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The combined effect of ascorbic acid (AsA) and hydrogen peroxide (H2 O2 ) on gel-forming ability and structural changes of lizardfish (LZ) and threadfin bream (TB) surimi were investigated. Addition of 0.15% AsA and 0.1% H2 O2 greatly increased breaking force and distance of LZ surimi by 300% and 55%, respectively. Combination of 0.2% AsA and 0.15% H2 O2 resulted in the maximum TB surimi gel improvement with 150% and 90% increase in breaking force and distance, respectively. Browning reaction obviously occurred when combined AsA and H2 O2 was added, due to ascorbic acid oxidation. Polymerization of myosin heavy chain via disulfide bonds was promoted, and the formation of disulfide bonds was involved through oxidation of sulfhydryl groups with increasing AsA and H2 O2 . Fourier-transform infrared (FT-IR) spectroscopy revealed a decrease in α-helix and an increase in β-sheet content as AsA and H2 O2 increased in both species. A decrease of band area of aliphatic (2,800 to 3,000 and 1,450 cm-1 ), aromatic (1,208, 757, and ratio 850/830 cm-1 ), and change of disulfide bonds (525 and 540 cm-1 ) suggested an increase in hydrophobic interactions and disulfide bonds with addition of these additives. Based on principal component analysis (PCA), textural characteristics were positively correlated with β-sheet content. Our study suggested that combination of AsA and H2 O2 greatly enhanced gelation of LZ and TB by increasing not only disulfide bonds but also hydrophobic interactions. PRACTICAL APPLICATION: The combined ascorbic acid and hydrogen peroxide can be used to improve gelation of two important tropical surimi species, namely threadfin bream and lizardfish surimi, without requirement of setting. The optimum concentration of each additive varied with fish species.

Meat quality attributes vary with chicken age. Understanding the relationship between poultry age and the quality of the meat would be beneficial for efficient poultry farming to meet market needs. The Korat hybrid chicken (KC) is a new crossbred chicken whose meat quality is distinct from that of commercial broiler (CB) chickens and has not been well characterized. In this study, we characterized the physico-chemical properties of KC meat and correlate the findings with Raman spectral data. The protein content of KC breast and thigh meat increased with age. The pH of thigh meat decreased, while the water-holding capacity of breast meat increased as the age of the chickens increased. The amount of cholesterol in breast meat decreased as the rearing period was extended. Inosine 5′-monophosphate and guanosine 5′-monophosphate of breast meat decreased as KC grew older. The shear force values of meat from older birds increased concomitantly with an increase in total collagen. Principle component analysis revealed that the meat quality of CB was greatly different from that of KC meat. High shear force values of KC meat at 20 wk of age were well correlated with an increase in the β-sheet structure (amide I) and amide III of collagen. Raman spectra at 3,207 cm−1 and relative α-helical content were negatively correlated with shear force values of KC breast meat. These could be used as markers to evaluate KC meat quality.

Carnosine enrichment of slow-growing Korat chicken (KRC) meat helps differentiate KRC from mainstream chicken. We aimed to investigate the effects of β-alanine and L-histidine supplementation on the carnosine synthesis in and quality and secondary structure of proteins in slow-growing KRC meat. Four hundred 21-day-old female KRC were used, and a completely randomized design was applied. The chickens were divided into 4 experimental groups: basal diet (A), basal diet supplemented with 1.0% β-alanine (B), 0.5% L-histidine (C), and 1.0% β-alanine combined with 0.5% L-histidine (D). Each group consisted of 5 replicates (20 chickens per replicate). On d 70, 2 chickens per replicate were slaughtered, and the levels of carnosine, anserine, and thiobarbituric acid reactive substances were analyzed. Biochemical changes were monitored using synchrotron radiation-based Fourier transform infrared microspectroscopy; 5 chickens per replicate were slaughtered, and the meat quality was analyzed. Statistical analysis was performed using ANOVA and principal component analysis (PCA). Group D chickens exhibited the highest carnosine meat content, followed by those in groups B and C. However, amino acid supplementation did not affect anserine content and growth performance. Higher carnosine levels correlated with increasing pH

