Probiotic expressing heterologous phytase improves the immune system and attenuates inflammatory response in zebrafish fed with a diet rich in soybean meal.

Although aquaculture is among the fastest growing food production sectors in the world, one of the bottlenecks for the continuity of its expansion is the dependence of animal protein on commercial feed formulations. Vegetable proteins are an alternative due to the low cost and high availability. However, this protein source is accompanied by a series of antinutritional and pro-inflammatory compounds, including phytate. Phytases can be added in feed for phytate degradation and increase nutrient availability. However, the use of purified phytases significantly increases the production costs. An interesting alternative is to use probiotics genetically modified as bioreactors for phytase production. In the present study, a strain of Bacillus subtilis secreting a fungal phytase was used to evaluate the effect of a feed with high content of soybean meal on zebrafish (Danio rerio). We analysed the condition factor (K) of fish, and the expression of genes related to the immune system, inflammatory response and oxidative. stress. The results obtained demonstrate that the transgenic probiotic was efficient in improving the fish condition factor, stimulating the immune system, reducing the inflammatory response and oxidative stress. Thus, probiotics acting as phytase bioreactors can be considered an interesting tool for the adaptation of commercial species to feed of lower cost.

Combination of phytase and citric acid, but not phytase alone, ensures regular rates of growth and bone mineralization in quails under severe phosphorus restriction.

An experiment was conducted to evaluate the effects of phytase (PHY), alone or in combination with citric acid (CA), on growing Japanese quails under moderate and severe phosphorus (P) restriction. For this, 504 one-day-old female quails were distributed in a completely randomised design with seven treatments and six replications. Treatments consisted of the following diets: (a) positive control (PC): formulated according to quails nutritional requirements; (b) negative control 40% (NC40 ): PC with 40% less aP; (c) NC40 +PHY; (d) NC40 +PHY+CA; (e) negative control 65% (NC65 ): PC with 65% less aP; (f) NC65 +PHY; (g) NC65 +PHY+CA. Phytase and CA were included at 300 FTU/kg and 40 g/kg, respectively. Performance, bone mineralization and plasma mineral concentration were evaluated at day 35; calcium (Ca) and P metabolism, between days 36 and 39. No significant differences in performance, plasma Ca and P were detected between quails fed PC or NC40 ; however, NC40 decreased tibia ash (p < 0.01). Treatment NC65 impaired daily weight gain (p = 0.01), tibia ash (p < 0.01), and tibia optical densitometry (p = 0.03), as well as modified plasma Ca (p < 0.01) and P (p = 0.03) concentrations when compared to PC. Both NC40 and NC65 reduced P excretion (p < 0.01). Phytase alone restored tibia ash of NC40 quails to regular rates. Nevertheless, only PHY and CA combined returned plasma P, tibia ash, tibia optical densitometry, and daily weight gain of quails fed NC65 to similar values as those observed in PC. In conclusion, PHY alone suppresses the negative effects of moderate P restriction in growing Japanese quails, but fails in this task under severe P restriction. In such a situation, only the combination of PHY and CA ensures regular rates of growth and bone mineralization. This concept should be better explored by producers in order to take full advantage of the reduction in P excretion achieved by lowering dietary P concentrations.

Nitrogen retention, energy, and amino acid digestibility of wheat bran, without or with multicarbohydrase and phytase supplementation, fed to broiler chickens.

