hic grap Peer-reviewed article no upplem o s ent M s er ie s Folate: sources , production and bioavailability SHALINI S. ARYA*, K. PAVITRA Shalini S. Arya *Corresponding author Institute of Chemical Technology, Food Engineering and Technology Department NM Parikh Marg, Matunga, Mumbai, 400 019, India

KEYWORDS: Folate; production; sources; bioavailability.

ABSTRACT: “Folate” is a generic term for forms of Vitamin B9 and their derivatives. Initially detected in brewer’s yeast and isolated from spinach, folates play a vital role in body functions like nucleic acid synthesis and RBC formation. Natural folates are preferable over synthetic forms since they have lesser side effects and are body-own forms; and also the metabolism of synthetic folic acid is very individual specifi c. Some physiological conditions like sickle cell disease and renal dialysis might increase cellular folate requirements. Folate defi ciency must be treated considering its metabolic inter-relationships with vitamin B12 and choline. The present review focuses on natural and synthetic folate sources, microbial production and bioavailability. Monographic supplement series:

INTRODUCTION folates are pteroylmonoglutamates. Pteroylpolyglutamates with up to 11 glutamic acid residues exist naturally. Folates is an essential nutrition component (important B Naturally occurring folates can have a variety of production vitamin) in the human diet, involved in many metabolic methods as follows (5). pathways, mainly in carbon transfer reactions such as purine and pyrimidine biosynthesis and amino acid inter-conversions. Folates exist as vitamers (one carbon folate derivatives) that are ployglutamates with varying oxidation states and substituents (1). Folates are important as they synthesize neurotransmitters by depleting excess homocysteine from the DIETARY SUPPLEMENTS: omega-3, proteins, vitamins blood, thereby benefi ting cardio vascular disease patients (2). The major sources of folates are green leafy vegetables, liver, beans and legumes, egg yolk, wheat germ, yeast, and folate fortifi ed breakfast cereal products.

CLASSIFICATION OF FOLATES

Folates exist in two forms- Naturally occurring folates are found in foods and in metabolically active forms in the human Figure 1. Structure of natural folates (reduced one carbon body (3). The synthetic form of folic acid is the folate found in substituents of polyglutamates) (10). supplements and fortifi ed foods. This is the more stable form and occurs rarely in foods or in the human body. FERMENTED FOODS

NATURALLY OCCURRING FOLATES Fermented foods are divided into three categories. - A groFOOD Fresh fruits, leafy green vegetables and legumes are some Dairy products natural sources rich in folate (4). Natural folates differ in the The elaboration of extent of the reduction state of the pteroyl group, nature fermented milks of the substituent on the pteridine ring and the number of containing elevated industry hi-tech glutamyl residues attached to the pteroyl group. They include levels of natural form of 5- methyltetrahydrofolate (5-MTHF), 5-formyltetrahydrofolate folates is a better suited (5-formyl-THF), 10 formyltetrahydrofolate (10-formyl-THF), alternative. According

