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Proteinase-producing halophilic archaea were isolated from Thai fish sauce collected from industrial fermentation tanks at various periods of fermentation. Five isolates namely, J-1-S4, J-1-S13, J-1-S22, 2 m-40-15-R2, and P-1-S8, were identified as Halobacterium salinarum with slightly different colony and morphological characteristics among isolates. Starters of five isolates were prepared and added to the anchovy mixed with 25% solar salt and fermented for 180 days at 30-35°C. Halophilic bacteria/archaea counts of inoculated samples were decreased and undetected after 120 days of fermentation. At 180 days of fermentation, α-amino group contents of inoculated fish sauce samples (856-1010 mM) and total nitrogen content (2.2%-2.5%) were higher than the control without archaea inoculation (p 0.05). All samples contained low amounts of biogenic amines, suggesting that all starters were not biogenic amine formers. The major volatile compound found in samples inoculated with H. salinarum P-1-S8 and H. salinarum 2 m-40-15-R2 was 3-methylbutanal, which contributes to the meaty note. Dimethyl disulfide, a compound that contributes to fecal note, was detected in all inoculated samples in a lower amount than in the commercial fish sauce (p 0.05). Thus, H. salinarum accelerated protein hydrolysis and produced desirable volatile compounds during fish sauce fermentation in a strain-specific manner.

Antioxidant activity and peptide profiles of fish sauce samples prepared under varied conditions, namely traditional process, starter culture addition, and a reduced salt process, were determined. ...

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.

The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptide...

The effects of various sous-vide (SV) cooking conditions (50-60℃, 30-60 min) on physicochemical properties related to the texture characteristics, protein structure/degradation, and sensory acceptability of tilapia fillet (Oreochromis niloticus) were investigated. With an increasing temperature and processing time of SV cooking, protein degradation (of both myofibrils and connective tissue) was more pronounced, as evaluated by the decrease in water- and salt-soluble proteins, total collagen, as well as the changes in the ratio of secondary protein structures (α-helix, β-sheet, β-turn, etc.), which were determined by synchrotron-FTIR (SR-FTIR). These degradations were associated with the improvement of meat tenderness, as estimated by shear force and texture profile analyzer (TPA) results. Among all SV conditions, using 60 ℃ for 45 min seems to be the optimal condition for tilapia meat, since it delivered the best results for texture characteristics and acceptability (p < 0.05). Moreover, principal component analysis (PCA) results clearly demonstrated that the highest texture-liking score of this condition was well associated with the intensity of β-sheets, which seem to be the crucial component that affected the texture of SV-cooked tilapia more so than other parameters. The findings demonstrated the potential of SR-FTIR to decipher the biomolecular structure, particularly the secondary protein structure, of SV-cooked tilapia. This technique provided essential information for a better understanding of the changes in biomolecules related to the textural characteristics of this product.

This study aims to shed light on the association between non-volatile and volatile compounds related to flavor/taste characteristics as well as sensory acceptability of Nile tilapia fillet (Oreochromis niloticus) cooked by various sous-vide (SV) conditions (50–60 ℃, 30–60 min), with fish cooked with boiling water used as control. Higher temperatures and longer processing times of SV cooking led to greater protein and lipid oxidation as indicated by the increase in total sulfhydryl (-SH), carbonyl, free fatty acid (FFA) contents as well as peroxide values (PV) and thiobarbituric acid reactive substance (TBARS) values. The differences in flavor/taste components including adenosine triphosphate (ATP)-related compounds, free amino acids (FAAs) and volatiles were also obtained, which directly affect sensory acceptability as evaluated by using the hedonic scale. Based on principal component analysis (PCA) results, the acceptability score was strongly correlated with inosine monophosphate (IMP) and acetoin, which seem to be the most crucial flavor enhancers for cooked tilapia. Among all samples, tilapia processed at 60 °C for 45 and 60 min, which contained significantly higher IMP and acetoin (p < 0.05) than others, had significantly higher flavor-liking and overall-liking scores, with a more than 7.5 meaning for high acceptability (p < 0.05), indicating the optimal SV conditions for tilapia fillet. Overall, the present finding indicated that the SV-cooking technique, at the optimal conditions, can improve the meat quality of cooked fish, in terms of flavor/taste characteristics, compared with traditional cooking (control).

