Methods For Determination Of Genetic Amines In Food And Amine-Producing Bacteria And Its Application

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.