(vigna racemosa), cowpea (vigna unguiculata)

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CHEMICAL CHANGES DURING OPEN AND CONTROLLED FERMENTATION OF WILD BEAN (VIGNA RACEMOSA), COWPEA (VIGNA UNGUICULATA) AND COMMON BEAN (PHASEOLUS VULGARIS) FLOURS

BY

DIFO VOUKANG HAROUNA

DEPARTMENT OF BIOCHEMISTRY AHMADU BELLO UNIVERSITY, ZARIA NIGERIA

APRIL 2013

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CHEMICAL CHANGES DURING OPEN AND CONTROLLED FERMENTATION OF WILD BEAN (VIGNA RACEMOSA), COWPEA (VIGNA UNGUICULATA) AND COMMON BEAN (PHASEOLUS VULGARIS) FLOURS

BY Difo Voukang HAROUNA, B.Sc Biochemistry (DSCHANG-CAMEROON) 2009 MSc/Scie/03790/2009- 2010

A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF A MASTER DEGREE IN BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY, FACULTY OF SCIENCE AHMADU BELLO UNIVERSITY, ZARIA NIGERIA

APRIL, 2013

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Declaration I hereby declare that the work in this thesis titled “Chemical Changes during Open and Controlled Fermentation of Wild Bean (Vigna racemosa), Cowpea (Vigna unguiculata) and Common Bean (Phaseolus Vulgaris) Flours” was performed by me in the Department of Biochemistry, under the supervision of Dr. E. Onyike and Prof. D. A. Ameh. The information derived from the literature has been duly acknowledged in the text and a list of references provided. No part of this work has been presented for another degree or diploma at any institution.

Difo, Voukang Harouna M.Sc/SCIE/03790/2009- 2010 ___________________

_____________ Signature

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______________ Date

Certification This thesis titled “Chemical Changes during Open and Controlled Fermentation of Wild Bean (Vigna racemosa), Cowpea (Vigna unguiculata) and Common Bean (Phaseolus Vulgaris) Flours’’ meets the regulations governing the award of the degree of Master of Science (M.Sc.) in Biochemistry of the Ahmadu Bello University, and is approved for its contribution to knowledge and literary presentation.

__________________________ Chairperson, Supervisory Committee

__________________

_______________

Signature

Date

Dr. E. Onyike

_______________________ Member, Supervisory Committee Prof. D. A. Ameh

_________________ Signature

_________________________ Head of Department Dr. H. M. Inuwa

__________________ Signature

___________________________ __________________ Dean, School of Postgraduate Studies Signature Prof. A. A. Joshua

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__________________ Date

_________________ Date

________________ Date

Dedication I wish to dedicate this work to my uncle, Alhadj Toukour Seini who has been my sole sponsor all through my academic pursuit and to my parents, Cheick Ali Difo and Hadja Rayta Rayhanatou for their moral and spiritual guidance.

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Acknowledgments I wish to express my thanks and sincere appreciations to my supervisory team, Dr. E. Onyike and Prof. D. A. Ameh for the scholarly, financial and moral support they gave me. I also wish to thank my uncle, Alhadj Issa for his financial and moral support during my master degree period in Nigeria. My profound gratitude goes to Mr. G. C. Njoku for the interest, concern and encouragement when it seemed all hopes were lost. He gave me the strength to continue at times of extreme difficulties and personally supervised some parts of this work. My gratitude also goes to Mr. Ndidi, Uche Samuel for he was always attending to me whenever I had statistical and/or English language comprehension problems. My regards to the chairman and members of the Biochemistry research committee for their effort in ensuring the successful completion of the defense of this thesis. My regards also go to Dr. I. O. Abdullahi and Prof. J. B. Ameh of the Department of Microbiology, ABU, Zaria for their assistance with great ideas in the microbiology aspect of this work. I also thank the laboratory staff of the Department of Biochemistry, ABU, Zaria, Nigeria for their assistance. I acknowledge the effort of Mr. Edward Adegbe of the Food Science and Technology Department (Institute of Agricultural Research, ABU, Zaria), Malam Musa Bashir of the MultiUser Science Research Laboratory, ABU, Zaria, and Malam Shitu, Mohammed Adamu of the Department of Microbiology, ABU, Zaria for their assistance in the practical aspect of this work whenever a method of analysis seems to be a little bit practically confusing to me.

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I sincerely acknowledge the role played by my friends, Ali Sani Suleiman and Abdullahi Abdul Malik Salman. Special thanks to my friends, Mr. John Walter and Mr. Kuduwah Jacob of the Department of Microbiology, ABU, Zaria, my class rep, Mr. Okoyomoh Kingsley and my other contemporaries whose words of encouragement and support helped in no small measure in seeing me through the period of this research. Finally, all praises and thanks to Almighty God, whose power it was to ensure that what went up definitely came down.

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Abstract The effects of open and controlled fermentation on the proximate composition, some mineral elements, antinutritional factors and flatulence- causing oligosaccharides of one wild bean (Vigna racemosa) and two cultivated beans (Vigna unguiculata and Phaseolus vulgaris) were studied. The aim of this work was to evaluate the effects of open and controlled fermentation on the proximate composition, anti-nutritional factors, some minerals and flatulence factors of Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris flours. The open fermentation was carried out using the microorganisms present in the atmosphere, while the controlled fermentation was carried out using Aspergillus niger as a starter. The two types of fermentation brought about more than 20% increase in the protein content of Vigna unguiculata but less than 20% increase in the case of Phaseolus vulgaris. In the case of Vigna racemosa, the protein content was increased by 12.41± 1.73% during open fermentation while it decreased by 29.42± 0.1% during controlled fermentation. The lipids, carbohydrates, crude fibre and ash content of the three types of bean were all reduced by less than 75% during the two types of fermentation, except the moisture content which showed an increase during controlled fermentation. Apart from calcium, the other elements (Fe, Na, Mg, Zn, and K) showed less than 90% reduction by the two types of fermentation in the three types of beans. The phytate, tannin, alkaloids, hydrogen cyanide, lectins, trypsin inhibitors and oxalate content of the three types of beans all showed more than 20% reduction by the two types of fermentation. The percentages of reduction due to controlled fermentation were higher than those of open fermentation in the eight antinutrients studied. So fermentation is an efficient method for detoxifying those antinutrients in both domesticated and wild beans studied even though it has some relatively reducing effects in

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some nutritional elements of the beans flour. Also it is noticeable that the controlled fermentation using Aspergillus niger as a starter is more efficient in the detoxification than open fermentation.

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Table of Contents Title…………………………..……………………………………………………….…….ii Declaration……………………………………………………………….………..……….iii Certification……………………………………………………………………...…………iv Dedication…………………………………………………………………..………………v Acknowledgments………………………………………………………………...………..vi Abstract…………………………………………………………………………...………viii Table of Content…………………………………………………………..…………………..….....x

List of figures……………………………………………………………………………..xiv List of tables………………………………………………………………...……………..xv List of Plates…………………………………………………………..………….………xvii List of Appendices………………………………………………………………….…...xviii

1.0 INTRODUCTION…………...…….…………………..…………...…….…………………...1 1.1 Statement of Research Problem ……………………………….…………...………….4 1.2 Justification…………………………..…………………….……………...…………….4 1.3 Null Hypothesis…………………………………….....…………………...……….……5 1.4 General Objectives………………….…………………………………..………………5 1.5 Specific Objectives……………………………………………………….……………...5

2.0 LITERATURE REVIEW……………………………………………………..……….…......7 2.1 Fermentation…………………………………….………......…………………………….…..7 2.1.1

Definition of Fermentation……………………………………..……….……..…....7

2.1.2 Classification of Fermentation………………………..………..……….……..……..7 2.1.3

Modes Of Fermentation Techniques ………………………………………………9

2.1.4

Fermentation Vessels……………………………….……………………………...13

2.1.5

Methods of Microbial Inoculation in Food Fermentations………….……….…….15

2.1.6

Nutritional Value of Fermented Foods……………………………………….……19

2.1.7

Health Effects of Fermented Foods………………………………………..………21

2.1.8

Toxins and Toxin- Producing Organisms in Fermented Foods……….…….……28

2.2 Biology of Aspergilus Niger………….…………………………..…………………………..29 x

2.2.1 Description of Aspergillus Niger colonies……………………….………………..29 2.2.2

Clinical Significance of A. Niger………….……………………………..………..30

2.2.3

Genomic of A. Niger……………….……………………………………………...30

2.2.4

Physiology of A. Niger…………….………………………………………………31

2.2.5

Industrial Applications of Aspergillus Niger………………………………….…..34

2.3 Biology of Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris………………....37 2.3.1

Botanical Study of the Three Plants……………………………………….………37

2.3.2

Chemical Composition of the Three Seeds………………………………………..39

2.3.3

Utilization of the Three Plants…………...………………………………………..43

2.4 Constraints Limiting Beans Utilization………………………..……………………….......45 2.4.1

Protein Quality……………………………………………………….……………45

2.4.2

Physical Constraints………………………………………………………..……...45

2.4.3

Biological Constraints……………………………………………………………..46

2.4.4

The Presence Of Flatulence Factors In Legumes………………………………….52

3.0 MATERIALS AND METHODS………………………………….………………….……..55 3.1 Materials……………………………………………………..………………………….……55 3.1.1 Collection And Preparation Of Samples……………………...…………………..…55 3.1.2 Chemicals……………………………….………………...…………………………58 3.2 Methods………………………………………………………..……………………...……...59 3.2.1 Open Fermentation………………………………………...…………..…………….59 3.2.2 Controlled Fermentation……………………………………………..………………59 3.2.2.1 Isolation of Aspergillus Niger From Bambara Nuts………………………….…....59 3.2.2.2 Selection of Simultaneous Tannin and Phytate Degrading Aspergillus Niger Isolate………………...…………………………………………………………...60 3.2.2.3 Preparation Of Inoculum……………………………………...…………………...61 3.2.2.4 Enzyme Extraction………………………………………………...………………61 3.2.2.5 Enzymatic Activity Assays………………………………………………………..61 3.2.3 Determination Of Proximate Composition………………….…………………...….…...62 3.2.3.1 Ash Content Determination………………………….…………………………….62 3.2.3.2 Determination of Moisture………………..………………………………………...62 xi

3.2.3.3 Determination of Crude Lipid Content………………………………..…………...63 3.2.3.4 Determination of Crude Fibre…………………………………………….……….63 3.2.3.5 Determination of Percentage Carbohydrate…………………………………….....64 3.2.3.6 Determination of Nitrogen Content and Crude Protein…………………………...64 3.2.4 Determination of Mineral Content..………………………………………….….……….67 3.2.5 Determination of Antinutritional Factors….………………………………..…………...67 3.2.5.1 Determination of Cyanide……………………………………………………...….67 3.2.5.2 Determination of Tannins………………………………………………………….67 3.2.5.3 Determination of Phytic Acid………………………………………………..……69 3.2.5.4 Determinition of Saponin………………………………………………..………...70 3.2.5.5 Determination of Trypsin Inhibitor Activity……………........................................70 3.2.5.6 Determination of Lectins………………………………...………………………...72 3.2.5.7 Determination of Alkaloids…………………………………..……………………73 3.2.5.8 Determination of Oxalate…...…………………………………………………..…..74 3.2.6 Determination of Flatulence Factors………..……………………….………….………..74 3.2.6.1 Defatting of Flour……………………………………………….…………...…….75 3.2.6.2 Extraction of Oligosaccharides……………………………………………………75 3.2.6.3 Separation of Oligosaccharides by Thin Layer Chromatography……...………….75 3.2.6.4 Quantitative Analysis of Sugars Using Phenol-Sulphuric Acid Method ….……...77

4.0 RESULTS…………..………………...…...………………………………………...………..79 4.1 Isolation, Identification and Screaning of Aspergillus Niger From Bambara nut.............79 4.1.1

Isolation…………………………………………………………………..………..79

4.1.2.

Identification……………………………………….……………………………...79

4.1.3

Screening……………………………………………………….………………….80

4.2 Proximate Composition of Raw and Fermented Beans Flours…...………………..…..…80 4.2.1

Proximate Composition of Raw and Fermented Vigna Ungulculata Flour ...…….80

4.2.2

Proximate Composition of Raw and Fermented Vigna Racemosa Flour………….83

4.2.3 Proximate Composition of Raw and Fermented Phaseolus Vulgaris Flour….………83 4.3 The Effects of Fermentation on the anti-Nutritional Factors of The Three Types of Beans…………………….……..…………………………………..……..…………83 xii

4.4 The Effect of Fermentation on the Mineral Content of the Three Types of Beans……………………………………………………….....………………………89 4.5 The Effect of Open and Controlled Fermentation on the Flatulence Factors of the Three Types of Beans ….………………………………………………………..…..93 4.5.1 Separation of the Oligosaccharides by Thin Layer Chromatography………………......93 4.5.2 The Effect of Open and Controlled Fermentation on the Raffinose and Stachyose Content of the Three Types of Beans .…………………………………………..……93 4.6 Enzyme Assay……………………...…………………………………...…………..………93

5.0 DISCUSSION…………..………..………………………………………..……..…………98 6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS……….………….…….102 6.1 Summary…….…………………………...…...……………………………………….102 6.2 Conclusion……………………………...…...…...…………………………………….103 6.3 Recommendations……………………..……..…...…………………………………..103 References……………………………………………....……………………...……………..…105 Appendices ……………………...…………………….……………………..………………….131

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List of Figures Figure 2.1: The diffusion of a weak organic acid into a microbial cell, and its dissociation yielding protons (H+) and potentially toxic anions (A-)………………..….28 Figure 2.2: Culture of Aspergillus niger ……………………………………..…………...30 Figure 2.3: Conidial head of A. niger …………………………………………..…………30 Figure 2.4: Vigna racemosa plant growing in the bush…………...………………………40 Figure 2.5: Phaseolus vulgaris plant…………………...………………………………….41 Figure 2.6: Vigna unguiculata plant growing in a farm…………………………..………41 Figure 2.7: Structural relationship of the raffinose family sugars ……….………………..54 Figure 4.1: Colony diameters of A. niger strain in tannic acid and sodium Phytate agar plate isolated from bambara nut of different seed coats …………………….…….81

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List of Tables Table 2.1

Pathogenic and food spoilage organisms…………………..………..…………..27

Table 4.1

The effect of Open and Controlled Fermentation on proximate composition of Vigna unguiculata .…………………………………………………..………....82

Table 4.2

The Effect of Open and Controlled Fermentation on Proximate Composition of Vigna racemosa ………...……..…………………………………..………….…84

Table 4.3

The Effect of Open and Controlled Fermentation on Proximate Composition of Phaseolus vulgaris ……………………….………………………………..….....85

Table 4.4

The Effect of Open and Controlled Fermentation on Antinutritional factors of Vigna unguiculata ……...………………………...……………………..……….86

Table 4.5

The Effect of Open and Controlled Fermentation on Antinutritional factors of Vigna racemosa …………………………………………………..……….…….87

Table 4.6

The Effect of Open and Controlled Fermentation on Antinutritional factors of Phaseolus vulgaris ……..……………………………………..…………………88

Table 4.7

The Effect of Open and Controlled Fermentation on the Mineral Content of Vigna unguiculata ...……………………………………………….…………...90

Table 4.8

The Effect of Open and Controlled Fermentation on the Mineral Content of Vigna racemosa …………………………..………………………..………..….91

Table 4.9

The Effect of Open and Controlled Fermentation on the Mineral content of Phaseolus vulgaris .…………...………………………………..……………..…92

Table 4.10

Effect of Open and Controlled Fermentation on Some flatulent factors of Vigna unguiculata ………………………………………………………….………..…94

Table 4.11

Effect of Open and Controlled Fermentation on Some flatulent factors of Vigna racemosa ……………….………………...……………………………......…….95

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Table 4.12

Effect of Open and Controlled Fermentation on Some flatulent factors of Phaseolus vulgaris ………..…………….…………………………..………..….96

Table 4.13

Phytase and Tannase activity after 48hrs of Aspergilus niger solid- state fermentation…………………………..…………………………..…………..….97

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List of Plates Plate I: Dried pods of Vigna racemosa…………………………………..……………...40 Plate II: Vigna racemosa plant growing on a fence……………………..………………40 Plate III: Matured dried seeds of Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris ……….…………………………………………………..…………….57 Plate IV: Mature Bambara nuts (Vigna subterranean) of three different color coats (cream, red and black)……………………..………..……………..………………..58 Plate V: Aspergillus niger isolated from bambara nuts of different color seed coats……………………………………………………………………..79

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List of Appendices Appendix 1: Standard Curve for Raffinose Determination…………………………...….131 Appendix 2: Standard Curve for Stachyose Determination………………………….…..132 Appendix 3: Standard Curve of Sodium Phosphate for Phytase Activity Assay.….…….133

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CHAPTER 1 INTRODUCTION Microorganisms used for fermentation process of food products are capable of growing on a wide range of substrates and can produce a remarkable spectrum of products and bioactive components that enhance the biofunctionality of food products and develop properties such as flavor (Yadav et al., 2012). Increasing prevalence of various chronic diseases i.e. obesity, diabetes, cardiovascular diseases and cancer that are associated with poor food habits, demands to develop new microbes for fermented food production that can enhance biofunctionality of foods against these life threatening health ailments (Yadav et al., 2012). The relatively recent advent of in vitro genetic manipulation has extended the range of products that may be produced by microorganisms and has provided new methods for increasing the yields of existing ones (Chambers and Pretorius, 2010). The term fermentation is derived from the Latin verb fervere, to boil, which describes the appearance of the action of yeast on extracts of fruit or malted grain during the production of alcoholic beverages. However, there are various schools of thought on the definition of fermentation. Therefore, in 1697, George Emst gave the first definite theory concerning the nature of fermentation as stated by Sena, (2012). He explained fermentation as being a violent internal motion of particles of a fermenting substance giving a loosening end of the constituents of the substance with the formation of new particles which are carbon dioxide and alcohol; carbon dioxide was liberated and the alcohol remains in liquid. Antoine – Laurent Lavoiser in 1789 deduced that there is a chemical nature occurring in fermentation, not the splitting of sugar into carbon dioxide and ethanol, although he claimed that the analysis of sugar is in error (Sena, 2012). In 1803, Louis Jacques Thenard in his turn observed that all fermentation