5,10-methylenetetrahydrofolate (5,10 methylene-THF), to a study (5), fermented - Table 1. Folate concentrations July/August 2012 5,10-methenyltetrahydrofolate (5,10-methenyl-THF), in dairy products (11). milk products contribute 5 formiminotetrahydrofolate (5-formimino-THF), signifi cantly to the 5,6,7,8-tetrahydrofolate (THF) and dihydrofolate (DHF), reference daily intake of folates. It is observed that Ropy as depicted in Figure 1. Most naturally occurring folates milk showed the highest folate concentration (110 + 20 µg/ - are pteroylpolyglutamates, containing two to seven mL) because of the presence of actively growing bacterial vol 23 n 4 glutamates joined in amide linkages to the γ-carboxyl strains, giving the milk its characteristic ropy appearance. of glutamate. The principal intracellular folates are These strains continued to produce folates into the milk matrix, pteroylpentaglutamates, while the principal extracellular causing the natural folate levels to rise (Table 1). XXIII Cereal based fermented products dominant producer, elevating folate levels in skim milk, while It is possible to increase the folate content in Egyptian lactobacilli have been found to deplete the available folate baladi bread using germinated wheat fl our from 30 μg/ 100 in the skim milk. It has been analysed experimentally that the g Dry Matter (DM) to 50 μg/ 100 g DM by parameters like adaptation of L. bulgaricus from a plant-associated habitat germination temperature and drying conditions (6). to the stable protein and lactose-rich milk environment through the loss of superfl uous functions and proto-co- Fermented vegetables operation with Streptococcus thermophilus makes it a It is possible to select lactic acid-producing starter cultures potent folate producer (13). that produce signifi cant amounts of 5-MTHF (almost doubling its concentration) during fermentation of vegetables (7). Optimization of the whole process of enhancing folate BIOAVAILABILITY OF NATURAL FOLATES concentrations in fermented vegetables is needed. On comparison of synthetic folic acid with natural dietary folates, synthetic folic acid has a substantially higher MICROBIAL PRODUCTION OF FOLATES bioavailability than natural folates, being rapidly absorbed across the intestine (16). The ability of de novo folate production is found in bacteria, Mammals are unable to synthesize a pteridine ring, one green plants, fungi and certain protozoa. The limitations of of the three groups of tetrahydrofolates; the other two use include the requirement of strict anaerobic conditions for groups are p-aminobenzoate and glutamate. They obtain folate production and possibilities of folate utilization by co- tetrahydrofolates from their diets or from microorganisms cultures. Folate produced in situ by the colonic microbiota in their intestinal tract. The intestinal absorption of folates is absorbed across the large intestine and incorporated into occurs at the monoglutamyl level. Many labile folates may the liver and kidney of piglets (8). be lost during residence in the acid peptic milieu of the stomach. For folate absorption, luminal digestion to the Native strains monoglutamyl form by an intestinal enzyme known as folate Some authors have isolated and identifi ed two new conjugase found in the brush border is the rate limiting step.

vol 23 n 4 This enzyme is an exopeptidase and is activated by zinc

- Lactobacillus strains from dairy products with an ability of high folate production and also potential to act as probiotics (17). A second enzyme, intracellular hydrolase, is found in (9). the lysosomes of intestinal cells, has a pH optimum of 4.5 In humans, folates produced by the microbiota in the small and is an endopeptidase (18). The function of the former is intestine are assimilated by the host (3). Folates produced believed to be primarily in the digestion of dietary folates July/August 2012

- in situ by the colonic microbiota are utilized by cells in the and that of the latter is still unknown. Monoglutamates are colonic epithelium. Research is required to determine if present in portal circulation, which is taken by the liver and these bacteria produce folates in the intestinal environment, converted into polyglutamate derivatives and stored or the form in which this folate occurs, the availability of this released into the blood. folate for transport and utilization by colonocytes from the In their reduced form cellular folates function conjugated industry hi-tech lumen; and the contribution of the intestinal microbiota to to a polyglutamate chain. The reduced forms are the total folate requirement of colonic epithelial cells. unstable chemically. They are easily split between the C-9 and N-10 bond to yield a substituted pteridine

groFOOD and p-aminobenzoylglutamate, which have no biologic A Metabolic engineering of production strains - The genes for folate biosynthesis have been identifi ed as activity (19). Substituting a carbon group at N-5 or N-10 Lactococcus (Lc) lactis and Lactobacillus (L) plantarum, decreases the tendency of the molecule to split, however, and it has been shown that L. bulgaricus also possesses the the substituted forms are also susceptible to oxidative folate biosynthesis genes. Metabolic engineering can be chemical rearrangements and, consequently, loss of activity used to increase folate levels in Lc. lactis, L. gasseri and L. (19). The folates found in food consist of a mixture of reduced reuteri (12). It has been shown that L. bulgaricus possesses all folate polyglutamates. the folate biosynthesis genes (13). Synthetic folates (Folic Acid) Production by co culture method Folic acid is itself not biologically active; its biological

The concurrent production of folate and Vitamin B12 with importance is due to tetrahydrofolate and other derivatives Lactic and Propionic Acid Bacteria was studied (14). L. after its conversion to dihydrofolic acid in the liver (20). plantarum SM39 (397 ng/mL extracellular folate) and P.

