A comparative study on the effects of fungal and bacterial phytase with or without citric acid on growth performance, serum mineral profile, bone quality, and nutrient retention in broilers.

Objective: Current research aimed to compare the effects of fungal and bacterial phytase with or without citric acid (CA) on growth performance, serum mineral profile, bone quality, and nutrient retention in birds given non-phytate phosphorus (nPP)-deficient diets. Materials and Methods: A total of 216 Indian River broiler chicks were disturbed into six groups, namely, i) positive control (PC), ii) negative control (NC) contained 0.2% lower nPP than that in the PC diet, iii) NC + fungal phytase (Aspergillus niger), iv) NC + fungal phytase with 2% CA, v) NC + bacterial phytase (Escherichia coli), and vi) NC + bacterial phytase with 2% CA. Results: Compared to the PC group, the NC group showed poor performance, serum phosphorus (P) content, P retention, and bone quality. However, with the inclusion of phytase, all these phenomena were improved. The addition of bacterial phytase showed better values compared with fungal phytase. The main effects of phytase were significant for the feed conversion ratio (FCR), metabolizable energy conversion ratio (MECR), and P retention. The addition of CA, either with fungal or bacterial phytases, did not show considerable beneficial effects on overall performances. However, the main effects of CA were significant on the FCR, MECR, and crude protein conversion ratio. Conclusion: Incorporating bacterial and fungal phytase into low-nPP diets enhanced the broiler's performance. The effects of bacterial phytase were more apparent than those of fungal phytase. However, the efficacy of phytase based on the source might relate to dose, and other factors need further investigation.

Effect of dietary calcium source, exogenous phytase, and formic acid on inositol phosphate degradation, mineral and amino acid digestibility, and microbiota in growing pigs.

The choice of the calcium (Ca) source in pig diets and the addition of formic acid may affect the gastrointestinal inositol phosphate (InsP) degradation and thereby, phosphorus (P) digestibility in pigs. This study assessed the effects of different Ca sources (Ca carbonate, Ca formate), exogenous phytase, and chemical acidification on InsP degradation, nutrient digestion and retention, blood metabolites, and microbiota composition in growing pigs. In a randomized design, eight ileal-cannulated barrows (24 kg initial BW) were fed five diets containing Ca formate or Ca carbonate as the only mineral Ca addition, with or without 1,500 FTU/kg of an exogenous hybrid 6-phytase. A fifth diet was composed of Ca carbonate with phytase but with 8 g formic acid/kg diet. No mineral P was added to the diets. Prececal InsP6 disappearance and P digestibility were lower (P ≤ 0.032) in pigs fed diets containing Ca formate. In the presence of exogenous phytase, InsP5 and InsP4 concentrations in the ileal digesta were lower (P≤0.019) with Ca carbonate than Ca formate. The addition of formic acid to Ca carbonate with phytase diet resulted in greater (P = 0.027) prececal InsP6 disappearance (87 vs 80%), lower (P = 0.001) InsP5 concentration, and greater (P ≤ 0.031) InsP2 and myo-inositol concentrations in the ileal digesta. Prececal P digestibility was greater (P = 0.004) with the addition of formic acid compared to Ca carbonate with phytase alone. Prececal amino acid (AA) digestibility of some AA was greater with Ca formate compared to Ca carbonate but only in diets with phytase (P ≤ 0.048). The addition of formic acid to the diet with Ca carbonate and phytase increased (P ≤ 0.006) the prececal AA digestibility of most indispensable AA. Exogenous phytase affected more microbial genera in the feces when Ca formate was used compared to Ca carbonate. In the ileal digesta, the Ca carbonate diet supplemented with formic acid and phytase led to a similar microbial community as the Ca formate diets. In conclusion, Ca formate reduced prececal InsP6 degradation and P digestibility, but might be of advantage in regard to prececal AA digestibility in pigs compared to Ca carbonate when exogenous phytase is added. The addition of formic acid to Ca carbonate with phytase, however, resulted in greater InsP6 disappearance, P and AA digestibility values, and changed ileal microbiota composition compared to Ca carbonate with phytase alone.

