【作者】 李志军；

【导师】 薛长湖； 吴永宁；

【作者基本信息】 中国海洋大学 ， 水产品加工及贮藏工程， 2007， 博士

【摘要】 本文建立了一种改进的丹磺酰氯柱前衍生RP-HPLC测定食品中生物胺的方法。方法以1,7-二氨基庚烷为内标,丹磺酰氯为衍生剂,通过优化试验确定了样品前处理和衍生条件。本法采用CAPCELL PAK C18色谱柱,荧光检测器(λex/λem=350/520)与紫外检测器(254nm)串联检测。以线性、精密度、检出限和回收率等为指标对方法进行了验证,结果表明, 8种标准生物胺在检测范围内,呈良好的线性相关(荧光,r=0.9981-0.9997;紫外,r=0.9981-0.9998)。荧光/紫外分别测定生物胺含量的RSD<10%。荧光检测器检出限:0.06-0.5mg/L,紫外检测器检出限:0.07-0.28mg/L。本方法测定葡萄酒中8种生物胺的平均回收率79.2-127.5%;啤酒中8种生物胺的平均回收率78.9-109.9%;酱油中8种生物胺的平均回收率75.2-107.5%;食醋中8种生物胺的平均回收率90.1-106.6%;水产品中8种生物胺的平均回收率除色胺(回收率57.7%,相对标准偏差19%)外为93.6-110.3%;乳制品中8种生物胺的平均回收率61.0-112.7%。加标回收的RSD<10%。本方法适合于不同基质食品中色胺,β-苯乙胺,腐胺,尸胺,组胺,酪胺,亚精胺和精胺等8种生物胺的测定。本文采用建立的HPLC方法全方位测定了几种常见食品中生物胺含量,初步掌握了我国常见食品中生物胺的含量状况。主要结果如下:1、测定国产葡萄酒和啤酒中生物胺含量:酒类样品中色胺未检出。干红葡萄酒中其它生物胺均有不同程度检出,腐胺检出率100%,其次为β-苯乙胺。干白葡萄酒中生物胺主要为腐胺,部分检出微量β-苯乙胺,其它生物胺未检出。啤酒中常见生物胺是腐胺、酪胺、亚精胺和尸胺,组胺含量低于2 mg/L。2、测定国产酱油和食醋中生物胺含量:酱油中除色胺检出率为72.5%,其它7种生物胺检出率100%,酪胺、尸胺、腐胺、β-苯乙胺和组胺是其主要生物胺成分,个别样品中生物胺总量较高。食醋中腐胺和尸胺检出率较高,β-苯乙胺、组胺和酪胺在食醋中普遍存在,组胺含量超过50 mg/L的样品达28.9%。本文还测定了部分其它中国传统调味品和发酵食品中生物胺含量。3、测定国产酸奶和奶酪中生物胺含量:酸奶中8种生物胺均有不同程度检出,但含量低,酪胺、腐胺、β-苯乙胺是酸奶中主要生物胺;奶酪中色胺、精胺、亚精胺未检出,β-苯乙胺和酪胺是其主要生物胺,但含量较低。4、水产品:鱼类和虾类样品中均有少量的精胺和亚精胺检出。新鲜鱼肉中生物胺种类少、含量低,但对于冷藏质量稍差的鱼肉,可检出生物胺种类和含量明显增加。新鲜对虾中除尸胺含量较高外,其余生物胺含量与新鲜鱼肉相似。虾酱中组胺、酪胺、腐胺和尸胺的含量普遍较高。鱼肉罐头样品中除色胺未检出外,其它生物胺均有少量检出,但罐头样品中生物胺总量较低。本文建立了检测产生物胺革兰氏阳性菌的聚合酶链式反应(PCR)体系。通过优化实验,确定了PCR反应的最佳条件,建立了一套分别检测产生组胺、酪胺和腐胺产生菌的单重PCR方法和可以同时检测产生组胺与酪胺、同时检测组胺与腐胺、同时检测酪胺与腐胺的二重PCR方法,并首次报道了以这三对特异性引物组合建立的一套同时检测组胺、酪胺和腐胺产生菌的复合PCR方法。对方法的敏感性、特异性作了验证。本方法具有简便、快速、特异性好等优点,为检测产组胺、产酪胺获产腐胺的革兰氏阳性菌提供了一种新的分子生物学手段。采用建立的PCR方法对23株酒类酒球菌和109株乳酸菌进行安全评价。结果:酒类酒球菌均为非组胺、酪胺和腐胺产生菌,无潜在的生物胺危害。乳酸菌均为非组胺和腐胺产生菌株,但有44株含有酪氨酸脱羧酶基因,为酪胺产生菌。这一结果与HPLC测定结果基本一致。本文对31株产酪胺乳酸菌PCR产物进行了测序,并对结果进行了比对分析。结果表明:以TD2/TD5为引物的PCR产物氨基酸序列中,均有酪氨酸脱羧酶蛋白序列中特有的高度保守序列LHVDAAY和PLP结合位点,证明31株菌的PCR扩增产物确是酪氨酸脱羧酶基因片段。酪氨酸脱羧酶基因及氨基酸序列具有一定程度的多态性。聚类分析结果表明:产酪胺乳酸菌仅具有菌种或菌株特异性,无菌属特异性。因此,酪氨酸脱羧酶基因不适于产酪胺菌的进化发育分析。更多还原

【Abstract】 In the thesis, an improved RP-HPLC method was developed for the quantitation of eight biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) in food. A fluorescence and ultraviolet detector, operated atλex/λem=350/520nm and at 254nm respectively, were connected in series and biogenic amines were monitored as their dansyl derivatives after pre-column derivatization, followed by diethyl ether clean-up and subsequent separation by reversed-phase high-performance liquid chromatography. The dansylamides were seprated on an Capcell PAK C18 MG (150×4.6 mm ID, participle size 5μm), using a 40-min gradient elution with a binary system of methanol-water and a flow rate of 1.5ml/min. Both detection modes were applied simultaneously to identify and quantify eight biogenic amines in food , after treatment by method of liquid-liquid extraction with equal volume of n-butanol-chloroform (1:1, v/v). 