Isolation and characterization of lactic acid bacteria from yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan.

AIMS: To isolate, characterize and identify lactic acid bacteria (LAB) in yan-tsai-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.

Weissellicin L, a novel bacteriocin from sian-sianzih-isolated Weissella hellenica 4-7.

AIM: To identify and characterize novel bacteriocins from Weissella hellenica 4-7. METHODS AND RESULTS: Weissella hellenica 4-7, isolated from the traditional Taiwanese fermented food sian-sianzih (fermented clams), was previously found to produce a bacteriocin active against Listeria monocytogenes and some other Gram-positive bacteria. Bacteriocin activity decreased slightly after autoclaving (121°C for 15 min), but was inactivated by protease K and trypsin. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 3205·6 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2 -KGFLSWASKATSWLVGP, by Edman degradation. The obtained partial sequence showed high homology with leucocin B-TA33a; however, at least two different residues were observed. No identical peptide or protein was found, and this peptide was therefore considered to be a novel bacteriocin produced by W. hellenica 4-7 and termed weissellicin L. CONCLUSIONS: The findings obtained in the current study suggest a novel bacteriocin produced by W. hellenica 4-7. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocins from Weissella remain rare, and this study is the second report of a bacteriocin produced by W. hellenica.

Screening and characterization of LAB-produced bacteriocin-like substances from the intestine of grey mullet (Mugil cephalus L.) as potential biocontrol agents in aquaculture.

AIM: To isolate and characterize bacteriocin-like inhibitory substance (BLIS)-producing lactic acid bacteria from the intestine of grey mullet. METHODS AND RESULTS: Inhibitory activity against at least one or more indicator strains was observed in one Enterococcus thailandicus, one Enterococcus faecium and two Lactococcus garvieae strains. Enterococcus faecium B3-8 and Ent. thailandicus B3-22 showed the greatest inhibitory activities against Listeria monocytogenes ATCC 19111 and were therefore further characterized. The results suggested that the inhibitory substances from the two strains showed similar characteristics with respect to sensitivity to heat and proteolytic enzymes. BLIS from Ent. thailandicus B3-22 was characterized by a broader inhibitory spectrum than that from Ent. faecium B3-8. SDS-PAGE revealed that the molecular size of partially purified BLISs from Ent. faecium B3-8 and Ent. thailandicus B3-22 was c. 5 and 3 kDa, respectively. The molecular mass of purified bacteriocin from Ent. thailandicus B3-22 was further determined to be 6319 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results indicated that BLIS from Ent. thailandicus B3-22 can effectively inhibit the growth of all tested L. garvieae strains. CONCLUSIONS: The findings obtained in this study suggest the potential use of Ent. thailandicus B3-22 as a biocontrol agent against pathogenic L. garvieae in the aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the characteristics of BLIS from Ent. thailandicus that showed potential for use as a biocontrol agent in the aquaculture.

Isolation and characteristics of lactic acid bacteria from koshu vineyards in Japan.

AIMS: To isolate, characterize and identify lactic acid bacteria (LAB) in the vineyards where koshu grapes, a primary wine grape cultivar in Japan, are grown. METHODS AND RESULTS: Sixty samples, including leaves, undamaged grape berries and soil under damaged berries, were collected at four koshu vineyards in Yamanashi Prefecture, Japan. One hundred and 15 acid-producing cultures were isolated from these samples, and the isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA (rDNA). Phenotypic and biochemical characteristics identified seven different bacterial groups (A to G). Lactococcus lactis ssp. lactis was the most abundant type of LAB distributed in three koshu vineyards, and Leuconostoc pseudomesenteroides was the most abundant LAB found in the remaining vineyard. Forty-six isolates produced bacteriocin-like inhibitory substances (BLIS) against the indicator strain Lactobacillus sakei JCM 1157(T). CONCLUSIONS: These results suggest that various LAB are distributed in koshu vineyards, of which a large number produce BLIS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the distribution and varieties of LAB that exist in koshu vineyards.

Effects of prebiotic oligosaccharides and trehalose on growth and production of bacteriocins by lactic acid bacteria.