Due to the overuse and abuse of antibiotics, several antibiotic resistant bacteria have emerged. Antimicrobial peptides (AMPs) have gained attention as alternative antimicrobial agents because of their unique mode of action that impedes bacterial resistance. Two novel antibacterial peptides were isolated from Alcalase-hydrolyzed chicken plasma by size exclusion and reverse-phase chromatography. They were identified by LC-MS/MS to be VSDH and CCCPKAF, which showed effective antibacterial activity toward Bacillus cereus DMST 5040, with varied modes of action. The peptide CCCPKAF caused cell membrane disintegration, as evidenced by propidium iodide (PI) uptake. In contrast, the peptide VSDH targeted intracellular molecules, including proteins and nucleic acids, as revealed by Synchrotron-based Fourier Transform Infrared (SR-FTIR). The secondary structure of intracellular proteins increased to a β-sheet structure concomitant with a decrease in the α-helix structure when exposed to 0.5 mM VSDH. Molecular docking analysis revealed that VSDH showed high binding affinity for the active sites of the various enzymes involved in DNA synthesis. In addition, it showed good affinity for a chaperone protein (Dnak), resulting in the misfolding of intracellular proteins. Nuclear magnetic resonance (NMR) and molecular dynamics simulations also indicated that VSDH chelated well with Mg2+, which could partly contribute to its antibacterial activity.

Changes in the lipid oxidation of silver carp (Hypophthalmichthys molitrix) stored in ice for 14 days and that of its respective washed mince were evaluated. Total lipid, phospholipid, polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid (MUFA) contents of the skin, belly flap and mince decreased as the storage time in ice increased. The washing process decreased the lipid contents but concentrated their phospholipid counterparts. The fish belly flap exhibited the highest thio-barbituric acid reactive substances (TBARS) value, while the mince had the lowest. 1-Hexanol, 1-octen-3-ol, and 1-hexanal were key volatile compounds detected in the belly flaps of fish stored for 7–14 days. Hexanal was the only major volatile compound found in washed mince prepared from fish stored for an extended period in ice, but in a much lower amount compared with that in the belly flap. FTIR (Fourier transform infra-red) spectra revealed a decrease in the number of cis double bonds, methylene groups and phosphate groups in lipids extracted from fish stored in ice for 7–14 days as compared with those extracted from fresh fish. Principle component analysis (PCA) revealed that the FT-Raman band at 1747 cm−1 could be a potential marker for tracking the degree of lipid oxidation in the belly flap of silver carp stored in ice. In addition, IR bands indicating phosphate group (925, 825 cm−1) in oil extracted from washed mince were correlated with the extent of the lipid oxidation of the raw material.

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.

The conformational changes of natural actomyosins prepared from fresh and freeze-thaw tilapia (Oreochromis niloticus) in the presence of Ca2+ or Mg2+ were investigated. The Ca2+-activated a...

The effect of NaCl (0.1 − 3.0 M) on self-assembly of silver carp myosin at 4 °C was investigated in terms of microstructure, conformation, intermolecular interactions, and particle size distribution. During setting at 4 °C, the self-assembly of myosin showed an obvious concentration dependence. At low concentrations ( 2+ -ATPase activity reached a maximum at 0.6 M NaCl. When the concentrations increased to >1.0 M, myosin further assembled into filaments dominated by hydrophobic interactions. Both rod-rod and head-head interactions contributed to the myosin filaments at high concentrations (1.0 − 3.0 M). The solubility and uniformity of the myosin assemblies decreased as the NaCl concentration increased from 1.0 to 3.0 M.

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.

