Wheat Phytase Research Articles

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.

In vitro degradation of phytate and lower inositol phosphates in soaked diets and feedstuffs

Abstract A series of in vitro experiments simulating liquid feeding were performed to evaluate the effect of microbial phytase addition, heat-treatment and soaking time on degradation of phytate and lower inositol phosphates when soaking compound wheat/soybean meal diets or the single feedstuffs wheat or soybean meal. The effect of phytase addition on phytate degradation was greatest in soybean meal, almost intermediate for wheat/soybean meal diets and not detectable in wheat, which might be due to a better accessibility to phytate in soybean meal compared with wheat. Heat-treatment seemed to enhance the accessibility between phytase and phytate, whereby phytate degradation was stimulated. Additionally, it was shown that wheat phytase is able to stimulate degradation of phytate in soybean meal. Independent of treatment, the amount of IP5–IP2 was extremely small in relation to phytate in both wheat and soybean meal, indicating that when one phosphate group is removed from the phytate complex, degradation of IP5–IP2 is completed. Consequently, it is anticipated that liquid feeding might result in a higher digestibility of plant P compared with dry feeding of pigs.

Influence of feeding level and dietary oil supplementation on apparent ileal and total tract digestibilities of phosphorus and calcium in pigs fed low-phosphorus diets supplemented with microbial or wheat phytase

Two experiments were conducted with pigs fitted with simple T-cannulas at the distal ileum to evaluate the effect of energy (ME) intake implemented by variation of feeding level (FL) (exp. 1) or dietary plant oil content (exp. 2), on apparent ileal (AID) and total tract digestibilities (ATTD) of phosphorus (P) and calcium (Ca), including measurements of lower myo-inositol phosphates at the ileal and fecal level. In exp. 1, 14 barrows (BW 7.3 kg) were fed one of two low-P corn-soybean meal diets providing either 600 U kg-1 microbial (Natuphos® 5000 G) or native wheat phytase at two FL (2.0 vs. 2.8 × ME maintenance requirement) according to a two-period cross-over design. The AID and ATTD of P and Ca were higher (P < 0.05) in pigs fed microbial phytase compared with wheat phytase. Furthermore, the recoveries of lower myo-inositol phosphates in the feces, but not in the ileal digesta, were higher (P < 0.05) when microbial instead of wheat phytase was added to the diet. Increasing the feed intake improved (P < 0.05) overall AID and ATTD of P, independent of the origin of the supplemental phytase. Additionally, the increase in FL improved (P < 0.05) AID of P in pigs fed wheat phytase. There was no effect (P > 0.05) of FL on ileal or fecal recovery of myo-inositol phosphates. In exp. 2, eight barrows (BW 20.3 kg) were fed a low-P corn-soybean meal diet providing 600 U kg-1 microbial phytase (Natuphos® 5000 G) supplemented without or with 6% plant oil according to a two-period cross-over design. There was no effect (P > 0.05) of oil supplementation on AID and ATTD of P or Ca. In conclusion, an increase in FL improves AID and ATTD of P in pigs fed diets supplemented with microbial or wheat phytase, while increasing energy density had no effect. Key words: Feeding level, phytase, phosphorus, calcium, pigs, digestibility

Influence of feeding level on apparent total tract digestibility of phosphorus and calcium in pigs fed low-phosphorus diets supplemented with microbial or wheat phytase

Eighteen pigs (initial body weight 11.3 kg) were used in a 5-week digestibility experiment to investigate the effects of supplemental microbial and native wheat phytase on apparent total tract digestibility (ATTD) of phosphorus (P) and calcium (Ca) at different feeding levels (FL) in a 3 × 3 factorial arrangement of treatments. A low-P (4.2 or 1.4 g/kg DM total or digestible P, respectively) corn–soybean meal diet, either containing 0 or 550 U/kg from microbial ( Penicillium) or wheat phytase, respectively, was fed at three FL (1.2-, 2.0- or 2.8-fold the ME maintenance requirement) in three consecutive periods. Pigs were kept in metabolism crates and feces and urine were quantitatively collected. Supplementation of the control diet with microbial phytase increased ( P < 0.001) ATTD of P and Ca by 22.6 and 18.3 percentage units, respectively. Similarly, but to a smaller extent, inclusion of wheat phytase increased ATTD of P ( P < 0.001) and Ca ( P = 0.005) by 7.4 and 4.9 percentage units, respectively. The overall ATTD of P and Ca were positively influenced by the level of feed intake ( P < 0.001). Increasing the FL improved the efficiency of microbial phytase ( P < 0.05) but not of wheat phytase ( P > 0.05) in rendering phytate P digestible. Phytase effects on ATTD of Ca were not affected ( P > 0.05) by an increase in FL.

