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

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

Dynamics of bone mineralization in primiparous sows as a function of dietary phosphorus and calcium during lactation.

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.

Insoluble-Phytate Improves Plant Growth and Arsenic Accumulation in As-Hyperaccumulator Pteris vittata: Phytase Activity, Nutrient Uptake, and As-Metabolism.

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.

Fluorescent ratiometric supramolecular tandem assays for phosphatase and phytase enzymes.

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.

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

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 CO2. 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 CO2 in the feed industry.

Effect of an Escherichia coli-derived phytase and a carbohydrase-protease cocktail derived from Bacillus spp. on performance, digestibility, bone mineralization and gut morphology in broilers fed different nutrient density diets.

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.

Endofungal bacteria and ectomycorrhizal fungi synergistically promote the absorption of organic phosphorus in Pinus massoniana.

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.

Screening and Identification of a Strain with Protease and Phytase Activities and Its Application in Soybean Meal Fermentation.

The aims of the study were to degrade the anti-nutritional factors (ANFs) such as phytic acid, glycinin, and ß-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 ß-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 ß-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 ß-conglycinin) in SBM by fermentation.

Ileal mineral digestibility and expression of nutrient transporter genes of broiler chickens in response to variable dietary total Ca and phytase supplementation are influenced by time on experimental diet and age of the birds.

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.

Digestible calcium equivalency of phytase and nutrient utilization of broiler chickens fed graded levels of limestone or phytase during the starter phase.

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.

Phytate-phosphorus and phytase on performance, bone characteristics, tissue and serum mineral concentration on broilers.

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.

The Impact of Various Organic Phosphorus Carriers on the Uptake and Use Efficiency in Barley.

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_b contributed to the reuse of P in barley.

Production of fungal phytases in solid state fermentation and potential biotechnological applications.

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.

Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica) / Purificación y caracterización bioquímica de fitasa de Bacillus cereus aislada del tracto gastrointestinal de caracol gigante africano (Achatina fulica)

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. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax 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.(AU)

Effect of dietary zinc source, zinc concentration, and exogenous phytase on intestinal phytate degradation products, bone mineralization, and zinc status of broiler chickens.

This study aimed to determine the effect of Zn source and dietary level on intestinal myo-inositol hexakisphosphate (InsP6) 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 InsP6 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.

Digestibility of calcium in calcium-containing ingredients and requirements for digestible calcium by growing pigs.

The concentration of Ca in plant feed ingredients is low compared with the requirement for pigs and most Ca in diets for pigs is provided by limestone and Ca phosphate. To determine digestibility values for Ca that are additive in mixed diets, the standardized total tract digestibility (STTD) of Ca needs to be calculated, and the STTD of Ca by growing pigs in most Ca-containing ingredients has been reported. Although Ca is an inexpensive nutrient compared with P and amino acids, excess Ca needs to be avoided because excess dietary Ca results in reduced P digestibility, reduced feed intake, and reduced growth performance of pigs. Recent data indicate that most diets produced for pigs in the United States and Europe contain ~0.20 percentage units more Ca than formulated, which likely is because of the use of limestone as a carrier in feed additives or as a flow agent in other ingredients. An excess of this magnitude without a corresponding excess of P will result in a reduction in daily gain of growing pigs by 50 to 100 g. Greater emphasis, therefore, needs to be placed on determining the concentration of Ca in diets for pigs. Microbial phytase increases the digestibility of both Ca and P and it is, therefore, important that the release of both Ca and P by phytase is considered in diet formulation. However, due to the relationship between Ca and P in postabsorptive metabolism, diets need to be formulated based on a ratio between digestible Ca and digestible P. To maximize average daily gain, this ratio needs to be less than 1.40:1.0 in diets for weanling pigs, and the ratio needs to be reduced as the body weight of pigs increases. In contrast, to maximize bone ash, the digestible Ca to digestible P ratio needs to increase from 1.67:1.0 in 11 to 25 kg pigs to 2.33:1.0 in finishing pigs. Gestating sows have reduced STTD and retention of Ca and P compared with growing pigs and formulation of diets for sows based on digestibility values obtained in growing pigs will result in inaccuracies in the provision of Ca and P. There is, however, a lack of data for the digestibility of Ca and P by gestating and lactating sows, and responses to microbial phytase by sows are not fully understood. There is, therefore, a need for research to generate more data in this area. In the present review, a summary of data for the digestibility of Ca in feed ingredients for pigs and estimates for the requirement for digestible Ca by growing and finishing pigs are provided.

