SEED PHYTATE, CROPS PERFORMANCE, ANIMAL AND HUMAN HEALTH, AND ENVIRONMENT POLLUTION

Phytic acid (inositol hexaphosphoric acid, InsP6), the predominant form of phosphorus storage, accounts for 65–85% of the total seed phosphorus. It is worth highlighting that phytic acid is a ubiquitous molecule, but we refer in this article to the effects of dietary phytic acids in the gut [1]. High concentration of phytic acid in foods limits micronutrient bioavailability as it binds minerals to form mixed salt phytin [2,3]. The undigested phytin excreted by humans and nonruminant animals significantly pollute soil and cause eutrophication in rivers and lakes [4]. Phytic acid is however critical for seed development, crops establishment and productivity [5]. Phytic acid contributes positively to human health as antidiabetic, antioxidant, anticancer or anti-inflammatory agent, prevent kidney stone formation, protect against atherosclerosis and coronary heart disease or in managing neurodegenerative diseases [6]. InsP6 molecule relative to citric acid exhibit significantly greater inhibitory effect against foodborne pathogens and therefore safe to use as for food preservation, food safety, and for greater shelf-life of the food products [7].

Globally about 2 billion people suffer from malnutrition causing substantial burden to society. Elevated CO₂ in atmosphere increases phytic acid content and reduces the concentrations of essential micronutrients such as Fe and Zn in the grains, resulting nutritionally inferior crop produce, which may further aggravate malnutrition amongst the people largely dependent on grain-based food products [8].

The main biological constraint before crop-genetic enhancers is how much phytate per se can be reduced that is not detrimental to plants and produce is beneficial to human and animal health. They face selecting for low phytate that may reduce overall nutritional quality of seed, as well as if it can be combined with increased grain Fe and Zn. Should they block the phytate biosynthetic pathway or degrade phytate in the developing seeds? Is there genetic variation for seed phytase activity to the extent that can be exploited to develop high seed phytase crops as alternative to fungal phytase to reduce feed cost? How to minimize the negative pleiotropic effects between low phytate—agronomic defects—human non-communicable diseases? Whether feed with varying phytate concentrations impact carcass characteristics of growing pigs or productivity of milking cows and poultry’s egg laying and quality? Whether any adverse effect on flavor and sensory characteristics of bread made from low phytate whole grains? Whether consumers prefer and willing to buying and eating low-phytate-based bakery products? This review article provides some answers to these questions by highlighting the advances in quantifying seed phytate or phytase activity, and how this knowledge assists developing low seed phytate cultivars.

LOW PHYTATE-INDUCED CHANGES IN SEED CHEMISTRY

Sparvoli and Cominelli [9] summarized low phytic acid (lpa) mutants originated through mutagenesis in grain crops with significant alteration in seed-phytate and metabolites, most with few exceptions associated with negative pleiotropic effects (e.g., low seed germination, stunted growth, reduced seed development and weight), thus with limited value to breeders. Crossbreeding between lpa mutants and wild types (WTs) showed predominance of crossing parents on the metabolite profile and imprinting of a specific mutation induced metabolite signature, consistently expressed in the homozygous lpa mutant offspring across generations, genetic backgrounds, and environments [10‒15]. Disruption of OsSULTR3;3 produced significant reduction in both phytic acid (PA) and total phosphorus (P) accompanied by changes in other metabolites and increased concentrations of nutritionally relevant compounds in two brown rice lpa-derived mutants [16].

Sakai et al. [17] noted no changes in total P and other minerals in mature seeds between two different lpa mutants and WT rice. They however observed significant changes in mineral localization within lpa seeds. P and K substantially decreased in the aleurone layer and diffused into the endosperm, while Zn and Cu localized in the narrower space around the aleurone layer. In WT, minerals were broadly distributed from the aleurone layer to the inner endosperm. The transgenic rice with the lpa phenotype also had similar pattern, indicating the role of phytate (InsP6) for the accumulation and distribution of minerals during seed development in rice.

The lpa mutants had no adverse effect on oil and protein contents or any individual amino acids and saturated fatty acids in soybean. Some mutants however consistently showed higher sucrose (47–86%) and lower raffinose (about 25%, 74–84%) and higher total isoflavone content (3039–4305 µg g⁻¹) than WT across environments [17,18].

The lpa1-1 mutation in maize exerted pleiotropic effects, unrelated to phytic acid biosynthesis, on seed density, ions content and on antioxidants in the seed. The flour contained high lignin and protein content, while total starch remained the same, but structure and granule sizes changed [19].

Reducing the seed phytate means more bioavailable minerals for absorption. For example, there were significantly higher Zn absorption by 47–52% in maize and 35–52% in rice as compared to WT [20]. Fe absorption from lpa beans was significantly higher than from their parental lines with normal phytate [21], while Ca absorption enhanced from low phytate maize-based tortillas as compared to WT [21].

Clearly, lpa mutants are useful genetic resource to enhance the bioavailability of minerals. However, some changes in seed chemistry and localization of minerals within lpa seeds were noted in cereal and grain legume crops, thus requiring careful selection of lpa mutants with no adverse impact on nutritional seed quality.

