigation of this is warranteci as folate is a vitamin that can provide many benefits to the diet. Preferences to consume beans with or without the liquid content found in canned beans will alter the percentage of macronutrients, folate and fiber contributeci to the diet per serving. Consuming the cmed beans with the liquid portion of the can will provide a slightly higher nunlent intake. Recent studies suggest that dry legumes may wntain a number of biologicaily active compounds referred to as phytonutrients. These phytonutrients which include trypsin inbibitors, phytates, haemagglutinins, saponllis and oligosaccharides, have traditiondy been labeled "anti-nutrients" because of theh ability to reduce the availability and utiIization of nutrients (Thompson, 1993). In the current study, these biologically active phytonutrients were predominantly seen in the raw legume seeds. Cooking and or processing reduced the levels of these phytonutnents. As a result, more efficient digestion and utilization of nutnents fkom the diet rnay occur. Varbus studies have indicated that as a result of the compositional changes that occw during processing, these phytonutrients occur at levels that may posses significant beneficial properties (Anderson and Wolf, 1995; Deshpaade et al., 1984; Limer, 1993; Thompson, 1993; Wang and Wixon, 1999). In the cunent study, the presence of phytonutrients was observed in both the raw and selected canned beans. DiEerent vatieties of beans, anâ the form in which the canned beans may be consumed, showed varying phytonutrient levels. The clinical applications of phytonutnents have received a great deal of interest in recent years. In particuiar, the phytonutrients present in soybeans have been studied extensively, with a number of reviews discussing their fidings (Anderson et al., 1995; Kennedy, 1995; Kushi et al., 1999; Liener, 1993; Messina, 1999; Messina and Bmes, 199 1; Thompson, 1993; Wang and Wixon, 1999 ). The current study indicates that certain phytonutrients are present in Ontario grown beam that were identifieâ in soybeans for exerting potential benefits. Results showed that the phytonutrient levels fiom the current study are generally lower than those found ui soy beans. The reason for this is not clear, however the fact that soy beans are oilseed, as opposed to grain legumes, may explain the diffaences. It is also possible that through commercial processing and canning beans with water, grain legumes undergo a higher level of leaching than oilseeds and are therefore unable to retain their phytonutrients to the same extent as soybeans. As there are a variety of methods for determining certain phytonutrients, it is possible that methods used in this analysis were different than those used in the literature for phytonutrient determination of soybeans. This could also explain compositional differences between soybeans and the baurs analysed in this study. Whether they exert the same protectiveness as what has been observed with soybean consumption, is still to be detennined. The second phase of this study was to investigate the effm of cornmerciaily pmcessed bean consumption, at different Ievels of intake, on the incidence of colonic preneoplasia in rats. nie results of this stuây show that canned Navy beam (rinsed fiom the tomato sauce) can significantly inhibit the incidence of AOM-induced colonic preneoplastic lesions in rats. AOM-induced colon cancer in male Fisher rats at a dose of 1Smg/kg of body weight is a co~nmonlyused mode1 for evaluating cancer promoting or inhibiting capacity of dietary components (HoIt et al, 1996). Colon cancer begins with the appearance of ACF, which sequentially develop into polyps and adenornas and ultimately carcinomas. Therefore ACF are reliable endpoint biomarkers of colon cancer for short-term animal studies (Bnice et al, 1993; Bird, 1995). In this stuây, protective trends were noticed in those animals that consumed the bean diets. Although only the 10% and 25% bean-fed groups showed a significant decrease in total AC, al1 ban-fed groups showed reductions in the incidence of total ACF and AC over the control-fed animais. The results suggest a stronger protective effect against colon cancer in those groups consuming 10% and 25% of their diet as beans followed by the 50% bean-fed diet. The reason for this is not clear. It is possible that the nutritional composition of each diet may be, in part, responsible for the observed results. Table 6.3.1 outiines the macronutnent composition of each diet. Although the protein content of îhe 50% bean diet is higher, the mean body weight of this group was significantly lower than the control, and lower than the other bean gmups. This may suggest that as the bean flou was quantitatively replacing the ADJ diet, the protein quality in the diet was beîoming