**6. Folic acid biofortification**

Biofortification is a promising and sustainable agriculture-based strategy to minimize Zn and Fe deficiency in dietary food substances [41]. Among the different strategies deployed, the plant breeding approach to develop biofortified crops and agronomic supplementation of micronutrients, such as foliar/soil application along with chemical fertilizers, have received maximum attention [42]. Breeding staple food crops for higher micronutrient contents, where the density of minerals and vitamins in food staples may be increased either through conventional plant breeding or using transgenic techniques. It is recognized as a nutrition-sensitiveagriculture intervention that can reduce vitamin and mineral deficiency [43]. Iron biofortification of beans, cowpea and pearl millet, zinc-biofortification of maize, rice, and wheat, and pro-vitamin A carotenoid-biofortification of cassava, maize, rice, and sweet potato are currently underway and at different stages of development [44, 45]. Results are promising for iron-biofortified crops, as partially iron-biofortified rice has improved the iron stores of reproductive-age women in the Philippines [46], iron-biofortified pearl millet has increased the iron stores and reversed iron deficiency in school children in India [47], and iron-biofortified beans have improved the iron stores in women in Rwanda [48]. The agronomic mode of biofortification includes the application of micronutrient fertilizer directly to the soil and/or foliar application. The agronomic biofortification is most suitable for staple crops with starch as the major component and is mainly practiced on crops such as rice, wheat, maize, sorghum, millet, and sweet potato and also on legumes. The foliar fertilization often results in more uptake of nutrients and ultimately efficient allocation in the edible plant parts [49]. Soil and foliar application combined together gives better results and has been shown as the most effective method for biofortification [42, 50]. Foliar application of micronutrients is generally much more effective in ensuring uptake into the plant because in such cases, immobilization of the nutrients in the soil can be avoided. Alternatively, microorganisms have been bioengineered to overproduce folates. *Bacillus subtilis* was modified at three different levels and an eightfold increase in folate levels was observed [51]. Metabolic engineering has also been developed in *Lactococcus lactis* leading to a more than threefold increase [52].

Several national health authorities have introduced mandatory food fortification with synthetic folic acid, which is considered as convenient fortificant, being cost efficient in production, more stable than natural food folate, and superior in terms of bioavailability and bio-efficacy. It has been reported that the mandatory folic acid fortification in such countries leads to significant increase in both serum and erythrocyte folate concentrations in all sex and age groups. Studies have shown that the mean serum folate concentration increased more than twofold (136%) and the mean erythrocyte folate concentration increased by 57%. The introduction of folic acid-fortified staple foods has effectively decreased the prevalence of NTD in the United States and Canada [53]. It was also observed that fortifying flour with iron and many water-soluble B group vitamins in the United States, Canada, and many other countries has resulted in preventing micronutrient deficiency conditions and is also a very cost-effective prevention of major neural tube defects *viz*., spina bifida and anencephaly, and also folate deficiency anemia. There are also reports showing that fortification with folic acid has led to a reduction in cases of heart diseases like strokes, which occur due to elevated homocysteine levels. According to the

published reports, around 50 countries have implemented the mandatory folic acid fortification program, including the United States and Australia.

In January 1998, the U.S. Food and Drug Administration (FDA) suggested the processing industries to add 140 μg folic acid/100 g to enriched bread, cereals, flours, corn meals, pasta, rice, and other grain products [54] to reduce NTDs. Because cereals and grains are used as staples and are widely consumed, these products have become important supplement of folic acid to the diet. The fortification program increased mean folic acid intakes in the United States by about 190 μg/ day [54]. Many other countries, including Costa Rica, Chile, and South Africa, have also established mandatory folic acid fortification programs [55, 56].
