Efficacy of the feed additive consisting of 6-phytase (produced by Komagataella phaffiiCGMCC 7.19) (Nutrase P) for chickens for fattening, other poultry for fattening or reared for laying and ornamental birds (Nutrex N.V.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the efficacy of 6-phytase produced by Komagataella phaffii CGMCC 7.19 (Nutrase P) as a zootechnical feed additive for chickens for fattening, other poultry for fattening or reared for laying and ornamental birds. In a previous opinion, the FEEDAP Panel concluded on the efficacy of Nutrase P for the target species at the level of 1,500 phytase units (FTU)/kg but could not conclude at the minimum recommended use level of 500 FTU/kg complete feed. The applicant has provided supplementary information, consisting in the statistical re-analysis of the long-term study assessed in the original opinion, in order to support the efficacy of the additive at the minimum recommended level of 500 FTU/kg complete feed. Considering the previously submitted studies and the re-analysis of the long-term study, the Panel concluded that the additive has a potential to be efficacious for chickens for fattening, other poultry for fattening or reared for laying and ornamental birds under the proposed conditions of use.

Safety and efficacy of the feed additive consisting of 6-phytase (produced by Aspergillus oryzaeDSM 33699) (RONOZYME® Hiphos GT/L) for poultry, pigs for fattening, weaned piglets and sows (DSM Nutritional Products Ltd).

The additive RONOZYME® Hiphos (GT/L) contains 6-phytase produced with a genetically modified strain of the filamentous fungus Aspergillus oryzae, it is currently authorised for poultry, pigs for fattening, weaned piglets and sows. The applicant has requested to change the production strain, substituting strain A. oryzae DSM 22594 for A. oryzae DSM 33699. RONOZYME® Hiphos (GT/L), manufactured with the production strain A. oryzae DSM 33699, did not give rise to safety concerns with regard to the genetic modification of the production strain. No viable cells of the production strain nor its recombinant DNA were detected in an intermediate product representative of both final forms of the additive. RONOZYME® Hiphos (GT/L) was considered safe for poultry, pigs for fattening, weaned piglets and sows at the recommended inclusion levels of 500-4,000 FYT/kg complete feed. The use of RONOZYME® Hiphos GT and L manufactured with the production strain A. oryzae DSM 33699 raised no concerns for consumers. In the absence of data on the final formulations, the Panel could not conclude on the potential of the additive to be irritant to eyes or skin, or a skin sensitiser. Due to the proteinaceous nature of the active substance, the additive was considered a respiratory sensitiser. The additive manufactured by A. oryzae DSM 33699 raises no safety concerns for the environment. The additive has the potential to be efficacious in poultry, pigs for fattening, weaned piglets and sows at 500 FYT/kg complete feed.

Safety and efficacy of a feed additive consisting of 6-phytase produced by Komagataella phaffiiCGMCC 7.370 (VTR-phytase powder/liquid) for all pigs and all avian species (Victory Enzymes GmbH).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of 6-phytase (VTR-phytase) as zootechnical feed additive for all pigs and all avian species. The additive VTR-phytase consists of 6-phytase and it is available in solid and liquid forms. VTR phytase (liquid/solid) was produced by a genetically modified strain of Komagataella phaffii (CGMCC 7.370). The genetic modification of the production strain does not give rise to safety concerns. Viable cells of the production strain and its DNA were not detected in the final products. The additive does not pose any safety concern regarding the production strain. VTR phytase (liquid/solid) produced by Komagataella phaffii CGMCC 7.370 is safe for all Suidae and all avian species at the proposed conditions of use. The use of both forms of the additive under assessment in animal nutrition under the proposed conditions of use raises no safety concerns for consumers or for the environment. The liquid VTR phytase and powder VTR phytase are non-irritant to skin or eyes but should be considered skin and respiratory sensitisers. The additive has the potential to be efficacious in laying hens at 1,000 U phytase/kg complete feed. The conclusion can be extrapolated to other birds for egg production or breeding. The FEEDAP Panel cannot conclude on the efficacy of all pigs or growing poultry species.

