The effect of germination on the phytase activity,phytate and total phosphorus contents of some Nigerian‐grown grain legumes

BACKGROUND: Grain legumes are under‐exploited as possible sources of phytase for the poultry industry. The current study was conducted to assess the effect of germination on phytase activities, phytate and total phosphorus content in samples of Nigerian‐grown grain legumes. The legumes screened were African yambean (AYB, Sphenostylis stenocarpa), lima bean (Phaseolus lunatus), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata) and groundnut (Arachis hypogea). RESULTS: Phytase activity was low in AYB, lima bean and pigeon pea but high in cowpea and groundnut. Phytate content ranged between 3.01 g kg^?1 and 8.95 g kg^?1 while total phosphorus content ranged between 2.63 g kg^?1 and 5.93 g kg^?1. The grain legumes with higher phytase activity recorded the lowest phytate and phosphorus content. During germination there was an initial 4‐fold to 35‐fold increase in phytase activity after 6–7 days of germination followed by a decrease until 10 days (P < 0.05). The increase in phytase activity during germination was accompanied by a significant reduction in phytate (P < 0.05) and a small but significant increase in total phosphorus. CONCLUSION: The increase in phytase activity and the accompanying decrease in phytate content could have a positive implication for the nutrition of poultry and ruminants and for the environment. Copyright © 2010 Society of Chemical Industry     

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.     

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.     

Phytins?ure in Getreide und Getreideerzeugnissen

Zusammenfassung Phytinsäure in Lebensmitteln wird für eine verringerte Bioverfügbarkeit von essentiellen Mineralstoffen verantwortlich gemacht; sie kann während der Verarbeitung durch Phytase teilweise abgebaut werden. Der durchschnittliche Phytinsäuregehalt betrug in Roggen 8,18 mg/g und im Mehl der Type 997 3,44 mg/g; im Ganzkornmaterial wurden durchschnittlich Phytaseaktivitäten von 3,7 U/g und im Mehl 2,6. U/g gefunden. In den beiden Kornhälften (quergeteilt) waren Phytat und Phytase etwa gleichmäßig verteilt. Während der dreitägigen Keimung blieb die Phytaseaktivität konstant, der Phytinsäuregehalt nahm um ein Drittel ab. Bei der Vermahlung und dem Schälen von Roggen wurden die größten Mengen an Phytinsäure und Phytase mit Schrot- und Grießkleien bzw. Schälfraktionen entfernt. Es wird daraus geschlossen, daß Substrat und Enzym in den gleichen morphologischen Strukturen, wenn auch intrazellulär getrennt, vorkommen. Küchentechnische Zubereitungsformen von Vollkornprodukten waren für eine Phytatreduzierung um so wirkungsvoller, 1.) je feiner das Getreide vermahlen war, 2.) je mehr Wasser zugegeben wurde und 3.) je länger Phytase im optimalen Temperaturbereich einwirken konnte. Extrusion von Vollkornschrot bewirkte erst bei einer hohen Temperatur ( 170 °C) eine 23%ige Reduktion, während Phytase schon bei 80 °C um etwa 80% geschädigt wurde.

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Phytic acid in cerealsI. Phytic acid and Phytase in rye and rye products

Summary Phytic acid in food is considered to be responsible for a reduced bioavailability of essential dietary minerals; its detrimental effects can be diminished by hydrolysis with phytase during processing. The average phytic acid content was 8.18 mg/g and 3.44 mg/g and average phytase activity was 3.7 U/g and 2.6 U/g in rye kernels and in flour (Type 997, 1.09 ash content), respectively. Phytate and Phytase were about equally distributed between the two kernel halves (cross sections). During the early stages of germination (3 days) phytase activity did not change, and phytic acid content was reduced to 67%. After milling most of the phytic acid and phytase activity were found in the bran fractions. It is concluded that substrate and enzyme are present in the same kernel structures but separate within the cells. Cooking of ground rye caused a phytate hydrolysis which was the more effective 1.) the smaller the particle sizes were, 2.) the more water was added, and 3.) the longer phytase worked at optimum temperature. Extrusion cooking of the rye whole flour at up to 100 °C did not influence the phytic acid level but caused a 23% reduction at 170 °C. Phytase activity was reduced by 80% by extrusion cooking at 80 °C.

