Enterococcus alishanensis sp. nov., a novel lactic acid bacterium isolated from fresh coffee beans.

A coccus-shaped organism, designated ALS3T, was isolated from fresh coffee cherries collected at a farm located in the Ali Mountain region of Taiwan. Sequence analysis of its 16S rRNA gene indicated that strain ALS3T belongs to the genus Enterococcus and has more than 98.5 % sequence similarity to Enterococcus pallens and Enterococcus hermanniensis. When comparing the ALS3T genome with these two type strains, the average nucleotide identity values and digital DNA-DNA hybridization values were 72.6-73.3 and 19.2 %, respectively. The G+C content of the genomic DNA from strain ALS3T was 35.6 mol%. Results of sequence analysis, together with enzymatic activities and characteristics of carbohydrate metabolism, indicated that strain ALS3T is distinct and represents a novel species, for which the name Enterococcus alishanensis sp. nov. is proposed. The type strain is ALS3T (=NBRC 109593T=BCRC 80605T).

Metschnikowia miensis f.a., sp. nov., isolated from flowers in Mie prefecture, Japan.

Four yeast strains (RIFY 10001T, RIFY 10002, RIFY 10003, and RIFY 10004) were isolated from flowers growing in fields of mustard and broad beans in Japan. Ascospore formation was not observed. Sequence analysis of the D1/D2 domain of the large subunit ribosomal RNA (LSU rRNA) gene of the four strains indicated that they belong to the genus Metschnikowia and are closely related to Metschnikowia hawaiiana strain CBS 9146T and Metschnikowia orientalis strain CBS 10331T. The D1/D2 domain of the LSU rRNA gene and internal transcribed spacer regions of strain RIFY 10001T were 85.7% identical to those of M. hawaiiana strain CBS 9146T. All four strains were distinguished from the M. hawaiiana strain CBS 9146T by their inability to ferment glucose. Hence, these four strains are novel species and were named as Metschnikowia miensis (holotype: RIFY 10001T; isotypes: NBRC 112445T = CBS 14749T).

Cellulomonas algicola sp. nov., an actinobacterium isolated from a freshwater alga.

An actinomycete strain, TKZ-21T, was isolated from a freshwater alga (Chetophoraceae) collected from the Takizawa River, Yamanashi, Japan, and examined using a polyphasic taxonomic approach. Cells were Gram-stain positive, aerobic, non-sporulating, motile, and coccoid or short rod-shaped. The strain grew in the presence of 0-4 % (w/v) NaCl, between pH 6-9.4, and over a temperature range of 15-40 °C, with optimum growth at 30 °C. The peptidoglycan type of strain TKZ-21T was A4ß, containing l-ornithine as diagnostic diamino acid and d-glutamic acid as the interpeptide bridge. The predominant menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, ninhydrin-positive glycolipid, and unidentified phospholipids. The major cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0, and the DNA G+C content was 75.6 mol%. On the basis of 16S rRNA gene sequence analysis, strain TKZ-21T was closely related to Cellulomonas fimi (98.5 % sequence similarity) and Cellulomonas biazotea (98.3 %). The genome orthoANI value between strain TKZ-21T and C. biazotea and C. fimi were 84.7 and 84.2 %, respectively. On the basis of fatty acid and MALDI-TOF MS profile analysis, phylogenetic analyses, genomic analysis, and phenotypic data, it is proposed that the isolate be classified as a representative of a novel species of the genus Cellulomonas, with the name Cellulomonas algicola sp. nov. The type strain is TKZ-21T (=NBRC 112905T=TBRC 8129T).

Lactobacillus musae sp. nov., a novel lactic acid bacterium isolated from banana fruits.

Two Gram-stain-positive, catalase-negative, rod-shaped, bacterial strains (313T and 311) were isolated from banana fruits in Taiwan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the highest similarity to both strains corresponded to the type strain of Lactobacillus nantensis (99.19 %), followed by Lactobacillus crustorum (98.99 %), Lactobacillus heilongjiangensis (98.59 %) and Lactobacillus farciminis (98.52 %). Phylogenetic analysis based on the sequences of two housekeeping genes, pheS and rpoA, revealed that these two strains were well separated from the Lactobacillus reference strains. DNA-DNA relatedness values revealed genotype separation of the two strains from the above four species. The DNA G+C content of strain 313T was 35.5 mol%. The strains were homofermentative and mainly produced l-lactic acid from glucose. The major cellular fatty acids of strain 313T were 18 : 1ω6c and/or 18 : 1ω7c, 16 : 0, and 19 : 1ω6c and/or 19 : 0 cyclo ω10c. Based on their physiological and genotypic characteristics, the isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillusmusae sp. nov. is proposed. The type strain is 313T=NBRC 112868T=BCRC 81020T).