Nutritional and Functional Roles of Phytase and Xylanase Enhancing the Intestinal Health and Growth of Nursery Pigs and Broiler Chickens.

This review paper discussed the nutritional and functional roles of phytase and xylanase enhancing the intestinal and growth of nursery pigs and broiler chickens. There are different feed enzymes that are currently supplemented to feeds for nursery pigs and broiler chickens. Phytase and xylanase have been extensively studied showing consistent results especially related to enhancement of nutrient digestibility and growth performance of nursery pigs and broiler chickens. Findings from recent studies raise the hypothesis that phytase and xylanase could play functional roles beyond increasing nutrient digestibility, but also enhancing the intestinal health and positively modulating the intestinal microbiota of nursery pigs and broiler chickens. In conclusion, the supplementation of phytase and xylanase for nursery pigs and broiler chickens reaffirmed the benefits related to enhancement of nutrient digestibility and growth performance, whilst also playing functional roles benefiting the intestinal microbiota and reducing the intestinal oxidative damages. As a result, it could contribute to a reduction in the feed costs by allowing the use of a wider range of feedstuffs without compromising the optimal performance of the animals, as well as the environmental concerns associated with a poor hydrolysis of antinutritional factors present in the diets for swine and poultry.

Safety and efficacy of a feed additive consisting of 6-phytase produced by Trichoderma reesei CBS 146250 (Axtra® PHY GOLD 30L, Axtra® PHY GOLD 30T, Axtra® PHY GOLD 65G) for all poultry species and all pigs (Danisco (UK) ltd).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of 6-phytase produced by the genetically modified strain Trichoderma reesei CBS 146250 (Axtra® PHY GOLD 30L, Axtra® PHY GOLD 30 T and Axtra® PHY GOLD 65G) as a zootechnical feed additive for all poultry species and all pigs. The FEEDAP Panel concluded that the genetic modification of the production strain does not give rise to safety concerns. Based on the no observed adverse effect level identified in a subchronic oral toxicity study in rats, the additive was considered safe for all poultry species and all pigs at the proposed conditions of use. The Panel also concluded that the use of the product as a feed additive does not give rise to concerns for consumers and the environment. Owing to the lack of data obtained with the final formulations, the Panel cannot conclude on the potential of the additive to be irritant to eyes or skin. Due to the proteinaceous nature of the active substance, it is considered a respiratory sensitiser. The panel concludes that the additive is efficacious in increasing the phosphorus utilisation when supplemented at 500 FTU/kg for all growing poultry species and all pigs, and at 300 FTU/kg in laying hens and other laying birds.

Effect of limestone solubility on mineral digestibility and bone ash in nursery pigs fed diets containing graded level of inorganic phosphorus or increasing dose of a novel consensus bacterial 6-phytase variant.

The effect of a novel consensus bacterial 6-phytase variant (PhyG) on total tract digestibility (ATTD) of minerals and bone ash was evaluated in pigs fed diets containing medium- and high-solubility limestone (MSL and HSL, 69.6 and 91.7% solubility, respectively, at 5 min, pH 3.0) in a randomized complete block design. For each limestone, eight diets were formulated: an inorganic phosphate-free negative control (NC) based on wheat, corn, soybean-meal, canola-meal and rice-barn [0.18% standardized total tract digestible (STTD) P and 0.59% Ca]; the NC supplemented with 250, 500, 1,000, or 2,000 FTU/kg of PhyG, and; the NC with added monocalcium phosphate (MCP) and limestone to produce three positive controls (0.33, 0.27, and 0.21% STTD P, and 0.75, 0.70, and 0.64% Ca, respectively; PC1, PC2, PC3). In total, 128 pigs (12.8 ± 1.33 kg, 8 pigs/treatment, housed individually) were adapted for 16 d followed by 4 d of fecal collection. Femurs were collected from euthanized pigs on day 21. Data were analyzed by one-way ANOVA with means separation by Tukey's test, and by factorial analysis (2 x 4: 2 levels of limestone solubility, 4 STTD P levels, and 2 × 5: 2 levels of limestone solubility, 5 PhyG dose levels). Phytase dose-response was analyzed by curve fitting. A consistent negative effect of HSL on ATTD P and Ca was observed in control diets (P < 0.001). Across phytase-supplemented diets, HSL reduced (P < 0.05) ATTD Ca and P (% and g/kg) compared with MSL. Across limestones, increasing phytase dose level increased (P < 0.05) ATTD P exponentially. Limestone solubility had no effect on bone ash, but PhyG linearly increased (P < 0.05) bone ash; 500 FTU/kg or higher maintained bone ash (g/femur) equivalent to PC1. In conclusion, ATTD P and Ca were reduced by a high compared with a medium soluble limestone, but the novel phytase improved ATTD P and Ca independent of limestone solubility.

