Isolation and in-vitro characterization of extracellular phytase producing bacterial isolates for potential application in poultry feed.

BACKGROUND: Phytase catalyses the breakdown of complex organic forms of phosphorous into simpler forms by sequential hydrolysis of phosphate ester bonds to liberate the inorganic phosphate. Supplementation of feeds with bacterial phytase therefore could enhance the bioavailability of phosphorus and micronutrients. Hence, the aim of this study was to isolate and characterize phytase producing bacteria from rhizosphere soil, fresh poultry excreta, and cattle shed to evaluate their potential in improving poultry feeds. Phytase producing bacteria were isolated using wheat bran extract medium. RESULTS: A total of 169 bacterial isolates were purified and screened for phytase activity. Out of these, 36 were confirmed as positive for phytase enzyme activity. The bacterial isolates were identified by cultural, morphological, and biochemical features. The isolates were also identified by using 16 S rRNA gene sequencing. The bacterial isolates (RS1, RS8, RS10 and RS15) were provided with gene bank database accession numbers of MZ407562, MZ407563, MZ407564 and MZ407565 respectively. All isolates increased phytase production when cultured in wheat bran extract medium (pH 6) supplemented with 1% (wt/v) galactose and 1% (wt/v) ammonium sulphate incubated at 50oC for 72 h. Proximate composition analysis after supplementation of phytase showed that phytase supplementation improved bioavailability of phosphorus, calcium, potassium and sodium in poultry feed. CONCLUSIONS: Overall, this study showed that the nutritional value of poultry feed can be improved using microbial phytase enzyme which reduces the cost of supplementation with inorganic phosphate.

A first model of the fate of dietary calcium and phosphorus in broiler chickens.

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

Toward standardized amino acid matrices for exogenous phytase and protease in corn-soybean meal-based diets for broilers.

A total of 468 male Ross 308 broilers were used in a digestibility study to determine the additivity of apparent or standardized amino acid (AA) digestibility values for corn, soybean meal (SBM), or a mixture of corn and SBM that were supplemented, or not, with either phytase, protease, or a combination of phytase and protease. These treatments generated a total of 12 experimental diets that were arranged in a 3 × 4 design. A nitrogen-free diet was also fed to estimate endogenous AA loss. Apparent and standardized AA digestibility values were assessed on day 28 posthatch. The apparent digestibility of AA in the complete diet was higher (P < 0.05) than expected based on the digestibility of the corn and SBM individually. However, this overestimation was corrected by the adjustment to standardized values. Importantly, addition of protease or the combination of protease and phytase increased (P < 0.05) the digestibility of AA in corn and SBM. Furthermore, these effects were arithmetically coherent with respect to the measured effects of the enzymes in the mixture of corn and SBM, even improving the additivity of AA digestibility values when assessed on an apparent basis. This study demonstrates that the effect of exogenous protease and phytase on AA digestibility in complete diets is predictable based on measurements made in individual ingredients. In addition to improving digestibility values per se, exogenous protease and phytase may enhance precision in least cost formulation systems.

Limestone particle size, calcium and phosphorus levels, and phytase effects on live performance and nutrients digestibility of broilers.

Limestone particle size (PS) affects its solubility and thus can influence broiler performance by altering the rate of calcium (Ca) release into the gastrointestinal tract. The objective of this research was to determine, using 2 × 2 × 2 factorial arrangement, the influence of PS (fine and coarse) and supplemented phytase (0 and 1,000 FYT/kg) in diets formulated with 2 Ca and Pi levels (positive control [PC]; negative control [NC]) on live performance, bone ash, and apparent ileal nutrients digestibility (AID). Starter PC: 0.9 Ca and 0.45 Pi; NC: 0.72 Ca and 0.03 Pi. Grower PC: 0.76 Ca and 0.38 Pi; NC: 0.58 Ca and 0.23 Pi. The 8 diets were assigned randomly to a total of 1,512 birds, with 21 birds per pen and 9 pens per treatment. The main effects of PS and Ca and Pi levels had no influence on feed intake (FI), body weight gain (BWG), or feed conversion ratio. Adding phytase improved BWG by 8 g and 50 g and FI by 25 g and 56 g at 0-14 D (P ≤ 0.05) and 0-35 D (P ≤ 0.05), respectively. Interaction between Ca and Pi levels and phytase improved BWG and FI for 0-14 D (P ≤ 0.05) and BWG during 15-28 D (P ≤ 0.05) for PC without phytase and for PC and NC with phytase when compared with NC without phytase. Birds fed PC without phytase, or either PC or NC with phytase were about 96 g heavier than NC without phytase. Birds fed either PC or NC diet with coarse limestone or PC with fine limestone gained approximately 14 g more (P ≤ 0.05) than birds fed NC with fine limestone for BWG at 0-14 D (P ≤ 0.05). Phytase increased tibia bone ash (14 D) by 1% (P ≤ 0.05). AID of Ca and Pi at 14 D was improved (P ≤ 0.05) by 66% when phytase was added to coarse limestone. Results indicate that phytase improved broiler performance without being affected by PS. Furthermore, phytase had greater influence on coarse limestone than on fine limestone for bone ash and AID. Ca and Pi levels were the most influential factors in determining bone ash although phytase inclusion could lead to an improvement during early days.

