STUDY OF THE KINETICS OF FORMATION OF BIOGENIC AMINES IN THE SARDINE AND MACKEREL DEPENDING ON THE MICROBIAL FLORA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. ElyounoussiCharifa

ElyounoussiCharifa 1.2 , Rachidi Abderrazzak 2 and Bekkali Mohammed 1 . 1. Laboratory of Aquatic Ecology and Environment. Research Team "Biodiversity, Pollution and Water Treatment, University Hassan II Faculty of Science Ain Chock, Casablanca, Morocco). 2. ONSSA (Regional Analysis Laboratory and Research, Casablanca Morocco). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (10), 739-748 740 (putrescine and cadaverine) or polyamines (spermine and spermidine) in function of the number of amine groups (Spano et al. 2010). The histamine, tyramine, putrescine and cadaverine, are formed from amino acids namely free histidine, tyrosine, ornithine, and lysine, respectively. Spermidine and spermine from putrescine (Zarei et al., 2011).The presence of biogenic amines in fish is source of concern for researchers, consumers, food companies and health authorities because of their toxicological effects. Biogenic amines were classified as potentially dangerous compounds, which can cause problems for the consumers (Halász et al., 1994;Santos, 1996), including facialcervical redness, rash, facial swelling , hot flashes, burning sensation in the throat, a taste of pepper in the mouth, itching, tingling of the skin. They are usually followed by type of headache disorders, heart palpitations, dizziness. Secondary symptoms, gastrointestinal, may appear: nausea, stomach pain, vomiting, diarrhea. (McLauchlin et al., 2006;Hungerford, 2010).In general, the incubation period is short, it ranges from minutes to hours. The symptoms usually disappear spontaneously within three hours. Exceptionally, they can last several days in the most severe cases. Biogenic amines are non-volatile compounds which are present in low concentrations in fresh fish but rapidly accumulate in the flesh after alteration by the infection of bacterial flora (Fernandez-Salguero and Mackie, 1987) formed by decarboxylation of amino acids. Although many biogenic amines were found in fish, only histamine, cadaverine and putrescine were found to be significant in the safety of fish and determination of its quality. Despite widely reported an association between histamine and Scombroid food poisoning, histamine alone seems insufficient to cause food toxicity. Putrescine and cadaverine were suggested to potentiate the toxicity of histamine. As regards the other hand deterioration only cadaverine was found to be a useful index of the initial stage of the decomposition of the fish. (Al Bulushi et al., 2009;Rezaei et al., 2007).
In most studies on the formation of biogenic amines in fish, researchers have focused on histamine poisoning and concluded that families Scombridae and Scomberesocideae are commonly involved in cases of histamine poisoning they contain high levels of free histidine in their muscle. Histamine is produced by bacteria which décarboxylate histidine to histamine in the fish under the action of enzymes of bacterial origin (Lehane and Olley, 2000;Taylor, 1986). Indeed, various Scombridae, including mackerel, tuna, bonito and saury, have been implicated in cases of histamine poisoning (Taylor, 1986;McLauchlin et al., 2006). However, non-scombroid fish that also contained high levels of free histidine in the muscle, such as sardines, anchovies, herring and merlin, has also been implicated in cases of poisoning with histamine (Taylor, 1986).
The accumulation of biogenic amines usually results from the decarboxylation of the amino acids by enzymes of bacterial origin, which is associated with hygiene. Therefore, the lack of hygiene is probably the main factor involved in the formation of these compounds. (Halász et al., 1994). Bacterial contamination may be derived from post-harvest contamination board fishing vessels at the treatment plant or in the distribution system.
In freshly caught fish, bacterial contamination is primarily found on the skin and gills from there, these organisms invade the muscle of fish and grow rapidly in response to a number of factors related to processing and storage conditions such as temperature, time, etc. In this case, it is important to identify some bacteria have the amino acid decarboxylase activity in order to estimate the risk of production of biogenic amines in seafood and prevent its accumulation in the product the sea (Ruiz-Capillas and Jiménez-Colmenero, 2010).
Fresh fish can be contaminated by a mixed bacterial population consisting of psychrotrophic bacteria, Gramnegative bacteria such as Enterobacteriaceae and Gram-positive bacteria such as lactic acid bacteria. The type of bacteria present in fish determines the type and amount of biogenic amines formed. Indeed, enterobacteria were often described as producers of high concentrations of biogenic amines in fish. (Ruiz-Capillas and Jiménez-Colmenero, 2010).
The objective of this study was to study the ability of bacteria isolated to produce biogenic amines (histamine, putrescine, cadaverine and tyramine) in fresh fish available for human consumption in Casablanca, Morocco, using chromatography high performance liquid with diode array detection (HPLC-DAD) reference method in accordance with Regulation No 2073/2005 and 11441/2007 that set the health conditions for the production and placing products on the market fishing.