The Korat chicken (KR), developed in Thailand, is a slow-growing breed developed as an alternative breed for Thai chicken producers. The growing interest in slow-growing chicken meat, due to its unique taste, distinct texture, health benefits, and higher broiler welfare have led to higher market demand for KR. However, its low feed efficiency (FE) has a significant negative impact on farm profitability. Understanding the molecular mechanism regulating FE allows for designing a suitable selection program and contributing to breeding more efficient chicken for poultry production. Thus, the objective of our study was to investigate the proteome differences and possible pathways associated with FE in male KR using a label-free quantitative proteomic approach. Seventy-five KR males were individually evaluated for FE, and duodenum samples from 6 animals (3 high-FE and 3 low-FE chickens) were collected at 10 wk of age for differential abundant proteins (DAPs), protein networks, functional enrichment, and pathway analyses. In this study, we found 40 DAPs significantly associated with FE pathways, including glycolysis/gluconeogenesis, peroxisome, oxidative phosphorylation, tight junction, and cysteine and methionine metabolism. Thus, variations in observed DAPs or genes related to DAPs could be interesting biomarker candidates for selection for higher feed utilization efficiency in chicken.

Chicken breed is one of the key factors that influence meat quality. The quality attributes of breast meat from commercial broiler (CB), Thai native chicken (NC, Leung Hang Khao), and the crossbred Korat chicken (KC) were investigated via synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy, Fourier transform Raman (FT-Raman) spectroscopy, and physicochemical analysis. The protein and carbonyl contents of KC and NC meats were higher than that of CB meat, but the lipid content was lower (P0.05). CB meat was characterized by high moisture, lightness (L*), and presence of taste-active nucleotides, namely, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP). Moreover, NC meat had the highest insoluble collagen and inosine contents (P0.05). The predominant protein secondary structures of KC and NC meats were β-turns and random coils, whereas α-helices were mainly found in CB meat. Based on principal component analysis, the meat quality and spectra were clearly separated by breeds. The high moisture and lipid content of meat corresponded to O-H stretching (3,203 cm

Effects of high-intensity ultrasound (HIU) treatments on gelation of threadfin bream (Nemipterus spp.) surimi at various NaCl contents (0.5, 1, and 2% NaCl) were investigated. Protein extractability at 0.5% NaCl was increased with the ultrasonic intensity (p

Red blood spot (RBS) commonly found in cooked chicken breast has caused severe economic loss as it is perceived as a sign of undercooked product. The objectives of this study were to investigate the cause of RBS as related to common ingredients used in marination, based on both chicken breast and isolated chicken hemoglobin (Hb) models. The effect of sodium chloride (NaCl), sodium tripolyphosphate (STPP), and glucose on thermal denaturation of Hb was investigated along with the extent of RBS formation in cooked marinated chicken breast. After vacuum tumbling for 65 min and subsequent storage at 4 °C for 20 hr, STPP and glucose were not absorbed into the center of chicken breast. However, Na+ was absorbed after 12 hr storage. The denaturation temperature (Td ) of isolated chicken Hb decreased to 65.8 °C in the presence of 1.5 M NaCl, while that of the control was 69.4 °C. STPP at pH 9 decreased Td of Hb to 61.4 °C. The alkaline pH induced by STPP destabilized the Hb structure. RBSs were observed at 100% incidence when cooked to core temperatures of 50 and 70 °C for 1 min. RBSs were completely eliminated at core temperature of 85 °C. The ingredients used during marination appeared to have a minimal effect on RBS formation due to their limited absorption into the chicken breast. The cooking temperature is a major factor governing RBSs, as it directly affects the denaturation of Hb.

