大菱鲆肠道中广谱拮抗活性乳酸菌的筛选及其细菌素鉴定

应用牛津杯法从大菱鲆肠道分离的乳酸菌中筛选出对革兰氏阳性和阴性细菌均具有抑制作用的菌株LP1-4，经生理生化反应和16S rRNA鉴定为植物乳杆菌（Lactobacillus plantarum）。通过温度、pH值和蛋白酶等因素分析，结果表明菌株LP1-4无细胞上清液（cell-free supernatant，CFS）中抑菌物质具有良好的热稳定性，在pH 2.5～5.0具有抑菌活性，且对蛋白酶敏感，初步判断为细菌素类抑菌物质。菌株LP1-4在最佳培养时间24 h时抑菌活性达到峰值，应用乙酸乙酯萃取后抑菌圈直径达25.42 mm。采用Labscale TFF System超滤分离和Sephadex G-25凝胶层析获得小于1.0 ku分子质量的细菌素。经高效液相色谱纯化分析，抑菌物质在液相分离条件下目标峰的保留时间为5.0 min。采用基质辅助激光解吸电离串联飞行时间质谱确定细菌素分子质量为0.854 ku，其氨基酸排列顺序为谷氨酸-天冬酰胺-赖氨酸-脯氨酸-丙氨酸-丙氨酸-脯氨酸-赖氨酸（ENKPAAPK）。本研究从大菱鲆肠道筛选具有广谱抗菌活性的菌株LP1-4，对研发控制水产品腐败菌和致病菌的乳酸菌生物防腐保鲜制剂具有潜在的应用价值。

Screening for Broad-Spectrum Antagonistic Lactic Acid Bacteria from Intestine of Turbot and Identification of Bacteriocin Produced by It

Strain LP1-4, with strong antagonistic activity against both Gram-negative bacteria and Gram-positive bacteria, was screened out of lactic acid bacteria (LAB) isolated from the turbot intestine using the Oxford cup method. LP1-4 was identified as Lactobacillus plantarum according to physiological and biochemical characteristics and 16S rRNA sequence analysis. The antimicrobial substances in the cell-free supernatant (CFS) of LP1-4 were heat stable and effective in the pH range of 2.5–5.0, but were sensitive to protease. These substances were identified as bacteriocins. The antibacterial activity of LP1-4 reached the peak after 24 h of culture, and the diameter of inhibition zone of the ethyl acetate extract was 25.42 mm. A bacteriocin with molecular mass less than 1.0 ku was purified from the extract by ultrafiltration using Labscale TFF system and Sephadex G-25 column chromatography. The retention time of the target peak in high performance liquid chromatography (HPLC) was 5.0 min. The molecular mass of the bacteriocin was estimated to be 0.854 ku by matrix-assisted laser desorption ionisation/time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). The amino acid sequence of this bacteriocin was Glu-Asn-Lys-Pro-Ala-Ala-Pro-Lys. In conclusion, LP1-4 has potential applications in controlling spoilage and pathogenic bacteria in aquatic products as a biological preservative.