ASSAY FOR IDENTIFYING lpa SEEDS IN MUTAGENIZED OR BREEDING POPULATIONS

A simple and cost-effective high throughput assay is needed to support breeding for low seed phytate crops. The currently available seed phytate assay protocols are destructive in nature and based on quantification of P in the flour obtained by milling the seeds [22‒24]. More recently, a seed density-based method directly identifies the putative lpa mutant seeds in a mutagenized maize (Zea mays) population. It is based on the principle that lpa seeds are lower in density than WT. The lpa seeds float in concentrated sugar solution (density 1.218–1.222 g cm⁻³) due to their lower density, unlike WT sibs that sink. This is non-disruptive, cost effective and timesaving assay that may be used to identify putative lpa seeds when screening mutagenized or early generation breeding populations [25]. In the absence of data available to demonstrate it could work with any lpa mutation in any species, the low-phytate phenotype should, however, initially be evaluated by FT-NIR for faster though destructive assessment [26] or nondestructive assessment by EDXRF [27], and later confirmed by wet seed chemistry in advanced generations.

GENES AND DIAGNOSTIC MARKERS ASSOCIATED WITH LOW SEED-PHYTATE OR HIGH SEED-PHYTASE ACTIVITY IN GERMPLASM POOLS

To date a number of low phytate mutants as well a few germplasms with high seed phytase activity were reported in cereal and food grain legume crops [9,25,28‒35]. Livestock’s raiser often adds fungal phytase to enhance feed value, however, it increases feed cost. Diagnostic markers are genetic tags (a string of DNA) which may be indirectly used to select for specific phenotypes (i.e., plant height, seed weight and quality or disease resistance, among other characteristics) in plant breeding. A few functionally characterized genes associated with low seed phytate, such as Pvmrp1 (orthologous to the lpa genes AtMRP5 and ZmMRP4) in common bean [18], dlMIPS in dolichos bean [36], Mf3, DH2 and CB5 in maize [37], OsITP5/6K-1, MIPS, and SPDT in rice [38‒40], ABCC5 and MIPS in soybean [41,42], and TaIPK1 in wheat [43], or their homologs in other crops, have potential to accelerate the development of food or feed crops low in seed phytate. In addition, the function of putative candidate genes associated with low-seed phytate or high seed-phytase activity in cereals and food grain legumes [44‒50] must be validated prior to use in crop breeding.

DEVELOPING LOW SEED PHYTATE BREEDING POPULATIONS/ADVANCED LINES AND CULTIVARS

Developing low phytate crops that yields well in optimal and stressed field environments is a significant breeding challenge. Plant breeders often face the dilemma about the extent of phytate to reduce in the grains that does not adversely impact growth and productivity of new cultivars but at the same time the produce is beneficial to human health. Progress in breeding or genetically engineered resource use efficient cultivars continues. While there are alternative approaches to deal with the problems posed by seed-derived dietary phytic acid, for example, use of phytase enzyme as feed additive or biofortification breeding. The proof of concept has been demonstrated that breeding for low (moderate reduction) phytate crops is achievable given adequate time and support. Populations at risk for iron and zinc deficiency, especially in developing economies, will benefit from production and consumption of low phytate grains, and find acceptance among farmers for sustainable agricultural production [51]. Both conventional (mutation or crossbreeding aided by applied genomics) and biotechnology (genetic engineering or gene editing) approaches have led to development of several low seed phytate lines yielding at par or 5–10% lower than the normal phytate cultivars as detailed herewith.

Physical and chemical mutagens were used to obtain stable low phytate mutant lines with moderate to severe reduction in seed phytate. When assessed the performance of sets of barley sib lines homozygous for WT or homozygous for one of the four independent lpa mutations (lpa1‐1, lpa2‐1, lpa3‐1, and M955) in ‘Harrington’, Bregitzer and Raboy [28] noted that WT sib sets yielded at par with ‘Harrington’, thereby suggesting presence or absence of lpa alleles as the major constraint to productivity in WT and low phytate lines. All mutations except lpa1‐1 yielded low in rainfed environments, thus suggesting that reduced stress tolerance accounts for low yield in other low phytate mutants. The lpa1-1 mutation being aleurone specific avoided yield loss under rainfed. The superior yield performance of lpa1‐1 was further confirmed when six low phytate barley mutants including lpa1‐1 were evaluated in multi-environment trials. lpa1‐1 had no adverse effects on yield while lpa4-1 resulted in low yield loss (3.5%) across all locations. In optimal environments, lpa1-1, lpa3-1, and lpa4-1 had yields similar to or within 5% range of that for WT sibling isolines [30]. The desirable effect of lpa1‐1—limited to aleurone layer—may be directly introduced in breeding programs to develop cultivars for stress-prone sites while lpa1-1, lpa3-1, and lpa4-1 may be exploited for developing barley cultivars adapted to optimal environments.

The lpa trait increase the nutritional quality of foods and feeds and improve P management in agriculture. These mutations are often accompanied by negative pleiotropic effects leading to agronomic defects —either seed viability and germination or plant growth and development or increase the resistance to cooking—limiting value to the breeders. Thus, achieving lpa mutants endowed with no negative pleiotropic effects on seed germination and field emergence or productivity is still a significant breeding challenge [52].

