Behaviour of plant‐derived extracellular phytase upon addition to soil

The behaviour of phytase after addition to three soil types with different sorption capacities was investigated. Phytase was collected from the roots of transgenic Arabidopsis thaliana that express a phytase gene from Aspergillus niger. Phytase activity in solution and on the solid phase of the soil was monitored over time. Phytase added to the solution phase of a soil suspension (1:20, w/v) was almost completely lost within 10 min in all soil types, while phytase in non-soil controls remained active in solution. Phytase activity lost from solution was recovered on the soil solid phase, suggesting rapid adsorption of the enzyme. Adsorption of phytase was less in soil taken from the rhizosphere of transgenic plants expressing phyA, indicating that the rhizosphere environment may help maintain phytase activity in solution. The activity of adsorbed phytase declined with time at a rate 2-4 times slower than that in the absence of soil. Adsorption of phytase in soils was highest at pH 4.5, which is below the reported isoelectric point (pI) of the Aspergillus phytase. As soil pH increased, adsorption decreased until, at pH 7.5, all phytase was in solution. Where phytase remained in solution, activity was maintained for at least 8 d. In contrast, the activity of adsorbed phytase was increasingly inhibited with time, particularly at low pH. By increasing the pH in soil suspensions, phytase that had remained active on the soil solid phase for 28 d was almost totally desorbed. Rapid immobilisation of phytase in soil may limit its capacity to interact with phytate, and this may compromise the ability of transgenic plants which exude phytase from their roots to acquire P from endogenous soil phytate.     

Oxidation of polyphenols in phytate-reduced high-tannin cereals: effect on different phenolic groups and on in vitro accessible iron.

After reduction of phytate with phytase, water slurries of two high-tannin cereal flours were incubated with polyphenol oxidase (mushroom tyrosinase), and the effects on different phenolic groups and on in vitro accessible iron were studied. Enzyme incubation was also performed after cooking, soaking, and germination of the cereals. Phytase incubation significantly decreased the phytate content, and incubation with polyphenol oxidase had a reducing effect on the total phenol content, as well as on the amount of catechol and resorcinol groups. The in vitro accessible iron increased when the cereals were incubated with phytase and polyphenol oxidase, and the highest accessibility of iron was obtained when the germinated samples were incubated. The results from this study imply that oxidation of polyphenols in high-tannin cereals, after reduction of phytate, may be used to increase the bioavailability of iron in foods prepared from these cereals.     

Influence of Added Enzymes and Bran Particle Size on Bread Quality and Iron Availability

The aim of this investigation was to study the influence of different bran proportions and particle sizes, addition of fungal phytase, and α‐amylase addition on bread quality and phytate levels, and how these treatments affect availability of iron to intestinal epithelial (Caco‐2) cells. Potential mineral contributions to dietary reference intakes and phytate‐to‐mineral molar ratios were also evaluated. Wheat bran supplementation significantly affected bread quality. Smaller bran particle size affected crumb firmness negatively, whereas the use of α‐amylase, in some cases in combination with phytase, could improve technological bread quality. The use of phytase in the formulation significantly reduced the level of phytates, and phytate hydrolysis also led to smaller bran particle size. Increasing the bran proportion used in the bread formulation increased the iron concentration in bread samples by 18.9%. Phytase addition proved to be a useful strategy to improve iron dialyzability; however, incomplete dephytinization still had an inhibitory effect on iron uptake, with the exception of samples formulated with 10% bran. The inhibitory effect of phytate could be predicted from the values of the phytate‐to‐iron ratios. Reduction of particle size did not improve iron availability or uptake by Caco‐2 cells.     