AIMS: To investigate the effects of two prebiotics and trehalose on the production of bacteriocins. METHODS AND RESULTS: Four carbohydrates [dextrose, fructo-oligosaccharides (FOS), raffinose, and trehalose] were used as the sole carbon source in a simple broth. Five bacteriocin-producing strains of bacteria, including those producing nisin, enteriocin, and other bacteriocins, were used, and their inhibitory activities when grown on each carbohydrate were determined. The inhibitory activity assay was performed using the agar well diffusion method, and Lactobacillus sakei JCM 1,157(T) was used as the indicator strain. Effective enhancement of bacteriocin production was observed with FOS and trehalose incubation. CONCLUSIONS: The results suggest that FOS and trehalose can effectively enhance the production of the five kinds of bacteriocins evaluated in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This study offers useful information for not only a new application of FOS and trehalose, but also the potential improvement of food preservation.

Characteristics of bacteriocin-like inhibitory substances from dochi-isolated Enterococcus faecium D081821 and D081833.

AIMS: To characterize bacteriocin-like inhibitory substances (BLIS) from two dochi-isolated Enterococcus faecium. METHODS AND RESULTS: Enterococcus faecium D081821 and D081833 were isolated from dochi (a traditional fermented food in Taiwan) and found to produce BLIS with inhibitory activities against Listeria monocytogenes, Clostridium perfringens, and Bacillus cereus. Strains D081821 and D081833 showed different growth temperatures and their BLIS showed different sensitivities to heat, proteolytic enzymes, and antibacterial spectra. Both BLIS were collected, partially purified, and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE showed that both partially purified BLIS were approximately 3.0 kDa in size. CONCLUSIONS: These results indicate that E. faecium D081821 and D081833 produce different BLIS with strong antibacterial actions against the tested pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggest that two different BLIS from dochi-isolated lactic acid bacteria have potential for use as food preservatives.

Characteristics and effects of temperature and surfactants on bacteriocin-like inhibitory substance production of soil-isolated Lactobacillus animalis C060203.

A soil isolate of Lactobacillus animalis was found to produce a bacteriocin-like inhibitory substance (BLIS) with a wide inhibitory spectrum against Gram-positive bacteria. The isolate exhibited high BLIS production in broth containing surfactants, such as Tween 20 and 80, but low BLIS production in the absence of surfactants. Culture temperature also had an effect on BLIS production. This is the first report to study the production and characteristics of L. animalis BLIS.

Isolation and characterization of lactic acid bacteria from suan-tsai (fermented mustard), a traditional fermented food in Taiwan.

AIMS: To isolate, characterize, and identify lactic acid bacteria (LAB) in suan-tsai (fermented mustard), a traditional fermented food in Taiwan. METHODS AND RESULTS: Suan-tsai samples were collected at five time points from a fixed fermenting bucket. Fifty cultures were isolated from suan-tsai samples, and isolates were divided into classes by phenotype and then into groups by restriction-fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified two different bacterial groups (A and B), and the results showed that Pediococcus pentosaceus was the most abundant LAB during the initial fermentation time. However, the more NaCl-tolerant species Tetragenococcus halophilus took the place of P. pentosaceus and became the most abundant LAB later. All isolates were grown in de Man, Rogosa, and Sharpe (MRS) broth containing 6% NaCl, but T. halophilus could grow only in MRS broth containing 10% NaCl. CONCLUSIONS: These results suggest that the LAB P. pentosaceus and T. halophilus play roles in the fermentation of suan-tsai. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the distribution and varieties of LAB that exist in the suan-tsai fermentation process.

Isolation and characterization of lactic acid bacteria from dochi (fermented black beans), a traditional fermented food in Taiwan.

AIMS: To isolate, characterize and identify lactic acid bacteria (LAB) in dochi (fermented black beans), a traditional fermented food in Taiwan. METHODS AND RESULTS: A total of 30 samples were collected from three different dochi producers and analysed after different periods of storage. Fifty-two cultures of LAB were isolated from dochi samples and the isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified six different bacterial groups (A-F) and showed that the majority of the isolates were homofermentative LAB. Enterococcus faecium was the most abundant of the dochi-isolated LAB. All isolated LAB were able to grow in MRS broth containing 6% NaCl, but only Enterococcus, Pediococcus and Tetragenococcus species could grow in MRS broth containing 10% NaCl. Furthermore, antibacterial activities of isolates were determined, and four isolates showed inhibitory activities against the indicator strain Lactobacillus sakei JCM 1157(T). CONCLUSIONS: These results suggest that Ent. faecium is the main LAB present during the fermentation of dochi. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the distribution and varieties of LAB that exist in the dochi fermentation process.