Materials and Methods:-
Sampling procedure:-The study focused on freshwater fish, sardine (Sardina pilchardus) and mackerel (Scomber scombrus), these fish were collected at different Casablanca outlets. At the time of landing, once collected, the samples were collected 741 aseptically in a cooler and transported directly to the laboratory. Soon as they arrive at the laboratory, all samples were subjected to a moderate washing followed unbref spin cycle the filter cloth to be washed free of the superficial mucus that would have helped too quickly to their poor appearance and odor.
Bacterial isolates:-Fish samples were taken from the part of the gills. The Enterobacteriaceae strains were isolated using the agar violet red bile glucose (VRBG) (Oxoid CM485) as described by (Pons-Sanchez et al., 2005). Production of oxidase was tested using oxidase disc (Fluka, 70439 oxidase test) and the production of catalase by suspending the cellular material in 3% hydrogen peroxide. Glucose metabolism was studied by the O / F test. All these tests were carried out for the initial classification of the strains isolated. Then, each of the selected individual bacterial colonies was extended repeatedly on agar plates using sterile loops to produce pure colonies. The isolates were identified according to the instructions of the API 20E reactions produced during the incubation period from 16h to 24 h, result in spontaneous color changes or revealed by the addition of reagents. The results were analyzed using the TM web Api Identification software API (bio Merieux). The identified strains are reported in Table 1.     The dosage of biogenic amines:-Sample preparation, mothers solutions and seeding dilutions were performed according to standard NM 08.0.101 (2008), in order to follow the kinetics of formation of biogenic amines: histamine, putrescine, cadaverine and tyramine products by the strains isolated from the gills of sardines and mackerel. The extraction of these amines is done after deproteinization step by perchloric acid 0.2 M are then labeled with dansyl chloride after the L-proline is added to neutralize excess dansyl chloride.

Results:-
The microbial flora of sardines and mackerel is reported in Table 1. The microflora of both species was found belonging to Gram-negative Enterobacteriaceae.
The average concentration of histamine and other biogenic amines produced by bacterial strains isolated from sardines and mackerel were estimated from samples in triplicate.
However, the variation in the ability to produce biogenic amines in different species is extremely wide, and this change was also observed between strains of the same species.
The concentrations of biogenic amines in function of incubation time, in Casablanca fish samples, are shown in Tables 3 and 4 and illustrated in Figures 2, 3, 4, 5, 6, 7, 8 and 9.
From the tables below, it may be noteworthy that the concentrations of histamine and putrescine, encountered in mackerel, are higher than sardines.

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The results showed that the maximum concentration of putrescine is 1038 mg / kg produced in Citrobacter Braakii A5 isolated mackerel. And the minimum concentration was cadaverine is 3.51 mg/kg produced in Enterobacter Cloaceae A1 isolated from sardines. Tables 3 and 4, putrescine was detected in all strains, with values ranging from 4.63 mg / kg to 1038 mg / kg after 32h of incubation. The highest concentrations of putrescine (1038 mg/kg in Citrobacter Braakii A5, 470.02 mg/kg in Raoultella Terrigena A8 and 433.27 mg/kg in Serratia Fonticola) have been found in the mackerel, and were greater than in the strains isolated from sardine, Enterobacter Cloaceae A1 (97.91 mg / kg), Escherichia Coli A2 (4.63 mg / kg) and Escherichia Coli A3 (8.93 mg / kg).

As shown in
The detection level of cadaverine was similar to that of putrescine and the maximum concentration of cadaverine was also found to occur in mackerel with a maximum value of 463 mg / kg in Raoultella terigena A8. In addition to the high level of cadaverine and putrescine, the histamine was also found to occur in mackerel with a maximum value of 384.92 mg / kg in Serratia Fonticola A6. And tyramine represents the lowest biogenic amine in the treated samples ( While the production of biogenic amines is a very complex phenomenon, depending on several variables, such as raw materials, processing conditions, micro-organisms of the growth kinetics and their proteolytic activities and decarboxylase, which interact with each Others (Gardini et al., 2001)   The results of Figures 3 and 4 showed that tyramine and putrescine remained at low levels in the 2 strains of Escherichia Coli during incubation. However, a significant increase of histamine and cadaverine were observed in these two strains. For example, the concentrations of histamine and cadaverine in Escherichia Coli A2 rose sharply to 230, 07 and 190.03 mg / kg for 32 heures incubation instead of 0 and 18.78 mg/ kg at the initial time respectively.
Significant differences were also observed among strains isolated the mackerel and are illustrated in Figures 5, 6 Results showed that all isolated strains are fish capable of producing at least four biogenic amines (Histamine, Putrescine, cadaverine and tyramine) (Ferencik, M 1970), and biogenic amine content was quite different in samples tested. These results confirm that the tested strains are capable of decarboxylating one or more amino acids, but the amine production differs between species. It depends not only on the species but also of the bacterial strain and environmental conditions (Lehane and Olley, 2000).

