ABSTRACT
Extraction, characterization and evaluation of bioactive constituents of eight indigenous legumes variously processed namely African breadfruit (Treculia africana) seeds, bambaranut (Vigna subterranean L.), red bean (Phaseolus vulgaris), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata L.), African yam bean (Sphenostylis stenocarpa) seed, African oil bean (Pentaclethra mycrophylla Benth.) seed and groundnut (Arachis hypogea L.) were studied using standard methods. The results of the chemical composition showed that legume samples vary significantly (p< 0.05) in all the parameters evaluated. Groundnut, African oil bean seed and African breadfruit had significantly higher protein, carbohydrate, fat and ash contents compared with other legumes. Also, groundnut, African oil bean and African breadfruit showed superiority in mineral and fibre abundance while, bambaranut had the lowest mineral and fibre contents. Linolenic acid is the most abundant fatty acids with values ranging from 38.78 – 84.57%. Percentage PUFA ranged from 40.15 – 48.97%. The total essential amino acids ranged from 24.11 – 66.67 mg/100 g. Extraction variables significantly (p<0.05) influenced the total phenolic yield of all the samples. Acetone extraction solvent gave the highest yield of total phenolic compounds in African breadfruit, African oil bean, African yam bean seed and groundnut; while ethanol extraction solvent gave the highest yield in bambaranut and cowpea; and methanol extraction solvent in red bean and pigeonpea. Processing methods applied significantly (p<0.05) influenced the total phenolic, tannin, anthocyanin, carotenoid and flavonoid contents of the samples. Pressure cooking exhibited a significant (p<0.05) reduction in the phenolic, tannin, anthocyanin, carotenoid and flavonoid contents in all the samples with increasing cooking time, however, there was minimal increase in the total phenolic and carotenoid contents of red bean, total anthocyanin of red bean and African oil bean. Roasting temperatures significantly (p<0.05) reduced the phenolic content of the samples except in bambaranut, red bean and African oil bean where there were increases with increasing cooking time. The tannin, anthocyanin, carotenoid and flavonoid contents were significantly (p<0.05) reduced with increasing roasting temperatures. Fermentation reduced the phenolic, tannin, anthocyanin, carotenoid and flavonoid contents of the samples with increasing fermentation time. However, red bean showed minimal increase with increasing fermentation time. There was significant (p<0.05) increase in the total phenolic content of all the samples with increasing germination time. But, tannin and flavonoid showed significant (p<0.05) reduction with increasing germination time. Germination significantly (p<0.05) reduced the carotenoid and anthocyanin levels in all the samples, however, red bean, pigeonpea and African oil bean showed increases with increasing germination time. Samples evaluated exhibited significantly (p<0.05) different antioxidant capacities. African oil bean and groundnut had the highest antioxidant activities 52.18% and 52.16%, respectively while, African yam bean seed was the lowest (19.85%). Similar trend was observed in the reducing power of the raw samples where groundnut, bambaranut and African breadfruit showed significantly (p<0.05) higher reducing power. Three (3) days germination significantly (p<0.05) increased the antioxidant capacities by 14.65%, 18.42%, 53.58%, 52.84%, 17.24%, 14.56%, 53.18% and 43.03% in African oil bean, bambaranut, cowpea, red bean, African breadfruit, groundnut, African oil bean and pigeonpea, respectively. The GC-MS analysis revealed the presence of diverse bioactive compounds which exhibit antitumor properties, antimicrobial properties, antidepressant, enzymes inhibition, bio stimulation, restoration of regular heartbeat.
