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Biofilm formation is a major challenge in the treatment of tuberculosis, leading to poor treatment outcomes and latent infections. The complex and dense extracellular polymeric substances (EPS) of the biofilm provides safe harbour for bacterium enabling persistence against anti-TB antibiotics. In this study, we demonstrated that rifampicin-encapsulated silk fibroin nanoparticles immobilized with antibiofilm enzymes can disrupt the Mycobacterium smegmatis biofilm and facilitate the anti-bacterial action of Rifampicin (RIF). The EPS of M.smegmatis biofilm predominantly comprised of lipids (48.8 ± 1.32 %) and carbohydrates (34.8 ± 4.70 %), similar to tuberculosis biofilms. Pre-formed biofilm eradication screening revealed that hydrolytic enzymes such as β-Glucosidase, Glucose oxidase, ɑ-Amylase, Acylase, and Phytase can exhibit biofilm eradication of M.smegmatis biofilms. The enzyme-mediated biofilm disruption was associated with a decrease in hydrophobicity of biofilm surfaces. Treatment with β-glucosidase and Phytase demonstrated a putative biofilm eradication by reducing the total carbohydrates and lipid composition without causing any significant bactericidal activity. Further, Phytase (250 μg/ml) and β-Glucosidase (112.5 ± 17.6 μg/ml) conjugated rifampicin-loaded silk fibroin nanoparticles (R-SFNs) exhibited an enhanced anti-bacterial activity against pre-formed M.smegmatis biofilms, compared to free rifampicin (32.5±7 μg/ml). Notably, treatment with β-glucosidase, Phytase and ɑ-amylase immobilized SFNs decreased the biofilm thickness by ∼98.84 % at 6h, compared to control. Thus, the study highlights that coupling anti-mycobacterial drugs with biofilm-eradicating enzymes such as amylase, phytase or β-glucosidase can be a potential strategy to improve the TB therapeutic outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships with individual or other organization that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Dietary phytate P (PP) concentration impacts Ca and P digestibility in broilers. Research was conducted to determine the impact of increasing concentration of dietary PP, with and without phytase, on broiler standardized ileal digestibility (SID) of Ca and P. Digestible (Dig) Ca and P were calculated by multiplying SID and the analyzed dietary Ca and P concentrations. The experiment was a factorial arrangement of 2 phytase (0 and 1,000 U/kg) and 4 PP (0.16, 0.23, 0.29, and 0.34%) concentrations. Treatments were fed for 36 h from 20 to 22 d of age (4 b/pen, n ≥ 7 replicate pens/treatment). Different ratios of corn and corn germ were used to achieve the desired PP concentrations. A limestone with 800 µm geometric mean diameter was used as the sole Ca source to achieve 0.7% Ca in the final diets (96% Ca from limestone). An additional diet was fed that was N, Ca- and P-free, for the determination of endogenous losses of each nutrient. Distal ileal digesta were pooled from all birds in a pen. There were no interactions between PP and phytase on SID Ca or Dig Ca from limestone. Irrespective of phytase inclusion, increasing PP from 0.16 to 0.34% decreased SID Ca from 53.8 to 38.1% (P < 0.05). The SID Ca averaged 41.5 and 51.4% in diets containing 0 and 1000 U phytase/kg, respectively, across all PP concentrations (P < 0.05). Interactions were seen between PP and phytase on SID and Dig P (P < 0.05) with SID P of 31.1, 24.0, 20.1, and 16.3% for broilers fed 0.16, 0.23, 0.29, and 0.34% PP diets without phytase, respectively. When phytase was included at 1000 U/kg, SID P was 89.9, 87.5, 73.9 and 60.4% for diets containing 0.16, 0.23, 0.29 and 0.34% PP, respectively (P < 0.05). Overall, phytase improved SID Ca and P independent of PP concentration. However, with increasing PP concentration, both SID Ca and P were negatively affected., Competing Interests: DISCLOSURES Authors have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to determine how different sources of Zn, Mn, and Cu in the feed without and with phytase affect prececal myo-inositol hexakisphosphate (InsP 6 ) breakdown to myo-inositol (MI), prececal P digestibility, bone mineralization, and expression of mineral transporters in the jejunum of broiler chickens. A total of 896 male broiler chicks (Cobb 500) were distributed to 7 diets with 8 replicate pens (16 birds per floor pen). Experimental diets were fed from day 0 to 28. Diets were without or with phytase supplementation (0 or 750 FTU/kg) and were supplemented with three different trace mineral sources (TMS: sulfates, oxides, or chelates) containing 100 mg/kg Zn, 100 mg/kg Mn, and 125 mg/kg Cu. Prececal InsP 6 disappearance and P digestibility were affected by interaction (phytase × TMS: P ≤ 0.010). In diets without phytase supplementation, prececal InsP 6 disappearance and P digestibility were greater (P ≤ 0.001) in birds fed chelated minerals than in birds fed sulfates or oxides. However, no differences were observed between TMS in diets with phytase supplementation. Ileal MI concentration was increased by exogenous phytase but differed depending on TMS (phytase × TMS: P ≤ 0.050). Tibia ash concentration as well as Zn and Mn concentration in tibia ash were increased by phytase supplementation (P < 0.010), but the Cu concentration in tibia ash was not (P > 0.050). Gene expression of the assayed mineral transporters in the jejunum was not affected by diet (P > 0.050), except for Zn transporter 5 (phytase × TMS: P = 0.024). In conclusion, the tested TMS had minor effects on endogenous phytate degradation in the digestive tract of broiler chickens. However, in phytase-supplemented diets, the choice of TMS was not relevant to phytate degradation under the conditions of this study., (© 2024. The Author(s).)

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.

1. This study assessed the effect of limestone particle size and microbial phytase incorporation on the fate of phosphorus (P) and calcium (Ca) along the gastrointestinal tract in 72 laying hens.2. Four experimental diets were formulated according to a 2 × 2 factorial arrangement to evaluate the effect of two coarse limestone (CL) inclusion. This included a mix (MIX) of 75% CL (2 - 4 mm) and 25% fine particles (FL, <0.5 mm) or 100% FL, in two different basal diets formulated without (MIX0 and FL0) or with 300 FTU of microbial phytase/kg (MIX300 and FL300).3. Contents of the crop, gizzard, duodenum, jejunum and ileum were collected to determine the mean retention time (MRT) of dry matter (DM), the recovery rate of Ca and P in each segment of the gastrointestinal tract and the apparent fractional digestibility coefficient (AD) of Ca and P in each intestinal segment.4. In hens fed FL, microbial phytase decreased the MRT of DM along the intestine ( p  < 0.05). In the crop and the gizzard, Ca recovery increased with MIX incorporation to a greater extent in hens fed without microbial phytase ( p  < 0.05). The mixed particle size incorporation decreased absorption kinetics of Ca in hens fed microbial phytase. The AD of P and the absorption kinetics of P were significantly decreased in hens receiving FL300, probably due to complex formation between Ca and phytic acid.5. This study showed that coarse limestone particles incorporation improved mineral utilisation along the digestive tract.

