DETOXIFICATION AND ANTI-NUTRIENT REDUCTION OF JATROPHA CURCAS SEED CAKE BY FERMENTATION USING BACILLUS SPECIES

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ABSTRACT

Jatropha curcas seed cake is a by-product generated from the oil extraction of J. curcas seed- a biodiesel producing plant‘s seed. Although, the seed cake contains a high level of protein, it has Phorbol ester and some anti-nutritional factors such as phytic acid, saponin, lectin and trypsin inhibitor making it not to be applied directly in the food or animal feed industries. This study was aimed at detoxifying the toxin and reducing the anti-nutritional factors in J. curcas seed cake by fermentation using Bacillus species. Three Bacillus strains (Bacillus coagulans, Paenibacillus macerans, Paenibacillus polymyxa) 1.0 × 10⁸ cells MacFarland‘s standard per 100ml were used in the study. The seed cake used for the detoxification was extracted both manually and with the use of a machine. This fermentation was carried out on 10g of seed cake in 100ml of distilled water for 5 days with submerged fermentation. Temperature (27⁰ C, 30⁰ C and 37⁰ C), pH (4.5, 6.5, 8.5) and Time (24 h, 48 h, 72 h, 96 h and 120 h) were also varied. After fermentation the toxin and anti-nutritional factor level was determined. Results showed that Paenibacillus macerans was able to degrade the toxin and reduce the anti-nutritional factors in the seed cake more than the other two. After fermentation phorbol ester A and B, phytic acid, saponin, lectin and trypsin inhibitor were reduced by 76.4 %, 99.3 %, 56.3 %, 43.6 %, 58.8 % and 64.9 % respectively. The reduction may be due to the activities of esterase, phytase and protease enzymes. Jatropha curcas seed cake was detoxified by bacterial fermentation using the three Bacillus strains and the rich protein fermented seed cake could be potentially used as animal feed.

viii

	TABLE OF CONTENT
		PAGE
Cover page		i
Fly leaf		ii
Title page		iii
Declaration		iv
Certification		v
Dedication		vi
Acknowledgement		vii
Abbreviation		viii
Abstract		ix
Table of Content		x
List of Table		xiv
List of Figures		xv
List of Plates		xvi
List of Appendices		xvii
CHAPTER ONE
1.0	INTRODUCTION	1
1.1	Background Information	1
1.2	Statement of Problem	3
1.3	Justification	4
1.4	Aim and Objectives	5
1.4.1	Aim	5
1.4.2	Specific objectives	5
1.5	Hypothesis Testing	5

CHAPTER TWO
2.0	LITERATURE REVIEW	6
2.1	Origin and Spread	6
2.2	Nomenclature and Taxonomy	7
2.3	Description	7
2.4	Uses of Jatropha	9
2.4.1	The Jatropha tree erosion control and improved water filtration	9
2.4.2	Livestock barrier and land demarcation	10
2.4.3	Fuelwood	10
2.4.4	Support for vanilla	10
2.4.5	Green manure	11
2.4.6	Plant extracts	11
2.4.7	Stem	11
2.4.8	Bark and roots	11
2.4.9	Leaves	12
2.4.10	Fruits and seeds	12
2.5	Toxicity and Invasiveness	12
2.5.1	Toxicity	12
2.5.2	Invasiveness	14
2.6	*Jatropha* Cultivation	14
2.6.1	Climate	14
2.6.2	Soil	15
2.6.3	Plant nutrition	16
2.6.4	Water requirement	17
2.6.5	Pests and diseases	17
2.6.6	Seed yield	18
2.6.7	Seed Harvest, processing and uses of Jatropha oil	20
2.7	*Jatropha* Oil	21
2.7.1	Properties of Jatropha oil	21
2.7.2	Uses of Jatropha oil	22
2.8	Properties and Uses of the Seed Cake	25

