Aspects of Enzymology and Biochemical Properties of Brevibacterium linens Relevant to Cheese Ripening: A Review1 FERGAL P. RATTRAY2 and PATRICK F. FOX Department of Food Chemistry, University College, Cork, Ireland ABSTRACT smear surface-ripened cheeses, such as Limburger, MuÈ nster, Brick, Tilsiter, and Appenzeller. The Brevibacterium linens is a major surface microor- metabolism and physiology of the microorganism de- ganism that is present in the smear of surface- termine its growth on smear surface-ripened cheeses ripened cheeses. The enzymology and biochemical and the effect of such growth on the characteristics of characteristics of B. linens influence the ripening and the cheese. Brevibacterium linens is a strictly aerobic final characteristics of smear surface-ripened cheeses. microorganism, with a rod-coccus growth cycle, with Proteolytic, peptidolytic, esterolytic, and lipolytic ac- ° tivities, which are of particular importance in the temperature growth optimum of 20 to 30 C. Brevibac- ripening process, are discussed in detail. This review terium linens is a halotolerant microorganism with also describes the production of volatile compounds, optimum growth at pH 6.5 to 8.5. The microflora of especially sulfur-containing ones, by B. linens, which smear surface-ripened cheeses is generally complex are thought to be important in respect to the flavor of and of a transient nature. Typically, ripening smear surface-ripened cheeses. The unique orange- progresses from a yeast and mold flora to a bacterial colored carotenoids and the factors effecting their flora, of which B. linens is a major component. Im- production by B. linens are also presented. The mediately after the manufacture of smear surface- catabolism of aromatic amino acids, bacteriocin ripened cheese, yeasts dominate the surface production, plasmids, and miscellaneous biochemical microflora; yeasts utilize the lactate present in the and physiological properties (peptidoglycan type, an- curd, leading to an increase in the pH that facilitates tibiotic resistance, insecticide degradation, and bio- the growth of bacteria such as Arthrobacter and technological applications) of B. linens are discussed. Micrococcus spp., but especially B. linens (99). Be- The problem associated with the current taxonomical cause of the brine salting of smear surface-ripened classification of B. linens strains caused by strain cheeses, only halotolerant microorganisms variation is evaluated. Finally, the application of B. predominate on the surface of the cheese. Brevibac- linens cell extracts or its proteolytic enzymes as terium linens is characterized by a relatively high salt cheese ripening acccelerants for semi-hard or hard tolerance (15%). The growth of B. linens on the sur- cheese varieties is considered. face is thought to be an essential prerequisite for the ( Key words: Brevibacterium linens, cheese, enzymol- development of the characteristic color, flavor, and ogy, biochemical properties) aroma of smear surface-ripened cheeses (1, 28, 56). The growth of B. linens is also stimulated by vitamin Abbreviation key: PHMB = p-hydroxy- production by the yeasts during growth (97, 98). The mercuribenzoate, NA = nitroanilide. major factors that influence the distinctive charac- teristics of smear surface-ripened cheeses and the INTRODUCTION number, type, and growth rate of the surface Brevibacterium linens has long been recognized as microflora are the physical and chemical characteris- an important dairy microorganism because of its tics intrinsic to the cheese (pH, water activity, redox ubiquitous presence on the surface of a variety of potential, composition, and size), the environmental parameters (ripening temperature, relative humid- ity) and the technological conditions during manufac- ture (ripening time, degree of mechanization, and Received February 12, 1998. Accepted December 8, 1998. microflora of cheese equipment). This review will 1Invited paper. cover the taxonomy, biochemistry, and enzymology of 2Corresponding author, present address: Fergal P. Rattray, Department of Physiology and Metabolism, Chr. Hansen A/S, B. linens with particular emphasis on its importance 10-12 Bùge AlleÂ, 2970 Hùrsholm, Denmark. in relation to bacterial smear surface-ripened cheeses. 