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’).     

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.     

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     

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.     

Dephosphorylation of phytate compounds by means of acid phosphatase from Aspergillus niger

A non-purified preparation of intracellular acid phosphatase (EC 3.1.3.2) from a waste mycelium of Aspergillus niger was utilised for dephosphorylation of phytate compounds present in food and feed ingredients. The enzymic hydrolysis of p-nitrophenylphosphate was used for assaying acid phosphatase activity, expressed in standard units (u). The hydrolysis of phytate phosphorus in wheat bran, soya bean meal and fully formulated feedstuffs for broilers (Galus galus; ‘Cornish’ × White Rock') was carried out at 40°C, a pH value of 4.5 and an enzyme dosage ranging from 12 to 30 u g^?1. Complete dephosphorylation of soya bean protein isolates was performed at 60°C, a pH value of 4.5 and an enzyme dosage of 12 u g^?1. In the gastrointestinal tract of broilers the in-vivo dephosphorylation of phytates present in feed was observed when the preparation of acid phosphatase was added to the diet.     

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.     

Effect of phytase inclusion and calcium/phosphorus ratio on the performance and nutrient retention of grower–finisher pigs fed barley/wheat/soya bean meal‐based diets

Two experiments were conducted to evaluate the effects of phosphorus (P) level and calcium (Ca)/total P (tP) ratio on the efficacy of microbial phytase. Experiment 1 examined the effects of P concentration and microbial phytase inclusion on mineral excretion and pig performance, while experiment 2 examined the effects of Ca/tP ratio and microbial phytase inclusion on mineral excretion and pig performance. In experiment 1, nutrient and mineral digestibility (n = 4) and growth performance (n = 12) were determined in pigs individually fed diets containing (T1) 5.5 g kg^?1 tP, 2.3 g kg^?1 available P (aP) and 8.0 g kg^?1 Ca, (T2) 5.5 g kg^?1 tP, 2.3 g kg^?1 aP, 8.0 g kg^?1 Ca and 750 FYT kg^?1 Peniophora lycii phytase, (T3) 4.3 g kg^?1 tP, 1.4 g kg^?1 aP and 8.0 g kg^?1 Ca and (T4) 4.3 g kg^?1 tP, 1.4 g kg^?1 aP, 8.0 g kg^?1 Ca and 750 FYT kg^?1 P lycii phytase. In experiment 2, nutrient and mineral digestibility (n = 4) and growth performance (n = 12) were determined in pigs individually fed diets containing (TT1) 4.3 g kg^?1 tP and 8.0 g kg^?1 Ca, (TT2) 4.3 g kg^?1 tP, 8.0 g kg^?1 Ca and 750 FYT kg^?1 P lycii phytase, (TT3) 4.3 g kg^?1 tP and 5.0 g kg^?1 Ca and (TT4) 4.3 g kg^?1 tP, 5.0 g kg^?1 Ca and 750 FYT kg^?1 P lycii phytase. All diets were formulated, using standard feeding values for the ingredients, to have similar concentrations of digestible energy (DE) and lysine. In experiment 1, pigs offered the low‐P diets had significantly lower P intake (P < 0.001), faecal P excretion (P < 0.05), Ca intake (P < 0.05) and faecal Ca excretion (P < 0.05) compared with pigs given the adequate‐P diets. These pigs also had significantly lower daily gain (P < 0.01), feed intake (P < 0.05) and feed conversion ratio (FCR) (P < 0.05). The inclusion of phytase in both the adequate‐ and low‐P diets increased the digestibility of energy (P < 0.05) and Ca (P < 0.01) but had no effect on pig performance. In experiment 2, lowering the Ca/tP ratio from 1.85:1 to 1.15:1 increased the DE content of the diet (P < 0.05). The inclusion of phytase increased (P < 0.05) the digestibility of protein (0.874 versus 0.840, SEM 0.009) and Ca (0.427 versus 0.380, SEM 0.019) as well as the DE content of the diet (14.47 versus 14.26 MJ kg^?1, SEM 0.073). There was a significant ratio × phytase interaction (P < 0.5) for P digestibility. Microbial phytase significantly increased P digestibility when added to the 1.15:1 ratio diet but had no effect when added to the 1.85:1 ratio diet. The inclusion of microbial phytase increased feed intake (2.16 versus 2.00 kg day^?1, SEM 0.05; P < 0.05) and weight gain (0.893 versus 0.818 kg day^?1, SEM 0.022; P < 0.05). Lowering the Ca/tP ratio resulted in a significant improvement in FCR (2.32 versus 2.40 kg kg^?1, SEM 0.03; P < 0.05). In conclusion, the beneficial effects of microbial phytase supplementation of pig diets are adversely affected by a wide Ca/tP ratio. © 2002 Society of Chemical Industry     

