Ronozyme® VP - 4a1603

Ronozyme® VP is currently authorised as feed additive for chickens for fattening and piglets (weaned) by Commission Regulation (EC) No 1259/2004 and Commission Regulation (EC) No 1811/2005 respectively. In the current application authorisation is sought under Article 10 (2) for Ronozyme® VP under the category/functional "zootechnical additives"/"digestibility enhancers". The authorisation is sought for the use of the feed additive for chickens for fattening and piglets (weaned). 

According to the Applicant, the active agent of Ronozyme® VP is endo-1,3(4)-beta-glucanase (glucanase) produced by Aspergillus aculeatus. The Applicant expressed the glucanase enzymatic activity in fungal beta-glucanase units (FBG), where one FBG is defined as "the amount of enzyme which under standard conditions (pH 5.0 and 30 °C) liberates glucose or other reducing carbohydrates at a rate corresponding to 1 μmol glucose per minute". 

The product is intended to be marketed as solid (CT) and liquid (L) formulations having a guaranteed minimum glucanase activity of 50 FBG / g and 120 FBG / ml respectively. The feed additive formulations are intended to be included through premixtures (solid) or directly in feedingstuffs (solid and liquid) to obtain a minimum activity of 10 FBG / kg feedingstuffs. 

For the quantification of the glucanase activity in the feed additive the Applicant provided a single-laboratory validated and further verified colorimetric method. Glucanase cleaves non-starch polysaccharides (NSP) releasing glycosylic moieties with reducing ends from beta-glucan. The reducing moieties are oxidized in an alkaline milieu by forming orange-yellow compounds with the 2-hydroxy-3,5-dinitrobenzoic acid. These orange-yellow compounds are measured at a wavelength of 530 nm and quantified against a validated Ronozyme® VP standard available from the Applicant upon request. 

For the quantification of the glucanase activity in premixtures and feedingstuffs the Applicant proposes a single-laboratory validated and further verified colorimetric method based on the quantification of the water soluble dyed fragments produced by the action of endo-1,3(4)-beta-glucanase on azo-barley beta-glucan substrate. The quantification of the glucanase activity is determined by using a standard curve of a certified Ronozyme® VP glucanase standard available from the Applicant upon request. 

Based on the satisfactory performance characteristics the EURL recommends for official control the colorimetric methods mentioned above for the quantification of the glucanase activity 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, as last amended by Regulation (EU) 2015/1761) is not considered necessary.

Recommended Method

as of:

Commission Implementing Regulation (EU) 2023/1333

For the quantification of 1,3(4)-beta- glucanase activity in the feed additive: 

• colorimetric method measuring coloured compound produced by the dinitro salicylic acid (DNSA) based on the enzymatic hydrolysis of beta- glucan at pH 5,0 and 50 °C.

For the quantification of 1,3(4)-beta- glucanase activity in premixtures and compound feed: 

• colorimetric method measuring water soluble dyed fragments based on the enzymatic hydrolysis of cross-linked azo-barley- glucan at pH 4,5 and 50 °C.