Biogenic amine contents in selected Egyptian fermented foods as determined by ion-exchange chromatography

Biogenic amines in foods may pose a public health problem. Therefore, histamine, tyramine, putrescine, cadaverine, spermine, and spermidine concentrations were measured in selected food items commonly consumed in Egypt. Foods examined were dairy products (blue cheese and Mesh cheese), meats (fermented and smoked cooked sausage), and fish (smoked and salted fermented fish [Feseekh], salted sardines, and anchovies). Egyptian fermented sausage had the highest concentration of total biogenic amines (2,482 mg/kg), followed by Mesh cheese (2,118 mg/kg) and blue cheese (2,084 mg/kg). The lowest concentration was found in smoked cooked sausage (111 mg/kg). Histamine was found at a high level (521 mg/kg) in Feseekh, and tyramine was highest (2,010 mg/kg) in blue cheese. These results indicate that some traditional Egyptian foods may pose a health risk due to the concentration of biogenic amines, especially histamine.     

Differences in biogenic amine patterns in fish obtained from commercial sources

Summary Using a high-performance liquid chromatography method, biogenic amines (histamine, cadaverine, putrescine, tyramine, spermidine and spermine) were determined in 16 samples of salted, smoked and freshly frozen fish obtained from commercial sources. The data were processed using principal component analysis. The results indicate that this multivariate statistical method greatly assisted the assessement of the quality of fish samples, particularily in connection with liquid chromatography. Some preliminary suggestions concerning selection of the Chromatographic data are described.

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Unterschiede im Gehalt an biogenen Aminen in Fisch aus dem Handel

Zusammenfassung Unter Anwendung der Hochleistungsflüssigchromatographie wurden die biogenen Amine (Histamin, Cadaverin, Putrescin, Tyramin, Spermidin und Spermin) in 16 Proben von gesalzenen, geräucherten und frisch gefrorenen Fischen aus dem Handel bestimmt. Die Daten wurden der Hauptkomponenten-Analyse unterworfen. Die Ergebnisse weisen darauf hin, daß die Multivarianz-Statistikmethoden zusammen mit der Flüssigchromatographie dem Analytiker hilft, die Qualität von Fisch zu erfassen. Einige vorläufige Hinweise, die die Auswahl der chromatographischen Daten betreffen, werden beschrieben.

     

Performance evaluation of a fluorescamine-HPLC method for determination of histamine in fish and fish products

A method for the quantification of histamine in fish and fish products using tandem solid-phase extraction and fluorescence derivatization with fluorescamine was previously developed. In this study, we improved this analytical method to develop an official test method for quantification of histamine in fish and fish products, and performed a single laboratory study to validate it. Recovery tests of histamine from fillet (Thunnus obesus), and two fish products (fish sauce and salted and dried whole big-eye sardine) that were spiked at the level of 25 and 50 μg/g for T. obesus, and 50 and 100 μg/g for the two fish products, were carried out. The recoveries of histamine from the three samples tested were 88.8-99.6% with good repeatability (1.3-2.1%) and reproducibility (2.1-4.7%). Therefore, this method is acceptable for the quantification of histamine in fish and fish products. Moreover, surveillance of histamine content in food on the market was conducted using this method, and high levels of histamine were detected in some fish products.     

Determination of biogenic amines with an electrochemical biosensor and its application to salted anchovies