Low phytic acid enhances bioavailability of micronutrients but may reduce seed germination and yield. Thus, improving seed yield and germination is the major breeding target while developing low phytate grain crops. High and low phytic acid maize populations isolated after three cycles of recurrent selection differed significantly for phytic acid concentration. The lpa population showed improved seed germination and bioavailable Fe was at par with lpa inbred lines. Recurrent selection for phytic acid level is thus a viable option for improving Fe bioavailability while maintaining good seed germination in lpa population [53]. Crossbreeding of lpa mutants with cultivars led to offspring combining high yield and low seed phytate in rice. Phytic acid content among the offspring was however dependent on environment and phytic acid contents of the crossing parents [54]. In a field test involving low phytate offspring derived after three successive backcrosses involving CX1834 (low-phytate) and B019 (normal-phytate) soybean lines, Spear and Fehr [54] identified one low phytate progeny with improved seed germination (equivalent to normal phytate) while other low phytate offspring had reduced germination. Low phytate (LP) soybean lines (56CX-1273; 56CX-1283) had field emergence and seed yield at par with cultivars (normal phytate, NP) ‘5002T’ and ‘Osage’, but with greater seed protein and ultrahigh soymeal protein. No crossover interaction or change in ranks for inorganic P (Pi) suggests that it is achievable to differentiate LPs from NPs irrespective of test environments [55]. The mutated gene may have also different effects in different species. The lpa1 mutant, the only characterized mutant in common bean, is devoid of negative pleiotropic effects normally associated with lpa mutants. It affects the PvMRP1, the gene coding for a putative tonoplastic phytic acid transporter [56]. Further, it was shown that loss of PvMRP1 is compensated by PvMRP2, however, its effect was seen in leaf tissues instead in seeds. The GUS activity in different organs of transgenic Arabidopsis thaliana and Medicago truncatula harboring 1.5 kb portion of the intergenic 5ˈ sequences of both PvMRP genes suggests a refined species-specific mechanism regulating gene expression for PvMRP genes [57].

Low phytate trait in rice has been successfully introgressed into otherwise high phytate ‘Super Basmati’ genetic background (lpa12-3, lpa111-1, lpa141, lpa56-3, lpa53-4, lpa99-2, lpa205-4), with improved seed germination and 4–18% yield advantage over parent cultivar [58].

In maize, a mutant plant with a lpa2-2 allele accumulates less phytic acid in seeds. The microsatellite marker ‘umc2230’ co-segregate with low phytate conferred by lpa2-2 alleles and may be deployed to develop low phytate maize [59]. Using umc2230 and marker-aided backcrossing, four low phytate maize lines with agronomic traits similar to its recurrent parent were bred [60]. A co-dominant cost-effective SNP marker for lpa1-1, caused by a single amino acid change from alanine to valine, differentiated maize inbred lines varying in phytic acid, and upon validation successfully distinguished homozygous dominant (WT) from homozygous recessive (lpa) or heterozygous genotypes at early vegetative phase [61].

A high throughput molecular marker assay detected a perfect association between recessive mutations in soybean genes homologous to the maize lpa1 linked with low seed phytate and identified for the first time a novel lpa2-b allele in a soybean line M766 which may be deployed in breeding for developing low phytate soybean germplasm. Two recessive MRP4/MRP5 mutant loci together confer low phytate phenotype in soybean unlike one single recessive mutant locus sufficient for expression of low phytate in maize. MIPS1 causes modifications to seed phosphorus and carbohydrate contents and improve nutritional value of soybean food and feed [62]. A Kompetitive allele specific PCR (KASP) assay-based marker BARCSOY_11_1495 was 93–97% effective in identifying low phytate mutation, which also differentiated advanced lines varying on seed phytate [63]. KASP assays are simple and cost effective and therefore may be deployed in soybean breeding.

To date a few low seed phytate cultivars such as barley ‘Herald’, ‘Clearwater’, and ‘Sawtooth’ [64‒66] and soybean ‘USDA-N6003LP’ and ‘N6202’ [67,68] have been released for cultivation in USA. These cultivars were developed by crossbreeding, and combine moderate reduction in seed phytate, multiple fold increase in Pi, adaptation, productivity (yielding at par or up to 90% of the control), seed quality, and good field emergence. ‘CDC Raezer’ and ‘CDC Limerick’ field peas combining low seed phytate and resistance to diseases have been released in Canada [69,70].

Quality refers to the variation in physical and chemical characteristics of seeds associated with low seed phytate. A multilocation evaluation of two lpa soybean mutants revealed that lpa did not affect oil and protein contents or protein and oil quality. However, Gm-lpa-TW75-1 had consistently higher sucrose (+47–86%) and low raffinose (−74–84%) contents than WT, while Gm-lpa-ZC-2 had higher total isoflavone content (+3039–4305 µg g⁻¹) than WT. Furthermore, F₃-derived lpa lines had total isoflavone content greater than WT, suggesting that lpa mutant lacks negative effects on seed quality [18].

The lpa soybean mutant Gm-lpa-TW-1-M relative to WT unraveled remarkable metabolic differences. The raffinose oligosaccharides and galactosyl cyclitols were reduced while sucrose and free amino acids were increased. Such induced metabolite changes were nearly unaffected by the crossbreeding involving lpa and WT and consistently expressed over generations and environments, indicating that this mutant is the ideal genetic resource to produce soybean seeds stably exhibiting low phytate trait with no adverse impact on seed metabolite changes in the offspring [10].

