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Keywords:

  • bioavailability;
  • deficiency;
  • folate;
  • folic acid;
  • health effects;
  • metabolic engineering;
  • microorganism;
  • S. thermophilus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

ABSTRACT:  Folate, a water-soluble vitamin, includes naturally occurring food folate and synthetic folic acid in supplements and fortified foods. Mammalian cells cannot synthesize folate and its deficiency has been implicated in a wide variety of disorders. A number of reviews have dwelt up on the health benefits associated with increased folate intakes and many countries possess mandatory folate enrichment programs. Lately, a number of studies have shown that high intakes of folic acid, the chemically synthesized form, but not natural folates, can cause adverse effects in some individuals such as the masking of the hematological manifestations of vitamin B12 deficiency, leukemia, arthritis, bowel cancer, and ectopic pregnancies. As fermented milk products are reported to contain even higher amounts of folate produced by the food-grade bacteria, primarily lactic acid bacteria (LAB), the focus has primarily shifted toward the natural folate, that is, folate produced by LAB and levels of folate present in foods fermented by/or containing these valuable microorganisms. The proper selection and use of folate-producing microorganisms is an interesting strategy to increase “natural” folate levels in foods. An attempt has been made through this review to share information available in the literature on wide ranging aspects of folate, namely, bioavailability, analysis, deficiency, dietary requirements, and health effects of synthetic and natural folate, dairy and nondairy products as a potential source of folate, microorganisms with special reference to Streptococcus thermophilus as prolific folate producer, and recent insight on modulation of folate production levels in LAB by metabolic engineering.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

Folates represent an essential nutrition component (important B vitamin) in the human diet, involved in many metabolic pathways, mainly in carbon transfer reactions such as purine and pyrimidine biosynthesis and amino acid interconversions. The daily recommended intake (DRI) as approved in European Union (EU) is 400 μg/d for adults (FAO/WHO 2002; IOM 2004). Since folate deficiency has been associated with the incidence of neural tube defects during the embryo development (Daly and others 1995; Kim 2004), higher intake (600 μg/d) is recommended for women before and during pregnancy. A low folate intake has also been associated with a number of health disorders, namely, Alzheimer's and coronary heart diseases; osteoporosis, increased risk of breast and colorectal cancer, poor cognitive performance, hearing loss, and so on have been attributed to the folate deficiency (Mason 1995; Morrison and others 1995; Boushey and others 1996; Ames 1999; LeBlanc and others 2007). Therefore, an exogenous supply of folic acid appears inevitable to prevent nutritional deficiency especially in view of the inability of mammalian cells to synthesize this vital biomolecule (Sarma and Duttagupta 1995).

Bioavailability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

The generic term “folate” includes the complete group of all folic acid derivatives, including the polylglutamates naturally present in foods and folic acid that is a synthetic folate form which is commonly used for food fortification and nutritional supplements. Bioavailability is defined as the proportion of a nutrient ingested that becomes available to the body for metabolic processes or storage. Natural food folates or pteroylpolyglutamates are hydrolyzed to pteroylmonoglutamate forms prior to absorption in the small intestine. The monoglutamate forms of folate, including folic acid, are transported across the proximal small intestine via a saturable pH-dependent process. Higher doses of folic acid are absorbed via a nonsaturable passive diffusion process (Shils and others 1994; Hendler and Rorvik 2001).

The bioavailability of ingested folate monoglutamates is significantly greater than that of folate polyglutamates presumably because of the requirement for hydrolysis of the latter (Shils and others 1994; Fitzpatrick 2003). The polyglutamate chain to which most of the natural folate is attached (McNulty and Pentieva 2004) hampers the bioavailability of dietary folate and so this polyglutamate chain must be removed, except for the proximal glutamate moiety, by the enzyme α-glutamyl hydrolase or human conjugase, present in the brush border of the small intestine (Reisenauer and Halsted 1987) and then it is absorbed and transported as a monoglutamate into the portal vein. Furthermore, folate-binding proteins from milk may act to increase efficiency of folate absorption by protecting dietary folates from uptake by bacteria in the gut, thus increasing absorption in the small intestine (Eitenmiller and Landen 1999). The enzyme responsible for hydrolysis (that is, folate conjugase) of folate polyglutamates may be specifically inhibited by food factors in yeast and beans and may be nonspecifically impaired at acid pH (Shils and others 1994; Fitzpatrick 2003).

The available data suggest that the polyglutamate form is 60% to 80% bioavailable compared with the monoglutamate form (Gregory 1995). The absorption efficiency of natural folates is approximately half from that of synthetic folic acid and thus relative bioavailability of dietary folates is estimated to be only 50% compared with synthetic folic acid (Sauberlich and others 1987; Forssen and others 2000) but there are still controversies (Gregory 1995; Sybesma and others 2003a; McNulty and Pentieva 2004). Folic acid taken on an empty stomach is twice as available as food folate, and folic acid taken with food is 1.7 times as available as food folate. For instance, 400 μg of folic acid taken on an empty stomach is equivalent to 470 μg of folic acid taken with food and is equivalent to 800 μg of food folate (Hendler and Rorvik 2001). Despite the large amount of information available on folate bioavailability, knowledge of this important part of folate nutrition is still described as fragmentary (Selhub and Rosenberg 1996; Gregory 1997; McNulty and Pentieva 2004).

