Lactococcus formosensis sp. nov., a lactic acid bacterium isolated from yan-tsai-shin (fermented broccoli stems) The Institute of Enology and Viticulture, University of Yamanashi, 1-13-1, Kitashin, Kofu, Yamanashi 400-0005, Japan A coccal-shaped organism, designated 516 T , was isolated from yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan. 16S rRNA gene sequencing results showed that strain 516 T had 98.9 % sequence similarity to that of the type strain Lactococcus garvieae NBRC 100934 T . Comparison of three housekeeping genes, rpoA, rpoB and pheS, revealed that strain 516 T was well separated from Lactococcus garvieae NBRC 100934 T . DNA-DNA hybridization studies indicated that strain 516 T had low DNA relatedness with Lactococcus garvieae NBRC 100934 T (46.1 %). The DNA G+C content of strain 516 T was 38.1 mol% and the major fatty acids were C 16 : 0 (22.7 %), C 19 : 0 cyclo v8c (17.9 %) and summed feature 7 (29.0 %). Based on the evidence, strain 516 T represents a novel species of the genus Lactococcus, for which the name Lactococcus formosensis sp. nov. is proposed. The type strain is 516 T (5NBRC 109475 T 5BCRC 80576 T ).

Aims: To isolate, characterize and identify lactic acid bacteria (LAB) in yantsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan. Methods and Results: A total of 226 LAB were isolated; 111 cultures were isolated from samples collected from seven different markets and 115 from six fresh broccoli samples. These isolates were characterized phenotypically and then initially divided into nine groups (r1 to r9) using restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Some isolates were further divided into four additional groups by other genetic analysis. The most common bacterial genera in yan-tsai-shin and fresh broccoli were Weissella, Lactococcus and Lactobacillus. Regional similarities in the LAB, with differences in diversity, were observed in this study. On the basis of phylogenetic analysis of 16S rRNA, rpoA, rpoB and pheS gene sequences, two strains were included in the genera Enterococcus and Lactococcus, respectively, and identified as potential novel species or subspecies. In addition, the novel enterococcal strain, and 33 L. lactis subsp. lactis and two Weissella cibaria strains were found to have bacteriocin-like inhibitory substance (BLIS) producing abilities. Conclusions: These results suggest that the LAB play important roles in the fermentation of yan-tsai-shin. Significance and Impact of the Study: This is the first report describing the distribution and varieties of LAB existing in yan-tsai-shin and fresh broccoli. In addition, two potential novel LAB species or subspecies and one potential novel BLIS were also found in this study.

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.