This paper describes the assembling and optimization of an electrochemical biosensor for the determination of biogenic amines (putrescine, cadaverine, histamine, tyramine, spermidine, spermine, tryptamine) commonly present in food products, and its application to salted anchovy samples. Variations of the amine content in anchovies during ripening time were measured both with the biosensor and ion chromatography with integrated pulsed amperometric detection (IC-IPAD). The probe is based on a platinum electrode which senses the hydrogen peroxide produced by the reaction catalysed by the enzyme diamine oxidase (DAO), purified from commercial seeds of cicer and immobilized on the electrode surface. Parameters such as enzyme immobilization and pH have been studied and optimised in order to obtain similar sensitivity for all the amines tested. The immobilization of the enzyme on a nylon-net membrane, using glutaraldehyde as cross-linking agent, and phosphate buffer at pH 8.0 were selected. The detection limit was 5 × 10⁻⁷ mol litre⁻¹. The linear range common to the amines tested was observed from 1 × 10⁻⁶ to 5 × 10⁻⁵ mol litre⁻¹. The effect of potentially interfering compounds was also evaluated. Underivatized biogenic amines such as putrescine, cadaverine, histamine, tyramine and spermidine were also detected with the IC-IPAD method. Changes in the concentration of biogenic amine content in salted anchovy samples, measured with the biosensor and IC-IPAD methods, exhibited the same trend and demonstrated that the biosensor is a useful tool to monitor the variation of the total amine content in fish during storage.     

六类鱼制品中生物胺的HPLC法测定

针对不同类型的鱼制品建立同时检测腐胺、尸胺、精胺、亚精胺、酪胺、苯乙胺、组胺和色胺8种生物胺含量的高效液相色谱(HPLC)法。应用该方法,测定了干燥制品、油炸制品、烘烤制品、发酵制品、混揉制品以及萃取制品6类鱼干制品中的生物胺含量。结果显示,萃取制品-鱼油中未检测出生物胺,其余5类产品中生物胺含量最高的为发酵制品(73.42~70.59 mg/kg)和混揉制品(55.48~49.88 mg/kg),其次为油炸制品(17.72~14.32 mg/kg),而鱼干制品(5.28~5.17 mg/kg)和烘烤制品(4.75~4.69 mg/kg)中的生物胺含量相对较少;另外,尸胺、组胺、精胺和亚精胺四种生物胺为除萃取制品之外的5类鱼制品中普遍存在的4种生物胺成分;其中尸胺是鱼制品中变化最大、影响最为明显的单体生物胺,检测产品中尸胺含量有利于监控、评判鱼干制品的质量。     

冷藏期内猪肉肌苷酸与生物胺两者变化关系的研究

采用高效液相色谱法测定生鲜猪肉的背最长肌在4℃温度条件下肌苷酸(IMP)和腐胺、尸胺、组胺、酪胺、亚精胺、精胺这六种生物胺含量的变化情况,从而进一步分析它们之间的相关性。结果表明,随着货架期的延长,IMP含量呈先上升后降低的趋势,并在第2d达到最高;精胺含量基本保持在4.0mg/kg左右,组胺的含量始终很低,其它的胺类物质都有所增加,尸胺的变化最为突出;虽然腐胺和亚精胺的含量比较低,但是仍然有明显的变化;酪胺变化也非常明显。从Person积差相关系数可以看出,IMP与其他指标相关系数呈负相关显著,有的指标是不显著的;在0.01水平上,IMP与亚精胺之间的负相关性最强,达到了-0.981;其次是尸胺与IMP之间,相关系数是-0.960,呈显著负相关;酪胺也与IMP显著负相关;IMP与腐胺、组胺、精胺的相关系数都不显著。因此,通过测定IMP含量变化可以预测猪肉新鲜度,且可作为猪肉保藏与加工过程中品质控制的重要指标之一。     

Biogenic amines in commercially produced Yulu,a Chinese fermented fish sauce

Seven biogenic amines were determined in 35 commercially produced Yulu samples from three provinces of China by pre-column derivatisation with dansyl chloride (Dns-Cl) and high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Putrescine, cadaverine, histamine and tyramine were the major biogenic amines (more than 100 mg kg^?1), while tryptamine, spermidine and spermine were regarded as minor biogenic amines (less than 25 mg kg^?1). Twenty samples contained more than 50 mg kg^?1 histamine (the limit for histamine in seafood products as suggested by the Food and Drug Administration). Twenty-one samples contained more than 100 mg kg^?1 tyramine and 10 contained more than 1000 mg kg^?1 total biogenic amines. This study provided data on biogenic amine levels in Chinese fermented fish sauce. The results suggested that biogenic amine content should be monitored in commercially produced Yulu.     

