| Summary: | Riboflavin, also known as vitamin B2, plays an important role in human cell metabolism and participates in various redox reactions and in energy utilization. In this study, 90 riboflavin-producing lactic acid bacteria (LAB) were screened out from pickle juices. The yields of riboflavin in these LAB were about 0.096−0.700 mg/L, and one strain, <i>Lactobacillus plantarum</i> RYG-YYG-9049, was found to produce the highest riboflavin content. Next, roseoflavin was used to induce the spontaneous mutation of RYG-YYG-9049, and selected roseoflavin-resistant colonies generally produced higher riboflavin contents, ranging from 1.013 to 2.332 mg/L. The No. 10 mutant, <i>L. plantarum</i> RYG-YYG-9049-M10, had the highest riboflavin content. Next, the molecular mechanism of enhancing riboflavin production in RYG-YYG-9049-M10 was explored, leading to the finding that roseoflavin treatment did not change the <i>rib</i> operons including the <i>ribA</i>, <i>ribB</i>, <i>ribC</i>, <i>ribH</i>, and <i>ribG</i> genes. Unexpectedly, however, this mechanism did induce an insertion of a 1059-bp DNA fragment in the upstream regulatory region of the <i>rib</i> operon, as compared to the wild-type RYG-YYG-9049. To the best of our knowledge, this is the first report that roseoflavin could induce an insertion of DNA fragment in LAB to increase riboflavin content, representing a new mutation type that is induced by roseoflavin. Finally, in order to fortify riboflavin content in soymilk, RYG-YYG-9049 and RYG-YYG-9049-M10 were used to ferment soymilk, and several fermentation parameters were optimized to obtain the fermented soymilk with riboflavin contents of up to 2.920 mg/L. In general, roseoflavin induction is an economical and feasible biotechnological strategy to induce riboflavin-overproducing LAB, and this strategy can be used to develop LAB-fermented functional foods that are rich in riboflavin.
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