[A simple indicator for ruling out inadequate phosphorus digestibility in growing pigs]. / Eine einfache Methode zum Ausschluss unzureichender Phosphorverdaulichkeit bei wachsenden Schweinen.

OBJECTIVE: Species differences in calcium and phosphorus metabolism can be expressed via dietary and faecal calcium/phosphorus ratios. The aim of the present meta-analysis was to use faecal Ca/P ratios as an indicator of phosphorus digestibility in growing pigs as a simple diagnostic tool. This tool can be useful in cases of suspected phosphorus deficiency but adequate dietary calcium and phosphorus contents. MATERIAL AND METHODS: Studies (n = 34) with phytase-supplemented (PHYT; n = 110) and non-supplemented control (CON; n = 106) diets were analysed for dietary intake, faecal excretion and apparent digestibility of calcium and phosphorus. A modified Lucas-test was used for both minerals plotting intake against faecal excretion (mg/kg body weight0.75). RESULTS: For calcium, there was no significant difference between PHYT and CON (p = 0.29) while in accordance with literature, the relative faecal phosphorus excretion was significantly lower in PHYT than CON (p < 0.01). Faecal calcium/phosphorus ratios were calculated and grouped according to the animals' body weight and apparent phosphorus digestibility (body weight ≤ 30 kg: apparent phosphorus digestibility ≤ 60 % and > 60 %; body weight > 30 kg: apparent phosphorus digestibility ≤ 40 % and > 40 %). CONCLUSION: Data distribution as displayed in a box plot shows that - given a dietary Ca/P ratio of > 1.2 - faecal Ca/P ratios of > 1.5 in pigs with a body weight of ≤ 30 kg and of > 1.2 in pigs with a body weight of > 30 kg indicate a high apparent digestibility of phosphorus of > 60 % and > 40 %, respectively. CLINICAL RELEVANCE: When faecal samples reveal a Ca/P ratio above the indicated thresholds, a low phosphorus digestibility is unlikely. No conclusion regarding the apparent phosphorus digestibility can be drawn from faecal Ca/P ratios below this threshold.

Application of the CEN/ISO Standard for Phytase Activity Measurements to a New Phytase Product: Determination of a Robust Conversion Factor by an Interlaboratory Study for Screening Feed Samples.

Background: Phytase-based preparations are important feed additives currently authorised in the European Union (EU). The European Standard (EN) and International Organization for Standardization (ISO) standard 30024 describes a harmonized method for the determination of phytase activity and is fit-for-purpose for official control of a group of phytase products. However, it is not suitable for the determination of the phytase activity of a new feed additive encoded as 4a16 in the EU Register of Feed Additives, to which a slightly different phytase activity definition has been attributed. Objective: To establish a robust conversion factor to support official control laboratories that apply the EN ISO method when monitoring feed products containing 4a16. Methods: The phytase activity of test materials was determined by the participants using the EN ISO and/or the "applicant" methods. Results: Robust relative SDs for repeatability and for reproducibility of the methods applied for the determination of the phytase activity in the materials containing the 4a16 feed additive ranged from 2.6 to 22% (EN ISO method) and from 2.4 to 39% (applicant method). Conclusions: The data obtained confirmed the performance characteristics published for other phytase-based feeds in the related standard methods. These results allowed us to estimate a factor of 2.68 to convert phytase activities measured with the EN ISO method into the enzyme activity measured with the applicant method. Highlights: The obtained conversion factor will allow EU official laboratories to screen feed samples supplemented with the 4a16 phytase by applying EN ISO Standard 30024.

