Preparation of endo-1,4-beta-xylanase, endo-1,3(4)-beta-glucanase and endo-1,4-beta-glucanase

In the current application an authorisation of a preparation of endo-1,4-beta-xylanase, endo-1,3(4)-beta-glucanase and endo-1,4-beta-glucanase is sought for the category / functional group 4(a) "zootechnical additives" / "digestibility enhancers" according to Annex I of Regulation (EC) No 1831/2003. The authorisation is sought for the use of the feed additive for poultry for fattening, poultry for laying, turkeys for fattening, piglets (weaned) and pigs for fattening. According to the Applicant, the feed additive contains three enzymes as active substances namely: endo-1,4-beta-xylanase (EC 3.2.1.8), endo-1,3(4)-beta-glucanase (EC 3.2.1.6) and endo-1,4-beta-glucanase (EC 3.2.1.4) produced by the Trichoderma reesei ATCC 74444 strain. The enzymatic activity for each of the three enzymes is expressed in international units (U), where one international unit (U) is the amount of enzyme (endo-1,4-beta-xylanase, endo1,3(4)-beta-glucanase or endo-1,4-beta-glucanase) that releases 1 micromole of reducing sugar (glucose or xylose) equivalents from wheat arabinoxylan, barley beta-glucan and carboxy-methyl-cellulose, respectively, per minute at pH 5.0 and 40 °C. The feed additive is intended to be marketed as solid (RONOZYME® MultiGrain (GT)) and as liquid (RONOZYME® MultiGrain (L)) formulations, having minimum activities of 2700 U / g (or U / ml) for endo-1,4-beta-xylanase, 700 U / g (or U / ml) for endo-1,3(4)-beta-glucanase, and 800 U / g (or U / ml) for endo-1,4-beta-glucanase. The feed additive is intended to be incorporated through premixtures or directly into compound feed (for the solid formulation) or directly sprayed onto the compound feed (for the liquid formulation). For pigs for fattening (new animal category) the Applicant recommends including the feed additive in compound feed in order to obtain minimum activities of 270, 70 and 80 U / kg compound feed for endo-1,4-beta-xylanase, endo-1,3(4)-beta-glucanase and endo-1,4-beta-glucanase respectively, while for the other animal categories the recommended activities are the same as specified in the Commission Implementing Regulation (EU) No 403/2013. For the determination of the endo-1,4-beta-xylanase, endo-1,3(4)-beta-glucanase and endo1,4-beta-glucanase activities in the feed additive the Applicant submitted a ring-trial validated colorimetric method based on the enzymatic hydrolysis of the respective substrates and the colour formation of the released reducing sugars with 3,5-dinitrosalicylic acid (DNS) at pH 5.0 and 40 °C. For the determination of the endo-1,4-beta-xylanase, endo-1,3(4)-beta-glucanase and endo1,4-beta-glucanase activities in premixtures and compound feed the Applicant submitted single-laboratory validated and further verified colorimetric methods based on the measurement of the soluble oligomeric cleavage products from the respective dye-labelled substrates. Based on the acceptable performance characteristics presented the EURL recommends for official control the ring-trial validated and the single-laboratory validated and further verified colorimetric methods submitted by the Applicant for the determination of the activities of endo-1,4-beta-xylanase, endo-1,3(4)-beta-glucanase and endo-1,4-beta-glucanase in the feed additive, premixtures and compound feed. 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.