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produced materials resembling brewers yeast which was used to ferment pure sugar and it eventually changed leaving a white man (i.e. foaming) (Sena, 2012). In physiological terms, fermentation is defined as the type of metabolism of a carbon source in which energy is generated by substrate level phosphorylation but to the Microbiologist, the term fermentation describes a form of energy yielding microbial metabolism in which an organic substance, usually, carbohydrate is incompletely oxidized and an organic carbohydrate acts as electron acceptor (Adams 1990). However to a biochemist, the term fermentation can be technically defined as the chemical transformation of organic compounds with the aid of enzymes particularly those made by microorganisms. Fermentation process is a process which involves the conversion of large molecules to small molecules or molecular oxidation/ reduction mechanisms mediated by selected micro-organisms (Yadav et al., 2012). Thus fermentation in food processing is defined as the conversion of carbohydrates to alcohol and carbondioxide or organic acids using yeast and/or bacteria, under anaerobic conditions (William and Dennis, 2011). The fermentation technology depends on the microbial components and produces different molecules from small laboratory scale to large industrial scale. Fermentation is also seen as one of the oldest and most economical methods of food production and preservation (Oyewole and Isah, 2012). Several experiments have demonstrated that fermentation of legumes enhances their nutritive value and antioxidant properties; reduces some anti-nutritional endogenous compounds such as phytic acid, and exerts beneficial effects on protein digestibility and biological value of legumes (Oyewole and Isah, 2012; Oboh and Lajide, 2012). Some anti-nutritional factors such as trypsin and cystatin inhibitors and lectins are heat-labile compounds and their negative effects are, therefore, markedly reduced by cooking (Adegunwa et al.,

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2012), while tannins and phytic acid are heat-stable compounds that retain negative effects on mineral and protein bioavailability after cooking (Ogun et al., 1989). The bean plant belong to the genus Vigna savi, (Willis, 1985) and the family Leguminosae-papilionoidae and the tribe Phaseoleae which is made up of about 80-100 species. They grow in the tropics and Asia (Mbagwu and Edeoga, 2006). The confusion and discrepancies recognized by different authorities in the estimation of the number of species is due to the perceived similarities in structural and reproductive biology of the bean plants (Mbagwu and Edeoga, 2006), and this lead to the recognition of 37 species by Daniel (1960), 25 species by Hutchinson and Dalziel (1954) and 22 species by Burkill (1995). Cowpea (Vigna unguiculata (L.) Walp.), Bambara groundnut (Vigna subterranae (L.) Verdc) and common bean (Phaseolus vulgaris, L.) belong to the sub-tribe phaseolinae, together with many other food legumes are important members of the tribe phaseolae (Felix et al., 2000) and are domesticated edible food legumes. However, Vigna racemosa, Vigna vexilata, Vigna vexilata macrosperma, Vigna luteola, Vigna oblongifolia, Vigna unguiculata dekindtiana, Vigna reticulata, Vigna ambasensis etc. are wild under- exploited Vigna spp. and are not edible (Carnovale et al., 1996). Vigna racemosa is readily found in abundance in the locality of Samaru- Zaria, Kaduna State, Nigeria and is commonly known as “waken ngizo” (Lizard beans) by Hausa people or “Bush Bean by other farmers of the locality. Therefore, the aim of the present study is to evaluate the chemical changes occurring in the seeds of Vigna racemosa, Vigna unguiculata, and Phaseolus vulgaris (black bean), subjected to open and controlled fermentation.

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1.1

Statement of Research Problems

Beans in general, are important sources of macronutrients, micronutrients and antioxidant compounds with a great potential for human and animal nutrition (Jesus et al., 2003), however, they contain several anti-nutritional factors which limit their consumption and affect the digestibility and bioavailability of nutrients (Bressani et al., 1993). Vigna racemosa like most other wild Vigna spp. contains some very important nutrients. However, it is not consumed by both human and animals due to its high level of antinutritional factors (Carnovale et al., 1996). 1.2

Justifications

Many developing countries in the world are living in abject poverty and are malnourished, which necessitates the sourcing for novel food sources. Considering the level of poverty and malnutrition in the world, especially the developing world (FAO, 2011), there is need to process hitherto wild beans which will make them available for consumption. Moreover, the chemical evaluation of wild under-exploited Vigna spp. seeds carried out on eight species of wild beans including Vigna racemosa by Carnovale et al., (1996), showed that all the accessions had high sulphur amino acid content (2.03- 3.63 g per 16 g N) and consequently high chemical score. There is therefore need to see if processing by fermentation can make Vigna racemosa eadible and comparable to known edible species. Fermentation of food has been found to reduce the risk of having food intoxication arising from toxicants found in food. In addition to this, several experiments have demonstrated that fermentation of legumes enhances their nutritive value and 4

antioxidant properties; reduces some anti-nutritional endogenous compounds such as phytic acid, and exerts beneficial effects on protein digestibility and biological value of legumes (Oyewole and Isah, 2012; Oboh and Lajide, 2012). Since Aspergillus niger has been proved to produce tannase (Dapiya et al., 2010) and phytase (Newkirk et al., 2001) , the solid- state fermentation using Aspergillus niger may be

a

promising

processing method

unveiling

the

usages

of

Vigna

racemosa,therefore the need for controlled fermentation. 1.3

Null Hypothesis

Fermentation does not reduce or eliminate the anti-nutritional factors and does not exert beneficial effects on proximate composition, mineral content and flatulence factors of Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris seeds. 1.4

General Objective

The general aim of the study is to evaluate the effects of fermentation on the proximate composition, anti-nutritional factors, some minerals and flatulence factors of Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris flours. 1.5 i.

Specific Objectives To isolate Aspergillus niger from three different varieties of bambara nuts based on the colour of their seeds coats (black, red and cream).

ii.

To screen Aspergillus niger for simultaneous high production of tannase and phytase to ferment beans’ flour.

iii.

To carry out proximate analysis on unfermented and fermented Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris seeds.

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iv.

To estimate the mineral elements in unfermented and fermented Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris seeds.

v.

To estimate the level of anti-nutritional factors in unfermented and fermented Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris seeds.

vi.

To estimate the level of flatulence factors in unfermented and fermented Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris seeds.

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CHAPTER 2 LITERATURE REVIEW 2.1

Fermentation

2.1.1 Definition of Fermentation From a biochemical point of view, fermentation is the process of deriving energy from the oxidation of organic compounds, such as carbohydrates, and using microbes as enzyme source and an endogenous electron acceptor, which is usually an organic compound. However, to the microbiologists, the term “fermentation” describes a form of energy-yielding microbial metabolism in which an organic substrate, usually a carbohydrate, is incompletely oxidized, and an organic carbohydrate acts as the electron acceptor (Adams, 1990).

Example of Chemical equation of fermentation of Glucose to ethanol: C6H12O6 + 2ATP + 2ADP + 4NADH ------> 2C2H5OH + 2CO2 +4ATP + 2H2O + 4NAD+ 2.1.2 Classification of Fermentation Fermentation processes may be classified in a number of different ways. The first systematic approach was proposed by Gale, (1947) who grouped microbiological processes in a series of type groups, oxidation, reduction, hydrolysis, etc. Such an arrangement though fundamentally attractive, is only suitable for specific reactions operating on specific substrates to yield specific products. Unfortunately, many commercially important fermentation processes cannot be so neatly described. Therefore Gale’s classification scheme was later on extended to a more detailed breakdown of ‘type reactions’ (Stodola, 1958). In this scheme, microorganisms, or more specifically their enzyme complements, are looked at as added means for controlled 7

organic synthesis. Again, this concept is not applicable to most of the fermentation processes now practised commercially—at least at the present level of knowledge regarding mechanisms. A different approach was proposed by Gaden, 1956. Here fermentation processes rather than specific reactions are grouped together and the overall free energy change involved is the basis for classification. The primary advantage of this scheme is technological; it coincides with the general classification of fermentation rate patterns suggested earlier. Experience has shown that fermentation processes fall more or less into three kinetic groups, which may be designated ‘types I to III’ for convenience (Mosier and Ladisch, 2009). i.

Type I: processes in which the main product appears as a result of primary

energy metabolism. Examples of this type of system are most common in the older branches of fermentation technology, such as, aerobic yeast propagation (mass propagation of cells in general), alcoholic fermentation, oxidation of glucose to gluconic acid, and dissimilation of sugar to lactic acid. ii.

Type II: processes in which the main product arises indirectly from reactions of

energy metabolism. In systems of this type, the product is not a direct residue of oxidation of the carbon source but the result of some side-reaction or subsequent interaction between these direct metabolic products. Examples include formation of citric and itaconic acids, and formation of certain amino acids iii.

Type III: processes in which the main product does not arise from energy

metabolism at all but is independently or accumulated by the cells. It is perfectly true that carbon, nitrogen, etc., provided in essential metabolites appear in product

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molecules but the major products of energy metabolism are CO2 and water. Antibiotic synthesis is a prime example of this type. Another important classification of fermentation could be done based on the source of microorganisms used in the process (as used in this work). Based on that classification, there is open fermentation, natural fermentation and controlled fermentation. a.

Open fermentation: the fermentation process which is performed in an open air

and uses the microorganisms present on the substrate (eg. food legumes) and those present in the atmosphere surrounding the fermentation vessel as fermenting microorganisms. b.

Natural fermentation: this is the fermentation process mostly operated in a

fermenter and is spontaneously and aseptically initiated with the microbiota naturally present on the substrates (Granito et al., 2002). c.

Controlled fermentation: this is the fermentation process that is controlled by the

use of specific cultures or starters from a batch of previously fermented products (Ibrahim et al., 2002) Microbial fermentation can also be classified based on the mode of operation of the bioprocess as batch fermentation, fed-batch fermentation and continuous fermentation. 2.1.3 Modes of Fermentation Techniques There are three main modes of fermentation techniques: batch, fed-batch and continuous. In industry, batch and fed-batch fermentation techniques have been used for the production of alcoholic beverages and fermented foodstuffs since before 3000 BC in Egypt and Sumeria (Mcneil and Harvey, 2008).

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Most fermentation processes were batch for much of human history, with fed-batch becoming common only with production of baker’s yeast and antibiotics and most industrial fermentation processes still operate as simple batch or fed-batch (Mcneil and Harvey, 2008) There are few continuous fermentation examples in industry: vitamin C, propionic acid and Quorn production. However, as a laboratory tool for studying the physiology of microorganisms, metabolomics, proteomics, etc., continuous fermentation techniques are well established and currently enjoying a resurgence of interest amongst researchers (Mcneil and Harvey, 2008). i. Batch Culture Fermentation This type of fermentation is also called a closed culture system because nutrients and other components are added in specific amounts at the start of the process and are not replenished once the fermentation has started. At the end of the process the product is recovered; then, the fermenter is cleaned, sterilized, and used for another batch process (Cinar et al., 2003). In the initial stages, microorganisms grow at a rapid rate in the presence of excess nutrients but as they multiply in large numbers they use up the nutrients. They also produce toxic metabolites which retard further growth of microorganisms during the later stages of the fermentation process. Batch fermentation is the simplest mode of operation, and is often used in the laboratory to obtain substantial quantities of cells or product for further analysis (Cinar et al., 2003).

10

ii.

Fed-Batch Culture Fermentation

Fed-batch culture is essentially similar to batch culture, and most fed-batches begin life with a straight forward batch phase; but unlike batch these cultures do not operate as closed systems (Mcneil and Harvey, 2008). In this process the nutrients and substrates are added at the start of the process and at regular intervals after the start. This is called controlled feeding. Inoculum is added to the fermentation vessel when microorganisms are in exponential growth phase and the fermenter is designed to accommodate the increasing volumes in order to maintain the system always in quasi-steady state. Fed-batch culture is controlled by feed-back control and control without feed-back (Hewitt et al., 2000). a. Feed-back control– The fermentation process is controlled by monitoring process parameters like dissolved oxygen content, carbon dioxide to oxygen ratio, pH, concentration of substrate, and concentration of the product. b. Control without feed-back– The substrates and nutrients are added at regular intervals. Fed-batch culture requires special equipment such as a reservoir which holds the nutrients, pH modifiers so that they can be added to the fermenter at regular intervals, and pumps to deliver culture medium aseptically to the fermenter. Fed-batch cultures can also be run in different ways, e.g. at a fixed volume where at a certain time point, a portion of the fermenter contents (consisting of spent medium, cells, product, and unused nutrients) is drawn off and replaced with an equal volume of fresh medium and nutrients (withdraw and fill), or at a variable volume where nothing is removed from the bioreactor during the time course of the process, with the cells and 11

product remaining within the vessel until the end of the fermentation period, and the addition of fresh medium and nutrients having the effect of increasing the culture volume (Mcneil and Harvey, 2008). iii.

Continuous Culture Fermentation

This method prolongs the exponential growth phase of microbial growth as nutrients are continually supplied and metabolites and other wastes are continually removed thus promoting continual growth of the microorganisms. Continuous culture fermentation is advantageous because of its high productivity. Historically, Continuous culture techniques have not been widely used in laboratory scale, but are more common in industry where these techniques are used for such processes as vinegar production, waste water treatment, ethanol production and single cell protein production (Hoskisson and Hobbs, 2005). Several control techniques can be used with continuous culture and the most commonly used continuous culture technique, the chemostat, operates on the basis of growth being restricted by the availability of a limiting substrate, while the turbidostat (another control technique) is operated under no limitations (Hoskisson and Hobbs, 2005). Another continuous cultivation technique includes the auxostat. a.

The Chemostat: This medium contains excess of all but one of the nutrients which

determine the rate of growth of the microorganism. At steady state of the chemostat the rate of input of medium into the fermenter is equal to the rate of output out of the fermenter. b.

The Turbidostat: This medium contains excess of all nutrients so the microbial

growth is at its maximum specific growth rate. The system consist of a photoelectric 12

cell which is a turbidity sensor that detects changes in turbidity of the contents in the fermenter and then controls the amount of medium fed to the fermenter. c.

The Auxostat: Auxostats use feed rate to control a state variable, such as pH or

dissolved oxygen, in continuous culture. The microorganisms themselves establish their own dilution rate, and auxostats tend to be much more stable than chemostats at higher dilution rates, especially at dilution rates approaching the maximum specific growth rate of the microorganism (Mcneil and Harvey, 2008). The high dilution rates exert a selection pressure upon the microbial population, which leads to more rapidly growing cultures. This method of continuous culture control is therefore ideal for such applications as high-rate propagation and detoxification of waste materials at maximum rate concentrations. 2.1.4 Fermentation Vessels Laboratory scale fermentations are carried out in shaker flasks, and flat bed bottles. Large scale fermentations are carried out in glass or stainless steel tank fermenters. A fermentation vessel should be cheap, not allow contamination of the contents, be nontoxic to the microorganism used for the process, be easy to sterilize, be easy to operate, be robust and reliable, allow visual monitoring of the fermentation process, allow sampling, and be leak proof. i.

Shaker Flasks: these are conical vessels made of glass and are available in

different sizes. The typical volume of these flasks is 250 ml. There are different types of shaker flasks, such as baffled, unbaffled or Erlenmeyer flask, and flying saucer. Shaker flasks are used for the screening of microorganisms and cultivation of microorganisms for inoculation. Shaker beds or shaker tables are used to allow oxygen transfer by their 13

continuous rotary motion. Baffled flasks are used to increase the oxygen transfer. Shaker flasks need to be plugged to prevent contamination with other microorganisms. Cotton-wool, polyurethane foam, glass, and synthetic plugs are commonly used. Fernwald shaker flasks and flat bed Thompson bottles are expensive and are not commonly used (Mosier and Ladisch, 2009). ii.

Stirred Tank Fermenters: These are the most commonly used fermenters.

They are cylindrical vessels with a motor driven agitator to stir the contents in the tank. The Top-entry stirrer (agitator) model is most commonly used because it has many advantages like ease of operation, reliability, and robustness. The Bottom-entry stirrer (agitator) model is rarely used. iii.

Air-Lift Fermenters: These fermenters do not have mechanical agitation

systems (motor, shaft, impeller blades) but contents are agitated by injecting air from the bottom. Sterile atmospheric air is used if microorganisms are aerobic and “inert gas” is used if microorganisms are anaerobic. This is a gentle method of mixing the contents and is most suitable for fermentation of animal and plant cell cultures since the mechanical agitation produces high shearing stress that may damage the cells. Air-lift fermenters are most widely used for large-scale production of monoclonal antibodies. Draft tubes are used in some cases to provide better mixing, mass transfer, and to reduce bubble coalescence by inducing circulatory motion (Mcneil and Harvey, 2008). iv.

Fixed Bed Fermenters: These are also called immobilized cell fermenters. The

cells are absorbed onto or entrapped in the solid surfaces like plastic beads, glass or plastic wool and solidified gels to render them immobile.

14

Fixed bed fermenters are most commonly used for waste water treatment and as biological filters in small aquarium water recycling systems and production of amino acids and enzymes. v.

Tower Fermenters: Tower fermenters are simple in design and easy to

construct. They consist of a long cylindrical vessel with an inlet at the bottom, an exhaust at the top, and a jacket to control temperature. They do not require agitation hence there are no shafts, impellers or blades. Tower fermenters are used for continuous fermentation of beer, yeast and SCP (Mcneil and Harvey, 2008). 2.1.5 Methods of Microbial Inoculation in Food Fermentations The fermentation bioprocess is the major biotechnological application in food processing. It is often one step in a sequence of food-processing operations, which may include cleaning, size reduction, soaking and cooking. Fermentation bioprocessing makes use of microbial inoculants for enhancing properties such as the taste, aroma, shelf-life, safety, texture and nutritional value of foods. Microbes associated with the raw food material and the processing environment serve as inoculants in spontaneous fermentations, while inoculants containing high concentrations of live micro-organisms, referred to as starter cultures, are used to initiate and accelerate the rate of fermentation processes in non-spontaneous or controlled fermentation processes. Microbial starter cultures vary widely in quality and purity (FAO, 2010). i.