freudenreichii DF 13 (1.6 µg/mL intracellular vitamin B12) were Folic Acid fortifi ed foods selected for the development of a batch co-culture process According to the Guidelines provided by the UNO and FAO in Yeast supplemented Whey Permeate Medium for high for fortifi cation (2006), grain fl our and enriched cereal grain and concurrent production of both vitamins. L. plantarum products have been the primary vehicles used to provide SM39 and P. freudenreichii DF13 showed a complementary folic acid, owing to the high dietary consumption of grains in DIETARY SUPPLEMENTS: omega-3, proteins, vitamins metabolism with higher folate yields in co-culture compared the form of noodles, bread and pasta. Studies on Folic acid to the sum of folate yields in single cultures. Highest vitamin fortifi cation of parboiled rice by multi- factorial analysis and yields of 8399 ± 784 ng/mL total folate and 751 ± 353 ng/mL kinetic investigation showed that both soaking and milling of

vitamin B12 were obtained in a two-step fermentation process parboiled rice was signifi cant factors in folic acid fortifi cation (three days anaerobic/four days aerobic) corresponding to (21). The optimum soaking time was deduced to be 1.97 h.

a folate-vitamin B12 mass ratio of 11 to 1. This ratio could be In 2006, the WHO and FAO have jointly published guidelines increased to physiological demands of 40 to 1 by adjusting for food fortifi cation with micronutrients, intended to make precursor concentrations. fortifi cation easier.

Streptococcus thermophilus: prolifi c folate producer Choice of fortifi cant S. thermophilus has a strain-specifi c ability of folate Folic acid is heat stable, but susceptible to the effects of

Monographic supplement series: production (15). S. thermophilus has been reported to be the oxidizing and reducing agents. Some fortifi cant loss could XXIV occur during processing. Folic acid has a light yellow colour, which is not carried over to fortified foods because it is added at such low levels, typically between 1.5 and 2.4ppm. There is some loss of the vitamin on exposure to light, and during cooking and baking (22). As folic acid concentrations in foods are diffi cult to measure, reported levels in fortified fl our and baked products are often subject to considerable assay error.

Folic acid supplements Folic Acid being water soluble demands supplementation in the body more frequently. According to the United States National Library of Medicine, a standard formulation of folic acid supplement tablet contains 400 mcg folic acid/ tablet that supply 100 percent daily value, as the Active Pharmaceutical Ingredient. It is recommended to complement folic acid supplements with Vitamin B12 supplements.

FOLATE DEFICIENCY IN INDIAN POPULATION

In a study (23), among the apparently normal urban South Indian elderly population, red cell folate levels were inversely correlated with plasma Homocysteine levels. Since the food consumption pattern is different between rural and urban populations in India- rural population thrives on traditional, region specifi c food, while urban population consumes a predominantly Western diet, there is a need for carrying out studies based Monographic supplement series: on food consumption pattern and further result interpretation.

Initiatives by Health Organizations to implement folate fortifi cation in India The India Flour Fortifi cation Network, under the guidance of Flour Fortifi cation Initiative (CDC, USA) ensures fortifi cation of wheat fl our with nutrients, including folates in the States of West Bengal, Gujarat, Madhya Pradesh, Tamil Nadu, Kerala, Chandigarh, Rajasthan, Punjab, Delhi and Andhra Pradesh. The Micronutrient Initiative (MI) - India has launched an Iron and Folic Acid (IFA) Program for preschool children, adolescent girls, pregnant and lactating women- wherein cereal fl ours are fortifi ed with folic acid pre mixes, to prevent anaemia and NTD. MI has also led national folic acid advocacy efforts in India. (Source: www.micronutrient.org) DIETARY SUPPLEMENTS: omega-3, proteins, vitamins

Indian Government initiatives As of February 2012, GAIN (Global Alliance for Improved Nutrition) and IHMR (Institute of Health Management) launched edible oil and wheat fl our fortifi ed with iron, folic acid, Vitamin A and Iodine. Efforts are on to serve fortifi ed Soya Dal to children through mid-day meal schemes, apart from launching mini-industrial plans to produce fortifi ed milk and lentils, for easy availability in open markets. (Source: www.gainhealth.org, www.iihmr.org).

Dietary Reference Intake (DRI) standards (The institute of medicine: national academy of sciences) The DRIs include a series of reference values for folate intake, including the Estimated Average Requirement (EAR), Recommended Dietary Allowance (RDA), Adequate Intake (AI) and the Tolerable Upper Intake Level (UL). The EAR is defi ned as the median usual intake of folate needed to meet the requirements of 50 percent of the population. The RDA is estimated from - the EAR by correcting for population variance and represents the average A groFOOD daily dietary intake level suffi cient to meet the nutrient requirement of approximately 98 percent of the population. The AI, defi ned as the quantity of folate consumed by a group with no evidence of folate inadequacy, was estimated when there were insuffi cient data on which to derive an industry hi-tech EAR. The UL pertains specifi cally to folic acid (rather than food folate) and is characterized as the maximum daily usual intake at which no risk of adverse health effects would be expected when consumed over long periods.

