Folate Production Research Articles

Ability of lactobacilli isolated from traditional cereal-based fermented food to produce folate in culture media under different growth conditions

Folates are essential vitamins (B9). Lactic acid bacteria (LAB) can be used to increase the folate levels in foods during fermentation. Here, 151 LAB strains isolated from fermented food were screened for folate production. Folates were extracted by heat treatment coupled with enzymatic extraction. Total, extra- and intracellular folate were quantified by microbiological assay. Most Lactobacillus plantarum and Lactobacillus fermentum strains synthesized folate, Pediococcus pentosaceus strains consumed folate, and Pediococcus acidilactici strains either consumed or produced. Seven strains were further examined for their ability to synthesize or consume folate during growth in rich folate medium (MRS) and folate-free medium (FACM). For most strains, the differences observed in MRS were attenuated in FACM. L. fermentum 8.2 and L. plantarum 6.2 produced the highest levels of folates in MRS (97 and 93 ng·mL−1) and FACM (29 and 44 ng·mL−1). In most cases, production reached a maximum from 9 or 11 h to 24 h. Incubations in FACM with mixed LAB cultures showed that folate production is not additive. We demonstrated how biosynthesis of folate by LAB depends on species, strains, and is highly influenced by time and medium composition. Characterized LAB strains can be further explored for novel folate bio-enriched functional food.

“Metformin-resistant” folic acid producing probiotics or folic acid against metformin’s adverse effects like diarrhea

Metformin, first line medication in the treatment of type2 diabetes by millions of patients worldwide, causes gastrointestinal adverse effects (i.e. diarrhea) in approximately 30% of patients, frequently leading to discontinuation. Interestingly, metformin was reported to increase life span in a microscopic worm, Caenorhabditis elegans, by decreasing folate and methionine production of bacteria that this worm uses as a food source. Metformin can be expected to have a similar effect on some microorganisms of human gut microbiota. This can disturb the balance of gut microbiota and cause gastrointestinal adverse effects by altering folate production of some types of bacteria and suppress their growth. Metformin resistant probiotics can be discovered or generated by artificial evolution/selection, and used to prevent these adverse effects. These patients can also be managed with folate supplementation.

Folate production and fol genes expression by the dairy starter culture Streptococcus thermophilus CRL803 in free and controlled pH batch fermentations

Folate production by Streptococcus thermophilus CRL 803 was examined during batch fermentations without pH control and at constant pH (6.0 and 5.0) at different incubation temperatures. Folate production was higher at 42 °C under both constant and non-constant pH fermentation conditions, the increase being more pronounced at constant pH 6.0. Folate production by S. thermophilus CRL 803 under different fermentation conditions was cell-growth-associated. These observations were supported by analysis at the transcriptional levels of fol genes in controlled pH of 6.0 and uncontrolled pH batch culture. At pH 6.0, the highest relative expression levels of folE, folQ, folK and folP genes (involved in de novo synthesis) were observed after 6 h of incubation.This is the first study on the expression of fol mRNA and their relation with folate synthesis by S. thermophilus, which is influenced by culture conditions.

Deleterious Effects of Chronic Folate Deficiency in the Ts65Dn Mouse Model of Down Syndrome.

Folate is an important B vitamin naturally found in the human diet and plays a critical role in methylation of nucleic acids. Indeed, abnormalities in this major epigenetic mechanism play a pivotal role in the pathogenesis of cognitive deficit and intellectual disability in humans. The most common cause of cognitive dysfunction in children is Down syndrome (DS). Since folate deficiency is very common among the pediatric population, we questioned whether chronic folate deficiency (CFD) exacerbates cognitive dysfunction in a mouse model of DS. To test this, adult Ts65Dn mice and their disomic littermates were chronically fed a diet free of folic acid while preventing endogenous production of folate in the digestive tract for a period of 8 weeks. Our results show that the Ts65Dn mouse model of DS was significantly more vulnerable to CFD in terms of plasma homocysteine and N5-methyltetrahydrofolate (5-MTHF) levels. Importantly, these changes were linked to degenerative alterations in hippocampal dendritic morphology and impaired nest building behavior in Ts65Dn mice. Based on our results, a rigorous examination of folate intake and its metabolism in individuals with DS is warranted.