Microbial phytase activity and their role in organic P mineralization

Plants respond to their external environment to optimize their nutrition and production potential to minimize the food security issues and support sustainable agriculture system. Phosphorus (P) is an important nutrient for plants and is involved in plant metabolic processes. It is mostly available as orthophosphate and has a tendency to form complexes with cations. It has low mobility in soil, thus becoming unavailable for plant uptake that causes a reduction in plant growth and yield. Besides free P, phytate is the major form of organic P in soil and plant tissues. Phytases obtained from different sources, that is, plants, animals, and microorganisms, catalyze the hydrolysis of phytate and release available forms of inorganic P. The knowledge of mechanisms involved in catalytic activity of phytase obtained from microorganisms in soil is limited. This review summarizes the role of microbial phytase in releasing organic P by hydrolysis of phytate and factors affecting its activity in the soil.     

Effects of copper source and concentration on in vitro phytate phosphorus hydrolysis by phytase

Five copper (Cu) sources were studied at pH 2.5, 5.5, and 6.5 to determine how Cu affects phytate phosphorus (PP) hydrolysis by phytase at concentrations up to 500 mg/kg diet (60 min, 40-41 degrees C). Subsequently, Cu solubility with and without sodium phytate was measured. Adding Cu inhibited PP hydrolysis at pH 5.5 and pH 6.5 (P < 0.05). This inhibition was greater with higher concentrations of Cu. Tri-basic copper chloride and copper lysinate inhibited PP hydrolysis much less than copper sulfate pentahydrate, copper chloride, and copper citrate (P < 0.05). A strong negative relationship was observed between PP hydrolysis and soluble Cu at pH 5.5 (r = -0.76, P < 0.0001) and 6.5 (r = -0.54, P < 0.0001). In conclusion, pH, Cu concentration, and source influenced PP hydrolysis by phytase in vitro and were related to the amount of soluble Cu and the formation of insoluble copper-phytin complexes.     

Methodological aspects of measuring phytase activity and phytate phosphorus content in selected cereal grains and digesta and feces of pigs

This study was to examine the time course of sample-specific linearity of intrinsic phytase hydrolysis in major cereal grains and in ileal digesta and fecal samples and to determine the time course of the microbial phytase-catalyzed hydrolysis of various sources of phytate for estimating phytate phosphorus (P) content. The intrinsic phytase activity in barley, corn, oat, and wheat samples was measured over multiple time points from 0 to 120 min at 1.5 mmol.L(-1) of sodium phytate at pH 5.5 and 37 degrees C. Time courses of hydrolysis of purified phytate and phytate associated with the cereal grain samples and the pig digesta and fecal samples were examined with the Natuphos microbial phytase over multiple time points from 0 to 48 h of incubation. The intrinsic phytase hydrolysis was linear (P < 0.05) for up to 120 min for the barley, corn, and wheat samples, whereas in the oat sample the hydrolysis was linear (P < 0.05) for only up to 30 min of incubation. The intrinsic phytase activities (phytase unit: mumol.kg(-1) of dry matter.min(-1)) for the barley, corn, and wheat samples were estimated to be 693, 86, and 1189 by linear regression analysis. Intrinsic phytase activity (412 phytase units) for the oat sample based on a 30-min incubation was considerably higher than the value (103 phytase units) determined from the 120-min incubation for the same oat sample. There were quadratic with plateau relationships (P < 0.05) between the hydrolytic release of inorganic P from various sources of phytate and the incubation time. The minimal incubation times required for the complete hydrolysis of phytate were estimated to be 4, 3, and 11 h for the purified phytate, the cereal grain samples, and the pig digesta and feces, respectively. It was concluded that multiple time point experiments need to be conducted to determine valid intrinsic phytase activity and phytate P content in samples through intrinsic and microbial phytase hydrolysis incubations.     

Use of fungal phytase to improve breadmaking performance of whole wheat bread.

The possible use of phytase as a breadmaking improver has been tested in whole wheat breads by adding different amounts of fungal phytase. The effect of phytase addition on the fermentation stage and the final bread quality was analyzed. The phytase addition shortened the fermentation period, without affecting the bread dough pH. Regarding the whole wheat bread, a considerable increase of the specific bread volume, an improvement of the crumb texture, and the width/height ratio of the bread slice were obtained. An in vitro assay revealed that the improving effect of phytase on breadmaking might be associated with the activation of alpha-amylase, due to the release of calcium ions from calcium-phytate complexes promoted by phytase activity. As a conclusion, phytase offers excellent possibilities as a breadmaking improver, with two main advantages: first, the nutritional improvement produced by decreasing phytate content, and second, all the benefits produced by alpha-amylase addition can be obtained by adding phytase, which promotes the activation of endogenous alpha-amylase.     

