Profile and bioavailability analysis of myo-inositol phosphates in rye bread supplemented with phytases: a study using an in vitro method and Caco-2 monolayers.

Commercial preparations of 6-phytase A alone and in combination with phytase B were used in rye breadmaking. Determination of bioavailability of myo-inositol phosphates from bread was performed by an in vitro digestion method followed by the measurement of an uptake by Caco-2 cells in culture. In bread supplemented with a combination of 6-phytase A and phytase B, a significant reduction in phytate content was observed from 3.62 µmol/g in the control to 0.7 µmol/g. Bioavailability of phytate estimated by an in vitro method simulating digestion in the human alimentary tract was 9% in the bread supplemented with phytase B, 7% (6-phytase A) and 50% in the control bread. In cell culture, the bioaccessibilities of inositol triphosphates from bread baked with the addition of 6-phytase A was higher by 36% as compared to the samples baked with phytase B and by 32% in breads baked with combination of both phytases.

The effect of microbial phytase and myo-inositol on performance and blood biochemistry of broiler chickens fed wheat/corn-based diets.

A total of 1,200 Ross broiler chickens were used in 2 separate feeding studies to explore the effect of myo-inositol (MYO) and phytase on performance and blood biochemistry of broilers fed diets formulated to be either adequate or insufficient in Ca and digestible P (dP). Supplementation of diets that were formulated to be insufficient in Ca and dP with MYO resulted in improved BW gain and feed conversion ratio in both experiments. However, these effects were most pronounced in the finisher phase, and moderate negative effects were observed during the starter period. Supplementation of the diet with microbial phytase improved BW gain and feed conversion ratio to a similar extent as was observed with MYO, and there was a degree of subadditivity between the 2 additives. Blood glucose concentrations were increased by both MYO and phytase, though possibly by different mechanisms, because insulin concentrations were not directly relatable to circulating glucose levels, especially when both MYO and phytase were applied simultaneously. The increase in blood glucose concentrations with MYO and phytase was most pronounced in the diet with a lower Ca and dP concentration. It can be concluded that dietary supplementation with MYO or phytase was effective in improving performance of commercial broiler chickens. However, further work is required to explore complex ontogenetic effects of MYO and possible involvement of both MYO and phytase in Na-dependent transport mechanisms.

Effect of inositol and phytases on hematological indices and α-1 acid glycoprotein levels in laying hens fed phosphorus-deficient corn-soybean meal-based diets.

The effects of feeding low nonphytate phosphorus (NPP) corn-soybean meal-based diets supplemented with myo-inositol at 0.1%, or with phytase B at 1,300 acid phosphatase units/kg, or with phytase B enriched in 6-phytase A at 300 phytase units/kg on the hematological indices and the α-1 acid glycoprotein (AGP) concentrations in the blood of Bovans Brown laying hens were investigated. The experimental design comprised also a negative control diet and an internal control diet that had the NPP content adjusted by the addition of 0.304 g of monocalcium phosphate per kg to reach the NPP level similar to that resulting from the combined action of both phytases. A total of sixty 50-wk-old hens were randomly assigned to the dietary treatments with 12 cage replicates of 1 hen, and fed the experimental diets until wk 62, when the blood samples were taken and analyzed for basic hematological indices and for AGP concentrations in sera. The hematological indices from all the experimental groups remained in a normal range; nevertheless, the statistically significant effects of diet on hemoglobin concentration (P = 0.003), erythrocyte counts (P = 0.035), the percentage of lymphocytes (P = 0.020), heterophils (P = 0.002), eosinophils (P = 0.023), and basophils (P = 0.001) in the leucocyte population, as well as on the heterophil to lymphocyte ratio (P = 0.003), were observed. The highest erythrocyte counts were characteristic for hens fed the diet supplemented with both phytase A and phytase B. The highest heterophil to lymphocyte ratios were found in blood of hens fed the diet supplemented with phytase B, whereas the highest basophil percentages and the highest AGP concentrations occurred in birds fed the negative control diet. A highly significant correlation was observed between AGP concentrations in sera and BW losses determined previously. The results indicate that the low-NPP corn soybean meal-based diets increased acute phase protein level in laying hens. Phytase B alone, and particularly in combination with phytase A, acted as a potent mediator of the response, whereas supplementary myo-inositol did not.

