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Biofortification of crops with nutrients: factors affecting

utilization and storage
Joana Dı́az-Gómez1, Richard M Twyman2, Changfu Zhu3,
Gemma Farré3, José CE Serrano4, Manuel Portero-Otin4,
Pilar Muñoz3, Gerhard Sandmann5, Teresa Capell3 and
Paul Christou3,6

Biofortification is an effective and economical method to Introduction

improve the micronutrient content of crops, particularly staples Nutrients in the human diet ultimately come from plants,
that sustain human populations in developing countries. but all our major food crops lack certain essential micro-
Whereas conventional fortification requires artificial additives, nutrients (vitamins and minerals) [1]. The endosperm of
biofortification involves the synthesis or accumulation of cereal staples such as rice, wheat and maize are the most
nutrients by plants at source. Little is known about the relative important source of calories for humans, providing 23%,
merits of biofortification and artificial fortification in terms of 17% and 10% of total global calories, respectively [2].
nutrient bioaccessibility and bioavailability, and much depends However, endosperm tissue lacks sufficient amounts of
on the biochemical nature of the nutrient, which can promote or vitamins (particularly vitamins A, E, C and folate) and
delay uptake, and determine how efficiently different nutrients minerals (particularly iron, zinc and selenium) [1,3]. Iron
are transported through the blood, stored, and utilized. Data and zinc deficiencies affect more than 50% of the human
from the first plants biofortified with minerals and vitamins population, resulting in poor growth and development, an
provide evidence that the way in which nutrients are presented impaired immune system, fatigue, muscle wasting, steril-
can affect how they are processed and utilized in the human ity and even death [2,3]. More than four million children
body. The latest studies on the effects of the food matrix, worldwide suffer from severe vitamin A deficiency
processing and storage on nutrient transfer from biofortified (VAD), including 250 000–500 000 per year who become
crops are reviewed, as well as current knowledge about partially or totally blind [4]. Women have a higher
nutrient absorption and utilization. demand for vitamin A during pregnancy, and currently
more than 20 million pregnant women in developing
Addresses countries suffer from VAD [4].
1
Department of Food Technology, University of Lleida—Agrotecnio
Center, Lleida, Spain
2 Strategies to address micronutrient deficiency include
TRM Ltd., York, UK
3
Department of Plant and Forestry Science, University of dietary diversification, nutritional supplements, fortifica-
Lleida—Agrotecnio Center, Lleida, Spain tion and biofortification [1–3]. A combination of
4
Department of Experimental Medicine, University of Lleida— approaches is likely to provide the greatest overall bene-
Biomedical Research Institute of Lleida (IRB Lleida), Lleida, Spain fit, but in some populations dietary diversification is
5
Biosynthesis group, Department of Molecular Biosciences,
J. W. Goethe University, Frankfurt, Germany
impractical and supplements are only suitable as short-
6
ICREA, Catalan Institute for Research and Advanced Studies, term interventions [2,3]. Fortification requires the addi-
Barcelona, Spain tion of nutrients to food products, for example, iodine is
added to table salt, and iron, zinc and folate are added to
Corresponding author: Christou, Paul ([email protected])
flour to make bread [2,3]. One major drawback of these
approaches is the limited stability of the additives, for
Current Opinion in Biotechnology 2017, 44:1–9 example, folate added to rice becomes more soluble at
This review comes from a themed issue on Plant biotechnology higher temperatures and is lost when the rice is boiled [2].
A second disadvantage is that additives can also affect the
Edited by Hans de Steur, Dominique van der Straten and
Teresa B Fitzpatrick quality of food, for example, iron additives are oxidized
over time and this has an impact on taste [3]. The third
and major limitation of conventional fortification is that it
is mainly suited to developed countries with the neces-
http://dx.doi.org/10.1016/j.copbio.2016.12.002 sary technical infrastructure and distribution networks,
0958-1669/Published by Elsevier Ltd. but is less appropriate for developing countries with their
extensive reliance on subsistence agriculture [2]. Biofor-
tification can address all three issues by facilitating the
development of nutrient-dense staple crops that can be
grown and distributed using existing agricultural practices
[3,5].

www.sciencedirect.com Current Opinion in Biotechnology 2017, 44:1–9

2 Plant biotechnology

Biofortification is well established in principle but there Box 2 The measurement of bioaccessibility and bioavailability.
are few practical examples of deployment thus far. Zinc- Nutrient bioaccessibility is usually determined in vitro, whereas
enriched rice and wheat have recently been deployed in nutrient bioavailability can be assessed either in vitro or in
Bangladesh and China, respectively; an orange sweet vivo. Solubility and dialysis assays measure bioaccessibility in vitro
potato rich in provitamin A carotenoids has been released based on the proportional solubility of a nutrient in a test sample, and
by equilibrium dialysis, respectively [23]. They only measure the
in Mozambique and Uganda; and provitamin A rich maize basic properties of nutrients and do not simulate the gut environ-
has been released in Zambia and Nigeria [5]. Golden Rice ment. Gastrointestinal models have been developed for the latter
II, the first transgenic biofortified crop engineered with purpose and can simulate parameters such as saliva flow, peristalsis
provitamin A carotenoids in the endosperm, has incurred and intestinal pH [23,63]. Samples can be collected at any time
during digestion, and bioavailability can be measured in addition to
multiple delays in terms of deployment. It is currently
bioaccessibility when coupled to intestinal cells. However, such
being backcrossed into locally adapted varieties in the models usually do not incorporate homeostatic mechanisms,
Philippines, Indonesia, India and Bangladesh [5]. Multi- intestinal bacteria and hepatic metabolism [23,62]. Caco-2 cells are
vitamin corn (registered as the protected variety epithelial cells derived from a human colonic adenocarcinoma and
Carolight1 in Spain) was developed by transforming an they behave very much like intestinal cells when grown on plastic
surfaces or Transwell inserts [23]. Caco-2 cells form a polarized
elite white-endosperm South African inbred line with monolayer that accurately models the physical and biochemical
four genes representing three different vitamin biosyn- barriers in the gut, thus providing a more realistic model of absorp-
thesis pathways, increasing the levels of b-carotene, other tion including the secretion of chylomicrons, although certain
carotenoids, vitamin C and folate [6]. Carolight1 also aspects are missing such as other epithelial cell types, mucin and
contains a Bacillus thuringiensis (Bt) gene making it pest biofilms [23,62].

