Somboon Tanasupawat

Lactobacillus acidipiscis sp. nov. and Weissella thailandensis sp. nov., isolated from fermented fish in Thailand

Eleven strains of homofermentative, rod-shaped lactic acid bacteria and five strains of heterofermentative, sphere-shaped lactic acid bacteria were isolated from fermented fish (pla-ra and pla-chom) in Thailand. They were identified as new species and named Lactobacillus acidipiscis sp. nov. and Weissella thailandensis sp. nov., respectively, on the basis of phylogenetic analysis of the 16S rRNA gene sequences, DNA relatedness and phenotypic characteristics. The type strain of L. acidipiscis is FS60-1T (= PCU 207T = NRIC 0300T = HSCC 1411T = JCM 10692T = TISTR 1386T) and the type strain of Weissella thailandensis is FS61-1T (= PCU 210T = NRIC 0298T = HSCC 1412T = JCM 10695T = TISTR 1384T).

Exopolysaccharide-producing lactic acid bacteria strains from traditional thai fermented foods : isolation, identification and exopolysaccharide characterization

Lactic Acid Bacteria (LAB) isolated from various traditional Thai fermented foods were screened for exopolysaccharides (EPS) production. From 104 isolates, two rod-shaped and five coccal-shaped LAB were able to produce EPS from sucrose on solid media. However, only the cocci were capable of producing EPS in liquid media and these were identified as Pediococcus pentosaceus. Pediococcus pentosaceus strains AP-1 and AP-3 produced EPS in high yield. In liquid media containing sucrose as carbon source, the amount of EPS produced by AP-1 and AP-3 strains was 6.0 and 2.5 g/L, respectively. The isolated and purified EPSs were chemically characterized. On the basis of sugar composition, methylation analysis and nuclear magnetic resonance spectroscopy, both the EPSs were shown to belong to the same dextran class. In particular, both EPSs differed from linear dextran by branching through 3,6-di-Osubstituted alpha-D-glucopyranosyl residues. The EPS from P. pentosaceus AP-3 was characterized by a relatively higher degree of branching and by a higher molecular weight than that from P. pentosaceus AP-1.

A Halophilic Serine Proteinase from Halobacillus sp. SR5-3 Isolated from Fish Sauce: Purification and Characterization

A halophilic bacterium was isolated from fish sauce, classified, and named Halobacillus sp. SR5-3. A purified 43-kDa proteinase produced by this bacterium showed optimal activity at 50 °C and pH 9–10 in 20% NaCl. The activity of the enzyme was enhanced about 2.5-fold by the addition of 20–35% NaCl, and the enzyme was highly stabilized by NaCl. It was found to be a serine proteinase related to either chymotrypsin or subtilisin. It absolutely preferred Ile at the P2 position of substrates. Thus, the enzyme was found to be a halophilic serine proteinase with unique substrate specificity.

Purification and Characterization of Serine Proteinase from a Halophilic Bacterium, Filobacillus sp. RF2-5

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 °C and pH 10–11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and α-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15–25% NaCl respectively using Suc–Ala–Ala–Phe–pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.

Proteinase-producing halophilic lactic acid bacteria isolated from fish sauce fermentation and their ability to produce volatile compounds

Halophilic lactic acid bacteria were isolated from fish sauce mashes fermented at 1 to 12 months. Seven out of sixty-four isolates were selected according to their proteolytic activity and growth at 25% NaCl for characterization and investigation of volatile compound production. All selected isolates were Gram-positive cocci with pairs/tetrads and grew at 0-25% NaCl, pH 4.5-9.0. Results of 16S rRNA gene sequence analysis showed 99% homology to Tetragenococcus halophilus ATCC 33315. The restriction fragment length polymorphism (RFLP) patterns of all isolates were also similar to those of T. halophilus ATCC 33315. These isolates were, thus, identified as T. halophilus. All isolates hydrolyzed fish protein in the medium containing 25% NaCl. Intracellular aminopeptidase of 7 isolates exhibited the highest activity of 2.85-3.67 U/ml toward Ala-p-nitroanilide (Ala-pNA). T.halophilus strains MS33 and M11 showed the highest alanyl aminopeptidase activity (P<0.05), and produced histamine in mGYP broth containing 5 and 25% NaCl in the level of 6.62-22.55 and 13.14-20.39 mg/100ml, respectively. Predominant volatile compounds of fish broth containing 25% NaCl inoculated with T. halophilus MS33 and MRC5-5-2 were 1-propanol, 2-methylpropanal, and benzaldehyde, corresponding to major volatile compounds in fish sauce. T.halophilus appeared to play an important role in volatile compound formation during fish sauce fermentation.

Staphylococcus piscifermentans sp. nov., from fermented fish in Thailand

New coagulase-negative staphylococci were isolated from fermented fish in Thailand. These organisms were differentiated from other Staphylococcus species on the basis of DNA relatedness and biochemical characteristics. Staphylococcus piscifermentans sp. nov. is described, and the type strain is strain SK03 (= NRIC 1817 = JCM 6057 = TISTR 824).

Gluconobacter japonicus sp. nov., an acetic acid bacterium in the Alphaproteobacteria.