Quinoa snack elaborated with Lactiplantibacillus plantarum CRL 1964 sourdough increases the mineral bioavailability in mice.

BACKGROUND: Consumption of pseudocereal-based foods decreased in phytate concentration can provide better nutrition concerning mineral bioavailability. This study aimed to evaluate the mineral bioavailability of quinoa sourdough-based snacks in a murine model. The mice were divided into five groups. One group was fed with basal snacks; three control groups received quinoa-based snacks made from non-fermented dough, dough without inoculum, and chemically acidified dough; and the test group (GF) received quinoa snacks elaborated from sourdough fermented by a phytase-positive strain, Lactiplantibacillus plantarum CRL 1964. Food intake, body weight, and mineral concentration in blood and organs (liver, kidney, and femur) were determined. RESULTS: Food consumption increased during the feeding period and had the highest (16.2-24.5%) consumption in the GF group. Body weight also increased during the 6-weeks of trial. The GF group showed higher (6.0-10.2%) body weight compared with the other groups from the fifth week. The concentrations of iron, zinc, calcium, magnesium, and phosphorus in blood, iron and phosphorus in the liver, manganese and magnesium in the kidney, and calcium and phosphorus in the femur increased significantly (1.1-2.7-fold) in the GF group compared to the control groups. CONCLUSION: The diet that includes quinoa snacks elaborated with sourdough fermented by phytase-positive strain L. plantarum CRL 1964 increased the concentrations of minerals in the blood, liver, kidney, and femur of mice, counteracting the antinutritional effects of phytate. This study demonstrates that the diminution in phytate content and the consequent biofortification in minerals are a suitable tool for producing novel foods. © 2024 Society of Chemical Industry.

Production, purification and characterization of phytase from Pichia kudriavevii FSMP-Y17and its application in layers feed.

INTRODUCTION: Phytase, recognized for its ability to enhance the nutritional value of phytate-rich foods, has has gained significant prominence. The production of this enzyme has been significantly boosted while preserving economic efficiency by utilizing natural substrates and optimizing essential factors. This study focuses on optimizing phytase production through solid-state fermentation and evaluating its effectiveness in enhancing nutrient utilization in chicken diets. OBJECTIVE: The objective is to optimize phytase production via solid-state fermentation, characterize purified phytase properties, and assess its impact on nutrient utilization in chicken diets. Through these objectives, we aim to deepen understanding of phytase's role in poultry nutrition and contribute to more efficient feed formulations for improved agricultural outcomes. METHODOLOGY: We utilized solid-state fermentation with Pichia kudriavzevii FSMP-Y17 yeast on orange peel substrate, optimizing variables like temperature, pH, incubation time, and supplementing with glucose and ammonium sulfate. Following fermentation, we purified the phytase enzyme using standard techniques, characterizing its properties, including molecular weight, optimal temperature and pH, substrate affinity, and kinetic parameters. RESULTS: The optimized conditions yielded a remarkable phytase yield of 7.0 U/gds. Following purification, the enzyme exhibited a molecular weight of 64 kDa and displayed optimal activity at 55 °C and pH 5.5, with kinetic parameters (Km = 3.39 × 10-3 M and a Vmax of 7.092 mM/min) indicating efficient substrate affinity. CONCLUSION: The addition of purified phytase to chicken diets resulted in significant improvements in nutrient utilization and overall performance, including increased feed intake, improved feed conversion ratio, enhanced bird growth, better phosphorus retention, and improved egg production and quality. By addressing challenges associated with phytate-rich diets, such as reduced nutrient availability and environmental pollution, phytase utilization promotes animal welfare and sustainability in poultry production.

Impact of phytase supplementation on nutrient variability, growth, and digestibility in weaned pigs fed a corn-SBM complex diet.