1,7-diaminoheptane was used as the internal standard. Linearity, repeatability, and recovery of the method were evaluated. In all cases a good correlation coefficient was obtained, ranging from 0.9981 to 0.9997 for fluorescence detection and from 0.9981 to 0.9998 for ultraviolet detection. The repeatability of the method was assessed by injecting ten repeated times each of three standard dansylamide solutions at low, medium and high concentration levels, during the same day. The relative standard deviations(RSDs) was less than 10% for both fluorescence and ultraviolet detection. The LOD ranging from 0.06 to 0.5mg/l for fluorescine detector, and from 0.07 to 0.28mg/l for ultraviolet. The recovery of the method ranged from 79.2 to 127.55 for wine, from 78.9 to 109.9% for beer, from 75.2 to 107.5% for soy sauce, from 90.1 to 106.6% for rice vinegar, and from 61.0 to 112.7% for yoghourt. In all cases, the RSD was less than 10%.A survey of biogenic amines in primary food in China as followed:1. Wine and beer:β-Phenylethylamine, Putrescine, Cadaverine, Histamine, Tyramine, Spermidine, and Spermine were found in the Chinese red wine samples. Most of the red wines presented low concentrations (less than 8mg/l). Tryptamine was not found in any of the red wine samples. Putrescine was detected in all samples (100%), followed byβ-Phenylethylamine (84.2%), Spermidine (60.5%), Histamine (57.8%), Tyramine (57.8%), Cadaverine (47.4%), and Spermine (36.8%). Putrescine andβ-Phenylethylamine were found in white wines, meanwhile the other biogenic amines were not found. The primary biogenic amines in Chinese beer were Putrescine, Tyramine, Spermidine, and Cadaverine. These beers presented contents lower than 2mg/l.2. Soy sauce and vinegar: The seven biogenic amines were found in all soy sauces samples, except Tryptamine. Tyramine, Cadaverine, Putrescine, andβ-Phenylethylamine were biogenic amines present in a high concentration. Several soy sauce samples showed very high concentration of biogenic amines (1982.52mg/kg.(ds)). The primary biogenic amines in Chinese rice vinegar samples were Putrescine, Cadaverine,β-Phenylethylamine, Histiamine, and Tyramine. 28.9% of these rice vinegar samples presented contents of Histamine higher than 50mg/l. In this thesis, partial other Chinese traditional seasonings and fermented foods were studied also.3.yoghourtand cheese: Most commercial Chinese yoghourts presented a low concentration of eight biogenic amines and Tyramine, Putrescine, andβ-Phenylethylamine were the major biogenic amines in Chinese yoghourt. In commercial Chinese cheese, Tryptamine, Spermidine, and Spermine were not found and the major biogenic amines wereβ-Phenylethylamine and Tyramine.4. Aquatic product: The content of biogenic amines was effected by the quality and the type of aquatic products. All fish samples and prawn samples presented a low content of spermidine and spermine. There were lower concentration and a few types of biogenic amines were found in fresh fish but it would increased when the quality of the fish decreasing. The content of biogenic amines of fresh prawn were as low as fresh fish, except cadaverine, which presented a higher concentration. Shrimp paste presented high contents of histamine, tyramine, putrescine, and cadaverine. Seven biogenis