Measurement of Folate in Foods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

The measurement of folates is complicated by the need to account for all its forms, which could easily include several dozen compounds if each form of the folate nucleus exists in all possible combinations with various polyglutamate chain lengths. Hence, the multiplicity of forms and the generally low levels in foods makes quantitative analysis of folate a difficult task (Arcot and Shrestha 2005). The assay of folates from foods generally involves 3 steps: liberation of folates from the cellular matrix; deconjugation from the polyglutamate to the mono- and diglutamate forms; and the detection of the biological activity or chemical concentration of the resulting folates. During the last 4 decades, numerous reports have been published (Eitenmiller and Landen 1999; Forssen and others 2000; LeBlanc and others 2007); regarding food folate content and the various methods of folate determination including: biological, microbiological, biospecific procedures (radiobinding or immuno assay), chromatographic, electrochemical, or spectrophotometric methods, some of which are carried out in combination with gel or high-pressure liquid chromatography (HPLC). Moreover, the sample preparation and extraction methods largely influence the amount of folate present in the extract, which is then assayed by a method of choice.

Microbiological assay (MA) serves as the traditional and most versatile method of folate analysis and is the only food folate method given official status by American Assn. of Analytical Chemists (AOAC). Microbiological methods of vitamin determination are based on the nutritional requirement of a microorganism for a certain vitamin. This allows the formulation of a basal medium that provides all of the growth requirements for the organism except for the vitamin to be assayed. When aliquots of the sample containing the vitamin being quantitated are added to the initially translucent medium, followed by inoculation with test organism, the organism reproduces in proportion to vitamin content, which can be measured photometrically (Voigt and Eitenmiller 1985).

The MA relies on the turbidimetric bacterial growth of Lactobacillus rhamnosus (Lactobacillus casei, ATCC 7469) which is the most commonly used and most accepted assay organism for folate analysis of natural products. It responds to natural folate forms present in foods, and does not respond to pteroic acid, a common folate degradation product. It has greater capacity for response to most native folates (α-glutamyl folate polymers) compared to the other assay organisms, although the response decreases as the number of glutamyl residues linked to the pteroyl group increases (Arcot and Shrestha 2005). Similarly, the biospecific techniques and HPLC do not respond well to longer chain derivatives of folate. To measure all the higher α-glutamyl folate polymers, these must be enzymatically deconjugated to folates with shorter glutamyl residues such as mono- or diglutamates prior to analysis. Once hydrolyzed these folate can support the growth of Lb. rhamnosus or be quantified by other methods. Treatment with conjugase alone is not much effective; thereby the use of additional enzymes, proteolytic, or amylolytic, has shown to liberate folate from foods (Pffeifer and others 1997; Tamura and others 1997; Rader and others 1998; Shrestha and others 2000; Johnston and others 2002). MA with an additional trienzyme extraction method in the order of α-amylase, protease, and finally conjugase along with the standard microbiological assay using Lb. rhamnosus as an assay organism in casein-based media for the estimation of folate was evaluated (Iyer and others 2009; Tomar and others 2009). Most current studies now determine food folate concentrations in response to growth of Lb. rhamnosus using 96-well microtiter plates. Although the MA is regarded as the premium method of folate assay, there are some associated problems such as stimulation or inhibition of Lb. rhamnosus growth by any nonfolate substance; folate contamination of reagents and glassware; variation in incubation time and temperature; sterilization procedure; inoculation volume, conjugase treatment; and pH of the assay medium (Wilson and others 1987; Tamura 1990).

The limitation of Lb. rhamnosus and other assay organism in differentiating folate derivatives in the folate extract has prompted the use of chromatographic techniques. These techniques involve 2 distinct steps: separation and purification of deconjugated extract, detection and quantification of eluted monoglutamates. HPLC is a popular technique for separation and purification of individual folates. They are generally based on reversed-phase chromatography with UV and/or fluorescence detection. Native reduced folate forms were separated and quantified by HPLC by several researchers (Vahteristo and others 1997; Kariluoto and others 2001; Jastrebova and others 2003). These methods have been applied to separate and detect the individual forms of folates, especially 5-methyl-tetra hydro folate (THF), 5-formyl-THF, and THF, mostly involving fluorescence detection after derivatization (Wigertz and Jagerstad 1995; Eitenmiller and Landen 1999; Poo-Prieto and others 2006). Several HPLC analysis of a range of foods extracted by tri-enzyme extraction was done using affinity chromatography columns prepared with immobilized FBP from milk (Pffeifer and others 1997; Ruggeri and others 1999). Only a few studies have been published on folates in milk and dairy products based on HPLC analyses (Wigertz and Jagerstad 1995; Vahteristo and others 1997; Konings 1999). So far, no HPLC method has been approved as suitable for food analysis in general, although intercalibration work is in progress (Finglas and others 1999). Extraction, enzymatic pretreatment, and sample clean up must be optimized to permit application to various types of foods. Although HPLC holds a high potential for future analysis of folates in food, it has several limitations. The limitations of HPLC are the rigorous sample clean-up procedure prior to final injection, lack of valid purification methods suitable for most of the food matrices, and the detectors that are unable to identify some of the folate derivatives. Also its complex sample extraction and purification procedures cause the loss of sensitive folate compounds which leads to the lower folate value (Arcot and Shrestha 2005). The most recent method for quantification of individual folate derivatives in foods and biological material is coupling HPLC with mass spectrometry (MS) along with liquid chromatographic (LC) systems. The primary advantage of LC-MS analysis is the ability to quantify the different folate forms, a superior specificity with often lower detection limits not obtainable by other conventional detection methods. Drawbacks for LC-MS methods used for food analysis are expensive equipment, absence of suitable commercially available internal standards, and so far, low sensitivities for some of the folate compounds.

