UNIVERSITI PUTRA MALAYSIA MICROBIOLOGICAL AND BIOCHEMICAL CHANGES OF FRESHWATER PRAWNS (MACROBRACHIUM ROSENBERGII) DURING STORAGE

Transcription

1 UNIVERSITI PUTRA MALAYSIA MICROBIOLOGICAL AND BIOCHEMICAL CHANGES OF FRESHWATER PRAWNS (MACROBRACHIUM ROSENBERGII) DURING STORAGE FATIMAH BINTI ABU BAKAR FSAS

2 MICROBIOLOGICAL AND BIOCHEMICAL CHANGES OF FRESHWATER PRAWNS (MACROBRACHIUM ROSENBERGII) DURING STORAGE By FA TIMAH BINTI ABU BAKAR Thesis Submitted in Fulfdment of the Requirement for the Degree of Doctor of Philosophy in the Faculty of Science and Environmental Studies Universiti Putra Malaysia March 2001

3 Lovingly for Abang, Ayong and 0 'meil, They matter more, above all else

4 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy. MICROBIOLOGICAL AND BIOCHEMICAL CHANGES OF FRESHWA TER PRAWNS (MACROBRACHIUM ROSENBERGII) DURING STORAGE By FATIMAH BINTI ABU BAKAR March 2001 Chainnan: Associate Professor Dr. Che Nyonya Abdul Razak Faculty: Science and Environmental Studies Tropical freshwater prawns are of considerable interest because of the importance of this commodity in international trade. They are one of the highest valued products in international fisheries trade and as such have good long term potential. Studies on the bacteriological and biochemical changes 'of pond water and cultured freshwater prawns were carried out to evaluate the relationship between water quality and microbiological changes during storage. Prawns were taken from three sampling sites viz: Site 1- Kg. Jumbang, Negri Sembilan; Site 2- Kg. Cangkat Tin, Perak and Site 3- Kg. Cenderiang, Perak. They were then stored at 3 different storage conditions: 1) 20 d at ambient (280 ± 2 C); 2) 10 d at 100 ± 2 C and 3) 16 d at iced storage (4 ± 2 C). Microbiological analysis was performed for total mesophilic and psychrophilic aerobic COWlts, proteolytic bacterial COWlts, histamine producing bacteria, cadaverine producing bacteria and putrescine producing bacteria in the prawns and pond water for the three sites using appropriate III

5 bacteriological agar media. Biochemical analysis was carried out for ph profiles using ph meter, freshness in terms of K-values from the test paper strips, amino acids profiles using the amino acid analyser, total volatile bases (TVB) and total volatile acids (TVA) from the distillation methods and biogenic amines viz; histamine, cadaverine and putrescine using the high performance liquid chromatography (HPLC) method. Results obtained showed that the microbiological quality of freshwater prawns was related to the micro flora of pond water where they were grown. The initial counts indicated the values were in the range of log 4+ CFU/g for all samples. Total mesophilic and psychrophilic counts of the head regions were higher than that of the body regions for all prawn samples and types of growth media tested. All samples showed an increase in counts with time and temperature of storage up to log 7+ CFU/g for mesophilic counts after 12 h at ambient, 6 d at looc and 12 d at iced storage. Site 2 samples had relatively higher counts as compared to that of the other two sites which correlated well with the levels determined in their pond water. Similar trends were observed for psychrophilic counts but at lower values for different types of media studied. Biochemical results showed similar trends of increment in values and were closely related to the microbiological data until spoilage set in. Effect of preservatives on quality changes, biogenic amines production and shelf life of prawns during iced storage were investigated by sensory, microbiological, physical and chemical analyses. Sensory results indicated that the whole prawns had a shelf life of about 20 d when treated with 2% sucrose, boric acid, lactic acid, sodium chloride, and sodium metabisulfite except for control IV

6 which had only 15 d shelf life. Boric acid, lactic acid and sodium metabisulfite managed to inhibit psychrophilic bacteria and biogenic amines formation in prawns while maintaining the mesophilic counts at lower levels during iced storage. Different analytical methods for the determination of histamine in prawns were evaluated. Use of enzymatic methods in the form of oxygen electrode and spectrophotometer offered excellent alternative methods for determination of histamine in freshwater prawns. The experimental results showed there was an increase of histamine measured by the oxygen electrode and very good correlation of above 0.98 with the HPLC analyser for all samples. Recovery percentages of about 100% for values of histamine between Ilg/g was observed. Similarly, use of spectrophotometer with coupled diamine oxidase and horseradish peroxidase in the system allowed a semi quantitative estimation of histamine in prawns. Correlation coefficients of r 2 = 0.99 were observed when compared to that of the HPLC method. Percentages of recovery of histamine in augmented samples were also about 100% for concentration of histamine between Ilg/g. v

