Food fortification is defined as the addition of one or more essential nutrients to a food, whether or not it is normally contained in the food, for the purpose of preventing or correcting a demonstrated deficiency of one or more nutrients in the population or specific population groups. Fortification therefore differs from enrichment, which is the process of restoring the nutrients to a food removed during refinement or production. Fortification commonly uses staple foods as vehicles to deliver micronutrients generally lacking or not contained insufficient concentration in the diet of a population. It has been implemented for a long period of time to achieve the successful control of vitamin A and D deficiencies, several B vitamins (thiamine, riboflavin, and niacin), as well as iodine and iron. Since the early 1940s, the fortification of cereal products with thiamine, riboflavin, and niacin has become a common practice. Foods from around the world have begun to be fortified with calcium, iron, phosphorus, and vitamins (especially A, B, C, and D), depending on the chemical composition of the basic foods.
Although the entire world population suffers from various nutritional deficiencies, people with low incomes are the most affected, particularly in developing countries, due to unsafe food consumption. Poverty, the lack of access to a variety of foods, and the lack of knowledge on appropriate dietary practices represent major drawbacks for socioeconomic development while also contributing to a vicious circle of underdevelopment. As such, long-term effects on health, learning, and productivity are significant, while they also generate a high level of social and public costs from reduced work capacity due to high rates of illness and disability.
METHODS OF FOOD FORTIFICATION
The four main methods of food fortification are:
1. Biofortification (i.e. breeding crops to increase their nutritional value which includes both plant breeding and genetic engineering)
2. Microbial biofortification and synthetic biology (i.e. addition of probiotic bacteria)
3. Commercial and industrial fortification (flour, rice, oils)
4. Home fortification (e.g. vitamin D drops)
The several types of food fortification are distinct because different techniques and procedures are used to fortify the target foods. Biofortification involves creating micronutrient dense staple crops using traditional breeding techniques and/or biotechnology. Using biotechnology (genetic engineering) to biofortify staple crops is more modern and has gained much attention in recent years. The most popular example of this approach is the transgenic ‘Golden Rice’ containing twice the normal levels of iron and significant amounts of beta-carotene. Microbial biofortification involves using probiotic bacteria (mostly lactic acid bacteria), which ferment to produce β-carotene either in the foods we eat or directly in the human intestine. Commercial and industrial fortification involves fortifying commercially available products such as flour, rice, cooking oils, sauces, butter etc. with micronutrients and the process occurs during manufacturing. Home fortification consists of supplying deficient populations with micronutrients in packages or tablets that can be added when cooking/consuming meal s at home (basically a merger of supplements and fortification).
Biotechnology and genetic modification techniques are being optimized for the production and development of healthy foods and improvement in the levels and activity of biologically active components in food plants (phytochemicals). Genetic engineering techniques have mostly been targeted at increasing yields of cash crops in the developing countries. However, the crops with improved food quality have gathered much less attention. Genetic modifications in plants include mutation breeding, improved conventional breeding, molecular breeding, transgenic breeding and somatic hybridization.
Gene marking and engineering techniques allow identifying the specific plant gene or genetic material that control nutrient contents. Such material is selected and used for developing varieties with higher micronutrient contents. Furthermore, it provides insight into the potential for application of transgenic technology in developing improved quality and functional foods for human nutrition and health. The production of increased levels of β-carotene (the precursor to vitamin A) in plants is especially important, as its precursor, lycopene has been shown to have physiological chemo-preventive effects with regard to various cancers. Furthermore, lycopene, commonly found in various carotenoid-containing plants such as tomatoes and carrots, is an essential ingredient in maintaining eye health and vision.
Biotechnology enables the selection of successful genotypes, isolation and cloning of favourable traits and the creation of transgenic crops for sustainable agriculture. The advent of biotechnological tools including marker-assisted selection and gene transfer across the species barrier has opened up novel opportunities for enhancing the seed-quality, disease and pest resistance, viral resistance, abiotic stress tolerance. The application of plant biotechnology to improve the nutritional content of staple food crops has perhaps the greatest potential to benefit global health. Genetic engineering methods can be used to increase the trace element content of staple foods such as cereals and legumes, which can be achieved by insertion of genes with the ability to produce the desired nutrients that are typically deficient. It may involve the identification and insertion from another source, or deletion of a gene to improve the desired trait like micronutrient density. This may be achieved by the introduction of genes that code for trace element-binding proteins, over expression of storage proteins already present or the expression of other proteins that are responsible for trace element uptake into plants.