Durancin L28-1A, a new bacteriocin from Enterococcus durans L28-1, isolated from soil.

AIMS: To isolate, characterize and identify bacteriocins from lactic acid bacteria in soil. METHODS AND RESULTS: Thirty-four acid-producing bacteria were isolated from 87 soil samples. Antibacterial activities were detected, and one strain, L28-1 produced a bacteriocin that was active against some Gram-positive bacteria. L28-1 was identified as Enterococcus durans by 16S rDNA sequence analysis and API50CHL. This bacteriocin did not lose its activity after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. The bacteriocin was purified by hydrophobic column chromatography, and Sep-Pak C(18). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the partially purified bacteriocin contained numerous protein bands. Two bands that displayed antibacterial activities were c. 3.4 and 2.5 kDa in size. In this work, the 3.4-kDa bacteriocin was analysed with N-terminal amino acid and DNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results indicated that the 3.4-kDa bacteriocin of Ent. durans L28-1 is a new natural enterocin variant. CONCLUSIONS: Enterococcus durans L28-1 produced a new bacteriocin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel bacteriocin that is produced by Ent. durans that has potential for use as a food preservative.

Isolation and identification of lactic acid bacteria from soil using an enrichment procedure.

AIMS: To survey, and identify and classify the ecological distribution of lactic acid bacteria from soil in Japan and Taiwan. METHODS AND RESULTS: Acid-producing bacteria were isolated from 68 soil samples, collected from Japan and Taiwan, in the rhizospheres of fruit trees, from the floor of a henhouse and around a horse farm. All isolates were identified by physiological and genetic tests. Thirty-two of the 54 isolates were identified as lactic acid bacteria (LAB), 16 as spore-forming lactic acid bacteria, five as Clostridium and one as Bacillus. These lactic acid bacteria represent five genera: Lactobacillus, Lactococcus, Enterococcus, Leuconostoc and Weissella. CONCLUSIONS: A high rate of isolating lactic acid bacteria was obtained from soil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that soil may be a common source for the isolation of lactic acid bacteria.

Time series analysis of aerobic bacterial flora during Miso fermentation.

AIMS: This article reports a microbiological study of aerobic mesophilic bacteria that are present during the fermentation process of Miso. METHODS AND RESULTS: Aerobic bacteria were enumerated and isolated from Miso during fermentation and divided into nine groups using traditional phenotypic tests. The strains were identified by biochemical analysis and 16S rRNA sequence analysis. They were identified as Bacillus subtilis, B. amyloliquefaciens, Kocuria kristinae, Staphylococcus gallinarum and S. kloosii. All strains were sensitive to the bacteriocins produced by the lactic acid bacteria isolated from Miso. CONCLUSIONS: The dominant species among the undesirable species throughout the fermentation process were B. subtilis and B. amyloliquefaciens. It is suggested that bacteriocin-producing lactic acid bacteria are effective in the growth prevention of aerobic bacteria in Miso. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful information for controlling of bacterial flora during Miso fermentation.

Widespread distribution of the bacteriocin-producing lactic acid cocci in Miso-paste products.

AIMS: A survey was conducted on the ecological distribution of bacteriocin-producing lactic acid bacteria in Miso-pastes, a typical fermented food in Japan. METHODS AND RESULTS Nine Miso-pastes were sampled for isolation of bacteriocin-producers. Almost all isolated enterococcal strains produced bacteriocins but no isolated tetragenococci did so. The bacteriocin-producing isolates were divided into nine groups by phenotypic tests. As the phenotypic characters were highly diverse, these strains could not be identified to species level on the basis of their phenotypes. The nine representative strains from each group were identified by 16S rRNA analysis. These bacteriocin-producers with one exception (Lactococcus sp.) were identified as strains of the Enterococcus faecium 'species group'. The bacteriocins of the nine strains were classified into five types according to their antibacterial spectral patterns and their SDS-PAGE profiles. The bacteriocins inhibited undesirable bacteria in Miso-pastes, such as Bacillus subtilis, but did not inhibit the useful Tetragenococcus halophila. CONCLUSIONS: The bacteriocin-producing lactic acid cocci were widespread at high frequencies in Miso-pastes. They were considered to play an important role in preventing the growth of undesirable bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that bacteriocin-producers act effectively as safe biopreservatives and may contribute to the biopreservation in Miso-pastes.

