COMPARISON OF PHYTOCHEMICALS AND THE ...

COMPARISON OF PHYTOCHEMICALS AND THE EFFECT OF PROCESSING IN SOME SELECTED EDIBLE BEANS IN SRI LANKA

By

Keerthana Sivakumaran

Thesis submitted to the University of Sri Jayewardenepura as the partial requirement for the award of Degree of Bachelor of Science (Special) in Food Science and Technology in 2016 i

DECLARATION The work described in this thesis was carried out by me as the fulfillment requirement for the special degree in Food Science and Technology under the supervision of Dr. Jagath Wansapala, Head of the Department, Department of Food Science and Technology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Dr. Theja Herath, Principal Research Scientist, Food Technology Section, Industrial Technology Institute and a report on this has not been submitted in whole or in part to any university or any other institution for another degree. ………………………………….. Date

………………………………….. K. Sivakumaran AS72697/2011/2012 AS2012740

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We certify that the above statement made by the candidate is true and this thesis is suitable for the submission to the University of Sri Jayewardenepura for the purpose of evaluation. …………………………………..

…………………………………..

Dr. Jagath Wansapala,

Dr. H. M. Theja Herath,

(Internal Supervisor)

(External Supervisor)

Head of the Department,

Principal Research Scientist,

Department of Food Science & Technology,

Food Technology Section,

Faculty of Applied Sciences,

Industrial Technology Institute,

University of Sri Jayewardenepura,

Bauddhaloka Mawatha,

Gangodawila, Nugegoda,

Colombo 7,

Sri Lanka.

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ACKNOWLEDGEMENT Foremost gratitude to my supervisor, Dr. Jagath Wansapala, Head of the department, Department of Food Science and Technology, Faculty of Applied Sciences, University of Sri Jayewardenepura for the continuous support of my research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. And I extend my gratitude for his support and encouragement given to conduct research at Industrial Technology Institute from beginning to end. Also my deepest gratitude goes to my external supervisor, Dr. H. M. Theja Herath, Principal Research Scientist, Food Technology Section, Industrial Technology Institute (ITI) for her invaluable assistance, encouragement and kind supervision. My sincere thanks also goes to Dr. Illmi Hewajulige, the Head of the Food Technology Section, Industrial Technology Institute for giving me the valid opportunity to carry out my research at industrial technology institute. Then my heartfelt thanks goes to all the members of academic staff of Department of Food Science and Technology, University of Sri Jayewardenepura for their encouragement & guidance to complete this research project successfully. Also I would like to offer my heartiest gratitude to Dr. Duminda Kuruppuarachchi Department of Decision Sciences, Faculty of Management Studies and Commerce, university of Sri Jayewardenepura for his valuable guidance and help to analyze my research data statistically. Eleven varieties of legumes were provided with the assistance of identification and selection from Grain legumes and oil crop research development centre -GLORDC, Agunakolapelessa by Mr. R. A. Ashoka Ranathunga, Research officer, Food Science & Technology Division, Grain Legumes and Oil Crops Research & Development Center, Angunakolapelessa is highly appreciated. Further financial support given to the ITI through Contract Project (PG 15/101) is highly acknowledged to my best. Then I greatly appreciate the guidance, encouragement and kind assistance of all the staff members of Food Technology Section, Industrial Technology Institute (ITI) and I very much grateful to Miss. Madara Samaranayake, Research scientist of ITI for given her kind and continuous support to carry out chemical analysis. Then my heartfelt thanks goes to my loving colleagues and friends who I met at ITI, for helping me in various ways to carry out my research successfully. My honorable mention goes to my loving parents for their blessing and enormous support through my research project.

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THIS THESIS IS AFFECTIONATELY DEDICATED TO MY BELOVED PARENTS AND TEACHERS

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TABLE OF CONTENTS DECLARATION .................................................................................................................................... ii ACKNOWLEDGEMENT ..................................................................................................................... iv LIST OF FIGURES ............................................................................................................................. viii LIST OF TABLES ................................................................................................................................. ix LIST OF ABBREVIATIONS AND SYMBOLS ................................................................................... x ABSTRACT........................................................................................................................................... xi CHAPTER 1 ........................................................................................................................................... 1 INTRODUCTION .................................................................................................................................. 1 1.1 Objectives ..................................................................................................................................... 2 1.1.1 General objectives .................................................................................................................. 2 1.1.2

Specific objectives .......................................................................................................... 2

CHAPTER 2 ........................................................................................................................................... 3 2. LITERATURE REVIEW ................................................................................................................... 3 2.1

Introduction to legumes .......................................................................................................... 3

2.1.1

Botanical description....................................................................................................... 4

2.1.2

Taxonomy and Classification.......................................................................................... 4

2.2

Legume verities and their cultivation practices ...................................................................... 5

2.2.1

Soya bean varieties.......................................................................................................... 6

2.2.2 Mung bean varieties ............................................................................................................... 7 2.2.3 Cowpea .................................................................................................................................. 8 2.2.4 Horse gram ............................................................................................................................. 9 2.3 Nutritional aspects of legumes .................................................................................................... 10 2.4 Anti-nutritional factors of legumes ............................................................................................. 11 2.5 Phytates ....................................................................................................................................... 12 2.5.1 Phytate as an anti-nutrient .................................................................................................... 14 2.5.2 Phytate and mineral interaction............................................................................................ 14 2.5.3 Effect of phytate on mineral availability .............................................................................. 15 2.5.4 Beneficial health effects of phytates .................................................................................... 16 2.5.5 Degradation of phytates ....................................................................................................... 16 2.5.6 Analytical method for phytates ............................................................................................ 18 2.5.7 Ion – exchange chromatography .......................................................................................... 20 2.6 Saponins ...................................................................................................................................... 22 2.6.1 Biological properties of saponins ......................................................................................... 23 2.6.2 Degradation of saponins...................................................................................................... 23 2.6.3 Analytical methods for saponins .......................................................................................... 24 2.7 Amylose ...................................................................................................................................... 26 vi

2.7.1 Amylose analytical techniques............................................................................................. 26 CHAPTER 3 ......................................................................................................................................... 27 MATERIALS AND METHODOLOGY .............................................................................................. 27 3.1 Sample procurement ................................................................................................................... 27 3.1.1 Sample preparation .............................................................................................................. 27 3.1.2 Preparation of defatted sample ............................................................................................. 27 3.1.3 Determination of moisture content ...................................................................................... 29 3.2 Quantitative determination of phytochemicals ........................................................................... 30 3.2.1 Establishment of a method to analyze phytates in legumes ................................................. 30 3.2.2 Determination of total phosphorus ....................................................................................... 34 3.2.3 Establishment of a method for the determination of saponin content .................................. 37 3.2.4 Amylose content determination ........................................................................................... 39 3.2.5 Processing of legumes .......................................................................................................... 40 3.2.6 Analysis of moisture content of processed samples ............................................................. 41 3.2.7 Determination of phytate in autoclaved samples ................................................................. 41 3.2.8 Determination of saponins in autoclaved samples ............................................................... 42 3.3 Data analysis ............................................................................................................................... 42 CHAPTER 4 ......................................................................................................................................... 43 RESULTS AND DISCUSSION ........................................................................................................... 43 4.1 Determination of moisture content ............................................................................................. 43 4.2. Determination of phytates .......................................................................................................... 46 4.2.1 Column recovery .................................................................................................................. 46 4.2.2 Phytate content ..................................................................................................................... 47 4.3 Determination of total phosphorus in legumes ........................................................................... 49 4.4 Phytate phosphorus ..................................................................................................................... 54 4.5 Saponin contents ......................................................................................................................... 57 4.6 Amylose content ......................................................................................................................... 60 4.7 Processing of legumes ................................................................................................................. 63 4.7.1 Moisture content determination in processed legumes ........................................................ 63 4.7.2 Phytates in processed legumes ............................................................................................. 64 4.7.3 Saponins in processed legumes ............................................................................................ 69 CONCLUSION ..................................................................................................................................... 71 RECOMMENDATION ........................................................................................................................ 72 BIBILIOGRAPHY ............................................................................................................................... 73 APPENDIX .................................................................................................................................... i -xxix

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LIST OF FIGURES

Figure 2.1 Cross sectional area of a typical mature legume seed .............................................. 4 Figure 2.2 Structure of phytic acid .......................................................................................... 12 Figure 2.3 Skeleton of steroid aglycone (left) and triterpene aglycone (right) ........................ 22 Figure 4.1 Variation of phytate content in legumes………………………....……………….48 Figure 4.2 Variation of phosphorus content among legume varieties ..................................... 51 Figure 4.3 Correlation curve Phytate vs Phosphorus ............................................................... 52 Figure 4.4 Phytate phosphorus content of legume varieties .................................................... 54 Figure 4.5 Correlation between phytate phosphorus and Phosphorus ..................................... 55 Figure 4.6 Phytate phosphorus and the total phosphorus ........................................................ 56 Figure 4.7 Saponin contents in eleven legume varieties .......................................................... 59 Figure 4.8 Amylose content of different legume varieties ...................................................... 62 Figure 4.9 Moisture contents of original legume samples and autoclaved samples ................ 64 Figure 4.10 Phytate contents in original and autoclaved samples ........................................... 68 Figure 4.11 Saponin contents in original and autoclaved samples .......................................... 59

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LIST OF TABLES Table 2.1 Main cultivated grain legume, their common name as well as species and distribution ................................................................................................................................. 5 Table 2.2 Recommended varieties in Sri Lanka ........................................................................ 6 Table 2.3 Composition of food legumes .................................................................................. 10 Table 2.4 List of anti-nutritional factors present in plants ....................................................... 11 Table 2.5 Phytate contents in some cereals and beans............................................................. 19 Table 2.6 Saponin content of some plants ............................................................................... 25 Table 2.7 Amylose content of plants ....................................................................................... 26 Table 4.1 Moisture content of legume varieties....................................................................... 43 Table 4.2 Recovery of the column ........................................................................................... 46 Table 4.3 Phytate content of legume varieties ......................................................................... 47 Table 4.4 Phosphorus content of 11 legume varieties ............................................................. 50 Table 4.5 Phytate Phosphorus content of legumes………………………......……………….54 Table 4.6 Saponin content of legumes…………………………………………………..….. 58 Table 4.7 Amylose content in legumes .................................................................................... 60 Table 4.8 Moisture content in autoclaved samples .................................................................. 63 Table 4.9 Phytate content in autoclaved samples……………………...........………………..66 Table 4.10 Saponin content in autoclaved legumes………………......………..…………….69

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LIST OF ABBREVIATIONS AND SYMBOLS

ANF - Antinutritional factors mg – milligram ml – milliliter mg/g – milligram oer gram psi – pounds per square inch GCMS - Gas chromatography-mass spectrometry HPLC - High performance liquid chromatography

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COMPARISON OF PHYTOCHEMICALS AND THE EFFECT OF PROCESSING IN SOME SELECTED EDIBLE BEANS IN SRI LANKA By Keerthana Sivakumaran ABSTRACT Grain legumes are important sources of proteins, minerals and vitamins for millions of people in the world, particularly in the developing countries. At the same time it comprises significant amount of ANFs which limit the functionality and bioavailability of proteins and minerals. Phytic acid which is one of the anti-nutritional factor (ANF) is a naturally occurring constituent of plant seeds, roots, tubers, and some fruits and vegetables where it acts as a storage form of phosphate. The major concern about the presence of phytate in the diet is its negative effect on mineral uptake such as Zn 2+, Fe 2+ / 3+, Ca 2+, Mg 2+. Phytate content in legumes can be reduced by different cooking methods and processing methods. Among which soaking and autoclaving are regarded as effective as well as common practices of domestic cooking. This study was carried out to determine the phytate content, saponins and amylose of some commonly consumed eleven legume varieties of Sri Lanka. Samples collection and identification were done at the Grain legumes and oil crop research development centre (GLORDC), Agunakolapelessa. The phytate content ranged from 2.600 + 0. 259 mg/g to 9.116 + 0. 608 mg/g. Pb01 variety of soya bean had the highest phytate content of 9.116 + 0. 608 mg/g and ANK black variety of Horse gram had the least phytate content 2.600 + 0. 259 mg/g. Saponin content ranged from 8.008 + 0. 699 mg/g in Pb 01 soyabean variety to 12.755 + 1.276 mg/g in MISB soyabean variety. Highest amylose content was present in MI 06, green gram variety with 22.580 + 0. 714 mg/100mg and the least in 8.709 + 0.129 mg/100mg in Pb 01. After cooking there was significant reduction in phytate and saponin contents ranging from 8-33% and 6-72%. There is a significant positive correlation (r=0.412) between the total phosphorus content and the phytate content of the samples irrespective of the varieties, Phytate phosphorus has a positive association with total phosphorus. (r = 0.512)

