Foodborne pathogens and their biofilms are considered as one of the most serious problems in human health and food industry. Moreover, safety of foods is a main global concern because of the increasing use of chemical food additives. Ensuring food safety enhances interest in discovery of new alternative compounds such as antimicrobial peptides (AMPs), which can be used as bio-preservatives in the food industry. In this study, the most important antimicrobial peptides of camel milk lactoferrin (lactoferrampin and lactoferricin) were recombinantly expressed in the form of chimeric peptide (cLFchimera) in a food-grade L. lactis strain. P170 expression system was used to express secreted cLFchimera using pAMJ1653 expression vector which harbors a safe (non-antibiotic) selectable marker. Peptide purification was carried out using Ni-NTA agarose column from culture medium with concentration of 0.13 mg/mL. The results of disk diffusion test revealed that cLFchimera had considerable antimicrobial activity against a number of major foodborne bacteria. Furthermore, this chimeric peptide showed strong and weak inhibitory effect on biofilm formation against P. aeruginosa, S. aureus E. faecalis, and E. coli, respectively. Antioxidant activity and thermal stability of the chimeric peptide was determined. The results showed that cLFchimera had antioxidant activity (IC50: 310 μ/mL) and its activity was not affected after 40 min of boiling. Finally, we evaluated the interaction of the peptide with LPS and DNA in bacteria using molecular dynamic simulation as two main intra and extra cellular targets for AMPs, respectively. Our in silico analysis showed that cLFchimera had strong affinity to both of these targets by positive charged residues after 50 ns molecular dynamic simulation. Overall, the engineered food-grade L. lactis generated in the present study successfully expressed a secreted chimeric peptide with antimicrobial properties and could be considered as a promising bio-preservative in the food industry.

中文翻译：

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用于生产抗菌肽的工程食品级乳酸乳球菌菌株的生成：体外和计算机评估

食源性病原体及其生物膜被认为是人类健康和食品工业中最严重的问题之一。此外，由于化学食品添加剂的使用不断增加，食品安全成为全球关注的主要问题。确保食品安全提高了人们对发现新的替代化合物的兴趣，例如抗菌肽（AMP），它可以在食品工业中用作生物防腐剂。在本研究中，骆驼奶乳铁蛋白最重要的抗菌肽（乳铁蛋白和乳铁蛋白）在食品级乳酸乳球菌菌株中以嵌合肽（cLFchimera）的形式重组表达。P170表达系统用于使用带有安全（非抗生素）选择标记的pAMJ1653表达载体来表达分泌的cLF嵌合体。使用 Ni-NTA 琼脂糖柱从浓度为 0.13 mg/mL 的培养基中进行肽纯化。纸片扩散试验结果表明，cLFchimera 对多种主要食源性细菌具有相当大的抗菌活性。此外，该嵌合肽分别对铜绿假单胞菌、金黄色葡萄球菌、粪肠球菌和大肠杆菌的生物膜形成显示出强和弱的抑制作用。测定了嵌合肽的抗氧化活性和热稳定性。结果表明，cLFchimera具有抗氧化活性（IC50：310 μ/mL），煮沸40 min后其活性不受影响。最后，我们使用分子动力学模拟分别作为 AMP 的两个主要细胞内和细胞外靶标，评估了肽与细菌中 LPS 和 DNA 的相互作用。我们的计算机分析表明，经过 50 ns 分子动力学模拟后，cLFchimera 通过带正电的残基对这两个靶标具有很强的亲和力。总体而言，本研究中产生的工程食品级乳酸乳球菌成功表达了具有抗菌特性的分泌嵌合肽，可被认为是食品工业中一种有前途的生物防腐剂。