Folate Production Research Articles

Comparative Genomics and In Vitro Experiments Provide Insight into the Adaptation and Probiotic Properties of Shouchella clausii

Shouchella clausii (S. clausii) has been marketed as an important commercial probiotic, displaying significant therapeutic effects on antibiotic-associated diarrhea and providing benefits to humans. This study aimed to explore the distribution, adaptation, and probiotic properties of S. clausii. Based on 16S rRNA gene analysis, 43 strains of S. clausii were isolated from 317 soil samples in China. Based on the genomic index of Average Nucleotide Identity (ANI) results, 41 strains were confirmed as S. clausii, while two strains, FJAT-45399 and FJAT-45335, were identified as potential novel species distinct from S. clausii. Combined phenotypic and genomic predictions indicated that S. clausii could survive under harsh conditions. Comparative genomics revealed that these isolates possess antibiotic resistance genes, as well as capabilities for bacteriocin and folate production, while lacking toxins and hemolytic activity. Hemolysis tests indicated that strain FJAT-41761 exhibited non-pathogenic γ-hemolytic activity, while also demonstrating resistance to multiple antibiotics, consistent with probiotic characteristics. These findings suggest that strain FJAT-41761 is safe and holds potential as a future probiotic.

Co-cultivation of selected Lactiplantibacillus plantarum strains with starter cultures under osmotic stress increases folate content and enhances product quality in fermented dairy

This study aimed to identify lactic acid bacteria (LAB) strains with high folate production potential to enhance the folate content in fermented dairy products. From 284 LAB strains sourced from fermented vegetables and dairy, six were selected for their outstanding folate production. Among these, Lactiplantibacillus plantarum TXZ2-26 and L. plantarum FJ2-3 demonstrated strong synergistic interactions with the starter culture, resulting in an increased total LAB cell count after co-cultivation. The effects of five different stress conditions on the growth, folate production, and expression of key folate-synthesis genes (folE, folP, folQ, folB, folK) in the two strains were further examined, revealing that the two strains demonstrated increased folate production under 3% NaCl stress. Finally, both unstressed and NaCl-stressed strains were used in the fermentation of goat and cow milk alongside the starter culture. The results revealed that the addition of L. plantarum TXZ2-26 and FJ2-3 significantly boosted the folate content of the fermented dairy products and improved their physicochemical properties, with the stressed strains exhibiting a more pronounced effect. These findings underscore the potential of L. plantarum TXZ2-26 and FJ2-3 as effective probiotics for producing folate-enriched fermented foods.

Screening of lactic acid bacteria for highly producing extra and intracellular folate and their potential use in production of bio-enriched yogurt

In this study, firstly, Microbial Assay (MA) was performed to evaluate the levels of intra and extracellular folate (Quatro folate) produced by 43 lactic acid bacteria strains isolated from traditional Iranian dairy products. The results obtained from strain screening showed that two strains of Lactobacillus delbrueckii subsp. bulgaricus, two strains of Streptococcus thermophilus, two strains of Lactobacillus delbrueckii subsp. lactis and one strain of Lactobacillus helveticus are highly folate producing. However, two strains of Lactobacillus delbrueckii subsp. bulgaricus produced higher amounts of folate (133.23 μg/mL and 102 μg/mL, respectively). In the second phase, eight yogurt samples were produced by using the two highest folate producing strains and compared with the control sample. The folate production in yogurt samples showed that Y8 sample has the highest amount of folate (117.93, 38.81 μg/mL determined by microbial and HPLC assay methods, respectively). It was found that the microbial assay method has the ability to measure different forms of folate derivatives compared to the HPLC method. The results exhibited that the isolates examined in this study showed the ability to produce extracellular folate at higher levels and can be used as bio-enrichment cultures to produce functional foods.

The use of propionic and lactic acid bacteria to produce cobalamin and folate in injera, an Ethiopian cereal-based fermented food