Genomic diversity and immunomodulatory activity of Lactobacillus plantarum isolated from dairy products.

In this study, we aimed to investigate some functional characteristics and the immunomodulatory properties of three strains of Lactobacillus plantarum of dairy origin which, in a previous screening, showed to be candidate probiotics. Genome sequencing and comparative genomics, which confirmed the presence of genes involved in folate and riboflavin production and in the immune response of dendritic cells (DCs), prompted us to investigate the ability of the three strains to accumulate the two vitamins and their immunomodulation properties. The ability of the three strains to release antioxidant components in milk was also investigated. Small amounts of folate and riboflavin were produced by the three strains, while they showed a good antioxidant capacity in milk with FRAP method. The immune response experiments well correlated with the presence of candidate genes influencing in DCs cytokine response to L. plantarum. Specifically, the amounts of secreted cytokins by DCs after stimulation with cells of Lp790, Lp813 and Lp998 resulted pro-inflammatory whereas stimulation with culture supernatants (postbiotics) inhibited the release of interleukin (IL)-12p70 and increased the release of the anti-inflammatory IL-10 cytokine. This study adds further evidence on the importance of L. plantarum in human health. Understanding how probiotics (or postbiotics) work in preclinical models can allow a rational choice of the different strains for clinical and/or commercial use.

Recombinant overexpression of dihydroneopterin aldolase catalyst potentially regulates folate-biofortification.

We aim to investigate the prospects of increased production of folate through the overexpression of heterologous dihydroneopterin aldolase catalyst. The gene encoding aldolase catalyst was cloned into an expression vector and the induced recombinant protein was purified through metal-affinity chromatography which appeared at 14 kDa position on polyacrylamide-gel. Remarkably, a periodic increase in the extracellular and intracellular folic acid concentration was observed at 4 h growth of induced recombinant DHNA samples than control in a pH-dependent manner. Maximum folate concentration was observed with at least twofold increase in induced recombinant samples at pH8.0 compared to the significant decline at 6 h growth. Consistently, heterologous overexpression of bacterial aldolase through Agrobacterium-mediated genetic transformation of tobacco led to more than 2.5-fold increase in the folate concentration in the transgenic leaves than control tissues. These data are veritable inspecting metabolic flux in both bacterial and plant systems, thus providing directions for future research on folate agri-fortification.

Measurements of Intra- and Extra-Cellular 5-Methyltetrahydrofolate Indicate that Bifidobacterium Adolescentis DSM 20083T and Bifidobacterium Pseudocatenulatum DSM 20438T Do Not Actively Excrete 5-Methyltetrahydrofolate In vitro.

Certain intestinal bifidobacteria have the ability to synthesize folates. In vitro experiments revealed a high production, cellular accumulation, and release of reduced folate vitamers like 5-methyltetrahydrofolate and tetrahydrofolate in folate-free medium (FFM). However, it is still unclear to which extent synthesized folates are polyglutamylated and probably not available for transport, and if they are actively released by excretion. To address these questions, we characterized intra- and extra-cellular pteroylmonoglutamates and polyglutamylated 5-methyltetrahydrofolate (5-CH3-H4PteGlu2−4) in Bifidobacterium adolescentis DSM 20083T and Bifidobacterium pseudocatenulatum DSM 20438T in vitro. Folates were measured by means of stable isotope dilution assays (SIDA) coupled with LC-MS/MS analysis using [2H4]-5-methyltetrahydrofolic acid, [2H4]-tetrahydrofolic acid, and [2H4]-5-formyltetrahydrofolic acid as internal standards. Cell viability was examined by fluorescence microscopy. Quantitation of folate production by B. adolescentis during the stationary phase revealed a linear increase of dead cells paralleled by increasing concentration of 5-formyltetrahydrofolate and 5-methyltetrahydrofolate (100% 5-CH3-H4PteGlu4) in FFM, whereas the intracellular concentrations of these vitamers remained constant. After 24 h, B. adolescentis (125 mg cells, wet weight) produced a total amount of 0.846 nmol 5-CH3-H4folate: 0.385 ± 0.059 nmol (46 ± 7%) and 0.461 ± 0.095 nmol (54 ± 11%) measured in the intracellular (viable cells; 52 ± 3% measured by fluorescence microscopy) and extracellular (lysed cells; 48 ± 3%) fraction, respectively. For B. pseudocatenulatum (124 mg cells, wet weight), 1.135 nmol 5-CH3-H4folate was produced after 24 h, and a similar proportionality between intra- and extra-cellular folate concentrations and viable/lysed cells was observed. These results indicate that the strains tested produce and accumulate 5-CH3-H4PteGlu4 for cellular metabolism, and that extracellular concentrations of the vitamer arise from cell lysis.