Key words: Mung bean, Cowpea, Soybean, Horse gram, phytates, saponins, amylose, processing, autoclaving, phosphorus, phytate phosphorus xi

CHAPTER 1 INTRODUCTION Phytochemicals can be defined as any compound found in plants (the ancient Greek word phyton means plant). Phytochemicals are certain non-nutritive plant chemicals which have some disease preventive properties. However, the term phytochemical is often used to describe a diverse range of biologically active compounds found in plants. Phytochemicals provide plants with colour, flavour and natural protection against pests. Numerous epidemiological studies have indicated that a diet rich in fruit and vegetables offers considerable health benefits to humans. Among these benefits are: 1. Reduction of the risk of developing many forms of cancer (lung, prostate, pancreas, bladder and breast). 2. Reduction of the risk of cardiovascular diseases. (Pankaja S. Chede, 2013) Legumes also known as dried beans and pulses are the edible seeds that grow in pods of annuals, biennials and perennials which are modified in many ways to facilitate their dispersal by animals, wind, and water. Legumes are simple, dry dehiscent fruit bearing pods containing one or more seeds, which split open by the two longitudinal lines into two halves at maturity. Legume plants belong to the family variously referred to as Fabaceae or Leguminosae within the order Fabales which is one of the largest and economically important families of the flowering plants.(León-López Liliana et al. 2013) . Cereals and pulses are the two main groups of grain crops that are grown around the world. Besides being a source of nutrients, foods, particularly plant foods, are a rich source of bioactive phytochemicals or bio nutrients or anti nutritive factors. Recent researches have associated the consumption of pulses with a decreased risk for a variety of chronic degenerative diseases such as cancer, obesity, diabetes and cardiovascular diseases. Pulse grains are rich source of protein, dietary fibre, complex carbohydrates, resistant starch and a number of vitamins and minerals viz., folate, potassium, selenium and zinc. In addition to the macronutrients, pulses contain a wide variety of nonnutritive bioactive components such as enzyme inhibitors, phytic acid, lectins, phytosterols, phenolic compounds and saponins (Christensen, 2010) There is a considerable interest in finding natural phytochemicals and antioxidants from plants due to their role in the treatment and/or prevention of various diseases. 1

Phytic acid which is one of the anti nutritional factor (ANF) is a naturally occurring constituent of plant seeds, roots, tubers, and some fruits and vegetables where it acts as a storage form of phosphate. Phytate has a strong binding capacity to form complexes with divalent minerals such as Ca2+, Mg2+,Fe2+,and Co2+ even at relatively low pH values and is thought to sequester these minerals in the digestive tract as well, making them unavailable for absorption.(Greiner et al. 2006). Several suitable processing practices such as soaking, cooking, germination, fermentation and autoclaving are employed to eliminate or reduce the levels of various antinutritional factors in grain legumes. (Chitra, 1994). Similarly, saponins which imparts the characteristic bitterness in foods, is a phytochemical with more beneficial effects such reducing cholesterol level in humans, showing anti cancer properties etc.

1.1 Objectives 1.1.1 General objectives 1. The main objective of the study was to determine the contents of phytate, saponin, amylose quantitatively and to examine the variability of those factors in some selected legume varieties cultivated in Sri Lanka.

1.1.2 Specific objectives 2. The specific objective of this study was to establish a method to analyze phytates, saponins and amylose in legumes 3. To determine the effect of processing on phytochemicals of the legumes 4. To determine the correlation between Phosphorus content and the phytate contents in legumes as well the correlation between the phytate phosphorus and the total phosphorus content.

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CHAPTER 2 LITERATURE REVIEW 2.1 Introduction to legumes Leguminosae is one of the largest and most important family of flowering plants constituting 650 to 750 genera, 18,000 to 19,000 species of herbs, climbers, shrubs and trees. This family broadly defined by the podded fruits (legumes). It is divided into four sub families as caesalpinoideae (2,800 species), mimosoideae (2,900), papilionoideae (14,000) and swartzioideae. Legumes belong within the order Fabales. Legumes are useful as human and animal food and soil-improving components of agricultural and agroforestry.(Ahmed & Hasan, 2014). Legumes also known as dried beans and pulses are the edible seeds that grow in pods of annuals, biennials and perennials which are modified in many ways to facilitate their dispersal by animals, wind, and water. Legumes are simple, dry dehiscent fruit bearing pods containing one or more seeds, which split open by the two longitudinal lines into two halves at maturity. Cereals and pulses are the two main groups of grain crops that are grown around the world. These two groups are the staple foods of most of the world population. Some part of the world might favour one over the other, but they are often consumed in a combination. Pulses are also known as “the poor man’s meat”, as they regularly replace meat, cheese, eggs and fish in the diet, especially during the harsh time of poverty. Besides being a source of nutrients, foods, particularly plant foods, are a rich source of bioactive phytochemicals or bionutrients or anti nutritive factors. The major classes of phytochemicals with disease-preventing functions are dietary fibre,

antioxidants,

detoxifying

agents,

immunity-potentiating

agents

and

neuropharmacological agents. Each class of these functional agents consists of a wide range of chemicals with differing potency. For example, antioxidant function is exhibited by some nutrients, such as vitamin E, vitamin C and provitamin A. Other phytochemicals that have antioxidant properties are carotenoids, phenolic compounds, flavonoids and isothiocyanates. Some of these phytochemicals have more than one function. Foods rich in these chemicals and exhibiting disease-protecting potential are called functional foods. (Shashi Kiran Misra, 2012).

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2.1.1 Botanical description A typical mature legume seed has a seed coat, the miniature plant or embryo as well as two cotyledons. Although some pulses do contain a distinct endosperm structure, in most pulses, the cotyledons represent the largest proportion of the dry seed, and they typically contain a reserve supply of starch and protein for growth. (Eashwarage, 2016)

Figure 2.1: Cross sectional area of a typical mature legume seed Source: Kadam et al, 1989

2.1.2 Taxonomy and Classification Kingdom Plantae (Plants) Sub kingdom Tracheobionta (Vascular plants) Super division Spermatophyta (Seed plants) Division Magnoliophyta (Flowering plants) Class Magnoliopsida (Dicotyledons) Subclass Rosidae Order Fabales Family Fabaceae or Leguminaceae (Pea family) 4

Genus Soybean- Glycine Pigeon Pea – Cajanus

Winged bean - Psophcarpus

Chick pea – Cicer

Beans - Phaseolus

Horse gram – Macrotyloma

Ground nut - Archis

Lentil – Lens

Pea - Pisum

Mung bean, Cowpea, Black gram, yard long bean – Vigna

2.2 Legume verities and their cultivation practices Table 2.1: Main cultivated grain legume, their common name as well as species and distribution Common name

Species

Distribution

Soyabean

Glycine max L

USA, Brasil, China, Argentina, Japan

Mung bean

Vigna radiate L

South Asia, china, India

Chick pea

Cicer arietinum

Mediterranean countries, South Asia

Black gram

Vigna mungo

India

Cowpea

Vigna unguiculata L

Africa, Asia, Mediterranean countries

Pea

Pisum sativum L

Europe, North america

Ground nut

Arachis hypogaea

Asia, Africa

Pigeon pea

Cajanus cajan L

India

Lentil

Lens culinaris

Turkey, Europe, Asia, Canada, USA

Source: Ildiko Schuster & Gajzago, 2004 There are varieties of legumes cultivated in Sri Lanka; Cowpea (Vigna unguiculate), mung bean (Vigna radiate wilczek), soybean (Glycine max L.), groundnut (Arachis hypogaea L.) and black gram (Vigna mungo L.). Dhal (Lens culinaris) and groundnut are the most popularly consumed legumes. Green gram and cowpea are the other important legumes. Soybean is the least preferred. (Hirakawacho & Chiyoda-ku, 2003).

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Table 1.2: Recommended varieties in Sri Lanka Legume

Recommended varieties

Soya bean

Pb 1, PM 13, PM 25, MISB 1

Mung

MI 5 (1982), Harsha (1990), Ari (1999), MI 6 (2004)

Cowpea

Bombay (1930), MI 35 (1962), Wijaya(1990), Waruni (1990), Dhawala (1997), MICP 1 (2011), ANKCP 1 (2014)

Black gram

MI 1(1965), Anuradha

Horse gram

ANK Brown, ANK Black

Source: Grain Legumes and Oil seed crop Research Development Center (GLORDC), Agunakolapelessa.

2.2.1 Soya bean varieties Glycine max, the soybean (also soya- or soja bean, formerly classified as Glycine soja), is an annual herbaceous plant in the Fabaceae (legume or bean family) that originated in southeastern Asia (including China, Japan, and Korea) that was domesticated more than 3,000 years ago for its edible seeds and young pods. (Bailey et al. 1976). At present soybean is one among the five major legumes cultivated in Sri Lanka, the others being cowpea, mung bean, black gram and ground nut. Soybean is recognized as a potential food crop which can bridge the gap between national needs and availability of protein. Soybean protein is considered as complete proteinsince it contains various essential amino acids required by the human body (V. Arulandy, 1995). In Sri lanka it is mostly cultivated in Anuradhapura and Matale district under irrigation in yala and as a rain fed crop in maha season. (Department of agriculture Sri Lanka, DAOSL, 2006). a) Pb01 It is an introduction from India and is a selection from variety Nanking. It shows an erect growth with a plant height of around 50-60 cm. It flowers in 25-35 days after planting. Flowers are purple in color. Pods are tan in color. Its medium sized seed have a thousand seed weight of about 155g. Seed are cream in color with a buff colored hilum.

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This matures in about 80-85 days after planting. Variety Pb-1 is suitable for rainfall uplands during the maha season as well as with supplementation irrigation during the yala season. Average yield 1700-2000 kg ha-1. b) MISB 1 Flowering occurs in 35-38 days after sowing. The variety matures in about 90 days after sowing. Mature pods are yellow in color. Number of pods per plant is 55 and number of seeds per pod is 2-3. A seed yield of 3000 - 3200 kg ha-1 can be expected from this variety. (Average yield-3 t/ha, Potential yield- >5 t/ha)

2.2.2 Mung bean varieties The mungbean, Vigna radiata (L.) Wilczek has been widely grown in southeast Asia, Africa, South America and Australia. Mung or green gram has been one of the most important grain legumes in the traditional farming systems of Sri Lanka. It has been one of the principal sources of cheap protein and its importance as a component if the Sri Lankan diet has grown over the years. Green gram not only contains a high percentage of easily digestible protein (23%), but it’s essential amino acid composition is also complementary to that of our staple diet, rice.(Ariyaratne, 1978). About 80% of mung bean crop cultivated during maha (Oct-Nov) season as rain fed uplands crop and rest is grown in yala (April-May) in paddy fields with supplementary irrigation. Land makes ridges or flat beds according to the water supply. (Department of agriculture Sri Lanka, DAOSL, 2006). a) MI 5 MI 5 was introduced to Sri Lanka in1982. This variety matures in about 50-65 days and average yield is 1.5 t/ha. Variety MI 5 seeds are susceptible for MYMV (white fly/thrips), CLS (Cercospora Leaf Spot). b) MI 6 MI 6 was introduced in 2004. This matures in about 55-58 days after planting and average yield is 1.8-2 t/ha. Variety MI 6 seeds are moderate resistance to MYMV (white fly/thrips).

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2.2.3 Cowpea Cowpea is an important legume crop in Sri Lanka. It is an inexpensive source of protein and a hardy crop well adapted to relatively dry environments. (Department of agriculture Sri Lanka, DAOSL, 2006). Cowpea verities Cowpea is mainly cultivated during Yala (Irrigated) - April and Maha (Rainfed), end of October to mid-November. Planting method is Rain-fed plant directly on flat or raised beds. During land preparation one ploughing followed by a harrowing ensures good weed control and a suitable seed bed. Cowpea can be grown on a wide range of soil, from predominantly sandy loam to clay ranging from acidic to basic (pH 4.5 - 8.0). As mung bean, space between rows should be 30-40 cm and space between plant in a rows should be 10cm15cm. Seed rate and depth of seeding are MI-1 – 35-40 kg/ha and 1-1.5cm respectively. (Grain legumes and oil crop research development centre -GLORDC, Agunakolapelessa) a) Bombay This also knows as ―Kalu Kaupi‖. Average yield is 1450 kg ha-1 (Maha 1.6-1.9 t/ha, Yala 1 t/ha) and suitable for Maha. Exhibits erect growth habit. Flowering occurs is about 40-50 days after planting. Flowers are purple in color. It possesses dark green pigmented long and pendulous pods. It matures in 75-90 days after planting seed are large and speckled grey brown in color. This variety is susceptible to root rot, bean fly, Pod borer and bruchids. b) Waruni Plant exhibits erect and determinate growth habit. It flowers in about 40 days after planting. It has bluish purple flowers. It bears long pendulous dark green pods. Medium sized seeds and reddish brown in color. It matures in 55 days after planting seed. It has a thousand seed weight of about 145g. Average yield is about 1.5 t/ha. Waruni is suitable for many cropping systems, tolerance to water stress, tolerance root rot, collar rot, leaf spots and YMV. c) Dhawala Dhawala exhibits a semi erect growth habit. Flowering occurs in 40-45 days after planting. Flowers are white in color. Pods are pendulous and green in color. Seed are large and cream colored with a black eye. It matures in 60-70 days after planting seed. Its thousand seed weight is 170g. Variety Dhawala is suitable for planting in will drained paddy lands during Yala season. Average yield 1600 kg ha-1.

