Phytate content and phytate degradation by endogenous phytase in pea (Pisum sativum)

In order to rapidly reduce the content of inositol tri–hexaphosphates in pea flour by action of the endogenous phytase, raw materials as well as incubation conditions have been evaluated. The phytate (inositol hexaphosphate) content was analysed in 27 pea varieties; the influence of storage time and the difference in phytate content between the germ and the cotyledon were determined. Furthermore, degradation of inositol phosphates by the endogenous phytase enzyme was studied in pea flour, germ and cotyledon. To find the maximum phytate degradation, the effects of temperature and pH during pea flour incubation were investigated. The most efficient phytate degradation in pea flour incubation was achieved at pH 7.5 and 45 °C. At this condition an almost complete degradation of phytate and a 66% reduction in the sum of inositol hexa‐, penta‐, tetra‐ and triphosphates were reached in 10 h. The storage time of pea seeds or removal of the germ did not have a major effect on the phytate content. Since several inositol pentaphosphate isomers were produced during phytate degradation, it can be concluded that peas contain several phytate‐degrading enzymes, or one phytate‐degrading enzyme with unspecific initial hydrolysation pattern. © 2001 Society of Chemical Industry.     

Phytate Reduction in Brown Beans (Phaseolus vulgaris L.)

Brown beans (Phaseolus vulgaris L.) were subjected to treatments to evaluate effects of pH, temperature, CaCl₂, tannase and fermentation on degradation of phytate. Soaking was performed at 21°C, 37°C and 55°C at pH 4.0, 6.0, 6.4, 7.0, and 8.0. Optimal conditions for phytate degradation were pH 7.0 and 55°C. After soaking 4, 8 or 17 hr at these conditions 79%, 87% and 98% of phytate was degraded, respectively. Addition of tannase enhanced reduction of phytate. Fermentation of presoaked whole beans resulted in reduction of 88% of phytate after 48 hr.     

Changes in Phytase Activity and Phytate During Germination of Two Fababean Cultivars

Two fababean cultivars were germinated over a 10-day period and the levels of phytate and activity of phytase monitored. In both cultivars there was a marked increase in phytase activity which was accompanied by a concomitant decrease in phytate. The enzyme level reached a maximum 6 days following germination and was much higher in Ackerperle compared to Diana. Nevertheless a marked reduction in phytate levels was evident at the end of 10 days which accounted for a decrease of 71.2% and 77.3% in Ackerperle and Diana, respectively.     

Phytate Reduction in Oats during Malting

To reduce phytate in oat products and thereby increase mineral bioavailability, optimal conditions for phytate degradation in oats were investigated. The effects of malting and incubation on phytate reduction in oats were determined and compared with phytate degradation in wheat, rye and barley. Studies of incubation temperatures showed an optimum for phytate reduction in oats between 37–40°C which differed from that in wheat (55°C). Malting of oats for 5 days at 11°C and subsequent incubation for 17 hr at 37–40°C reduced phytate by 98%. Addition of malted rye also reduced the phytate content of oats to low levels.     

Degradation of Phytate in Foods by Phytases in Fruit and Vegetable Extracts

ABSTRACT The ability of phytases from fresh fruits and vegetables to degrade phytate in foods was determined in vitro. In contrast to the more consistent presence of phytase in vegetables, only a few fruits contained detectable phytase activity. The highest phytase activities were 121 and 97 nmol min^‐1 g^‐1 in scallion leaves and avocado fruit, respectively. Avocado fruit also contained 0.14% phytate on a wet‐weight basis. Crude phytase extracts were incubated with ground corn tortillas or refried beans at 37 °C. Most of the phytate was degraded within 30 min by a 4‐fold excess by dry weight of scallion leaves. Avocado extracts incubated alone metabolized most of their endogenous phytate within 2 h.     

Changes in Phytase Activity and Phytate During the Germination of Six Canola Cultivars

Six canola cultivars were germinated for 8-days and the levels of phytate and phytase activity monitored. Of these, the cultivar Regent exhibited the lowest level of phytate as well as the highest phytase activity. Germination of Regent for up to 2-days resulted in the reduction of phytate from 1.26% to 0.85% without any deleterious changes in the quality or quantity of the oil, based on the negligible levels of free fatty acids produced.     

Phytate Hydrolysis by Phytase in Cereals; Effects on In Vitro Estimation of Iron Availability

Phytate (inositol hexaphosphate) hydrolysis by endogenous and exogenous phytases was studied for their effect on increasing iron availability in cereals. Wheat bran and whole meal flours of rye and oats were soaked at optimal conditions for phytase activity (55°C, pH 5) for different time intervals. Phytate and its degradation products were determined by HPLC and related to iron solubility under simulated physiological conditions. Small amounts of phytate (< lμmol/g) had a strong negative effect on iron solubility. When inositol hexa- and pentaphosphates of wheat bran and rye flour were completely hydrolyzed by activating endogenous phytase, iron solubility under simulated physiological conditions increased from 3 to 53% (wheat) and 5 to 21% (rye). Addition of wheat phytase to uncooked oatmeal increased iron solubility from 4 to 11 and in precooked to 18%, while endogenous phytase of uncooked oatmeal had less effect on phytate digestion and iron solubility.     

