Department of Biology, The Gandhigram Rural Institute (Deemed to be University), Gandhigram, 624302 Dindigul, Tamil Nadu, India.
Department of Biology, The Gandhigram Rural Institute (Deemed to be University), Gandhigram, 624302 Dindigul, Tamil Nadu, India.
Int J Biol Macromol. 2022 Nov 1;220:291-306. doi: 10.1016/j.ijbiomac.2022.08.087. Epub 2022 Aug 17.
The current study aimed to screen bacteriocin producing LAB from different dairy products and evaluation of their biological properties. Initially, 12 (4-chess, 4-curd, and 4-yohurt) LAB species were isolated and only 4 isolates alone were selected based on their clear yellow halo zone around the colonies in the selective medium. The selected 4 isolates were identified based on their morphological and biochemical characteristics. Among them, the strain CH3 have showed better antimicrobial effects on selected human pathogens. The isolated strain CH3 were further identified as Lactococcus lactis strain CH3 (MZ636710) by SEM imaging and 16 s rRNA molecular sequencing. Bacteriocin was extracted from L. lactis strain CH3 and partially purified using 60 % ammonium sulphate and then completely purified by G-50 column chromatography. The purified bacteriocin showed a specific activity of 5859.37 AU/mg in 24.7 % of recovery and 10.9-fold purification. The molecular weight of bacteriocin was 3.5 kDa as observed in SDS-PAGE. The bacteriocin showed sensitivity to proteolytic enzymes and resistance to high temperature, wide range of pH, organic solvents and detergents. FT-IR spectral studies of bacteriocin detected the existence of OH/NH-stretching, CH, and COC and CO bonds. NMR spectrum showed one doublet and 4 various singlet peaks at different ppm, indicating the occurrence of six amino acids in the structure of purified bacteriocin. The purified bacteriocin have shown stronger antimicrobial and anti-biofilm activity against selected human pathogens at 100 μg/mL. SEM showed the evidence of structural deformation and loss of membrane integrity of bacterial cells treated with bacteriocin. Bacteriocin exhibited greater DPPH radical scavenging potential with an EC value of 12.5 μg/mL. Bacteriocin have not shown significant toxicity on normal human dermal fibroblast (NHDF) cells (83.2 % at 100 μg/ mL). Furthermore, in silico studies using molecular modeling and docking were performed to know the proteins involved in antimicrobial action. The results suggests that bacteriocin could be an alternative to combat AMR pathogens and more suitable for food and dairy industries to preserve food without contamination.
本研究旨在从不同乳制品中筛选产细菌素的 LAB，并评估其生物学特性。最初，从 12 种（4 种奶酪、4 种凝乳和 4 种酸奶） LAB 种中分离出细菌，仅根据选择性培养基中菌落周围清晰的黄色晕圈，选择了 4 种单独的分离株。根据形态学和生化特征对所选 4 株分离株进行鉴定。其中，菌株 CH3 对选定的人类病原体表现出更好的抗菌效果。通过 SEM 成像和 16s rRNA 分子测序，进一步鉴定分离株 CH3 为乳球菌乳亚种 CH3（MZ636710）。从乳球菌乳亚种 CH3 中提取细菌素，并使用 60%硫酸铵部分纯化，然后通过 G-50 柱色谱完全纯化。纯化的细菌素在 24.7%的回收率和 10.9 倍的纯化度下具有 5859.37AU/mg 的比活度。SDS-PAGE 观察到细菌素的分子量为 3.5kDa。细菌素对蛋白酶敏感，对高温、宽 pH 值范围、有机溶剂和洗涤剂有抗性。细菌素的 FT-IR 光谱研究检测到 OH/NH 伸缩、CH 和 COC 和 CO 键的存在。NMR 谱在不同 ppm 处显示一个双峰和 4 个不同的单峰，表明纯化的细菌素结构中存在 6 种氨基酸。在 100μg/mL 时，纯化的细菌素对选定的人类病原体具有更强的抗菌和抗生物膜活性。SEM 显示了用细菌素处理的细菌细胞结构变形和膜完整性丧失的证据。细菌素对 DPPH 自由基具有更强的清除潜力，EC 值为 12.5μg/mL。细菌素对正常人皮肤成纤维细胞（NHDF）细胞无明显毒性（在 100μg/mL 时为 83.2%）。此外，还进行了使用分子建模和对接的计算机模拟研究，以了解参与抗菌作用的蛋白质。结果表明，细菌素可能是对抗 AMR 病原体的一种替代方法，更适合食品和乳制品行业在不污染的情况下保存食品。
