Wheat Phytase Research Articles

Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity.

The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dosedependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

Effect of long-chain inorganic polyphosphate treated with wheat phytase on interleukin 8 signaling in HT-29 cells.

ObjectiveThis study was performed to investigate the potential effect of wheat phytase on long-chain inorganic polyphosphate (polyP)-mediated interleukin 8 (IL-8) signaling in an intestinal epithelial cell line, HT-29 cells.MethodsCell viability and the release of the pro-inflammatory cytokine IL-8 in HT-29 cells exposed to polyP1150 (average of 1,150 phosphate residues) treated with or without wheat phytase were measured by the EZ-CYTOX kit and the IL-8 ELISA kit, respectively. Also, the activation of cellular inflammatory factors NF-κB and MAPK (p38 and ERK 1/2) in HT-29 cells was investigated using ELISA kits.ResultsPolyP1150 negatively affected the viability of HT-29 cells in a dose-dependent manner. However, 100 mM polyP1150 dephosphorylated by wheat phytase increased cell viability by 1.4-fold over that of the intact substrate. Moreover, the 24 h exposure of cells to enzyme-treated 50 mM polyP1150 reduced the secretion of IL-8 and the activation of NF-κB by 9% and 19%, respectively, compared to the intact substrate. PolyP1150 (25 and 50 mM) dephosphorylated by the enzyme induced the activation of p38 MAPK via phosphorylation to 2.3 and 1.4-fold, respectively, compared to intact substrate, even though it had little effect on the expression of ERK 1/2 via phosphorylation.ConclusionWheat phytase could attenuate polyP1150-induced IL-8 release in HT-29 cells through NF-κB, independent of MAP kinases p38 and ERK. Thus, wheat phytase may alleviate inflammatory responses including hypercytokinemia caused by bacterial polyP infection in animals. Therefore, wheat phytase has the potential as an anti-inflammatory therapeutic supplement in animal husbandry.

Phytate degradation and phosphorus utilisation by broiler chickens fed diets containing wheat with increased phytase activity

ABSTRACT 1. The objective of this study was to investigate wheat genotypes bred for increased intrinsic phytase activity for InsP6 disappearance and the formation of lower inositol phosphates in such wheat-fed broiler chickens. The influence of monocalcium phosphate (MCP) supplementation on these characteristics and the utilisation of P and Ca were also determined. A three-step in vitro assay and a broiler trial were performed. 2. In the 63 wheat genotypes tested in vitro, phytase activity varied from 1900 FTU/kg to 5200 FTU/kg, and InsP6 disappearance increased with higher phytase activity of wheat in a linear manner. The addition of MCP significantly reduced in vitro InsP6 disappearance by one-third, independent of the inclusion level of wheat in the feed. When exogenous phytase was added to wheat, in vitro InsP6 disappearance increased independently of the phytase activity of the wheat used. 3. In the broiler trial, four wheat genotypes with phytase activities between 2400 and 3700 FTU/kg were included at 400 g/kg in diets with and without MCP. The diets were not pelleted. Separately, wheat 1, without MCP, was tested with the addition of exogenous phytase. Unsexed Ross 308 broiler chickens were allocated to 72 metabolic units of 10 birds each and assigned one of the nine diets. Mineral utilisation was measured based on excreta collection from 20 to 23 d of age. Digesta from the crop and terminal ileum were collected on d 24. 4. In the crop and ileum, InsP6 disappearance was not affected by the wheat genotypes, but the addition of MCP significantly decreased InsP6 disappearance. Precaecal P disappearance was significantly reduced by the addition of MCP, with wheat genotypes also exerting an effect. Wheat genotypes and the addition of exogenous phytase significantly affected P utilisation. Exogenous phytase had no effect on InsP6 disappearance in the crop but did up to the terminal ileum, the precaecal InsP6 and P disappearance increased with the addition of exogenous phytase. 5. Although the intrinsic wheat phytase activity exerted distinct effects on in vitro InsP6 disappearance, no such effect was found in the broiler trial. The addition of MCP significantly inhibited InsP6 degradation in vitro and in vivo.