Like in many developing countries, the traditional Ethiopian diet relies mainly on starchy staple foods and often lacks sufficient animal-sourced foods which are crucial for cobalamin intake. Furthermore, the concentration of folate in traditionally prepared injera, an Ethiopian cereal-based fermented staple food, is highly variable and injera contains biologically inactive corrinoids. This study aimed to improve the cobalamin and folate content of injera by using cobalamin-producing Propionibacterium freudenreichii and folate-producing Lactiplantibacillus plantarum strains, both individually and combined. Since injera is fermented using backslopping, we also assessed the ability of these strains to produce cobalamin and folate consistently across successive fermentation batches. Changes in the microbial ecosystem were monitored using real-time PCR. The theoretical contribution of the injera prepared using the selected strains to the cobalamin and folate intake of children and women of reproductive age was also calculated. Results showed that using the selected bacterial strains individually increased cobalamin (up to 19.2 μg/100 g of dry matter) and folate (up to 180.2 μg/100 g of dry matter) levels in the injera dough over several backslopping fermentation batches. Regular consumption of the injera with enhanced vitamin content produced using each strain alone would be capable of fulfilling the entire recommended nutrient intake for cobalamin and up to 29 % of the recommended intake for folate for children and women of reproductive age. However, when the strains were used together, the production of both vitamins was reduced. The presence of certain common endogenous bacterial species and genera exhibited significant variability, highlighting the complex response of the native microbiota to the different inoculation strategies employed. Future experiments should consider selecting a microbial consortium comprising non-competing microorganisms to ensure the simultaneous production of cobalamin and folate in fermented foods.

Power-to-Vitamins: producing folate (vitamin B9) from renewable electric power and CO2 with a microbial protein system

We recently proposed a two-stage Power-to-Protein technology to produce microbial protein from renewable electric power and CO2. Two stages were operated in series: Clostridium ljungdahlii in Stage A to reduce CO2 with H2 into acetate, and Saccharomyces cerevisiae in Stage B to utilize O2 and produce microbial protein from acetate. Renewable energy can be used to power water electrolysis to produce H2 and O2. A drawback of Stage A was the need for continuous vitamin supplementation. In this study, by using the more robust thermophilic acetogen Thermoanaerobacter kivui instead of C. ljungdahlii, vitamin supplementation was no longer needed. Additionally, S. cerevisiae produced folate when grown with acetate as a sole carbon source, achieving a total folate concentration of 6.7 mg per 100 g biomass with an average biomass concentration of 3 g l-1. The developed Power-to-Vitamin system enables folate production from renewable power and CO2 with zero or negative net-carbon emissions.

Integrating fruit by-products and whey for the design of folate-bioenriched innovative fermented beverages safe for human consumption

Global concerns over folate deficiency, the risks of excessive synthetic folic acid consumption, and food loss implications for environmental sustainability and food security drive needs of innovative approaches that align food by-product valorisation with folate bio-enrichment. This study explored the use of three fruit by-products extracts (grape, passion fruit, and pitaya) and whey to develop a folate bio-enriched fermented whey-based beverage. Three strains (Lacticaseibacillus rhamnosus LGG, Bifidobacterium infantis BB-02, and Streptococcus thermophilus TH-4) were tested for folate production in different fermentation conditions in modified MRS medium and in a whey-based matrix prepared with water extracts of these fruit by-products. B. infantis BB-02 and S. thermophilus TH-4, alone and in co-culture, were the best folate producers. The selection of cultivation conditions, including the presence of different substrates and pH, with grape by-product water extract demonstrating the most substantial effect on folate production among the tested extracts, was crucial for successfully producing a biofortified fermented whey-based beverage (FWBB). The resulting FWBB provided 40.7 μg of folate per 100 mL after 24 h of fermentation at 37 °C, effectively leveraging food by-products. Moreover, the beverage showed no cytotoxicity in mouse fibroblast cells tests. This study highlights the potential for valorising fruit by-products and whey for the design of novel bioenriched foods, promoting health benefits and contributing to reduced environmental impact from improper disposal.

Neuroprotective Effects Exerted by a Combination of Selected Lactic Acid Bacteria in a Mouse Parkinsonism Model under Levodopa-Benserazide Treatment.

Alterations of the microbiota-gut-brain axis has been associated with intestinal and neuronal inflammation in Parkinson's disease (PD). The aim of this work was to study some mechanisms associated with the neuroprotective effect of a combination (MIX) of lactic acid bacteria (LAB) composed by Lactiplantibacillus plantarum CRL2130 (riboflavin overproducing strain), Streptococcus thermophilus CRL808 (folate producer strain), and CRL807 (immunomodulatory strain) in cell cultures and in a chronic model of parkinsonism induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in aged mice, and under levodopa-benserazide treatment. In vitro, N2a differentiated neurons were exposed to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and treated with intracellular bacterial extracts or with conditioned media from BV-2 cells exposed to the bacterial extracts. In vivo, motor skills, tyrosine hydrolase (TH) in brain and cytokine concentrations in serum and in brain were evaluated. The study of the faecal microbiota and the histology of the small intestine was also performed. The results showed that the neuroprotective effect associated with LAB MIX administration did not interfere with levodopa-benserazide treatment. This effect could be associated with the antioxidant and immunomodulatory potential of the LAB selected in the MIX, and was associated with the significant improvement in the motor tests and a higher number of TH + cells in the brain. In addition, LAB MIX administration was associated with modulation of the immune response. LAB administration decreased intestinal damage with an increase in the villus length /crypt depth ratio. Finally, the administration of the LAB MIX in combination with levodopa-benserazide treatment was able to partially revert the intestinal dysbiosis observed in the model, showing greater similarity to the profiles of healthy controls, and highlighting the increase in the Lactobacillaceae family. Different mechanisms of action would be related to the protective effect of the selected LAB combination which has the potential to be evaluated as an adjuvant for conventional PD therapies.

