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SPOILAGE INDICATORS FOR DETERMINING TUNA AND MAHI-MAHI QUALITY
AND SAFETY
By
JING BAI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2018
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© 2018 Jing Bai
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To my family, faculty advisors, and my friends who teach me how
to love this wonderful world
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ACKNOWLEDGMENTS
First of all, I would like to express my sincere appreciation to
my committee chair,
Dr. Paul J. Sarnoski for his patient advice and encouragement in
the past three years.
He puts his trust in students and can see the true potential of
his students. He cares so
much about students’ projects and provides insightful advice
about the research. I also
sincerely appreciate my committee members, Dr. Renée M.
Goodrich-Schneider, Dr.
Shirley M. Baker, Dr. Naim Montazeri, and Dr. George L. Baker
for spending their
precious time to provide valuable guidance and aid through this
process. I would like to
thank all the faculty members for imparting knowledge to me, all
the staff members for
helping me prepare paperwork, and order laboratory supplies in
the Food Science and
Human Nutrition Department at UF.
I would especially like to thank the Yeoman Fellowship Fund and
the Seafood
Industry Research Fund (SIRF) for supporting my research.
I would like to express special thanks to my great family,
including my husband,
my parents, my parents in-law and my little son, for their
support and constant
encouragement. I express my gratitude to Yangyang Song, my
husband, for supporting
every decision I make and helping me solve any problems I meet.
I am extremely
grateful to my parents for teaching me to develop integrity and
telling me how to face
the challenge in the life. I am not afraid of difficulties in
the life because I understand my
family always stands behind me and silently support me.
I would like to thank my colleagues La’Oshiaa Reed, Stephen
Koltun, Robert
Nusbaum, Yaozhou Zhu and Ying Fan in our lab for providing aid
in my projects and
making my stay in UF much more pleasurable. I also would like to
express my gratitude
to all my friends in my life for their warm love and endless
encouragement.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS
..................................................................................................
4
LIST OF TABLES
............................................................................................................
7
LIST OF FIGURES
..........................................................................................................
8
ABSTRACT
.....................................................................................................................
9
CHAPTER
1 INTRODUCTION
....................................................................................................
11
2 LITERATURE REVIEW
..........................................................................................
14
Economics of Tuna and
Mahi-Mahi.........................................................................
14 Aquaculture of Tuna and Mahi-Mahi
.......................................................................
15 Fish Spoilage
..........................................................................................................
16 Amino Acids and Fish Spoilage
..............................................................................
19 Biogenic Amine Concerns
.......................................................................................
20 Histamine in Tuna and Mahi-Mahi
..........................................................................
22 Effect of Spoilage on Fish Volatile Compounds
...................................................... 24
Colorimetric Strips for the Detection of Volatile
Amine............................................ 27 GC Methods to
Determine the Aroma Profile of Seafood as Chemical Indicators
of Spoilage
...........................................................................................................
30 HPLC and UHPLC Methods for Biogenic Amine Detection
.................................... 34 ELISA Detection of
Histamine
.................................................................................
37
3 A RAPID UHPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF
AMINO ACIDS AND BIOGENIC AMINES IN TUNA AND MAHI-MAHI
................... 40
Digest......................................................................................................................
40 Background Information and Objectives
.................................................................
40 Materials and
Methods............................................................................................
43
Fish Samples and Preparation
.........................................................................
43 Standards and Reagents
..................................................................................
43 Extraction and Derivatization
............................................................................
44 Determination of Biogenic Amines
...................................................................
45 Method Validation
.............................................................................................
46 Histamine ELISA Test Kit
.................................................................................
46
Results and
Discussion...........................................................................................
47 Method Development
.......................................................................................
47 Linearity and Sensitivity
....................................................................................
49 Recovery and Repeatability
..............................................................................
49 Resolution and Theoretical Plates
....................................................................
51
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Application to Different Spoilage Grade of Mahi-Mahi (Coryphaena
hippurus) and Yellowfin Tuna (Thunnus albacares)
...................................... 51
Summary
................................................................................................................
58
4 AROMA PROFILE CHARACTERIZATION OF MAHI-MAHI AND TUNA FOR
DETERMINING SPOILAGE USING PURGE AND TRAP GAS CHROMATOGRAPHY-MASS
SPECTROMETRY (PT-GC-MS) ............................. 66
Digest......................................................................................................................
66 Background Information and Objectives
.................................................................
67 Materials and
Methods............................................................................................
70
Fish Samples and Preparation
.........................................................................
70 Standards and Reagents
..................................................................................
70 Extraction Procedures
......................................................................................
71 Purge and Trap Conditions
...............................................................................
72 GC-MS Analysis
...............................................................................................
72 Calculations
......................................................................................................
72
Results and
Discussion...........................................................................................
73 Summary
................................................................................................................
82
5 DETERMINING QUALITY ATTRIBUTES OF MAHI-MAHI AND TUNA BY
OPTIMIZED COLORIMETRIC STRIPS
..................................................................
90
Digest......................................................................................................................
90 Background Information and Objectives
.................................................................
91 Materials and
Methods............................................................................................
93
Fish Samples and Preparation
.........................................................................
93 Standards and Reagents
..................................................................................
94 Colorimetric Strip Method
.................................................................................
95 Determination of Biogenic Amines and Free Amino Acids by
UHPLC
(Conducted in Chapter 3)
..............................................................................
96 Determination of Aroma Profile by PT-GC-MS (Conducted in Chapter
4) ........ 96 Statistical Analysis
............................................................................................
97
Results and
Discussion...........................................................................................
97 Summary
..............................................................................................................
106
6 CONCLUSION
......................................................................................................
112
APPENDIX: EXTERNAL STANDARD PREPARATION FOR UHPLC METHOD
........ 115
LIST OF REFERENCES
.............................................................................................
116
BIOGRAPHICAL SKETCH
..........................................................................................
133
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LIST OF TABLES
Table page 3-1 Linearity for different amino acids and amines
analyzed by UHPLC .................. 59
3-2 Percentage recoveries of three biogenic amines
................................................ 60
3-3 The number of theoretical plates, and resolution of biogenic
amines ................. 60
3-4 Amino acids and biogenic amines (mg/kg) in seven grades of
mahi-mahi (M) ... 61
3-5 Amino acids and biogenic amines (mg/kg) in seven grades of
tuna (T) ............. 62
3-6 ELISA results of mahi-mahi (M) and tuna (T) calculated by
using the standard provided in kit and standard prepared in lab.
....................................... 63
3-7 Pearson correlation coefficients (r) between ELISA results
of mahi-mahi (M) and tuna (T) calculated by using the standard
provided in the kit, standard prepared in lab and histamine results
from UHPLC method. .............................. 64
4-1 Volatile compounds associated with spoilage in seven grades
of mahi-mahi calculated by internal standard method, (ng/g) fish
sample. ............................... 85
4-2 Volatile compounds associated with spoilage in seven grades
of tuna calculated by internal standard, (ng/g) fish sample.
............................................ 86
4-3 Biogenic amines contents in seven grades of mahi-mahi and
tuna sample (ng/kg) calculated by spiking standard method and
external standard method.
..............................................................................................................
87
4-4 Flavor descriptors of volatile compounds associated with
spoilage in mahi-mahi and tuna samples. Pearson correlation
coefficients between levels of volatile compounds with increasing
spoilage grade of mahi-mahi and tuna ....... 89
5-1 Linearity of rose bengal strips and BPB strips
.................................................. 109
5-2 Volatile biogenic amines in seven grades of mahi-mahi
samples detected by rose bengal and BPB strips for fish samples
(n=5) ........................................... 109
5-3 Volatile biogenic amines in seven grades of tuna sample
calculated by rose bengal and BPB strips for fish samples (n=5)
................................................... 109
5-4 Pearson correlation coefficients (r) between methods for
mahi-mahi (n=3)...... 110
5-5 Pearson correlation coefficients (r) between methods for
tuna (n=3) ............... 111
A-1 Concentrations of each amino acid in five levels of external
standard solutions and stock solution, mg/L
solution.......................................................
115
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LIST OF FIGURES Figure page 2-1 Global catches of albacore,
bigeye, skipjack and yellowfin data from 1960 to
2016. Data from WCPFC (2016).
.......................................................................
39
3-1 Chromatographic separations of Mahi-mahi grade 1 sample
spiked with 10ppm of each amino acid and biogenic amine standards..
............................... 65
4-1 Example of a chromatogram (mahi-mahi grade 7).
............................................ 84
5-1 Biogenic amine cocktail standard solutions reacted with rose
bengal strips. Photo courtesy of author.
.................................................................................
108
5-2 Biogenic amine cocktail standard solutions reacted with BPB
strips. Photo courtesy of author.
............................................................................................
