Bifidobacterium animalis ssp. animalis DSM 16284, Ligilactobacillus Salivarius DSM 16351 and Enterococcus faecium DSM 21913

In the current application an authorisation is sought for the microbial product Biomin^® C3  under the category 'zootechnical additives', functional group 'gut flora stabilizers' according to Annex I of Regulation (EC) No 1831/2003. Specifically, authorization is sought for Biomin^® C3, which is composed of three microorganism strains, Bifidobacterium animalis ssp. animalis DSM 16284, Enterococcus faecium DSM 21913 and Lactobacillus salivarius ssp. salivarius DSM 16351¹. The feed additive is intended to be mixed at a total viable count ranging from 1x10⁸ CFU/kg to 1x10⁹ CFU/kg of feedingstuffs for chickens for fattening.

For the enumeration of Bifidobacterium animalis ssp. animalis DSM 16284, Enterococcus faecium DSM 21913 and Lactobacillus salivarius ssp. salivarius DSM 16351 strains in feed additive, premixtures and feedingstuffs, the applicant proposed a ring-trial validated spread plate methods developed by Leuschner et al. All three methods are currently available as CEN method: EN 15785; EN 15788; EN 15787. The performance characteristics of the CEN methods, for plate enumeration of the three active substance in premixtures and feedingstuffs, after log₁₀ conversion, are:

- a standard deviation for repeatability (S_r) ranging from 0.12 to 0.24 log₁₀ CFU/g,

- a standard deviation for reproducibility (S_R) ranging from 0.23 to 0.41 log₁₀ CFU/g; and

- a limit of quantification (LOQ) of 10⁵ CFU/g.

Based on these acceptable performance characteristics, the CRL recommends for official control the following CEN methods, EN 15785; EN 15788; EN 15787, for the enumeration of Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp., in feed additive, premixtures and feedingstuffs.

Molecular methods were used by the applicant for strain identification. The CRL recommends for official controls pulsed field gel electrophoresis (PFGE), a generally recognised standard methodology for microbial identification.

Further testing or validation is not considered necessary.

¹New name: Ligilactobacillus salivarius DSM 16351.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2024/1200

Identification:

• DNA sequencing methods 

  or 

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

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

• Bifidobacterium animalis ssp. animalis DSM 16284: 

  spread plate method EN 15785

• Ligilactobacillus salivarius DSM 16351: 

  spread plate method EN 15787 

• Enterococcus faecium DSM 21913: 

  spread plate method EN 15788