MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY STUDYING HISTAMINE IN SEAFOOD PRODUCTS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SIZE 14-15 SUPERVISORS STUDENT Msc DO TAN KHANG LE ANH VU Student code: 3082568 Session: 34 (2008-2013) Can Tho, 2013 APPROVAL SUPERVISORS STUDENT Msc DO TAN KHANG LE ANH VU Can Tho, May 6, 2013 PRESIDENT OF EXAMINATION COMMITTEE CONTENTS Contents Abstract Introduction Materials and methods 2.1 Materials 2.2 Methods 2.2.1 Optimization of histamine extracted solvent 2.2.2 Comparison of dansylation procedure and benzoylation procedure in histamine analysis in fish products 2.2.3 Changes in histamine and microbial analyses in tuna muscle by time and temperature 2.2.4 Study the histamine content in some samples of seafood products 2.2.5 Statistical analysis Results and dicussion 3.1 Optimization of histamine extracted solvent 3.2 Comparison of dansylation procedure and benzoylation procedure in histamine analysis in fish products 3.3 Changes in histamine and microbial analyses in tuna muscle by time and temperature 3.4 Study the histamine content in some samples of seafood products Conclusions and Suggestion 4.1 Conclusions 4.2 Suggestion References i i ii 3 3 6 9 11 15 21 25 25 25 26 ABSTRACT The study was undertaken to asses the effect of different solvents in histamine analysis in seafoods The results showed that histamine in seafood samples was efficiently extracted with perchloric acid 0.6M and trichloroacetic acid 6% Moreover, the commonly applied HPLC method to determine biogenic amines in seafoods after dansylation was compared with an alternative benzoylation procedure Results showed a significant difference in analysis efficiency among these two procedures for histamine quantitation in tuna samples The effects of storage time and temperature at 0, 4oC on the development of histamine and microbial in tuna were also studied Tuna stored at 4oC results in higher histamine level than tuna stored at 0oC Sample stored at 0oC contained potentially toxic histamine concentrations (49.1 mg/kg after 11 days of storage), whereas when stored at 4oC, the sample contained highly toxic histamine concentrations (161.29 mg/kg after days of storage) An increase overtime of microbial population was observed in all samples tested Bacteria counts increased from 4.54 log10 cfu/g to 5.88 log10 cfu/g in the samples stored at 0°C, while for the samples stored at 4°C, an increase was shown dramatically in counts from 4.54 log10 cfu/g to 8.88 log10 cfu/g The histamine levels in some common seafoods were also reported Keywords: HPLC, histamine, seafoods, extracted solvents, temperature storage ii INTRODUCTION Currently histamine poisoning is one of the most common threats to the health of consumers Histamine poisoning occurs after consuming seafoods containing high level of either histamine or other biogenic amines This foodborne intoxication was originally called “scombroid poisoning” because it was primarily associated with the consumption of fishes belonging to Scombridae and Scomberesocidae families such as tuna, mackerel, bonito, bluefish, and the like These species contain high levels of free histidine in their muscle that is decarboxylased to histamine When the conditions for the development of bacteria are favorable (for example when the fish are kept in improper temperatures), decarboxylase enzyme produced by the bacteria will metabolize histidine to histamine Other biogenic amines produced during the development of bacteria may increase the toxicity of histamine The toxicity of histamine depends on the ability of individuals to metabolize normal dietary intakes of histamine In some places of the world, histamine poisoning accounted for the largest proportion of cases of poisoning associated with fish and fish products (FAO/WHO, 2012) In Vietnam, histamine poisoning often occurs and the fish usually associated with these cases are tuna and mackerel (Ministry of Fisheries, 2003) Normally, after catching, fish has very low levels of histamine, but this amount increases with the decomposition of fish Thus, histamine has also been used as an indicator to assess the quality of the fish US Food and Drug Administration (USFDA, 2012) has set this histamine level at 50 mg/kg, above which it is considered a potential health hazard Considering the importance of histamine in fish and fish products for legal, toxicological, and quality purposes, it is essential to have accurate analytical methods Histamine in