The study was conducted to determine the effects of multicarbohydrase (MC) preparation (700 U α-galactosidase, 2,200 U galactomannanase, 3,000 U xylanase, and 22,000 U ß-glucanase per kg of diet) and phytase (Phy, 500 FTU per kg of diet) supplementation on the nutritive value of wheat bran (WB) in broiler chicks. Trial 1 determined retention of nutrients and apparent metabolizable energy corrected by nitrogen (AMEn). One reference diet (RD) protein-free (85% corn based) was fortified to determine the WB nutrient retention coefficient. Trial 2 determined standardized ileal digestibility (SID) of AA, when pancreas and liver were weighed. An additional group of bird was fed with an RD with 5% casein-corn starch diet, fortified with vitamins and minerals to quantify the endogenous fraction and determine SID of AA. For each trial, the test diets were made by mixing RD and WB 7:3 (wt/wt) and fed without or with MC or Phy or combination. Male broilers (Cobb 500), 245 d old, were allocated to five treatments to give seven replicates (seven birds/cage). The birds were fed a commercial diet from day 0 to10 followed by Trial 1 diets from day 11 to 18 and finally Trial 2 diets from day 19 to 21. Excreta samples were collected on days 15-18 and all birds were slaughtered on day 21 for ileal digesta. There was an interaction (P < 0.05) between MC and Phy on retention of DM, N, P, and AMEn. An interaction (P < 0.05) was also observed on SID of Arg, His, Leu, Lys, Phe, Thr, Val, Asp, Cys, Glu, and Ser. Responses of MC plus Phy supplementation were higher (P < 0.05) on overall SID of AA by 6.05% (75.18 to 94.26%), compared with responses for MC (2.35%; 72.04 to 88.97) or Phy (3.46%; 73.27 to 92.13). Liver and pancreas weights were affected (P < 0.05) by the single MC supplementation. The MC and Phy combination may be an effective strategy to improve AA utilization of WB in broiler chickens.

Interaction between xylanase and phytase on the digestibility of corn and a corn/soy diet for broiler chickens.

An experiment was carried out to evaluate the digestibility and metabolizability of corn and a corn/soy-based diet with the inclusion or not of xylanase and/or phytase in broilers. In the trial, 1,120 broiler chicks were distributed according to a completely randomized experimental design, consisting of 16 treatments, with 10 replicates of 7 birds each. Treatments were evaluated following a factorial arrangement (4 × 2 × 2), with 4 xylanase levels (zero, 50, 100, or 150 fungal ß-xylanase units/kg; FXU), 2 phytase levels (zero or 1,000 phytase units/kg; FTU), and 2 diets (corn/soy or pure corn). The same basal diets were fed from one to 14 d post hatch for all birds, after which the experimental diets were provided until d 25. All birds were euthanized on d 25 for collection of ileal contents. Samples of feed, excreta, and ileal digesta were analyzed for determination of apparent ileal digestibility. The effect of xylanase on the coefficient of apparent dry matter metabolizability and apparent metabolizable energy was increased by the presence of phytase in the complete diet but not in the diet based on pure corn resulting in a diet*phytase*xylanase interaction (P < 0.01; P < 0.001, respectivaly). Equivalent effects were observed for the apparent coefficient of ileal protein digestibility in which xylanase effects were potentiated by the presence of phytase only in the complete diet, resulting in a significant 3-way interaction. In corn there was a limitation in improving digestibility when we added increasing levels of xylanase with phytase. Otherwise in the corn/soy-based diets, the enzymes were potencialized when they were added together. The fact that the effect of xylanase was enhanced by the presence of phytase in complete diets but not in pure corn may be associated with differences in substrate (arabinoxylan and/or phytate) concentration and presentation, diet nutrient balance, or other factors. It can be concluded that the interactive effects of xylanase and phytase can be substantial but may depend on the characteristics of the diet fed.

Efficiency of microbial phytase supplementation in diets formulated with different calcium:phosphorus ratios, supplied to broilers from 22 to 33 days old.