Effect of folA gene in human breast milk-derived Limosilactobacillus reuteri on its folate biosynthesis.

Folate supplementation is crucial for the human body, and the chemically synthesized folic acid might have undesirable side effects. The use of molecular breeding methods to modify the genes related to the biosynthesis of folate by probiotics to increase folate production is currently a focus of research. In this study, the folate-producing strain of Limosilactobacillus reuteri B1-28 was isolated from human breast milk, and the difference between B1-28 and folA gene deletion strain ΔFolA was investigated by phenotyping, in vitro probiotic evaluation, metabolism and transcriptome analysis. The results showed that the folate producted by the ΔFolA was 2-3 folds that of the B1-28. Scanning electron microscope showed that ΔFolA had rougher surface, and the acid-producing capacity (p = 0.0008) and adhesion properties (p = 0.0096) were significantly enhanced than B1-28. Transcriptomic analysis revealed that differentially expressed genes were mainly involved in three pathways, among which the biosynthesis of ribosome and aminoacyl-tRNA occurred in the key metabolic pathways. Metabolomics analysis showed that folA affected 5 metabolic pathways, involving 89 different metabolites. In conclusion, the editing of a key gene of folA in folate biosynthesis pathway provides a feasible pathway to improve folate biosynthesis in breast milk-derived probiotics.

Potential probiotic characteristics and genomic analysis of a new folate-producing lactic acid bacteria Lactiplantibacillus plantarum ZFM55.

Folate is crucial for maintaining health, but humans are unable to synthesize folate and need to obtain it from food. Lactiplantibacillus plantarum can produce the necessary vitamin B for the human body, including folate. Whole genome sequencing technology can clarify the physiological characteristics of folate production in Lactiplantibacillus plantarum. In order to explore new Lactiplantibacillus plantarum that produce folate, the folate production and probiotic characteristics of Lactiplantibacillus plantarum ZFM55 isolated from infant feces were investigated, and whole genome sequencing was performed. The folate synthesis ability of Lactiplantibacillus plantarum ZFM55 were measured, and its total folate production was 299.72 ± 28.81 ng mL-1. Subsequently, its probiotic properties were explored. The antibacterial test showed that its inhibition zone diameter against Staphylococcus aureus and Salmonella typhimurium was 15.5 ± 0.82 mm and 13.88 ± 0.98 mm, respectively. The tolerance test results indicated that it maintained good activity in simulated gastrointestinal tract and bile salt environments. In vitro intestinal simulation experiments had confirmed that Lactiplantibacillus plantarum ZFM55 can increase the abundance of beneficial bacteria such as Bifidobacteria in the intestine and inhibit the growth of harmful bacteria such as Escherichia_Shigella. Genomic sequencing indicated that the genetic material of Lactiplantibacillus plantarum ZFM55 contains one chromosome and three plasmids, and it has 20 genes related to folate synthesis, which explains its ability to produce folate. This study reports a new potential probiotic that produces folate, and provides ideas for exploring probiotics with specific probiotic characteristics. © 2024 Society of Chemical Industry.

Effect of fruit by‐products and orange pectin on folate (vitamin B9) production by selected starter and probiotic strains

SummaryThis study selected the best folate‐producing strains in folate‐free medium among five commercial starter and probiotic strains and evaluated the utilisation of fruit by‐products (pitaya, passion fruit and grape), modified pectin from orange albedo (MP) and other carbohydrate sources for growth and folate production by the selected strains. The three selected strains [Lacticaseibacillus (L.) rhamnosus GG, Bifidobacterium (B.) infantis BB‐02, and Streptococcus (S.) thermophilus TH‐4] grew equally well in m‐MRS broth containing these products or carbohydrates, reaching populations above 7 log CFU mL−1 in 24 h. Production of folate was strain‐dependent. L. rhamnosus GG showed the highest folate production when supplemented with fruit by‐products and MP, achieving the utmost folate concentration after 24 h in the presence of grape by‐product (101.2 ± 11.2 ng mL−1). The MP has also influenced positively the production of folate by this strain. The study evidenced that fruit by‐products can be explored as substrates for bio‐enriched food product development using selected folate‐producing strains.