Biospecific procedures or ligand binding methods are sensitive, rapid, and specific methods of folate analysis that can be used as an alternative to HPLC and microbiological assays (Finglas and Morgan 1994; Arcot and Shrestha 2005). They are broadly divided into 2 groups: first, those based on the use of naturally occurring vitamin binding proteins with either radiolabels (radio-labeled protein binding assay, RPBA) or enzyme labels (enzyme protein binding assay, EPBA); second, those based on the specific interaction of an antibody with its antigen, for example, the radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA) (Finglas and Morgan 1994). Most radioassay/enzyme-labeled protein binding assay procedures are competitive binding assays, based on competition between radiolabeled/enzyme-labeled and unlabeled folate compounds for a FBP followed by determination of radioactivity/enzymatic activity associated with protein-bound and free species of labeled folate, which reflects the concentration of unlabeled folate present in the sample extract (Andrew-Kabilafkas 2001; Arcot and Shrestha 2005).

Immunoassays are highly sensitive and specific as a result of the interaction of an antibody molecule with its target, a high affinity interaction that occurs even in complex matrices. The principle of the immunoassay is almost the same as EPBA except that antibodies are used in place of naturally occurring vitamin binding proteins (Finglas and Morgan 1994). ELISA principle is based on recognition of folate with a polyclonal antibody raised against folic acid. ELISA performed on a microtitration plate format is fast proving to be well suited to routine food analysis (Finglas and Morgan 1994; Arcot and others 2002). ELISA-based kits containing these antibodies are now commercially available in the market. Only a very limited number of studies have reported folate concentrations in milk, using this method (Andersson and Oste 1994; Wigertz and Jagerstad 1995).

A comparative correlation study of MA, ELISA, and HPLC with UV detection was done for the estimation of folate in milks from different Indian milk species in our lab (Iyer and others 2009). Although there existed a good correlation among estimated average values of folate from different milks by all these methods yet MA was found to be the best, highly efficient, sensitive, and reproducible method which gave the total folate content of the sample analyzed (Iyer and others 2009). Despite the speed and convenience of these assays, their application to food analysis is limited due to a varying affinity for different forms of folate and its failure to analyze total folate content in foods. In particular, different forms of folate show very low affinities and the binding is strongly pH dependent. Hence, these give a much lower response to folate derivatives other than folic acid, thereby underestimating the natural folate content of foods. Thus, it appears that no folate analytical method is perfect. The choice of a particular method is largely determined by the nature and purpose of analysis, for example, food composition, nutritional intervention, regulatory purpose, and to a lesser degree by the resources available, assay time and cost, and analysts themselves.

Folate Deficiency and Requirements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

Folate, an important B-group vitamin, participates in many metabolic pathways such as DNA and RNA biosynthesis and amino acid interconversions. This is involved in essential functions of cell metabolism such as DNA replication, repair and methylation, and synthesis of nucleotides, amino acids, and some vitamins. Mammalian cells cannot synthesize folate; therefore, an exogenous supply of this vitamin is necessary to prevent nutritional deficiency. Although folate is omnipresent in a normal human diet, folate deficiencies still occur frequently, even in well-developed countries (Konings and others 2001; O'Brien and others 2001).

Folate deficiency is developed in the presence of malnutrition, due to low intake of folate-containing foods, or as a result of severe alcoholism. A more important risk factor is malabsorption, especially for diseases affecting either intestinal pH or the jejunal mucosa, for example, celiac disease (Murray 1996). Malabsorption syndromes, including Crohn's disease, tropical sprue, and gluten sensitive enteropathy can result in folate deficiency secondary to inadequate absorption of the vitamin (Shils and others 1994; Hendler and Rorvik 2001; Fitzpatrick 2003). In addition, the following drugs can cause folic acid depletion: oral contraceptives, anticonvulsants, H-2 receptor antagonists, barbiturates, cholestyramine, anti-inflammatory drugs, methotrexate, aspirin, antacids, and alcohol (Murray 1996; Arcot and Shrestha 2005). Secondary folate deficiency (also giving rise to megaloblastic anemia) may be due to vitamin B12 deficiency (Fishman and others 2000). Hence folate deficiency in humans is associated with several health problems, such as anemia, cancer, cardiovascular diseases as well as neural tube defects in newborns.

