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PURPOSE: For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36µL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. CONCLUSION: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

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Male broiler chickens (384), Cobb 500, were housed in metabolic cages to assess the efficacy of phytase in diets with low and high phytate-phosphorus on the performance, bone physical characteristics, tissue and serum mineral deposits. Birds were distributed in four treatments with a 2 x 2 factorial arrangement in a completely randomized block design. Experimental diets based on maize-soybean meal were T1 - diet low phytate-phosphorus; T2 - diet low phytate-phosphorus and phytase (500 FTU/kg); T3 - diet high phytate-phosphorus; T4 - diet high phytate-phosphorus and phytase (500 FTU/kg). Feed intake, body weight, weight gain and feed conversion ratio were assessed. Two left tibias per experimental unit were analyzed for physical characteristics and mineral concentration; a section of skinless breast muscle and blood were collected to measure the concentration of calcium, phosphorus and sodium. Results showed interaction between bone stiffness and serum calcium. The inclusion of phytase in diets with low and high phytate-phosphorus did not alter performance, bone resistance and flexibility, mineral deposits in the tibia and breast muscle, but increased bone stiffness after 22 days of age. It also provided a higher serum calcium rate in broilers fed diets with low phytate-phosphorus up to 32 days of age.

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Iron is routinely supplemented in broiler feeds intending to prevent dietary deficiencies. The present research was conducted with the objective of assessing Fe requirements of broilers when fed supplemental phytase. A total of 1,280 1-d-old male Cobb × Cobb 500 were distributed in a 2 × 5 factorial arrangement (phytase-supplemented feeds × 5 graded increases of supplemental Fe) in 80 battery cages, eight replications of eight chicks each. The trial was replicated once. Chicks were fed a Fe-deficient diet without phytase (Fe analyzed at 31.30 ± 3.79 mg/kg) from placement to 7 d and then randomly distributed into battery cages with corresponding dieting treatments with or without phytase and graded increases of supplemental Fe. Feeds were formulated with corn and soybean meal (SBM), laboratory-grade calcium carbonate, and phosphoric acid; therefore, the vast majority of dietary Fe originated from corn and SBM (analyzed diet had 53.3 ± 1.41 mg/kg Fe). Phytase was added in excess to the producer recommendation of 1,000 FYT (4,452 ± 487 FYT/kg analyzed) such that phytate degradation was expected to be maximized. Supplemental Fe was from laboratory-grade ferrous sulfate heptahydrate (FeSO47H2O) which was increasingly added to the feeds (analyzed Fe in the supplemented feeds were: 53.3 ± 1.41, 65.5 ± 0.59, 77.2 ± 1.97, 87.6 ± 1.72, 97.7 ± 1.33 mg/kg). There were no interactions between phytase and dietary Fe for any response throughout the study (P > 0.05). Supplementing phytase had no effects on Fe intake or Fe excretion, as well as on hematocrit (Ht), hemoglobin (Hb), ferritin, Fe contents in the liver or thigh muscle color (P > 0.05). However, phytase-supplemented feeds produced better live performance as well as higher ileal digestible energy and Fe digestibility (P < 0.05). No effects were found for dietary Fe in live performance at day 28 (P > 0.05). On the other hand, increasing dietary Fe led to linear increases in Fe retention and excretion, Fe contents in livers, as well as Ht and Hb at 14 d (P < 0.05). Quadratic responses (P < 0.05) were observed for Hb at 21 d, serum ferritin on days 14, 21, and 28 (maximum responses were 83.3, 104.0, 91.9, and 88.3 mg/kg Fe, respectively). In conclusion, supplementing Fe adding to a total of 97.7 mg/kg dietary Fe did not affect live performance traits. However, the average of Fe-related blood parameters was maximized at 91.9 mg/kg dietary Fe. Supplementing phytase provided a significant increase in Fe digestibility.

Iron is routinely supplemented in broiler feeds to prevent possible dietary deficiencies. We carried out an experiment with the objective of evaluating the Fe requirements of broilers fed with the exogenous enzyme phytase. From the eighth day, a total of 1,280 male broilers were distributed in a combination of diets supplemented with phytase or not and 5 graded increases in dietary Fe. Diets were formulated with corn and soybean meal, laboratory-grade calcium carbonate, and phosphoric acid. Phytase was added in excess (4,452 ± 487 FYT/kg analyzed) to facilitate complete degradation of dietary phytate. Laboratory-grade ferrous sulfate heptahydrate was increasingly added to the feeds to provide Fe. Iron in the experimental diets was present at 53.3 ± 1.41, 65.5 ± 0.59, 77.2 ± 1.97, 87.6 ± 1.72, 97.7 ± 1.33 mg/kg. Supplementing diets with phytase resulted in enhanced live performance, along with increased digestibility of ileal energy and Fe. Linear increases in Fe retention and excretion, hepatic Fe contents, and serum ferritin were observed with the progressive increase in dietary Fe. The supplementation of a total of 97.7 mg/kg of Fe in diets was found to have no significant impact on live performance traits. However, the Fe-related blood parameters reached their maximum levels at a dietary Fe level of 91.9 mg/kg. Phytase supplementation provided a significant increase in the digestibility of Fe and other nutrients evaluated.