Functional Properties and Antioxidant Potentials of Kunun-zaki (A Non-Alcoholic Millet Beverage) Produced using Pediococcus pentosaceus and Pichia kudriavzevii

ABSTRACT Kunun-zaki is an African fermented millet beverage, rich in lactic acid bacteria (LAB) and yeasts. The indigenous LAB and yeasts were evaluated for the in vitro production of exopolysaccharide, folate, and riboflavin. Selected strains were employed as starters in the production of kunun-zaki and the antioxidant potentials were determined. Pediococcus sp. MK13 showed a high profile of exopolysaccharide, folate, and riboflavin of 797 mg/L, 21.42 µg/mg, and 0.023 mg/g, respectively, while Pichia sp. UK7 produced 352 mg/L exopolysaccharide, 45.9 µg/mg folate, and 0.018 mg/g riboflavin. Kunun-zaki from unmalted millet fermented with a consortium of Pediococcus pentosaceus MK13 and Pichia kudriavzevii UK7 (UPPK) had the highest DPPH scavenging activity of 90%. Kunun-zaki with unmalted millet containing a single culture of Pichia kudriavzevii UK7 had the highest phenolic (928.93 GAE µg/mg) and flavonoid (810.17 GAE µg/mg) contents. The UPPK had the highest overall acceptability (7.36). In conclusion, all the starter-fermented kunun-zaki possessed health relevant functional benefits.

Complete genome sequence of the probiotic Bifidobacterium adolescentis strain iVS-1

ABSTRACT Bifidobacterium adolescentis iVS-1 is a human-isolated strain known to possess several probiotic properties. Here, its genome was completely sequenced to examine genes associated with lactose metabolism and other potentially beneficial traits, such as the production of folate and gamma-aminobutyric acid (GABA).

Cereal-based fermented foods as a source of folate and cobalamin: The role of endogenous microbiota

Folate (vitamin B9) and cobalamin (vitamin B12) deficiencies potentially affect millions of people worldwide, leading to different pathologies. In Ethiopia, the diet is characterized by high consumption of fermented cereal-based foods such as injera, a good source of folate but not of cobalamin, which is only found in foods of animal origin that are rarely consumed. Some of the bacteria responsible for the fermentation of cereals can synthesize cobalamin, but whether or not fermented cereal food products contain cobalamin remains underexplored. The objective of this study was to assess the folate and cobalamin content of injera collected from various households in Ethiopia at different stages of production. Global (16S rRNA gene sequencing) and specific (real-time PCR quantification of bacteria known for folate or cobalamin production) bacterial composition of these samples was assessed. UPLC-PDA was used to identify the cobalamin to see whether the active or inactive form was present. Surprisingly, teff flour contained 0.8 μg/100 g of cobalamin, most probably due to microbial contamination from the environment and the harvesting process. While fermentation increased the folate and cobalamin content in some households, their levels decreased in others. Conversely, cooking consistently reduced the level of the vitamins. Fresh injera contained, on average, 21.2 μg/100 g of folate and 2.1 μg/100 g of cobalamin, which is high, but with marked variation depending on the sample. However, the form of cobalamin was a corrinoid that is biologically inactive in humans. Injera fermentation was dominated by lactic acid bacteria, with significant correlations observed between certain bacterial species and folate and cobalamin levels. For example, a high proportion of Fructilactobacillus sanfranciscensis, a known folate consumer, was negatively correlated with the folate content of injera. On the contrary, Lactobacillus coryniformis, known for its cobalamin synthesis ability was present in high proportion in the cobalamin-rich samples. These findings highlight the complex interrelationship between microorganisms and suggest the involvement of specific bacteria in the production of folate and cobalamin during injera fermentation. Controlled fermentation using vitamin-producing bacteria is thus a promising tool to promote folate and cobalamin production in fermented food.

