Inositol phosphate degradation by the action of phytase enzyme in legume seeds

The kinetics of inositol phosphate degradation during the action of naturally occurring endogenous phytase for up to 90 min in pea and lentil flours has been studied, and compared with the addition of commercial phytase enzyme. In raw lentils IP₆, IP₅, IP₄ and IP₃ were present, whilst in peas only the presence of IP₆ and IP₅ was observed. Endogenous phytases were activated when legume flour was suspended in acidified water at pH 5.5 and 37 °C, and significant differences between lentils and peas were found. IP₆ suffered a sharp reduction in lentils and peas (81–91 and 73–93%, respectively), this reduction being slightly more pronounced after the addition of commercial phytase. The content of IP₅ decreased in lentils (48–69%), and increased in peas, except when commercial phytase acted for the first 30 min, after which a reduction was found (23%). The content of IP₄ generally decreased in lentils, except when endogenous phytase acted for 30 min when an increase was observed. However, in peas, IP₄ appeared in high concentrations up to 60 min by the action of both endogenous and exogenous phytases. The content of IP₃, on the other hand, did not change greatly in lentils. In peas it was not detected after the action of endogenous phytase enzyme and it appeared in a large amount after the action of commercial phytase. In order to obtain legume flour with low IP₆ and IP₅ contents and notable IP₄ and IP₃ contents, the action of naturally endogenous phytase for 30 min in lentils is recommendable, as well as the addition of commercial phytase enzyme for 60 min in peas.     

Monitoring the stepwise phytate degradation in the upper gastrointestinal tract of pigs

The degradation and formation of inositol phosphates as affected by microbial phytase and gastrointestinal enzyme activities during the passage of phytate through the stomach and small intestine were studied in two experiments with four barrows and three collection periods. The degradation and formation of inositol phosphates were measured at the duodenal and ileal sites using Cr‐NDR, TiO₂ and Co‐EDTA as indigestible markers. In experiment 1, the effect of graded doses of Aspergillus niger phytase (0, 150 and 900 FTU Natuphos^® kg^?1), added to a maize–soybean meal‐based diet with very low intrinsic phytase activity on the degradation of phytate and the formation of inositol phosphates during digestion in the stomach and small intestine was investigated. In experiment 2, three different mixtures of inositol phosphates, produced by Aspergillus niger phytase, containing mainly high, intermediate and low phosphorylated inositol phosphates, were added to the same maize–soybean meal‐based diet as used in experiment I. The fate of the inositol phosphates during digestion in the stomach and small intestine was studied. Experiment 1 showed that the extent of phytate degradation was dependent of the graded dietary phytase activities. At high phytase activity (900 FTU kg^?1 of diet), strong phytate degradation occurred and the once hydrolysed phytate was rapidly dephosphorylated to lower inositol phosphates (mainly inositol di‐ and triphosphates). Intermediate inositol phosphates, such as inositol tetraphosphates, were quantitatively unimportant in duodenal and ileal digesta. At a phytase activity of 150 FTU kg^?1 of diet, a broader spectrum of intermediate inositol phosphates was determined, which was probably due to a slower breakdown of phytate. Experiment 2 showed as a predominant result that lower inositol phosphates such InsP₄ and InsP₃ were degraded, whereas InsP₂ accumulated in the duodenal and ileal digesta. No substantial disappearance of phytate from the stomach and small intestine was found when high concentrations of soluble phytate were added to the diet, which indicates that no substantial phytate absorption occurs in the upper part of the pig gut. Copyright © 2005 Society of Chemical Industry     

THE CHARACTERISTICS OF SOYBEAN PHYTASE

Soybean phytase was extracted with 2% CaCl₂ and partially purified by ammonium sulphate fractionation followed by dialysis in 0.01 M tris-maleate buffer, pH 6.5. The enzyme showed an optimum pH of 4.8 and optimum temperature of 60°C. The phytase was partially inhibited at high substrate concentration, with an optimum substrate concentration at 20 mM and a K_m value of 2.4 × 10^-3 M. V_max was 0.22 μmole P_i liberated/min/mL enzyme. The inactivation and activation energies for the hydrolysis of phytic acid were approximately 47,000 cal/mole and 11,100 cal/mole, respectively. Enzyme activity was inhibited by about 25%, 23% and 22% in the presence of 10^-3 M Zn⁺⁺, Cu⁺⁺ and Hg⁺⁺, respectively, and was also decreased by about 85% in the presence of 10^-1 M N-ethylmaleimide and sodium fluoride. Reducing and chelating agents at concentrations up to 10^-1 M inhibited activity by about 50% and by more than 90%, respectively.     