Effects of inositol, inositol-generating phytase B applied alone, and in combination with 6-phytase A to phosphorus-deficient diets on laying performance, eggshell quality, yolk cholesterol, and fatty acid deposition in laying hens.

Phytase B, a product of Aspergillus niger phyB gene expressed in Trichoderma reesei, which increased myo-inositol concentrations in 20 mM sodium phytate solution 7.5-fold during 120-min incubation, a combination of phytase B with 6-phytase A, and pure myo-inositol were tested as feed supplements in Bovans Brown laying hens. In the 2-factorial experiment (2×5), birds from wk 50 to 62 were fed 2 basal diets, corn-soybean (CSM) or wheat-soybean (WSM), using 12 one-hen cages per treatment. For both basal diets, the dietary treatments included negative control (0.08% nonphytate P in CSM, 0.13% nonphytate P in WSM; NC); internal control groups, NC+0.04% nonphytate P from monocalcium phosphate, MCP (IC); NC+0.1% of myo-inositol (Inos), NC+phytase B at 1,300 units of phytase B-acid phosphatase activity (AcPU)/kg (PhyB), NC+phytase B at 1,300 AcPU/kg+6-phytase A at 300 FTU/kg (PhyA+B). Feed intake, laying performance, and eggshell quality were determined. The total lipid and cholesterol contents as well as fatty acid profile were assessed in egg yolks collected from hens fed CSM diets, as was fatty acid profile. The hens fed the WSM diet consumed significantly more feed, laid a higher mass of eggs daily with higher mean weights, and had a higher hen-day egg production than the birds receiving the CSM diets. Similarly, higher values for yolk weights, shell weights, shell thickness, shell density, and breaking strengths were determined in the eggs laid by the hens fed the WSM diets. In hens fed either the CSM diets with phytase B alone, or in combination with 6-phytase A, enhanced feed intakes, egg mass, and hen-day egg production were recorded. Phytases also enhanced the eggshell quality parameters in the hens fed both variants of the diets. Phytase B alone, or in combination with 6-phytase A, reduced the total lipid and cholesterol concentrations in egg yolks collected from the hens fed the CSM diets, whereas the combination of both phytases improved the n-6:n-3 polyunsaturated fatty acids ratio. In the CSM diets, the supplemental myo-inositol suppressed feed intakes, reduced egg production, had no effect on eggshell quality and reduced the deposition of eicosanoid fatty acids in yolks. When comparing the effects of feeding MCP, myo-inositol, and phytases on the nutritional quality of the eggs laid by the hens fed phosphorus-deficient CSM diets, it seems that the enhancements in nutritional quality cannot be attributed solely to higher phosphorus level or higher concentrations of myo-inositol.

Towards complete dephosphorylation and total conversion of phytates in poultry feeds.

The rate of phytate P removal from feed (level of dephosphorylation, DL) and the extent to which the molecule of phytic acid is deprived of phosphate moieties (conversion degree, CD) were studied in vitro and in a feeding trial with broilers fed corn-soybean diets. In the in vitro model, phytase A asymptotically increased DL and CD. Phytase B influenced DL only at low dosages of phytase A [0 or 250 phytase activity units (FTU)/kg], but it enhanced CD irrespective of phytase A activity. In the feeding trial, 3-phytase A and 6-phytase A (at 750 FTU/kg) exerted similar effects on broiler performance and similarly influenced bone mineralization, P retention, and Ca retention. Phytase B [6,400 acid phosphatase activity units (ACPU)/kg] enhanced feed intake, BW gain (BWG), toe ash, and P retention but not the retention of Ca. Myo-inositol fed at 0.1% significantly increased BWG, but it reduced P retention. Under conditions of a higher CD (excess of phytase B), 3-phytase A was more effective in enhancing performance than 6-phytase A, but it reduced Ca retention. Lower phytase B activities (0 to 3,200 ACPU/kg) with added 6-phytase A were more necessary for optimal growth of chickens than for enhanced P and Ca retention (4,800 to 6,400 ACPU/kg). The efficacy of both forms of phytase A and phytase B depended on the Ca level in feed. There is enough evidence to conclude that myo-inositol phosphates resulting from simultaneous action of 3-phytase A and phytase B affect bird physiology differently than intermediates accumulated by the action of 6-phytase A and phytase B.