7 Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah. PERUBAHAN MIKROBIOLOGI DAN BIOKIMIA UDANG AIR TA WAR (MACROBRACHIUM ROSENBERGII) SEMASA PENYIMPANAN Oleh FATIMAH BINTI ABU BAKAR Mac 2001 Pengerusi: Fakulti: Profesor Madya Che Nyonya Abdul Razak, Ph.D. Sains dan Pengajian Alam Sekitar Minat yang mendalam terhadap udang air tawar tropika menyebabkan komoditi ini penting dalam perdagangan antarabangsa. 1a merupakan satu daripada produk bermutu tertinggi dalam pasaran perikanan antarabangsa dan justeru itu mempunyai potensi jangka panjang yang baik. Kajian tentang perubahan bakteriologi dan biokimia ke atas air kolam dan udang air tawar peliharaan telah dijalankan daripada tiga tapak persampelan iaitu: Tapak 1 - Kg. Jumbang, Negri Sembilan; Tapak 2 - Kg. eangkat Tin, Perak dan Tapak 3 - Kg. eenderiang, Perak. Sampel udang hidup daripada tiga tapak berbeza dibawa balik ke makmal dalam beg polietina beroksigen sebelum disimpan selama 20 j pada suhu bilik (28 ± 2 e), 10 h pada 10 ± 2 e dan 16 h pada simpanan berais (4 ± 2 e). Analisis mikrobiologi dijalankan bagi jumlah kiraan aerob mesofilik dan psikrofilik, kiraan bakteria proteolitik, bakteria penghasil histamina, bakteria penghasil kadaverina dan penghasil putresina dalam udang dan air kolam untuk ketiga-tiga tapak dengan menggunakan medium agar bakteriologi yang Vl

8 sesum. Analisis biokimia telah dijalankan bagi profil ph menggunakan meter ph, kesegaran daripada aspek nilai- K menggunakan strip kertas ujian, profil asid amino menggunakan penganilisis asid amino, jumlah bes meruwap dan jumlah asid meruap melalui kaedah penyulingan dan amin biogenik seperti; histamina, kadaverina dan putresina menggunakan kaedah kromatografi cecair berprestasi tinggi (HPLC). Keputusan diperolehi menunjukkan mutu mikrobiologi udang air tawar adalah berhubungkait dengan mikroflora air kolam di mana udang membiak. Kiraan awal menunjukkan nilai bakteria adalah dalam julat log 4+ CFU/g bagi kesemua sampel. Jumlah kiraan aerob mesofilik dan psikrofilik terdapat pada bahagian kepala adalah lebih tinggi berbanding dengan bahagian badan udang bagi kesemua sampel dan jenis medium pertumbuhan yang diuji. Semua sampel mempamerkan peningkatan dalam bilangan mengikut masa dan suhu penstoran sehingga mencapai log 7+ CFU/g bagi mengikut kiraan mesofilik selepas 12 j pada suhu bilik, 6 h pada looc dan 12 h pada penyimpanan ais. Sarnpel dari Tapak 2 mempunyai bilangan bakteria yang lebih tinggi berbanding dengan dua tapak yang lain, di mana ini berkait rapat dengan bilangan yang diperolehi dalarn air kolarn masing-masing. Pola yang sarna dilihat bagi kiraan psikrofilik tetapi pada nilai yang lebih rendah bagi jenis medium berbeza yang dikaji. Keputusan uj ian biokimia juga menunjukkan corak peningkatan yang sarna dalam nilai dan berkait rapat dengan data mikrobiologi sehingga mencapai tahap kerosakan. Kesan pengawet ke atas perubahan mutu dan hayat simpan udang semasa penyimpanan berais telah dikaji melalui analisis penilaian deria, mikrobiologi, fizikal dan kimia. Keputusan penilaian deria menunjukkan bahawa udang mempunyai hayat Vll