Implications of Folate Deficiency on Health

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

Anemia

Body stores are relatively small and labile, so temporary reduction of dietary intake can produce short-term (less than 1 mo) deficiency of folate which is first manifested in erythrocytes and bone marrow cells and produces characteristic effects on the red blood cells (RBC) parameters, as these cells have a relatively high turnover rate. A low folate level lead to reduction in de novo DNA and RNA biosynthesis necessary for normal cell division and protein/enzyme synthesis and thus leads to prolongation of the synthesis phase of cell division which results in abnormal red cell precursors and hence cells become megaloblastic (enlarged). Also as folates function as a carbon carrier in the formation of heme, the iron containing nonprotein portion of hemoglobin (Hoffbrand and others 2006) therefore without enough folate, the body can not make or maintain new blood cells leading to anemia. As per the study by Dugdale (2006) short-term folate deficiency produces characteristic effects on the RBC parameters that is, mean cell volume of RBC increases and hemoglobin decreases. Similarly, Lynn and others (2006) has also observed that folate deficiency is associated with nutritional anemia in Lebanese women of childbearing age as anemia not related to iron deficiency was partly explained by plasma folate deficiency. Risk factors are a poor diet, overcooking food, alcoholism, having a history of malabsorption diseases, and pregnancy. Oral or intravenous folic acid supplements may be taken on a short-term basis until the anemia has been corrected, or in the case of poor absorption by the intestine, replacement therapy may be lifelong (Murray 1996).

Pregnancy and neural tube defects

Another important cause of folate deficiency is an increased requirement. Pregnancy is associated with a marked acceleration in 1-carbon transfer reactions, including those required for nucleotide synthesis and thus cell division, which is the basis for the substantial increase in folate requirements during pregnancy. Thus, as pregnancy doubles the need of dietary folates, recommendations in most countries are therefore set to 400 μg dietary folates/d (Yates and others 1998). Low maternal folate status has been associated with premature birth, low birth-weight and increased risk of neural tube defects (NTDs) in the offspring. NTDs result due to incomplete development of the central nervous system and its closely related surrounding structures during the early stages of pregnancy (Wald and others 1991; Van Der Put and others 2001). The most common NTDs are spina bifida (spinal cord at the lumbar vertebra not covered with bone) and anencephaly (no brain) (Brody and Shane 2001). Data also indicate that folic acid could potentially reduce the risk of miscarriage. Researchers in Sweden, where the grain supply has not been fortified with folic acid, found that folate deficiency was associated with a 50% increase in risk of early miscarriage (George and others 2002). Other data suggest that folic acid supplementation before conception may have the potential to reduce the frequency of Down's syndrome. Some mothers of infants with Down's syndrome have abnormal metabolism of folate, as well as mutations in folate genes, features also seen in neural tube defects (James and others 1999; Barkai and others 2003). Folic acid is also important for lactating women. Due to the demands of breastfeeding on the mother's folic acid stores, the RDA (recommended daily allowance) for lactating women in the United States is 500 μg DFE day.

Depression

Chronic, severe folate deficiency, has also been associated with different neurological problems, including stroke, depression, and Alzheimer's disease (Luchsinger and others 2007; Treon and others 2008). There is a reported high incidence of folate deficiency in some psychiatric patients, including those with depression, dementia, and schizophrenia (Shils and others 1994). Recent research indicates that folate deficiency and low folate status are linked to depression, persistent depressive symptoms, and poor antidepressant response (Coppen and Bailey 2000; Morris and others 2003). One double-blind placebo controlled trial found that both depressed and schizophrenic patients receiving 5 mg of methylfolate daily experienced significant improvements in clinical and social recovery (Godfrey and others 1990). Furthermore, a randomized placebo controlled trial found that only 0.5 mg of folic acid significantly improved the antidepressant action of fluoxetine (Coppen and Bailey 2000). It has been hypothesized that since folic acid deficiency is associated with reduced brain levels of S-adenosyl L-methionine, a key player in mood enhancement, supplemental folic acid might be helpful in some with depression.

Cognitive function/Alzheimer's disease

Homocysteine has emerged as an independent risk factor for stroke, vascular disease affecting the brain and Alzheimer's disease (Weir and Scott 1998; Morris 2002). Nevertheless, since improvement of folate status lowers homocysteine levels, the hypothesis that folate supplementation may lower the risk of the various forms of neuropsychiatric dysfunction.

Information has been emerging regarding a connection between homocysteine metabolism and cognitive function, from age-related memory loss to vascular dementia and Alzheimer's disease. Supplementation with vitamins B12, B6, and folic acid, all of which are important in homocysteine metabolism, has been effective in enhancing cognitive performance in older adults (Calvaresi and Bryan 2001). Furthermore, clinical research suggests that subjects with low levels of folate have a higher risk of developing Alzheimer's disease (Wang and others 2001), and increased homocysteine and reduced folate levels have been associated with neurodegeneration (Shea and others 2002). Laboratory data show that homocysteine induces direct neurotoxicity (Shea and others 2002). Also, in a mouse model of Alzheimer's disease, investigators found a decreased number of neurons and elevated levels of homocysteine in the blood and brain of mice with a folic acid deficient diet. As the folate deficient mice suffered more DNA damage in nerve cells than the folate fed mice, the researchers hypothesize that increased levels of homocysteine in the brain may cause damage to the DNA and folic acid's ability to lower homocysteine levels may reduce the same (Kruman and others 2002). Thus further prospective and controlled clinical trials are necessary to better understand the preventive and therapeutic effect of folic acid in neurodegeneration, Alzheimer's disease, and cognitive enhancement.

