Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia.

Sustainable aquafeed production requires fishmeal replacement, leading to an increasing use of plant-derived ingredients. As a consequence, higher levels of antinutritional substances, such as non-starch polysaccharides and phytate, are present in aquafeeds, with negative effects on fish performance, nutrient digestibility and overall gut health. To alleviate these negative effects, providing exogenous digestive enzymes and/or probiotics can be an effective solution. In this study, we tested the effect of dietary supplementation of enzymes (phytase and xylanase) and probiotics (three strains of Bacillus amyloliquefaciens) on nutrient digestion kinetics and volatile fatty acid content along the gut, and the distal gut microbiome diversity in Nile tilapia. Chyme volatile fatty content was increased with probiotic supplementation in the proximal gut, while lactate content, measured for the first time in vivo in fish, decreased with enzymes along the gut. Enzyme supplementation enhanced crude protein, Ca and P digestibility in proximal and middle gut. Enzymes and probiotics supplementation enhanced microbial interactions as shown by network analysis, while increased the abundance of lactic acid bacteria and Bacillus species. Such results suggest that supplementation with exogenous enzymes and probiotics increases nutrient availability, while at the same time benefits gut health and contributes to a more stable microbiome environment.

Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

Interactions between phytase and xylanase enzymes in male broiler chicks fed phosphorus-deficient diets from 1 to 18 days of age.

A total of 735 one-day-old male broiler chicks were used to evaluate the interactions between different levels of phytase and xylanase enzymes on performance and bone mineralization. Basal nonphytate P (nPP)-deficient diets (0.15%) were supplemented with different levels of phytase [0X, 1X, 2X, 3X, and 4X of recommended level (X = 500 phytase units per kg of feed)] alone or in combination with 3 levels of a xylanase preparation [0X, 1X, and 2X of recommended level (X = 0.1 g per kg of feed; a mixture with predominantly xylanase activity)]. A standard curve was developed using different levels of nPP (0.15 to 0.45%) to estimate the P equivalency of each enzyme combination. Treatments were replicated with 7 pens of 5 chicks each. The findings indicated that reducing dietary nPP level had a severely negative influence on bird performance and bone ash content. Results also showed that increasing levels of phytase significantly (P < 0.05) improved BW, feed intake, feed conversion ratio, mortality, and toe and tibia bone ash contents in a dose-dependent fashion. The P equivalency of phytase was also dose dependent, with estimates of 0.08, 0.11, 0.15, and 0.19 for 1X, 2X, 3X, and 4X supplementation levels of phytase, respectively. Xylanase preparation at 1X level failed to influence measured criterion; however, increasing the level of xylanase (2X) resulted in deteriorating BW and feed conversion ratio. The P equivalency of xylanase preparation at 1X and 2X was estimated as 0.010 and 0.014%. There were no interactions between phytase and xylanase preparation (P > 0.05). In conclusion, phytase was effective in improving bird performance and bone mineralization; however, xylanase supplementation failed to enhance phytase efficacy.

Interactive effects of phytase and xylanase supplementation with extractable salt-soluble protein content of corn in diets with adequate calcium and nonphytate phosphorus fed to broilers.

The objective was to determine the effects of extractable salt-soluble protein content of corn (PS) and exogenous enzyme supplementation on N, starch, and energy digestibility in broilers fed diets adequate in Ca and nonphytate P. Broilers were randomly distributed into floor pens (6 replicate pens per treatment) with 28 birds per pen at 1 d of age. Treatments consisting of 4 sources of corn varying in PS (A, 58.1; B, 54.2; C, 53.7; and D, 30.6 mg of BSA equivalent values) with or without phytase (0 and 1,000 phytase units/kg) and xylanase (0 and 16,000 units of xylanase activity/kg) were randomly assigned to each pen. Different sources of corn were provided from 1 to 9 and 24 to 29 d of age. However, enzyme treatments were provided throughout the experiment. From 1 to 9 d of age, no interactions were observed. Apparent ileal N digestibility (AIND) and apparent ileal digestible energy (IDE) of diets with the lowest PS (based on corn D) were lower (P ≤ 0.05) than those of diets with a higher PS. Phytase increased (P ≤ 0.01) AIND and IDE by 5 and 16%, respectively, and xylanase exerted the opposite effect (P ≤ 0.03). From 24 to 29 d of age, phytase and xylanase in combination resulted in reduced (P ≤ 0.05) AIND of diets with a low PS (based on corn D) compared with the basal diet in broilers. Broilers fed diets with the highest or lowest PS (based on corn A or D) had lower (3-way interaction; P ≤ 0.05) IDE when phytase and xylanase were supplemented in combination compared with either enzyme alone. In conclusion, responses to exogenous enzyme supplementation are not constant and are influenced by the source of ingredients as well as the age of broilers. The magnitudes of the responses to phytase on nutrient and energy digestibility were greater at 9 compared with 29 d of age.