different foods, including fish and fish products, have traditionally been determined by means of standard chromatographic techniques such as thin layer chromatography, gas chromatography, capillary electrophoresis, flow injection analysis, and high performance liquid chromatography (HPLC) Histamine has a very important role in the food industry In recent years, many studies appear to study on histamine due to large potential applications of this amine in the assessment of quality seafood and seafood products In addition, high levels of histamine in seafood is also a health hazard for consumers.For these reason, the thesis "Studying histamine in seafood products by high performance liquid chromatography" has been carried out Thesis objectives: - Selecting the appropriate solvent for extraction process and analysis procedure to determine histamine in seafoods by high performance liquid chromatography - Studying the development of histamine in seafood over time and storage temperature - Quantification of histamine content in some common seafoods 2 MATERIALS AND METHODS 2.1 Materials - Seafood products were purchased in supermarkets in Can Tho City - Reagents: Methanol, ethanol, perchloric acid (PCA), tricholoro acetic acid (TCA), NaOH, H3PO4, dansyl chloride, benzoyl chloride, HCl, histamine dihydrochloride, KH2PO4… 2.2 Methods 2.2.1 Optimization of histamine extracting process - Preparation of samples: Fresh tuna (Thunnus albacares) was preserved at 4oC for week before analysis to make sure there was large amount of histamine in tuna samples - Extraction of Histamine: • Extraction using 0.6M perchloric acid (PCA): Extraction was carried out following the method of Cseinati and Forgacs (1999) A 10 g sample was homogenized with 20 ml 0.6M PCA for 10 The homogenate was centrifuged at 7000 rpm for 10 at 4°C, after which the supernatant was made up to 50 ml with 0.6M PCA, filtered and stored at 0°C until use • Extraction using trichloroacetic acid 6%: Samples extraction were done according to the method of Yung-Hsiang et al (2001) A g sample was homogenized with 20 ml trichloroacetic acid (TCA) 6% for 10 The homogenates were centrifuged (10.000 g, 10 min, 4oC) and filtered The filtrates were then placed in volumetric flasks, and TCA was added to a final volume of 20 ml and stored at 0°C until use • Extraction using methanol (99%): A modified method of Lin et al (1976) was used for the process of methanol extraction A 10g sample was homogenized with 50 ml methanol then transferred to a volumetric flask, after which it was immersed in a water bath at 60ºC for 15 The sample was cooled and then centrifuged at 7000 rpm for 10 at 4oC The supernatant was decanted, filtered and stored at 0°C until use - Determination of Histamine:  Histamine determination by benzoylation procedure:  Derivation of extracted solutions using benzoyl chloride: The benzoyl derivatives of all samples were prepared according to a method of Hwang et al (1997) One milliliter of 2M sodium hydroxide and 10 µl of benzoyl chloride were added sequentially to ml of samples extracted solution.The resulting solution was vortex, mixed and allowed to stand at 70oC for 20 Benzoylation was stopped by cooling the test tubes in an ice bath for 30 min, and the mixed solution was extracted with 3ml of diethylether After centrifugation at 3000 rpm, the supernatant was filtered through a 0.45 µm filter  Separation of Histamine with HPLC: Histamine in test samples were determined with a Hitachi liquid chromatograph (Hitachi, Tokyo, Japan), consisting of a Model L-6200 pump, a Rheodyne Model 7125 syringe loading sample injector, a Model L-4000 UV-Vis detector (set at 254 nm), and a Model D-2500 Chromato-integrator A Lichrospher 100RP-18 reversed-phase column (125x4.6 mm, E Merck, Damstadt, Germany) was used for separation The gradient elution program began with 50:50 (v/v) methanol:water at a flow rate of 0.8 ml/min for the first 0.5 min, followed by a linear increase to 85:15 methanol:water (0.8ml/min) during the next 6.5 The methanol:water mixture was held constant at 85:15 (0.8ml/min) for and then decreased to 50:50 (0.8ml/min) during the next  Histamine determination by dansylation procedure:  Derivation of extracted solutions using dansyl chloride: Derivation was carried out following the method of Earola et al (1993) A 1ml sample extract was made alkaline by adding 200 µl of 2N NaOH A 300 µl saturated sodium bicarbonate and ml Dns-Cl was added to the alkaline extract The reaction