Cardiovascular disease

Over the last few years, several studies have reported beneficial effects of folates on endothelial function, a surrogate end point for cardiovascular risk. Evidence suggests that adequate folate consumption is important for the prevention of cardiovascular disease. In addition, a growing body of evidence based on epidemiologic studies suggests an inverse association between folate intake measured by dietary questionnaire or serum folate level and risk of cardiovascular disease (Loria and others 2000; Voutilainen and others 2001; Bazzano and others 2002; Wald and others 2002). This association is based on the fact that plasma homocysteine levels increase in response to inadequate folate intake. And so elevated plasma homocysteine levels are recognized as an independent risk factor for coronary heart disease. These findings are supported by other clinical studies, which indicate that hyperhomocysteinemia is positively associated with cardiovascular disease (Boushey and others 1995; Selhub and others 1995). The Framingham Heart Study showed that the higher the blood level of homocysteine, the greater the degree of narrowing of carotid arteries to the rain, which increases the likelihood of strokes (Selhub and others 1995). Serum homocysteine can be lowered by folic acid supplementation (Brouwer and others 1999; Wald and others 2001). Based on the findings of Wald and others (2002), a decrease in serum homocysteine of 3 μmol/L (achievable by daily intake of about 0.8 mg folic acid) could reduce the risk of ischemic heart disease by 16%, deep vein thrombosis by 25%, and stroke by 24%. Hence, adequate intake of folate reduces homocysteine levels in the blood (Danesh and Lewington 1998; Verhoef and others 1998; Ashfield-Watt and others 2001; Verhaar and others 2002) and thus may play a role in the prevention of cardiovascular disease.

The mechanism by which hyperhomocysteinemia might increase the risk of vascular disease is unclear, but several hypotheses have been proposed (Boushey and others 1995; Selhub and others 1995; Hendler and Rorvik 2001). Additional well-designed controlled clinical trials are needed to conclusively prove the efficacy of folic acid in the prevention and treatment of cardiovascular disease.

Cancer

A low folate status has also been associated with elevated risk of cancer (Glynn and Albanes 1994; Mason 1995). Many epidemiologic, animal, and human studies suggest that folate status modulates carcinogenesis. Although these observations have been made in a number of tissues (for example, breast, pancreas, stomach, lung, cervix, esophagus), the data are clearly most compelling for the colorectal cancer, which represents the 2nd leading cause of death due to malignancies (Choi and Mason 2002; Leahy and others 2005). An increasing number of epidemiologic studies suggest that higher intakes of folate either from dietary sources or from supplements may reduce the risk of this cancer (Van Guelpen and others 2006; Pompei and others 2007). Folic acid from dietary sources alone was associated with a modest reduction in the risk of colon cancer (Giovannucci and others 1998).

A functional folate deficiency due to folate antagonists/metabolic inhibitors generally shows a selective toxicity for rapidly growing tumors because of the increased rate of DNA synthesis in these tumors (Brody and Shane 2001). Folate deficiency is related to the misincorporation of uracil for thymidine during DNA synthesis and to an increased frequency of chromosomal breaks (Giovannucci 2002). These aberrations are reported to be normalized following supplementation with folic acid. Giovannucci (2002) provides a summary of several other possible mechanisms that may play a role in the prevention of colon cancer with folate. Interestingly, research suggests that a diet low in methionine (methyl group donor) and folate and high in alcohol may increase the risk of colon cancer 2- to 5-fold. Two recent studies have found a link between folic acid intake and childhood leukemia. One of the key enzymes central to the processing of folic acid (that is, methy tetra hydro folate reductase [MTHFR]) works to break down and reduce levels of folate. Some people inherit a variant MTHFR gene, which makes the enzyme inactive and higher levels of folic acid occur as a result. Also in a study it was found that the use of folic acid during pregnancy almost halved the risk to children of developing leukemia (Thompson and others 2001). While the data in regard to folate and cancer risk is intriguing, further well-controlled studies are necessary to define the optimal dosage, duration, timing, and the appropriate target population for folate chemoprevention and treatment.

Arsenic genotoxicity

Besides these, the effect of dietary folate deficiency on arsenic genotoxicity has been studied in vitro and in animals. It has been proposed that arsenic exerts its toxic effects, in part, by perturbing cellular methyl metabolism. Methyltransferase activity, which requires folate as a key nutrient, is essential for phase II detoxification of arsenic and arsenic-based pesticides (Levin 1999). In vitro research suggests that folic acid protects embryo fibroblasts from sodium arsenite cytotoxicity in a dose-dependent manner (Ruan and others 2000). Although data in animals indicate no significant protection against arsenate-induced neural tube defects following folic acid supplementation (Ferm and Hanlon 1986), other animal research found that folate deficiency enhances arsenic-induced clastogenesis using a mouse peripheral blood micronucleus assay at doses of 5 mg/kg and higher (McDorman and others 2002).

