alpha-amylase EC 3.2.1.1 (4a21)

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2015/47

Determination of alpha-amylase:

- Colorimetric method based on the quantification of the dyed fragments produced by the action of alpha-amylase on red starch substrates

Executive Summary

Ronozyme RumiStar (CT) (L) is a product for which authorisation as feed additive is sought under the category "zootechnical additives", functional groups 4(a) "digestibility enhancers" according to Annex I of Regulation (EC) No 1831/2003. The active agent of Ronozyme RumiStar (CT) (L) is α-amylase (E.C. 3.2.1.1), produced by a strain of Bacillus Licheniformis (DSM 21564). The authorisation for dairy cows in the lactation period is requested. The enzymatic activity of the α-amylase is expressed in “KNU” units. According to the Applicant, one KNU is defined as the amount of enzyme that releases 6 μmol p-nitrophenol per minute from 1.86 mM ethylidene-G7-p-nitrophenyl-maltoheptaoside at pH 7.0 and 37 °C.

The product is intended to be marketed in two formulations: (i) as Coated Thermo tolerant granulate (CT) with a minimum activity of 160 KNU/g, and (ii) as Aqueous Liquid (L) with a minimum activity of 240 KNU/g. The Applicant recommended α-amylase activity in complete feedingstuffs ranging from 300 to 600 KNU/kg.

For the determination of the activity of α-amylase in the feed additive, premixtures and feedingstuffs, the Applicant proposed two single-laboratory validated and further verified colorimetric methods. In all methods the activity of the samples is calibrated against reference enzyme standards with known activity determined at the definition conditions of the activity unit. The following performance characteristics for the determination of α-amylase in feed additives, premixtures and feedingstuffs, were recalculated by the CRL: - for feed additives, a relative standard deviation for repeatability (RSD_r) ranging from 1.6 to 3.3 %, a relative standard deviation for intermediate precision (RSD_ip) ranging from 3.5 to 9.1 %; - for premixtures, RSD_r ranging from 3.9 to 6.5 %, RSD_ip ranging from 3.6 to 6.5 %; - for feedingstuffs, RSD_r ranging from 4.1 to 7.2 %, RSD_ip ranging from 5.1 to 13.6 %, a recovery rate (R_Rec) ranging from 92.6 to 114.4 %, a limit of detection (LOD) and quantification (LOQ) of 2 and 7 KNU/kg feedingstuffs, respectively. Based on the satisfactory performance characteristics mentioned above, the CRL recommends for official control the methods submitted by the applicant for the determination of α-amylase 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.