Soybean seeds low in phytate, high in sucrose, and low in raffinosaccharides are highly desirable from a nutritional and environmental standpoint. Seed phytate levels are not reduced significantly after mips mutation in double mutant mrp-l/mrp-n as revealed by the analysis of recombinant inbred lines (RILs) resulting from crossing two soybean lpa mutants bearing mips mutation on chromosome 11 (VP-5089), and mrp-n plsu mrp-1 mutations on chromosomes 3 and 19, respectively. The mutant mrp-l/mrp-n reversed, however, the desired high sucrose/low stachyose/low raffinose of homozygous soybeans for the mips/mips). The homozygous mips/mrp-l/mrp-n had 7.7% sucrose, 4.2% stachyose and 0.94% g^-1 in dry seed. The myo-inositol flux in mips seeds may be changed by blocking phytic acid transport—which is affected by mrp-l/mrp-n—from the cytoplasm site where its synthesis occurs to the storage site, thus restoring to the levels noticed in the wild type the raffinosaccharides [71]. Such results demonstrates the effects of these low phytic acids related to mutations regulating seed phytate and oligosaccharides.

Hard-to-cook (HTC) defects of lpa beans relative to the wild types is due to presence of lectin phytohemagglutinin L (PHA-L) in seed. The lack of phytic acid perturb Ca distribution that as a consequence alters the cooking time and the stability of a bean storage protein, a lectin, causing severe gastrointestinal discomforts [72]. Whether lpa beans provide more bioavailable iron than a biofortified bean? The biofortified and lpa beans provide more bioavailable iron (421 and 431 µg vs 278 µg) than control beans (regular iron and PA concentrations) to reduce iron deficiency in healthy Rwandese women with low iron status [73]. Thus, adverse gastrointestinal effects of reduced iron bioavailability should be investigated to factor while developing biofortified or lpa beans.

A pronounced effect on phytic acid and metabolite profile was reported when two lpa rice mutants each carrying myo-inositol kinase mutated genes OsMIK or OsMRP5 were intercrossed. The phytic acid content in resulting double mutant was reduced by −63% relative to −26% or −47% in single mutant, while the metabolite profile was predominated by the OsMIK mutation effect. Crossbreeding of two single-gene lpa rice mutants may generate offspring with significant reduction in phytic acid and a mutant gene-specific metabolite signature [74].

In summary significant progress have been achieved that generating lpa mutants and subsequently transferred the lpa trait into elite genetic background, which may be further used as a genetic resource for crossbreeding with the aim of releasing newly bred cultivars. Availability of genetic tags (DNA markers) and associated high throughput genotyping assay to breeding programs in National Agricultural Research Systems (NARS) may accelerate development and release of low seed phytate cultivars in low- and middle-income countries (LMICs).

GENETICALLY MODIFIED LOW SEED PHYTATE CROPS

Breeding for low phytate either through induced mutations or crossbreeding has been frustrating due to negative pleiotropic effects between low phytate and desirable agronomic traits, including seed yield and germination. Alternative to both is either blocking the phytate biosynthetic pathway or degrading phytate in developing seeds to produce low phytate crops through genetic engineering.

Maize lpa1 mutants are defective in MRP ATP-binding cassette (ABC) transporter that is highly expressed in embryos, but also in immature endosperm, germinating seed, and vegetative tissues. Silencing this transporter in an embryo-specific manner produced maize transgenic seeds that were low in phytic acid and high in Pi that germinate well, and were not associated with reduction in seed weight. Furthermore, suppressing the homologous soybean MRP gene also produced low-phytic acid seed, paving the door that this technology may be possibly applied in other crops as well [74].

RNAi-mediated down-regulation of OsITP/6K-1, which altered the transcripts of essential phytic acid pathway genes without affecting the transcript of other OsITPK homologs, led to 46% reduction in seed phytate and 3-fold increase in Pi. The low phytate seeds exhibited 1.3- and 1.6-folds increase in Fe and Zn in polished seed endosperm, respectively [39], while RNAi-mediated embryo-specific downregulation of ITPK-2 produced rice seeds in relation to WT reduced phytic acid by 37%, nearly 3-fold increase in Pi, and increased iron and zinc in polished rice grains. Importantly, the reduced phytic acid content had no adverse impact on seed germination or seedling growth of transgenic rice [75]. A decrease in IPK1 transcript levels in RNAi-mediated silencing of IPK1 at T₄ generation wheat seeds resulted in 28–56% low phytate, and significant increases in Pi, and Fe and Zn. However, lowering seed phytate adversely impacted seeds spikelet⁻¹ [43]. More importantly, the silencing of these genes in homozygous transgenic lines did not lead to any defect in seed development including low germination or overall agronomic performance as often evidenced in mutagenesis. CRISPR/Cas9-mediated disruption of TaIPK1A gene in cv. ‘Borlaug 2016’ reduces phytic acid and improves iron and zinc accumulation in wheat grains [76], while knocking out three functional paralogs of BnITPK resulted low phytate mutants with an increased Pi in the canola (rapeseed) grade spring cultivar ‘Haydn’ [77]. These are thus important genetic resources for the development of biofortified cultivars of wheat and rapeseed.

The targeted disruption of the expression of MIPS1 using seed specific promoter in soybean resulted in T₄ generation lines with 39–41% reduction in seed phytate as compared to WT. Significantly Fe, Zn and Ca, respectively, increased by 15–22%, 7–11% and 10–15% in low phytate lines [78]. Transgenic lines expressing inositol polyphosphate 6-/3-/5-kinase gene specific RNAi construct in seeds of ‘Pusa-16’ soybean cultivar had exceptionally low seed phytate levels, moderate accumulation of Pi, and elevated mineral contents [79].

Clearly, a few transgenes are now available as well new mutants through CRISPR/Cas9 mediated gene editing for targeted reduction of seed phytate without adverse impact on plant growth, development and reproduction in cereals and grain legumes.