Hence, inadequate folate status is associated with an increased risk for chronic diseases having a negative impact on human health. Although folate is present in various foods constituting our ordinary diet, yet is insufficient to meet RDA, which makes us vulnerable to folate deficiency. This problem can be addressed either by fortifying foods with folic acids or by use of folate rich foods and fermentation fortification by employing folate synthesizing food-grade bacteria to increase the in situ folate levels in fermented foods (LeBlanc and others 2007). Many reviews have shown the health benefits associated with increased folate intakes and by consequence many countries now possess mandatory folate enrichment programs. Methods such as fortification have proven to be very useful in reducing health problems associated with folate mal-intake (Wald and others 1991). Lately, a number of studies have shown that high intakes of folic acid, the chemically synthesized form of folate (tolerable upper intake level, 1000 μg/d as per Natl. Academies of Science), can cause adverse health effects as highlighted by the FDA (1996) such as the masking of the early hematological manifestations of vitamin B12 deficiency, leukemia, arthritis, bowel cancer, and ectopic pregnancies (Sweeney and others 2007; Wright and others 2007). Further concerns include the potential to promote cancer (Charles and others 2004; Kim 2004) and the recent hypothesis that exposure of the fetus to excess folic acid may favor the selection of the methylentetrahydrofolate polymorphism, associated with a range of debilitating illnesses (Lucock and Yates 2005). However, natural folates do not cause such adverse health effects in individuals (Scott 1999; LeBlanc and others 2007; Wright and others 2007). For these reasons, many researchers have been critically evaluating the dietary sources of folates and looking for novel methods to increase concentrations of naturally occurring folate variants in foods. It was proposed that these problems could be circumvented by consumption of a mixed diet containing folate-rich foods.

Dietary Sources of Folate

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

Folate is present in most foods such as legumes (beans, nuts, peas, and so on), leafy greens (such as spinach), citrus, some fruits, vegetables (broccoli, cauliflower), liver, and (fermented) dairy products (Eitenmiller and Landen 1999). Among dairy products, milk and fermented dairy products represent important dietary sources of folates (Lin and Young 2000). Milk is a well-known source of folate. Food composition tables and review papers based on microbiological assay report total folate values for milk in the range of 5 to 7 μg/100 g (Forssen and others 2000) with 5-methyl-THF as the major form (Muller 1993; Finglas and others 1999). These are present in both free form and bound to folate binding proteins (Arcot and Shrestha 2005).

Many dairy products, however, are processed using microbial fermentations in which folate can be synthesized, significantly (Lin and Young 2000) increasing folate concentrations in the final product. Therefore, fermented milk products like yogurt (100 μg/L), are reported to contain even higher concentrations of folate than nonfermented milk (20μg/L). The elevation of folate level can be attributed to the production of additional folates by bacteria. Therefore, production and consumption of folates by the microorganisms will be probably the most detrimental factors in determining the final folate level in various fermented foods, such as yogurt, kefir, bread, and fermented milk products. Yogurt and buttermilk contain approximately 13.7 and 7.5 μg 100/g folate, respectively, with approximately 80% to 90% of the total folate appearing in the 5-formyl-THF form (Muller 1993; Vahteristo and others 1997).

The U.S. Dept. of Agriculture (USDA) recommends the consumption of at least 3 servings of milk products as part of a healthy daily diet. Taking into account this recommendation, and considering that a normal serving consists of 240 mL, currently available fermented milk products could contribute significantly to the reference daily intake of folates (up to 23% of RDI). Besides fermented dairy products, microorganisms are capable of increasing folate content in a wide variety of other nondairy foods as wine, beer, rye, bread, sourdough, and fermented vegetables (Jagerstad and others 2004, 2005; Leroy and De Vuyst 2004; Kariluoto and others 2006).

However, due to the potential risks of fortification with folic acid, fermented milks containing elevated levels of natural form folates seem to be more rational for fortification purposes (Scott 1999). Fermented milks are considered as a potential matrix among dairy products, for folate fortification because folate binding proteins of milk improve folate stability and the bioavailability of both 5-methyltetrahydrofolate and folic acid may be enhanced (Jones and Nixon 2002; Aryana 2003; Verwei and others 2003). Hence, proper strain selection and use of folate-producing microorganisms is an interesting strategy to increase “natural” folate levels in foods. Fermentation fortification, therefore, is a novel concept with a potential to increase folate content in food products by number of ways: (1) bacterial strain selection, (2) delivery engineering, and (3) metabolic engineering (LeBlanc and others 2007).

Folate Produced by Food-Grade Microorganisms

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

The property of folate biosynthesis can be found in 2 of the 3 domains of the phylogenetic tree. The exception is the domain of the Archaea. Species in this domain use tetrahydromethanopterin (THMPT) as 1 carbon carrier instead of THF. However, exceptions of the ability to synthesize folate can also be found within the group of the Bacteria and Eukarya. However, many bacteria synthesize this cofactor by themselves from simple precursors but some auxotrophic bacteria, including many LAB, have a strict growth requirement for folic acid (Lin and Young 2000; Hugenholtz and Smid 2002). The ability of de novo folate production is not only found in bacteria but also in green plants, fungi, and certain protozoa through the folate biosynthesis pathway.