Simultaneous Analysis of Biogenic Amines in Canned Fish by HPLC

A simple HPLC method was developed for simultaneous determination of putrescine, cadaverine, tryptamine, 2-phenylethylamine, spermidine, spermine, histamine, tyramine, and agmatine. Amines were derivatized with benzoyl chloride and separated well on Lichrosphere are 100 RP-18 column using a gradient elution system with a mixture of methanol and water in 10 min. Amine content of canned fish was detected by this method, and except for anchovie products most samples contained the nine amines at low levels. This method would be useful for detection of amines in fish.     

Relation of biogenic amines with microbial and sensory changes of whole and filleted freshwater rainbow trout (Onchorynchus mykiss) stored on ice

The biogenic amine (BA) content of whole and filleted rainbow trout was monitored during ice storage for a period of 18 days and related to respective microbial and sensorial changes occurring during the same period. Eight amines, namely, putrescine, cadaverine, tyramine, spermidine, tryptamine, beta-phenylethylamine, spermine, and histamine, were determined. Agmatine was not detected in any of the fish samples. In all cases, concentration of BAs was higher (P < 0.05) in filleted compared with whole trout samples. Pseudomonads, H2S-producing bacteria, and, to a lesser extent, Enterobacteriaceae were the dominant microorganisms in both whole and filleted trout. Higher populations (P < 0.05) of these microorganisms were present in filleted trout compared with whole fish samples. Of the BAs determined, concentration of putrescine, cadaverine, spermidine, tryptamine, and beta-phenylethylamine increased steeply in both whole and filleted trout between days 15 and 18 of storage when pseudomonads and H2S-producing bacteria reached approximately 10(6) to 10(7) CFU/g. For the rest of the BAs, including tyramine, histamine, and spermine, a stepwise increase was recorded throughout the entire storage period. Interestingly, Enterobacteriaceae counts remained below 10(6) throughout the entire storage period, accounting for the lower production of histamine. A putrescine value of 13 to 14 mg/kg and a spermidine value of approximately 7 mg/kg for both the whole and filleted trout obtained after 12 and 9 days, respectively, may be proposed as the upper limit for spoilage initiation (freshness indicator) of fresh rainbow trout based on sensorial and microbiological (total viable count of 10(6) to 10(7)) data. With respect to other amines determined, both tyramine and spermine may also be proposed as freshness indicators preferably for whole trout, whereas tryptamine, beta-phenylethylamine, histamine, and cadaverine produced only during later stages of storage are not suitable as freshness indicators of either whole or filleted trout.     

Simultaneous determination of eight underivatised biogenic amines in fish by solid phase extraction and liquid chromatography-tandem mass spectrometry

Biogenic amines on fish tissue are formed as a result of bacterial contamination and spoilage during storage. A new method based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using a triple quadrupole (QqQ) analyser was developed for the analysis of eight biogenic amines (cadaverine, histamine, phenylethylamine, putrescine, spermine, spermidine, tyramine and tryptamine) in fish tissues. Sample preparation was performed by extraction with trichloroacetic acid 5% and solid phase extraction clean up with STRATA X cartridge. The MS/MS method was validated and compared with a method based on the analysis of dansyl derivatives by LC and fluorescence detector (FD). MS/MS achieved higher sensitivity (from 0.02 mg kg⁻¹ for spermidine and phenylethylamine to 0.2 mg kg⁻¹ for spermine) when compared to FD (from 1 mg kg⁻¹ for putrescine and tyramine to 4 mg kg⁻¹ for histamine); MS/MS method showed higher precision too, with intraday relative standard deviations (RSDs) from 1% to 4% with respect to those obtained with FD method (from 3% to 17%).Recovery study was conducted at two different fortification levels and the average ranged from 71% to 93% for all of the studied compounds with RSDs lower than 18%. Matrix-matched standards were used to counteract matrix effect observed in MS/MS determination. The applicability of the method was demonstrated by the analysis of biogenic amines in fish obtained from commercials of Valencia.     