Comparison of the efficacies of a novel aspergillus niger mycelium with separate and combined effectiveness of phytase, acid phosphatase, and pectinase in dephosphorylation of wheat-based feeds fed to growing broilers.

Efficacies of phytase, phosphorolytic enzymes (phytase + acid phosphatase), an enzymic "cocktail" (phytase + acid phosphatase + pectinase + citric acid), a novel Aspergillus niger (fungal) mycelium (FM), and FM enriched in phytase and antioxidants were investigated in growing broilers (Days 1 to 21) fed wheat-based diets. Broilers were fed the following seven diets at 0.69% Ca: 1) a negative control diet, 0.17% nonphytate P (NPP); 2) Diet 1 + 750 phytase units/kg diet; 3) Diet 1 + 750 phytase units + 3,156 units acid phosphatase/kg diet; 4) Diet 1 + 750 phytase units + 3,156 acid phosphatase units + 1,900 units of pectinase/g diet + 3% citric acid; 5) Diet 1 + 4% FM; 6) Diet 1 + 4% FM + 1,300 phytase units + 2% ascorbic acid and 1% of glucose oxidase; and 7) a positive control diet (Diet 1 + 0.24% NPP from dicalcium phosphate). The dietary treatments were fed to four pen replicates of eight birds each. Prior to feed formulation, mycelium and antioxidants dosages were optimized on Diet 1 by an in vitro technique and an experimental design module of a statistical software package. Phytase addition increased BW gain (BWG), feed intake, and P retention. Subsequent addition of acid phosphatase resulted in further increases in BWG, feed intake, and toe ash and reduced digesta viscosity; however, neither P nor Ca retention were improved. Body weight gain and feed intakes superior to those found in chicks fed Diet 7 were observed in birds receiving the cocktail of enzymes (Diet 4) or FM. Chicken fed Diet 6 had the highest percentage of toe ash and retained 76 and 51% of P and Ca, respectively. Supplementation of wheat-based 0.17% NPP diets with FM increased bursa of Fabricius weights and reduced the intestinal surface covered by Peyer's patches.

Effects of phosphorolytic and cell wall-degrading enzymes on the performance of growing broilers fed wheat-based diets containing different calcium levels.

A study was conducted to determine the cumulative effects of phosphorolytic enzymes, cell wall-degrading enzymes, and citric acid and Ca levels on feed intake, BW gain (BWG), feed conversion, intestinal viscosity, and toe ash of broilers (d 1 to 21) fed wheat-based diets. Broilers were fed the following six diets at either 0.59, 0.69, or 0.79% Ca: 1) a negative control (NC) diet, 0.17% available P; 2) NC + 750 phytase units/kg diet; (3) phytase + 3,156 units of acid phosphatase/kg diet; 4) phytase + acid phosphatase + 1,900 units of pectinase/g diet; 5) phytase + acid phosphatase + pectinase + 3% citric acid; and (6) NC plus 0.24% available P. The 18 dietary treatments were fed to four pen replicates of eight birds each. Phytase addition at the low Ca level increased BWG, improved feed intake and conversion and toe ash, and reduced intestinal viscosity and ileal length. Subsequent addition of acid phosphatase, at 0.69% Ca, resulted in increases in BWG, 42%; feed intake 32%; feed conversion 7.5%; and toe ash, 63% over the NC diet. Pectinase addition produced further improvements in 21-d BWG and feed intake at 0.59 and 0.79% Ca, increased toe ash in chicks fed 0.79% Ca, and reduced intestinal viscosity. Supplementation of wheat-based 0.17% available P diets with phytase and acid phosphatase and with appropriate concentrations of pectinase, citric acid, and Ca significantly improved BWG, feed intake and conversion and intestinal viscosity over the 0.41% available P diets. Bone mineralization of chicks fed phytase + acid phosphatase and 0.69% Ca and those fed phytase + acid phosphatase + pectinase + citric acid and 0.59% Ca was similar to that of chicks fed the 0.41% available P diets.