Low digestibility of phytate phosphorus, their impacts on the environment, and phytase opportunity in the poultry industry.

Phosphorus is an essential macro-mineral nutrient for poultry, needed for the body growth, development of bones, genomic function, good quality flesh, and eggs production. The imbalance of organic phosphorus sources in the diet mostly affect the phosphorus digestibility, reduces the poultry performance and health, and increases the environmental pollution burden. A study was reviewed to estimate the low phytate phosphorus digestibility of ingredients in poultry diet and their impacts on environmental ecosystem and opportunity of phytase supplementation. Plant ingredients mostly used in poultry diets are rich in phytate phosphorus. The phytate phosphorus digestibility and utilization is low in the gut of birds which leads to decrease other nutrients digestibility and increase excessive excretion of phosphorus with additional nutrients in the manure. When that manure applied to the lands containing excessive residual phosphorus and additional nutrients which pollute soil, groundwater disturbed the entire ecosystem. This issue is developed by poultry due to lack of digestive enzyme phytase which promotes the phytate phosphorus during digestion and reduces the excessive losses of phosphorus in excreta. To overcome this matter, the addition of mostly exogenous phospho-hydrolytic phytase enzymes in the diet, i.e. Escherichia coli, Peniophora lycii, Aspergillus niger, and Ficum, are the possible ways to increase the digestibility and utilization of phytate phosphorus and promote the stepwise release of phosphorus from phytate and significantly decrease phosphorus excretion. The aim of this review is to highlight the role of phytase supplementation in the poultry feeding, improvement of phytate phosphorus digestibility with performance, and reduction of phosphorus pollution from the environment.

Microfluidic Platform for Multimodal Analysis of Enzyme Secretion in Nanoliter Droplet Arrays.

High-throughput screening of cell-secreted proteins is essential for various biotechnological applications. In this article, we show a microfluidic approach to perform the analysis of cell-secreted proteins in nanoliter droplet arrays by two complementary methods, fluorescence microscopy and mass spectrometry. We analyzed the secretion of the enzyme phytase, a phosphatase used as an animal feed additive, from a low number of yeast cells. Yeast cells were encapsulated in nanoliter volumes by droplet microfluidics and deposited on spatially defined spots on the surface of a glass slide mounted on the motorized stage of an inverted fluorescence microscope. During the following incubation for several hours to produce phytase, the droplets can be monitored by optical microscopy. After addition of a fluorogenic substrate at a defined time, the relative concentration of phytase was determined in every droplet. Moreover, we demonstrate the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to monitor the multistep conversion of the native substrate phytic acid by phytase secreted in 7 nL droplets containing 50-100 cells. Our method can be adapted to various other protocols. As the droplets are easily accessible, compounds such as assay reagents or matrix molecules can be added to all or to selected droplets only, or part of the droplet volume could be removed. Hence, this platform is a versatile tool for questions related to cell secretome analysis.

Subsequent bone and metabolic responses of broilers to high-non-phytate phosphorus diets in the starter period.

1. An experiment was performed to elucidate the subsequent effects of high-non-phytate phosphorus (NPP) diets on growth performance, blood metabolites, bone characteristics and P retention of broilers fed on low-NPP grower diets. The 42-d study was designed as a 2 × 2 × 2 + 1 factorial, which included two starter NPP concentrations (4.5 and 5.5 g/kg; d 0-21), two grower NPP concentrations (1.5 and 2.3 g/kg; d 22-42), with or without phytase (1000 FTU/kg), with a reference diet containing an adequate NPP concentration over the course of the trial. 2. In the starter period, growth performance and P retention were not affected by experimental diets. The high-NPP diet increased plasma P concentration, increased tibia ash and tibia P contents and decreased plasma alkaline phosphatase (ALP) activity at d 21. 3. No significant interaction was observed between NPP concentrations in the starter and grower periods and phytase. The main effect data indicated that the increase in NPP concentration in the starter diets had no effects on growth performance in the grower period and overall. The high-NPP diet in the early stage of growth reduced plasma P concentration, plasma ALP activity and tibia ash content at d 42. The main effect data also showed that exogenous phytase increased body weight gain in the grower period and overall. 4. It can be concluded that feeding increased NPP diets have no effects on growth performance in the starter period. This feeding strategy results in negative effects on plasma P concentration and bone ash content at d 42. Also, exogenous phytase is effective in improving growth performance, bone characteristics and apparent P retention of growing broilers fed diets that are inadequate in phosphorus.