Concentration of Ca in most plant feed ingredients is low compared with the requirement for pigs and dietary Ca is, therefore, mostly provided by limestone and calcium phosphates. Although Ca is an inexpensive nutrient compared with P and amino acids, excess dietary Ca may result in reduced P digestibility, feed intake, and growth performance of pigs. Excretion of P from pigs is increased if dietary Ca is provided above the requirement, which may increase environmental pollution. Therefore, determination of the digestibility of Ca in dietary sources of Ca and formulation of diets based on the ratio between digestible Ca and digestible P are needed to reduce Ca and P excretions. This review provides a summary of values for the digestibility of Ca in feed ingredients and also provides estimates for the requirement for digestible Ca by weanling and growing-finishing pigs. Summarized data from experiments that determined the requirement for digestible Ca demonstrated that there are linear correlations between body weight of growing-finishing pigs and digestible Ca to digestible P ratios needed to maximize growth or bone ash.

Effect of dietary calcium concentration and exogenous phytase on inositol phosphate degradation, mineral digestibility, and gut microbiota in growing pigs.

Variations in the dietary Ca concentration may affect inositol phosphate (InsP) degradation, and thereby, P digestibility in pigs. This study assessed the effects of dietary Ca concentration and exogenous phytase on InsP degradation, nutrient digestion and retention, blood metabolites, and microbiota composition in growing pigs with ileal cannulation. In a completely randomized row-column design with four periods, eight ileal-cannulated barrows (initial body weight 27 kg) were fed four corn-soybean- and rapeseed meal-based diets containing 5.5 or 8.5 g Ca/kg dry matter (DM), with or without 1,500 FTU of an exogenous hybrid-6-phytase/kg diet. No mineral P was added and the P concentration in the feed was 4.8 g P/kg DM. Prececal InsP6 disappearance in pigs fed diets containing exogenous phytase was lower (P = 0.022) with additional Ca than without. Concentrations of InsP2-4 isomers and myo-inositol in the distal ileal digesta and prececal P digestibility were greater (P < 0.001) with exogenous phytase than without exogenous phytase. In feces, InsP6 disappearance was lower (P < 0.002) and concentration of InsP5 and InsP4 isomers was higher (P ≤ 0.031) with additional Ca compared to without additional Ca. The prececal amino acid digestibility, energy digestibility, and hindgut disappearance of energy did not differ. The Shannon diversity index of the microbiota in the distal ileal digesta and feces was similar among the diets but was lower in the distal ileal digesta than in the feces (P < 0.001). Permutation analysis of variance revealed no dietary differences between the bacterial groups within the ileal digesta and fecal samples (P > 0.05). In conclusion, additional Ca reduced the effect of exogenous phytase on prececal InsP6 degradation. Endogenous InsP degradation was impaired by additional Ca only in the hindgut but the abundance of bacterial genera in feces was not affected.

The dietary calcium concentration can influence the release of phosphorus from phytate in growing pigs. This study assessed the effects of dietary calcium and exogenous phytase on inositol phosphate (InsP) degradation and nutrient digestibility in ileal-cannulated, growing pigs. The phosphorus, calcium, and myo-inositol concentrations in the blood, microbiota composition in the ileal digesta and feces, and volatile fatty acid concentrations in the feces were also evaluated. Additional dietary calcium decreased prececal inositol hexakisphosphate (InsP6) disappearance, but only with exogenous phytase. Concentrations of InsP2-4 isomers and myo-inositol in the ileal digesta and prececal phosphorus digestibility were greater with exogenous phytase, but not affected by dietary calcium concentration. In contrast, fecal InsP6 disappearance was lower and the concentration of InsP4-5 isomers in feces was greater with additional dietary calcium. Regarding microbiota, the Shannon diversity index was lower in ileal digesta than in feces but was unaffected by dietary calcium concentration or exogenous phytase. In conclusion, dietary calcium concentration is relevant for phytate disappearance in feces, but not in the ileal digesta. However, when exogenous phytase is used, the dietary calcium concentration is important because prececal phytate degradation is changed.

Interactions of zinc with phytate and phytase in the digestive tract of poultry and pigs: a review.

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.

The effects of dietary microbial 6-phytase on growth parameters, intestinal morphometric properties and selected intestinal genes expression in rainbow trout (Oncorhynchus mykiss, Walbaum 1876).

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.

Trait stacking simultaneously enhances provitamin A carotenoid and mineral bioaccessibility in biofortified Sorghum bicolor.

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.