This study evaluated the effects of different nutrient matrices, with or without phytase supplementation, on growth performance, nutrient digestibility, ileal amino acid (AA) digestibility, and blood inositol in pigs fed a complex diet based on corn-soybean meal. Four hundred newly weaned cross-bred (Landrace × Yorkshire × Duroc) 21-day-old piglets of initial body weight 6.35 ± 1.91 kg were allotted to one of the five dietary treatments: Control (CNT), a corn-soybean-based standard diet; negative control 1 (NC1), a standard diet with reduced available phosphorus (Av.P) (-0.125%), metabolizable energy (ME) (-40 kcal), and crude protein (CP) (-0.3%); NC1 with 500 phytase units per kilogramme (FTU/kg) (N1P5); negative control 2 (NC2), a standard diet with greater reduction of Av.P (-0.150%), ME (-55 kcal), and CP (-0.45%,); and NC2 with 1000 FTU/kg (N2P10). Piglets were housed in a random arrangement based on sex and body weight and data were analyzed as a randomized complete block design using analysis of variance. Results showed that the body weight and average daily gain of the NC2 treatment were lower (p < 0.05) compared to NC2. Gain to feed ratio was greater (p < 0.05) in the CNT and N1P5 treatments compared to the NC1, NC2, and N2P10 treatments. The CP digestibility was higher (p < 0.05) in N1P5 and N2P10 treatments compared to other treatments. Moreover, the digestibility of phosphorus and calcium was higher (p < 0.05) in N1P5 and N2P10 treatments than in CNT, NC1, and NC2 treatments. The digestibility of non-dispensable AA; histidine, isoleucine, leucine, phenylalanine, and valine were increased (p < 0.05) in N1P5 and N2P10 than in CNT, NC1, and NC2 treatments. Nevertheless, the digestibility of dispensable AA, glutamic acid, was higher (p < 0.05) in N1P5 and N2P10 treatments than in CNT, NC1, and NC2 treatments. Blood myo-inositol concentration was higher (p < 0.05) in N1P5 and N2P10 treatments compared to CNT, NC1, and NC2 treatments in phase 2. These results demonstrated enhanced outcomes under conditions of moderate deficiency, whereas more pronounced deficiencies necessitated increased phytase dosages to observe significant improvements. The efficacy of phytase was evident in its ability to elevate average daily gain, gain to feed ratio, phosphorus and calcium, CP, AA, and blood myo-inositol.

Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients.

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T50 30) was increased from 55.12°C to 62.44°C, and Tm value was increased from 48.36°C to 53.18°C. Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzymesubstrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

Effects of graded levels of dietary microbial 6-phytase on performance, intestinal histomorphology, caecal microbial population and short-chain fatty acid composition of Lohmann white-classics.

1. This study was conducted to determine the effects of graded levels of phytase on the performance, egg quality and gut health of white laying hens.2. Treatments consisted of a negative control (NC) diet containing 0.14% available phosphorus (avP), positive control (PC) diet containing 0.35% avP provided via dicalcium phosphate (DCP) and DCP replaced in the PC by with three graded levels of phytase derived from Komagataella phaffii at 500 (PC-500), 750 (PC-750) and 1000 (PC-1000) FTU/kg which provided 0.176%, 0.188% and 0.200% of avP, respectively.3. Egg production, feed intake, feed conversion ratio and jejunal morphometry were negatively affected in NC-fed birds (p < 0.05). Considering the whole period, birds fed a diet supplemented with graded levels of phytase shared the same egg production and feed intake levels with PC birds (p < 0.05). Feed conversion ratio was significantly lowered by 4.9%, 1.6% and 7.6% in hens fed on diets PC-500, PC-750 and PC-1000, respectively compared to those fed the PC (p < 0.05).4. Neither of the dietary treatments affected cracked eggs, dirty eggs, eggshell breaking strength and eggshell thickness. Dietary supplementation of phytase significantly increased villus surface area by 15%, 36% and 40% in PC-500, PC-750 and PC-1000 birds, respectively compared to PC (p < 0.05).5. A significant increase in lactobacillus count was observed in line with increasing the level of phytase (p < 0.05). Dietary treatments had no effect on the caecal coliform or aerobic populations. Furthermore, phytase supplementation significantly increased the concentrations of total caecal short-chain fatty acid (SCFA; p < 0.01).6. In conclusion, along with improving performance parameters, the inclusion of phytase in laying hen diets can ameliorate intestinal morphology and stimulate caecal microflora and increase SCFA concentrations.