Phytase in non‐ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors

This review focuses on phytase functionality in the digestive tract of farmed non‐ruminant animals and the factors influencing in vivo phytase enzyme activity. In pigs, feed phytase is mainly active in the stomach and upper part of the small intestine, and added phytase activity is not recovered in the ileum. In poultry, feed phytase activities are mainly found in the upper part of the digestive tract, including the crop, proventriculus and gizzard. For fish with a stomach, phytase activities are mainly in the stomach. Many factors can influence the efficiency of feed phytase in the gastrointestinal tract, and they can be divided into three main groups: (i) phytase related; (ii) dietary related and (iii) animal related. Phytase‐related factors include type of phytase (e.g. 3‐ or 6‐phytase; bacterial or fungal phytase origin), the pH optimum and the resistance of phytase to endogenous protease. Dietary‐related factors are mainly associated with dietary phytate content, feed ingredient composition and feed processing, and total P, Ca and Na content. Animal‐related factors include species, gender and age of animals. To eliminate the antinutritional effects of phytate (IP₆), it needs to be hydrolyzed as quickly as possible by phytase in the upper part of the digestive tract. A phytase that works over a wide range of pH values and is active in the stomach and upper intestine (along with several other characteristics and in addition to being refractory to endogenous enzymes) would be ideal. © 2014 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Effect of exogenous phytase on feed inositol phosphate hydrolysis in an in vitro rumen fluid buffer system

Three in vitro experiments using a rumen fluid buffer system were performed to investigate the effect of addition of 4 experimental phytases (Phy1, Phy2, Phy3, and Phy4) compared with no addition of phytase on feed inositol phosphate hydrolysis in wheat and rapeseed cake to determine which of the 4 phytases was most suitable under rumen-like conditions. The feedstuffs were incubated with a mixture of physiological buffer, ruminal fluid, and exogenous phytase at pH 6.2, after which the samples were incubated for different periods. Incubations were stopped using HCl, and the samples were analyzed for inositol phosphates via high performance ion chromatography. Addition of phytase (Phy1) resulted in enhanced degradation of myo-inositol hexakisphosphate (InsP₆) in rapeseed cake, whereas addition of exogenous phytase did not improve the degradation of InsP₆ in wheat. Only rapeseed cake was therefore used subsequently. All 4 phytases increased degradation of InsP₆ in rapeseed cake in the in vitro system, and degradability of InsP₆ increased with higher incubation time and higher phytase dosages, independent of phytase. Addition of 2 units of phytase per gram of substrate of the phytases Phy1, Phy2, Phy3, and Phy4 led to an undegraded InsP₆ content of 56, 49, 70, and 18%, respectively, when incubated with rapeseed cake for 6 h, indicating that Phy2 and Phy4 were the most effective phytases. However, Phy2 had a higher specific activity than Phy4, as 60% of the original InsP₆ content was remaining after 3 h when 5 mg of enzyme protein per gram of substrate of Phy2 was added to rapeseed cake, whereas 150 mg of enzyme protein per gram of substrate of Phy4 was necessary to achieve a similar result. Therefore, Phy2 appeared to be most applicable under rumen-like conditions.     

Dephytinization of food stuffs by phytase of Geobacillus sp. TF16 immobilized in chitosan and calcium-alginate

In this work, Geobacillus sp. TF16 phytase was separately immobilized in chitosan and Ca-alginate with the efficiency of 38% and 42%, respectively. These enzymes exhibited broad substrate specificity. Maximal relative phytase activity was measured at pH 5.0 and 95°C and pH 3.0 and 75°C for chitosan and Ca-alginate, respectively. The enzymes were highly stable in a wide pH and temperature range. Values of K_m and V_max were determined as 2.38 mM and 3401.36 U/mg protein for chitosan, and 7.5 mM and 5011.12 U/mg protein for Ca-alginate. The immobilized enzymes showed higher resistance to proteolysis. After 4 h incubation, hydrolysis capacities of chitosan- and Ca-alginate immobilized enzymes for soymilk phytate were calculated as 24% and 33%, respectively. The chitosan- and Ca-alginate immobilized phytases conserved its original activity after 8 and 6 cycles of reuse, respectively. The features of the enzymes were very attractive and they might be useful for some industrial applications.     

