Folates are essential micronutrients, and folate deficiency still occurs in many countries. Lactic acid bacteria (LAB) are known to be able to synthesize folates during fermentation, but the folate production is strain-dependent and influenced by the fermentation medium, presence of a folate precursor, and fermentation time. This study aimed to screen extracellular folate-producing LAB from local food sources and evaluate the factors influencing their folate biosynthesis during milk fermentation. The selection of folate-producing LAB was based on their ability to grow in folate-free medium (FACM), with folate concentrations quantified by microbiological assay. Growth of the 18 LAB in FACM varied between isolates, with only 8 isolates growing well and able to synthesize extracellular folate at relatively high concentrations (up to 24.27 ng/mL). The isolates with highest extracellular folate levels, Lactobacillus fermentum JK13 from kefir granules, Lactobacillus plantarum 4C261 from salted mustard, and Lactobacillus rhamnosus R23 from breast milk, were applied to milk fermentation. The last two isolates were probiotic candidates. The three isolates consumed folate when it was present in the milk, and its consumption was in line with their growth. The availability of folate precursors affected the amount of folate consumed, but did not lead to increased folate concentrations in the medium after 72 h fermentation. The results of this study indicate that these isolates cannot be utilized for producing folate in folate-containing milk, as it shows feedback inhibition on folate biosynthesis.

Introduction Folate or vitamin B9 is a water-soluble vitamin composed of several conjugated molecules namely pteridine ring, paraaminobenzoic acid (PABA), and glutamic acid. 1,2 Folate is required for normal cell fission and growth. It functions as a cofactor involved in various metabolic reactions in the body, such as synthesis, repair, DNA methylation reactions; nucleotide synthesis; and amino acid metabolism. 3,4 Folate is needed in a certain amount, especially during pregnancy and lactating period. The daily recommended intake of folate are 65 µg/d of dietary folate equivalents (DFEs) for 0-6 month infants as adequate intake (AI), 80 µg DFE for 7-12 month infants (AI), 150 µg DFE for 1-3 year old children as recommended dietary allowance (RDA), 200 µg DFE for 4-8 year old children (RDA), 300 µg DFE for 9-13 year old children (RDA), 400 µg DFE for 14-18 year old teens (RDA), 400 µg DFE for adults 19 years old and older (RDA), 500 µg DFE for lactating women (RDA), and 600 µg DFE for pregnant women (RDA). 5 If folate requirement is not sufficiently met, the body will experience folate deficiency and trigger various diseases such as anemia, neural tube defects, homocysteinemia, cardiovascular disease, and cancer. 4,6-9 Folate cannot be produced in the body, therefore, it must be obtained from food intake. It is naturally present in various types of food, such as cereals, fruits, vegetables, spices, nuts, eggs, and cheese. 10 However, natural folate has unstable properties, and its content is easily reduced during washing and processing. 11,12 Alternatively, folic acid, a synthetic form of folate, is generally chosen as the main source in fulfilling the daily needs as a food fortificant and food supplement due to its stability. 13,14 However, the metabolic process of folic acid in the body is relatively slow hence the body is not able to completely convert folic acid in large quantities. This results in a substantial accumulation of unmetabolized folic acid in cells. High levels of unmetabolized folic acid in the blood will cause a variety of metabolic disorders, such as masking symptoms of vitamin B12 deficiency, cognitive impairment, reducing the immune system, and cancer. 15,16 The emergence of health problems related to the use of synthetic folate has prompted many researchers to look for other, more stable, safer and more efficient sources of natural folate. Lactic acid bacteria (LAB) are known to produce folate both intracellularly and extracellularly. 12,17-19 Intracellular folate is in the intact cells, while extracellular folate is secreted to the growth medium. Extracellular folate is in the form of monoglutamate, thus it has a higher bioavailability compared to intracellular folate which is in the form of polyglutamate. Polyglutamate requires an enzymatic conjugation process

Research background. Lactic acid bacteria (LAB) are known to produce folate. However, this ability is highly strain-dependent. Folate synthesis in specific LAB strains is affected by the availability of folate, which can be consumed by LAB under certain conditions. Moreover, differences in folate synthesis capabilities are related to the presence of folate biosynthesis-related genes and regulation of this pathway. Experimental approach. As basic information to better understand the regulation of folate biosynthesis between LAB species and strains, folate biosynthetic genes were screened and identified in folate-producing and non-folate-producing LAB isolates from various local food sources in Indonesia. The extracellular folate productivity levels of the isolates were analyzed using high-performance liquid chromatography with a diode array detector (HPLC-DAD). Result and conclusions. Eleven of the thirteen LAB isolates tested had all of the eight genes involved in folate biosynthesis (folE, folQ, folB, folK, folP, folC1, folA, and folC2). Furthermore, these isolates produced extracellular folate ranging from 10.37 to 31.10 µg/mL. In contrast, two non-folate-producing isolates lacked several folate biosynthetic genes, such as folQ, folP, and folA, and possibly corresponded to their inability to synthesize folate de novo. Phylogenetic tree construction revealed that the folate biosynthetic genes (excluding folK and folP) from six distinct species of folate-producing LAB isolates were monophyletic with homologous genes from other LAB species in the database. Novelty and scientific contribution. In this study, the distribution of folate biosynthetic genes in various LAB species was determined. The findings from this research support the use of folate biosynthesis marker genes in the genotypic screening for folate-producing LAB.