Encapsulating enzymes in metal-organic frameworks is a common practice to improve enzyme stability against harsh conditions. However, the synthesis of enzyme@MOFs has been primarily limited to small-scale laboratory settings, hampering their industrial applications. Spray drying is a scalable and cost-effective technology, which has been frequently used in industry for large-scale productions. Despite these advantages, its potential for encapsulating enzymes in MOFs remains largely unexplored, due to challenges such as nozzle clogging from MOF particle formation, utilization of toxic organic solvents, controlled release of encapsulated enzymes, and high temperatures that could compromise enzyme activity. Herein, we present a novel approach for preparing phytase@MIL-88 A using solvent-free spray drying. This involves atomizing two MOF precursor solutions separately using a three-fluid nozzle, with enzyme release controlled by manipulating defects within the MOFs. The physicochemical properties of the spray dried particles are characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Leveraging the efficiency and scalability of spray drying in industrial production, this scalable encapsulation technique holds considerable promise for broad industrial applications., Competing Interests: Declaration of competing interest All authors declare no competing interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fishmeal and fish oil have been the main sources of protein and fatty acid for aquaculture fish. However, their increasing price and low sustainability have led the aquafeed industry to seek sustainable alternative feedstuffs to meet the nutritional requirements of fish and improve their health and performance. Plant proteins have been successfully used to replace fishery derivatives in aquafeeds, but the presence of anti-nutritional substances is a potential drawback of this approach. Thus, it has been reported that phytate breakdown can be caused by feed supplementation with exogenous phytase. The inclusion of microalgae has been proposed to improve gut functionality in fish fed diets with a high vegetable protein content. The aim of this study was to evaluate the effect on the growth and gut microbiota of European seabass (Dicentrarchus labrax) juveniles of a diet containing a blend of microalgae (Arthrospira platensis and Nannochloropsis gaditana) and different concentrations of phytase. An 83-day feeding trial was conducted, comprising four experimental diets with 2.5% microalgae and 500, 1,000, 2,000, or 10,000 phytase units (FTU)/kg feed and a microalgae- and phytase-free control diet. At the end of the trial, a significantly increased body weight was observed in fish fed the diet with the highest phytase concentration (10,000 FTU/kg) versus controls, although the gut bacterial composition did not differ from controls in alpha or beta diversity with either majority (Weighted UniFrac) or minority bacterial strains (Unweighted UniFrac). In comparison to the control group, the groups fed diets with 1,000 or 2,000 FTU/kg diets had a lower alpha diversity (Shannon's diversity index), while those fed diets with 500 FTU/kg or 1,000 FTU/kg showed distinct clusters in beta diversity (involving minority ASVs). According to these findings, the diet containing the 2.5% microalgae blend with 10,000 FTU/kg may be useful to increase the aquafeed quality and sustain the growth performance of juvenile European seabass., (© 2024. The Author(s).)

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(OD 600 ) 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 OD 600 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., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Biological enzymes are multifunctional macromolecules that can perform hundreds of reactions simultaneously. An enzyme must possess specific characteristics to meet industrial needs, such as stability over a wide pH and temperature range and high specific activity. A phytase and xylanase mixture is generally added to poultry feed to improve the bird's health and productivity. Despite this, animal farmers have noticed no difference in productivity, and a leading cause is the high temperature at which feed is pulverized, which inactivates enzymes. A thermo-stable enzyme system can overcome these hitches. Commonly, coatings and immobilization reduce losses caused by physical-chemical factors in feed processing and digestion. To this end, we engineered the multifunctional xylanase-phytase domains on a single polypeptide fused by a helical linker. First, the ideal linker sequence was chosen by computing each selected linker's root mean square deviation (RMSD). The selected helical linker provides sufficient structural flexibility for substrate binding and product release evaluated by molecular docking and molecular dynamic simulation studies. Furthermore, a domain-domain interaction has stabilized the bridging partners, attaining the thermal optima for xylanase and phytase at 90 °C. Even at the above-optimal temperature (100 °C), the recombinant PLX was relatively stable and retained 64.2% and 59.2% activity for xylanase and phytase, respectively, when surveyed for ten hours. So far, to this date, this is the highest degree of thermostability achieved by any recombinant phytase or xylanase.Communicated by Ramaswamy H. Sarma.

The digestive characteristics of plant proteins are crucial for their nutritional value and utilization efficiency. In this study, an in vitro semi-dynamic digestion model was employed to investigate the gastric digestion process of soybean protein after treatment with phytase. The results found that phytase treatment reduced the phytate content in soybean proteins (22.83 ± 0.09 to 8.72 ± 0.07 mg/g), shifted its isoelectric point towards the alkaline range by 1 pH unit, and significantly improved its solubility at pH 4.0. Particularly for protein sample treated with phytase after acid precipitation, the formation of aggregates during digestion was weakened, resulting in a significantly higher digestion rate compared to untreated SPI, with digestion being at least 15 min faster than SPI. This study provides a strategy for preparing soybean protein with faster digestion and weaker clot-forming ability during digestion, which offers insights for the application of soybean protein in clinical nutrition products and specialized medical foods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

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 ( T 50 30 ) was increased from 55.12°C to 62.44°C, and T m 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 enzyme-substrate 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.

1. A study was conducted to assess the possibility of totally replacing supplemental phosphorus sources in White Leghorn (WL) layer diets (aged 28 to 45 weeks of age) with microbial phytase supplementation. One thousand commercial layers (HyLine White) of 28 weeks of age were housed in California cages fitted in open-sided poultry shed at the rate of 20 layers in each replicate. Ten replicates were randomly allotted to each treatment, and the respective diet was fed from 28 to 45 weeks of age.2. A control diet (CD) containing the recommended levels of non-phytate phosphorus (3.6 g/kg NPP) and four other test diets (2-5) having sub-optimal levels of NPP (2.4, 2.0, 1.6 and 1.2 g/kg), but with supplemental microbial phytase (600 FTU/kg) were prepared and fed for the trial duration.3. The layers fed with lower levels of NPP with phytase had the same laying performance as the group fed the CD. Egg production, feed efficiency, egg mass, shell defects, egg density, shell weight, shell thickness, ash content and breaking strength of the tibia and sternum were not affected by feeding the lowest concentration of NPP (1.2 g/kg) plus microbial phytase.4. Phytase supplementation in diets with sub-optimal levels of NPP (2.4, 2 and 1.6 g/kg) significantly improved the Haugh unit score compared to those fed the CD.5. It was concluded that supplemental phosphorus can be completely replaced with microbial phytase (600 FTU/kg) in a diet without affecting egg production, shell quality or bone mineral variables in WL layers (28 to 45 weeks).

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

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., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The study aims to statistically optimize the phytase production by Penicillium oxalicum PBG30 in solid-state fermentation using wheat bran as substrate. Variables viz. pH, incubation days, MgSO 4 , and Tween-80 were the significant parameters identified through the Plackett-Burman design (PBD) that majorly influenced the phytase production. Further, central composite design (CCD) method of response surface methodology (RSM) defined the optimum values for these factors i.e., pH 7.0, 5 days of incubation, 0.75% of MgSO 4 , and 3.5% of Tween-80 that leads to maximum phytase production of 475.42 U/g DMR. Phytase production was also sustainable in flasks and trays of different sizes with phytase levels ranging from 394.95 to 475.42 U/g DMR. Enhancement in phytase production is 5.6-fold as compared to unoptimized conditions. The in-vitro dephytinization of feed showed an amelioration in the nutritive value by releasing inorganic phosphate and other nutrients in a time-dependent manner. The highest amount of inorganic phosphate (33.986 mg/g feed), reducing sugar (134.4 mg/g feed), and soluble protein (115.52 mg/g feed) was achieved at 37 °C with 200 U of phytase in 0.5 g feed for 48 h. This study reports the economical and large-scale production of phytase with applicability in enhancing feed nutrition.