Phytase Mediated Beneficial Impact on Nutritional Quality of Biofortified Wheat Genotypes

Background: Biofortification has been proposed as an intervention towards alleviation of micronutrient deficiency in the population of developing countries. However, the presence of anti- nutritional factor phytic acid in staple cereals chelates divalent cations and decreases their bioavailability for monogastric animals. Thus, the use of phytase enzyme for hydrolysing phytate-P and enhancing the amount of free divalent cations is of great importance. Methods : In this study, two phytases i.e. APF1 phytase from fungal source and commercial wheat phytase were supplemented with flours of biofortified wheat genotypes and their impact on food quality parameters was accessed. Since commercial wheat phytase is costly, it was used as known phytase to compare the application of APF1 phytase. The phytic acid content was reduced in the range of 70 to 84% with APF1 phytase and 79 to 89% with the wheat phytase as compared to untreated samples, respectively. In contrast to phytate, the dialyzability of important micronutrients Fe and Zn enhanced in the range of 21.9 to 48% and 39.5 to 96% with APF1 phytase and, 6.10 to 30% and 23.2 to 81% with wheat phytase, over untreated samples, respectively. Results and Discussion: A decrease in tannin content was observed in the range of 8 to 23% and 7 to 23% after treatment with APF1 and wheat phytase, respectively. The phytase treatment has resulted in increased soluble protein content and inorganic phosphate content to different level over untreated samples. Conclusion: The study revealed that APF1 phytase was comparatively more effective for enhanced nutritional quality of wheat flour through phytase supplementation for its food based applications.

Wheat phytase can alleviate the cellular toxic and inflammatory effects of lipopolysaccharide.

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1–3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

Addition of Whole Wheat Flour During Injera Fermentation Degrades Phytic Acid and Triples Iron Absorption from Fortified Tef in Young Women

Iron deficiency is a major public health concern in Ethiopia, where the traditional diet is based on tef injera. Iron absorption from injera is low due to its high phytic acid (PA) content. We investigated ways to increase iron absorption from FeSO4-fortified tef injera in normal-weight healthy women (aged 21-29 y). Study A (n=22) investigated the influence on fractional iron absorption (FIA) from FeSO4-fortified injera of 1) replacing 10% tef flour with whole wheat flour (a source of wheat phytase), or 2) adding an isolated phytase from Aspergillus niger. Study B (n=18) investigated the influence on FIA of replacing FeSO4 in tef injera with different amounts of NaFeEDTA. In both studies, the iron fortificants were labeled with stable isotopes and FIA was calculated from erythrocyte incorporation of stable iron isotopes 14 d after administration. In study A, the median (IQR) FIA from the 100% tef injera meal was 1.5% (0.7-2.8%). This increased significantly (P<0.05) to 5.3% (2.4-7.1%) on addition of 10% whole wheat flour, and to 3.6% (1.6-6.2%) on addition of A. niger phytase. PA content of the 3 meals was 0.62, 0.20, and 0.02g/meal, respectively. In study B, the median (IQR) FIA from the 100% tef injera meal was 3.3% (1.1-4.4%) and did not change significantly (P>0.05) on replacing 50% or 75% of FeSO4 with NaFeEDTA. FIA from tef injera by young women was very low. NaFeEDTA was ineffective at increasing iron absorption, presumably due to the relatively low EDTA:Fe molar ratios. Phytate degradation, however, greatly increased during tef fermentation on addition of native or isolated phytases. Replacing 10% tef with whole wheat flour during injera fermentation tripled FIA in young women and should be considered as a potential strategy to improve iron status in Ethiopia.