COMBINING HIGH IRON AND ZINC DENSITY WITH LOW PHYTATE TO ENHANCING MICRONUTRIENTS BIOAVAILABILITY

Widespread malnutrition, caused by deficiency of Fe and Zn, afflicts more than two billion people globally. A high phytate diet is associated with reduced absorption of Fe and Zn in the gastrointestinal tract [80]. Molar ratios of Phytate:minerals are used to estimate micronutrient bioavailability. Phyt:Fe molar ratio above 1 causes negative effect on Fe absorption, while a Phyt:Zn molar ratio above 15 cause low Zn bioavailability [81,82]. Biofortification has been successful to improving seed Fe and Zn contents in grain crops [83]. While breeding for low seed phytate is often (with few exception) mirrored with negative pleiotropic effects [5], combining it with seed biofortification another dimension of conflict for the plants and human health [9,84].

Double mutant offspring originating from a cross between high- and low-density mutants displayed in grains significantly higher Zn and low phytic acid contents, and lowest molar ratio of phytic acid to Zn across all environments. Such progenies contained relatively high content of bioavailable Zn in brown rice [85]. The homozygous lpa and WT offspring and double lpa mutants originating from crosses involving MIPS1 and IPK1 soybean mutants with WT or crossing the mutants among themselves revealed that lpa trait of the MIPS1 mutant was not altered by crossbreeding with a WT cultivar. The lpa offspring had InsP6 reductions of about 44% compared to WT. IPK1 offspring had up to 12.4 mg g⁻¹ accumulation of specific InsP3-InsP5 reduction compared to the progenitor lpa mutant (4.7 mg g⁻¹). The extent of InsP6 reduction (43–71%) depended on the WT crossing parents, with double mutants exhibiting up to 87% InsP6 reductions. IPK1 offspring had substantial accumulations of specific InsP3-InsP5 isomers (up to 12.4 mg g⁻¹) compared to the progenitor lpa mutant (4.7 mg g⁻¹), while the extent of InsP6 reduction (43–71%) depended on the WT crossing parent. Double mutants exhibited up to 87% InsP6 reductions accompanied by moderate accumulations of InsP3-InsP5 (2.5 mg g⁻¹). Crossbreeding therefore modulates the amounts of both InsP6 and InsP3-InsP5 contents in lpa soybean mutants and improve their nutritional quality [86]. In wheat, gamma rays induced mutants showed more than two-fold higher grain Fe and Zn concentrations, 1.5-fold lower phytic acid concentration, and 3.8- and 6.6-folds lower Phyt:Fe and Phyt:Zn molar ratios compared to WT [87]. Thus, valuable genetic resources for use in breeding to select for increased Fe and Zn concentrations and lower phytic acid, a win-win combination, to minimize the risk of micronutrients malnutrition in humans.

BAKING AND NUTRITIONAL QUALITY, NUTRIENT RETENTION, IN VIVO BIOAVAILABILITY AND ABSORPTION OF MINERALS

Biofortification often alters the flavor, taste, appearance, and other feature of the foods, which may limit the consumer acceptance. Equally important is whether poor consumers prefer and are willing to buy nutritious foods. A few surveys and choice preferences experiment from Asia and Africa regions revealed that poor consumers prefer nutritionally enhanced (Fe, Zn, or Vitamin A) food. Food with little or no effect on taste and appearance is regarded more positively than that with significantly altered characteristics. However, willingness to pay for nutritional attributes is small [5,88].

The lpa hard wheat-derived germplasm was not associated with detrimental effects on flour protein concentration, dough mixing properties, or bread loaf volume, but showed substantial increase in flour ash concentration relative to WT wheat. Higher flour ash is an indication of higher proportion of aleurone and bran fragments and Asian noodles prepared from such flour have dull appearance. The lpa germplasm had significant redistribution of minerals from the bran to the endosperm. The lpa soft wheat had greater sodium carbonate and sucrose, which is an indication that lpa wheat had greater apparent starch damage or pentosan content than its WT sibs [89].

A comparative study involving WT and lpa lines in the genetic background of ‘Grandin’ red spring wheat revealed no differences in total P concentration, however, the distribution of P between phytic acid and Pi was altered [90]. This research shows that Pi in lpa grain was up to 340% of WT grain, while phytic acid phosphorus in lpa grain reduced to as low as 65% of the concentration in WT grain. Moreover, such differences in P were also reflected in flour: Pi in break and reduction flours of lpa wheat was 3 to 4 times the concentration in break and reduction flours from WT wheat, with total P concentration in lpa flour 20% greater than in WT flour. Furthermore, mineral concentrations in bran and shorts of lpa and WT wheats were similar. However, magnesium (Mg) concentrations in lpa break and reduction flours were significantly greater than in WT flour. The lpa wheat had little effect on concentrations of other minerals, while lpa grain flour had on average greater ash (4.38 g kg⁻¹) than WT grain flour ash (3.86 g kg⁻¹). The protein content of lpa and WT flours was similar. The lpa flour had a longer time to mixograph peak and greater mixograph peak height than WT flours, while bread loaf volume between lpa and WT flours was similar. Thus, lpa-derived germplasm can produce flours with greater Pi and Mg concentration and little effect on bread flour functionality.