Fermentation and enzyme treatment of tannin sorghum gruels: effects on phenolic compounds, phytate and in vitro accessible iron

The presence of polyphenols and phytate in cereal products has been shown to interfere with the bioavailability of minerals such as iron. In the present study, we added enzymes (wheat phytase and mushroom polyphenol oxidase) during fermentation of tannin sorghum gruels prepared from flour with or without addition of 5% flour of germinated tannin-free sorghum grains (power flour), and investigated the effects on phenolic compounds, phytate and in vitro accessible iron. Assayable phenolic compounds were significantly reduced by fermentation, with high reductions observed in gruels with added enzymes. Fermentation of the gruels with addition of enzymes reduced (on average) total phenols by 57%, catechols by 59%, galloyls by 70% and resorcinols by 73%. The phytate content was significantly reduced by fermentation (39%), with an even greater effect after addition of power flour (72%). The largest reduction of phytate (88%) was, however, obtained after addition of phytase. The in vitro accessible iron was 1.0% in the sorghum flour and it increased after fermentation with power flour and/or with enzymes. The highest in vitro accessibility of iron (3.1%) was obtained when sorghum was fermented with addition of power flour and incubated with phytase and polyphenol oxidase after the fermentation process.

Moderate Decrease of pH by Sourdough Fermentation Is Sufficient To Reduce Phytate Content of Whole Wheat Flour through Endogenous Phytase Activity

Whole wheat bread is an important source of minerals but also contains considerable amounts of phytic acid, which is known to impair their absorption. An in vitro trial was performed to assess the effect of a moderate drop of the dough pH (around 5.5) by way of sourdough fermentation or by exogenous organic acid addition on phytate hydrolysis. It was shown that a slight acidification of the dough (pH 5.5) with either sourdough or lactic acid addition allowed a significant phytate breakdown (70% of the initial flour content compared to 40% without any leavening agent or acidification). This result highlights the predominance of wheat phytase activity over sourdough microflora phytase activity during moderate sourdough fermentation and shows that a slight drop of the pH (pH value around 5.5) is sufficient to reduce significantly the phytate content of a wholemeal flour. Mg "bioaccessibility"of whole wheat dough was improved by direct solubilization of the cation and by phytate hydrolysis.

Enzymatic Hydrolysis of Organic Phosphorus in Swine Manure and Soil

Organic phosphorus (Po) exists in many chemical forms that differ in their susceptibility to hydrolysis and, therefore, bioavailability to plants and microorganisms. Identification and quantification of these forms may significantly contribute to effective agricultural P management. Phosphatases catalyze reactions that release orthophosphate (Pi) from Po compounds. Alkaline phosphatase in tris-HCl buffer (pH 9.0), wheat (Triticum aestivum L.) phytase in potassium acetate buffer (pH 5.0), and nuclease P1 in potassium acetate buffer (pH 5.0) can be used to classify and quantify Po in animal manure. Background error associated with different pH and buffer systems is observed. In this study, we improved the enzymatic hydrolysis approach and tested its applicability for investigating Po in soils, recognizing that soil and manure differ in numerous physicochemical properties. We applied (i) acid phosphatase from potato (Solanum tuberosum L.), (ii) acid phosphatases from both potato and wheat germ, and (iii) both enzymes plus nuclease P1 to identify and quantify simple labile monoester P, phytate (myo-inositol hexakis phosphate)-like P, and DNA-like P, respectively, in a single pH/buffer system (100 mM sodium acetate, pH 5.0). This hydrolysis procedure released Po in sequentially extracted H2O, NaHCO3, and NaOH fractions of swine (Sus scrofa) manure, and of three sandy loam soils. Further refinement of the approach may provide a universal tool for evaluating hydrolyzable Po from a wide range of sources.

