EVALUATION OF EFFECTS OF CAPSAICIN IN SUPPRESSION OF PLANT PATHOGENS IN TOMATOES.

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ABSTRACT

Chilies contain an active component known as capsaicin. It is hydrophobic and is the main capsaicinoid. Due to this component, chilies have obtained certain bioactive components such as antioxidants, phenolic and anti-microbial properties. Based on the mentioned characteristics, chilies exhibit bio pesticide characteristics which can be used to control pest and diseases in crops. In this study, four varieties of chilies were used, including habanero, bird’s eye, cayenne and bullet chilies. This was to assess the potential utilization of capsaicin in control of late blight fungal disease and bacteria speck in tomatoes
To determine the capsaicin content in the four varieties, extraction of capsaicin from both oven- dried and fresh chilies was conducted using three different solvents at different amounts namely 100ml, 150ml and 200ml which included, methanol 99%, a mixture of methanol 99% and acetone and ethanol 99%. Evaluation of the antioxidant and total phenolic composition of capsaicin from the four varieties was analyzed using the Dipheny1-1-picrtyl-hydrazyl (DPPH) method and the absorbance at λmax 760nm measured on a spectrophotometer respectively. Determination of the sensitivity of capsaicin on late blight and bacterial speck in tomatoes was conducted using the agar dilution method. Moreover, determination of the antimicrobial properties of capsaicin from the four varieties of chilies was conducted by examining the minimum inhibitory concentration and the minimum bactericidal concentration.
From extraction using different solvents at different amounts, the ideal solvent was said to be ethanol at 200ml giving an ideal extraction ratio of 1:10. Fresh habanero, bird’s eye, cayenne and bullet chilies had the highest amount of capsaicin respectively at an amount of 23.09 mg/g, 12.36mg/g, 7.37mg/g and 3.80mg/g respectively. Oven died habanero, bird’s eye, cayenne and bullet chilies had the highest amount of capsaicin respectively at an amount of 16.84mg/g, 9.60mg/g, 6.39mg/g and 3.21mg/g respectively. Therefore, fresh chilies had a higher amount of capsaicin than oven dried chilies.
A positive correlation was observed between the amount of capsaicin present in the chili varieties and the bioactive components available in the chilies. Total phenolic was highest in both fresh and oven- dried habanero, bird’s eye, cayenne and bullet chilies respectively, having fresh chilies with a higher total phenolic than oven-dried chilies. Total phenolic ranged between 2923mg/g- 352mg/g in fresh chilies and 2274.5mg/g – 117mg/g in oven-dried chilies.
Similarly, to total phenolic, there was a correlation between the amount of capsaicin in a variety and antioxidant activity in the same. Antioxidant activity was highest in both fresh and oven- dried habanero, bird’s eye, cayenne and bullet chilies respectively, having fresh chilies with a higher antioxidant activity than oven-dried chilies. Antioxidant activity ranged between 50%- 86% in fresh chilies and 46%-74% in oven-dried chilies.
Capsaicin from fresh chilies had a higher inhibition than oven-dried chilies. Bacterial speck was more susceptible to capsaicin than late blight across all varieties. Zone of inhibition for bacterial speck ranged between 0.96cm – 2.48cm in fresh chilies and 0.63cm – 1.95cm in oven- dried chili having habanero with highest zone of inhibition and bullet chili the lowest. Zone of inhibition for late blight ranged between 0.68cm – 2.22cm in fresh chilies and 0.56cm – 1.75cm in oven-dried chili having habanero with highest zone of inhibition and bullet chili the lowest as well.
Capsaicin from fresh chilies had a higher MBC than oven-dried chilies. Late blight was more susceptible to the capsaicin than bacterial speck across all varieties. Minimum bactericidal concentration for late blight ranged between 0.01% - 0.18% in the first tube and 0.21% - 0.53% in the second tube incubate, using fresh chilies and 0.02% - 0.20 in the first tube and 0.29% - 0.55% in the second tube incubated, using oven-dried chili having habanero with highest minimum bactericidal concentration and bullet chili the lowest. Minimum bactericidal concentration for bacterial speck ranged between 0.01% - 0.19% in the first tube and 0.26% - 0.55% in the second tube incubate, using fresh chilies and 0.02% - 0.21% in the first tube and 0.30% - 0.61% in the second tube incubated, using oven-dried chili having habanero with highest minimum bactericidal concentration and bullet chili the lowest.as well.
From the study, capsaicin from habanero exhibited the most preferred characteristics of bio pesticides from the four varieties tested. A positive correlation between amount of capsaicin and the efficiency of the bio active components characteristics as a bio pesticide was observed. Thus, the higher the amount of capsaicin, the higher the efficacy of capsaicin as a bio pesticide.
More study of the efficacy of capsaicin in diseases in tomatoes under in vivo environment is recommended to evaluate the influence of environmental conditions in the bio pesticide properties.