Dephytinising efficacy of wheat phytase in enhancing the bioaccessibility of minerals in high phytate foods

Iron deficiency anemia and zinc deficiency are major public health problems in developing world which are attributable to the poor bioavailability due to the presence of phytic acid in plant foods. Dephytinisation by enzymatic application is identified to be ideal approach, however the commercial availability of phytases from food sources is scarce. This prompted us to undertake the investigation on the dephytinisation of the high phytate food matrices by utilising the indigenous phytase of wheat. Attempts were made to enhance the phytase activity by various approaches. Phytase activity of wheat was increased by fivefold on sprouting (96 h). The native phytase of wheat activated by incubation at optimum conditions of pH and temperature has shown an increase in the activity as a function of time. Isolation and partial purification of phytase from sprouted wheat further increased the activity by 1.5 fold. Thermostability of phytase was enhanced by additives such as sodium citrate, calcium chloride and magnesium sulphate. Addition of phytase activated wheat flour to soya flour enhanced the bioaccessible iron by 1.7 fold. Partially purified phytase enhanced the bioaccessible iron by 1.9 fold and zinc by 2.4 fold in defatted soya flour reflecting dephytinisation. Investigation on complete purification of phytase are warranted.

Potential immune-modulatory effects of wheat phytase on the performance of a mouse macrophage cell line, Raw 264.7, exposed to long-chain inorganic polyphosphate.

ObjectiveThis experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP.MethodsNitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit.ResultsP700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 μM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700.ConclusionIn animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.

Effect of fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa.

BackgroundQuinoa is a pseudocereal with relatively high content of proteins and minerals that also contains mineral inhibitors such as phytate. The aim of the present study was to evaluate lactic acid fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa. Various processes were evaluated, and quinoa grains were dry‐roasted, milled, and fermented, either with or without the addition of wheat phytase or activated quinoa phytase (added as back‐slop starter), for 10 hr. In other processes, raw quinoa flour was fermented for 10 hr or 4 hr and dry‐roasted. Hedonic sensory evaluation was then performed to evaluate the acceptability of the fermented flours prepared as porridges.ResultsThe combined dry roasting and fermentation processes significantly (p < .05) degraded phytate between 30% and 73% from initial content. The most effective process was fermentation of raw quinoa flour followed by dry roasting, which improved the estimated zinc and iron bioavailability. Particularly, estimated zinc bioavailability improved from low (Phy:Zn 25.4, Phy·Zn:Ca 295) to moderate (Phy:Zn 7.14, Phy·Zn:Ca 81.5). Phytate degradation was mainly attributed to the activation of endogenous phytase during fermentation. Dry roasting was effective in improving the sensory attributes of the fermented quinoa flour. Porridge made with raw quinoa flour fermented for 4 hr and dry‐roasted was more favorable to overall acceptability than that which was fermented for 10 hr and dry‐roasted.ConclusionFermentation of quinoa flour for 4 hr followed by dry roasting was successful in improving both nutritional and sensory attributes of the final product.

Catalytic properties of wheat phytase that favorably degrades long-chain inorganic polyphosphate.

ObjectiveThis study was conducted to determine catalytic properties of wheat phytase with exopolyphosphatase activity toward medium-chain and long-chain inorganic polyphosphate (polyP) substrates for comparative purpose.MethodsExopolyphosphatase assay of wheat phytase toward polyP75 (medium-chain polyP with average 75 phosphate residues) and polyP1150 (long-chain polyP with average 1150 phosphate residues) was performed at pH 5.2 and pH 7.5. Its activity toward these substrates was investigated in the presence of Mg2+, Ni2+, Co2+, Mn2+, or ethylenediaminetetraacetic acid (EDTA). Michaelis constant (Km) and maximum reaction velocity (Vmax) were determined from Lineweaver-Burk plot with polyP75 or polyP1150. Monophosphate esterase activity toward p-nitrophenyl phosphate (pNPP) was assayed in the presence of polyP75 or polyP1150.ResultsWheat phytase dephosphorylated polyP75 and polyP1150 at pH 7.5 more effectively than that at pH 5.2. Its exopolyphosphatase activity toward polyP75 at pH 5.2 was 1.4-fold higher than that toward polyP1150 whereas its activity toward polyP75 at pH 7.5 was 1.4-fold lower than that toward polyP1150. Regarding enzyme kinetics, Km for polyP75 was 1.4-fold lower than that for polyP1150 while Vmax for polyP1150 was 2-fold higher than that for polyP75. The presence of Mg2+, Ni2+, Co2+, Mn2+, or EDTA (1 or 5 mM) exhibited no inhibitory effect on its activity toward polyP75. Its activity toward polyP1150 was inhibited by 1 mM of Ni2+ or Co2+ and 5 mM of Ni2+, Co2+, or Mg2+. Ni2+ inhibited its activity toward polyP1150 the most strongly among tested additives. Both polyP75 and polyP1150 inhibited the monophosphate esterase activity of wheat phytase toward pNPP in a dose-dependent manner.ConclusionWheat phytase with an unexpected exopolyphosphatase activity has potential as a therapeutic tool and a next-generational feed additive for controlling long-chain polyP-induced inappropriate inflammation from Campylobacter jejuni and Salmonella typhimurium infection in public health and animal husbandry.

