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EVALUATION OF THE NUTRITIONAL, AMINO-ACID PROFILE, OIL AND TOXICOLOGICAL FACTORS OF SAND BOX (HURA CREPITAN) SEEDS
ABSTRACT
The nutritional, amino acid profile, oil and toxicological factors of sandbox (Hura crepitan) seeds were evaluated. Four treatments of the seed flour samples (raw, cooked, roasted and fermented) were produced and analyzed for proximate composition, mineral, vitamins, anti-nutrients, and amino acids using standard methods. The seed oil was extracted from the flour samples and characterized using standard techniques. Albino rats were fed with the seed flour samples compounded with the normal feed control) and toxicological indicators were tested using standard methods. The seed flour samples had moisture content range of 8.34% (roasted) to 12.60% (fermented), fat contents of 31.87% (cooked) to 38.13% (roasted), ash contents of 2.09% (cooked) to 3.52% (raw), protein content range of 15.46% (cooked) to 23.97% (raw) crude fibre content of 3.67% (cooked) to 6.55% (fermented) and carbohydrate content of 19.72% (fermented) to 34.16% (cooked). There were no significant (p>0.05) difference in the proximate composition of the seed flour samples. Four minerals: Potassium (2.240 – 2108.0mg/kg), sodium (114.0 – 974.4mg/kg) phosphorus (219.0-367.54mg/kg) and magnesium (82.02 – 128.0mg/kg) were predominant in the seed-flour samples. There were significant (P<0.05) differences between the sodium and potassium contents of raw or cooked seed flour and roasted or fermented seed flour. The major vitamins found in the seed flour were vitamin E (4386.44 – 4578.29g/100g), vitamin A (3054.48 – 3248.86g/100g) and vitamin C (64.00 – 197.20mg/100g). The anti-nutrients of concern in the seed-flour were flavonoids (17.50% in raw), alkaloid (6.20% in raw), tannin (5.24% in raw), and cyanogenic glycoside (1.76% in raw). Fermentation and moist cooking were found to be more effective in the reduction of the anti-nutrients. Twenty amino acids were identified in the seed flour. The three major ones were arginine (3.25g/100g in cooked and 8.05g/100g in fermented), glutamic acid (6.05g/100g in cooked and 10.2g/100g in fermented) and valine (8.03g/100g in raw and 8.58g/100g in fermented). The limiting amino acid is methionine with a chemical score of 44.52%. It was observed that Hura crepitan seed flour yielded up to 38.13% in oil and the oil was golden yellow in colour and remained liquid at room temperature (280C – 310C), with iodine values of 120.5 to 171.32 and smoke point range of 2090C – 2240C. Thus it is a semi drying oil good for frying and conversion to alkyd resins in paint industry. The rats fed with fermented and cooked seed flour had safe serum levels of alkaline phosphate, aspartate aminotransferase, alanine aminotransferase, albumin, urea, creatinine, triglycerides, cholesterol, high density lipoprotein, and low density lipoprotein, electrolytes that did not differ significantly (P<0.05) in relation to the control fed rats. Thus its consumption has no ill effect on the rats. The major fatty acids in the oil were oleic (28.59%), linoleic (25.12%) and lauric (14.67%). This suggested that the oil could be substituted for vegetable oils like soya bean oil or groundnut oil which have similar fatty acid composition. Hura crepitan seed is a good source of nutrient and oil waiting to be tapped.