resistant [7]. Biofortification is a sustainable approach Bioaccessibility and bioavailability can be investigated in vivo in
which can bring nutritious staple crops to populations humans or animal models, including complete balance studies
(which estimate the bioaccessible or bioavailable fraction by com-
that are difficult to supply with supplements or fortified paring ingested and excreted quantities) and tissue analysis
food products, and once the crop is developed there are no (including plasma/serum fractions). Humans are more appropriate for
recurring costs other than those associated with normal in vivo studies caused by the well-documented differences in
agriculture. However, it is necessary to consider the intestinal absorption mechanisms among rats, chickens and humans
[64] although such methods are laborious and expensive and
efficiency of nutrient delivery by biofortified crops com-
restricted by ethical constraints [63].
pared to other interventions in order to determine the
long-term benefits of this approach. Data from the first
biofortified crops are now available to allow such compar-
isons (Boxes 1 and 2).
consumption of minced/chopped spinach rather than
Fate of nutrients produced in plants whole leaves both as raw tissue [8] and after microwaving
The fate of organic nutrients in plant tissues is highly [9]. Dietary fibers such as cellulose, lignin, pectin and
dependent on their solubility and their affinity for the alginate do not appear to affect folate bioavailability [10].
constituents of the plant tissue matrix. Baking causes the loss of endogenous bread folates
(40%) as well as added synthetic folic acid (30–60%).
Folate Furthermore, the bread matrix inhibits folate absorption
Folate is soluble in water and is easily released from the [11].
matrix, thus plasma folate levels are higher following the
Carotenoids
In contrast, the bioavailability of fat-soluble nutrients
Box 1 Glossary. appears to be much more dependent on associations with
 Supplementation is the oral delivery of micronutrients in the form
matrix components and other dietary constituents, as
of pills, or powdered formulations that are dissolved before admin- shown for the six major dietary carotenoids (b-carotene,
istration [3]. a-carotene and b-cryptoxanthin with provitamin A activ-
 Fortification is the practice of deliberately increasing the content of ity, lycopene, lutein and zeaxanthin without provitamin A
an essential micronutrient and thus improving the nutritional quality of
activity) [12]. Carotenoids are associated with proteins in
food, for example, iron and zinc added to flour, and iodine added to
table salt [3,61]. many green leafy vegetables, whereas in carrots and
 Biofortification is the process by which the nutritional quality of tomatoes they are also stored in a semi-crystalline form
food crops is improved through agronomic practices, conventional [13,14]. Cooking, food processing, and the enzymatic
plant breeding, or modern biotechnology [61]. processes during digestion weaken the cell walls and
 Bioaccessibility is the amount of an ingested nutrient that is
released from the food matrix in the gastrointestinal tract and
disrupt the protein–carotenoid complexes, promoting
becomes available for absorption [23,62]. release and increasing bioavailability [15]. The bioavail-
 Bioavailability is the amount of an ingested nutrient that is avail- ability of carotenoids appears to depend on food particle
able for utilization or for storage, including gastrointestinal digestion, size, with more efficient absorption from smaller food
absorption, metabolism, tissue distribution, and bioactivity [23,62].
particles produced by homogenization, grinding, or
milling. The bioavailability of carotenoids after release

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 Biofortification of crops with nutrients Dı́az-Gómez et al. 3