amines were found in canned fish, except Tryptamine, but the concentration of biogenic amines in canned fish were very low.Formation of biogenic amines may occur in fermented foods and beverages due to the amino acid decarboxylase activities of Gram-positive bacteria. This study describes a series simple and rapid simplex- and multiplex-PCR methods to determine the ability to produce histamine, tyramine and putrescine by bacteria. In a screening of primers, three pairs of primers based on sequences from histidine, tyrosine, ornithine decarboxylases of Gram-positive bacteria were selected. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases(HDC) of Gram-positive bacteria, 1133-bp DNA fragment from bacterial tyrosine decarboxylases(TDC), and 1700bp-bp DNA fragment from bacterial ornithine decarboxylases(ODC). A novel multiplex PCR method for the simultaneous detection of bacteria, which potentially produce histamine, tyramine, and putrescine is proposed in the present work. The methods were successfully applied to several biogenic amine-producing bacteria strains, even when DNAs of several target organisms were included in the same reaction. No amplification was observed with DNA from nonproducing lactic acid bacteria(LAB) strain in food. The use of this molecular tool for early and rapid detection of Gram-positive biogenic amine-producing bacteria is of interest in evaluating the potential of cultured indigenous strains to produce biogenic amines in a fermented food product as well as to validate the innocuity of potential starter strains in the food industry.The safety of twenty three Oenococcus oeni strains and one hundred and nine LAB strains was evaluated by the PCR assay. The results of the experiments showed that 367-bp fragments and 1700-bp fragments were not amplified from the DNA of one hundred and thirty two supplied strains. 1133-bp fragments were amplified from the DNA of forty four strains in one hundred and nine LAB strains. It indicated that they were not histamine-, tyramine-, and putrescine-producing strain in twenty three Oenococcus oeni strains and not histamine- and putrescine-producing strain in one hundred and nine lactic acid bacreia strains. The forty four LAB strains were potentially tyramine-producing LAB. The results of PCR assay were consistent with that of determination of the production of biogenic amines by HPLC.The PCR products of thirty one in forty four tyramine-positive LAB strains were sequenced. Blasting the gene sequences and the protein sequences to GenBank or each other showed that the sequences are highly homology and they all contained the highly conserved LHVDAAY motif and PLP attachment site. It indicated that the amplication fragments of PCR were partial tdc gene. The result of phylogenetic analysis of the amino acid sequences of TDC from tyramine-positive LAB strains suggests that tyramine production is specific to species or strain and the TDC gene cannot be used for the universal phylogenetic analysis of tyramine-positive bacteria.更多还原