The effect of steeping on soluble phosphorus levels in wheat, wheatfeed and soyabean meal

Much of the phosphorus in feed ingredients is unavailable to the pig because it is locked up in phytic acid and phytates, which the pig cannot digest. Raw ingredients that have not been heat-treated contain endogenous phytases which can be activated by steeping in water prior to inclusion in liquid feed for pigs. This results in an increase in soluble phosphorus due to the hydrolysis of phytic acid and phytates, and hence an increase in the phosphorus available to the pig. However, with heat-treated ingredients such as soyabean meal (SBM) endogenous enzymes are destroyed during processing. In liquid feed systems steeping combinations of raw materials may have an advantageous affect on the availability of phosphorus as the endogenous phytases of the cereal component of the diet may hyrolyse phytates present in SBM. In wheat the phytate phosphorus and phytase is concentrated in the aleurone layer of the grain (Eeckhout and De Paepe 1994). Therefore, the addition of wheatfeed to liquid feed may increase the amount of endogenous wheat phytase present and hence the amount of soluble P released during steeping. The amount of phosphorus that is available to the pig from these raw ingredients is 30%, 42% and 66% of the total phosphorus for SBM, wheat and wheatfeed respectively (Ewing 1997). The objective of this study was to determine the effect of steeping SBM with wheat, wheatfeed or a combination of the two on the release of soluble phosphorus.

Additivity of the effect of cereal and microbial phytases on apparent phosphorus absorption in growing pigs fed diets with marginal P supply

Cereal phytases originating from wheat and rye or in combination with supplemented microbial phytase (Natuphos ®) each were added to phytase-inactivated cereal-based diets to determine whether phytases of different origin exhibit additivity in their response on coefficient of apparent phosphorus (P) absorption in growing pigs. Thirty castrates with an initial body weight of 16.5 kg were kept in stainless steel metabolism crates during a 20 days experimental period. The animals were allocated to six dietary treatments. The basal diets (Diets 1, 3 and 5) were devoid of phytase from wheat and/or rye due to complete inactivation of plant phytase in these grains by microwave treatment. Total phytase activity in the experimental diets was calculated to amount in total to 150 U/kg originating in equal parts from wheat and microbial phytases (Diet 2) or rye and microbial phytases (Diet 4) or wheat and rye phytases (Diet 6). The diets were fed twice daily at a rate of 375 g per meal. An adaptation period of 10 days to the diets and experimental conditions was followed by a quantitative collection of faeces and urine during 10 days. The supplementation of phytase-inactivated basal diets with combinations of microbial and wheat phytases (Diet 2), microbial and rye phytases (Diet 4) as well as with wheat and rye phytases (Diet 6) increased ( P<0.05) coefficient of apparent P absorption by 0.179, 0.150 and 0.058 in the wheat-, rye- and wheat–rye-based diets, respectively. The linear regression model was used for predicting the additivity of the effect of each of the intrinsic (cereal) and extrinsic (microbial) phytases on apparent P absorption in growing pigs. It was concluded that different sources of phytases exhibit linear additivity in their response on apparent P absorption in growing pigs, provided that existing differences in equivalency values between cereal and microbial phytases, obtained under similar experimental conditions, are taken into consideration.

Comparative evaluation of the efficacy of cereal and microbial phytases in growing pigs fed diets with marginal phosphorus supply

AbstractTwo experiments with a total of 76 growing pigs (average initial body weight 16.6 kg) were conducted to compare the efficacy of cereal phytases (wheat and rye) and supplemented microbial phytase (Natuphos®). Using the slope ratio technique, the dose–response relationship between five levels of phytase (0, 50, 100, 150 and 200 U kg−1) and the apparent absorption of phosphorus (P) within each source of phytase was calculated. Graded phytase levels in the diets were obtained by adding increasing amounts of microbial phytase or phytase‐containing wheat (Exp 1) or rye (Exp 2) to phytase‐inactivated basal diets at the expense of phytase‐inactivated wheat (Exp 1) or rye (Exp 2). Except for wheat phytase, addition of phytase to the basal diets increased (P &lt; 0.05) apparent P absorption, with microbial phytase being more efficient (P &lt; 0.05) than cereal phytase. There were no significant differences in apparent P absorption between the wheat‐ or rye‐based diets when either microbial or cereal phytases were supplemented from 0 to 200 U kg−1. It could be derived from the results of this study, by means of regression analysis, that the efficacy of cereal phytases was 40% compared to microbial phytase.© 2002 Society of Chemical Industry

Hydrolysis of phytic acid by intrinsic plant and supplemented microbial phytase (Aspergillus niger) in the stomach and small intestine of minipigs fitted with re-entrant cannulas. 3. Hydrolysis of phytic acid (IP6) and occurrence of hydrolysis products (IP5, IP4, IP3 and IP2).