Numerous researchers have reported that LAB, such as the industrial starter bacteria Lactococcus lactis, S. thermophilus, and Leuconostoc species have the ability to synthesize folate (Lin and Young 2000; Hugenholtz and Smid 2002; Crittenden and others 2003; Papapstoyiannidis and others 2006) whereas many Lactobacilli species happen to consume folate (Hugenholtz and Kleerebezem 1999; Smid and others 2001; Sybesma and others 2003a). The ability to produce folate can differ remarkably between different LAB (2 to 214 μg/L folate). The production and consumption of folates by the microorganisms applied will be probably the most important factor determining the folate level. It is now known that not only the yogurt starter cultures and L. lactis have the ability to produce folate but also this important property exists in other LAB species as Lb. acidophilus, Leuconostoc lactis, Bifidobacterium longum, and some strains of Propionibacteria, well-known vitamin B12 producer, can also produce large amounts of folate (Lin and Young 2000; Hugenholtz and Smid 2002; Crittenden and others 2003; Sybesma and others 2003b). Majority of folate produced by L. lactis and Leuconostoc spp. being intracellular is not excreted into the milk and hence is lesser bioavailable while S. thermophilus produces folate extracellularly during milk fermentation. Similarly, probiotic microorganisms as Propionibacteria spp. (Hugenholtz and Smid 2002; Holasova and others 2004) and Bifidobacteria spp. (Crittenden and others 2003; Pompei and others 2007) are also well-known folate producer but majority of folate produced is intracellular and in the case of Propionibacteria it also consumes folate from the medium. Moreover, the major limitations of use of these organisms for biofortification of folate include their requirement of strict anaerobic conditions for folate production and possibilities of folate utilization by co-cultures when used as adjunct starter.

Besides LAB, Saccharomyces cerevisiae also has high folate content ranging from 1 to 4 mg/100 g yeast (Witthöft and others 1999). The main forms are H4-folate and 5-CH3-H4 folate in exclusively polyglutamylated forms (Hjortmo and others 2005, 2008; Kariluoto and others 2006). Folate content in different strains of S. cerevisiae shows large differences among strains that clearly indicate the importance of choosing the proper starter culture when optimizing folate content in yeast-fermented food (Hjortmo and others 2008).

A recent study provided new perspectives on the specific uses of folate-producing probiotics such as bifidobacteria and propionibacteria, to prevent the localized folate deficiency that is associated with premalignant changes in the colonic epithelia. The oral administration of probiotic strains may confer a more efficient protection against inflammation and cancer, both by exerting the beneficial effects and by delivering folate to colonic-rectal cells. Folate produced in situ by the colonic microbiota is absorbed across the large intestine and incorporated into the liver and kidneys (Camilo and others 1996; Dudeja and others 1997; Asrar and O'Connor 2005). Increased intestinal Bifidobacterium populations, induced by consumption of human breast milk, have been correlated with an enhanced folate status in rats (Krause and others 1996). It is therefore possible that probiotic bacteria active in the intestinal tract may be able to contribute to the folate requirement of colonic epithelial cells though further research is required in this area.

Among all these food-grade bacteria and dairy starters, S. thermophilus has been adjudged as the best folate producer that produces folate extracellularly in the milk during fermentation.

Streptococcus Thermophilus: Prolific Folate Producer

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

S. thermophilus is known to produce folic acid during growth in milk, which is a functional attribute. S. thermophilus has a strain-specific ability of folate production (Rao and others 1984; Smid and others 2001; Tomar and others 2009) and has been reported to produce higher quantity of folate in comparison to other LAB, majority of which is excreted into milk (Crittenden and others 2003; Papapstoyiannidis and others 2006). However, great differences have been observed in the production ability of individual strains. By application of different S. thermophilus strains used in yogurt productions, the folate contents ranging from 2 up to 15 μg/100 g were found (Smid and others 2001). S. thermophilus has been reported to be the dominant producer, elevating folate levels in skim milk, while lactobacilli have been found to deplete the available folate in the skim milk. Yet fermentations with mixed cultures showed that folate production and utilization by the cultures is additive (Crittenden and others 2003). Sybesma and others (2003a) reported highest folate production by S. thermophilus strains as high as 214 μg/L. Later, Holasova and others (2004) showed about more than 6-fold increase in the 5-MTHF content by S. thermophilus. Tomar and others (2009) found culture NCDC177 (35 μg/mL) to be the best folate producer among the S. thermophilus cultures available at Natl. Collection of Dairy Cultures, Natl. Dairy Research Inst., Karnal (Haryana, India).

Folate Biosynthesis Pathway and Folate-Related Reactions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

Folate is a tripartite molecule, composed of a pterin residue, Para Amino Benzoic Acid (PABA) and a γ-linked glutamate moiety. The folate biosynthetic pathway consists of a series of reactions (8 consecutive steps) in which these 3 structural elements are modified and coupled via several steps into the biological active cofactor tetrahydrofolate. These 3 elements reflect the 3 pathways (pterin, PABA, and glutamate pathway) that are involved in the synthesis of the building blocks of folate. The pterin pathway reflects the conversion of guanidine tri phosphate (GTP) by GTP cyclohydrolases (I or II). The pterin proportion is made by the conversion of GTP synthesized in the purine biosynthesis pathway. The PABA originates from chorismate and is synthesized by same biosynthesis pathways as required for the aromatic amino acids while the 3rd component glutamate is normally taken up from the medium. The tetrahydrofolate that is produced is converted in the 1-carbon pathway into 1-carbon carriers involved in several reactions in the synthesis of purines, pyrimidines, and so on. Another remarkable feature of the folate biosynthesis pathway is that folate is involved in the synthesis of its own precursor, GTP.