Permeability of angiotensin I-converting enzyme (ACE) inhibitory peptides (KPLLCS, ELFTT, and KPLL) identified from the in vitro gastrointestinal digest of cooked chicken breast was investigated using the Caco-2 cell model system. KPLLCS was originally the most effective ACE inhibitor (IC50 0.37 μM), but it was degraded during permeation through Caco-2 cells. Among these peptides, KPLL showed the highest permeability in intact form, and partially degraded to KP and LL, which still showed ACE inhibitory activity after permeation. ACE inhibitory activity of permeated KPLL was highest of 56%. KPLL and KP possessed a mixed inhibitor characteristic, while LL was a non-competitive inhibitor. Based on molecular docking, K at N-terminus of KPLL is a key structure contributing to ACE inhibition as it can occupy the active site of ACE. Determining the structural stability and degree of peptide transport across epithelial cell is required to confirm their potential as ACE inhibitors.

Although silver carp bones (SCB) are generated as waste, they are a natural source of calcium and have the potential to be a food ingredient. When the SCB had been soaked in hot sodium hydroxide (0...

Nine novel peptides from tilapia hydrolysate were isolated and identified to be EKL, EKP, HKPA, ELSC, ALSC, ASLCH, SLCH, LPGYF, and LEVPGY. In addition, six fragments from in silico gastrointestinal digestion of the identified peptides, including EK, KPA, SC, CH, PGY, and EVPGY, were synthesized. The most effective parent and digested peptides showing ABTS · + scavenging capacity were LPGYF and SC, respectively. All C- and Y-containing peptides were more effective than ascorbic acid (AsA). In contrast, K-containing peptides exhibited less antioxidant activity. All 15 peptides showed potent intracellular reactive oxygen species scavengers in the AAPH-induced HepG2 cell oxidative stress model. In addition, the digested peptides SC, CH, and PGY up-regulated the expression of CAT and SOD1 in HepG2 cells. The peptide PGY was the most effective cellular antioxidant. Thus, tilapia peptides could be potent nutraceutical products to reduce cellular oxidative stress.

The aim of this study was to illustrate the effect of high intensity ultrasound (HIU) pre-treatment modes (HIU pre-treatment before salt-chopping (HIUBC), after salt-chopping (HIUAC) and after stuffing into polyvinylidene chloride casing (HIUAS)) on the gelation properties of silver carp surimi. HIUBC facilitated the protein dispersion and more compact microstructures formation, which was beneficial to obtaining the highest water-holding capacity (WHC) of HIUBC gels. Meanwhile, the puncture properties of HIUBC gels were significantly improved via the formation of more e-(γ-Glu)-Lys and S–S bonds. HIUAC and HIUAS promoted the formation of e-(γ-Glu)-Lys bonds, but also induced the protein degradation, as evidenced by the decreased solubility and relatively higher TCA-soluble peptides. Thus, no obvious improvement of puncture properties and WHC was observed for HIUAC or HIUAS gels. In conclusion, HIUBC was beneficial to facilitating protein dispersion, thereby improving gelation properties of surimi during heat-induced gelling process. However, HIUAC and HIUAS hardly influenced gelation properties of surimi. This might provide an effective method of HIU pre-treatment for promoting gelation properties of surimi.

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.

Biological and chemical dynamics were investigated during fermentation of fish sauce inoculated single and combined starter cultures, Virgibacillus sp. SK37 and Tetragenococcus halophilus MS33. At 120–150 days of fermentation, cell numbers of Virgibacillus sp. SK37 and T. halophilus MS33 were lower as detected by qPCR in fish sauce inoculated with a single culture compared with the sample inoculated with combined cultures. In addition, qPCR revealed that the sequential inoculation of Virgibacillus sp. SK37 followed by the addition of T. halophilus MS33 after 1 month (SK37_1M + MS33) showed bacterial counts that were approximately 0.5–1 LogCFU/mL higher than the single starter culture treatments. α-Amino contents of fish sauce added simultaneous co-cultures (MS33 + SK37) and SK37_1M + MS33 were 1160.41 and 1185.71 mM, respectively. SK37_1M + MS33 showed a lower amount of histamine than the control ( P Virgibacillus sp. SK37 or T. halophilus MS33 showed higher amounts of total glutamic acid and greater total amounts of amino acids than the control ( P , P

