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FUNGAL CONTAMINATION OF SPICES USED IN LOCAL MALAYSIAN CUISINES

SYAMILAH BINTI NORDIN

UNIVERSITI SAINS MALAYSIA JUNE 2021

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PUSAT PENGAJIAN TEKNOLOGI INDUSTRI UNIVERSITI SAINS

MALAYSIA

BORANG PENYERAHANDISERTASI MUTAKHIR SATU (1) NASKAH

Nama penyelia:

Bahagian:

Saya telah menyemak semua pembetulan/pindaan yang dilaksanakan oleh Encik/Puan/Cik

mengenai disertasinya sebagaimana yang dipersetujui oleh Panel Pemeriksa di Viva Voce-nya.

2. Saya ingin mengesahkan bahawa saya berpuashati dengan pembetulan/pindaan yang dilaksanakan oleh calon.

Sekian, terima kasih.

(Tandatangan dan cop) Tarikh

20/08/2021 Dr. Norlia binti Mahror

Bahagian Teknologi Makanan

Syamilah binti Nordin

DR. NORLIA MAHROR Senior Lecturer Food Technology Division School of Industrial Technology

Universiti Sains Malaysia 11800 Minden, Pulau Pinang

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FUNGAL CONTAMINATION OF SPICES USED IN LOCAL MALAYSIAN CUISINES

by

SYAMILAH BINTI NORDIN

A dissertation submitted in the partial fulfilment of the requirements for the degree of Bachelor of Technology (B.Tech) in the field of Food Technology.

School of Industrial Technology Universiti Sains Malaysia

June 2021

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DECLARATION BY AUTHOR

This dissertation is composed of my original work and contains no material previously published or written by another person except where due reference has been made in the text. The content of my dissertation is the result of work I have carried out since the commencement of my research project and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution.

SYAMILAH BINTI NORDIN JUNE 2021

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ACKNOWLEDGEMENTS

Alhamdulillah, all praise and gratitude to Allah, The Creator, The Greatest, The Most Kind and Merciful, who has guided, inspired, and given me the strength to finish my thesis writing.

My deepest appreciation and thanks to my supervisor, Dr Norlia Mahror for her kindness and guidance throughout my journey to do the research and the writing of the thesis. This thesis would not have been possible without her guidance.

Besides my supervisor, my appreciation goes to all the lecturers who have guided me and given the lectures regarding the thesis writing. Next, my particular gratitude also goes to the lab assistant, Mr. Ghoni, for assisting and guiding me a lot during my lab work.

I would like to thank my partner, Nurul Afifah Samsudin for helping me a lot during the research. I also thank my fellow coursemates especially, Nur Ili Hasbullah and Nurshatila Nasrudin for their kind words and support. I also would like to acknowledge my friend, Nurul Nadiah Sahire for her guidance and support throughout my thesis writing. Finally, I would like to thank my mum, Rijah binti Anton, my dad, Nordin bin Habibuding and my siblings for their endless support and love they have given to me.

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TABLE OF CONTENT

DECLARATION BY AUTHOR ii

ACKNOWLEDGEMENTS iii

TABLE OF CONTENT iv

LIST OF FIGURES vi

LIST OF TABLES vii

LIST OF ABBREVIATIONS viii

LIST OF APPENDICES ix

ABSTRAK x

ABSTRACT xii

1.1 Research background 1

1.2 Rationale of the study 2

1.3 Objectives 3

CHAPTER 2 LITERATURE REVIEW 4

2.1 Spices 4

2.1.1 Worldwide production of spices 6

2.1.2 Types of spice 7

2.1.2a Cinnamon 7

2.1.2b Clove 8

2.1.2c Star anise 9

2.1.2d Coriander seed 10

2.1.2e Pepper 11

2.1.2f Fennel 13

2.1.2g Cumin 14

2.1.2h Mixed spices 15

2.2 Fungal contamination in spices 16

2.2.1 Aspergillus spp. 17

2.2.2 Penicillium spp. 19

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v

2.3 Mycotoxins 19

CHAPTER 3 MATERIALS AND METHODS 22

3.1 Sample collection 22

3.2 Media and reagents 22

3.3 Preparation of agar 23

3.4 Fungal enumeration 23

3.4.1 Dilution plating 23

3.4.2 Direct plating 24

3.5 Fungal identification 24

CHAPTER 4 RESULTS AND DISCUSSION 25

4.1 Fungal enumeration 25

4.1.1 Ground spices 25

4.1.2 Whole spices 30

4.2 Identification of Aspergillus spp. and Penicillium spp. 32

4.3 Prevalence of Aspergillus spp. in ground spices 38

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 40

REFERENCES 41

APPENDICES 48

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LIST OF FIGURES

Figure Page

2.1 Cinnamaldehyde chemical structure 8

2.2 Eugenol chemical structure 9

2.3 Anethole chemical structure 10

2.4 d-linalool chemical structure 12

2.5 Piperine chemical structure 13

2.6 Cuminaldehyde chemical structure 15

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LIST OF TABLES

Table Page

2.1 List of some commonly used spices 6

2.2 Occurrence of toxigenic fungi in spices 19

4.1 Total fungal counts in different types of ground spices 29 4.2 Percentage of fungal infection in different types of whole

spices

32

4.3 Morphological characteristics of the species isolated from different types of spices

34

4.4 Isolation frequency of Aspergillus spp. in ground spices 40

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LIST OF ABBREVIATIONS

ADM Aspergillus Differentiation Medium

AFs Aflatoxins

BPW Buffered peptone water CFU Colony forming units CYA Czapek Yeast Autolysate DG18 Dichloran-glycerol Agar