compromised. This would be in good agreement with pubiished literature, which reports bean protein to be somewhat deficient in sulfiu-containhg amino acids, such as methionine. The possibility of a poor quality protein intake in the 50% bean fed group is mer supported by significantly higher mean PER and fmd intake values, at the end of the study. Based on this information, the 50% bean-fed group ideally should not have experienced significantly lower body weights. If in fact, the 50% bean-fed group did suffer a methionine deficiency, this would help to explain why the least protective effect was obswed in this grop, as a methionine deficiency may produce the alteration in DNA methylation, which is characteristic of tumors and premalignant tissue (World Research Fund, 1997). Another possibility that may help to explain why the 50% bean-fed group showed the least protective effect against AOM-induced colonic preneoplasia may be related to the micronutrient composition of the diets. Table 6.3.2. (page 98) ,outlines the micronutrient composition of each diet. As the bean flour was quantitatively replaceci by the AIN diet, the micronutnent content of the bean diets lessen, with the most pronounced differences seen in the 50% diet. The current analysis of folate indicates that the folate in canned beans is almost completely eliminated during processing. nierefore, the only folate being supplied to the animals is that which was provideci by the AIN diet. As a result of the AIN diet king substituted for bean flour, the 50% bean-fed group consumeci a substantially lesser amount of folate. Low folate intake has been associated with increased risks of colon adenorna and colon cancer (Freudenheim et al, 199 1). Animal studies have also shown that calcium may be a promising agent to reduce the risk of colon cancer (Hyman et al, 1998). Hyman et al (1998), showed that calcium supplements modestly reduced recurrence of colon adenornas in a recently reportai randomized trial. The calcium composition of the 50% bean-fed group was considerably lower than al1 other groups. This may suggest that the lack of sufficient calcium intake rnay have contributeci to the iesser protective effect. It is important to point out that the animais were placed on their respective diets at only 5 weeks of age. The 50% bean fed group consumed a considerable lower micronutrient intake during their growth stage and in adulthood compared to the other groups. Perhaps the low micronutrient intake during their growth stage and adulthood had some sort of an effect on the animals natural ability to ward off disease. The curent andysis found that the Navy beans used to quantitatively replace the AIN diet contained various phytonutrients. Studies have shown that very high intakes of these wmponents rnay produce adverse effects by reducing the availability of nutrients and thus causing growth inhibition (Thompson, 1993). The higher level of phytonutrients in the 50% bean diet coupled with already compromised micronutrient intalces, may have also been respoasible for the 50% bean-fed group to show the least protective effect. Al1 three bean-fed groups showed a significant decrease in the large size ACF, with the greatest reduction occurring in the10% and 25% gmups, followed by the 50% bean-fed group. This is of great importance because large foci are suggested to have more growth potential over smaller foci, hence they are more aggressive (BVd, 1995). Aggressive foci respond diffefently to growth factors or promoters than less aggressive foci allowing thnn to get prornoted more rapidly to the next step in carcinogenesis. While the decrease in total ACF in the bean-fed groups is not significant, the pronounced reductive effect seen in ACF of increasing size (Fig. 6.4.2.) suggests that beam rnay slow the growth of ACF detaining them hmgrowing into bigger foci. A number of components within legumes have been suggested for playing a role in the prevention of cancer, which may also provide some explanation for the protective trend that was observed. Dry beans also contain some of the nchest sources of total dietary fibre (Hughes et al, 1997; Hughes and Swanson, 1989). Bean fibre contains considerable amounts of insoluble fibre hown for diluting carcinogens or bile acid promoters (Jenkins et al, 1987). insoiuble fibre also results in increases in fecal output. In this study, there was a significant increase in fecal output in di beau-fed groups at the end of the study compared to the control-fed group. It is suggested that smail fecal masses may be associated with bowel disease, which can weaken the intestinal wall, and allow components such as mutigens or carcinogens to become more highly concentrateci in the luminal contents (Burkitt, 1973). It is possible that the insoluble fibres fiom the beans used in