is favored by the presence of fats because carotenoids are The role of the food matrix, food processing
incorporated into lipid droplets before entering the and storage
micelles, whereas soluble fibers, sterols and stanols Food matrix
inhibit the absorption of carotenoids [13,16–18]. The The major role of the food matrix in terms of nutrient
inhibitory effect of fibers may reflect the higher viscosity bioaccessibility and bioavailability is to trap the nutrients
of fibrous solutions, the formation of gel aggregates, the within cells or subcellular compartments, and to provide
incomplete hydrolysis of triacyl glycerols, or carotenoid constituents that interact chemically with specific nutri-
aggregation [19]. Carotenoids are lipophilic and may also ents to either encourage or delay their release, leading to
compete with plant sterols and stanols for solubilization in their classification as absorption promoters and inhibitors
mixed micelles [18]. (Table 1). Lipid food components increase the bioacces-
sibility of fat-soluble nutrients, so cooking methods that
preserve fats (e.g., frying) tend to outperform methods
Vitamin E that disperse them (e.g., boiling) in terms of promoting
Vitamin E comprises eight fat-soluble molecules (a, b, g the bioaccessibility of nutrients such as b-carotene, as
and d tocopherol and the corresponding tocotrienols, with recently shown for biofortified cassava [30]. Similarly,
a-tocopherol possessing the greatest biological activity) b-carotene bioaccessibility increased by threefold–five-
and like carotenoids its bioavailability is therefore highly fold in a transgenic biofortified sorghum line when the
dependent on interactions with the food matrix [20,21]. lipid content was increased from 5% to 10% [31]. Inhi-
Accordingly, the bioaccessibility of vitamin E varies bitors such as phytate, oxalate and polyphenols reduce
extensively in different types of food, ranging from the bioaccessibility of iron and zinc by forming insoluble
0.47% in apple to almost 100% in banana, white bread complexes. Transgenic maize, rice and sorghum with
and lettuce. Interestingly, the bioaccessibility of lower phytate levels in the seeds have been developed
a-tocopherol was similar to that of g-tocopherol when to address this issue [32]. Biofortification is advantageous
sourced from almonds, wheat germ, cheese or hazelnuts, for iron nutrition because plants can be engineered to
but a-tocopherol was more bioaccessible than g-tocoph- maximize bioaccessibility. In contrast, standard fortifica-
erol when sourced from banana, bread, lettuce and milk. tion is achieved using sparingly soluble iron compounds
This may reflect the food matrix effect, which determines to avoid an undesirable metallic taste, but the bioavail-
the location of tocopherols, their physicochemical state, ability of such compounds is low [33]. Agronomic inter-
and the co-presentation of absorption effectors such as ventions are short-term strategies that focus on the use of
fibers, fats, sterols and stanols [22]. soil and foliar mineral fertilizers, but regular applications
are required [34]. In maize, rice and wheat, foliar fertili-
zation achieves higher levels of zinc accumulation than
Calcium and iron soil fertilization [35]. Mineral biofortification is most
The bioavailability of minerals is affected by the food efficient when cereals are not consumed as flours, for
matrix, intrinsic chemical properties such as the oxidation example, rice grain. Accordingly, zinc in rice grains bio-
state and counter-ion, and also by co-presented food fortified using zinc-rich fertilizer is absorbed to a similar
substances, because all of these factors can affect solubil- extent as the same rice variety fortified artificially with
ity [23]. Calcium must be solubilized before it can be zinc immediately before consumption [36].
absorbed. The extracellular calcium concentration
depends on intestinal absorption, kidney reabsorption Food processing
and bone resorption/formation, which are regulated by Food processing can enhance the bioaccessibility and
the calcium sensing receptor (CaSR) located in the para- bioavailability of nutrients by removing inhibitors or
thyroid gland [24]. The absorption of calcium is highly releasing nutrients from the food matrix (Table 2) but
dependent on the abundance of phytate and oxalate, it can also reduce nutritional value. For example, most
which can combine with calcium to form insoluble com- cereal grains are dehulled and milled before consump-
plexes [25,26]. Calcium also forms complexes with tion, causing significant losses of minerals [32] and certain
proteins, so cooking can help to release calcium for vitamins [37]. Carotenoid levels tend not to be affected by
absorption, but the cooking method is important because light milling, but greater losses are caused by heavy
the soluble calcium leaches into water used for boiling, milling [38,39]. Genetic engineering strategies that
but is retained during baking [27]. Vitamin D is also promote the accumulation of nutrients in the endosperm
required for calcium absorption [28]. Similarly, iron in rather than the bran or husk are therefore useful to
meat and fish is relatively easy to absorb because of its increase the nutritional value of polished grains [40], as
favorable oxidation state and its storage in the form of shown by the expression of enzymes that promote the
ferritin–iron complexes that release the mineral readily, synthesis of phytosiderophores in rice, leading to the
whereas some dietary proteins (such as albumin, casein, modulation of endogenous metal transporter gene expres-
phosvitin and conglycinin) and certain plant polyphenols sion and the mobilization of zinc and iron from the bran to
can reduce the bioavailability of iron [23,29]. the endosperm [41]. Cooking and thermal food processing

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4 Plant biotechnology

Table 1

Relationship between micronutrients and the food matrix

Carotenoids [14,26]
 In photosynthetic plant tissues, carotenoids are bound to proteins in the inner chloroplast membrane, whereas in other tissues, such as fruits and
endosperm, they are mainly found in chromoplasts. Carotenoids accumulate in the plastoglobuli of chloroplasts and chromoplasts, but in the latter
they are also deposited as crystals
 Cell walls, carotenoid–protein complexes and fibers trap carotenoids and inhibit absorption
 The type and amount of fat can influence carotenoid absorption by promoting the excretion of bile salts, enhancing micelle formation and
carotenoid solubilization
 Xanthophylls are more hydrophilic than carotenes and are thus easier to absorb
 Carotenoid cis isomers are more easily absorbed than trans isomers due to their greater polarity and solubility

Iron and zinc [32,33,49,65]

 Iron and zinc are chelated by other food components for transport (e.g., nicotianamine, peptides, proteins and organic acids). Proteins can also
enhance absorption
 Heme iron and organic zinc complexes are more readily absorbed than non-heme iron and inorganic zinc salts. The absorption of non-heme iron
and zinc from plant-based foods can be enhanced by consuming meat, poultry, fish or seafood in the same meal
 Phytate, oxalate, phenolic compounds and fibers form insoluble complexes with iron and zinc. The efficiency of complex formation depends on the
chemical properties of the mineral, the pH and the presence of other compounds. Phytate binds preferentially to calcium and iron, limiting their
bioavailability but increasing zinc bioavailability
 Organic acids (such as ascorbate and citrate) and cysteine promote iron absorption. EDTA can promote iron and zinc absorption
 Calcium can compete with iron for intestinal absorption; its effect on zinc has not been determined

Water-soluble vitamins [14,65]

 Vitamins that form complexes in the food matrix are absorbed less efficiently than free vitamins
 Vitamins B1, B3, B6 and B9 can become trapped in the insoluble part of the food matrix in certain plant foods, reducing their bioaccessibility
 Dietary fiber does not have a significant impact on the absorption of water-soluble vitamins