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This variety suitable for many cropping systems such as well drained paddy lands in yala and susceptible to YMV. Dhawala mostly cultivates in-between Yala & Maha or Maha – late November. d) MICP 1 This variety exhibits erect and determinate growth. It matures in 66-70 days after planting seed. Seeds are medium and cream color. It has a thousand seed weight of 130g. Average yield 1500 kg ha-1. This variety is suitable for many cropping systems such as well drained paddy lands in yala. e) ANKCP 1 It matures in 60-66 days after planting seed. Average yield is 1500 kg ha-1. Pods are pale straw colour. Seeds are pale brown color and rhomboid shape. This variety is recommended for 3rd season cultivation also and 75% harvest from 1st pick

2.2.4 Horse gram Horse gram [Macrotyloma uniflorum (Lam) Verde] is a dry land legume crop grown mainly on marginal soils. Although rich in proteins (20 %), due to less acceptable taste and flavor of cooked products, it is consumed only by the farming community and low-income groups. Thus, it has remained an underutilized food legume. Such grain legumes are however, potential sources for preparation of protein products like concentrates and isolates. The residue left over after separation of proteins can be further processed to obtain starch. (Chavan et al. 2010). a) ANK Black (Anguna Black Kollu) Average yield of ANK black is 900 kg/ha and can be harvested 85-95 days after planting. b) ANK Brown (Anguna Brown Kollu) Average yield of ANK brown is 800 kg/ha and can be harvested 95-100 days after planting (department of agriculture – performance report, 2014)

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2.3 Nutritional aspects of legumes Legumes are the richest source of nutrients (protein, starch, minerals and vitamins) and important health protective compounds (phenolics, inositol phosphates and oligosaccharides). Legume proteins are composed of several thousand specific proteins. About 70 to 80% of the crude protein in legumes seeds is storage protein. Legume seeds accumulate large amount of proteins during their development. The main protein fractions are albumin and globulin. The albumin fraction has a well-balance amino acid profiles and is relatively rich in sulfur containing amino acid (methionine and cysteine), whereas, the globulin fraction differ in their amino acid composition, molecular weight of protein sub units and physico-chemical properties. Proteins are present in pea and beans up to 20% and up to 40 % in soybean and lupin. The protein is rich in lysine, and is therefore complementary to cereals in lysine balance. The oil content of legume (except soy and lupin) is 1-2%, the oil is composed mainly of polyunsaturated fatty acids. (Schuster-gajzágó, 2015).

Table 2.3: Composition of food legumes Legumes

Variety

Moisture Protein Ash Fiber

Fat

Carbohydrates

Reference

Mung

MI 5

11.99

25.99

3.96

5.55

1.54

62.97

(Gunathilake

MI 6

11.48

26.56

3.95

5.01

1.24

51.17

et al. 2016)

Soya

Pb 1

9.7

36.56

6.14

7.93

22.01

18.11

bean

MISB 1

11.0

39.70

6.35

7.93

21.17

15.29

Cowpea

Bombay

11.05

24.98

3.43

4.36

1.81

52.22

Waruni

11.1

25.03

3.78

6.84

1.51

58.76

Dhawala

9.5

22.84

3.62

5.06

1.72

54.37

MICP

6.81

25.22

4.3

3.04

1.86

51.79

ANKCP

10.99

24.90

4.10

5.75

2.03

57.24

11.81

21.96

3.56

-

0.78

61.89

1 Horse

ANK

gran

brown ANK

(Eashwarage 2016)

11.72

24.19

3.57

-

0.85

59.66

black Source: Gunathilake et al. 2016 & Eashwarage, 2016

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2.4 Anti-nutritional factors of legumes To avoid predation, sedentary species (plants, fungi and bacteria) synthesize a range of low and high molecular weight compounds. These secondary metabolites play a role in defense against herbivores, insects, pathogens or adverse growing conditions. (Santosh Khokhar et al. 2002) Legumes are rich sources of antinutritional factors. Table 2.2 List of anti-nutritional factors present in plants Low molecular weight anti nutritional

Protein anti nutritional factors and toxins

factors β-ODAP*

(nonprotein

amino

acids), Allergens

Lathyrogens Cyanogens

Alpha-amylase inhibitors,

Flatulence factors – alpha galactosides, Lectins oligosaccharides e.g: raffiinose Glucosinolates

Lipase inhibitors

Mycotoxins

Protease inhibitors

Phytic acid

Transmissible

spongiform

encelopathies

(TSE) saponins

Toxins (bacterial, mushroom, marine)

Polyphenols and tannins

Viruses

Alkaloids, gossypol, oestragenic factors, sinapins Source: Santosh Khokhar et al. 2009 Some physical processes involves in processing legumes include autoclaving at temperatures above 1000C , blanching to inactivate endogenous enzymes and avoid cooking, ordinary cooking, extrusion, roasting at 1200C – 2500C, soaking or chemical processing (treatment with thiols, sulphide, Cu salts, ascorbic acid).

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2.5 Phytates Phytic acid (phytate; myo-inositol 1,2,3,4,5,6, hexakisphosphate) is the primary source of inositol and storage phosphorus in plant seeds contributing ~ 70% of total phosphorus.(Greiner et al. 2006). Lolas and Markakis, 1975 stated that phytate accounts for 80% of the total phosphorus in most legumes. Phytate which is a naturally compound formed during maturation of plant seeds and grains is a common constituent of plant-derived foods.(Greiner et al. 2006).

Figure 1.2 Structure of phytic acid Source: (Liyan Chen, 2013) Inositols with 4, 5 or 6 phosphate groups are common in the seed of many of our grain legume and can reach concentration higher than 10% of dry matter. In diacotyledons seeds such as legumes, nuts and oilseeds, phytates are found closely associated with proteins and is often isolated or concentrated with protein fraction of these foods, which are the stores for phosphate and mineral nutrients that are important for plant nutrition and especially vulnerable during germination (Bora, 2014). The abundance of phytic acid in cereal grains is a concern in the foods and animal feeds industries because the phosphorus in this form is unavailable to monogastric animals due to a lack of endogenous phytases; enzymes specific for the dephosphorylation of phytic acid. In addition, the strong chelating characteristic of phytic acid reduces the bioavailability of other essential dietary nutrients such as minerals (e.g. Ca2+ ,Zn2+,Mg2+, Mn2+, Fe2+/3+,proteins and amino acids.(García-Estepa et al. 1999). Phytic acid is hydrolysed enzymatically by phytases, or chemically to lower inositol phosphates such as inositol pentaphosphate (IP5), inositol tetraphosphate (IP4), inositol triphosphate (IP3) and possibly the inositol di- and monophosphate during storage, fermentation, germination, food processing and digestion in the human gut. Only IP6 and IP5 have a negative effect on 12

bioavailability of minerals, the other hydrolytic products formed have a poor capacity to bind minerals or the complexes formed are more soluble. Phytic acid is the principal storage forms of phosphorus in plant seeds. In animal cells, myoinositol polyphosphates are ubiquitous, and phytic acid (myoinositol hexakisphosphate) is the most abundant, with its concentration ranging from 10 to 100 µM in mammalian cells, depending on cell type and developmental stage. The interaction of phytic acid with specific intracellular proteins has been investigated in vitro, and these interactions have been found to result in the inhibition or potentiation of the physiological activities of those proteins. The best evidence from these studies suggests an intracellular role for phytic acid as a cofactor in DNA repair by nonhomologous end-joining. Other studies using yeast mutants have also suggested intracellular phytic acid may be involved in mRNA export from the nucleus to the cytosol. There are still major gaps in the understanding of this molecule, and the exact pathways of phytic acid and lower inositol phosphate metabolism are still unknown. As such, the exact physiological roles of intracellular phytic acid are still a matter of debate. Phytic acid may be considered a phytonutrient, providing an antioxidant effect. Phytic acid's mineral binding properties may also prevent colon cancer by reducing oxidative stress in the lumen of the intestinal tract. Researchers now believe phytic acid, found in the fiber of legumes and grains, is the major ingredient responsible for preventing colon cancer and other cancers. Phytates also have the potential to be used in soil remediation, to immobilize uranium, nickel and other inorganic contaminants. Phytic acid is also protective against bowel cancer which is why a diet high in unprocessed grains is recommended to prevent it. (Shashi Kiran Misra, 2012) The daily intake of phytate for humans on vegetarian diets, on an average, is 2000–2600 mg whilst, for inhabitants of rural areas in developing countries, on mixed diets, it is 150–1400 mg. usually legume based food (cooked) items contain higher amounts phytate than do cerealbased food items. Few food items, such as sesame seeds (toasted), soy protein concentrate, rice (unpolished and cooked), maize bread (unleavened) and peanuts have exceptionally high amounts of phytate (Dahiya, 2016). Phytates rapidly accumulates in grains and seeds during their ripening period and maturation accompanied by other substances such as starch, protein and lipids. The accumulation site of phytates in grains and seeds is within the subcellular single membrane particles, aleurone grains or protein bodies. The aleurone grains are located in the aleurone cells of monocotyledonous seeds such as cereals. The aleurone grains of rice are composed of two major parts: high phytate 13

containing particle and surrounding coat that consists of protein and carbohydrate. In dicotyledonous seeds such as legumes and other seed globoids are located within the proteinaceous matrix of protein bodies. In many seeds and grains phytates accumulates during seed development and reached its highest level at seed maturity. (Alagiyawanna, 2008). Because of its complex nature and its interaction with proteins and minerals, it is becoming increasingly important from nutrition point of view.(Chitra 1994) 2.5.1 Phytate as an anti-nutrient The major concern about the presence of phytate in the diet is its negative effect on mineral uptake. Minerals of concern in this regard would include Zn 2+, Fe 2+ / 3+, Ca 2+, Mg 2+, Mn 2+, and Cu 2+.(Greiner et al. 2006)

2.5.2 Phytate and mineral interaction Phytate forms complexes with numerous divalent and trivalent metal cations. (Erdman, 1979). Stability and solubility of the metal cation- phytate complexes depends on the individual cation, the pH-value, the phytate:cation molar ratio, and the presence of other compounds in the solution. Phytate has six reactive phosphate groups and hence regarded as a chelating agent. A cation can bind to one or more phosphate group of a single phytate molecule or bridge two or more phytate molecules. Most phytates tend to be more soluble at lower compared to higher pH-values. Solubility of phytates increase at pH-values lower than 5.5–6.0 with Ca2+, 7.2–8.0 with Mg2+ and 4.3–4.5 with Zn2+ as the counter ion. In contrast, ferric phytate is insoluble at pH values in the range of 1.0 to 3.5 at equimolar Fe3+: phytate ratios and solubility increases above pH 4. Two cations, when present simultaneously, act jointly to increase the quantity of phytate precipitation. Depending on Zn2+: phytate molar ratio, Ca2+ ions enhances the formation of Calcium-Zinc phytate, by adsorbing Zn2+ ions into phytate. For high Zn2+: phytate molar ratios, Ca2+displaces Zn2+from phytate binding sites and increases its solubility. The amount of free Zn2+is directly proportional to the Ca2+ concentration. For low Zn2+: phytate molar ratios, Ca2+ potentiate the precipitation of Zn2+ as phytate. Thus, higher levels of Ca2+result in a more extensive precipitation of the mixed phytates. Mg2+ also has been shown in vitro to potentiate the precipitation of Zn2+in the presence of phytate, however, Mg2+ has been found to exert a less pronounced effect on Zn2+ solubility than Ca 2+

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2.5.3 Effect of phytate on mineral availability The formation of insoluble metal cation-phytate complexes at physiological pH-values is regarded as the major reason for a poor mineral availability. Zn2+ was reported to be the essential mineral most adversely affected by phytate. Human studies indicated that phytate inhibits Ca2+ absorption, but the effect of phytate on Ca2+ availability seems to be less pronounced compared to that on the availability of iron and particularly Zn2+. This may be due to the relatively high Ca2+ content of plant-based foods, the capability of the bacterial flora in the colon to de- phosphorylate phytate and the fact, that Ca 2+ could be absorbed from the colon. Relatively few studies have dealt with the effects of phytate on dietary Cu 2+, Mn 2+ and Mg 2+ utilisation. Phytate has been shown to decrease their bioavailability. Phytic acid has been linked to the inhibition of digestive enzymes such as protease (O'Dell and de Boland, 1976), trypsin (Sing11 end Krikorian, 1982) and pepsin (Knuckles et al., 1985). O'Dell and de Boland (1976) investigated the extent of phytate-protein interaction in aqueous extracts of a high lysine and a commercial hybrid corn germ, soybean flakes and sesame meal. The authors suggested that phytate interacts with some proteins to form insoluble products or complexes, which are not easily dissociated by electrophoresis at a high pH. Digested phytate does not affect protein digestibility, while it has been reported to have found an improvement in amino acid availability with decreasing levels of phytate. This difference may be at least partly due to the use of different protein sources. The inhibitory effect increases with the number of phosphate residues per myo-inositol molecule and the myo-inositol phosphate concentration. This inhibition may be due to the non-specific nature of phytate- protein interactions, the chelation of calcium ions which are essential for the activity of trypsin and αamylase, or the interaction with the substrates of these enzymes. The inhibition of proteases may be partly responsible for the reduced protein digestibility. Phytate has also been considered to inhibit α-amylase in vivo as indicated by a negative relationship between phytate intake and blood glucose response. Therefore, food rich in phytate has been considered to have great nutritional significance in the prevention and management of diabetes mellitus.