2.8.1	Livestock feed	25
2.8.2	Organic fertilizer	27
2.8.3	Fuel	27
2.9	Using the Fruit Shells and Seed Husks	27
2.10	Phorbol Esters	30
2.10.1	Phorbol ester toxicity	31
2.10.2	Detoxification	34
2.11	*Bacillus*	37
2.11.1	Bacillus coagulans	38
2.12	*Paenibacillus*	41
2.12.1	Paenibacillus macreans	42
2.12.2	Paenibacillus polymyxa	43
2.13	Optimization	44
CHAPTER THREE
3.0	MATERIALS AND METHODS	45
3.1	Collection of Samples	45
3.2	*Isolation of Bacillus* Species**	45
3.3	Morphological Identification	46
3.3.1	Gram staining	46
3.3.2	Endospore staining	46
3.4	Biochemical Characterization	47
3.4.1	Microgen Kit for Bacillus ID	47
3.5	Raw Material and Proximate Analysis	47
3.5.1	Moisture content	48
3.5.2	Ash content	48
3.5.3	Crude protein	48
3.5.4	Crude fibre	49
3.5.5	Digestible carbohydrate	50
3.6	Analysis for Toxin and Anti-nutritional factors	50
3.6.1	Phorbol ester	50
3.6.2	Phytic acid	50

3.6.3	Lectin	51
3.6.4	Trypsin inhibitor	51
3.6.5	Saponin	51
3.7	Submerged Fermentation	52
3.8	Optimization of Detoxification Conditions	53
CHAPTER FOUR
4.0	RESULTS	54
CHAPTER FIVE
5.0	DISCUSSION	81
5.1	Proximate Analysis	81
5.2	Phytochemical Analysis	81
5.3	Effect of the Isolates on Phorbol Ester Detoxification	82
5.4	Effect of the Isolates on Phytic Acid Reduction	83
5.5	Effect of the Isolates on Saponin Reduction	84
5.6	Effect of the Isolates on Lectin Reduction	84
5.7	Effect of the Isolates on Trypsin Inhibitor Reduction	85
5.8	Effect of pH, Temperature and Time on Detoxification of
	Phorbol Esters by Isolates	85
CHAPTER SIX
6.0	SUMMARY, CONCLUSION AND RECOMMENDATION	87
6.1	Summary	87
6.2	Conclusion	88
6.3	Recommendation	88
REFERENCES		90
APPENDICES		105

xii

LIST OF TABLES

Tables		Page
Table 4.1	Proximate Composition of Dry Jatropha Curcas Seed Cake	55
Table 4.2	Percentage Reduction of Fermented Jatropha Curcas Seed Cake	57
Table 4.3	Effect of Isolates on Reduction of Phorbol Ester During
	Fermentation of Jatropha Curcas Seed Cake	59
Table 4.4	Effect of Isolates on Reduction of Phytic Acid During Fermentation
	of Jatropha Curcas Seed Cake	60
Table 4.5	Effect of Isolates on Reduction of Saponin During Fermentation
	of Jatropha Curcas Seed Cake	61
Table 4.6	Effect of Isolates on Reduction of Lectin During Fermentation
	of Jatropha Curcas Seed Cake	63
Table 4.7	Effect of Isolates on Reduction of Trypsin Inhibitor During
	Fermentation of Jatropha Curcas Seed Cake	64
Table 4.8	Effect of Temperature on Reduction of Phorbol Ester by Isolates
	During Fermentation of J. curcas Seed Cake.	65
Table 4.9	Effect of Fermentation Period on Reduction of Phorbol Ester on
	isolates During Fermentation of J. curcas Seed Cake	67
Table 4.10	Effect of pH on Reduction of Phorbol Ester by Isolates During

xiii

Fermentation of J. curcas Seed Cake

	LIST OF FIGURES
Figures		Page
Figure 4.1	Phytochemical Content of Dry J. Curcas Seed Cake Processed
	Using Different Oil Extraction Methods	56
Figure 4.2	Phorbol Ester Concentration at 27⁰C and pH 4.5	69
Figure 4.3	Phorbol Ester Concentration at 27⁰C and pH 6.5	71
Figure 4.4	Phorbol Ester Concentration at 27⁰C and pH 8.5	72
Figure 4.5	Phorbol Ester Concentration at 30⁰C and pH 4.5	73
Figure 4.6	Phorbol Ester Concentration at 30⁰C and pH 6.5	75
Figure 4.7	Phorbol Ester Concentration at 30⁰C and pH 8.5	76
Figure 4.8	Phorbol Ester Concentration at 37⁰C and pH 4.5	77
Figure 4.9	Phorbol Ester Concentration at 37⁰C and pH 6.5	79
Figure 4.10	Phorbol Ester Concentration at 37⁰C and pH 8.5	80

