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Folate: sources , production and bioavailability

Article  in  Agro Food Industry Hi Tech · September 2012

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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 specific. Some physiological conditions like sickle cell disease and renal dialysis might increase cellular folate
requirements. Folate deficiency 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: DIETARY SUPPLEMENTS: omega-3, proteins, vitamins - AgroFOOD industry hi-tech - July/August 2012 - vol 23 n 4
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
blood, thereby benefiting 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
fortified 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 fortified 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.

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
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), Table 1. Folate concentrations to a study (5), fermented
5,10-methenyltetrahydrofolate (5,10-methenyl-THF), in dairy products (11). milk products contribute
5 formiminotetrahydrofolate (5-formimino-THF), significantly 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
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).

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 Cereal based fermented products
It is possible to increase the folate content in Egyptian
baladi bread using germinated wheat flour from 30 μg/ 100
g Dry Matter (DM) to 50 μg/ 100 g DM by parameters like
germination temperature and drying conditions (6).
Fermented vegetables
It is possible to select lactic acid-producing starter cultures
that produce significant amounts of 5-MTHF (almost doubling
its concentration) during fermentation of vegetables (7).
Optimization of the whole process of enhancing folate
concentrations in fermented vegetables is needed.
MICROBIAL PRODUCTION OF FOLATES
The ability of de novo folate production is found in bacteria,
green plants, fungi and certain protozoa. The limitations of
use include the requirement of strict anaerobic conditions for
folate production and possibilities of folate utilization by co-
cultures. Folate produced in situ by the colonic microbiota
is absorbed across the large intestine and incorporated into
the liver and kidney of piglets (8).
Native strains
Monographic supplement series: DIETARY SUPPLEMENTS: omega-3, proteins, vitamins - AgroFOOD industry hi-tech - July/August 2012 - vol 23 n 4
Some authors have isolated and identified two new
Lactobacillus strains from dairy products with an ability of
high folate production and also potential to act as probiotics
(9).
In humans, folates produced by the microbiota in the small
intestine are assimilated by the host (3). Folates produced
in situ by the colonic microbiota are utilized by cells in the
colonic epithelium. Research is required to determine if
these bacteria produce folates in the intestinal environment,
the form in which this folate occurs, the availability of this
folate for transport and utilization by colonocytes from the
lumen; and the contribution of the intestinal microbiota to
the total folate requirement of colonic epithelial cells.
Metabolic engineering of production strains
The genes for folate biosynthesis have been identified as
Lactococcus (Lc) lactis and Lactobacillus (L) plantarum,
and it has been shown that L. bulgaricus also possesses the
folate biosynthesis genes. Metabolic engineering can be
used to increase folate levels in Lc. lactis, L. gasseri and L.
reuteri (12). It has been shown that L. bulgaricus possesses all
the folate biosynthesis genes (13).
Production by co culture method
The concurrent production of folate and Vitamin B12 with
Lactic and Propionic Acid Bacteria was studied (14). L.
plantarum SM39 (397 ng/mL extracellular folate) and P.
freudenreichii DF 13 (1.6 µg/mL intracellular vitamin B12) were
selected for the development of a batch co-culture process
in Yeast supplemented Whey Permeate Medium for high
and concurrent production of both vitamins. L. plantarum
SM39 and P. freudenreichii DF13 showed a complementary
metabolism with higher folate yields in co-culture compared
to the sum of folate yields in single cultures. Highest vitamin
yields of 8399 ± 784 ng/mL total folate and 751 ± 353 ng/mL
vitamin B12 were obtained in a two-step fermentation process
(three days anaerobic/four days aerobic) corresponding to
a folate-vitamin B12 mass ratio of 11 to 1. This ratio could be
increased to physiological demands of 40 to 1 by adjusting
precursor concentrations.
Streptococcus thermophilus: prolific folate producer
S. thermophilus has a strain-specific ability of folate
production (15). S. thermophilus has been reported to be the
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dominant producer, elevating folate levels in skim milk, while
lactobacilli have been found to deplete the available folate
in the skim milk. It has been analysed experimentally that the
adaptation of L. bulgaricus from a plant-associated habitat
to the stable protein and lactose-rich milk environment
through the loss of superfluous functions and proto-co-
operation with Streptococcus thermophilus makes it a
potent folate producer (13).
BIOAVAILABILITY OF NATURAL FOLATES
On comparison of synthetic folic acid with natural dietary
folates, synthetic folic acid has a substantially higher
bioavailability than natural folates, being rapidly absorbed
across the intestine (16).
Mammals are unable to synthesize a pteridine ring, one
of the three groups of tetrahydrofolates; the other two
groups are p-aminobenzoate and glutamate. They obtain
tetrahydrofolates from their diets or from microorganisms
in their intestinal tract. The intestinal absorption of folates
occurs at the monoglutamyl level. Many labile folates may
be lost during residence in the acid peptic milieu of the
stomach. For folate absorption, luminal digestion to the
monoglutamyl form by an intestinal enzyme known as folate
conjugase found in the brush border is the rate limiting step.
This enzyme is an exopeptidase and is activated by zinc
(17). A second enzyme, intracellular hydrolase, is found in
the lysosomes of intestinal cells, has a pH optimum of 4.5
and is an endopeptidase (18). The function of the former is
believed to be primarily in the digestion of dietary folates
and that of the latter is still unknown. Monoglutamates are
present in portal circulation, which is taken by the liver and
converted into polyglutamate derivatives and stored or
released into the blood.
In their reduced form cellular folates function conjugated
to a polyglutamate chain. The reduced forms are
unstable chemically. They are easily split between the
C-9 and N-10 bond to yield a substituted pteridine
and p-aminobenzoylglutamate, which have no biologic
activity (19). Substituting a carbon group at N-5 or N-10
decreases the tendency of the molecule to split, however,
the substituted forms are also susceptible to oxidative
chemical rearrangements and, consequently, loss of activity
(19). The folates found in food consist of a mixture of reduced
folate polyglutamates.
Synthetic folates (Folic Acid)
Folic acid is itself not biologically active; its biological
importance is due to tetrahydrofolate and other derivatives
after its conversion to dihydrofolic acid in the liver (20).
Folic Acid fortified foods
According to the Guidelines provided by the UNO and FAO
for fortification (2006), grain flour and enriched cereal grain
products have been the primary vehicles used to provide
folic acid, owing to the high dietary consumption of grains in
the form of noodles, bread and pasta. Studies on Folic acid
fortification of parboiled rice by multi- factorial analysis and
kinetic investigation showed that both soaking and milling of
parboiled rice was significant factors in folic acid fortification
(21). The optimum soaking time was deduced to be 1.97 h.
In 2006, the WHO and FAO have jointly published guidelines
for food fortification with micronutrients, intended to make
fortification easier.
Choice of fortificant
Folic acid is heat stable, but susceptible to the effects of
oxidizing and reducing agents. Some fortificant loss could
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 difficult to measure, reported levels in fortified flour 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 specific food, while urban population consumes a
predominantly Western diet, there is a need for carrying out studies based
on food consumption pattern and further result interpretation.

