植物乳杆菌D1501发酵黄浆水的抑菌活性及其中细菌素的分离与鉴定

Antimicrobial Activity of Soy Whey Fermented by Lactobacillus plantarum D1501 and Purification and Identification of Bacteriocin from It

将植物乳杆菌D1501(Lactobacillus plantarum D1501)接种于黄浆水中,37℃静置培养24 h后制成酸浆。抑菌活性测定表明,该方法制备的酸浆对供试的革兰氏阳性菌、革兰氏阴性菌具有明显的抗菌活性,对假丝酵母有弱的抑菌活性,但对酿酒酵母和2株霉菌没有抑制作用。经中和及酶解实验推测酸浆中的抑菌物质为细菌素。将经过30、10、3 kDa超滤分离得到的无细胞超滤液旋蒸浓缩定容后,采用RESOURCE-Q阴离子交换柱和300SB-C18反相层析柱依次纯化,得到色谱纯度细菌素物质,命名为Lp100。该细菌素经液相色谱-串联质谱法分析,确定分子质量约为1434.90 Da,氨基酸序列为MCCKVLLLLSRR。最终细菌素比活力为7247.00 IU/mg,纯化倍数为79.94倍,回收率为1%。

Lactobacillus plantarum D1501 was inoculated into soy whey and cultivated under static conditions at 37℃for 24 h to prepare fermented soy whey(FSW).Antimicrobial tests showed that FSW had obvious antimicrobial activity against both Gram-positive and Gram-negative bacteria tested,and had weak antimicrobial activity against Candida,but exhibited no inhibitory effect on Saccharomyces cerevisiae or two mold strains tested.The bacteriostatic substance in FSW was speculated to be bacteriocin by enzymatic hydrolysis and neutralization experiments.The cell-free neutral supernatant of L.plantarum D1501 was separated by 30,10 and 3 kDa ultra filtration tubes sequentially and concentrated by rotary evaporation.Then it was subjected to RESOURCE-Q anion exchange chromatography and reverse-phase chromatography with 300SB-C18 for further purification.The purity of the purified bacteriocin was determined to be high by high performance liquid chromatography(HPLC),and it was named as Lp100.The molecular mass of Lp100 was determined by LC-MS/MS to be approximately 1434.90 Da,and its amino acid sequence was MCCKVLLLLSRR.The specific activity of the purified product was 7247.00 IU/mg with 79.94-fold purification and 1%recovery.