108
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Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
SPOILAGE INDICATORS FOR DETERMINING TUNA AND MAHI-MAHI
QUALITY
AND SAFETY
By
Jing Bai
August 2018
Chair: Paul J. Sarnoski Major: Food Science
The consumption of spoiled fish containing high levels of
histamine result in the
highest incidence of illness from fish poisoning. Tuna (Thunnus
albacares) and mahi-
mahi (Coryphaena hippurus) are two major fish species
responsible for histamine
poisoning in the United States. The main purpose of this
research was to develop
spoilage indicators for determining tuna and mahi-mahi quality
and safety. A reversed-
phase ultra-high performance liquid chromatography (UHPLC)
method, purge and trap
gas chromatography-mass spectrometry (PT-GC-MS), and two color
strip methods
were developed and optimized to be used for determining fish
spoilage. The rapid
UHPLC method developed in this study could identify and quantify
dansylated amino
acids, histamine and other biogenic amines that can act as
co-indicators of histamine
(scombroid) poisoning in tuna and mahi-mahi fish sample
simultaneously within 17.5
minutes. This UHPLC method showed good linear response,
sensitivity, resolution,
percentage recovery, repeatability, and number of theoretical
plates. Twenty aroma
compounds in mahi-mahi and sixteen volatile compounds in tuna
associated with fish
spoilage could be determined by this purge and trap GC-MS method
without a
derivatization procedure. Volatile compounds identified as key
spoilage indicators of
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tuna and mahi-mahi were amines (dimethylamine, trimethylamine,
isobutylamine, 3-
methylbutylamine, and 2-methylbutanamine), alcohols
(2-ethylhexanol, 1-penten-3-ol
and isoamyl alcohol, ethanol), aldehydes (2-methylbutanal,
3-methylbutanal,
benzaldehyde), ketones (acetone, 2,3-butanedione, 2-butanone,
acetoin) and dimethyl
disulfide. A rose bengal strip, and a bromophenol blue strip
created in this study
produced standard curves with good linearity and also showed
uniform colorimetric
response to volatile amines. The colorimetric strips were
validated by investigating the
correlation of the results obtained by colorimetric strips with
the increasing spoilage
grades of fish, and results obtained by histamine-specific ELISA
kit, UHPLC and PT-
GC-MS and satisfactory correlations were obtained. The three
detection methods
developed in this study can be used to monitor the quality
changes of mahi-mahi and
tuna.
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CHAPTER 1 INTRODUCTION
Tuna is one of the top five consumed seafood species and
accounts for around 5%
of fisheries and aquaculture production for human consumption in
the world (Paquotte,
2003). The annual global catch of tuna in the ocean (wild
caught) increased from
698,260 tonnes in 1960 to 4,857,709 tonnes in 2016 (WCPFC,
2016). Mahi-mahi
(Coryphaena hippurus) is found mostly in tropical regions and
most of the catch occurs
in the Pacific Ocean. The annual landings of mahi-mahi have
increased 7.5 folds in last
60 years (Whoriskey et al., 2011). One of the largest consumers
of mahi-mahi is the
United States (Hunter, 2013).
Fish spoilage means any change in the condition of fish that
leads to fish
becoming less palatable or even toxic. These changes include
off-flavors formation,
amino acids changing, texture deterioration, discolorations, the
decrease of nutritional
value and other alterations in fish (Ashie et al., 1996). During
fish spoilage, toxic
biogenic amines, including histamine, cadaverine, and putrescine
may be produced in
certain fish species (Bulushi et al., 2009). The highest
incidence of illness from fish
poisoning is associated with the consumption of time and
temperature abused
scombroid fish that contain significant amounts of histamine
(Morrow et al. 1991). Two
major fish species responsible for histamine poisoning in the
United States are from
tuna and mahi-mahi (Ahmed, 1991).
The colorimetric strip is a cost-effective method that has been
widely used in
food analysis. This method is based on the principle that
chemical indicators or
bioactive sensors adhered to the treated papers could change
color when reacting with
the specific compound in food. The pH paper is used in a wide
range of food laboratory
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to industrial applications and the paper changes color under the
influence of hydroxide
or hydrogen ions in the food system. The other major
applications of colorimetric strips
in food science are the detection of heavy metals, testing milk
pasteurization,
determination of toxins and foodborne pathogens. In 2016, a new
kind of indicator strip
combining bromophenol blue (BPB) was developed to detect
degradation levels of
seafood (Dole et al., 2016).
Gas chromatography-mass spectrometry (GC/MS) is an instrument
that is mostly
used to identify and quantify volatile and semi-volatile
compounds in seafood (Duflos et
al., 2006; Grimm et al., 2000; Wong et al., 1967). Separation of
components is based on
the principle that different compounds have different strengths
of interaction with the
stationary phase. Mass spectrometry can sensitively identify
molecular weight of
fragment molecules. Volatile compounds have been used as
indicators for the quality
assessment of seafood products (Wierda et al., 2006; Soncin et
al., 2008; Alasalvar et
al., 2005).
Ultra-high performance liquid chromatography (UHPLC) is an
advanced type of
separation technology. UHPLC has the same principle as
high-performance liquid
chromatography (HPLC) in that the separation of compounds is
dependent on the
compound affinity between mobile and stationary phases.
Comparing with regular LC
system, UHPLC can be operated under pressure as high as 120 MPa,
be packed with
silica column with smaller particle size, separates molecules
faster with a higher
resolution, and needs less mobile phase (Wu et al., 2001). UHPLC
has been widely
used in determining free amino acids and biogenic amines
associated with fish spoilage
(Jia et al. 2012; Simat et al., 2011).
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The main purpose of this research was to develop new or refined
spoilage
indicators for determining tuna and mahi-mahi quality and
safety. A rapid UHPLC
method was developed to identify and quantify amino acids,
histamine and other
biogenic amines that can act as co-indicators of histamine
(scombroid) poisoning in
tuna and mahi-mahi fish samples. A GC-MS method was set up to
determine the aroma
profile of mahi-mahi and tuna for chemical indicators of
spoilage. Rapid detection strips
were developed and optimized to change color uniformly and give
good linearity. The
correlations between the colorimetric strips with UHPLC, GC, and
ELISA were reported.
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CHAPTER 2 LITERATURE REVIEW
Economics of Tuna and Mahi-Mahi
Tuna is a kind of saltwater fish and belongs to Scombridae
family. Around 50
species fall into the category of tuna and the five major
species of tuna for consumption
are albacore (Thunnus alalunga), bigeye (Thunnus obesus),
bluefin (Thunnus thynnus),
skipjack (Katsuwonus pelamis) and yellowfin (Thunnus albacares)
(Vinas, 2009). Tunas
are widely distributed in oceans around the world. Tuna plays an
important role in
seafood international trade and accounts for around 5% of
fisheries and aquaculture
production for human consumption in the world (FAO, 2010). The
annual global catch of
tuna has tended to increase and the global catch of four major
tuna species from 1960
to 2014 are shown in Figure 2-1 (WCPFC, 2016). There are three
major parts of the
global tuna market: sashimi market, fresh and frozen tuna, as
well as canned tuna
(Jimenez-Toribio et al., 2010). The United States is the second
largest importer of tuna
after Japan (FAO, 2010). The top five species of fresh tuna
imported to the USA are
yellowfin, bigeye, albacore, bluefin, and skipjack. Fresh and
frozen tuna, canned tuna,
are imported into the United States with an estimated amount of
287,440 tonnes per
year (FAO, 2017).
Mahi-mahi, which also named as common dolphinfish (Coryphaena
hippurus), is
a migratory pelagic fish and is found mostly in the tropical
regions of the world. About
sixty countries are known for mahi-mahi landings and most of the
catch occurs in the
Pacific Ocean. Peru, Taiwan Province of China, Indonesia and
Ecuador are the major
countries where mahi-mahi are landed the most. Nearly 60% of
mahi-mahi imported into
the United States are from Peru and Ecuador. Global landings of
mahi-mahi increased
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from 7000 tonnes in 1950 to 103,000 tonnes in 2013 (FAO, 2016).
The United States is
one of the largest consumers of mahi-mahi (Hunter, 2013).
Aquaculture of Tuna and Mahi-Mahi
During the last decades, aquatic farming has been a fast-growing
food
production industry powered by technological impulsion.
Currently, bluefin tuna
(Thunnus thynnus) is the dominate species in tuna aquaculture.
Bluefin tuna is a
valuable tuna species that can be sold through the selective
sushi and sashimi market
(Tseng et al., 2012). The global catch of wild caught bluefin
tuna decreased from 89,000
tones in 1980 to 42,000 tonnes in 2011 due to unsustainable
fishing decreasing wild
stocks (Metian et al., 2014). To meet the continuous high demand
of bluefin tuna,
aquaculture of this species has been under development over the
past thirty years.
Large-scale commercial bluefin tuna aquaculture started in the
1980s, and now
accounts for 18% of global bluefin tuna production (Metian et
al., 2014). The
Mediterranean region, Mexico, Australia, and Japan are major
regions that perform
bluefin tuna aquaculture. Japan is the largest importer of
farmed bluefin tunas. Before
2007, almost all of the Mediterranean farmed Atlantic bluefin
were exported to the
Japanese market. However, after Mexico began to farm Pacific
bluefin at a lower
production cost, Mexican farmed bluefin has been competitive in
the Japanese market
(FAO 2010).
Mahi-mahi has been considered as a promising candidate species
for
commercial aquaculture production due to its world-wide
consumption. The aquaculture
development of the mahi-mahi began in 1981, and circular tanks
were used to stock the
fish (Lee and Ostrowski, 2001). However, raising mahi-mahi on a
large commercial-
scale is not achieved by far due to the technological
challenges. Mahi-mahi would be
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sexually mature after six months, and mono-sex culture is best
to achieve a high growth
rate of fish. However, there is no current solution to this
problem. The size of mahi-mahi
harvested at six months is not comparable with the wild fish,
which weight is generally
above 5 kg. New techniques are needed to make the farmed
mahi-mahi to have
equivalent quality with the wild mahi-mahi already in the
market.
