A collaborative study of a method for enumeration of probiotic enterococci in animal feed

Abstract

Aims: Validation of an enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic enterococci used as feed additives.

Methods and results: Twenty laboratories in 12 European countries carried out a collaborative study. A plate count method using bile esculin azide (BEA) agar was used. Precision data in terms of repeatability (r) and reproducibility (R) of the method using different feeding stuffs and three inoculation levels were determined. Enterococci were present in the samples as a single component or in mixtures with other probiotic feed additives. The enumeration of enterococci on BEA agar showed a relative standard deviation (RSD)r of 1.5-3.6% and an RSD(R) between 2.9 and 7.4%. BEA agar was selective for enterococci in the presence of other probiotic micro-organisms such as pediococci, lactobacilli and yeast.

Conclusions: For routine analysis of viable enterococci concentrations in feeding stuffs, the use of BEA is recommended. This methodology is not applicable for mineral feeds.

Significance and impact of the study: An official control method for enumeration of authorized probiotic enterococci in feeding stuffs was validated. The results are intended for consideration for adoption as CEN and ISO standards.