In a comparative study of Fe, Zn, and phytate retention after preparing common household recipes Hummel et al. [3] noted greater average Fe retention (77–91%) after boiling than Zn (41–84%) or phytate (50–86%). Soaking led to a significant decrease in Zn and phytate after boiling and refrying, but no change in Fe. The lpa beans relative to other group of beans had not shown a consistent pattern of difference in Fe and phytate retention. However, lpa beans had significantly lower retention of Zn compared to conventional and biofortified beans. This result suggests that combining the lpa plus increased Fe and Zn may further enhanced the nutritional benefits of biofortified beans, by decreasing its Phyt:Fe and Phyt:Zn molar ratios [3].

Bioavailability of minerals is compromised by high phytate in the food. Seed grains constitute the major source of dietary energy. About 80% of the seed phosphorus in food grains is in the form of phytate. Phytate binds minerals such as Ca, Fe and Zn and therefore these minerals are unavailable for absorption in nonruminant animals including human digestive tract. Maize is the major source of dietary food to millions of people in developing countries. Hambidge et al. [91] noted greater mean Ca absorption, by 42.8% (0.50 ± 0.03), from tortilla meals prepared from low-phytate as compared to high-phytate maize tortilla meals (0.35 ± 0.07). The molar ratios of phytic acid to Zn in polenta prepared from low- and high-phytate maize were 17:1 and 36:1, which respectively corresponded with fractional absorption of Zn to 0.30 ± 0.13 and 0.17 ± 0.11 [92]. In another experiment involving tortilla prepared from low phytate maize hybrids and their WT isohybrids revealed that fractional absorption of Zn from tortilla is positively related to the extent of phytate reduction in low-phytate hybrids [93].

A study involving Swedish women under free-living conditions and single meal intervention of eating whole grain rye bread with high or low phytate revealed no change in any of the Fe status biomarkers detected after 12 weeks of intervention in high-phytate-bread group, while in low- phytate-bread group, both ferritin and total body Fe were decreased, indicating that single meal intervention increase in Fe bioavailability from dephytinization of cereals [94]. In low phytate pea lines, bioavailable Fe as assessed by cell culture bioassay was reported 1.4–1.9-fold higher than that of the normal phytate cultivars [95]. However, it should be noted that several factors contribute to iron absorption and human body adapts to absorbing higher percentages of sources of iron over time; any conclusion obtained from a short period may give a false measure too low a bioavailability [96]. Although positive effect of low phytate diets on Ca, Fe and Zn bioavailability were unfolded, a long-term assessment, both in vivo and on healthy subjects, are needed to assess bioavailability and absorption of minerals from diets differing in phytate concentrations.

Crop biofortification has resulted in development and release of high Fe biofortified beans in Africa (http://www.harvestplus.org). The consumption of biofortified-, lpa-, and non-biofortified-beans among Rwandese women with low Fe status in the body showed that biofortified- and lpa-beans provided more bioavailable Fe than non-biofortified beans. The total amount of Fe absorbed from biofortified and lpa-beans was 50% greater than non-biofortified beans. The lpa-beans were, however, hard to cook and their consumption caused adverse gastrointestinal symptoms among most who ate them [73].

An in vivo study involving red jungle fox (Gallus gallus), Warkentin et al. [97] noted that certain biofortified low phytate field pea cultivars provided greater Fe bioavailability and modest increase in Fe status, had significant effect on gut microbiota and duodenal brush border membrane (BBM) functionality. BBM is the major site of nutrients transport and absorption in the small intestine [98].

Natto, a traditional food prepared from fermented soybean and popular dish amongst Japanese, prepared from low phytate (LP) soybean revealed improved color, lower in stickiness, hardness, and taste, but with greater Pi concentrations than those prepared from normal-phytate (NP) natto. Ca, Mg, and K concentrations were the same between LP and NP natto. A slightly higher protein in NP natto than that in LP natto. Thus, improved qualities of LP-soybean natto may increase its acceptability overseas and thereby increase its commercial value and contribute to sustainable food and nutritional security to people in Southeast Asia [99].

In brief, no significant changes were noted in bread functionality from grains differing in seed phytate contents. Noodles prepared from low phytate grains had, however, dull appearance due to high ash content in flour that may limit its consumption, while improved quality of natto from low-phytate soybean may be commercialized. Greater nutrition retention and absorption was noticed in household recipes made from low-phytate flours. Biofortified- and lpa-beans provided more bioavailable Fe than non-biofortified beans. Consumption of low-phytate bean however causes stomach discomfort. Subjects eating low-phytate products showed significant effect on gut microbiota and BBM functionality. Overall, consumption of low phytate grains (or flours) may contribute healthy livelihood to inhabitants in LMICs.

