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/100 ml, 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.     

Improvement of Fish Sauce Quality by Strain CMC5‐3‐1: A Novel Species of Staphylococcus sp.

Staphylococcus sp. CMC5‐3‐1 and CMS5‐7‐5 isolated from fermented fish sauce at 3 to 7 mo, respectively, showed different characteristics on protein hydrolysis and volatile formation. These Gram‐positive cocci were able to grow in up to 15% NaCl with the optimum at 0.5% to 5% NaCl in tryptic soy broth. Based on ribosomal 16S rRNA gene sequences, Staphylococcus sp. CMC5‐3‐1 and CMS5‐7‐5 showed 99.0% similarity to that of Staphylococcus piscifermentans JCM 6057^T, but DNA–DNA relatedness was <30%, indicating that they were likely to be new species. DNA relatedness between these 2 strains was only 65%, suggesting that they also belonged to different species. The α‐amino group content of 6‐month‐old fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 was 740.5 mM, which was higher than that inoculated by the strain CMS5‐7‐5 (662.14 mM, P < 0.05). Histamine was not produced during fermentations with both strains. Fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 showed the highest content of total glutamic acid (P < 0.05). The major volatile compound detected in fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 was 2‐methypropanal, contributing to the desirable dark chocolate note. Staphylococcus sp. CMC5‐3‐1 could be applied as a starter culture to improve the umami and aroma of fish sauce.