Draft Genome Sequence of Weissella koreensis KCTC 3621T

Weissella koreensis is a Gram-positive, rod-shaped, nonmotile, and facultative anaerobic species belonging to the lactic acid bacteria (LAB). The members of this species have been repeatedly isolated from kimchi (a traditional Korean fermented food) and are known for their beneficial effects on human and animal intestinal microflora through producing various clinically important amino acids such as γ-aminobutyric acid and ornithine. Here we report the genome sequence of the type strain of W. koreensis (KCTC 3621^T) to provide taxonomic and functional insights into the species.     

Lactic acid bacteria associated with wine grapes from several Australian vineyards

AIMS: The detection and isolation of lactic acid bacteria by enrichment methods from wine grapes cultivated in vineyards located in New South Wales, Australia. METHODS AND RESULTS: Enrichment cultures in de Man, Rogosa and Sharpe (MRS) broth, MRS + ethanol (5%), MRS broth supplemented with 15% (v/v) tomato juice (MRST), pH 5.5 and 3.5 and autoenrichment in grape juice homogenate were used to detect lactic acid bacteria on wine grapes. Bacteria were isolated from enrichment cultures by plating onto MRS and MRST agar and identified by 16S rDNA sequence analysis and phenotypical methods. A molecular method, PCR-denaturing gradient gel electrophoresis (DGGE) was also used to examine the bacteria that developed in enrichment cultures. Species of Lactobacillus, Enterococcus, Lactococcus and Weissella were detected in enrichments by plating and PCR-DGGE. Other bacteria (Sporolactobacillus, Asaia, Bacillus ssp.) were also found in some enrichment cultures. The principal malolactic bacterium, Oenococcus oeni, was not isolated. CONCLUSIONS: The incidence and populations of lactic acid bacteria on wine grapes were very low. Damaged grape berries showed a greater presence of these bacteria than undamaged berries. The diversity of bacterial species isolated from the grapes was greater than those previously reported and represented both lactic acid bacteria and nonlactic acid bacteria. Some of these bacteria (i.e. Lactobacillus lindneri, Lactobacillus kunkeei) could be detrimental to wine production. Oenococcus oeni was not found on grapes, but its recovery could be obscured by overgrowth from other species. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria are significant in wine production because they conduct the malolactic fermentation and cause stuck or sluggish alcoholic fermentation and wine spoilage. This study investigates wine grapes as a potential source of these bacteria.     

Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S–23S rRNA gene intergenic spacer region

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S–23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR–RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S–23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.     

Isolation and characterization of a novel lactic acid bacterium for the production of lactic acid

We isolated a novel lactic acid bacterium from a Korean traditional fermented food, soybean paste. The newly isolated strain, dubbed RKY2, grew well on glucose, sucrose, galactose, and fructose, but it could not utilize xylose, starch, or glycerol. When the partially amplified 16S rDNA sequence (772 bp) of the strain RKY2 was compared with 10 reference strains, it was found to be most similar toLactobacillus pentosus JCM 1588^T, with 99.74% similarity. Therefore, the strain RKY2 was renamedLactobacillus sp. RKY2, which has been deposited in the Korean Collection for Type Cultures as KCTC 10353BP.Lactobacillus sp. RKY2 was found to be a homofermentative lactic acid bacterium, because its end-product from glucose metabolism was found to be mainly lactic acid. It could produce more than 90 g/L of lactic acid from MRS medium supplemented with 100 g/L of glucose, with 5.2 g L⁻¹ h⁻¹ of productivity and 0.95 g/g of lactic acid yield.     

Isolation and analyses of uranium tolerant <Emphasis Type="Italic">Serratia marcescens</Emphasis> strains and their utilization for aerobic uranium U(VI) bioadsorption

Enrichment-based methods targeted at uranium-tolerant populations among the culturable, aerobic, chemo-heterotrophic bacteria from the subsurface soils of Domiasiat (India’s largest sandstone-type uranium deposits, containing an average ore grade of 0.1 % U₃O₈), indicated a wide occurrence of Serratia marcescens. Five representative S. marcescens isolates were characterized by a polyphasic taxonomic approach. The phylogenetic analyses of 16S rRNA gene sequences showed their relatedness to S. marcescens ATCC 13880 (≥99.4% similarity). Biochemical characteristics and random amplified polymorphic DNA profiles revealed significant differences among the representative isolates and the type strain as well. The minimum inhibitory concentration for uranium U(VI) exhibited by these natural isolates was found to range from 3.5–4.0 mM. On evaluation for their uranyl adsorption properties, it was found that all these isolates were able to remove nearly 90–92% (21–22 mg/L) and 60–70% (285–335 mg/L) of U(VI) on being challenged with 100 μM (23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solutions, respectively, at pH 3.5 within 10 min of exposure. his capacity was retained by the isolates even after 24 h of incubation. Viability tests confirmed the tolerance of these isolates to toxic concentrations of soluble uranium U(VI) at pH 3.5. This is among the first studies to report uranium-tolerant aerobic chemoheterotrophs obtained from the pristine uranium ore-bearing site of Domiasiat.     

