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

Abstract

A collaborative study comprising 20 laboratories in 12 European Countries to validate an enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic lactobacilli used as feed additives was carried out. A spread plate method following BS ISO 15214:1998 using four different agars, MRS, acidified MRS, MRS with triphenyl tetrazolium chloride (TTC) and LAMVAB (a selective medium) was validated. Precision data in terms of repeatability ( r) and reproducibility ( R) of the method for each medium using different feeding stuffs with a high and a low inoculation level were determined. Lactobacilli were present in the samples in mixtures with other probiotics. The enumeration of lactobacilli on all agars showed a relative standard deviation (RSD r ) between 1.2% and 3.4% and a RSD R between 2.2% and 5.2%. MRS agar was preferred, followed by acidified MRS and MRS+TTC agar. LAMVAB gave about 0.5 log 10 cfu g −1 lower counts than the MRS media. For routine analysis the use of MRS agar is recommended depending on the presence and concentration of other probiotics such as enterococci, pediococci and yeast, acidified MRS or MRS+TTC agar are recommended. Pediococci and yeast were able to grow on LAMVAB. The methodology is not applicable to mineral feed. The results are intended for consideration for adaptation as CEN and ISO standards.