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The objective of this study was to evaluate the effect of a novel consensus bacterial 6-phytase variant (PhyG) on egg productivity, eggshell quality, and body composition of laying hens fed inorganic phosphate-free diets with reduced energy and nutrients from 23 to 72 wk of age. Five treatments were randomly assigned, performing 28 replicates per treatment with 4 hens each, totaling 560 Hy-Line W80 birds. A positive control (PC) feed was formulated to contain adequate levels of energy and nutrients. A negative control (NC) feed was formulated without added inorganic phosphate (0.12% nonphytic phosphorus [nPP]) and reduced in Ca, Na, dig AA, and metabolizable energy in comparison with PC feed. Phytase was supplemented in the NC feed at 0, 300, 600, and 900 FTU/kg of feed. The responses evaluated were performance, egg quality, economic analysis, body composition, and tibia composition. Data were analyzed by a 2-factor (diet and age) repeated measure analysis. Overall, the feed intake, hen-day egg production, egg mass, and egg revenue were reduced by the complete removal of dicalcium phosphate (DCP) (P < 0.05). Supplement phytase in the NC diet elicits a positive response on each one of those variables. Laying hens consuming the NC feed with 900 FTU/kg of phytase produced more eggs per hen-housed compared with the phytase dosages of 300 and 600 FTU/kg. Body composition was not affected by dietary nPP, Ca, Na, dig AA, and energy reductions (P > 0.05). At 72-wk-old, tibia ash was reduced in hens consuming the NC diet vs. PC (P < 0.05) and no difference was observed between hens supplemented with phytase and the PC feed. Margin over feeding cost increased in a dose-dependent manner with phytase supplementation. Supplementation with 900 FTU/kg of phytase is recommended to improve the number of eggs produced per hen-housed and the number of marketable eggs produced through 23 to 72 wk of age, under this dietary setting.

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The effects of ß-mannanase supplementation in metabolizable energy (ME)-reduced diets containing xylanase-phytase were investigated on growth performance, fecal score, ultra-sounded backfat thickness and loin depth, blood profile, apparent total tract digestibility (ATTD), digesta passage rate, and fecal microbiome in grower pigs (n = 40, 26.09 ± 0.96 kg) randomly assigned within 4 treatments: a control diet containing isolated phytase and xylanase valued at 40 kcal of ME/kg (CD0), CD0 + ß-mannanase (0.3 g/kg valued at 30 kcal of ME/kg) (CD70), CD0 + ß-mannanase (0.3 g/kg valued at 45 kcal of ME/kg) (CD85), and CD0 + ß-mannanase (0.3 g/kg valued at 60 kcal of ME/kg) (CD100). Growth performance was not affected in pigs fed ME-reduced diets containing ß-mannanase. Pigs with CD100 had lower serum IL-1ß concentration, and higher IL-10 was observed in pigs on CD0 than those fed ß-mannanase. Coefficients of ATTD, and ATTD of DM and CP were higher in animals fed CD85 or CD100. Pigs with CD85 had higher alpha diversity richness but lower Firmicutes:Bacteroidota ratio. Acidaminococcaceae and Ruminococcaceae were more abundant in pigs fed CD0, but lower for Christensenellaceae NSJ-63 and NSJ-63 sp014384805. Pigs in CD85 showed higher Bacteroidaceae and Prevotella abundance, and lower for Streptococcaceae and Streptococcus. In conclusion, supplementation of ß-mannanase in diets containing xylanase-phytase saved 85 to 100 kcal of ME/kg by supporting growth performance and improving nutrient digestibility in grower pigs.

