Summary bachelor of science thesis the advanced program in biotechnology

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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 1. Introduction 2. 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 3. 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 4. Conclusions and Suggestion 4.1 Conclusions 4.2 Suggestion References i i ii 1 3 3 3 3 6 6 7 8 9 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 7 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 1. 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 1 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 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 1 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 min. The homogenate was centrifuged at 7000 rpm for 10 min 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 5 g sample was homogenized with 20 ml trichloroacetic acid (TCA) 6% for 10 min. 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. 3 • 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 min. The sample was cooled and then centrifuged at 7000 rpm for 10 min 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 2 ml of samples extracted solution.The resulting solution was vortex, mixed and allowed to stand at 70oC for 20 min. 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 4 min, followed by a linear increase to 85:15 methanol:water (0.8ml/min) during the next 6.5 min. The methanol:water mixture was held constant at 85:15 (0.8ml/min) for 5 min and then decreased to 50:50 (0.8ml/min) during the next 2 min.  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 2 ml Dns-Cl was added to the alkaline extract. The reaction mixture was transferred to a 40°C incubator for 45 min. Residual Dns-Cl was removed by adding 100 µl ammonia. After centrifugation for 30 min 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 5 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). 5 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, 4 and 7 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 1 2nd 3rd 4th 7th day day day day day nd 0oC Zero th th 2 4 7 day day day 6 th 9 11th day day - Determination of Histamine: Histamine was determined by using optimum histamine analysis procedure, which derived in experiment 2.2.2. - Indicator for Assessment: Histamine concentration (mg/kg). - Microbial Analysis: The total viable count (TVC) test was done according to the method of Guinn et al., (1999). - Indicator for Assessment: Microbial population (log10 cfu/g). 2.2.4 Study the histamine content in some samples of seafood products - Preparation of Samples: Seven seafood samples were purchased from local supermarkets in Can Tho City, descriptions of each sample are given in Table 3. Table 3: Description of seafood samples. Samples Fresh tuna Producer/Provider Expired day Co.op Mart supermarket Fresh amberjack Metro supermarket Fresh mackerel Metro supermarket Canned mackerel Co.op Mart supermarket 11/2014 Canned tuna Co.op Mart supermarket 8/2015 Dried scomber Vinatext supermarker 9/2013 Dried shirmp Vinatext supermarker 11/2013 - Determination of Histamine: Histamine was determined by using optimum histamine analysis procedure, which derived in experiment 2.2.2. - Indicator for Assessment: Histamine concentration (mg/kg). 7 2.2.5. Statistical analysis methods The experiment was conducted in triplicates. The results were reported as mean values ± standard deviation. The ANOVA test was used for data analysis. The differences between the mean values were considered significant when p<0.05. All data were analysed using Statgraphic Centurion XV statistical package for windows (Statgraphics Centurion XV, Manugistics, Rockville, USA, 2009). 8 Inc., 3. RESULTS AND DISCUSSIONS 3.1 Optimization of histamine extracting process Complete and selective extraction of target analytes from complex food samples is of great importance in food analysis. Solid samples are most frequently extracted with acidic solvents which also act as deproteinisation agent during the liquid solid extraction. Because of the high content of fat in fish samples, extraction solvents that can remove fat or protein are frequently selected for achieving high extraction efficiency. In this study, perchloric acid (0.6M), trichloroacetic acid (6%, m/v), methanol (99%) was tested as extraction solvents for the samples. Results revealed that histamine concentrations of different levels were obtained after the extraction of samples with different solvents (Figure 4). 9 Figure 4: Mean of histamine concentrations in tuna samples extracted by using Met - methanol; PCA - perchloric acid (0.6M) and TCA - trichloroacetic acid (6%). As shown, the highest histamine concentration was from 0.6M perchloric acid for samples analysed by dansylation procedure. The samples extracted by trichloroacetic acid 6% and analysed by benzoylation procedure resulted in higher yield of histamine concentrations as compared to the samples extracted in all the extract medium. These results coincide with those of BenGriggrey et al. (2001), who suggested perchloric acid and trichloroacetic acid are more effective than methanol, acetonitrile and acetone in extracting histamine in fish products. RuizCapillas and Moral (2001) also reported that PCA and TCA are 10 highly efective biogenic amine extractors for fish and fish products because of their efect on protein precipitation. Since most of the biogenic amines present in fish are in bound form, the histamine concentration was low when extracted by methanol (Shalaby et al., 1996). In this study, histamine concentrations obtained in the tuna fish was quite high in the acid fraction, suggesting that histamine in fish are acid solube. Because perchloric acid and trichloroacetic acid provided good peak shape and effective separation, it was selected as the optimized extraction solutions in the following experiments. The suitable acid for extraction of histamine from fish samples and analysed by dansylation procedure was 0.6M perchloric acid, while trichloroacetic acid 6% was suitable acid for extraction histamine from fish samples analysed by benzoylation. 