Application of the simplex method for optimization of chromatographic analysis of biogenic amines in fish

The aim of this study was to optimize the separation of biogenic amines with ion-exchange high-performance liquid chromatography, using the modified simplex method. A modified chromatographic response function was used, and two methodological parameters were selected as simplex variables: the proportion of the organic solvent 2-propanol in the mobile phases, added as a new mobile phase, and the variation of the gradient curve at one specific step in the elution program. In optimum conditions the correct resolution and separation of the biogenic amines (tyramine, β-phenylethylamine, histamine, tryptamine, putrescine, cadaverine, agmatine, spermidine and spermine) were completed in 16 min, reducing the analysis time by 9 min compared with the original method. The regression coefficients were higher than 0.99 in all the amines, except for spermine (0.989). The detection limits varied between 0.06 mg/L, for histamine and agmatine, and 0.22 mg/L, for tryptamine, while retention time repeatability, determined as a relative standard deviation, was between 4.64% for histamine and 11.95% for tryptamine. The repeatability variation for the amine concentrates was found between 0.71% in cadaverine and 3.23% for tryptamine. Applying the optimized method to samples of refrigerated sardine and sea bream demonstrated the effectiveness of the method in these fish species.     

Effect of delayed gutting on biogenic amine contents during ripening of European anchovies

The effect of delayed gutting on biogenic amine formation during ripening of European anchovies (Engraulis encrasicholus) was studied. The main difference between the two processes was the use of gutted or ungutted fish in the first step of ripening. Biogenic amines and microbial counts were studied in seven batches of samples taken during manufacturing from fresh anchovies and from anchovies throughout ripening. The formation of amines commonly related to fish spoilage (histamine, tyramine, cadaverine, putrescine and agmatine) was significantly higher in the first step of ripening of batches in ungutted fish. In contrast, no differences were observed between the content of the natural polyamines spermine and spermidine.     

Limosilactobacillus fermentum FSCBAD033控制带鱼加工下脚料发酵鱼露中生物胺含量研究

目的 控制带鱼加工下脚料低盐发酵鱼露中的生物胺含量。方法 以耐盐性高效生物胺降解乳酸菌Limosilactobacillus fermentum FSCBAD033为功能发酵剂控制带鱼加工下脚料发酵鱼露中的生物胺含量,并研究其对鱼露发酵过程中的pH值、NaCl含量、总可溶性氮(Total Soluble Nitrogen,TSN)含量、氨基酸态氮(Amino Acid Nitrogen,AAN)含量、挥发性盐基氮(Total Volatile Basic Nitrogen,TVB-N)含量以及发酵结束时感官风味的影响。结果 与Control组相比,接种L. fermentum FSCBAD033发酵剂对鱼露发酵过程中的NaCl含量影响不大(p>0.05),但可促进pH值下降以及TSN和AAN含量上升,并显著抑制TVB-N的产生(p<0.05)；显著降低发酵结束时鱼露中52.38%尸胺、40.21%组胺、45.44%酪胺、23.74%腐胺、52.67%苯乙胺和43.26%总生物胺(p<0.05),对含量较低的色胺、精胺和亚精胺影响不大；此外,接种L. fermentum FSCBAD033发酵剂可增强发酵结束时鱼露的鲜味和肉味,减弱氨味、腥味和臭味,整体提升鱼露感官风味。结论 L. fermentum FSCBAD033可作为带鱼加工下脚料低盐发酵的功能发酵剂,用于生产低盐、营养丰富、感官风味好且生物胺含量低的高品质鱼露产品。     

发酵香肠中生物胺的研究

本文采用RP-HPLC法测定了发酵香肠中的生物胺(苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺、精胺)的含量。样品经0.4mol/L高氯酸溶液提取,丹磺酰氯衍生,流动相为乙腈和水,采用梯度洗脱,流速为1mL/min,紫外检测波长为254nm。该方法检测限为:腐胺、尸胺、亚精胺、酪胺和精胺为0.1μg/mL,组胺0.5μg/mL,苯乙胺0.05μg/mL。回收率分别为苯乙胺86.71%、腐胺88.88%、尸胺94.55%、组胺87.57%、酪胺83.67%、亚精胺88.55%、精胺94.91%。结果表明发酵香肠中生物胺的种类及含量因香肠的品种而异,7种生物胺平均总量为13.40mg/100g,变异范围为7.83~19.13mg/100g。本法简便、快速、灵敏、可靠。     