This investigation was performed to obtain a promising phytase enzyme producing yeast. In this regard, the PSM was used to isolate the phytase-producing Hanseniaspora guilliermondii S1 (MG663578) from sugarcane juice. The SSF optimum conditions for phytase generation were optimized using (OVAT) one-variable-at-a-time strategy using both Box-Behnken design and shake flask method (g/100 ml: 0.05 yeast extract, 0.15 Peptone, 0.05 malt extract 0.50 dextrose, pH 5.8 and 28ᵒC). The protein model developed was shown to be adequate for phytase production (91% accuracy), with the greatest phytase productivity in shake flask with substrate jack fruit seed powder being 395 ± 0.43 U/ml compared to 365U/ml for the BBD projected value. Crude Phytase was partially purified with a protein recovery of 43%, revealing a molecular weight of 120 kDa. It had an enzyme kinetic value of Km 3.3 mM and a Vmax of 19.1 mol/min. The 3D structure of PhyS1 amino acid sequences (PhyS1. B99990002) was simulated using Modeler 9.23, and the validated result revealed that 86.7% were in the favored region by Ramachandran plot. The SAVES server verified the 3D PDB file as satisfactory, and the model (in.pdb format) was uploaded in the PMDB database with the accession number ID: PM0082974. At the lab level, Hanseniaspora guilliermondii S1 (MG663578) producing phytase exhibited successful plant growth promotion activity in Ragi - CO 19 (Eleusine coracana L.) and Rice -Navarai - IR 64 (Oryza sativa L.). As a result, a phytase-based formulation for sustainable agriculture must be developed and tested on a large scale in diverse geographical areas of agricultural lands to determine its effect and potential on plant development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The need for industrially and biotechnologically significant enzymes, such as phytase, is expanding daily as a result of the increased use of these enzymes in a variety of operations, including the manufacture of food, animal feed, and poultry feed. This study sought to characterize purified phytase from A. awamori AFE1 isolated from longhorn beetle for its prospect in industrial applications. Ammonium sulfate precipitation, ion-exchange chromatography, and gel-filtration chromatography were used to purify the crude enzyme obtained from submerged fermentation using phytase-producing media, and its physicochemical characteristics were examined. The homogenous 46.8-kDa phytase showed an 8.1-fold purification and 40.7% recovery. At 70 C and pH 7, the optimum phytase activity was noted. At acidic pH 4-6 and alkaline pH 8-10, it likewise demonstrated relative activity of 88-95% and 67-88%, respectively. It showed 67-70% residual activity between 30 and 70 C after 40 min, and 68-94% residual activity between pH 2 and 12 after 2 h. The presence of Hg + , Mg 2+ , and Al 3+ significantly decreased the enzymatic activity, whereas Ca 2+ and Cu 2+ enhanced it. Ascorbic acid increased the activity of the purified enzyme, whereas ethylenediaminetetraacetic acid (EDTA) and mercaptoethanol inhibited it. The calculated values for K m and V max were 55.4 mM and1.99 μmol/min/mL respectively. A. awamori phytase, which was isolated from a new source, showed unique and remarkable qualities that may find use in industrial operations such as feed pelleting and food processing.

The capacity of combinations of feed enzymes, natural betaine and a probiotic, combined with alternative plant-based ingredients, to totally replace soybean meal (SBM) in a broiler diet was evaluated. Day-old Ross 308 males (2,574) were assigned to 9 treatments (13 pens/treatment, 22 birds/pen) in a completely randomized design. All diets were pelleted and fed ad libitum in 4 phases: starter, grower, finisher 1, finisher 2 (0-10, 10-21, 21-35, and 35-42 d of age, respectively). Treatments included: 1) control diet containing SBM (SBM control), supplemented with phytase (PhyG), at 2,000, 1,500, 1000 and 1,000 FTU/kg in each phase and xylanase (X) at 750 U/kg, [crude protein (CP): 23.5%, 22.0%, 20.2% and 19.3% in each phase]; 2) to 5), alternative (ALT), SBM-free diets, containing the same CP level as the control ("CP high"), supplemented with PhyG as in the control, protease (P, 800 U/kg) and in 2) xylanase (750 U/kg) (ALT+PhyG+P+X), 3) xylanase-β-glucanase (XB, 1,200 U/kg and 152 U/kg) (Alt+PhyG+P+XB), 4) XB plus betaine (800 g/ton) (ALT+PhyG+P+XB+Bet), and 5) XB plus a probiotic [150,000 colony forming units (CFU)/g] (ALT+PhyG+P+XB+Prob); 6) to 9) as treatments 2) to 5) but with CP reduced by -2.0 to -1.5% points vs. control ('CP low'). Final (d 42) BW and overall (d 0-42) feed conversion ratio (FCR) of birds fed the SBM control exceeded breeder objectives (+3.8% and -1.9%, respectively). Overall FCR was reduced and d 42 BW increased in birds fed "low" vs. "high" CP (P < 0.01). Overall FCR and feed intake were not different in ALT+PhyG+XB+P+Bet and ALT+PhyG+XB+P+Prob vs. the control, whereas final BW was reduced (P < 0.05) in all ALT treatments but close to breeder objectives (98.3%) in ALT+PhyG+XB+P+Prob. Feed costs of this treatment were similar to the control. Total replacement of SBM with alternative plant-based ingredients in a CP-low diet supplemented with hydrolytic enzymes and probiotics can achieve growth performance outcomes close to commercial breeder objectives., Competing Interests: DISCLOSURES Abiodun Bello, Leon Marchal and Yueming Dersjant-Li are employees of Danisco Animal Nutrition & Health (IFF), a global supplier of enzymes., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phytases [myo-inositol(1,2,3,4,5,6) hexakisphosphate phosphohydrolases] are phytate-specific phosphatases not present in monogastric animals. Nevertheless, they are an essential supplement to feeding such animals and for human special diets. It is crucial, hence, the biotechnological use of phytases with intrinsic stability and activity at the acid pHs from gastric environments. Here we use Metadynamics (METADY) simulations to probe the conformational space of the Aspergillus nidulans phytase and the differential effects of pH and glycosylation in this same space. The results suggest that strategic combinations of pH and glycosylation affect the stability of native-like conformations and alternate these structures from a metastable to a stable profile. Furthermore, the protein segments previously reported as more thermosensitive in phytases from this family present a pivotal role in the conformational changes at different conditions, especially H2, H5-7, L8, L10, L12, and L17. Also, the glycosylations and the pH-dependent charge balance modulate the mobility and interactions at these same regions, with consequences for the surface solvation and active site exposition. Finally, although the glycosylations have stabilized the native structure and improved the substrate docking at all the studied pHs, the data suggest a higher phytate receptivity at catalytic poses for the unglycosylated structure at pH 6.5 and the glycosylated one at pH 4.5. This behavior agrees with the exact change in optimum pH reported for this enzyme, expressed on low or high glycosylating systems. We hope the results and insights presented here will be helpful in future approaches for rational engineering of technologically promising phytases and intelligent planning of their heterologous expression systems and conditions for use.Communicated by Ramaswamy H. Sarma.

1. This study determined the effect of dietary Zn concentration and source in phytase-supplemented diets on bone mineralisation, gastrointestinal phytate breakdown, mRNA -level gene expression (in jejunum, liver and Pectoralis major muscle) and growth performance in broiler chickens.2. Male Cobb 500 broilers were housed in floor pens (d 0-d 21) to test seven treatments with six replicate pens (12 birds per pen). Diets were arranged in a 2 × 3 + 1-factorial arrangement. The experimental factors were Zn source (Zn-oxide (ZnO) or Zn-glycinate (ZnGly) and Zn supplementation level (10, 30 or 50 mg/kg of diet). A maize-soybean meal-based diet without supplementation and formulated to contain 28 mg Zn/kg (analysed to be 35 mg Zn/kg), served as a control.3. Zinc source and level did not influence ( p  > 0.05) bone ash concentration and quantity or mineral concentrations in bone ash. Tibia thickness was greater in the treatment ZnO10 than in the treatments ZnO30 and ZnGly50 (Zn level × Zn source: p  = 0.036), but width and breaking strength were not affected.4. Pre-caecal P digestibility and concentrations of phytate breakdown products in the ileum, except for InsP 5 , were not affected by Zn source or level. Only the expression of EIF4EBP1 (eukaryotic translation initiation factor 4E-binding protein 1) and FBXO32 (F-box only protein 32) in Pectoralis major muscle was affected by source, where expression was increased in ZnO compared to ZnGly diets ( p  < 0.05).5. In conclusion, Zn level and source did not affect gastrointestinal phytate degradation and bone mineralisation in phytase-supplemented diets. The intrinsic Zn concentration appeared to be sufficient for maximum bone Zn deposition under the conditions of the present study but requires validation in longer-term trials.

Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from Turicimonas muris , TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the Pichia system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70°C. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60°C. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80°C heat treatment without significant loss of or with increased activity.

The objective of the current study was to assess the impact of dietary phytase supplementation on Labeo rohita fingerlings and to examine the effects on growth, nutrient digestibility and chemical characteristics of diets containing rice protein concentrate (RPC) as a major protein source. Six experimental diets were made, i.e., a positive control (fishmeal-based diet with no phytase), FM0; a negative control (RPC-based diet with no phytase), RPC0; and four supplemental phytase levels (250, 500, 1000, and 2000 FTU/kg). Fingerlings with an average weight of 9.42 ± 0.02 grams (mean ± SD) were randomly distributed into six experimental groups of three replicates, each containing 25 fish per tank (75 liters of water), provided with experimental diets at a rate equivalent to 5% of their body weight for 90 days, and uneaten feed was collected after 2 hours to determine feed consumption. The feces were collected before feeding to estimate digestibility. Phytase in combination with the RPC-based diet significantly (p < 0.05) enhanced phytate phosphorus in vitro hydrolysis; growth performance; nutrient (crude protein, crude fat, moisture and gross energy) and mineral (P, Ca, Mg, Na, K, Zn, Mn and Cu) digestibility; digestive enzyme (protease, lipase and amylase) activity; and mineral deposition up to 1000 FTU/kg phytase. However, the hepatosomatic and viscerosomatic indices and carcass composition were not influenced (p > 0.05) by phytase supplementation. Increasing phytase supplementation in the RPC-based diets led to a significant (p < 0.05) decrease in the serum biochemical parameters (alkaline phosphatase activity, aspartate aminotransferase, alanine aminotransferase), which resulted in improved liver health. In conclusion, phytase-supplemented RPC-based diets improved the growth, mineral/nutrient digestibility, digestive enzymes, serum biochemistry, and mineral deposition of L. rohita fingerlings up to 1000 FTU/kg. Broken line regression analysis revealed that the optimum phytase concentration in the RPC-based diet for L. rohita was 874.19 FTU/kg., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Khizar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

To maximize the efficiency of dietary P utilization in swine production, understanding the mechanisms of P utilization in lactating sows is relevant due to their high P requirement and the resulting high inorganic P intake. Gaining a better knowledge of the Ca and P quantities that can be mobilized from bones during lactation, and subsequently replenished during the following gestation, would enable the development of more accurate P requirements incorporating this process of bone dynamics. The objective was to measure the amount of body mineral reserves mobilized during lactation, depending on dietary digestible P and phytase addition and to measure the amount recovered during the following gestation. Body composition of 24 primiparous sows was measured by dual-energy x-ray absorptiometry 2, 14, 26, 70 and 110 days after farrowing. Four lactation diets were formulated to cover nutritional requirements, with the exception of Ca and digestible P: 100% (Lact100; 9.9 g Ca and 3.0 g digestible P/kg), 75% (Lact75), 50% without added phytase (Lact50) and 50% with added phytase (Lact50 + FTU). The gestation diet was formulated to cover the nutritional requirements of Ca and digestible P (8.2 g Ca and 2.6 g digestible P/kg). During the 26 days of lactation, each sow mobilized body mineral reserves. The mean amount of mobilized bone mineral content (BMC) was 664 g, representing 240 g Ca and 113 g P. At weaning, the BMC (g/kg of BW) of Lact50 sows tended to be lower than Lact100 sows (-12.8%, linear Ca and P effect × quadratic time effect) while the BMC of Lact50 + FTU sows remained similar to that of Lact100 sows. During the following gestation, BMC returned to similar values among treatments. Therefore, the sows fed Lact50 could recover from the higher bone mineral mobilization that occurred during lactation. The P excretion was reduced by 40 and 43% in sows fed Lact50 and Lact50 + FTU, respectively, relative to sows fed Lact100. In conclusion, the quantified changes in body composition during the lactation and following gestation of primiparous sows show that bone mineral reserves were mobilized and recovered and that its degree was dependent on the dietary P content and from phytase supplementation during lactation. In the future, considering this potential of the sows' bone mineralization dynamics within the factorial assessment of P requirement and considering the digestible P equivalency of microbial phytase could greatly limit the dietary use of inorganic phosphates and, thus, reduce P excretion., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Phytate, the principal P storage in plant seeds, is also an important organic P in soils, but it is unavailable for plant uptake. However, the As-hyperaccumulator Pteris vittata can effectively utilize soluble Na-phytate, while its ability to utilize insoluble Ca/Fe-phytate is unclear. Here, we investigated phytate uptake and the underlying mechanisms based on the phytase activity, nutrient uptake, and expression of genes involved in As metabolisms. P. vittata plants were cultivated hydroponically in 0.2-strength Hoagland nutrient solution containing 50 μM As and 0.2 mM Na/Ca/Fe-phytate, with 0.2 mM soluble-P as the control. As the sole P source, all three phytates supported P. vittata growth, with its biomass being 3.2-4.1 g plant -1 and Ca/Fe-phytate being 19-29% more effective than Na-phytate. Phytate supplied soluble P to P. vittata probably via phytase hydrolysis, which was supported by 0.4-0.7 nmol P min -1 g -1 root fresh weight day -1 phytase activity in its root exudates, with 29-545 μM phytate-P being released into the growth media. Besides, compared to Na-phytate, Ca/Fe-phytate enhanced the As contents by 102-140% to 657-781 mg kg -1 in P. vittata roots and by 43-86% to 1109-1447 mg kg -1 in the fronds, which was accompanied by 21-108% increase in Ca and Fe uptake. The increased plant As is probably attributed to 1.3-2.6 fold upregulation of P transporters PvPht1;3/4 for root As uptake, and 1.8-4.3 fold upregulation of arsenite antiporters PvACR3/3;1/3;3 for As translocation to and As sequestration into the fronds. This is the first report to show that, besides soluble Na-phytate, P. vittata can also effectively utilize insoluble Ca/Fe-phytate as the sole P source, which sheds light onto improving its application in phytoremediation of As-contaminated sites.

Ratiometric fluorescent assays have a built-in correction factor which enhances assay accuracy and reliability. We have developed fluorescent ratiometric supramolecular tandem assays for phosphatase and phytase enzymes using a mixture of three molecular components. One of the molecules is a tetra-cationic fluorescence quencher called CalixPyr which can bind and quench the polyanionic pyrene fluorophore, CMP, that emits at 430 nm. Polyphosphates can disrupt the CMP/CalixPyr complex and alter the fluorescence intensity (responsive signal). CalixPyr has no effect on the fluorescence emission of cationic pentamethine cyanine fluorophore, cCy5, which emits at 665 nm and acts as a non-responsive reference signal. The continuous ratiometric fluorescent assay for alkaline phosphatase monitored hydrolytic consumption of adenosine triphosphate (ATP). The continuous ratiometric fluorescent assay for phytase activity monitored hydrolytic consumption of phytate. With further development this latter assay may be useful for high throughput assessment of phytase activity in individual batches of fortified animal feed. It is likely that the three-molecule mixture (CMP, CalixPyr, cCy5) can become a general assay platform for other enzymes that catalyse addition/removal of phosphate groups from appropriate molecular substrates.