Exploring the biotechnological potential of novel soil-derived Klebsiella sp. and Chryseobacterium sp. strains using phytate as sole carbon source.

Phosphorus (P) is essential for biological systems, playing a pivotal role in energy metabolism and forming crucial structural components of DNA and RNA. Yet its bioavailable forms are scarce. Phytate, a major form of stored phosphorus in cereals and soils, is poorly bioavailable due to its complex structure. Phytases, enzymes that hydrolyze phytate to release useable phosphorus, are vital in overcoming this limitation and have significant biotechnological applications. This study employed novel method to isolate and characterize bacterial strains capable of metabolizing phytate as the sole carbon and phosphorus source from the Andes mountains soils. Ten strains from the genera Klebsiella and Chryseobacterium were isolated, with Chryseobacterium sp. CP-77 and Klebsiella pneumoniae CP-84 showing specific activities of 3.5 ± 0.4 nkat/mg and 40.8 ± 5 nkat/mg, respectively. Genomic sequencing revealed significant genetic diversity, suggesting CP-77 may represent a novel Chryseobacterium species. A fosmid library screening identified several phytase genes, including a 3-phytase in CP-77 and a glucose 1-phosphatase and 3-phytase in CP-84. Phylogenetic analysis confirmed the novelty of these enzymes. These findings highlight the potential of phytase-producing bacteria in sustainable agriculture by enhancing phosphorus bioavailability, reducing reliance on synthetic fertilizers, and contributing to environmental management. This study expands our biotechnological toolkit for microbial phosphorus management and underscores the importance of exploring poorly characterized environments for novel microbial functions. The integration of direct cultivation with metagenomic screening offers robust approaches for discovering microbial biocatalysts, promoting sustainable agricultural practices, and advancing environmental conservation.

Effects of enzyme supplementation on growth performance, digestibility of phosphorus, femur parameters and fecal microbiota in growing pigs fed different types of diets.

A 42-days study was conducted to evaluate the effects of different dietary types (corn-or wheat-soybean meal-based diet) and phytase (Phy) or a multi-carbohydrase and phytase complex (MCPC) supplementation on growth performance, digestibility of phosphorus (P), intestinal transporter gene expression, plasma indexes, bone parameters, and fecal microbiota in growing pigs. Seventy-two barrows (average initial body weight of 24.70 ± 0.09 kg) with a 2 × 3 factorial arrangement of treatments and main effects of diet type (corn-or wheat-soybean meal-based-diets) and enzyme supplementation (without, with Phy or with MCPC). Each group was designed with 6 replicate pens. The MCPC increased (p < 0.05) average daily gain (ADG) and final body weight (BW). A significant interaction (p = 0.01) was observed between diet type and enzyme supplementation on apparent total tract digestibility (ATTD) of P. The ATTD of P was higher (p < 0.05) in wheat soybean meal-based diets compared to corn-soybean meal-based diets. Compared with the corn-soybean meal-based diet, the relative expression of SLC34A2 and VDR genes in the ileum and SLC34A3 in jejunum of growing pigs fed the wheat-soybean meal based diet was lower (p < 0.05). The MCPC significantly reduced (p < 0.05) the relative expression of TRPV5 and CALB1 genes in the ileum and increased the expression of CALB1 in the duodenum compared to control diet. The phytase increased (p < 0.05) the relative expression of SLC34A1 gene in the duodenum in comparison to control diet and MCPC-supplemented diet. The Ca and P contents in plasma from pigs fed corn-soybean meal-based diet were higher (p < 0.05) than those from pigs fed wheat-soybean meal-based diet, and the parathyroid hormone (PTH) and calcitonin (CT) concentrations were lower (p < 0.05) than those fed wheat-soybean meal-based diet. The content of Ca and P in the femur and the bone strength of pigs in the corn-soybean meal group were significantly higher (p < 0.05) than those in the wheat-soybean meal groups. The phytase increased (p < 0.05) the Ca and P content and bone strength of the femur. Additionally, diet type and both enzymes significantly improved fecal microbial diversity and composition. Taken together, diet type and exogenous enzymes supplementation could differently influence the growth performance, utilization of phosphorus, intestinal transporter gene expression, bone mineralization and microbial diversity and composition in growing pigs.

