A PCR method was developed to detect spores of Bacillus sporothermodurans in 1, 10, and 100 ml of raw milk. Two primers were derived from a unique sequence after subtractive hybridization of B. sporothermodurans DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B. sporothermodurans. Specific identification was proven on a large collection of Bacillus strains and on strains from relevant taxa. The detection of B. sporothermodurans in raw milk is based on activation, germination, and outgrowth of the spores, followed by PCR identification. Spores from 10 and 100 ml were concentrated by centrifugation after chemical extraction of the milk components. The total test takes 28 h. The detection limits are 9, 0.4, and 0.22 CFU/ml for 1, 10, and 100 ml, respectively. Bacillus sporothermodurans is a gram-positive bacterium which produces highly heat-resistant endospores (7, 11, 17). These exceptionally heat-resistant endospores may survive sterilizing and ultrahigh temperature (UHT) treatment. UHT-treated and sterilized milk products are considered commercially sterile when after incubation for 15 days at 30°C the total count is Յ10 CFU/0.1 ml (1). B. sporothermodurans can reach up to 10 5 CFU/ml in sterilized and UHT-treated milk products and therefore causes the problem of nonsterility. The bacterium is considered nonpathogenic (8, 21) and causes in certain circumstances some spoilage effects (12). B. sporothermodurans is characterized by phenotypic tests, 16S rRNA sequencing, and molecular typing techniques (12, 17). Pettersson et al. (17) showed the occurrence of two different types of B. sporothermodurans. These two types differed phenotypically and genotypically. The collection of B. sporothermodurans strains, described by Klijn et al. (12), was homogeneous on the basis of 16S rRNA sequences, randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic (REP) fingerprinting patterns. All phenotypic and genetic characterization methods described till now for B. sporothermodurans are quite time-consuming and can be applied only on pure bacterial cultures. Application of PCR offers the opportunity to develop specific identification methods which can be applied for bacterial detection in complex food matrices like raw milk (15). Sensitive detection can be obtained even in the presence of a background flora of 10 5 CFU/ml (9). In this paper, a PCR identification method for B. sporothermodurans is presented. Specific sequences of B. sporothermodurans are obtained by subtractive hybridization, which allows the removal of homologous sequences of two different strains so that unique sequences for the target organism can be isolated (3). The unique sequence of B. sporothermodurans forms the basis of the specific PCR identification method. The specificity of this method was tested on pure bacterial strains and on raw-milk samples. The analysis of raw-milk samples was necessary because false-positive reactions can be obtained due to the presence of n...

A direct detection method for Clostridium tyrobutyricum spores in up to 100 ml of raw milk is presented. The bacterial spores are concentrated by centrifugation after chemical extraction of the milk components. The vegetative cells are selectively lysed, and their DNA is digested and washed away. Afterwards, the DNA is liberated from the spores by microwave treatment. For the identification of the C. tyrobutyricum DNA, a two-step PCR method with two nested pairs of primers is used. The primers were derived from the 16S-23S rRNA spacer region of C. tyrobutyricum, and the specificity of each of them for C. tyrobutyricum is demonstrated. The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk.

No abstract

No abstract

A simple method is described to detect, within 2 h, complete failure of the starter due to bacteriophages in the manufacture of Cheddar cheese. This method is based on the observation that about 105 disturbing bacteriophages per ml, which cause complete failure of the starter, inhibit the normal impedance decrease brought about by growth of lactic starter bacteria, as recorded in the Bactometer 32 Microbial Monitoring System.