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

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.

To establish the HTC defect development, the cooking kinetics of seeds of ten bean accessions (belonging to seven common bean market classes), fresh and conventionally aged (35 °C, 83% RH, 3 months) were compared to those obtained after soaking in specific salt solutions (in 0.1 M sodium acetate buffer at pH 4.4, 41 °C for 12 h, or 0.01 M CaCl2 at pH 6.2, 25 °C for 16 h and subsequently cooking in CaCl2 solution, or deionised water). The extent of phytate (inositol hexaphosphate, IP6) hydrolysis was evaluated to better understand the role of endogenous Ca2+ in the changes of the bean cooking kinetics. A significant decrease in the IP6 content was observed after conventional ageing and after soaking in a sodium acetate solution suggesting phytate hydrolysis (release of endogenous Ca2+). These changes were accompanied by an increase in the cooking time of the beans. Smaller changes in cooking times after soaking in a sodium acetate solution (compared to conventionally aged beans) was attributed to a lower ionisation level of the COOH groups in pectin (pH 4.4, being close to pKa value of pectin) limiting pectin Ca2+ cross-linking. In beans soaked in a CaCl2 solution, the uptake of exogenous cations increased the cooking times (with no IP6 hydrolysis). The change in cooking time of conventionally aged beans was strongly correlated with the extent of IP6 hydrolysis, although two groups of beans with low or high IP6 hydrolysis were distinguished. Comparable trends were observed when soaking in CaCl2 solution (r = 0.67, p = 0.14 or r = 0.97, p = 0.03 for two groups of beans with softer or harder texture during cooking). Therefore a test based on the Ca2+ sensitivity of the cooking times, implemented through a Ca2+ soaking experiment followed by cooking can be used as an accelerated test to predict susceptibility to HTC defect development during conventional ageing. On the other hand, a sodium acetate soaking experiment can be used to predict IP6 hydrolysis of conventionally aged bean accessions and changes of cooking times for these bean accessions (with exception of yellow bean-KATB1).

Utilization of common beans is greatly hampered by the hard-to-cook (HTC) defect induced by ageing of the beans under adverse storage. Large bean-to-bean variations exist in a single batch of beans. Therefore, a texture-based bean classification approach was applied in this detailed study on beans with known textures, to gain in-depth insights into the role of the pectin-cation-phytate mechanism in relation to the texture changes during subsequent cooking of Red haricot fresh and aged beans. For the first time, a correlation between the texture (exhibited after cooking) of a single bean seed before ageing (fresh) and its texture after ageing was established. Furthermore, scanning electron microscopy coupled with energy dispersive spectrometry (SEM-EDS) based in situ cell wall associated mineral quantification revealed that the cell wall associated Ca concentration was significantly positively correlated with the texture of both fresh and aged cooked Red haricot bean cotyledons, with ageing resulting in a significant enrichment of Ca at the cell wall. These additional Ca cations originate from intracellular phytate hydrolysis during ageing, which was shown to affect the texture distribution of aged beans during cooking significantly. The relocation of the mineral cations from the cell interior to the cell wall occurs mainly during storage rather than subsequent soaking of the cotyledons. In addition, the pectin-cation-phytate hypothesis of HTC was further confirmed by demethylesterification of the cell wall pectin and increased pectin-Ca interactions upon ageing of the cotyledons, finally leading to HTC development of the cotyledon tissue.

In this study, two chemical bean seed hardening methods were used to investigate the changes in cooking behavior associated with Ca2+ transport and phytate hydrolysis to better understand their role in the pectin-cation-phytate hypothesis. The texture evolution of fresh and hardened red kidney beans was evaluated, hardening being induced by soaking or in a CaCl2 solution (0.01 M, 0.05 M, 0.1 M) or sodium acetate buffer (0.1 M, pH 4.4, 41 °C). The beans soaked in a CaCl2 solution at higher concentrations or in sodium acetate buffer for a longer time exhibited a delayed cooking behavior. This study also explored the bio-chemical changes (calcium content in different bean substructures, phytate content and the pectin degree of methylesterification (DM) in the cotyledons) occurring in the beans during chemical hardening and cooking. The Ca2+ concentrations in the whole beans and cotyledons of beans soaked and cooked in CaCl2 solutions significantly increased while inositol hexaphosphate IP6 content showed no significant changes. This indicates that the delayed texture drop in this case results from the influx of exogenous Ca2+ in the cotyledons and seed coats during cooking while the IP6 was not hydrolyzed and did not release endogenous Ca2+. For beans soaked in sodium acetate buffer, phytate profiling showed increased hydrolysis of IP6 with longer soaking time, suggesting the migration of endogenous Ca2+ released from phytate hydrolysis contributing to the delayed cooking of these beans. These results indicate that both an exogenous Ca2+ influx during soaking and cooking and an endogenous Ca2+ replacement resulting from phytate hydrolysis can play an important role in the hardening of beans. In neither of the cases, a significant change in pectin DM was observed during chemical hardening, therefore limiting the delayed cooking to the role of Ca2+ transport. The outcome of both cases is inline with the basic principles of the pectin-cation-phytate hypothesis whereby pectin DM changes are hardly involved and different mechanisms of release/transport are involved.