Modeling the efficiency of phosphorus utilization in growing pigs.

Microbial phytase has been used to reduce P excretion from swine to mitigate environmental pollution. The objective of the study was to quantify the effect of feeding a low-P phytase-supplemented diet on growth and P utilization in growing pigs using mathematical models. A total of 20 weaned piglets (BW = 6.5 kg) housed in metabolism cages were randomly assigned to a standard diet (STD) or P-amended diet containing reduced P content and supplemented with phytase (AMN) with 10 pigs/diet. Body weight and feed consumption were recorded weekly so complete growth and cumulative P intake (cPI) curves could be modeled. A function with fixed point of inflexion (Gompertz) and a variable point of inflexion (generalized Michaelis-Menten) were considered in determining bioequivalence by analyzing BW vs. age relationships, whereas the monomolecular function was used to describe BW vs. cPI. All functions were incorporated into a nonlinear mixed effects model, and a first-order autoregressive correlation structure was implemented to take into account repeated measures. There was no difference between the 2 groups in final BW when the Gompertz equation was fitted (176 vs. 178 kg with SE of 7 kg for the STD and AMN, respectively) or the rate parameter (0.0140 vs. 0.0139 with SE of 0.0004 for the STD and AMN, respectively). The generalized Michaelis-Menten equation also showed a similar trend. When BW was expressed as a function of cPI the derivative with respect to cPI represented P efficiency, so it was possible to analyze the expected difference of the 2 diets in using P for BW gain and express it as a continuous function of cPI. The analysis showed through the entire growth period the difference in P efficiency was different from zero. On average, 56 g of supplemented inorganic P was consumed by a pig fed the AMN to reach market weight. In contrast, 309 g of supplemented inorganic P was consumed by the group fed the STD to reach similar BW. It would depend on other factors, but feeding pigs the AMN can result in economic benefit. Pigs fed the AMN excreted 19% less P compared with those fed the STD. In conclusion, nonlinear mixed model analysis (with repeated measures) was suitable for growth and efficiency analysis and showed that pigs fed the AMN consumed less than 20% of the inorganic P and performed as well as those fed the traditional inorganic P supplemented diet. The implications for mitigating P pollution, especially in areas where P loading is already problematic, are substantial.

Phosphorus equivalency value of an Escherichia coli phytase in the diets of White Pekin ducks.

Male White Pekin ducks at 7 d posthatch were used in a 10-d experiment to determine the equivalency value of an Escherichia coli phytase (Phyzyme XP) in corn-soybean meal-based mash diets. Two hundred fifty-six ducks were grouped by weight into 8 blocks of 8 cages with 4 ducks per cage. The 8 diets consisted of a P-adequate positive control corn-soybean meal formulated to contain CP, Ca, total P, and nonphytate P at 220, 8, 6.6, and 4 g/kg, respectively; a low-P negative control (NC) corn-soybean meal basal diet formulated to contain CP, Ca, total P, and nonphytate P at 220, 6.5, 3.9, and 1.3 g/kg, respectively; NC diet plus 0.5, 1.0, or 1.5 g of inorganic P from monosodium phosphate/kg; and NC diet plus E. coli phytase at 500, 1,000, or 1,500 units/kg. Ducks were provided ad libitum access to water and experimental diets. Feeding the low-P NC diet to ducks reduced (P<0.01) BW gain, feed intake, G:F, tibia ash, ileal digestibility, and utilization of P. Supplementation of the NC diet with inorganic P or phytase linearly improved (P<0.01) final weight, BW gain, feed intake, G:F, tibia ash, and ileal P digestibility. There were linear increases (P<0.01) in the utilization of P from 33.8 to 42% or from 33.8 to 46.6% as dietary added inorganic P or phytase increased from 0 to 1.5 g/kg or 0 to 1,500 units/kg, respectively. Linear regression equations for the tibia ash as the dependent variable and supplemental intake of inorganic P and phytase or supplemental level of inorganic P and phytase as independent variables were used to generate P equivalency values of phytase. The mean P equivalency values of phytase from linear regression equations derived from ducks fed a low-P NC diet supplemented with graded levels of inorganic P or phytase from d 7 to 17 posthatch for 500, 1,000, and 1,500 phytase units/kg of diet were 0.453, 0.847, and 1.242 g/kg of diet, respectively. The results of this study showed that this phytase was efficacious in hydrolyzing phytate P for bone mineralization and growth of ducks.

