Alpha-galactosidase (EC 3.2.1.22); Endo-1,4- beta-glucanase (EC 3.2.1.4)

In the current application authorisation is sought for Biogalactosidase BL (AlphaGal BL) under the category “zootechnical additives”, functional groups 4(a), according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought to use Biogalactosidase BL as a digestibility enhancer for chicken for fattening. The additive is intended to be marketed as a solid formulation. 

Biogalactosidase BL consists of two active agents: (i) α-galactosidase (EC 3.2.1.22) produced by Sacharomyces cerevisiae (CBS 615.94) and (ii) endo-1,4-beta-glucanase (EC 3.2.1.4) produced by Aspergillus niger (CBS 120604). The enzymatic activities of alpha-galactosidase and endo-1,4-beta-glucanase are expressed in enzyme units (U). According to the applicant (i) one alpha-galactosidase unit (U) is the quantity of enzyme which liberates 1 μmol of p-nitrophenol per minute from p-nitrophenyl-alpha-galactopyranoside (pNPG) at pH = 5.0 and 37^oC, whereas (ii) one endo-1,4-beta-glucanase unit (U) is the quantity of enzyme which liberates 1 mg of reducing sugar (glucose equivalent) per minute from beta-glucan at  pH = 5.0 and 50^oC. Biogalactosidase BL has an alpha-galactosidase and endo-1,4-beta-glucanase target activity of 1000 U/g and 5700 U/g.  The product is intended to be mixed into premixtures and/or feedingstuffs to obtain activity levels of 50 to 100 U alpha-galactosidase /kg and 285 to 570 U endo-1,4-beta-glucanase /kg in complete feedingstuffs.

For the determination of alpha-galactosidase the applicant proposes two single laboratory validated and further verified colorimetric methods for the analysis: - in feed additives; and in premixtures and feedingstuffs. These methods are based on the production of p‑nitrophenol (pNP) and D-galactose from p-nitrophenyl-alpha-galactopyranoside (pNPG) at pH = 5.0 at 37^oC.  The following performance characteristics were reported: (a) relative standard deviations for repeatability (RSD_r) ranging from 2.7 to 10 %;  (b) relative standard deviations for reproducibility (RSD_R) ranging from 3 to 6 %; (c) recovery rates (R_Rec) ranging from 81 to 103 %; and a limit of quantification (LOQ) of 10 U/kg feedingstuffs. 

For the determination of the activity of endo-1,4-beta-glucanase in the feed additive the applicant proposes a single laboratory validated and further verified colorimetric method, based on the hydrolyses of a barley beta-D-glucan at 50^oC and pH = 5.0 to release reducing sugars reacting with 3.5-dinitro salicylic acid (DNS). As for the determination of the enzyme activity of endo-1,4-beta-glucanase in the premixtures and feedingstuffs the applicant proposes another single laboratory validated colorimetric method using azurine cross-linked barley glucan substrate. 

The following performance characteristics were reported: (a) RSD_r ranging from 3.3 to 10 %; (b) RSD_R ranging from 1.3 to 15 %; (c) R_Rec around 100 %; and a limit of detection (LOD) and a limit of quantification (LOQ) of 66 and 132 U/kg feedingstuffs, respectively.

Based on the above mentioned performance characteristics, the CRL recommends for official control the single laboratory validated and further verified colorimetric methods submitted by the applicant for the determination of alpha-galactosidase and endo-1,4-beta-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

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2015/2382

- Commission Implementing Regulation (EU) 2024/781

For the determination of alpha-galactosidase in the feed additive, premixtures and compound feed:

• Colorimetric method measuring p-nitrophenol released by action of alpha-galactosidase from p-nitrophenyl-alpha-galactopyranoside substrate

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

• Colorimetric method measuring water soluble dye released by action of endo-1,4-beta-glucanase from azurine-crosslinked barley glucan substrate