# _Applied_
## 4 Discussion  
Lactic acid bacteria (LAB) are a group of microorganisms widely used in the industry for the production of fermented foods. In addition, LAB are used as probiotics in various functional products due to their various properties with health benefits [24]. One of the health benefits of LAB includes supplying vitamins to the host through _de novo_ synthesis [50; 54]. Vitamin B2, also known as riboflavin, is not synthesized in the human body and must be supplied through ingestion [1]. Accordingly, many researchers have discovered LAB strains that overproduce riboflavin and conducted research to manufacture riboflavin-enriched foods using these strains [18; 19; 20; 21]. Roseoflavin is an analog of riboflavin, which is toxic to microorganism as it cannot be used for the synthesis of FAN and FMN, the co-enzyme forms of riboflavin. Therefore, it has been reported that roseoflavin can be used to increase riboflavin production in microorganisms by inducing mutations in RFN element upstream of the rip operon [20; 21]. Thus, LAB that are inherently resistant to roseoflavin have the potential to overproduce riboflavin. In this study, LAB that overproduces riboflavin was isolated from Kimchi, a known fermented vegetable with high vitamin B2 content. LAB strains overproducing riboflavin were selected through riboflavin-free CDM plates containing roseoflavin. Of the LAB isolated from 26 Kimchi samples, 46 strains were able to grow without riboflavin, with 18 strains producing riboflavin at a detectable level in CDM (Table S1). There were five strains capable of forming colonies even in an environment in which roseoflavin was present at a high concentration (150 ug/mL), and only one strain produced very high riboflavin regardless of the presence or absence of riboflavin (Figure 2). The species of the finally selected strain was \(L\)_plantarum_, which was named HY7715. HY7715 produced significantly higher riboflavin than the \(L\)_plantarum_ type strain NCDO1752, which is known to grow without riboflavin (Figure 3A). This riboflavin-overproducing ability of HY7715 was predicted to be due to high _ribA_, _ribB_, _ribC_, _ribH_, and _ribG_ gene expression (Figure 3B). Interestingly, HY7715 and NCDO1752 showed only one nucleotide difference in the rib operon regulatory region, which was consistent with previous studies [19; 21] that a single nucleotide mutation alters the operon regulation ability, resulting in increased riboflavin production (Figure 3C). The riboflavin overproduction ability of HY7715 was maintained for at least 30 days even in the absence of selective pressure (Figure 4). Moreover, the riboflavin-overproducing ability of HY7715 was maintained even in mass culture, a prerequisite for industrialization (Figure 6A). This suggested that HY7715 could become an industrially valuable LAB in the area of riboflavin biosynthesis. Riboflavin deficiency often occurs when nutritional intake is unbalanced because riboflavin is not synthesized in the body and the remaining riboflavin that has not been converted to a coenzyme is excreted in the urine [2; 5; 8]. There have been previous studies to improve riboflavin deficiency with fermented soymilk using riboflavin overproducing LAB strains [16]. EGRAC, one of the indicators of riboflavin deficiency, was found to be improved, but riboflavin uptake and excretion in vivo were not confirmed in that study. Therefore, in this study, it was confirmed that the intake of HY7715, a new riboflavin-overproducing strain, can increase riboflavin levels in-vivo. Mice that were pre-supplied with HY7715 showed high levels of riboflavin in plasma and urine even when riboflavin deficiency was induced through nutrient restriction (Figure 8A,B). The fact that riboflavin was excreted in the urine even when riboflavin was not supplied suggested that HY7715 produces and supplies enough riboflavin in the mouse body.