Identification Of Biogenic Amines Produced By Lactic Acid Bacteria And Detection Biogenic Amines In Food

Biogenic amines produced by microorganism are alkaline, low-molecular organic compounds. Proper amount biogenic amines have physiological activity, while excessive intake may bring about health lesion. Lactic acid bacteria with decarboxylase activity, through decarboxylation of amino acids reaction, may produce biogenic amines which have the potential security risks.In this paper, 61 strain lactic acid bacteria were screened by a decarboxylase medium, then Using PCR method experiments were performed to amplify the genomic DNA of lactic acid bacteria in order to detect amino acid decarboxylase gene. The molecular method detected potential biogenic amine risk formation in food before the amine was produced Finally, RP-HPLC analysis biogenic amines produced in decarboxylase medium and the food matrix.The reserch provided the method to evaluate the safety of Lactic acid bacteria more accurately and ensure their applications in food more securely. The results as follows:1. According to the biogenic amine metabolism characters of LAB, amino acid decarboxylase medium was used to screen BA-produced bacteria from 61 lactic acid bacterias produce amines. Adding amino acids precursors: histidine, tyrosine, ornithine, lysine to the medium, after a pyridoxal 5'-phosphate induced in a poor-nutrient and low-acidic condition, LAB with decarboxylase activity can produce biogenic amines. The result was showed by the color changing of indicator. Two histidine-positive bacteria, 16 strains tyrosine-positive ware found. Cadaverine and putrescine were not detected among 61 lactic acid bacteria.2. Aligning histidine decarboxylase and tyrosine decarboxylase gene sequences, 61 lactic acid bacteria genomic DNA as template, 3 lactic acid bacteria with a histidine decarboxylase gene, 22 lactic acid bacteria with a tyrosine decarboxylase gene were successfully amplified in use of primers PHDC1/PHDC2 and P1-rev/P2-for, The PCR products of 3 histidine-positive and 3 tyramine-positive LAB strains were sequenced. Blasting the gene sequences and the protein sequences to GenBank, It indicated that the amplication fragments of PCR were partial hdc and tdc gene.The results comparing with the microbiological assay results was basically consistent. The molecular method detect potential biogenic amine risk formation in food before the amine was produced.PCR methods offer the advantages of speed, sensitivity, simplicity and specific detection of amino acid decarboxylase genes.3. The biogenic amines were extracted by 10% trichloroacetic acid, benzoyl chloride derivation. Five biogenic amine standard curves were established. The regression equation are: histidine: y = 44859x + 51524; tyramine: y = 44859x + 51524; putrescine y = 63114x + 65892; cadaverine: y = 63114x + 65892. It was linear over the range of concentrations between 1mg/L and 50 mg/L (R>0.995). The detection limits were 0.08mg/L for histamine and tyramine, 0.10mg/L for putrescine and cadaverine. It has good stability, high sensitivity and high recovery rate.4. Potential 25 LAB producing biogenic amines in liquid decarboxylase culture medium and four kind of leavened food systems ware analyzed by HPLC. The result showed that, 3 strains histamine positive LAB produced histamine 6.99 mg/L-69.46 mg/L in decarboxylase media. 22 tyramine-positive LAB produced tyramine 4.06 mg/L-70.10 mg/L. In four kinds of food systems, 25 LAB produced the lowest biogenic amine in the fermented milk, highest content was less than 4mg/L. In the fermentation vegetables, the fermentation grape, in the fermentation bouillon, tyramine contents were less than 20mg/L, the histamine contents were less than 6 mg/L. All productions were lower than FDA limit standard request in food, the the tyramine content less than 100 mg/L and histamine content less than 50mg/L .