25
UNIVERSITI PUTRA MALAYSIA MICROBIOLOGICAL AND BIOCHEMICAL CHANGES OF FRESHWATER PRAWNS (MACROBRACHIUM ROSENBERGII) DURING STORAGE FATIMAH BINTI ABU BAKAR FSAS 2001 35
UNIVERSITI PUTRA MALAYSIA
MICROBIOLOGICAL AND BIOCHEMICAL CHANGES OF FRESHWATER PRAWNS (MACROBRACHIUM ROSENBERGII)
DURING STORAGE
FATIMAH BINTI ABU BAKAR
FSAS 2001 35
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
Lovingly for Abang,
Ayong and 0 'meil,
They matter more, above all else ........... .
11
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
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 l OoC 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
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 1 00% for values of histamine between 0-200 Ilg/g was observed. S imilarly,
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 r2 = 0.99 were observed when compared to that of the
HPLC method. Percentages of recovery of histamine in augmented samples were
also about 1 00% for concentration of histamine between 0-200 Ilg/g.
v
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: Profesor Madya Che Nyonya Abdul Razak, Ph.D.
Fakulti: 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 panj ang 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), 1 0 h pada 1 0° ± 2°e dan
1 6 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
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 1 2 j pada suhu bilik, 6 h pada lOoC dan 1 2 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 j enis medium berbeza
yang dikaji. Keputusan ujian 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
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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 1 5 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
1 00% bagi nilai-nilai histamina di antara 0-200 )lg/g dicerap. Sej aj ar dengan ini,
penggunaan spektrofotometer melalui sistem gandingan enzim diamine oksidase dan
peroksidase horseradish memberi anggaran separa kuantitatif kandungan histamina
dalam udang. Koefisien korelasi melebihi r2 = 0.99 adalah diperolehi apabila
perbandingan dibuat dengan kaedah HPLC. Peratus pengesanan semula histamina
dalam sampel yang ditambah piawai adalah juga disekitar 1 00% bagi kepekatan
histamina di antara 0-200 )lg/g.
Vlll
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 co
supervisor, 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 of this study.
IX
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
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 1981. 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 2001
Xl
. �
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.
xu
AINIIDERlS, Ph.D. Professor, Dean of Graduate School, Universiti Putra Malaysia
Date:
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
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TABLE OF CONTENTS
Page
DEDICATION . . . ... . .. ...... ..... ... . .. .. ... ... . . .. ... ... . .. ... .. . .. ... . .. ... .. 11 ABSTRACT . .. , ............................ " .............. , .... ,. . .. . .. . .. ... . . 111 ABSTRAK.... ..... ......... . . . . .. . . . . . . . ... ........ . . . ..... .. .. .. ... . . . .. ... . . . . VI ACKNOWLEDGEMENTS............ ..... .. .. . .. ..... . ... . .. ... .. ..... ... .. IX APPROV AL SHEETS .. " ......................... " ........ , .. , ... . .. . .. . .. . . Xl DECLARATION FORM . .. . .. . .... . . . ....... ...... ... ... .. . ..... ... ... .. . .. .. Xlll LIST OF TABLES... . .. . .. . . .... ........... . . . .... . . . . .. . .. .. . .. .... ... .. ..... XVlll LIST OF FIGURES.. .... . .. ... . . . . . ...... .. ... ..... ... ... ... ... .. . .. . ..... .. . . xx LIST OF ABBREVIATIONSINOTATIONS/ GLOSSARY OF TERMS . ... ........ ... ... . . .. ... ... . . . ... . . . . . . . .... . .. ..... xxv
CHAPTER
I INTRODUCTION ..... ... .. . ... .. .. . . .. .. ...... ... . . .. ... .. .. . 1
II LITERA TUREREVIEW .. . . . .. . . . ... ... . . . .. ... . . . . ..... , . ... 5 Freshwater Prawn Industry and Aquaculture. . .. . .. . .. . .. . . 5
The Freshwater Prawn Industry . .... . . .. ... . . . .. . . . . 5 Aquaculture and Life Cycles of Freshwater Prawns . .. . .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Composition of Freshwater Prawns.... .. ..... .. . . . . 7
Changes Occurring in Prawns During Storage .. .. . ... .... . . 8 Biochemical Changes. ... . .. . . .. . .. . . . . .. ... ..... ... ..... . .. .. . 9 Physical Parameters .. .. . .... .. .... ... .. ... ... .. . .. . .. ... ...... . 9