3.2. Comparison of dansylation procedure and benzoylation procedure in histamine analysis in fish products Amongst the available analytical techniques, HPLC is by far the most frequently used to separate and quantify histamine. Since histamine present in the food samples neither show an adequate absorption, nor exhibit significant fluorescence, derivatisation has to be performed in order to increase the sensitivity needed for a subsequent UV, VIS or fluorescence detection (Onal, 2007). In this work the amines were determined by HPLC using two procedures – dansylation procedure and benzoylation procedure – based on pre-column derivatisation. In both methods, the reversed-phase column was used. Typical chromatograms of dansyl chloride and benzoyl chloride derivatives of the standard and of the extract of sample are shown in Figure 5. As shown, 11 histamine are well separated in the nearly 3 min runtime for benzoyl procedure, where as this seperation time was 5 min for dansyl procedure. The peaks of histamine were satisfactorily resolved with good peak resolution, sharpness, and symmetry, and are consistent with previous report (Hwang et al., 1997). However, the histamine levels determined on six test samples revealed the significant difference in analysis efficency between two procedures (Figure 6). In general the histamine values analysed by benzoylation procedure were higher than the histamine levels determined by dansylation procedure (5 of 6 test samples). The histamine values determined by benzoylation procedure that had lower histamine values from dansylation procedure was 52.51 (mg/kg) on the third test. The dansylation procedure result was 52.96 (mg/kg) for this test. However, this difference was not statistically significant. 12 Figure 5: Typical chromatograms of the histamine standard and samples. (A), (C) Histamine standard (1 µg/ml); (B), (D) tuna sample at day 7 of storage time. (A) and (B): chromatography derived by benzoylation procedure; (C) and (D): chromatography derived by dansylation procedure. Figure 6: Mean of histamine concentrations in tuna samples determined by dansylation procedure and benzoylation procedure. 13 The results of the experiment clearly represented that benzoyl procedure was more accurate than dansylation procedure in histamine quantitation in fish. This is related to the fact that benzoyl chloride forms stable compounds after reaction with both primary and secondary amino groups and the products are more stable than those formed using dansyl chloride. Moreover, the long derivatisation time is also an evident drawback of dansylation procedure, as it contributed to decrease the stable of flouresence compounds. Beside of that, ammonia has to be used after the derivatisation in order to remove an excess of the dansyl reagent. Without this step, the by-products such as dansylamide (Dns-NH2), dansyl sulphonic acid (Dns-OH) and dansyl hydrazine (Dns-N2H3) most likely appear in the chromatogram (De Mey et al., 2011) and the excess of dansyl chloride may coelute with histamine. Unfortunately, an addition of 100 µl concentrated ammonia and incubation for 30 min at room temperature, as suggested by Earola et al. (1993), could not totally remove the excess of dansyl chloride. As a result, low amounts of the by-products can still be detected in the chromatogram and the peak of histamine does not remain unaffected. In contrast to the dansylation procedure, benzoylation is less frequently used for the determination of biogenic amines in fish and fish products. However, fast benzoylation gives an opportunity to reduce the analysis time. From the literature it comes out that the benzoyl derivatisation used to be performed at the temperatures ranging from room temperature to 70oC. At the room temperature, the derivatisation needs an overnight 14 incubation, while at the increased temperatures the incubation time can range between 15 (Krause et al., 1995) and 30 min only (Chen et al., 2003). In most cases, an incubation period of 20 min at 70oC was recommended (Romero et al., 2000). In that way, the highest available yields of the studied analytes were obtained and as the excess peak of the benzoyl reagent was separated from the analytes, no problems were encountered with quantification thereof. In contrast to dansylation (which shows severe decomposition of dansylated amines at the temperatures higher than 65oC), have mentioned that at higher temperatures the peak ascribed to the excess of benzoyl reagent decreased, without the loss of the peak areas of the analytes. Another advantage of using benzoyl chloride is the simple way to stop the derivatisation reaction. While an addition of irritating ammonia is necessary to remove an excess of dansyl chloride, an easy cooling step, for example, an ice bath for 30 min, is sufficient to stop the benzoylation reaction. Since benzoylation procedure provided better results as well as faster and easier to conduct than dansylation procedure, it was selected as the main procedure in the following experiments. 3.3. Changes in histamine and microbial analyses in tuna muscle by time and temperature The amount of histamine produced by the fish is strongly influenced by temperature, time, storage conditions and fish spieces (Lehane and Olley, 1999). This experiment was designed to investigate the changes of histamine and microbial population in tuna in two different storage temperature (0oC and 4oC). 15 - 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 7 – 8. 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 (p<0.05) in relation to the storage time. A significant increase in the histamine value is shown until 7th day which was above FDA standard on the 3rd day (>50 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 (p<0.05) between the mean concentration of histamine during the storage procedure, except for histamine concentration on the 11th day. However, histamine does not exceed the safe limits (50 mg/kg) at the end of the life of the experiment. 16
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