Spontaneous Inoculation of Fermentation Processes

In many developing countries, fermented foods are produced primarily at the household and village level, using spontaneous methods of inoculation (FAO, 2010). Spontaneous fermentations are largely uncontrolled. A natural selection process, however, evolves in 15

many of these processes which eventually results in the predominance of a particular type or group of micro-organisms in the fermentation medium. A majority of African food-fermentation processes make use of spontaneous inoculation. Major limitations of spontaneous fermentation processes include their inefficiency, low yields of product and variable product quality. While spontaneous fermentations generally enhance the safety of foods owing to a reduction of pH, and through detoxification, in some cases there are safety concerns relating to the bacterial pathogens associated with the raw material or unhygienic practices during processing (FAO, 2010). ii.

“Appropriate” Starter Cultures as Inoculants of Fermentation Processes

“Appropriate” starter cultures are widely applied as inoculants across the fermented food sector, from the household to industrial level in low-income and lower-middleincome economies. These starter cultures are generally produced using a backslopping process which makes use of samples of a previous batch of a fermented product as inoculants (Holzapfel, 2002). Appropriate starter cultures are widely applied in the production of fermented fish sauces and fermented vegetables in Asia and in cereal or grain fermentations in African and Latin American countries. The inoculation belt used in traditional fermentations in West Africa serves as a carrier of undefined fermenting micro-organisms, and is one example of an appropriate starter culture (Holzapfel, 2002). It generally consists of a woven fibre or mat or a piece of wood or woven sponge, saturated with “high”-quality product of a previously fermented batch. It is immersed into a new batch, in order to serve as an inoculant. The inoculation belt is used in the production of the indigenous East African (Kenyan) fermented porridges, “uji” and “mawe”, as well as in the production of the Ghanaian beer, “pito”.

16

Iku, also referred to as iru (a West African fermented Legume), is yet another example of an “appropriate” starter culture produced by backslopping. This starter culture is produced from concentrated fermented ‘’dawadawa’’ (Nigerian fermented legume product), mixed with ground unfermented legumes, vegetables such as pepper, and cereals, such as ground maize. It is stored in a dried form and is used as an inoculant in ‘’dawadawa’’ fermentations in West Africa (Holzapfel, 2002). iii.

Defined Starter Cultures as Inoculants of Fermentation Processes

Few defined starter cultures have been developed for use as inoculants in commercial fermentation processes in developing countries. Nevertheless, according to FAO, (2010) the past ten years have witnessed the development and application of laboratoryselected and pre-cultured starter cultures in food fermentations in a few developing countries. These developments have taken place primarily in Asian countries. “Defined starter cultures” consist of single or mixed strains of micro-organisms (Holzapfel 2002). They may incorporate adjunct culture preparations that serve a food-safety and preservative function. Adjunct cultures do not necessarily produce fermentation acids or modify texture or flavour, but are included in the defined culture owing to their ability to inhibit pathogenic or spoilage organisms. Their inhibitory activity is due to the production of one or several substances such as hydrogen peroxide, organic acids, diacetyl and bacteriocins (Hutkins, 2006). By and large, defined cultures are produced by pure culture maintenance and propagation under aseptic conditions. They are generally marketed in a liquid or powdered form or else as a pressed cake. Loog-pang, a defined culture marketed in Thailand in the form of a pressed rice cake, consists of Saccharomyces cerevisiae, Aspergillus oryzae or Rhizopus sp. and Mucor. Loog-pang has a shelf life of 2–3 days at 17

ambient temperature and 5–7 days under refrigerated conditions. Ragi cultures are commercially produced by the Malaysian Agricultural Research and Development Institute by mixing a culture inoculum which generally consists of Rhizopus oligosporus with moistened sterile rice flour, and incubating it at ambient temperature for four days. This starter has a shelf life of two weeks under refrigerated conditions (Merican and Quee-Lan, 2004). It is widely used as an inoculant in the production of traditional Malaysian fermented foods. The use of DNA-based diagnostic techniques for strain differentiation can allow for the tailoring of starter cultures to yield products with specific flavours and/or textures. Random amplified polymorphic DNA (RAPD) techniques have been applied in, for example, Thailand, in the molecular typing of bacterial strains and correlating the findings of these studies to flavour development during the production of the fermented pork sausage, nham. The results of these analyses led to the development of three different defined starter cultures which are currently used for the commercial production of products having different flavour characteristics (Valyasevi and Rolle, 2002). iv.

Genetically Modified (GM) Starter Cultures

To date, no commercial GM micro-organisms that would be consumed as living organisms exist (FAO, 2010). Products of industrial GM producer organisms are, however, widely used in food processing and no major safety concerns have been raised against them. Rennet which is widely used as a starter in cheese production across the globe is produced using GM bacteria. Thailand currently makes use of GM Escherichia coli as an inoculant in lysine production. Many industrially important enzymes such as α-amylase, gluco-amylase, lipase and pectinase and bio-based fine chemicals, such as lactic acid, amino acids, antibiotics, nucleic acid and polysaccharides, are produced in 18

China using GM starter cultures. Other developing countries which currently produce enzymes using recombinant micro-organisms include Cuba, Brazil, India, and Argentina (FAO, 2010). 2.1.6 Nutritional Value of Fermented Foods Generally, a significant increase in the soluble fraction of a food is observed during fermentation. The quantity as well as quality of the food proteins as expressed by biological value, and often the content of water soluble vitamins is generally increased, while the antinutritional factors show a decline during fermentation (Oyewole and Isah, 2012). Fermentation results in a lower proportion of dry matter in the food and the concentrations of vitamins, minerals and protein appear to increase when measured on a dry weight basis (Adams, 1990). Different fermentation methods have been shown to have increasing effects on the proximate composition and a decreasing effect on the antinutrients content of food legumes (Ojokoh et al., 2012; Ari et al., 2012). So fermentation causes significant changes in food composition as follow: i.

Proteins

The protein efficiency ratio (PER) of wheat was found to increase on fermentation, partly due to the increase in availability of lysine. A mixture of wheat and soybeans in equal amounts would provide an improved pattern of amino acids. The fermentation process rose the PER value of the mixture to a level which was comparable to that of casein (Hesseltine and Wang, 1980). Fermentation may not increase the content of protein and amino acids unless ammonia or urea is added as a nitrogen source to the fermentation media (Reed, 1981). The relative nutritional value (RNV) of maize increased from 65% to 81% when it was germinated, and fermentation of the flour made of the germinated maize gave a further increase in RNV to 87% (Lay and Fields, 1981). 19

Fermentation of legumes for making ‘’dhokla’’ and fermentation of millet for making ‘’ambali’’ did not show any improvement in the values reported for PER, TD, BV and NPU in relation to the unfermented products (Aliya and Geervani, 1981). So fermentation has an important effect on the protein content of foods. ii.

Vitamins

During fermentation certain micro-organisms produce vitamins at a higher rate than others do. The content of thiamine and riboflavin in dhokla and ambali was about 50% higher after fermentation. The availability of some selected B- vitamins was significantly enhanced by fermentation by between 71.2- 94.2% (Ochanda et al., 2010). The levels of vitamin B12, riboflavin and folacin were increased by lactic acid fermentation of maize flour, while the level of pyridoxine was decreased (Murdock and Fields, 1984). Fermented whole onion plant retained 97% of vitamin A activity, while fermented egg plant only retained 34% of the vitamin A activity (Speek et al., 1988). Kefir made from ten different kefir grain cultures showed significant (>20%) increase for pyridoxine, cobalamin, folic acid and biotin and reduction exceeding 20% for thiamine, riboflavin, nicotinic acid, and pantothenic acid depending on the culture used. iii.

Minerals

The mineral content is not affected by fermentation unless some salts are added to the product during fermentation or by leaching when the liquid portion is separated from the fermented food (Zamora and Fields, 1979). Sometimes, when fermentation is carried out in metal containers, some minerals are solubilised by the fermented product, which may cause an increase in mineral content. In an investigation carried out by Olakunle and Adebola, (2012), there was no significant change in the mineral composition of locust beans during natural fermentation. 20

iv.

Antinutrients

Phytate content in bread was lowered when the amount of yeast or the fermentation time was raised (Harland and Harland, 1980). Phytate content in locust bean seeds was lowered from 0.51 mg/g to 0.31 mg/g by fermentation (Eka, 1980). Natural lactic fermentation of maize meal decreased phytate phosphorus by 78% (Chompreeda and Fields, 1984). In bambaranut milk, tannin content could be reduced by fermentation (Obizoba and Egbuna, 1992). A more recent investigation has shown that fermentation significantly reduces antinutritional factors like phytate, oxalate, tannin, saponin and cyanide using inoculums of aspergillus niger on Chrysophyllum albidum seeds (Adeyemi et al., 2012) 2.1.7 Health Effects Of Fermented Foods 2.1.7.1 Probiotic Effect of Fermented Food In the late 19th century, microbiologists identified microflora in the gastrointestinal (GI) tract of healthy individuals that differed from those found in diseased individuals. These beneficial microflora found in the GI tract were termed probiotics. Probiotics, literally meaning ‘for life’, are micro-organisms proven to exert health-promoting influences in humans and animals (Marteau et al. 1995). Most probiotics fall into the group of organisms known as lactic acid-producing bacteria and are normally consumed in the form of yogurt, fermented milks or other fermented foods. Some of the beneficial effect of lactic acid bacteria consumption include: (i) improving intestinal tract health; (ii) enhancing the immune system, synthesizing and enhancing the bioavailability of nutrients; (iii) reducing symptoms of lactose intolerance, decreasing the prevalence of allergy in susceptible individuals; and (iv) 21

reducing risk of certain cancers (Parvez et al., 2006). The mechanisms by which probiotics exert their effects are largely unknown, but may involve modifying gut pH, antagonizing pathogens through production of antimicrobial compounds, competing for pathogen binding and receptor sites as well as for available nutrients and growth factors, stimulating immunomodulatory cells, and producing lactase (Parvez et al., 2006). Recent scientific investigation has supported the important role of probiotics as a part of a healthy diet for human as well as for animals and may be an avenue to provide a safe, cost effective, and ‘natural’ approach that adds a barrier against microbial infection. 2.1.7.2 Flatulence Reducing Effect of Fermentation During fermentation of the beans for preparation of ‘’Tempe’’, the trypsin inhibitor is inactivated, and the amount of several oligosacharides which usually cause flatulence are significantly reduced (Hesseltine, 1983). Bean flour inoculated with Lactobacillus and fermented with 20% moisture content, showed a reduction of the stachyose content (Duszkiewicz-Reinhard et al., 1994). 2.1.7.3 Anticholesterolemic Effects of Fermented Food Hepner et al. (1979) reported hypercholesteremic effect of yoghurt in human subjects receiving a one-week dietary supplement. Studies on supplementation of infant formula with Lactobacillus acidophilus showed that the serum cholesterol in infants was reduced from 147 mg/ml to 119mg/100 ml (Harrison and Peat, 1975). In an in vitro study, the ability of 23 strains of lactic acid bacteria isolated from various fermented milk products to bind cholesterol was investigated. No cholesterol was found inside the cells (Taranto et al., 1997). Poppel and Schafsma (1996) have also reported the ability of yoghurt to lower the cholesterol level in serum by controlled human trials. Possible 22

role of lactic acid bactera in lowering cholesterol concentration and various mechanisms by which it may be possible has been discussed by Haberer et al (1997). Brigidi et al (1993) have cloned a gene encoding cholesterol oxidase from Streptomyces lividans into Bacillus, Lactobacillus and E. coli. 2.1.7.4 Effect on Transit Time, Bowel Function and Glycemic Index The transit time for 50% (t50) of the gastric content was significantly reduced for regular unfermented milk (42±10 min) in comparison with a fermented milk product indigenous to Sweden called "långfil" or ropy milk (62±14 min). Another study (Wilhelm, 1993) reports increase in transport time and improved bowel function in patients with habitual constipation. The number of defecations per week increased from three during control period to seven using conventional fermented milk and fifteen when acidophilus milk was served. Regular unfermented milk also gave significantly higher increase in glycemic index curve than fermented milk product called långfil (Strandhagen et al., 1994). Liljeberg et al (1995) have shown that presence of acid, especially acetic or lactic acid would lower the glycemic index in breads to a significant level. Koji which is prepared from Aspergillus oryzae and beni-koji made from Monascus pilosus were found to express rises in blood pressure (Tsuji et al., 1992). 2.1.7.5

Anti- carcinogenic Effects of Fermented Food

There are interesting data on anticarcinogenic effect of fermented foods showing potential role of lactobacilli in reducing or eliminating procarcinogens and carcinogens in the alimentary canal (Reddy et al., 1983; Shahani, 1983; Mital and Garg, 1995). The enzymes β-glucuronidase, azoreductase and nitroreductase, which are present in the intestinal canal, are known to convert procarcinogens to carcinogens (Goldin and Gorbach, 1984). Oral administration of Lb rhamnosus GG was shown to lower the 23

faecal concentration of b-glucuronidase in humans (Salminen et al., 1993) implying a decrease in the conversion of procarcinogens to cancinogens. Fermented milk containing Lactobacillus acidophilus given together with fried meat patties significantly lowered the excretion of mutagenic substances compared to ordinary fermented milk with Lactococcus fed together with fried meat patties (Lidbeck et al., 1992). The process of fermentation of foods is also reported to reduce the mutagenicity of foods by degrading the mutagenic substances during the process. Lactic acid bacteria isolated from dadih, traditional Indonesian fermented milk, were found to be able to bind mutagens and inhibit mutagenic nitrosamines. 2.1.7.6 Food Safety Aspects of Fermented Foods It has been estimated that more than 13 million infants and children under five years of age die annually in the tropical regions of the world. After respiratory infections, diarrhoea diseases are the commonest illnesses and have the greatest negative impact upon the growth of infants and young children. The causes of diarrhoea have traditionally been ascribed to water supply and sanitation (Motarjemi et al., 1993). Foods prepared under unhygienic conditions and frequently heavily contaminated with pathogenic organisms play a major role in child mortality through a combination of diarrhoea diseases, nutrient malabsorption, and malnutrition. All food items contain microorganisms of different types and in different amounts. Which microorganisms that will dominate depends on several factors, and sometimes microorganisms initially present in very low numbers in the food, for example lactic acid bacteria (LAB), will outnumber the other organisms inhibiting their growth. In contrast to fermented meat, fish, dairy and cereal products, fermented vegetables have not been recorded as a significant source of microbial food poisoning (Fleming and McFeeters, 1981).

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2.1.7.7 Effect of Fermentation on Pathogenic Organisms Over a study period of nine months, a group of children fed with lactic acid fermented gruel had a mean number of 2.1 diarrhoea episodes compared to 3.5 for the group fed with unfermented gruel (Lorri and Svanberg, 1994). Although Salmonella, Campylobacter, Shigella, Vibrio, Yersinia and Escherichia are the most common organisms associated with bacterial diarrhoea diseases, other enterotoxigenic genera, including Pseudomonas, Enterobacter, Klebsiella, Serratia, Proteus, Providencia, Aeromonas, Achromobacter and Flavobacterium, have also been reported (Nout et al., 1989). In addition, it was found that there was no significant difference between the behaviour of the pathogens in fermented porridge or acid-supplemented non fermented porridge, which implies that the anti-microbial effect is due to presence of lactic and acetic acids at reduced pH, and that other anti-microbial substances do not play a detectable role (Nout et al., 1989). Similarly, Adams (1990) suggested that lactic acid bacteria are inhibitory to many other microorganisms when they are cultured together, and this is the basis of the extended shelf life and improved microbiological safety of lactic-fermented foods. Lactobacillus species can produce a variety of metabolites, including lactic and acetic acids which lower pH, that are inhibitory to competing bacteria, including psychrotrophic pathogen (Breidt and Fleming, 1997). This effect could be due to a combination of many factors as shown in Table 2.1. The inhibition by organic acids has been attributed to the protonated form of these acids, which are uncharged and may therefore cross biological membranes (Figure 2.1). The resulting inhibition of growth may be due to acidification of the cytoplasm and/or accumulation of anions inside the cell (Adams, 1990; Russel, 1992; Breidt and Fleming, 1997). The ability of an acid to inhibit bacteria depends principally on the pKa of the

25

acid: the higher the pKa of the acid, the greater the proportion of undissociated acid, and the more inhibitory the acid is likely to be. On this basis, one would expect acetic acid (pKa = 4.75) to be a more effective antimicrobial agent than lactic acid (pKa = 3.86) (Adams, 1990).

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Table 2.1: Pathogenic and food spoilage organisms Products

Main Target Organism

Organic Acid Lactic Acid

Putrefactive and Grambacteria, some fungi

negative

Acetic Acid

Putrefactive bacteria, clostridia, some yeasts and some fungi

Hydrogen peroxide

Pathogens and spoilage organisms, especially in protein- rich foods

Enzymes Lactoperoxidase system with hydrogen peroxide

Pathogens and spoilage bacteria (milk and dairy products)

Lysozyme (by recombinant DNA)

Undesired Gram-positive bacteria

Low-molecular-weight metabolites Reuterin

Wide spectrum of bacteria, yeasts, and molds

Diacetyl Gram-negative bacteria Fatty acids Different bacteria Bacteriocins Nisin Other

Some LAB and Gram-positive bacteria, notably endospore-formers Gram-positive bacteria, inhibitory spectrum according to producer strain and bacteriocin type

Culled from Breidt and Fleming, (1997)

27

Intracellular pH > pKa

Ac- + H+

HAc

Ac- + H+

HAc Extracellular pH ≤ pKa

Figure 2.1: The diffusion of a weak organic acid into a microbial cell, and its dissociation

yielding protons (H+) and potentially toxic anions (A-). Culled from

Adams, (1990) 2.1.8 Toxins and Toxin- Producing Organisms in Fermented Foods Lactic starter cultures were found to be effective in preventing the formation of botulin toxin, even in the absence of nitrate (Shahani, 1983). No aflatoxin production was reported in Tempe and miso prepared using Rhizopus oligosporus and Aspergillus oryzae on soya bean, chickpea and horsebean (Paredes-López and Harry, 1988). Aspergillus flavus grown in broth had a lower aflatoxin production when 10% cell free supernatant culture fluid from lactobacilli was added. This effect could not be explained on the basis of pH or competition (Karunaratne et al., 1990). Studies, mainly with Aspergillus oryzae, have shown no traces of aflatoxin production in traditional mouldfermented products (Wang and Hesseltine, 1981). However when an aflatoxin producing strain was inoculated at the same time, large amounts of aflatoxin was found. The aflatoxin production of Aspergillus parasiticus was studied and found to increase in the presence of Lactococus lactis (Luchese and Harrigan, 1990). In contaminated peanut 28

press-cake, Rhizopus oligosporus and Neurospora sitophila were found to reduce the aflatoxin content by 50 and 60% respectively (Paredes-López and Harry, 1988). Aspergillus niger has also been used to ferment many food items with significant effect as in the case of this work. 2.2

Biology of Aspergillus niger

The Aspergillus fungus was first recognized as an organism in 1729 by Micheli. The genus Aspergillus is found worldwide and consists of more than 180 officially recognized species, and comprises a particularly important group of filamentous ascomycete species. Although it includes Aspergillus fumigatus, which is the major filamentous fungal pathogen of humans (Brookman and Denning, 2000; Latge, 1999). However most of the members are useful microorganisms in nature for degradation of plant polysaccharides (de Vries, 2003; de Vries et al., 2000), and they are important industrial microorganisms for the large-scale production of both homologous and heterologous enzymes (Fawole and Odunfa, 2003; Wang et al., 2003a; Wang et al., 2003b). Among them, Aspergillus oryzae and Aspergillus niger are on the Generally Recognized as Safe (GRAS) list of the Food and Drug Administration (FDA) in the United States (Tailor and Richardson, 1979). A. fumigatus is the most common infectious cause of human mortality and a major allergen (Estey, 2001; Martino, 2002). 2.2.1 Description of Aspergillus niger Colonies On Czapek dox agar, colonies of A. Niger consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heads (Ellis, 2006). Conidial heads are large (up to 3 mm x 15-20 µm in diameter), globose, dark brown, becoming radiate and tending to split into several loose columns with age. Conidiophores are smooth-walled, hyaline or turning dark towards the vesicle. Conidial 29

heads are biseriate with the phialides borne on brown, often septate metulae. Conidia are globose to subglobose (3.5-5.0 µm in diameter), dark brown to black and roughwalled.