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     

Canola Phytase: Isolation and Characterization

Two phytase isoenzymes were isolated from 8-day germinated canola cv Regent. Gel filtration chromatography of an ammonium sulfate fractionated extract on Sephadex G-100 produced one peak with phytase activity. The phytase fraction was separated into two isoenzymes by DEAE-cellulose chromatography. The optimum pH was 4.5–5.0 and 5.0 for the phytase isoenzymes 1 and 2, respectively. Both isoenzymes exhibited maximum activity at 50°C. Km values at pH 5.0 were 0.36 and 0.25 mM for phytase 1 and 2 isoenzymes, respectively, while molecular weight determination showed both fraction were identical with a molecular weight of 70,100 ± 4,000 daltons.     

Phytate content is reduced and β‐glucanase activity suppressed in malted barley steeped with lactic acid at high temperature

The effect of different steeping conditions on phytate, β‐glucan and vitamin E in barley during malting was studied by a full factorial experiment with three variables (steeping temperature, barley variety and steeping additions). Addition of lactic acid to the steeping water induced a reduction of phytate during steeping and germination, especially in combination with the high steeping temperature (48 °C). Furthermore, lactic acid and high temperature steeping inhibited β‐glucanase development, resulting in a well‐preserved β‐glucan content after germination. When steeping was conducted without addition of lactic acid, the low steeping temperature (15 °C) promoted development of both phytase and β‐glucanase activity during germination. A slightly higher level of tocopherols and tocotrienols was observed in samples steeped at 15 °C than in samples steeped at 48 °C. However, addition of lactic acid reduced the amount for both temperatures. When lactic acid bacteria were added to the steeping water none of the parameters studied differed from samples steeped with water only. The results show the possibility of combining phytate degradation with a preserved β‐glucan content during malting and can thus be of interest for development of cereal products with improved nutritional value. Copyright © 2004 Society of Chemical Industry     

Lactic Fermentation of Non-Tannin and High-Tannin Cereals: Effects on In Vitro Estimation of Iron Availability and Phytate Hydrolysis

The effect on iron availability estimated in vitro and phytate hydrolysis was investigated in non-tannin and high-tannin cereals, lactic fermented as flour/water slurries or gruels. A natural starter culture initiated fermentation and addition of germinated flour and phytase in the fermentation process was tested. Lactic fermentation of nontannin cereals with added flour germinated sorghum seeds or wheat phytase increased iron solubility from about 4% up to 9 and 50%, respectively. Soaking flour in water before adding starter culture had a similar effect. The increase in soluble iron was strongly related to enzymatic deeradation of phytate (p<0.001). The reduction of inositol hexa- and pentaphosphates was about 50% with added germinated flour. Reduction was > 90% after soaking the flour prior to fermentation and almost complete with 50 mg phytase added. High-tannin cereals showed a minor increase in soluble iron after fermentation, ascribed to the inhibitory effect of tannins (both on iron solubility and on enzymatic hydrolysis of phytate). Lactic-fermented cereal foods have a potential in developing countries to improve iron nutrition.     

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.     

Enzymatic Reduction of Phytate in Whole Wheat Breads

The presence of phytate in flour may be responsible for reduced bioavailability of iron, magnesium, zinc, and calcium from bread. The effect of various concentrations of commercial phytase or phosphatase added to whole wheat flour-yeast doughs on their phytate and nonphytate phosphorus content has been investigated. By using 2.0% (flour basis) of phytase and 0.11% phosphatase the initial phytate phosphorus concentration of the dough was reduced to 1/8 and 1/12 of its initial values, respectively. Storage of the whole wheat breads for up to 96 hr at room temperature showed further significant reduction of phytate phosphorus. The phytate phosphorus content of yeast leavened whole wheat breads decreased during 2 hours of dough fermentation, baking and the subsequent 48 hours of storage at room temperature from 24 mg/100g dough (dry matter) to 1.7 mg/100g bread (dry matter); the phytate phosphorus continued to decrease and after 96 hours storage it was 0.6 mg/100g bread.     

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.     

Effects of Germination and Dietary Calcium on Zinc Bioavailability from Peas

Bioavailability of zinc was determined by rat weight gain and tibia Zn response from whole pea sprouts (WPS), heat treated peas (HTP) and purified diets (PD1 and 2) supplemented with Zn and phytate for similar phytate/Zn molar ratios (phy/Zn). With diets adequate Ca (0.75%), weight gains from WPS (phy/Zn = 24) and HTP (phy/Zn = 39) were equivalent or better to a nonphytate control (NPC). Tibia Zn decreased in order from: NPC, WPS, PD1, HTP and PD2. At the 1.5% Ca level reduced growth responses from WPS and HTP indicated interaction between Ca and phytate. Zn availability from peas may be superior to that from grains and other legumes previously reported. The decreased phytate and increased phytase resulting from germination may explain the enhanced responses from pea sprouts.     