methods such as pasteurization can destroy heat-sensitive additives in the standard commercial maize diet [46].
organic nutrients such as folate and B6 group vitamins, Transgenic biofortified cassava also preserves the bioac-
and the cooking method can also encourage leaching, but cessibility of provitamin A carotenoids after processing,
the bioaccessibility of other nutrients can increase when with a greater efficiency of b-carotene transfer to micelles
they are released from the plant matrix by cooking. (30–45%) than non-transgenic cassava (27–31%) [47]. In
Transgenic biofortified cassava provides sufficient bio- contrast, the transfer of b-carotene to micelles in trans-
available vitamin B6 after cooking: the leaves and roots genic sorghum was less efficient (1–5%) than in non-
retain, respectively, ninefold and fourfold more non- transgenic sorghum (6–11%) [31]. Several studies have
phosphorylated B6, than non-transgenic cassava [42]. highlighted the importance of genotype-specific effects
Transgenic biofortified rice meets folate requirements on the retention of carotenoids during identical proces-
even after cooking losses of 45% (100 g of rice contains sing treatments, probably reflecting differences in the
500 mg of folates) [43]. Interestingly, only 43% of the food matrix [30,38,45,47]. The impact of cooking on
original content of provitamin A carotenoids was retained the retention of b-carotene also varies according to the
in fortified rice grains after cooking, whereas iron, zinc, genotype [30,38,45], and genotype has a greater
folic acid and vitamin B12 levels usually remained above effect on the quantity of b-carotene in the micelle frac-
80% of the original value [44]. In contrast, b-carotene tions than on the retention of b-carotene after processing
was retained when biofortified maize [38,39] and [47].
biofortified pumpkin [45] were cooked (>72% and
>78%, respectively) suggesting that biofortification pre- Storage
vents the loss of provitamin A carotenoids during cooking The stability of nutrients during storage is also an impor-
more effectively than standard fortification, probably tant consideration because biofortified maize loses a
caused by the food matrix effect. In hens fed on trans- greater quantity of carotenoids during post-harvest stor-
genic biofortified maize, provitamin A carotenoids are age than during cooking [38]. As discussed above for
preferentially diverted to the liver, whereas non-provita- cooking and processing, genotype has an important
min A carotenoids accumulate in the egg, in some cases impact on carotenoid stability during storage [48]. More-
doubling the initial concentration in the feed. When over, maize genotypes which lose more carotenoids
non-provitamin A carotenoids were supplied as intrinsic during drying tend to lose less during storage [49].
components of the transgenic biofortified maize, these Biofortified cassava was more susceptible to carotenoid
nutrients were more efficiently absorbed than carotenoid losses during storage than white cassava with added red

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 Biofortification of crops with nutrients Dı́az-Gómez et al. 5

Table 2

Effect of processing on the micronutrient content of food

Carotenoids [14,26,49,66]
Drying Can reduce carotenoid levels but this depends on the drying method, the temperature/time combination and the genotype of the
plant source
Storage Can cause the loss of carotenoids, but this depends on the crop species, genotype and storage conditions
Milling Increases carotenoid bioavailability because the food particle size is reduced
Blanching Enhances carotenoid retention because it inactivates peroxidases that can lead to the formation of undesirable colors and
flavors, and carotenoids are released from carotenoid–protein complexes
Fermentation Does not usually affect carotenoid retention, and can also remove inhibitors and/or favor the accumulation of nutritional
promoters
Nixtamalization Defined as soaking maize grains in an alkaline solution, which can reduce carotenoid levels. Bioaccessibility depends more on
the subsequent processing/cooking method
Heating Can increase bioaccessibility by releasing carotenoids from plant tissues and disrupting carotenoid–protein complexes,
although this depends on the plant source and the cooking method. Boiling and steaming retain more carotenoids than baking
and frying
Exposure to light and long-term heating induces trans-to-cis isomerization resulting in the loss of provitamin A activity, although
photoxidation is the main factor responsible of carotenoid isomerization
Iron and zinc [32,49,65,67]
Dehulling Reduces the level of inhibitors that prevent mineral uptake. This process removes the bran, reducing the amount of fiber and
phenolic compounds, but phytate levels still remain high because phytate is also present in the germ. Significant quantities of
minerals can also be lost, for example, up to 50% of the iron in some grains, whereas zinc losses are more variable
Milling Degrades the cell wall, allowing minerals to interact with other components. Iron, zinc and phytate levels are reduced by milling,
but the remaining iron and zinc is more bioavailable
Soaking Can reduce phytate levels by solubilizing phytate or activating endogenous phytases. However, blanching and soaking also
cause the leaching of minerals
Fermentation Can degrade phytate through the action of microbial phytases. Fermentation can also enhance iron and zinc absorption because
low-molecular-weight organic acids are produced during this process. The phytate content is reduced more during fermentation
than during cooking
Nixtamalization Can reduce iron absorption by competing with calcium, although it may also improve zinc and iron absorption by reducing the
phytate content
Heating Can enhance mineral absorption by softening the cell walls and removing inhibitors. Minerals are heat-stable, although losses
can occur due to leaching. The bioaccessibility of iron is affected more than zinc by the cooking method
Water-soluble vitamins [14,49,65]
Drying Can result in the loss of vitamins, especially air drying which promotes oxidation
Storage Does not appear to affect water-soluble vitamins, except B vitamins in long-term storage, and vitamin C due to oxidation during
storage
Dehulling The grain components are separated, resulting in significant losses of certain B vitamins that accumulate in the bran and germ
Milling
Soaking Reduces the levels of water-soluble vitamins by leaching
Blanching Inactivates enzymes that oxidize vitamin C but also encourages the loss of vitamin C by leaching
Fermentation Can increase the bioavailability of certain B vitamins (e.g., B2 and B3)
Nixtamalization Can reduce the content of certain B vitamins (e.g., B1 and B3), but in some cases the remaining quantity can become more
bioavailable (e.g., B3)
Cooking Can release vitamins from the food matrix but can also destroy heat-sensitive vitamins (B1, B2 and C), although this depends on
temperature/time combinations. The greatest losses during cooking occur due to vitamins leaching into the water, so steaming
is preferred to boiling

palm oil, suggesting that fatty acids can prevent caroten- Downstream behavior of absorbed nutrients
oid degradation [50]. Transgenic biofortified rice with Nutrient supplements and fortified foods are provided in
improved folate stability was recently reported to main- well-controlled doses to avoid toxicity. One concern
tain folate levels for 4 months when stored at 28  C [43]. about biofortification is that dosing would be more diffi-
The sequestration of carotenoids in chromoplasts, which cult to control, but recent studies have shown that the
act as a metabolic sink, can enhance carotenoid levels uptake of nutrients from biofortified crops is regulated at
during storage, as recently shown for transgenic potatoes the level of absorption from the gut, and also at the
(cv. Désirée) in cold storage for 5 months [51]. Neverthe- cellular level and by the modulation of storage reservoirs,
less, when the Phureja cultivar was used as the parental based on the abundance of nutrients already in the body
genotype (high carotenoid content in tubers) instead of and the demand for certain nutrient molecules [53].
Désirée (low carotenoid content in tubers), there were no
significant changes in total carotenoid levels during cold Each vitamin and mineral has a specific transporter that
storage [52]. facilitates its uptake from the gut, but some unrelated