15

2.5.4 Beneficial health effects of phytates Though intake of phytate is reported to cause adverse health effects on human consumption. Dietary phytate was reported to prevent kidney stone formation, protect against diabetes mellitus, caries, atherosclerosis and coronary heart disease as well as against a variety of cancers. The levels of phytate and its dephosphorylation products in urine, plasma and other biological fluids are fluctuating with ingestion or deprivation of phytate in the human diet Furthermore, calcium phytate was capable of lowering blood Pb2+ levels. Thus, phytate seems to be a helpful means to counteract acute oral Pb2+ toxicity. The effect of calcium phytate on acute Cd2+ toxicity is still discussed controversially, but the majority of studies point to an improved Cd2+ absorption in the presence of phytate. This may result in a Cd2+ accumulation in liver and kidney. (Greiner et al. 2006)

2.5.5 Degradation of phytates Degradation of phytates occur during food processing or in the gastrointestinal tract. Degradation can be of two types enzymatic or non enzymatic processes. Enzymatic degradation occur as a result of biological processing and preparation of plant food/feed during steeping, malting, hydrothermal processing, fermentation and adding phytase. In the gastrointestinal tract, phytase hydrolyses inisitol-6-phosphate to myo inositol and to inorganic phophates via immediate myo-inisitol phophates. (Alagiyawanna, 2008) Germination of legume seeds lead to pronounced metabolic changes and the structural profile of various compounds are altered. Germination increases the digestibility, shortens the cooking time and enhances the nutritive value of legumes. (Chitra 1994) Soaking of pulses, which is an integral part of cooking, results in reduction in phytic acid content. According to soaking of dry beans in water for 12 hours at 240C resulted in slight decrease in phytate and greater losses had been observed when they were soaked in NaHCO3 and mixed salt solutions. These chemicals affect the seed coat permeability thus leading to greater losses. Phytic acid is said to have an effect over the cooking time, where higher the phytate content the shorter is the cooking time of dehusked seeds. Khokar and Chauhan reported that cooking of soaked seeds lower phytic acids by 7-11% in chickpea and 6-9% in black gram. 16

Reddy et al. (1978) did not notice any breakdown of phytate phosphorus during cooking of black gram seeds and cotyledons. They observed some losses in total phosphorus and phytate phosphorus during short time cooking, due to leaching of these components into the cooking water. lyer et a1 1980 also investigated the efiects of soaking in mixed salt solution and distilled water on phytic acid of the Great Northern, pinto and kidney beans. They reported a reduction of 8.7% to 69.6% in phytate content of the above three beans. Akinyele observed a decrease of 21 and 46 percent phytic acid on cooking two cowpea varieties. Autoclaving has been proved to reduce phytic acid content in legume grains such as chickpea, black gram, moth bean, cowpea, black gram, amphidiploids and mung bean. Two hours of autoclaving reduced approximately 70% of soy phytate. Kumar observed that insoluble complexes between phytate and either components were formed during cooking and hence the phytic acid content is decreased. Sprouting was found to be instrumental in lowering the phytic acid content of mung bean, cowpea, and lima bean. Horse gram and moth bean, peanut, chick pea, and black gram reported 28-34% reduction in phytic acid. Cooking of sprouts reduced the phytic acid content of chickpea grains. Roasting reduces the phytate content. (Reddy and Sathe, 2002)

17

2.5.6 Analytical method for phytates Analysis of phytates is relied upon indirect quantification, as there is no specific reagent available for determination of phytates. Determination of phytates rely upon quantification of Inisitol or Phosphate is used as indirect quantification tool or the stoichiometric relationship between phytates with some cation that is easy to measure. Phytates for an insoluble complex (precipitate) with Ferric ions in dilute acid solutions, which is the basis of many analytical methods. Determination of phytates is based on the analysis of phosphorus or iron in the isolated ferric phyate or based on the determination on residual iron in the solution after the precipitation of Ferric phytates from a known concentration of ferric salt in acidic solution. Harland and Oberlease in 1977 introduced an ion exchange chromatographic method with stepwise gradient elution for quantification of inositol-6-phosphate. The elute is collected, digested to convert organic phosphate into inorganic phosphate which is measured and inositol6-phosphate equivalent is calculated. With the identified draw backs and later on with modifications, this method has been adopted as an AOAC method. Reddy, (Ed.), Sathe, (Ed.) in 2002 suggested an HPLC method for the separation of inositol-6phosphate from inositol with C-18 stationary phase and aqueous Potassium Hydrogen phosphate or Sodium acetate mobile phase. This method is said to have some disadvantages like poor retention in the column resulting poor resolution.

18

Table 2.3 Phytate contents in some cereals and beans Cereal/bean

Phytate %

Wheat

0.39-1.35

Corn

0.75-2.22

Oat

0.42-1.16

Barley

0.38-1.16

Rye

0.54-1.46

Sorghum

0.67-1.35

Common millets

0.50-0.70

Pearl millets

0.18-0.99

Brown rice (long grain)

0.84-0.99

Polished rice (long grain)

0.34-0.60

White rice (enriched)

0.23

Rice (Basmati)

0.06

Winged beans

0.63-2.67

Soy beans

1.00-2.22

Peas

0.222 – 1.22

Lentils

0.27-1.05

Peanuts

1.05-1.76

Cowpeas

0.37-1.45

Green gram

0.59-1.10

Black gram

0.72-1.46

Lima beans

0.23-2.52

Source: Reddy, (Ed.), Sathe, (Ed.) (2002)

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2.5.7 Ion – exchange chromatography Ion chromatography separation is based on ionic (or electrostatic) interactions between ionic and polar analytes, ions present in the eluent and ionic functional groups fixed to the chromatographic support. The two distinct mechanisms are, a ion exchange due to competitive ionic binding (attraction) and ion exclusion due to repulsion between similarly charged analyte ions and the ions fixed on the chromatographic support. This chromatography is one of the most important adsorption techniques used in the separation of peptides, proteins, nucleic acids and related biopolymers which are charged molecules in different molecular sizes and molecular nature. Mobil phases consist an aqueous buffer system into which the mixture to be resolved. The stationary phase usually made from inert organic matrix chemically derivative with ionizable functional groups (fixed ions) which carry displaceable oppositely charged ion. Ions which exist in a state of equilibrium between the mobile phase and stationary phases giving rise to two possible formats, anion and cation exchange are referred to as counter ion. Exchange‐ able matrix counter ions may include protons (H+ ), hydroxide groups (OH- ), single charged mono atomic ions (Na+ , K+ , Cl- ), double charged mono atomic ions (Ca2+, Mg2+), and polyatomic inorganic ions (SO4 2-, PO4 3-) as well as organic bases (NR2H+ ) and acids (COO- ) (Acikara, 2013). Cations are separated on cation-exchange resin column and anions on an anion exchange resin column. The analyte ions and similarly charged ions of the eluent compete to bind to the oppositely charged ionic functional group on the surface of the stationary phase. In cation exchange chromatography; S-X-C + + M+↔ S-X-M+ + C+ In this process the cation M+ of the eluent replaced with the analyte cation C+ bound to the anion X- which is fixed on the surface of the chromatographic support. In anion exchange chromatography; S-X+A - + B-↔ S-X+B - + AThe anion B- of the eluent replaced with the analyte cation A- bound to the positively charged ion X+ on the surface of the stationary phase. The adsorption of the analyte to the stationary phase and desorption by the eluent ions is repeated during their journey in the column, resulting in the separation due to ion-exchange.

20

Ion exchange chromatography, which is also known as adsorption chromatography, is a useful and popular method due to its; high capacity, high resolving power, mild separation conditions, versatility and widespeared applicability, tendency to concentrate the sample, relatively low cost. This technique has been used for the analyses of anions and cations, including metal ions, mono- and oligosaccharides, alditols and other polyhydroxy compounds, aminoglycosides (antibiotics), amino acids and peptides, organic acids, amines, alcohols, phenols, thiols, nucleotides and nucleosides and other polar molecules. It has been successfully applied to the analysis of raw materials, bulk active ingredients, counter ions, impurities, and degradation products, excipients, diluents and at different stages of the production process as well as for the analysis of production equipment cleaning solutions, waste streams, container compatibility and other applications.

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2.6 Saponins Saponins are secondary metabolites synthesized by many different plant species. Their name is derived from Latin word “sapo” meaning soap, due to their surfactant properties which allows forming stable soap-like foam upon shaking in aqueous solution. (Moghimipour & Handali, 2015). It acts as a chemical barrier or shield in the plant defense system to counter pathogens and herbivores. Therefore, it is found in plant tissues that are most vulnerable to fungal or bacterial attack or insect predation (Cheok et al. 2014). Saponins are amphiphilic compounds, with the presence of a lipid-soluble aglycone and water-soluble chain(s) in their structure. They are divided into two groups: Steroidal saponins, which occur as glycosides in certain pastures plants and triterpenoid saponins, which occur in soybean and alfalfa. Saponins are glycosides containing a polycyclic aglycone molecule of either C27 steroid or C triterpenoid (collectively termed as 30 sapogenins) attached to a carbohydrate. Structurally saponins in food exist as glycosides, with a hydrophobic triterpenoid or steroid (sapogenin) group linked to water-soluble sugar residues. Saponins on hydrolysis yield an aglycone known as sapogenin (Das et al. 2012). Saponin =

Sugar + Sapogenin Glycine + Aglycone

The main types of steroid aglycones include the spirostan, furostan, and nautigenin derivatives whereas oleanan derivatives comprise the more common triterpenoid aglycones. The amount and type of sugar residues vary between saponin species, the most common being glucose, glucuronic acid, arabinose, rhamnose, xylose, and fucose attached at either the C-3 position (monodesmoside saponins) or on both the C-3 and C- 22 position (bidesmoside saponins). Saponins are characterized by a bitter taste and foaming properties. (Shashi Kiran Misra, 2012).