The phytate and phytase of soybean tempeh

Soybeans were fermented into tempeh by Rhizopus oligosporus NRRL 2710. The phytic acid content of soybeans was reduced by about one-third as a result of this fermentation, while an equivalent amount of phosphate was released in the tempeh. The reduction of phytic acid was due to phytase elaborated by the mould of the fermentation. The pH optimum of this enzyme was 5.6 and the K_m of the phytase-phytate reaction was 0.28 × 10^?3 M.     

Production of phytase by Aspergillus ficuum and reduction of phytic acid content in canola meal

Production of the phytase (EC 3.1.3.8) from Aspergillus ficuum in a submerged batch process was inhibited by high concentrations of glucose. The inhibition was overcome by applying a fed batch technique in the production of the enzyme. Tests carried out at different oxygen concentrations revealed that aeration had a beneficial effect on the production of the enzyme. The enzyme showed an optimum pH and temperature of 5·0 and 60°C, respectively. Preincubation of the enzyme preparation at 60°C resulted in relatively fast denaturation of the enzyme. Upon storage at 4°C it lost only 15% of its activity in 5 weeks. Aspergillus ficuum also produced phytase when grown on canola meal by a solid state technique. The enzyme catalysed degradation of the phytic acid present in the meal and completely eliminated it, rendering the commodity more suitable for animal feed. An apparent 10% increase in protein content of the canola meal was noted as a result of the growth of the microorganism.     

CHARACTERIZATION OF PHYTASE ACTIVITY IN LUPIN SEED

Lupin phytase (myo-inositol hexaphosphate phosphohydrolase) and the degradation of its substrate phytic acid were studied during seed germination. Phytase and acid phosphatase activities were detected in nongerminated seeds and increased from days 1 to 8 of germination, while the concentration of phytic acid decreased during the same period. Phytase was extracted with 2% CaCl₂ solution and partially purified by ammonium sulfate fractionation and HPLC-gel permeation chromatography. Substrate specificites were determined using pnitrophenylphosphate or phytic acid. The pH and temperature optima for the fraction obtained by gel permeation were 5.0 and 50C respectively, while the Km and V _max values were 0.33 mM and 0.13 μmol·mL^?1·min^?1, respectively. It was not possible to separate phytase from phosphatase by use of gel permeation chromatography due to similarities in apparent molecular mass, however resolution of the two enzymes was achieved by DEAE-cellulose chromatography.     

Phytase Activity from Lactobacillus spp. in Calcium-Fortified Soymilk

Abstract: The presence of phytate in calcium-fortified soymilk may interfere with mineral absorption. Certain lactic acid bacteria (LAB) produce the enzyme phytase that degrades phytates and therefore may potentially improve mineral bioavailability and absorption. This study investigates the phytase activity and phytate degradation potential of 7 strains of LAB including: Lactobacillus acidophilus ATCC4962, ATCC33200, ATCC4356, ATCC4161, L. casei ASCC290, L. plantarum ASCC276, and L. fermentum VRI-003. Activity of these bacteria was examined both in screening media and in calcium-fortified soymilk supplemented with potassium phytate. Most strains produced phytase under both conditions with L. acidophilus ATCC4161 showing the highest activity. Phytase activity in fortified soymilk fermented with L. acidophilus ATCC4962 and L. acidophilus ATCC4161 increased by 85% and 91%, respectively, between 12 h and 24 h of fermentation. All strains expressed peak phytase activity at approximately pH 5. However, no phytate degradation could be observed.     

Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa)

A phytate-degrading enzyme (myo-inositol hexakisphosphate phosphohydrolase) has been purified about 5,400-fold from germinated oat seedlings to apparent homogeneity. The molecular mass of the native monomeric enzyme was estimated to be about 67 kDa. Optimal pH for degradation of phytate was 5.0 and the optimal temperature 38 °C. Kinetic parameters for the hydrolysis of Na-phytate are K_M 30 µM and k_cat 356 s⁻¹ at 35 °C and pH 5.0. The oat phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyses phytate in a stepwise manner. The first hydrolysis product was identified as D /L -l(1,2,3,4,5) P₅. © 1999 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF CANOLA SEED (BRASSICA sp.) PHYTASE

