一种利用RT-HPLC分析乳酸菌产生物胺的方法

A RT-HPLC Method for the Determination of Biogenic Amine Produced by Lactic Acid Bacteria

具有脱羧酶活性的乳酸菌可通过氨基酸的脱羧反应产生具有潜在安全风险的生物胺。本文利用脱羧酶培养基初步筛查61株乳酸菌产生物胺情况,再通过RT-HPLC法测定其在发酵液和发酵乳中的生物胺含量。用10%的三氯乙酸提取样品中的生物胺,采用苯甲酰氯衍生处理后,以甲醇/水为流动相,进行梯度洗脱,流速0.8mL/min,紫外检测器波长为254nm。结果显示,组胺和酪胺得到良好的分离,在给定的浓度范围内呈现良好的线性关系(R2>0.995)。在发酵液和发酵乳中添加生物胺混合标准溶液,平均回收率为97.92%-101.14%,相对偏差RSD<5%。结果表明,发酵液与发酵乳中生物胺的RT-HPLC法,是一种快捷、稳定、灵敏度高的检测方法,其与脱羧酶培养基法结合可以准确地实现对乳酸菌产生物胺的评价。

Biogenic amines are produced by some lactic acid bacteria of decarboxylase activities which are potential risk factors for lactic acid bacteria-fermented foods. In this paper, the biogenic amine-producing capacities of 61 lactic acid bacteria strains were analyzed on a decarboxylase medium. Then the methodology of RT-HPLC determination for biogenic amines in fermented broth and milk was studied. The biogenic amines in test samples were extracted by 10% trichloroacetic acid and then derived with benzoyl chloride. The HPLC analysis for the samples were conducted as the following condition： mobile phase was a gradient elution program of methanol and water, the flow rate was 0.8 mL/min. UV detector was used in the final determination on 254 nm. The results showed that biogenic amines were separated well on such condition. The method was in a linear way for the amines studied at given concentrations （R2〉 0.995）. The average recovery ratios ranged from 97.92%-101.14% for all samples. The RSDs were less than 5%. It can be seen that the RT-HPLC method was rapid, stable, high sensitive. Combination of this HPLC method and microbial primary screening on a decarboxylase medium could evaluate the biogenic amine-producing capacity of lactic acid bacteria more accurately.