Figure 2.2 Skeleton of steroid aglycone (left) and triterpene aglycone (right) Source: (Jiayi, 2016) 22

2.6.1 Biological properties of saponins Saponins have been found having pharmaceutical properties of hemolytic, mol-luscicidal, antiinflammatory, antifungal or antiyeast, antibacterial or antimicrobial, antiparasitic, antitumor, and antiviral and it is discovered scientifically of having pharmaceutical properties of antioxidant, immunological adjuvant activities. The beneficial effect of saponins intake inplasmacholesterol for human is another important factor that contributes to the continuous sorting of saponins. Saponins have been used in foods as natural surfactant and serve as preservative in controlling microbial spoilage of food. (Cheok et al. 2014). Certain evidences show that saponins provide neuro protective effects on attenuation of central nervous system disorders, such as Parkinson’s disease, stroke, Huntington’s disease, and Alzheimer’s disease. Furthermore some in vivo studies have shown that saponins tumorinhibitory effects and antifungal activity. (Jiayi, 2016) The presence of saponins in soybean has attracted considerable interest owing to both health benefits and adverse sensory characteristics. Soybean saponins are heat-stable glycosides and comprise a hydrophobic aglycone (triterpenoid sapogenin) linked to one or more hydrophilic mono- or oligosaccharide moiety. Soyasaponins are classified into three groups (A, B, and E) based on their respective aglycone moieties. Group A acetylated saponins present in soybean, are implicated as the compounds mostly responsible for the undesirable taste while group B and E saponins have health benefits. Group B and E saponins are indicated to possess inhibitory activity against the infection of human immunodeficiency virus (HIV) and the activation of the Epstein-Barr virus early antigen. Recent in vitro studies suggest that saponin B and E have hypocholesterolemic, immuno-stimulatory, anticarcinogenic, antioxidative, anti- tumour, antivirus, anti-hepatitic, anti-diabetic, and hepatoprotective effects.(Rupasinghe & Jackson, 2004)

2.6.2 Degradation of saponins Saponin content reduces with the increase in soaking time of the grains in water. The losses occur as a result oof leaching out of these antinutrients into soaking water under the influence of concentration gradient. Ireland reported that saponins survived cooking and food processing. On the other hand, reduction in saponin levels during cooking of moth bean, chick pea and black gram and pigeon pea was observed. 23

Autoclaving of soaked seeds cause a great reduction iin saponin levels ranging from 33-39% in chickpea and 20-27% in black gram. Increased losses of saponins were reported with increased period of pressure cooking. Hence saponins are heatlabile. Germination and sprouting has been reported to cause reduction in saponin levels, where sprouting is found to be more effective than soaking or ordinary cooking and autoclaving, in reducing saponin content in chick pea (44-51%) and black gram (37%-40% losses). Longer the period of germination, more was the loss in saponin content. The reduction in saponin level during germination may also be due to enzymatic degradation. 2.6.3 Analytical methods for saponins There are colourimetric methods for quantification of total saponins whereas chromatographic methods that analyse individual saponins. The colourimetric method uses vanillin as the colourization reagent under strong acidic conditions to develop colour. The results are then measured using UV- spectrophotometer. The most commonly used chromatographic technique is HPLC coupled with UV detector. One major difficulty in HPLC determination is most saponins possess weak isolated olefinic chromophores that absorbs at short wavelengths and with weak absorbance coefficients. In order to overcome this problem, analytical methods have been developed using evaporative light scattering detector (ELSD) coupled with HPLC. (Ha et al. 2006) Other chromatographic methods for saponins analysis include low-pressure column chromatography (LPCC), gas chromatography (GC), and ultra performance liquid chromatography (UPLC). Gas chromatography usually combined with MS for saponins determination. (Oleszek, 2002) Compared with HPLC, UPLC is more rapid, sensitive and has higher resolution Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method has some draw backs since some saponins cannot be detected as they cannot be ionized. And, this method cannot be used for quantification of saponins. (Cheok et al. 2014)

24

Table 2.4 Saponin content of some plants

Plant

Saponin content, g/kg

Chickpea (Cicer arietinum)

2.3-60

Soy bean (Glycine max)

5.6-56

Dry bean (Phaseolus vulgaris)

4.5-21

Mung bean (Phaseolus aureus)

0.5-5.7

Broad bean (Vicia faba)

3.5

Lentil (Lens culinaris)

1.1-5.1

Green pea (Pisum sativum)

1.1-1.8

Peanut (Arachis hypogaea)

0-16

Asparagus (Asparagus officinalis)

15

Sesame seeds (Sesamum indicum)

3

Oats (Avena sativa)

1-1.3

Source: S.S.Deshpande, 2002

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2.7 Amylose Starches from different botanical sources are unique in their chemical compositions, morphologies and functionalities. This is due to differences in amylose (AM)/amylopectin (AP) ratio, genotype, soil type (during growth) and intensity of radiation of the sun during growth. The legume starches are known for their high amylose content and consequent tendency to undergo retrogradation and syneresis, these idiosyncrasies tend to limit their applications in the food industry. In contrast, cereal starches possess lower AM content and smaller granules.(Omodunbi Ashogbon & Temitope Akintayo, 2013). The difficulty in the isolation of starches from legume have been attributed to the presence of a highly hydrated fine fiber fraction which is derived from the cell wall enclosing the starch granules. 2.7.1 Amylose analytical techniques Amylose is commonly determined in cereal starches by the potentiometric, amperometric or colourimetric measurement of the iodine binding capacity of the amylose with the resultant formation of amylose-iodine inclusion complexes.

These methods may have some

uncertainties. Amylopectin-iodine complexes also form, and these reduce the concentration of free iodine measured by the non-colourimetric methods and may absorb at similar wavelengths to amylose-iodine complexes in colourimetric methods. These complexes lead to an overestimation of the amylose. (Megazyme, 2015).

Table 2.5 Amylose content of plants Plant Horse gram (macrotyloma uniflorum)

Amylose content 32.14%

Reference (Marimuthu & Krishnamoorthi, 2013)

Mung (Vigna radiate L.)

29.9-33.6%

(Kaur et al. 2011)

Soybean (Glycine max)

19-22%

(Stevenson et al. 2006)

Cowpea (Vigna unguiculata)

15.09-25.54%

(Hamid et al. 2014)

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CHAPTER 3 MATERIALS AND METHODOLOGY 3.1 Sample procurement In this study, two varieties from mung bean (MI5 and MI6), five varieties from cowpea (ANKCP1, MICP1, Bombay, Wauni and Dhawala), two varieties from soybean (Pb01and MISB1) and two varieties from horse gram (ANK black, ANK brown) recommended by the Department of Agriculture were selected for comparison of anti-nutritional factors and phytochemicals. These eleven legume varieties are obtained from Angunakolapelessa, Grain Legumes and Oil Seed Crops Research and Development Centre, which is the main agriculture research centre located in Southern Dry Zone. Obtained samples were stored in the cold room at 100C till further usage.

3.1.1 Sample preparation Collection of the legume samples were by random sampling method which has been carried out by the research officers of Grain Legumes and Oil Seed Crops Research and Development Centre, Agunakolapelessa. All varieties were collected from same field with same environment conditions. Cleaned and dried whole legume seeds were ground with a RETSCH S/S CROSS BEATER Hammer Mill Sk1 to pass a 0.5 mm (500 μm) sieve prior to chemical composition measurement.

3.1.2 Preparation of defatted sample The SOXTHERM principle simplifies and accelerates the traditional soxhlet method is adopted and approved by the AOAC. Usually the solvent extraction step alone takes six hours while soxtherm takes only two hours and six samples can be analyses at same time. By immersing the sample in hot solvent the extractable material is removed more quickly than the traditional method. The solvent is re-circulated to rinse the extractable material into the

27

bottom of the beaker. Finally the solvent is removed by evaporation automatically and recovered for safe disposal or reuse. 3.1.2.1 Materials:- Automatic extraction systems Soxtherm (C. GERHARDT GMBH & CO. KG · Analytical Systems) Electronic Balance, at least 1 mg sensitivity (METTLER TOLEDO AG204) Electrical drying oven to be operated at 105°C ± 1°C (MEMMERT NLE 500) 33x80mm, cellulose extraction thimbles, extraction beaker (glass) and holders for thimbles Cotton wool free of fat Fume cupboard Desiccator with silica gel desiccant Heating water bath (MEMMERT) Measuring cylinder, 250 ml (Borosilicate glass) Tongs 3.1.2.2 Reagent:-

Petroleum ether boiling point 60-80ºC (Analytical grade)

3.1.2.3 Method:Legume seed flour (5.00 g, n=3) was accurately weighed and transferred into extraction thimble. A fat free cotton plug was carefully placed on the top of the sample to avoid splitting. Extraction beaker was rinsed with petroleum spirit and dried in an oven at 105ºC for 30 min. After it was cooled in a desiccator, weight of the beaker was measured. Sample containing extraction thimble was placed in an extraction beaker using holders and 150ml of petroleum ether was added. The tube was fixed in the soxtherm automatic extraction system and the 28

extraction was carried out at 150°C for 2.5h. The extraction process was performed in 5 programmable steps which ensure complete extraction of the samples. Then extraction beaker was removed from the extraction unit and placed in a heating water bath for evaporate residue solvent. After that beaker was placed in an oven at 105ºC and dried the contents for two hours and weighed was measured after cooling. The beaker was replaced in the oven for 30 minutes, cooled and reweighed until its content reach to constant weight. 3.1.3 Determination of moisture content Moisture content of the ground legume sample was determined. All the calculations were done in dry weight basis. 3.2.3.1 Materials: Electronic Balance, at least 1 mg sensitivity (METTLER TOLEDO AG204) Electrical drying oven to be operated at 105°C ± 1°C (MEMMERT NLE 500) Moisture Dishes (Aluminum) Air tight desiccator with silica gel desiccant

3.2.3.2 Procedure: Legume seed flour (2.00 g, n=3) was accurately weighed into previously cleaned, dried, and weighed moisture dishes. Then the uncovered dish with the lid alongside was dried in the oven maintained at 105°C for five hours. After that the dish was covered while in the oven and transferred to the desiccator and weighed soon after the sample reaches to room temperature (30°C±2). The process of drying, cooling and weighing was repeated within thirty minutes intervals until the difference between the two consecutive weighing was 1mg. Finally the loss of weight was recorded. Percent moisture content was calculated using the following equation.

29

3.2.3.3 Calculation: Moisture (% by mass) =

weight (dish + dried sample) – weight of empty X 100 Weight of the sample

3.2 Quantitative determination of phytochemicals Clean and dry whole legume seeds were ground with a RETSCH S/S CROSS BEATER Hammer Mill Sk1 to pass a 0.5mm (500μm) sieve for determining phytochemicals.

3.2.1 Establishment of a method to analyze phytates in legumes AOAC 986.11 Anion exchage method was chosen to analyze phytates in legumes and the method was studied for its recovery using a standard phytate solution. Phytate extracted from duplicate test portions of dried legume flour using dilute HCl, mixed with Na2EDTA-NaOH solution and placed on an ion exchange column. The phytate which is eluted with 0.7M NaCl is wet digested with a mixture of acids to release inorganic P which is measured colourimetrically. 3.2.1.1 Materials:

Analytical balance (Mettler – Toledo) Glass column equipped with valve 0.7mm x 30cm Kjeldahl digestion flask, 250ml (Borosilicate glass) Kjeldhal digestion rack (VELP Scientifica DK 20) Volumetric flasks 25ml (Borosilicate glass) Volumetric flasks 100ml (Borosilicate glass) Volumetric flasks 250ml (Borosilicate glass) Volumetric flasks 1liter (Borosilicate glass) Beakers 100ml (Borosilicate glass) 30

UV- Spectrophotometer. 3.2.1.2 Reagents:

Anion exchange resin (AG 1- X 4 Chloride form, 100-200 mesh) Hydrochoric acid (Analytical grade) Sodium Chloride (Laboratory reagent grade) Phosphate standard solution (Potassium Hydrogen Phosphate), Ammonium molybdate (Laboratory reagent grade) Concentrated H2SO4 (Analytical grade) 1-ammino-2-naphthol-4-sulfonic acid (Laboratory reagent grade) Disodium ethylenediaminetetraacetate Na2EDTA (Laboratory reagent grade) Sodium hydroxide (Laboratory reagent grade) Sodium phytate (Laboratory reagent grade)

3.2.1.3 Solutions

Hydrochoric acid 2.4%(w/v) 0.1M Sodium Chloride 0.7M Sodium Chloride Phosphate standard solution - 80µg/ml 5M Sulphuric acid Molybdate solution (2.5% ammonium molybdate in 0.5M H2SO4) Sulfonic acid reagent (0.16g 1-ammino-2-naphthol-4-sulfonic acid, 1.92g Na2SO3, 9.60g NaHSO3) Na2EDTA-NaOH solution (10.23g of Na2EDTA in 7.5g NaOH) Sodium phytate stock solution solution 375µg/ml Sodium phytate working standard solution 2.8µg/ml

31

3.2.1.4 Procedure 3.2.1.4.1 Preparation of standard phosphate curve Standard phosphate solution (Primary standard, 80µg/ml) was prepared and a series of standard solution were prepared by pipetting 0.5, 1.0, 2.0, 4.0 and 8.0ml from the primary standard to 50ml volumetric flasks. Water (20ml) was added to the flasks and mixed well. Then 2ml of molybdate solution and 1 ml of Sulfonic acid solution were added to it, mixed well and diluted to the volume. After 15 minutes absorbance were measured at 640nm. 3.2.1.4.2. Preparation of the column A glass column (about 0.7mm x 30cm) with a valve was used to prepare the ion exchange column. About 3ml of water was added into the empty mounted column and water slurry of 0.9g resin was added to it. After the resin has been formed the column was washed with 15ml of 0.7M NaCl and then with 15ml of distilled water. 3.2.1.4.3 Testing the column recovery A standard phytate solution (375 µg/ml) was prepared and it was diluted to prepare a working standard solution. The working standard solution was diluted to prepare a standard solution (2.8 µg/ml) which was used to check the recovery of the column. Standars phytate solution 1ml and I ml of Na2EDTA-NaOH were added into a 25ml volumetric flask, mixed well and diluted to the volume. This solution was transferred into the column and the elute was discarded. Then the column was eleted with 15ml of distilled water and 0.1M NaCl respectively. The elutes were discarded. Finally the column was eluted with 15ml of 0.7M NaCl and the fraction was collected to a digestion tube. Then 0.5ml of concentrated H2SO4 and 3ml of concentrated HNO3 were added to the tube and digested on a kjeldal rack at 2500C until yellow fumes come out. The boiling was continued till clear solution was obtained. When the flask was cooled 10ml of distilled water was added. It was heated for 10 minutes at low heat. After cooling the contents of the tube was transferred to a 50ml volumetric flask. Then 2ml of molybdate solution and 1ml sulfonic acid were added and mixed well. After 15 minutes absorbance was measured at 640nm. Duplicate was done with the standard phytate solution to test the recovery of the column. Same procedure was repeated with 1.5g, 1.7g and 2.0g of resin. The percentage of the recovery was calculated as follows;

32

% of recovery

=

Quantity obtained in the analysis

X 100

Actual quantity in the solution

3.2.1.4.4 Analysis of phytates in legumes Eleven legume varieties, Soya (Pb01, MISB01), Cowpea (Waruni, Dhawala, MICP01, ANKCP01, Bombay), Horse gram (ANK black, ANK brown), Green gram (MI 05, MI06) were used in the analysis.