Effect of Diamines, Polyamines and Tuna Fish Extracts on the Binding of Histamine to Mucin In Vitro

Binding of [ring-2-¹⁴C]-histamine to mucin was studied. Maximum binding of 2.5 μmol histamine/μmol mucin was observed at a histamine concentration of 0.5 mg/ml. Histamine binding was decreased by 46.5% by spermine, 25.3% by spermidine, 9.5% by cadaverine, and 11.1% by putrescine. These maximum effects were observed at molar ratios of histamine to polyamine of 1:1 for spermine and spermidine, 1:2 for cadaverine, and 1:2.3 for putrescine. The relative effects on histamine binding to mucin of extracts from wholesome canned tuna fish and a mixture of spermine, spermidine, cadaverine and putrescine were also examined. Although the total neutral aqueous extract of tuna had little effect on histamine-mucin binding (5.4% inhibition), the extract of bases from tuna and the prepared base mixture showed maximum inhibition of histamine binding to mucin of 23.1% and 21.4%, respectively. Certain other components of tuna extracts (NaCl, aspartic acid and glutamic acid) did not decrease histamine-mucin binding under the conditions used.     

Levels of biogenic amines in retail market fermented meat products

The qualitative and quantitative profile of biogenic amines (BA) in 50 samples of dry fermented sausages sold in Greek retail markets were determined by HPLC. Putrescine, cadaverine, tryptamine, β-phenylethylamine spermidine, spermine were analysed by UV detection after pre-column derivatization with benzoyl chloride, whereas tyramine and histamine were analysed by fluorescence detection after post-column derivatization with o-phthaldialdehyde (OPA). With the exception of spermidine and spermine a wide variation of BA levels was observed among the samples. Of the BA examined, tyramine, putrescine, histamine and cadaverine showed high concentrations ranging from: 0 to 510 mg/kg (median: 197.7 mg/kg), 0 to 505 mg/kg (median: 96.5 mg/kg), 0 to 515 mg/kg (median: 7.0 mg/kg) and 0 to 690 mg/kg (median: 3.6 mg/kg), respectively. The histamine content of 28% of the samples exceeded the toxicity limit of 100 mg/kg set for histamine in some fish species. Levels of tryptamine and β-phenyl-ethylamine never exceeded 50 and 29 mg/kg, respectively. Results of the present study suggest that the amounts of BA in dry fermented sausages, sold in Greek retail markets, may pose a potential health risk for sensitive individuals or for those undergoing classical monoamine oxidase inhibiting (MAOI) drug therapy.     

市售东海鱼鲞生物胺及相关质量指标分析

为研究零售期东海鱼鲞的生物胺潜在风险及生物胺与品质指标的相关性,以浙江地区的4种鱼鲞（鳗鱼、大黄鱼、鲳鱼和马鲛鱼）为研究对象,对理化指标（水分含量、NaCl、pH、TVB-N和POV）、微生物指标（菌落总数）和8种生物胺进行测定分析。结果表明,4类鱼鲞的理化、微生物和生物胺指标各不相同。水分含量为25.6%～59.7%,NaCl含量为3.4%～24.1%,pH为6.08～6.97,TVB-N为7.7～56.4 mg/100 g,菌落总数为3.43～5.67 lg CFU/g,POV为0.04～1.76 g/100 g,其中50%样品超过水产干制品POV限值。鱼鲞样品的主要生物胺为亚精胺、组胺、尸胺和腐胺。15%的样品组胺含量>100 mg/kg,超过FDA和/或欧盟的限量标准,其中黄鱼鯗B4的组胺含量达到了1405.88 mg/kg。全部样品的酪胺含量均<100 mg/kg,符合FDA限量标准。生物胺总量在96.05～2164.64 mg/kg之间,其中15%的样品>1000 mg/kg,超过FDA限量标准。相关分析表明,部分生物胺指标与菌落总数和NaCl含量呈中度或高度相关（P<0.05）,相关系数r分别为0.727～0.975和?0.828～?0.970。综上,东海鱼鯗,包括以非高组胺鱼为原料的产品,可能存在生物胺超标风险及其他质量问题。本研究为鱼鲞类水产干制品的品质评价和安全控制提供了基础数据和参考依据。     