9 simpan selama lebih kurang 20 h apabila dirawat dengan 2% sukrosa, asid borik, asid laktik, sodium klorida, dan sodium metabisulfit kecuali sampel kawalan yang mana hayat simpannya hanya 15 h. Asid borik, asid laktik dan sodium metabisulfit berupaya merencat pertumbuhan bakteria psikrofilik dan pembentukan amin biogenik dalam udang sementara memelihara bilangan mesofilik pada aras yang lebih rendah.. semasa pen]'lmpanan also Penggunaan kaedah enzim melalui kaedah biosensor dalam bentuk elektrod oksigen dan spektrofotometer menawarkan kaedah altematif yang baik untuk menentukan kandungan histamina dalam udang. Keputusan ujikaji menunjukkan peningkatan histamina semasa penyimpanan diukur melalui signal elektrod oksigen menggunakan enzim diamine oksidase. Korelasi tinggi melebihi 0.98 setanding dengan kaedah HPLC bagi kesemua sampel dan peratus pengesanan semula kira-kira 100% bagi nilai-nilai histamina di antara )lg/g dicerap. Sejajar dengan ini, penggunaan spektrofotometer melalui sistem gandingan enzim diamine oksidase dan peroksidase horseradish memberi anggaran separa kuantitatif kandungan histamina dalam udang. Koefisien korelasi melebihi r 2 = 0.99 adalah diperolehi apabila perbandingan dibuat dengan kaedah HPLC. Peratus pengesanan semula histamina dalam sampel yang ditambah piawai adalah juga disekitar 100% bagi kepekatan histamina di antara )lg/g. Vlll

10 ACKNOWLEDGEMENTS I wish to express my sincere gratitude and thanks to my supervisor, Associate Professor Dr. Che Nyonya Abdul Razak of the Department of Biochemistry and Microbiology for her guide, confidence and encouragement during the course of this study and all along I have known her since. I wish to thank also my co-supervisor Professor Dr. Abu Bakar Salleh for his guidance and giving me the encouragement I needed to complete this study. To my other cosupervisor, Associate Professor Dr. Mahiran Basri, I wish to thank you for the support and encouragement. My thanks and appreciation is due to Dr. Nazamid Saari for his help and lending me his spectrophotometer. To the Faculty of Food Science and Biotechnology, UPM, I am grateful for the facilities in carrying out the research work. My thanks and great appreciation are also due to my graduate student, N ga Kea Soon for his help in formatting this thesis; Encik Mohd Reza Hussein for helping me with the purchasing of chemicals and equipment; Shirlene and Puan Asmarani Abdullah for typing part of the tables in this thesis. I also wish to extend my sincere thanks to all the staff of the Department of Food Science for whatever help they have rendered me one way or the other during the course ofthis study. IX

11 To the late Professor Terugishe Motohiro and wife of Japan, whom I missed, who also had supplied me with some of the literature for this study, I wish God bless you. In loving memory of my late husband, Allahyarham Ahmad Ariffin, whom I dearly missed, who sacrificed a lot and help me in the early part of my work, and wished to see me successful and happy all my life, " May Allah Cucuri Rahmat ke atas Roh mu ". To my husband, Mohd Razali Sahudin, I am utmost grateful and I highly appreciate you for the confidence, being supportive, understanding and loving all the way and for spending time and much more for driving me to get my live prawn samples at various remote places whenever I needed to. My appreciation is not complete unless I extend it to both my children, Ayong and O'meil for being very understanding and putting up with my "busyness". Last but not least, my thanks goes to Professor Tan Sri Datuk Dr. Syed Jalaludin Syed Salim for giving me the opportunity for this study and to the Malaysian Government and Universiti Putra Malaysia for granting me the study leave and sponsoring the work. x

12 I certity that an Examination Committee met on 28th March 2001 to conduct the final examination of Fatimah binti Abu Bakar on her Doctor of Philosophy thesis entitled "Microbiological and Biochemical Changes of Freshwater Prawns (Macrobrachium rosenbergii) During Storage" in accordance with Universiti Pe$nian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Norhani binti Abdullah, Ph.D. Associate Professor Jabatan Biokimia dan Mikrobiologi Fakulti Sains dan Pengajian Alam Sekitar Universiti Putra Malaysia (Chairman) Che Nyonya binti Abdul Razak, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member) Abu Bakar bin Salleh, Ph.D. Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member) Mahiran binti Basri, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member) Aminah binti Abdullah Ph.D. Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (Independent Examiner). GHAZALI MOHA YIDIN, Ph.D. Pro sorl Deputy Dean of Graduate School, Universiti Putra Malaysia Date :0 2,\AM Xl