A selected probiotic strain of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> CM33 isolated from breast-fed infants as a potential source of β-galactosidase for prebiotic oligosaccharide synthesis

Lactic acid bacteria from healthy breast-fed infants were isolated and screened for β-galactosidase production in MRS broth. Among 49 isolates that exhibited the yellow clear zone on MRS agar supplemented with bromocresol blue, the isolate CM33 was selected as being the highest β-galactosidase producer and was identified as Lactobacillus fermentum based on its morphological characteristics and 16S rDNA nucleotide sequence. L. fermentum CM33 exhibited a good survival rate under the simulated stomach passage model, comparable to known probiotic strains L. gallinarum JCM2011 and L. agilis JCM1187. L. fermentum CM33 was antagonistic to pathogenic bacteria Listeria monocytogenes, Escherichia coli 0157:H7, Salmonella typhi, and Salmonella enteriditis, using the well diffusion method. In addition, the selected lactobacilli exhibited a high growth rate when cultivated in modified MRS containing commercial galactooligosaccharide (GOS) as a sole carbon source, as well as in glucose. A preliminary study on the enzymatic synthesis of oligosaccharide using crude β-galactosidase revealed the capability for oligosaccharide synthesis by the transgalactosylation activity.     

Novel Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented cabbage product

To determine the dominant microorganisms involved in kimchi fermentation and to examine their effect on kimchi fermentation, we randomly isolated and characterized 120 lactic acid bacteria from kimchi during a 5-day fermentation at 15°C. Leuconostoc citreum was dominant during the early and mid-phases of kimchi fermentation whereas Lactobacillus sake/Lactobacillus curvatus or Lactobacillus brevis were found during later stages. Eighty-two out of 120 isolates (68%) were identified as Leuconostoc citreum by means of a polyphasic method, including 16S rDNA sequencing and DNA/DNA hybridization. A few Weissella confusa-like strains were also isolated during the mid-phase of the fermentation. Strain IH22, one of the Leuconostoc citreum isolates from kimchi, was used as an additive to evaluate growth and acid production in kimchi fermentation. This strain was consistently over 95% of the population in IH22-treated kimchi over a 5-day fermentation, while heterogeneous lactic acid bacteria were observed in the control kimchi. The pH in IH22-treated kimchi dropped rapidly but was stably maintained for 5 days, compared to its slow and prolonged decrease in the control kimchi. These results indicate that Leuconostoc citreum IH22 dominates over and retards the growth of other lactic acid bacteria in kimchi, suggesting it can be used as a bacterial starter culture to maintain the quality of kimchi for prolonged periods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determining the Suitability of Lactobacilli Antifungal Metabolites for Inhibiting Mould Growth

Summary In recent years, public concern about indoor mould growth has increased dramatically in the United States. In this study, lactic acid bacteria (LAB), which are known to produce antimicrobial compounds important in the biopreservation of food, were evaluated to determine if the same antimicrobial properties can be used to inhibit mould fungi that typically colonize wood. Based on biomass measurement, cell-free supernatants from Lactobacillus casei subsp. rhamnosus and Lactobacillus acidophilus grown in deMan Rogosa Sharpe (MRS) broth inhibited 95–100% growth of three mould fungi and one stain fungus associated with wood-based building materials. Lactic acid and four unknown compounds ⩽ kDa molecular weight were fractionated from the culture supernatant by thin layer chromatography and high-performance liquid chromatography. Antifungal activity, which was attributed to one or more unknown metabolites, was retained during heating and neutralization. A 1:2 dilution of L. casei supernatant inhibited 100% growth of all test fungi.     

Isolation and identification of lactic acid bacteria from sediments of a coastal marsh using a differential selective medium

Aims:  To identify lactic acid bacteria (LAB) colonies isolated from sediments of a coastal marsh by the reduction of 2,3,5‐triphenyltetrazolium chloride (TTC) in MRS medium. Methods and Results:  Single colonies isolated from sediments of a coastal marsh by enrichment in MRS broth were selected from MRS‐TTC plates and classified according to colony phenotype based on TTC reduction. A total of 37 colonies grouped in seven different phenotypes were identified by analysis of its 16S ribosomal gene sequence. Most isolates belonged to the Firmicutes phylum, mainly to orders Bacillales and Lactobacillales. LAB were represented by 20 isolates, 15 of which belong to the genus Weissella. Conclusions:  Enrichment in MRS was highly selective for the isolation of bacteria belonging to phylum Firmicutes. Several different phenotypes were developed by LAB and must be considered during LAB isolation based on TTC reduction. Significance and Impact of the Study:  To our knowledge, this is the first study aimed at determining a relationship between colony phenotype from TTC reduction and a partial identification of isolates based on 16S ribosomal gene sequence similarities. Besides, this is the first report of isolation of W. cibaria from environmental samples.     