Hydrolysis of phytate in the stomach and the small intestine as influenced by intrinsic plant (wheat) and supplemented microbial phytase (Aspergillus niger) were investigated with six minipigs (40-50 kg initial body weight) fitted with re-entrant cannulas in the duodenum, 30 cm posterior to the pylorus (animals 1, 4, 5 and 6) and ileocecal re-entrant cannulas, 5 cm prior the ileocecal junction (animals 1, 2 and 3), respectively. Dietary treatments were as follows: (1) diet 1, a corn-based diet [43 U phytase/kg dry matter (DM)]; (2) diet 2, diet 1 supplemented with microbial phytase (818 U/kg DM); and (3) diet 3, a wheat-based diet (1192 U/kg DM). At 07 30 h and 19 30 h, each animal was fed 350 g diet mixed with 1050 ml de-ionized water. Digesta were collected continuously and completely during a 12-h period after feeding. Mean hydrolysis rates of IP6 in the stomach as measured at the proximal duodenum of animals 1, 4, 5 and 6 were 9.0, 77.2 and 66.2% for diet 1, 2 and 3, respectively. Microbial phytase was much more effective in phytate hydrolysis than wheat phytase. Mean IP6 hydrolysis rates of the respective diets in the stomach and small intestine as measured at the distal ileum of animals 1, 2 and 3 were 19.0, 62.6 and 64.6% and were lower than treatment means of the stomach only. Differences existed between experimental animals with respect to their ability to hydrolyse IP6 in the stomach independent of the presence and source of dietary phytase. Considerable amounts of hydrolysis products occurred in both the duodenal and ileal digesta when diets 2 and 3 were fed; however, only traces were determined after ingestion of diet 1. Independent of dietary treatment, four IP5 isomers were detected, but in different amounts.

Hydrolysis of phytic acid by intrinsic plant and supplemented microbial phytase (Aspergillus niger) in the stomach and small intestine of minipigs fitted with re-entrant cannulas. 2. Phytase activity.

Hydrolysis of phytate in the stomach and the small intestine as influenced by intrinsic plant (wheat) and supplemented microbial phytase (A. niger) were investigated with six minipigs (40-50 kg initial BW) fitted with re-entrant cannulas in the duodenum, 30 cm posterior to the pylorus (animals 1, 4, 5, and 6) and ileocecal re-entrant cannulas, 5 cm prior the ileocecal junction (animals 1, 2, and 3), respectively. Dietary treatments were as follows: (1) diet 1, a corn-based diet (43 U phytase/kg DM); (2) diet 2, diet 1 supplemented with microbial phytase (818 U/kg DM) and (3) diet 3, a wheat-based diet (1192 U/kg DM). At 0730 and 1930 per animal 350 g diet mixed with 1050 ml de-ionized water were fed. Digesta were collected continuously and completely during 12 h after feeding. In the duodenal digesta, 70% of the microbial phytase (diet 2) and 45% of the wheat phytase (diet 3) were recovered within 12 h after ingestion of the phytases, whereas only negligible amounts were detected in the digesta of pigs fed the phytase-poor corn-based diet 1. Most phytase activity passed through the stomach within the first hour after feeding. Microbial phytase activity at pH 2.8 was less sensitive to acidic pHs, such as those found in the stomach, than phytase activity at pH 5.3. Phytase activities in the digesta of the distal ileum did not depend either on source or amount of dietary phytase activity.

Enzymatic characterization of organic phosphorus in animal manure.