13 This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. AINIIDERlS, Ph.D. Professor, Dean of Graduate School, Universiti Putra Malaysia Date: xu

14 I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or currently for any other degree at UPM or other institutions. F Xlll

15 TABLE OF CONTENTS Page DEDICATION ABSTRACT...,... "...,..., ABSTRAK VI ACKNOWLEDGEMENTS IX APPROV AL SH EETS.. "... "...,..,... Xl DECLARATION FORM Xlll LIST OF TABLES LIST OF FIGURES xx LIST OF ABBREVIATIONSINOTATIONS/ GLOSSARY OF TERMS xxv CHAPTER I INTRODUCTION II LITERA TUREREVIEW ,... 5 Freshwater Prawn Industry and Aquaculture The Freshwater Prawn Industry Aquaculture and Life Cycles of Freshwater Prawns Composition of Freshwater Prawns Changes Occurring in Prawns During Storage Biochemical Changes Physical Parameters Temperature Time Chemical Parameters " 10 Nucleotides Indole ph Total Volatile Bases and Total Volatile Acids Amino Acids Biogenic Amines Free Fatty Acids Microbial Changes Microbial Count...,..,..."..,.., Microbial Flora Other Deteriorative Changes...,..,..." Texture Deterioration Off-flavour Development Shell Discolouration XVlll xiv

16 Bacterial Production of Histamine, Putrescine and Cadaverine Toxicity of Histamine, Putrescine and Cadaverine Effects of Preservatives on Shelf life of Prawns Review on the Methods of Determination of Biogenic Amines Amino Acid Decarboxylation..., Trimethylamine Oxide Conversion..., 34 Thermal Amino Acid Decomposition Biochemical and Sensory Properties of Biogenic Amine Methods of Determination of Biogenic Amines in Prawns Chromatographic Methods Enzymatic Methods The Enzyme / Oxygen Electrode METHODOLOGY Materials......, 42 Aquaculture of Prawns and Pond Water Quality Sampling of Prawns Biochemical Analyses Determination of ph Determination of Total Volatile Acids (TVA)... Determination of Total Volatile Bases (TVB)... K-values Determination of Amino Acids Drying, Redrying and Derivatization for Samples and Standard Amino Acids Determination of Biogenic Amines Preparation of Standard Amine Solution..., Preparation of Standard Curves Sample Preparation and Amine Extraction..., Derivatization of Standard Amine Solution HPLC Conditions Chromatographic Conditions...,..." Reproducibility and Recovery Studies on Biogenic Amine Production in Prawns Microbiological Analysis Effect of Preservatives on Shelf Life and Biogenic Amines Production in Prawns Sample Preparation and Treatment Biochemical Analyses of Preserved Prawns ph K-values Biogenic Amines Microbiological Analyses of Preserved Prawns Sensory Evaluation xv

17 Alternative Methods for Detennination of Biogenic Amines in Freshwater Prawns Enzymatic Methods Preparation of Polarographic Method/Oxygen Electrode Sample Preparation.... ' ".... Colorimetric MethodJUse of Spectrophotometer... Sample preparation. '"...,...,... Standard Histamine Solutions Reliability and Reproducibility Studies IV RESULTS... Pond Water Quality and Soil Type Studies... Microbiological Quality of Pond Water.... Growth of Prawns and Feeding Regime Biochemical Changes... ph... K-values... Total Volatile Acids... Total Volatile Bases... Amino Acids... Biogenic Amines Microbial Changes... Total Aerobic Counts... Mesophilic Counts... Psychrophilic Counts... Effect of Preservatives on Shelf Life of Prawns... Biochemical Analysis of Preserved Prawns... Microbiological Quality of Preserved Prawns... Sensory Evaluation of Preserved Prawns... Comparison of Different Enzymatic Methods for the Determination of Histamine in Freshwater Prawns... Polarographic Method/Oxygen Electrode... Colorimetric MethodlUse of Spectrophotometer V...,., " DISCUSSION.. ' " Pond Water Quality and Soil Types Studies... Microbiological Quality of Pond Water.... Growth of Prawns and Feeding Regime... Biochemical Changes... ph... K-va1ues... Total Volatile Acids Total Volatile Bases... Amino Acids... Biogenic Amines..., XVI