xiv

LIST OF PLATES

Plate

Page

Plate 2.1

Jatropha curcas Seed Cake From NARICT Zaria

	LIST OF APPENDICES
Appendix		Page
Appendix I	Gram Staining Characteristics of the Isolates	105
Appendix II	Endospore Staining Characteristics of the Isolates	106
Appendix III	Biochemical Test Characteristics of the Isolates	107

xvi

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background Information

Jatropha curcas is a species of flowering plant in the Euphorbiaceae family. It is native to the American tropics, especially Mexico and Central America (Janick and Robert, 2008). It is cultivated in tropical and subtropical regions around the world, becoming naturalized in some areas. The specific epithet, "curcas", was first used by the Portuguese doctor Garcia de Orta more than 400 years ago with uncertain origin. Common names includes Barbados Nut, Purging Nut, Physic Nut and JCL (J. curcas Linnaeus), whereas ―Lapalapa‖ (Yoruba) ―Binidazugu‖ (Hausa) and ―Owulo idu‖ (Ibo) in Nigeria. It is a multipurpose tree because of industrial and medicinal uses.

J. curcas is a poisonous, semi-evergreen shrub or small tree, reaching a height of 6 m (Janick and Robert, 2008). It is resistant to a high degree of aridity, allowing it to be grown in deserts. The seeds contain an average of 34.4% oil (Achten et al., 2008) with a range between 27-40% (Achten et al., 2007). Besides the economic potential of processing the oil to produce high-quality biodiesel fuel usable in a standard diesel engine, the seeds also contain the highly poisonous toxalbumin curcin.

Bacillus is a genus of Gram-positive, rod-shaped (bacillus) bacteria and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes (oxygen reliant), or facultative anaerobes (having the ability to be aerobic or anaerobic). They test positive for the enzyme catalase when there has been oxygen used or present (Turnbull, 1996). Ubiquitous in nature, Bacillus includes both free-living (non-parasitic) and parasitic pathogenic species.

Under stressful environmental conditions, the bacteria can produce oval endospores and thus remain in a dormant state for very long period of time. (Madigan and Martinko, 2005). The main habitat of endospore-forming Bacillus organisms is the soil. B. subtilis strains secrete enzymes, such as amylase, protease, pullulanase, chitinase, xylanase, lipase, and esterase are produced commercially and their production represents about 60% of the commercially produced industrial enzymes (Morikawa, 2006).

Esterases and lipases catalyze the hydrolysis of ester bonds and are widely distributed in animals, plants and microorganisms. In organic media, they catalyze reactions such as esterification, intesterification and transesterification (Kawamoto et al., 1987). Esterases differ from lipases mainly on the basis of substrate specificity and interfacial activation (Long, 1971). Esterases are found in plants, animals and microbes, but the majority of industrially produced esterase are derived from microbial sources. This is because they can be engineered for production of esterase with desirable properties for industrial need. The microbial sources include bacteria, fungi, yeasts and actinomycetes (Torres et al., 2005). The applications of esterases are found in various fields, including inorganic synthesis process.

Paenibacillus is a genus of facultative anaerobic, endospore-forming bacteria, originally included within the genus Bacillus and then reclassified as a separate genus in 1993 (Ash et al., 1993). Bacteria belonging to this genus have been detected in a variety of environments such as soil, water, rhizosphere, vegetable matter, forage and insect larvae, as well as clinical samples (Lal and Tabacchioni, 2009: McSpadden-Gardener, 2004: Montes et al., 2004: Ouyang et al., 2008).