Changes in Phytate Content in Whole Meal Wheat Dough and Bread Fermented with Phytase‐Active Yeasts

The degradation of inositol hexakisphosphate (IP6) was evaluated in whole meal wheat dough fermented with baker's yeast without phytase activity, different strains of Saccharomyces cerevisiae (L1.12 or L6.06), or Pichia kudriavzevii with extracellular phytase activity to see if the degradation of IP6 in whole meal dough and the corresponding bread could be increased by fermentation with phytase‐active yeasts. The IP6 degradation was measured after the dough was mixed for 19 min, after the completion of fermentation, and in bread after baking. Around 60–70% of the initial value of IP6 in the flour (10.02 mg/g) was reduced in the dough already after mixing, and additionally 10–20% was reduced after fermentation. The highest degradation of IP6 was seen in dough fermented with the phytase‐active yeast strains S. cerevisiae L1.12 and P. kudriavzevii L3.04. Activity of wheat phytase in whole meal wheat dough seems to be the primary source of phytate degradation, and the degradation is considerably higher in this study with a mixing time of 19 min compared with earlier studies. The additional degradation of IP6 by phytase‐active yeasts was not related to their extracellular phytase activities, suggesting that phytases from the yeasts are inhibited differently. Therefore, the highest degradation of IP6 and expected highest mineral bioavailability in whole meal wheat bread can be achieved by use of a phytase‐active yeast strain with less inhibition. The strain S. cerevisiae L1.12 is suitable for this because it was the most effective yeast strain in reducing the amount of IP6 in dough during a short fermentation time.

Phytase-mediated mineral solubilization from cereals under in vitro gastric conditions.

Enzymatic dephosphorylation of phytic acid (inositol hexakisphosphate) in cereals may improve mineral bioavailability in humans. This study quantified enzymatic dephosphorylation of phytic acid by measuring inositol tri- to hexakisphosphate (InsP3-6) degradation and iron and zinc release during microbial phytase action on wheat bran, rice bran and sorghum under simulated gastric conditions. InsP3-6 was depleted within 15-30 min of incubation using an Aspergillus niger phytase or Escherichia coli phytase under simulated gastric conditions, with the two enzymes dephosphorylating cereal phytic acid at similar rates and to similar extents. Microbial phytase-catalyzed phytate dephosphorylation was accompanied by increased iron and zinc release from the cereal substrates. However, for wheat bran at pH 5, the endogenous wheat phytase activity produced mineral release equal to or better than that of the microbial phytases. No increases in soluble cadmium, lead or arsenic were observed with microbial phytase-catalyzed phytate dephosphorylation. Microbial phytase treatment abated phytate chelation hence enhanced the release of iron and zinc from phytate-rich cereals under simulated gastric conditions. The data infer that acid-stable microbial phytases can help improve iron bioavailability from phytate-rich cereal substrates via post-ingestion activity. © 2015 Society of Chemical Industry.