Intraspecific diversity of Oenococcus oeni isolated during red wine-making in Japan.

Using molecular and chemotaxonomic techniques, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium isolated during red wine-making in Japan. The results confirmed high values of DNA-DNA relatedness and strong similarity among 16S rDNA sequences of the isolates with the O. oeni-type strain. Pulsed-field gel electrophoresis (PFGE) by NotI identified four patterns among the strains. Three different patterns of lactate dehydrogenase mobility were seen and there was a strong correlation between PFGE pattern and mobility. The present results suggest that the different strains of O. oeni comprise one species, and that variations in the genomic profiles of the different strains of O. oeni, including Japanese isolates are well correlated.

MUP1, high affinity methionine permease, is involved in cysteine uptake by Saccharomyces cerevisiae.

Using a mutant defective in cysteine uptake, which is resistant to a toxic analog of cysteine, allylglycine, we searched for a gene that complements the defect in cysteine uptake in a yeast genomic library and found a DNA fragment causing the recovery of cysteine uptake and sensitivity to allylglycine. The gene in the fragment was identical to MUP1, the high affinity methionine permease gene. We conclude that Mup1 is a major permease in cysteine uptake.

The est1 regulation depends on the oxygen concentration in Acetobacter pasteurianus.

The regulation mechanism for expression of the ethanol inducible esterase gene, est1, was investigated in A. pasteurianus. Deletion analysis of the 5' non coding region of est1 showed that the FNR-binding consensus sequence is important in the induction of est1 by ethanol. Cells grown under oxygen starvation produced esterase-1 in not only the presence but also the absence of ethanol. These results suggest that the induction of est1-expression depends on the oxygen concentration, and the gene may be induced by a FNR-like factor activated by a decrease in the intracellular oxygen concentration.

A gene encoding phosphatidylethanolamine N-methyltransferase from Acetobacter aceti and some properties of its disruptant.

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Purification and characterization of intracellular alpha-L-rhamnosidase from Pseudomonas paucimobilis FP2001.

alpha-L-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca2+ and remained stable for several months when stored at -20 C. The optimum pH was 7.8; the optimum temperature was 45 degrees C. The Km, V(max) and k(cat) for p-nitrophenyl alpha-L-rhamnopyranoside were 1.18 mM, 92.4 microM x min(-1) and 117,000 x min(-1), respectively. Examination of the substrate specificity using various synthetic and natural L-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far.

Role of intracellular esterases in the production of esters by Acetobacter pasteurianus.

Esters are the major flavor compounds produced by Acetobacter sp. during vinegar production. The two genes encoding the esterases in the bacteria were disrupted, and the effects of the disruptions studied. When cultured in the presence of ethanol, the est1 gene-disrupted mutant (DE1K) did not produce any ethyl acetate or isoamyl acetate. However, the disruption of est2 did not affect the ester production. Ethyl acetate production by N-23 (pME122P) and DE1K (pME122P), which contain est1, was 1.7-fold higher than that by the wild type, N-23. On analyzing the relationship between ethyl acetate production and the extracellular ethanol and acetic acid concentrations, we found that the highest amount of ethyl acetate was produced when the molar ratio of ethanol and acetic acid was 1:1. These results indicate that the ester production by Acetobacter sp. is mostly catalyzed by the intracellular esterase, esterase-1, with ethanol and acetic acid used as the substrates.

A permease exhibiting a dual role for lysine and biotin uptake in Saccharomyces cerevisiae.

Among Saccharomyces cerevisiae strains each defective in one of 11 amino acid permeases, a lysine permease disruptant (DK) exhibited about 2-fold reductions in maximum cell density and fermentation ability compared to the parent in a synthetic medium. These unusual properties of DK were found to result from the requirement of biotin for growth, in contrast to the parent whose growth was not dependent on external biotin. The rate of 14C-labeled biotin uptake and the intracellular free biotin content of DK were 2-2.5 fold lower than in the parent. We suggest that lysine permease in S. cerevisiae has the ability to transport both lysine and biotin.