Screening and Spontaneous Mutation of Pickle-Derived Lactobacillus plantarum with Overproduction of Riboflavin, Related Mechanism, and Food Application

Abstract

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, Lactobacillus plantarum 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, L. plantarum 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 rib operons including the ribA, ribB, ribC, ribH, and ribG genes. Unexpectedly, however, this mechanism did induce an insertion of a 1059-bp DNA fragment in the upstream regulatory region of the rib 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.

Keywords: Lactobacillus; fermented soymilk; pickle juice; roseoflavin mutation; vitamin B2.

Figure 1

The chemical structures of riboflavin (A) and its analog roseoflavin (B).

Figure 2

Bacterial colonies obtained upon incubation of the diluted mixture of 11 different pickle juices on MRS plate (A) and chemically defined medium (CDM) plate (B).

Figure 3

The concentration of riboflavin produced by the RYG-YYG-9049 strain and its ten mutant strains. Values with different lowercase letters indicate statistical significance (p < 0.05).

Figure 4

(A) Alignment of the rib operon regulatory region of RYG-YYG-9049 and its mutant strain. The predicted −10 and −35 recognition sequences and ribosomal binding sites (RBS) are boxed. The ellipsis represents the increased gene sequence of the mutant strain. The ribG start codon is boxed with solid lines. The RFN element is indicated by the arrows below. (B) The inserted 1059-bp gene sequence.

Figure 5

(A) pH value of fermented soymilk by the RYG-YYG-9049 wild strain and its No. 10 mutant strain under different conditions. (B) Changes of riboflavin value in soymilk that was inoculated with the bacteria at different time and temperatures.