The current study aimed to quantify the digestible calcium (Ca) equivalency of a new phytase (HiPhorius) in broiler chickens. A total of 1,152 male Cobb 500 broiler chickens were used in an experiment in which 8 diets consisting of graded levels of Ca supplied with limestone or graded levels of the phytase were fed. The 8 dietary treatments were based on a corn-soybean meal diet containing 5.1 g/kg of Ca and 5.1 g/kg of phosphorus (P) as negative control (NC); the NC + 1.3, 2.6, or 3.9 g/kg of Ca from limestone; and the NC + 500, 1,000, 2,000, or 4,000 FYT/kg of phytase. Birds were fed the experimental diets for 3 d (from d 7 to 10) or 14 d (from d 7 to 21) to determine apparent ileal digestibility (AID) and apparent total tract retention (ATTR) of dry matter (DM), CP, Ca, and P. In the 10-day-old birds, increasing the levels of Ca decreased the AID of P and Ca (linear, P < 0.05). Increasing the levels of phytase quadratically improved (P < 0.05) the AID of Ca and P. The AID of DM and CP in the younger birds quadratically increased (P < 0.05) as the levels of Ca increased. There were linear and quadratic effects (P < 0.05) of increasing phytase level for the AID of DM and a linear effect (P < 0.05) for the AID of CP. In the 21-day-old birds, increasing Ca levels resulted in a linear decrease (P < 0.05) in the AID of CP and P, and a quadratic decrease (P < 0.05) in the ATTR of CP. Increasing phytase levels linearly and quadratically (P < 0.05) improved the AID and ATTR of CP, P, and the ATTR of Ca. The analyzed phytase activity in the diets supplemented with phytase ranged from 1,520 to 7,661 FYT/kg. The digestible Ca equivalence for dietary phytase at 1,520 to 7,661 FYT/kg ranged from 1.55 to 2.02 g/kg in the 10-day-old birds fed for 3 d and from 0.52 to 0.64 g/kg in 21-day-old birds fed for 14 d. The results showed the reduction in Ca level that could be accommodated by phytase supplementation, which is markedly different between the younger and older birds. Feeding duration influenced the impact of phytase supplementation on Ca and P digestibility, with better efficacy of phytase observed in the 10-day-old birds fed for 3 d. Also, the results showed the extra-phosphoric effects of phytase on the utilization of other essential nutrients such as protein and Ca., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ectomycorrhizal fungi (ECMFs) that are involved in phosphorus mobilisation and turnover have limited ability to mineralise phytate alone. The endofungal bacteria in the ectomycorrhizal fruiting body may contribute to achieving this ecological function of ECMFs. We investigated the synergistic effect and mechanisms of endofungal bacteria and ECMF Suillus grevillea on phytate mineralisation. The results showed that soluble phosphorus content in the combined system of endofungal bacterium Cedecea lapagei and S. grevillea was 1.8 times higher than the sum of C. lapagei and S. grevillea alone treatment under the phytate mineralisation experiment. The S. grevillea could first chemotactically assist C. lapagei in adhering to the surface of S. grevillea. Then, the mineralisation of phytate was synergistically promoted by increasing the biomass of C. lapagei and the phosphatase and phytase activities of S. grevillea. The expression of genes related to chemotaxis, colonisation, and proliferation of C. lapagei and genes related to phosphatase and phytase activity of S. grevillea was also significantly upregulated. Furthermore, in the pot experiment, we verified that there might exist a ternary symbiotic system in the natural forest in which endofungal bacteria and ECMFs could synergistically promote phytate uptake in the plant Pinus massoniana via the ectomycorrhizal system., (© 2023 John Wiley & Sons Ltd.)

Two experiments were conducted to determine the impact of Ca, phytase, sampling time, and age on the digestibility (AID) of Ca and P and the expression of their transporters. Cobb 500 male chicks (N = 600) were used in each experiment and allocated to cages with 10 (Exp 1, 8-11 d) or 5 (Exp 2, 21-24 d) birds/cage and 10 (Exp 1) or 20 (Exp 2) reps/treatment. Treatments were a 2 × 3 factorial arrangement, with low (LOW) or standard (STD) Ca level and 3 phytase (PHY) levels (0, 300, or 3,000 FYT/kg). Ileal digesta were collected at 8, 12, 24, 48, and 72 h, and jejunum tissues at 12, 48, and 72 h after the start of feeding experimental diets. In Exp 1, there was no effect of Ca or phytase on the AID of Ca at 8, 12, or 24 h. Phytase increased the AID of P (P < 0.05) at all time points, and the magnitude was influenced by Ca. At 12 h, the mRNA level of P (NaPi-IIb) and Ca (CaSR) transporters was greatest in the LOW diets without phytase (Ca × PHY, P ≤ 0.06). In Exp 2, the STD diet decreased the AID of Ca and P (P < 0.05) at 8, 24, 48, or 72 h. Phytase increased the AID of Ca (P < 0.05) at 8, 12, and 24 h, and decreased the AID of Ca (quadratic, P < 0.05) in the STD diet (48 h). The AID of P (P < 0.05) increased with phytase at all sampling times. At 48 h, 3,000 FYT/kg decreased (P < 0.05) mRNA expression of NaPi-IIb and Ca transporter ATP2B1 in the STD diet (Ca × PHY, P < 0.05). In conclusion, to avoid adaptation of broilers to Ca and P deficiencies, the optimal time on experimental diets is ≤ 48 h for young broilers and ≤ 24 h in older birds due to up- or down-regulation of Ca and P transporters in response to dietary Ca, P, and phytase., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The aims of the study were to degrade the anti-nutritional factors (ANFs) such as phytic acid, glycinin, and β-conglycinin and improve the values of soybean meal (SBM). Firstly, in this study, a strain PY-4B which exhibited the best enzymatic activities of protease (403.3 ± 17.8 U/mL) and phytase (62.9 ± 2.9 U/mL) was isolated and screened among the isolates. Based on the analysis of physiological and biochemical characteristics and 16S rDNA sequence, the strain PY-4B was identified and named as Pseudomonas PY-4B. Next, Pseudomonas PY-4B was applied to fermentation of SBM. The results showed that the contents of glycinin and β-conglycinin were decreased by 57-63%, and the phytic acid was remarkably degraded by 62.5% due to the fermentation of SBM by Pseudomonas PY-4B. The degradation of glycinin and β-conglycinin resulted in increase of contents of water-soluble proteins and amino acids in fermented SBM. Moreover, Pseudomonas PY-4B exhibited no hemolytic activity and slight inhibitory effect on the growth of pathogen Staphylococcus aureus and the wide range of pH tolerance (3 to 9). In summary, our study indicates that isolated strain Pseudomonas PY-4B is a safe and applicable strain and has the ability to effectively degrade the ANFs (phytic acid, glycinin, and β-conglycinin) in SBM by fermentation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The objective of this study aimed to investigate the effectiveness of phytase alone and in combination with multi-carbohydrase in available phosphorus (AP) and energy-deficient diets on growth performance, nutrient digestibility, tibia traits, and carcass quality of broilers. A total of 288 1-d-old Ross 308 broiler chicks were randomly assigned to one of 6 dietary treatments in a completely randomized design, with each treatment having 6 replicates and 8 birds per cage. The treatments were as follows: 1) positive control with adequate nutrition (PC); 2) 0.20% AP-deficient diet without phytase (NC-1); 3) 0.25% AP-deficient diet without phytase (NC-2); 4) NC-1 diet plus 500 FTU/kg phytase (NCP-1); 5) NC-2 diet plus 750 FTU/kg phytase (NCP-2); and 6) NC-2 with 100 kcal/kg ME deficient diet plus 1,000 FTU/kg phytase and multi-carbohydrase: 2,200 U/kg galactomannanase, 30,000 U/kg xylanase, 22,000 U/kg β-glucanase, and 700 U/kg α-galactosidase (NCPM). Broilers in the PC group and those fed enzyme-supplemented diets exhibited greater (P < 0.05) growth performance in terms of body weight, average daily gain, and feed conversion ratio, along with enhanced tibia mineralization through the ash, calcium (Ca), phosphorus (P) composition, and tibia breaking strength compared to NC diets throughout the study. Among the treatments, broilers assigned to the NCPM group showed greater (P < 0.05) levels of energy and P digestibility on days 21 and 35. Concerning carcass characteristics, the leg meat yield was greater (P < 0.05) in broilers fed the NCP-2 diet compared to all other treatments on days 21 and 35. Supplementation of phytase at doses of 500 FTU/kg and 750 FTU/kg effectively recovers AP deficiencies of 0.20% and 0.25%, respectively, in broiler diets without compromising the growth performance of broilers. Additionally, combining multi-carbohydrase and phytase led to AP and energy loss recovery, which improved tibia mineralization and nutrient digestibility through their synergistic interaction., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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, 8 ileal-cannulated barrows (24 kg initial BW) were fed 5 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Background: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO 2 ., Results: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB - 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml., Conclusion: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO 2 in the feed industry., (© 2023. The Author(s).)