the current study may have in part been responsible for their protective effect. Dry beans also contain soluble fibre, which appears to be fmented into SCFA such as butyrate, thus lowering colonic pH and influencing bacterial metabolism (Fleming et al, 1985). Because the current stuây did not anal yze the insoluble and soluble fibre components of the bans used in this study, we can only speculate that they are present, and are exerting some sort of beneficial effwt. In addition to a good source of dietary fibre, dry beans also contain a number of biologically-active oompounds referred to as phytochemicals. Phytochemicals such as saponins, phytic acid, protease inhibitors, polyphenols, lectins and oligosaccharïdes al1 have demonstrated to have anticarcinogenic properties (Thompson, 1993). Very little is known about the phytochemicals in dry beans and the levels at which they may exert protective effects against chronic disease. In conclusion, the results of this study suggest that canned Navy beans have a beneficial effect on AOM-induced colonic prmeoplastic markers of colon cancer. However, bean consumption in Western diets still remains low. Further clinical trials are required not only to revive dry beans consumption as a healthy part of the daily diet, but to also encourage its inclusion into the diet as a healthfid food known for its protective effsts against disease. CHAPTER 8 - SUMMARY- Overaiî Objective: The overail objective of this thesis was to investigate the nutritional and phytonutrient composition of selected Ontario grown beans, and study th& effect in azoxymethane- induced preneoplasia in rats. Results form the ment study found canneci Navy beans to contain anticarcinogenic phytonutnent compounds capable of iohibiting chernicdy-induced preneoplastic lesions in the rat colon. Specific Objectives: a) Nutritional composition of rmv Ontario grown Kidney, Fmand Nmbeans With regard to the raw legumes, the data hmthe present analysis was found to be in close agreement with other published data. Moisture values for raw legumes were generally in good agreement with data tabulated in the USDA H=dbook No. 8, except for Fava beans, which showed slightly higher values than the published data. The protein, fat and ash values determineci for the samples in the current analysis were also in good agreement with the data provided in Handbook No. 8. The slight variations among lots of raw legumes may be prllnarily caused by differences among legurne samples and environmental conditions. ii) Values reported here for totai dietary fiber for raw beans were much less than totai dietary fibre values reported in Handbook No. 8. 6) Nuîrirional composin'on ofcanned Onaria grown Kidney, Fava and Navy beans. i) Data for moisîure, protein, fat, ash and carbohydrate for cannd Fava and Kidney beans (homogenized with liquid contents in the cm)were generally in good agreement with data provided in Handbook No. 8 (Appendix). There were discrepancies noted fiom cment andysis and that of Handbook No. 8 with regard to the canned navy beans (homogenized with tomato sauce). These discrepancies may be a factor of the beans being cooked in tomato sauce instead of water. For the samples that were rinsed and drained nom the liquid contents in the can, the poximate values for protein, fat, carbohydrate and ash (with the exception of moisttm) appear to be slightly greater than that of the beans analyzed with the liquid contents fkom the can. ii) The fiber content of the select& canned beans were generally in good agreement to those reported in Handbook No. 8. iii) The percentage of folate remaining following processing was much lower then values provided by Handbook No. 8. With the exception of the Fava bean, which showed a 6 1- 77% reduction in foiate, virtuaiiy 100% of folate was lost after processing of canned Navy and Kidney beans. C) Phytonutrknt composin'on of raw Ontbrio grown Wney, Fova and N;.y &ans. i) The cumnt study indicated that the phytonutrients trypsin inhibitors, phytates, latins, saponins and FOS were present in the selected raw Ontario grown beans chosen for this study. à') Phytonutrient composition of canned Ontcrn'o gram Kidnqy, Faw and Nqbeans i) The ment study indicated that phytonutnents remained following commercial processing. Results hmthe this andysis also showed that the phytonutrient levels hm canned Navy, Kidney and Fava beans where generaily Iower than literature values provideci for Soy beans. The values hmthe ment study also indicate a higher loss of phytonutnents when water contents Wrn the can was removed, suggesting leaching of I phytonutrients dmgprocessing. e) Nutritional andphytonutrient composilrn of raw and canned Ontario grown Kirbtcy. Favu and Novy beans cooked at differernt time intervllk i) The tune / treated analysis of Navy beans