mixture was transferred to a 40°C incubator for 45 Residual Dns-Cl was removed by adding 100 µl ammonia After centrifugation for 30 at 2500 rpm, the supernatant was filtered through a 0.45 µm filter  Separation of Histamine with HPLC: Histamine in test samples were determined with a Shimadzu Prominence HPLC apparatus (Shimadzu, Kyoto, Japan) equipped with a SPD-M20A diode array detector (set at 254 nm) and two binary gradient pumps (Shimadzu LC-10AT), auto sampler (SIL 20AC), column oven (CTO-20AC), and a communication bus module (CBM20A) with valve unit FCV-11AL was used The column was a reverse-phase, Spherisorb Si C18 pH-St, 250x4.6 mm (Phenomenex, Macclesfield, Cheshire, UK) The mobile phase was 95:5 (v/v) acetonitrile : water, delivered at a flow rate of 1.0 ml/min - Indicator for Assessment: Histamine concentration (mg/kg) 2.2.2 Comparison of dansylation procedure and benzoylation procedure in histamine analysis in fish products - Preparation of samples: Fresh tuna (Thunnus albacares) was preserved at 4oC and taken at the appropriate times (zero, 1, 2, 3, and days during storage procedure) to make sure there was a wide range of amount of histamine in tuna samples - Extraction of Histamine: Histamine in fish samples was extracted by using optimum solvent derived from previous experiment (2.2.1) - Determination of Histamine: Histamine was determined by using benzoylation and dansylation procedure – as described in 2.2.1 - Indicator for Assessment: Histamine concentration (mg/kg) 2.2.3 Changes in histamine and microbial analyses in tuna muscle by time and temperature - Preparation of samples: Fresh tuna (Thunnus albacares) was preserved at 4oC and 0°C Samples were taken at the appropriate times (Table 2) and stored at -20°C to minimise bacterial activity that could influence the results prior to analysis For all samples, the histamine and microbiological analysis were assayed collectively to optimise efficiency and to limit possible day-today variability of analyses Table 2: Sample taken times of tuna stored at 4oC and 0oC Temperature Sample Taken Times st 4oC Zero 2nd 3rd 4th 7th day day day day day nd 0oC Zero th th day day day th 11th day day - Effect of storage time and temperature on histamine level The average of histamine concentrations of tuna during the time – temperature storage procedure are presented in Figures – As shown, in tuna samples stored at 4oC, the increases of histamine varied from as low as 36.93 mg/kg to as high as more than 161.29 mg/kg The histamine concentrations showed significant difference (p50 mg/kg) In tuna samples stored at 0oC, the results revealed a steady in histamine levels during storage procedure, although it was not as dramatic as at 4oC The obtained results also showed a significant difference (p4.4ºC for more than 4h after the initial chilling, also it was found that some of bacteria species can produce histamine in toxic levels even at to 5°C ( FDA, 2001 and Dalgaard et al., 2008) USFDA (2012) considered that the best quality fish has histamine value less than 10 mg/kg, while 22 histamine values between 10 to 30 mg/kg are accepted as middle quality and 30-50 mg/kg histamine value is critical as it is close to the level of FDA regulation (50 mg/kg) According to the above mentioned conclusion the results showed that these fish represented the critical state regardless the other impact factors (Craven et al., 2001) Canned mackerel and canned tuna have frequently associated with histamine poisoning, especially for products that stored for a long time (Taylor, 1986) Histamine are stable to thermal processing, and therefore the presence of histamine in canned products indicates that the fish has been microbially spoiled before heating The canned mackerel and canned tuna samples analysed in this investigation all had low amounts of histamine (1 and mg/kg, respectively) These low results would indicate that when the fish were caught they were handled and processed in an appropriate manner The results is also concide with FernándezSalguero and Mackie (2000), who reported very low levels (less than mg/kg) of histamine in canned tuna, mackerel, and sardine The highest histamine concentrations were obtained from dried scomber and dried shrimp, which were 58.2 and 61.7 mg/kg, respectively These results were above the maximum permitted histamine level (50 mg/kg), which indicate that the quality of these samples may be a serious problem for public health In Asia and in particular those areas with