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The effect of Virgibacillus sp. SK37, together with reduced salt content, on fish sauce quality, particularly free amino acids and odor-active compounds, was investigated. Virgibacillus sp. SK37 was inoculated with an approximate viable count of 5 log CFU/mL in samples with varied amounts of solar salt, for example, 10, 15, and 20% of total weight. Eighteen selected odorants were quantitated by stable isotope dilution assays (SIDA), and their odor activity values (OAVs) were calculated. Samples prepared using 10% salt underwent spoilage after 7 days of fermentation. The viable count of Virgibacillus sp. SK37 was found over 3 months in the samples containing 15 and 20% salt. However, acceleration of protein hydrolysis was not pronounced in inoculated samples at both 15 and 20% salt. Virgibacillus sp. SK37, together with salt contents reduced to 15-20%, appeared to increase the content of 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, acetic acid, and 2-methylpropanoic acid. However, only aldehydes were found to have an effect on the overall aroma of fish sauce based on high OAVs, suggesting that the inoculation of samples with Virgibacillus sp. SK37 under reduced salt contents of 15-20% likely contributed to stronger malty or dark chocolate notes.

A NaCl-activated proteinase produced by Virgibacillus sp. SK33 was purified to homogeneity using phenyl-Sepharose and Sephadex G-75 with a yield of 12% and purification of 2.6-fold. A single protein was detected at approximately 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, three subunits with molecular weights of 27,858, 33,918, and 35,368 Da were obtained from MALDI-TOF mass spectra, implying that the enzyme was a heterotrimer. The isoelectric point of the proteinase was 5.4. Optimum catalytic activity was at 55 degrees C and pH 7.5. The enzyme showed serine characteristics as it was completely inhibited by phenylmethanesulfonyl fluoride. The purified proteinase showed broad specificity toward oxidized insulin B including Gln4, Cys7, Glu13, Ala14, Leu15,17, Tyr16,26, Arg22, Phe24,25, and Lys29. Dominant cleavage sites of the enzyme were Tyr16-Leu17 and Phe25-Tyr26, indicating that it preferably hydrolyzed aromatic amino acids located on the P1 site. Among various substrates studied, the enzyme hydrolyzed anchovy protein to the greatest extent at 4 M NaCl. Activity increased with either CaCl2 or NaCl concentration with the maximum 2-fold increase at either 50 mM CaCl2 or 4 M NaCl. The enzyme was also highly stable up to 500 mM CaCl2 or 4 M NaCl. The proteinase showed high stability in various organic solvents (25%, v/v) including dimethylsulfoxide, methanol, acetonitrile, and ethanol. Results of peptide mass fingerprint and de novo peptide sequencing showed that the purified proteinase is a novel proteinase. The proteinase from Virgibacillus sp. SK33 could have a potential application in high ionic strength environments and aqueous-organic solvent systems.

Comprehensive analysis of the potent odorants in Thai premium fish sauce samples was accomplished by use of complementary volatile isolation methods combined with gas chromatography-olfactometry (GC-O) and GC-mass spectrometry. Odorants of intermediate and low volatility were determined by direct solvent extraction/solvent-assisted flavor evaporation (DSE-SAFE) and aroma extract dilution analysis (AEDA). Meanwhile, static headspace dilution analysis (SHDA) and headspace solid-phase microextraction (H-SPME) were used to determine the highly volatile odorants. Results of AEDA indicated the importance (log3FD factor≥6) of five acidic odorants (butanoic acid, 3-methylbutanoic acid, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone, and 2-phenylacetic acid) and four neutral/basic odorants (3-methylbutanal, (Z)-1,5-octadien-3-one, phenylacetaldehyde, and o-aminoacetophone). Results of SHDA indicated the predominant (log3FD factors≥5) headspace odorants were methanethiol, 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, dimethyl trisulfide, 3-(methylthio)propanal, and butanoic acid. Concentrations for 21 odorants were determined by stable isotope dilution analysis (SIDA), and their odor-activity values (OAVs) were calculated. Among these, methanethiol, 2-methylpropanal, 3-methylbutanal, dimethyl trisulfide, 3-(methylthio)propanal, and butanoic acid had the highest OAVs (>500). Results of aroma recombination and omission studies revealed the importance of acids, aldehydes, and sulfur-containing compounds to the overall aroma of the Thai fish sauce.

Structural changes and dynamic rheological properties of sarcoplasmic proteins from striped catfish ( Pangasius hypophthalmus ) treated by various pH-shift processes were investigated. Isoelectric precipitation of acid-extracted sarcoplasmic proteins led to the lowest solubility in water. Sarcoplasmic proteins were unfolded after extremely acidic and alkaline extraction, exposing tryptophan and aliphatic residues. The alpha-helical structure was converted to beta-sheet following acidic extraction, whereas alkaline treatment did not disturb the alpha-helical structure of sarcoplasmic proteins. Disulfide formation, hydrogen bonding via tyrosine residues, and hydrophobic interactions occurred under extreme pH extraction. Acidic extraction induced denaturation and aggregation of sarcoplasmic proteins to a greater extent than did alkaline treatment. Hydrophobic interactions via aliphatic and aromatic residues were formed during isoelectric precipitation. Sarcoplasmic proteins were partially refolded after isoelectric precipitation followed by neutralization. Sarcoplasmic proteins prepared from an alkaline pH-shift process readily aggregated to form a gel at 45.10 degrees C, whereas higher thermal denaturation temperatures (>80 degrees C) and gel points ( approximately 78 degrees C) were observed in acid-treated sarcoplasmic proteins. The pH condition used for extraction, precipitation, and neutralization greatly affected structural changes of sarcoplasmic proteins, leading to different thermal and dynamic rheological properties.

Fish liver transglutaminase (FTG), a Ca(2+)-dependent enzyme, exhibits different characteristics from the Ca(2+)-independent microbial transglutaminase (MTG), leading to potential differences in their substrate specificity and reactivity. The ability of these enzymes to catalyze isopeptide bond formation by incorporating 5-(biotinamido)pentylamine (BPNH2) into peptides derived by tryptic digestion of threadfin bream (TB)-myosin was investigated to identify reaction sites and substrate specificity using a peptidomic strategy. BPNH2 was incorporated into TB-myosin peptides to a greater extent by MTG than FTG. Peptides derived from TB-myosin heavy chain (MHC) shared highest similarity to amberjack-MHC on the basis of a Mascot database search. Amino acid sequences and modification sites of BPNH2-tagged peptides were identified by tandem mass spectrometry based on the amberjack-MHC sequence. The BPNH2 modification sites catalyzed by both TGases were at the myosin rod. Most of the BPNH2 peptides contained charged amino acids (E, R, K) at the glutaminylamide site of reactive glutamine (Q*). The alpha-acrylamide site of Q* contained E, F, or L on peptides catalyzed by both enzymes, I, Q, or A on peptides catalyzed only by FTG, and V on a peptide catalyzed only by MTG. These results demonstrate the different structural requirements for glutaminyl substrates between these two enzymes.