Chicken blood has limited utilization despite its high protein content. Production of a blood hydrolysate exhibiting angiotensin I-converting enzyme (ACE)–inhibitory activity would be means of valorizing chicken blood. The optimized conditions used to produce chicken blood corpuscle hydrolysate (BCH) by Alcalase were 51.1°C, 4% enzyme, and pH 9.6 for 6 h, resulting in a 35.8% degree of hydrolysis and 37.7% ACE inhibition at a peptide concentration of 0.2 mg/mL. The permeate of a 1-kDa membrane, BCH-III, showed a 2.5-fold increase in ACE inhibition compared with that of BCH. BCH-III was resistant to in vitro gastrointestinal digestion, whereas the BCH digesta exhibited an increased ACE-inhibitory activity after digestion. Both BCH and BCH-III were rich in hydrophobic amino acids. A single administration of BCH and BCH-III to spontaneously hypertensive rats at concentrations of 600 and 100 mg/kg, respectively, lowered the systolic blood pressure by −57.7 and −70.9 mmHg, respectively, 6 h after oral administration compared with the control group. The blood pressure–lowering effect of the 600 mg/kg BCH dose was comparable with that of the 100 mg/kg BCH-III dose after 4 wk of oral administration. Both BCH and BCH-III could be developed for use as nutraceutical products with antihypertensive effects.

Modulation of inflammation-related immune response on THP-1 macrophages of protein hydrolysates derived from tilapia mince, casein and pea protein, were investigated. The protein substrates were hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinase. The degree of hydrolysis (DH) of casein was observed to be the highest throughout the course of hydrolysis. When challenging THP-1 macrophages, tilapia mince hydrolysate (TMH) enhanced innate immunity through induction of IL-1β and COX-2 expression. Anti-inflammatory activity was observed in casein hydrolysate (CH) and pea protein hydrolysate (PPH) by attenuating lipopolysaccharide- (LPS) induced pro-inflammatory gene expression in THP-1 macrophages. CH suppressed IL-1β, IL-6, IL-8, TNF-α and COX-2, while PPH reduced LPS-induced IL-6 and TNF-α responses. In addition, CH and PPH showed stronger suppression of LPS-induced pro-inflammatory gene expression compared with non-hydrolyzed casein and pea protein. These results suggest that TMH, CH and PPH prepared from V. halodenitrificans SK1-3-7 proteinase are potential functional food ingredients with immunomodulatory activity.

Effects of high intensity ultrasound (HIU) on physicochemical properties of tilapia (Oreochromis niloticus) actomyosin in low NaCl concentrations were investigated. The protein content extracted in low NaCl concentrations (0.1–0.3 M NaCl) increased with increasing HIU intensity up to 20.62 W/cm2 (p

The effects of in vitro gastrointestinal (GI) digestion on the antioxidative activity of hydrolysates prepared from chicken blood plasma and red blood cell (RBC) by pepsin and thermolysin were investigated. The pepsin-hydrolyzed plasma (PHP) showed the highest scavenging activity of ABTS radicals (P < 0.05). RBC and plasma hydrolysates prepared by pepsin were hydrolyzed by GI proteases to a greater extent than hydrolysates prepared by thermolysin as evidenced by MALDI-TOF mass spectra. The antioxidative activity of all digesta increased compared to their respective parent hydrolysates, and PHP digesta showed the highest activity (P < 0.05). The digesta of PHP and thermolysin-hydrolyzed plasma showed cytoprotective properties in a dose-dependent manner, and 100 μg/mL of PHP digesta exhibited the highest protection of HepG2 cells against tert-butyl hydroperoxide (P < 0.05). Based on dichloro-dihydro-fluorescein diacetate assay, PHP digesta exhibited the greatest intracellular reactive oxygen species scavenging activity of approximately 71% at 100 μg/mL (P < 0.05). The peptide sequencing of PHP digesta revealed that they contained less than 10 amino acid residues, with an average hydrophobicity of 18.6. Chicken blood plasma is a better protein source for protein hydrolysates, and their digesta showed higher antioxidant activity compared to RBCs.