Production of galactooligosaccharide by β-galactosidase fromKluyveromyces maxianus varlactis OE-20

A galactooligosaccharide (GalOS)-producing yeast, OE-20 was selected from forty seven strains of yeast growing in Korean traditionalMeju (cooked soybean) and the yeast was tentatively identified asKluyveromyces maxianus varlactis by its morphology and fermentation profile. A maximum yield of 25.1%(w/w) GalOS, which corresponds to 25.1 g of GalOS per liter, was obtained from the reaction of 100 g per liter of lactose solution at 30°C, pH 7.0 for 18 h with an intracellular crude β-galactosidase. Glucose and galactose were found to inhibit GalOS formation. The GalOS that were purified by active carbon and celite 545 column chromatography were supplemented in MRS media and a stimulated growth was observed of some intestinal bacteria. In particular the growth rate ofBifidobacterium infantis in the GalOS containing MRS broth increased up to 12.5% compared to that of the MRS-glucose broth during a 48 h incubation period.     

Identification and characterization of lactic acid bacteria in ragi tape

One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.     

Phenotypic and phylogenetic analysis of lactic acid bacteria isolated from forage crops and grasses in the Tibetan Plateau

A total of 140 lactic acid bacteria (LAB) strains were isolated from corn, alfalfa, clover, sainfoin, and Indian goosegrass in the Tibetan Plateau. According to phenotypic and chemotaxonomic characteristics, 16S rDNA sequence, and recA gene PCR amplification, these LAB isolates were identified as belonging to five genera and nine species. Corn contained more LAB species than other forage crops. Leuconostoc pseudomesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus brevis, and Weissella paramesenteroides were dominant members of the LAB population on alfalfa, clover, sainfoin, and Indian goosegrass, respectively. The comprehensive 16S rDNA and recA-based approach effectively described the LAB community structure of the relatively abundant LAB species distributed on different forage crops. This is the first report describing the diversity and natural populations of LAB associated with Tibetan forage crops, and most isolates grow well at or below 10°C. The results will be valuable for the future design of appropriate inoculants for silage fermentation in this very cold area.     

Cytotoxic, antioxidative, and ACE inhibiting activities of dolsan leaf mustard juice (DLMJ) treated with lactic acid bacteria

This study was performed to know whether there is any change of physiological activity in DLMJ which is inoculated by lactic acid bacteria. Lactic acid bacteria were isolated from Dolsan leaf mustard Kimchi (DLMK) at 20°C. In the optimum ripening period, the population ofLeuconostoc andLactobacilli in the DLMK were found to be high. TheLeuconostoc, Lactobacilli andLactococci strains were identified asLeuconostoc mesenteroides., Leuconostoc gelidum, Weissella confusa, Lactobacillus plantarum, Lactobacillus raffinolactis, Lactococcus lactis andWeissella confusa using the Biolog system. The most predominant strain which was isolated from DLMK wasWeissella confusa. As the results of the phylogenetic analysis using 16s rDNA sequence, theWeissella confusa turned out to beWeissella kimchii, with 99.0% similarity. To investigated the change of physiological activity in DLMJ by lactic acid bacteria, 7 predominant strains inoculated to DLMJ (Dolsan Leaf Mustard Juice). The cytotoxicity was found to be under 19.55% all cases Also, the antioxidative activity of the DLMJ treated with lactic acid bacteria was very low, which might have been due to the reduced antioxidative phytochemicals during the preparation of the sterile, sample. The ACE inhibiting activity of DLMJ by inoculation withWeissella kimchii was shown to be the highest (94.0%). This could be that the degradation of sulfur containing materials in DLMJ byWeissella kimchii gave rise to ACE inhibiting activity.     

Rice straw fermentation using lactic acid bacteria

To efficiently utilize rice straw and lessen its disposal problem on the environment, a lactic acid bacteria community, SFC-2 was developed from natural fermentation products of rice straw by continuous enrichment with the MRS-S broth (MRS broth with sucrose), and used to accelerate the fermentation of air-dried straws. The SFC-2 could rapidly lower the pH of the broth and produce high levels of lactic acid. Using a combination of plate isolation, denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing, the microbial composition of the SFC-2 was classified into Lactobacillus, mainly comprised of L. fermentum, L. plantarum and L. paracacei. An evaluation of the fermentation effect of SFC-2 on rice straw showed that it lowered the pH and significantly (P<0.05) increased lactic acid concentration in the straw. Further analysis with DGGE indicated that L. plantarum, L. fermentum and L. paracasei were the dominant species during fermentation.     