Germinated Altex and Westar (Brassica napus) and Candle and Tobin (B. campestris) cultivars of Canola were screened for phytase activity. On the basis of this preliminary screening, 7-day germinated Altex seedlings were selected as a source for isolation and characterization of phytase. Partial purification of a crude extract (FI) by acetone precipitation resulted in an 8-fold increase in phytase activity. Ion-exchange chromatography of the partially purified preparation (FII) yielded two fractions (FIIIA and FIIIB) both of which demonstrated phytase and phosphatase activities. Further purification by gel filtration chromatography resulted in two fractions (FIVA1 and FIVA2) from fraction FIIIA and two fractions (FIVB1 and FIVB2) from fraction FIIIB. Fraction FIVB1 demonstrated both phytase and phosphatase activities, FIVA2 and FIVB2 demonstrated phosphatase activity but no phytase activity and FIVA1 showed phytase but no phosphatase activity. Fraction FIVB1, which showed highest phytase activity (5.3 IU/mg protein), had the following characteristics: temperature optimum of 50°C, pH optimum of 5.2, K_m of 0.36 mM and relative activity for pyrophosphate 232 times higher than for phytate.     

Physicochemical properties of soy protein prepared by enzyme‐assisted countercurrent extraction

Synchronous improvements of both nutritional properties and the flavour of protein by optimising preparation processes are highly challenging. The prehydrolysis of soy meals by cocktail enzyme (β‐glucosidase, phytase and acid protease) treatment and subsequent countercurrent extraction were designed based on similar hydrolysis conditions. The composition, flavour volatiles and physicochemical properties of soy proteins were investigated. Compared to alkaline extraction and acid precipitation, enzyme‐assisted countercurrent extraction significantly increased protein yield and carbohydrate content, accompanied by a decrease in protein purity. This protein exhibited larger molecular weight distribution, less flavour volatiles, higher thermal stability and surface hydrophobicity, as evidenced by higher denaturation temperature (T_d) and enthalpy change (ΔH) of protein. The conversion of isoflavone glycosides to aglycone and the partial degradation of phytic acid were observed for enzyme‐prepared soy proteins. These results suggest a feasible protocol for producing a nutrient‐improved soy protein with excellent flavour and thermal stability.     

Peniophora lycii phytase is stabile and degrades phytate and solubilises minerals in vitro during simulation of gastrointestinal digestion in the pig

BACKGROUND: Microbial phytases (EC 3.1.3) are widely used in diets for monogastric animals to hydrolyse phytate present in the feed and thereby increase phosphorus and mineral availability. Previous work has shown that phytate solubility is strongly affected by calcium in the feed and by pH in the gastrointestinal (GI) tract, which may have an effect on phytase efficacy. An in vitro model simulating the GI tract of pigs was used to study the survival of Peniophora lycii phytase and the effect of the phytase on phytate degradation, inositol phosphate formation and mineral solubilisation during in vitro digestion of a 30:70 soybean meal/maize meal blend with different calcium levels. RESULTS: The phytase retained 76 and 80% of its initial activity throughout the gastric in vitro digestion. Total phytate hydrolysis by P. lycii phytase was in the same range at total calcium levels of 1.2 and 6.2 mg g^?1 dry matter (DM), despite very large differences in phytate solubility at these calcium levels. However, at 11.2 and 21.2 mg Ca g^?1 DM, phytate hydrolysis was significantly lower. The amount of soluble mineral was generally increased by P. lycii phytase. CONCLUSION: Stability of P. lycii phytase during gastric digestion was not found to be critical for phytate hydrolysis. Furthermore, original phytate solubility was not an absolute requirement for phytate degradation; phytate solubility seemed to be in a steady state, allowing insoluble phytate to solubilise as soluble phytate was degraded. This is new and interesting knowledge that adds to the current understanding of phytate–phytase interaction. Copyright © 2007 Society of Chemical Industry     

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

BACKGROUND: 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. RESULTS: Endogenous wheat phytase effectively degraded wheat InsP₆ + 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. CONCLUSION: 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. Copyright © 2011 Society of Chemical Industry     

Rapid Purfication of β -Galactosidase (Aspergillus Niger) from a Commercial Preparation

β-Galactosidase (A. niger) was purified from a commercial source in order to study the protein nature of the enzyme and some of its kinetic properties. The enzyme was rapidly purified by acetone precipitation, gel filtratior, and affinity chromatography. The specific activity of the purified enzyme was twice as high as that found in previous studies. The K_m and V_max for o-nitrophenyl β-D-galactopyranoside were 2.02 mM and 345 μmoles/min/mg protein respectively at pH 4.5 and 37°C. The procedure described yields a highly active enzyme which may be suitable for immobilization and hydrolysis of lactose. The molecular weight of the enzyme was 117,000 and the isolectric point was 4.9. The enzyme appears to be a glycoprotein and may contain multiple molecular forms.     