Fish Spoilage
Fish spoilage can happen rapidly after fish landing and various
components
break down or new compounds form during this process. The three
main types of fish
spoilage mechanisms are microbial, enzymatic, and chemical
(Ghaly et al., 2010). Lipid
oxidation, protein degradation and the decrease of other
valuable molecules are major
concerns of fish spoilage (Clancy et al. 1995; Ghaly et al.,
2010). Every year, almost 10
to 12 million tonnes of fish are lost due to spoilage accounting
for ten percent of the total
fish production (FAO, 2010). Fish spoilage is considered as an
important aspect of food
safety.
The growth and metabolism of microbes is considered as a main
reason leading
to the spoilage of fish. Higher levels of free amino acids and
trimethylamine oxide exist
in fish materials than other types of meat and these substances
are good microbial
substrates (Gram, 2002). Fish has an immune system to prevent
bacteria from invading
the fish tissue but the immune system stops after fish death.
Bacteria then can enter the
fish through the skin and contaminate the body cavity, belly,
gill tissue, and kidney and
proliferate freely (Fraser and Sumar, 1998). Microorganisms with
amino acid
decarboxylase activity can produce biogenic amines, and
sulphides with unpleasant
odors (Ghaly et al., 2010). Histamine is produced in raw fish by
the reaction of the
bacterial histamine decarboxylase. Histamine is produced by
gram-positive lactic acid
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bacteria in fermented products, such as wine, aged cheeses, and
fish sauce. However,
in raw fish tissue, histamine is produced by gram-negative
enteric bacteria such as
Enterobacter aerogenes, Morganella morganii and Pseudomonas
aeruginosa
(Hungerford, 2010). These bacteria, which produce histamine in
fish, generally exist in
the saltwater environment and are naturally present on the
external surfaces and inside
of live fish (Visciano et al., 2012). There are two routes of
synthesis of putrescine in fish.
Arginine can indirectly produce putrescine by arginine
decarboxylase via agmatine. Also,
putrescine can be formed from arginine by arginine deiminase,
ornithine
carabamoyltransferase and ornithine decarboxylase (Prester
2011). The genus
Staphylococcus is the major bacteria producing putrescine in
fish tissue (Wunderlichova
et al., 2014). Free lysine can produce cadaverine by lysine
decarboxylase and it has
been known that various species of bacteria have lysine
decarboxylase activity. A
research study showed that ninety-two percentage of mesophilic
bacteria with
decarboxylase activity isolated from mahi-mahi had lysine
decarboxylase activity (Frank
et al., 1985). Shewanella putrifaciens, Photobacterium
phosphoreum and Vibrionacaea
are the major bacteria responsible for the production of
trimethylamine (TMA), which
has a strong fishy odor (Ghaly et al., 2010). Beyond producing
amines, the growth of
microbes, such as Shewanella putrifaciens and Pseudomonas
perolens can also lead
the formation of short-chain carbonyls, alcohols, esters, sulfur
compounds and others
with unpleasant odors (Duflos et al., 2010; Jorgensen et al.,
2001).
Enzymatic spoilage is another basic mechanism of fish spoilage.
The muscle
tissue and gut of fish contain endogenous enzymes and these
enzymes lead to autolytic
reactions in spoiled fish. Autolytic enzymes in fish influence
the texture of fish tissues,
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18
such as tenderization and flesh softening. The changes of
texture shorten the lifetime of
the fish product and have negative effects on the quality. The
accumulations of
hypoxanthine and formaldehyde as a result of postmortem
endogenous enzymatic
activity influence the textural quality of fish. After fish
death, glycogen is hydrolyzed to
lactic acid and this glycolysis process is a result of
endogenous enzymatic activity. Due
to the accumulation of lactic acid, the pH of fish meat falls,
and the fish tissue is more
susceptible to bacterial growth. Proteolytic enzymes widely
exist in muscle and the
viscera of fish, and belly bursting can be caused by these
enzymes (Ghaly et al., 2010).
Muscle proteins can be hydrolyzed by endogenous proteolytic
enzymes after the death
of fish and the rigor mortis was observed. As the process of
fish spoilage continues,
rigor resolves, and the muscle of fish become becomes limp
(Cheret et al., 2007;
Olafsdottir et al., 1997). The degradation of proteins produces
free amino acids and
peptides, which can be used as the nutrition source for
microbial growth. Lipid oxidation
in fish tissue is also influenced by endogenous enzymes, such as
lipoxygenase and
peroxidase, and unpleasant odors are formed during this process
as well (Hultmann et
al., 2004).
Chemical spoilage is the third mechanism of fish spoilage and
mainly includes
oxidative rancidity and non-enzymatic browning. Fish tissue is
rich in unsaturated fatty
acids that can be oxidized during fish spoilage. Hydroperoxides
are produced during the
propagation procedure in lipid oxidation and then break down to
form compounds with
unpleasant flavors. Research showed that the non-enzymatic lipid
oxidation of tuna
meat continued during frozen storage, and frozen tuna had higher
peroxide values than
fresh tuna (Tanaka et al., 2016). The Maillard reaction
involving amino acids, peptides
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19
and reducing sugars in fish is a non-enzymatic browning and
leads color changes and
formation of specific flavors in spoiled fish that has been
cooked (Ashie et al., 1996).
The autoxidation of myoglobin to metmyoglobin, is also
responsible for the browning
discoloration in spoiled fish (Genigeorgis, 1985).
Amino Acids and Fish Spoilage
Seafood materials are rich in free amino acids and peptides that
are produced
from autolysis of fish muscle proteins and are important
substrates or catalysts for
reactions pertaining to fish spoilage (Fraser and Sumar, 1998).
The protein content in
tuna is around twenty-three percent of the wet weight basis and
is the source of free
amino acids (Peng et al., 2013). Essential amino acids are those
amino acids that
cannot to be synthesized in humans and thus need to be obtained
from diet. Free
tyrosine, tryptophan, threonine, cystine, valine, lysine,
methionine, isoleucine, leucine,
phenylalanine are essential amino acids that have been
identified in tuna (Sen, 2005).
Mahi-mahi is a rich source of protein and contains eighteen
amino acids, including
alanine, arginine, aspartic acid, cystine, glutamic acid,
glycine, histidine, isoleucine,
leucine, leucine, methionine, phenylalanine, proline, serine,
threonine, tryptophan,
tyrosine, valine, which have been determined in mahi-mahi to
participate in building
muscle protein in fish (Ostrowski et al., 1989). Free histidine,
ornithine, lysine, and
glutamine was identified and quantified in mahi-mahi by Antoine
et al. (2002). However,
research about investigating the other major free amino acids in
mahi-mahi has not
been studied.
Amino acids are essential components and play the central role
in metabolic
pathways. Oxidative rancidity is a non-enzymatic mechanism that
produces
hydroperoxides and has been considered as a major cause of fish
spoilage for a long
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20
time (Ghaly et al., 2010). In this process, unsaturated fatty
acids or triglycerides in fish
are oxidized and "rancid" odors, flavors are released. Amino
acids have been found to
catalyze this oxidation reaction alone or in association with
specific trace metal ions
(Ashie, 1996). Amino acids and peptides in fish also participate
in non-enzymatic
browning, such as the Maillard reaction, and cause discoloration
of fish muscle (Ocano-
Higuera, 1992). Amino acids are also precursors of some
substances, such as biogenic
amines. Biogenic amines are organic bases and are produced in
fish by microbial
decarboxylation of amino acids or by transamination of amino
acids (Zhai et al., 2012).
Many biogenic amines have biological activity and can influence
physiological functions
in human body. Histamine, cadaverine, and putrescine are
biologically active amines
and have been widely studied due to their toxicity (FDA, 2011).
During spoilage,
deamination of amino acids produce ammonia, and deamination of
sulfur-containing
amino acids form sulfur compounds which give unpleasant
off-odors to seafood
(Herbert and Shewan, 1975).
Biogenic Amine Concerns
Among the biogenic amines found to occur during the spoilage
process of fish,
only histamine, cadaverine, and putrescine are considered as
significant markers of
food quality (Bulushi et al., 2009). It has been found that
though decarboxylation,
histamine, cadaverine, and putrescine are produced from the free
amino acids histidine,
lysine, and arginine, respectively (Prester, 2011). Histamine
has a heterocyclic structure,
cadaverine and putrescine have an aliphatic structure (Mohamed
et al., 2009). Although
the toxicological levels of individual biogenic amines are
difficult to establish, a
maximum level of total amines has been proposed as 750–900 mg/kg
(Ladero et al.,
2010).
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21
Histamine is identified as the major natural chemical
responsible for fish
poisoning. Histamine intoxication was first found originating
from the family Scombridae
(Lehane and Olley, 2000). One characteristic of scombroid fish
is that these fish contain
a high amount of histidine, which is the precursor of histamine.
