factors influencing the biogenic amine formation in salt cured fishes of tuticorin

111 Pages·2017·1.34 MB·English

“FACTORS INFLUENCING THE BIOGENIC AMINE
FORMATION IN SALT CURED FISHES OF
TUTICORIN”

Thesis submitted in part fulfillment of the requirements for the Degree
Master of Fisheries Science in Fish Quality Assurance and Management
to the Tamil Nadu Fisheries University, Nagapattinam

T. SURYA, B.F.Sc.,
ID. No. MFT15075 (FQM)

FISH QUALITY ASSURANCE AND MANAGEMENT
FISHERIES COLLEGE AND RESEARCH INSTITUTE
TAMIL NADU FISHERIES UNIVERSITY
THOOTHUKUDI- 628 008
2017
CERTIFICATE
This is to certify that the thesis entitled, “Factors influencing the biogenic
amine formation in salt cured fishes of Tuticorin”, submitted in part fulfillment of the
requirements for the award of the degree of Master of Fisheries Science in Fish
Quality Assurance and Management to the Tamil Nadu Fisheries University,
Nagapattinam is a record of bonafide research work carried out by Ms. T. Surya
under my supervision and guidance and that no part of this thesis has been
submitted for the award of any degree, diploma, fellowship or other similar prizes
and that the work has not been published in part or full in any scientific or popular
journal or magazine.

Place: Thoothukudi Dr. G.JEYASEKARAN
Date : Chairman

Place:
Date:

EXTERNAL EXAMINER

APPROVED BY
Chairman : Dr. G. JEYASEKARAN
Director of Research i/c

Members : Dr. R. JEYA SHAKILA
Professor & Head

Members : Dr. N. NEETHISELVAN
Professor

Place : Thoothukudi
Date :
Dedicated to
My Parents
And
Teachers
ACKNOWLEDGEMENT
I express my hearty gratitude to my chairman Dr. G. Jeyasekaran, Director
of Research i/c, Tamil Nadu Fisheries University, Nagapattinam for his expert
guidance, constant encouragement and providing necessary infrastructure and
resources to accomplish my research work.
I have great pleasure in expressing my deep sense of gratitude to my
Advisory Committee member Dr. R. Jeya Shakila Professor & Head, Department
of Fish Quality Assurance and Management, for her advice and many insightful
discussions and suggestions for the successful completion of this work.
I am greatly indebted to the Advisory Committee member Dr. N.
Neethiselvan, Professor, Department of Fishing Technology and Fisheries
Engineering, for his guidance and encouragement throughout the period of study.
I wish to express my sincere thanks to Mrs. R. Shalini, Mrs. V. Alamelu
Assistant Professors, Department of Fish Quality Assuarance and Management for
their ready help rendering in many ways during the period of this study on
encouragement.
I owe my sincere gratitude to Dr. G. Sugumar, Dean, Fisheries College and
Research Institute for providing necessary facilities to carry out this study.
I wish to express my special thanks to Mr. B. Sivaraman, M. Palani Kumar,
M. Manikandan, M. Essakirajan, R. Udhay Kumar A. Danasekaran, Mahesh
Kumar, Mrs. Rajeshwari, Mrs. Vimala, for rendering their help, encouragement
and profuse support in bringing out the thesis.
My special thanks to Mr. R. Kumaresan, Assistant Librarian, Mrs.
Ezhilrani, Library Assistant and staff of the library for their valuable help in the
collection of reference material.
I record my heartfelt thanks to Ms. N. Asha Hema Malini, Lidiya Wilwet,
A. Priya tharshini, C. Rajeswari, S. V. Anushalini, S. Deepika, M. Renuha devi,
S. Jerusha, Reshma Ramesh, U. Arisekar, K. Vijay, K. Masilan, E. Prabu and
L. Vinoth kumar for their kindly assistance in completing this research work. I also
extend my heartfelt thanks to my juniors Dinesh Kumar and S. Sundhar.
I wish to express my special thanks to all my seniors and juniors for their
sincere help and encouragement. I have great pleasure in expressing my gratitude
to Tamil Nadu Fisheries University, Nagapattinam for providing PG Merit
Scholarship during the tenure of my study.
It is worthy to mention the love, patience and everlasting support from my
Parents Mr. M. Thamizhselvan, Mrs. P. Chitra and my family, for the successful
completion of the thesis.

