Endo- 1,4-beta- xylanase (EC 3.2.1.8), protease (EC 3.4.21.62) and alpha- amylase (EC 3.2.1.1)

In the current application authorisation is sought under Article 4 (1) for Axtra® XAP 104 TPT under the category/functional group (4 a) "zootechnical additive"/"digestibility enhancers", according to the classification system of Annex 1 of Regulation (EC) No 1831/2003. The authorisation is sought for the use of the feed additive for chickens for fattening and reared for laying, laying hens and all minor poultry species.

According to the Applicant, Axtra® XAP 104 TPT is a preparation containing endo-1,4-β-xylanase, α-amylase and protease. The Applicant expressed the enzyme activities in different units defined as follows: 

- one unit of endo-1,4-β-xylanase activity (UX) is the amount of enzyme, which liberates 0.48 micromoles per minute of reducing sugars, expressed as xylose equivalents, from a wheat arabinoxylan substrate at pH 4.2 and 50 °C; 

- one unit of α-amylase activity (UA) is the amount of enzyme required to release, in the presence of an excess of α-glucosidase, 0.20 micromoles per minute of glucosidic linkages, expressed as p-nitrophenol equivalents, from a maltoheptasoide substrate at pH 8.0 and 40 °C; and 

- one unit of protease activity (UP) is the amount of enzyme which liberates 2.3 micrograms per minute of phenolic compounds, expressed as tyrosine equivalents, from a casein substrate at pH 10.0 and 50 °C 

According to the Applicant, Axtra® XAP 104 TPT has a guaranteed minimum enzyme activity of 20000 UX/g endo-1,4- β-xylanase, 2000 UA/g α-amylase and 4000 UP/g protease. The product is intended to be incorporated directly in feedingstuffs or through premixtures with the following proposed minimum enzyme activities in feedingstuffs: 1000 UX/kg for endo-1,4-β-xylanase; 100 UA/kg for α-amylase and 2000 UP/kg for protease. 

For the quantification of the active substances in the feed additive, premixtures and feedingstuffs the Applicant submitted three single-laboratory validated and further verified colorimetric methods, based on the enzymatic hydrolysis: 

- by xylanase of an azurine cross-linked wheat arabinoxylan substrate at pH 4.2 and 50 °C for the determination of endo-1,4- β-xylanase; 

- by amylase of an azurine cross-linked starch polymer substrate at pH 8.0 and 40 °C for the determination of α-amylase; and 

- by protease of a dyed cross-linked casein substrate at pH 10.0 and 50 °C for the determination of protease. 

Based on the performance characteristics available the EURL recommends for official control the proposed single-laboratory validated and further verified colorimetric methods for the quantification of the three enzymes 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) 2023/1713

For the quantification of endo-1,4-beta-xylanase in the feed additive, premixtures and compound feeds: 

• Colorimetric method, based on the enzymatic hydrolysis by xylanase of an azurine cross-linked wheat arabinoxylan substrate at pH 4,2 and 50 °C

For the quantification of protease in feed additive and premixtures and compound feeds: 

• Colorimetric method based on the enzymatic hydrolysis by protease of a dyed cross-linked casein substrate at pH 10,0 and 50 °C

For the quantification of alpha-amylase in feed additive, premixtures and compound feeds: 

• Colorimetric method based on the enzymatic hydrolysis by amylase of an azurine cross- linked starch polymer substrate at pH 8,0 and 40 °C