FOOD FORTIFICATION THROUGH GENETIC ENGINEERING (BIOFORTIFICATION)
In contrast to plant breeding, the techniques of genetic engineering allow the transfer of heritable traits between completely unrelated species. In recent years, genetic engineering techniques have been used to introduce new traits into commercially important plants thereby producing combinations of features which could not been achieved by traditional breeding. Several key factors play an important role for successful genetic transformation of crop plants including the development of reliable tissue culture and regeneration methods, preparation of gene constructs with suitable promoters, efficient transformation techniques, recovery and multiplication of transgenic plants, characterization of transgenic plants for the introduced traits and transfer of transgenes into elite cultivars by conventional plant breeding methods. Genetic engineering methods have been used for enhancement of micronutrients in staple crops. These are a few examples on micronutrient enrichment in staple crops through transgenic approaches.
The greatest part of the iron in the human body is found in erythrocytes as haemoglobin, where its main function is to carry oxygen from the lungs onto the tissues. Iron deficiency causes anaemia, the most common and widespread nutritional disorder in the world and a public health problem in both industrialized and non-industrialized countries. As a component of myoglobin, a protein that supplies oxygen to the muscles, iron supports metabolism. Additionally, iron is necessary for growth, development, normal cellular function, and the synthesis of hormones and the connective tissue.
The most important way to improve the iron content in crops is through the enhancement of the absorption, transport and accumulation of iron. The absorption rate of iron in plant food source is lower than 10%. For example it is, 1% in rice, 3% in corn and black bean, 4% in lettuce, and 5% in wheat (Cheng and Hardy 2003). This is due to the fact that many inhibitory factors in plant food source impair the absorption of iron. Such factors include phytic acid, oxalic acid, and carbonate, which form an insoluble salt with iron. Certain amino acids (e.g., cysteine) and proteins also have an important role in determining the availability and uptake of Fe during digestion. A cysteine-rich metallothionein-like protein has been expressed in rice endosperm, resulting in a nearly seven-fold increase in cysteine fraction content of the seed, which had a positive impact on Fe uptake. Iron absorption is influenced by the presence of phytates and tannins in grains such as maize. Modified maize in which phytates have been reduced by insertion of the phytase gene resulted in greater availability of iron, despite no change in overall content of iron (Raboy 1996; Mendoza et al 1998; Bouis 2003). The increased iron absorption from low-phytic acid maize is particularly applicable in areas where maize and its products are staple foods. This also applies to other grain cereals where iron absorption is compromised by the presence of phytates and other anti-nutritional components. Transgenic wheat a expressing a high level of phytase has been developed with Aspergillus niger phytase gene for obtaining higher availability of iron and zinc (Brinch-Pedersen et al2000) and rice (Lucca et al 2001a,b). Samuelsen et al (1998) showed that the transgenic tobacco plant expressing a yeast ferric reductase gene increased leaf iron content by 50%. Similarly, increasing the concentration of the storage proteins like phytoferritin and metallothionein could increase the content of Fe and Zn absorption, respectively.