Use of Phytases in Ethanol Production from E‐Mill Corn Processing

Effects of phytase addition, germ, and pericarp fiber recovery were evaluated for the E‐Mill dry grind corn process. In the E‐Mill process, corn was soaked in water followed by incubation with starch hydrolyzing enzymes. For each phytase treatment, an additional phytase incubation step was performed before incubation with starch hydrolyzing enzymes. Germ and pericarp fiber were recovered after incubation with starch hydrolyzing enzymes. Preliminary studies on phytase addition resulted in germ with higher oil (40.9%), protein (20.0%), and lower residual starch (12.2%) contents compared to oil (39.1%), protein (19.2%), and starch (18.1%) in germ from the E‐Mill process without phytase addition. Phytase treatment resulted in lower residual starch contents in pericarp fiber (19.9%) compared to pericarp fiber without phytase addition (27.4%). Results obtained led to further investigation of effects of phytase on final ethanol concentrations, germ, pericarp fiber, and DDGS recovery. Final ethanol concentrations were higher in E‐Mill processing with phytase addition (17.4% v/v) than without addition of phytase (16.6% v/v). Incubation with phytases resulted in germ with 4.3% higher oil and 2.5% lower residual starch content compared to control process. Phytase treatment also resulted in lower residual starch and higher protein contents (6.58 and 36.5%, respectively) in DDGS compared to DDGS without phytase incubations (8.14 and 34.2%, respectively). Phytase incubation in E‐Mill processing may assist in increasing coproduct values as well as lead to increased ethanol concentrations.     

Phytase and acid phosphatase activities in plant feedstuffs

A total of 183 samples representing 24 feedstuffs were analyzed for total phosphorus, phytate phosphorus content, phytase (Phy), and acid phosphatase (AcPh) activities with the objective to predict the capacity to hydrolyze phytic acid and to contribute to formulating environmentally adequate diets for monogastric animals. Of the cereals and cereal byproducts analyzed, only rye (5147 U kg(-)(1); 21 955 U g(-)(1)), wheat (1637 U kg(-)(1); 10 252 U g(-)(1)), rye bran (7339 U kg(-)(1); 56 722 U g(-)(1)), and wheat bran (4624 U kg(-)(1); 14 106 U g(-)(1)) were rich in Phy and AcPh activities. Legume seeds and oilseeds contained negligible Phy activity and a moderate amount of AcPh activity, except for kidney bean (33 433 U g(-)(1)) and full-fat linseed meal (13 263 U g(-)(1)). On the other hand, a significant linear regression between phytate phosphorus (y) and total phosphorus (x) was observed in cereal byproducts (R(2) = 0. 95; y = 0.8458x - 0.0367; P < 0.001) and oil seeds (R(2) = 0.95; y = 0.945x - 0.20; P < 0.001). Phy and AcPh were positively correlated with respect to phytate phosphorus in cereals, cereal byproducts, and other byproducts and negatively correlated in legume seeds and oilseeds. Except for cereals, the highest correlation between enzyme activities and phytate phosphorus was found for phytase. It is not possible to predict Phy and AcPh activities from phytate phosphorus content by linear and quadratic regressions. Finally, only highly significant and positive correlation was found between Phy and AcPh activities for cereals, cereal byproducts, and oilseeds.     