Monographic supplement series: DIETARY SUPPLEMENTS: omega-3, proteins, vitamins - AgroFOOD industry hi-tech - July/August 2012 - vol 23 n 4
Initiatives by Health Organizations to implement folate fortification in India
The India Flour Fortification Network, under the guidance of Flour Fortification
Initiative (CDC, USA) ensures fortification of wheat flour 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 flours are fortified 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)

Indian Government initiatives

As of February 2012, GAIN (Global Alliance for Improved Nutrition) and
IHMR (Institute of Health Management) launched edible oil and wheat flour
fortified with iron, folic acid, Vitamin A and Iodine. Efforts are on to serve
fortified Soya Dal to children through mid-day meal schemes, apart from
launching mini-industrial plans to produce fortified 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 defined 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
daily dietary intake level sufficient to meet the nutrient requirement of
approximately 98 percent of the population. The AI, defined as the quantity
of folate consumed by a group with no evidence of folate inadequacy,
was estimated when there were insufficient data on which to derive an
EAR. The UL pertains specifically 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
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
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
fortification negative effects, while also
maintaining nutrition benefits (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-efficient of variation
Monographic supplement series: DIETARY SUPPLEMENTS: omega-3, proteins, vitamins - AgroFOOD industry hi-tech - July/August 2012 - vol 23 n 4

The production methods vary for the two, and **Value for women nursing one infant.
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).
REFERENCES AND NOTES
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143(1-2), pp. 41-47 (2010). 4(2), pp. 52-59 (2007).
2. H.J. Blom et al., Journal of Inherited Metabolic Disorders, 34(1), pp. 26. M.S. Morris, American Journal of Clinical Nutrition, 91, pp. 1733-
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
(2008).
5. J.G. LeBlanc et al., Communicating Current Research and
Educational Topics and Trends in Applied Microbiology,, 1, pp. 329-
339 (2007).
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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).
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9(33), pp. 5383-5391 (2011).
10. L. Taiz et al., Plant Physiology (5 e), Sinauer Associates
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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
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9279 (2006).
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).
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18. N. Tyagi et al., Journal of Orthopedic Research, 29,, pp. 1511-1516
(2011).
19. R. Blakely, North Holland Research Monographs Frontiers of Biology,
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Sciences of the United States of America, 106(36),, pp. 15424-15429
(2009).

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