The introduced Naat-A (nicotianamine synthase) gene with a 35S promoter in rice was shown to result into some iron-efficient rice strains in calcareous soil (Takahashi 2003). Nicotianamine synthase stimulates the production of siderophores in the roots that improves the uptake of iron in the plants. Overexpression of zinc transporter protein from Arabidopsis with the ubiquitin promoter has been reported to increase the uptake and transport of zinc and iron in transgenic barley plants (Ramesh et al 2004). Goto et al (1999) reported a two to three-fold increase in iron in transgenic rice expressing the soybean ferritin gene. The transgenic tobacco plants expressing the same gene under influence of a constitutive promoter showed approximately 30% more iron in the leaves than that of non-transgenic leaves (Goto et al2000). To increase iron content in rice seeds, the ferritin gene from Phaseolus vulgaris was expressed in rice endosperm under the control of the glutelin promoter that resulted in over two-fold increase in the iron content (Lucca et al 2002). Similarly, Vasconcelos et al (2003) expressed the ferritin gene again under the control of the endosperm-specific glutelin promoter and demonstrated an increase of Fe and Zn content not only in the whole grain, but also in the polished grains. Liu et al (2004), observed the iron content in the milled transgenic rice with ferritin gene from soybean under rice glutein promoter to be up to 64% higher than that of the untransformed rice. Anai et al (2003), constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA and introduced into rice plants and found that alpha-linolenic acid content of the transgenic seeds increased dramatically up to ten-folds than that of the control, and transgene was stably inherited in the next progenies. The expression of recombinant humanlactoferritine (rHLF) in rice endosperm produced not only 5 g rHLF per kg dehusked rice grains, but also increased by about two-folds Fe content (Nandi et al 2002).
Vitamin A is the name of a group of retinoids soluble in fats, including retinol, retinal, and retinyl esters. Vitamin A represents an essential nutrient, thus necessary in small amounts for the normal functioning of the visual system in human beings, maintenance of cell growth function, epithelial cell integrity, immune function, and reproduction. Provitamin A or β–carotene is one of plant carotenoids, a major precursor for vitamin A which is generally thought to be the most important for humans. In addition to being a precursor for vitamin A, β-carotene is an important antioxidant that helps to prevent harmful free radical damage in the body. Dietary carotene has about half of the biological activity of vitamin A because of the low efficiency of carotene to retinol conversion. Vitamin A deficiency is more widespread in parts of countries where rice or wheat or cassava is the staple food when compared to areas where the major staple is yellow maize, millet or sweet potato, all of which provide considerable amounts of pro-vitamin A. In tomato, seven major biosynthetic steps, and more than 20 genes, have been well characterized in the synthesis of carotenoids. Hauge and Trost (1928) described a major gene for carotene content in maize, and designated it the Y (yellow) locus that is incompletely dominant. The enormous progress made in the cloning of β-carotene genes has opened up the possibility of modifying and engineering the carotenoid biosynthetic pathway in plants, especially in food crops, considering the importance of carotenoids like β-carotene in human nutrition and health. Several approaches have been used to increase the level of β-carotene in some key crop plants:
Engineering β-carotene biosynthetic pathway into the rice endosperm was novel in the sense that it was aimed at a tissue that was totally devoid of the pathway. Carotenoids do not accumulate in the rice endosperm; however, the general precursor geranylgeranyl pyrophosphate (GGPP) is present in this tissue. Burkhardt et al (1997), for the first time demonstrated that it is possible to engineer β-carotene biosynthetic pathway in a non-photosynthetic, carotenoid-lacking plant tissue by transforming a japonicarice variety T309 with daffodil phytoene synthase gene driven by a seed-specific promoter from glutenin (Gt1), where the transgenic plants accumulated phytoene in the endosperm. This result was extended to the ultimate β-carotene accumulation by Ye et al (2000), who introduced three genes, a daffodil phytoene synthase (psy1) gene under the control of seed specific Gt1 promoter, an phytoene desaturase gene (crtI) from Erwinia uredovera (codes for enzymes mediates four desaturation steps, for which two plants enzymes are required) driven by CaMV 35S promoter, and daffodil lycopene β-cyclase (lcy) driven by CaMV 35S promoter into T309 japonica rice line. This resulted in the accumulation of carotenoids (surprisingly, mostly β-carotene) in the endosperm. Interestingly, they were also able to produce β-carotene only by introducing the phytoene synthase and phytoene desaturase activities in the absence of heterologous β-cyclase. This work was further extended to several widely grown indica rice varieties from different eco-geographical regions of Asia (Datta et al 2003). Recently, it has been observed that source of the phytoene synthase gene plays an important role in alleviating β-carotene level and thought to be the rate limiting and major regulatory step for carotenoid biosynthesis. Paine et al (2005), developed golden rice 2 by introducing the maize psy1 gene in combination with the crtI (carotene desaturase) gene and observed an increase of up to 23-folds of total ß-carotene when compare to the golden rice 1.