Hydrolysis of precipitated phytate by three distinct families of phytases

While genetically modified plants that secrete histidine acid phosphatases (HAPs), β-propeller phytases (BPPs) and purple acid phosphatases (PAPs) have been shown to assimilate soluble phytate, little is known about whether these plants have the ability to hydrolyze precipitated phytate. In this study, the ability of representative members of these three classes of phytases to hydrolyze metal-phytate salts and to hydrolyze phytate adsorbed to aluminum precipitates was compared. All three phytases were able to hydrolyze Ca²⁺-, Mg²⁺-, and Mn²⁺-phytates, but were unable to hydrolyze Al³⁺-, Fe²⁺-, Fe³⁺-, Cu²⁺-, and Zn²⁺-phytates. When these ions were present, the hydrolysis of Ca²⁺-phytate was prevented. Citrate was more potent than malate and oxalate in solubilizing some of these phytate salts for enzyme hydrolysis. Phytate adsorbed to aluminum precipitates was resistant to all three enzymes, except when organic acids were added (citrate>oxalate>malate). While increasing concentrations of organic acids were inhibitory to enzyme activity (oxalate >citrate>malate), PAP was more resistant to citrate than HAP. As desorption of phytate from a solid surface by organic acids is essential for phytase activity, the genetic engineering of plants that enhances the secretion of both citrate and phytases from the root may be a feasible approach to improving soil phytate assimilation.     

Treating Thin Stillage and Condensed Distillers Solubles with Phytase for Production of Low‐Phytate Coproducts

Fuel ethanol production from grains is mainly based on dry‐grind processing, during which phytate is concentrated about threefold in distillers dried grains with solubles (DDGS), a major coproduct. To reduce phytate in DDGS, Natuphos and Ronozyme industrial phytase preparations were used to treat commercially made thin stillage (TS). Changes in phosphorous (P) profile were monitored, and effects of reaction temperature, time, and enzyme concentration were investigated. Results showed that at a temperature ≤60°C for Natuphos phytase (≤70°C for Ronozyme phytase) and a concentration ≤4.8 FTU/mL of TS for Natuphos phytase (≤48 FYT/mL for Ronozyme phytase), a complete phytate hydrolysis (phytate P decreased to 0) could be achieved within 5–60 min of enzymatic treatment. Reduction in phytate P was generally accompanied by increase in inorganic P, whereas total P remained relatively unchanged. When condensed distillers solubles (CDS), the concentrated form of TS, was used as the substrate, phytate hydrolysis by each of the two enzyme preparations was as effective as on TS. Because a previous study from the author's laboratory showed that all types of P are mostly concentrated in TS and CDS but much less in distillers wet grains, phytase treatments of TS and CDS described in the present study can be an effective means in producing low‐phytate DDGS.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

日粮中添加植酸酶对鸡粪中不同形态磷含量的影响

由于植物性饲料中大部分磷不能被动物吸收利用,生产上往往添加一定无机磷,容易造成禽畜粪中的磷累积,提高环境磷污染风险。为保证种鸡日粮磷需求前提下降低鸡粪磷排放量,设计0、300、500IU植酸酶用量与0．4％、0．3％、0．2％有效磷含量的不同组合日粮进行种鸡饲养试验,研究了不同日粮对鸡粪磷形态的影响。结果显示,添加植酸酶对鸡粪中总磷、有机磷和无机磷含量影响未达显著水平,但显著降低鸡粪植酸磷含量。鸡粪总磷、植酸磷、有机磷和无机磷含量则随日粮有效磷含量增加而显著提高。其中,以0．2％有效磷日粮处理鸡粪总磷、有机磷和无机磷含量最低,0．2％有效磷＋500IU植酸酶日粮处理鸡粪植酸磷含量最低。因此,在生产上降低有效磷含量的同时添加适量植酸酶可以降低鸡粪磷盈余。     

The importance of lactic acid bacteria for phytate degradation during cereal dough fermentation