Safety evaluation of the food enzyme 3-phytase from the non-genetically modified Aspergillus niger strain PHY93-08.

The food enzyme 3-phytase (myo-inositol-hexakisphosphate 3-phosphohydrolase EC 3.1.3.8) is produced with the non-genetically modified Aspergillus niger strain PHY93-08 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in nine food manufacturing processes. Since residual amounts of food enzyme-total organic solids (TOS) are removed in two of the food manufacturing processes, dietary exposure was calculated only for the remaining seven processes. It was estimated to be up to 0.763 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2560 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 3355. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

A New Biosynthetic 6-Phytase Added at 500 Phytase Unit/kg Diet Improves Growth Performance, Bone Mineralization, and Nutrient Digestibility and Retention in Weaned Piglets and Growing-Finishing Pigs.

Two experiments were performed to evaluate the effect of a biosynthetic 6-phytase added at 500 phytase unit (FTU)/kg diet on growth performance, bone mineralization, and nutrient digestibility and retention in weaned piglets and growing-finishing pigs. Experiments were performed on 90 weaned male and female piglets with an average initial body weight (BW) at 7.7 ± 0.73 kg, 26 days of age) and 300 male and female growing pigs (initial BW: 21.0 ± 3.44 kg) for 43 and 98 days in experiments 1 and 2, respectively. In each experiment, the animals were assigned to one of three treatments according to a randomized complete block design. The treatments consisted of a positive-control (PC) diet formulated to meet nutrient requirements; a negative-control (NC) diet reduced similarly in calcium (Ca) and digestible P by 0.15 and 0.12% points in phases 1 and 2, respectively, in piglets and by 0.14, 0.11, and 0.10% points, respectively, in phases 1, 2, and 3 in growing-finishing pigs, compared with PC diet; and a NC diet supplemented with the new 6-phytase at 500 FTU/kg diet (PHY). The dietary P and Ca depletion reduced (p < 0.05) the final BW (-11.9%; -7.8%,), average daily gain (ADG, -17.8%; -10.1%), average daily feed intake (ADFI, -9.9%; -6.0%), gain-to-feed (G:F) ratio (-8.9%; -4.6%), and apparent total tract digestibility (ATTD) of P (-7.7% points; -6.7% points) in nursery piglets and growing pigs, respectively. It also decreased (p < 0.001) P and Ca retention by 6.1 and 9.4% points, respectively, in nursery pigs and ash, P, and Ca contents in metacarpal bones by 18.4, 18.4, and 16.8%, respectively, in growing pigs. Compared to animals fed the NC diet, phytase supplementation improved (p < 0.001) the final BW (+7.7%; +11.3%), ADG (+12.5%; +15.0%), G:F ratio (+8.4%; +5.8%), ATTD of Ca (+10.8% points; +7.2% points), and ATTD of P (+18.7% points; +16.6% points) in weaned piglets and growing pigs, respectively. In addition, phytase also increased (p < 0.001) P and Ca retention by 6.1 and 9.4% points, respectively, in nursery pigs and ash, P, and Ca contents in metacarpal bones by 17.7, 15.0, and 15.2%, respectively, in growing pigs. The final BW, ADG, G:F ratio, and bone traits in animals fed the NC diet supplemented with phytase were comparable to animals fed the PC diet. This finding indicates the ability of this novel biosynthetic phytase to restore performance and bone mineralization by improving the availability of P and Ca in piglets and growing pigs fed P- and Ca-deficient diets.