Other non-scombroid
fish species, such as mahi-mahi, are also implicated in
scombroid poisoning (FDA,
2005). Some bacteria have been found to produce histamine in
fish samples at a
temperature as low as 0 °C and this phenomenon makes it
difficult to prevent histamine
formation in fish products (Hungerford, 2010). Research pointed
out that histamine did
not distribute uniformly in spoiled fish (Lehane and Olley,
2000). Histamine content that
exceeds a concentration of 50 ppm (5 mg/100g) in tuna and
mahi-mahi represented the
decomposition in these fish (FDA, 2005). According to the fish
and fishery products
hazards and controls guidance (FDA, 2011), illness-causing fish
mostly contains more
than 200 mg/kg histamine. Putrescine and cadaverine can
potentiate histamine toxicity
by inhibiting the intestinal histamine-metabolizing enzymes and
diamine oxidase
(Bulushi et al., 2009; Visciano et al., 2012).
Scombroid poisoning can cause allergy-like symptoms and the
onset of
scombroid poisoning is rapid, which range is from several
minutes to 3 hours (Bulushi et
al., 2009). After ingestion of spoiled fish containing more than
100 ppm of histamine, the
person might have symptoms including oral numbness, headache,
dizziness,
palpitations, difficulty in swallowing and some allergy-like
symptoms (Bulushi et al.,
2009; Hungerford, 2010). However, some people are sensitive to
the biogenic amines
and even ingesting a low amount of histamine can lead to the
onset of symptoms.
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22
Histamine, putrescine, and cadaverine are considered essential
spoilage
markers of fish products due to ability to show the microbial
contamination and
degradation reactions during storage. The amount of histamine
and putrescine of
mackerel (S. scombrus) increased after storage of fish samples
at 22 °C for 12 hours.
These two biogenic amines were considered as quality markers of
mackerel (Prester et
al., 2009). The formation of histamine, putrescine, and
cadaverine in herring was
observed after the fish samples were stored at 10 °C for two
days (Mackie et al., 1997).
Histamine, putrescine, and cadaverine also accumulated in
sardines after a storage
period at 4 °C and these three biogenic amines were identified
as quality indicators of
sardines (Sardina pilchardus) (Ozogul et al., 2006). Histamine
content in sardine
increased to 620 ppm after the sardine samples stored at 25 °C
for 24 hours (Visciano
et al., 2007).
Histamine in Tuna and Mahi-Mahi
Tuna and mahi-mahi have been considered as two major sources of
scombroid
poisoning (Ahmed, 1991; FDA, 2011). Histidine is a precursor of
histamine formation
and the muscle tissue of tuna and mahi-mahi contains large
amounts of histidine
(Bulushi et al., 2009). Free histidine levels in tuna are around
7 g/kg and in mahi-mahi
are around 5 g/kg (Antoine et al., 1999). Histamine production
in fish depends on the
level of endogenous histidine in the fish, the presence of
bacterial histidine
decarboxylase, and the environmental conditions (Visciano et
al., 2012). The
decarboxylase enzymes produced in spoiling fish by certain
bacteria can convert amino
acids to biogenic amines (Lehane and Olley, 2000). Freshly
caught fish have the low
level of histamine, usually are less than 0.1 mg/100g (Auerswald
et al., 2006).
Histamine is formed from free histidine by bacterial histidine
decarboxylases in fish
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23
tissue, usually when fish is exposed to elevated temperatures
after the catch (Visciano
et al., 2012). There are 112 species of bacteria that have been
identified as histidine-
decarboxylating bacteria (Taylor et al., 1978). The family
Enterobacteriaceae, the
genera Clostridium and Lactobacillus are the major bacteria
families responsible for
histidine decarboxylation (Lehane and Olley, 2000).
Histidine-decarboxylating bacteria
are present at a great proportion of the microbial population
when fish spoil. Lehane and
Olley (2000) found that 31% of isolates from decomposing
skipjack tuna and 7% of
isolates from spoiled mahi-mahi growing at warm temperature were
histidine-
decarboxylating bacteria. Decarboxylase enzymes produced by
endogenous bacteria
are insignificant when compared with those produced by exogenous
sources (Lehane
and Olley, 2000). Due to histidine-decarboxylating bacteria
growing rapidly when the
temperature is near 32.2 °C, high temperature spoilage is
identified as the main reason
leading to accumulation of histamine in fish (FDA, 2011).
Research showed that after twelve-day storage at 7 °C, the
histamine
concentration in mahi-mahi increased from 0 mg/100g to 160
mg/100g, while the
histidine concentration decreased from 400 mg/100g to 180
mg/100g (Antoine et al.,
2002). Histamine amount in both red and white muscle of tuna
increased after storage
in a controlled environment for 33 days, and histidine amount of
these fish samples
decreased (Ruiz-Capillas and Moral, 2004). The amount of
histamine, cadaverine,
putrescine of tuna increased after a storage period, and these
biogenic amines were
considered as hygienic quality markers of tuna (VecianaNogues et
al., 1997). Rossi et
al. (2002) reported that after 48 hours storage at room
temperature, the levels of
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24
histamine, cadaverine, and putrescine in Skipjack (Katsuwonus
pelamis) increased to
1533, 649 and 60 mg/kg, respectively.
Effect of Spoilage on Fish Volatile Compounds
The volatile profile is one essential quality parameters of fish
meat, and it can
reflect the organoleptic characteristic of the fish product
(Edirisinghe et al., 2007).
Volatile compounds produced in fish meat are mainly based on
microbial action,
enzymatic action, lipid oxidation and other chemical reactions.
Several specific alcohols,
carbonyls, acids, amines, sulfur compounds, aldehydes, and
ammonia have been
identified as spoilage indicators of fish products due to the
content of these compounds
changing during fish spoilage (Ashie et al.,1996; Duflos et al.,
2006).
Short-chain carbonyls, alcohols, and esters can generate due to
the microbial
spoilage, enzymatic or non-enzymatic lipid oxidation. Ethanol,
2,3-butanediol 1-penten-
3-ol, 3-methyl-1-butanol, 1-butanol, and 1-octen-3-ol were
alcohols, which content in
tested fish samples increased during fish spoilage and response
for the pungent,
alcoholic and creamy odor (Leduc et al., 2012; Duflos et al.,
2005; Olafsdottir et al.,
2005). The level of ethanol reached 314 mg/kg in pink salmon
(Oncorhynchus
gorbuscha) after three days storage at 10 °C due to the
microorganisms utilizing
carbohydrates (Himelbloom et al., 2013). The accumulation of
branched-chain alcohols
in spoiled fish is reported as the result of degradation of
amino acids (Rehbein and
Oehlenschlager, 2009). The aldehydes 3-methylbutanal and
2-methylbutanal the fishy
odor and have been identified as fish spoilage indicator due to
their formation in whiting,
mackerel and cod flesh during spoilage (Duflos et al., 2005).
Ethyl acetate and ethyl
butanoate are two esters determined in spoiled fish and
contribute fruity and sweet
odors (Iglesias et al., 2009; Olafsdottir et al., 2005).
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25
Volatile amines, such as trimethylamine (TMA), dimethylamine
(DMA), and
isobutylamine, are identified as essential spoilage indicators
due to their gradual
accumulation during fish spoilage and the contribution of
characteristic fishy odor.
Trimethylamine oxide (TMAO) has been widely found in marine fish
and is used as an
osmoregulant by fish to avoid dehydration and balance changing
salt levels from the
environment (Gram et al., 2002). Gram-negative bacteria can
reduce TMAO to
trimethylamine (TMA) to obtain energy. TMAO is non-odorous,
however, TMA is a
volatile compound with a very low odor threshold and a stale
fish odor. The level of TMA
in fish has been identified as an indicator of microbial
deterioration in fish (Fraser and
Sumar, 1998). TMA has been used as an effective marker to
distinguish the fresh and
spoiled fish samples (Leduc et al., 2012; Bene et al., 2001;
Ghaly et al., 2010).
Trimethylamine oxide in fish fillets can be also reduced to
dimethylamine (DMA) during
spoilage of fish. The concentration of DMA in freshly caught
fish is as low as 2 ppm.
DMA starts to accumulate automatically from TMAO by the activity
of endogenous
enzymes in the very early stage of spoilage (Chan et al., 2006).
DMA has an ammonia-
like odor, and the amount of DMA has been accepted as an index
for fish freshness.
Research showed that the content of DMA increased in albacore
tuna after frozen
storage (Ben-gigirey et al., 1999).
Other major volatile amines produced during spoilage of fish are
ammonia and
isobutylamine. Ammonia usually already present in freshly caught
fish and accumulates
during fish storage by deamination of amino acids. Isobutylamine
has fishy type odor
and is commonly considered as the microbial degradation product.
This amine is
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26
produced by decarboxylation of valine during fish spoilage
(Gruger et al., 1972; Eskin,
2013; Gill et al., 1983).
Sulfur compounds are considered as another key component of the
volatile
compounds formed in fish spoilage process (Gram et al., 2002).
Sulfur compounds are
originally in low concentrations in the fish body and accumulate
after fish landing (Duflos
et al., 2006). Sulfur compounds have extremely low thresholds
and give out very
unpleasant odors. The generation of sulfur compounds in fish is
mainly by microbial
enzymatic activity (Ashie et al., 1996). Plenty of
sulfur-containing amino acids, peptides
exist in fish tissue and the degradation of these compounds
during spoilage process
produce the odorous sulfur compounds. Hydrogen sulfide, dimethyl
disulfide, dimethyl
trisulfide and methanethiol are sulfur compounds commonly found
in spoiled fish (Kawai
et al., 1996). Methylethyl disulfide, 3-(methylthio)-propanal,
1-(methylthio)-propane, 2-
methyl-3-furanthiol are sulfur compounds that also be found in
spoiled tuna (Varlet and
Fernandez, 2010).