1999 J Dairy Sci 82:891±909 891 892 RATTRAY AND FOX TAXONOMY OF BREVIBACTERIUM LINENS ences in maximum growth temperature and salt toler- ance (117). Another area of difference between the Classification of the genus Brevibacterium has two proposed homology groups is that pigment presented taxonomists with problems because of its production is light-dependent for strain ATCC 9172 close morphological similarity to other genera, such (type strain and member of DNA-DNA homology as Arthrobacter, Caseobacter, Corynebacterium, and group 1) but is light-independent for other strains Rhodococcus. The genus Brevibacterium was proposed (including strain ATCC 9175, DNA-DNA homology initially by Breed (14), with B. linens as the type group 2) (94). Foissy (41), using an electrophoretic species, and was recognized in the seventh edition of zymogram technique, examined intra- and extracellu- Bergey's Manual of Determinative Bacteriology (15); lar enzymatic activities in 15 strains of B. linens.On no mention was made of coryneform morphology. the basis of the protein bands, it was possible to However, later research showed that B. linens had divide the 15 strains into three biotypes. By use of a coryneform morphology and showed a rod-coccus cycle similar electrophoretic zymogram technique, Sùrhaug similar to that of Arthrobacter globiformis (95, 110). (118) studied intracellular dipeptidase activities in 6 In the eighth edition of Bergey's Manual of Deter- strains of B. linens. There were considerable differ- minative Bacteriology (106), the genus Brevibac- ences in the number of dipeptidases observed for the terium was listed as incertae sedis because of a num- various strains in addition to differences in substrate ber of reports indicating its close similarity to the specificity, further indicating the heterogeneity be- genus Arthrobacter and because the coryneform mor- tween B. linens strains. Further heterogeneity within phology was overlooked by Breed (14); da Silva and the species was evident in another electrophoretic Holt (23), Davis and Newton (24), and Bousfield zymogram study (29) of intracellular esterase activi- (10) even proposed that B. linens should be reclassi- ties in 18 strains of B. linens; the number of esterase fied as Arthrobacter linens. bands varied from 2 to 6, depending on the strain. It Later numerical taxonomic (66, 114) and is, however, necessary to mention that the expression chemotaxonomic (74, 93, 111) studies illustrated the of these various enzymatic activities may be depen- heterogeneity of the group and also indicated that B. dent on the growth medium and environment, and, linens was a distinct taxon that should form the basis therefore, any attempted classification of B. linens of a redefined genus Brevibacterium, as was sug- strains based on these reports should be treated with gested initially by Yamada and Komagata (128) and caution. later by Jones (65), Keddie and Cure (73), and Sharpe et al. (117). On the basis of these and further Characteristics of the Genus studies, Collins et al. (21) redefined the genus Brevibacterium Brevibacterium (Breed), which forms the basis of the genus in the first edition of Bergey's Manual of Sys- Brevibacterium spp. exhibit a marked rod-coccus tematic Bacteriology (67). cycle during growth on complex media; during the At present, the genus Brevibacterium contains the exponential phase, the cells are morphologically rod- type species, B. linens, and the species Brevibacterium shaped, but, as the cells enter the stationary phase of iodinum, B. casei, and B. epidermidis. However, the growth, they become coccoid-shaped. Both rod and situation is further complicated by studies of DNA- coccoid forms are Gram-positive, but some strains and DNA homology (40), which indicate heterogeneity older colonies decolorize readily. Some important bio- exists within the species B. linens; DNA-DNA homol- chemical and morphological properties of Brevibac- ogy studies have shown that those strains presently terium spp. include the following: no endospore forma- designated as B. linens constitute at least two distinct tion, nonmotile, optimum growth temperature of 20 to species. Of the strains studied by Fiedler et al. (40), 30 or 37°C (depending on species and strain), ob- ATCC 9172 (the type strain), ATCC 19391, ATCC ligate aerobes, slight or no acid production from glu- 9864, and strain B3 represent one homology group; cose, extracellular proteinase production, catalase- this group represents the species B. linens because it positive, cell-wall peptidoglycan contains meso- contains the type strain, ATCC 9172. A second homol- diaminopimelic acid (DAP) as the diamino acid, ab- ogy group contains strains ATCC 9175, AC 251, AC sence of arabinose in the cell wall, absence of mycolic 252, AC 474, and B4. acids, and large amounts of dehydrogenated menaqui- The presence of at least two DNA-DNA homology none (67). groups within the species B. linens is further indi- In the first edition of Bergey's Manual of Systematic cated by considerable differences in the nutritional Bacteriology (67), a number of species are mentioned requirements of different strains (94) and by differ- that almost certainly are not members of the genus Journal of Dairy Science Vol. 82, No. 5, 1999 REVIEW: