Endo-1,4-beta-xylanase produced by Trichoderma reesei (CBS 114044) - 4a8i

Econase® XT is the trade name of a feed additive containing as active substance endo-1,4-beta-xylanase (EC 3.2.1.8) produced from Trichoderma reesei (CBS 114044). This feed additive is currently authorised by different Commission Regulations under the category/functional 4(a) "zootechnical additives"/"digestibility enhancers" according to the classification system of Annex I of Regulation (EC) No 1831/2003 (feed additive identification number 4a8). In the current application a renewal of the feed additive authorisation under Article 14 of the Regulation (EC) No 1831/2003 is requested for different avian and porcine species. 

The endo-1,4-beta-xylanase activity is expressed in BXU units, where "one BXU is the amount of enzyme, which liberates one nanomole per second of reducing sugars, expressed as xylose equivalents, from birch xylan at pH 5.3 and 50 °C". The feed additive is intended to be marketed as light-brown powder formulations (Econase XT 25; Econase XT 5 P and Econase XT P) or as brown liquids (Econase XT 25L and Econase XT L) with minimum activities ranging from 160000 BXU/g to 4000000 BXU/g. Endo-1,4-beta-xylanase (4a8) is intended to be incorporated directly or through premixtures at a minimum endo-1,4-beta-xylanase activity in feedingstuffs of 8000, 16000 or 24000 BXU/kg depending on the target species. 

For the quantification of the activity of endo-1,4-beta-xylanase in the feed additive and premixtures the Applicant submitted a single-laboratory validated and further verified spectrophotometric method, based on the formation of reducing sugars reacting with 3,5-dinitrosalicylic acid (DNS) at pH 5.3 and 50 °C while for the feedingstuffs the Applicant submitted a different single-laboratory validated and further verified spectrophotometric method based on the quantification of water soluble dye fragments produced at pH 5.0 and 50 °C by the action of endo-1,4-β-xylanase on commercially available azurine cross-linked wheat arabinoxylan substrates from Megazyme. For the feed additive and premixtures external calibration is performed using a commercially available xylose standard, while for feedingstuffs external calibration is carried out using a xylanase standard with known enzyme activity and subjected to the same experimental conditions than the feedingstuffs samples. For all matrices the measurements are performed by spectrophotometry at 540 nm. 

According to the results provided by the Applicant in the frame of the respective validation and verification studies, relative standard deviations for repeatability (RSD_r) and for intermediate precision (RSD_ip) ranging from 2.1 to 8.9 % and from 4.1 to 7.2 %, respectively, were obtained for the quantification of the activity of endo-1,4-beta-xylanase in the feed additive, premixtures and feedingstuffs.

Based on the performance characteristics available the EURL recommends for official control these methods for the quantification of the total endo-1,4-beta-xylanase activity in these three matrices. 

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) 2023/1332

Characterisation of the active substance in the additive and the premixture: 

reducing sugar assay for endo-1,4- beta-xylanase by colorimetric reaction of dinitrosalicylic acid reagent on reducing sugar yield at pH 5,3 and 50 °C.

Characterisation of the active substance in the compound feed: 

colorimetric method measuring water soluble dye released by the enzyme from azurine crosslinked wheat arabinoxylan substrate.