Effects of a Novel Consensus Bacterial 6-Phytase Variant on Growth Performance and Bone Ash of Broilers Fed Complex Diets Highly Deficient in Minerals, Digestible Amino Acids and Energy through 42 Days of Age.

An experiment was conducted to evaluate the effects of increasing the dose of a novel consensus bacterial 6-phytase variant expressed in Trichoderma reesei (PhyG) in broilers fed complex diets highly deficient in minerals, dig AA, and energy. Diets were a nutrient-adequate control (PC); a nutrient-reduced control (NC) formulated with a reduction in available P (avP) by 0.199%, Ca by 0.21%, crude protein by 0.72-1.03%, dig Lys by 0.064-0.084%, Na by 0.047%, and ME by 87.8 kcal/kg, respectively; and NC supplemented with PhyG at 500, 1000, and 2000 FTU/kg feed. BW was decreased and FCR increased in the NC vs. PC, while the PhyG treatments were similar to the PC. Carcass yield and bone ash were also maintained with PhyG supplementation. Phytase provided economic benefit on a feed cost per kg of weight basis for 1 to 35 d; the cost reductions equated to USD 0.006, 0.016, and 0.02/kg BWG at 500, 1000, and 2000 FTU/kg. In conclusion, this trial demonstrated that supplementation with a novel consensus phytase variant in diets highly deficient in minerals, dig AA, and energy maintained growth performance and provided economic benefit, with production benefits being maximized at inclusion levels of 2000 FTU/kg.

Nutritional and physiological responses to dietary phosphorus levels and phytase in pullets and laying hens.

The objective of this study was to determine the effects of dietary available phosphorus (P) levels and dietary phytase added into the very low-P diet on the performance, mineral balance, odor emission, and stress responses in growing pullets and laying hens during 13 to 32 wk of age. One hundred sixty-eight pullets (Hy-Line Brown) were randomly assigned into 1 of 4 dietary treatments with 7 replicates of 6 birds each. Experimental diets were formulated to contain 3 graded P levels at 0.25, 0.35, and 0.45% during 13 to 15 wk (phase 1), 0.25, 0.35, and 0.45% during 16 to 18 wk (phase 2), and 0.20, 0.30, and 0.40% during 19 to 32 wk (phase 3). In addition, dietary phytase (500 FTU/kg matrix values) was added into the very low-P diets (0.20% during 13-15 wk, 0.25% during 16-18 wk, and 0.20% during 19-32 wk) to meet the nutritional adequacy with standard P diets. In all phases, decreasing dietary P levels did not affect (P > 0.05) growth, laying performance, and egg qualities. Decreasing dietary P levels linearly increased the relative duodenal and oviduct weights (P < 0.05), and quadratically increased the relative ovary weight in pullets (P = 0.016). Dietary phytase lowered (P = 0.021) the relative duodenal weight compared with the very low-P diet. Tibia breaking strength and tibia Mg contents in pullets were linearly lowered (P < 0.05) as dietary P levels decreased. Dietary phytase tended to increase (P = 0.091) tibia breaking strength and significantly increased (P = 0.025) tibia Mg content compared with the very low-P diet. Dietary P levels and dietary phytase affected (P < 0.05) ileal crypt depth and ileal villus height: crypt depth ratio in pullets. Decreasing dietary P levels linearly decreased (P < 0.01) crude fat digestibility and P excretion in both pullets and laying hens. Dietary phytase reversed (P < 0.05) the very low-P diet-mediated decrease of crude fat digestibility in pullets and laying hens. Dietary P levels and dietary phytase affected (P < 0.05) odor emission including ammonia in pullets and total volatile fatty acids in laying hens. Finally, lowering dietary P levels increased (P < 0.01) yolk corticosterone concentrations and the increased corticosterone concentration by the very low-P diet was reversed by dietary phytase. Collectively, our study shows that decreasing dietary P levels induced nutritional and physiological responses in pullets and laying hens and these P-mediated negative effects were mitigated by dietary phytase.

