该研究采用高效液相色谱结合手性色谱柱的方法,建立了一种对乳酸菌发酵液中L/D-乳酸定性定量检测的分析方法。该方法中在1.0~ 400.0 μg/mL范围中,L/D-乳酸质量浓度与检测结果均呈良好线性关系,L/D-乳酸检出限为0.25 μg/mL,定量限为0.80 μg/mL。基于2株乳酸菌的发酵液对该方法进行验证,结果表明发酵液中L/D-乳酸得到良好分离,该方法精密度、重复性良好,回收率为90.0%~103.7%;在不同稀释度的发酵液中线性相关系数R2均在0.999以上;稳定性的相对标准偏差5%以下;对涵盖4属8种的18株可用于食品的乳酸菌菌株产生L/D-乳酸的情况进行分析,结果表明乳酸菌产L/D-乳酸含量具有菌株差异性,同种乳酸菌产D-乳酸比例具有相同趋势。利用该方法对乳酸菌发酵液中L/D-乳酸的定性定量检测,结果准确稳定。该研究为食品用菌种的安全性评价中菌株代谢产物D-乳酸的检测提供了有效数据支撑和实践参考。
In this study, an analytical method for the qualitative and quantitative determination of L/D-lactic acid in the fermentation broth of lactic acid bacteria was established by HPLC combined with chiral column. In this method, the concentration and detection results of L/D-lactic acid showed a good linear relationship in the range of 1.0-400.0 μg/mL. The limit of detection was 0.25 μg/mL, the limit of quantification was 0.80 μg/mL. The method was verified based on fermentation broth of two strains of lactic acid bacteria, the results showed that L/D-lactic acid in fermentation broth were well separated, and the method had good precision and repeatability, and the recovery rate was between 90.0% and 103.7%. The linear correlation coefficient R2 were more than 0.999 in fermentation broth with different dilution degrees, and relative standard deviation of stability less than 5%. The production of L/D-lactic acid by 18 strains of lactic acid bacteria that can be used in food, including 8 species and 4 genera, was analyzed, the results showed that the content of L/D-lactic acid produced by lactic acid bacteria was different among strains, and the proportion of D-lactic acid had the same trend in species. The qualitative and quantitative determination of L/D-lactic acid in lactic acid bacteria fermentation broth by this method was accurate and stable. This study provides effective data support and practical reference for the detection of D-lactic acid, the metabolite of strains, in the safety evaluation of microbial food cultures.
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