Dietary effects on inositol phosphate breakdown in the crop of broilers

ABSTRACTThe effect of diets differing in enzyme supplements, mineral phosphorus (P) and microwave wheat treatment on phytate hydrolysis and lower inositol phosphate isomers (InsPs) appearance in broiler crops was studied. The broilers (16- and 15-day-old) were assigned to 48 pens of 15 or 20 birds each (n = 8 pens per treatment) in Experiments 1 and 2, respectively. In Experiment 1, birds received a low-P wheat-soybean meal diet where the wheat was either microwave treated or not. These diets were offered without further supplementation or with added phytase (500 FTU/kg diet), alone or in combination with a xylanase (16,000 BXU/kg diet). In Experiment 2, two maize-soybean meal-based diets were fed, without or with monocalcium phosphate supplementation. Furthermore, these diets were offered without further supplementation or with phytase at 500 or 12,500 FTU/kg diet. On day 23 or 24 (Experiments 1 and 2, respectively), crop digesta were pooled per pen, freeze-dried and analysed for InsPs and the marker TiO2. Microwaving reduced the intrinsic phytase activity and InsP6 hydrolysis, but increased the concentration of Ins(1,2,3,4,5)P5 and Ins(1,2,4,5,6)P5 in the digesta of crop (Experiment 1). Microwave treatment significantly interacted with enzyme supplementation for Ins(1,2,5,6)P4 concentration, indicating a synergistic effect of intrinsic and supplied phytase in the crop. Xylanase tended to support phytase hydrolysis in diets with microwave-treated wheat. Phytase addition increased InsP6 hydrolysis up to 79% (Experiment 2). Thus, wheat phytase activity can cause high InsP6 hydrolysis in the crop. Treatment differences in lower InsPs indicated that hydrolysis of the first InsP6 phosphate group is not the only step in the degradation cascade in the crop of broilers that is influenced by dietary factors.

Mechanisms and Pathways of Phytate Degradation: Evidence from Oxygen Isotope Ratios of Phosphate, HPLC, and Phosphorus‐31 NMR Spectroscopy

Phytate, the salt of myo-inositol hexakisphosphate, and its partially dephosphorylated products are commonly present in the environment, but their origins and bioavailability are not well understood. This research applied phosphate O isotope ratios (δ18OP) in combination with nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC) methods to characterize the kinetics and pathways of phytate degradation by wheat phytase and the O isotopic composition during progressive degradation of phytate. Our results show that phytate degradation undertakes two pathways: d-inositol-1,2,3,5,6-pentakisphosphate, d-inositol-1,2,5,6-tetrakisphosphate, d-inositol-1,2,6-trisphosphate, d-inositol-1,2-bisphosphate, d-inositol-1-phosphate; and d-inositol-1,2,4,5,6-pentakisphosphate, d-inositol-1,2,5,6-tetrakisphosphate, d-inositol-1,5,6-trisphosphate. The first pathway is similar to that previously known, while the other one is a new pathway. Isotope results show that the cleavage of the P–O bond during phytate degradation is accompanied by the introduction of one O atom solely from water to the released inorganic orthophosphate. Interestingly, isotopic compositions of all phosphate moieties in K phytate used in this study were found to be the same. This means that the original source of phytate can be tracked from its partially dephosphorylated products. Overall, these results provide improved insights into the mechanisms and pathways of phytate degradation and highlight the importance of an isotopic tool that can potentially be used for tracking phytate source and its degradation products in the environment.

Dephytinisation with Intrinsic Wheat Phytase and Iron Fortification Significantly Increase Iron Absorption from Fonio (Digitaria exilis) Meals in West African Women