Volatile acids, such as formic acid, acetic acid, and propionic
acids, are produced
from the breakdown of certain amino acids and atmospheric
oxidation of lipids
(Olafsdottir et al., 2005; Koutsoumanis et al., 1999). These
volatile acids also contribute
to the odor of spoiled fish. The formation of acetic acid in cod
(Gadus morhua) was
observed after a ten-day spoilage process, and this formation
was associated with
microbial activity (Duflos et al., 2006). The concentration of
formic acid and acetic acid
in smoked salmon increased with storage time (Hansen et
al.,1995). Terpenes are
compounds already exist in freshly caught fish, and some
specific terpenes accumulate
during the spoilage process and contribute to odor change of
fish products. Limonene
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27
concentration in seabream and Baltic herring increased during
frozen storage (Iglesias
et al., 2009; Aro et al., 2003). Beyond the limonene
concentration increasing, α-pinene,
3-carene also accumulated during the spoilage of seabream
(Alasalvar et al., 2005).
Alkanes and alkenes also produced in some specific fish types,
such as mackerel, as
the storage time increases (Dulos et al., 2006). The volatile
profile is a typical feature of
food, and the volatile compounds difference between fresh and
spoiled fish can be used
as chemical fingerprints to reflect the relative compounds
changes.
Colorimetric Strips for the Detection of Volatile Amine
More research has been focused on using several amine sensitive
dyes to
determine volatile amines (Rakow et al., 2005; Steiner et al.,
2010; Kuchmenko et al.,
2011). Amine sensitive dyes are colorimetrically responsive to
volatile bases produced
during food spoilage and simultaneously change their color.
Metalated
tetraphenylporphyrins, pH indicators and highly solvatochromic
dyes are three families
of chemically responsive dyes, which have been used to determine
biogenic amines
(Rakow et al., 2005). Solvatochromic dyes are a category of
chemical compounds that
change color depending on the polarity of the solvent dissolving
the dye. The electronic
structure of solvatochromic dyes usually contains a strong
zwitterionic component, and
electron donating and withdrawing groups are at the opposite
ends of the molecule
(Reichardt, 1994). As the solvent polarity increases, a
bathochromic shift occurs with
positive solvatochromic dyes and a hypos-chromic shift occurs to
negative
solvatochromic dyes (Cartwright, 2016). The commonly reported
class of
solvatochromic dyes includes azobenzenes, thiazines, pyridinium
N-phenolate betaine
dyes, and merocyanines (Atwood et al., 2017).
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28
Bromophenol blue is sulfonated hydroxy-functional
triphenylmethane dye and is
a commonly used pH indicator dye with a low pKa (Mills et al.,
1995). As an acid-base
indicator, bromophenol blue will lose a proton when the pH of
the environment around
this indicator is higher than the pKa of the dye. This
displacement changes the
electronic distribution within the molecule and the indicator
changes its color from yellow
to blue (Flores, 1978). Bromophenol blue has been known can
react with basic amines
(Kuchmenko et al., 2011). Bromophenol blue was found to react
with volatile biogenic
amines produced by a cod sample and showed a dramatic color
change from yellow to
blue (Miller et al., 2006). Bromophenol blue also has been used
as a color indicator to
assess the freshness of guava. As the volatile compounds
produced during developing
of guava, pH in the package headspace decreased and bromophenol
blue changed its
color from blue to green (Kuswandi et al., 2012).
Rose bengal is a xanthene dye with photophysical properties and
has played a
significant role in photobiology and dye-sensitized oxygenation
(Lamberts and Neckers,
1984). Rose bengal changes its color based on a protonation and
deprotonation
reaction. The dye is in the lactone form and transparent when
exposed to a low pH
environment; while it changes to its quinoid form with pink
color as pH increases
(Akerlind et al., 2011; Schoolaert et al., 2016). A rapid
colorimetric method was built by
using rose bengal to quantify anhydrous caffeine and
chlorphenoxamine hydrochloride
simultaneously in a pharmaceutical (Amin et al., 1995). Rose
bengal has been used to
detect the existence of amines and ammonia based on the
principle that amines have
basicity and can neutralize rose bengal (Paczkowski et al.,
1985). A monitoring tape
using rose bengal as the indicator to determine the ammonia gas
content in the air has
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29
been developed (Nakano et al., 1994). Rose bengal was used as a
xanthene dye in an
indicator device to react with the volatile amines produced by
biological agents in food.
The principle of this indicator device was that rose bengal
would change its color from
transparent to pink when exposed to the volatile amines in
tested food (Miller et al.,
2006).
Filter paper can be used to absorb dye solution to make
colorimetric strips. After
the strips are exposed to the headspace of samples, dye on the
strips will react with
volatile biogenic amines and change color (Dole et al., 2016). A
colorimeter is an
instrument that measures color, and it is used to measure the
indicator strips. A L*a*b*
system is a cylindrical coordinate system and is used to
quantify the color of strips. b* is
the yellow/ blue coordinate and the more positive the b* value
means more yellow hue
is present; the more negative the b* value means more blue hue
is present. a* value
presents the red/green coordinate, which negative value means
green and positive
value means red. A series of standard cocktails with different
concentrations can be
used to quantify the volatile amines in tested samples.
In previous research, BPB strips were found to correlate
volatile biogenic amine
content with quality grades for mahi-mahi (Dole et al., 2016).
However, the BPB strip
method is a broad detection method for the class of volatile
biogenic amines and as a
result the BPB strips were not in total agreement with the
results from ELISA, which
measure a specific analyte, in this case histamine (Dole et al.,
2016). The BPB strips
developed in Phase I did not have a good uniformity of color
change and the linearity of
the standard curve from BPB method was low (Dole et al., 2016).
The indicator device
containing rose bengal, which used to detect the biogenic amines
in food, was more
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30
complicated and with a higher cost than colorimetric strips
(Miller et al., 1999). Rapid
assays should be developed and optimized to produce an accurate,
sensitive, low cost
and timesaving method to detect fish spoilage.
GC Methods to Determine the Aroma Profile of Seafood as Chemical
Indicators of Spoilage
Gas Chromatography-Mass Spectrometry (GC-MS) is a highly
effective analytical
instrument to separate, identify and quantify volatile and
semi-volatile chemicals from a
complex food matrix (Lambropoulou et al., 2007; Sandra et al.,
2003; Wang et al., 1999).
In gas chromatography, the mobile phase is the gas that moves
through the column,
and the stationary phase is a polymer film that coats the column
filling or the column
wall (Abraham et al., 1999). Compounds passing through the
column have different
strengths of interaction with the stationary phase. The compound
having a larger
interaction with the stationary phase needs a longer time to
interact with the column and
migrates through the column later than other compounds (Sneddon
et al., 2007). Mass
spectrometry can provide the “fingerprint” information of a
molecule including its
molecular weight, structure or elemental composition. This
principle of this technique is
that molecules in samples are converted into ions as in the gas
phase with or without
fragmentation and then are distinguished by their mass-to-charge
ratio (m/z). The major
application of GC-MS includes identification and quantification
of food composition, food
additives, aroma components, transformation products (Simko et
al., 2002; Wishart,
2008; Bianchi et al., 2007). It can also detect a variety of
contaminants, such as
pesticides, packaging materials, and toxins (Tanaka et al.,
2000).
Purge and trap method is a dynamic headspace extraction method
commonly
connected to GC-MS or gas chromatography-olfactometry (GC/O).
The purge and trap
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31
method was established and firstly applied in analytical
techniques in the 1970s (Snow
and Slack, 2002). For the purge and trap process, inert gas goes
through the sample
and volatile analytes are stripped from the sample. Volatile
compounds are then re-
focused on a trap and then thermally desorbed onto a GC. The
development of this
dynamic extraction method improves the detection levels of
analytical instrumentation
and provide accurate and precise analysis. Comparing with the
static headspace
method, purge and trap method has a lower limit of detection
(LOD) value and can be
more sensitive (Lucentini et al., 2005; Beltran et al., 2006).
This dynamic extraction
method was able to extract volatile compounds in a higher amount
than solid-phase
microextraction (SPME) (Povolo et al., 2003). A trap containing
certain adsorbent resins
is able to remove water from the volatile compounds introduced
onto GC. A purge and
trap method was equipped to GC/MS to concentrate volatile
compounds of menhaden
fish oil and twenty-nine compounds, including aldehydes,
ketones, and carboxylic acids,
were able detected (Hsieh et al., 1989). A Tenax column was used
to trap volatile
compounds after the fish sauce was purged for sixteen minutes
and twenty-three
compounds were determined by GC-MS (Fukami et al., 2002). Purge
and trap is widely
used in collecting and concentrating volatile components from
fish meat, such as
gilthead sea bream, pink salmon, sockeye salmon, and atlantic
salmon (Girard et al.,
2000; Alexi et al., 2017; Jonsdottir et al., 2008).
Amine columns usually have low or mid polarity phases and are
designed for
determining amines and other specific basic chemical compounds
without any complex
derivatization procedure. Using amine columns in GC-MS can
improve the response for
the basic compounds and also prevent tailing of these analytes.
