Enumeration of probiotic bacilli spores in animal feed: Interlaboratory study

spores or a blank that contained vegetative probiotic bacteria only. For pretreatment A, the repeatability relative standard deviation (RSDr) was 2.9% for the low level and 2.5% for the high. The reproducibility relative standard deviation (RSDR) values were 7.8 and 5.9%, respectively. Pretreatment B revealed RSDr values of 1.1 and 1.0%, and RSDR values of 5.8 and 3.4%, respectively. The heat treatment (pretreatment B) of feed samples had better precision data, resulted in higher viable bacilli counts, and was more effective in deactivating vegetative background flora. It is therefore recommended for adoption for official control purposes and for CEN and ISO standards. P robiotics are viable microorganisms used in animal nutrition as feed supplements to produce beneficial effects in the host animal (1). Probiotic bacilli have effects on a variety of animals and are therefore added to animal feedstuffs (2–9). Methods for the official control of probiotic bacilli used as feed additives were developed within a European Community project (SMT4 CT98-2235) to enable independent quality control of these products. For acceptance of standards by the Comite Europeen de Normalisation (CEN), methods must be validated by means of an interlaboratory study. The method developed in this study was based on reports in the literature and on available standard methods that suggest heat treatment to inactivate vegetative cells before bacterial spore enumeration in feedstuffs (10, 11). The heat pretreatmentisroutinelyusedinindustriallaboratoriesandinvolves heating the initial suspension of the feed sample at 80C for 10 min. The project partner of the European Union project with responsibility for the development of a probiotic bacilli enumeration method suggested an ethanol pretreatment. This required initial suspension of the feedstuff sample in50%ethanolandincubationatroomtemperatureonarotary shaker for 1 h. The ethanol treatment has been described as an alternative for spores of Clostridium botulinum, which can be either heat-sensitive or -resistant (12). Both methods used nonselective tryptone soy agar (TSA) and incubated at 37C for 16–24 h under aerobic conditions. MethodprecisiondatahavenotbeenavailableforeitherpretreatmentenumerationmethodsexceptforBacilluscereus(13). The 2 pretreatment methods for enumeration of bacilli spores were therefore validated according to a protocol conforming to the International Harmonized Protocol for Collaborative Studies (14). Data from the interlaboratry study were used to calculate the repeatability (r) and reproducibility (R) of the methods in relation to feed and premixtures for both pretreatments using nonselective TSA (15). The precision data of both methods were compared and, according to their performance in the trial, one method was selected for Official Control Method, and is intended for submission to CEN for inclusion in