Low iron and high phytic acid content make fonio based meals a poor source of bioavailable iron. Phytic acid degradation in fonio porridge using whole grain cereals as phytase source and effect on iron bioavailability when added to iron fortified fonio meals were investigated. Grains, nuts and seeds collected in Mali markets were screened for phytic acid and phytase activity. We performed an iron absorption study in Beninese women (n = 16), using non-dephytinised fonio porridge (FFP) and dephytinised fonio porridge (FWFP; 75% fonio-25% wheat), each fortified with 57Fe or 58Fe labeled FeSO4. Iron absorption was quantified by measuring the erythrocyte incorporation of stable iron isotopes. Phytic acid varied from 0.39 (bambara nut) to 4.26 g/100 g DM (pumpkin seed), with oilseeds values higher than grains and nuts. Phytase activity ranged from 0.17±1.61 (fonio) to 2.9±1.3 phytase unit (PU) per g (whole wheat). Phytic acid was almost completely degraded in FWFP after 60 min of incubation (pH≈5.0, 50°C). Phytate∶iron molar ratios decreased from 23.7∶1 in FFP to 2.7∶1 in FWFP. Iron fortification further reduced phytate∶iron molar ratio to 1.9∶1 in FFP and 0.3∶1 in FWFP, respectively. Geometric mean (95% CI) iron absorption significantly increased from 2.6% (0.8–7.8) in FFP to 8.3% (3.8–17.9) in FWFP (P<0.0001). Dephytinisation of fonio porridge with intrinsic wheat phytase increased fractional iron absorption 3.2 times, suggesting it could be a possible strategy to decrease PA in cereal-based porridges.

Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry

Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His6 tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His6 tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide.

The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin

Phytases increase utilization of phytate phosphorus in feed. Since wheat is rich in endogenous phytase activity it was examined whether wheat phytases could improve phytate degradation compared to microbial phytases. Moreover, it was investigated whether enzymatic degradation of phytate is influenced by the matrix surrounding it. Phytate degradation was defined as the decrease in the sum of InsP₆ + InsP₅. Endogenous wheat phytase effectively degraded wheat Ins₆ + InsP₅ at pH 4 and pH 5, while this was not true for a recombinant wheat phytase or phytase extracted from wheat bran. Only microbial phytases were able to degrade InsP₆ + InsP₅ in the entire pH range from 3 to 5, which is relevant for feed applications. A microbial phytase was efficient towards InsP₆ + InsP₅ in different phytate samples, whereas the ability to degrade InsP₆ + InsP₅ in the different phytate samples ranged from 12% to 70% for the recombinant wheat phytase. Wheat phytase appeared to have an interesting potential. However, the wheat phytases studied could not improve phytate degradation compared to microbial phytases. The ability to degrade phytate in different phytate samples varied greatly for some phytases, indicating that phytase efficacy may be affected by the phytate matrix.

Effect of heat-treatment, phytase, xylanase and soaking time on inositol phosphate degradation in vitro in wheat, soybean meal and rapeseed cake

An in vitro method was used to evaluate the degradation of myo-inositol hexakisphosphate (InsP 6) in non-heat-treated wheat (NHW), heat-treated wheat (HW), soybean meal (SBM) or rapeseed cake (RSC) soaked separately or in combination. The feedstuffs were soaked in water (20 °C) and samples were collected after 2, 4, 8 and 24 h. The following hypotheses were tested: (1) phytase addition to soaked NHW or HW improves InsP 6 degradation, (2) phytase and xylanase addition to soaked NHW or HW improves InsP 6 degradation further compared solely with phytase, (3) phytase addition to SBM or RSC improves InsP 6 degradation, and (4) soaking of SBM or RSM together with NHW (ratio 1:1) stimulates InsP 6 degradation from SBM or RSC due to the activity of wheat phytases, whereby phytase addition results in no further InsP 6 degradation. Additionally, degradation of lower inositol phosphates ( myo-inositol pentakisphosphate– myo-inositol bisphosphate or InsP 5–InsP 2) was studied to verify if InsP 6 degradation leads to an accumulation of InsP 5–InsP 2. In NHW or HW addition of phytase alone or with xylanase had no significant effect on InsP 6 degradation. However, InsP 6 degradation was influenced by the interaction between heat-treatment and soaking time (P≤0.001). This was mainly due to a smaller proportion of non-degraded InsP 6-P at 24 h in HW compared with NHW (0.13 vs. 0.47) (P≤0.001) possibly caused by structural changes imposed by the heat-treatment. In SBM, RSC, SBM/NHW or RSC/NHW, the InsP 6 degradation was affected by the interaction between phytase addition and soaking time (P≤0.001) as phytase reduced the proportion of non-degraded InsP 6-P at 2, 4, 8 or 24 h. Soaking of NHW, SBM or RSC (without phytase) separately resulted in a limited InsP 6 degradation, whereas a pronounced InsP 6 degradation occurred when RSC or SBM were soaked together with NHW (without phytase) because wheat phytases degraded InsP 6 in SBM or RSC. However, addition of phytase to SBM/NHW or RSC/NHW increased further the degradation of InsP 6 indicating that endogenous wheat phytases were not sufficient. The formation of InsP 5–InsP 2-P was very small (40–180 μg/kg DM) during the degradation of InsP 6 indicating a rapid and almost complete degradation of these compounds. In conclusion, the effect of phytase on InsP 6 degradation during soaking depends on the feedstuff and processing of the feedstuff. InsP 6 degradation results in a negligible accumulation of InsP 5–InsP 2. Soaking of plant feedstuffs prior to feeding of pigs may improve P digestibility compared with dry feeding, but studies with pigs are required to clarify this.