An experiment was conducted with broilers from 22 to 33 days of age to evaluate the efficiency of six microbial phytases supplemented in diets (1500 FTU/kg) that were formulated with three different calcium:available phosphorus (Ca:P(avail)) ratios (4.5:1.0, 6.0:1.0 and 7.5:1.0). A positive control diet without phytase was formulated with a Ca:P(avail) ratio of 7.5:3.4 to meet the nutritional requirements of the broilers. The P and ash contents of the tibia, magnesium in the plasma, performance, balance and retention of phytate phosphorus (P(phyt)), intake of total P and nitrogen (N), nitrogen-corrected apparent metabolizable energy and apparent digestibility of dry matter of the diets were not influenced (p > 0.05) by the type of phytase or the dietary Ca:P(avail) ratio. However, there was an interaction (p < 0.05) between the phytase type and the Ca:P(avail) ratio for the retention coefficients of total P, Ca and N. Phytase B resulted in the highest Ca deposition in the tibia (p < 0.01). Phytases D, E and F reduced the Ca concentrations in the tibia (p < 0.01) and plasma (p < 0.05). Phytase D increased the P level in the plasma and decreased the total P excretion (p < 0.01). Phytases E and F increased Ca excretion, while phytase A reduced it (p < 0.01). Regardless of the phytase type, increasing the dietary Ca:P(avail) ratio reduced (p < 0.05) the plasma P concentration and the excretion of total P and N and, conversely, increased (p < 0.05) the plasma concentration, intake and excretion of Ca. For the rearing period evaluated, it is possible to reduce the P(avail) of the diet to 1.0 g/kg when Ca is maintained at 7.5 g/kg, and the diet is supplemented with 1500 FTU of phytase A, C, D or E/kg. This diet allows the maintenance of performance and adequate bone mineralization, and it improves the Ca, total P and P(phyt) utilization in addition to reducing the excretion of N and P into the environment.

Digestibility marker and ileal amino acid digestibility in phytase-supplemented soybean or canola meals for growing pigs.

Two experiments using soybean meal (SBM) or canola meal (CM) were conducted to investigate whether the choice of digestibility marker influenced the apparent ileal digestibility (AID) or standardized ileal digestibility (SID) of N and AA in diets supplemented with phytase. In each experiment, 18 barrows fitted with T-cannulas at the ileocecal junction were assigned to 3 diets consisting of a N-free diet to determine endogenous losses of N and AA, a semipurified diet (SBM in Exp. 1 or CM in Exp. 2), and the semipurified diet supplemented with phytase at 1,000 phytase units/kg. Three digestibility markers including acid-insoluble ash (AIA), chromic oxide (Cr2O3), and titanium dioxide (TiO2) were added to each diet at 3 g/kg. Each diet was fed for 7 d, consisting of a 5-d adjustment and a 2-d collection of ileal digesta. In both studies, basal ileal endogenous losses determined with Cr2O3 as a digestibility marker were lower (P<0.01) than with those determined with AIA or TiO2 digestibility markers. Using SBM as the protein source in Exp. 1, there was no interaction between phytase and digestibility marker on AID or SID of AA. The AID of N and AA in SBM using AIA as a digestibility marker tended to be lower (P<0.1) compared with Cr2O3 or TiO2 digestibility markers. Phytase supplementation increased (P<0.001) the AID of Ca and P. The use of AIA or Cr2O3 digestibility marker tended to be associated with lower (P<0.1) SID values compared with TiO2. Phytase did not affect the SID of N or any AA in SBM except for Met, for which there was an increase (P<0.05) with phytase supplementation. Using CM as the protein source in Exp. 2, there were significant interactions between digestibility marker and phytase. Phytase supplementation had effects (P<0.01) on AID or SID when Cr2O3 or TiO2 was used as the digestibility marker. With Cr2O3 or TiO2 as the digestibility marker in the CM diets, phytase supplementation increased (P<0.05) the SID of N and all AA (except Trp). There was no SID of N or AA response to phytase supplementation of CM when AIA was used as a digestibility marker. In contrast, there were no clear improvements in AA digestibility from phytase supplementation for SBM. Phytase effects on AID or SID of AA were dependent on the digestibility marker used in diets when CM was used as the protein source but not when SBM was used as the protein source. Therefore, AA digestibility response to phytase supplementation may depend on the protein being evaluated as well as the choice of digestibility marker.