Fumonisin B1 esterase (3.1.1.87) produced by Komagataella phaffii NCAIM (P) Y001485

Executive Summary

In the current application an authorisation of fumonisin B1 esterase (3.1.1.87) produced by Komagataella phaffii NCAIM (P) Y001485 is sought under Article 4 and under the category/functional group 1(m) "technological additives"/"substances for reduction of the contamination of feed by mycotoxins: substances that can suppress or reduce the absorption, promote the excretion of mycotoxins or modify their mode of action" according to Annex I of Regulation (EC) No 1831/2003. The authorisation is sought for the use of the feed additive for all pigs (piglets, pigs for fattening, sows and minor growing and reproductive porcine species). According to the Applicant, the feed additive contains as active substance fumonisin esterase (EC 3.1.1.87) which is produced by Komagataella phaffii NCAIM Y001485.
The activity of fumonisin B1 esterase is expressed in fumonisin esterase units (U), where “one U unit is the amount of enzyme that releases one μmol of hydrolyzed fumonisin B1 (HFB1) per minute from 100 μM of fumonisin B1 in 50 mM phosphate buffer pH 6.0 at 37 °C”. The feed additive is intended to be marketed as solid preparation having a guaranteed minimum activity of fumonisin B1 esterase corresponding to 1200 U/g. The marketed product (Free Yeast® F) is intended to be incorporated through premixtures or directly into feedingstuffs. The Applicant recommended to include the feed additive in feedingstuffs in order to obtain its content ranging from 50 to 300 g/ton feedingstuffs.
For the quantification of the fumonisin B1 esterase activity in the feed additive, premixtures and feedingstuffs the Applicant submitted a single-laboratory validated and further verified method based on the selective quantification of the reaction product HFB1 by High Performance Liquid Chromatography coupled with fluorescence detection (HPLC-FLD).
The following method performance characteristics were derived from the validation and verification studies:
- for the feed additive in a range of enzyme activity between 167 and 1667 U/g: a relative standard deviation for repeatability (RSDr) ranging from 1.8 to 5.1 %, a relative standard deviation for intermediate precision (RSDip) ranging from 4.1 to 9.4 % and a recovery rate (RRec) ranging from 88 to 102 %;
- for premixtures in a range of enzyme activity between 417 and 4167 U/kg: a RSDr ranging from 1.6 to 7.6 %, a RSDip ranging from 4.7 to 9.8 % and a RRec ranging from 89 to 109 %; and
- for feedingstuffs in a range of enzyme activity between 21 and 208 U/kg: a RSDr ranging from 3.1 to 4.1 %, a RSDip ranging from 3.4 to 4.4 % and a RRec ranging from 90 to 101 %.

Furthermore, a limit of detection (LOD) and a limit of quantification (LOQ) of 0.21 and 0.67 U of fumonisin B1 esterase /kg of feedingstuffs, respectively, were reported by the Applicant.
Based on the performance characteristics available, the EURL recommends for official control the single-laboratory validated and further verified HPLC-FLD method, based on the selective quantification of the reaction product HFB1 produced at pH 6.0 and 37 °C by the action of fumonisin B1 esterase (3.1.1.87) to fumonisin B1, for the quantification of the fumonisin B1 esterase activity in the feed additive, premixtures and feedingstuffs.
Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005, as last amended by Regulation (EU) 2015/1761) is not considered necessary.