Microbial phytase is added to commercial pig diets to increase phosphorus (P) availability and reduce P excretion. It is known that an excess of calcium (Ca), mostly sourced from limestone, can affect phytase efficacy. However, less is known about the impact of limestone quality. This study investigated the effect of a medium- compared to a high-soluble limestone (MSL and HSL, respectively), in combination with increasing dose levels of a novel phytase (PhyG), on mineral digestibility and bone mineralization in young pigs. Without phytase, total tract digestibility of P was lower with HSL than MSL, indicating a negative effect of more soluble limestone on mineral digestibility. Increasing the phytase dose increased digestibility of P with either limestone, and reduced the negative effect of HSL at high dose. Bone mineralization was unaffected by limestone but markedly increased by phytase. At 1,000 FTU/kg, PhyG released an estimated 1.89 or 2.32 g/kg of digestible P from monocalcium phosphate in diets containing MSL and HSL, respectively based on bone ash content. The results demonstrate the efficacy of PhyG in young pig diets whilst indicating that limestone solubility can affect phytase efficacy.

Safety and efficacy of a feed additive consisting of 6-phytase (produced by Komagataella phaffii DSM 23036) (OptiPhos®) for poultry for fattening, chickens reared for laying, laying hens, turkeys reared for breeding, weaned piglets, pigs for fattening and sows for the renewal of their authorisation and for the new use in breeding hens and turkeys, ornamental birds, suckling piglets and minor pig species for fattening and breeding (Huvepharma EOOd).

OptiPhos® is the trade name of the feed additive under assessment containing 6-phytase produced by a genetically modified strain of Komagataella phaffii. It is authorised for use in chickens for fattening and reared for laying, turkeys for fattening and reared for breeding, other avian species for fattening, laying hens, weaned piglets, pigs for fattening and sows. This scientific opinion concerns the request for the renewal of the authorisation of the additive for the species/categories for which there is an authorisation and the extension of use to breeding hens, turkeys for breeding purposes, ornamental birds, minor pig species for fattening or breeding and suckling piglets for the period when solid feed is given. The applicant provided evidence that the additive currently in the market complies with the conditions of the authorisation. There is no new evidence that would lead the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) to reconsider previous conclusions that the additive remains safe for the animal species/categories, the consumer and the environment under the authorised conditions of use. This conclusion applies also to the new target species/categories for which a request of extension of use is made. The additive is not irritant to skin or eyes but should be considered a respiratory sensitiser. There was no need for assessing the efficacy of the additive for the authorised species/categories. The additive has the potential to be efficacious at 125 OTU/kg complete feed in all poultry species other than turkeys, ornamental birds, pigs for fattening, sows and minor pig species for fattening or breeding; and in turkeys for fattening, reared for breeding and for breeding purposes, weaned piglets and suckling piglets (for the period when solid feed is given) at 250 OTU/kg feed.

Effect of a novel consensus bacterial 6-phytase variant in grower pigs fed corn-soybean meal-based diets formulated with a full nutrient matrix and no added inorganic phosphorus.

