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

 

 

ix


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

 

 

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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

 

 

xi


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

68

 

 

 

 

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


26


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

xv


 

 

 

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.

 

1


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).

 

 

2


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

 

3


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.

 

 

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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

 

Ha - 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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