Diversity of Lactic Acid Bacteria Associated with Banana Fruits in Taiwan.

Banana is a popular fruit worldwide. The lactic acid bacteria (LAB) microflora in banana fruits has not been studied in detail. A total of 164 LAB were isolated from banana fruits in Taiwan. These isolates were initially divided into nine groups (r1 to r9) using restriction fragment length polymorphism analysis and 16S ribosomal DNA sequencing. Isolates belonging to Lactobacillus plantarum group were further divided into three additional groups using multiplex PCR assay targeting the recA gene. The most common bacterial genera found in banana fruits were Lactobacillus and Weissella. The distribution of LAB indicated that, in most cases, neighboring regions shared common strains, but there were still some differences between regions. On the basis of phylogenetic analysis of 16S rRNA, rpoA, and pheS gene sequences, two strains included in the genera Lactobacillus were identified as potential novel species or subspecies. In addition, a total 36 isolates were found to have bacteriocin-producing abilities. These results suggest that various LAB are associated with banana fruits in Taiwan. This is the first report describing the distribution and varieties of LAB associated with banana fruits. In addition, one potential novel LAB species was also found in this study.

Purification and characterization of plantaricin Y, a novel bacteriocin produced by Lactobacillus plantarum 510.

Lactobacillus plantarum 510, previously isolated from a koshu vineyard in Japan, was found to produce a bacteriocin-like inhibitory substance which was purified and characterized. Mass spectrometry analysis showed that the mass of this bacteriocin is 4,296.65 Da. A partial sequence, NH2- SSSLLNTAWRKFG, was obtained by N-terminal amino acid sequence analysis. A BLAST search revealed that this is a unique sequence; this peptide is thus a novel bacteriocin produced by Lactobacillus plantarum 510 and was termed plantaricin Y. Plantaricin Y shows strong inhibitory activity against Listeria monocytogenes BCRC 14845, but no activity against other pathogens tested. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was completely inactivated by protease K. Furthermore, trypsin-digested bacteriocin product fragments retained activity against L. monocytogenes BCRC 14845 and exhibited a different inhibitory spectrum.

Lactococcus formosensis sp. nov., a lactic acid bacterium isolated from yan-tsai-shin (fermented broccoli stems).

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 C16 : 0 (22.7 %), C19 : 0 cyclo ω8c (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) ( = NBRC 109475(T) = BCRC 80576(T)).

Diversity of lactic acid bacteria associated with fresh coffee cherries in Taiwan.

A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substance-producing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812.

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.

Lactococcus taiwanensis sp. nov., a lactic acid bacterium isolated from fresh cummingcordia.

One coccal strain, designated 0905C15(T), was isolated from fresh cummingcordia, which is the main ingredient of pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan. 16S rRNA gene sequencing results showed that strain 0905C15(T) had 98.22-98.82 % sequence similarity to that of the type strains of four Lactococcus lactis subspecies (L. lactis subsp. lactis BCRC 12312(T), L. lactis subsp. cremoris BCRC 12586(T), L. lactis subsp. hordniae BCRC 80474(T) and L. lactis subsp. tructae BCRC 80475(T)). Comparison of two housekeeping genes, recA and rpoB, revealed that strain 0905C15(T) was well separated from the reference strains of the genus Lactococcus. DNA-DNA hybridization studies indicated that strain 0905C15(T) had low DNA relatedness to the four Lactococcus lactis subspecies (9.7-15.24 %). The DNA G+C content of strain 0905C15(T) was 39.6 mol %. Based on the evidence, strain 0905C15(T) represents a novel species of the genus Lactococcus, for which the name Lactococcus taiwanensis sp. nov. is proposed. The type strain is 0905C15(T) ( = NBRC 109049(T) = BCRC 80460(T)).

Enterococcus saccharolyticus subsp. taiwanensis subsp. nov., isolated from broccoli.

A coccal strain isolated from fresh broccoli was initially identified as Enterococcus saccharolyticus; however, molecular identification and phenotypic traits did not support this identification. DNA-DNA hybridization with the type strain of E. saccharolyticus (76.4 % relatedness), DNA G+C content (35.7 mol%), phylogenetic analysis based on 16S rRNA, pheS and rpoA gene sequences, rep-PCR fingerprinting and profiles of cellular fatty acids, whole-cell proteins and enzyme activities, together with carbohydrate metabolism characteristics, indicated that this strain is distinct and represents a novel subspecies, for which the name Enterococcus saccharolyticus subsp. taiwanensis subsp. nov. is proposed. The type strain is 812(T) ( = NBRC 109476(T) = BCRC 80575(T)). Furthermore, we present an emended description of Enterococcus saccharolyticus and proposal of Enterococcus saccharolyticus subsp. saccharolyticus subsp. nov. (type strain ATCC 43076(T) = CCUG 27643(T) = CCUG 33311(T) = CIP 103246(T) = DSM 20726(T) = JCM 8734(T) = LMG 11427(T) = NBRC 100493(T) = NCIMB 702594(T)).