Real-time quantitative polymerase chain reaction (qPCR) methods were developed for the quantification of Virgibacillus sp. SK37 and Tetragenococcus halophilus MS33, which were added as starter cultures in fish sauce fermentation. The PCR assays were coupled with propidium monoazide (PMA) treatment of samples to selectively quantify viable cells and integrated with exogenous recombinant Escherichia coli cells to control variabilities in analysis procedures. The qPCR methods showed species-specificity for both Virgibacillus halodenitrificans and T. halophilus as evaluated using 6 reference strains and 28 strains of bacteria isolated from fish sauce fermentation. The qPCR efficiencies were 101.1% for V. halodenitrificans and 90.2% for T. halophilus. The quantification limits of the assays were 10(3) CFU/mL and 10(2) CFU/mL in fish sauce samples with linear correlations over 4 Logs for V. halodenitrificans and T. halophilus, respectively. The matrix effect was not observed when evaluated using fish sauce samples fermented for 1-6 months. The developed PMA-qPCR methods were successfully applied to monitor changes of Virgibacillus sp. SK37 and T. halophilus MS33 in a mackerel fish sauce fermentation model where culture-dependent techniques failed to quantify the starter cultures. The results demonstrated the usability of the methods as practical tools for monitoring the starter cultures in fish sauce fermentation.

Protein hydrolysate from frame, bone and skin (FBSH) of threadfin bream was prepared using Virgibacillus sp. SK33 proteinase and fractionated using sequential ultrafiltration membranes with molecular weight cut-offs (MWCO) of 30, 5 and 1 kDa, respectively. Four fractions, namely FBSH-I (>30 kDa), FBSH-II (5-30 kDa), FBSH-III (1-5 kDa), and FBSH-IV (

Korat-chicken breast and thigh were subjected to heating at 70, 100 or 121 °C for 30 min and simulated in vitro gastrointestinal digestion. At 70 or 100 °C heating, digests of breast possessed higher ACE inhibitory activity than those of thigh. The highest ACE inhibitory activity was found in the digest of breast cooked at 70 °C (B/H-70), whereas breast heated at 121 °C (B/H-121) exhibited the lowest. The 1-kDa permeate of the B/H-70 digest revealed higher permeability through colorectal adenocarcinoma monolayers and ACE inhibitory activity than did B/H-121. Among nine transported peptides, APP derived from myosin showed the highest ACE inhibition, with a non-competitive characteristic (Ki 0.93 μM). Molecular docking showed that APP interacts with ACE via hydrogen bonds, electrostatic and van der Waals interactions. In conclusion, mild thermal treatment of chicken breast resulted in a higher amount of transported peptides, exerting higher ACE inhibitory activity, which could lead to potential health benefits.

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.

The angiotensin I-converting enzyme (ACE) inhibitory activity of protein hydrolysates from tilapia muscle fractions, namely mince (M), washed mince (WM), and sarcoplasmic protein (SP), were investigated. Each fraction was hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases for up to 24 h. After 8 h of hydrolysis, the M hydrolysate (48% degree of hydrolysis (DH)) showed the highest ACE inhibitory activity, with an IC50 value of 0.54 mg/ml, while the SP hydrolysate exhibited the lowest DH and ACE inhibition. In vitro gastrointestinal digestion reduced the ACE inhibitory activity of the M hydrolysate but enhanced its transport across Caco-2 cell monolayers. The transported peptides were found to contain 3–4 amino acid residues showing strong ACE inhibition. The novel ACE inhibitory peptide with the highest inhibition was found to be MCS, with an IC50 value of 0.29 μM. Therefore, tilapia mince hydrolyzed by V. halodenitrificans proteinases contained ACE inhibitory peptides that are potentially bioavailable.