Amine columns are
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32
also able to separate the natural compounds having the oxygen
groups easily
influenced by hydrogen bonding. A SPME-GC-MS method with
Rtx-Volatile amine
column was able to determine trimethylamine (TMA) and
dimethylformamide in the
headspace above solid hexamethylene triperoxide diamine (HMTD)
without a
deactivation procedure (Steinkamp et al., 2016). Trimethylamine
and dimethylsulfide in
marine sediments were also analyzed by using a GC-MS method with
Rtx-Volatile
amine column, which was base-deactivated (Zhuang et al., 2017).
GC with flame
ionization detector (GC-FID) using cold on-column injection with
an Rtx-5 amine column
was able to identify and quantify putrescine and cadaverine in a
standard solution
without any derivatization procedure (Bonilla et al., 1997). A
Rtx-5 amine column was
also used in a GC system coupled to a nitrogen-phosphorus to
detect the ephedrines in
urine samples simultaneously (Eenoo et al., 2001). Volatile
amines C1 to C9, including
dimethylamine, trimethylamine, monoethylamine, isopropylamine
and others, in
standard solution was able separated and detected by GC-FID with
Rtx-5 Amine
column or PoraPLOT Amines column (Abalos et al., 2001).
Over the years, different samples extraction methods and column
types have
been used to detect spoilage of seafood products by GC-MS.
Volatile compounds
considered as spoilage indicator of cold smoked salmon were
identified by using three
different GC-MS methods. All these three GC-MS methods used
purge and trap method
as dynamic headspace collection and DB-5 MS column or DB-1701
column were used
to separate volatile compounds (Jorgensen et al., 2001; Joffraud
et al., 2001; Jonsdottir
et al., 2008). A SPME-GC-MS method using a ZB-Wax column was
developed to
determine the storage influence on volatile compounds of fresh
king salmon (Wierda et
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33
al., 2006). GC-MS method equipped with CAR/PDMS fiber to extract
volatile
compounds and Rtx-WAX column was able to investigate the
spoilage indicators of sea
bream and prawn (Soncin et al., 2008). Effect of storage on sea
bream was identified by
a GC-MS method using a Tenax trap and a WCOT fused silica column
and spoilage
markers were determined (Alasalvar et al., 2005). Volatile
compounds of European
seabass produced during storage were able to be detected by
using the GC-MS method
with dynamic headspace extraction and RTX-5 column (Leduc et
al., 2012). Freshness
markers of whiting were detected using a GC-MS method containing
CAR/PDMS fiber
and a BPX5 capillary column (Duflos et al., 2010). Volatiles
identified as spoilage
markers of yellowfin tuna was also detected by an SPME-GC-MS
method (Edirisinghe
et al., 2007). The GC-MS methods mentioned above were able to
detect alcohols, acids,
aldehydes, alkanes, ketones, trimethylamine and sulfur
compounds.
Amines in seafood, such as isobutylamine, 3-methylbutylamine,
and 2-
methylbutylamine, have been identified as spoilage markers of
seafood product and
give off fishy odor (Gill et al., 1983; Eskin, 2013; Mayr and
Schieberle, 2012). However,
these amines have not been able to be detected by using GC-MS
without derivatization
because short chain amines have high polarity, basic character,
and high aqueous
solubility. Preparation of amine derivatives is usually a
necessary step to increase the
volatility of compounds when using GC to analyze amines
(Staruszkiewicz et al., 1981;
Rogers et al., 1997; Du et al., 2001). For example, amines
including putrescine and
cadaverine in salmon were able detected by using an SPME-GC-MS
method with on-
fiber derivatization procedures (Awan et al., 2008). A new
simplified and accurate GC-
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34
MS method should be developed to separate and determine amines
and other spoilage
markers of fish products without any complex derivatization
step.
HPLC and UHPLC Methods for Biogenic Amine Detection
Ultra-high performance liquid chromatography (UHPLC) is another
used
instrument to measure amino acids and biogenic amines in seafood
products. When the
mobile phase passes through the column, components in mobile
phase have varying
strengths of interaction with the stationary phases. During an
LC separation run, the
composition of the mobile phase is often changed to alter the
phase partitioning of each
compound between the mobile phases and stationary phases. This
is called gradient
elution. The elution time of each compound is dependent on the
relative strengths of its
interaction with the mobile and stationary phase.
Reversed-phase columns (typically C18) are usually used as
stationary phases
and polar mobile phases, such as aqueous ammonium acetate,
acetonitrile, formic acid,
or mixtures of these solvents, are usually used for the
separation of biogenic amines
(Erim 2013). For example, an HPLC with C18 column and
fluorescence detection was
used study biogenic amines in canned tuna fish, and mackerel
(Peng et al., 2008). An
HPLC method with fluorescence derivatization used the C18 column
to separate eight
different amines, including histamine, putrescine, cadaverine
and others, in wines
(Busto et al., 1997). HPLC with C18 column was used to detect
seven biogenic amines
in beer (Tang et al., 2009). A C18 column with 1.8 μm particles
was applied in a UHPLC
method and was able to separate putrescine, cadaverine,
histamine, tyramine and other
four biogenic amines in fish and chicken samples (Dadakova et
al., 2009).
For fish and fish products, aqueous trichloroacetic acid (TCA)
is a major solvent
used to extract free amino acid and biogenic amines due to its
good protein precipitation
-
35
capacity (Hwang et al., 1997). Research showed that five
biogenic amines were able to
be extracted from homogenized fish and fishery products by using
a 5% TCA solution
(Shakila et al., 2001). Histamine, putrescine, tyramine, and
spermidine were extracted
from canned tuna sample by TCA solution, and then their
concentrations in canned tuna
were calculated (Zarei et al., 2011). Five free amino acids and
six biogenic amines were
extracted by TCA solution from tuna muscle tissue and quantified
by HPLC (Ruiz-
Capillas et al., 2004). TCA solution was used to extract
biogenic amines from fish tissue
and eight biogenic amines, including putrescine, cadaverine,
histamine, were able to be
determined in this extraction solution (Sagratini et al.,
2012).
A derivatization step is required for HPLC because most of the
biogenic amines
are lack of chromophore. Dansyl chloride and o-phthaldialdehyde
(OPA) are two major
derivatives used in HPLC methods (Malle et al. 1996;
VecianaNogues et al., 1997;
Salazar et al., 2000). Amino groups of free amino acids and
biogenic amines can react
with dansyl chloride and stable derivatives with a chromophore
can be formed. Due to
their fluorescent characteristics, the dansyl derivatives can be
determined using UV
detection. Dansyl chloride was used for derivatize biogenic
amines in the extraction
solution of fish and fishery product (Shakila et al., 2001).
Eight biogenic amines in
several types of fish and fish products were derivatized by
dansyl chloride and then
identified by HPLC system equipped with fluorescence detector
(Zhai et al., 2012). The
reagent o-phthaldialdehyde can react with amines and free amino
acids to produce
fluorescent products. Histamine, putrescine, and cadaverine in
tuna fish reacted with o-
phthaldialdehyde and produced stable derivatives (Rossi et al.,
2002). Free amino acids
in fish, including lysine, histidine, and others, were reacted
with o-phthaldialdehyde
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36
(OPA) to be able detected by a fluorescence detector (Antoine et
al., 1999). Nine
biogenic amines in canned yellowfin tuna, including histamine,
putrescine, and
cadaverine, were derivatized by dansyl chloride in pre-column
derivatization method
and were derivatized by o-phthaldialdehyde (OPA) in a
post-column derivatization
method (Simat and Dalgaard, 2010).
The HPLC technique is sensitive, reproducible and the most
useful for
simultaneous detection of free amino acids and biogenic amines
related to fish spoilage
indicators by far (Onal et al., 2007). Ten biogenic amines in
tuna considered as hygienic
quality indicators were monitored by using HPLC method equipped
with a C18 column
and involving a post-column derivatization procedure
(VecianaNogues et al., 1997).
Four free amino acids in mahi-mahi, which are considered as the
precursors of biogenic
amines, were reacted with o-phthaldialdehyde (OPA) and then
analyzed by HPLC
method with C18 column (Antoine et al., 2002). Dansylated amines
in fish and fishery
products were separated and detected by an HPLC method with a
good linearity and
sensitivity (Shakila et al., 2001). Eight biogenic amines as
spoilage markers in
fermented fish products reacted with dansyl chloride and were
able to be determined by
HPLC (Kose et al., 2012). Thirteen amino acids in mountain trout
reacted with 9-
fluorenylmethyl chloroformate were detected, and quantified by
HPLC system (Gunlu et
al., 2014). The simultaneous detection of biogenic amines and
amino acids can also be
achieved by HPLC technique. Histamine, cadaverine, tryptamine,
tyramine and their
precursor amino acids were derivatized by dansyl chlorides and
were able detected
simultaneously by an HPLC-UV detection method (Mazzucco et al.,
2010).
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37
The UHPLC technique is an evolved instrument that can be
operated under
higher pressure than a regular HPLC system. This characteristic
of UHPLC is that
columns with smaller particle sizes are used than in regular
HPLC. Due to the smaller
particle size packed in the LC column, the UHPLC has better
efficiency and resolution
than regular HPLC. UHPLC has been used in biogenic amines
detection to decrease
analysis time. UHPLC was applied to determine seven biogenic
amines in Bokbunja
wines and produced good linearity for calibration curves of
standards (Jia et al. 2012).
