antimicrobial properties of essential oils againgst bulk-tank milk isolated bacteria

114 Pages·2017·2.91 MB·English

ANTIMICROBIAL PROPERTIES OF ESSENTIAL OILS
AGAINGST BULK-TANK MILK ISOLATED BACTERIA

TSHEGOFATSO NHABE

Dissertation submitted in fulfillment of the requirement for the degree

MASTER OF HEALTH SCIENCE

ENVIRONMENTAL HEALTH

In the

Department of Life Science

At the

Central University of Technology, Free State

Supervisor: Dr N.J. Malebo (Ph.D. Microbiology)

Bloemfontein
South Africa
2017

© Central University of Technology, Free State
DECLARATION OF INDEPENDENT WORK

I, Tshegofatso Nhabe, hereby declare that this research project submitted to the Central University
of Technology, Free State for the degree MASTER OF HEALTH SCIENCE: ENVIRONMENTAL
HEALTH is my own work and has not been submitted before to any institution by myself or any
other person in fulfillment of the requirements for the attainment of any qualification.

Signature Date
.......................... .....................

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© Central University of Technology, Free State
ACKNOWLEDGEMENTS

 I would first like to thank God for giving me the strength to finish this degree and for seeing
me through it all.
 Secondly, Dr N.J Malebo; my supervisor for her willingness to accept me as a student. I
could not have asked for a more supportive and knowledgeable advisor, both in and outside
the laboratory setting. Through all of my fears and doubts, she saw something and always
believed in me. The encouragement I have received from her to develop and trust my own
critical thinking skills has been invaluable, and I am profoundly grateful that she made the
decision to mentor me.
 Every member of the Department of Life Sciences has my heartfelt thanks. Nothing could
have been done without the aid I received from my instructors, fellow classmates, and
friends.
 My family, especially my grandmother; she has been a constant source of aid especially
these past two years. I am so thankful that I did not have to go through this period of time
without her. I could not ask for a more loving and supportive grandmother and continually
thankful for the blessing that she is.
 I am highly indebted to Central University of Technology, Free State for providing a
wonderful opportunity to study my Master‟s degree in Environmental Health and awarding
me a National Research Fund student grant.
 Lastly, I would like to thank the Department of Education for providing the financial grant
throughout the study period.

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© Central University of Technology, Free State
TABLE OF CONTENTS
CHAPTER ONE
LITERATURE REVIEW: ANTIMICROBIAL PROPERTIES OF ESSENTIAL
OILS AGAINST BULK-TANK ISOLATED BACTERIA
Page
Summary 3
1. Introduction 5
1.1 Psychotropic bacteria 5
1.2 Coliforms 6
1.3 Spore forming bacteria 7
2. Possible sources of contamination 7
2.1 Microbial contamination within the udder 9
2.2 Equipment 10
3. Antibiotics 11
3.1.1 Antibiotic resistance 13
3.1.2 Implications of antibiotic resistant bacteria in milk 15
4. Strategies for control 16
5. Alternative antimicrobials 17
5.1 Mode of action of Essential Oils 19
5.2 Susceptibility of gram-negative and gram-positive bacteria 21
6. Artemisia afra (Lengana) 23
6.1 Morphology and Traditional uses 24
6.2. Biological activity of Artemisia afra 25
7. Rationale 25
8. References 27
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© Central University of Technology, Free State
CHAPTER TWO
QUANTIFICANTS AND IDENTIFICATION OF BULK-TANK ISOLATED
BACTERIA
Page
2.1 Abstract 39
2.2 Introduction 40
2.3 Materials and methods 42
2.3.1 Sample collection 42
2.3.2 Isolation and identification of bacteria 43
2.4 Confirmation of microbial isolates 43
2.4.1 MALDI TOF MS 43
2.5 Results and Discussion 45
2.6 Conclusion 55
2.7 References 57

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© Central University of Technology, Free State
CHAPTER THREE
ANTIBIOTIC RESISTANCE PROFILES OF BULK-TANK ISOLATED
BACTERIA
Page
3.1 Abstract 64
3.2 Introduction 65
3.3 Materials and methods 67
3.4 Results and Discussion 68
3.5 Conclusion 72
3.6 References 73

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© Central University of Technology, Free State
CHAPTER FOUR
ANTIMICROBIAL PROPERTIES OF ARTESIMIA AFRA AGAINST BULK-
TANK ISOLATED BACTERIA
Page
4.1 Abstract 78
4.2 Introduction 80
4.3 Materials and methods 81
4.3.1 Essential oil extraction and characterization 82
4.3.2 Bio-assay preparation 82
4.3.3 Scanning Electron Microscopy and Transmission Electron Microscopy 82
4.4 Results and Discussion 83
4.5 Conclusion 90
4.6 References 91

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CHAPTER FIVE
CONCLUSIONS AND RECOMMENDATIONS

Page
5.1 Introduction 96
5.2 General discussion 97
5.3 Conclusions and recommendations 100
5.4 References 103

VII

© Central University of Technology, Free State
LIST OF FIGURES
Chapter 1 Page
Figure 1: Possible sources of contamination 9
Figure 2: Mechanisms of action and target sites of essential oils on microbial cells
21
Figure 3: Live plant of A. afra (Lengana) 23
Chapter 4
Figure 1: Pseudomonas rhodesiae bio-assay 87

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LIST OF TABLES
Chapter 1 Page
Table 1: Major antibiotics grouped according to their mechanisms of action and their
chemical structures 13
Chapter 2
Table 1: Standard plate counts of samples taken in August 2015 51
Table 2: Standard plate counts of samples taken in November 2015 52
Table 3: MALDI TOF MS direct colony and Extraction method isolates 54
Chapter 4
Table 1: Chemical composition of A. afra oil 83
Table 2: SEM of gram negative bacteria treated with A. afra oil and their controls
88
Table 3: TEM gram negative bacteria treated with A. afra oil and their controls
89

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