The below mentioned statistics are approved by the FAO/ WHO, and apart - July/August 2012 from these, certain countries have their RDA values based on occurrence of folate related disorders like NTD and the regional diet patterns. To determine the additional quantity of folate needed during lactation, the average volume of milk produced (0.78 L) was multiplied by the folate - concentration of breast milk (85 μg/L), and this quantity was multiplied by vol 23 n 4 a bioavailability correction factor of 2. This amount (133 μg) was added to the EAR for non pregnant, non lactating women to derive the RDA (500 μg/day DFE). XXV From Table 2, it is clear that the DRI is determined by the stage of growth, the gender and the form of intake, i.e., folate/ folic acid.

Synthetic folic acid on health: pros and cons A folic acid controlled release formulation using mesoporous particles might release fortifi cation negative effects, while also maintaining nutrition benefi ts (24). It has been reported that periconceptional supplementation with folic acid prevents NTD, congenital heart diseases, oral clefts and pre term births (10). At the same time, unmetabolized synthetic folic acid can cause cancer, depression and cognitive impairment (17).

CONCLUSION

Folates and folic acid are hence understood to vary in their mechanism of action, due to Table 2. DRI Values as recommended by IOM. bio availability differences, that can be co- related to differences in their chemical forms. *Co-effi cient of variation The production methods vary for the two, and **Value for women nursing one infant.

vol 23 n 4 these factors are suggested to be considered - while deciding the DRI for folates. In the succeeding part, the 21. K. Kam et al., Journal of Food Engineering, 108(1), pp. 238-243 role of folates in health is discussed in detail. (2011). 22. A.K. Srestha et al., Food Chemistry, 130(2), pp. 291-298 (2011). 23. V. Shobha et al., Indian Journal of Medical Research, 132, pp. 432-449 (2011). July/August 2012

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industry hi-tech 75-81 (2011). 1744 (2010). 3. Food and Nutrition Board, Institute of Medicine, Folate, pp. 196-305 (1998). 4. L.H. Allen, Food Nutrition Bulletin, 29(2 supplement), pp. S20-S34

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- 5. J.G. LeBlanc et al., Communicating Current Research and Educational Topics and Trends in Applied Microbiology, 1, pp. 329- 339 (2007). 6. M. Hefni et al., LWT- Food Science and Technology, 44(3), pp. 706-712 (2011). 7. M. Jagerstad et al., Food Science and Technology, 37, pp. 603-611 (2004). 8. S. Aufreiter et al., American Society for Nutrition, 141(3), pp. 336-372 (2011). 9. M. G. Dana et al., African Journal of Biotechnology, 9(33), pp. 5383-5391 (2011). 10. L. Taiz et al., Plant Physiology (5 e), Sinauer Associates Inc (2010). 11. L. Alm, Journal of Dairy Science, 65(3), pp. 353-359 (1982). 12. A. Wegkamp et al., Applied and Environmental Microbiology, 70(5), pp. 3146-3148 (2004). 13. M. van de Guchte et al., Proceedings of the National Academy of Sciences of the United States of America, 103(24), pp. 9274- 9279 (2006). DIETARY SUPPLEMENTS: omega-3, proteins, vitamins 14. S. Hugenschmidt, A dissertation submitted to ETH ZURICH for the degree of Doctor of Sciences, (2006). 15. S.K. Tomar et al., Milchwissenshaft, 64(3), pp. 260-263 (2009). 16. G.E. Mullin, Nutrition in Clinical Practice, 26(1), pp. 84-87 (2011). 17. T.B. Shafi zadeh et al., The Journal of Nutrition, 137, pp. 1149-1153 (2007). 18. N. Tyagi et al., Journal of Orthopedic Research, 29, pp. 1511-1516 (2011). 19. R. Blakely, North Holland Research Monographs Frontiers of Biology, 13, North Holland Publishing Company (1969). 20. W.S. Bailey et al., The proceedings of the National Academy of Sciences of the United States of America, 106(36), pp. 15424-15429

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