Temperature. ... .. .. . . . . . . .. .. . . .. ..... . . . .. . .. . .. ... . . . 9 Time.. .. .... ...... ... . . ...... .... . . . . . . . . . . . . . . . . . . . . . . . . . 1 0
Chemical Parameters .. . . ... .. . .. ...... .. ... ..... .. . .. . . . .. . . . " 1 0 Nucleotides. .. .. .. . .. . .... ... ... . . ...... ..... ... .... ... . 1 0 Indole ... . . . .. .. ... .. ..... . .... . .. .. . .. . . .. . . . . . . .. ... . . .. 14 pH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 14 Total Volatile Bases and Total Volatile Acids.. ... 15 Amino Acids. . .............. . .. . . ... . ..... .. . ..... ... ... 15 Biogenic Amines .. . .. .. . .. ... .. ....... . .. .. . .......... . 15 Free Fatty Acids ... . . .. . .. ... ... ..... . . ....... .. . . ..... 1 6 Microbial Changes.... ... .. . . ....... ....... ... . .... .. .. 17 Microbial Count ... ... , . . , . . . . . . . . . . . . . " . . , . . , . . . . . . . . . 1 8 Microbial Flora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9
Other Deteriorative Changes . .. ... , . . , . .. . . .. .. . " . .. . .. . .. . . .. 22 Texture Deterioration .. ... . . . .. . ... .. . .. . .. ... .. ... . . . . 22 Off-flavour Development ... . . . .. . .. ... . . . .. . .. . ... . .. 23 Shell Discolouration . .. ..... ... ...... ... ..... ...... .. ... 23
xiv
Bacterial Production of Histamine, Putrescine and Cadaverine........................................................ 24 Toxicity of Histamine, Putrescine and Cadaverine......... 27 Effects of Preservatives on Shelf life of Prawns. ............ 29 Review on the Methods of Determination of Biogenic Amines... . ........................................................ 3 1
Amino Acid Decarboxylation ............. , ..... . .. ... 32 Trimethylamine Oxide Conversion .................. , 34 Thermal Amino Acid Decomposition.......... ...... 35
Biochemical and Sensory Properties of Biogenic Amine... 35 Methods of Determination of Biogenic Amines in Prawns .. ........... ......... ......... .... ......... ...... ...... .... 37
Chromatographic Methods............................ 38 Enzymatic Methods .. ...... ........ .................... 39 The Enzyme / Oxygen Electrode...................... 4 1
11 1 METHODOLOGY.. ... ..... .... ..... .... .. . .. . .. .... .. . .. . .. . ... 42 Materials ...... .................................................... , 42 Aquaculture of Prawns and Pond Water Quality............ 42 Sampling of Prawns. ....... ................. ..................... 42 Biochemical Analyses........................................... 44
Determination of pH. ..................... .............. 44 Determination of Total Volatile Acids (TVA)....... 44 Determination of Total Volatile Bases (TVB)....... 45 K-values................................................... 46 Determination of Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . 47 Drying, Redrying and Derivatization for Samples and Standard Amino Acids.............. .. . ......... ... 48 Determination of Biogenic Amines....... ........... . 48 Preparation of Standard Amine Solution ......... , . .. 49 Preparation of Standard Curves..... ........ ........... 49 Sample Preparation and Amine Extraction ... ,. .. .... 49 Derivatization of Standard Amine Solution. . .. ... . .. 50 HPLC Conditions........ ................................ 5 1 Chromatographic Conditions . . . . . . . . , . . . . . . . . . . . " . . . . . 5 1 Reproducibility and Recovery Studies on Biogenic Amine Production in Prawns................ ......... . . 5 1
Microbiological Analysis....................................... 52 Effect of Preservatives on Shelf Life and Biogenic Amines Production in Prawns.................................. 53
Sample Preparation and Treatment................... 53 Biochemical Analyses of Preserved Prawns......... 54
pH................................................ 54 K-values.......................................... 54 Biogenic Amines..... ....................... .... 54
Microbiological Analyses of Preserved Prawns..... 54 Sensory Evaluation....................................... 55
xv
IV
V
Alternative Methods for Detennination of Biogenic Amines in Freshwater Prawns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of Polarographic Method/Oxygen
56 56
Electrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Sample Preparation . . . .... . .. . . . . . . . ... . . . . . . ' " . . . . . ... . Colorimetric MethodJUse of Spectrophotometer . . . Sample preparation. '" . . . . . . . . . . . . . , . . . . . . , . . . . . . . . . . . . . Standard Histamine Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . Reliability and Reproducibility Studies . . . . . . . . . . . . . . .