The release of encrypted angiotensin converting enzyme (ACE) inhibitory peptides upon simulated in vitro gastrointestinal (GI) digestion of Korat crossbred (KC) and commercial broiler (BR) breast meat varied with cooking methods. Digestas of KC breast meat heated at 70 °C for 0.5 h (H-0.5) showed the highest ACE inhibitory activity, while digestas of KC chicken heated at 121 °C for 1 h showed the lowest inhibitory activity. ACE inhibitory activity varied with the protein digestibility. After a series of chromatographic techniques, we identified KPLLCS, ELFTT, and KPLL, novel potent ACE inhibitor peptides whose sequences were homologous to that of myosin and whose IC50 values were 0.37, 6.35 and 11.98 μM, respectively. ELFTT and KPLL were resistant to plasmin hydrolysis for up to 60 min. Therefore, KC chicken meat heated at H-0.5 could serve as a source of ACE inhibitory peptides upon GI digestion.

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.

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 (P < 0.05). Thus, the digesta of KC breasts subjected to AC-15 provides not only nutritional value but also antioxidant activity at the cellular level.

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.

Changes in chemical parameters of tropical fish used for surimi production—namely, threadfin bream (TB; Nemipterus spp.), lizardfish (LZ; Saurida spp.), and goatfish (GF; Upeneus spp.)—during ice storage and their respective washed mince were investigated. Total volatile basic nitrogen (TVB-N), trimethylamine (TMA), hypoxanthine (Hx), and total biogenic amine (BA) content of mince increased with storage time. Crude protein of fish and their respective washed mince reduced as storage time of fish was prolonged. Principal component analysis (PCA) revealed that washed mince produced from fish stored in ice for 7–12 days showed chemical characteristics that were distinct from those of washed mince prepared from spoiled fish. The TMA, TVB-N, and Hx content of washed mince appeared to be sufficient raw material freshness indicators in these three species.

Production of protein hydrolysate as nutraceuticals is typically based on the activity of the hydrolysate, which might not yield the optimal activity under physiological condition due to structural modification of peptides upon gastrointestinal (GI) digestion. This study systematically compared the chemical and cellular antioxidant activities of the in vitro digesta of tilapia protein and its hydrolysates prepared with various degree of hydrolysis (DH) by Alcalase. The enzymes used in the in vitro GI digestion analysis significantly contributed to the peptide content, Trolox equivalent antioxidant capacity (TEAC), and oxygen radical absorbance capacity (ORAC). Proteins and all hydrolysates were slightly digested by pepsin but hydrolyzed extensively by pancreatin. Both hydrolysate and digesta predominantly scavenged free radicals via hydrogen atom transfer (HAT). The antioxidant activities of the hydrolysates increased with the increasing DH up to 16 h of hydrolysis. However, the digesta of 10-h hydrolysate displayed the highest chemical and HepG2 cellular antioxidant activities, while the protein digesta displayed the lowest. Principal component analysis (PCA) showed that the TEAC of the digesta was positively correlated with the cellular antioxidant activity (CAA). Therefore, the production of protein hydrolysate should be optimized based on the activity of the hydrolysate digesta rather than that of hydrolysates.