Rosati et al (2000) were able to enhance the conversion of β-carotene from lycopene, which is normally present in high amounts in tomato fruit, by transforming with β-lcy gene from Arabidopsis driven by fruit-specific promoter. They also reported an increase in the total carotenoid level in the fruit. In another study, while the expression of crtI gene driven by CaMV 35S promoter was shown to increase the lycopene content of transgenic tomato fruits, unexpectedly this also resulted in 50% decrease in total carotenoids, mainly at the expense of lycopene while β-carotene increased by about three folds (Romer et al 2000). Fray et al (1995) reported overexpression of tomato fruit phytoene synthase by transforming phytoene synthase cDNA (psy1) under the control of CaMV 35S promoter in tomato, which led to a reduction in the levels of gibberellin A1 (GA1) and chlorophyll. This result highlighted the complexities of manipulating carotenogenic pathway, as the increased flux towards the direction of one product may affect the other essential metabolites, thereby affecting the phenotype. However, Fraser et al (2002), transformed tomato with phytoene synthase (crtB) gene from E. uredovora driven by fruit-specific tomato polygalacturonase promoter and reported a 2-4 fold higher carotenoid levels in the primary transformants, whereas, phytoene, lycopene, β-carotene and lutein levels also increased by two-folds. This suggests that manipulating carotenogenic pathway in a seed-specific, rather than constitutive manner could be the right option. There are also reports of transgenic tomatoes containing carotenoids that are not normally present in the fruit. Zeaxanthin and β-cryptoxanthin containing fruits have been produced through the expression of two cDNAs: the Arabidopsis β-Lcy and Capsicum β-carotene hydroxylase (β-Chy), both with the tomato Pds promoter (Dharmapuri et al 2002).
The generation of transgenic maize with enhanced pro-vitamin A content in their kernels due to the overexpression of the bacterial genescrtB (phytoene synthase) and crtI (phytoene desaturase) under the control of a ‘super g-zein promoter’ for endosperm-specific expression was reported by Aluru et al (2008). Data showed an increase of total carotenoids of up to 34-folds with a preferential accumulation of β-carotene in the maize endosperm. Zhu et al (2008) reported combinatorial nuclear transformation – a novel method for the rapid production of multiplex-transgenic plants and for modifying a complex metabolic pathway in maize. They introduced five carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. Distinct metabolic phenotypes were observed, which also allowed the identification of complementing rate-limiting steps in the pathway. This process allowed the generation of plants with extraordinary levels of β-carotene and other carotenoids, including complex mixtures of hydroxyl carotenoids and keto-carotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes. Naqvi et al (2009) transformed elite inbred South African transgenic corn plants in which the levels of vitamins were shown to increase specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-folds the normal amount of β-carotene, 6-folds the normal amount of ascorbate, and double the normal amount of folate where the trait was found to be stable at least through to the T3 homozygous generation.
The introduction of the crtB (phytoene synthase) gene from E. uredovora in Brassica napus under the control of seed-specific napin promoter was shown to result in a 50-folds increase in the total carotenoid level (Shewmaker et al 1999). Surprisingly, the predominant compounds accumulating in the seeds were α- and β-carotene, and not lutein, which is the predominant carotenoid in non-transformed control seed. While sterol levels remained same, tocopherol and chlorophyll levels were significantly reduced in the transgenic seed. Ravanello et al (2003) tested three gene construct carrying the additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene β-cyclase (crtY) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. The transgenic seeds from two genes construct including bacterial crtB and the plant lycopene β-cyclase showed an increase in the levels of total carotenoid which was similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of β- to & carotene compared to the original construct. However, the β – to α-carotene ratio increased from 2:1 to 3:1 when a three-gene construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. It allows further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values. In 2008, Yu et al used RNAi approach for carotenoid enhancement by down regulating the lycopene epsilon cyclise gene and found increased levels of β-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein.
Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls, lutein, antheraxanthin, violaxanthin, and xanthophyll esters. However, none of these carotenoids have provitamin A activity. Romer et al(2002), observed dramatic increase in the zeaxanthin content and the total tuber carotenoid content up to 5.7-folds due to the down-regulation of zeaxanthin epoxidase in the tubers. Similarly, Diretto et al (2006), silenced the lycopene ε-cyclase (ε-LCY) by Agrobacterium mediated transformation of an antisense fragment of this gene under the control of the patatin promoter. The transgenic tubers thus produced showed a significant increase in β-carotenoid levels where the β-carotene showed a maximum increase of about 14-folds while the total carotenoids increased up to 2.5-folds. Interestingly, these changes were not accompanied by a decrease in lutein, thereby suggesting that ε-LCY is not rate limiting for lutein accumulation. Subsequently, the non-heme β-carotene hydroxylases CHY1 and CHY2 in the tuber were also silenced where CHY silenced tubers showed more dramatic changes in carotenoid content than ε-LCY silenced tubers, with β-carotene increasing up to 38-folds and total carotenoids up to 4.5-folds. These changes were accompanied by a decrease in the immediate product of β-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, violaxanthin and neoxanthin. Together with ε-cyclization of lycopene, β-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis (Diretto et al 2006).
Metabolic engineering of plant carotenoids has been achieved through different strategies such as “push strategies”, in which a gene encoding a rate-limiting step in the pathway is overexpressed, and “block strategies”, relying on the silencing of a biosynthetic step situated immediately downstream of the compound whose levels are to be increased. Transgenic potato plants have been produced by expressing Erwinia uredovora crtB gene encoding phytoene synthase under the control of tuber-specific promoter (Ducreux et al 2005). In these tubers, violaxanthin, lutein, antheraxanthin, and β-carotene were found as major carotenoids. Diretto et al (2006), transformed potato with three genes of bacterial mini pathway, phytoene synthase (crtB), phytoene desaturase (crtI) and lycopene β-cyclase (crtY) from Erwinia, under tuber specific or constitutive promoter. Expression of all three genes, under tuber specific promoter, resulted in tubers with a deep yellow (‘‘golden’’) phenotype without any adverse leaf phenotypes.
Legumes provide an excellent combination of nutrients for a balanced human diet. They alone contribute 33 % of the dietary protein needs of humans (Graham and Vance 2003). For most of the legumes, the major carotenoids detected include β-carotene, lutein and cryptoxanthin. Lutein is found in most of the legumes and is clearly the major carotenoid, followed by other carotenoids like β-carotene and cryptoxanthin. Among the legumes, lutein can make up for over 60% of the sum of the total carotenoids, whereas β-carotene and cryptoxanthin were detected in (<20%) low proportions (Siong et al 1995). In the case of groundnut, maximum concentration of carotenoid occurs in the immature kernels and diminishes as the seeds advances to maturity (Pattee et al 1969). Since plant breeders have not yet found any wild and mutant lines with high β-carotene content in the groundnut to be used in breeding, genetic engineering may be the only approach to enhance β-carotene in this crop.
Keeping in the view the limitations of breeding to produce high β-carotene groundnut, studies have been carried out to enhance its provitamin A of the β-carotene content to combat malnutrition through genetic transformation (Sharma and Anjaiah 2000). Preliminary results indicated 100-folds increase in β-carotene content as compared to the untransformed plants in the case of groundnut. In-house cloning was carried out for the amplification of the phytoene synthase gene from cDNA of the maize (Zm psy1) and it was fused with constitutive promoter or the oil body-specific oleosin promoters (Sharma KK unpublished results). Following molecular characterization, and on the basis of total carotenoids and HPLC data, 14 transgenic events were selected and advanced to T2 generation. The β-carotene levels ranged from 0.02-0.72 μg/gm in T1 seeds which is a decent 70-folds increase than the untransformed control plants. Molecular and biochemical data showed the stability of gene in advanced generation with enhanced β-carotene levels.