Discussion:-
Fish is one of the most perishable foods, mainly due to the action of microorganisms that occur on the surface of freshly caught fish. Determining the microbiological quality at a very important role in maintaining the high quality of fishery products.
A large population of microbial flora were isolated and identified to have the ability to produce biogenic amines, Morganella morganii, Enterobacter aerogenes and Raoultella planticola (Björnsdóttir-Butler et al., 2009). In this study, strains were isolated and identified, which is similar to previous findings, such as Escherichia coli, Citrobacter braakii, Serratia fonticola, Raoultella terigena and Enterobacter cloaceae, the microbial flora was found Gram-negative Enterobacteriaceae of the genus has Using the API 20 E galleries ( Table 1).
The biogenic amine content in Casablanca fish samples are shown in (Table 2 and 3). The result showed that the fish contained at least four biogenic amines and biogenic amine content was quite different depending on the species. It depends not only on the species but also of the bacterial strain (Lehane and Olley, 2000).
Putrescine and cadaverine has no direct toxicological effects on human health. However, it has been proved that the presence of putrescine and cadaverine in aquatic products can potentially promote the toxicological effects of histamine and tyramine, inhibiting histamine metabolizing enzymes, such as monoamine or diamine oxidase and histamine methyl transferase (Smith, 1980;Stratton et al., 1991). In addition, the appearance of putrescine and cadaverine in food can react with nitrite to form heterocyclic carcinogenic nitrosamines, which are among the most important human carcinogens (Park et al, 2010.Santos, 1996.
In this study, the maximum concentrations of Putrescine and cadaverine were observed in mackerel, with 1038 values and 463 mg / kg, which were much higher than 4.63 and 3.51 mg / kg, respectively, in the sardine.
The largest quantities of histamine presented earlier in fish species were 1270 mg / kg in mackerel (Shalaby, 1996) and 399 mg / kg in amberjack (Auerswald et al., 2006). Note that in this study, however, the species of fish contained much lower levels of histamine that previous reports (Shalaby, 1996) and similar to the reports (Auerswald et al., 2006) with a maximum value of (384.92 mg / kg) in mackerel, as presented in Table 4, that is a level six times greater than the legal limit (50 mg / kg) suggested by the Food and Drug Administration for scombrids and related products (US FDA, 2001), and is also much higher than the acceptable level (100 kg / mg) fixed for scombrids fish by the EU (EEC, 1991 It is essential to pay attention to tyramine in aquatic products because of its toxic effects on the human body, the concentrations of tyramine in all studied samples were much lower than the suggested limit of 100 mg / kg (Santos, 1996; Ten Brink et al., 1990). Low levels of tyramine were also observed in the octopus and squid (Kim et , 2009), which was a higher concentration than the standard (100 mg / kg) to human health suggested by (Brink et al., 1990;. Santos, 1996). However, this high level of tyramine in fresh fish was rarely recorded.
The formation of biogenic amines in fish depends on various factors such as the contents of free amino acids gradually increase with extended storage times due to proteolysis by both endogenous and exogenous proteases (Makarios-Laham and Lee, 1993) and the presence of bacteria producing decarboxylases. Thus, biogenic amines can be formed by microbial decarboxylation of free amino acids. However, levels of biogenic amines formed can be heavily influenced by the storage conditions.

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In the present study significant increases (p <0.05) of the biogenic amines histamine, putrescine and cadaverine (as shown in the figures) are observed in all samples during incubation at 20 ° C for 32heures. The results show that significant increases (p <0.05) putrescine and cadaverine were held in all strains studied after incubation, and these two amines become the dominant biogenic amines in these aquatic products. Similar changes in putrescine and cadaverine were observed in slices of barramundi (Bakar et al., 2010) and Spanish mackerel (Middlebrooks et al., 1988).
The results show that the biogenic amines, including putrescine, cadaverine, histamine and tyramine are found to be strongly correlated with the presence of enteriobacteria in the fish.
The production and accumulation of biogenic amines has been registered in the exponential phase between 8 to 32 h. However, the maximum production appears to be variable and depends on the species of fish, bacterial strains of the same species and the amine considered.
However, several samples of fish showed relatively high levels of histamine (up to 384.92 mg / kg), putrescine (up to 1038 mg / kg), cadaverine (up to 457.56 mg / kg) and tyramine (up to 201.9 mg / kg). In addition, we found that the concentrations of biogenic amines can greatly increase during storage at temperatures above 4 ° C. Therefore, it must raise safety concerns, given the high levels not only for the production of histamine but also to putrescine, cadaverine, and tyramine in the species of fish.