The capacity of a novel consensus bacterial 6-phytase variant (PhyG) to entirely replace dietary inorganic phosphorus (Pi) source in grower pigs fed diets with reduction of calcium (Ca), net energy (NE), and digestible amino acids (AA) was evaluated, using growth performance and apparent total tract digestibility (ATTD) of nutrients as outcome measures. A total of 352 mixed-sex pigs (initial BW 23.4 kg) were randomized to 4 treatments, 8 pigs/pen, and 11 pens/treatment. Diets were corn-soybean meal-based and formulated by phase (grower 1, 25 to 50 and grower 2, 50 to 75 kg BW). The positive control diet (PC) provided adequate nutrients and a negative control diet (NC) was formulated without Pi (1.2 g/kg ATTD P) and reduced in Ca (-0.12 to -0.13 percentage points), NE (-32 kcal/kg), and digestible essential AA (-0.004 to -0.026 percentage points) vs. PC. Two further treatments comprised the NC plus 500 or 1,000 FTU/kg of PhyG. Data were analyzed by ANOVA, mean contrasts and orthogonal polynomial regression. Nutrient reductions in the NC reduced (P < 0.05) average daily gain (ADG) during grower 1 and overall (73 to 136 d of age), increased (P < 0.05) feed conversion ratio (FCR) during grower 1 and overall and tended to reduce (P < 0.1) average daily feed intake (ADFI) during grower 2 and overall, vs. PC. Phytase supplementation improved (P < 0.05) FCR during grower 1, ADG during grower 2 and overall, ATTD of DM and P, and tended to improve DE (P = 0.053) in a linear dose-dependent manner. PhyG at 1,000 FTU/kg resulted in growth performance (all measures, all phases) equivalent to PC. The findings demonstrate that PhyG at 1,000 FTU/kg totally replaced Pi in complex grower pig diets containing industrial co-products, compensated a full nutrient matrix reduction and maintained performance.

Safety assessment of a novel thermostable phytase.

A novel 6-phytase (Phytase TSP, trade name OptiPhos® PLUS) with improved thermostability has been developed for use in animal feed. The safety of the new phytase was evaluated by testing for genotoxicity and subchronic toxicity. In in vitro and in vivo genotoxicity assays Phytase TSP concentrate was not mutagenic and did not induce biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes. In a subchronic toxicity study, male and female rats administered 100, 500 or 1000 mg/kg body weight/day of Phytase TSP concentrate via oral gavage for 90 days had no mortalities, and no treatment-related effects on body weight, food consumption, clinical observations or ophthalmology. Furthermore, there were no changes in haematology, clinical chemistry, urinalysis, gross pathology, organ weights or histopathology that could be attributed to the test article. Several endpoints exhibited statistically significant effects, but none was dose-related or considered to be of toxicological relevance. Based on these results, Phytase TSP concentrate (OptiPhos® PLUS) was not genotoxic and the No Observed Adverse Effect Level (NOAEL) for male and female rats was 1000 mg/kg body weight/day.

Assessment of the application for renewal of authorisation of 6-phytase produced by Trichoderma reeseiCBS 122001 as a feed additive for pigs and poultry, from Roal Oy.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the assessment of the application for renewal of authorisation of 6-phytase produced by Trichoderma reesei CBS 122001 (brand name: Finase® EC). The applicant has provided evidence that the additive currently in the market complies with the existing conditions of authorisation. The Panel concludes that the additive remains safe for poultry for fattening, breeding and laying, and all pigs, the consumer and the environment under the authorised conditions of use. Regarding user safety, the Panel reiterates that the additive is not a skin or eye irritant or sensitiser but should be considered a potential respiratory sensitiser. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation. These conclusions also apply to the new proposed liquid formulation Finase® EC 5 L.

Safety and efficacy of Nutrase P (6-phytase) for chickens for fattening, other poultry for fattening, reared for laying and ornamental birds.