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 . . .
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
58 58 59 60 60
62 62 62 65 66 66 68 68 71 73 77 86 86 91 96 105 105 109 109
114 114 115
128 128 129 129 130 130 132 133 134 136 138
Microbial Changes . . . . . . . . . . . . . . ......................... 141 Mesophilic Aerobic Bacteria . . .. . . . . . . . . . . . . . . . . . . . . . . . 141 Psychrophilic Counts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 46
Effect of Chemical Preservatives on Shelf Life of Prawns. 147 Biochemical Analysis of Preserved Prawns . . . . . . . . ... 147 Microbiological Quality of Preserved Prawns . . . . . .... 149 Sensory Evaluation of Preserved prawns . . . . . . . . . . . .... 150
Comparison of Different Enzymatic Methods for the Determination of Histamine in Freshwater Prawns . . . . . . . . . . . 1 5 1
Polarographic Method/Oxygen Electrode . . . . . . . . . . . . . 151 Colorimetry MethodIU se of Spectrophotometer. . . .. 153
V CONCLUSION . . . . . . . . . . . ... . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 1 56
VII BIBLIOGRAPHY . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . , . .. . .. 159
APPENDICES . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . ; .............. 171
APPENDIX A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 APPENDIX B . . . .. . . . . . . . . . . . . : .................................... 175 APPENDIX C . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...... ................... 182 BIODATA OF THE AUTHOR
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Table
1
2
3
4
5
6
7
8
9
1 0
1 1
1 2
LIST OF TABLES
Page
Bacterial species possessing lysine or ornithine decarboxylase... 26
Biogenic amines and their amino acid precursors .. .. ....... .. ..... 34
Some physical characteristics and aquaculture studies of pond water and prawns sampled from the three sites... . . . . . . . . . . . 63
Microbial flora of water sampled from the three sites with respect to number , colony types and size grown on different agar media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
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 . . . . . . . . . . . . . . . . . . . . . 98
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 ..... ... .. ... .. . , ., . . 1 00
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..................................... 1 0 1
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 02
Psychrophilic counts of iced stored prawn body samples (log CPU/g) treated with different preservatives. . . . . . . . . . . . . . . . . 1 1 0
Psychrophilic counts of iced stored prawn head samples (log CPU/g) treated with different preservatives................. 1 1 0
1 3 Sensory evaluation of ice-stored M. rosenbergii treated with different preservatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 3
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Table
14
15
16
Page
Recovery percentage of histamine during storage of M rosenbergii at different temperature using the oxygen electrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 16
Recovery percentage of histamine during storage of M rosenbergii at different temperature using the HPLC. . . . . . 1 18
Comparison of the recovery percentage of histamine during ambient storage of M rosenbergii by the enzymatic method using the colorimetry and the HPLC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
17 Comparison of the recovery percentage of histamine during 100 C storage of M rosenbergii by the enzymatic method using the spectrophotometer and the HPLC. . . . . . . . . . . . . . . . . . . . . 126
18 Comparison of the recovery percentage of histamine during iced storage of M rosenbergii by the enzymatic method using the spectrophotometer and the HPLC . . . . . . . . . . . . . . . . . . . . . 127
XIX
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 ). l ODe, ( c ) iced storage . . .. . ... .. 67
2. K value (%) profile of M rosenbergii for all samples from three sites stored at different temperature ( a ). Ambient ( b ) . 1 0De, ( c ) iced storage . . ... . . ........ , .............. , . . . . . . . . ... 69
3 . Total volatile acids (TVA) profile M rosenbergii for all samples from three sites stored at different temperature. ( a ) . Ambient ( b ) . lODe, ( c ) iced storage . . . . . ... . .. . ..... . . . .. . .. 70
4. Total volatile bases (TVB) profile of M rosenbergii for all samples from three sites stored at different temperature. ( a ). Ambient ( b ). 1 0oe, ( c ) iced storage ........ ..... , . . . . . .. . . .. 72
5 . Amino acids profile of M rosenbergii during iced storage sampling for 1 6 days for all three sites ( a ). Site 1 , ( b ). Site 2, ( c ) Site 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
6 . 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 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
7 . Amino acids profile of M rosenbergii during 1 0° e storage sampling for 1 0 days for all three sites ( a ). Site 1 , ( b ) . Site 2, ( c ) Site 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
8. 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 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
9 . Amount of biogenic amines (putrescine, cadaverine and histamine) produced from the body regions of M rosenbergii during 1 0° e storage for 1 0 days for all three sites ( a ). Site 1 , ( b ) . Site 2, ( c ) Site 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
1 0. 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 . . . . . . . . . . . . . . . . . . , . . .. . ..... . . , . .. . . . . . . . . . . . . . .. 79
xx
Figure
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 ...... , . . . . . . . " . . . . . .. . . . . . . . . . . . . . . . . . . . . . . , . . , . .
