Bacillus subtilis CNCM I-4606, CNCM I-5043, CNCM I-4607 and Lactococcus lactis CNCM I-4609

In the current application an authorisation is sought under Article 4(1) for the preparation of Bacillus subtilis CNCM I-4606, Bacillus subtilis CNCM I-5043, Bacillus subtilis CNCM I-4607 and Lactococcus lactis CNCM I-4609 (MixBaLac) under the category/ functional group 1(n) ‘technological feed additive’/‘hygiene condition enhancer’, according to Annex I of Regulation (EC) No 1831/2003. The authorisation is sought for the use of the feed additive for all animal species. 

According to the Applicant, the feed additive contains as an active substance the viable spores of Bacillus subtilis (CNCM I-4606, CNCM I-5043, CNCM I-4607) and live cells of Lactococcus lactis (CNCM I-4609). The feed additive contains a minimum content of 2x10⁹ Colony Forming Units (CFU) of total Bacillus subtilis /g feed additive and a minimum content of 2x10⁹ CFU of Lactococcus lactis /g feed additive. 

The feed additive is intended to be used directly into feedingstuffs or through premixtures at a minimum dose of 1x10⁹ CFU of total Bacillus subtilis /kg (or L) complete feedingstuffs and at a minimum dose of 1x10⁹ CFU of Lactococcus lactis /kg (or L) complete feedingstuffs.

For the identification of Bacillus subtilis CNCM I-4606, Bacillus subtilis CNCM I-5043, Bacillus subtilis CNCM I-4607 and Lactococcus lactis CNCM I-4609, the EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised methodology for genetic identification of bacterial strains. 

For the enumeration of the total Bacillus subtilis (CNCM I-4606, CNCM I-5043 and CNCM I-4607) in the feed additive, premixtures and feedingstuffs the Applicant submitted the ring-trial validated spread-plate CEN method EN 15784. 

Based on the performance characteristics available, the EURL recommends this method for official control for the enumeration of the total Bacillus subtilis (CNCM I-4606, CNCM I-5043 and CNCM I-4607) in the feed additive, premixtures and feedingstuffs. 

For the enumeration of Lactococcus lactis (CNCM I-4609) in the feed additive, premixtures and feedingstuffs the Applicant proposed the ISO 15214 pour-plate method dedicated for the enumeration of mesophilic lactic acid bacteria in food and feed. 

Based on acceptable applicability data of the method, the EURL recommends it for official control for the enumeration of Lactococcus lactis (CNCM I-4609) 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) 2024/764

Enumeration in the feed additive, premixtures and compound feeds of the total Bacillus subtilis CNCM I-4606, CNCM I-5043 and CNCM I-4607:

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

Enumeration in the feed additive, premixtures and compound feeds of the Lactococcus lactis CNCM I-4609:

• Poured plate method on MRS agar (EN 15214)

Identification of Bacillus subtilis CNCM I-4606, CNCM I-5043 and CNCM I-4607 and of the Lactococcus lactis CNCM I-4609:

• Pulsed Field Gel Electrophoresis (PFGE) CEN/TS 17697 

  or 

• DNA sequencing methods