Fig. 2.2: Culture of Aspergillus niger. (Ellis, 2006)

Fig. 2.3: Conidial head of A. niger. (Ellis,

2006)

2.2.2 Clinical Significance of A. niger Aspergillus niger is one of the most common and easily identifiable species of the genus Aspergillus, with its white to yellow mat later bearing black conidia. This is the third most common species associated with invasive pulmonary aspergillosis and is also often a causative agent of aspergilloma and is the most frequently encountered agent of otomycosis (Ellis, 2006). 2.2.3 Genomic of A. niger Genomics involves the ‘‘determination and use of genome sequences of organisms to identify genes and non-coding but potentially functionally important regions of the genome’’ (Bennett et al., 2001; Hofmann et al., 2003). The genome size of most 30

filamentous fungi is estimated to be 30–40 Mb, encoding 9000—13,000 genes (Machida, 2002). Molecular and genetic studies of Aspergillus species tend to concentrate on four thoroughly investigated species: Aspergillus nidulans, A. niger, A. oryzae, and A. fumigatus and other closely related species. By using the Bacterial Artificial Chromosome (BAC) technique, the genome of a derivative of the enzyme-producing strain of A. niger NRRL 3122 (ATCC 22343, CBS 115989) was sequenced. The project was completed in 2001 by the Gene Alliance (Geleen, Netherlands, and Hilden, Germany), a division of DSM (Amsterdam, Netherlands). The genome size is 35.9 Mb, containing 14,097 predicted genes (Archer and Dyer, 2004). A draft of the wildtype ATCC strain 9029 was sequenced by Integrated Genomics. The Pacific Northwest National Laboratory has purchased the sequence, and it is available to researchers upon request. Genencor has access to the A. niger genome sequence data of Integrated Genomics (Machida, 2002). The Joint Genome Institute (JGI) initiated a sequencing program for A. niger ATCC strain 1015, a citric acid producer, in December 2004 as part of the United States Department of Energy Genome Program, with participation of the Pacific Northwest National Laboratory and Oakridge National Laboratory. 2.2.4 Physiology of A. Niger 2.2.4.1 The Life Cycle of Aspergillus Niger Most Aspergillus species, except A. fumigatus, have both sexual and asexual processes for spore duplication. Some, such as A. nidulans, are also able to reproduce via a parasexual cycle. The asexual cycle is the primary means for cell dispersion and protects the fungal genome in unfavorable conditions, and secondary metabolite production is frequently 31

associated with these developmental processes. Sporulation produces conidia, containing the haploid, uninucleated asexual spores. Vegetative growth is initiated by germination of the spore, with formation of tubular hyphae, growing in a polar fashion by apical extension and branching to form a network of mycelium, which acquires nutrients from the environment. Conidiation involves many common developmental themes, including temporal and spatial regulation of gene expression, cell specialization, and intercellular communication. The asexual developmental pathway in Aspergillus spcies is very well characterized at the molecular level (Adams, 2002; Andrianopoulos, 1994; Guest et al., 2004; Prade, 1993). The parasexual cycle as a recombination process during mitosis offers the genetic benefits of meiosis through a mitotic pathway (Pontecorvo, 1953). It provides a physiological vehicle to increase the production of different metabolites and products of Aspergillus, including citric acid (Sarangbin et al., 1994) and recombinant proteins (Bodie et al., 1994) and for generation of robust strains to resist toxic products (Antier et al., 1993; Kirimura et al., 1992). The technique of parasexual analysis can be used to map gene orders and assign new genes to the haploid chromosome. The sexual cycle is initiated from differentiation of aggregations of vegetative mycelia to hulle cells or promodia, which further develops in to cleistothecia, which contain asci. Ascospores are produced by meiosis. Some genes related to sexual reproduction have been cloned. The gene veA was identified as a negative regulator of sexual development, and the gene product VeA is a positive activator of sexual development (Kim et al., 2002). Asexual and sexual reproduction can coexist in Aspergillus. Offspring from both pathways have the same genotype (Bruggeman et al., 2003) 32

2.2.4.2 Physiological Responses and the Signal Transduction of A. niger The capacities of microbial species to survive and respond physiologically to changes in their environment enable these species to exist under a broad range of conditions. This property is also important in exploiting these organisms in industrial processes and in gaining perspectives into the relationships between culture conditions, cell growth, and productivity. Aspergillus species can respond to a variety of changes including pH, osmotic, and oxidative stresses. Signal transduction systems mediate environmental change and developmental processes of the cell such as reproductive cycles and structure differentiation, thus producing metabolites related to these physiological conditions (Calvo et al., 2002). Gene regulation in response to ambient pH ensures the production of proteins and metabolites appropriate to the prevailing environmental pH; for example, acid and alkaline phosphatases are produced at the corresponding pHs. The products of the palA, B, C, F, H, and I constitute a signaling pathway which, under alkaline conditions, results in proteolytic conversion of the three-zinc-finger-wide domain transcription factor PacC (pacC) to its functional form. It binds to the DNA consensus sequence 50-GCCARG-30, activates the expression of alkaline-expressed genes, and represses acid-expressed genes (Vautard-Mey et al., 2003). 2.2.4.3 Carbon and Nitrogen Metabolism in A. niger Aspergillus uses a wide variety of substrates for growth, and can switch between several different biochemical pathways for the assimilation of these various substrates (Hintz et al., 1995). Processes for production of the pathway enzymes are under genetic regulation.

33

Carbon and nitrogen catabolism are subject to catabolite repression (Dzikowska et al., 2003). Aspergillus likely has an extensive array of uptake systems associated with regulatory mechanisms. There is significant research on citrate and succinate production related to modeling of flux networks and stochiometry. The presence of the preferred C and N-substrates glucose and ammonia will suppress the production of enzymes for utilizing other resources. Genomic, biochemical, and physiological information were used to identify the central carbon metabolism in A. niger and to reconstruct the metabolic network, and established a stoichiometric model involving 284 metabolites and 335 reactions (David et al., 2003). The reactions found to be essential for growth on different carbon sources involved the major metabolic pathways, including the TCA cycle and oxidative phosphorylation (for all carbon sources), the pentose phosphate shunt (for pentoses), gluconeogenesis (for glycerol and acetate), and the glyoxylate pathway (for acetate). 2.2.5 Industrial Applications of Aspergillus Niger i.

Citric Acid Production:

Overproducing mutant cultures of A. niger capable of fermenting corn and potato starches have been developed (Haq et al., 2003a) and investigated for citric acid production using by-products of sugar industries (Haq et al., 2003b). Strain improvement programs focusing on some of the aforementioned metabolic steps and the participating

enzymes

include

mutation

and

molecular

genetics

strategies.

Overexpression of phosphofructokinase (PFK) by A. niger will lead to a high citric acid accumulation.

34

ii.

Extracellular Enzymes Production:

Due to their high capacity for producing and secreting extracellular enzymes, Aspergilli play an important role in production of industrial enzymes (de Vries et al., 1999a, b; Lockington et al., 2002). Aspergillus species are also important microorganisms in the fermented food industry and produce a variety of amylases and proteases (MacKenzie et al., 2000; Petersen et al., 1999). Aspergillus species, especially GRAS-designated strains, produce and secrete a variety of industrial enzymes including a-amylases, glucoamylases, cellulases, pectinases, xylanases and other hemicellulases, and proteases. Enzymes degrading cellulose, hemicellulose, pectin, and other plant polysaccharides are typically complexes of a variety of enzymes having different substrate specificities with respect to the nature of the substrates and linkages they attack and also the locations of these linkages (endo- or exo-) within polysaccharides. Genomic data from DSM related to A. niger indicates that only a fraction of the enzymes secreted by that organism have thus far been characterized (Martens et al., 2005). For example, database mining of the A. niger genome resulted in the identification of 12 new starch-modifying enzymes (Yuan et al., 2005). iii.

Biotransformations:

Biotransformations exploit the versatility and high reaction rates achievable with enzymes under mild conditions to catalyse reactions that are highly region- and stereospecific (Ward, 1991). This high selectivity is particularly useful in implementing synthesis or modification of complex chemical structures into bioactive molecules or their

precursors.

Aspergillus

species

have

been

employed

extensively

for

implementation of specific steroil hydroxylations, oxidations, hydrolysis, esterification, 35

isomerisations, and racemic resolutions. In addition, Aspergillus species have been employed in biotransformations of alicyclic insecticides, aromatic and phenoxy herbicides, organophosphorus and other pesticides, cyclic hydrocarbons, terpenes, and alkaloids. iv.

Environmental Applications of A. niger

Biosorption of heavy metals by A. niger biomass has been investigated by different laboratories. The effect of copper (II), lead (II), and chromium (VI) ions on the growth and bioaccumulation properties of A. niger was studied as a function of initial pH and metal ion concentration (Dursun, 2003; Dursun et al., 2003). Although fungal growth is affected by the metal ions, copper (II) and lead (II) can be effectively removed with a maximum specific uptake capacity of 15.6 and 34.4 mg/ g biomass, respectively. Optimum pH for biosorption of copper (II) and lead (II) from aqueous solutions by A. niger is 5.0 and 4.0, respectively (Dursun, 2003). Biosorption processes are gaining considerable interest in replacing or supplementing the conventional dye wastewater treatment processes. A. niger biomass immobilized in a polysulphone matrix in the form of spherical beads can effectively remove Acid Blue 29, Basic Blue 9, Congo Red, and Disperse Red 1 from aqueous solutions (Fu and Viraraghavan, 2003). v.

Other Industrial Products of A. niger

Production of gluconic acid and oxalic acid by A. niger has been reported (Liu et al., 2003; Rymowicz and Lenart, 2003). A mutant strain of A. niger could produce 68 g/l of oxalic acid in a 7-days fermentation using crude rapeseed oil and post-refining fatty acids (Rymowicz and Lenart, 2003). When production of gluconic acid was tested under submerged semisolid surface and solid-state surface fermentation, highest level of gluconic acid (106 g/l dm_3) with 94.7% yield under solid-state fermentation condition 36

(Singh et al., 2003). Single-cell oil production (Neelagund et al., 2003; Singh, 1992) from A. niger, A. nidulans and Aspergillus sydowii and single-cell protein production from A. niger (Kuhad et al., 1997; Singh et al., 1991) have been reported, but neither is commercially viable. 2.3

Biology Of Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris

The bean plants belong to the genus Vigna savi, (Willis, 1985) and the family Leguminosae-papilionoidae and the tribe Phaseoleae which is made up of about 80-100 species that are tropical especially in Africa and Asia (Mbagwu and Edeoga, 2006). Genus Vigna includes more than 80 species that are grouped into 6 subgenera Vigna, Ceratotropis, Plectotropis, Sigmoidotropis, Lasiosporon, and Haydonia. The subgenus Macrorynchus that was previously placed in genus Vigna was transferred to genus Wajira recently (Thulin et al. 2004). Subgenus Vigna or African Vigna comprises of 39 species (Maxted et al. 2004) and includes 2 agriculturally important species viz. Vigna unguiculata (cowpea) and Vigna subterranea (Bambara groundnut). 2.3.1 Botanical Study of the Three Plants (Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris) Cowpea (Vigna unquiculata L. Walp) is a diploid species (2n = 2x = 22) (Diaga, 2011) and a dicotyledonous leguminous food crops (Ogbemudia et al., 2010). It belongs to the kingdom Plantae, division (Magnoliophyta), class (Magnollopsida), order (Fabales), family Fabaceae formally (Leguminiseae), Sub-family (Faboideae), genus (Vigna) and species (unguiculata) (Ogbemudia et al., 2010). Vigna unguiculata has 11 subspecies that includes ssp. unguiculata having the cultivated forms (var. unguiculata) and wild forms (var. spontanea) and 10 wild perennial subspecies (Pasquet 1999; Maxted et al., 2004). The perennial subspecies have been grouped into 5 that are allogamous viz. ssp. baoulensis, ssp. burundiensis,

37

ssp. letouzeyi, ssp. aduensis, and ssp. pawekiae, and 5 autogamous viz. ssp. dekindtiana, ssp. stenophylla, ssp. tenuis, ssp. alba, and ssp. pubescens. The cultivated forms of V. unguiculata are further divided into 5 groups: Unguiculata, Biflora, Sesquipedalis, Textilis, and Melanophthalmus (Pasquet 2000; Maxted et al., 2004). Vigna racemosa, with many other bean plants are wild under- exploited Vigna spp. and are not cultivated (Carnovale et al., 1996). The wild bean (Vigna racemosa) belongs to the kingdom Plantae, order (Fabales), family Fabaceae formally (Leguminiseae), Subfamily (Faboideae), genus (Vigna) and species (racemosa). Vigna racemosa plant is a blue-flowered species with a chromosome number of 2n=22 (Maréchal et al., 1978). Palynological and anatomical studies on the roots, histochemical studies (Mbagwu and Edeoga, 2006) and Pollen Grain Micro Sculpturing and its Systematic Application (Wael and Taha Kasem, 2009) were carried out on Vigna racemosa and many other wild vigna spp.. in order to classify them and show their relationship. Very few studies have been done to reveal the nutritional aspect of Vigna racemosa seeds. The proximate composition and the antinutritional factor content of Vigna racemosa were unveiled by Carnovale et al., (1996) which found that Vigna racemosa accessions contain about (223- 244g/kg) of protein, (53- 63HA) of lectins, (6.178.60g/kg) of phytic acid and (11.54- 13.21g/kg) of tannins. Common bean usually refers to food legumes of the genus Phaseolus, family Leguminosae, subfamily Papilionoideae, tribe Phaseoleae, and subtribe Phaseolinae. The genus Phaseolus contains some 50 wild-growing species distributed only in the Americas (Asian Phaseolus have been reclassified as Vigna) (Gepts, 2001). The genus also contains five domesticated species: in decreasing order of importance, common bean (Phaseolus vulgaris L.), lima bean (P. lunatus L.), runner bean (Phaseolus coccineus L.), tepary bean (P. acutifolius A. Gray), and year bean (P.

38

polyanthus Greenman), with distinct adaptations and reproductive systems: mesic and temperate, predominantly self-pollinated; warm and humid, predominantly selfpollinated; hot and dry, cleistogamous; cool and humid, outcrossing; and cool and humid, outcrossing, respectively. Lima bean is phylogenetically more distant from the other domesticated species, which are sibling species and constitute a syngameon. The principal species economically and scientifically is common bean and is originated in Latin America where its wild progenitor (P. vulgaris var. mexicanus and var. aborigineus) has a wide distribution ranging from northern Mexico to northwestern Argentina (Gepts, 2001). All species of the genus are diploid and most have 22 chromosomes (2n = 2 x = 22) and a few species show an aneuploid reduction to 20 chromosomes. The genome of common bean is one of the smallest in the legume family at 625 Mbp per haploid genome (Gepts, 2001). 2.3.2 Chemical Composition of The Three Types of Beans Seeds The chemical composition of cowpea is similar to that of most edible legumes which ranges from 19.97 – 25.4g/100g for protein, 0.87 – 3.5g/100 for fat, 60.8 – 66.4g/100g for carbohydrates, 5.0 – 6.9g/100g for the crude fibre. The ash content ranges from 3.1 – 3.9g/100g and the moisture 8.31 – 10.64g/100g. The vitamin contents are 0.41 – 0.99mg/100g thiamine, 0.29 – 0.76mg/100g riboflavin, 2.15 – 3.23mg/100g niacin (Elias et al., 1964; Ritchey et al., 1976; IAR Report, 1994). Thus most of its nutritional value is provided by proteins and carbohydrates (Bressani, 1985). The seed protein can be separated into 4 differents types – albumin, globulins, prolamins and glutelins. The predominant and most characteristic type of protein found in legumes (peas and beans) is the globulins which constitute about 80g/100g of the total proteins. The globulin type can be further fractionated into legumin and vicilin (Milner, 1974).