an extensive coastline and high ambient temperatures such as Vietnam, the use of fermentation as a preservation method for fish has been practised from the earliest of times (Beddows, 1985) Drying process may occur ranging from 1-2 days to over 2-3 weeks 23 seafoods GDL causes decreasing of pH in dry seafoods, which results in increasing decarboxylase activity of bacteria Amino acid decarboxylase activity is stronger in an acidic environment, the optimum pH being between 4.0 and 5.5 In these conditions bacteria are more strongly encouraged to produce these enzymes, as a part of their defence mechanisms against the acidity In addtion to this, the effect of drying process on histamine content (Beddows, 1985) and microorganisms containing decarboxylating enzymes may be present (Blood, 1976) During drying process, complex bacterial activity occurs and therefore it could be expected that high levels of histamine would be found in these products Moreover, the attention is paid to the influence of glucono-δ-lactone (GDL) on the production of histamine in dry where, Veciana-Nogues et al (1997) and Ganowiak et al., (1991) found that thermal process increased the amount of histamine as result of protein breakdown In the same direction, Pan (1985) reported that the optimal temperature for histidine decarboxylase are 55°C, therefore, it may be probable that during the come-up time in thermal processing, there was added degradation by endogenous and microbial enzymes to produce histamine 24 CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions - The optimum solvents for extraction of histamine were perchloric acid 0.6M and trichloroacetic acid 6% - The benzoylation procedure was more effective than the dansylation procedure in histamine analysis of seafoods - Freezing of fish at 0°C significantly reduces histamine formation than freezing at 4°C temperature For cold storage temperatures, 0°C is recommended, since it will increase the shelf life of the fish than 4°C temperature Though deterioration of the fish sample was obvious from the microbiological analysis, it was faster in the 4°C stored fish samples than in the 0°C stored fish samples - Seven samples of seafood products obtained from supermarket in Can Tho city were analysed for histamine content Analysis of these samples showed that two samples of dried scomber and dried shirmp did not comply with the allowed maximum permitted level of 50 mg/kg histamine as stated in the FDA Standard 4.2 Suggestions - Using the obtained procedure to investigate histamine level in fresh seafoods and seafood products - Studying the levels of other biogenic amines in seafoods 25 REFERENCES Vietnamese Ministry of Fisheries 2003 Đảm bảo chất lượng thủy sản NXB Nông nghiệp, Hà Nội English Baranowski, B.L et al 1990 Efects of storage time and temperature on the microfora and amine development in Spanish mackerel (Scomberomorus maculatus), J Food Sci., 53, 1024 Beddows, M.H 1985 Biogenic amines: Their importance in foods Int J Food Microbiol., 29, 213 Ben-Gigirey, B., J.M.V.B de Sousa and T.G Villa 2000 Characterization of biogenic amine-producing Stenotrophomonas maltophilia strains isolated from white muscle of fresh and frozen albacore tuna International Journal of Food Microbiology, 57: 19-31 Chen, J., R Hesterberg, W Lorenz, U Schmidt, M Crombach and C.D Stahlknecht 2003 Inhibition of human and canine diamine oxidase by drugs used in an intensive care unit: relevance for clinical side effects? Inflammation Research, 16(3-4): 91-94 Craven, C., K Hilderbrand, E Kolbe, G Sylvia, M Daeschel, B Gloria and H.J An 2001 Understanding and controlling histamine formation in troll-caught albacore tuna: a review and update of preliminary findings from the 1994 season Oregon State University Sea Grant, Oregon Publication No ORESU-T-01-001 26 Cseinati and Forgacs, 1999 Histamine food poisoning: an update Fish Technology News, 11: 3-5, Dalgaard, M., T.K, Smith, and A Harris 2006 High performance liquid chromatographic method for detection of biogenic amines in feedstufs, complete feeds and animal tissues J Agric Food Chem., 48, 1708 De Mey, J.E., W.R Hutkins and S.L Taylor 2011 Biogenic amines in cheese and other fermented foods: a review Journal of Food Protection, 54: 460-470 Du, W.X 1976 Development of biogenic amines in yellowfn tuna (Tunnus albacore): Efect of storage and correlation with decarboxylase-positive bacterial flora J Food Sci., 67, 292 Earola, M., F Szlam, J.T Holden, K Yamaguchi, D Denson and