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Veröffentl.-Nr. 5342 der Bundesforschungsanstalt für Getreide-und Kartoffelverarbeitung, Detmold     

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.     

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’).     

Changes in phytate and minerals during germination and cooking of fenugreek seeds

The effects of germination, cooking and roasting on the phytic acid content, total phosphorus, water soluble inorganic phosphorus and mineral content of fenugreek seeds have been estimated. After 96 h germination, the dry weight of fenugreek seeds decreased while total ash content increased. Phytase and phosphatase activity of the ungerminated and germinated seeds have been assayed. It is observed that during germination the phytic acid values diminish and the water soluble inorganic phosphorus values increase. Phytase activity which is absent in the ungerminated seeds originates after germination and the phosphatase activity is increased in the germinated seeds. Heat treatment practised in cooking and roasting fenugreek seeds has less effect on phytate elimination than does germination. Changes in calcium, magnesium, iron, manganese, copper and zinc are found to be dependent on the loss of dry weight which occurs during processing of fenugreek seeds.     

Effect of heat treatments on the phytic acid content of maize products

Processing of maize (Zea mays L fresh and dry) for the production of various traditional products results in the loss of phytic acid. Fresh mature corn contains less phytic acid (1·71 g kg^?1) than dry corn (7·15–7.60 g kg^?1). The loss of phytic acid varies from 18·1 to 46·7% for fresh maize and from 11·5 to 52·6% for dry maize respectively among the heat treatments given. From a nutritional viewpoint, consumption of maize as chapati and after roasting in a sand bath or on charcoal will improve the availability of minerals.     

PURIFICATION AND CHARACTERIZATION OF A PHYTASE FROM SPELT

Four soluble phytases were identified in germinating spelt. Although numerous purification strategies were applied, none of the four phytases could be purified to homogeneity. The purest phytase preparation, called D21, contained a phytase (major component) and an acid phosphatase (APH) (minor component). The phytase behaves like a monomeric protein of a molecular mass of about 68 kDa and shows a broad substrate specificity. Optimal pH for degradation of phytate was 6.0 and the optimal temperature 45C. Kinetic parameters for the hydrolysis of Na-phytate were K_M 400 μmoll^?1 and k_cat 368s^?1 at pH 6.0. The spelt phytase D21 degrades phytate stepwise.     

Wheat Phosphorus Availability: 1—In VitroStudy; Factors Affecting Endogenous Phytasic Activity and Phytic Phosphorus Content

Phytasic activity and phytic P content, which are important factors in determining the availability of P, were measured in 56 wheat samples. The different agronomic and genetic factors which could have an influence on these two variables were studied, specifically N and/or P fertilisation, date of harvest, preharvest sprouting and variety of French wheat. Phytasic activity ranged from 206 to 775mUg⁻¹, with an average of 508mUg⁻¹ and a standard deviation of 109mUg⁻¹. Only the variety of wheat appeared as a significant factor explaining the endogenous phytasic activity ( P =0·006). The phytic P content varied between 0·92 and 2·80gkg⁻¹ DM, with an average of 2·18gkg⁻¹ and a standard deviation of 0·35gkg⁻¹ DM. None of the factors studied significantly affected the phytic P content of the wheat. This content was correlated with the total P content ( r =0·56; P< 0·05). The activity of the endogenous phytase was independent of the quantity of its substrate, the phytic phosphorus of the kernels of wheat.     

