6-phytase EC 3.1.3.26

In the current two applications (FAD-2012-0015 and FAD-2012-0033) authorisation is sought under article 4(1) for Quantum Blue 5 L, 10 L, 5 G and 40 P under the category/functional group 4(c) "zootechnical additives"/"substances which favourable affect the environment", according to the classification system of Annex I of Regulation (EC) No 1831/2003.

According to the Applicant, the active agent of all Quantum Blue products is 6-phytase (EC 3.1.3.26) produced by the strain Trichoderma reesei (CBS 126897). The authorisation is sought for the use of the feed additive for a variety of animal species. According to the Applicant, Quantum Blue 5 L and 10 L are liquid preparations with a guaranteed minimum enzyme activity of 5000 and 10000 FTU/g, respectively, while Quantum Blue 5 G and 40 P are a granulate (G) or powder (P) preparation with a guaranteed minimum enzyme activity of 5000 and 40000 FTU/g, respectively. Quantum Blue preparations are intended to be used in premixtures and/or complete feedingstuffs to obtain minimum enzyme activities for 6-phytase of 150 and 250 FTU/kg feedingstuffs depending on the target species. The dry products can be mixed with the final feed or with the mineral premixtures, while the liquid product is to be sprayed onto feed pellets.

The Applicant used the enzyme activity units as defined in the EN ISO 30024: - One 6-phytase unit (FTU) is the amount of enzyme which liberates 1 micromole of inorganic phosphate from sodium phytate in one minute at 37°C and pH 5.5.

For the determination of the activity of 6-phytase in the feed additive, premixtures and feedingstuffs, the Applicant proposed an in-house developed and validated colorimetric method measuring the inorganic phosphate released by the enzyme from the sodium phytate substrate. On the request of the EURL the Applicant applied the internationally recognised ring-trial validated colorimetric CEN method (EN ISO 30024) for the determination of 6-phytase in the feed additive, premixtures and feedingstuffs samples containing Quantum Blue products and provided performance characteristics similar to those reported in the EN ISO 30024 standard: - a precision (repeatability and reproducibility) ranging from 1.2 to 5.5 %; - a recovery rate ranging from 80.4 to 102.4 %; and - a limit of quantification of 101 FTU/kg feedingstuffs.

Based on the experimental evidence and the performance characteristics presented, the EURL recommends for official control the EN ISO 30024 method, for the determination of the activity of the 6-phytase 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) is not considered necessary.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2025/142

For the determination of 6-phytase in the feed additive, premixtures and compound feed:

• Colorimetric method based on the enzymatic reaction of 6-phytase on the phytate – EN ISO 30024