Microbial biodiversity, quality and shelf life of microfiltered and pasteurized extended shelf life (ESL) milk from Germany, Austria and Switzerland.

Information on factors limiting the shelf life of extended shelf life (ESL) milk produced by microfiltration and subsequent pasteurization is very limited. In this study, three different batches of ESL milk were analyzed at different stages of the production process and during storage at 4 °C, 8 °C and 10 °C in order to evaluate the changes in bacterial cell counts, microbial diversity and enzymatic quality. Additionally, detailed biodiversity analyses of 250 retail ESL milk packages produced by five manufacturers in Germany, Austria and Switzerland were performed at the end of shelf life. It was observed that microfiltration decreased the microbial loads by 5-6 log10 units to lower than 1 CFU/mL. However, bacterial counts at the end of shelf life were extremely variable and ranged between <1 and 8 log10 CFU/mL. 8% of all samples showed spoilage indicated by cell counts higher than 6 log10 CFU/mL. The main spoilage groups of bacteria were Gram-negative post-process recontaminants (Acinetobacter, Chryseobacterium, Psychrobacter, Sphingomonas) and the spore formers Paenibacillus and Bacillus cereus, while other spore formers and Microbacterium spp. did not reach spoilage levels. Paenibacillus spp. and B. cereus apparently influenced enzymatic spoilage, as indicated by increased free fatty acid production, pH 4.6 soluble peptide fractions and off-flavors. In some cases, enzymatic spoilage was observed although microbial counts were well below 6 log10 CFU/mL. Thirteen B. cereus isolates were characterized for their toxin profiles and psychrotolerance. Hbl, nhe, and cytK toxin genes were detected in ten, thirteen, and four isolates, respectively, whereas the ces gene was always absent. Interestingly, only three of the thirteen isolates could be allocated to psychrotolerant genotypes, as indicated by the major cold shock cspA gene signature. Generally, large discrepancies in microbial loads and biodiversity were observed at the end of shelf life, even among packages of the same production batch. We suggest that such unexpected differences may be due to very low cell counts after ESL treatment, causing stochastic variations of initial species distributions in individual packages. This would result in the development of significantly different bacterial populations during cold storage, including the occasional development of high numbers of pathogenic species such as B. cereus or Acinetobacter.

Sphingobacterium lactis sp. nov. and Sphingobacterium alimentarium sp. nov., isolated from raw milk and a dairy environment.

Four non-fermenting, rod-shaped, Gram-staining-negative bacterial strains, designated WCC 4512(T), WS 4555, WCC 4521(T) and WS 4556, were isolated from raw milk and the dairy environment. Phylogenetic analyses based on 16S rRNA and groEL gene sequences demonstrated the affiliation of the four strains to two distinct clusters within the class Sphingobacteriia, phylum 'Bacteroidetes'. Strains WCC 4512(T) and WS 4555 showed the highest 16S rRNA gene sequence similarity to the type strain of S. daejeonense (97.3 and 97.2%, respectively), whereas strains WCC 4521(T) and WS 4556 were most closely related to S. composti LMG 23401(T) (97.6% 16S rRNA gene sequence similarity). The DNA G+C contents of strains WCC 4512(T) and WCC 4521(T) were 44.2 and 39.3 mol%, respectively. The major cellular fatty acids and the presence of menaquinone MK-7 as the predominant quinone for both strains WCC 4512(T) and WCC 4521(T) supported their affiliation to the genus Sphingobacterium. DNA-DNA hybridization experiments between strain WCC 4512(T) and S. daejeonense LMG 23402(T) and between strain WCC 4521(T) and S. composti LMG 23401(T) revealed DNA relatedness values of 2% (repetition, 3%) and 8% (repetition, 17%), respectively. On the basis of phenotypic and genetic properties, as well as phylogenetic distinctiveness, it is suggested that the four strains represent two novel Sphingobacterium species with strain WCC 4512(T) (=DSM 22361(T)=LMG 25272(T)) as the type strain of Sphingobacterium lactis sp. nov. (WS 4555 is a reference strain of S. lactis) and strain WCC 4521(T) (=DSM 22362(T)=LMG 25273(T)) as the type strain of Sphingobacterium alimentarium sp. nov. (WS 4556 is a reference strain of S. alimentarium).

Bavariicoccus seileri gen. nov., sp. nov., isolated from the surface and smear water of German red smear soft cheese.

The phylogenetic position and physiological characters of six hitherto-unknown lactic acid bacterial isolates, which form part of the surface microbiota of German red smear soft cheese, are reported. The coccoid cells are aerotolerant, Gram-positive, catalase-negative and non-motile. The cell-wall peptidoglycan contains alanine, glutamic acid, lysine and aspartic acid and is of the A4alpha type (l-Lys-d-Asp). The sequences of the 16S rRNA, groEL and rpoB genes of the six isolates are identical and reveal that these isolates represent an independent lineage within the radiation of the family Enterococcaceae in the phylum Firmicutes. Their closest phylogenetic neighbour is the lactic acid bacterium Atopobacter phocae M1590/94/2(T), with which they share 94.9 % 16S rRNA gene sequence similarity; representatives of other genera such as Granulicatella, Carnobacterium and Trichococcus are more distantly related. DNA-DNA hybridization studies reveal that the six isolates are members of a single species, and this is confirmed by similarities in biochemical characteristics. The six isolates were assigned four different groups by Fourier-transform infrared and randomly amplified polymorphic DNA typing. Therefore, it is formally proposed that these isolates should be classified in a single novel species of a novel genus and be named Bavariicoccus seileri gen. nov., sp. nov. The type strain of Bavariicoccus seileri is WCC 4188(T) (=DSM 19936(T) =CCUG 55508(T)).