Abstract
A 90-day subchronic oral toxicity study was conducted to evaluate the safety of a consensus bacterial phytase variant 6-phytase (PhyG) for use as an animal feed additive. This phytase is produced by fermentation with a fungal (Trichoderma reesei) production strain expressing a biosynthetic variant of a consensus bacterial phytase gene assembled via ancestral reconstruction with sequence bias for the phytase from Buttiauxella sp. Rats were administered PhyG daily via oral gavage at dose-levels of 0 (distilled water), 250, 500 or 1000 mg total organic solids (TOS)/kg bodyweight (bw)/day (equivalent to 0, 112,500, 225,000 and 450,000 phytase units (FTU)/kg bw/day, respectively). No test article-related adverse effects were observed. A no-observed-adverse-effect level (NOAEL) for PhyG was established as 1000 mg TOS/kg bw/day, the highest test concentration. Based on this NOAEL and an estimate of broiler consumption determined from the proposed inclusion of the phytase in feed at the maximum recommended level (4000 FTU/kg), a margin of safety value of 1613 was calculated. Results of in vitro genotoxicity testing and in silico protein toxin evaluation further confirmed PhyG to be non-genotoxic and not likely to be a protein toxin upon consumption. These data support the safety of PhyG as an animal feed additive.
Highlights
Pigs and poultry diets are typically based on cereals and oilseeds in which up to 70–80 % of the phosphorus (P) content is bound to phytate [1]
The experimental phytase, PhyG, was developed and manufactured by DuPont Nutrition and Biosciences (Wilmington, DE, USA) as described below. This variant was developed via ancestral reconstruction based on available bacterial phytase sequences, with sequence bias for Buttiauxella sp phytase. This resulted in a consensus biosynthetic bacterial phytase sequence with high- melting temperature, which was subjected to further protein engineering with multiple amino acid substitutions and screening for improved enzymatic performance
Recombinant DNA technologies have enabled the use of host microbial organisms as vehicles (‘production strains’) for producing large quantities of phytases identified as having desirable characteristics, by insertion of the candidate phytase gene into the host genome followed by expression using an appropriate promoter system [31]
Summary
Pigs and poultry diets are typically based on cereals and oilseeds in which up to 70–80 % of the phosphorus (P) content is bound to phytate (the salt of phytic acid, myo-inositol hexaphosphate; IP6) [1]. Phytate is poorly utilized by monogastric animals (pigs, poultry) due to a lack of endogenous phytase activity. Phytase (myo-inositol hexakisphosphate phosphohydrolase) is commonly added to commercial pig and poultry diets to improving the bioavailability of P from phytate. Since the first generation of microbial phytase commercialized in the 1990s, new generation phytases have been developed [11]. These new phytases have increased bioefficacy and may possess other desirable characteristics such as more active in upper gastro-intestinal tract to achieve rapid and complete phytate hydrolysis, increased substrate affinity/specificity and/or enhanced thermal stability to better withstand the high temperatures associated with the pelleting process. New phytases with further enhanced activity and more desirable biochemical and thermal properties continue to be sought
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