Bacillus licheniformis ATCC 53757 and its protease EC 3.4.21.19 (4d12)

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2016/898

Identification and enumeration of Bacillus licheniformis ATCC 53757 in the feed additive, premixtures and feedingstuffs:

Identification:

- Pulsed field gel electrophoresis (PFGE)

Enumeration:

-Spread plate method using tryptone soya agar - EN 15784

Quantification of protease in the feed additive, premixtures and feedingstuffs:

- Colorimetric method measuring para-nitroaniline (pNA) released by the enzymatic reaction of protease on Suc-Ala-Ala- Pro-Phe-pNA substrate at 37 °C

Executive Summary

In the current application authorisation is sought under article 4(1) for Cibenza® EP150 under the category/functional group 1(d) "zootechnical additives"/"other zootechnical additives", according to the classification system of Annex I of Regulation (EC) No 1831/2003. The authorisation is sought for the use of the feed additive for chickens for fattening, chickens reared for laying, and minor avian species (ducks, geese, pigeons and other game birds) for meat production. Cibenza® EP150 is a dry preparation containing two active substances: a protease (EC 3.4.21.19) and viable spores of non-genetically modified Bacillus licheniformis (strain PWD-1, ATCC 53757). The Applicant stated the following minimum activity/concentration in the product: 6x105 U/g for protease and 1x109 Colony Forming Units (CFU)/g for Bacillus licheniformis. The Applicant defined the protease activity units (U) as the amount of protease that liberates 1 micromole of para-nitroaniline (pNA) from the Succinyl-Ala-Ala-Pro-Phe-pNA (C30H36N6O9) substrate per minute at pH 8.0 and 37°C. Cibenza® EP150 is intended to be used in premixtures and feedingstuffs, with the minimum activity/concentration in feedingstuffs of: 3x105 U/kg for protease and 5x108 CFU/kg for Bacillus licheniformis, respectively.

For the identification and characterisation of the strain Bacillus licheniformis ATCC 53757 the Applicant studied biochemical parameters and applied DNA sequencing, together with Pulsed Field Gel Electrophoresis (PFGE). The EURL recommends for official control PFGE, the generally recognised standard methodology for microbial identification that is currently being evaluated by the CEN Technical Committee 327 to become European Standards.

For the enumeration of spores of Bacillus licheniformis ATCC 53757 in feed additive, premixtures and feedingstuffs, the Applicant submitted the ring-trial validated CEN spread plate method (EN 15784). However, this method is not applicable to mineral feeds composed mainly of minerals and containing at least 40 % crude ash. During the review process one of the National Reference Laboratories identified the ring-trial validated VDLUFA method 28.2.2 for the enumeration in mineral feed. Based on the performance characteristics available, the EURL recommends for official control the EN 15784 and VDLUFA methods.

For the quantification of the protease activity in the feed additive, premixtures and feedingstuffs the Applicant submitted a single-laboratory validated and further verified colorimetric methods based on the quantification of para-nitroaniline (pNA) released by the action of protease on the synthetic peptide substrate (Succinyl-Ala-Ala-Pro-Phe-pNA). Based on the satisfactory experimental evidence provided the EURL recommends for official control the colorimetric methods submitted.

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.