Keywords: anti-nutrients, amino acids, cooked, fermented, sandbox seed
TABLE OF CONTENTS
Cover page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v-xi
List of Tables xii-xiii
List of Figures xiv
List of Plates xv
Abstract xvi
CHAPTER ONE INTRODUCTION
1.1 Background of the Study 1
1.2 Statement of the Problem 4
1.3 The Objectives of the Work 4
1.4 Justification 5
1.5 Scope of the Study 5
CHAPTER TWO LITERATURE REVIEW
2.1 Characterization and Uses of Hura crepitan
(Sand box) Tree 6
2.1.1 Uses of Hura crepitan (sand box) tree 7
2.2 Oil Seeds 7
2.3 Processing of Oil Seeds 7
2.3.1 Oil Extraction 8
2.3.2 Modification of Vegetable Oil 8
2.4 Post-processing Deterioration 9
2.5 Nutrients in Foods 10
2.5.1 Carbohydrates 10
2.5.2 Protein 11
2.5.3 Fat 11
2.5.4 Vitamins 12 2.5.5 Minerals 14
2.5.6 Water 15
2.6 Amino Acids 15
2.7 Nutritional Status of Oil Seeds 17
2.8 Anti-nutrients of Related Seeds and their Oil 20
2.9 Fats and Oils 24
2.9.1 Classes of Fats/Oils 24
2.9.2 Relation of Class of Fat/Oil to Health 26
2.10 Some Popular Vegetable Oils 27
2.10.1 Palm Oil 27
2.10.1.1 Characteristics of Palm Oil 27
2.10.1.2 Palm Oil Products 28
2.10.1.3 Uses of Palm Oil 28
2.10.2 Palm Kernel Oil 30
2.10.2.1 Uses of Palm Kernel Oil 32
2.10.3 Groundnut Oil 33
2.10.3.1 Characteristics 33
2.10.3.2 Merits of Peanut Oil 33
2.10.4 Castor Oil 34
2.10.4.1 Characteristics 34 2.10.4.2 Uses of Castor Oil 35
2.10.5 Coconut (Cocos nucifera linn) Oil 36
2.10.5.1 Characteristics 36
2.10.5.2 Uses of Coconut Oil 36
2.10.6 Soybean Oil 37
2.10.6.1 Characteristics 37
2.10.6.2 Uses of Soybean Oil 38
2.11 Microorganisms Involved in Spoilage of Oil Seeds 38
2.11.1 Types of spoilage caused by microorganisms in oil seeds 39
2.12 Toxicology 40
2.12.1 Testing methods 41
2.13 Haematology Tests 43
2.14 Summary of Review of Literature 45 CHAPTER THREE
MATERIALS AND METHODS
3.1 Collection of Raw Material 47
3.2 Processing of the Hura crepitan Seeds 47
3.2.1 Determination of the Proximate Composition of the
Hura crepitan Seed Flour 48
3.2.1.1 Determination of Moisture Content of
Hura crepitan flour samples 48
3.2.1.2 Determination of the Fat Content of
Hura crepitan Flour Samples 49
3.2.1.3 Determination of Ash content of Hura
crepitan flour samples 50
3.2.1.4 Determination of Protein Content of Hura
crepitan flour Samples 50
3.2.1.5 Determination of Crude Fibre 52
3.2.1.6 Determination of Carbohydrate Content 53
3.3 Determination of the Mineral Composition of the
Huracrepitan Seed 53
3.3.1.1 Determination of Mineral Elements: Calcium,
Potassium and Sodium 53
3.3.1.2 Determination of Phosphorous 54
3.3.1.3 Determination of Se, Mg, Pb, Mn, Fe, Ni, Zn Using
Buck 200 AAS 55
3.4 Determination of Vitamin Contents of Hura crepitan
Seed Flour 55
3.4.1 Determination of Vitamin B2 (Riboflavin) 55 3.4.2 Determination of Vitamin B1 (Thiamin) 56
3.4.3 Determination of Niacin 57
3.4.4 Determination of Vitamin E (Tocopherol) 57
3.4.5 Determination of Vitamin A 58
3.4.6 Determination of Ascorbic Acid (Vitamin C) 59
3.5 Determination of Anti-nutrient Contents of Hura
crepitan Seed Flour 60
3.5.1 Determination of Alkaloid Content 60
3.5.2 Determination of Tannin Content 60
3.5.3 Content Determination of Saponin 61
3.5.4 Determination of Flavonoid Content 62
3.5.5 Determination of Cyanogenic Glycoside Content 62
3.5.6 Determination of Oxalate Content 63
3.5.7 Determination of the Phenol Content of Hura crepitan 64
3.5.8 Determination of the Phytate Content of the Hura
crepitan Seed Flour 65
3.6 Determination of Amino Acids of Hura crepitan
Seed Flour 67
3.7 Determination of the Physico-chemical Properties
of the Hura crepitan Seed oil 70
3.7.1 Determination of the Percentage of Oil in Hura
crepitan (Sand Box) Seed 70
3.7.2 Determination of the Acid Value 70
3.7.3 Determination of the Saponification Value 71
3.7.4 Determination of Iodine Value 72
3.7.5 Determination of the Peroxide Value 72