A total of 882 pigs (PIC TR4 × [Fast LW × PIC L02]; initially 33.2 ± 0.31 kg) were used in a 112-d study to evaluate the effects of different bones and analytical methods on the assessment of bone mineralization response to changes in dietary P, phytase, and vitamin D in growing pigs. Pens of pigs (20 pigs per pen) were randomized to one of five dietary treatments with nine pens per treatment. Dietary treatments were designed to create differences in bone mineralization and included: 1) P at 80% of NRC (2012) standardized total tract digestible (STTD) P requirement, 2) NRC STTD P with no phytase, 3) NRC STTD P with phytase providing an assumed release of 0.14% STTD P from 2,000 FYT/kg, 4) high STTD P (128% of the NRC P) using monocalcium phosphate and phytase, and 5) diet 4 with additional vitamin D3 from 25(OH)D3. On day 112, one pig per pen was euthanized for bone, blood, and urine analysis. Additionally, 11 pigs identified as having poor body condition which indicated a history of low feed intake (unhealthy) were sampled. There were no differences between treatments for final body weight, average daily gain, average daily feed intake, gain to feed, or bone ash measurements (treatment × bone interaction) regardless of bone ash method. The response to treatment for bone density and bone mineral content was dependent upon the bone sampled (density interaction, P = 0.053; mineral interaction, P = 0.078). For 10th rib bone density, pigs fed high levels of P had increased (P < 0.05) bone density compared with pigs fed NRC levels with phytase, with pigs fed deficient P, NRC levels of P with no phytase, and high STTD P with extra 25(OH)D3 intermediate, with no differences for metacarpals, fibulas, or 2nd ribs. Pigs fed extra vitamin D from 25(OH)D3 had increased (P < 0.05) 10th rib bone mineral content compared with pigs fed deficient P and NRC levels of P with phytase, with pigs fed industry P and vitamin D, and NRC P with monocalcium intermediate. Healthy pigs had greater (P < 0.05) serum Ca, P, vitamin D concentrations, and defatted bone ash than those unhealthy, with no difference between the two health statuses for non-defatted bone ash. In summary, differences between bone ash procedures were more apparent than differences between diets. Differences in bone density and mineral content in response to dietary P and vitamin D were most apparent with 10th ribs., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Enzyme combinations, particularly phytase (PHY) with various carbohydrases and proteases, are utilized in commercial broiler production to enhance nutrient and energy bioavailability., Objective: A feeding study was undertaken to determine whether the efficiency of an Escherichia coli-derived PHY and a feed enzyme complex (FEC) derived from Bacillus spp. containing carbohydrase and protease as main activities in broiler chickens is dependent on diet quality. A total of 900 male one-day-old broiler chickens (Ross 308) were assigned to a 2 × 3 factorial arrangement of the treatments with 2 different nutrient density diets, standard nutrient diet (SN diet) and a low-nutrient diet (LN diet; -100 kcal/kg for AMEn and -5% for crude protein [CP] and limiting amino acids), and 3 enzyme treatments (control [no enzymes], PHY and PHY + FEC). Each treatment group was composed of 6 replicates of 25 birds each., Results: The LN diet caused a decrease in performance index, tibia length and diameter, tibia calcium content and jejunal villus surface area (VSA). The interaction effects between diet and enzyme supplementation were observed (p < 0.05) on overall average daily gain (ADG), performance index, tibia ash content and jejunal villus height (VH) and VSA, with the favourable benefits of PHY + FEC treatment being more pronounced in the LN diets. Regardless of dietary nutrient density, supplementation with PHY alone or combined with FEC enhanced (p < 0.05) final body weight, overall ADG and jejunal villus height (VH)/crypt depth, with the highest values observed in the PHY + FEC group. The PHY + FEC treatment also improved (p < 0.05) overall feed conversion ratio, apparent ileal digestibility of dry matter, organic matter, CP, and energy, and tibia phosphorus content compared to the control treatment., Conclusions: The results indicate that the simultaneous addition of PHY and FEC to the LN diets improved the growth rate, bone mineralization and gut morphology., (© 2024 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

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.

Organic phosphorus (OP) is an essential component of the soil P cycle, which contributes to barley nutrition after its mineralization into inorganic phosphorus (Pi). However, the dynamics of OP utilization in the barley rhizosphere remain unclear. In this study, phytin was screened out from six OP carriers, which could reflect the difference in OP utilization between a P-inefficient genotype Baudin and a P-efficient genotype CN4027. The phosphorus utilization efficiency (PUE), root morphological traits, and expression of genes associated with P utilization were assessed under P deficiency or phytin treatments. P deficiency resulted in a greater root surface area and thicker roots. In barley fed with phytin as a P carrier, the APase activities of CN4027 were 2-3-fold lower than those of Baudin, while the phytase activities of CN4027 were 2-3-fold higher than those of Baudin. The PUE in CN4027 was mainly enhanced by activating phytase to improve the root absorption and utilization of Pi resulting from OP mineralization, while the PUE in Baudin was mainly enhanced by activating APase to improve the shoot reuse capacity. A phosphate transporter gene HvPHT1;8 regulated P transport from the roots to the shoots, while a purple acid phosphatase (PAP) family gene HvPAPhy&#95;b contributed to the reuse of P in barley.

A 60-day feeding trial was conducted to study the hematology, liver, and intestinal histoarchitecture of Labeo rohita fed with a combination of exogenous enzymes, essential amino acids, and essential fatty acids to DORB (De-oiled rice bran) based diets. Three treatments viz., T1 [DORB + phytase and xylanase (0.01% each)], T2 [DORB + phytase (0.01%) + xylanase (0.01%) + L-lysine(1.4%) + L-methionine (0.4%) + EPA and DHA (0.5%)] and T3 [DORB + phytase (0.01%), xylanase and cellulase (0.075%) + L-lysine (1.4%) +L-methionine (0.4%) + EPA and DHA (0.5%)] were used in the present study. Serum total protein, albumin content and A/G ratio varied significantly ( p  < 0.05) among groups. Globulin content did not vary significantly among groups ( p  ≥ 0.05). The Hb content, RBC and MCV count varied significantly ( p  < 0.05) whereas MCH, MCHC content, WBC and lymphocyte count did not vary significantly among groups ( p  > 0.05). The liver and intestine examination revealed no visible alteration and showed normal histo-architecture. Based on the finding it is concluded that DORB supplemented with exogenous enzymes, essential amino acids and essential fatty acids with phytase (0.01%), xylanase and cellulase (0.075%), L-lysine (1.4%), DL-methionine (0.4%) and EPA and DHA (0.5%) improves the health of L. rohita .