12. 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 3 ...................................................
13. 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 s ites 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 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16. 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 3 ....................................................
17. Amount of cadaverine in prawn body samples treated with d ifferent preservatives under icing conditions ........................
18. Amount of cadaverine in prawn head samples treated with different preservatives under icing conditions ........................
19. Amount of putrescine in prawn body samples treated with different preservatives under icing conditions ... , . . . . . . . . . . " . . . .. . . .
20. Amount of putrescine in prawn head samples treated with different preservatives under icing conditions ........................
21. pH of prawn body samples treated with different preservatives
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83
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87
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89
107
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108
under icing conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III
22. pH of prawn head samples treated with different preservatives under icing conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 111
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Figure
23. Freshness evaluation of M. rosenbergii treated with different preservatives as determined by K-value (%) .......................
24. 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 ............................................
26. Correlation between amount of histamine in M rosenbergii determined by oxygen electrode compared to that of HPLC during iced storage for 16d ......................................
27. Correlation between amount of histamine in M. rosenbergii determined by spectrophotometer compared to that of HPLC during ambient storage for 20 h .................. , .....................
28. Correlation between amount of histamine in M. rosenbergii determined by spectrophotometer compared to that ofHPLC during 10 C storage for 10 d ...........................................
29. 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 ..........................................................
31. Sampling site 2 at Kg. Cangkat Tin, Tanjung Tualang, Perak ......................................................................
32. Sampling site 3 at Kg. Cenderiang, Tapah, Perak ..................
33 . Chromatogram of cadaverine, putrescine and histamine standards from HPLC chart recorder .................................
34. Chromatogram of cadaverine, putrescine and histamine from the prawn sample as seen on the HPLC
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120
120
121
122
122
122
172
173
174
176
chart recorder............................................................ 177
35a Response recorded from the histamine standard using the oxygen electrode ... , ............................. , . .. . .. . .. . .. . . . . .. 178
35b Response recorded for histamine from the prawn sample using the oxygen electrode...... .. ..... .. ... .... .. .... .. .... .. .. . ... . .. 179
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Figure Page
36a Histamine producing bacteria isolated from M. rosenbergii seen as deep purple colonies on Modified Niven's agar. .......... 180
36b Proteolytic bacteria isolated from M. rosenbergii produced colonies with clear zones on Skim Milk agar. ....................... 181
37. Cadaverine standard curve (0-80 ppm) using HPLC method ...... 183
38. Cadaverine standard curve (0-800 ppm) using HPLC method .... 184
39. Histamine standard curve (0-125 ppm) using HPLC method ..... 185
40. Histamine standard curve (0-1250 ppm) using HPLC method .... 186
41. Putrescine standard curve (0-150 ppm) using HPLC method ..... 187
42. Putrescine standard curve (0-1200 ppm) using HPLC method ... 188
43. Histamine standard curve (0-50 ppm) using spectrophotometer method ..... " . . . . . .. . . . " . . , . . . . . . . . . . . . . . . . . . . . . . . 189
44. Histamine standard curve (0-250 ppm) using spectrophotometer method ............................................. 190
45. Histamine standard curve (0-500 ppm) using spectrophotometer method ............................................. 191
46. Histamine standard curve (0-100 ppm) using Oxygen electrode method .......................................... , . . . 192
47. Histamine standard curve (0-200 ppm) using Oxygen electrode method .............................................. 193
48. Histamine standard curve (0-500 ppm) using Oxygen electrode method .............................................. 194
49. Histamine standard curve (0-1000 ppm) using Oxygen electrode method .............................................. 195
50a Control prawn samples on the 15th day after treatment with Ice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
SOb Prawn samples on the 15th day after treatment with 2% NaCl. ... 197
SOc Prawn samples on the 15th day after treatment with 1% lactic acid ......................................................................... 198
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Figure
50d Prawn samples on the 15th day after treatment with 2% boric
Page
acid........................................................................ 199
SOe Prawn samples on the lSth day after treatment with 2% sucrose. 200
50f Prawn samples on the 15th day after treatment with 2% sodium metabisulphite........................................................... 201
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