P and Ca digestibility is increased in broiler diets supplemented with the high-phytase HIGHPHY wheat.

Around 70% of total seed phosphorus is represented by phytate which must be hydrolysed to be bioavailable in non-ruminant diets. The limited endogenous phytase activity in non-ruminant animals make it common practice to add an exogenous phytase source to most poultry and pig feeds. The mature grain phytase activity (MGPA) of cereal seeds provides a route for the seeds themselves to contribute to phytate digestion, but MGPA varies considerably between species and most varieties in current use make negligible contributions. Currently, all phytases used for feed supplementation and transgenic improvement of MGPA are derived from microbial enzymes belonging to the group of histidine acid phosphatases (HAP). Cereals contain HAP phytases, but the bulk of MGPA can be attributed to phytases belonging to a completely different group of phosphatases, the purple acid phosphatases (PAPhy). In recent years, increased MGPAs were achieved in cisgenic barley holding extra copies of barley PAPhy and in the wheat HIGHPHY mutant, where MGPA was increased to ~6200 FTU/kg. In the present study, the effect of replacing 33%, 66% and 100% of a standard wheat with HIGHPHY wheat was compared with a control diet with and without 500 FTU of supplemental phytase. Diets were compared by evaluating broiler performance, ileal Ca and P digestibility and tibia development, using nine replicate pens of four birds per diet over 3 weeks from hatch. There were no differences between treatments in any tibia or bird performance parameters, indicating the control diet did not contain sufficiently low levels of phosphorus to distinguish effect of phytase addition. However, in a comparison of the two wheats, the ileal Ca and P digestibility coefficients for the 100% HIGHPHY wheat diets are 22.9% and 35.6% higher, respectively, than for the control diet, indicating the wheat PAPhy is functional in the broiler digestive tract. Furthermore, 33% HIGHPHY replacement of conventional wheat, significantly improved Ca and P digestibility over the diet-supplemented exogenous phytase, probably due to the higher phytase activity in the HIGHPHY diet (1804 v. 1150 FTU). Full replacement by HIGHPHY gave 14.6% and 22.8% higher ileal digestibility coefficients for Ca and P, respectively, than for feed supplemented with exogenous HAP phytase at 500 FTU. This indicates that in planta wheat PAPhys has promising potential for improving P and mineral digestibility in animal feed.

Contribution of intestinal- and cereal-derived phytase activity on phytate degradation in young broilers.

There is little consensus as to the capability of poultry to utilize dietary phytate without supplemental phytase. Therefore, an experiment was conducted to examine the extent to which endogenous phytase of intestinal and cereal origin contributes to phytate degradation in birds aged 0 to 14 d posthatch. Ross 308 broilers (n = 720) were fed one of 4 experimental diets with differing dietary ingredient combinations and approximate total phytate levels of 10 g/kg, dietary phytase activity analyzed at 460 U/kg, dietary calcium (Ca) levels of 11 g/kg, and nonphytate-phosphorus (P) levels of 4 g/kg. Broiler performance, gizzard, duodenum, jejunum and ileum pH, Ca and P digestibility and solubility, amount of dietary phytate hydrolyzed in the gizzard, jejunum, and ileal digesta phytase activity were analyzed at d 4, 6, 8, 10, 12, and 14 posthatch. Intestinal endogenous phytase activity increased significantly (P < 0.001) between d 4 and 6, resulting in increased phytate hydrolysis in the gizzard (P = 0.003), jejunum (P < 0.001), and ileum (P < 0.001). Phytase activity and phytate hydrolysis continued to increase with age, with a greater phytase activity and associated increase in phytate hydrolysis and mineral utilization between d 10 and 12. Gizzard and jejunum Ca and P solubility and ileal Ca and P digestibility increased significantly (P < 0.001), and gastrointestinal pH decreased significantly (P < 0.001) between d 4 and 6. By d 14, phytase activity recovered in the ileum was approximately 45 U/kg. There were strong correlations between phytase activity measured in the ileum and phytate hydrolyzed in the gizzard (r = 0.905, P < 0.001), jejunum (r = 0.901, P = 0.023), and ileum (r = 0.938, P = 0.042). This study shows intestinal- and dietary-derived endogenous phytase activity is responsible for phytate-P hydrolysis in broilers.