This study aimed to determine the effect of Zn source and dietary level on intestinal myo-inositol hexakisphosphate (InsP 6 ) disappearance, intestinal accumulation of lower InsP and myo-inositol (MI), prececal mineral digestibility, bone mineralization, and Zn status of broilers without and with exogenous phytase in the feed. Male Ross 308 broilers were allocated in groups of 10 to 8 treatments with 8 pens each. Experimental diets were fed from d 7 to d 28 and contained 33 mg/kg dry matter plant-intrinsic Zn. Experimental factors were phytase supplementation (0 or 750 FTU/kg) and Zn source (none [0 mg/kg Zn], Zn-sulfate [30 mg/kg Zn], Zn-oxide [30 mg/kg Zn]). Additional treatments with 90 mg/kg Zn as Zn-sulfate or Zn-oxide and phytase were included to test the effect of Zn level. No Zn source or Zn level effects were observed for ADG, feed conversion ratio, prececal P digestibility, intestinal InsP 6 disappearance, and bone ash concentration. However, those measurements were increased by exogenous phytase (P < 0.001), except the feed conversion ratio, which was decreased (P < 0.001). Ileal MI concentrations were affected by phytase × Zn source interaction (P < 0.030). Birds receiving exogenous phytase and Zn supplementation had the highest MI concentrations regardless of exogenous Zn source, whereas MI concentrations were intermediate for birds receiving exogenous phytase only. Exogenous phytase and exogenous Zn source increased the Zn concentration in bone and blood of broilers (P < 0.001). In conclusion, measures of exogenous phytase efficacy were not affected by phytase × Zn source interaction. Further studies are needed to rule out an effect from Zn sources other than those tested in this study and to investigate the effect of Zn supplementation on endogenous phosphatases. The missing effect of increasing Zn supplementation from 30 to 90 mg/kg in phytase-supplemented diets gives reason to reconsider the Zn supplementation level used by the industry., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Phytase supplementation is gaining importance in animal nutrition because of its effect on phosphorus (P) digestibility and the increasing relevance of P for sustainable production. The potential inhibitors of phytase efficacy and phytate degradation, such as calcium (Ca) and zinc (Zn), have been a subject of intense research. This review focuses on the interactions of Zn with phytate and phytase in the digestive tract of poultry and pigs, with an emphasis on the effects of Zn supplementation on phytase efficacy and P digestibility. In vitro studies have shown the inhibitory effect of Zn on phytase efficacy. However, relevant in vivo studies are scarce and do not show consistent results for poultry and pigs. The results could be influenced by different factors, such as diet composition, amount of Zn supplement, mineral concentrations, and phytase supplementation, which limit the comparability of studies. The chosen response criteria to measure phytase efficacy, which is mainly tibia ash, could also influence the results. Compared to poultry, the literature findings are somewhat more conclusive in pigs, where pharmacological Zn doses (≥ 1000 mg kg -1 Zn) appear to reduce P digestibility. To appropriately evaluate the effects of non-pharmacological Zn doses, further studies are needed that provide comprehensive information on their experimental setup and include measurements of gastrointestinal phytate degradation to better understand the mechanisms associated with Zn and phytase supplements. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Phytases are important enzymes used for eliminating the anti-nutritional properties of phytic acid in food and feed ingredients. Phytic acid is major form of organic phosphorus stored during seed setting. Monogastric animals cannot utilize this phytate-phosphorus due to lack of necessary enzymes. Therefore, phytic acid excretion is responsible for mineral deficiency and phosphorus pollution. Phytases have been reported from diverse microorganisms, however, fungal phytases are preferred due to their unique properties. Aspergillus species are the predominant producers of phytases and have been explored widely as compared to other fungi. Solid-state fermentation has been studied as an economical process for the production of phytases to utilize various agro-industrial residues. Mixed substrate fermentation has also been reported for the production of phytases. Physical and chemical parameters including pH, temperature, and concentrations of media components have significantly affected the production of phytases in solid state fermentation. Fungi produced high levels of phytases in solid state fermentation utilizing economical substrates. Optimization of culture conditions using different approaches has significantly improved the production of phytases. Fungal phytases are histidine acid phosphatases exhibiting broad substrate specificity, are relatively thermostable and protease-resistant. These phytases have been found effective in dephytinization of food and feed samples with concomitant liberation of minerals, sugars and soluble proteins. Additionally, they have improved the growth of plants by increasing the availability of phosphorus and other minerals. Furthermore, phytases from fungi have played an important roles in bread making, semi-synthesis of peroxidase, biofuel production, production of myo-inositol phosphates and management of environmental pollution. This review article describes the production of fungal phytases in solid state fermentation and their biotechnological applications., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

This study investigated the effects of dietary 6-phytase, produced by a genetically modified Komagataella phaffii, on growth performance, feed utilisation, flesh quality, villus morphometric properties, and intestinal mRNA expression in rainbow trout. Six iso-nitrogenous, iso-lipidic, and iso-caloric diets were formulated and fed to triplicate groups of juvenile rainbow trout weighing 32.57 ± 0.36 g (mean ± SD) for 90 days. The dietary treatments included two positive controls (PC), one formulated with 400 g/kg of fish meal named T1, and the other formulated with 170 g/kg of fish meal plus 1% avP derived from monocalcium phosphate named T2. The remaining dietary treatments consisted of a negative control (NC) formulated with 170 g/kg of fish meal (T3), NC+ 750, NC+ 1500, and NC+ 3000 OTU/kg levels of phytase designated as T4, T5, and T6 diets respectively. Compared to T1, weight gain (WG) increased by 16.29, 13.71 and 11.66% in T4, T5 and T6, respectively (p < 0.05). Feed conversion ratio (FCR) was lowered by 3.2 and 0.8% in T4 and T5 compared to T1 (p < 0.05). WG, feed intake (FI), FCR, final body length, bone ash, bone ash P, and intestinal morphometry were negatively affected in T3 fed fish (p < 0.05). Whole-body fish nutrient, bone ash, bone ash phosphorus (P) compositions and mucosal villus morphometric properties improved in rainbow trout fed diets supplemented with phytase dose ranging from 750-3000 OTU. Bone ash increased by 6.12% in T5 compared to T1 (p < 0.05). Phytase inclusion enhanced the profitability of feeding juvenile rainbow trout such diets as it reduced the feed price and economic conversion rate. Dietary inclusion of phytase down-regulated mRNA expression of genes responsible for fatty acid synthesis and lipogenesis in juvenile rainbow trout. Dietary phytase up-regulated the mRNA expression of genes (SLC4A11 and ATP1A3A) responsible for nutrient uptake and down-regulated intestinal expression of MUCIN 5AC-like genes (mucus secreting genes) in juvenile rainbow trout. Along with improving performance parameters, the inclusion of phytase in rainbow trout diet containing plant-based protein sources, can preserve intestinal morphology by regulating the mRNA expression of genes responsible for fatty acid synthesis, lipogenesis and nutrient uptake and transport., (© 2023 The Authors. Journal of Animal Physiology and Animal Nutrition published by Wiley-VCH GmbH.)