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6 Plant biotechnology

nutrients can also share the same transporter, as shown for must be confirmed before they can be widely deployed.
the sodium-dependent multivitamin transporter that can The bioavailability of nutrients is partly dependent on
mobilize pantothenic acid, biotin, a-lipoic acid and iodine the intrinsic qualities of each nutrient molecule and partly
[54]. In this context, the transport of one nutrient can be dependent on their presentation in the context of the food
inhibited in a concentration-dependent manner by other matrix.
compounds that share the same transporter. Fat-soluble
compounds are also mobilized by lipid transporters that The major difference between biofortification and stan-
vary in specificity. For example, carotenoids are absorbed dard fortification is that the latter involves additives that
via scavenger receptors (class B type 1 and Niemann-Pick are mixed with the food, whereas biofortification embeds
type C1-like 1) that are selective for particular carotenoid the nutrients inside plant cells. The bioencapsulation of
molecules such as lutein [55]. Some nutrients can only be nutrients in this manner can prevent them from leaching
absorbed as a complex with a ligand that is secreted into during cooking and processing, as shown by the direct
the gut. For example, intrinsic factor is secreted by gastric comparison of b-carotene levels after cooking fortified
parietal cells to absorb cobalamin (vitamin B12), poly- and transgenic biofortified rice, but can also enhance the
glutamyl folates must be processed by glutamate carboxy- binding of nutrients to plant proteins and fibers, as shown
peptidase II, and the pancreatic secretion of g-glutamyl for iron and other minerals. The full value of biofortified
hydrolase is necessary to release folate for absorption [56]. crops can therefore be realized only by combining the
adoption of biofortified varieties with the most appropri-
The intestinal uptake of nutrients is adaptively regulated ate food preparation and cooking methods to maximize
by the substrate level in the diet and depends primarily the bioavailability of different nutrients. Moreover, cook-
on the number of transporters in the apical and basolateral ing and storage losses could be reduced by growing crops
cell membranes of endothelial cells. For thiamin, this in which the nutrients are more stable (e.g., transgenic
involves the transcriptional regulation of thiamin trans- folate-biofortified rice). Biofortified crops can help to
porter-2 [57]. The production of nutrient-specific trans- alleviate micronutrient deficiency in at-risk populations
porters is regulated at the level of transcription. High in a sustainable manner. Some biofortified crops (e.g.,
levels of nutrient bioavailability lead to the suppression of rice, maize, cassava and pumpkin) achieve better results
transcription and starvation causes the transporter gene to than others (e.g., sorghum), but rural populations are
be induced. In some cases, specific epigenetic changes accustomed to eating staple crops commonly harvested
have been observed in the promoter of the transporter in their area, so biofortification strategies must be tailored
gene, for example, the oversupply of riboflavin leads to for different communities to achieve the greatest
the epigenetic suppression of the riboflavin transporter improvements in nutritional health.
gene [58].
Acknowledgements
The transport of nutrients after absorption may also be Research at the University of Lleida is supported by MINECO, Spain
regulated. For example, fat-soluble vitamins, carotenoids (BIO2014-54426-P; BIO2014-54441-P, FEDER funds), the Catalan
and v-3 fatty acids (particularly docosahexaenoic and Government (2014 SGR 1296 Agricultural Biotechnology Research Group)
and Recercaixa. J. Dı́az-Gómez thanks the University of Lleida for a
eicosapentaenoic acids) are transported in lipid vesicles pre-doctoral grant.
that require chylomicron assembly and secretion, and
these processes are inhibited when the corresponding References and recommended reading
nutrients are plentiful [59]. Similarly, iron is exported Papers of particular interest, published within the period of review,
from enterocytes via ferroportin and its distribution is have been highlighted as:
limited by the availability of transferrin. Finally, specific  of special interest
intracellular sensors of nutrient bioavailability may regu-  of outstanding interest
late tissue distribution. For example, the absorption of
1. Zhu C, Naqvi S, Gómez-Galera S, Pelacho AM, Capell T,
iron is inhibited by the regulatory protein hepcidin which Christou P: Transgenic strategies for the nutritional
is stored in macrophages. Interestingly, hepcidin synthe- enhancement of plants. Trends Plant Sci 2007, 12:548-555.
sis is sensitive to both circulating iron and intracellular 2. Rawat N, Neelam K, Tiwari VK, Dhaliwal HS: Biofortification
iron stores because the macrophages communicate with of cereals to overcome hidden hunger. Plant Breed 2013,
132:437-445.
hepatocytes to regulate hepcidin release via multiple
indicator proteins, including transferrin and transferrin 3. Gómez-Galera S, Rojas E, Sudhakar D, Zhu C, Pelacho AM,
Capell T, Christou P: Critical evaluation of strategies for mineral
receptor-2 [60]. fortification of staple food crops. Transgenic Res 2010,
19:165-180.
Conclusions 4. Bai C, Twyman RM, Farré G, Sanahuja G, Christou P, Capell T,
The biofortification of staple crops was envisaged as a Zhu C: A golden era—pro-vitamin A enhancement in diverse
crops. In Vitro Cell Dev Biol—Plant 2011, 47:205-221.
sustainable strategy to deliver nutritious food to popula-
5. Saltzman A, Birol E, Bouis HE, Boy E, De Moura FF, Islam Y,
tions that are unsuitable for other intervention measures, Pfeiffer WH: Biofortification: progress toward a more
but the bioavailability of nutrients in biofortified crops nourishing future. Glob Food Secur 2013, 2:9-17.