ASSESSING IMPACT OF PHYTATE ON HUMAN HEALTH

Although phytate may affect the bioavailability of trace elements, dietary phytate may prevent formation of kidney stones, or protect humans against diabetes mellitus, caries, atherosclerosis, coronary heart maladies, and various cancers [6,100‒105]. Hence, phytates provides an example of being both good and bad for human nutrition and health, particularly for vegans and vegetarians who eat a lot of high-phytate diets based on cereals and legume grains or nuts. Further, it has been reported that the majority of the InsP6 found in organs and tissues has a dietary origin and is not a consequence of endogenous synthesis, as evidenced from rat to human, and health benefits linked to extracellular InsP6 relate to dietary InsP6 [106,107]. However, Irvine and colleagues demonstrated that InsP6 does not and cannot exist in mammalian serum or urine. Therefore, any physiological effects of dietary InsP6 can only be due either to its actions in the gut as a polyvalent cation chelator or to inositol generated by its dephosphorylation by gut microflora [1]. A purification protocol based on Titanium dioxide (TiO₂) beads bind inositol phosphates which enabled Wilson et al. [108] quantify InsP6, its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. They further visualized InsP6, InsP7 and InsP8 in Dictyostelium extracts and a variety of mammalian cell lines and tissues, as well InsP6 in human plasma and urine. Hence, active InsP6 phosphatase is noted in human plasma but undetectable in either fluid, thereby challenging reports that InsP6 is found in human biofluids and the related advice of using it as supplement in diets. A capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) was used to analyze complex mixtures of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) with high sensitivity using stable isotope labeled (SIL) for matrix-independent quantitative assessment. SIL-CE-ESI-MS accurately monitored InsPs and PP-InsPs in wild type and knockout mammalian cell lines and in model organisms arising from compartmentalized cellular synthesis pathways by feeding cells with either [¹³C₆]-myo-inositol or [¹³C₆]-D-glucose. It thus provides evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential SIL-CE-ESI-MS to dissect inositol phosphate metabolism and signalling [109].

ASSESSING THE IMPACT OF LOW SEED PHYTATE (or HIGH PHYTASE) FEED ON NONRUMINANT ANIMALS

Phytic acid reduces the bioavailability of minerals in case of monogastric livestock’s, such as pigs and poultry, as they lack phytase enzyme in their digestive tract. Livestock’s raisers often add fungal or bacterial-phytase to enhance feed value. Assessing the impact of low phytate- or high phytase-based diets on nonruminants may help livestock’s farmers develop efficient and cost-effective feeds to optimize animals’ health and productivity.

There are various factors affecting feed phytase efficiency in gastrointestinal tract on non-ruminant or monogastric domestic animals such as pig (mainly in the stomach and upper small intestine) and poultry (mostly in upper part of digestive tract). The antinutritional effects of phytate (InsP6) are eliminated through hydrolysis as quickly it is in the upper part of the digestive tract. They are related to the phytase itself, the diet or the animal [110]. Type, optimum pH and resistance to endogenous protease are the factors associated to the phytate per se, while the dietary phytate content, feed ingredient composition and feed processing, and total P, Ca and Na content are those factors influencing its activity. Species, gender and age are the animal related factors. As indicated by Dersjant-Li [110] both phytate content and activity vary between crops over farming seasons and across regions due to changes in climate or weather and physical environments. Dus˘ková et al. [111] indicated that phytic acid P amounts were 112, 153 and 185 g kg⁻¹ total P in the faeces of young pigs (40–60 kg) after feeding them with supplemental phytase (490 FTU kg⁻¹), finishing pigs’ and non-lactating sows, respectively, whereas the excreta of laying hens had 362 g kg⁻¹ total P (or 23.7 g phytic acid kg⁻¹ dry matter). Schlemmer et al. [112] found that phytate degradation was nearly completed throughout the whole gut and very similar in non-heated or extruded diets, but total P availability was higher in the former. Contrarily, poultry research showed incomplete phytate degradation (remarkably in broiler chickens) even if augmented with phytase [113,114]. Thus, addressing phytate in animal feces should minimize the risk of eutrophication of water ways and water bodies in LMICs, while such an effort in developed economies will reduce the feed cost and phosphorus pollution.

A comparative feeding experiment on growing swine’s fed on low- and normal-phytate maize feed revealed that low-phytate maize feed with reduced levels of Pi supplementation had no adverse effects on pig performance, bone strength, or carcass characteristics, which suggests that P in low phytate maize is available and adequate to growing-finishing swine. The improved carcass characteristics in pigs fed with low phytate feed further suggests more nutrients bioavailable to absorption in the digestive tract [115]. In another feeding experiment involving NutriDense (ND) and NutriDense Low-Phytate (NDLP) maize hybrids relative to yellow dent (YD) maize in swine diets, Hastad et al. [116] noted 4.5% and 2.5% greater metabolizable energy (ME) of ND and NDLP than that of YD maize in growing swine, while in finishing pigs, it was 5.3% greater than that of YD maize, suggesting ND or NDLP-based maize feed resulted in enhanced feed efficiency. ND and NDLP hybrids are a high-protein and high-oil type or a high-protein, high-oil and low-phytate maize hybrids.

In an experiment involving growing and finishing pigs fed with five maize-soybean meal-based diets, positive control (PC, supplemented with Pi and Ca), negative control (NC, with 0.13% and 0.11% reduction in available P and Ca, respectively), and three NC diets supplemented with bacterial phytase of 1000, 2000, 3000 FTU kg⁻¹ in the feed, da Silva et al. [117] observed that supplementing increasing levels of phytase to a maize- and soybean meal-based diet with Pi and Ca reduction improved daily weight gain and feed intake of growing pigs, which maintained even in finishing pigs.

A study on supplementation of very low protein diets with maize-phytase in pigs decreased the adverse effects of such diets on weight gain and protein efficiency ratio, increased Ca and P digestibility, improved bone characteristics and produced diverse effects on fecal bacterial population, suggesting that maize-phytase (4000 FTU kg⁻¹ phytase vs 2000 FTU kg⁻¹ phytase) is potentially useful for enhancing the growth performance of weaned pigs fed with low-protein diets and should be considered for sustainable swine production [118].