Effects of phytase supplementation in mature horses fed alfalfa hay and pelleted concentrate diets.

An experiment was conducted to study P digestibility in mature horses because of the growing environmental concerns regarding P runoff and previous equine research focused mostly on young and growing animals or used ponies as a model. Phytase supplementation of swine and poultry diets can result in greater phytate-P digestibility, leading to a decreased need for inorganic P supplementation and a decrease in P excreted to the environment; this, however, has not been demonstrated in the horse. Six mature Arabian geldings were fed 6 diets consisting of pelleted concentrate and alfalfa hay. The concentrates consisted mainly of soybean hulls, ground corn, wheat midds, broken rice, and beet pulp, and phytase was added to the concentrates accordingly before pelleting. There were 3 diet types: control (concentrate and hay), high P (greater P concentrate and hay), and forage only, and each diet type included 1 phytase-supplemented diet and 1 non-phytase-supplemented diet, resulting in 6 total diets. Phytase supplementation for the forage only diet was accomplished by feeding a nominal amount of concentrate formulated solely as a vehicle for the phytase. Horses had unrestricted access to water throughout the experiment. Using a Latin square design, all horses received all diets over a period of 12 wk. In each week, the new diet was fed for 11 d of diet acclimation, which was followed by a 3-d total collection of feces and urine for each horse. There was no effect (P < 0.05) of phytase supplementation on P output in the urine or feces, resulting in no differences in P apparent digestibility. Analysis of the feed and feces for phytate revealed a 93% average disappearance rate of phytate, indicating that horses are highly capable of degrading phytate and that phytase supplementation was not beneficial. Thus, the results indicate that mature horses are able to maintain a near 0 P balance, with adequate P provided in the diet even as phytate, and increased P intakes above requirement may result in increased potentially detrimental outputs to the environment.

Enhanced phytase production from Achromobacter sp. PB-01 using wheat bran as substrate: prospective application for animal feed.

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB-01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X-100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB-01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non-starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro-industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium.

Evaluation of a highly soluble calcium source and phytase in the diets of broiler chickens.

An experiment was conducted to evaluate the influence of a highly soluble Ca (HSC) source on performance, bone ash and mineralization, and apparent ileal digestibility of Ca, P, N, and energy in Ross 708 broiler chickens. Dietary Ca was supplied by the HSC source and monocalcium phosphate to provide 4 levels of Ca (0.45, 0.60, 0.75, or 0.90%). Available P (aP) was maintained at 0.32% in all the diets. Each diet was supplemented with 0, 500, or 2,500 U/kg of phytase as a 4 × 3 factorial. An additional diet was formulated using limestone to contain 0.90% Ca and 0.45% aP as a positive control (PC). Diets were fed to 7 replicate cages of 8 birds per cage from d 0 to 21 posthatch. Feed intake (FI) was linearly (P ≤ 0.05) reduced, and BW gain was quadratically (P ≤ 0.05) reduced as Ca from HSC increased from 0.45 to 0.90%. Phytase supplementation linearly (P ≤ 0.05) increased FI and BW gain and improved feed conversion ratio. Tibia ash and P weights were lower in birds fed 0.90% Ca from HSC compared with broilers fed 0.90% Ca from limestone. Phytase supplementation increased tibia ash and P weights in broilers fed 0.60, 0.75, or 0.90% HSC (Ca level × phytase P ≤ 0.05). Phytase supplementation linearly (P ≤ 0.05) increased tibia Ca weight. Reducing HSC from 0.90 to 0.45% or increasing phytase supplementation linearly (P ≤ 0.05) improved ileal P digestibility. Ileal Ca digestibility increased linearly only in broilers fed 0.45% Ca from HSC as phytase supplementation increased (Ca level × phytase P ≤ 0.05). Nitrogen or energy digestibility were quadratically (P ≤ 0.05) influenced as HSC or phytase increased. In conclusion, feeding HSC with phytase allowed for reductions in dietary Ca while maintaining broiler performance and bone ash. In addition, P digestibility and P in the bones were increased in broilers fed reduced dietary Ca in the presence of phytase.