Phytate forms insoluble precipitates with various cations that are recalcitrant to digestion in poultry. Dietary supplementation with exogenous phytase has been shown to improve phytate solubility and digestibility and, in turn, improve animal growth performance. Although the kinetics of phytate hydrolysis by exogenous phytase are well described in vitro, the progression of the reaction in vivo is still not well defined. The aim of the present study was, therefore, to monitor the kinetic variation of myo-inositol (myo-Ins) levels in both circulation and feather following exogenous phytase supplementation. In experiment 1, 4 week-old male broilers were individually housed with ad libitum access to water and a standard commercial diet. Birds were maintained under environmental temperature of 24°C and 30% RH. Birds were cannulated in the cutaneous ulnar vein on the right wing and remained untouched for 3 days. On the day of the experiment, birds were randomly divided into three body weight-matched groups and fed either the control diet, the control diet-supplemented with myo-Ins or Ronozyme HiPhos (0.06%, DSM Nutritional Products, Switzerland) for 10 h. In the experiment 2, birds were fed only HiPhos for 30 h. Growing feathers and blood were collected at baseline and then every 2 h for 10 h (experiment 1) and 30 h (experiment 2) post-prandially. Plasma and feather myo-Ins levels were determined by UHPLC-MS/MS. The relative expression of inositol polyphosphate-1-phosphatase (INPP1), inositol hexakisphosphate kinase 1-3 (IP6K1-3), inositol-3-phosphate synthase (ISYNA), and multiple inositol-polyphosphate phosphatase 1 (MNPP1) genes in blood and feathers was determined by real-time qPCR using 2-ΔΔCt method. Plasma and feather myo-Ins levels were significantly increased by HiPhos at 6 h to 8 h post-prandial. The mRNA abundances of INPP1, IP6K1, and ISYNA in the circulation were significantly down regulated at all periods compared to the baseline levels. IP6K2, IP6K3, and MINPP1 gene expression, however, was up regulated at 8 h post-prandial and then returned to the baseline levels. In feathers, the expression of INPP1 was induced at 8 h post-prandial and remained higher compared to the baseline. The expression of IP6K2, IP6K3, and MINPP1 was down regulated during the first 10 h and then returned to baseline levels for the rest of the post-prandial period. Taken together, our data show that phytase modulates the expression of genes associated with myo-Ins metabolism and generates release of myo-Ins in both circulation and feather at 6-10 h post-feeding. Feather myo-Ins concentration could be used as a non-invasive method to monitor phytate hydrolysis in practice.

Two experiments (Exp.) with ileally cannulated growing barrows were conducted. The concentrations of positional inositol phosphate (InsP) isomers in ileal digesta and feces were determined, as well as the prececal and total tract phytate (InsP6) hydrolysis, and digestibility of dry matter, P, Ca, nitrogen, and gross energy. Prececal amino acid (AA) digestibility and digestive enzyme activities in ileal digesta were also studied. In both Exp., pigs had an initial body weight (BW) of 28 kg and were completely randomized to a Double Latin Square Design with eight pigs, four diets, and three periods of 12 d each. Feces and ileal digesta were collected for 5 d and 2 d, respectively. Pigs were housed individually in stainless steel metabolic units. Water was available ad libitum and feed was provided two times daily at an amount of 4% of mean BW. In Exp. 1, pigs received a corn-soybean meal (SBM)-based diet that was supplemented with 0, 750, 1,500, or 3,000 FTU of a microbial phytase/kg diet. In Exp. 2, pigs were allotted to a 2 × 2 arrangement of diets based on corn and SBM or an SBM-rapeseed cake (RSC) mix and phytase supplementation at 0 or 1,500 FTU/kg of diet. In ileal digesta of pigs fed without the phytase supplement, the dominating InsP isomers beside InsP6 were InsP5 isomers. The InsP pattern in ileal digesta changed with the inclusion of microbial phytase in both Exp., as there was a remarkable increase in Ins(1,2,5,6)P4 concentration (P < 0.001). In both Exp., the myo-inositol concentration in ileal digesta was greater upon phytase addition (P < 0.001). Without phytase supplementation, prececal and total tract P digestibility were low, whereas hardly any InsP6 was excreted in feces. There was no difference between prececal and total tract P digestibility values. For most AA studied in Exp. 2, prececal digestibility was lower (P < 0.01) when the diet contained RSC. However, phytase supplementation did not significantly affect prececal AA digestibility in both Exp. The present study showed that InsP6 disappearance by the end of the ileum can be increased up to around 90% in SBM- and SBM-RSC-based diets when microbial phytase is supplemented, but prececal P digestibility hardly exceeded 60%. The study confirms that pigs cannot benefit from a remarkable InsP6 degradation in the hindgut.