Formation of biogenic amines during the maturity process of raw meat products, for example of cervelat sausage

The raw meat product cervelat sausage with a diameter of 90 mm was investigated during maturation and storage over a period of about 12 weeks. The examination was carried out by ion exchange chromatography (amino acid analyzer) and included the amines ethylamine, propylamine, butylamine, putrescine, histamine, cadaverine, tyramine, β-phenylethylamine, spermidine and spermine. In addition, the starter bacteria cultures varied according to manufacturer. Over the duration of investigation, five different production batches were observed in parallel. During the maturity process and storage, after 87 days, tyramine (58.9 mg kg⁻¹) showed a clear dominance, followed by spermine (34.1 mg kg⁻¹), putrescine (26.1 mg kg⁻¹) and spermidine (8.7 mg kg⁻¹), if starter bacteria culture 1 was used. Other amines were not detected. The use of starter bacteria culture 2 led, after 87 days, to the highest content of putrescine (244.1 mg kg⁻¹), followed by tyramine (119.2 mg kg⁻¹), histamine (52.9 mg kg⁻¹), spermine (37.5 mg kg⁻¹) and spermidine (9.2 mg kg⁻¹). With the use of starter bacteria culture 3, for spermine and spermidine a comparable behavior was shown to that with culture 1 or 2. After 87 days, putrescine reached the highest value of 484.2 mg kg⁻¹, followed by tyramine (119.4 mg kg⁻¹) and histamine (111.0 mg kg⁻¹). Cadaverine (36.9 mg kg⁻¹) could also be determined. As an accompanying investigation, the parameters pH, water activity (a_w) and microbial count (lactobacilli, enterobacteria, staphylococci and yeast) were determined. An investigation of the sections center-middle-edge showed that the content of amines increases to its highest values in the middle. The edge showed the lowest content. This behavior correlates with the a_w distribution of a raw sausage.     

盐渍鱼酶解制备鱼露及挥发性风味物质的研究

在对Alcalase2.4L和Papain双酶分步酶解盐渍鱼酶解过程研究的基础上酶解鲜鱼和半年盐渍鱼制备鱼露,运用感官分析和顶空固相微萃取(HS-SPME)结合气质联用(GC-MS)法对其风味进行比较分析。结果表明:Alcalase2.4L、Papain双酶分步酶解盐渍鱼酶解效果较好,先加Papain6h后再加Alcalase2.4L效果较好,但鲜味氨基酸的比例不如先加Alcalase2.4L后加Papain高;与鲜鱼酶解鱼露相比,虽然盐渍鱼使蛋白酶的酶解效果有所降低,但盐渍鱼酶解鱼露的风味较好,较接近传统鱼露的风味,且含有挥发性酸类,但具有鱼露特征香味的挥发性风味物质没有检出,说明需进一步发酵才能达到传统成熟鱼露的风味。     

Histamine contents of salted seafood products in Taiwan and isolation of halotolerant histamine-forming bacteria

Fifty-seven salted seafood products sold in the fishing village stores in Taiwan, including salted fish product, salted mollusc product and salted shrimp product, were tested to determine the occurrence of histamine and histamine-forming bacteria. Although the average content of each of nine different biogenic amines in all samples was less than 5.0 mg/100 g, 10.5% (6/57) of tested samples had the histamine content greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration. One histamine-producing bacterial strain, capable of producing 78.5 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH), was identified as Bacillus megaterium. The B. megaterium isolate was a halotolerant bacterium which grew well to an elevated NaCl concentration of 15% in TSBH medium. Besides, it had a consistent ability to produce >300 ppm of histamine at 10% NaCl concentration in TSBH medium after 72 h.