Endo-1,4- beta-glucanase EC 3.2.1.4 (4a1616)

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2015/2305

For the determination of endo-1,4- beta-glucanase in feed additive, premixtures and feedingstuffs:

- colorimetric method based on the quantification of water soluble dyed fragments (azurine) produced by the action of endo-1,4-beta-glucanase on azurine-crosslinked cellulose

Executive Summary

In the current application authorisation is sought under articles 4(1) (new species and formulation) and 10(2) (re-evaluation of the additive already authorised under council directive 70/524/EEC) for Hostazym C under the category "zootechnical additives", functional groups 4(a) "digestibility enhancers" according to Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for the use of the feed additive for chickens for fattening, weaned piglets, and other birds for fattening except turkeys. The active agent of Hostazym C is endo-1,4-b-glucanase (E.C. 3.2.1.4), produced by the strain Trichoderma longibrachiatum.

According to the Applicant, enzymatic activity of the active agents is expressed in “CU” units, where: One CU of endo-1,4-b-glucanase is the amount of enzyme that liberates 0.128 micromoles of reducing sugars (glucose equivalents) from barley beta-glucan per minute at pH 4.5 and 30 °C.

The feed additive is intended to be marketed in two microgranulate formulations (Hostazym C 2000 and 7000) and one liquid formulation (Hostazym C 2000). Hostazym C 2000 (microgranulate and liquid) and Hostazym C 7000 have a endo-1,4-b-glucanase minimum activity of 2000 and 7000 CU/g, respectively. The Applicant proposed the following minimum activities for endo-1,4-b-glucanase in complete feedingstuffs: 350 CU/kg for weaned piglets; 500 CU/kg for chickens for fattening and for other birds for fattening except turkeys.

For the determination of the activity of endo-1,4-b-glucanase in the feed additive, premixtures and feedingstuffs, the Applicant proposed a single-laboratory validated and further verified colorimetric method based on the quantification of water soluble dyed fragments produced by the action of endo-1,4-b-glucanase on commercially available azurine crosslinked cellulose. 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 method performance characteristics for feedingstuffs were recalculated by the EURL, based on the experimental data provided by the Applicant: - a relative standard deviation for repeatability (RSDr) ranging from 8.03 to 9.66 %; - a relative standard deviation for intermediate precision (RSDip) ranging from 8.03 to 9.76 %; - a recovery rate (RRec) ranging from 96 to 100 %, and - a limit of quantification (LOQ) of 57.6 CU/kg, which is well below the minimum activity proposed by the Applicant. Based on the satisfactory performance characteristics mentioned above, the EURL recommends for official control the single-laboratory validated and further verified method submitted by the Applicant for the determination of endo-1,4-b-glucanase 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.