Six-month-old (FS6) and 12-month-old (FS12) Thai fish sauces were separated by Sephadex-G25 desalting columns into 3 fractions, namely, F1 (peptides/Maillard reaction products), F2 (peptides/NaCl) and F3 (peptides/amino acids). The F3 fractions from both samples showed the highest chemical and cellular antioxidant activities and could inhibit ACE (P

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

Enterococcus faecium CN-25 was isolated from a traditionally Thai fermented fish roe, som-kai-pla, and was found to possess enterocin A and B genes. Among the 30 microorganisms tested, the produced bacteriocin exhibited antagonistic activity against Listeria monocytogenes TISTR 1327. The bacterium maximally produced bacteriocin at a concentration of 1828 AU/ml at the beginning of the stationary phase (at 18 h) in the inexpensive rice bran medium. The antilisterial activity of the compound was not affected by heating at 60–100 °C for up to 30 min. High antilisterial activity remained at pH 2. Partially purified CN-25 bacteriocin derived from E. faecium CN-25 completely inhibited the growth of L. monocytogenes at the minimum concentration of 2.38 μg/ml. The compound had no effect on the efflux of ATP from L. monocytogenes but triggered the intracellular hydrolysis of ATP, as evidenced by a decrease in the luminescence of total ATP. The bacteriocin also depleted the transmembrane electrical potential (ΔΨ) but had no effect on the transmembrane pH gradient (ΔpH). Therefore, the partially purified CN-25 peptide may be useful for improving the product safety of fermented fish products.

Angiotensin I-converting enzyme (ACE) inhibitory activity of tilapia mince (TM) hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases with a 48% degree of hydrolysis showed the highest ACE inhibitory activity with an IC 50 of 0.54 mg/mL. After ultrafiltration (UF) and chromatographic separation via anion exchange, cation exchange and size exclusion chromatography, the fraction with the highest ACE inhibitory activity with an IC 50 of 0.15 mg/mL was obtained. The peptides showed uncompetitive inhibition characteristics with an inhibition constant (K i ) of 0.18 mg/mL. The peptides showed high thermostability at 100 and 121 °C, as well as pH stability at a wide pH range of 2–10, and also maintained ACE inhibitory activity after in vitro gastrointestinal digestion. The most potent novel ACE inhibitory peptide identified was MILLLFR with an IC 50 of 0.12 µM. TM hydrolysate prepared by V. halodenitrificans SK1-3-7 proteinases could serve as a source of peptides with ACE inhibitory activity.

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 hydrolysis of milk proteins by the recombinant AprX-SK37 protease and the changes in the rheological properties of the milk gel generated with AprX-SK37 and glucono-δ-lactone (GDL) were investigated. The AprX-SK37 and rennet selectively hydrolysed κ-casein to yield a 16-kDa band, while subtilisin hydrolysed all of the casein components. Milk treated only with AprX-SK37 formed softer gel. Storage modulus (G′) values of the combined gels increased with GDL concentrations up to 7 g/L. High tan δ was observed in the combined gel at 8.75 g/L GDL alongside syneresis. AprX-SK37 is a promising milk-clotting enzyme when combined with an optimal GDL concentration.

Muscle protein is a valuable source of energy and essential amino acids, which are needed for growth and maintenance of physiological functions. It also provides peptides known as bioactive peptides exerting some health benefits, including antihypertension, antioxidant activity, immunomodulatory activity, and improving brain function, among others. Bioactive peptides can be generated from digestion or enzymatic reaction. Cooking condition is found to critically affect digestibility of chicken breast and release of bioactive peptides. Extreme thermal treatment at 121°C/15 min reduces simulated gastrointestinal digestion (GI) of chicken breast. In addition, release of peptides inhibiting angiotensin converting enzyme (ACE) that regulates blood pressure via renin–angiotensin system decreases. This implies that extreme thermal process of muscle food lower nutritional values and available ACE inhibitor peptides. Enzymatic hydrolysis is also an effective means to produce bioactive peptides, providing appropriate protease(s) is applied under the optimal condition. Hydrolysates or bioactive peptides would likely be modified through GI digestion. Thus, changes of bioactivities upon GI digestion should be taken into consideration for optimization of protein hydrolysate production. Structural changes of bioactive peptides further take place during transepithelial transport. This would definitely affect bioactivities of peptides reaching the target organ in either positive or negative manner. To further develop functional food or nutraceutical products, efficacy of bioactive peptides should be tested in vivo to assure their health benefits.