Interaction of phytate with protein and minerals in a soybean–maize meal blend depends on pH and calcium addition

In order to investigate possible interactions of phytate with protein and minerals in simplified animal diets, studies were conducted on the solubility of endogenous phytate, protein and essential minerals in a soybean–maize meal blend within a physiological relevant pH range. The blend was mixed with water for 10 min and then allowed to incubate at 40 °C (30 min) after adjustment of the pH. Finally, soluble phytate, protein, zinc, manganese and iron were determined. Phytate and mineral solubility was highly influenced by pH whereas protein solubility was less affected. Addition of 5 g Ca²⁺ kg^?1 drastically reduced the solubility of phytate, zinc, manganese and iron at pH above 4.4, indicating that the formation of insoluble phytate–mineral complexes is increased in the presence of calcium. The action of pepsin increased the solubility of protein and phytate at pH below 4, indicating that insoluble phytate–protein complexes are present at low pH. Calcium had the same solubilising effect as pepsin at pH 2–4 but to a lesser degree. Copyright © 2007 Society of Chemical Industry     

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     

Calcium Additives and Sprouted Wheat Effects on Phytate Hydrolysis in Whole Wheat Bread

Whole wheat bread loaves were subjected to treatments of different fermentation periods, different sources and levels of calcium, and the addition of sprouted wheat. Phytate losses increased with increased fermentation time. Increasing the calcium level inhibited phytate hydrolysis when the calcium was provided by nonfat dry milk, CaCl₂, or nonfat yogurt, whereas phytate hydrolysis in loaves supplemented with CaCO₃ remained nearly constant. Milk-derived calcium exerted the greatest inhibition of phytate hydrolysis. The addition of sprouted wheat decreased absolute phytate losses. A comparison of phytate losses in yeasted vs nonyeasted loaves suggested that endogenous wheat phytase was quantitatively more important than yeast phytase during breadmaking.     

Estimation of the Bioavailability of Iron and Phosphorus in Cereals using a Dynamic In Vitro Gastrointestinal Model

A recently developed in vitro gastrointestinal model was evaluated for the estimation of the bioavailability of Fe and phosphorus and its correlation with biovailability in vivo. In vitro experiments were carried out without and with phytase supplementation (750 FTU kg^-1 feed) using rapeseed, sunflowerseed, wholewheat and white wheat flour. Phytase addition during in vitro digestion of rapeseed and sunflowerseed resulted in markedly increased dialysability of iron (67% and 20%) and phosphorus (31% and 66%). The release of free phosphorus during digestion of wholewheat and white wheat flour in the in vitro gastrointestinal model was observed to be correlated with the endogenous phytase activity in wheat. Comparison with different in vivo studies revealed that the in vitro gastrointestinal model could be used for a relative estimation of the bioavailability of Fe and phosphorus. © 1997 SCI.     

Bioavailability of Calcium Citrate Malate Added to Microbial Phytase-Treated, Hydrothermally Cooked Soymilk

Effects of microbial phytase on bioavailability of calcium added to corn/soy diets were investigated in chickens. No effect (P > 0.17) of phytase was found for weight gain, feed intake, tibia/body weight, ash%, and ash Ca% when calcium citrate malate (CCM) was added to corn/soybean meal and corn/hydrothermally cooked (HTC) soymilk diets. Using calcium carbonate in corn/HTC soymilk diets, means for weight gain, feed intake, tibia/body weight, and ash% were less (P < 0.05) without phytase. We hypothesize that CCM is less amenable to the formation of calcium phytate complexes than are other calcium salts and, therefore, should be more effective for fortification of soymilk products.     

Sourdough fermentation of wheat fractions rich in fibres before their use in processed food

Fibre‐rich fractions of wheat are an important source of minerals but also contain considerable amounts of phytic acid, known to impair mineral absorption. This study explores the efficiency of wheat bran sourdough fermentation on phytate hydrolysis and mineral solubility, in comparison with whole‐wheat flour. In vitro trials were performed to assess the consequences of the addition of calcium carbonate (CaCO₃), an alkalinising salt, on phytic acid breakdown and mineral bioavailability during sourdough fermentation. Sourdough fermentation was found effective for solubilising minerals in whole‐wheat flours but was less effective with bran. In addition, sourdough acidity was blunted by the addition of CaCO₃, whereas degradation of phytic acid remained effective. Despite extensive breakdown of phytic acid (almost 70%), the addition of calcium exerted a very negative effect on zinc solubility. In conclusion, a pre‐fermentation process of whole cereals or bran, in suitable conditions of hydration, allows degradation of the major part of phytic acid and optimal mineral bioavailability. Copyright © 2007 Society of Chemical Industry     

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.     