Other associated carotenoids including lutein, zeaxanthin and β-cryptoxanthin have been also found to increase by 10-30 folds in transgenic as compared to control. Several transgenic events of groundnut were found to accumulate high levels of β-cryptoxanthin (0.51-9.45 μg/g). Semi-quantitative RT-PCR was performed at different seed stages of control and selected transgenic events indicated that the products of lycopene cyclase and phytone synthase genes were found to be present in mature seeds of transgenic plants and absent in the control, whereas phytoene desaturase was present in both types, although the level of expression varied at different stages of seed development. The second generation transgenic events carrying the Zm psy1 and tomato β-lycopene cyclase gene have also been developed where, β-carotene levels were enhanced multi folds (0.75- 5.5 μg/g) when compared to the untransformed controls (0.01-0.03 μg/g) (Sharma KK unpublished results).
At ICRISAT, initially a single psy1 gene from maize was used to develop transgenic pigeon pea for enhanced level of β-carotene using the Zm psy1 gene driven by the oleosin promoter through Agrobacterium-mediated genetic transformation. Over 140 putative transgenic pigeon pea events with maize psy1 were developed and characterized at the molecular level for the integration and expression of the transgenes (Sharma KK unpublished results). Total carotenoids content in seeds from the primary T0 putative transgenic pigeon pea plants were estimated spectrophotometrically and two-fold increases in total carotenoid content were observed in several transgenic events over the non-transgenic (control) pigeon pea plant. These 11 events showed 2 to 3-folds increases in β-carotene levels (6-11 μg/g in transgenic events, in contrast to 2 μg/g in the untransformed control) evidenced using HPLC analysis. Studies also indicated that the transgenic pigeon pea events had much higher lutein content over the controls amongst the individual carotenoids. Besides β-lycopene cyclase gene was cloned from tomato and used in combination with the psy1 gene under the control of CaMV35S promoter to further improve β-carotene levels in transgenic groundnut. Efforts are underway to develop marker-free pigeon pea transgenic plants carrying both maize psy1 and tomato β-lyc genes to meet the target levels of β-carotene in this important pulse crop (Sharma KK unpublished results).
Transgenic Arabidopsis over-expressing D-galacturonic acid reductase (GalUR) driven by constitutive 35S CaMV promoter has demonstrated its ability to increase vitamin C levels by at least 2-3 folds over non-transgenic control (Agius et al2003), This is also possible in other plant species where D-galacturonic acid is present in all plant species as cell wall component of pectins. Chen et al (2003), observed that overexpression of DHAR (dehydroascorbate reductase) in plants increase the level of ascorbic acid through improved ascorbate recycling, a DHAR cDNA from wheat was isolated and expressed in tobacco and maize, where DHAR expression was increased up to 32- and 100-folds, respectively. The increase in DHAR expression increased foliar and kernel ascorbic acid levels 2- to 4-folds and significantly increased the ascorbate redox state in both tobacco and maize. In addition, the level of glutathione, the reductant used by DHAR, also increased. These results demonstrate that increasing the expression of the enzyme responsible for recycling ascorbate can elevate the content of vitamin C in plants.
ADVANTAGES AND LIMITATIONS OF FOOD FORTIFICATION
Being a food based approach food fortification has several advantages over other interventions as it does not necessitate a change in dietary patterns of the population, can deliver a significant proportion of the recommended dietary allowances for a number of micronutrients on a continuous basis, and does not call for individual compliance. It could often be dovetailed into the existing food production and distribution system, and therefore, can be sustained over a long period of time.
If consumed on a regular and frequent basis, fortified foods will maintain body stores of nutrients more efficiently and more effectively than will intermittent supplements. Fortified foods are also better at lowering the risk of the multiple deficiencies, an important advantage to growing children who need a sustained supply of micronutrients for growth and development, and to women of fertile age who need to enter periods of pregnancy and lactation with adequate nutrient stores.
The limitations of food fortification are also well known: food fortification alone cannot correct micronutrient deficiencies when large numbers of the targeted population, either because of poverty or locality, have little or no access to the fortified food, when the level of micronutrient deficiency is too severe, or when the concurrent presence of infections increases the metabolic demand for micronutrients. In addition, various safety, technological and cost considerations can also place constraints on FF interventions. Thus proper food fortification programme planning not only requires assessment of its potential impact on the nutritional status of the population but also of its feasibility in a given context. Further, it needs to be controlled by appropriate legislation.