Endo-1,4-beta- xylanase (EC 3.2.1.8), Endo-1,4-beta- glucanase (EC 3.2.1.4), Xyloglucan- specific endo- beta-1,4-glucanase (EC 3.2.1.151)

In the current application an authorisation of a preparation of endo-1,4-beta-xylanase, endo-1,4-beta-glucanase and xyloglucan-specific-endo-1,4-beta-1,4-glucanase is sought under Article 4 under 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 species, ornamental birds and piglets (weaned and suckling). 

According to the Applicant, the feed additive contains three active substances: (i) endo-1,4-beta-xylanase (EC 3.2.1.8), (ii) endo-1,4-beta-glucanase (EC 3.2.1.4) and (iii) xyloglucan-specific-endo-beta-1,4-glucanase (EC 3.2.1.151), which are produced by Trichoderma citrinoviride B-125 (DSM 33578). 

The activity of: (i) endo-1,4-beta-xylanase is expressed in endo-pentosanase units (EPU), where one EPU unit is the amount of enzyme, which liberates 0.0083 micromoles of reducing sugars (xylose equivalents) from oat spelt xylan per minute at pH 4.7 and 50 °C; (ii) endo-1,4-beta-glucanase is expressed in cellulase units (CU), where one CU unit 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; and (iii) xyloglucan-specific-endo-beta-1,4-glucanase is expressed in xyloglucanase unit (XGU), where one XGU unit is the amount of enzyme that releases low-molecular fragments from dyed xyloglucan in amount equal to the amount of such fragments liberated from 1 unit enzyme standard under the conditions of the assay (50 °C and pH 4.5). The feed additive is intended to be marketed as solid and liquid formulations having the following guaranteed minimum activities: 15000 EPU/g for endo-1,4-beta-xylanase, 1000 CU/g for endo-1,4-beta-glucanase and 1000 XGU/g for xyloglucan-specific-endo-beta-1,4-glucanase. The solid product is intended to be incorporated through premixtures or directly into feedingstuffs while the liquid formulation should be applied after the pelleting process by spraying it on the pellets to obtain a minimum content for endo-1,4-beta-xylanase, endo-1,4-beta-glucanase and xyloglucan-specific-endo-beta-1,4-glucanase respectively of 1500 EPU, 100 CU and 100 XGU/kg feedingstuffs. 

For the quantification of the endo-1,4-beta-xylanase activity in the feed additive, premixtures and feedingstuffs the Applicant submitted single-laboratory validated and further verified colorimetric methods based on the quantification of water soluble dyed fragments produced at pH 4.7 and 50 °C, by the action of endo-1,4-beta-xylanase on commercially available azurine cross-linked wheat arabinoxylan substrates from Megazyme. 

For the quantification of the endo-1,4-beta-glucanase activity in the feed additive, premixtures and feedingstuffs the Applicant submitted 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-beta-glucanase on commercially available azurine cross-linked cellulose substrate from Megazyme. 

For the quantification of xyloglucan-specific-endo-beta-1,4-glucanase activity in the feed additive, premixtures and feedingstuffs the Applicant submitted a single-laboratory validated and further verified colorimetric method based on the quantification of soluble dyed labelled fragments produced at pH 4.5 and 50°C by the action of xyloglucan-specific-endo-beta-1,4-glucanase on commercially available xyloglucan substrate from Megazyme. 

Based on the acceptable performance characteristics presented the EURL recommends for official control the single-laboratory validated and further verified colorimetric methods submitted by the Applicant for the determination of the activity of endo-1,4-beta-xylanase, endo-1,4-beta-glucanase and xyloglucan-specific-endo-beta-1,4-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, as last amended by Regulation (EU) 2015/1761) is not considered necessary.

Recommended Method

as of:

-Commission Implementing Regulation (EU) 2023/1169

-Commission Implementing Regulation (EU) 2024/2180

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

• colorimetric method measuring water soluble dye released by action of endo-1,4-beta-xylanase from azurine cross-linked wheat arabinoxylan substrate.

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

• 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.

For the determination of xyloglucan- specific endo-beta-1,4-glucanase activity in the feed additive, premixtures and compound feed: 

• colorimetric method based on the quantification of soluble dyed labelled fragments produced by the action of xyloglucan-specific endo- beta-1,4-glucanase on xyloglucan substrate.