TABLE
OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List
of Tables xii
List
of Figures xiii
Abstract xiv
CHAPTER 1: INTRODUCTION 1
1.1 Background
of the Study 1
1.2 Statement
of the Problem 3
1.3 Justification
of the Study 4
1.4 Objectives
of the Study 6
CHAPTER 2: LITERATURE
REVIEW 7
2.1 Legumes
7
2.2 Some
Traditional Legume Cooking Technologies 9
2.2.1 Soaking 9
2.2.2 Dehulling 10
2.2.3 Cooking 10
2.2.4 Fermentation 11
2.2.5 Sprouting 12
2.2.6 Toasting 13
2.3 Legumes
Involved in this Study 13
2.3.1 African
breadfruit (Treculia africana) 13
2.3.2 Bambaranut 14
2.3.3 Red
bean 15
2.3.4 Pigeon
pea 15
2.3.5 Cowpea 16
2.3.6 African
yam bean seed 17
2.3.7 African
oil bean seed 19
2.3.8 Groundnut 20
2.4 Classification
of Phytonutrients 21
2.4.1 Terpenes
21
2.4.2 Polyphenols 23
2.4.3 Carotenoids 25
2.4.4 Glucosinolates
(GLN) 27
2.4.5 Lectins 27
2.4.6 Alkaloids 27
2.4.7 Chlorophyll 28
2.4.8 Betalains 28
2.4.9 Capsaicinoids 29
2.4.10 Organosulfur compounds (OSCs) 29
2.5 Bioactive
Compounds in Legumes 30
2.5.1 Phenolic
acids 30
2.5.2 Isoflavones 31
2.5.3 Dietary
fibre 32
2.5.4 Tannins 35
2.5.5 Saponins 35
2.5.6 Anthocyanins 36
2.5.7 Lignans 36
2.5.8 Coumestans 37
2.5.9 Catechin
and epicatechin 37
2.5.10 Phytic
acid 37
2.6 Methods
for Extraction of Phytochemicals 38
2.6.1 Conventional
extraction methods 38
2.6.2 Infusions 38
2.6.3 Maceration 38
2.6.4 Soxhlet
extraction 39
2.6.5 Steam
and hydro-distillation 40
2.7 Factors Affecting Extraction Methods 40
2.7.1 Solvent 40
2.7.2 Temperature 41
2.7.3 Time 42
2.8 Biochemical Pathways of Important
Phytochemicals 42
2.8.1 Shikimate pathway 43
2.8.2 Polyketide pathway 43
2.8.3 Isoprenoid pathway 46
2.9 Biological Function of Phenolic
Phytochemicals 48
2.9.1 Antioxidant property 48
2.9.2 Modulation of cellular physiology 49
2.9.3 Anticarcinogenic and antimutagenic
properties 49
2.9.4 Management and prevention of cardiovascular
diseases (CVD) 50
CHAPTER 3: MATERIALS AND
METHODS 51
3.1 Material 51
3.2 Sources
of the Raw Materials 51
3.3 Preparation
of the Raw Materials 51
3.4 Methods
52
3.4.1 Proximate
analyses 52
3.4.2 Minerals
content determination 52
3.4.3 Determination
of dietary fibre 53
3.4.4 Fatty
acids determination 54
3.4.5 Amino
acids determination 54
3.5 Extraction
of Total Phenolic Compounds 55
3.6 Extraction
Variables for Total Phenolic Compounds 56
3.6.1 Extraction
solvent type evaluation 56
3.6.2 Extraction
solvent concentration evaluation 56
3.6.3 Extraction
time evaluation 57
3.6.4 Extraction
temperature evaluation 57
3.7 Processing
Methods 57
3.7.1 Cooking
of the legume samples 57
3.7.2 Roasting
of the legume samples 57
3.7.3 Fermentation of the samples 58
3.7.2 Germination
of the samples 58
3.8 Determination
of Some Bioactive Compounds 60
3.9 Characterization
of Bioactives 61
3.9.1 Gas
chromatography mass spectrophometry (GC- MS) analysis 61
3.9.2 Identification
of Components 61
3.10 In Vitro Antioxidant Evaluation of the
Phytochemicals 61
3.10.1 Reducing
power assay 61
3.10.2 1,1-Diphenyl-2-picrylhydrazyl
method (DPPH●) assay 62
3.11 Experimental
Design 62
3.12 Data
and Statistical Analysis 63
CHAPTER 4: RESULTS AND DISCUSSION 64
4.1 Proximate Composition of Legume Samples 64
4.2 Mineral Composition of Legume Samples 67
4.3 Fibre Profile of Legume Samples 71
4.4 Fatty Acid Profile of Legume Samples 73
4.5 Amino Acid Profile of Legume Samples 76
4.6 Summary of the Amino Acid Composition of
the Samples 78
4.7 Solvent Types Evaluation on Total
Phenolic Content of AOB, ABF, GGN
and AYB 80
4.8 Solvent Types Evaluation on Total Phenolic
Content of BBN, CPB, RBS
and PGP 83
4.9 Acetone Concentration Evaluation on
African Oil Bean Total Phenolic
Content 85
4.10 Effect of Acetone Concentrations on African
Breadfruit Total Phenolic
Content 87
4.11 Acetone Concentration Evaluation on African
Yam Bean Seed Total
Phenolic 89
4.12 Acetone Concentration Evaluation on
Groundnut Total Phenol 91
4.13 Effect
of Ethanol Concentrations (20 – 100%, v/v) on Bambaranut Total
Phenolic Content 93
4.14 Ethanol Concentration Evaluation on Cowpea
Total Phenolic Content 95
4.15 Effect
of Methanol Concentrations (20 – 100%, v/v) on Red Bean Total
Phenolic Content 97