The efficacy of an enzymic cocktail and a fungal mycelium in dephosphorylating corn-soybean meal-based feeds fed to growing turkeys.

A study was conducted to determine the efficacy of phytase, an enzymic cocktail, and a waste Aspergillus niger mycelium to hydrolyze phytate present in corn-soybean meal diets. One hundred turkey poults were assigned to dietary treatments for 2 wk (days 7 to 21). Dietary treatments included: 1) NRC (1994) diet (NRC), with recommended concentration of 0.6% available P (aP) and 1.2% Ca; 2) Phytase diet (PHYT), 1,000 units phytase/kg diet, 0.16% aP, and 0.84% Ca; 3) cocktail diet (COC), 1,000 units of phytase/kg diet plus acid phosphatase (100 units/g of diet), acid protease (42 units/g of diet), pectinase (2.94%), 0.16% aP, and 0.84% Ca; 4) Fungal mycelium diet (MYC), 5% mycelium, 0.16% aP, and 0.84% Ca; and 5) a positive control diet (CTRL+), 0.42% aP, and 0.84% Ca. Turkeys fed the PHYT diet consumed less feed and gained less weight but retained more P than poults fed the CTRL+ or NRC diets. Poults fed the COC diet performed as well as poults fed CTRL+ or NRC diets but retained more P (77%) and Ca (68%). Poults fed the MYC diet retained 79% P, gained the most weight, and were more efficient than poults fed any other dietary treatment. In vitro P release from experimental diets correlated well (R = 0.906) with P retention as observed in the feeding trial. Compared with the diet containing phytase as the sole supplemental enzyme, both the enzymic cocktail and fungal mycelium enhanced performance, bone mineralization, and retention of P and Ca in growing turkeys.

An in vitro procedure for studying enzymic dephosphorylation of phytate in maize-soyabean feeds for turkey poults.

An in vitro method was developed to predict inorganic P release from maize-soyabean poultry feeds containing supplemental phytase (EC 3.1.3.8), and to quantify the effect of acid phosphatase (EC 3.1.3.2), fungal protease (EC 3.4.23.6) and Aspergillus niger cellulase (EC 3.2.1.4) on phytate dephosphorylation. Pepsin (EC 3.4.23.1) and pancreatin digestion periods were preceded by a 30 min pre-incubation at pH 5.25 to simulate digestion in the crop of poultry. Pancreatin digestion was carried out in dialysis tubing, with a ratio of about 1:25 (v/v) between the digesta and dialysing medium, to simulate gradient absorption from the duodenum. The feed:water ratio was kept within physiological limits and a constant proportion of feed weight to digestive enzymes was maintained. There was a linear response to increasing dosages of phytase up to 1000 phytase units (FTU)/kg feed, and to increasing phosphate concentration in feeds. In vivo validation was performed with growing turkeys (1-3 weeks) fed on diets containing 12 g Ca/kg and 0, 500 or 1000 FTU phytase/kg in a factorial arrangement with 0, 1, 2 or 3 g supplemental phosphate/kg (from KH2PO4). After a simple transformation (variable/in vitro P = f (in vitro P)), amounts of P hydrolysed from feed samples by in vitro digestions correlated with 3-week body-weight gain (R 0.986, P < 0.0001), toe ash (R 0.952, P < 0.0001), feed intake (R 0.994, P < 0.0001) and feed efficiency (R 0.992, P < 0.0001). The dephosphorylating ability of phytase in vitro was significantly enhanced (P < 0.05) by the addition of acid phosphatase. Fungal acid protease and Aspergillus niger cellulase also enhanced the dephosphorylation process in vitro.

Relationship between citric acid production and accumulation of phytate-degrading enzymes in Aspergillus niger mycelia.

The ability of eight strains of Aspergillus niger to produce citric acid by the solid surface method were found to correlate with their capabilities to synthesize intracellular enzymes which degrade phytates (phytase and acid phosphatase). Another high correlation was observed between phytase and acid phosphatase activities bound to the cell walls of mycelia.