MEA Malt Extract Agar

OTA Ochratoxin A

PDA Potato Dextrose Agar

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LIST OF APPENDICES

Appendix Page

Appendix A Ground spices sample description 47

Appendix B Halal certified samples packaging 49

Appendix C Fungal contamination in ground spices 50

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PENCEMARAN KULAT DALAM REMPAH YANG DIGUNAKAN DALAM MASAKAN TEMPATAN DI MALAYSIA

ABSTRAK

Rempah ratus banyak digunakan dalam pelbagai masakan di Malaysia untuk meningkatkan rasa dan aroma makanan. Walau bagaimanapun, rempah ratus terdedah kepada jangkitan kulat kemudian pencemaran mikotoksin sekiranya kaedah penyimpanannya sesuai untuk pertumbuhan kulat. Oleh itu, kajian ini bertujuan untuk menentukan kelaziman kulat mikotoksigenik dalam rempah yang digunakan dalam masakan tempatan Malaysia dan untuk mengenalpasti spesies-spesies kulat berdasarkan ciri-ciri morfologi. Sebanyak 48 sampel yang terdiri daripada serbuk rempah dan rempah penuh telah dibeli secara rawak di pasar runcit di Pulau Pinang. Tahap pencemaran oleh kulat di dalam serbuk rempah dan rempah penuh masing-masing ditentukan dengan menggunakan kaedah pencairan dan kaedah langsung. Berdasarkan penemuan daripada kaedah langsung, jintan putih (4.0 × 103 CFU/g) mencatatkan jumlah kulat tertinggi, diikuti dengan lada hitam (3.6 × 103 CFU/g) dan biji ketumbar (3.3 × 103 CFU/g).

Sementara itu, jangkitan kulat tertinggi untuk rempah penuh ialah biji ketumbar (91%) diikuti oleh lada hitam (90%) dan lada putih (87%). Sebaliknya, bunga lawang dan cengkeh bebas daripada pencemaran kulat. A. niger adalah spesies utama yang terdapat dalam serbuk campuran kurma dan serbuk kari dengan kekerapan pengasingan ialah masing-masing sebanyak 72% dan 65%. A. niger digambarkan sebagai koloni hitam dengan miselium yang tidak ketara pada Czapek Yeast Autolysate (CYA) dan Malt Extract Agar (MEA). Selain itu, A. carbonarius, A. chavelieri, dan A. tamarii juga diasingkan dari rempah ratus yang lain seperti lada hitam dan biji ketumbar dengan kekerapan pengasingan yang lebih rendah. A. niger adalah kulat mikotoksigenik yang

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biasanya menghasilkan okratoksin A, dan ia diklasifikasikan sebagai karsinogen yang berpotensi kepada manusia (Kumpulan 2B). Penemuan ini mungkin menunjukkan risiko pendedahan okratoksin kepada pengguna kerana rempah ratus biasanya digunakan dalam pelbagai masakan tempatan di Malaysia.

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FUNGAL CONTAMINATION OF SPICES USED IN LOCAL MALAYSIAN CUISINES

ABSTRACT

Spices are widely used in various cuisines in Malaysia to enhance the flavour and aroma of the cuisines. However, spices are susceptible to fungal infection and subsequent mycotoxin contamination if the storage conditions are favorable for the fungal growth.

Thus, this study aimed to determine the prevalence of mycotoxigenic fungi in spices used in Malaysian local cuisines and to identify the species based on morphological characteristics. A total of 48 samples that consist of ground spices and whole spices were purchased randomly at the retail market in Penang. The level of fungal contamination in ground and whole spices were analyzed using dilution plating method, and direct plating method, respectively. Based on the direct plating results, cumin (4.0 × 103 CFU/g) recorded the highest total fungal load, followed by black pepper (3.6 × 103 CFU/g) and coriander seed (3.3 × 103 CFU/g). Meanwhile, the highest incidence of fungal infection for the whole spices was recorded by coriander seed (91%) followed by black pepper (90%) and white pepper (87%). In contrast, star anise and cloves were free from fungal contamination. A. niger was the predominant species found in kurma mix and curry powder with the isolation frequency of 72% and 65% respectively. A. niger is described as black colonies with inconspicuous mycelium on Czapek Yeast Autolysate (CYA) and Malt Extract Agar (MEA). In addition, A. carbonarius, A. chavelieri, and A. tamarii were also isolated from other spices such as black pepper and coriander seeds with lower isolation frequency. A. niger is the most common mycotoxigenic fungi that produce ochratoxin A, which is classified as a potential human carcinogen (Group 2B). These

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findings might indicate the risk of ochratoxin exposure to the consumers since spices are commonly used in various local cuisines in Malaysia.

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