1.2 Statement of Problem

The seeds of J. curcas contain oil, which can be used as a renewable biodiesel source and applications in the manufacture of soaps and cosmetics (Makkar et al., 1998). J. curcas seed cake is a by-product generated from the oil extraction of J. curcas seeds in a biodiesel processing plant. It has high protein content of approximately 50 -60% (Haas and Mittelbach, 2000) and could be used in animal feeds and also as protein hydrolysate. However, it contains the phorbol esters, which are toxic compounds, and the anti-nutritional factors such as trypsin inhibitors, phytic acids, lectins and saponins.

Phorbol esters are the most potent tumor promoters known. They exhibit a remarkable ability to amplify the effect of a carcinogen but are themselves not carcinogenic (Wender et al., 1998). The seeds from J. curcas had been reported to be orally toxic to humans, rodents and ruminants of which phorbol esters had been identified as the main toxic agent (Becker and Makkar, 1998). Pure phorbol esters can kill when administered in microgram quantities (Heller, 1996). Ingestion of phorbol esters (LD₅₀ for mice: 27mg/kg body mass) can cause lung and kidney damage, resulting in fatality (Li et al., 2010).

Detoxification of toxin is necessary for J. curcas seed meal utilization, after which the detoxified seed cake may be used as animal feed and its protein hydrolysate (fermented liquid) as plant growth promoter. Biological detoxification of J. curcas seed cake has not been widely studied. However, toxins in cotton seed were successfully detoxified by microbial fermentation (Zhang et al., 2006).

Despite its intrinsic advantages, J. curcas seed like soybean seed has the problem of antinutritional factors. In addition to thermos-labile lectins and trypsin inhibitors, J. curcas

contains toxic lipo-soluble but thermo-stable phorbol esters (Heller, 1996: Makkar and Becker, 1997). Phorbol esters have to be removed or lowered to levels that do not elicit a toxic response from animals in order for the J. curcas seed meal to be used as an ingredient in livestock feeds. Makkah and Becker (1997) reported that phorbol esters were highly soluble in ethanol, giving some possibility of detoxification of the meal.

1.3 Justification

J. curcas seed cake is well adapted to grow in marginal areas with low (480mm) rainfall and poor soils. In such areas, it grows without competing for space with food crops (Gaydou et al., 1982: Heller, 1996). J. curcas seed meal (10-20g Kg^-1 residual oil) has a crude protein content ranging from 580-640g Kg-¹ of which 90% is true protein (Makkar et al., 1997: Makkar and Becker, 1997). The plant‘s ability to thrive in marginal areas and its high crude protein makes it an attractive complement and or substitute to soybean meal as a protein source in livestock feeds. The use of J. curcas will reduce the competition between man and livestock for soybean that is currently prevailing since soybean is used in both livestock and human feeds. Phorbol esters are the major impediment to the wide commercial use of Jatropha meal as feedstock. During extraction of oil from Jatropha seed, 70-75% of Phorbol esters associate with the oil and 25-30% remain strongly bound to the matrix of seed meal (Wink et al., 1997). The Phorbol esters have been found to be responsible for skin-irritant effects and tumor promotion (Wink et al., 1997). J. curcas seed cake is mainly used as manure and can be made more useful when detoxified and hence its use in animal feeds. Thus, this research was set to achieve this following aim and objectives.

1.4 Aim and Objectives

1.4.1 Aim

The aim of this study was to detoxify and reduce the anti-nutritional factors in J. curcas seed cake by fermentation using Bacillus species.

1.4.2 Specific Objectives

The specific objectives of this study were to:

1. Isolate and identify Bacillus species from the soil and kilishi.

2. Determine the proximate composition and phytochemical factors of non-fermented and fermented J. curcas seed cake.

3. Determine the reduction of phorbol esters and anti–nutritional factors in J. curcas seed cake by fermentation using Bacillus species.

4. Determine the optimal environmental condition for the detoxification of the Jatropha curcas seed cake.

1.5Hypothesis Testing

H₀ - Bacillus coagulans, Paenibacillus polymyxa and Paenibacillus macerans have no effect on the detoxification and anti-nutritional factors reduction in J. curcas seed cake

H_a - B. coagulans, P. polymyxa and P. macerans have effect on the detoxification and anti-nutritional factors reduction in J. curcas seed cake

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