Information on the forms of P present in animal manure may improve our ability to manage manure P. In most investigations of manure P composition, only inorganic and total P are determined, and the difference between them is assigned as organic P. In this study, we explored the possibility of identifying and quantifying more specific organic P forms in animal manure with orthophosphate-releasing enzymes. Pig (Sus scrofa) manure and cattle (Bos taurus) manure were first sequentially fractionated into water-soluble P, NaHCO3-soluble P, NaOH-soluble P, HCl-soluble P, and residual P. The fractions were separately incubated with wheat phytase, alkaline phosphatase, nuclease P1, nucleotide pyrophosphatase, or their combinations. The released orthophosphate was determined by a molybdate blue method. Part of the organic P in those fractions could be identified by the enzymatic treatments as phytate (i.e., 39% for pig manure and 17% for cattle manure in water-soluble organic P), simple phosphomonoesters (i.e., 43% for pig manure and 15% for cattle manure in NaOH-soluble organic P), nucleotide-like phosphodiesters (2-12%), and nucleotide pyrophosphate (0-4%). Our data indicate that the enzymatic treatment is an effective approach to identify and quantify the organic P forms present in animal manures.

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli

Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via d/ l-Ins(1,2,3,4,5)P 5, d/ l-Ins(2,3,4,5)P 4, d/ l-Ins(2,4,5)P 3 or d/ l-Ins(1,2,4)P 3, d/ l-Ins(1,2)P 2 or Ins(2,5)P 2 or d/ l-Ins(4,5)P 2 to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the d-Ins(1,2,3,5,6)P 5 isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F 1 and F 2 from wheat bran and the myo-inositol phosphates produced by fraction F 2. Biochim. Biophys. Acta 302, 326–328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via d-Ins(1,2,3,4,5)P 5, d-Ins(2,3,4,5)P 4, d-Ins(2,4,5)P 3, Ins(2,5)P 2 to finally Ins(2)P (notation 6/1/3/4/5).

Inhibitory effect of wheat fibre extract on calcium absorption in Caco-2 cells: evidence for a role of associated phytate rather than fibre per se.

In spite of the strong evidence for the beneficial health effects of dietary fibres, one of the potential nutritional disadvantages of high fibre diets is the adverse effect on the bioavailability of micronutrients, especially minerals and trace elements. With regard to Ca, there is considerable evidence that phytate, which is associated with fibre in many foods, such as cereals and soya products, inhibits Ca absorption. However, there is some doubt as to whether fibres per se have an influence on Ca absorption. Therefore, the purpose of this study was to investigate the effect on Ca absorption of two cereal-based fibre extracts (wheat bran and barley hull). In addition, in order to distinguish between the effect of the fibre components per se and the associated phytate content in these fibre extracts, we investigated the effect of dephytinised wheat and barley fibre extracts and the effect of phytate, as sodium phytate at levels present in the fibre extracts, on Ca absorption. Ca absorption was assessed in Caco-2 cells, as a model for studying Ca absorption in humans. The effect of wheat and barley fibre extracts, dephytinised wheat and barley fibre extracts, cellulose, and of sodium phytate on transepithelial 45Ca transport and 45Ca uptake was studied in differentiated Caco-2 cells grown on permeable filter supports. Wheat and barley fibre extracts were dephytinised with wheat phytase. Wheat fibre extract had a 3.2-fold higher phytate content (48.0 mmol/kg) than barley fibre extract (15.1 mmol/kg). Enzymatic dephytinisation of both fibre extracts reduced the phytate content to undetectable levels. The rate of transepithelial 45Ca transport across Caco-2 cell monolayers and the uptake of 45Ca into Caco-2 cells were unaffected by cellulose or barley fibre extract. On the other hand, inclusion of wheat fibre extract in the Ca transport buffer (50 g fibre/l) significantly reduced the rate of 45Ca transport (by 17 and 19% respectively) and the uptake of 45Ca (by 24 and 25% respectively) relative to a fibre-free buffer and a control fibre (cellulose) transport buffer. Increasing the phytate concentration of the transport buffer from 0 to 2 mM (a level close to that in the wheat fibre containing buffer) significantly reduced the rate of 45Ca transport (by 16%) and 45Ca uptake (by 26%). Dephytinisation of the wheat fibre extract removed its inhibitory effects on 45Ca transport and e.g. 45Ca uptake. The results from the present study in Caco-2 cells suggest that it is the phytate in wheat fibre extract which is the major inhibitory factor of Ca absorption and that wheat fibre per se has little if any inhibitory effect on Ca absorption. In addition, the results of this study support the usefulness of Caco-2 cells for investigating the effects of dietary factors on the cellular uptake and transepithelial intestinal transport of Ca.

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.