39

Figure 2.4: Vigna racemosa plant growing in the bush. Culled from www.westafricanplants.senckenberg.de

PlateI: Dried pods of Vigna racemosa

PlateII: Vigna racemosa plant growing on a

fence

40

Figure 2.5: Phaseolus vulgaris plant. Culled from http: davesgarden.com/members

Figure 2.6: Vigna unguiculata plant growing in a farm. Culled from www.google.com/images

41

However Prolamins and glutelins constitute the bulk of the proteins of most cereals. Since prolamins contain only small amount of lysine, the poor quality of many cereals is often attributed to their prolamin content (Kakade, 1974). The lysine content of cowpea is relatively high, making it an excellent impower of the protein quality of cereal grains (Bressani et al., 1973). However, cowpea proteins as in the case of other legumes is deficient in sulphur – containing amino acids particularly methionine (Onayemi and Potter, 1976). Variability in protein content from 19.79 – 25.4g/100g was influenced by genotype as well as by environmental factors (Bliss, 1975). The total carbohydrate content in seeds of legumes (cowpea) varies from 50.66 to 68g/100g, with starch contributing as much as 32 – 48g/100g. Total soluble sugars content is from 13.75 to 19.75g/100g, amylase content ranges from 20.88 – 48.72g/100g while amylopectin varies from 11.42 to 36.58g/100g (Srinivasa, 1976; Ritchey et al., 1976; Arora and Das, 1976; Reddy et al., 1984). Sucrose content ranged from 0.36 – 1.4g/100g, raffinose content ranged from 1.26 – 4.12g/100g while stachyose content wass from 1.21 – 4.98g/100g (Onigbinde and Akinyele, 1983) Verbascose content was about 0.9g/100g average, while xylose was 1.67mg/g, fructose about 0.72mg/g average. Galactose, glucose and maltose are in trace amounts (Phillip and Abbey, 1989). The chemical evaluation of wild under-exploited vigna spp. seeds carried out by Canovale et al., (1996) shown that the protein content of vigna racemosa seeds ranges from 223 to 244g/kg for the accessions used; the trypsin inhibitors content ranges from 53 to 63TIU/mg protein; the lectin content ranges from 2000 to 5500g/ml; the phytic acid content ranges from 6.17 to 8.60g/kg and the tannin content ranges from 11.54 to 13.21g/kg. In the same study, it has been shown that vigna racemosa as many other wild vigna spp. has a high suphur amino acids content which is better than the cultivated

42

vigna spp. The chemical composition of the vigna racemosa seeds is not yet well documented. Dry common bean (Phaseolus vulgaris L) is a legume widely consumed throughout the world and it is reconigzed as the major source of dietary protein in many LatinAmerican and African countries. A large variability exists in common bean seeds; color and size are two important quality characteristics for the consumers. Seed size and weight depend on genetic variations, cultivar and environmental conditions (González de Mejía et al., 2005). The seed color of beans is determined by the presence and concentration

of

flavonol

glycosides,

anthocyanins,

and

condensed

tannins

(proanthocyanidins) (Beninger and Hosfield, 2003; Aparicio-Fernández et al., 2005). Recently, common bean is gaining increasing attention as a functional or nutraceutical food, due to its rich variety of phytochemicals with potential health benefits such as fiber, polyphenolic compounds, lectins, unsaturated fatty acids, trypsin inhibitors, phytic acid, among others (Guzmán-Maldonado and Paredes-López, 1998). 2.3.3 Utilizations of the Three Plants (Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris) In tropical Africa cowpeas are primarily used in the form of dry seed cooked as a pulse in a large variety of dishes (Aykrod and Doughty, 1982). Preference is for brown, white or cream seeds with a small eye and wrinkled or rough seed coat (Williams, 1974). In households, in Nigeria, they are cooked alone, with yam, maize or rice; steamed as in moin-moin; fried as in bean cake (akara) or made into bean soup “gbegiri” (Aykrod and Doughty, 1982; Williams, 1984). In many areas of West and East Africa, the tender leaves are cooked like spinach or as a relish. Generally the grower also likes to obtain some dry-seed yields from the plucked crop. Green beans or cut green pods used as a 43

vegetable are of secondary importance. Cowpeas are also grown for fodder, ground cover or green manure but to a much lesser extent than for pulse. In Asia, the pulse uses of cowpeas are important primarily in the drier regions such as India where increasing amount of the crop is used as “dhal” (split decorticated grains) (Aykroyd and Doughty, 1982). In the more humid areas of south east and southern China, the long bean is the predominant form and is used as cut green beans. The plant tissues of wild vigna spp. are used for human consumption (seeds and tubers), for animal feed (leaves, pods) and for their medicinal properties (roots) (Padulosi and Ng, 1990). Most Vigna savi species such as V. unguiculata, V. radiata and V. racemosa are called the poor- man's meat owing to their uses as a primary protein source (Chopra and Swamy, 1975). Common bean is the most important legume worldwide for direct human consumption and the crop is consumed principally for its dry (mature) beans, shell beans (seeds at physiological maturity),and green pods (Gepts, 2001). When consumed as seed, beans constitute an important source of dietary protein (22% of seed weight) that complements cereals for over half a billion people mainly in Latin America. Annual production of dry beans is around 15 million tonnes and average yield is700 kg/ ha, although yields in certain countries reach 2000±3000 kg/ ha. The largest producers of dry beans are Brazil, Mexico, China, and the USA. Annual production of green beans is around 4.5 million tonnes, with the largest production around the Mediterranean and in the USA. Common bean was used to derive important principles in genetics. Mendel used beans to confirm his results derived in peas. Johannsen used beans to illustrate the quantitative nature of the inheritance of certain traits such as seed weight. Sax established the basic

44

methodology to identify quantitative trait loci (for seed weight) via co-segregation with Mendelian markers (Seed color and color pattern). 2.4

Constraints Limiting Beans Utilization

2.4.1 Protein Quality One of the primary constraints militating against cowpea is the protein quality. Cowpea protein as in the case of other legume proteins is deficient in sulphur containing amino acids with a level of 1.2g/100g and 1.8g/100g protein for methionine and cysteine respectively. The primary deficiency of methonine and cysteine in many instances is further complicated by a secondary deficiency of tryptophan (Evans and Bandemer, 1967). This makes cowpea protein inferior when compared to protein from animal source. However the protein quality can be improved by supplementation with methionine (Onayemi and Potter, 1976). Cowpea is very rich in lysine and the amino acid profiles complement those of the cereal proteins to give a high protein quality (Bressani et al., 1974). 2.4.2 Physical Constraints The processing of cowpea for preparation of bean products like moin-moin, bean cake (akara), bean soup (gbegiri) poses some problems. The seed coat has to be removed before it can be ground into paste. This means steeping the cowpea seed in water to loosen the seed coat or divising other means for removing the coat. Observation have shown that removing cowpea seed coat can be tedious and time consuming especially for those who use cowpea to prepare dishes on a large scale. For instance, it take as long as 90 minutes soaking time to remove the seed coat of a variety of cowpea (named 45

Sampea 6 : 1696) (IAR Report, 1994). Therefore the ease with which the coat can be removed is an important factor. Cowpeas require some cooking before they are eaten. This thermal process provides tenderization of the cotyledon which increase product palatability, digestibility and inactivates endogenous toxic factors that would markedly limit the final nutritional value (Sefa-Dede et al., 1978; Uebersax and Ruengsakulrach, 1989). Investigations showed that cooking time which is usually long varies for different varieties. Another physical constraint associated with cowpea consumption is the storage induced hard-tocook defect. This term indicates a resistance of seeds to softening during cooking. This phenomenon is normally associated with certain legumes stored under high temperature and high humidity. The hard-to-cook seeds require longer cooking time than soft seeds and decrease the nutritional quality (Tuan and Pillips, 1991). 2.4.3 Anti-nutritional Factors (Biological Constraints) of Beans Like other legumes, cowpea can synthesize a variety of undesiderable substances termed antinutrients that are known to exert deleterious effect when ingested by man and animals. These include: (a)

Trypsin Inhibitors

These are naturally occurring proteinase inhibitors capable of combining with trypsin and other serine proteinase to form an inactive complex. The trypsin inhibitor – trypsin complex are almost certainly held by secondary rather than primary bonds (Laskowski and Laskowski, 1954). According to Richardson, (1977) trypsin inhibitors are proteins which have the peculiar property of forming reversible stoichiometric protein-protein complex with various proteolytic enzymes thus bringing about competitive inhibition of their catalytic function. 46

These plant proteinase inhibitors are generally small proteins having molecular weight under 50,000 and more commonly less than 20,000 (Vogel, et al., 1966). A number of inhibitors from several legumes have minimum molecular weights of below 10,000 and are often present as dimmers or tetramers (Kakade and Simons, 1970). Nearly all plant inhibitors inhibit enzymes of animal or microbial origins having trypsin like - or chymotrypsin – like specificities. Trypsin inhibitor is an important serine proteinase inhibitor of the digestive tract of animals. It soon became apparent however that many of the so-called trypsin inhibitors were also inhibitory to the related enzyme chymotrypsin (Vogel et al., 1966). In some cases the reactive site of inhibitor was the same for both enzymes but several of the other inhibitors were demonstrated to be “double-headed” or polyvalent, that is, containing different reactive site for the independent inhibitor of the two proteolytic enzymes. Sometimes the trypsin and chymotrypsin inhibitors are strictly specific for these two enzymes but other examples have subsequently been shown to inhibit a range of other serine proteinase (elastase, thrombin, plasmin and kallikrein) (Bidlingmayer et al., 1972; Richardson, 1977). Recently a proteinase inhibitor of trypsin and chymotrypsin was isolated from potato tuber (Solanum tuberosum L.). The molecule consists of one polypeptide chain with molecular weight of 17 kilo Dalton. The inhibitor interacts with trypsin or chymotrypsin in 1:1 molar ratio and the enzyme-inhibitor complex undergoes substrate – dependent dissociation. The inhibitor does not suppress the activity of subtilisin and pancreatic elastase. The formation of “triple” complex containing one trypsin molecule and one chymotrypsin molecule indicates that the inhibitor has two independent active centres for these enzymes (Revina et al., 1996). The physiological functions of trypsin

47

inhibitors include: functioning as a regulatory agents in controlling endogenous proteinase and being as a storage proteins (Ryan, 1973), serving as a plant protetion (Green and Ryan, 1972), having therapeutic application (Richardson, 1977; Silverstone, 1985), and finally having colostrum protection effects (Laskowski and Laskowski, 1954). The exact nutritional significance of trypsin inhibitor is difficult to assess (Pusztai, 1967). If the diet of rats and chicks contain high levels of these compounds, the protein in the diet is poorly utilized and tissue growth and repaires are impaired. This is due to either the limited availability of amino acid methionine or to the inactivation of proteases (Rachis and Gumbman, 1981). The inactivation of proteases resulted in excessive enzyme secretion and pancreatic hypertrophy (Bender, 1987). Metabolic disturbance in cysteine and methionine utilization was also suggested (Pusztai, 1967). The nutritional value of many leguminous seeds has been reported to improve during germination (Collins and Sanders, 1976) and in most cases following controlled heat treatment (Liener and Kakade, 1969; Ogun et al., 1989). Both processes inactivate and reduce the levels of these inhibitors. The level of trypsin inhibitor in cowpea is intermediate with an activity ranging from 16.5 -35.3 Tiu/mg sample (Elkowicz and Sosulski, 1982; Nnanna and Phillips, 1990). Ogu et al., (1989) observed a reduction from an average of 27.6 to 10.7 Tiu/mg sample when 4 cultivars of cowpea were soaked in hot water. (b)

Phytohaemagglutinins

Phytochaemagglutinins are proteins or glycoproteins which can bind to specific carbohydrate residue in cell membrane and capable of agglutinating red blood cells (Lis and Sharon, 1973; Goldstein and Hayes, 1978). The fact that these protein fractions 48

were capable of agglutinating red cells gave them the name haemagglutinins. It was also established that relative haemagglutinins activities of various seed extracts were quite different when tested with erythrocytes from different animals. This specificity toward specific types of blood cells led to coining the word lectin (Latin, legere – to choose). The action of lectin is due to the binding to intestinal mucosal cells causing malfunction, disruption and lesion in the small intestine. These actions interfere with the absorption of nutrient from the guts (Liener, 1974; Jaffe, 1980). In vitro experiment with intestinal loops taken from rats fed this lectin revealed 50% decrease in the rate of absorption of glucose across the intestinal walls compared to controls (Liener, 1974). Ricin, the lectin of castor bean is extremely toxic, about 100 times more toxic than most of the other bean lectins (Jaffe, 1969). Concanavalin A, the lectin from Jack bean injected into animals caused the agglutination of red blood cells, followed by hemolysis and finally death (Edmundson et al., 1971). However, the level in cowpea was observed to be low (Elkowicz and Sosulki, 1982). Since the toxicity of these compounds is dramatically reduced by cooking with moist heat, their presence in human diets is of little cause for concern. Toxicity is also reduced because many toxic compounds are destroyed or neutralized in a normal digestive tract and most are poorly absorbed. Poor absorption means that lectins reach the colon in a biological intact form and thus can have a beneficial effect. They appear to protect the human body against colon cancer either by causing hyper-secretion of intestinal mucus or by exerting a direct toxic effect on tumor cells (Liener, 1980). (c)

Tannins

The term tannins referred originally to substances with the ability to tan leather (Haslam, 1979). It is now generally used to include any naturally occurring soluble 49

compound of high molecular weight (500 – 3,000) and containing a large number of phenolic hydroxyl groups (one to two hundred molecular weight), to enable it to form an effective cross-link with proteins and other molecules (Bate-Smith and Swain, 1962; Kumar and Singh, 1984). The hydrolysable and condensed tannins are two groups of the compound widely distributed in the plant kingdom which are differentiated by their structure and reactivity towards hydrolytic agents (Haslam, 1966). However, the principal forage tannins are usually of a condensed type (Mcleaod, 1974). The presence of condensed tannins has been demonstrated in legumes and pasture species (Burns et al., 1967). In most legumes and cereals, the seed coat or outer portions contain fairly high amount of tannins (Rao and Prabhavathi, 1982). Tannins react with protein to form protein-tannin complexes (Haslam, 1981). Proteins which strongly associate with tannins are rich in the amino acid proline, relatively large and hydrophobic, posses conformationally open and flexible structure (Geneviena et al., 1994). Tannins have a harsh astringent taste and produce in the palate a feeling of roughness, dryness and constriction. The primary reaction whereby astringency develops is via precipitation of proteins and mucopolysaccharides in the mucous secretion (Goldstern and Swain, 1963; Geneviena et al., 1994). The deleterious effect of dietary tannins on the feeding of the insects, larvae and animals on tannin rich plants is quantitatively defensive. Tannins repel predators by virtue of their strong astringent taste and because of their anti-nutritional characterisatics once ingested. Their role is elegantly summarized as plant chemical defense (Bate-Smith, 1973). Unpalatability due to astringent tannins leads to reduction in the voluntary food intake in ruminants (Mcleod, 50

1974). Tannins also diminish the permeability of the gut wall by reacting with the outer cellular layer of the gut, so reducing absorption. The reduced digestibility of the tannin rich feeds can be explained on the basis of inhibiting digestive enzymes (Bressani and Elias, 1979; Griffith and Mosley, 1980). In the body, they bind proteins, precipitate the proteins of the epithelium, penetrate through the superficial cell and cause liver damage (Mehansho et al., 1987). A 3-5% solution of tannin is toxic orally and retards growth. Tannin inhibits virtually every digestive enzyme and reduces the bioavailability of iron and vitamin B12 (Liener, 1980; Butler, 1989). Tannins are both mutagenic and carcinogenic (Stolz, 1982). Cowpea are fairly high in tannins (Laurena et al., 1987) Ogun et al., (1989) reported a range of 0.08 – 0.15g/100g dry weight for four cultivars of cowpeas. Polyphenols (condensed tannins) are heat stable and are located mainly in the seed coat of cowpeas and other legumes. The level of tannins in cowpeas can be significantly reduced by decortications (Desphande et al., 1982) and a complete elimination was observed when the seed coat of cowpeas was removed (Ogun et al., 1089). (d)

Phytates

Phytates represent a complex class of naturally occurring compounds that can significantly influence the function and nutritional properties of food (Maga, 1982). The compound phytic acid can be commonly called myo-inositol hexaphosphoric acid or scientifically 1, 2, 3, 4, 5, 6 – hexakis (dihydrogen phosphate) myo-inositol (IUPAC1UB, 1968). The term phytin implies a calcium – magnesium salt of phytic acid. The phytate ion complexes with di - and trivalent metallic ions (Zn2+, Ca2+, Mg2+, Fe2+, and Fe3+) and is the principal storage form of phosphorus in many kinds of seeds.

51

The ability of phytic acid to complex with metals is one of the main nutritional concerns associated with phytates. Phytic acid and their derivatives can bind essential dietary minerals, thus making them insoluble and biologically unavailable or only partially available for consumption. This problem becomes especially important when two or more cations are present, in that a synergistic binding effect can occur. This is particularly true for zinc and calcium and also zinc and copper. The greatest impact of phytates relative to human nutrition is its reduction in zinc bioavailability (Newkirk, and Classen, 2001). The physiological roles of phytates in seeds have been suggested to include, serving as a phosphorus store (Hall and Hodges, 1966), an energy store (Biswas and Biswas, 1965) and an activator of dormancy (Sobolev and Rodionova, 1966). Phytate serve as source of phosphate, cations and myo-inositol during germination due to the action of phytase (Mayer, 1956); Darbre and Noris, 1957; Williams, 1970). The dry matured beans and cowpeas contain approximately 1g/100g dry weight (Makower, 1969; Ogun et al., 1989) and more than 90% of the phytic acid in beans is soluble in water. Proper processing or preliminary soaking, particularly when the soaking water is changed can eliminate the action of phytates considerably (Ogun et al., 1989). 2.4.4 The Presence of Flatulence Factors in Legumes Flatulence is one of the constraints limiting the wider acceptability and utilization of legume seeds in humans. This phenomenon has been attributed mainly to the presence in legume of alpha-galacto-oligosaccharides of the raffinose family (French, 1954; Gross, 1962). The alpha-galactoligosaccharides of the raffinose family constitute of sugars related to raffinose by the fact of having one or more alpha-D-galactopyranosyl group in their structures. These D-galactosyl groups are found in nature joined to sugars 52

such as D-glucose, to sucrose, to certain polysaccharides and to a few non-sugars such as glycerol and inositol (French, 1954). These members include mainly raffinose, stachyose, verbascose and ajugose. The structural relationship shows that the group increase from lower to the highest series by the successive addition of one alpha-Dgalactopyranosyl group to sucrose (Fig. 2.2). According to Hardinge et al., (1965) soybean has the highest levels of raffinose (1.9g/100g) and stachyose (5.2g/100g) while chickpea (Cicer arietium) and Pigeon peas (Cajanus cajan) have the highest levels of verbascose (4.2g/100g) for 10 investigated legume seeds. About 0.48g/100g of highest oligosaccharide which was confirmed to be ajugose is contained in some Nambyquarea variety of groundnuts (Arachis hypogea) (Tharanathan et al., 1976). The presence of ajugose has also been reported in horsebeans (Vicia faba), smooth peas (Pisum sativum), wrinkled peas (Pisum sativum) and lupines (Lupinus species) (Cerning-Beroard and Filiatre, 1976). In their own investigation, Onigbinde and Akinyele, (1983) reported the levels of raffinose to range from 1.26 – 4.19g/100g, and for stachyose to range from 1.21 – 4.98g/100g for 20 varieties of cowpeas used for their study. Phillip and Abbey, (1989) reported an average value of 3.76mg/g for verbascose in cowpeas.

