# _Applied_
## 1 Introduction  
Riboflavin, also known as vitamin B2, is a water-soluble vitamin found in a variety of nutritional sources. It is not synthesized by the human body and must be obtained through consumption of a variety of animal and plant foods, including beans, meat, and dairy products [1]. The main active forms of vitamin B2 are flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which act as coenzymes in mitochondrial metabolism and as hydrogen carriers in biological redox reactions [2]. Most riboflavin is absorbed in the proximal small intestine and is mainly stored in the liver, spleen, kidney and heart muscles in the form of FAD [3, 4]; the riboflavin that is not absorbed or stored is excreted in the urine [5]. The daily recommended intake (RDI) of riboflavin is 1.3 mg/d and 1.1 mg/d, respectively, for men and women, to maintain good health [3, 6]. Riboflavin is continuously excreted in the urine, making its deficiency relatively common when the vitamin intake is insufficient [5, 7]. Riboflavin deficiency may be related to maladaptive conditions, including inadequate dietary intake and wasting disease. Symptoms associated with riboflavin deficiency (ariboflavinosis) include stomatitis, cehiloss, and genital rash of the mouth and tongue. It has also been associated with visual impairments, such as night blindness and psychological disorders, including migraine headaches, mild anemia, and depression [8, 9, 10]. Riboflavin deficiency is relatively rare in developed countries but remains a serious health problem in developing and industrialized countries as deficiency can occur in up to 50% of the elderly or young population [6, 11].  
To prevent riboflavin deficiency, many countries, including the United States, have included this vitamin in essential fortification programs in mass consumption foods. However,vegetarians or people who rarely consume dairy products are still exposed to riboflavin deficiency [12; 13; 14]. Therefore, some of the beneficial microorganisms, including lactic acid bacteria (LAB), have been proposed as alternative means to increase the concentration of group B vitamins in foods based on their de novo biosynthetic ability [15; 16]. Riboflavin-producing LAB have been obtained from various ecological environments, including dairy products, human gastrointestinal tract, grains, and fermented vegetables [15; 17; 18; 19; 20]. In addition, roseoflavin, a toxic analogue of riboflavin, is often used to induce spontaneous mutations to obtain LAB strains with high riboflavin productivity [19; 20; 21; 22; 23; 24].  
Several studies have reported on manufacturing foods with increased riboflavin, such as bread, pasta, and soy milk, using LAB that overproduces riboflavin [18; 19; 20], and reports on improving riboflavin deficiency using fermented soy milk rich in riboflavin are also available [16]. There have also been studies on the treatment of gastrointestinal diseases using riboflavin overproducing LAB [25; 26; 27]. However, there is not enough evidence on whether consuming these foods can resolve riboflavin deficiency, and there is no research on whether riboflavin is absorbed in vivo by directly ingesting riboflavin-overproducing LAB. Therefore, this study was conducted to discover a new LAB strain with high riboflavin productivity in order to develop beneficial probiotic strain that can be used as alternative vitamin B2 supplements. For this, a LAB strain with high riboflavin production capacity was selected using chemically defined medium (CDM) containing roseoflavin, and the molecular mechanism of riboflavin overproduction was verified through _rib_ operon gene analysis. In addition, the potential of this LAB as a probiotic was confirmed through simulated gastrointestinal track (GIT), and the production of riboflavin in vivo was confirmed using riboflavin deficiency animal model. Finally, the biological safety of this strain was verified through antibiotic resistance test, hemolytic reaction test and whole-genome analysis.