Effect of Phytate and Partially Hydrolyzed Phytate on in vitro Protein Digestibility

The effects of sodium phytate and partially hydrolyzed sodium phytate (0 - 82% hydrolyzed) on pepsin digestion of casein and bovine serum albumin were evaluated by an in vitro procedure using dialy-sates of pepsin digestion over a period of 0 – 23 hr. The inhibitory effect of phytate differed with substrate and increased with dose level. At the highest phytate level, the digestion of casein and bovine serum albumin was reduced by 14% and 7%, respectively. The inhibitory effect of the phytate was inversely correlated with the degree of phytate hydrolysis. Hydrolysis for 16 hr almost eleminated the inhibitory effect of phytate.     

Does the pelleting process affect the nutritive value of a pre‐starter diet for suckling piglets? Ex vivo studies on mineral absorption

BACKGROUND: The effects of pelleting on the extent of the Maillard reaction (MR) and on calcium, magnesium and zinc solubility and absorption were analysed in a conventional pre‐starter diet for suckling piglets. Development was tested measuring colour, absorbance (280/420 nm), fluorescence, residual free lysine, furosine, hydroxymethylfurfural (HMF) and furfural contents before and after pelleting. Fluorescence, absorbance and mineral solubility were also measured after in vitro digestion of diets. The effects on mineral absorption were tested using Caco‐2 cells. RESULTS: MR indexes confirmed the development of the reaction during the pelleting of this particular diet compared with the meal diet. The CIE‐Lab colour parameters showed a decrease in luminosity (L*) and progress of the colour to the red zone (a*) in the pelleted diet. A 36% decrease in free lysine content was observed. Significant correlations were observed between fluorescence intensity and furosine levels, HMF and furfural. The pelleting process did not modify calcium and magnesium solubility after in vitro digestion, but soluble zinc increased. The efficiency of calcium and zinc transport across Caco‐2 cell monolayers was greater in the pelleted diet. CONCLUSIONS: Evidence of MR development is shown, resulting in various nutritional consequences. Optimisation of pelleting could result in a better formulation of diets for feedstuffs. Copyright © 2010 Society of Chemical Industry     

Effect of tannic acid on in vitro enzymatic hydrolysis of some protein sources

The pH‐stat system has been used to assess the effect of tannic acid (TA) on solubility and in vitro enzyme hydrolysis of different proteins. Added TA (from 10 to 50 g kg^?1) decreased the extent of hydrolysis of bovine serum albumin. Enzymic hydrolysis of casein, pea meal, soybean meal, and haemoglobin (HB) was increased, as measured by total amino acids released and by the degree of hydrolysis. SDS‐PAGE confirmed the results of the in vitro enzymatic hydrolysis. These findings suggest that, under in vitro conditions, when simulating the gastrointestinal environment of domestic mammals, the negative effects of TA described from in vivo experiments are not necessarily due to reduced hydrolysis of proteins. Copyright © 2003 Society of Chemical Industry     

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.     

Improving mineral availability in soymilk by dephosphorylation of phytic acid using an alkaline phytase from Bacillus amyloliquefaciens DS11

To enhance mineral availability in soymilk, the alkaline phytase from Bacillus amyloliquefaciens DS11 was applied to degrade the phytic acid in soymilk, and resulting effects on mineral profiles were assessed. Nearly 60% of phytic acid was degraded within 25 min by treatment with 0.090 unit/mg of phytate, whereas most phytic acid was removed in 125 min using 0.18 unit/mg of phytate. Compared to a control, the free calcium, magnesium, phosphorus, and iron contents significantly (p<0.05) increased by 1.9, 1.8, 4.0, and 4.0×, respectively, after 125 min of reaction using 0.18 unit/mg of phytate. Free minerals were released from the phytate complex by enzymatic dephosphorylation, resulting in enhancement of mineral availability in soymilk. Alkaline phytase DS11 has a great potential for enhancing the mineral availability in neutral pH phytate-rich foods, like soymilk.     

Assessing Chemical Form of Calcium in Wheat, Spinach, and Kale

Spinach, wheat and kale represent a broad range in absorption of calcium from foods (5–40%). These plants were intrinsically labeled with ⁴⁵Ca and examined for ⁴⁵Ca solubility characteristics, including specific enzyme treatments, subcellular distribution, and in vitro bioa-vailability. Solubility was <2% for spinach, ～40% for wheat and 70% for kale, depending on the solvent. Solubility paralleled the in vitro⁴⁵Ca bioavailability results of 0.3±0.l%, 26.4±5.2%, and 76.3 ±1.2% for these three plant foods. Spinach calcium exists largely as calcium oxalate which is not easily dissociated. Phytase treatment of wheat indicated that a major portion of calcium was bound to phytate. Knowledge of the chemical form of calcium in plants can help in designing processing procedures to improve calcium absorption.