Lactic acid fermentation of cereal flours resulted in a 100 (rye), 95-100 (wheat), and 39-47% (oat) reduction in phytate content within 24 h. The extent of phytate degradation was shown to be independent from the lactic acid bacteria strain used for fermentation. However, phytate degradation during cereal dough fermentation was positively correlated with endogenous plant phytase activity (rye, 6750 mU g(-1); wheat, 2930 mU g(-1); and oat, 23 mU g(-1)), and heat inactivation of the endogenous cereal phytases prior to lactic acid fermentation resulted in a complete loss of phytate degradation. Phytate degradation was restored after addition of a purified phytase to the liquid dough. Incubation of the cereal flours in buffered solutions resulted in a pH-dependent phytate degradation. The optimum of phytate degradation was shown to be around pH 5.5. Studies on phytase production of 50 lactic acid bacteria strains, previously isolated from sourdoughs, did not result in a significant production of intra- as well as extracellular phytase activity. Therefore, lactic acid bacteria do not participate directly in phytate degradation but provide favorable conditions for the endogenous cereal phytase activity by lowering the pH value.     

Optimal conditions for phytate degradation, estimation of phytase activity, and localization of phytate in barley (cv. Blenheim)

Using a multivariate experimental design, optimal conditions for phytate degradation were found to be pH 4.8 and 57 degrees C in barley flour (cv. Blenheim) and pH 5.2 and 47 degrees C in a crude extracted phytase from barley. Three methods for measuring phytase activity in raw and hydrothermally processed barley were compared. Incubation at pH 5 and 55 degrees C for 60 min did not give significantly different results (p > 0.05), whereas incubation at pH 5 and 50 degrees C for 10, 20, 30, and 60 min gave significantly different results (p < 0.001) between methods. The change in microstructure of phytate globoids during hydrothermal processing showed that the degradation was highest in the scutellum cells and less in the aleurone layer.     

Differential interaction of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates

The effect of the soil environment on the mobility, stability and catalytic activity of phytase from two sources was compared, as these factors have important implications for the efficacy of enzyme function in soil. Phytase from an ascomycete fungus (Aspergillus niger) and a basidiomycete fungus (Peniophora lycii) was added to soil suspensions from three contrasting soils and activities in the solution and solid phase were monitored. The two enzymes were compared because the P. lycii phytase was known to have greater specific activity and a more acidic isoelectric point (pI) than A. niger and therefore predicted to have different adsorption characteristics. When added to soil suspensions buffered at pH 7.5, both phytases remained in solution in all of the soils. In contrast at near natural soil pH (pH 5.5), only the P. lycii phytase remained in solution, while the A. niger phytase was rapidly adsorbed to the soil solid phase. The extent of this adsorption was reduced, however, in a soil-dependent manner by prior addition of bovine serum albumin (BSA) to the soil suspensions. At the natural pH of the soil, the stability of the P. lycii phytase in soil solution was improved under sterile conditions, whereas degradation of the A. niger phytase was unaffected. Subsequently, P. lycii phytase was shown to be more effective at hydrolysing myo-inositol hexakisphosphate added to the soil. Moreover, the P. lycii phytase also hydrolysed more organic phosphate that was endogenous to a range of soils. This research indicates that the physicochemical properties of fungal phytases affect their mobility and temporal stability and their capacity to hydrolyse inositol phosphates in soil environments.     

Characterization of phytase activity from cultivated edible mushrooms and their production substrates

Phytase is used commercially to maximize phytic acid degradation and to decrease phosphorus levels in poultry and swine manure. To determine phytase content in edible mushrooms, basidiomata of Agaricus bisporus and three specialty mushrooms (Grifola frondosa, Lentinula edodes, and Pleurotus cornucopiae) and spent mushroom substrate (SMS) were surveyed. Enzyme activity ranged from 0.046 to 0.074 unit/g of tissue for four A. bisporus types (closed and open whites and closed and open browns) grown at The Pennsylvania State University's Mushroom Test Demonstration Facility (MTDF). The addition of various nutrient supplements to phase II mushroom production substrate did not alter phytase activity in A. bisporus. Portabella mushrooms (open brown) obtained from a commercial farm had significantly higher levels of phytase activity (0.211 unit/g of tissue) compared to A. bisporus grown at the MTDF. Of the specialty mushrooms surveyed, maitake (G. frondosa) had 20% higher phytase activity (0.287 unit/g of tissue) than commercial portabella mushrooms. The yellow oyster mushroom (P. cornucopiae) ranked second in level of phytase activity (0.213 unit/g of tissue). Shiitake (L. edodes) contained the least amount of phytase in basidiomata (0.107 unit/g of tissue). Post-crop steam treatment (60 degrees C, 24 h) of SMS reduced phytase activity from 0.074 to 0.018 unit/g. Phytase was partially purified from commercially grown portabella basidiomata 314-fold with an estimated molecular mass of 531 kDa by gel filtration chromatography. The optimum pH for activity was 5.5, but appreciable phytase activity was observed over the range of pH 5.0-8.0. Partially purified A. bisporus phytase was inactivated following a 10-min incubation at > or =60 degrees C.     

