NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation

ABSTRACT:  Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 °C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca²⁺ activated the enzyme activity but reduced enzyme stability at 65 °C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.     

Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation

Abstract: Cell‐associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris‐maleate (pH 7) at 37 °C for 2 h. Major cell‐associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32‐kDa proteinase showed strong amidolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐AMC. Activity of all cell‐associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65‐kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal‐dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell‐associated proteinases from a moderate halophile, Virgibacillus sp. Practical Application: The cell‐associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell‐associated proteinases are key factors contributing to protein‐degrading ability at high salt environment of Virgibacillus sp. SK 33.     

Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures

ABSTRACT:  A means to accelerate fish sauce fermentation without adversely affecting fish sauce quality was investigated. Starter cultures prepared from Virgibacillus sp. SK33, Virgibacillus sp. SK37, and Staphylococcus sp. SK1-1-5 were added separately to anchovy that was hydrolyzed by 0.25% Alcalase at 60 °C for 2 h followed by 0.5% Flavourzyme at 50 °C for 4 h. The mixtures were then adjusted to contain 25% solar salt and incubated at 35 °C for 4 mo. α-Amino contents of all inoculated samples were higher than the control (without the addition of starter culture) during the course of fermentation. After 4-mo fermentation, the samples inoculated with Staphylococcus sp. SK1-1-5 contained the highest α-amino content of 733.37 ± 13.89 mM while that of the control was 682.67 ± 3.33 mM. Amino acid profiles of inoculated samples showed similar patterns to that of commercial product fermented for 12 mo, with glutamic, aspartic, and lysine being predominant amino acids. Virgibacillus sp. SK33 appeared to decrease histamine content of fish sauce by 50% when compared to the control. Volatile compounds analyzed by GC–MS of all inoculated samples fermented for 4 mo exhibited a similar pattern to those of the 12-mo-old commercial product. Samples inoculated with Staphylococcus sp. SK1-1-5 produced higher levels of volatile fatty acids and showed similar sensory characteristics to the commercial fish sauce fermented for 12 mo. Staphylococcus sp. SK1-1-5 is a potential strain that can be applied to produce fish sauce with overall sensory characteristics of traditional fish sauce in shorter time.     

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.     

Spent brewery yeast sludge as a single nitrogen source for fibrinolytic enzyme production of Virgibacillus sp. SK37

The objectives of this study were to investigate the suitable food industrial byproducts and conditions affecting proteinase production of Virgibacillus sp. SK37 as well as to evaluate fibrinolytic activity of secreted proteinases. Among 5 food industrial wastes including soybean pomace, rice bran, mungbean protein, spent brewery yeast sludge (Ys), and fish sauce sludge, Ys was the best nitrogen source for proteinase production from Virgibacillus sp. SK37. The highest level of proteinase production was obtained in the medium containing 1% Ys, 2.5% NaCl, pH 7.5 at 40°C, which was about 1.7 times higher than the commercial yeast extract medium. Virgibacillus sp. SK37 proteinases completely hydrolyzed fibrinogen within 50 min with Aα-chain being the first target and followed by Bβ- and Γ-chains. Virgibacillus sp. SK37 could be a potential source of fibrinolytic enzyme that can be developed as a functional food ingredient.     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.