Development of Genus/Species-Specific PCR Analysis for Identification of <Emphasis Type="Italic">Carnobacterium</Emphasis> Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes. Received: 25 July 2001 / Accepted: 19 October 2001     

Thermococcus peptonophilus sp. nov., a fast-growing,extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents

Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean. These strains were cocci, obligately anaerobic Archaea about 0.7–2 μm in diameter. Optimum growth conditions for OG-1 and SM-2 were at 85–90°C (range 60–100°C), pH 6 (range pH 4–8), a NaCl concentration of 3% (range 1–5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005–5%). Elemental sulfur stimulated the growth rate fourfold. Ammonium slightly stimulated growth. Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids. OG-1 showed the fastest growth rate within the genus Thermococcus. Growth was inhibited by rifampicin. The DNA G+C content was 52 mol%. Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus. OG-1 and SM-2 were different than the described Thermococcus species. We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. Received: 8 March 1995 / Accepted: 24 May 1995     

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man–Rogosa–Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.     

Medium components effecting bacteriocin production by two strains of Lactobacillus plantarum ST414BZ and ST664BZ isolated from boza

Bacteriocins ST414BZ and ST664BZ, produced by Lactobacillus plantarum, inhibited the growth of a number of lactic acid bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae. Optimal production of bacteriocin ST664BZ (12 800 AU/mL) was recorded in MRS broth with an initial pH of 6.0 and 6.5. Bacteriocin ST414BZ was produced in MRS broth at lower pH values, ranging from 6.5 to 5.0. Low levels of bacteriocin activity were produced in BHI, M17, 10% (w/v) soy flour and 10% (w/v) molasses, suggesting that specific nutrients are required for optimal production. Bacteriocin ST414BZ production doubled (from 12 800 to 25 600 AU/mL) in MRS broth with tryptone as sole nitrogen source, or when glucose was replaced with maltose. Bacteriocin ST664BZ production, on the other hand, was less influenced by changes in nitrogen content, but increased two-fold (to 25 600 AU/mL) when glucose was replaced with sucrose, maltose or mannose, or when MRS broth was supplemented with 2.0 g/L KH₂ PO₄. Enrichment of MRS broth with vitamins B₁₂, B₁ or C did not stimulate production of the two bacteriocins. Growth in the presence of DL-6,8-thioctic acid increased bacteriocin ST664BZ production to 25 600 AU/mL. Concluded from these results, optimal levels of bacteriocins ST414BZ and ST664BZ will be produced in boza enriched with tryptone and maltose.     

First record of Pythium grandisporangium in Japan

Pythium grandisporangium was isolated from roots of common reed and seawater in a saline marsh in Osaka Bay for the first time in Japan. Morphological features and rDNA ITS sequences were described on isolates of the species. The isolates grew on corn meal agar containing 0–9% sea salts, with 1–3% being the ideal range for growth. The isolates produced no disease on roots of common reed, but they colonized root surfaces in in vitro inoculation studies.     

Antifungal lactic acid bacteria with potential to prolong shelf-life of fresh vegetables

AIM: The aim of this study was to isolate and identify antifungal lactic acid bacteria from fresh vegetables, and evaluate their potential in preventing fungal spoilage of vegetables. METHODS AND RESULTS: Lactic acid bacteria from fresh vegetables were enriched in MRS (de Man Rogosa Sharpe) broth and isolated by plating on MRS agar. All the isolates (359) were screened for activity against Aspergillus flavus of which 10% showed antifungal activity. Potent antifungal isolates were identified by phenotypic characters and confirmed by partial 16S rRNA gene sequencing. These were screened against additional spoilage fungi viz. Fusarium graminearum, Rhizopus stolonifer, Sclerotium oryzae, Rhizoctonia solani, Botrytis cinerea and Sclerotinia minor by overlay method. Most of the isolates inhibited wide range of spoilage fungi. When fresh vegetables were inoculated with either cell suspension (10(4) cells ml(-1)) or cell-free supernatant of Lact. plantarum, followed by application of vegetable spoilage fungi (A. flavus and F. graminearum, R. stolonifer, B. cinerea each with 10(4) conidia ml(-1)) the vegetable spoilage was significantly delayed than control. CONCLUSIONS: Fresh vegetables constitute a good source of lactic acid bacteria with ability to inhibit wide range of spoilage fungi. Such bacteria can be applied to enhance shelf-life of vegetables. In the present study, we report for the first time the antifungal activity of Weissella paramessenteroides and Lact. paracollinoides isolated from fresh vegetables, against wide range of food spoilage fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Fresh vegetables can be used as a source of antifungal lactic acid bacteria. Their exploitation as biopreservative will help in prolonging shelf-life of fresh vegetables.