The role of epigenetic modifications in the formation of heterogeneous phenotypes in diabetes mellitus (a literature review)

This review article provides a summary and update on the role of epigenetic mechanisms in predisposition and progression of diabetes, analyzes the data concerning the cause-and-effect relationship between epigenetic changes and the emergence of distinct metabolic phenotypes. Extensive genetic research has enabled the isolation of a group of genes associated with a high risk of developing diabetes. However, numerous data point to the key role of so-called epigenetic modifications in the interaction between genes and the environment, which arise during ontogenesis based on the existing genotype under the influence of external factors. These modifications do not affect the primary DNA sequence, but influence gene expression through chemical modification and alteration of the secondary structure of DNA molecules and chromatin. Epigenetic mechanisms can program pathological phenotypes in subsequent generations. The main molecular mechanisms of epigenetic modifications are DNA methylation, histone and miRNA modification. Changes in the expression of genes that ensure the synthesis of key enzymes and regulatory molecules lead to disruption in the main signaling metabolic pathways. Deregulation of genes responsible for inflammatory, atherosclerotic and other pathological processes, in particular, leads to endothelial dysfunction and development of diabetic complications, such as cardiovascular diseases, diabetic nephropathy, retinopathy, neuropathy. Hyperglycemia, oxidative stress, inflammatory factors are known as mediators in the pathogenesis of type 2 diabetes and its complications. Since epigenetic modifications are reversible, the methylation process can be influenced by exercise, dietary, lifestyle changes and pharmacological agents such as methyl group donors. For example, S-adenosylmethionine, through participation in methylation reactions, can modulate the folate cycle function and production of homocysteine, an endothelium-toxic substance. Thus, the study of molecular modifications in chromatin structure and the features of activation and inhibition of various signaling pathways is a pressing task, the resolution of which will enable a deeper understanding of the pathogenesis of diabetes and the development of approaches to correct metabolic disorders.

The major role of Listeria monocytogenes folic acid metabolism during infection is the generation of N-formylmethionine.

Folic acid is an essential vitamin for bacteria, plants, and animals. The lack of folic acid leads to various consequences such as a shortage of amino acids and nucleotides that are fundamental building blocks for life. Though antifolate drugs are widely used for antimicrobial treatments, the underlying mechanism of bacterial folate deficiency during infection is unclear. This study compares the requirements of different folic acid end-products during the infection of Listeria monocytogenes, a facultative intracellular pathogen of animals and humans. The results reveal the critical importance of N-formylmethionine, the amino acid used by bacteria to initiate protein synthesis. This work extends the current understanding of folic acid metabolism in pathogens and potentially provides new insights into antifolate drug development in the future.

Potential folate-producing strains and their applications in biofortification of fermented Moringa oleifera leaves powder

Folate is an essential vitamin for the human body and must be obtained exogenously. It is recognized that some microorganisms are capable of producing folates during growth, revealing possibilities for innovations of natural folate biofortified foods. The aim of this study was to identify potential folate-producing strains to enhance folate content in fermented Moringa oleifera leaves powder (MOLP). Although most of the tested strains exhibited folate production, there were significant differences among them in terms of both quantity and distribution, whether intracellular or extracellular. 10 strains with potential for folate production were chosen for enhancing the folate content of MOLP. The two most promising strains, Lactiplantibacillus sp. LP1 and Saccharomyces cerevisiae Y-J-1, demonstrated high folate levels up to 1940 and 2115 μg/100 g dry matter, respectively. These levels were 1.6 and 1.8-fold the initial value in MOLP. Additionally, the fermentation of MOLP not only enhanced its nutritional quality but also preserved its significant bioactivity. This study provides the initial understanding of how selected microorganisms can naturally enrich folates in fermented MOLP, presenting an alternative solution to address the widespread issue of folate deficiency while promoting human health.

A computational perspective towards the identification of promising lead molecules against 6-hydroxy-methyl dihydropterin pyrophosphokinase (HPPK) from Acinetobacter baumannii

Acinetobacter baumannii is an ESKAPE pathogen that causes endocarditis, pneumonia, blood infections, urinary tract infections, and several other illnesses. In addition, it is mainly responsible for nosocomial infection-related mortality. Gram-negative A. baumannii bacterium (AYE Strain) has high MDR and XDR levels. Due to its function in synthesizing purines and amino acids, folic acid is a significant molecule necessary for the growth of bacteria. The metabolic pathway of folate production is therefore a potential therapeutic target to inhibit bacterial growth. In the current study, the three-dimensional model of 6-Hydroxy-methyl dihydropterinpyrophosphokinase (HPPK) was predicted and subsequently processed through a virtual high throughput screening (vHTS) against compounds from Enamine HTSC library, that could bind to its active site. Three lead candidates (Z73322064, Z354558542, and Z906123504) and a control molecule (7,8 dihydro-7,7-dimethyl-6-hydroxymethlypterin; Accession Number: DB02278) were identified using several screening criteria namely estimated binding affinity, estimated inhibition constant, drug-like properties, ADME properties, mode of binding, and interaction patterns of the screened compounds. The physiological behavior of ligand binding on the HPPK enzyme was then studied using molecular dynamics simulations of apo and ligand bound complexes. This study proposed the following three molecules: Z73322064, Z354338542, and Z906123504 as promising lead candidates against the substrate-binding site of the HPPK enzyme from A. baumannii using global, essential dynamics studies along with MM/PBSA based binding free energy analysis. Communicated by Ramaswamy H. Sarma