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To reduce P excretion and increase the sustainability of poultry farms, one needs to understand the mechanisms surrounding P metabolism and its close link with Ca metabolism to precisely predict the fate of dietary P and Ca and related requirements for birds. This study describes and evaluates a model developed to estimate the fate of Ca and P consumed by broilers. The Ca and P model relies on three modules: (1) digestion of Ca and P; (2) dynamics of Ca and P in soft tissue and feathers; and (3) dynamics of body ash. Exogenous phytase affects the availability of Ca and P; thus, to predict the absorption of those minerals, the model also accounts for the effect of phytase on Ca and P digestibility. We used a database to estimate the consequences of dietary Ca, P, and phytase over feed intake response. This study followed a four-step process: (1) Ca and P model development and its coupling with a growth broiler model; (2) model behavior assessment; (3) sensitivity analysis to identify the most influential parameters; and (4) external evaluation based on three databases. The proportion of P in body protein and the Ca to P ratio in bone are the most sensitive parameters of P deposition in soft tissue and bone, representing 91 and 99% of the total variation. The external evaluation results indicated that body water and protein had an overall mean square prediction error (rMSPE) of 7.22 and 12.3%, respectively. The prediction of body ash, Ca, and P had an rMSPE of 7.74, 11.0, and 6.56%, respectively, mostly errors of disturbances (72.5, 51.6, and 90.7%, respectively). The rMSPE for P balance was 13.3, 18.4, and 22.8%, respectively, for P retention, excretion, and retention coefficient, with respective errors due to disturbances of 69.1, 99.9, and 51.3%. We demonstrated a mechanistic model approach to predict the dietary effects of Ca and P on broiler chicken responses with low error, including detailed simulations to show the confidence level expected from the model outputs. Overall, this model predicts broilers' response to dietary Ca and P. The model could aid calculations to minimize P excretion and reduce the impact of broiler production on the environment. A model inversion is ongoing that will enable the calculation of Ca and P dietary quantities for a specific objective. This will simplify the use of the model and the feed formulation process.

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Phosphate release from inorganic and organic phosphorus compounds can be enzymatically mediated. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation, and diagnostic analysis. Here, we describe a functional metagenomics approach enabling rapid identification of genes encoding these enzymes. The target genes are detected based on small- and large-insert metagenomic libraries derived from diverse environments. This approach has the potential to unveil entirely new phosphatase families or subfamilies and members of known enzyme classes that hydrolyze phosphomonoester bonds such as phytases. Additionally, we provide a strategy for efficient heterologous expression of phosphatase genes.

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Abstract The growth of aquaculture sector is strongly dependent upon the continuous supply of inexpensive fish feed with balanced nutritional profile. However, fish meal (FM) is unable to satisfy this demand due to its scarce supply and high cost. In order to test the potential of cottonseed meal (CSM) as a fish meal replacer, a feeding trial of 12 weeks was conducted to check growth performance and proximate composition of Labeo rohita fingerlings. The protein ration of the test feed was satisfied by replacing FM with CSM at 0, 25, 50 and 75%. Sixteen test diets viz., TD1 (control), TD2, TD3, TD4, TD5, TD6, TD7, TD8, TD9, TD10, TD11, TD12, TD13, TD14, TD15 and TD16 were supplemented with citric acid (CA; 0 and 2.5%) and phytase (PHY; 0 and 750 FTU/kg) in a completely randomized design with 3×3 factorial arrangement. The highest weight gain (11.03g), weight gain% (249.21%), specific growth rate (1.39) and best feed conversion ratio (1.20) were recorded by fish fed with TD12. Furthermore, the same level increased the crude protein (59.26%) and fat (16.04%) being significantly different (p<0.05) than that of control. Conclusively, the addition of acidified phytase (CA; 2.5%, PHY; 750 FTU/kg) in TD12 (CSM=50%) led to the improved growth and proximate composition of L. rohita fingerlings.

Resumo O crescimento do setor de aquicultura é fortemente dependente do fornecimento contínuo de rações baratas para peixes com perfil nutricional equilibrado. Porém, a farinha de peixe (FM) não consegue atender a essa demanda devido à sua escassa oferta e alto custo. Com o objetivo de testar o potencial da farinha de semente de canola (MSC) como substituto da farinha de peixe, um ensaio alimentar de 12 semanas foi conduzido para verificar o desempenho de crescimento e a composição centesimal de alevinos de Labeo rohita. A ração de proteína da ração teste foi satisfeita substituindo FM por CSM em 0, 25, 50 e 75%. Dezesseis dietas de teste, viz., TD1 (controle), TD2, TD3, TD4, TD5, TD6, TD7, TD8, TD9, TD10, TD11, TD12, TD13, TD14, TD15 e TD16 foram suplementadas com ácido cítrico (CA; 0 e 2,5%) e fitase (PHY; 0 e 750 FTU / kg) em um delineamento inteiramente casualizado com arranjo fatorial 3 × 3. O maior ganho de peso (11,03g), % de ganho de peso (249,21%), taxa de crescimento específico (1,39) e melhor taxa de conversão alimentar (1,20) foram registrados por peixes alimentados com TD12. Além disso, o mesmo nível aumentou a proteína bruta (59,26%) e a gordura (16,04%), sendo significativamente diferente (p <0,05) do controle. Conclusivamente, a adição de fitase acidificada (CA; 2,5%, PHY; 750 FTU / kg) em TD12 (CSM = 50%) levou a um melhor crescimento e composição próxima de alevinos de L. rohita.