4.16 Methanol Concentrations Evaluation on
Pigeon Pea Total Phenolics 99
4.17 Influence
of Extraction Time Variable on Total Phenolic Content of AOB,
ABF, GGN and AYB 101
4.18 Influence
of Extraction Time Variable On Total Phenolic Content of BBN,
CPB, RBS and PGP 104
4.19 Extraction
Temperature Evaluation on Total Phenolic Content of AOB,
ABF, GGN, and AYB 107
4.20 Extraction
Temperature Evaluation on Total Phenolic Content of BBN,
CPB, RBS and PGP 110
4.21 Effects of Processing Methods on Total
Phenolic Content 113
4.22 Effects of Processing Methods on Total
Tannin 119
4.23 Effects of Processing on Anthocyanin
Content 123
4.24 Effect of Processing Methods on Carotenoid
Content 128
4.25 Effect of Processing Methods on Flavonoid
Content 132
4.26 Antioxidant Capacity and Reducing Power of
the Raw a 3-day
Germinated Samples 136
4.27 GC-MS Analysis Result for ABF, BBN, RBS and
PGP 139
4.28 GC-MS Result for Cowpea Bean and African
Yam Bean Seed 141
4.29 GC-MS Result for African Oil Bean and
Groundnut 144
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS 146
5.1 Conclusion 146
5.2 Recommendations 148
References 150
Appendix 182
LIST
OF TABLES
2.1 Constituents
of dietary fibre according to the definition of the American
Association of Cereal Chemists 34
4.1 Proximate
composition of legume samples 66
4.2 Mineral
composition of legume samples 70
4.3 Fibre
profile of legume samples 72
4.4 Fatty
acid profile of legume samples 74
4.5 Amino
acid profile of legume samples 76
4.6 Summary
of amino acid composition of the legume samples 79
4.7 Effect
of processing methods on total phenolic content 116
4.8 Effect
of processing methods on total tannin content 122
4.9 Effect of processing methods on
anthocyanin content 126
4.10 Effect
of processing
methods on total carotenoid content 131
4.11 Effect
of processing methods on total flavonoid content 135
4.12 Antioxidant capacity and reducing power of
the raw and 3-day
germinated samples 138
4.13 GC-MS analysis of some of the legumes 139
4.14 GC-MS analysis of CPB and AYB 143
4.15 GC-MS analysis of AOB and GGN 145
LIST
OF FIGURES
2.1 Examples of terpenes with established
functions in nature 22
2.2 Chemical structures of the main classes of
polyphenols 24
2.3 Some chemical structures of carotenoids 26
2.4 Polyketide biosynthetic pathway leading to
anthraquinones 45
2.5 Overview of terpenoids biosynthesis in plants
showing the basic stages
of
the process and major products 47
3.1 Generalized processing flow chart 59
4.1 Effect of solvent type on total phenolic
content of AOB, ABF, GGN and
AYB
82
4.2 Effect of solvent types on total phenolic
content of BBN, CPB, RBS and
PGP 84
4.3 Effect of acetone concentrations on African
oil bean total phenolic content 86
4.4 Effect of acetone concentrations on
African breadfruit total phenolic
content 88
4.5 Effect of acetone concentrations on
African yam bean seed total
phenolic content 90
4.6 Effect
of acetone concentrations on groundnut total phenolic content 92
4.7 Effect
of ethanol concentrations on bambaranut total phenolic content 94
4.8 Effect
of ethanol concentrations on cowpea bean total phenolic content 96
4.9 Effect
of methanol concentrations on red bean total phenolic content 98
4.10 Effect
of methanol concentrations on pigeon pea total phenolic content 100
4.11 Effect of extraction
time on total phenolic content of AOB, ABF, GGN
and AYB 103
4.12 Effect of
extraction time on total phenolic content of BBN, CPB, RBS
and PGP 106
4.13 Effect of
extraction temperature on total phenolic content of AOB, ABF,
GGN and AYB 109
4.14 Effect of
extraction temperature on total phenolic content of BBN, CPB,
RBS and PGP 112
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Legumes
are cherished globally as a sustainable, readily available and cheap
alternative protein sources. In the tropics, they are the second most important
food crops after cereals (Apata and Ologhobo, 1997; Ubom, 2007; Annor et al., 2014). Indigenous legumes both
wild and cultivated are therefore important affordable alternative protein
sources most especially to low income earners of the developing countries where
the grains constitute part of the daily staple food.