53

Figure 2.7: Structural relationship of the raffinose family sugars.

54

CHAPTER 3 MATERIALS AND METHODS 3.1

Materials

3.1.1 Collection And preparation Of Samples Vigna racemosa seeds were harvested around Zaria city, a city located in Kaduna state (Northern Nigeria). The matured fruits were harvested with their pods, sun-dried and the seeds were removed from the pods, threshed and winnowed, then free from broken seeds, dust, and other foreign materials to obtain clean seeds. Matured Vigna unguiculata and Bambara nuts (Vigna subterranean) seeds were purchased from local farmers in Zaria, Kaduna State, Nigeria and matured Phaseous vulgaris (black bean) from local farmers in Ngaoundere, Adamaoua region, Cameroon. Those seeds were taken to the laboratory of Bichemistry Department, Ahmadu Bello University, Zaria, Nigeria where they were picked clean of all debris and broken seeds. The plants were identified at the Herbarium of the Department of Biological Science, Ahmadu Bello University-Zaria, Nigeria where the following voucher numbers 6903, 1461, and 2008 were assigned to Vigna racemosa, Vigna unguiculata and Phaseolus vulgaris respectively. The seeds were then stored in a plastic container at room temperature (27o – 30oC) for subsequent analysis. .

55

(a)

Vigna unguiculata seeds

(b)

Vigna racemosa seeds

56

(c) Phaseolus vulgaris seeds Plate III (a, b, c): matured dried seeds of Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris

(a) Cream coloured seeds coat bambaranuts

57

(b) Red coloured seeds coat bambaranuts

( c) Black coloured seed coat bambaranuts Plate IV (a, b, c): Mature Bambara nuts (Vigna subterranean) of three different color coats (cream, red and black). 3.1.2 Chemicals All chemicals and reagents were of analytical grade, they include: Chloroform, acetic acid, aniline, acetone, silica gel, petrolium ether, sulphuric acid, sodium acetate, Folin Ciocalteu reagent (1 N): Commercially available Folin Ciocalteu reagent (2N) was 58

diluted with an equal volume of distilled water, sodium carbonate (20%), standard tannic acid solution (0.5mg/ml) etc. 3.2

Methods

3.2.1 Open Fermentation Raw beans were washed with distilled water and dried in an oven at 55oC for 24h. After drying, bean samples were grinded in a laboratory bench mill (Thomas-WILEY, Laboratory mill, Model 4, Arthur H. Thomas Company, Philadelphia, PA., U.S.A.) and sieved, and the 1 mm fraction were collected. The bean flour was suspended in distilled water at 300 g/l concentration as found to be the optimal concentration for fermentation by Dablado et al., (2002). The suspension was allowed to ferment naturally with the microorganisms present in the seeds and in the surrounding atmosphere for 48 hours. After the fermentation, the microbial growth was terminated by drying at 55o C in oven for 24 h (Fadahunsi, 2009) and re-ground using the laboratory bench mill. 3.2.2 Controlled Fermentation About 250 g of each type of ground bean was weighed into 500ml flat bottom flask and autoclaved at 121oC for 15min. Moisture content of the samples was adjusted to 25% before aseptic inoculation with spore suspension of Aspergillus niger, containing 1.064 x 107 spores/25 g of flour (Bhat et al., 1997), and incubated at room temperature (29 ± 3oC) for 48 h. After the fermentation, the fungal growth was terminated by drying at 55 o C in oven for 24 h (Fadahunsi, 2009) and re-ground using kitchen blender. 3.2.2.1

Isolation of Aspergillus Niger from Bambaranuts

Cleaned and sorted Bambara nuts were intermittently moistened and allowed to grow mouldy. Aspergillus niger was isolated from the mouldy Bambara seeds by standard 59

procedures as adopted by Pang and Ibrahim (2004) and identified according to the method of Ellis (2006). The isolation of fungi was carried out on potato dextrose agar (PDA) using three varieties of bambaranuts based on the color of their seeds coats (black, red and cream). One g of each of the samples was suspended in 10 ml of sterile distilled water and shaken vigorously for 10 min. Later, 1.0 ml of the resulting liquid was spread on the surface of PDA using an L-shaped glass rod and incubated at 37ºC for 5-7 days. The fungal isolates formed were subcultured to purity and examined for phytase and tannase activities. The identification of Aspergillus niger was made by the simple microscopic method of Ellis (2006). It was observed that the isolate possessed distinct conidiophores terminated by a swollen vesicle bearing flask-shaped phialides. The spores showed black coloration and were produced in long chains from the ends of the phialides. Growth on Czapek dox agar showed that colonies consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heeds (Ellis, 2006) 3.2.2.2

Selection of Simultaneous Tannin and Phytate Degrading

Aspergillus Niger Isolate Screening for simultaneous tannin and phytic acid degrading Aspergillus niger isolates was carried out by simple agar plate method; an enrichment culture technique in minimum medium as adopted by Gustavo et al. (2001). The selection medium (g/l); NaNO3-3.00, KH2PO4-1.00, MgSO4. 7H2O-0.50, FeSO4. 7H2O-0.01, agar- 30.0 was autoclaved at 121oC for 15 min and supplemented with 1% phytic acid (10ml) and 1% tannic (10ml) previously filter-sterilized through Whatman filter paper No1 with PH- 4.0 adjusted using 100 mM NaOH. Strains of Aspergillus niger isolated were then point 60

inoculated and incubated at 30oC for 96 h and the diameters (mm) of the colonies were measured at 24 h intervals. 3.2.2.3

Preparation of Inoculum

The Aspergillus niger spore inoculum was prepared from the isolate that exhibited highest tannin and phytate utilization potential, by adding 10 mL of sterile distilled water containing 2.5% Tween 80 (polyoxyethlene sorbitan monoolate) to a fully sporulated slant culture. The spores were dislodged by vigorous shaking and spores number

estimated

by

direct

microscopic

enumeration

using

cell-counting

hemocytometer (Neubauer chamber; Merck, S.A., Madrid, Spain). The volume of spores’ suspension was adjusted to 1.064 x107 spores/ml and the harvested Aspergillus niger spores were centrifuged at 2500 rpm for 2 min, washed in sterile distilled water and re-centrifuged. The washed cells were then used as inoculums singly in the fermentation of the three species of beans (Dapiya et al., 2010). 3.2.2.4

Enzyme Extraction

After incubation, acetate buffer (pH 5.0, 0.02 M) was added, and the flasks were shaken at 200 rpm for 1h. The solution was filtered and centrifuged at 10,070 g for 30 min at 4 0 C (Centrifuge Beckman J2-21, Beckman-Coulter, Inc., Fullerton, CA, USA). The supernatant was assayed for phytase and tannase activity. The material that was retained on the filter was dried in an oven and used for proximate, antinutrients, mineral and flatulent factors analysis. 3.2.2.5 Enzymatic Activity Assays Phytase and tannase activities were evaluated according to Lee et al. (2005) and Mondal et al. (2001), using sodium phytate and tannic acid as substrates, respectively. One unit 61

of enzymatic activity was defined as the amount of enzyme needed to release 1µM of phosphate per min when evaluating phytase and the amount of enzyme needed to release 1 µM/min of residual tannic acid when evaluating tannase. 3.2.3 Determination of Proximate Composition 3.2.3.1

Ash Content Determination

The term ash refers to the residue left after combustion of the oven dried sample and is a measure of the total mineral content. Determination of the ash content was done according to (AOAC, 1990). Three different crucibles preheated in a muffle furnace at about 525 oC. Each crucible was cooled in a desiccotor and weighed. Approximately 1.0g of each sample was weighed into different crucible. The crucible and their contents were transferred into a muffle furnace set at 525oC and allowed to stay for 1 hour. The weights of crucible and content were taken and recorded. The percentage ash was calculated using the expression below

3.2.3.2 Determination of Moisture The method employed for the determination of moisture content in the sample based on the measurement of the loss in weight due to drying at a temperature of about 105oC as describe by, AOAC, 1980). A watch glass was washed and dried in an oven at about 105oC, it was cooled and weighed empty.

62

About 2.0g of sample was weighed into a clean watch glass. The watch glass and its content were dried in an air circulated oven at about 105oC to constant weight. The watch glass and its content was cooled in a desiccators and reweighed. The percentage moisture was obtained using the expression

3.2.3.3 Determination of Crude Lipid Content The lipid content was similarly determined by the procedure described in AOAC (1990). A clean dry round bottom flask containing antibumping granules was used. Exactly 210cm3 of petroleum ether (60 – 80oC) was poured into the flask fitted with Soxhlet extraction unit. The weighed sample was transferred into the thimble, which was already fixed into the Soxhlet extraction unit. Cold water was put on to circulate. The heating mantle was switched on and heating rate adjusted until the solvent was refluxed at a steady rate. Extraction was carried out for eight hours. The sample was removed and dried to constant weight in an oven and reweighed.

Where the weight of lipid extracted was given to be the loss in weight of the sample after extraction and drying with oven. 3.2.3.4

Determination of Crude Fibre

Crude fibre was determined by the method of AOAC 1990. Two grams of each type of ground beans flour sample was placed in a round bottom flask. 100ml of 0.25M H2SO4

63

was added and the mixture boiled under reflux for 30minutes. The insoluble matter was washed several times with hot water until it is acid free. Thereafter, it was transferred into a flask containing 100ml of hot 0.312M NaOH solution. The mixture was boiled again under reflux for 30 minutes and filtered under suction; the insoluble residue was washed with hot water until it is base free. It was dried to constant weight in an oven at 1000 cooled in a decicator and incinerated in a furnace at 5500C for 2 hours. It was put off and allowed to cool down. It was removed cooled in a desiccators and weighed (C3). The crude fibre was calculated as loss in weighed on ashing. Weight of the original sample = W

3.2.3.5

Determination of Percentage Carbohydrate

The percentage carbohydrate was obtained by difference Percentage carbohydrate = 100 – (% moisture + %protein + %fat + %ash) 3.2.3.6

Determination of Nitrogen Content And Crude Protein

The kjeldahl method of AOAC, (1990), was used. a. Principle It is based on the principle that proteins are major compounds containing nitrogen, they include amino acids, purines, ammonium salts, and vitamins B1. Nitrogen is used as an index of the protein termed crude protein as distinct from true protein.

64

b. Steps for Determination A. Mineralization steps of organic substance in boiling sulphuric acid. 2H2SO4

2SO2

+ 2H2O + O2

COOH 2R

CH

+

O2

2 CO2

+ 2H2O

+ 2 NH3

NH2 2NH3

+

H2SO4

(NH4)2SO4

B. Distillation steps of ammonium sulphate after alkalization of the boric acid solution (NH4)2SO4

+

NaOH

2Na2SO4

NH3 + H2O H3BO3

+

2H2O

+ 2NH3

NH4OH

+ NH4OH

(NH4)3BO3

+

3H2O

C. Titration of ammonium with hydrochloric acid of standardized concentration (NH4)3BO3 + HCl

3NH4Cl

+

H3BO3

c. Procedure Exactly 20g of the sample was weighed into 100ml Kjeldahl flask and a few antibumpimg granules were added. One gram of the mixed catalyst (CuSO4 and K2SO4 in the ratio 8:1 respectively) and 15ml of concentrated sulphuric acid was added. The flask was placed on a Kjeldahl digestion rack and heated until a clear solution was

65

obtained. At the end of digestion the flask was cooled and the sample was quantitatively transferred to 100ml volumetric flask and made up to the mark with distilled water. 10ml of the digest was pipette into Markham semi micro nitrogen steel tube. Ten milliliters (10ml) of 40% NaOH solution was added cautiously. The sample was steam distilled liberating ammonia into a 100ml conical flask containing 10ml of 4% boric acid and a drop of methyl blue indicator until colour changed from pink to green. Thirty milliliters (30ml) volume of sample was collected. The content of the conical flask was titrated with 0.1M HCL. The end point was indicated by a colour change from green to pink and the volume (v) of the acid for each distillate was noted. Percentage nitrogen per sample was calculated using the expression below

Where M = Molarity of HCL 14 = atomic weight of Nitrogen 100 = total volume of digest 100 = % conversion 10 = volume of digest taken 1000 = to convert to liter The crude protein was calculated as % crude = 6.25× %N 6.25 is the conversion factor since it is assumed that protein contains 16% nitrogen. 66

3.2.4 Determination of Mineral Content The following minerals: magnesium, calcium, zinc, iron, potassium, and sodium were determined using atomic absorption spectrophotometry as described by AOAC, (1990). Two grams of the sample was digested with 23ml of a mixture of HNO3 (SG. 1.57) H2SO4 (SG. 1.84) and HCIO4 (7:8:8 by volume). Then the cooled digest was diluted to 50ml with distilled water, filtered through a Whatman 45 filter paper and made up to 100ml with distilled H2O in a glass volumetric flask. This final solution was for atomic absorption spectrophotometry. 3.2.5 Determination of Antinutritional Factors 3.2.5.1

Determination of Cyanide

The cyanide content was determined according to the method of AOAC (1984). Two grams of the sample was weighed into a flask and 100ml of distilled water added to it and allowed to hydrolyse for 1hr. 10ml of 2.5% NaOH was measured and carefully poured into the sample holder. The soxhlet apparatus was set up and was distilled into the sample holder containing the 2.5% NaOH until about 70ml was collected. It was carefully transferred to a 100ml volumetric flask and the sample holder rinsed with distilled water successively and also poured into volumetric flask. It was made up to the mark. Twenty-five milliliters (25ml) of the distillate was pipetted into a conical flask, 2ml of 6M NH4OH was added and 0.5ml of 10% KI solution was added, it was titrated with 0.02M AgNO3 to a first turbid colour (1ml of 0.02 M AgNO3 = 1.08 mg cyanide).

67

3.2.5.2

Determination Of Tannins

i. Principle: The tannin content was estimated spectrophotometrically by Folin-Denis method. The method is based on oxidation of the molecules containing a phenolic hydroxyl group. The tannin and tannin- like compounds reduce phosphotungungustomolybdic acid in alkaline solution to produce a highly coloured blue solution; the intensity of which is proportional to the amount of tannin and can be estimated against standard tannic acid solution at wavelength of 725 nm. ii.

Sample Preparation And Extraction Of Tannins:

The sample was dried at 55+1°C and ground to pass through a sieve of 1mm diameter. Tannins extraction was done using 400mg ground sample in conical flask with 40 ml diethyl ether containing 1% acetic acid (V/V) and mixed to remove the pigment material. Carefully discarded the supernatant after 5 minute and 20 ml of 70% aqueous acetone was added and sealed the flask with cotton plug covering with aluminum foil and kept in electrical shaker for 2 hours for extraction. Then it was filtered through Whatman filter paper No. 1 and sample was kept in refrigerator at 4°C until analysis. iii.

Standard Calibration Curve Preparation:

From the stock solution of tannic acid (0.5 mg/mI) using 0. 10, 20, 30, 40 and 50 µL in test tubes and volume was made to 1.0 ml. It gives a tannic acid concentration of 0, 5, 10, 15, 20 and 25µg respectively. Then 0.5 ml Folin reagent and 2.5 ml 20% sodium carbonate was added and the whole content was mixed properly and after 40 minutes, absorbance reading was taken at 725nm in spectrophotometer.

68

iv.

Method:

Tannin was estimated according to the procedure of Makkar et al., (1993). Fifty microlitres of tannins extract for each sample was taken in test tube and the volume made up to 1 .0 ml with distilled water. Then, 0.5 ml Folin Ciocalteu reagent was added and mixed properly. Then 2.5 ml 20% sodium carbonate solution added mixed and kept for 40 minutes at room temperature. Optical density was taken at 725 nm in spectrophotometer and concentration was estimated from the standard curve. v.

Calculation: %

.Where: An = absorbance of test sample As = absorbance of standard tannic acid C = concentration of standard tannic acid (mg/ml) Df= dilution factor =Vex/ Va W = weight of test sample (mg) Vex = total volume of extract; Va= volume of extract analyzed 3.2.5.3

Determination of Phytic Acid

The phytic acid was determined using the procedure described by Lucas and Markakas (1975).

69

This entails the weighing of 2.0g each of raw and processed Beans flour samples into 250 mLs conical flask. 100 ml of 2% hydrochloric acid was used to soak each sample in the conical flask for 3 hrs and then filtered through a double layer of hardened filter papers. Fifty mililitres of each filtrate was placed in 250 mLs beaker and 107 ml of distilled water added in each case to give proper acidity. Ten mililitres of 0.3% Ammonium thiocyanate solution was added into each solution as indicator and titrated with standard iron chloride solution, which contained 1.95mg iron per ml. The end point was slightly brownish- yellow color persisting for 5 min. The percentage phytic acid was calculated using the formula:

Where: Y = titre value x 1 .95mg. 3.2.5.4

Determinition of Saponin

Saponins were determined by the gravimetric method of Brik et al., (1963), as modified by Hudson and El-Defrawi (1979). Exactly 2g of dry ground sample was weighed into a thimble and transferred into the Soxhlet extraction chamber fitted with a condenser and a round flat bottomed flask. Some quantity of acetone enough to cause a reflux was poured into the flask for 3 hours by heating the flask on a hot plate and the solvent distilled off. This was the first extraction. Then enough methanol was added to the extract. The saponin was then exhaustively extracted for three hours by heating the flask on a hot plate after which the final and initial weights of the saponin was noted.