18 Microbial Changes Mesophilic Aerobic Bacteria Psychrophilic Counts Effect of Chemical Preservatives on Shelf Life of Prawns. 147 Biochemical Analysis of Preserved Prawns Microbiological Quality of Preserved Prawns Sensory Evaluation of Preserved prawns Comparison of Different Enzymatic Methods for the Determination of Histamine in Freshwater Prawns Polarographic Method/Oxygen Electrode Colorimetry MethodIU se of Spectrophotometer V CONCLUSION.... VII BIBLIOGRAPHY APPENDICES , ; APPENDIX A APPENDIX B : APPENDIX C BIODATA OF THE AUTHOR XVll

19 LIST OF TABLES Table Page Bacterial species possessing lysine or ornithine decarboxylase Biogenic amines and their amino acid precursors Some physical characteristics and aquaculture studies of pond water and prawns sampled from the three sites Microbial flora of water sampled from the three sites with respect to number, colony types and size grown on different agar media Mesophilic counts of Site 1 samples for body and head regions of M. rosenbergii during storage at different temperature as plated on different agar media Psychrophilic counts of Site 1 samples for body and head regions of M. rosenbergii during storage at different temperature as plated on different agar media Mesophilic counts of Site 2 samples for body and head regions of M rosenbergii during storage at different temperature as plated on different agar media , 99 Psychrophilic counts of Site 2 samples for body and head regions of M rosenbergii during storage at different temperature as plated on different agar media ,., Mesophilic counts of Site 3 samples for body and head regions of M rosenbergii during storage at different temperature as plated on different agar media Mesophilic counts of Site 3 samples for body and head regions of M rosenbergii during storage at different temperature as plated on different agar media Psychrophilic counts of iced stored prawn body samples (log CPU/g) treated with different preservatives Psychrophilic counts of iced stored prawn head samples (log CPU/g) treated with different preservatives Sensory evaluation of ice-stored M. rosenbergii treated with different preservatives XVlll

20 Table Page Recovery percentage of histamine during storage of M rosenbergii at different temperature using the oxygen electrode Recovery percentage of histamine during storage of M rosenbergii at different temperature using the HPLC Comparison of the recovery percentage of histamine during ambient storage of M rosenbergii by the enzymatic method using the colorimetry and the HPLC Comparison of the recovery percentage of histamine during 100 C storage of M rosenbergii by the enzymatic method using the spectrophotometer and the HPLC Comparison of the recovery percentage of histamine during iced storage of M rosenbergii by the enzymatic method using the spectrophotometer and the HPLC XIX

21 LIST OF FIGURES Figure Page l. ph of body and head regions profile of M. rosenbergii for all samples from three sites stored at different temperature ( a). Ambient ( b ). lode, ( c ) iced storage K value (%) profile of M rosenbergii for all samples from three sites stored at different temperature ( a ). Ambient ( b ). 10De, ( c ) iced storage...,..., Total volatile acids (TVA) profile M rosenbergii for all samples from three sites stored at different temperature. ( a). Ambient ( b ). lode, ( c ) iced storage Total volatile bases (TVB) profile of M rosenbergii for all samples from three sites stored at different temperature. ( a ). Ambient ( b ). 10oe, ( c ) iced storage..., Amino acids profile of M rosenbergii during iced storage sampling for 16 days for all three sites ( a). Site 1, ( b ). Site 2, ( c ) Site Amino acids profile of M rosenbergii during ambient storage sampling for 20 hours for all three sites ( a ). Site 1, ( b ). Site 2, ( c ) Site Amino acids profile of M rosenbergii during 10 e storage sampling for 10 days for all three sites ( a). Site 1, ( b ). Site 2, ( c ) Site Amount of biogenic amines (putrescine, cadaverine and histamine) produced from the body regions of M rosenbergii during ambient storage for 20 hours for all three sites ( a ). Site 1, ( b ). Site 2, ( c ) Site Amount of biogenic amines (putrescine, cadaverine and histamine) produced from the body regions of M rosenbergii during 10 e storage for 10 days for all three sites ( a ). Site 1, ( b ). Site 2, ( c ) Site Amount of biogenic amines (putrescine, cadaverine and histamine) produced from the body regions of M rosenbergii during iced storage for 16 days for all three sites. ( a ). Site 1, ( b ). Site 2, ( c ) Site 3...,..., xx