Susceptibility of wheat and Aspergillus niger phytases to inactivation by gastrointestinal enzymes

The activity of wheat and Aspergillus niger phytases was determined following preincubation for 60 min at 37 degrees C alone or in the presence of pepsin or pancreatin to examine their ability to survive in the gastrointestinal tract. At pH 3.5 both phytases were stable, but at pH 2.5 wheat phytase rapidly lost activity. Following preincubation at pH 3.5 in the presence of 5 mg of pepsin/mL, A. niger phytase retained 95% of its original activity, whereas only 70% of the wheat phytase activity was recovered. The stability of A. niger phytase in the presence of pepsin was the same at pH 2.5 as at pH 3.5. Results similar to those with pepsin at pH 3.5 were obtained following preincubation of the phytases in the presence of pancreatin at pH 6.0.     

Quantitative analysis of phytate globoids isolated from wheat bran and characterization of their sequential dephosphorylation by wheat phytase

Wheat phytase was purified to investigate the action of the enzyme toward its pure substrate (phytic acid - myo-inositol hexakisphosphate) and its naturally occurring substrate (phytate globoids). Phytate globoids were purified to homogeneity from wheat bran, and their nutritionally relevant parameters were quantified by ICP-MS. The main components of the globoids were phytic acid (40% w/w), protein (46% w/w), and several minerals, in particular, K > Mg > Ca > Fe (in concentration order). Investigation of enzyme kinetics revealed that K(m) and V(max) decreased by 29 and 37%, respectively, when pure phytic acid was replaced with phytate globoids as substrate. Time course degradation of phytic acid or phytate globoids using purified wheat phytase was followed by HPIC identification of inositol phosphates appearing and disappearing as products. In both cases, enzymatic degradation initiated at both the 3- and 6-positions of phytic acid and end products were inositol and phosphate.     

Effect of Specific Mechanical Energy on Protein Bodies and α-Zeins in Corn Flour Extrudates

Zeins, the storage proteins of corn, are located in spherical entities called protein bodies. The disruption of protein bodies and zein release during extrusion may influence the texture of corn-based extruded foods. In this work, chemical and microscopic studies were conducted on corn flour that had been extruded under mild to extreme conditions to determine the specific mechanical energy (SME) required to break apart protein bodies and release α-zein, and to assess changes in protein-protein interactions. Transmission electron microscopy with immunolocalization of α-zein revealed that starch granules and protein bodies remained intact under mild processing conditions (SME 35–40 kJ/kg), but under harsher conditions, protein bodies were disrupted and α-zein was released. At SME ≈100 kJ/kg, protein bodies appeared highly deformed and fused together with the α-zein released, whereas at higher SME, protein bodies were completely disrupted and α-zein was dispersed and may have formed protein fibrils. Protein in extrudates was less soluble in urea and SDS than in unprocessed corn flour, but it was readily extracted with urea, SDS, and 2-ME. This was likely due to protein aggregation upon processing due to a prevalence of hydrophobic interactions and disulfide bonds. This research directly relates SME during extrusion to chemical and structural changes in corn proteins that may affect the texture of corn-based, ready-to-eat food products.