PCR screening of an African fermented pearl-millet porridge metagenome to investigate the nutritional potential of its microbiota

Cereals are staple foods in most African countries, and many African cereal-based foods are spontaneously fermented. The nutritional quality of cereal products can be enhanced through fermentation, and traditional cereal-based fermented foods (CBFFs) are possible sources of lactic acid bacteria (LAB) with useful nutritional properties. The nutritional properties of LAB vary depending on the species and even on the strain, and the microbial composition of traditional CBFFs varies from one traditional production unit (TPU) to another. The nutritional quality of traditional CBFFs may thus vary depending on their microbial composition.As the isolation of potentially useful LAB from traditional CBFFs can be very time consuming, the aim of this study was to use PCR to assess the nutritional potential of LAB directly on the metagenomes of pearl-millet based fermented porridges (ben-saalga) from Burkina Faso. Genes encoding enzymes involved in different nutritional activities were screened in 50 metagenomes extracted from samples collected in 10 TPUs in Ouagadougou.The variability of the genetic potential was recorded. Certain genes were never detected in the metagenomes (genes involved in carotenoid synthesis) while others were frequently detected (genes involved in folate and riboflavin production, starch hydrolysis, polyphenol degradation). Highly variable microbial composition - assessed by real-time PCR - was observed among samples collected in different TPUs, but also among samples from the same TPU. The high frequency of the presence of genes did not necessarily correlate with in situ measurements of the expected products. Indeed, no significant correlation was found between the microbial variability and the variability of the genetic potential. In spite of the high rate of detection (80%) of both genes folP and folK, encoding enzymes involved in folate synthesis, the folate content in ben-saalga was rather low (median: 0.5μg/100g fresh weight basis). This work highlighted the limit of evaluating the nutritional potential of the microbiota of traditional fermented foods by the only screening of genes in metagenomes, and suggests that such a screening should be completed by a functional analysis.

Probiotic-loaded microcapsule system for human in situ folate production: Encapsulation and system validation

This study focused on the use of a new system, an alginate|Ɛ-poly-l-lysine|alginate|chitosan microcapsule (APACM), able to immobilize a folate-producing probiotic, Lactococcus lactis ssp. cremoris (LLC), which provides a new approach to the utilization of capsules and probiotics for in situ production of vitamins. LLC is able to produce 95.25±26μg·L−1 of folate, during 10h, and was encapsulated in the APACM. APACM proved its capacity to protect LLC against the harsh conditions of a simulated digestion maintaining a viable concentration of 6logCFU·mL−1of LLC. A nutrients exchange capacity test, was performed using Lactobacillus plantarum UM7, a high lactic acid producer was used here to avoid false negative results. The production and release of 2g·L−1 of lactic acid was achieved through encapsulation of L. plantarum, after 20h. The adhesion of APACM to epithelial cells was also quantified, yielding 38% and 33% of capsules adhered to HT-29 cells and Caco-2 cells, respectively.

Potential probiotic Pichia kudriavzevii strains and their ability to enhance folate content of traditional cereal-based African fermented food

With the aim of selecting starter cultures with interesting probiotic potential and with the ability to produce folate in a food matrix, yeast strains isolated from fermented cereal-based African foods were investigated. A total of 93 yeast strains were screened for their tolerance to pH 2 and 0.3% of bile salts. Pichia kudriavzevii isolates gave the best results. Selected P. kudriavzevii strains were tested for survival to the simulated human digestion and for adhesion to Caco-2 cells. Moreover, presence of folate biosynthesis genes was verified and production of extra and intra-cellular folate determined during growth in culture medium. 31% of yeast strains could tolerate pH 2, while 99% bile salts. Survival rate after simulated digestion ranged between 11 and 45%, while adhesion rate between 12 and 40%. Folate production was mainly intracellular, maximum after 24 h of growth. To be closer to traditional cereal-based fermentations, a P. kudriavzevii strain with good probiotic potential was co-inoculated with Lactobacillus fermentum strains in a pearl millet gruel. This resulted in in situ folate production that peaked after 4 h. The use of strains with both probiotic and nutritional enrichment properties may have a greater impact for the consumers.

