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Twelve strains of homofermentative lactobacilli and two strains of heterofermentative lactobacilli were isolated from kefir grains by using R-CW agar medium. The physiological and biochemical characteristics, DNA guanine-plus-cytosine contents, and levels of DNA-DNA relatedness of these isolates and previously described lactobacilli were compared. Our results indicated that two new species, Lactobacillus kefirgranurn and Lactobacillus parakefir, could be distinguished. The type strain of L. kefirgranurn sp. nov. is GCL 1701 (= JCM 8572), and the type strain of L. parakefir sp. nov. is GCL 1731 (= JCM 8573). The microorganisms in kefir grains have been studied by many workers (6, 11, 14, 16-18). Kandler and Kunath (7) described Lactobacillus kefir, a new species of heterofermen- tative lactobacilli isolated from kefir grains and kefir. Toba et al. (21) developed a medium for isolation of bacteria which produce a capsular polysaccharide that previously had been named kefiran by Kooiman (10) and La Riviere and Kooiman (11). Fujisawa et al. (4) also studied bacteria isolated from kefir grains and described a new species, Lactobacillus ke- firanofaciens. The fact that there have been many studies shows MATERIALS AND METHODS Bacterial strains. Twelve homofermentative strains and two heterofermentative strains were isolated from kefir grains obtained from E. C. Hansen's Laboratory in Copenhagen, Denmark. Reference Lactobacillus strains were obtained from the Japan Collection of Microorganisms, Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, Japan. Isolation of bacteria from kefir grains. Active kefir grains were rinsed and diluted with sterile anaerobic diluting solution

Lactobacillus kefirgranum sp. nov. and Lactobacillus parakefir sp. nov., Two New Species from Kefir Grains