Current Opinion in Biotechnology 2017, 44:1–9 www.sciencedirect.com

 Biofortification of crops with nutrients Dı́az-Gómez et al. 7

6. Naqvi S, Zhu C, Farré G, Ramessar K, Bassie L, Breitenbach J, carotenoids, folate, iron, magnesium, polyphenols, zinc, and
 Perez Conesa D, Ros G, Sandmann G, Capell T, Christou P: vitamins B6, B12, D, and E. Front Physiol 2012, 3:1-22.
Transgenic multivitamin corn through biofortification of This is a brief review article covering the major methods for the analysis of
endosperm with three vitamins representing three distinct nutrient bioaccessibility and bioavailability.
metabolic pathways. Proc Natl Acad Sci U S A 2009,
106:7762-7767. 24. Zhang C, Miller CL, Brown EM, Yang JJ: The calcium sensing
This was the first report to describe a biofortified food crop in which receptor: from calcium sensing to signaling. Sci China Life Sci
different organic nutrients were improved simultaneously (specifically, 2015, 58:14-27.
vitamin A and other carotenoids, vitamin C and folate). Despite the losses
that could occur during processing, nutrients provided as intrinsic com- 25. Liang J, Han B-Z, Nout MJR, Hamer RJ: In vitro solubility of
ponents could be more stable due to the food matrix effect. calcium, iron and zinc in relation to phytic acid levels in rice-
based consumer products in China. Int J Food Sci Nutr 2010,
7. Zanga D, Capell T, Zhu C, Christou P, Thangaraj H: Freedom-to- 61:40-51.
operate analysis of a transgenic multivitamin corn variety.
Plant Biotechnol J 2016, 14:1225-1240. 26. La Frano MR, de Moura FF, Boy E, Lönnerdal B, Burri BJ:
 Bioavailability of iron, zinc, and provitamin A carotenoids in
8. Castenmiller JJ, van de Poll CJ, West CE, Brouwer IA, biofortified staple crops. Nutr Rev 2014, 72:289-307.
 Thomas CM, van Dusseldorp M: Bioavailability of folate from A comprehensive overview of the bioavailability of organic and inorganic
processed spinach in humans. Effect of food matrix and nutrients in biofortified crops (maize, rice, cassava, beans, wheat, sweet
interaction with carotenoids. Ann Nutr Metab 2000, 44:163-169. potatoes, and pearl millet) produced by Harvest Plus.
An important study which addressed the bioavailability of folate in
spinach prepared in different ways, and one of only a few studies to 27. Repo-Carrasco-Valencia RAM, Encina CR, Binaghi MJ, Greco CB,
look at interactions between nutrients rather than single nutrients in de Ferrer PAR: Effects of roasting and boiling of quinoa,
isolation. The addition of dietary fiber to food containing processed kiwicha and kañiwa on composition and availability of
spinach did not affect the plasma folate response. Moreover, carotenoids minerals in vitro. J Sci Food Agric 2010, 90:2068-2073.
added to the control diet did not affect folate levels.
28. Heaney R: Vitamin D and calcium interactions: functional
9. van het Hof KH, Tijburg LBM, Pietrzik K, Weststrate JA: Influence outcomes. Am J Clin Nutr 2008, 88:541S-544S.
of feeding different vegetables on plasma levels of
carotenoids, folate and vitamin C. Effect of disruption of the 29. Collings R, Harvey LJ, Hooper L, Hurst R, Brown TJ, Ansett J,
vegetable matrix. Br J Nutr 1999, 82:203-212. King M, Fairweather-Tait SJ: The absorption of iron from
whole diets: a systematic review. Am J Clin Nutr 2013,
10. Ristow KA, Gregory J 3rd, Damron BL: Effects of dietary fiber on 98:65-81.
the bioavailability of folic acid monoglutamate. J Nutr 1982,
112:750-758. 30. Berni P, Chitchumroonchokchai C, Canniatti-Brazaca SG, De
 Moura FF, Failla ML: Impact of genotype and cooking style on
11. Öhrvik V, Öhrvik H, Tallkvist J, Witthöft C: Folates in bread: the content, retention, and bioaccessibility of b-carotene in
retention during bread-making and in vitro bioaccessibility. biofortified cassava (Manihot esculenta Crantz)
Eur J Nutr 2010, 49:365-372. conventionally bred in Brazil. J Agric Food Chem 2014,
62:6677-6686.
12. Fernández-Garcı́a E, Carvajal-Lérida I, Jarén-Galán M, Garrido- This study considers interacting factors such as genotype and prepara-
Fernández J, Pérez-Gálvez A, Hornero-Méndez D: Carotenoids tion method rather than solely measuring the total nutrient content of the
bioavailability from foods: from plant pigments to efficient raw product. In addition to the retention of large amounts of b-carotene
biological activities. Food Res Int 2012, 46:438-450. after cooking, the bioaccessibility of lipids also increased, particularly
after frying compared to boiling.
13. Hoffman R, Gerber M: Food processing and the Mediterranean
diet. Nutrients 2015, 7:7925-7964. 31. Lipkie TE, De Moura FF, Zhao ZY, Albertsen MC, Che P,
14. Ball GFM: Vitamins in Foods. Analysis, Bioavailability and Stability.  Glassman K, Ferruzzi MG: Bioaccessibility of carotenoids from
CRC Press; 2006. transgenic provitamin A biofortified sorghum. J Agric Food
Chem 2013, 61:5764-5771.
15. Murador DC, da Cunha DT, de Rosso VV: Effects of cooking In this article, the bioaccesibility of carotenoids in transgenic sorghum
techniques on vegetable pigments: a meta-analytic approach was found to be lower than in non-transgenic sorghum, showing that
to carotenoid and anthocyanin levels. Food Res Int 2014, sorghum performs less well than other biofortified crops. However, an
65:177-183. increase in the lipid content should help to increase the bioaccessibility of
provitamin A carotenoids.
16. Goltz SR, Campbell WW, Chitchumroonchokchai C, Failla ML,
Ferruzzi MG: Meal triacylglycerol profile modulates 32. Raes K, Knockaert D, Struijs K, Van Camp J: Role of processing
postprandial absorption of carotenoids in humans. on bioaccessibility of minerals: influence of localization of
Mol Nutr Food Res 2012, 56:866-877. minerals and anti-nutritional factors in the plant. Trends Food
Sci Technol 2014, 37:32-41.
17. Lakshminarayana R, Baskaran V: Influence of olive oil on the
bioavailability of carotenoids. Eur J Lipid Sci Technol 2013, 33. Moretti D, Biebinger R, Bruins MJ, Hoeft B, Kraemer K:
115:1085-1093.  Bioavailability of iron, zinc, folic acid, and vitamin A from
fortified maize. Ann N Y Acad Sci 2014, 1312:54-65.
18. Yonekura L, Nagao A: Intestinal absorption of dietary An important benchmark study on the bioavailability of both organic and
carotenoids. Mol Nutr Food Res 2007, 51:107-115. inorganic nutrients in maize prepared by standard fortification, allowing
the comparison of biofortified varieties in subsequent studies.
19. Yonekura L, Nagao A: Soluble fibers inhibit carotenoid
micellization in vitro and uptake by Caco-2 cells. Biosci 34. Carvalho SMP, Vasconcelos MW: Producing more with less:
Biotechnol Biochem 2009, 73:196-199. strategies and novel technologies for plant-based food
biofortification. Food Res Int 2013, 54:961-971.
20. Nesaretnam K: Multitargeted therapy of cancer by tocotrienols.
Cancer Lett 2008, 269:388-395. 35. Joy EJM, Stein AJ, Young SD, Ander EL, Watts MJ, Broadley MR:
Zinc-enriched fertilisers as a potential public health
21. DellaPenna D, Mène-Saffrané L: Vitamin E. In Advances in
intervention in Africa. Plant Soil 2015, 389:1-24.
Botanical Research. Edited by Rébeillé F, Douces R. Elsevier Inc.;
2011:179-227. 36. Brnic M, Wegmüller R, Melse-Boonstra A, Stomph T, Zeder C,
22. Reboul E, Richelle M, Perrot E, Desmoulins-Malezet C, Pirisi V, Tay F, Hurrell R: Zinc absorption by adults is similar from
Borel P: Bioaccessibility of carotenoids and vitamin E from intrinsically labeled zinc-biofortified rice and from rice
their main dietary sources. J Agric Food Chem 2006, fortified with labeled zinc sulfate. J Nutr 2016, 146:76-80.
54:8749-8755.
37. Dunn ML, Jain V, Klein BP: Stability of key micronutrients added
23. Etcheverry P, Grusak MA, Fleige LE: Application of in vitro to fortified maize flours and corn meal. Ann N Y Acad Sci 2014,
 bioaccessibility and bioavailability methods for calcium, 1312:15-25.