Nutrase P is available in powder, granulated, thermostable granulate and liquid forms. It is based on a 6-phytase produced by a genetically modified strain of K. phaffii. The production strain contains four copies of the ampicillin resistance gene and one copy of a bleomycin resistance gene. Although Nutrase PTS 10,000 (thermostable granulate) and Nutrase PG 10,000 (granulate) do not raise concern regarding the presence of viable cells of the production strain and its recombinant DNA, uncertainties remain on the presence of viable cells and DNA of the production strain in Nutrase PL 10,000 (liquid) and Nutrase PD 100,000 (powder). The FEEDAP Panel cannot conclude on the safety of the additive, in any form, for the target species for which application is made due to major limitations in the study in chickens for fattening provided. Considering the production strain and the results obtained in the genotoxicity studies, the Panel concludes that additive does not pose a toxicological safety concern regarding the consumers of the products obtained from animals receiving the additive. The additive, in its all formulations, is not a skin or eye irritant and is not dermal sensitiser. However, owing to the proteinaceous nature of the active substance it should be considered a respiratory sensitiser. The active substance of the additive is a protein and as such would not raise concerns for the environment. Considering that the production strain harbours AMR genes and there is uncertainties regarding the presence of viable cells and DNA of the production strain in Nutrase PL 10,000 (liquid) and Nutrase PD 100,000 (powder), the Panel cannot conclude on the safety of these two formulations of the additive for the target species, consumer, user and environment. Owing to the lack of data, the FEEDAP Panel cannot conclude on the efficacy of the additive.

Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to weaned piglets fed a corn-soybean meal-based diet without added inorganic phosphate.

The utility of a next generation biosynthetic bacterial 6-phytase (PhyG) in restoring bone ash, bone phosphorus (P) content and performance in piglets depleted in P was evaluated. A total of 9 treatments were tested as follows. Treatment 1, a negative control (NC) diet; treatments 2, 3, 4, NC supplemented with 250, 500 or 1,000 FTU/kg of PhyG; treatments 5, 6, NC supplemented with 500 or 1,000 FTU/kg of a commercial Buttiauxella sp phytase (PhyB); treatments 7, 8, 9, NC supplemented with monocalcium phosphate (MCP) to provide 0.7, 1.4 and 1.8 g/kg digestible P, equating to a digestible P content of 1.8, 2.5 and 2.9 g/kg. The latter constituting the positive control (PC) diet with adequate P and calcium (Ca). The NC was formulated without inorganic P (1.1 g digestible P/kg) and reduced in Ca (5.0 g/kg). Additional limestone was added to treatments 7 to 9 to maintain Ca-to-P ratio between 1.2 and 1.3. A total of 162 crossed Pietrain × (Large White × Landrace) 21-d-old piglets (50% males and 50% females) were fed adaptation diets until 42 d old and then assigned to pens with 2 pigs/pen and 9 pens/treatment in a completely randomized block design. Piglets were fed mash diets based on corn and soybean meal ad libitum for 28 d. At the end of the study, one piglet perpen was euthanized and the right feet collected for determination of bone strength, bone ash and mineral content. Compared with the PC, the NC group had reduced average daily gain (ADG) and increased feed conversion ratio (FCR) during all growth phases and overall, and at d 28 (70 d old) NC pigs had bones with reduced ash, Ca and P content (P < 0.05). The PhyG at 250 FTU/kg improved bone ash vs. NC. Increasing PhyG dose linearly or quadratically improved bone ash, ADG and FCR (P < 0.05). At ≥ 500 FTU/kg, both PhyG and PhyB maintained ADG and FCR equivalent to PC. Linear regression analysis was done to compare the measured response parameters to increasing digestible P from MCP. Based on this analysis it was shown that PhyG and PhyB at 1,000 FTU/kg could replace 1.83 and 1.66 g/kg digestible P from MCP in the diet, respectively, on average across metacarpi bone ash, ADG or FCR. These findings suggest that the biosynthetic phytase is highly effective in the tested dietary setting.

Safety and efficacy of APSA PHYTAFEED® (6-phytase) as a feed additive for laying hens and other laying birds.

