SPOILAGE INDICATORS FOR DETERMINING TUNA AND ......mahi (Coryphaena hippurus) are two major fish...

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

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


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


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

16

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

17

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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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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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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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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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.

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

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

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).

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

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

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).

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

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

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

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

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

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-

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

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).

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

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.

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

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

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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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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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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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

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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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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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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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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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

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