www.sciencedirect.com Current Opinion in Biotechnology 2017, 44:1–9

8 Plant biotechnology

38. Mugode L, Ha B, Kaunda A, Sikombe T, Phiri S, Mutale R, Davis C, maize (Zea mays L.) genotypes during controlled postharvest
 Tanumihardjo S, De Moura FF: Carotenoid retention of storage. J Agric Food Chem 2016, 64:2727-2736.
biofortified provitamin A maize (Zea mays L.) after Zambian
traditional methods of milling, cooking and storage. J Agric 49. Suri DJ, Tanumihardjo SA: Effects of different processing
Food Chem 2014, 62:6317-6325.  methods on the micronutrient and phytochemical contents of
This article evaluates the impact of different home-cooking techniques, maize: from A to Z. Compr Rev Food Sci Food Saf 2016,
as well as milling and storage, on the retention of provitamin A carote- 15:912-926.
noids in conventional biofortified maize varieties released in Zambia. A recent review article which summarizes the changes in the micronu-
trient content (e.g. provitamin A carotenoids, xanthophylls, anthocyanins
39. Pillay K, Siwela M, Derera J, Veldman FJ: Provitamin A and other polyphenols, water-soluble vitamins, and minerals) of maize
 carotenoids in biofortified maize and their retention during products after applying different processsing methods (e.g. drying, sto-
processing and preparation of South African maize foods. rage, milling, soaking, heating and nixtamalization).
J Food Sci Technol 2014, 51:634-644.
This study examines the retention of provitamin A carotenoids in biofor- 50. Bechoff A, Chijioke U, Tomlins KI, Govinden P, Ilona P, Westby A,
tified maize varieties after milling and cooking. Boy E: Carotenoid stability during storage of yellow gari made
from biofortified cassava or with palm oil. J Food Compos Anal
40. De Steur H, Blancquaert D, Strobbe S, Lambert W, Gellynck X, Van 2015, 44:36-44.
Der Straeten D: Status and market potential of transgenic
biofortified crops. Nat Biotechnol 2015, 33:25-29. 51. Li L, Yang Y, Xu Q, Owsiany K, Welsch R,
Chitchumroonchokchai C, Lu S, Van Eck J, Deng XX, Failla M,
41. Banakar R, Alvarez Fernández Á, Abadı́a J, Capell T, Christou P: Thannhauser TW: The Or gene enhances carotenoid
The expression of heterologous Fe (III) phytosiderophore accumulation and stability during post-harvest storage of
transporter HvYS1 in rice increases Fe uptake, translocation potato tubers. Mol Plant 2012, 5:339-352.
and seed loading and excludes heavy metals by selective Fe
transport. Plant Biotechnol J 2016 http://dx.doi.org/10.1111/ 52. Campbell R, Morris WL, Mortimer CL, Misawa N, Ducreux LJM,
pbi.12637. Morris JA, Hedley PE, Fraser PD, Taylor MA: Optimising
ketocarotenoid production in potato tubers: effect of genetic
42. Li K-T, Moulin M, Mangel N, Albersen M, Verhoeven-Duif NM, background, transgene combinations and environment. Plant
Ma Q, Zhang P, Fitzpatrick TB, Gruissem W, Vanderschuren H: Sci 2015, 234:27-37.
Increased bioavailable vitamin B6 in field-grown transgenic
cassava for dietary sufficiency. Nat Biotechnol 2015, 33:1029- 53. Jeong J, Guerinot ML: Biofortified and bioavailable: the gold
1032. standard for plant-based diets. Proc Natl Acad Sci U S A 2008,
105:1777-1778.
43. Blancquaert D, Van Daele J, Strobbe S, Kiekens F, Storozhenko S,
 De Steur H, Gellynck X, Lambert W, Stove C, Van Der Straeten D: 54. Quick M, Shi L: The sodium/multivitamin transporter: a
Improving folate (vitamin B9) stability in biofortified rice multipotent system with therapeutic implications. Vitam Horm
through metabolic engineering. Nat Biotechnol 2015, 2015, 98:63-100.
33:1076-1078.
A comprehensive study which considers methods to increase folate 55. Sato Y, Suzuki R, Kobayashi M, Itagaki S, Hirano T, Noda T,
stability and demonstrates that the presence of folate-binding proteins Mizuno S, Sugawara M, Iseki K: Involvement of cholesterol
(FBP) achieves higher and more stable concentrations of folate in membrane transporter Niemann–Pick C1-like 1 in the
biofortified rice grains under storage conditions. intestinal absorption of lutein. J Pharm Pharm Sci 2012, 15:256-
264.
44. Wieringa FT, Laillou A, Guyondet C, Jallier V, Moench-Pfanner R,
 Berger J: Stability and retention of micronutrients in fortified 56. Shafizadeh TB, Halsted CH: Gamma-glutamyl hydrolase, not
rice prepared using different cooking methods. Ann N Y Acad glutamate carboxypeptidase II, hydrolyzes dietary folate in rat
Sci 2014, 1324:40-47. small intestine. J Nutr 2007, 137:1149-1153.
This article explores the retention of nutrients (vitamin A, iron, zinc, folic