The effect of thermal treatments on chemical and cellular antioxidant activities of chicken breasts subjected to in vitro gastrointestinal digestion was investigated. Breast of Korat crossbred chicken (KC) and commercial broiler (BR) were cooked under various conditions, namely heating at 70 °C for 30 min (H-0.5) and 24 h (H-24), autoclaving (AC) at 121°C for 15 min (AC-15) and 60 min (AC-60). Protein digestibility decreased upon the extreme thermal treatment of AC-60. The H-0.5 improved metal chelating activity of KC digesta, FRAP, and anti-liposome oxidation of BR digesta. Digesta of BR/H-0.5 and KC/AC-15 at 50 μg/mL exhibited the highest cytoprotective effect against tert-butyl hydroperoxide (TBHP)-induced oxidative damage of HepG2 cells. In addition, the KC/AC-15 digesta at a concentration as low as 12.5 μg/mL inhibited intracellular TBHP-induced reactive oxyfen species (ROS) production (P0.05). Thus, the digesta of KC breasts subjected to AC-15 provides not only nutritional value but also antioxidant activity at the cellular level.

Proteinase inhibitory activity of sarcoplasmic protein (SP) extracted from common carp (Cyprinus carpio) muscle and its gel-improving ability were investigated. SPs displayed 89% and 54% inhibitory activity toward trypsin at 40 and 65 °C, respectively. Protein bands with molecular mass of 69, 50, 44, 41, and 35 kDa appeared on trypsin inhibitory activity staining under nonreducing condition when incubated at 40 °C, while 2 protein bands at 54 and 35 kDa were observed at 65 °C. Addition of SP at 0.18 g protein/100 g increased textural properties of threadfin bream surimi gel. However, when SP was added in combination with various CaCl2 concentrations (0.1% to 0.5%) it did not further improve textural properties as compared to the addition of SP alone. Retention of myosin heavy chain of threadfin bream surimi was greater with the addition of SP. These results indicated that the gel-enhancing effect of common carp SP was due to the inhibitory activity toward endogenous trypsin-like proteinases in threadfin bream surimi. Practical Application: Sarcoplasmic protein from common carp muscle could be used as a functional protein ingredient that minimizes muscle proteolysis and improves textural properties of surimi containing trypsin-like endogenous proteinases.

Fish sauce production relies on a natural fermentation process requiring 12-18 months for process completion. Virgibacillus sp. SK37 has been shown to be a potential strain for fish sauce acceleration. However, hydrolytic activity of proteinases bound at cell surface of this strain has not been well elucidated. Addition of 0.2 % CaCl(2) (w/w) in conjunction with starter cultures of Virgibacillus sp. SK 37 increased protein hydrolysis as measured by α-amino group content throughout fermentation (P0.05). Cell-bound proteinases from Virgibacillus sp. SK 37 were extracted into a free form by incubating the washed cells in Ca(2+)-free buffer at 37 °C for 2 h. Cell-bound proteinases revealed molecular mass of 19, 20, 22, 32, 34, and 44 kDa based on a synthetic peptide zymogram. The proteinases showed subtilisin-like serine characteristics with the highest activity at 50 °C and pH 8 and 11. Activity of the extracted proteinases increased ~4 times at ≥100 mM CaCl(2). In addition, CaCl(2) enhanced thermal stability of the extracted proteinases. Enzymes showed proteolytic activity in either the absence or presence of 10 and 25 % NaCl toward fish muscle, soy protein isolate, and casein substrates. Cell-bound proteinases were likely to play an important role in protein hydrolysis during fish sauce fermentation.

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.

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 degrees C for 2 h followed by 0.5% Flavourzyme at 50 degrees C for 4 h. The mixtures were then adjusted to contain 25% solar salt and incubated at 35 degrees C for 4 mo. alpha-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 alpha-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.

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.

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 was30%, 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, P0.05). 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 (P0.05). 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.