Supplementation of alkaline phytase (Ds11) in whole-wheat bread reduces phytate content and improves mineral solubility

Abstract: In this study, alkaline phytase was added to whole‐wheat bread and the phytate content and mineral profiles were compared to commercially available acidic phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline phytase DS11 and a 35% reduction was observed with acidic phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe²⁺ by the phytase treatment. The Fe²⁺ content increased 10‐fold at pH 7 and 8‐fold at pH 5 with alkaline phytase DS11. Alkaline phytase DS11 was shown to be effective at phytate reduction in whole‐wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.     

Effect of frying various legumes under optimum conditions on amino acids,in vitro protein digestibility,phytate and oligosaccharides

The effects of optimum conditions of frying soybean, broad bean and Polish pea on proximate analysis and amino acid composition were investigated. Maximum water absorption of 192.93, 129.05 and 148.68% for soybean, broad bean and Polish pea was reached after soaking periods of 16.41, 17.84 and 16.45 h respectively. Sensory evaluation of different temperatures (150, 170 and 190 °C) and frying times (5, 10 and 15 min) for preparing soybean, broad bean and Polish pea indicated that there was a significant difference (P < 0.01) between treatments. The most acceptable fried soybean, broad bean or Polish pea was obtained after frying at 190 °C for 5 min. All the processed legumes under investigation had higher protein digestibility as measured in vitro and minimum reduction in total amino acid composition. The optimum frying process is an effective method for lowering phytate content (myo‐inositol hexaphosphate) by 89.77, 67.70 and 82.88% respectively as well as causing considerable loss of total oligosaccharides by 64.35, 16.16 and 49.44%. © 2000 Society of Chemical Industry     

Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1

A fungus producing high levels of phytase was isolated from air and identified as Cladosporium sp. The phytase production was stimulated by phytate in the medium used. The maximum production of phytase (108 U/ml) occurred in a medium containing 1.0 g of phytate per 100 ml. The phytase was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration. Based on SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 32.6 kDa, and the narrow protein band indicated that this phytase is not glycosylated. The phytase has an optimum pH of 3.5, and an optimum temperature of 40 degrees C. The phytase activity was stimulated by 2-mercaptoethanol and dithiothreitol, and inhibited by Ba2+, Pb2+, iodoacetate, p-chloromercuribenzoate and phenylmethylsulfonyl fluoride. The phytase displayed high affinity for phytate and the Km was 15.2+/-3.1 microM. NMR analyses (1D and 2D) indicated that the end hydrolysis product of phytate was myo-inositol 1,2,5-triphosphate.     

The effects of germination,fermentation, and fermentation additives on the phytate content of rice flour

The phytate contents in nine rice cultivars (Oryza sativa cv.) grown in Korea were investigated and the effects of germination, fermentation, and fermentation additives on the phytate content of rice flour were studied. In nondehulled paddy rice, the phytate content was the highest in Goamibyeo and the lowest in Hanareumbyeo. In milled rice, the phytate content was the highest in Suweon517 and the lowest in Manmibyeo. For Goamibyeo, which had high phytate contents both before and after milling, there was a significant (p<0.05) reduction in the phytate content of paddy rice after germination. The phytate content reduction occurred when germinated paddy flour was fermented at 50°C for 24 h. Paddy rice phytate was completely removed when ammonium sulfate was added to germinated paddy rice flour, which was then allowed to ferment at 50°C for 24 h.     

Biochemical changes in phosphorus compounds and in the activity of phytase and α-amylase in the rice (Oryza sativa) grain during germination

Changes in the phytic acid, inorganic phosphorus and ATP contents, and in the activity of phytase and α-amylase in rice (Oryza sativa L) grains were determined during 18 days of germination in a dark room. The effect of phytic acid on α-amylase activity was studied in vitro. Rice grains immersed in sterilised deionised water at 14^°C germinated on the fifth day. Phytase activity, detected in the ripening rice grains, increased linearly until the eighth day and reached a maximum on the tenth day. There was a marked decrease in phytate and an increase in inorganic phosphorus accompanying germination. There was a good inverse correlation between the levels of both phytase activity and inorganic phosphorus, and phytate breakdown. α-Amylase activity was detected on the fourth day and increased markedly from the 12th to the 16th day of germination. ATP level increased from the second to the fourth day and slightly decreased from the fourth to the eighth day; it increased rapidly again from the eighth to the 18th day of germination. α-Amylase activity was influenced by both pH and phytic acid concentration in the assay system. At 75 mM phytic acid, α-amylase activity was lowered by 23%, 93% and 52% at pH 4–0, 5–0 and 6–0 respectively. When the enzyme, phytate and Ca²⁺ were incubated together at pH 5–0, the inhibition of α-amylase by phytic acid was markedly decreased by addition of Ca²⁺. The chemical affinity of Ca²⁺ for phytic acid was higher in the reaction at pH 5–0 than in those at pH 4–0 and pH 6–0, and over 98% of Ca²⁺ in the reaction system was precipitated as Ca-phytate.