Antioxidant peptides of mungbean meal hydrolysed by Virgibacillus sp. SK37 proteinases (VH), Alcalase (AH) and Neutrase (NH) were investigated. The antioxidant activities based on 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) radical-scavenging, ferric-reducing antioxidant power (FRAP) and metal chelation of VH were comparable to those of NH. VH was purified using ultrafiltration, ion exchange and gel filtration chromatography. The purified peptides (F37) from VH, which had the highest specific antioxidant activity, consisted of four peptides containing an arginine residue at their C-termini. In addition, the ABTS radical-scavenging activity of the purified peptides (F42) at 0.148 mg/ml was comparable to that of 1 mM of butylated hydroxytoluene (BHT). These two fractions were stable over a wide pH (4–10) and temperature (25–121 °C) range. Virgibacillus sp. SK37 proteinase is a potential processing-aid for the production of a mungbean meal hydrolyzate with antioxidant properties.

Small-scale mud carp (SC, Cirrhina microlepis) and common carp (CC, Cyprinus carpio) are underutilized freshwater fish. The objective of this study was to elucidate the gel-forming ability of mince and washed mince from these species in relation to washing cycles and CaCl2 addition. Protein loss of up to 67% was observed after three-washing cycles of both species, rendering relatively low yield. About 85–90% of total fat was removed from SC flesh after three-washing cycles, but only two-washing cycles were sufficient for fat removal in CC mince. Mince and washed mince of both species did not exhibit severe proteolysis with low autolytic activity at 65°C. SC exhibited superior gel-forming ability to CC. Two- and three-washing cycles in conjunction with a 40°C-preincubation resulted in the highest textural properties of SC and CC, respectively. CaCl2 increased the breaking force of CC and SC mince gels up to 65 and 95% at 0.3 and 0.5% CaCl2, respectively, as compared to without CaCl2. However, it showed neg...

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 conditions of isolating protein from tilapia frame by-products (TFB) by a pH-shift process were investigated along with their physicochemical and gelling properties. The minimum solubility of TFB protein was observed at pH 5.5 and gradually increased at both acid and alkaline sides. The highest solubility was observed at pH 2 and 12. Protein solubility of TFB increased as the ratio of alkaline extraction medium and extraction time increased. The highest protein extraction was found at the ratio of ground sample to extraction medium of 1 to 9 and extraction time of 15 min. The maximum protein recovery was found at pH 12 with 19.19%. The color of all protein isolate samples obtained from either acid or alkaline extraction was dark brown. Whiteness of a TFB protein isolate extracted at pH 2 was the highest with a value of 14.69. An alkaline pH-shift process effectively removed fat as much as 95%. Breaking force and deformation of recovered protein gel from an alkaline pH-shift were higher than those from...

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.

Threadfin bream sarcoplasmic proteins (TBSP) were identified by 2-dimensional polyacrylamide gel electrophoresis and the major proteins were likely be pyruvate kinase, creatine kinase, aldolase, and carbonic anhydrase. Emulsifying and rheological properties of emulsion stabilized by TBSP at pH 3, 7.5, and 12 were investigated. Surface hydrophobicity of TBSP increased with pH. Emulsifying activity index was highest at pH 3 followed by pH 12 and pH 7.5. The mean droplet diameter ( d 3,2 ) was in the order of pH 7.5 > pH 3 > pH 12 at oil volume fraction ranging from 0.1 to 0.5. A decrease of oil volume fraction resulted in a reduction of oil droplet diameter. At oil volume fraction of 0.5, TBSP-stabilized emulsion at pH 7.5 and 12 exhibited gel-like characteristics, while liquid emulsion was observed at pH 3, based on visual observation and dynamic rheological analysis. Storage modulus (G′) values of emulsion at all studied pH values increased with temperature, implying the development of protein gel network upon heating. The gel-like emulsion at pH 12 absorbed more protein and water than at pH 7.5. Protein retention and water holding capacity of emulsion increased with oil volume fraction. Hydrophobic interactions between proteins and oil droplets were likely responsible for the development of a gel-like emulsion.