Transcriptional and physiological analyses uncover the mineralization and uptake mechanisms of phytic acid in symbiotically grown Vicia faba plants.

Legume-rhizobia symbiosis requires high phosphorus (P) in the form of ATP to convert atmospheric nitrogen (N) into ammonia. The fixed ammonia is converted to NH4+ by H+-ATPase via protonation. To the best of our knowledge, most of these research works resort to using only inorganic P (Pi) to the neglect of the organic P (Po) counterpart. As it stands, the potential regulating roles of plasma membrane (PM) H+-ATPases during legume-rhizobia symbiosis in response to phytic acid supply and how it alters and modulates the regulation of PM H+-ATPases remain obscure. To contribute to the above hypothesis, we investigate the mechanisms that coordinately facilitate the growth, uptake, and transcript expression of PM H+-ATPase gene isoforms in response to different P sources when hydroponically grown Vicia faba plants were exposed to three P treatments, viz., low- and high-Pi (2.0 and 200 µM KH2PO4; LPi and HPi), and phytic acid (200 µM; Po) and inoculated with Rhizobium leguminosarum bv. viciae 384 for 30 days. The results consistently reveal that the supply of Po improved not only the growth and biomass, but also enhanced photosynthetic parameters, P uptake and phosphatase activities in symbiotically grown Vicia faba relative to Pi. The supply of Po induced higher transcriptional expression of all PM H+-ATPase gene isoforms, with possible interactions between phosphatases and H+-ATPase genes in Vicia faba plants when exclusively reliant on N derived from nodule symbiosis. Overall, preliminary results suggest that Po could be used as an alternative nutrition in symbiotic crops to improve plant growth.

Dietary inclusion of phytase and stimbiotic decreases mortality and lameness in a wire ramp challenge model in broilers.

RESEARCH HIGHLIGHTS: Wire ramp model reproducibly induced lameness/BCO in broilers.Treatments did not affect growth, but phytase with stimbiotic significantly reduced BCO.Phytase increased circulating inositol, and wire flooring decreased bone inositol.

Identification of phytase producing bacteria from acidifying Tithonia diversifolia: Potential for ruminant feed development.

Phytate content in feed ingredients can negatively impact digestibility and palatability. To address this issue, it is necessary to study microbes capable of breaking down phytate content. This study aimed to isolate and characterize phytase-producing bacteria from decaying materials rich in phytic acid. The research was conducted in several stages. The first stage involved isolating phytase-producing bacteria from the acidification of Tithonia diversifolia using growth media containing Na-phytate. Bacterial isolates that produced clear zones were then tested for their activity and ability to produce several enzymes, specifically phytase, cellulase, and protease. The next step was to test the morphological characteristics of the bacterial isolate. The final stage of bacterial identification consisted of DNA isolation, followed by PCR amplification of the 16S rRNA gene, DNA sequence homology analysis, and construction of a phylogenetic tree. Based on research, three isolates were found to produce clear phytase zones: isolates R5 (20.3 mm), R7 (16.1 mm) and R8 (31.7 mm). All isolates were able to produce the enzymes phytase (5.45-6.54 U/ml), cellulase (2.60-2.92 U/ml), and protease (22.2-23.4 U/ml). Metagenomic testing identified isolate R7 and R8 as Alcaligenes faecalis and isolate R5 as Achromobacter xylosoxidans. The isolation and characterization of phytase-producing bacteria from Tithonia diversifolia acidification resulted in the identification of two promising candidates that can be applied as sources of phytase producers. Phytase-producing bacteria can be utilized to improve digestibility and palatability in animal feed.