Structural changes and functional properties of threadfin bream (Nemipterus sp.) sarcoplasmic proteins (TB-SP) subjected to various pH conditions (pH 3, 5, 6.3, 9, and 12) after subsequent pH readjustment to pH 7 were investigated. Fourier transform infrared spectroscopy revealed the loss of alpha-helical and beta-sheet structures of TB-SP after being subjected to pH 3 or pH 12 treatments. The extent of structural and conformational changes of TB-SP subjected to pH 3 was greater than alkaline pHs (pH 9, 12) and pH 5, respectively. The water holding capacity of lyophilized TB-SP treated at pH 3 and pH 12 increased about 6.5- and 5.4-fold, respectively, as compared to the crude counterpart. Both acid and alkaline pH treatments increased fat absorption capacity of lyophilized sample about 2-fold, but drastically decreased its solubility. The water soluble fraction of extremely acidic (pH 3-->7) and alkaline (pH 12-->7) samples exhibited higher oil binding capacity as measured by diphenylhexatriene fluorescence and emulsifying activity. A gel-like structure was formed when water-soluble fraction of crude TB-SP and those subjected to moderate pHs (pH 5, 9) at 2 mg/mL was prepared for the emulsion containing 50% oil (v/v). Functional properties of TB-SP varied, depending on the pH-adjustment process applied.

Thailand is the second largest surimi producer in the world and 50% of surimi is produced from threadfin bream. During surimi processing, sarcoplasmic proteins are removed through water washing and discarded in the waste stream. This study was aimed at investigating the proteinase inhibitory activity of sarcoplasmic proteins.Sarcoplasmic proteins from threadfin bream (TBSP) exhibited inhibitory activity toward trypsin but did not inhibit papain and chymotrypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition stained by trypsin inhibitory activity revealed three protein bands of molecular mass of 95, 41 and 37 kDa. Inhibitory activity of TBSP reached a maximum when subjected to 45 degrees C and completely disappeared at 60 degrees C. The breaking force and deformation of lizardfish surimi gel with added TBSP and pre-incubated at 37 degrees for 20 min increased with additional levels of TBSP (P0.05). Trichloroacetic acid-oligopeptide content of lizardfish surimi gel with added TBSP decreased with the addition of 4 g kg(-1) TBSP (P0.05). Retention of myosin heavy chain (MHC) increased when TBSP concentration was increased. TBSP effectively protected MHC from proteolysis at 37 degrees C to a similar extent as egg white powder, but efficacy of TBSP was not observed at 65 degrees C.TBSP could be applied to reduce proteolytic degradation of lizardfish surimi or other surimi associated with trypsin-like proteinase, rendering an improvement in surimi gelation set at 37-40 degrees C.

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.

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

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.

Biochemical and conformational changes of purified sardine myosin were investigated at various pHs. The purity of myosin, as determined by SDS-PAGE, was approximately 94.6%. One major band at 205 kDa, corresponding to myosin heavy chain, and 3 light chains at 31, 24, and 23 kDa were observed on the SDS-PAGE gel. The greatest myosin protein solubility was observed at pH 7 and remained constant up to pH 11. Sardine myosin showed no solubility at pHs 2.5 to 5.0. Three endothermic peaks were obtained for samples prepared at pHs 7 and 10, while no peaks were shown for pH 2 samples, indicating chemical denaturation of myosin occurred before thermal treatment. The greatest Ca(2+)-ATPase activity was observed at pH 7, while no activity was observed between pHs 2 and 5 and at pH 11. Total sulfhydryl content was not measured at pHs 2.5 to 4 while the greatest measure was obtained for samples at pH 5.5. Surface hydrophobicity was not detected from pHs 2.5 to 5.0; thereafter the content remained consistent through pH 11. Storage modulus, indicating the elastic element of myosin gels, was minimally affected at pH 2, indicating myosin was chemically denatured before the temperature sweep treatment. However, at pH 10, the thermal exposure of myosin, as evidenced by dynamic thermograms with deeper valleys at 40 to 60 degrees C, was noted, indicating myosin was not damaged by adjustment to pH 10 and therefore was still able to undergo thermal gelation.