3.7.6 Determination of the Refractive Index 73
3.7.7 Determination of the Density Value 74
3.7.8 Determination of Smoke Point 74
3.7.9 Determination of the Colour of Oil 74
3.7.10 Determination of Fatty Acid Constituents of Hura
crepitan Seed Oil 75
3.7.11 Determination of the Storage Stability of the
Hura crepitan Seed Oi 76
3.8 Toxicological Assessment of Sand Box (Hura crepitan)
Seeds 76
3.8.1 Animal Grouping and Feeding Plan 76
3.8.2 Collection and Preparation of Serum 78
3.8.3 Determination of Alkaline Phosphatase (ALP) 78
3.8.4 Determination of Aspartate Aminotransferase (AST) 79
3.8.5 Alanine Amino-Transferase (ALT) 80 3.8.6 Determination of Albumin 80 3.8.7 Determination of Bilirubin (BIL) 81
3.8.8 Determination of Urea 82
3.8.9 Determination of Creatinine (CREA) 83 3.8.10 Determination of Triglycerides (TRIGS) 84
3.8.11 Determination of Serum Cholesterol (CHOL) 85
3.8.12 Determination of Low Density Lipoprotein (LDL)
Cholesterol 86
3.8.13 Determination of High Density Lipoprotein (HDL)
Cholesterol 87
3.8.14 Determination of Sodium 88
3.8.15 Determination of Potassium 89
3.8.16 Determination of Chloride 90
3.8.17 Determination of Total Protein (TP) 91
3.8 Hematological Tests 92
3.8.1 Estimation of Haemoglobin 92
3.8.2 Red Blood Cell Count (RBC) 93
3.8.3 White Blood Cell Count (WBC) 93
3.9 Histopathological Test 94
3.10 Microbial Analysis 95
3.10.1 Media Preparations 95
3.10.2 Preparation of Sample Dilutions and
Inoculation of Plates 95
3.10.3 Isolation of Pure Cultures 96
3.10.4 Identification of Isolated Bacteria Cultures 96
3.10.4.1 Spore Staining Test 97
3.10.4.2 Gram Staining 97 3.10.4.3 Catalase Test 98
3.10.4.4 Oxidation/Fermentation Test 98
3.10.4.5 Indole Test 99
3.10.4.6 Motility Test 99
3.10.4.7 Gelatin Hydrolysis Test 100
3.10.4.8 Methyl Red Test 100
3.10.4.9 Coagulate Testing 100
3.10.4.10 Identification of Fungal Colonies 100 CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Results 102
4.2 Discussion 137
4.2.1 Proximate Composition of Hura crepitan Seed
Flour Samples 137
4.2.2 Mineral Contents of Hura crepitan Seed Flour Samples 141
4.2.3 Vitamins in Hura crepitan Seed Flour Samples 144
4.2.4 Anti-nutrients in Hura crepitan Seed Flour Samples 145
4.2.5 Amino Acid Profile of the Hura crepitan 147
4.2.6 Chemical Properties of Hura crepitan Seed Oil 149
4.2.7 Physical Properties of Hura crepitan Seed Oils
Samples 151
4.2.8 The Fatty Acid Profile of Hura crepitan Seed Oil 153
4.2.9 Values of Deterioration Indicators in Hura crepitan Oil Under Storage Condition (Darkness and Light)
for 180 days (6 months) 154
4.2.10 Toxicological Indicators in rats fed with
Hura crepitan flour samples 157
4.2.10.1 Liver Function Test Indicators 157
4.2.11.2 Indicators of Kidney Function Status 163
4.2.10.3 Indicators of Heart Function Status 165
4.2.10.4 Electrolytes in the Serum of Rats Fed with Hura
crepitan Seed Flours Sodium (Na+) 169
4.2.10.5 Serum Protein Levels 172
4.2.11.1 Red Blood Cell (RBC) Counts of Rats Fed on Hura
crepitan Flour Samples 172
4.2.11.2 White Blood Cell (WBC) Counts of Rats Fed on
Hura crepitan Flour Samples 173
4.2.11.3 Blood Hemoglobin Levels (Values) in Rats Fed on
Hura crepitan Flour Samples 174
4.2.12 Weights of the Animals (ALBINO RATS) 174
4.2.13 The pictures of histologic sections of kidney and liver 175
4.2.14 The Microflora of Stored Hura crepitan Seed Flour 175
CHAPTER FIVE CONCLUSION AND RECOMMENDATION
5.1 Conclusion 178
5.2 Contributions to Knowledge 179
5.3 Recommendations 180
5.4 Suggestions for Further Studies 180
REFERENCES 181
APPENDIX 203
CHAPTER ONE INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The sandbox tree (Hura crepitan), also known as ‘possum wood’ and ‘jabillo’ in Spanish is an evergreen tree of the spurge (Euphorbiaceae) family. It is native to tropical regions of North and South America in Amazon Rainforest (PIER, 2005). It is recognized by the many dark, pointed spines on its brown bark.