 
Table of Contents
 
DECLARATION ii
Plagiarism Declaration Form for Students iii
ACKNOWLEDGEMENT iv
DEDICATION v

CHAPTER ONE: INTRODUCTION
1.1 Background Information 1
1.2 Statement of the problem 2
1.3 Justification 3
1.4 Aim 4
1.5 Purpose of the study 4
1.6.1 General Objective; 4
1.6.2 Specific Objectives; 4
1.7 Research questions 5

CHAPTER 2: LITERATURE REVIEW
2.1 Chili 6
2.1.1 Nutritional composition 6
2.1.2 Bioactive components in chili 6
2.1.2.1 Capsaicin in chilli 7
2.2 Antimicrobial properties in capsaicin 8
2.2.1 Antifungal properties 8
2.2.2 Antibacterial properties 8
2.2.3 Insecticidal properties 8
2.2.4 Antioxidant activity 8
2.3. Tomato 9
2.3.1 Overview 9
2.3.2 Common Diseases in tomatoes 10
2.3.2.1 Bacteria Spot 10
2.3.2.2. Bacterial Speck 11
2.3.2.3 Bacterial Canker 11
2.3.2.4. Early Blight 12
2.3.2.5. Late Blight 12
2.3.2.6 Fusarium wilt 12
2.3.2.7. Damping off 13
2.3.3. Synthetic pesticides in tomatoes 13
2.3.4. Negative impacts of use synthetic of pesticide in tomato production 14
2.4 Knowledge Gap 15

CHAPTER THREE: DETERMINATION OF THE CAPAICIN CONTENT OF CHILI VARIETIES GROWN IN KENYA
ABSTRACT 16
3.1. INTRODUCTION 17
3.2. EXPERIMENTAL DESIGN 17
3.3. MATERIALS AND METHODS 18
3.3.1. STUDY SITE 18
3.3.2. MATERIALS 18
3.3.3. METHODOLOGY 18
3.3.3.3.2. Extraction with methanol 99% and acetone 19
3.4. RESULTS AND DISCUSSION 20
3.5. CONCLUSION 30
3.6. RECOMMENDATIONS 30

CHAPTER FOUR: EVALUATION OF THE ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC COMPOSITION OF CAPSAICIN
ABSTRACT 31
4.1. INTRODUCTION 32
4.2. EXPERIMENTAL DESIGN 33
4.3. MATERIALS AND METHODS 33
4.3.1. MATERIALS 33
4.3.2 METHODOLOGY 33
4.4.2. ANTIOXIDANT ACTIVITY ON FRESH AND OVEN-DRIED CHILIES 38
4.5. CONCLUSION 41
4.6. RECOMMENDATION 41
CHAPTER FIVE: DETERMINATION OF ANTIMICROBIAL PROPERTIES OF CAPSAICIN ON LATE BLIGHT AND BACTERIAL SPECK IN TOMATOES 42
ABSTRACT 42

5.1. INTRODUCTION
5.2. EXPERIMENTAL DESIGN 46
5.3. MATERIALS AND METHODS 47
5.3.1. MATERIALS 47
5.3.2. PREPARATION OF CHILI EXTRACT 47
5.4. RESULTS 50
5.5. DISCUSSIONS 51
5.5.1. Effect of capsaicin extracted from chili varieties on the in vivo inhibition zones against bacterial speck and late blight of tomato 51
5.5.2. Effect of chili varieties on the minimum inhibitory concentration against bacterial speck and late blight of tomato 52
5.6. CONCLUSION 54
5.7. RECOMMENDATIONS 54