However, a UHPLC method that can detect the major free amino
acids and biogenic
amines simultaneously has not been developed. A new UHPLC method
is needed to be
able to investigate the content change of biogenic acids and
their precursor amino acids
during fish spoilage. Also, the spoilage effect on the major
amino acids in fish should be
studied to monitor the quality change of fish product.
ELISA Detection of Histamine
Enzyme-linked immunosorbent assay (ELISA) is a biochemical assay
technique
used to detect substances such as peptides and antibodies. An
antigen (the specific
antigen is usually proprietary knowledge) is immobilized on a
solid surface and a
specific antibody binds to the antigen. A substance is added
(usually a chromophore) to
give a detectable signal after the preceding reaction. ELISA has
been used to measure
histamine in food, and this method was based on a color-change
reaction (Serrar et al.,
1995). The AOAC (No. 070703), The Neogen Veratox® test kit
(Neogen Corp, Lansing,
MI) has been validated as a quantitative ELISA test to determine
histamine in tuna.
The AOAC (No. 070703) method has good reproducibility, and can
be rapidly,
and sensitively performed in the research laboratory (Lupo et
al., 2011). It is known that
ELISA test kits are good technology for disease outbreak
investigatory studies. The
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38
detection ranges for this AOAC (No. 070703) is up to 50 ppm and
no false positive or
negative result was found when using this kit to test a wide
range of histamine standard
solutions (Hungerford et al., 2012). Histamine content in
several fish products, including
bonito, salmon, mackerel, herring and others, were quantified by
the Neogen Veratox®
ELISA kit and good recoveries of histamine were observed (Kose
et al., 2011). The
AOAC (No. 070703) was also used to detect the histamine
concentration in tuna and
mahi-mahi samples and the ELISA results correlated with the
sample spoilage grade.
However, using an ELISA kit in routine safety detection of large
number samples in an
industrial process can be expensive (Lehane and Olley, 2000).
Also, the reagents used
for ELISA kit are easily degraded and should be stored
refrigerated at 2-8 ºC no longer
than twelve months.
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39
Figure 2-1. Global catches of albacore, bigeye, skipjack and
yellowfin data from 1960 to 2016. Data from WCPFC (2016).
0
1,000,000
2,000,000
3,000,000
4,000,000
5,000,000
6,000,000
1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2014
2016
Glo
bal catc
hes (
tones)
Year
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40
CHAPTER 3 A RAPID UHPLC METHOD FOR THE SIMULTANEOUS
DETERMINATION OF AMINO
ACIDS AND BIOGENIC AMINES IN TUNA AND MAHI-MAHI
Digest
Tuna and mahi-mahi are two major fish species responsible for
histamine
poisoning in the United States. The purpose of this research was
to develop a rapid
Ultra-High Performance Liquid Chromatography (UHPLC) method to
identify and
quantify amino acids, histamine and other biogenic amines that
can act as co-indicators
of histamine (scombroid) poisoning in tuna and mahi-mahi. In
this reversed-phase
UHPLC method, amino acids and biogenic amines were extracted
from homogenized
mahi-mahi (Coryphaena hippurus) and yellowfin tuna (Thunnus
albacares) using
aqueous 5% trichloroacetic acid (TCA) and were derivatized with
dansyl chloride. The
dansylated compounds were separated using a C18 reversed phase
column with 1.3
µm particle size and then detected by an ultraviolet (UV)
detector. The modified UHPLC
method could determine ten amino acids and four biogenic amines
simultaneously in
mahi-mahi (Coryphaena hippurus) and yellowfin tuna (Thunnus
albacares) within 17.5
minutes. This UHPLC method showed good linear response,
sensitivity, resolution,
recovery, repeatability, and the number of theoretical plates.
The UHPLC method
developed in this study is a rapid and accurate method to
monitor quality changes of
mahi-mahi and tuna by inspecting the changes of amino acids and
biogenic amines.
Background Information and Objectives
Fish spoilage is defined as any undesirable changes of fish that
happen rapidly
after fish landing and involves the breakdown of various organic
compounds or
formation of new molecules (Ashie et al., 1996; Ghaly et al.,
2010). The major concern
of fish spoilage includes lipid oxidation, protein degradation
and the decline of other
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41
nutritional components in fish (Clancy et al. 1995; Prester,
2011). Fish spoilage is
considered as the main issue that causes fish loss and each year
there are around 10
to 12 million tonnes of fish that are lost due to the spoilage
(FAO, 2010). The autolysis
of fish muscle proteins results in the formation of a large
number of free amino acids
and polypeptides, which are essential substrates or catalysts
for reactions pertaining to
fish spoilage (Ghaly et al., 2010). Studies also reported that
several free amino acids
were related to the flavor of fish (Fraser and Sumar, 1998;
Ghaly et al., 2010; Ruiz-
Capillas and Moral, 2004). Arginine, glutamic acid, glycine,
alanine, phenylalanine,
isoleucine, leucine, lysine, histidine, and tyrosine have been
identified as major free
amino acids in tuna and mahi-mahi (Ruiz-Capillas and Moral,
2004; Sen, 2005; Chong,
2014).
Biogenic amines are organic bases and can be produced during the
process of
fish spoilage by microbial decarboxylation of free amino acids
or by transamination of
free amino acids (Zhai et al., 2012). Even though several
biogenic amines are produced
during fish spoilage, only histamine, cadaverine, and putrescine
are identified as the
chemical indicators of fish quality and safety (Bulushi et al.,
2009). Histamine
(scombroid) poisoning, which is associated with the consumption
of spoiled scombroid
fish containing significant amounts of histamine, is identified
as the highest incidence of
illness from fish poisoning (Morrow et al. 1991). Histamine
toxicity can be potentiated by
cadaverine and putrescine due to their inhibiting ability to the
intestinal histamine-
metabolizing enzymes and diamine oxidase (Bulushi et al., 2009;
Visciano et al., 2012).
Histamine, cadaverine, and putrescine can be synthesized by the
decarboxylation of
free histidine, lysine, and arginine, respectively (Prester,
2011). The formation of these
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42
three amines in spoiled fish depends on the content of their
endogenous precursor free
amino acids, the presence of bacterial decarboxylase and the
environmental conditions
(Visciano et al., 2012). Tuna and mahi-mahi are two major
sources of histamine
poisoning in the United States due to high levels of histidine
existing in their muscle
tissue (Ahmed, 1991; Bulushi et al., 2009).
High-performance liquid chromatography (HPLC) is a sensitive,
reproducible
instrument to identify and quantify free amino acids and
biogenic amines associated
with fish spoilage (Onal et al., 2007; VecianaNogues et al.,
1997; Antoine et al., 2002;
Shakila et al., 2000; Kose et al., 2012; Gunlu et al., 2014;
Mazzucco et al., 2010). Ultra-
high performance liquid chromatography (UHPLC) is an evolved
separation technology
that has the same principle with HPLC that the compound affinity
between mobile and
stationary phases determine the separation of compounds on the
column. The UHPLC
instrument can be operated under very high pressures produced by
a column with small
particle size and has better efficiency and resolution than
traditional HPLC. The UHPLC
technology has been applied in simultaneous detection of
biogenic amines and amino
acids in fermented food products, such as wine and cheese, to
reduce the elution time
and improve the resolution (Jia et al., 2011). A UHPLC-MS/MS
method was developed
by He et al. (2016) to simultaneously determine nine biogenic
amines and their
precursors in cheese, red wine, and fish meat.
However, a rapid and simple UHPLC method that could
simultaneously detect
the major free amino acids and biogenic amines related to fish
spoilage has not been
developed. A new UHPLC method was needed to be able to
simultaneously investigate
the content change of biogenic acids and the major amino acids
in fish products.
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43
Materials and Methods
Fish Samples and Preparation
The mahi-mahi (Coryphaena hippurus) and yellowfin tuna (Thunnus
albacares)
analyzed in this research were caught commercially from South
Pacific waters. More
than five sensory experts in the Food and Drug Administration
(FDA) and National
Marine Fisheries Service (NMFS) applied the sensory grading
system provided in the
FDA Office of Regulatory Affairs (ORA) Laboratory Manual (FDA,
2013) to evaluate the
fish filets of mahi-mahi (Coryphaena hippurus) and yellowfin
tuna (Thunnus albacares)
into seven grades. The grading system used by FDA/NMFS experts
depended on
olfaction and were graded 1 to 7 to represent their quality.
Grade 1 represented high
quality, while grade 7 represented very poor quality fish.
The individually packaged and graded fish filets from FDA/NMFS
were shipped
overnight, received frozen on dry ice, and then stored in a -20
ºC freezer until analysis
was performed. For each grade of fish samples, vacuum packaged
frozen samples
were defrosted overnight at room temperature, and then were
chopped and
homogenized by a blender (Total Blend Classic, Blendtec, Orem,
UT) to perform
chemical analysis.
Standards and Reagents
All chemicals used in this study were of analytical grade or
higher. Amino acid
mix standards, L-Alanine, L-Arginine, L-Glutamic acid, Glycine,
L-Isoleucine, L-Leucine,
L-Lysine, L-Phenylalanine, L-Histidine, L-Tyrosine, histamine,
cadaverine, sodium
bicarbonate, and formic acid solution were supplied by
Sigma–Aldrich (St. Louis, MO).