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg 2+ and Zn 2+ enhanced phytate hydrolysis by the enzyme, while Na + showed mild inhibition but Hg 2+ severely inhibited the enzymatic activity. K m and V max were estimated to be 0.11 mM and 55.6 μmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Phosphorous actively participates in numerous metabolic and regulatory activities of almost all living organisms including animals and humans. Therefore, it is considered as an essential macronutrient required supporting their proper growth. On contrary, phytic acid (PA), an antinutritional substance, is widely known for its strong affinity to chelate essential mineral ions including PO 4 3- , Ca 2+ , Fe 2+ , Mg 2+ , and Zn 2+ . Being one the major reservoir of PO 4 3- ions, PA has great potential to bind PO 4 3- ions in diverse range of foods. Once combined with P, PA transforms into an undigested and insoluble complex namely phytate. Produced phytate leads to a notable reduction in the bioavailability of P due to negligible activity of phytases in monogastric animals and humans. This highlights the importance and consequent need of enhancement of phytase level in these life forms. Interestingly, phytases, catalyzing the breakdown of phytate complex and recycling the phosphate into ecosystem to its available form, have naturally been reported in a variety of plants and microorganisms over past few decades. In pursuit of a reliable solution, the focus of this review is to explore the keynote potential of bacterial phytases for sustainable management of phosphorous via efficient utilization of soil phytate. The core of the review covers detailed discussion on bacterial phytases along with their widely reported applications viz. biofertilizers, phosphorus acquisition, and plant growth promotion. Moreover, meticulous description on fermentation-based strategies and future trends on bacterial phytases have also been included., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including sorghum ( Sorghum bicolor ). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Model porridges made from transgenic events were evaluated for carotenoid and mineral content as well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 μg g -1 DW) compared to null segregants (4.2 μg g -1 DW) and wild-type control (Tx430; 3.7 μg g -1 DW). Phytase activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg g -1 DW), altered the profile of inositol phosphate catabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1), and zinc (19.0 to 4.9) in engineered material, suggesting improved mineral bioaccessibility. Improved phytate : mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were over 23 times greater in transgenic events compared to corresponding null segregants and wild-type controls. A 200 g serving of porridge made with these transgenic events provide an estimated 53.7% of a 4-8-year-old child's vitamin A estimated average requirement. These data suggest that combinatorial approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.

With the increasing demand for P fertilizer for world food production, the use of soil organic P fraction via mineralization could become an important P resource in agricultural soils. However, the predominant organic P species, phytic acid, has been considered rather recalcitrant to mineralization due to its active interaction with dissolved metals like Ca 2+ in soil pore water. Calcium ions can be an inhibitor to many phytases, yet the mechanism was not clear. The objective of this study was to understand the effects of Ca 2+ (aq) on the phytase activity and inhibitory mechanisms using batch degradation kinetic experiments, Nuclear Magnetic Resonance (NMR) spectroscopy, Saturation Transfer Difference (STD) NMR, and Circular dichroism (CD) spectroscopy. The phytase activity followed Michaelis-Menten kinetics and increased Michaelis constant K m and decreased V max with Ca 2+ addition were observed at pH 6. Therefore, mixed inhibition was the inhibition mechanism which was likely a result of the allosteric effect of Ca 2+ . The near-UV CD spectra supported phytase secondary conformational change upon the interaction between Ca 2+ and the enzyme. It was found that phytase initially reacted with the D/L-3 phosphate of phytic acid at pH 6. At pH 8, the overall phytase activity decreased, yet the effect of Ca 2+ on phytase activity was the opposite of that of pH 6. Enhanced phytase activity with Ca 2+ addition was attributed to the structural change of phytic acid upon the Ca 2+ complexation, which was confirmed by NOE spectra. The Ca 2+ -phytic acid complex might be a more favorable substrate than the free phytic acid. Unlike the findings from pH 6, Ca 2+ didn't induce significant changes in either the near- or far-UV region of the CD spectra at pH 8. Furthermore, P5 was found to be the target of phytase at pH 8. The study revealed the pH-specific effects of Ca 2+ on the mineralization of phytic acid., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Inert digestibility index markers such as titanium dioxide are universally accepted to provide simple measurement of digestive tract retention and relative digestibility in poultry feeding trials. Their use underpins industry practice: specifically dosing regimens for adjunct enzymes added to animal feed. Among these, phytases, enzymes that degrade dietary phytate, inositol hexakisphosphate, represent a billion-dollar sector in an industry that raises ca. 70 billion chickens/annum. Unbeknown to the feed enzyme sector, is the growth in cell biology of use of titanium dioxide for enrichment of inositol phosphates from extracts of cells and tissues. The adoption of titanium dioxide in cell biology arises from its affinity under acid conditions for phosphates, suggesting that in feeding trial contexts that target phytate degradation this marker may not be as inert as assumed. We show that feed grade titanium dioxide enriches a mixed population of higher and lower inositol phosphates from acid solutions. Additionally, we compared the extractable inositol phosphates in gizzard and ileal digesta of 21day old male Ross 308 broilers fed three phytase doses (0, 500 and 6000 FTU/kg feed) and one inositol dose (2g/kg feed). This experiment was performed with or without titanium dioxide added as a digestibility index marker at a level of 0.5%, with all diets fed for 21 days. Analysis yielded no significant difference in effect of phytase inclusion in the presence or absence of titanium dioxide. Thus, despite the utility of titanium dioxide for recovery of inositol phosphates from biological samples, it seems that its use as an inert marker in digestibility trials is justified-as its inclusion in mash diets does not interfere with the recovery of inositol phosphates from digesta samples., Competing Interests: The feeding trials described in this study were commissioned at Nottingham Trent University by AB Vista. AB Vista had no role in conduct of the trial, data collection and analysis, decision to publish or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Sprigg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Development of exogenous enzymes is one of the most important discoveries in animal nutrition. The supplementation of exogenous enzymes in broiler diets allows for supplying nutrient deficiencies and to decrease endogenous losses., Objectives: The effects of phytase (Hostazym and Phyzyme) and xylanase (Ronozyme) enzymes were investigated on growth performance and Mucin2 gene expression in broilers., Methods: A completely randomized design was applied, including 7 treatments, 4 replicates and 25 birds per replicates. A total of 700 male Ross (308) broiler chickens were fed with similar diets supplemented by Hostazym and Phyzyme (500 and 1000 FTU/kg) and Ronozyme (100 and 200 EXU/kg). Weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) were determined for three phases and entire rearing period. On 42 days of age, four birds per replicate were slaughtered. Total RNA was extracted from jejunum samples, and Mucin2 gene expression was measured by real-time PCR., Results: Phytase and xylanase enzymes had a significant effect (p < 0.05) on traits (WG and FCR) in grower and finisher phases and whole rearing period, but FI was not affected by enzymes (p > 0.05). Carcass (74.13 g) and breast (27.76 g) weights by Hostazym (1000 FTU/kg) were higher than other treatments (p < 0.05). Weight of liver, bursa and spleen were significantly influenced by enzymes (p < 0.05). Likewise, bursa and spleen weights in Hostazym (1000 FTU/kg feed) and Ronozyme (200 EXU/kg feed) were significantly higher than other treatments (p < 0.05). Mucin2 gene expression was affected by enzymes in whole treatments. The lowest amount of Mucin2 gene expression belonged to Ronozyme (200 and 100 EXU/kg), and the highest was belonging to Hostazym (1000 FTU/kg)., Conclusions: Phytase enzymes have higher effect on broiler performance and Mucin2 gene expression compared to xylanase. High doses of Hostazym (1000 FTU/kg feed) could be supplemented in broiler chicken diets to improve optimum growth and feed efficiency., (© 2023 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)