The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato.

The sweet potato sporamin promoter was used to control the expression in transgenic potato of the E. coli appA gene, which encodes a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The sporamin promoter was highly active in leaves, stems and different size tubers of transgenic potato, with levels of phytase expression ranging from 3.8 to 7.4% of total soluble proteins. Phytase expression levels in transgenic potato tubers were stable over several cycles of propagation. Field tests showed that tuber size, number and yield increased in transgenic potato. Improved phosphorus (P) acquisition when phytate was provided as a sole P source and enhanced microtuber formation in cultured transgenic potato seedlings when phytate was provided as an additional P source were observed, which may account for the increase in leaf chloroplast accumulation (important for photosynthesis) and tuber yield of field-grown transgenic potato supplemented with organic fertilizers. Animal feeding tests indicated that the potato-produced phytase supplement was as effective as a commercially available microbial phytase in increasing the availability of phytate-P to weanling pigs. This study demonstrates that the sporamin promoter can effectively direct high-level recombinant protein expression in potato tubers. Moreover, overexpression of phytase in transgenic potato not only offers an ideal feed additive for improving phytate-P digestibility in monogastric animals but also improves tuber yield, enhances P acquisition from organic fertilizers, and has a potential for phytoremediation.

Phosphorus equivalence of microbial phytase in turkey diets as influenced by calcium to phosphorus ratios and phosphorus levels.

Male day-old turkey poults (n = 768) were fed 0, 300, 600, or 900 U of phytase/kg of a corn-soybean diet in combination with four Ca:total P (tP) ratios of 1.1, 1.4, 1.7, and 2.0:1, and two levels of nonphytate P (nP) of 0.27 and 0.36% in a 21-d trial. Dietary Ca:tP ratios were obtained by varying defluorinated phosphate and limestone at the expense of cornstarch. The calculated dietary percentage of phytate P was 0.266 for all diets. Phytase additions linearly increased (P < 0.05) BW gain, feed intake, gain:feed, toe ash content, and apparent retentions of Ca and P at each Ca:tP ratio and nP level, but the response was influenced by dietary Ca:tP ratios and P levels. The detrimental effect (P < 0.02) of widening the Ca:tP ratio was observed for all measurements at each phytase and P level, and was greatest at lower phytase and P levels. Widening the Ca:tP ratio from 1.4 to 2.0 decreased the phytase efficacy by 7.4 and 4.9%, respectively, for 0.27 and 0.36% nP diets, which was close to the decrease in the phytase activity in vitro by 7.5 and 6.7%, respectively. The largest responses to supplemental phytase were achieved when poults were fed diets with 600 and 900 U of phytase/kg diet, respectively, for 0.36 and 0.27% nP, and for Ca:tP ratios ranging from 1.1 to 1.4:1. Second-order translog equations were generated for the phytase, Ca:tP ratio, and P effect, and nonlinear and linear equations for the phytase and Ca:tP ratio effect. Based on an assessment for the R2 and P values of equations, BW gain, feed intake, toe ash content, and P retention were sensitive measurements of the response to phytase addition. Equivalent equations were developed to determine the P equivalency of supplemental phytase. About 652 and 963 U of phytase were equivalent to 1 g nP, respectively, for 0.27 and 0.36% nP diets in turkey poults from hatch to 21 d of age.