Supplementation with fruit and okara soybean by-products and amaranth flour increases the folate production by starter and probiotic cultures

The ability of two starter cultures (Streptococcus (S.) thermophilus ST-M6 and St. thermophilus TA-40) and eleven probiotic cultures (St. thermophilus TH-4, Lactobacillus (Lb.) acidophilus LA-5, Lb. fermentum PCC, Lb. reuteri RC-14, Lb. paracasei subsp. paracasei, Lb. casei 431, Lb. paracasei subsp. paracasei F19, Lb. rhamnosus GR-1, and Lb. rhamnosus LGG, Bifidobacterium (B.) animalis subsp. lactis BB-12, B. longum subsp. longum BB-46, and B. longum subsp. infantis BB-02) to produce folate in a modified MRS broth (mMRS) supplemented with different fruit (passion fruit, acerola, orange, and mango) and okara soybean by-products and amaranth flour was investigated. Initially, the folate content of each vegetable substrate was determined: passion fruit by-product showed the lowest folate content (8±2ng/mL) and okara the highest (457±22ng/mL). When the orange by-product and amaranth flour were added to mMRS, all strains were able to increase folate production after 24h of fermentation. B. longum subsp infantis BB-02 produced the highest concentrations (1223±116ng/mL) in amaranth flour. Okara was the substrate that had the lowest impact on the folate production by all strains evaluated. Lb. acidophilus LA-5 (297±36ng/mL) and B. animalis subsp. lactis BB-12 (237±23ng/mL) were also able to produce folate after growth in mMRS containing acerola and orange by-products, respectively. The results of this study demonstrate that folate production is not only strain-dependent but also influenced by the addition of different substrates in the growth media.

Lactic acid fermentation as a tool for increasing the folate content of foods

ABSTRACTFolate is an essential micronutrient involved in numerous vital biological reactions. The dietary consumption of naturally occurring vitamin B9 is often inadequate in many countries, and supplementation or fortification programs (using synthetic folic acid) are implemented to alleviate folate deficiency. Other food-based alternatives are possible, such as the use of lactic acid bacteria (LAB) to synthesize folate during fermentation. Many studies have been conducted on this topic, and promising results were reported for some fermented dairy products. However, in other studies, folate consumption by LAB or rather low folate production were observed, resulting in fermented foods that may not significantly contribute to the recommended B9 intake. In addition, the optimum conditions for folate biosynthesis by LAB are still not clear. The aim of this review was thus to (i) clarify the ability of LAB to produce folate in food products, (ii) check if the production of folate by LAB in various fermented foods is sufficient to meet human vitamin B9 requirements and (iii) suggest ways to optimize folate production by LAB in fermented food products.

Folate bio-fortification of yoghurt and fermented milk: a review

Folate, an essential vitamin, plays an important role in human life for the synthesis of nucleotides, vitamins and some amino acid. This vitamin cannot be synthesized by humans and must be obtained exogenously to prevent folate deficiency, neural tube defects (NTDs) and other related diseases. Folic acid, the chemically synthesized form of folate, is used for fortification and supplementation, but it can cause adverse effects such as vitamin B12 deficiency in high intake. Therefore, novel methods to increase concentrations of naturally occurring folate in foods have grabbed the interest of researchers. The application of bacterial cultures in food fermentation is a novel strategy to increase “natural” folate levels. Dairy products, especially yoghurt, are an appropriate choice for bio-fortification of folate as they contain folate-binding protein which improves folate bioavailability. This study will review the folate production by probiotic bacteria in fermented milk based on various strains and culture conditions.