Purification and Catalytic Properties of a Phytase from Scallion (Allium fistulosum L.) Leaves

Levels of phytase comparable to that in wheat germ were detected in a variety of fresh vegetables. Scallion leaf phytase was purified 31 000-fold to an activity of 500 μmol min-1 mg-1 at 37 °C and had a Km of 200 μM for inositol hexakisphosphate and maximum activity at pH 5.5. The enzyme had a temperature optimum of 51 °C and was inactivated by preincubation for 10 min at 65 °C. The product specificity appeared similar to that of wheat phytase and was not altered by a 10-fold excess of magnesium. Vegetable phytases may be an alternative to seed and microbial phytases for use in the processing of food and feed products. Keywords: Phytase; phosphatase; phytate; inositol phosphates; Allium

Analysis of Inositol Mono- and Diphosphate Isomers Using High-Performance Ion Chromatography and Pulsed Amperometric Detection

A rapid and sensitive high-performance ion chromatography method for separation and quantitative determination of inositol mono- and diphosphate (IP1−IP2) isomers using pulsed amperometric detection is described. The method involves extraction of samples with HCl, separation of inositol phosphates from the crude extract by anion-exchange chromatography, NaAc gradient elution in an NaOH environment to perform isomer separation on a high-performance anion-exchange column, and detection with pulsed amperometric detector. The applicability and sensitivity of the method is illustrated by measurement of the content of IP1 and IP2 in foods, human ileal contents, and enzymatic hydrolysis products of phytate (inositol hexaphosphate). The major IP1 and IP2 isomers formed during phytate hydrolysis with wheat phytase were shown to be Ins(2)P, dl-Ins(1)P, and dl-Ins(1,2)P2 and with Aspergillus niger phytase, Ins(2)P and dl-Ins(1,2)P2. Keywords: Inositol mono- and diphosphate isomers; high-performance ion chromatography; pulsed amperometric detection

Reduction in the Levels of Phytate During Wholemeal Bread Making; Effect of Yeast and Wheat Phytases

Abstract No difference in wheat phytase activity was observed when different types of acid were used to adjust the pH of wholemeal wheat flour suspensions to pH 5·0, the optimum for wheat phytase. When whole wheat bread was made without additives or after adjustment of the dough pH with acetic acid or lingonberry (traditional ingredients in bread making in Sweden), 64%, 96% and 83%, respectively, of the initial phytate was hydrolysed. A small but significant difference between breads with and without yeast or with deactivated yeast was found, indicating that yeast contributed some phytase activity under the conditions of bread making (pH 5·3–5·8 and 30–37°C). The optimum pH of yeast phytase was found to be 3·5. The isomers of IP 5 formed with purified wheat phytase or yeast phytase were studied using sodium phytate as substrate. Wheat phytase formed 1,2,3,4,5-IP 5 whereas yeast phytase formed 1,2,4,5,6-IP 5 . Determination of the isomers of inositol pentaphosphate demonstrated that the reduction in phytate levels in bread compared with wholemeal flour resulted from both wheat and yeast phytase activities.

Dietary Aspergillus niger Phytase Increases Iron Absorption in Humans

Phytate is an inhibitor of iron absorption that can be removed before the intestinal site of absorption by microbial phytase, thereby increasing iron absorption from a meal. The effects of two kinds of dietary phytase, cereal phytase and microbial phytase from Aspergillus niger, on iron absorption were investigated. Iron absorption was measured from single meals containing white wheat rolls supplemented with wheat bran with or without phytase activity (expt. 1) and phytase-deactivated wheat bran with or without addition of microbial phytase from A. niger (expt. 2). Each experiment had 10 subjects and two different radio iron tracers: 55Fe and 59Fe were used for comparison of the absorption from the test meals in each experiment. No differences in iron absorption were found between meals containing wheat bran with or without phytase activity. Addition of microbial phytase to the meal containing phytase-deactivated wheat bran increased iron absorption from 14.3 ± 2.6% to 26.1 ± 3.8% (P < 0.0001). Two pH optima, one at pH 2.0 and one at pH 6.0, were found for A. niger phytase at 37°C, but activity occurred at all pH values between 1.0 and 7.5. The results suggest that effective and complete degradation of phytate occurred in the stomach when A. niger phytase was given with the meal. This may be explained by high activity of microbial phytase at physiological pH conditions of the stomach, whereas wheat phytase has a different pH optimum.