CHAPTER SIX; GENERAL CONCLUSIONS AND RECOMMENDATION
6.1: GENERAL CONCLUSIONS 55
References 56
 


ABBREVIATIONS

DPPH- Diphenyl-1-picryl-hydrazyl EU – European Union
KEPHIS – Kenya Plant health Inspectorate Service MBC- Minimum bactericidal concentration
MIC – Minimum inhibitory concentration MRLs – Maximum residue levels
PCPB – pest control product board PHI – pre harvest interval
PTC – plasma total cholesterol RNS – Reactive nitrogen species ROS – Reactive oxygen species SMO – Singlet molecular oxygen WHO – World Health Organization




 
CHAPTER ONE
INTRODUCTION

1.1 Background Information

Chilies contain an active component known as capsaicin. It is hydrophobic and is the main capsaicinoid (Adiellsson, 2009). It has been shown to contain bioactive compounds that have potential antimicrobial and antifungal properties. Further, it is also very rich in antioxidants and is attributed to the ability to act as a scavenger of singlet molecular oxygen (SMO), reactive oxygen species (ROS), peroxyl radicals and reactive nitrogen species (RNS) (Derbalah A, 2019). It has gradually been adapted to control diseases in various crops but the dosage of each of these bioactive compounds required to inhibit the development of a disease and microbial growth is yet to be established (Gervais et al., 2008).

Tomato (Solanum lycopersicum) is from the nightshade family and originated from South America in the 16th century. It is known to be rich in various nutrients that play a great role in the functioning of the body systems. Fiber aids in our digestive health and regular bowel movements. Vitamin C which is an essential nutrient acts as an antioxidant, potassium aids in blood pressure control and heart disease prevention, Vitamin K1 helps in blood clotting and bone health while Folate aids in normal tissue growth and cell functions. It is also rich in carotenoid that is responsible for the red pigmentation in tomatoes and considered an antioxidant as well, beta carotene which is an antioxidant and is converted into vitamin A in our bodies. Naringenin is a flavonoid that aids in decreasing inflammation. Chlorogenic acid is a powerful antioxidant and is known to lower blood pressure (Bjarnadottir, 2019) and many other beneficial components. All these beneficial traits in tomatoes are dependent on the physiological and phenological traits in the tomato. If any of the traits is compromised, the levels of the beneficial traits are reduced or depleted ( Vliet, 2017).

Tomatoes constitutes 98 % of the vegetables usually consumed at household level and 82% of the most consumed vegetable from high end to low level consumer in Kenya ranking as the highest consumed vegetable (Dijkstra, 2015). This shows the importance of tomatoes in Kenya.

From a report released by Kenya Plant Health Inspectorate Service (KEPHIS) in 2017, the major challenges faced in tomato production is plant pest and diseases and high pesticide residues ranking at 54% and 48% respectively. Many of these diseases occur both in open field and controlled environment and are classified as either fungal, bacterial or viral. Some of the major diseases include bacterial canker, bacterial speck, bacterial spot, bacterial wilt, buckeye rot, early blight, fusarium wilt, gray mold, late blight, tomato mosaic virus, tomato spotted wilt, tomato yellow leaf curl among others (Jones , et.,al 2014). These diseases have posed to be the largest threat to tomato production in Kenya. Diseases are most prevalent in the tropics (savary, 2014)and damage ranges from reduced plant vigor to plant death by interfering with plant physiological processes therefore the plant losses its ability to synthesize its bio chemical properties and nutritive qualities (Muhammad, 2014). Therefore, the range of diseases attacking tomatoes is large and needs effective disease control methods. The increased demand of tomatoes on account of population explosion and increased consumption frequency of tomatoes has compelled the use pesticides for better crop protection (Tomer, 2013). Continuous use and accumulation of synthetic pesticides have led to pesticide toxicity which has resulted to oxidative stress on plants as a result of the generation of reactive oxygen species (ROS) (Xia et al., 2009). Several cases of synthetic pesticide poisoning are recorded every year. Risks of poisoning depend on toxicity, dose and period of exposure. Pesticide toxicity may cause neurological and psychiatric complications, brain tumors, cancers, spontaneous abortions, stillbirths, and birth defects. This has led to use of bio pesticides as an alternative since they are able to breakdown naturally causing no toxicity. Therefore, the extract chili can be used as an alternative to synthetic pesticides to control diseases and increase the level of antioxidants in tomatoes (Sawhney et al., 2016).