Phytate: impact on environment and human nutrition. A challenge for molecular breeding

Phytic acid (PA) is the primary storage compound of phosphorus in seeds accounting for up to 80% of the total seed phosphorus and contributing as much as 1.5% to the seed dry weight. The negatively charged phosphate in PA strongly binds to metallic cations of Ca, Fe, K, Mg, Mn and Zn making them insoluble and thus unavailable as nutritional factors. Phytate mainly accumulates in protein storage vacuoles as globoids, predominantly located in the aleurone layer (wheat, barley and rice) or in the embryo (maize). During germination, phytate is hydrolysed by endogenous phytase(s) and other phosphatases to release phosphate, inositol and micronutrients to support the emerging seedling. PA and its derivatives are also implicated in RNA export, DNA repair, signalling, endocytosis and cell vesicular trafficking. Our recent studies on purification of phytate globoids, their mineral composition and dephytinization by wheat phytase will be discussed. Biochemical data for purified and characterized phytases isolated from more than 23 plant species are presented, the dephosphorylation pathways of phytic acid by different classes of phytases are compared, and the application of phytase in food and feed is discussed.

Effect of wheat type grinding, heat treatment and phytase supplementation on growth efficiency and nutrient utilization of wheat-based diets for broilers

This experiment studied the effect of phytase and processing (heat treatment, grind size) on performance and nutrient utilization of broilers fed diets from 1 to 21 d that included hardred spring (HRS) and durum wheat with three grind sizes (470, 560, 630 µm) with or without heat treatment (oven dry at 80°C for 15 h) before supplementation with phytase (Natuphos® 0 or 600 FTU kg-1). All 24 diets contained 0.5 g Avizyme 1302 kg-1 and were formulated (80% wheat, 20% basal diet) to supply nutrients necessary to meet the requirements of broiler chicks, except for a low level of nonphytate phosphorus (P, 2.7 g kg-1). The medium ground HRS-based diet produced significantly better feed conversion ratio (FCR) and body weights compared with the other two grind sizes. Heat treatment significantly improved feed conversion ratio and apparent metabolizable energy (AME), protein, and P digestibility of diets. It also significantly decreased feed intake, but produced no differences in 21 d body weight. Supplementation with phytase significantly improved AME, protein, and P digestibility of test diets. The AME of diets was influenced by wheat type, processing and grind size. Phytase increased N and P digestibility. Improvements in N and P digestibility by phytase and heat treatment were dependent on wheat type and grind size.The present results suggest an advantage of both phytase and xylanase in durum- and HRS-based diets. Heat processing destroyed endogenous phytase and xylanase in wheat-based diets, and increased the levels of soluble non-starch polysaccharides. Combined phytase, heat treatment, and grind size may synergistically improve N and P digestibility. Key words: Wheat source, grind size, heat treatment, phytase, digestibility, broiler