Putrescine was purchased from MP Biomedicals (Santa Ana, CA).
Dimethylamine,
dansyl chloride were obtained from ACROS Organics (Geel,
Belgium). Sodium
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44
hydroxide, HPLC-grade acetonitrile, HPLC-grade water, ammonium
hydroxide,
trichloroacetic acid, and hydrochloric acid were supplied by
Fisher Chemical (Pittsburgh,
PA).
Individual free amino acid and biogenic amine solutions were
prepared
separately by dissolving reagent into 0.1M HCl. Then these
individual standard
solutions were diluted at various levels for separation of the
compounds on the UHPLC
for determination of retention time.
Individual free amino acids were 2.5 μmoles/L in 0.1M HCl in the
purchased
amino acids mix standard solution (Sigma–Aldrich, St. Louis,
MO). The amino acid mix
standard was diluted to achieve an approximately 200 ppm stock
solution (amino acid
quantities differed because of molarity to ppm conversion). The
stock biogenic amine
cocktail was 600 mg/L for each amine and was prepared by
dissolving cadaverine,
putrescine, histamine, and dimethylamine in 0.1M HCl. The stock
solutions were then
diluted to produce concentrations suitable for UHPLC
analysis.
For quantitation, external calibration curves were utilized.
Five different
concentrations of biogenic amines cocktail containing
cadaverine, putrescine, histamine
were used: 5, 10, 50, 100, 200 mg/L. Five different
concentration levels of the amino
acids mix standard with dimethylamine (5, 10, 50, 100, 200 mg/L)
were used for
quantitation.
Extraction and Derivatization
The extraction procedure of Zhai et al. (2012) with
modifications was used in this
study. In brief, 3 g homogenized fish sample was added in a 15
mL centrifuge tube with
10 mL of 5% trichloroacetic acid (TCA) and then was vortexed for
15min. The centrifuge
tube spun at 5000 g at 4 ºC for ten minutes. After the extract
was removed, the
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45
remaining solid was extracted using 10 mL of 5% TCA again by the
same procedures
as above, and the supernatant was collected. Both supernatants
were combined and
passed through a Whatman No. 1 filter paper.
The derivatization procedure of Simat et al. (2011) with
modifications was
applied. An amount of 300 μL 2 mol/L NaOH solution and 300 μL of
saturated NaHCO3
solution were added to 1 mL of filtered fish extract or 1 mL of
standard solution. Dansyl
chloride was dissolved in acetone to archive 1% (w/v)
concentration, and 2 mL of this
solution was added the resulting solution and then was protected
from light and
incubated for 60 min at 40 ºC in a water bath (Isotemp 220,
Fischer Scientific,
Pittsburgh, PA). Excess dansyl chloride was removed by adding
120 μL of an NH4OH
solution (4 mol/L) and then the solution was stored away from
light for 1 hour. The
solution was collected and filtered by a 0.2 μm PTFE membrane
(Phenomenex,
Torrance, CA) before LC injection. For each grade of mahi-mahi
or tuna, the extraction
and derivatization procedure were performed in triplicate.
Determination of Biogenic Amines
Quantification of the amino acids and biogenic amines was
carried out by using
an Agilent 1290 Infinity Series UHPLC System. A Kinetex® 1.3 µm
C18 Column, 50 x
2.1 mm was used for separation. The mobile phase for this UHPLC
method was water
with 0.1% formic acid(A) and acetonitrile with 0.1% formic
acid(B). The flow rate was 0.5
mL/min and the column temperature was 30 ºC. The elution
gradient was: 0 min 5% B;
1 min 5% B; 5 min 41% B; 11 min 68% B; 14 min 95% B; 14.5 min
95% B; 14.6 min 5%
B; 17.5 min 5% B. The injection volume was 10 μl. The analytes
were detected at a
wavelength of 254 nm.
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46
Method Validation
The sensitivity of this optimized UHPLC method was investigated
by determining
the limit of detection (LOD) and the limit of quantification
(LOQ). For the peak of each
amino acid and biogenic amine, the calculated signal to noise
ratio was determined by
the software of this UHPLC instrument. The LOD value was 3 times
the calculated
signal to noise ratio and was converted to concentration units
by using the
concentration of the compound. The calculation of LOQ was
similar to LOD and was
calculated as 10 times of the signal to noise ratio.
The percentage recoveries of cadaverine, putrescine, and
histamine were
determined by spiking biogenic amine cocktails in triplicate
into homogenized grade 1 of
mahi-mahi and tuna samples verified to contain biogenic amines
below detable levels,
to achieve the final concentration of each amine as 50 mg/kg
fish. Then the spiked fish
samples were extracted, derivatized, and the detected biogenic
amines levels were
compared with the theoretical amount.
To determine the repeatability of this UHPLC method, six
injections of a single
fish extraction with 10 mg/kg of each free amino acid and
biogenic amine were
compared, and the percentage relative standard deviation (%) for
each amine and free
amino acids were reported.
The resolution (R) and the number of theoretical plates for
histamine, cadaverine,
and putrescine were calculate using the formulas provided by
Moldoveanu and David
(2017).
Histamine ELISA Test Kit
An AOAC-validated ELISA test kit supplied from Neogen
Corporation (Lansing,
MI) was also used to quantify histamine levels in mahi-mahi
(Coryphaena hippurus) and
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47
yellowfin tuna (Thunnus albacares) in this study. Fish sample
preparations, dilutions,
and test kit procedures followed the kit instructions. The
standard provided by the
ELISA kit and histamine standards prepared in the lab were used
to build calibration
curves separately to quantify histamine levels in fish samples.
For each grade of mahi-
mahi or tuna, the ELISA test was performed in triplicate.
Results and Discussion
Method Development
To obtain a rapid, sensitive UHPLC method with good resolution,
different
extraction procedures, components of the mobile phase, elution
gradients, column
temperatures and the wavelengths of the detector were compared.
Figure 3-1 shows
the chromatographic separations of the fourteen dansylated
compounds in a mahi-mahi
grade 1 sample spiked with 10 mg/kg of each amino acid and
biogenic amine standard.
The reversed-phase UHPLC method developed in this study was able
to separate ten
amino acids and four biogenic amines from tuna and mahi-mahi
samples within 17.5
minutes.
The HPLC instrument has been reported as a sensitive and
accurate tool for the
determination of free amino acids in mahi-mahi (Coryphaena
hippurus), bigeye tuna
(Parathunnas mebachi) and flounder (common flounder) (Antoine et
al., 1999; Ruiz-
Capillas and Moral, 2004). Detection of derivatized biogenic
amines in fish products by
using HPLC or UHPLC with various detectors have been documented
(Veciana-Nogues
et al., 1997; Shakila et al., 2001; Kose et al., 2012; Simat et
al., 2011). He et al. (2016)
reported a UHPLC-MS/MS method that could simultaneously extract
and analyze
tyramine, histamine, tryptamine, putrescine, agmatine,
spermidine, cadaverine,
spermine, phenylethylamine and their precursor amino acids in
red wine, cheese, and
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48
fish. Among the biogenic amines detected in the study conducted
by He et al. (2016),
only histamine, putrescine and cadaverine were considered as
fish spoilage indicators
and also other major amino acids in fish were not determined in
this method. The
reversed phase UHPLC method developed in our study focused on
rapidly separating
and determining the major amino acids in fish and the biogenic
amines as fish spoilage
indicators without the use of costly tandem mass spectrometry.
Leucine and
dimethylamine (DMA) could not be separated and quantified by
this modified UHPLC
method. Due to its high-volatility, DMA could be determined by
other analytical
instruments such as gas chromatography-mass spectrometry (Chan
et al., 2006).
LC columns with small particle size (1.3 μm) used in this
reversed-phase UHPLC
method improved resolution, prevented peak broadening and
reduced the elution time.
The particle size of the LC column is of primary importance when
choosing a stationary
phase and decreasing particle size can increase separation
efficiency (Tuzimski et al.,
2015). Several studies have focused on optimizing
chromatographic separation of
biogenic amines by reducing the particle size of LC column.
Previously, Simat and
Dalgaard (2011) reduced the particle size of the C18 column from
5 µm to 1.8 µm in a
pre-column derivatization HPLC method and was able to reduce the
elution time used
for separating nine amines in seafood from 29 minutes to 12
minutes. Jia et al. (2011)
reduced the particle size of the C18 column to 1.7 µm in an
LC-Q-TOFMS method to
lower the elution time for separating 23 amino acids and 7
biogenic amines in beer,
cheese, and sausage. The method reported by Cai et al. (2016)
used a C18 column
with 1.8 µm particle size in HPLC–MS/MS to separate five
isotope-coded derivatized
biogenic amines in rice wine within 8 minutes. However, column
back pressure is
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49
inversely proportional to the square of the column particle
diameter (Harris et al., 2017).
Due to that the UHPLC instrument can be operated under higher
pressure, a UHPLC
instrument is needed when using LC column with very small
particle size.
Linearity and Sensitivity
For this developed UHPLC method, the linearity of each amino
acid and amines
was studied by using the calibration curve obtained from 5
different concentration
levels. Calibration curves of the dansylated amino acids and
amines showed excellent
linearity with a coefficient of determination (r