Following a request from the European Commission, the EFSA Panel on Additives and products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of APSA PHYTAFEED ® 20,000 GR/L (6-phytase) as a zootechnical feed additive for laying hens and other poultry species for laying. The additive is a preparation of 6-phytase produced by a genetically modified strain of Komagataella phaffii and has been previously assessed by the FEEDAP Panel in the context of three applications for its use in different species/categories. The Panel concluded in those opinions that the production strain is safe, and that the use of the additive as a feed additive would raise no safety concerns for the consumers and the environment. The additive was also considered not to be irritant to skin or eyes or a dermal sensitiser but it should be considered as a respiratory sensitiser. The Panel considered that the new use in laying hens and other poultry species for laying would not modify the previously drawn conclusions with respect to the consumers, users and the environment. A tolerance trial in laying hens and a subchronic oral toxicity study were made available to support the safety of the additive for the target species/categories subject of this new application; from the results obtained, the Panel concluded that the additive is safe for laying hens at the recommended level of use (300 U/kg feed) with a wide margin of safety and therefore the conclusion was extrapolated to other laying birds. The FEEDAP Panel concluded that the additive has the potential to be efficacious in laying hens at the level of 300 U/kg feed and this conclusion was extrapolated to other laying birds.

Modification of the terms of the authorisation of Natuphos® E as a feed additive for chickens for fattening or reared for laying/breeding.

The additive Natuphos® E presents 6-phytase produced by a genetically modified strain of Aspergillus niger. The additive is currently authorised in the EU for use as a feed additive for chickens for fattening, chickens reared for laying, pigs for fattening, sows, minor porcine species for fattening or for reproduction, turkeys for fattening, turkeys reared for breeding, all other avian species (excluding laying birds) and weaned piglets. The authorisation for chickens for fattening or reared for laying is at a minimum content of 750 FTU/kg feed. The applicant has requested to lower this dose to 125 FTU/kg feed. The Panel considered a total of three short-term trials and five long-term trials. In two short-term trials, the birds that received 125 FTU/kg feed showed a significantly better retention of phosphorus from the diets. Similarly, in two long-term trials, the birds that received 125 FTU/kg feed showed a significantly better performance. Consequently, the Panel concluded that the additive has a potential to improve the performance/phosphorus retention of the birds at the dose of 125 FTU/kg feed.

Safety and efficacy of Natuphos® E (6-phytase) as a feed additive for laying hens, minor poultry and other avian species for laying.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of Natuphos® E (6-phytase) as a feed additive for laying hens, minor poultry and other avian species for laying. The additive Natuphos® E consists of 6-phytase (phytase; Enzyme Commission Number 3.1.3.26) and is intended to be used as a feed additive for laying hens, minor poultry and other avian species for laying as a zootechnical additive, functional group of digestibility enhancers. This additive was previously assessed by the FEEDAP Panel in 2017 for avian and porcine species. The production strain of the phytase present in the product is a genetically modified strain of Aspergillus niger. Based on the previous opinion, the FEEDAP Panel concluded that the genetic modification of the production strain does not give rise to safety concerns. The production strain and its DNA were not detected in the concentrate used to formulate the products. The FEEDAP Panel previously concluded that the additive was safe for the target species, consumers and the environment when used at 200 FTU/kg feed. The additive Natuphos® E is not considered to be toxic by inhalation or irritant for skin or eye; however, it should be regarded as a dermal sensitiser and a potential respiratory sensitiser. The additive has the potential to be efficacious in improving the performance and/or the phosphorus utilisation in laying hens at 200 FTU/kg feed; the conclusions drawn in laying hens can be extrapolated to all minor poultry and other avian species for laying.

Safety and efficacy of Natuphos® E (6-phytase) as a feed additive for avian and porcine species.

Natuphos® E is a feed additive that contains a 6-phytase available in powder, granulated and liquid forms which is intended to be used as a feed additive for avian and porcine species. The production strain of the phytase present in the product is a genetically modified strain of Aspergillus niger. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the genetic modification of the production strain does not give rise to safety concerns. The production strain and its DNA were not detected in the concentrate used to formulate the products. Based on the tolerance studies provided, the Panel concluded that the additive is safe for the target species under the conditions of use with a wide margin of safety. The Panel also concluded that the use of the product as a feed additive does not give rise to concerns for consumers. Evidence was provided showing that the additive is not toxic by inhalation or irritant for skin or eyes, however, it should be regarded as a dermal sensitiser and a potential respiratory sensitiser. The use of the additive as a feed additive poses no risks to the environment. Based on the efficacy studies provided, the Panel concluded that the additive has the potential to be efficacious in chickens for fattening, turkeys for fattening, piglets, pigs for fattening and sows. These conclusions were extended to chickens reared for laying, turkeys reared for breeding and extrapolated to minor poultry species and other avian species for fattening and to the point of lay and to minor porcine species. The Panel considered that there was insufficient information to conclude on the efficacy in laying hens.