[Tannins, phytic phosphorus, phytase activity in the seed of 12 sorghum grain hybrids (Sorghum bicolor (L) Moench)]. / Contenido de taninos, fósforo fítico y actividad de fitasas en el grano de 12 hibridos de sorgo granífero (Sorghum bicolor (L) Moench).

In order to evaluate the variability in concentration of secondary metabolites among grains from different sorghum hybrids (Sorghum bicolor (L) Moench) and provide some information for rational use in animal feeds, grain samples were collected from 12 hybrids (Criollo 1, Criollo 27, Chaguaramas III, Chaguaramas VII, Cristiani Burkard H-8046-2, Guanipa 95, Himeca 101, Himeca 400, Pioneer 81-G67, Pioneer 83-G88, Pioneer 86-P42 y Tecsem 120) sowed on 2008 in the Turén municipality, Portuguesa State-Venezuela. Using standardized analytical methods, hybrids showed polyphenolics compounds contents (0.92 +/- 0.37% TaE), total (0.61 +/- 0.29% TaE) and condensed tannins (0.95 +/- 0.65% LeuE), phytic phosphorus (0.17 +/- 0.03%) and intrinsic phytase activity (12.3 +/- 6.74 U/kg) in the ranges described in the literature, with an important variation among hybrids that must be considered for their inclusion levels in the formulation of balanced feeds, as well as plant breeding programs.

Differences in phytase activity and phytic acid content between cultivated and Tibetan annual wild barleys.

The Qinghai-Tibetan Plateau in China is considered to be one of the original centers of cultivated barley. At present, little is known about the phytase activity (Phy) or phytic acid content (PA) in grains of Tibetan annual wild barley. Phy and PA were determined in grains of 135 wild and 72 cultivated barleys. Phy ranged from 171.3 to 1299.2 U kg(-1) and from 219.9 to 998.2 U kg(-1) for wild and cultivated barleys, respectively. PA and protein contents were much higher in wild barley than in cultivated barley. Tibetan annual wild barley showed a larger genetic diversity in phytase activity and phytic acid and protein contents and is of value for barley breeding. There is no significant correlation between phytase activity and phytic acid or protein content in barley grains, indicating that endogenous phytase activity had little effect on the accumulation of phytic acid.

Effect of dietary supplementation with copper sulfate or tribasic copper chloride on the growth performance, liver copper concentrations of broilers fed in floor pens, and stabilities of vitamin E and phytase in feeds.

An experiment was conducted using a total of 840, 1-day-old, Arbor Acres commercial male broilers to compare copper (Cu) sulfate and tribasic Cu chloride (TBCC, Cu(2)(OH)(3)Cl) as sources of supplemental Cu for broilers fed in floor pens. Chicks were randomly allotted to one of seven treatments for six replicate pens of 20 birds each, and were fed a basal corn-soybean meal diet (10.20 mg/kg Cu) supplemented with 0, 100, 150, or 200 mg/kg Cu from either Cu sulfate or TBCC for 21 days. Chicks fed 200 mg/kg Cu as TBCC had a higher (P < 0.05) average daily gain (ADG) than those consuming other diets. Liver Cu contents of broilers fed diets supplemented with TBCC were numerically lower (P > 0.05) than those of broilers fed diets supplemented with Cu sulfate. The vitamin E contents and the phytase activities in the feed fortified with TBCC were higher (P < 0.01) and numerically higher (P > 0.05) compared with those in the feeds fortified with Cu sulfate stored at room temperature, respectively. The vitamin E contents in liver and plasma of broilers fed diets supplemented with TBCC were higher (P < 0.05) than those of birds fed diets supplemented with Cu sulfate. This result indicates that TBCC is more effective than Cu sulfate in improving the growth of broilers fed in floor pens, and it is chemically less active than Cu sulfate in promoting the undesirable oxidation of vitamin E in feeds.

Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies.

AIMS: To isolate, clone and express a novel phytase gene (phy) from Bacillus sp. in Escherichia coli; to recover the active enzyme from inclusion bodies; and to characterize the recombinant phytase. METHODS AND RESULTS: The molecular weight of phytase was estimated as 40 kDa on SDS-polyacrylamide gel electrophoresis. A requirement of Ca(2+) ions was found essential both for refolding and activity of the enzyme. Bacillus phytase exhibited a specific activity of 16 U mg(-1) protein; it also revealed broad pH and temperature ranges of 5.0 to 8.0 and 25 to 70 degrees C, respectively. The K(m) value of phytase for hydrolysis of sodium phytate has been determined as 0.392 mmol l(-1). The activity of enzyme has been inhibited by EDTA. The enzyme exhibited ample thermostability upon exposure to high temperatures from 75 to 95 degrees C. After 9 h of cultivation of transformed E. coli in the bioreactor, the cell biomass reached 26.81 g wet weight (ww) per l accounting for 4289 U enzyme activity compared with 1.978 g ww per l producing 256 U activity in shake-flask cultures. In silico analysis revealed a beta-propeller structure of phytase. CONCLUSIONS: This is the first report of its kind on the purification and successful in vitro refolding of Bacillus phytase from the inclusion bodies formed in the transformed E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient and reproducible protocols for cloning, expression, purification and in vitro refolding of Bacillus phytase enzyme from the transformed E. coli have been developed. The novel phytase, with broad pH and temperature range, renaturation ability and substrate specificity, appears promising as an ideal feed supplement. Identification of site between 179th amino acid leucine and 180th amino acid asparagine offers scope for insertion of small peptides/domains for production of chimeric genes without altering enzyme activity.

Estimating equivalency values of microbial phytase for amino acids in growing and finishing pigs fitted with steered ileo-cecal valve cannulas.

Ten crossbred barrows (48.3 +/- 2.3 kg of initial BW) fitted with steered ileo-cecal valve cannulas were used to investigate the effects of supplemental microbial phytase on the apparent ileal digestibilities (AID) of AA, Ca, P, N, and DM, and the apparent total tract digestibilities of Ca, P, N, and DM. All diets were corn-soybean meal-based, and contained 0.44% Ca and 0.40% total P. Diets 1, 2, and 3 contained 12.0, 11.1, and 10.2% CP, respectively. Diets 4 and 5 had the same ingredient composition as diet 3, plus 250 and 500 U/kg phytase (Natuphos), respectively. Pigs were randomly allotted to 1 of 5 dietary treatments in a paired 5 x 5 Latin square with an extra period to test for carryover effects. Each 14-d period consisted of a 7-d adjustment followed by a 3-d total collection, a 12-h ileal digesta collection, a 3-d readjustment, and a second 12-h ileal digesta collection. Pigs were housed individually in metabolism pens (1.2 x 1.2 m). Water was supplied ad libitum, and feed was supplied at a level of 9% of the metabolic BW (BW(0.75)) per day in 2 equal daily feedings. As the dietary CP concentration increased, the AID of CP and all AA measured increased linearly (P < 0.05) with the exception of proline. In addition, the apparent total tract digestibilities (grams per day) and retention of N (grams per day) increased linearly (P < 0.01) with increasing CP levels. Supplementing diets with phytase increased the AID of Ca (P < 0.01), P (P < 0.001), CP (P = 0.07), and the AA (P < 0.10) Gly, Ala, Val, Ile, Thr, TSAA, Asp, Glu, Phe, Lys, and Arg. Protein and phytase response equations were generated for those AA affected (P < 0.10) by both CP level and phytase supplementation. Based on these equations, 500 U/kg of phytase can replace 0.52 percentage units of the dietary CP, which includes a 0.03 percentage unit improvement in Lys AID. The results of this study show that supplementing pig diets with microbial phytase improves CP and AA digestibilities in addition to Ca and P digestibilities.

Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control.

This study examined the feasibility of using the promoter of the pig parotid secretory protein (PSP) gene for expression of the phytase transgene in mouse models. The pig parotid secretory protein gene is specifically expressed at high levels in the salivary glands. The 10-kb upstream promoter region of the gene necessary for tissue-specific expression has been identified. We have constructed phytase transgenes composed of the appA phytase gene from Escherichia coli driven by the upstream promoter region of the pig PSP gene with a 3' tail of either bovine growth hormone or the pig PSP gene polyadenylation signal. Transgenic mouse models with the construct showed that the upstream region of the pig PSP gene is sufficient for directing the expression of phytase transgenes in the saliva. Expression of salivary phytase reduced fecal phytate by 8.5 and 12.5% in 2 transgenic mouse lines, respectively. These results suggest that the expression of phytase in salivary glands of monogastric animals offers a promising biological approach to relieve the requirement for dietary phosphate supplements and to reduce phosphorus pollution from animal agriculture.