Genome shuffling of Lactobacillus plantarum for improving antifungal activity

Many species of lactic acid bacteria have the ability to inhibit the growth of spoilage fungi in food and feed products. Genome shuffling is a powerful process that could efficiently improve desirable industrial phenotypes of microbial strains through recursive protoplast fusion. In our study, the antifungal activity of Lactobacillus plantarum IMAU10014 was improved by genome shuffling. The starting populations were obtained via ultraviolet irradiation and diethyl sulphate mutagenesis and subjected to recursive protoplast fusion with Lactobacillus helveticus IMAU40097. The optimal conditions for protoplast formation and regeneration were found to be 15 mg/ml lysozyme and 5 μg/ml mutanolysin for 40 min at 37 °C. The protoplasts were inactivated under ultraviolet light for 30 min or heated at 60 °C for 120 min. After three rounds of genome shuffling, the inhibitory activity of strain F3C2 and F3A3 were improved to 192% and 200%, respectively, compared to the wild-type strain. The shuffled strains have a broad spectrum of antifungal activity and are promising candidates for bio-preservatives. It was also revealed that lactic acid has a positive correlation with antifungal activity. Antifungal activity was not related only to lactic acid, phenyllactic acid or any other single inhibitory substance, but rather to a combination of such substances.