Gross composition,amino acid,phytic acid and trace element contents of thirteen cowpea cultivars and their nutritional significance

Thirteen cultivars of cowpea (Vigna unguiculata (L) Walp) were analysed for their proximate composition, amino acid, trace element and phytic acid contents. Crude protein values ranged from 206·8 to 283·8 g kg^?1 DM. The mean contents (g kg^?1) of other major nutrients were ether extract, 18·6; ash, 38·8; total dietary fibre, 121·8 and carbohydrates, 573·4. When compared with the provisional amino acid scoring pattern of FAO, all cultivars were low in methionine and high in lysine, isoleucine, leucine and phenylalanine plus tyrosine. Values for threonine and valine were variable compared with the pattern. The range of values for the chemical score was 0·61–0·74. Phytic acid values ranged from 5·10 to 10·27 g kg^?1, and the phytate: zinc molar ratios were all higher than that (15:1) above which zinc deficiency can be induced. Most of the trace elements showed wide variation in their occurrence among the cultivars. The values for zinc and iron showed less variation. The toxic elements mercury and selenium were present in varying amounts in more than half of the cultivars while tin was found in only one variety.     

Investigations on the trypsin inhibitor,hemagglutinin, phytic and tannic acid contents of cowpea Vigna unguiculata

Ten cultivated varieties of mature dry beans Vigna unguiculata were analysed for trypsin inhibitor (TI) and hemagglutinating activities, phytic acid, phytic acid-phosphorus and tannic acid. The respective concentrations were: 19·6–28·2 TUI mg^?1 protein,¹ 33·5–98·9 HU mg^?1 protein,² 280–331 mg 100 g^?1, 131–200 mg 100 g^?1 and 0·42–0·78 g 100 g^?1 dry weight. Phytic acid-phosphorus as a percentage of total phosphorus was highest in Farv-13 (49·9) and lowest in Samaru local (29·8). Considerable variability in hemagglutinating activity was evident among the different varieties as indicated by the high percentage co-efficient of variation. Some of these differences may be genetic and may provide an opportunity for the genetic development of cowpea strains with superior protein quality, low in hemagglutinin content.     

Characterization of polyphenol oxidase from Uapaca kirkiana fruit

Polyphenol oxidase (PPO), the enzyme responsible for the postharvest spoilage of fruits, was extracted and purified from Uapaca kirkiana peel and pulp by ammonium sulfate precipitation and dialysis. Further purification of peel PPO was carried out by gel filtration chromatography. Optimum pH values were 7 and 8 for peel and pulp PPO, respectively. The optimum temperatures for peel and pulp PPO were 45 and 35 °C, respectively. Inhibition studies of the PPO enzyme were performed using citric acid, sodium azide, sodium metabisulfite and thiourea. The most effective inhibitors were sodium azide and citric acid for both peel and pulp PPO. V_max and K_m values were 13.63 units min^?1 and 4.923 mmol L^?1, respectively, for peel PPO and 14.03 units min^?1 and 5.43 mmol L^?1, respectively, for pulp PPO. Three isoenzymes of Uapaca kirkiana PPO were detected by polyacrylamide gel electrophoresis. One of the isoenzymes could be identified as having a molecular weight of 26 625 Da. Copyright © 2005 Society of Chemical Industry     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Determination of phytic acid in feeds and faeces of pigs and poultry by capillary isotachophoresis

A method for phytic acid determination in the feeds and faeces of pigs and poultry has been developed on the basis of capillary isotachophoresis. Phytic acid was extracted by 0.95 M HCl and separated from interfering compounds by iron precipitation. Complete formation of ferric phytate required 7 mol FeCl₃ mol⁻¹ phytic acid. Residual Fe³⁺ was estimated colorimetrically by the tiron reagent, and ferric phytate was dissolved in 1.5 M NaOH at 9 mol NaOH mol⁻¹ Fe precipitated. Analyses were carried out using an electrolyte system with Cl⁻ as the leading anion, bis‐tris‐propane, and 2‐morpholinoethanesulphonic acid as the terminating anion. The recovery of phytic acid (added to hen faeces) using this procedure was 962 ± 24 g kg⁻¹. The limit of determination of phytic acid was 0.3 µmol ml⁻¹ extract. The amount of phytic acid in feeds ranged from 8.3 to 10.8 g kg⁻¹ on a dry matter basis. Phytic acid P represented 112 g kg⁻¹ total P in faeces of young pigs (40–60 kg) fed a feed with supplemental phytase (490 U kg⁻¹), 153 g kg⁻¹ total P in faeces of finishing pigs and 185 g kg⁻¹ total P in faeces of non‐lactating sows. Excreta of laying hens contained 23.7 g phytic acid kg⁻¹ dry matter (362 g kg⁻¹ total P). The isotachophoretic method is sufficiently simple and reproducible to be used for routine analyses of feeds and faeces. © 2000 Society of Chemical Industry     

Chemical characteristics and antioxidant activities of polysaccharide purified from the seeds of Plantago asiatica L.