Effect of indigenous lactic acid bacteria isolated from goat milk and cheeses on folate and riboflavin content of fermented goat milk

The aim of the present study was to isolate riboflavin- and folate-producing lactic acid bacteria (LAB) from raw goat milk and cheeses, identify them and evaluate their capability to increase the content of these vitamins in fermented goat milk, envisaging potential application for development of novel bioenriched goat milk products. From 179 LAB isolates obtained, 151 (84%) were capable to produce at least one of these vitamins. The average production of total folate and riboflavin in vitamin-free media was 138 ng/ml and 364 ng/ml, respectively. Based on RAPD-PCR and 16S rDNA sequencing, 19 different genetic profiles were obtained and 7 species were identified, with predominance of Streptococcus thermophilus (7), Weissella paramensenteroides (6), and Lactococcus lactis (4). Seven isolates that produced folate and riboflavin above the average were tested for vitamins production in UHT goat milk. Five isolates were capable to increase four to six fold the original amount of folate in the milk in 24 h. Folate content in milk fermented with Lc. lactis FP368 for 24 h was 313 ng/ml that could provide 19% of the recommended daily intake of this vitamin. In addition, St. thermophilus FP268 increased the folate concentration in the milk almost four fold in only 6 h.

Technological properties of Lactic acid bacteria isolated from raw cereal material

Lactic Acid Bacteria (LAB) were isolated from cereals and seeds from Argentinean markets. Colonies representing different morphological appearances were isolated and differentiated based on phenotypic characteristics. Fifty strains were identified by 16S rRNA gene sequencing analysis obtaining LAB from genera Enterococcus, Lactobacillus and Pediococcus. Technological and nutritional characteristics (acidifying capacity, antimicrobial production, proteolytic activity, folate production) were analyzed. Lactobacillus pentosus ES124 and Lactobacillus plantarum ES137 presented high production of folate (61 and 57 ng/mL, respectively) as did Enterococcus mundtii ES63 which reached a total folate production of 70 ng/mL. Six LAB strains produced bacteriocin-like inhibitory substances (BLIS) with antilisterial activity. Eight strains were selected for use as sourdough starters and in situ applications based on their important technological properties.

Folates in Fruits and Vegetables: Contents, Processing, and Stability.

Folates play a key role in human one-carbon metabolism and are provided by food. It is well established that folates are beneficial in the prevention of neural tube defects and cardiovascular and neurodegenerative diseases. Fruits and vegetables, and especially green vegetables, are the main sources of folates. In parallel, fruits and vegetables, with high contents of folates, are mostly consumed after processing, such as, canning, freezing, or home-cooking, which involve folate losses during their preparation. Hence, it is important to know the percentage of folate losses during processing and, moreover, the mechanisms underlying those losses. The current knowledge on folate losses from fruit and vegetables are presented in this review. They depend on the nature of the respective fruit or vegetable and the respective treatment. For example, steaming involves almost no folate losses in contrast to boiling. Two main mechanisms are involved in folate losses: (i) leaching into the surrounding liquid and (ii) oxidation during heat treatment, the latter of which depending on the nature of the vitamer considered. In this respect, a vitamer stability decreases in the order starting from folic acid followed by 5-HCO-H4 folate, 5-CH3 -H4 folate, and, finally, H4 folate. Further studies are required, especially on the diffusion of the vitamers in real foods and on the determination of folate degradation products.

Milk fermented with selected strains of lactic acid bacteria is able to improve folate status of deficient rodents and also prevent folate deficiency

Folate deficiencies are common in many parts of the world. The use of folate producing food grade microorganisms has been proposed as a more natural alternative to fortification with the chemical folic acid. The aim of this study was to evaluate the effectiveness of a novel fermented milk elaborated with adequately selected folate producing lactic acid bacteria. This product was tested using both a rodent depletion–repletion model and a complete deficient model. The folate bio-enriched fermented milk was able to increase plasma folate concentrations and decrease homocysteine levels. This is the first report demonstrating that a naturally folate bio-enriched fermented milk, elaborated with folate-producing starter cultures, not only is effective to improve folate status but can also prevent folate deficiency. Consumers also would obviously benefit from this type of product because they could increase their folate intakes while consuming foods that form part of their normal diets.

Differences in folate production by bifidobacteria of different origins.

