The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.The most significant period of cheese production is probably the ripening process, during which starter and nonstarter bacteria, chymosin, and the indigenous milk enzymes develop the organoleptic and textural properties of the cheese. This is particularly true for surface-ripened cheese, due to the variety and abundance of microorganisms on its surface. Surface-ripened cheeses can be divided into mold ripened, e.g., Camembert and Brie, and bacteria ripened, e.g., Limburger, Tilsit, Brick, and Münster. The latter cheeses are sometimes called washed-rind cheeses because of the extensive washing given to the surfaces of these cheeses during ripening. They are also called smear cheeses, because of the glistening appearance of the surface, or red-smear cheeses, due to the production of a red carotinoid by Brevibacterium linens, which is thought to be the most common bacterium occurring on the surface of the cheese. For this reason, B. linens is often deliberately inoculated onto the surface of the cheese during the early days of ripening, either as a commercial preparation or a...

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Phenotypic and phylogenetic studies were performed on 11 strains of a Microbacterium-like organism isolated from the surface of a smear-ripened cheese. The isolates were Gram-positive, catalase-positive, facultatively anaerobic, oxidase-negative, non-spore-forming, non-motile, small, slender rods and grew in 12 % (w/v) NaCl. Chemotaxonomic investigation revealed that all the isolates belonged unambiguously to the genus Microbacterium. They contained type B1 peptidoglycans with L-lysine as the diamino acid and glycolyl acyl types ; rhamnose and galactose were the cell wall sugars. The GMC content ranged from 69 to 72 mol %. The major menaquinones were MK-11 and MK-12 and the major fatty acids were anteiso C15 :0 and C17 :0 and iso C16 :0. Phylogenetic analysis of the 16S rRNA sequences of four isolates showed that they represented a new subline in the genus Microbacterium, with Microbacterium barkeri as their nearest phylogenetic neighbour. M. barkeri showed the highest sequence similarity to the isolates ; however, DNA-DNA hybridization showed that the isolates had only 38 % chromosomal similarity to M. barkeri. Based on the phylogenetic and phenotypic distinctiveness of the isolates, it is proposed that they be classified as a new Microbacterium species, for which the name Microbacterium gubbeenense sp. nov. is suggested. The type strain has been deposited as LMG S-19263 T ( l NCIMB 30129 T ). The GenBank accession number for the 16S rDNA sequence of the type strain is AF263563.