3.2.1.4.5 Extraction of phyate from legumes Initially, 2.0g of the ground powder was added into an Erlenmeyer flask and 40ml of 2.4% HCl was added into it. The flask was kept on a magnetic stirrer and extracted for 3 hours at room temperature. After 3 hours, the solution was extracted through Whatman number 1 filter paper 3.2.1.4.6 Determination of phytates Filtrate (1.0ml) was pipetted out into a 25ml volumetric flask and 1ml of Na2EDTA-NaOH was added and diluted to the volume. The same procedure in recovery of the column (3.2.1.4.3) was followed. Testing for phytates in each legume variety was done in duplicates. A blank was prepared with 1 ml of 2.4% HCl and 1ml of Na2EDTA-NaOH solution in a 25ml volumetric flask. 3.2.1.4.7 Calculation: Calculations for typical standard curve ml, standard solution

Micrograms of

Absorption of 640 nm

Phosphorus

Concentration/A (K)

0.25

20

0.047

425.531

0.50

40

0.093

430.107

1.00

80

0.158

506.329

2.00

160

0.325

492.307

33

4.00

320

0.665

“Mean K”

Phytate, mg/g sample =

481.203 467.09

‘mean K’ x A x 40 0.282 x 1000 x w

A

= Absorbance

W

= Weight of the sample

Mean K

= Standard P (µg)/A/n (Standards)

Phytate

= 28.2% P

3.2.1.4.8 Determination of phytate phosphorus Phytate phosphorus content was determined using the standard curve for phosphorus.

3.2.2 Determination of total phosphorus AOAC method 995.11 Phosphorus (total) in foods method was used to analyse the phosphorus content in eleven legume varieties. Here, the total phosphorus content of the legume sample (ground powder) was quantified. Product is dry ashed to remove organic materials. The acid soluble phosphate residue forms a blue complex with Sodium molybdate in the presence of ascorbic acid as the reducing agent. Intensity of blue colour is measured spectrophotometrically.

34

3.2.2.1 Materials: Analytical balance (Mettler – Toledo) Crucibles Volumetric flasks (borosilicate glass) – 500ml, 250ml, 100ml, 50ml Hot plate Water bath Metal basket Muffle furnace UV- Spectrophotometer (Shimadzu UV-1601) 3.2.2.2 Reagents: Concentrated Hydrochoric acid (Analytical grade) Concentrated Sulphuric acid (Analytical grade) Zinc oxide (Laboratory reagent grade) Potassium hydroxide (Laboratory reagent grade) Sodium molybdate (Laboratory reagent grade) Ascorbic acid (Laboratory reagent grade) Potassium acid phosphate (Laboratory reagent grade) 3.2.2.3. Solutions: Potassium hydroxide – 50% w/v. about 50g of KOH was dissolved in 50ml water.

Sodium molybdate solution – to a 500ml of volumetric flask, 140ml of concentrated H2SO4 was added and the solution was allowed to cool to room temperature. Then 12.5 g of Sodium molybdate was added and mixed. The solution was diluted to the mark. Ascorbic acid solution – 5g of ascorbic acid was dissolved in 100ml of volumetric flask and the solution was diluted to the mark. Molybdate-ascorbic acid solution – immediately before use, 25ml of sodium molybdate solution and 10ml of ascorbic acid solution was transferred to a 100ml volumetric flask, the solution was mixed and diluted to mark. 35

Phosphorus stock standard solution – 1.0mg P/ml. KH2PO4 was dried at 1010C for 2 hours and desiccated. 1.0967g of dried KH2PO4 was dissolved in distilled water 250ml volumetric flask, diluted to the mark with water and mixed well. Phosphorus working standard solution – 0.01 mg P/ml. about 5.0ml of the stock solution was transferred to a 500ml volumetric flask and diluted to the mark with distilled water.

3.2.2.4 Preparation of standard curve for Phosphorus From the Phophorus working standard solution 1.0,2.0, 3.0 , 4.0, 5.0 ml of were pipetted out to prepare standard solutions of 0.0, 0.01, 0.02, 0.03, 0.04, 0.05 mg P into 50ml volumetric flasks. Water (15ml) was added to each flasks. 20ml of molybdate – ascorbic solution was added to each flasks and swirled. The flasks were loosely stoppered and placed in a metal basket. The metal basket was placed in vigorously boiling water bath for 15 minutes. Then the flasks were cooled under the tap water and diluted to the volume with deionized water. Absorbance was measured at 823nm. 3.2.2.5 Total Phosphorus content of legumes Flour (1.5g) was weighed into a crucible and 0.5g of Zinc oxide was added and mixed. Then the samples were ashed in the muffle furnace at 5500C for 4 hours. Then the crucibles were removed from the furnace and let to cool. To the cold crucibles, 5ml of water, and 5ml of concentrated HCl were added. The crucibles were covered with watch glass and boiled for 5 minutes in a water bath. The contents of the crucibles were filtered into a 100ml volumetric flask and rinsed the crucibles and watch glass with hot water through the filter into the flask. After cooling the flask to room temperature, 50% KOH was added until the solution was slightly opalescent. HCl was added until the opalescent disappears and extra 2 drops were also added. The solution was cooled to room temperature and diluted to the volume with water. Then 10ml of the solution was transferred into a 100ml volumetric flask and diluted to the mark. Then 5 ml of the diluted solution was transferred to a 50ml volumetric flask and 15ml of deionized water was added. Then 20ml of molybdate ascorbic acid solution was added and swirled. The flasks were loosely stoppered and placed in a metal basket. The metal basket was placed in vigorously boiling water bath for 15 minutes. Then the flasks were cooled under the tap water and diluted to the volume with deionized water. Absorbance was measured at 823nm.

36

3.2.2.6 Calculation P, g/100g = 100 x (V2/V1) x P W Where V1 = volume of the solution used in the colour reaction,ml V2 = volume of the volumetric flask containing ash test portion, 100ml P = amount of phosphorus from standard curve corresponding to the absorbance of the analyte, mg W = weight of the test portion, mg

3.2.3 Establishment of a method for the determination of saponin content Double solvent extraction gravimetric method was used to determine the saponin content from the legumes. The method has been adapted from (G.N. & V. 2009) 3.2.3.1 Materials: Analytical balance (Mettler – Toledo) Electrical drying oven to be operated at 105°C ± 1°C (MEMMERT NLE 500) Seperation funnel (100ml) Water bath Magnetic stirrer Beakers 100ml (Borosilicate glass) Air tight desiccator with silica gel desiccant 3.2.3.2. Reagents: Ethyl alcohol (Laboratory regaent grade) Sodium chloride (Laboratory reagent grade) Diethyl ether n-butanol solution 37

Ethyl alcohol – 20% 3.2.2.3 Solutions 20% (v/v) ethanol 5%(w/v) NaCl 3.2.3.3 Procedure: 3.3.3.3.1 Extraction of saponin from ground sample About 2 g of plant sample was dispersed in 20 ml of 20% ethanol. The suspension was heated over a hot water bath for 4 h with con-tinuous stirring at about 55ºC. The mixture was filtered using whatman number 1 filter paper and the residue re-extracted with another 20 ml of 20% ethanol. The combined extracts were reduced to 40 ml over water bath at about 90ºC. 3.2.3.3.2 Determination of saponin The concentrate was transferred into a 100 ml separating funnel and 10 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 6 ml of n-butanol extracts were washed twice with 1 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the sample were dried in the oven at 1050C into a constant weight. The saponin content was calculated in percentage. 3.2.3.4 Calculation:

% saponin =

W2 – W1 x 100 W

W = weight of sample used W1 = weight of empty evaporating dish W2 = weight of the dish + saponin extract

38

3.2.4 Amylose content determination

Simple Iodine-Colourimetric method was used for determination of amylose content. This method has been adopted from AACC. 3.2.4.1 Materials: UV-visible spectrophotometer (Model: Shimadzu UV-1601) Analytical balance (Mettler – Toledo) 100ml volumetric flasks (Borosilicate glass) 3.2.4.2 Reagents: Standard potato amylose (brand – Sigma) Acetic acid (Analytical grade) 3.2.4.2 Solutions: Standard potato amylose (brand – Sigma), 1N Acetic acid, 1N NaOH, 2% I2/KI solution, 95% Ethyl alcohol 3.4.3 Procedure: 3.2.4.3.1 Preparation of standard amylose curve Standard potato amylose (40mg) was precisely measured into an Erlenmeyer flask (100ml). ethy alcohol (95%, 1ml), NaOH (1N, 9ml) were added to the flask and boiled to gelatinize for 10 minutes in boiling water bath. The solution was cooled to room temperature and was transferred into a volumetric flask (100ml) with two successive washings. Then the contents were diluted to the mark with distilled water. The aliquots of standards amylose solution (1ml, 2ml, 3ml, 4ml, 5ml) were transferred to five separate volumetric flasks (100ml). aliquots of acetic acid (1N; 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml) were added separately to each volumetric flasks followed by addition of Iodine/Potassium Iodide solution (2ml). The contents of each flask were diluted to 100ml mark with distilled water. Meanwhile blank was prepared without sample with other same conditions. After stabilizing the samples at 300C for 20 minutes, the absorption was measured 620nm using UV- spectrophotometer.

39

3.2.4.3.2 Determination of amylose in legumes Powdered sample of the legume variety (particle size 0.5mm, 100mg) was precisely measured into an Erlenmeyer flask (100ml). ethy alcohol (95%, 1ml), NaOH (1N, 9ml) were added to the flask and boiled to gelatinize for 10 minutes in boiling water bath. The solution was cooled to room temperature and was transferred into a volumetric flask (100ml) with two successive washings. An aliquot (5ml) was transferred into a volumetric flask (100ml). acetic acid (1N, 1ml) and Iodine/Potassium Iodide (2ml) were added. The solution of each flask were diluted to 100ml mark with distilled water. Meanwhile blank was prepared without sample with other same conditions. After stabilizing the samples at 300C for 20 minutes, the absorption was measured 620nm using UV- spectrophotometer.

3.4.4 Calculation: % amylose = Cs x 100/5 x 100 Where Cs is the concentration corresponding to the absorbance value for the sample from the standard curve for amylose.

3.2.5 Processing of legumes The eleven varieties of legumes that has been used for the earlier analysis were processed to determine the variation in the phytochemical content. 3.2.5.1 Materials: Analytical balance (Mettler-Toledo) Autoclave (SAKURA steam sterilizer 2595) Snackmaster dehydrator – American harvest TM Cotton plug Conical flasks 250ml (Borosilicate glass)

3.2.5.2 Solutions: Distilled water

40

3.2.5.3 Soaking Initially, 20-25g of the legume seeds were weighed into a beaker. The weighed sample was cleaned with tap water, soaked in distilled water of 1:10 ratio (sample/distilled water) for 10 hours. 3.2.5.4 Autoclaving Then, the sample was rinsed several times in water and autoclaved at 1210 C for 15 minutes. After autoclaving the samples were dried using a domestic dehydrator at 400C till constant weight was obtained. 3.2.5.4 Sample preparation After complete drying the samples were grounded into powder using hammer mill (0.5mm sieve size). The samples were stored in the cold room at 100C until further use.

3.2.6 Analysis of moisture content of processed samples 3.2.6.1 Materials: Moisture Analyzer 3.2.6.2: Procedure The moisture content was determined using the moisture analyzer. At first, 5g of the sample was weighed to the pan, the lid was closed, final moisture content was determined and displayed in 10 to 12 minutes.