1.2 Statement of the problem

Chilies are rich in antibacterial and antifungal properties (savary, 2014). Heavy investment on the efficient uses of synthetic products in relation to anti-microbial properties. However, very limited data and information of use of natural antibacterial and antifungal extracts or products in control of microbial growth in tomatoes despite its contribution to nutrition and health (Tsimbiri, 2015).

Bioactive compounds in chili are underutilized and can be used to develop bio products with anti-microbial properties (Mitem S.E, et al., 2018). There are very few products which have been developed from chilies while they have the ability to develop a potential bio product.

Kenya chili production is only 1% of the horticultural production. Production mainly targets export market with local consumption being as low as 6% of the total production (KEPHIS, 2016), this shows that there is low utilization of chili in Kenya as human food and commercial product.

This study will therefore explore ways of utilizing chilies as a functional food and antimicrobial in making bio pesticides in tomato production.

1.3 Justification

According to World’s Healthiest Foods, tomatoes stands out in terms of the mix of phytonutrients components such as anti-inflammatory, antioxidants, minerals, vitamins and many more. Besides the various benefits, it is the most consumed vegetable in Kenya proving to be readily available and affordable to most of the people in Kenya (Dijkstra, 2015). According to World Health Organization (WHO), due to the growing population and demand, the need to grow tomatoes has risen but the crop experiences wide range of diseases leading to economic losses, low yields, inability to meet demand and high pricing.

In attempt to control these diseases, high levels of pesticide residues have occurred as pre harvest intervals (PHI) are not observed due to demand. This has led to the loss of the phytonutrients and bio compounds in tomatoes leaving the consumer not benefiting from these properties but experiencing health deterioration caused by pesticide residues. From a study carried out recently, tomatoes ranked highest on having pesticide residues. The tests detected an average of 7.1 pesticides on every conventionally grown tomato sample collected, with a maximum of 19 different pesticides or breakdown products on a single sample. Four pesticides, one insecticide and three fungicides were responsible for the bulk of the residues detected on tomatoes. This came along with lower levels of nutrients and bioactive compounds in the samples (Tomer, 2013).

Several millions of cases of synthetic pesticide poisoning are registered every year. Risks of poisoning depend on toxicity, dose and period of exposure (Alexandra , 2009). Pesticide toxicity may cause neurological and psychiatric complications, brain tumors, cancers, spontaneous abortions, stillbirths, and birth defects. (Adams, 2009) Pesticides are detrimental to the environment and produce considerable damage to ecosystems. They may be harmful to non-target species, pollute air, water and soil. Pesticides considerably affect natural biological equilibrium, diminish biodiversity, reduce nitrogen fixation, contribute to the disappearance of pollinators, threaten fish, and destroy bird and animal habitats (Tomer, 2013). This has led to recommendation of bio pesticides which naturally breakdown and help in maintaining the beneficial properties of tomatoes and also aid in reducing the levels of pesticide residues and observing MRLs.

Chilies have been recently used as a bio pesticide due to various active compounds which are been known to be antifungal and antibacterial. However, there is limited knowledge on the dosage of the optimum concentration required to inhibit growth of the fungus and reduce microbial activity (Gervais et al., 2008). The results of this study will help give a recommended dosage of the optimum concentration of extract chili and the level of antioxidants in the extract chili which will also be beneficial to tomatoes.

1.4 Aim.

To assess the potential utilization of capsaicin in control of major fungal and bacterial plant pathogens in tomatoes.

1.5 Purpose of the study.

To contribute to the reduction of pathogenic contamination in tomatoes with bioactive compounds in chili.

1.6 Objectives

1.6.1 General Objective;

To evaluate the antimicrobial and antioxidant properties of bioactive compounds in capsaicin for use in the suppression of plant pathogens in tomatoes.

1.6.2 Specific Objectives;

i. To determine the capsaicin content of chili.
ii. To evaluate the total phenolic composition of capsaicin and antioxidant activity.
iii. To determine the suppressive effect of capsaicin on late blight and bacterial speck in tomatoes.
iv. To determine the antimicrobial (antibacterial and antifungal) properties of capsaicin.

1.7 Research questions

i. What is the capsaicin content of chili?
ii. What is the total phenolic composition of capsaicin and antioxidant activity?
iii. What concentration of capsaicin inhibits the growth of late blight and bacterial speck in tomatoes?
iv. What is the antimicrobial (antibacterial and antifungal) properties of capsaicin?

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