Folate Biosynthesis Genes in Lactic Acid Bacteria

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

To modulate the folate content in fermented products it is crucial to have knowledge of the genes involved in folate biosynthesis. To date, L. lactis is the only species for which all folate biosynthesis genes are annotated. Folate gene cluster is a cluster of 5 genes of approximate size of 10 KB, present on chromosome. This folate gene cluster contains folA, encoding dihydrofolate reductase, folB, predicted to encode dihydroneopterin aldolase, folK, and folE, encoding the biprotein 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine pyrophospho-kinase and GTP cyclohydrolase I, folP predicted to encode dihydropteroate synthase, and folC encoding the bifunctional protein folate synthetase and polyglutamyl folate synthetase. The other genes present in the folate gene cluster clpX, ysxc, and ylgG are not likely to be involved in folate biosynthesis. The gene folE, encoding GTP cyclohydrolase I, was always identified as an independent gene. The location of the folate genes on the chromosome can be a relevant factor for metabolic engineering purposes, since organization of the genes in a single gene cluster facilitates metabolic engineering strategies.

Metabolic Engineering of Production Strains

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

Metabolic engineering is used to alter metabolite or protein levels in the cell. This can be done by overexpression or disruption of metabolic or regulatory genes. In LAB, several metabolic pathways have been rerouted or overexpressed; for example, those for alanine, riboflavin, sorbitol, mannitol, and folate production. Traditionally, a single pathway is engineered and many aspects of this pathway are investigated. However, the development of metabolic models and functional genomic tools can help to investigate impact of metabolic engineering on the overall performance of the host organism. Thus, folate production levels in lactic acid bacteria can be modulated by gaining insight in the genes, pathways, and metabolites that are involved.

The genes for folate biosynthesis have been identified in some LAB such as Lc. lactis and L. plantarum, and it has been shown that L. bulgaricus also possesses the folate biosynthesis genes. However, some LAB cannot synthesize folate because some of the genes involved in folate biosynthesis may be lacking in the genome for environmental and nutritional reasons. Metabolic engineering can be used to increase folate levels in Lc. lactis, L. gasseri, and L. reuteri (Sybesma and others 2003b; Wegkamp and others 2004). The folate-consuming L. gasseri was transformed into a folate producer by the transfer of a broad-host-range plasmid containing the folate gene cluster from Lc. lactis (Wegkamp and others 2004). Similarly, Lc. lactis strains have been modified to produce intracellularly folates with a short glutamyl tail length (average polyglutamyl tail length of 3) or with a long polyglutamyl tail length (average polyglutamyl tail length of 8), which generates an increased retention of folate in the cells (Sybesma and others 2003a). These strains were evaluated in animal models to determine if the folate glutamyl tail length affects bioavailability. In contrast to monoglutamyl folate, polyglutamyl folates cannot be transported across the cell membrane (Sybesma and others 2003a) Hence, the release of intracellular polyglutamyl folate depends on the disruption of the cells during passage through the gastrointestinal tract. The clear responses of lactococcal cells added to a folate free diet of deficient rodents on the folate levels in organs and blood indicate that these cells lyse after consumption and that the bacterial folate becomes available for absorption in the gastrointestinal tract of the rat (LeBlanc and others 2009). This study provided the 1st animal trial with food containing living bacteria that were engineered to increase the intracellular accumulation of folate or to change the average polyglutamyl tail length compared to a wild-type lactococcal strain. The finding endorsed that Lc. lactis could be used to deliver and release folates in the gastrointestinal tract. Thus knowledge of the folate biosynthesis genes and the metabolic pathway in several folate-producing LAB enabled us to modulate the folate levels many folds.

Future Prospects

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References

Further research, exploration, and development of folate-producing LAB and yeast strains and combinations of them for food applications should be encouraged. More needs to be learnt about folate forms produced and the interactions between food matrix and folate-producing microorganisms. By a careful testing of the folate production ability of microbial strains used in the production of fermented milk starters, formulations may be optimized for natural enhancement of the folate content in food products. Essential for applying this fermentation fortification in food processes is to have knowledge on the genes and pathways that are involved in folate production. Therefore, the increase in folate levels in yogurts and fermented milks is possible through judicious selection of the microbial species through bioprospecting native strains of folate-producing microbes from different niche (ethnic foods, fruits and vegetables, vegetation, and so on) and cultivation conditions. The food industry should now take the next step to use this information for selecting folate-producing strains as part of their starter cultures to produce fermented products with elevated levels of this essential vitamin. Such products would provide economic benefits to food manufacturers since increased “natural” folate concentrations would be an important value-added effect without increasing production costs. Consumers would obviously benefit from such products since they could increase their folate intakes while consuming products that form part of their normal lifestyle. Further, the proper selection of probiotic folate-producing strains provides a strategy for the development of novel functional foods with increased nutritional value.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Bioavailability
  5. Measurement of Folate in Foods
  6. Folate Deficiency and Requirements
  7. Implications of Folate Deficiency on Health
  8. Dietary Sources of Folate
  9. Folate Produced by Food-Grade Microorganisms
  10. Streptococcus Thermophilus: Prolific Folate Producer
  11. Folate Biosynthesis Pathway and Folate-Related Reactions
  12. Folate Biosynthesis Genes in Lactic Acid Bacteria
  13. Metabolic Engineering of Production Strains
  14. Future Prospects
  15. References
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