3.2.7 Determination of phytate in autoclaved samples Phytate content of the legume samples were determined using the anion-exchange method of AOAC 986.11 described in 3.2.1 Establishment of a method to analyze phytates in legumes

41

3.2.8 Determination of saponins in autoclaved samples

Saponin content of the autoclaved legume samples were determined using double solvent gravimetric method described in 3.2.2 Establishment of a method for the determination of saponin content.

3.3 Data analysis All the datas were analysed using non parametric tests. The data were statistically evaluated by kruskal wallis test for k-independent samples by using SPSS 16.0 software and significant differences between medians were determined by Mann-whitney U test. The correlation between phosphorus and phytate contents were determined using Spearman’s rho correlation test. Wilcoxan’s signed rank test was used to determine the significance difference between phytates and saponins before and after processing. All test procedures were made at 5% significant level.

42

CHAPTER 4 RESULTS AND DISCUSSION As since all the data points obtained were either duplicates (n=2) or triplicates (n=3) a normal distribution cannot be assumed. As such all the data analysis have been done using non parametric data analysis methods using SPSS. For performing ANOVA the distribution should be normal, variances of multiple groups should be equal and the data should be independent. 4.1 Determination of moisture content Moisture content of each legume seed was determined according to the oven drying method and results of analysis are given in 3.1.3 In present study moisture content of all three legume species (Mung bean, cowpea and soybean) ranged from 6.81 ± 0.05% to 11.99 ± 0.48%. The highest value was obtained from mung bean MI 5 (11.99 ± 0.48%) and the lowest for cowpea MICP 1 (6.81 ± 0.05%). Table 4.1 Moisture content of legume varieties Legume

Variety

% Moisture content

Pb01

9.24 + 0.63

MISB 01

9.57 + 0.37

Waruni

11.05 + 0.06

MICP 01

6.81 + 0.05

Bombay

11.05 + 0.39

Dhawala

9.5 + 0.05

ANKCP 01

10.99 + 0.1

MI 05

11.99 + 0.48

MI 06

11.48 + 0.21

ANK black

11.72 + 0.17

ANK brown

11.81 + 0.86

Soya bean

Cowpea

Mung bean

Horse gram

43

There is a significant difference P 0.05) was found between Pb 1 (9.24 ± 0.63%) and MISB 1 (9.57 ± 0.37%) in their moisture content. These observations are in agreement with those reported by Joshi et al. (2015), as their findings the moisture content for full fat seed flour ranged between from 8.54% to 10.20%. When consider the mean values of horse gram, no significant difference (P > 0.05) was found between ANK Black (11.72 ± 0.17%) and ANK Brown (11.81 ± 0.86%) in their moisture content. These observations are in agreement with those reported by Sreerama et al., (2011). as their findings the moisture content for horse gram was 6.8 ± 0.2. According to the Marimuthu & Krishnamoorthi, (2013), it was 6.72 ± 0.03. According to Kalidass & Mohan, (2012), the moisture content ranged between10.2 – 13.0. Present finding regarding moisture content of mung bean, cowpea, soybean and horse gram are in conformity with values described in previous literature, however slight variations may be due to genotype and environmental conditions (Qayyum et al., 2012). 44

% MOISTURE 14 11.99 12

Moisture %

10

11.05 9.24

11.05

9.57

8

10.99

11.48

11.72

11.81

MI 06

ANK black

ANK brown

9.5 6.81

6 4 2 0 Pb01

MISB 01 Waruni MICP 01 Bombay Dhawala ANKCP 01

Soyabean

MI 05

Cowpea Legume variety

Mung bean

Horse gram

Figure 4.1: Variation in moisture content among legume species

Figure 4.1 represents the variation in moisture content among the legume species

45

4.2. Determination of phytates Phytic acid, a major component of all legumes, has a tremendous potential for binding positively charged molecules such as cations or proteins. The interaction between phytic acid and minerals such as iron, zinc, calcium, magnesium etc., leads to the formation of complexes that are insoluble at intestinal pH and, hence, biologically unavailable for absorption (Erdman, 1979) 4.2.1 Column recovery Recovery of the column was determined using a standard phytate solution. Table 4.2: Recovery of the column Amount of resin

% of recovery

1.0000g

55.00%

1.5000g

79.41%

1.7000g

84.00%

2.0000g

96.00%

According to the AOAC 986.11 method for phytate determination 0.5g of the resin must be used for the analysis. But when the column has been tested for the recovery it gave lower results of recovery as such to improve the recovery the amount of resin was increased quantitatively and the recovery percentage was calculated. Best result was obtained for 2.0000g of resin where the recovery was 96.00%. In the above method 2.4% HCl was used to extract the phytates from dry food samples. The extract from HCl is mixed with EDTA-NaOH solution and placed in the ion exchange column. Phytate is eluted with 0.7M NaCl and the elute was wet digested with a mixture of HNO3H2SO4 to release inorganic phosphorus which is measured colourimetrically. Amount of phytae in original test solution is calculated as hexaphosphate equivalent. A graph of Absorbance (at 640nm) versus concentration (mg/ml KH2PO4) was used to determine the concentration from absorbance values of the inorganic phosphorus containing solutions. The same curve was used in determining the recovery values.

46

4.2.2 Phytate content Phytate contents were determined according to AOAC 986.11 method. During the study phytate content of the eleven legume varieties have been analyzed. All the calculations were done in dry weight basis. Table 4.3: Phytate content of legume varieties Legume Soyabean

Cowpea

Mung bean

Horse gram

Variety

Phytates mg/g dry basis

% Phytate

Pb01

9.116 + 0.608

0.9116 + 0.060

MISB 01

8.189 + 0.031

0.8189 + 0.003

Waruni

3.561 + 0.140

0.3561 + 0.014

MICP 01

6.088 + 0.193

0.6087 + 0.019

Bombay

3.651 + 0.446

0.3651+ 0.045

Dhawala

6.881 + 1.232

0.6881 + 0.012

ANKCP 01

5.495 + 0.027

0.5495 + 0.003

MI 05

4.440 + 0.958

0.444 + 0.096

MI 06

3.909 + 0.087

0.390 + 0.009

ANK black

3.470 + 0.259

0.347 + 0.026

ANK brown

5.372 + 0.546

0.537 + 0.055

Values are given as means with standard with each determination performed in duplicates For the above list of datas, Kruskal wallis test for k-independent samples at 95% was carried out among the four legume groups. Where, P < 0.05, so there is a significant different in medians of the legume groups, Soyabean, Cowpea, Mung and Horsegram. Thereafter MannWhitney test for 2 independent sample was carried out for Horsegram and soyabean which had the highest difference in mean ranks. The P < 0.05 indicated the significant difference between the 2 groups, horsegram and soyabean. Thereafter the test for k-independent samples within the groups (Pb01 9.116 + 0.608 , MISB 8.189 + 0.031, ANK black 3.470 + 0.259, ANK Brown 5.372 + 0.546 mg/g) gave P>0.05 therefore there is no significant difference between the varieties of soyabean and horsegram. The values for phytates in soyabean is significantly higher than those of other varieties. Pb01 exhibits the highest phytate content of 9.116 + 0.608

47

mg/g followed by MISB with 8.189 + 0.031mg/g, while minimum was found to be in ANK black with 3.470 + 0.259 mg/g. There is no significance difference P>0.05 within soya varieties Pb01 and MISB at 5% significance level. Same way there is no significant difference among Waruni 3.561 + 0.140 mg/g, MICP01 6.088 + 0.193 mg/g, Bombay 3.651 + 0.446 mg/g, Dhawala 6.881 + 1.233 mg/g and ANKCP01 5.495 + 0.027 mg/g. the medians of MI05 4.440 + 0.958 mg/g and MI06 3.909 + 0.087 mg/g are equal at 5% significance interval. The medians of ANK black 3.470 + 0.259 mg/g and ANK brown 5.372 + 0.546 mg/g show no significance difference P>0.05. Similar observations on the phytate content of different cowpea varieties have been reported by several investigations. According to Reddy and Sathe, (2002), the phytate content for soyabean can range from 1.00 – 2. 22%, green gram 0.59-1.10%, Cowpea 0.37-1.45%. While Chitra, (1994) reported that phytic acid was reported to be high in soya bean varieties 36.4 mg/g followed by black gram13.7 mg/g, pigeon pea 12.7 mg/g and Mung bean 12.0 mg/g and chick pea 9.5 mg/g. Raboy et al. (1984) reported a mean phytic acid concentration of 17.6 g/kg for 38 soybean lines [Glycine mnx (L.)], with lines ranging from 13.9-23.0 g/kg. Farinu and Ingrao, (1991) reported that phytic acid content of cowpea showed large varietal differences. Phytic acid contents of thirteen cowpea cultivars varied from 5.1 g/kg to 10.27 g/kg According to Deshpande et al., 1982 the phytate content of legumes varies from 0.40 to 2.0% depending upon the species and the variety and most of it is present in the outer aleurone layers of the cotyledons or the endosperm. Reddy et al., 1989 stated that phytate rapidly accumulates

(%) phytates

1.2 1

0.9116 0.8189

0.8

0.6881 0.6087

0.5495

0.6

0.537 0.444

0.4

0.3561

0.3651

0.39

0.347

0.2 0 Legumes variety

Figure 4.2: Variation of phytate content among legume varieties 48

in seeds during the ripening period, accompanied by other storage substances such as starch and lipids. N.R.Reddy et al, 2000 reported that during soyabean maturation the phytate content increased from 0.87% to 1.26% dry weight basis. In legumes, phytate is distributed throughout the cotyledon and located within the subcellular inclusions of protein bodies. It has been stated that 99% of the phytate in dry peas was in the cotyledons and 1% in the embryo axis. Phytate phosphorus represents about 65% of the total phosphorus in the cotyledons and 20% of the total phosphorus in the embryo axis. The hull or seed coat fraction contain little or no phytate. Present finding regarding phytate content of mung bean, cowpea, soybean are in conformity with values described in previous literature, however slight variations may be due to genotype and environmental conditions (M. M. N. Qayyum et al, 2012) variety or cultivar, climatic conditions, location, irrigation conditions, type of soil, and year during which they are grown (Bassiri and Nahapetian, 1977)

4.3 Determination of total phosphorus in legumes

AOAC method 995.11 Phosphorus (total) in foods method was used to analyse the phosphorus content in eleven legume varieties. Legume sample is dry ashed to remove any organic compounds. Acid soluble phosphate forms a blue complex with Na2MoO4 in the presence of ascorbic acid as the reducing agent. Intensity of the blue colour is determined spectrophotometrically. Phytic acid is the principal form of phosphorus in many seeds and that about 40-80% of the total phosphorus contents of dry legume seeds are in the form of phytic acid phosphorus (Lolas and Markakis, 1975). Ologhobo (1989) analysed six varieties of soybean [Glycine max (L.)], for phytic acid and total phosphorus content. There did not appear to be much variation between the soybean varieties in phytic acid and phytic-phosphorus which ranged between 0.32-0.44% and 0.09-0.12% respectively. Phytic acid and phytic-phosphorus represented 62.2-83.9% and 17.9-23.5% of total phosphorus respectively. Phosphorus content in eleven legume varieties were determined to find the correlation between phosphorus and phytates in legumes.

49

Table 4.4: Phosphorus content of 11 legume varieties Legume

Soya bean

Cowpea

Mung bean

Horse gram

Variety

Phosphorus mg/100g dry

Pb01

basis

MISB 01

573.695 + 3.366 654.937 + 0.046

Waruni

443.185 + 0.00

MICP 01

441.438 + 1.772

Bombay

544.714 + 1.893

Dhawala

377.697 + 0.309

ANKCP 01

427.447 + 0.000

MI 05

373.085 + 0.637

MI 06

405.625 + 3.161

ANK black

284.489 + 4.660

ANK brown

275.042 + 1.435

There is a significant difference P0.05 of phosphorus content within the two varieties of soya bean respectively. There is no significant difference P>0.05 between Waruni 443.185 + 0.0 mg/100g 0, MICP 01 441.438 + 1.772 mg/100g, Bombay 544.714 + 1.893 mg/100g, Dhawala 377.697 + 0.309 mg/100g, ANKCP01 427.447 + 0.00 mg/100g. Same way there is no significant difference P >0.05 between MI05 373.085 + 0.637 mg/100g and MI06 405.625 + 3.161 mg/100g and ANK black 284.489 + 4.660 mg/100g and ANK brown 275.042 + 1.435 mg/100g.

50

Phosphorus content Vs Legume variety 800

Phosphorus mg/100g

700 600 500 400

654.937 573.695

544.714 443.185441.438

427.447 377.697

405.625 373.085 284.489275.042

300 200 100 0

Legume variety

Figure 4.1: Variation of phosphorus content among legume varieties

This chart represents the variation in the phosphorus content among legume varieties, where Soyabean varieties have the highest phosphorus content and Horse gram has least phosphorus content.