Five strains, NBRC 3271(T), NBRC 3272, NBRC 3263, NBRC 3260 and NBRC 3269 were examined genetically, phylogenetically, phenotypically and chemotaxonomically. The DNA G+C contents of the five strains were 55.1-56.4 mol%. The five strains had low levels of DNA-DNA hybridization of 13-51 % to the type strains of Gluconobacter frateurii, Gluconobacter thailandicus, Gluconobacter oxydans, Gluconobacter cerinus, Gluconobacter albidus and Gluconobacter kondonii and formed a cluster that was separate from the type strains of the six Gluconobacter species given above in phylogenetic trees based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences. The five strains weakly produced dihydroxyacetone from glycerol, but not 2,5-diketo-d-gluconate or a water-soluble brown pigment from d-glucose and contained ubiquinone-10. The five strains were assigned as representing a novel species of the genus Gluconobacter, for which the name Gluconobacter japonicus sp. nov. is proposed. The type strain is NBRC 3271(T) (=BCC 14458(T)=strain 7(T), K. Kondo). Cells of the type strain are motile by means of polar flagella and the DNA G+C content is 56.4 mol%.

Natrinema gari sp. nov., a halophilic archaeon isolated from fish sauce in Thailand

Two Gram-negative, rod-shaped, halophilic archaea, designated strains HIS40-3(T) and HDS3-1, were isolated from anchovy fish sauce (nam-pla) collected from two different locations in Thailand. The two strains were able to grow at 20-60 degrees C (optimum 37-40 degrees C), at 1.7-5.1 M NaCl (optimum 2.6-3.4 M NaCl) and at pH 5.5-8.5 (optimum pH 6.0-6.5). Hypotonic treatment with less than 1.7 M NaCl caused cell lysis. The major polar lipids of the isolates were C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, two glycolipids and one unidentified lipid. The DNA G+C contents were 64.0-65.4 mol%. In addition to phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strains HIS40-3(T) and HDS3-1 were related most closely to species of the genus Natrinema. Levels of 16S rRNA gene sequence similarity between strains HIS40-3(T) and HDS3-1 and the type strains of recognized Natrinema species were 99.1-96.6 %. The two novel strains could be distinguished from recognized Natrinema species on the basis of low levels of DNA-DNA relatedness and differences in whole-cell protein patterns and phenotypic properties. Levels of 16S rRNA gene sequence similarity and DNA-DNA relatedness between the two strains were 99.7 and 77.7 %, respectively, suggesting that they should be classified as representing a single species. Based on these taxonomic data, strains HIS40-3(T) and HDS3-1 are considered to represent a novel species of the genus Natrinema, for which the name Natrinema gari sp. nov. is proposed. The type strain is HIS40-3(T) (=BCC 24370(T) =JCM 14663(T) =PCU 303(T)).

Cohnella thailandensis sp. nov., a xylanolytic bacterium from Thai soil.

A xylanolytic bacterium, strain S1-3(T), was isolated from soil collected in Nan province, Thailand. It was characterized taxonomically based on phenotypic characteristics and 16S rRNA gene sequence comparison. The strain was a Gram-stain-positive, facultatively anaerobic, spore-forming, rod-shaped bacterium. It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The major menaquinone was MK-7. Iso-C₁₆:₀ (39.5 %) and anteiso-C₁₅:₀ (26.8 %) were predominant cellular fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lysyl-phosphatidylglycerol were the major polar lipids. The DNA G+C content was 53.3 mol%. Phylogenetic analysis using 16S rRNA gene sequences showed that strain S1-3(T) was affiliated to the genus Cohnella, and was closely related to Cohnella ginsengisoli GR21-5(T) and Cohnella thermotolerans CCUG 47242(T) with 95.7 and 95.3 % sequence similarity, respectively. Strain S1-3(T) could be clearly distinguished from related species of the genus Cohnella by its physiological and biochemical characteristics as well as by its phylogenetic position. Therefore, the strain represents a novel species of the genus Cohnella, for which the name Cohnella thailandensis sp. nov. is proposed. The type strain is S1-3(T) (=KCTC 22296(T) =TISTR 1890(T) =PCU 306(T)).

Bacillus siamensis sp. nov., isolated from salted crab (poo-khem) in Thailand

A Gram-positive, endospore-forming, rod-shaped bacterium, strain PD-A10(T), was isolated from salted crab (poo-khem) in Thailand and subjected to a taxonomic study. Phenotypic and chemotaxonomic characteristics, including phylogenetic analyses, showed that the novel strain was a member of the genus Bacillus. The novel strain grew in medium with 0-14 % (w/v) NaCl, at 4-55°C and at pH4.5-9. The predominant quinone was a menaquinone with seven isoprene units (MK-7). The major fatty acids were anteiso-C₁₅:₀ and anteiso-C₁₇:₀. Polar lipid analysis revealed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysylphosphatidylglycerol, glycolipid and unknown lipids. The DNA G+C content was 41.4 mol%. The 16S rRNA gene sequence similarities between strain PD-A10(T) and Bacillus amyloliquefaciens NBRC 15535(T), Bacillus subtilis DSM 10(T), Bacillus vallismortis DSM 11031(T) and Bacillus mojavensis IFO 15718(T) were 99.5, 99.4, 99.4 and 99.2 %, respectively. Strain PD-A10(T) showed a low degree similarity of rep-PCR fingerprints and low DNA-DNA relatedness with the above-mentioned species. On the basis of the data gathered in this study, strain PD-A10(T) should be classified as representing a novel species of the genus Bacillus, for which the name Bacillus siamensis sp. nov. is proposed. The type strain is PD-A10(T) (=BCC 22614(T)=KCTC 13613(T)).