J.H Levy 1993 Mechanisms of non-immunological histamine and tryptase release from human cutaneous mast cells Anesthesiology, 92(4): 1074-1081 FAO/WHO 2012 FAO/WHO Expert Meeting on the Public Health Risks of Histamine and Other Biogenic Amines from Fish and Fishery Products Meeting Report FAO Headquarters, Rome, Italy USFDA 2001 Fish and fisheries products hazards and controls guidance 3rd edition U.S FDA Center for Food Safety and Applied Nutrition, Maryland Fernandez-Salguero, K and T Mackie 2000 Changes in the number of halotolerant histamine-forming bacteria and 27 contents of non-volatile amines in sardine meat with addition of NaCl Bull Jpn Soc Sci Fish., 59, 123 Fran, N.K and H.H., Yoshinaga 1987 Rapid method for detection of histamine-producing bacteria Int J Food Microbiol., 5, 137 Gabowiak, L.H et al 1991 Quality changes in sardines (Sardina pilchardus) stored in ice and at ambient temperatures Food Microbiol., 13, 123 Gram, L 1985 Fish spoilage bacteria–problems and solutions Curr Opin Biotechnol., 13, 262 Guin, G.J., M.P Oria and L Douglas 1997 Potential hazards in cold-smoked fish: biogenic amines Journal of Food Science, 66: 1088-1099 Hwang, R.R., J.H Orr and W.W Wallis 1997 Histamine formation in fish: microbiological and biochemical conditions In: Chemistry and Biochemistry of Marine Food Products (Martin, R.E., Flick, G.J., Hebard, C.E., and Ward, D.R., eds.), Avi Publishing Company, Connecticut pp 39-50 Kim, L.V., H.H., Huss, and P., Dalgaard 2000 The efect of biogenic amine production by single bacterial cultures and metabiosis in cold-smoked salmon J Appl Microbiol., 89, 920 Kim, S., J Noda, and K Takama 1990 Growth and polyamine production of Alteromonas ssp in fish meat extracts under modifed atmosphere Bull Fac Fish Hokkaido Univ., 41, 213 28 Krause, C.I., M Chen, J.A Koburger, W.S Otwell and M.R Marshall 1995 Bacterial growth and histamine production on vacuum packaged tuna Journal of Food Science, 55(1): 59-63 Lin, S., C Imark and M Kneubuhl 1976 Biogenic amines in foods: histamine and food processing Inflammation Research, 48(6): 296-300 Lopez-Sabater, O., F.P, Nnenna, and N Christopher 1996 Mobiological assessment of stored Tilapia guineesis Afric J Food Sci., 5(4):242-247 Onal, A 2007 A review: Current analytical methods for the determination of biogenic amines in foods Food Chem., 103, 1475 Romero, A., M Melac, G Pauli and N Frossard 2002 Comparative activity of cetirizine and mizolastine on histamine-induced skin wheal and flare response at 24 h British Journal of Clinical Pharmacology, 53(3): 250-254 Ruiz-Capillas, C and A Moral 2001 Production of biogenic amines and their potential use as quality control indices for hake (Merluccius merluccius L.) stored in ice J Food Sci.: 66, 1030 Shalaby, A.R 1996 Significance of biogenic amines to food safety and human health Food Research International, 29(7): 675 – 690 29 Sims, G.G., G Farn, and R.Y York 1992 Quality index for tuna correlation of sensory attributes with chemical indices J Food Sci., 57, 1112 Staruszkiewicz, W.F andP.L., Rogers 2001 Performance of Histamine Test Kits for Applications to Seafood Presentation on October 26, 2001 4th World Fish Inspection & Quality Control Congress, Vancouver, BC Taylor, S.L 1986 Histamine food poisoning: toxicology and clinical aspects CRC Critical Reviews in Toxicology, 17(2): 91-128 Veciana-Nogues, M.T., A Marine Font and M.C Vidal Carou Biogenic amines as hygienic quality indicators of tuna Relationships with microbial counts, ATP related compounds, volatile and organo-leptic changes J Agric Food Chem., 45, 2036, 1997 Wendakoon, H., K Shiomi, and T Kikuchi 1990 Agmatine as a potential index for freshness of common squid J Food Sci., 52, 936 Yung-Hsiang, S.B., M.K Mukundan and K.A Kumar 2001 A simple and rapid method for colorimetric determination of histamine in fish flesh Food Control, 16(5): 465-472 30 ... determine histamine in seafoods by high performance liquid chromatography - Studying the development of histamine in seafood over time and storage temperature - Quantification of histamine content... procedure in histamine analysis in fish products 2.2.3 Changes in histamine and microbial analyses in tuna muscle by time and temperature 2.2.4 Study the histamine content in some samples of seafood products. .. amine in the assessment of quality seafood and seafood products In addition, high levels of histamine in seafood is also a health hazard for consumers.For these reason, the thesis "Studying histamine