Diversity of lactic acid bacteria in sian-sianzih (fermented clams), a traditional fermented food in Taiwan.

BACKGROUND: Sian-sianzih (fermented clams) is a popular traditional fermented food in Taiwan. The lactic acid bacteria (LAB) microflora in sian-sianzih have not been studied in detail. In this study, LAB from sian-sianzih were isolated, characterized and identified. RESULTS: A total of 186 cultures of LAB were isolated from seven sian-sianzih samples and 29 cultures were isolated from its main raw substrate: clams. The identification results revealed up to 11 distinct bacterial species belonging to five genera in sian-sianzih, and three species belonging to two genera in clams. The most common bacterial genera in sian-sianzih were Lactobacillus and Weissella, followed by Leuconostoc, Pediococcus and Lactococcus. A regional similarity in LAB, with differences in diversity, was observed in the current study. On the other hand, Lactococcus lactis subsp. lactis was the most common species found in raw clam samples. The results also suggested that greater LAB diversity could be observed in wild clams than in cultured ones. Furthermore, antibacterial activities of the isolates were determined, and one Weisella hellenica strain showed inhibitory activity against the indicator strain Lactobacilluas sakei JCM 1157(T) . A sensory assessment of seven sian-sianzih samples was also performed and the results indicated that diversity of LAB has a great effect on its aroma and taste formation. CONCLUSION: The results demonstrate that various LAB species are distributed in sian-sianzih and have a great effect on the flavor of sian-sianzih.

Isolation and characterisation of lactic acid bacteria from jiang-gua (fermented cucumbers), a traditional fermented food in Taiwan.

BACKGROUND: Jiang-gua (fermented cucumbers) is a popular traditional fermented food in Taiwan. The microflora of lactic acid bacteria (LAB) in jiang-gua have not been investigated in detail. In this study, LAB from jiang-gua were isolated, characterised and identified. RESULTS: A total of 103 LAB were isolated; 70 cultures were isolated from jiang-gua samples and 33 cultures were isolated from its raw substrate, cucumber. These isolates were mainly characterised phenotypically and then divided into seven groups (A-G) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. The isolates were identified as Enterococcus casseliflavus, Leuconostoc lactis, Leuconostoc mesenteroides, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactococcus lactis subsp. lactis, Weissella cibaria and Weissella hellenica. The antibacterial activities of the isolates were determined and 11 Lc. lactis subsp. lactis strains showed inhibitory activity against the indicator strain Lactobacillus sakei JCM 1157(T) . CONCLUSION: Heterofermentative W. cibaria and Leu. lactis were the major LAB found in jiang-gua samples without soy sauce. In soy sauce-added samples, homofermentative L. pentosus and L. plantarum were the most abundant LAB. In addition, the results also suggested that HhaI and RsaI restriction enzymes could be applied to distinguish W. hellenica and Weissella paramesenteroides.

Isolation and characterisation of lactic acid bacteria from yan-jiang (fermented ginger), a traditional fermented food in Taiwan.

BACKGROUND: Yan-jiang (fermented ginger) is a popular traditional fermented food in Taiwan. The lactic acid bacteria (LAB) microflora in yan-jiang has not been studied in detail. In this study, LAB from yan-jiang were isolated, characterised and identified. RESULTS: A total of 176 LAB were isolated; 160 cultures were isolated from yan-jiang samples and 16 cultures were isolated from raw ginger. These isolates were characterised phenotypically and then divided into nine groups (A to I) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Lactobacillus sakei and Lactococcus lactis subsp. lactis were the major LAB found in the initial 2 days of fermentation without pickled plums; these species were mostly replaced by Weissella cibaria and L. plantarum after 3 days of fermentation. In the fermentation bucket with added pickled plums, W. cibaria was the most abundant LAB found during fermentation. The antibacterial activities of the isolates were determined. Twenty-four Lc. lactis subsp. lactis and 19 W. cibaria strains showed inhibitory activity against the indicator strain L. sakei JCM 1157(T) . CONCLUSION: Results demonstrate that various LAB species were more numerous when fermentation was carried out without pickled plums. LAB also had effects on the aroma and flavour of yan-jiang.

Isolation and characterization of lactic acid bacteria from jiang-sun (fermented bamboo shoots), a traditional fermented food in Taiwan.