Influence of the action of exogenous enzymes on the polyphenolic composition of pea: Effect on the antioxidant activity

The polyphenolic composition of green pea (Pisum sativum, variety Esla) was determined by HPLC-DAD and HPLC-(ESI)MS procedures. The action of the enzymes tannase, α-galactosidase, phytase and viscozyme on pea flours, at a semi-pilot scale, was assayed for the polyphenolic composition. Different modifications in the initial concentration of polyphenolics occur depending on the enzyme, but a general decrease was observed in all of them. Free radical scavenging activity of the methanolic extracts, determined by the DPPH test, was also evaluated. The treatments with phytase, viscozyme, α-galactosidase or tannase produced a decrease in the antioxidant activity when compared to raw peas. The results of the analysis of principal components to examine the relationship between antioxidant activity (EC₅₀) and concentrations of polyphenolics in each of the pea samples showed that the hydroxycinnamic compounds are the most related to antioxidant activity.     

Simultaneous application of phytase and xylanase to broiler feeds based on wheat: in vitro measurements of phosphorus and pentose release from wheats and wheat-based feeds†

An in vitro procedure that simulated digestion in growing broilers was tested to predict phosphorus availability and arabinoxylan hydrolysis in samples of nine wheat varieties and in a wheat-based diet. Amounts of dialysable phosphorus freed from wheat samples correlated with activities of endogenous phytase (R = 0.913; p < 0.0001), whereas amounts of pentoses released were correlated with viscosities of the digested samples (R = 0.899; p < 0.0001). Differences in phosphorus release resulting from graded levels of microbial phytase added to feeds that were either autoclaved or not autoclaved revealed a decreasing role of endogenous phytase in dephosphorylation as levels of microbial phytase supplementation grew. Amounts of pentoses released from feeds containing two different xylanase preparations reflected literature data on different in vivo efficacies of those preparations. Simultaneous addition of phytase and xylanase affected phosphorus release in a manner that depended upon the form of xylanase preparation used (liquid or powder). There was a positive influence of acid protease on both phytate and arabinoxylan hydrolysis in feeds supplemented with phytase. Effects observed by the in vitro procedures corresponded to in vivo phenomena described in the literature. © 1999 Society of Chemical Industry     

In-vitro and in-vivo dephosphorylation of rapeseed meal by means of phytate-degrading enzymes derived from Aspergillus Niger

A meal of ‘double low’ rapeseed (var ‘Jantar’) was subjected to phytate hydrolysis using enzyme preparations derived from a mycelium of Aspergillus niger which contained phytase (EC 3.1.3.8) and acid phosphatase (EC 3.1.3.2) activities. The complete conversion of myo-inositol hexa- and pentaphosphates present in rapeseed meal to lower phosphate esters of myo-inositol was accomplished at 40°C, a pH value of 4.5, phytase dosage (in phytase units (PhytU)) 0.1 PhytU g^?1 accompanied by acid phosphatase activity 37.1 units g^?1, in 1 h. Under these conditions, complete dephosphorylation was observed in 4 h. Decreasing the pH value to 3.0 caused a rise in the amount of inorganic phosphorus released, while increasing to 5.5 resulted in substantial reduction in the reaction rate. Purification of phytase to a specific activity 0.375 PhytU mg^?1 of protein exhibited a negative influence upon the yield of rapeseed dephosphorylation. The substitution of calcium phosphate for a preparation of phytase in feed containing rapeseed meal did not cause significant differences in the body weight gain or in tibia mineralisation of broilers (Gains galus, ‘Astra B’).     

Control of amylase and protease activities in a phytase preparation by ampholyte-free preparative isoelectric focusing for unrefined cereal-containing bread

Direct addition of a food additive-grade Aspergillus niger phytase preparation to 30% brown rice flour-added bread ingredients reduced the bread myo-inositol hexaphosphate (IP₆) content. The phytase preparation had protease and amylase activities. Addition of the crude phytase preparation induced bread crust collapse. Protease-free high- and low-amylase phytase fractions were prepared using preparative ampholyte-free isoelectric focusing (autofocusing). Addition of the protease-free low-amylase phytase fraction did not significantly affect loaf volume and did not collapse the crust. Addition of the protease-free high-amylase phytase fraction significantly increased loaf volume approximately 1.5× in the presence of amylase at 193–1029 U. However, addition of 1029 and 2058 U of amylase induced partial and whole bread crust collapse, respectively. Therefore, removal of protease and control of amylase activities in the phytase preparation are crucial in the preparation of brown rice flour-added bread with a low myo-inositol phosphate (IP₆) and good swelling property.