Previous research has shown that phytases derived from Escherichi coli are more effective on nutrient digestibility and reduction of P excretion than fungal phytase in broiler chicks and in pigs [119,120]. The pigs fed on phytase + low-phytate relative to adequate-P diets were more efficient at reducing the P and Ca excretion and restoring average daily gain. The P released by phytase was absorbed and contributed to improved bone growth, greater rates of tissue accretion, and increased body weight, but no change in tissue P concentrations. This, however, has limit beyond which the excess P is excreted in urine [121].

A feeding experiment involving broiler chicken feeds containing grains from low-phytate barley and maize isolines or near isogenic WT barley and maize, Jang et al. [122] noted greater mean growth and bone responses for barley compared to maize diets, greater for 60% grain inclusion compared to 40%, and higher for low phytate compared to WT grains. The low phytate and P-supplemented type-grain diets were comparable. The chickens fed with low-phytate-based diets relative to WT or P-supplemented diets, respectively, exerted 33% and 43% less P in the environment. Young broiler chickens fed on low phytate pea-based diet showed lower weight gain and feed intake but greater bone strength than those fed on normal pea or soybean meal [123].

More recently, super dosing of phytase in the chicken’s diet improved bone development and morphometric measurements of the femur and tibiotarsus in broilers of 1 to 21 d of age. The phytase super dosing also improved mineral matter and the protein digestibility coefficients, which probably relate to phytate degradation and enhanced myo-inositol availability [124]. Continuous application of phytase in the feed however may deliver more or less than expected benefits therefore its judicious use is recommended as to date our knowledge on how phytase enzyme interact with feed ingredients is limited [125].

An assessment of five type of poultry feed, positive control (PC) and negative control (NC) diets with recommended P levels in layer diets and NC diet supplemented with three super doses (10,000, 20,000, 30,000 FTU kg⁻¹ feed) of phytase, revealed that a diet containing 20,000 FTU kg⁻¹ phytase had positive effect on egg production rate, but dietary treatments including 20,000 FTU phytase had no beneficial effect on egg quality as measured by variation in egg weight, egg mass, eggshell strength, eggshell color, egg yolk color, and egg protein quality (Haugh unit) in laying hens [126]. Rice bran is often added in poultry feed. However, it is poor source of plant proteins but contain highest amount of phytate-bound phosphorus. The phytate-bound phosphorus is poorly utilized by poultry. Laying hens fed with maize-soybean-based feed supplemented with 250 FTU Kg⁻¹ microbial phytase and 25% rice bran had improved performance, egg composition, and shell quality [127].

Clearly, low phytate- or high phytase-based feeds are not only efficient in P utilization and growth and productivity of nonruminant livestock’s, but also for reduction of P excreta to minimize environmental pollution. Enabling policy environments for taxing high phytate animal waste may encourage more investment on private agricultural research programs to deal with excess phytate in food and feedstocks [128].

ASSESSING BENENFIT/COST OF PURSUING BREEDING FOR LOW PHYTATE CROPS

Very recently and after four decades of research, Raboy explains the pros of breeding low phytate crops from a social and economic perspective [51]. It has been indicated that about 20% reduction in soil P mining during rice farming—achieved by reducing seed total P through breeding—could save growers several hundred million US dollars annually in fertilizer inputs [129]. In this regard, the barley cultivar ‘Herald’ [64] exhibited up to 15% reduction in seed total P due to the lpa1-1 mutant. Such a cultivar may allow limiting the use of non-renewable rock phosphate reserves.

CONCLUDING REMARKS

The SDG2—known as Zero Hunger—aims to end hunger by both achieving food security and improving nutrition while promoting sustainable agriculture. Boosting nutrition sustainably through healthy diets is therefore key for succeeding in this endeavor. In this regard, people eating mostly grain crops (cereals and legumes) that are rich in phytic acid are affected by Fe and Zn deficiency because this compound binds to them to form salts that their body excretes. Hence, breeding low phytate crops appears as an interesting approach for tackling this mineral shortcoming in the human diets, thus reducing human malnutrition. Indeed, the genetic enhancement of crop using various germplasm sources and breeding methods, as described above, allows developing nutritious low-phytate cultivars, particularly in cereals such as barley, maize and rice, or legumes such as soybean. Combining a lpa seed phenotype with increased Fe and Zn is an achievable breeding target, while food products prepared from low phytate flour has similar functionality and acceptance as those from normal phytate flour. However, a long-term assessment must be pursued to assess bioavailability and absorption of minerals from diets differing in phytate concentrations. Phytic acid, though antinutrient, plays an indispensable role directly or indirectly in preventing human diseases. The medicinal significance of phytic acid outweighed its negative impact. Hence, there is a critical need for LMICs to improve on the dietary pattern of its people in addition to nutrition education. Reducing a nutrient in the diet may be risky rather than increasing the amount. Hence, pro- and cons- should be carefully investigated before developing low-seed phytate crops, especially in LMICs. A moderate reduction of seed phytate should be beneficial to untangle negative complexity involving environment-food/feed production-animal/human nutrition triangle.

AUTHOR CONTRIBUTIONS

Sangam L. Dwivedi and Rodomiro Ortiz conceptualized the review structure, conducted literature search, wrote and approved the manuscript for submission.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

ACKNOWLEDGEMENTS

Sangam L. Dwivedi acknowledges the contribution of Ramesh Kotana, Senior Manager, Indian School of Business (ISB), Hyderabad, India for arranging reprints on seed phytate as valuable literature resources that helped him draft his part of contribution to this manuscript. We are grateful to the anonymous reviewers for making very useful suggestions on improving the manuscript.