Phytase is commonly used as a feed enzyme in monogastric animals to increase the bioavailability of phytate phosphorus and other nutrients. The accumulation of myo-inositol phosphate intermediates during phytate degradation in various segments of the gastrointestinal tract (GIT) is poorly understood. The aim of this study was to determine the efficacy of Buttiauxella spp. phytase in degrading the phytate in corn, soybean meal, and complete corn-soybean meal diet to myo-inositol phosphate esters (IP1-IP5) and completely dephosphorylated myo-inositol rings using an in vitro model of the poultry upper GIT. Our results show that the phytase hydrolyzes phytate efficiently to small IP esters, whereas the myo-inositol level remains constant between control and phytase treatments. Although the in vitro digestion model does not incorporate all factors that govern phytate hydrolysis, it is a valuable tool for evaluating phytase efficacy at various enzyme doses and with different feed ingredients.

The aim of the study was to observe the effects of dietary available phosphorus (aP) and calcium (Ca), with regular or super doses of phytase, on phytate hydrolysis and subsequent influences on broiler growth performance and nutrient utilization. In a 2 × 3 factorial design, 384 Ross-308 broilers were allocated to one of 6 dietary treatments with 8 replicates in a randomized complete block design for 21 days. Diets were nutritionally adequate (positive control, PC) or marginally deficient in aP and Ca (negative control, NC), with 0, 500 or 1,500 FTU/kg phytase. Bird and feed weights were recorded on d 0 and 21, excreta were collected on d 19 and 20, and gizzard and ileal contents were collected on d 21. Body weight gain (P < 0.01) increased linearly with phytase in the PC and quadratically in the NC. There was an interactive effect on ileal DM, N, and P utilization, increasing quadratically with phytase supplementation in the NC, but there was no phytase influence in the PC (P < 0.05). Phytase linearly increased copper (P < 0.001) and linearly decreased Ca (P < 0.05) utilization in the ileum. Phytase decreased ileal (IPx, inositol x-phosphate) IP6 and IP5 and increased inositol (quadratic, P < 0.001) but had no effect on IP4 or IP3. The influence of the dietary aP was more apparent on the hydrolysis of phytate and phytate esters after the ileum, with increasing (linear and quadratic, P < 0.05) IP4 and IP3 content in the excreta of birds fed the NC or PC when phytase was added. Phytate hydrolysis improves the growth potential of birds fed NC diets, allowing them to match the growth performance of birds fed PC diets and improve nutrient utilization. These results indicate that dietary Ca and aP concentrations can be reduced when phytase is supplemented. It also may be beneficial to apply the enzyme nutrient matrix to other nutrients in the diet to maintain an optimal balance of nutrients in the digesta.

The objective was to characterise degradation of myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) and formation of inositol phosphate (InsP) isomers in different segments of the broiler digestive tract. Influence of an Aspergillus niger (PhyA) and two Escherichia coli-derived (PhyE1 and PhyE2) phytases was also investigated. A total of 600 16-d-old broilers were allocated to forty floor pens (ten pens per treatment). Low-P (5·2 g/kg DM) maize-soyabean meal-based diets were fed without (basal diet; BD) or with a phytase added. On day 25, digesta from different digestive tract segments were pooled per segment on a pen-basis, freeze-dried and analysed for P, InsP isomers and the marker TiO2. InsP6 degradation until the lower ileum (74 %) in BD-fed birds showed a high potential of broilers and their gut microbiota to hydrolyse InsP6 in low-P diets. Different InsP patterns in different gut segments suggested the involvement of phosphatases of different origin. Supplemented phytases increased InsP6 hydrolysis in the crop (P < 0·01) but not in the lower ileum. Measurements in the crop and proventriculus/gizzard confirmed published in vitro degradation pathways of 3- and 6-phytases for the first time. In the intestinal segments, specifically formed InsP4-5 isomers of supplemented phytases were still present, indicating further activity of these enzymes. Myo-inositol tetrakisphosphate (InsP4) accumulation differed between PhyE1 and PhyE2 compared with PhyA in the anterior segments of the gut (P < 0·01). Thus, the hydrolytic cleavage of the first phosphate group is not the only limiting step in phytate degradation in broilers.