in the cument study indicated that as cooking continued, bans took on more water, and the macronutrient and phytonutrient contribution progressively declined. fi The effects of canned Nqbans on co1onicpreneoplasia in rats i) With regard to the effect of canned Navy bean consumption on AOM induced colonic preneoplasia in rats, protective trends were observed in those animais that consumed the bean diets. ii) Although only the 10% and 25% bean-fed groups showeû a significant deçnase in total AC, a11 beau-fed groups showed reductions in the incidence of total ACF and AC over the control-fed animals. iii) With regard to the size of the ACF, ali three bean-fed groups showed a significant decrease in the large size ACF, with the greatest reduction occhgin the 10% and 25% bean-fed groups. iv) In this study, there was a significant increase in fecal output in ail bean-fed groups at the end of the shidy compared to the control-fed group. One possible explanation may be a result of the increased amount of insoluble fibre in the bean-containhg diets. V) It is possible that the phytonutrient components in beans may have contributed to the lower incidence of preneoplasia in the animais which consumed the bean diets over the control. CHAPTER 9 - CONCLUSION - 9.1 CONCLUSION It was hypothesized that raw and canneci Ontario grown beans wntain substantial amounts of macronutrients, micronutrients and phytonutrients that can be affecteci during processing and may also have an effect on wlonic preneoplasia. In order to test this hypothesis, the following specific objectives were undertaken: A) to investigate both the nutritional and phytonutrient composition of raw and mxnmercially processeci canned Navy, Kidney and Fava beans and B) investigate the effects of canned Navy bean consumption on the development of preneoplastic lesions in the colons of azoxyrnethane- treated F344 Fisher rats. The macro- and micro- nutrient and phytonutrient content of canned beans, homogenized with and without the liquid content found in the cm, stiil contained a respectable amount of these components even aiter processing, when compareci to their raw equivalents. The nutrient and phytonutrient content of canned bans homogenized with the liquid content nom the cm appeared to be slightly higher than those beans that were not homogenized with the liquid contents, suggesting leaching. From this we can conclude that the utilization of beans with the liquid content fkom the can will provide a slightly higher level of nutrients and phytonutrients to the diet. Although the liquid content fiom the canned Navy beans (tomato sauce) were rinsed fkom the beans fed to the rats, the consumption of these Navy beans still showed a significant protective effect against the development of aberrant crypt foci and aberrant crypts in chemically induced colonic preneoplasia in the rats. In conclusion, this study supports the hypothesis as the resuits demonstrate that Navy, Kidney and Fava beans contain anticarcinogenic phytonutrient compounds capable of inhibiting chemically-induced preneoplasîic lesions in the rat colon. CHAPTER 10 - FUTUREDlRECTIONS- 10.1 FUTURE INVESTIGATIONS Legumes are globaiiy important dietary components which contain appreciable quantities of nutrients and phytonutrients. It is therefore ixnportant to develop a thorough understanding of the nutritional disease preventing benefits of their nutnents and phytonutrients that could not be answered in this study. To accomplish this, several aspects need to be investigated and evaluated. A) Focus on individual fùnctional components in beans to determine which components are specifically proàucing beneficial effects and the extent of their benefits. B) Evaluate the protein quality (amino acid profile) of beans and understand their role in preventing chronic diseases. C) Evaluate the micronutrient composition of bans and detemine their role in the prevention of various chronic diseases. Other areasfor future studîes are al- listed: D) Study the effect of agricultural practices and indicate differences in phytonutrient composition of beans. E) Anaiyze and compare the nutrient and phytonutrient composition ofbeans processed using traditionai and modem methods. F) Dietary intervention studies ushg clinical models to investigate the role of beans in the prevention of cardiovascular disease. G) Animal studies to fiirther understand the role of beans in initiation, progression and tumorogenesis stages of colon caucer. H) Long terni animal studies using cancer as the end point to study the role of beans in the prevention of colon cancer. 1) Short and long temi human intervention studies to investigate the role of bans in the prevention of colon cancer, cancer of other sites and cardiovascular disease. 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