Bacillus velezensis ATCC PTA-6737

In the current application authorisation is sought for the microbial feed additive Bacillus subtilis ATCC PTA-6737 (Bacillus subtilis PB6)¹ under the category ‘zootechnical additives’, functional group 'gut flora stabilisers' according to Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the use of Bacillus subtilis PB6 as a gut flora stabiliser for chickens for fattening. The feed additive has a minimum of 1x10¹⁰ colony-forming units (CFU) per gram of viable spores of Bacillus subtilis ATCC PTA-6737 as active agent in maltodextrin carrier. The feed additive is intended to be mixed into complete feedingstuffs at a final concentration ranging from 1x10⁷ to 5x10⁷ CFU/kg of feedingstuffs. 

For the enumeration of Bacillus subtilis ATCC PTA-6737 in the feed additive, premixtures and feedingstuffs, the applicant proposes the draft CEN method - prEN 15784:2008 E – an internationally recognised spread plate method. This method was ring-trial validated using the premixtures and feedingstuffs samples containing Bacillus subtilis spores. The performance characteristics of the draft CEN method reported after logarithmic transformation of measured values (CFU) are: 

- For the premixtures: (1) a standard deviation for repeatability (s_r) of 0.09 log₁₀ and (2) a standard deviation for between-laboratory reproducibility (s_R) of 0.32 log₁₀. 

- For the feedingstuffs: (1) a s_r of 0.07 log₁₀ and (2) a s_R of 0.35 log₁₀. 

The applicant used the above mentioned spread plate method to analyse the various matrices containing Bacillus subtilis ATCC PTA-6737 spores and reported the following results: (a) 1x10⁹ to 1.5x10¹¹ CFU/g of feed additive; (b) 1x10⁷ to 1.5x10⁹ CFU/kg for premixtures and (c) 1x10⁷ to 1.5x10⁸ CFU/kg for feedingstuffs. The results obtained for feed additive and premixtures are considered acceptable; this method is therefore recommended for official controls for the feed additives and premixtures in the frame of the authorisation. 

As regards feedingstuffs, the CRL notes that the limit of quantification reported by the applicant upon request (LOQ = 1x10⁷ CFU/kg feedingstuffs) is identical to the minimum dose proposed and is below the LOQ reported in the draft CEN method (2x10⁷ CFU/kg). On the basis of the available information, the draft CEN method is recommended for official control of the feedingstuffs containing Bacillus subtilis PB6 at the dosages above the LOQ reported by CEN. Below 2x10⁷ CFU/kg the CRL is not able to conclude on the suitability of this method for official control purposes. 

Molecular methods were used by the applicant for identification of the active agent. For official controls pulsed field gel electrophoresis (PFGE), a generally recognised standard methodology for microbial identification, is recommended.

Further testing or validation is not considered necessary.

¹New name: Bacillus velezensis ATCC PTA-6737

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2023/366

- Commission Implementing Regulation (EU) 2024/3166

Enumeration: 

• Spread plate method on tryptone soya agar (EN 15784)²

Identification: 

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

  or 

• DNA sequencing methods²

²The text is according to Commission Implementing Regulation (EU) 2024/3166