International Journal of Systematic and Evolutionary Microbiology (2001), 51, 45–49 Printed in Great Britain Lactobacillus cypricasei sp. nov., isolated from Halloumi cheese Paul A. Lawson, 1 Photis Papademas, 1 Carmen Wacher, 1,2 Enevold Falsen, 3 Richard Robinson 1 and Matthew D. Collins 1 Author for correspondence : Paul A. Lawson. Tel : ›44 118 9357000. Fax : ›44 118 9357222. e-mail : p.a.lawson!reading.ac.uk 1 Department of Food Science and Technology, University of Reading, Reading RG6 6AP, UK 2 Departamento de Alimentos and Biotecnologı ! a, Facultad de Quı ! mica, Universidad Nacional Auto ! noma de Me ! xico, Mexico 3 Culture Collection, Department of Clinical Bacteriology, University of Go $ teborg, Go $ teborg S-413 46, Sweden Four strains of a hitherto unknown bacterium isolated from Halloumi cheese were compared by using phenotypic and phylogenetic studies. Comparative 16S rRNA gene sequencing demonstrated that the strains were identical to each other and represent a new subline within the genus Lactobacillus. The unknown bacterium was readily distinguished from other described Gram- positive catalase-negative taxa by means of biochemical tests and electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Lactobacillus cypricasei sp. nov. The type strain of L. cypricasei is CCUG 42961 T (fl CIP 106393 T ). Keywords : Lactobacillus cypricasei, Halloumi cheese, 16S rRNA, taxonomy, phylogeny INTRODUCTION The lactic acid bacteria possess a large number of metabolic properties that are responsible for their successful use as starter cultures in the commercial production of fermented dairy, meat and vegetable products and beverages (Hammes et al., 1992 ; Stiles & Holzapfel, 1997 ; Wibowo et al., 1985). The genus Lactobacillus represents the largest group of rod- shaped organisms within the lactic acid bacteria : currently, over 60 species are recognized. Some members of this group of organisms are important in the generation of particular flavours and in other ripening processes associated with specific cheeses (Elsoda, 1993 ; Elortondo et al., 1998 ; Fitzsimons et al., 1999; Hong et al., 1998). Halloumi cheese originated in Cyprus, but its appeal has spread worldwide. It is semi-hard, elastic, has no obvious skin}rind, has a close texture with no holes and is easily sliced. The colour varies from white (when a mixture of ovine}caprine milk is used) to yellowish (when bovine milk is the main ingredient). A feature of the production method of this particular cheese is that no starter cultures are used ; rather, the flavour and texture depend solely on the indigenous microflora of the milk (Papademas & Robinson, 1998). During the ................................................................................................................................................. Abbreviation : MRD, maximum recovery diluent. The GenBank accession number for the 16S rRNA gene sequence of strain CCUG 42961 T is AJ251560. course of a study to determine the microflora of Halloumi cheese, organisms corresponding to Lacto- bacillus brevis, Lactobacillus pentosus, Lactobacillus plantarum and Enterococcus faecium were recovered. In addition, a hitherto unknown Lactobacillus-like bacterium was isolated. In this article, we report the characteristics of this unknown lactic acid bacterium and the results of a polyphasic taxonomic study. On the basis of the results of this study, a new Lactobacillus species, Lactobacillus cypricasei sp. nov., is described. METHODS Bacterial strains and cultivation. Strains LMK1, LMK2 and LMK3 were isolated from Halloumi cheese in which ovine milk from a dairy located in the province of Paphos, Cyprus was used as the major ingredient. Strain LMD2 was isolated from Halloumi cheese made from ovine milk from a dairy located in the province of Nicosia, Cyprus. The cheese- making processes were as described previously (Papademas & Robinson, 1998). Two blocks of cheese of each type were unwrapped and cut in half. One portion was then shredded with a grater and 10 g weighed into a stomacher bag. The cheese was then blended with 90 ml maximum recovery diluent (MRD ; catalogue no. CM 733 ; Oxoid, Unipath) and serial dilutions made to 10’ using MRD. Duplicate aliquots from each dilution were put onto MRS agar (De Man et al., 1960) and incubated aerobically at 37 C for 72 h. Total colony counts were taken and the plates examined with a hand-lens in order to establish the dominant colony types. Five colonies each of the predominant types were taken and subcultured on MRS agar. Strains LMK1, LMK2, LMK3 01526 # 2001 IUMS 45