Safety and efficacy of OPTIPHOS® (6-phytase) as a feed additive for finfish.

The additive OPTIPHOS ® is a preparation of 6-phytase that is authorised for use in avian species, weaned piglets, pigs for fattening and sows. The applicant seeks for an extension of use of the product to finfish, at a dose range between 250 and 1,000 OTU/kg feed. The FEEDAP Panel concluded in a previous opinion that there are no concerns for consumer safety and no risks for the environment are expected from the use of the product as feed additive. Moreover, it was concluded that the additive is not a skin/eye irritant or a skin sensitiser, but has the potential to be a respiratory sensitiser. The Panel considered that the new use of the additive would not change the previous conclusions regarding the safety for the consumer, user and environment. The results of a tolerance study performed in juvenile rainbow trout (Oncorhynchus mykiss) showed no negative effects of the additive on the fish when offered up to 100 times the maximum recommended dose. Therefore, the Panel concluded that the additive is safe for the rainbow trout and extrapolated this conclusion to all finfish. The Panel evaluated three efficacy trials, two performed in rainbow trout and one in Atlantic salmon (Salmo salar). In these studies, the performance of the fish was monitored as well as phosphorus digestibility and phosphorus retention parameters. In the three trials, the performance and the phosphorus retention were improved by the additive at the lowest tested dose (in rainbow trouts at 250 OTU/kg feed and in Atlantic salmon at 500 OTU/kg feed). The FEEDAP Panel concluded that the additive has a potential to be efficacious in rainbow trout and salmon at 500 OTU/kg. The Panel extrapolated the conclusion to all finfish species.

Optimization of Recombinant Expression of Synthetic Bacterial Phytase in Pichia pastoris Using Response Surface Methodology.

BACKGROUND: Escherichia coli phytase is an acidic histidine phytase with great specific activity. Pichia pastoris is a powerful system for the heterologous expression of active and soluble proteins which can express recombinant proteins in high cell density fermenter without loss of product yield and efficiently secrete heterologous proteins into the media. Recombinant protein expression is influenced by expression conditions such as temperature, concentration of inducer, and pH. By optimization, the yield of expressed proteins can be increase. Response surface methodology (RSM) has been widely used for the optimization and studying of different parameters in biotechnological processes. OBJECTIVES: In this study, the expression of synthetic appA gene in P. pastoris was greatly improved by adjusting the expression condition. MATERIALS AND METHODS: The appA gene with 410 amino acids was synthesized by P. pastoris codon preference and cloned in expression vector pPinkα-HC, under the control of AOX1 promoter, and it was transformed into P. pastoris GS115 by electroporation. Recombinant phytase was expressed in buffered methanol-complex medium (BMMY) and the expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic assay. To achieve the highest level of expression, methanol concentration, pH and temperature were optimized via RSM. Finally, the optimum pH and temperature for recombinant phytase activity was determined. RESULTS: Escherichia coli phytase was expressed in P. pastoris under different cultivation conditions (post-induction temperature, methanol concentration, and post-induction pH). The optimized conditions by RSM using face centered central composite design were 1% (v/v) methanol, pH = 5.8, and 24.5°C. Under the optimized conditions, appA was successfully expressed in P. pastoris and the maximum phytase activity was 237.2 U/mL after 72 hours of expression. CONCLUSIONS: By optimization of recombinant phytase expression in shake flask culture, we concluded that P. pastoris was a suitable host for high-level expression of phytase and it can possess high potential for industrial applications.