The Hura crepitan tree (plate 1.1) is reported to grow up to 30m (100ft) high in the Amazon region of South America with large ovate leaves which are papery thin (Adewale et al., 2014). This tree was introduced by travelers to Nigeria, though they do not grow up to 30m in height and their leaves are not as wide as observed in the Amazon region. This may be because of climatic conditions and type of soil. This tree grows up to 10m high in Nigeria. They are planted as shade trees along the walkways in church compounds, parks, cities and schools.
The fruits are tyre shaped capsules (plate 1.3), 3cm to 4cm in thickness with a diameter of 5cm to 6cm, with sixteen carpels arranged radially around the central axis (Adewale et al., 2014). When ripe and dry, capsules explode to disperse some seeds up to 14metres away. Hence, the name ‘Dynamite’ tree, because of the explosive sound of the ripe dry fruit as it splits into segments. The seeds are circular and flattened about 2cm in diameter with smooth brown colour.
This tree offers a wide range of uses, for instance, the plant secretes yellowish milky latex used by Amerindians to produce poisoned darts (Jones, 2007; Charles et al., 2007). In Nigeria, presently, the Hura crepitan tree is seen as an ornamental tree used to provide shade along walkways and within large premises such is its present relevance within the campus of the Federal University of Technology, Owerri (FUTO).
Plate 1.1: Friuts of Hura crepitan Tree
Plate 1.2: Seed of Hura crepitan Plate 1.3: Hura crepitan Matured Dried Friut of
1.2 STATEMENT OF THE PROBLEM
The use of wild plants and seeds in different localities provide optimum source of nutrients especially in times of food scarcity (Hamza, 2002), yet there is only scanty information on the nutritional and anti-nutritional qualities of some of these wild plants and seeds. Studies on such plants and seeds may help in identifying the values and potentials of the plant or seed. Also without investigation into the toxicological potentials of the seed or plant, the use of such materials in pharmaceutical or food preparations could have prolonged side effects that may occasionally be fatal./;
Hura crepitan seed is one of such seed that need to be studied. Within the campus of Federal University of Technology, Owerri (FUTO), some of the seeds are crushed by vehicles along the concrete roads. A closer look and touch on the crushed seeds reveal that the seed contain some reasonable quantity of oil. However, there is little information on the oil and antinutrient of the seeds.
1.3 THE OBJECTIVES OF THE WORK
The main objective of this work is therefore to evaluate the nutritional, amino-acid profile, oil characteristics and toxicological factors of sand box (Hura crepitan) seeds.
The specific objectives of this study are to:
- Determine the proximate composition of the seed flour
- Determine the vitamin and mineral contents of the seed flour
- Evaluate the amino acid profile of the seed flour
- Evaluate the anti-nutritional factors in the seed flour
- Extract and characterize the Hura crepitan seed oil vi. Assess the toxicological effect of the seed flour on albino rats.
1.4 JUSTIFICATION
It is envisaged that a good nutrient-balance, in the seed flour with regards to protein, amino acid and polyunsaturated fatty acid oil will reveal the potential of the seed for inclusion in human and animal diet.
There is an increasing need to search for oil from non-conventional and under-utilized tropical plant sources, sandbox seed (plate 1.2) is one potential example. The study may provide oils that could be used in foods, chemical industries, pharmaceutical industries and cosmetic industries. Thus it is envisaged that this study when completed may expose another oil seed with a good potential for food and industrial uses. Also Raw Materials Research and Development Council (RMRDC) have been fostering researches which will lead to development of our raw materials, oil seeds inclusive. The edible oils that are produced in commercial quantities are often used for non-edible industrial products such as biopesticides which are ecologically more tolerable than conventional chemical insecticides (Ajayi and Oderinde, 2002).
1.5 SCOPE OF THE STUDY
The study is aimed at carrying out a research on Hura crepitan seed with the hope of acquiring information on the seed and consequently its potential uses. Therefore the study will investigate the proximate composition, mineral and vitamin contents, amino acid profile, antinutrients contents of the Hura crepitan seed flour, the extraction and characterization of the oil, heptatoxic effects of the seed flour on test animals (albino rats) and microorganisms associated with the spoilage of the seed.
EVALUATION OF THE NUTRITIONAL, AMINO-ACID PROFILE, OIL AND TOXICOLOGICAL FACTORS OF SAND BOX (HURA CREPITAN) SEEDS