Production of cleavage-resistant phytase transgenic pigs by handmade cloning.

With the rapid development of intensive animal husbandry in the livestock industry, large quantities of manure waste containing phytate phosphorus are being generated. Phytase can effectively solve the problem of high phosphorus pollution in the feces of monogastric animals. Enviropig, which produces phytase in the salivary glands and secretes the enzyme in the saliva, were first generated in 1999. However, phytase is easily inactivated during digestion. To address this problem, cleavage-resistant phytase transgenic pigs were generated using handmade cloning in this study. Transgene construction was improved and three cell lines carrying Cafp were obtained. In total, 810 blastocysts were generated and 712 good-quality were transferred into six recipients. Fourteen piglets were born, of which six survived after weaning. Polymerase chain reaction and sequencing results showed that seven (three live and four dead) of the fourteen piglets carried Cafp. Phytase activity in the saliva of the six live cloned pigs was tested at four months of age, and only one pig had 0.155 FTU/mL enzyme activity. The other five pigs may not have been activated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, representing a 25.4% decrease in fecal emission compared to the average of controls. Immunohistochemical results on the three Cafp-positive pigs that died after six months of age showed that the transgene was only expressed in parotid glands, confirming tissue-specific gene expression. In conclusion, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to managing phosphorus nutrition and environmental pollution in animal husbandry.

Improvement of the recombinant phytase expression by intermittent feeding of glucose during the induction phase of methylotrophic yeast Pichia pastoris.

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.

Novel 4th-generation phytase improves broiler growth performance and reduces woody breast severity through modulation of muscle glucose uptake and metabolism.

The objective of the present study was to determine the effect of a novel (4th generation) phytase supplementation as well as its mode of action on growth, meat quality, and incidence of muscle myopathies. One-day old male broilers (n = 720) were weighed and randomly allocated to 30 floor pens (24 birds/pen) with 10 replicate pens per treatment. Three diets were fed from hatch to 56- days-old: a 3-phase corn-soy based diet as a positive control (PC); a negative control (NC) formulated to be isocaloric and isonitrogenous to the PC and with a reduction in Ca and available P, respectively; and the NC supplemented with 2,000 phytase units per kg of diet (NC + P). At the conclusion of the experiment, birds fed with NC + P diet were significantly heavier and had 2.1- and 4.2-points better feed conversion ratio (FCR) compared to birds offered NC and PC diets, respectively. Processing data showed that phytase supplementation increased live weight, hot carcass without giblets, wings, tender, and skin-on drum and thigh compared to both NC and PC diets. Macroscopic scoring showed that birds fed the NC + P diet had lower woody breast (WB) severity compared to those fed the PC and NC diets, however there was no effect on white striping (WS) incidence and meat quality parameters (pH, drip loss, meat color). To delineate its mode of action, iSTAT showed that blood glucose concentrations were significantly lower in birds fed NC + P diet compared to those offered PC and NC diets, suggesting a better glucose uptake. In support, molecular analyses demonstrated that the breast muscle expression (mRNA and protein) of glucose transporter 1 (GLUT1) and glucokinase (GK) was significantly upregulated in birds fed NC + P diet compared to those fed the NC and PC diets. The expression of mitochondrial ATP synthase F0 subunit 8 (MT-ATP8) was significantly upregulated in NC + P compared to other groups, indicating intracellular ATP abundance for anabolic pathways. This was confirmed by the reduced level of phosphorylated-AMP-activated protein kinase (AMPKα1/2) at Thr172 site, upregulation of glycogen synthase (GYS1) gene and activation of mechanistic target of rapamycin and ribosomal protein S6 kinase (mTOR-P70S6K) pathway. In conclusion, this is the first report showing that in-feed supplementation of the novel phytase improves growth performance and reduces WB severity in broilers potentially through enhancement of glucose uptake, glycolysis, and intracellular ATP production, which used for muscle glycogenesis and protein synthesis.

Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella Phaffii.

BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.