| dc.description.abstract | Folate-producing lactic acid bacteria (LAB) can act as folate-efficient producers or overproducers. Folate-efficient bacteria will only produce folate as their needs and only if folate is not available in the medium, whereas folate-overproducing bacteria can produce folate beyond their needs, even though there is early folate in the medium. However, a microorganism must have an efficient metabolic system; hence, finding folate-overproducing bacteria might be challenging. This work aimed to determine key genes for folate production and obtain folate-producing LAB. In particular, the objectives of this research were: (1) mapping the folate biosynthesis genes in diverse LAB species and identifying their role in the ability of LAB to produce folate; (2) finding potential folate biosynthesis marker genes in the genotypic screening for folate producers; (3) identifying the types of folate and the distribution of intra- and extracellular folate; and (4) applying stress exposure to methotrexate (a folate analogue) and the addition of enhancer compounds (PABA, glutamate, PABA-glutamate, CaCl2, and ascorbic acid) for effectively increasing extracellular folate production in LAB.
Of 13 LAB isolates from Indonesian fermented foods (tempeh, tapai, fermented mustard, kefir granules) and breast milk, eleven isolates had all of the eight genes involved in folate biosynthesis (folE, folQ, folB, folK, folP, folC1, folA, and folC2) and could produce extracellular folate (ranging from 10.37 to 31.10 µg/mL). In contrast, two LAB isolates lacked several folate biosynthesis genes (folQ, folP, and folA) and could not produce extracellular folate. The folQ gene, which was not found in non-folate-producing isolates, could potentially be used as a marker for folate biosynthesis. Phylogenetic tree analysis showed that the folate biosynthesis genes (except folK and folP) of the LAB isolates in this study were on the same branch as the folate biosynthesis genes of the various LAB isolates from the database, supported by a higher percentage of conserved sites than the variable sites. The folK and folP genes may be non-essential genes in the folate biosynthesis pathway, and further research is needed to study their contribution level to de novo folate biosynthesis.
Production of extracellular folate by selected LAB isolates was further evaluated in folate-free media and analyzed using the HPLC-DAD method with three individual-form folate standards, i.e., tetrahydrofolate (THF), 5-methyl-tetrahydrofolate (5-MTHF), and folic acid. Although the three folate standards could be detected with good peak separation, only the 5-MTHF peak was detected in extracellular folate samples of LAB isolates. Another peak that was only found in samples of folate-producing isolates was also suspected to be a folate peak and had a much higher area than the 5-MTHF peak, and further quantified as other folates, although further analysis is needed to confirm the form of folate. Five LAB isolates (R23, JK13, BK27, S2SR08, and NG64) produced 10% of 5-MTHF, while the other 7 LAB isolates (4C261, R12, R15, BD2, JK16, BG7), which included the reference strain (WCFS1), produced a 5-MTHF content of only 6%, and the rest are other folates. The highest extracellular folate producer isolates, namely Lacticaseibacillus rhamnosus R23 from ASI and Limosilactobacillus fermentum JK13 from kefir granules produced a much higher proportion of extracellular folate (95%) than their intracellular folate, with an optimum incubation time of 24 hours. The two LAB isolates can be utilized for the production of extracellular folate.
Increased production of extracellular folate was carried out on the isolates that produced the highest extracellular folate (R23 and JK13) through stress exposure to methotrexate as a folate analogue with a concentration of 2.5 mg/L. The increase in extracellular folate production was further carried out in isolate R23 by adding various enhancers (PABA, glutamate, PABA-glutamate, CaCl2, and ascorbic acid) with the best concentrations from the results of previous studies, namely 100 μmol/L PABA, 100 μmol/ L glutamate, 100 μmol/L PABA: glutamate (1:1 ratio), 100 mg/L CaCl2, and 0.2% ascorbic acid. However, using these two methods did not increase extracellular folate production. Exposure to methotrexate stress can cause changes in the morphology of BAL colony cells to become elongated to form filaments (filamentation).
This research resulted in several novelties, namely (1) mapping of folate biosynthesis genes in folate-producing and folate-consuming LAB isolates, which indicated that the presence of folate biosynthesis genes contributed to the ability of LAB to synthesize folate; (2) The folQ gene has potential as a marker gene for genotypic screening of folate-producing LAB isolates; (3) Two high folate-producing isolates were obtained, namely L. rhamnosus R23 from ASI and L. fermentum JK13 from kefir granules, with a much higher proportion of extracellular folate than intracellular folate so that it could be utilized for extracellular folate production; (4) 5-methyl-tetrahydrofolate is a form of folate produced by isolates R23 and JK13, where this form of folate has high bioavailability so that it can be an added value from the utilization of the two isolates for extracellular folate production; (5) Formation of bacterial filaments may be one mechanism of resistance of folate-producing LAB in response to exposure to methotrexate that has not been reported before, although this mechanism may work synergistically with other mechanisms. | id |
