ISOLATION, BIOCHEMICAL CHARACTERIZATION AND MOLECULAR IDENTIFICATION OF RIBOFLAVIN OVERPRODUCING LACTIC ACID BACTERIA FROM DAIRY PRODUCTS

Abstract

Background: Vitamins are daily need of every individual and their deficiency can result in serious illness. Riboflavin (vitamin B2) is one of the essential vitamins required for normal functioning of human body. Some strains of Lactic Acid Bacteria (LAB) belonging to the genus Lactobacillus and Lactococcus have the potential to produce riboflavin in excess. Since LAB are generally regarded as safe (GRAS), therefore, could be used for the production of riboflavin enriched products. Methods: To estimate riboflavin concentration in market milk and yogurt, 20 samples were collected and riboflavin contents were determined spectrophotometrically. For isolation of potential riboflavin overproducers, 20 milk and yogurt samples were collected from four major cities of Pakistan and cultured on MRS agar. Gram positive and catalase negative colonies were selected for further studies. DNA was extracted from these isolates and 16S rDNA gene was amplified by PCR followed by DNA sequencing for identification at species level. The isolates identified as belonging to genus Lactobacillus or Lactococcus were then grown on Riboflavin Assay Medium (RAM), to test their riboflavin overproducing ability. The potential riboflavin overproducers were then tested for their safety in terms of hemolysis and antibiotic resistance. Finally, potential riboflavin overproducers found to be safe were then used for yogurt preparation. The yogurt thus obtained was then tested for riboflavin contents and comparison was done with market yogurt samples. Results: The mean riboflavin concentration observed in the market milk and yogurt samples was 2.515 ± 0.65 mg/L and 2.54 ± 0.573 mg/L respectively. To isolate potential riboflavin overproducers, 20 samples were spread on MRS agar. From the colonies thus obtained, 78 isolates were selected for Gram staining and catalase reaction. The results showed that 19 isolates were Gram positive and catalase negative (a typical characteristic of Lactic Acid Bacteria). Extracted DNA and PCR showed clear bands on gel. The sequencing results confirmed 7 LAB strains. Safety tests confirmed that these strains are safe and could be used for production of edible products. Four strains were found suitable for yogurt preparation and the highest riboflavin content of the prepared yogurt was 6.3 mg/L. Conclusion: Riboflavin overproducing LAB can be isolated from dairy products. They have the potential to double the content of riboflavin in the edible products to fulfill the necessary daily requirements of human body