70

3.2.5.5

Determination of Trypsin Inhibitor Activity

This was done using the spectrophotometric method, described by Amtfield et al., (1985). A measured weight (10g) of the test sample was dispersed in 50ml of 0.5 M Nacl solution and stirred for 30 min at room temperature. It was centrifuged and the supernatant filtered through Whatman No 42 filter paper. The filtrate was used for the assay. Standard trypsin was prepared and used to treat the substrate solution (N-

- benzoyl -

Dl – arginine – p – anilide; BAPA). The extent of inhibition was used as a standard for measuring the trypsin inhibitory activity of the test sample extract. Into a test tube containing 2ml of extract and 10ml of the substrate (BAPA) 2ml of the standard trypsin solution was added. Also 2ml of the standard trypsin solution was added in another test tube containing only 10ml of the substrate. The latter served as the blank. The content of the tubes were allowed to stand for 30min and then the absorbances of the solution measured spectrophotometrically at 410nm wavelength. One trypsin activity unit inhibited is given by an increase of 0.01 absorbance unit at 410nm.

Where Au = Absorbance of test sample As = Absorbance of standard (uninhibited) sample

71

F = Experimental factor given as F= Vf / Va x 1/ W Vf = Total volume of extract Va = Volume of extract analysed W = Weight of sample analysed 3.2.5.6

Determination of Lectins

The hemagglutinin content was determined using the spectrophotometric method of Onwuka (2005). One gram of each sample was dispersed in a 10ml normal saline solution buffered at pH 6.4 with a 0.01M phosphate buffer solution. The solution was allowed to stand at room temperature for 30min and then centrifuge to obtain the extract. To 0.1ml of the extract diluents in a test tube, 1ml of trypsinized rabbit blood was added. Control with the test tube containing only the blood cells was mounted and both tubes were allowed to stand 4 hours at room temperature. 1ml of normal saline was added to all the test tubes and allowed to stand for 10min after which the absorbance is read at 620nm. The test tube containing only the blood cells and normal saline serves as blank. The result is expressed as haemagglutinin units per milligram of the sample. Hemagglutinin unit/mg = (b-a) x F 72

Where b = absorbance of test sample solution a = absorbance of the blank control F = experimental factor given by: F = (1/W x Vf/Va) D Where W = weight of sample Vf = total volume of extract Va = volume of extract used in the assay D = dilution factor (if any) The dilution factor D = 1ml to 10ml and 0.1ml out of the 10ml. That is 10x 10/ 0.1 = 100ml. 3.2.5.7

Determination of Alkaloids

The gravimetric method of Horbone (1980) was adopted. Five grams of each sample were dispersed into 50ml of 10% acetic acid solution in ethanol. The mixture was shaken well and allowed to stand for 4 hours before filtering. The filtrate was evaporated to one quarter (1/4) of its original volume. Drop wise conc. NH4OH was added to precipitate the alkaloids. The precipitate was filtered off with a weighed filter paper. The precipitate was dried on the filter paper in the oven at 690 C for 30min and reweighed.

73

By weight difference, the weight of alkaloids determined and expressed by:

Where W2 = weight of the precipitate before drying W1 = weight of the precipitate after drying W = weight of the sample 3.2.5.8

Determination of Oxalate

Oxalate was determined by using of Oke (1969). The total oxalic acid of the powdered samples was determined by weighing 2g into a 250ml flask. Then 190 ml distilled water and 10 ml of 6M hydrochloric acid were added. The mixture was warmed for 1hour on boiling water bath, cooled, transferred into a 250 ml volumetric flask, diluted to volume and filtered. Four drops of methyl red indicator were added followed by concentrated ammonia till the solution turned faint yellow. It was then heated to 100oC, allowed to cool and filtered to remove precipitate containing ferrous ions. The filtrate was boiled and 10 ml of 5% calcium chloride was added with constant stirring. It was then allowed to stand overnight. The mixture was filtered through Whatman No 4 filter paper. Then the precipitate was washed several times with distilled water and transferred to a beaker and 5 ml of 25% sulphuric acid was added to dissolve the precipitate. The resultant solution was maintained at 80oC and titrated against 0.5% potassium permanganate until the pink color persisted for approximately one minute.

74

A blank was also run for the test sample. From the amount of potassium permanganate used, the oxalate content of the unknown sample sample was calculated using the equation below: 1 ml potassium permanganate = 2.24mg oxalatee 3.2.6 Determination of Flatulence Factors Flatulence causing oligosaccharides (mainly stachyose and raffinose) were evaluated as described by Onyenekwe et al., (1999). 3.2.6.1

Defatting of Flour

About 20g of the dried milled flour was wrapped in a whatman filter paper, fastened tightly with wire to prevent leakage of flour and placed in an extraction thimble (30 x 100cm). The thimble was placed in a soxhlet extractor containing petroleum (bp. 40 to 60oC) and the oil extracted for 6hrs (the bean cake flour was extracted for 24hrs). The wrapped flour was then removed and dried to a constant weight in an oven at 60 oC to remove residual petroleum spirit (Hymowitz et al., 1972). 3.2.6.2

Extraction of Oligosaccharides

Five grams of samples were heated (60oC) with 50ml of 80% ethanol for 1 hour on a hot plate with magnetic stirring. After centrifugation (5000 G), residues were extracted twice using 20ml of ethanol and centrifuged each time at 5000 G for 10 minutes. Extracts (supernatants) were combined. Five ml of 10% lead acetate was used to precipitate non-carbohydrate compounds (proteins) from the extract and centrifuged for 10 minutes at 5000G. The excess lead was removed from the extrat by adding 0.2g sodium carbonate. After centrifuging at 5000G for 10 minutes, the volume was

75

concentrated to 35ml. This is a slight modification of the method used by Borejszo and Khan, (1992). 3.2.6.3

Separation of Oligosaccharides by Thin Layer Chromatography

(Qualitative Analysis). Forty grams of gel (Silica gel) and 95ml of distilled water were manually shaken in a glass stoppered flask for 1min. The slurry was transferred to a spreader and a layer of 0.5mm thick was deposited on clean glass plates. The plates were allowed to dry overnight at room temperature, and then were used without further preparation, except for scoring into lanes 3cm in width. However, if the plates are not used overnight and stored, the plates are activated by heating in an oven for 30 min at 110oC before use (Stahl and Kaltenbach, 1965).This is a slight modification of the method described by Onyenekwe et al., (1999). Thirty microlitres of sugar extract were applied to the plates using a micro pipette (Cole-parmar, Chicago) Application were made in small increments (less than 1µL) under a stream of warm air from a heat gun to facilitate rapid drying and thus minimize spot diffusion. Ascending chromatography was conducted in closed glass tank lined with Whatman No. 4 paper saturated with the developing solvent. (The solvent system was chloroformacetic acid – water (3.0; 3.5; 0.5v/v). The solvent front was run to a distance of 15cm from the origin. This is a modification of the De Stafanis and Ponte, (1968) procedure to facilitate the proper separation of the sugars. After a run, the plates were dried with the acid in an oven and were spread with the detecting reagent. 76

The spotting agent containing diphenylamine was used. The reagent was made up by dissolving 1g di[henyllamine and 1ml aniline in 100ml of acetone; Prior to spraying, 10ml of the acetone solution was mixed with 1ml 85% phosphoric acid. The sprayed plates were heated for 10 min at 130oC. The sugars appeared as dark grey or brown spots (Stahl and Kaltenbach, 1965). 3.2.6.4

Quantitative Analysis of Sugars Using Phenol-Sulphuric Acid

Method For quantitation, the kieselgel area (2 x 2cm) corresponding to each oligosaccharide (Raffinose and stachyose) spot was scraped from unsprayed duplicate plates and eluted with 3ml of distilled water for 2 hrs and filtered to give the sugar solution. One ml of sugar solutions was pipette into a series of test tubes of internal diameter between 16 and 20 mm and 1ml of 5% phenol was added to each tube. Then 5ml of conc. H2SO4 was added rapidly, the stream of acid being directed against the surface rather than the side of the tubes in order to obtain good mixing. The tubes were allowed to stand for 10 min, and then they were shaken and placed for 20 min in a water bath at 28oC before reading were taken. The measured at 490nm. Blanks were prepared by substituting 1ml of the eluent of (2 x 2cm) area of the gel that does not contain sugar for that containing sugar. 

Preparation of Standard Curve for the Determination of the Sugars

A standard solution of 100μg/ml of raffinose, and 100μg/ml of stachyose were used for the preparation of the standard curves. The standard solutions were serially diluted in a series of test tubes to give concentrations of 6.25, 12.50, 25.00, 50.0 and 75.0μg/ml. The colour was developed 77

using phenol-sulphuric acid assay method. In the blank 1ml of sugar was substituted with 1ml of distilled water. The absorbance at 490nm was read off using a spectrophotometer. The data obtained were used to plot the separate standard curved for the determination of raffinose and stachyose (Appendix I and II). 

Calculation of concentration using the standard curve

Thirty microlitres of the sugar extracts were spotted, area (2x2cm) corresponding to each oligosaccharide spot was scraped from the unsprayed duplicate plate and eluted with 3ml of distilled water. One milliliter of the filtrate was used for the analysis which is equivalent to the oligosaccharide contained in 10ul of the extract. If the absorbance of the oligosaccharide in 10μl of extract gave a concentration say Y μg from the standard curve. 0.01ml of extract gave a concentration Yμg ..35ml of extract will give a concentration of g = 3500Y μg 5g of the sample contains 3500Y μg

..100g of sample will contain = 70,000Y μg% = 0.07Y g/100g

78

CHAPTER 4 RESULTS 4.1

Isolation, Identification and Screening of Aspergillus Niger from Bambara

Nuts 4.1.1 Isolation Different colonies (isolates) were observed on the PDA plats that were spread with the liquid from three types of Bambara nuts (Plate V).

Plate V: Aspergillus niger isolated from Bambara nuts of different color seed coats 4.1.2

Identification

The black isolate was identified based on the structure morphologies as observed under the light microscope. It was observed that the isolate possessed distinct conidiophores terminated by a swollen vesicle bearing flask-shaped phialides. The spores showed

79

black coloration and were produced in long chains from the ends of the phialides. Growth on Czapek dox agar showed that colonies consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heeds (Ellis, 2006). Based on these characteristics, the black colored isolate was identified to be Aspergillus niger. 4.1.3

Screening

The Aspergillus niger isolate from the three type of bambara nuts were capable of exhibiting simultaneous phytase and tannase activities on the agar plate supplemented with 1% tannic acid and sodium phytate and the diameters of the clear zones (colony) are noted in fig.4.1. The isolate from red colored seed coat of Bambaranut showed the highest diameter of growth at each interval of 12 hrs. 4.2

Proximate Composition of Raw and Fermented Beans Flours.

4.2.1 Proximate Composition of Raw and Fermented Vigna ungulculata Flour The table 4.1 shows the proximate composition of raw and fermented Vigna ungulculata flour. It was observed that the protein content increased by more than 20% due to the two types of fermentation, while the lipid, ash, moisture, fiber and carbohydrate were decreasing.

80

White Bambara nut seeds Red Bambara nut Seeds Black Bambara nut Seeds

70 59.9

60

Colony Diameters (mm)

50.3

50

50 40.3 40 35

34.66

36

29

30

26 22

20

18

17

10 0

0

0

0 0

24

48

72

96

Incubation Time (hour)

Figure 4.1: Colony diameters of A. niger strain in tannic acid and sodium phytate agar plate isolated from Bambara nut of different seed coats

81

Table 4.1: The effect of Open and Controlled Fermentation on proximate composition of Vigna unguiculata*

Processing methods

%Protein

%Lipid

%Moisture

%Ash

%Fibre

%Carbohydrate

Raw

24.49± 0.58

9.5± 0.21

5.45± 0.10

4.49± 0.15

4.45± 0.029

51.77± 0.88

Open Fermented (OF)

30.58± 0.90

7.1± 0.12

4.61± 0.047

3.96± 0.017

3.68± 0.044

50.14± 0.71

%Change due to OF**

24.81± 1.3↑

25.18± 2.3↓

15.28± 2.8↓

11.49± 2.6↓

17.23± 0.6↓

3.14± 1.04↓

Controlled Fermented (CF)

29.82± 0.057

3.92± 0.044

6.09± 0.012

1.65± 0.029

3.17± 0.015

55.35± 0.087

%Change due to CF**

21.87± 2.68↑

58.71± 1.34↓

11.88±1.78↑

63.10± 1.87↓

28.68± 0.79↓

6.97±1.74↑

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase

82

4.2.2

Proximate Composition of Raw and Fermented Vigna Racemosa Flour

The table 4.2 shows the proximate composition of raw and fermented Vigna racemosa flour and the percentages of change due to the two types of fermentation. It was observed that the protein content increased by less than 20% as affected by open fermentation. However that protein content decreased by 29.42+0.1% during controlled fermentation. The controlled fermentation affected the lipid, moisture, ash and carbohydrate by more than 20%, while open fermentation affected these parameters by less than 20% apart from the moisture content (Table 4.2). 4.2.3

Proximate Composition of Raw and Fermented Phaseolus Vulgaris Flour

Table 4.3 shows the proximate composition of raw and fermented phaseolus vulgaris flour and the percentages of change due to the two types of fermentation. Less than 20% (19.07+2.37% and 9.86 + 0.81%) increase due to the two types of fermentation was observed. Lipid and moisture were affected by more than 20% during the two types of fermentation. 4.3

The Effec of Fermentation on the Anti-Nutritional Factors of the Three Types

of Beans Table 4.4, table 4.5 and table 4.6 shows the anti-nutritional factors content as well as the percentage s of changes due to open and controlled fermentation of Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris flours respectively. From the three tables it was observed that all the anti-nutrients studied here were reduced by the two types of fermentation on the three types of beans.

83

Table 4.2: The Effect of Open and Controlled Fermentation on Proximate Composition of Vigna racemosa*

Processing methods

%Protein

%Lipid

%Moisture

%Ash

%Fibre

%Carbohydrate

Raw

27.12± 0.44

7.4± 0.2

3.91± 0.067

2.77± 0.135

2.82± 0.039

55.88± 0.49

Open Fermented (OF)

30.49± 0.84

6.68± 0.25

2.79± 0.030

2.43± 0.090

2.3± 0.058

55.31± 0.73

%Change due to OF**

12.41± 1.73↑

9.76±1.62↓

28.67±1.82↓

12.28±1.12↓

18.56±1.02↓

1.02±0.44↓

Controlled Fermented (CF)

19.14± 0.40

2.75±0.017

4.75± 0.028

1.91± 0.030

2.80± 0.029

68.65± 0.38

%Change due to CF**

29.42± 0.10↓

62.77± 1.25↓

21.46± 2.09↑

30.79± 2.26↓

0.93± 0.41↓

22.88±0.87↑

*: Values are means ± standard error of the mean for triplicate samples **: ↓ means percentage decrease; ↑ means percentage increase.

84

Table 4.3: The Effect of Open and Controlled Fermentation on Proximate Composition of Phaseolus vulgaris*

Processing methods

%Protein

%Lipid

%Moisture

%Ash

%Fibre

%Carbohydrate

Raw

27.75±0.30

7.63± 0.16

3.29± 0.067

3.38± 0.055

3.2± 0.058

54.75± 0.43

Open Fermented (OF)

33.05± 0.97

6.003± 0.28

2.58± 0.034

3.08± 0.049

2.9± 0.029

52.3± 1.17

%Change due to OF**

19.07± 2.37↑

21.40± 2.02↓

21.66± 0.65↓

9.06± 0.50↓

9.33± 1.44↓

4.49± 1.44↓

Controlled Fermented (CF)

30.48± 0.22

2.53± 0.018

5.66± 0.047

2.65± 0.018

3.08± 0.044

55.59± 0.13

%Change due to CF**

9.86± 0.81↑

66.77±0.78↓

72.12± 2.07↑

21.73± 1.54↓

3.63± 0.46↓

1.55± 0.55↑

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increa

85

4.4: The Effect of Open and Controlled Fermentation on Antinutritional factors of Vigna unguiculata*

Processing methods

%Phytate

%Tannin

%Alkaloids

(× 10-5)

%Saponin

%Lectines

(× 10-5)

Hydrogen Cyanide (mg/100g)

(× 10-5)

Trypsin inhibitors (TIU/g)

%Oxalate (× 10-10)

0.187± 0.0033

8600 ± 8.82

0.77± 0.015

25000± 2900

0.013± 0.00058

213300± 3300

0.844± 0.0079

222 ± 1.2

Open Fermented (OF)

0.103± 0.0033

5420 ± 5.77

0.46± 0.0088

18300± 2000

0.009± 0.00033

140000± 5800

0.447± 0.0038

135 ± 0.577

%Change due to OF**

44.64± 1.52↓

36.78± 0.077↓

40.92± 1.22↓

26.56± 0.87↓

28.11± 1.68↓

34.42± 1.89↓

47.09± 0.61↓

39.28± 0.37↓

Controlled Fermented (CF)

0.0604± 0.0002

420 ± 15

0.18± 0.015

42 ± 1.45

0.004± 0.00015

12.3± 1.45

0.0028± 0.00015

59.7 ± 0.882

%Change due to CF**

67.59± 0.49↓

95.06± 0.17↓

77.21± 1.47↓

99.83± 0.0186↓

65.03± 1.45↓

99.99± 0.00071↓

99.67± 0.014↓

73.17± 0.29↓

Raw

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase. For tannin, saponin, lectins and oxalate, the %change for OF and CF are not multiplied by 10 -5 or 10-10.

86

Table 4.5: The Effect of Open and Controlled Fermentation on Antinutritional factors of Vigna racemosa*

Processing methods

%Phytate

%Tannin

%Alkaloids

%Saponin

(× 10-5)

%Lectines

(× 10-5)

Hydrogen Cyanide (mg/100g)

%Oxalate

(× 10-5)

Trypsin inhibitors (TIU/g)

(× 10-10)

0.306± 0.0037

19440 ± 5.77

1.898± 0.0042

86700± 3300

0.067± 0.00058

26000± 5800

1.21± 0.0079

472 ± 1

Open Fermented (OF)

0.203± 0.0033

14420 ± 5.77

1.85± 0.0058

53300± 2700

0.044± 0.00033

210000± 5800

0.93± 0.0059

193± 0.333

%Change due to OF**

33.49± 0.29↓

25.82 ± 0.052↓

2.53± 0.72↓

38.52± 0.98↓

33.82± 0.75↓

19.25± 0.43↓

23.66± 0.83↓

59.18± 0.16↓

Controlled Fermented (CF)

0.090± 0.00015

1500 ± 15

1.33± 0.044

64± 1.73

0.013± 8.82E -05

24± 2.03

0.0067± 0.00012

14.4± 0.0333

%Change due to CF**

70.40± 0.35↓

92.40± 0.073↓

29.76± 2.20↓

99.93± 0.0015↓

80.55± 0.046↓

99.99± 0.00098↓

99.44± 0.011↓

96.94± 0.00058↓

Raw

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase. For tannin, saponin, lectins and oxalate, the %change for OF and CF are not multiplied by 10 -5 or 10-10.

