Iram Daraksha, Kindarle Uday Arun, Sansi Manish Singh, Meena Sunita, Puniya Anil Kumar, Vij Shilpa
Antimicrobial Peptides, Biofunctional Probiotics & Peptidomics Laboratory, Dairy Microbiology Division, ICAR-National Dairy Research Institute, Karnal, India.
Biofunctional Peptidomics & Metabolic Syndrome Laboratory, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, India.
J Food Biochem. 2022 Dec;46(12):e14450. doi: 10.1111/jfbc.14450. Epub 2022 Oct 13.
Antimicrobial peptides (AMPs) are emerging as promising novel drug applicants. In the present study, goat milk was fermented using Lactobacillus rhamnosus C25 to generate bioactive peptides (BAPs). The peptide fractions generated were separated using ultrafiltration membranes with molecular weight cut-offs of 3, 5, and 10 kDa, and their antimicrobial activity toward Gram-positive and Gram-negative bacteria was investigated. Isolated AMPs were characterized using RP-HPLC and identified by LC-MS/MS. A total of 569 sequences of peptides were identified by mass spectrometry. Out of the 569, 36 were predicted as AMPs, 21 were predicted as cationic, and out of 21, 6 AMPs were helical peptides. In silico analysis indicated that the majority of peptides were antimicrobial and cationic in nature, an important factor for peptide interaction with the negative charge membrane of bacteria. The results showed that the peptides of <5 kDa exhibited maximum antibacterial activity against E. faecalis, E. coli, and S. typhi. Further, molecular docking was used to evaluate the potent MurD ligase inhibitors. On the basis of ligand binding energy, six predicted AMPs were selected and then analyzed by AutoDock tools. Among the six AMPs, peptides IGHFKLIFSLLRV (-7.5 kcal/mol) and KSFCPAPVAPPPPT (-7.6 kcal/mol), were predicted as a high-potent antimicrobial. Based on these findings, in silico investigations reveal that proteins of goat milk are a potential source of AMPs. This is for the first time that the antimicrobial peptides produced by Lactobacillus rhamnosus (C25) fermentation of goat milk have been identified via LC-MS/MS and predicted as AMPs, cationic charges, helical structure in nature, and potent MurD ligase inhibitors. These peptides can be synthesized and improved for use as antimicrobial agents. PRACTICAL APPLICATIONS: Goat milk is considered a high-quality source of milk protein. According to this study, goat milk protein is a potential source of AMPs, Fermentation can yield goat milk-derived peptides with a broad antibacterial activity spectrum at a low cost. The approach described here could be beneficial in that the significant AMPs can be synthesized and used in the pharmaceutical and food industries.
抗菌肽正成为有前景的新型药物候选物。在本研究中，使用鼠李糖乳杆菌C25发酵山羊奶以产生生物活性肽（BAPs）。使用截留分子量为3、5和10 kDa的超滤膜分离产生的肽段，并研究它们对革兰氏阳性菌和革兰氏阴性菌的抗菌活性。使用反相高效液相色谱法（RP-HPLC）对分离出的抗菌肽进行表征，并通过液相色谱-串联质谱法（LC-MS/MS）进行鉴定。通过质谱法共鉴定出569个肽序列。在这569个序列中，36个被预测为抗菌肽，21个被预测为阳离子肽，在这21个中，6个抗菌肽为螺旋肽。计算机模拟分析表明，大多数肽本质上具有抗菌性和阳离子性，这是肽与细菌负电荷膜相互作用的一个重要因素。结果表明，分子量小于5 kDa的肽对粪肠球菌、大肠杆菌和伤寒沙门氏菌表现出最大的抗菌活性。此外，使用分子对接来评估有效的MurD连接酶抑制剂。基于配体结合能，选择了6个预测的抗菌肽，然后通过AutoDock工具进行分析。在这6个抗菌肽中，肽IGHFKLIFSLLRV（-7.5 kcal/mol）和KSFCPAPVAPPPPT（-7.6 kcal/mol）被预测为高效抗菌剂。基于这些发现，计算机模拟研究表明山羊奶蛋白是抗菌肽的潜在来源。这是首次通过LC-MS/MS鉴定出鼠李糖乳杆菌（C25）发酵山羊奶产生的抗菌肽，并预测其为抗菌肽、具有阳离子电荷、天然螺旋结构以及有效的MurD连接酶抑制剂。这些肽可以合成并改良用作抗菌剂。实际应用：山羊奶被认为是优质的乳蛋白来源。根据这项研究，山羊奶蛋白是抗菌肽的潜在来源，发酵可以低成本产生具有广泛抗菌活性谱的山羊奶衍生肽。这里描述的方法可能是有益的，因为重要的抗菌肽可以合成并用于制药和食品工业。