One-carbon metabolism and microbial pathogenicity.

One-carbon metabolism (OCM) pathways are responsible for several functions, producing a number of one-carbon unit intermediates (formyl, methylene, methenyl, methyl) that are required for the synthesis of various amino acids and other biomolecules such as purines, thymidylate, redox regulators, and, in most microbes, folate. As humans must acquire folate from the diet, folate production is a target for antimicrobials such as sulfonamides. OCM impacts the regulation of microbial virulence such that in a number of instances, limiting the availability of para-aminobenzoic acid (pABA), an essential OCM precursor, causes a reduction in pathogenicity. Porphyromonas gingivalis, however, displays increased pathogenicity in response to lower pABA levels, and exogenous pABA exerts a calming influence on heterotypic communities of P. gingivalis with pABA-producing partner species. Differential responses to pABA may reflect both the physiology of the organisms and their host microenvironment. OCM plays an integral role in regulating the global rate of protein translation, where the alarmones ZMP and ZTP sense insufficient stores of intracellular folate and coordinate adaptive responses to compensate and restore folate to sufficient levels. The emerging interconnections between OCM, protein synthesis, and context-dependent pathogenicity provide novel insights into the dynamic host-microbe interface.

Screening, isolation and biotechnological potentials of foodborne Lactobacillus fermentum strains MT903311 and MT903312

Vitamins are an essential food source with excellent roles in the cellular metabolism and other essential nutrients required in food intake but cannot be synthesized by humans. There have been reports of some lactic acid bacteria (LAB) abilities with probiotic activities to produce food-grade vitamins. Our study aimed to investigate lactic acid bacteria (LAB) possessing antimicrobial activities and extracellular production of folate from different Nigerian fermented foods. LAB was assayed for their antimicrobial activities against clinical isolates of Escherichia coli and Salmonella typhimurium and their extracellular production of essential vitamins. Among the 43 isolates of LAB, two strains of Lactobacillus fermentum showed the highest inhibitions against the test bacteria and demonstrated the highest concentrations of extracellular vitamins production. The range of vitamins produced at 24 h was between 12.23 and 801.79 μg/ml, while the highest vitamin production of 801.79 and 310.55 μg/ml was observed for folate and vitamin B12 respectively, the lowest production was for B1+B2. Consistent vitamin production was typical with only L. fermentum MT903311 and L. fermentum MT903312, so were their antimicrobial activities. The L. fermentum strains isolated in this study could be exploited and applied in food products to substitute synthetic vitamin enrichment and fortification.

Folate production and its distribution during growth of lactic acid bacteria isolated from fermented food and breast milk

Production of folate by lactic acid bacteria (LAB) isolated from tempe, fermented mustard, kefir, tapai, and breast milk was analyzed after 24 h of growth in folate-free media. The LAB isolates with the highest folate production were further investigated for folate distribution in intracellular and extracellular cells in a folate-free medium for 48 h. The two main forms of folates, i.e., tetrahydrofolate (THF) and 5-methyl-tetrahydrofolate (5-MTHF), as well as folic acid, were detected and quantified using the HPLC-DAD method with each folate standard as calibration curves. Two folate peaks were found in the spent medium of folate-producing isolates; one of them was identified as 5-MTHF, while the other peak was identified as other folates. Lacticaseibacillus rhamnosus R23 from breast milk and Limosilactobacillus fermentum JK13 from kefir granules were the selected folate-producing isolates, with the production of total folates at 98.37 mg/ml and 85.67 mg/ml, respectively, after 24 h of incubation. The two isolates showed nearly similar patterns of intra- and extracellular folate distribution; that is, extracellular folate excreted into the media has a much higher proportion than intracellular folate. Therefore, the two LAB isolates can be utilized for extracellular folate production.