57. Nabokina SM, Subramanian VS, Valle JE, Said HM:
acid, and vitamin B12) in fortified rice produced using different techniques
Adaptive regulation of human intestinal thiamine uptake by
(hot extrusion, cold extrusion, and coating) and different cooking meth-
extracellular substrate level: a role for THTR-2 transcriptional
ods (absorption method with or without soaking, washing before cooking,
regulation. Am J Physiol Gastrointest Liver Physiol 2013, 305:
cooking in excess water, and frying rice before cooking).
G593-G599.
45. Ribeiro EMG, Chitchumroonchokchai C, de Carvalho LMJ, de
 Moura FF, de Carvalho JLV, Failla ML: Effect of style of home 58. Subramanian VS, Ghosal A, Kapadia R, Nabokina SM, Said HM:
cooking on retention and bioaccessibility of pro-vitamin A Molecular mechanisms mediating the adaptive regulation of
carotenoids in biofortified pumpkin (Cucurbita moschata intestinal riboflavin uptake process. PLoS One 2015, 10:
Duch.). Food Res Int 2015, 77:620-626. e0131698.
This study considers the retention of provitamin A carotenoids in five 59. Wang Y, Lin Q, Zheng P, Li L, Bao Z, Huang F: Effects of
biofortified pumpkin varieties prepared by steaming, boiling in water or eicosapentaenoic acid and docosahexaenoic acid on
boiling in sugar syrup. The study stands out because the bioaccessibility chylomicron and VLDL synthesis and secretion in Caco-2
of provitamin A carotenoids in cooked pulp was determined by measuring cells. Biomed Res Int 2014, 2014:684325.
the transfer to mixed micelles during in vitro digestion and confirmed by
their accumulation in Caco-2 cells. 60. Evstatiev R, Gasche C: Iron sensing and signalling. Gut 2012,
61:933-952.
46. Moreno JA, Dı́az-Gómez J, Nogareda C, Angulo E, Sandmann G,
 Portero-Otin M, Serrano JCE, Twyman RM, Capell T, Zhu C, 61. World Health Organization (WHO). e-Library of Evidence for
Christou P: The distribution of carotenoids in hens fed on Nutrition Actions. http://www.who.int/elena/titles/biofortification/
biofortified maize is influenced by feed composition, en/. [Accessed 18 October 2016].
absorption, resource allocation and storage. Sci Rep 2016,
6:35346. 62. Carbonell-Capella JM, Buniowska M, Barba FJ, Esteve MJ,
A very recent article describing how carotenoids in hen feed are taken up Frı́gola A: Analytical methods for determining bioavailability
and distributed in the tissues of hens, including their eggs. Carotenoids in and bioaccessibility of bioactive compounds from fruits and
biofortified maize are taken up and distributed more efficiently than vegetables: a review. Compr Rev Food Sci Food Saf 2014,
carotenoids provided as feed supplements, indicating a food matrix 13:155-171.
effect.
63. Cardoso C, Afonso C, Lourenço H, Costa S, Nunes ML:
47. Failla ML, Chitchumroonchokchai C, Siritunga D, De Moura FF, Bioaccessibility assessment methodologies and their
Fregene M, Manary MJ, Sayre RT: Retention during processing consequences for the risk-benefit evaluation of food. Trends
and bioaccessibility of b-carotene in high b-carotene Food Sci Technol 2015, 41:5-23.
transgenic cassava root. J Agric Food Chem 2012,
60:3861-3866. 64. Sanahuja G, Farré G, Berman J, Zorrilla-López U, Twyman RM,
Capell T, Christou P, Zhu C: A question of balance: achieving
48. Ortiz D, Rocheford T, Ferruzzi MG: Influence of temperature appropriate nutrient levels in biofortified staple crops. Nutr Res
and humidity on the stability of carotenoids in biofortified Rev 2013, 26:235-245.

Current Opinion in Biotechnology 2017, 44:1–9 www.sciencedirect.com

 Biofortification of crops with nutrients Dı́az-Gómez et al. 9

65. Gibson RS: The role of diet- and host-related factors in A recent review article comparing the retention of provitamin A carote-
nutrient bioavailability and thus in nutrient—based dietary noids (after processing, cooking, and storing) of three biofortified crops
requirement estimates. Food Nutr Bull 2007, produced by Harvest Plus.
28:S77-S100.
67. Pereira EJ, Carvalho LMJ, Dellamora-Ortiz GM, Cardoso FSN,
66. De Moura FF, Miloff A, Boy E: Retention of provitamin A Carvalho JLV: Effect of different home-cooking methods on the
 carotenoids in staple crops targeted for biofortification in bioaccesibility of zinc and iron in conventionally bred cowpea
Africa: cassava, maize and sweet potato. Crit Rev Food Sci Nutr (Vigna unguiculata L. Walp) consumed in Brazil. Food Nutr Res
2015, 55:1246-1269. 2016, 60:29082.

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