The terms “legumes” and “pulses” are used
interchangeably in that, all pulses are considered legumes but not all legumes
are considered as pulses (El-Taby, 1992; Singh et al., 2016). According
to FAO (2002); Doghari et al. (2009) and Singh et
al. (2016) the term “pulse” is exclusively for dry and edible seeds of
leguminous plant. The term excludes other legumes classified as oil crops such
as soybean, peanut etc. and those harvested green for food such as green beans,
peas and sprouts. The latter are classified as vegetables (Subuola et al.,
2012). Some of the common legumes grown in the tropics include
cowpea, soybean, pigeonpea, African yam bean, bambaranut, kidney bean and lima
bean (Apata and Ologhobo, 1997; Fasoyiro, et
al., 2006). In Nigeria, cowpea, groundnut and soybean are the major legumes
consumed while; in West Africa they constitute the major commercial cash crops
(Annor et al., 2014; Ade-Omowaye et al., 2015). Lesser grown and
underutilized legumes in Nigeria include African breadfruit, African yam bean
seed, bambaranut, African oil bean, pigeonpea among others (Ene-Obong
and Carnovale, 1992). They are grossly underutilised attributed to their
characteristic beany flavour; high antinutrient/phytochemical content and lack
of scientific information on their food potentials and possible food
applications.
Legumes
have a special place in proper human nutrition because they contain more than 2
times protein than cereals depending on the type (Reyes-Moreno et al., 1993; Annor et al., 2014). Generally, legumes are good sources of complex
carbohydrates which have been shown to be beneficial in the prevention and
management of heart related diseases and diabetes. These functions are possibly
attributed to their appreciable amount of water-soluble fibre content (Rice-Evans
et al., 1997). They also serve as a
large reservoir of bioactive compounds most especially the phenolics (Hu, 2003;
Jacobs and Gallaher, 2004; Enujiugha, 2010). Furthermore, legumes are also good
sources of vitamins such as thiamine, riboflavin, niacin; vitamins e.g.
pyridoxine and folic acid; minerals such as calcium, iron, copper, zinc,
phosphorus, potassium and magnesium and are excellent sources of
polyunsaturated fatty acids such as linoleic
and linolenic acids (Annor et al., 2014).
Food
processing locally applied on legumes include dehulling, soaking, germination,
fermentation, cooking among others. These yield edible products with high
nutritional quality and improved physiological benefits (Xu and Chang, 2008; Chew et al., 2011; Tan et al.,
2013).
Initially, plant bioactives such as total free
phenolics, tannins, phytic acid were considered as antinutritional substances
and their presence in food/feed materials was considered to be undesirable from
the nutritional point of view (Hollman and Katan, 1999; Emilio, 2007; Weston, 2010;
Aluko, 2011; Awika, 2011; Vadivel and Biesalski,
2012a). However, with increased researches, new findings have now shown that
the so call antinutrients confer beneficial physiological functions on human
health at certain dosage. Essentially, these bioactive compounds have been
demonstrated to possess many favourable medicinal properties including
potential antioxidant activity (Siddhuraju and Manian, 2007; Randhir et al., 2008). These bioactive compounds
are either positively or negatively affected by different food processing
methods. In line with that Fernandez-Orozco et al. (2009); Tarzi et al.
(2012) studied the effect of germination on phytochemical profile and antioxidative
potential of some conventional legumes. Furthermore, Chew et al. (2011); Doss et al.
(2011) and Vadivel and Biesalski (2012a); Tan et al. (2013) and Salem et al.
(2014) investigated the effects of some
traditional processing such as soaking, cooking, germination and roasting on
phytochemical content and antioxidant activities of some common and wild
legumes indigenous to Asia. Despite the potentials of lesser known legumes as
alternative protein sources and the beneficial roles of bioactive compounds,
there is dearth of scientific information on their nutritional profile and
bioactive potentials, hence, the thrust of this study. This study is an attempt
to profile the nutritional composition and bioactive constituents; evaluate the
influence of local processing methods on the bioactive compounds and to assess
the antioxidant and reducing power of lesser known legumes.