BACKGROUND: A water‐soluble polysaccharide from the seeds of Plantago asiatica L. (P. asiatica L. polysaccharide, PLP) was extracted with hot water and purified by gel filtration chromatography. The chemical characteristics of PLP were determined by high‐performance gel permeation chromatography (HPGPC) and Fourier transform infrared (FTIR) spectroscopy. In addition, the antioxidant activities of PLP in vitro were evaluated using various test systems, including scavenging of 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) radicals, scavenging of superoxide radicals generated by 1,2,3‐phentriol autoxidation, scavenging of hydroxyl radicals and inhibition of lipid peroxidation. RESULTS: The molecular weight of PLP was determined by HPGPC to be about 1894 kDa. PLP contained 29.2 g kg^?1 protein and 145.8 g kg^?1 uronic acid. The FTIR spectrum of PLP also revealed typical characteristics of a polysaccharide containing protein and uronic acid. Moreover, the results showed that PLP possessed antioxidant activities, but lower than those of ascorbic acid. CONCLUSION: PLP is an acid protein‐bound polysaccharide of high molecular weight, but its structure needs further study. The present results suggest that PLP could potentially be used as a natural antioxidant. Copyright © 2009 Society of Chemical Industry     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

In vitro bioavailability of calcium and iron from selected green leafy vegetables

The objective of the present investigation was to analyze the relative influence of oxalic acid, phytic acid, tannin and dietary fiber on in vitro availability of iron and calcium from green leafy vegetables (GLV). Thirteen GLV were selected and analyzed for iron, calcium, oxalic acid, phytic acid, tannin and dietary fiber contents using standard methods. The bioavailability of calcium and iron in the GLV was estimated by equilibrium dialysis. Oxalic acid content was less than 1 g kg^?1 in four greens and ranged between 1.22 to 11.98 g kg^?1 in the remaining. Dietary fiber ranged from 19.5 to 113.7 g kg^?1. Tannin content ranged between 0.6138 and 2.1159 g kg^?1 with the exception of two GLV that had 0.1332 and 14.8619 g kg^?1. Four GLV were found to have approximately 40% bioavailable iron, while the others were in the range of 6–30%. In vitro available calcium was less than or equal to 25% in eight GLV and between 34% and 52% in five GLV. Multiple regression analysis revealed that these factors together accounted for 53% (r² = 0.53) and 45% (r² = 0.45) inhibition of iron and calcium absorption, respectively. These findings infer that calcium and iron availability is influenced by the constituents present in the GLV. Copyright © 2006 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.     

Dephytinisation of Sangak and Barbari bread made from different extraction rate flours increases iron and zinc bioaccessibility in Caco‐2 cells

Bread is a staple food in many countries and an important source of iron and zinc. The bioavailability of these minerals is generally low because of the content of phytic acid. Traditional Iranian breads were prepared with flours of different extraction rates, Sangak at 93% and Barbari at 82%. Breads were dephytinised by addition of Aspergillus niger phytase during in vitro digestion. The effect upon iron and zinc bioaccessibility in the Caco‐2 cell model was investigated. The cellular uptake of iron and zinc was lower from Sangak, compared to Barbari, despite higher mineral content in Sangak. Dephytinisation of both breads increased iron uptake in the Caco‐2 cells (0.65 vs. 1.64 in Sangak and 0.77 vs. 1.97 ng mg^?1 protein in Barbari). Zinc uptake increased from 0.98 to 2.8 in Sangak and from 1.4 to 2.9 ng mg^?1 protein in Barbari. Thus, dephytinisation substantially improves iron and zinc bioaccessibility.