BACKGROUND: In this study, lactic acid bacteria (LAB) were isolated, characterized and identified from jiang-sun (fermented bamboo shoots; a traditional fermented food in Taiwan). Samples were collected at seven time intervals from a fixed fermenting bucket during the fermentation process of jiang-sun and its initial ingredients, dochi (fermented soybeans) and bamboo shoots. RESULTS: A total of 57 LAB cultures were isolated; 42 cultures were isolated from jiang-sun samples and 15 cultures were isolated from dochi and bamboo shoots. These isolates were characterized phenotypically and then divided into three groups (A-C) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Alteration of microbial populations during the fermentation process was observed. While various LAB were found in the dochi and bamboo shoots, it was mostly replaced by Lactobacillus plantarum after 1 day of fermentation. Furthermore, the antibacterial activities of the isolates were determined, and one Enterococcus faecium strain showed inhibitory activity against all the indicator strains. CONCLUSION: Results suggest that L. plantarum is the main LAB present during the fermentation of jiang-sun. To our knowledge, this is the first report describing the distribution and varieties of LAB that exist in the jiang-sun fermentation process.

Isolation and characteristics of lactic acid bacteria isolated from ripe mulberries in Taiwan.

The objective of this study was to isolate, characterize, and identify lactic acid bacteria (LAB) from ripe mulberries collected in Taiwan. Ripe mulberry samples were collected at five mulberry farms, located in different counties of Taiwan. Eighty-eight acid-producing cultures were isolated from these samples, and isolates were divided into classes first by phenotype, then into groups by restriction fragment length polymorphism (RFLP) analysis and sequencing of 16S ribosomal DNA (rDNA). Phenotypic and biochemical characteristics led to identification of four bacterial groups (A to D). Weissella cibaria was the most abundant type of LAB distributed in four mulberry farms, and Lactobacillus plantarum was the most abundant LAB found in the remaining farm. Ten W. cibaria and one Lactococcus lactis subsp. lactis isolate produced bacteriocins against the indicator strain Lactobacillus sakei JCM 1157(T). These results suggest that various LAB are distributed in ripe mulberries and W. cibaria was the most abundant LAB found in this study.

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing Leuconostoc pseudomesenteroides 607 Isolated from Persimmon.

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05 Da and a partial N-terminal amino acid sequence of NH2-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97 Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.

Searching for bacteriocin-producing lactic acid bacteria in soil.

A survey was conducted on the isolation and characterization of bacteriocin-producing lactic acid bacteria in soil. Forty-two acid-producing bacterial strains were isolated from 55 soil samples collected in Yamanashi prefecture, Japan. Investigation of antibacterial activities of isolates revealed that three isolates, Lactobacillus animalis C060203, Enterococcus durans C102901 and Leuconostoc mesenteroides subsp. mesenteroides C060204, showed antibacterial activities against the indicator strain of Lactobacillus sakei JCM 1157T. Bacteriocin from Enterococcus durans C102901 showed different characteristics from the known durancin L28-1A, produced by Enterococcus durans L28-1. Furthermore, this is the first report of a bacteriocin being produced by Lactobacillus animalis. Viewing from the species, bacteriocins from strains C102901 and C060203 showed high possibilities for the novel substances. These significant findings suggest that soil may be a common source for the isolation of novel bacteriocin-producing lactic acid bacteria.

Draft Genome Sequence of Streptococcus orisasini SH06, Isolated from a Healthy Thoroughbred Gastrointestinal Tract.

Streptococcus orisasini SH06 was isolated from a healthy thoroughbred gastrointestinal tract. Here, we report the draft genome sequence of this organism. This paper is the first published report of the genomic sequence of S. orisasini.

Detection and Characterization of Weissellicin 110, a Bacteriocin Produced by Weissella cibaria.

BACKGROUND: Weissellicin 110 is the only bacteriocin reported in Weissella cibaria up to now. This bacteriocin represents several unique features. This is the first report on the gene sequence that encodes for the bacteriocin. OBJECTIVES: Providing a rapid detection method to isolate the weissellicin 110 encoding gene and determination of the bacteriocin distribution were the objectives. MATERIALS AND METHODS: Bacteriocin from W. cibaria 860106 was purified and analyzed using mass spectrometry for proteins sequencing. The draft genome sequence of W. cibaria 860106 was generated using next generation sequencing. PCR was applied to detect the weissellicin 110 encoding gene. RESULTS: The molecular weight and partial protein sequence were obtained for the bacteriocin from W. cibaria 860106. An open reading frame (ORF) was identified as weissellicin 110 from the draft genome sequence. PCR primers were designed to amplify the weissellicin 110 encoding gene and these primers detected sequences from other 27 BLIS-producing W. cibaria strains previously isolated from either various Taiwanese fermented foods or the respective raw materials. CONCLUSIONS: The genetic information of weissellicin 110 was obtained, enabling rapid detection of the weissellicin 110 encoding gene. Results suggest that weissellicin 110 producing W. cibaria strains are widely distributed inTaiwanese fermented foods.