1.3
STATEMENT OF THE PROBLEM
In recent years, research efforts have
been intensified on the possibilities of utilization of natural sources of
bioactives for the dietary management of certain chronic diseases due to
attendant deleterious effects associated with the use of synthetic chemicals.
In this respect, local and underutilised legumes would be excellent natural sources to exploit. Before now, phytochemicals
such as total free phenolics, tannins, terpenoids, phytic acid among others were
considered as antinutritional substances and their presence in food/feed was considered
to be undesirable. Research findings have proven the health beneficial role of these
phytochemicals which exhibit bioactivity in the body. Notably, these bioactive
compounds have been demonstrated to possess potent favourable
medicinal/physiological functions (Siddhuraju and Manian 2007; Randhir et al.
2008). Due to their health beneficial effects, the availability of such
bioactives in the diet has been advocated in recent years by both the food
scientists, nutritionists and consumers. This has led into a need to process
foods with specific health benefits such as functional foods. Most of the
common/local processing methods have been reported to affect the levels of
bioactive compounds such as polyphenolics, tannin, catechins etc. Furthermore,
to be able to feed the rapidly increasing population in Nigeria and Africa in
general, there is need to nutritionally profile lesser known legumes. In that
regard, research studies have been ongoing in presenting lesser known legumes
and their suitability in different food applications. Therefore, it has become
important to provide scientific data on the effects of some food processing
methods on their bioactive potentials and profile their nutritional
composition.
1.3 JUSTIFICATION OF THE
STUDY
Legumes
serve as a large reservoir of bioactive compounds most especially the phenolics
and these bioactives have been positively implicated in the management of
degenerative diseases (Silva et al., 2007 and Singh et al., 2016). This has led to increased
research efforts on the possibilities of exploiting locally available and natural
sources of bioactives for the dietary management of those diseases.
The
rapidly increasing population of the third world countries calls for increase
researches in providing alternative food sources. There are thousand lesser
known plant food sources that might substantially add to the array of available
nutrients most especially the protein need (Nah and Chau, 2010). The lesser
known legumes which are readily available and cheap, well adapted to extreme
environmental conditions and highly resistant to drought, diseases and pest
infestation are alternative sources to exploit.
Food
processing methods such as fermentation, germination, cooking etc. have shown
to affect both the nutritional composition and phytochemical profile. The main
cause of phytochemical loss in food is high temperature degradation. But, for
lipophilic ones such as carotenoids found in tomatoes, they might remain stable
or increase in content upon application of high temperature which help in liberating
them from cellular membranes (Dewanto et
al., 2002; Palermo et al., 2014).
Other processing techniques like mechanical processing can also liberate carotenoids
and other phytochemicals from the food matrix thereby increasing their
bioavailability (Palermo et al.,
2014; Hotz, and Gibson, 2007). While in some cases, food processing is important
in the elimination/reduction of phytotoxins or antinutrients. For example, in
communities where cassava is the major staple, traditional practices which
involve some local processing such as soaking, cooking, fermentation, etc. are
necessary to avert poisoning from cyanogenic glycosides present in unprocessed
cassava (FAO,
2002).
In
that regard, research studies have been ongoing in presenting lesser known
legumes and their suitability in different food applications. Fasoyiro et al. (2006) assessed the proximate,
mineral and antinutrient of four lesser grains found in Nigeria. The
antioxidant properties of some commonly consumed and underutilised legumes in
Nigeria were reported (Oboh, 2006). In the same vain, Ade-Omowaye et al. (2015) profiled the nutritional composition
of nine underexploited legumes indigenous to Southwest Nigeria; while, James et al. (2016) assessed the potentials of
protein concentrate from seven legumes indigenous to Northern Nigeria for
different food applications. It is therefore important to assess the effects of
different processing methods on some bioactive compounds in lesser known
legumes; evaluate their antioxidant potentials as well as profile their
nutritional composition. This will establish their bioactive and nutrient
potentials as alternative food sources to be exploited.
1.4 OBJECTIVES
OF THE STUDY
The
broad objective of the study is to profile the nutritional composition and to
assess the effect of different processing methods on bioactive compounds of
selected lesser local legumes. The specific objectives of the study are to:
(i)
Determine the chemical composition of the raw
samples
(ii)
Evaluate the effect of solvent type,
solvent concentration, extraction time and extraction temperature variables on
total phenolic yield
(iii)
Assess the effects of different processing
methods on some bioactive constituents
(iv)
Determine the antioxidant capacity and
reducing power of the raw and three days germinated samples
(v)
Characterize bioactive compounds in the
raw samples.
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