(T. Surya)
ABSTRACT

Title : Factors influencing the biogenic amine
formation in salt cured fishes of Tuticorin
Name : T. Surya
Degree : M.F.Sc. (FQM)
Chairman : Dr. G. Jeyasekaran
Department : Fish Quality Assurance and Management
College : Fisheries College and Research Institute,
Thoothukudi
Year and University : 2017, Tamil Nadu Fisheries University
This study was undertaken to examine the quality of the commercial
salt cured fishes of Tuticorin. The commercial salt cured fishes had moisture
content ranging from 12.78 to 58.8%, water activity from 0.60 to 0.89, salt content
from 1.0 to 23.4 % and TVB-N content from 20.47 to 389.54 mg/100g. Biogenic
amine content was also examined for the samples using UPLC-TUV with newly
developed method. Of the 50 samples tested 60 % of the commercial salt cured
fishes had histamine exceeding prescribed limit of 20 mg/100g. The salt cured
seerfish and sardine collected in November had higher halotolerant bacterial count
of 6.49 and 5.69 log cfu/g, respectively. The halotolerant bacteria isolated from salt
cured seerfish and sardine were S. arlettae, S. gallinarum, S. sciuri and S. lentus.
Salinicoccus salsiraiae, Bacillus thuringiensis, B. megaterium, B.
paralicheniformis, B. pumilus, B. licheniformis, B. safensis, Providencia
alcalifaciens and Providencia vermicola. All the species under Bacillus had more
ability to form cadaverine & histamine and less ability to form tyramine.
Eventhough, the isolated Staphylococcus possessed the ability to form the
important biogenic amines, it formed the cadaverine and tyramine at higher levels.
Providencia alcalifaciens isolated from salt cured seerfish had the ability to form
histamine and cadaverine. Providencia vermicola, Bacillus thuringiensis and were
found to be the producers of histamine, cadaverine, putrescine and tyramine.
This study also dealt with the development of best protocol for the salt
curing of fish in whole & eviscerated forms after 0, 6, 9, 12 &15 h delayed process.
Laboratory prepared salt cured sardines were also examined for quality
parameters. Moisture content of laboratory prepared salt cured sardines of both
the forms on the final day of drying ranged from 10.76 to 13.76 %. The low moisture
content correlated well with the low halotolerant bacterial count on its final day of
drying. Water activity of laboratory prepared salt cured sardines ranged from 0.73
to 0.85. The 15 h delayed salt cured whole sardines had higher histamine contents
(156.827 mg/100g), cadaverine (405.984 mg/100g), putrescine (160.495 mg/100g)
and tyramine (86.294 mg/100g) on the final day of drying. The delayed processing
results in higher biogenic amine contents. Whole sardines had higher biogenic
amines and halotolerant bacterial count than the eviscerated sardines. The TVB-N
content of 15 h delayed whole and eviscerated salt cured sardines was 358.87 and
229.77 mg/100g, respectively, which were above the threshold level of 200
mg/100g. The cadaverine formation was several manifold higher than the other
amines. The sardines processed up to 9 h delay yielded a satisfactory salt cured
products with respect to biogenic amines and other quality parameters.
CONTENTS
Sl.No. TITLE Page
No.
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 5-19
2.1. Significance of biogenic amines in food poisoning 5
2.2. Limits of biogenic amines in seafood 7
2.3. Incidence of foodborne poisoning due to biogenic amines 8
2.4. Amine forming bacteria 10
2.4.1. Isolation and Identification of biogenic amine formers 11
from salt cured fishes
2.5. Occurrence of biogenic amines in salt cured fishes 15
2.6. Analysis of biogenic amines 17
2.7. Quality of salt cured fishes 19
3. MATERIALS AND METHODS 20- 32
3.1. Materials 20
3.1.1. Raw material 20
3.1.2. Reagents for different analysis 20
3.1.2.1. Salt content analysis 20
3.1.2.2. Total volatile base nitrogen (TVB-N) analysis 20
3.1.2.3. Total halotolerant bacterial count enumeration 21
3.1.2.4. Biogenic amine formers isolation 21
3.1.2.5. Biogenic amine analysis 21
3.1.2.6. Dansylation of amines 22
3.1.2.7. DNA extraction 22
3.1.2.8. Polymerase Chain Reaction 23
3.1.2.9. Agarose gel electrophoresis 23
3.2. Equipments 25
3.3. Methods 26
3.3.1. Preparation of salt cured products 26
3.3.2. Moisture content analysis 27
3.3.3. Water activity analysis 27
3.3.4. Salt content analysis 27
3.3.5. Analysis of Total Volatile Base Nitrogen (TVB- N) 28
3.3.6. Enumeration of Total halotolerant bacterial count 28
3.3.7. Isolation of biogenic amine formers 28
3.3.8. Sardine infusion broth to test amine forming ability 29
3.3.9.1. Preparation of biogenic amine standards 29
3.3.9.2. Extraction of biogenic amine 29
3.3.9.3. Extraction of biogenic amine from bacterial culture 30
3.3.9.4. Dansylation of amines 30
3.3.9.5. Biogenic amine analysis 30
3.3.10.1. DNA extraction 31
3.3.10.2. Amplification 31
3.3.10.3. Agarose gel electrophoresis 32
3.3.11. Sequencing of the PCR products 32
3.3.12. Statistical analysis 32
4 RESULTS 33- 65
4.1. General quality of commercial salt cured fishes of Tuticorin 33
4.2. Biogenic amine content of commercial salt cured fishes of 44
Tuticorin
4.3. Isolation and identification of biogenic amine forming 49
bacteria from selected salt cured fishes
4.3.1. Enumeration of total halotolerant bacterial count 49
4.3.2. Isolation and identification of halotolerant biogenic amine 50
formers
4.3.3. Amine forming ability of selected biogenic amine 53
forming bacteria
4.4. The effect of processing protocol on formation of amines in 56
salt cured sardines
4.4.1 Moisture content 57
4.4.2. Water activity 57
4.4.3. Salt content 57
4.4.4. TVB-N content 58
4.4.5. Total halotolerant bacterial count 58
4.4.6. Biogenic amine content 59
5 DISCUSSION 66-77
5.1. General quality of commercial salt cured fishes of Tuticorin 66