51

Y = 0.015 * x -1 r = 0.403

Figure 4.4 Correlation curve Phytate vs Phosphorus In beans, phytate phosphorus constitutes a major portion of the total phosphorus. Of the total phosphorus phytate phosphorus accounts for 50.0% to 70.0% in soybeans, 69.0% in green grams, 29.8% to 50.0% in cowpeas. (N.R.Reddy, 2000). But according to Ologhobo, (1989) who analysed six varieties of soybean [Glycine max (L.)], for phytic acid and total phosphorus content. There did not appear to be much variation between the soybean varieties in phytic acid and phytic-phosphorus which ranged between 0.32-0.44% and 0.09-0.12% respectively. Phytic acid and phytic-phosphorus represented 62.2-83.9% and 17.9-23.5% of total phosphorus respectively. Ologhobo and Fetuga (1984) observed significant differences in phytate anion, total phosphorus and phytate phosphorus in several Nigerian varieties of cowpeas, lima beans and soybeans. Their results indicated that the soybean dry seeds were the richest source of phytate (1.47% dry weight basis) followed in descending order by cowpeas (1.37%) and lima beans (0.88%). The ratio of phytate phosphorus as percentage of total phosphorus was highest in soybeans and lowest in lima beans.

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Phytic acid content of eighteen varieties of mature dry limabeans (Phaseolus lunntus) ranged between 0.76-1.14% showing considerable variation among the varieties (Ologhobo and Fetuga, 1982). This study further indicated that phytic acid phosphorus represented 31.3-59.4% of total phosphorus with an average of 47.2%. These results are partly consistent with a view that phytic acid is the principal form of phosphorus in many seeds and that about 40-80% of the total phosphorus contents of dry legume seeds are in the form of phytic acid phosphorus (Lolas and Markakis, 1975). A correlation is a simple statistic that explains whether there's a relationship or association between any two variables. The resulting statistic from a correlation equation is called a correlation coefficient. There is a significant, P>0.05, positive correlation between phosphorus and phytate contents with the correlation coefficient of 0.408. Which means with the increase in phosphorus content the phytate content increases noticeably. According to the previous findings, there was a significant positive correlation (r = 0.99) between phytic acid and total phosphorus content in all the legumes.(Chitra, 1994). Raboy et nl. (1984) reported a mean phytic acid concentration of 17.6 g/kg for 38 soybean lines [Glycine mnx (L.)], with lines ranging from 13.9-23.0 g/kg. Among the soybean lines studied, phytic acid and seed total phosphorus were highly and positively correlated (r = 0.94) The magnitude of correlation coefficient obtained in the analysis was low one of the reason could be the prolonged storage of legumes could had led to activation of phytase enzyme at high humidity and high temperature conditions which can lead to significant loss in phytates. According to Chitra, 1994 the decrease in phytic acid after 12 months of storage was the highest in chickpea (56-67%) stored at 25 and 37°C and the lowest in soybean (29%) stored at 25 and 37°C. Dhal samples of all the legume species stored at higher temperatures (i.e 25 and 37°C) exhibited a greater loss in phytic acid than samples stored at 5°C. In a warm, moist environment, there would be increased metabolic activity, phytase activation, and membrane degradation. On a broad level, phytate synthesis can be regulated in two ways. First, by the amount of photoassimilates and phosphorus translocated to the grain. Second, by the partitioning of these substrates among different pools and competing metabolic pathways in the developing grain. As an example of the first case, it has been shown that phytate levels are correlated with the supply of P to the plant and with the content of inorganic phosphorus in leaves (Raboy and

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Dickinson 1984; Raboy and Dickinson, 1993), which ultimately leads to increased translocation of P to the grain. 4.4 Phytate phosphorus Table 4.2 Phytate phosphorus content of legume varieties Legume

Soya bean

Cowpea

Mung bean

Horse gram

Variety

Pb01

Phytate Phosphorus

Phytate P as a % of

mg/100g dry basis

total phosphorus 58.314

MISB 01

334.545 + 13.397 286.233 + 0.922

Waruni

117.787 + 4.629

26.577

MICP 01

241.258 + 7.653

54.653

Bombay

135.447 + 4.576

24.866

Dhawala

254.643 + 23.27

67.419

ANKCP 01

192.136 + 0.954

44.949

MI 05

131.827 + 28.484

35.334

MI 06

119.321 + 6.996

29.417

ANK black

103.056 + 10.255

36.225

ANK brown

159.119 + 19.095

57.853

43.704

There is a significant difference P0.05 in phytate phosphorus contents. Same way there is no significant difference P>0.05 between ANK black 103.056 + 10.255 mg/100g and ANK brown 159.119 + 19.095 mg/100g varieties and MI05 131.827 + 28.484 mg/100g and MI06 119.321 + 6.996 mg/100g of Mung bean varieties.

0.75 x – 100 R = 0.512

Figure 4.5: Correlation between phytate phosphorus and Phosphorus The above graph represents the association between phosphorus content and the phytate phosphorus content among the eleven legume varieties. The correlation coefficient is 0.512, which is positive which means with the increase in the phosphorus content phytate phosphorus content increases, there is a significant correlation P>0.05 between the two variables. Figure represents higher the total phosphorus content higher the phytate phosphorus. An experiment conducted by (Cossa et al. 1999) where for 100 selected maize samples phytate phosphorus and phosphorus contents were determined where the correlation coefficient was 0.70.

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Though some of the results of phytate phosphorus and total phosphorus obtained deviates from that reported in the literature, could be due to certain reasons such as variations in the environmental factors such as locations, irrigation conditions, type of soil, fertilizer applications, year of growing the cultivar etc. The sample that has been used for the analysis was stored in the cold room till further usage which could had again led to loss in phytates with storage time.

Phytate Phosphorus Vs P

1000.000 900.000 800.000 700.000 600.000 500.000 400.000 300.000 200.000 100.000 0.000

Phosphorus mg/100g

Pb 01 MISB Waru MICP Bomb Dhaw ANKC MI 05 MI 06 ANK ANK (defat 01(de ni 01 ay ala P 01 Black Brow ted) fatted n ) 573.695654.937443.185441.438544.714377.697427.447373.085405.625284.489275.042

Phytate_Phosphorus mg/100g 334.545286.233117.787241.258135.447254.643192.136131.827119.321103.056159.119

Legume variety Phytate_Phosphorus mg/100g

Phosphorus mg/100g

Figure 4.6: Phytate phosphorus and the total phosphorus

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4.5 Saponin contents Saponins are common in a large number of plants and plant products. It is having important role in human and animal nutrition. Saponins have biological role as membranepermeabilising, immunostimulant and hypocholesterolaemic properties and it has found to have significant affect growth and feed intake in animals. These compounds have been observed to kill protozoans, to impair the protein digestion and the uptake of vitamins and minerals in the gut and to act as hypoglycemic agent. These compounds thus affect animals in both positive and negative ways. (Das et al. 2012) For the quantification of saponin the method called double solvent gravimetric extraction has been used, where 20% ethanol has been used initially to dissolve all the saponins because saponins are soluble in aqueous phase, while alcohol prevents foaming of the sample due to the presence of saponins. Diethyl ether was used to remove the organic and fatty mater present in the concentrated alcoholic extract of saponins. Thereafter butanol was used to remove all the saponins from the aqueous phase and evaporated to dryness to obtain the precipitate. Table 4.6: Saponin contents of legumes Legume

Legume variety

Saponin mg/g dry

% Saponins

basis Soyabean

Cowpea

Green gram

Horse gram

Pb01

8.008 + 0.699

0.800 + 0.069

MISB 01

12.755 + 0.833

1.276 + 0.083

Waruni

7.060 + 1.038

0.706 + 0.104

MICP 01

9.872 + 3.560

0.987 + 0.356

Bombay

9.249 + 0.748

0.925 + 0.075

Dhawala

8.756 + 0.599

0.876 + 0.060

ANKCP 01

8.437 + 0.960

0.843 + 0.096

MI 05

11.828 + 0.658

1.183 + 0.066

MI 06

12.588 + 1.328

1.258 + 0.133

ANK black

11.516 + 0.775

1.152 + 0.078

ANK brown

10.060 + 0.727

1.006 + 0.699

Highest saponin content was present in MISB01 12.755 + 0.833 mg/g followed by Pb01 8.008 + 0.699. among cowpea varieties MICP 01 has the highest saponin content 9.872 + 3.560 mg/g. 57

among mung bean varieties MI 06 has12.588 + 1.328 mg/g. ANK black 11.516 + 0.775 mg/g has the highest saponin content among horse gram varieties. Kruskal wallis test for k-independent samples was done for four legume varieties, soyabean, cowpea mungbean and horsegram. There is significant difference P0.05 as such there is no significant difference between the two legume groups. Same way there was no significant difference among the legume groups when Mann whitney test for independent samples was performed pairwise. When k-independent samples test was done among the 2 varieties of soyabean Pb01 8.008 + 0.699 mg/g and MISB01 12.755 + 0.833 mg/g there is a significant difference P < 0.05 existing between the 2 groups. Same way there is a significant difference existing among the varieties of cowpea Waruni 7.060 + 1.038 mg/g, MICP01 9.872 + 3.560 mg/g, Bombay 9.249 + 0.748 mg/g, Dhawala 8.756 + 0.599 mg/g , ANKCP 01 8.437 + 0.960 mg/g. there is no significant difference among the saponin content of mung bean varieties MI 05 11.828 + 0.658 mg/g and MI 06 12.588 + 1.328 mg/g and horsegram varieties ANK black 11.516 + 0.775 mg/g and ANK brown 10.060 + 0.727 mg/g. Within grain legumes, the saponin content varies between 0.5% and 5% dry weight, with soybean being the most important dietary source(Santosh Khokhar & Richard K Owusu Apenten n.d.) According to (Loren Cordain, 2015) soyabean saponin concentration can range upto 4040 mg/kg, green gram upto 500 mg/kg and kidney beans upto 3500 mg/kg. according to D.E. Okwu and B.O. Orji, 2007, a research conducted on phytochemical composition of three cultivars of Vigna unguiculata and Glycine max grown in Nigeria saponins ranged between (0.11-0.23 mg 100 g-1), which is contradictory to that stated earlier by Loren Cordain, 2015. According to S.M Bala, 2012 saponin content of cowpea ranges from 0.02 to 0.08%. Price et al, 1986 states that soybean saponin content can range from 5.6 to 56 g/kg, mung bean 0.5 – 5.7 g/kg. From the literature cited it has been stated to use 20g or 5 g of sample, but for the analysis 2g of test portion has been used, which could had led for some experimental errors, as it’s a gravimetric method. 58

1.8

1.614

1.6 1.4

(%) saponins

1.2

1.183

1.132 0.987

1 0.8

0.925

1.258 1.152 1.006

0.876 0.843

0.706

0.6 0.4 0.2 0

Legume variety

Figure 4.7: Saponin contents in eleven legume varieties This figure represents the variation in the saponins contents across selected edible legume varieties.

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4.6 Amylose content

Starch is the major storage polysaccharide of higher plants and is deposited in partially crystalline granules varying in morphology and molecular structure between and within plant species. Besides being a major plant metabolite, starch is also the dominating carbohydrate in the human diet.(Kaur et al. 2011). Many of the properties of cereal starches that determine their suitability for particular end-uses are dependent upon their amylose/ amylopectin ratios. These properties include gelatinisation and gelation characteristics, solubility, the formation of resistant starch, and, for rice, the cooking and textural characteristics of whole grains. Thus, the measurement of the amylose content of starches is an important quality parameter for starch processing. (Megazyme, 2015) Table 4.7 Amylose content in legumes Legume

Variety

Amylose mg/100mg dry

% amylose

basis Soya bean

Cowpea

Green gram

Horse gram

Pb01

8.705 + 0.129

8.705 + 0.129

MISB 01

8.988 + 0.175

9.329+ 0.175

Waruni

20.852 + 0.164

20.641 + 0.164

MICP 01

18.557 + 0.408

18.557 + 0.408

Bombay

21.019 + 0.189

21.019 + 0.189

Dhawala

20.597 + 0.215

20.597 + 0.215

ANKCP 01

20.059 + 0.250

20.059 + 0.250

MI 05

22.235 + 0.913

22.488 + 0.913

MI 06

22.580 + 0.714

22.580 + 0.714

ANK black

20.098 + 0.043

20.098 + 0.043

ANK brown

19.227 + 0.043

19.227 + 0.043

Mung bean has the highest amylose content where MI06 variety has the highest of 22.580 + 0.632 mg/100g followed by MI 06 22.235 + 0.803 mg/100g. While Soyabean varieties have

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the lowest amylose content with Pb01 8.705 + 0.100 mg/100g having the lowest amylose content. According to Kruskal wallis test conducted for k-independent samples there is a significant difference P