Bifidobacteria are known to produce folate, a vital nutrient for humans. Previous studies have suggested that the ability to produce folate is strain dependent, but further adequate evaluation is needed. In this study, a total of 44 strains, including 12 species and 7 subspecies, of bifidobacteria were investigated for the production of folate during cultivation in medium containing essential levels of folate for growth of the tested strains. An in vitro assay showed that all strains of human-residential bifidobacteria (HRB) were able to produce folate, whereas most strains of non-HRB were not, with the exception of the B. thermophilum and B. longum ssp. suis strains. The differences in the in vivo production of folate by HRB and non-HRB were confirmed using mono-associated mice. The fecal folate concentrations, blood levels of hemoglobin and mean corpuscular volumes were significantly higher in the mice colonized with a folate producer, B. longum subsp. longum, compared with mice colonized with a nonproducer, B. animalis subsp. lactis. Our results confirmed the differences in folate production between HRB and non-HRB strains and suggested the benefit of HRB to hosts from the perspective of potential folate delivery.

Vitamin paradox in obesity: Deficiency or excess?

Since synthetic vitamins were used to fortify food and as supplements in the late 1930s, vitamin intake has significantly increased. This has been accompanied by an increased prevalence of obesity, a condition associated with diabetes, hypertension, cardiovascular disease, asthma and cancer. Paradoxically, obesity is often associated with low levels of fasting serum vitamins, such as folate and vitamin D. Recent studies on folic acid fortification have revealed another paradoxical phenomenon: obesity exhibits low fasting serum but high erythrocyte folate concentrations, with high levels of serum folate oxidation products. High erythrocyte folate status is known to reflect long-term excess folic acid intake, while increased folate oxidation products suggest an increased folate degradation because obesity shows an increased activity of cytochrome P450 2E1, a monooxygenase enzyme that can use folic acid as a substrate. There is also evidence that obesity increases niacin degradation, manifested by increased activity/expression of niacin-degrading enzymes and high levels of niacin metabolites. Moreover, obesity most commonly occurs in those with a low excretory reserve capacity (e.g., due to low birth weight/preterm birth) and/or a low sweat gland activity (black race and physical inactivity). These lines of evidence raise the possibility that low fasting serum vitamin status in obesity may be a compensatory response to chronic excess vitamin intake, rather than vitamin deficiency, and that obesity could be one of the manifestations of chronic vitamin poisoning. In this article, we discuss vitamin paradox in obesity from the perspective of vitamin homeostasis.

Biosynthesis and cellular content of folate in bifidobacteria across host species with different diets

BackgroundBifidobacteria, one of the most common bacteria of the intestinal tract, help establish balance in the gut microbiota and confer health benefits to the host. One beneficial property is folate biosynthesis, which is dependent on species and strains. It is unclear whether the diversity in folate biosynthesis is due to the adaptation of the bifidobacteria to the host diet or whether it is related to the phylogeny of the animal host. To date, folate production has been studied in the bifidobacteria of omnivorous, and a few herbivorous, non-primate hosts and humans, but not in carnivores, non-human primates and insects. In our study we screened folate content and composition in bifidobacteria isolated from carnivores (dog and cheetah), Hominoidea omnivorous non-human primates (chimpanzee and orangutan) and nectarivorous insects (honey bee). ResultsBifidobacterium pseudolongum subsp. globosum, a species typically found in non-primates, was isolated from dog and cheetah, and Bifidobacterium adolescentis and Bifidobacterium dentium, species typically found in humans, were respectively obtained from orangutan and chimpanzee. Evidence of folate biosynthesis was found in bifidobacteria isolated from non-human primates, but not from the bifidobacteria of carnivores and honey-bee. On comparing species from different hosts, such as poultry and herbivorous/omnivorous non-primates, it would appear that folate production is characteristic of primate (human and non-human) bifidobacteria but not of non-primate.Isolates from orangutan and chimpanzee had a high total folate content, the mean values being 7792 μg/100 g dry matter (DM) for chimpanzee and 8368 μg/100 g DM for orangutan. The tetrahydrofolate (H4folate) and 5-methyl-tetrahydrofolate (5-CH3–H4folate) distribution varied in the bifidobacteria of the different animal species, but remained similar in the strains of the same species: B. dentium CHZ9 contained the least 5-CH3–H4folate (3749 μ/100 g DM), while B. adolescentis ORG10 contained the most (8210 μg/100 g DM). ConclusionOur data suggest a correlation between phylogenetic lineage and capacity of folate production by bifidobacteria, rather than with dietary type of the host.