Bacillus subtilis FERM BP-07462, Enterococcus lactis FERM BP-10867 and Clostridium butyricum FERM BP-10866

In the current application an authorisation is sought under Article 4(1) for a preparation containing Bacillus subtilis TO-A, Enterococcus faecium T-110 and Clostridium butyricum TO-A (BIO-THREE®)¹, under the category / functional group 4(b) 'zootechnical additives' / 'gut flora stabilisers', according to 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, turkeys for fattening, turkeys reared for breeding and all minor avian species to slaughter or to point of lay. 

According to the Applicant, the feed additive contains as active agents viable spores of the non-genetically modified microorganism (non-GMM) B. subtilis TO-A, non-GMM E. faecium T-110 and viable spores of non-GMM C. butyricum TO-A. The feed additive is intended to be marketed as dry or liquid preparations containing a minimum content of 1x10⁷ Colony Forming Unit (CFU) B. subtilis TO-A /g, 1x10⁸ CFU E. faecium T-110 /g and 1x10⁷ CFU C. butyricum TO-A /g and to be used directly in complete feedingstuffs or in complementary feeds and in water at a minimum dose of 2.0x10⁶ CFU B. subtilis TO-A; 2.0x10⁷ CFU E. faecium T-110 and 2.0x10⁶ CFU C. butyricum TO-A /kg complete feedingstuffs and of 1.0x10⁶ CFU B. subtilis TO-A, 1.0x10⁷ CFU E. faecium T-110 and 1.0x10⁶ CFU C. butyricum TO-A /l of water. 

For the identification of the three target strains the EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised methodology for the genetic identification of bacterial strains. 

For the enumeration of Bacillus subtilis TO-A and Enterococcus faecium T-110 in the feed additive, premixtures, feedingstuffs and water the Applicant proposed the ring-trial validated spread plate methods EN 15784 and EN 15788, respectively, while for the enumeration of Clostridium butyricum TO-A in the feed additive, premixtures feedingstuffs and water, the Applicant proposed the pour plate method ISO 15213. Furthermore, the Applicant provided evidences of the suitability of all the mentioned methods for the enumeration of these microorganisms in the mentioned matrices. 

Based on the performance characteristics reported and the applicability evidences provided by the Applicant, the EURL recommends for official control the internationally recognised standard methods EN 15784, EN 15788 and ISO 15213 for the enumeration of Bacillus subtilis TO-A, Enterococcus faecium T-110 and Clostridium butyricum TO-A respectively in the feed additive, premixtures, feedingstuffs and water.

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.

¹New names: Bacillus subtilis FERM BP-07462, Enterococcus lactis FERM BP-10867 and Clostridium butyricum FERM BP-10866.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2024/2185

Identification: 

• DNA sequencing methods or Pulsed Field Gel Electrophoresis (PFGE) – CEN/TS 17697

Enumeration in the feed additive, premixtures, compound feed and water of: 

Bacillus subtilis FERM BP-07462: 

• Spread plate method on tryptone soya agar (EN 15784)

Enterococcus lactis FERM BP-10867: 

• Spread plate method using bile esculin azide agar (EN 15788)

Clostridium butyricum FERM BP-10866: 

• Pour plate method using iron sulfite agar (ISO 15213)