Endo-1,4- beta-xylanase EC 3.2.1.8 and Endo-1,3(4)- beta-glucanase EC 3.2.1.6 (4a22)

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2015/661

- Commission Implementing Regulation (EU) 2015/2304

For the quantification of endo-1,4-beta- xylanase activity:

- viscosimetric method based on decrease in viscosity produced by action of endo-1,4-beta-xylanase on the xylan containing substrate (wheat arabinoxylan).

For the quantification of endo-1,3(4)- beta-glucanase activity:

- viscosimetric method based on decrease in viscosity produced by action of endo-1,3(4)-beta-glucanase on the glucan substrate barley betaglucan at pH = 5,5 and 30 ºC.

Executive Summary

In the current application authorisation is sought under article 4(1) for Rovabio® Spiky, under the category/functional 4(a) "zootechnical additives"/"digestibility enhancers" according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the use of the feed additive for chickens for fattening; chickens reared for laying and minor poultry species (for fattening and reared for laying). According to the Applicant, the feed additive contains two active substances: endo-1,4-beta-xylanase (EC 3.2.1.8) and endo-1,3 (4)-beta-glucanase (EC 3.2.1.6), produced from Talaromyces versatilis (IMI 378536) and (DMS 26702), respectively. The enzymatic activity for both enzymes is expressed by the Applicant in viscosity or in DNS units, where: - One xylanase (or beta-glucanase) viscosity unit (U) is defined as the amount of xylanase (or beta-glucanase) that hydrolizes wheat arabinoxylan (or barley beta-glucan), reducing the solution viscosity, in order to change the relative fluidity by one dimensionless unit per minute, at pH 5.5 and 30 oC; - One xylanase (or beta-glucanase) DNS unit corresponds to the amount of xylanase (or beta-glucanase) which liberates from the birchwood xylan (or barley beta-glucan) one micromol of xylose (or glucose) per minute at 50 oC and pH 4.0 (or pH 5.0). The product is intended to be marketed in powder (Rovabio Spiky AP) and liquid (Rovabio Spiky LC) formulations, having the following guaranteed minimum activities: - for xylanase: 22000 U/g or 2300 DNS units/g and 5500 U/ml or 570 DNS units /ml, and - for beta-glucanase: 15200 U/g or 1600 DNS units /g and 3800 U/ml or 400 DNS units /ml. The carrier in the solid formulation is wheat flour, while sorbitol, potassium sorbate and demineralized water are used for the liquid formulation. The feed additive is intended to be incorporated into premixtures and/or complete feedingstuffs to obtain a minimum xylanase and beta-glucanase activity of 1100 and 760 U/kg, respectively. For the quantification of xylanase and beta-glucanase activities in the feed additive, premixtures and feedingstuffs, the Applicant submitted a single-laboratory validated and further verified viscosimetry methods. Furthermore, the Applicant submitted alternative colorimetric (DNS) methods for the quantification of xylanase and beta-glucanase activities in the feed additive. In the viscosimetry methods the endo-1,4-beta-xylanase (or the endo-1,3 (4)-beta-glucanase) catalyses the hydrolysis of xylosidic (or glycosidic) bonds in the wheat arabinoxylan (or barley beta-glucan) substrate to yield xylose (or glucose) and reduces consequently the viscosity of the sample solution. The colorimetric methods are based on the enzymatic hydrolysis of the birchwood xylan (or barley beta-glucan) and the consequent colour formation of released sugar with 3,5-dinitrosalicylic acid (DNS) at pH 4.0 (or pH 5.0) and 50 oC. Based on the acceptable performance characteristics the EURL recommends for official control the validated and further verified viscosimetry methods for the determination of xylanase and beta-glucanase in the feed additive, premixtures and feedingstuffs together with the alternative colorimetric (DNS) methods for the determination of xylanase and beta-glucanase in the feed additive. 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.