87

Table 4.6: The Effect of Open and Controlled Fermentation on Antinutritional factors of Phaseolus vulgaris*

Processing methods

%Phytate

%Tannin

%Alkaloids

%Saponin

(× 10-5)

%Lectines

(× 10-6)

Hydrogen Cyanide (mg/100g)

%Oxalate

(× 10-6)

Trypsin inhibitors (TIU/g)

(× 10-10)

0.233± 0.0033

12400± 8.82

1.31± 0.026

433000± 33000

0.04± 0.00058

2330000± 33000

1.07± 0.0059

575 ± 0.577

Open Fermented (OF)

0.139± 0.001

10400± 56

1.023± 0.0088

223000± 23000

0.029± 0.00058

1830000± 37000

0.077± 0.0050

158 ± 0.333

%Change due to OF**

40.39± 1.26↓

16.15± 0.50↓

21.84± 1.13↓

48.67± 1.33↓

27.49± 1.28↓

21.73± 0.51↓

92.77± 0.46↓

72.58± 0.083↓

Controlled Fermented (CF)

0.0603± 0.00012

440± 10

0.85± 0.029

187± 8.82

0.0024± 0.00012

120± 8.82

0.0024± 0.00015

58 ± 0.000

%Change due to CF**

74.16± 0.38↓

96.46± 0.083↓

35.09± 2.17↓

99.96± 0.0050↓

93.99± 0.38↓

99.99± 0.00038↓

99.78± 0.013↓

89.91± 0.010↓

Raw

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase. For tannin, saponin, lectins and oxalate, the %change for OF and CF are not multiplied by 10-5 , 10-6 or 10-10.

88

The highest percentage of reduction is 38.52+0.98 (saponin), 44.64+1.52 (phytate), and 92.77+0.46 (trypsin inhibitor) in Vigna unguiculata, Vigna racemosa and Phoseolus vulgaris respectively during open fermentation. During controlled fermentation, the highest percentages of reduction were 99.99+0.00098 (lectins), 99.99+0.00071 (lectins) and 99.99+0.00038 (lectins) in Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris respectively. 4.4

The Effect of Fermentation on the Mineral Content of the Three Types of

Beans The Fe, Ca, Na, Zn, Mg, and K content as well as their percentages of change after open and controlled Fermentation in Vigna unguiculata, Vigna racemosa and Phaseolus vulgaris are presented in table 4.7, 4.8 and 4.9 respectively. The six elements were reduced by fermentation. The highest percentage of reduction was observed in the calcium content (more than 90%) in the three types of beans by both open and controlled fermentation.

89

Table 4.7: The Effect of Open and Controlled Fermentation on the Mineral Content of Vigna unguiculata*

Processing Methods

Fe (ppm)

Ca (ppm)

Na (ppm)

Zn (ppm)

Mg (ppm)

K (ppm)

Raw

2.35± 0.00058

611.113± 0.0088

16.77± 0.015

0.69± 0.00058

20.08± 0.0058

106.61± 0.012

Open Fermented (OF)

2.26± 0.0018

11.19± 0.0012

5.73± 0.015

0.535± 0.0012

14.16± 0.0088

63.97± 0.012

%Change due to OF**

4.028± 0.071↓

98.169± 0.00022↓

65.85± 0.070↓

22.42± 0.24↓

29.50± 0.024↓

39.99± 0.0053↓

Controlled Fermented (CF)

1.61± 0.0015

9.196± 0.0029

7.90± 0.0015

0.53± 0.0015

12.43± 0.0012

13.50± 0.015

%Change due to CF**

31.60± 0.079↓

98.50± 0.00046↓

52.876± 0.035↓

23.28± 0.27↓

38.09± 0.016↓

87.33± 0.015↓

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase

90

Table 4.8: The Effect of Open and Controlled Fermentation on the Mineral Content of Vigna racemosa*

Processing Methods

Fe (ppm)

Ca (ppm)

Na (ppm)

Zn (ppm)

Mg (ppm)

K (ppm)

Raw

2.39± 0.0023

468.75± 0.0088

14.35± 0.0058

0.778± 0.00058

23.64± 0.0058

119.60± 0.015

Open Fermented (OF)

1.43± 0.0018

9.545± 0.0028

4.23± 0.015

0.563± 0.0012

15.67± 0.012

104.65± 0.012

%Change due to OF**

40.19± 0.048↓

97.96± 0.00064↓

70.55± 0.089↓

27.635± 0.130↓

33.70± 0.036↓

12.50± 0.021↓

Controlled Fermented (CF)

0.330± 0.00088

8.622± 0.0017

3.40± 0.0012

0.334± 0.0021

13.58± 0.0012

15.80± 0.015

%Change due to CF**

86.20± 0.045↓

98.161± 0.00039↓

76.31± 0.018↓

57.070± 0.260↓

42.55± 0.0091↓

86.79± 0.011↓

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase

91

Table 4.9: The Effect of Open and Controlled Fermentation on the Mineral content of Phaseolus vulgaris* Processing Methods

Fe (ppm)

Ca (ppm)

Na (ppm)

Zn (ppm)

Mg (ppm)

K (ppm)

Raw

2.488± 0.0018

370.407± 0.012

8.99± 0.0058

0.819± 0.00058

22.36± 0.0088

132.32± 0.012

Open Fermented (OF)

1.856± 0.0037

10.647± 0.0015

4.85± 0.023

0.435± 0.0012

17.68± 0.0058

66.08± 0.012

%Change due to OF**

25.398± 0.160↓

97.126± 0.00043↓

46.05± 0.24↓

46.89± 0.18↓

20.94± 0.057↓

50.06± 0.012↓

Controlled Fermented (CF)

1.31± 0.021

16.960± 0.0018

3.60± 0.0012

0.424± 0.0023

21.61± 0.0015

19.53± 0.017

%Change due to CF**

47.35± 0.840↓

95.421± 0.00033↓

59.95± 0.019↓

48.19± 0.32↓

3.38± 0.045↓

85.24± 0.014↓

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase

92

4.5

The Effect of Open and Controlled Fermentation on the Flatulence Factors of

the Three Types of Beans 4.5.1

Separation of the Oligosaccharides by Thin Layer Chromatography

Thin layer chromatography revealed the presence of raffinose, stachyose, two spots above raffinose (suspected to be of lower moleculer weight than raffinose) and two spots below stachyose which were suspected to be a higher molecular weight alpha-galactoside (verbascose and ajugose) in the ethanol extract of all the varieties of bean investigated. 4.5.2

The Effect of Open and Controlled Fermentation on the Raffinose and

Stachyose Content of the Three Types of Beans The raffinose and stachyose content of the three varieties of beans were all reduced by fermentation. The percentages of reduction are presented in table 4.10; table 4.11 and table 4.12. 4.6 The Enzyme Activity Assay The phytase and tannase activities are presented in table 4.13. The phytase activity using Vigna racemosa as the substrate after 48 hours of Aspergillus niger solid-state fermentation showed the highest activity (

),

While the tannase activityin Vigna unguiculata as substrate after the 48 hours of Aspergillus niger solid- state fermentation was the highest ( (Table 4.13).

93

)

Table 4.10: Effect of Open and Controlled Fermentation on Some flatulent factors of Vigna unguiculata*

Processing methods

Raffinose (g/100g)

Stachyose (g/100g)

Raw

2.035± 0.137

9.569± 0.87

Open Fermented (OF)

0.305± 0.021

3.872± 0.121

%Change due to OF**

84.81± 1.71↓

59.04± 2.84↓

Controlled Fermented (CF)

0.517± 0.010

0.059± 0.0048

%Change due to CF**

74.29± 2.11↓

99.37±0.049↓

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase

94

Table 4.11: Effect of Open and Controlled Fermentation on Some flatulent factors of Vigna racemosa*

Processing methods

Raffinose (g/100g)

Stachyose (g/100g)

Raw

1.85± 0.021

12.325± 0.80

Open Fermented (OF)

1.24± 0.069

4.30± 0.094

%Change due to OF**

33.11± 2.96↓

64.90± 1.70↓

Controlled Fermented (CF)

0.75± 0.043

1.519± 0.028

%Change due to CF**

59.47± 2.20↓

87.59± 0.62↓

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase

95

Table 4.12: Effect of Open and Controlled Fermentation on Some flatulent factors of Phaseolus vulgaris*

Processing methods

Raffinose (g/100g)

Stachyose (g/100g)

Raw

5.74± 0.36

9.692± 0.68

Open Fermented (OF)

0.152± 0.014

4.21± 0.24

%Change due to OF**

97.32± 0.38↓

56.45± 1.44↓

Controlled Fermented (CF)

1.062± 0.030

0.410± 0.017

%Change due to CF**

81.30±1.61↓

95.71± 0.43↓

*: Values are means ± standard error of the mean for triplicate samples. **: ↓ means percentage decrease; ↑ means percentage increase.

96

Table 4.13: Phytase and Tannase activity in the medium after 48hrs of Aspergilus niger solid- state fermentation* Substrate

Phytase activity (U)**

Tannase activity (U)**

Vigna unguiculata

b

b

Vigna racemosa

c

a

Phaseolus vulgaris

a

a

*: Values are mean ± standard error of mean (SEM) for three replicates and values with different superscripts are significantly different from each other at P < 0.05. Data were analysed using the One-way Analysis of Variance (ANOVA) and the Duncan multiple range test was used for determination of difference between means using the SPSS 17 software package. **: One unit of enzymatic activity was defined as the amount of enzyme needed to release 1µmol of phosphate per min when evaluating phytase and the amount of enzyme needed to release 1 µmol/min of residual tannic acid when evaluating tannase.

97

CHAPTER 5 DISCUSSION Different colonies (isolates) were observed on the Potato Dextrose Agar (PDA) plates that were spread with the liquid from the three types of Bambara nuts. The black-colored colonies were observed on the three Bambara nuts varieties, while red-colored colonies were observed on the cream- and red-colored seed coat Bambara nuts (Plate III). The green colonies were observed on the black-colored seed coat Bambara nuts. The black-colored colonies were identified as Aspergillus niger based on the structural morphologies as observed under the light microscope. It was observed that the isolate possessed distinct conidiophores terminated by a swollen vesicle bearing flask-shaped phialides (Pitt and Samson, 2000). The black-colored colonies consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heads (Ellis, 2006). The red and green color isolates that appeared on the PDA plates may be other Aspergillus sp. or other type of fungi or microorganism. Bradoo et al (1996) reported formation of a clear zone around the mycelium, suggesting tannase activity. However, the observation of this clear zone is difficult, therefore, measurement of colony diameter was applied since direct measurement of the colony diameter is a good indicator of the ability of tannic acid and phytic acid utilization as carbon sources which could be attributed to the tannase and phytase activities in the medium (Pinto et al., 2001). The Aspergillus niger isolated from the red-colored seed coat Bambara nuts showed the highest colony diameter in the selection medium and was

98

selected as the highest simultaneous producer of phytase and tannase (Fig. 4.1) and assayed for enzymatic activity by Solid State Fermentation (SSF). Vigna unguiculata and phaseolus vulgaris showed an increase in their protein content due to fermentation. It was observed that the protein content of Vigna unguiculata increased by more than 20% due to the two types of fermentation, while the lipid, ash, moisture, fiber and carbohydrate decreased. That increase could be due to the increase in the biomass brought about by the fermenting microorganisms. It could also be thought that the biomass present in Vigna unguiculuta is more than that of Phaseolus vulgaris by considering the percentage increase in the protein content. It has also been shown that the increase in the protein susceptibility to proteolytic enzymes is due to partial protein denaturaion (as a result pH change and other factors) during fermentation (CZarneka et al; 1998). Vigna racemosa showed a little increase (less than 20%) in the protein content during open fermentation which could also be due to the above mentioned reasons. However controlled fermentation decreased its protein content. This could be due to the metabolism of Aspergillus niger with respect to other compounds present in Vigna racemosa. Aspergilus niger might have produced some compounds capable of interfering with protein content of vigna racemosa. The lipid, carbohydrate, fibre, ash and moisture content were reduced in the three types of beans during the open and controlled fermentation which is consistent with earlier works (Martin-cabrejas et al, 2004, Granito et al, 2002). The reduction in these parameters could be due to the metabolism by the microorganisms in the fermentation medium. The increase in the moisture content during controlled fermentation could be due to the fact that 24 hours was not sufficient to ensure complete drying of the bear paste after fermentation. 99

Table 4.4, Table 4.5 and table 4.6 show that phytate, tannin, lectins, saponins, hydrogen cyanide, trypsin inhibitors and oxalate were all reduced by the two types of fermentation. The reduction of these complex and toxic molecules was attributed to degradation by microorganisms (Madeira et al., 2011). The higher percentages of reduction of anti-nutrtional factors observed in controlled fermentation could be attributed to the fact that the presence of more than one microorganism in open fermentation might have resulted in competition. An undesired microorganism is often the faster growing species and consumes the fermentation media components but does not give the desired product. The mineral elements evaluated in the present study (Fe, Ca, Na, Zn, K, Mg) were all reduced by the two types of fermentation (tables 4.7, 4.8, 4.9). Irrespective of leaching in fermentation water, mineral utilization could be taking place by microorganisms responsible for the 48 hours fermentation (Zamora and Fields, 1979). Moreover, reduction in ash content and minerals by leaching in soaking or cooking water has been revealed (Kazamas and Fields, 1981).The reduction in the mineral content during fermentation could also be attributed to the effect of concentration due to the increase in biomass. The level of raffinose and stachyose were reduced by the two types of fermentation in the three types of beans. In the domesticated beans, open fermentation reduced raffinose more than controlled fermentation, while it is the contrast in Vigna racemosa. However, the stachyose level was more reduced by controlled fermentation than by open fermentation. Since these oligosaccharides are fermented by intestinal bacteria (Granito et al; 2001), the

100

present finding is of great interest, suggesting that a simple processing method like fermentation can be employed in order to reduce flatulence-causing factors. Table 4.13 shows the phytase and tannase activity in the medium during the 48 hours of Aspergillus niger solid state fermentation in the three beans. The SSF was selected because of reports that the production of the enzymes increased by some folds in SSF when compared to liquid surface and submerged fermentations (Lekha and Lonsane, 1997). The assay of both enzymes using three bean flours show that phytase activity ranges between and

U while tannase activity

ranges between

and

U

(Table 4.13), which is consistent with the reports of Madeira et al. (2011), Roopesh et al. (2006) and Vassilev et al. (2007)

101

CHAPTER 6 SUMMARY, CONCLUSION AND RECOMMENDATIONS 6.1 Summary This work has revealed that: i.

The strain of Aspergillus niger isolated from red seed coats of bambara nuts has the highest potential of simultaneous production of phytase and tannase.

ii.

Open fermentation increased the protein content by 12.41% and 24.81% for Vigna racemosa and Vigna unguiculata respectively, while it generally decreased their antinutritional factors content by more than 15%. Open fermentation also reduced the mineral content and flatulent factors by more than 20% for all the beans used in this study.

iii.

Controlled Fermentation using Aspergillus niger as a starter, increased the protein content by 9.86% and 21.87% for domesticated beans (Phaseolus vulgaris and Vigna unguiculata), while it reduced the protein content of Vigna racemosa by 24.42%. The antinutrients were generally reduced by more than 25% for all the beans. In addition, the mineral content and the flatulence factors were all reduced by more than 20% and 50% respectively.

6.2 Conclusion In conclusion, fermentation is an efficient method for detoxifying tannins, phytates, alkaloids, saponins, hydrogen cyanide, trypsin inhibitors, lectins and oxalate in domesticated and wild beans even though it reduces relatively some of the nutrient elements of the beans. The present research work has also shown that controlled fermentation using Aspergillus niger as a starter is more efficient in detoxifying the above mentioned antinutrients in both wild and domesticated beans compared to open fermentation. 6.3 i.

Recommendations There is still need for further studies on the effect of fermentation on other antinutrients

present but not assessed in this work. ii.

Vigna racemosa as well as many other wild beans need to be maintained and preserved in order to find out their usages and add more varieties to the known edible beans . This can reduce the competition between humans and animals for beans protein.This can also maintain biodiversity and add potential food source alternative.

iii.

Other processing methods need to be employed on wild beans in general in order to

further study the effect of processing on the level of reduction of antinutrients. iv.

Studies such as the evaluation of the biological value (BV), the protein efficiency ratio (PER), the relative nutritional value (RNV) and the chronic toxicity studies should be done on this wild bean and result compared to the cultivated ones in order to ascertain its safety.

103

v.

Genetic engeneering methods can also be used to generate a microorganism that has a high potential of reducing antinutrients in this wild bean or to produce a safer variety of the wild bean.

104

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Appendix 1: Standard Curve for Raffinose Determination

0.7

0.6

y = 0.0029x + 0.0037 R² = 0.9985

Absorbance (nm)

0.5

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Linear (Series1)

0.2

0.1

0 0

50

100

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Concentration (µg/ml)

130

Appendix 2: Standard Curve for Stachyose Determination

0.8 y = 0.0036x - 0.0008 R² = 0.9985

0.7

Absorbance (nm)

0.6 0.5 0.4 Series1

0.3

Linear (Series1)

0.2 0.1 0 0

-0.1

50

100

150

200

250

Concentration (µg/ml)

131

Appendix 3: Standard Curve of Sodium Phosphate for Phytase Activity Assay

0.14 y = 6.7337x R² = 0.9725

Absorbance (nm)

0.12

0.1

0.08

0.06 Series1 Linear (Series1)

0.04

0.02

0 0

0.005

0.01

0.015

0.02

Concentration (g/ml) Appendix 3: Standard Curve of sodium Phosphate for Phytase Activity Assay

132