Enterococcus lactis DSM 7134 and Lacticaseibacillus rhamnosus DSM 7133

In the current application authorisation is sought under Article 10(2) for Provita LE, consisting of two active agents: Enterococcus faecium DSM 7134¹ and Lactobacillus rhamnosus DSM 7133², under the category/functional group 4(b), ''zootechnical additives/gut flora stabilisers'', according to the classification system of Annex I of Regulation (EC) No 1831/2003. The feed additive will be marketed in a powdered form containing a minimum concentration of 7x10⁹ CFU/g of Enterococcus faecium DSM 7134 and 3x10⁹ CFU/g of Lactobacillus rhamnosus DSM 7133, with dextrose anhydrous, maltodextrin and whey powder as carriers. Specifically, authorisation is sought for the use of the feed additive for calves for rearing and it is intended to be mixed at a total concentration of the two microorganisms ranging from 1x10⁹ to 5x10⁹ CFU/kg of complete feedingstuffs.

For the enumeration of the two microorganisms in the feed additive, premixtures and feedingstuffs the Applicant proposed two internationally recognised ring-trial validated spread plate method issued by the European Committee for Standardization (CEN): (i) for the enumeration of Enterococcus spp (EN 15788), using Bile Esculin Azide Agar and for enumeration of Lactobacillus spp (EN 15787), using MRS agar. The performance characteristics reported for CEN methods after logarithmic transformation (CFU) are: 

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

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

- a limit of detection (LOD) of 1x10⁵ CFU/kg feedingstuffs.

Based on the performance characteristics presented, the EURL recommends for official control, the two CEN methods (EN 15788 and EN 15787) for the determination of Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133 in the feed additive, premixtures and feedingstuffs.

Molecular methods were used by the Applicant to identify the active agent in the feed additive. The EURL recommends instead for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised standard methodology for microbial identification. 

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.

¹New name:Enterococcus lactis DSM 7134.

²New name: Lacticaseibacillus rhamnosus DSM 7133

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2024/1187

Identification: 

• DNA sequencing methods 

  or

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

Enumeration in feed additive and compound feed of: 

• Enterococcus lactis DSM 7134: spread plate method using bile esculin azide agar (EN 15788)

   

• Lacticaseibacillus rhamnosus DSM 7133: spread plate method using MRS agar (EN 15787)