22 Figure Page 11. Amount of biogenic amines (putrescine, cadaverine and histamine) produced from the head regions of M. rosenbergii during ambient storage for 20 hours for all three sites ( a ). Site 1, ( b ). Site 2, ( c ) Site 3..., " ,.., Amount of biogenic amines (putrescine, cadaverine and histamine) produced from the head regions of M. rosenbergii during 10 C storage for 10 days for all three sites ( a ). Site 1, ( b ). Site 2, ( c ) Site Amount of biogenic amines (putrescine, cadaverine and histamine) produced from the head regions of M. rosenbergii during iced storage for 16 days for all three sites. ( a ). Site 1, ( b ). Site 2, ( c ) Site 3..., , " 14. Total mesophilic and psychrophilic aerobic counts of body and head regions of M rosenbergii for the three (3) sampling sites stored at ambient storage..., ' " 15. Total mesophilic and psychrophilic aerobic counts of body and head regions of M rosenbergii for the three (3) sampling sites stored at 10 C storage.. ( a ). Site 1, ( b ). Site 2, ( c ) Site Total mesophilic and psychrophilic aerobic counts of body and head regions of M rosenbergii for the three (3) sampling sites stored at iced storage. (a). Site 1, ( b ). Site 2, ( c ) Site Amount of cadaverine in prawn body samples treated with different preservatives under icing conditions Amount of cadaverine in prawn head samples treated with different preservatives under icing conditions Amount of putrescine in prawn body samples treated with different preservatives under icing conditions..., " Amount of putrescine in prawn head samples treated with different preservatives under icing conditions ph of prawn body samples treated with different preservatives under icing conditions III 22. ph of prawn head samples treated with different preservatives under icing conditions XXI

23 Figure Page 23. Freshness evaluation of M. rosenbergii treated with different preservatives as determined by K-value (%) Correlation between amount of histamine in M. rosenbergii determined by oxygen electrode compared to that of HPLC during ambient storage for 20 h..., ,..., 25. Correlation between amount of histamine in M rosenbergii determined by oxygen electrode compared to that of HPLC during 10 C storage for 10 d Correlation between amount of histamine in M rosenbergii determined by oxygen electrode compared to that of HPLC during iced storage for 16d Correlation between amount of histamine in M. rosenbergii determined by spectrophotometer compared to that of HPLC during ambient storage for 20 h..., Correlation between amount of histamine in M. rosenbergii determined by spectrophotometer compared to that ofhplc during 10 C storage for 10 d Correlation between amount of histamine in M rosenbergii determined by spectrophotometer compared to that of HPLC during iced storage for 16 d..., 30. Sampling site 1 at Kg. Jumbang, Galau, Kuala Pilah, Negri Sembilan Sampling site 2 at Kg. Cangkat Tin, Tanjung Tualang, Perak Sampling site 3 at Kg. Cenderiang, Tapah, Perak Chromatogram of cadaverine, putrescine and histamine standards from HPLC chart recorder Chromatogram of cadaverine, putrescine and histamine from the prawn sample as seen on the HPLC chart recorder a 35b Response recorded from the histamine standard using the oxygen electrode...,..., Response recorded for histamine from the prawn sample using the oxygen electrode XXll

24 Figure Page 36a 36b Histamine producing bacteria isolated from M. rosenbergii seen as deep purple colonies on Modified Niven's agar Proteolytic bacteria isolated from M. rosenbergii produced colonies with clear zones on Skim Milk agar Cadaverine standard curve (0-80 ppm) using HPLC method Cadaverine standard curve (0-800 ppm) using HPLC method Histamine standard curve (0-125 ppm) using HPLC method Histamine standard curve ( ppm) using HPLC method Putrescine standard curve (0-150 ppm) using HPLC method Putrescine standard curve ( ppm) using HPLC method Histamine standard curve (0-50 ppm) using spectrophotometer method... " ".., Histamine standard curve (0-250 ppm) using spectrophotometer method Histamine standard curve (0-500 ppm) using spectrophotometer method Histamine standard curve (0-100 ppm) using Oxygen electrode method..., Histamine standard curve (0-200 ppm) using Oxygen electrode method Histamine standard curve (0-500 ppm) using Oxygen electrode method Histamine standard curve ( ppm) using Oxygen electrode method a Control prawn samples on the 15 th day after treatment with Ice SOb Prawn samples on the 15 th day after treatment with 2% NaCl SOc Prawn samples on the 15 th day after treatment with 1% lactic acid XXlll

25 Figure Page 50d Prawn samples on the 15 th day after treatment with 2% boric acid SOe Prawn samples on the ls t h day after treatment with 2% sucrose f Prawn samples on the 15th day after treatment with 2% sodium metabisulphite XXIV