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Abstract Fishmeal; being a limited and costly feed ingredient is continuously been substituted with locally available plant proteins. However, the occurrence of anti-nutritional factors in plant meal suppresses its potential to be fully replaced. Therefore, in this study we aimed to study the synergistic effects of dietary additives like citric acid and phytase enzyme supplementation on growth performance and nutrient digestibility of Cirrhinus mrigala fingerlings. Canola meal (CM) was used as a test ingredient to replace fishmeal (FM) as; 0%, 25%, 50% and 75%. These four diets were further supplemented by varying levels of phytase (0 and 750 FTU kg-1) and citric acid (0% and 2.5%) to formulate total sixteen test diets as T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, T13, T14, T15 and T16. Each treatment contained three replicates; applied to fish groups having 15 fingerlings each; following 3×3 factorial arrangement. 1% of chromic oxide was added as an inert marker. Maximum weight gain% (288%) and the lowest value of FCR (1.07) were recorded when fish was fed on diet T12 as compared to fish fed control diet (T1). Similarly, optimum nutrient digestibility values such as crude protein (77%), crude fat (84%) and gross energy (70%) were noted on same level. It was concluded that 50% canola meal can optimally replace fishmeal when supplemented with phytase and citric acid at the levels of 750 FTU kg-1 and 2.5%, respectively.

Resumo A farinha de peixe, por ser um ingrediente alimentar limitado e caro, é continuamente substituída por proteínas vegetais disponíveis localmente. No entanto, a ocorrência de fatores antinutricionais na farinha de plantas suprime seu potencial de ser totalmente substituída. Portanto, neste estudo objetivamos estudar os efeitos sinérgicos de aditivos dietéticos como ácido cítrico e suplementação com enzima fitase sobre o desempenho de crescimento e digestibilidade de nutrientes de alevinos de Cirrhinus mrigala. A farinha de canola (CM) foi usada como ingrediente de teste para substituir a farinha de peixe (FM) como: 0%, 25%, 50% e 75%. Essas quatro dietas foram suplementadas por níveis variados de fitase (0 e 750 FTU kg-1) e ácido cítrico (0% e 2,5%) para formular um total de 16 dietas de teste como T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, T13, T14, T15 e T16. Cada tratamento continha três repetições; aplicado a grupos de peixes com 15 alevinos cada; seguindo o arranjo fatorial 3 × 3. 1% de óxido crômico foi adicionado como um marcador inerte. % de ganho de peso máximo (288%) e o valor mais baixo de FCR (1,07) foram registrados quando os peixes foram alimentados com dieta T12 em comparação com peixes alimentados com dieta controle (T1). Da mesma forma, valores ótimos de digestibilidade de nutrientes, como proteína bruta (77%), gordura bruta (84%) e energia bruta (70%) foram anotados no mesmo nível. Concluiu-se que 50% da farinha de canola pode substituir de forma ideal a farinha de peixe quando suplementada com fitase e ácido cítrico nos níveis de 750 FTU kg-1 e 2,5%, respectivamente.

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A trial was conducted to evaluate phosphorus release capacity in different commercial phytases throught performance and bone characteristics of broilers. A total of 2,400-day-old male Cobb 500® chicks were assigned in a completely randomized design with 12 dietary treatments ((1 to 5: with increasing levels of non-phytate phosphorus (NPP) (0.20; 0.25; 0.30; 0.35 and 0.40%) without phytase); and 6 to 12: with 0.20% supplemented with different commercial phytases), 8 replicates and 25 birds per pen. There was a linear increase (P<0.05) in the feed intake and body weight gain and a linear improvement (P<0.05) in the feed conversion ratio of broilers fed increasing NPP. The tested phytases did not release similar amounts of phytic phosphorus. Based on body weight gain response, 500 FTU/kg of diet releases a minimum and maximum of 0.074 to 0.152%, 0.062 to 0.157 and 0.059 to 0.169% of PP among the studied phytases in the periods from 1 to 21, 1 to 35 and 1 to 42 days, respectively. In conclusion, the commercial phytases released different concentrations of phytate phosphorus from the diets. Thus, is necessary to have a better method to evaluate phytase activity in order to avoid subnormal levels of available P in diets.

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