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.

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.

Gelation characteristics of tropical surimi, namely threadfin bream (TB), bigeye snapper (BS), goatfish (GF) and lizardfish (LF) prepared in the absence and presence of 10 g kg−1 egg white proteins were evaluated using either ohmic (OH) or water bath (WB) heating. LF and GF surimi exhibited higher endogenous proteolytic activity than BS and TB. Ohmic heating markedly minimized proteolysis of LF and GF surimi as evidenced by a reduction of trichloroacetic acid (TCA)-soluble oligopeptide content of gels and more retention of myosin heavy chain (MHC). Ohmic heating increased breaking force and deformation of TB and BS surimi by 1.3 and 1.6 times, respectively, as compared to water bath heating. However, TB surimi gels heated by a higher applied voltage gradient of 16.7 V cm−1 exhibited lower breaking force than those heated at 6.7 V cm−1. Gels heated ohmically contained lower total sulfhydryl concentration, indicating the greater extent of disulfide bond formation as compared to gels heated in a 90 °C water bath. The rapid heating method with shorter heating time could improve water holding capacity and preserve color of tropical surimi gels when compared to water bath heating.

The conformational changes and rheological properties of soluble sarcoplasmic proteins isolated from striped catfish (Pangasius hypophthalmus), treated at various pHs (2–12), were investigated. Isoelectric point of striped catfish sarcoplasmic proteins was determined to be pH 5. SDS–PAGE of sarcoplasmic proteins treated at various pHs, showed molecular masses ranging from 11 to 97 kDa. Most sarcoplasmic proteins, regardless of treated pHs, showed a molecular mass of 43 kDa. A decrease in total sulfhydryl content was observed when the pH was shifted away from 6, indicating disulfide formation at pH lower and higher than 6. Gradual increases of S0-ANS and S0-PRODAN were observed as pH increased from 6 to 12, indicating the unfolding of sarcoplasmic proteins during alkaline extraction. DSC thermograms of sarcoplasmic proteins treated at pH 5–9 exhibited an exothermic transition peak, probably due to disulfide bond formation, and/or hydrophobic interactions, which was highly related to the onset temperature of G′ rising. Gel network formation of sarcoplasmic proteins did not take place at extreme pHs ( 9) where proteins were highly charged while the viscoelastic properties of sarcoplasmic proteins were observed at pH 5.5–9. The highest G′ value at 90 °C was observed at pH 5.5 and 8 (P ⩽ 0.05). The gel point, a temperature at which G′ = G″, increased to higher temperature as pH was shifted away from 7.

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 Ca 2+ , Mg 2+ and Sr 2+ 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.

BACKGROUND: 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. RESULTS: 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 °C and completely disappeared at 60 °C. The breaking force and deformation of lizardfish surimi gel with added TBSP and pre-incubated at 37° for 20 min increased with additional levels of TBSP (P < 0.05). Trichloroacetic acid–oligopeptide content of lizardfish surimi gel with added TBSP decreased with the addition of 4 g kg−1 TBSP (P < 0.05). Retention of myosin heavy chain (MHC) increased when TBSP concentration was increased. TBSP effectively protected MHC from proteolysis at 37 °C to a similar extent as egg white powder, but efficacy of TBSP was not observed at 65 °C. CONCLUSION: 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 °C. Copyright © 2009 Society of Chemical Industry

The cross-linking of β-lactoglobulin (BLG) was efficiently catalysed by microbial transglutaminase (MTG) but not by fish (threadfin bream) liver transglutaminase (FTG). BLG cross-linking was inhibited by 2 mM 5-(biotinamido) pentylamine (BPNH2) and MTG incorporated BPNH2 into BLG ∼5 times more than was FTG. The glutaminyl sites for the incorporation of BPNH2 into BLG by FTG and MTG were identified using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS analyses showed that MTG and FTG incorporated 4 and 1 residues of BPNH2 per molecule of BLG, respectively. The BPNH2-tagged BLG was digested by trypsin and BPNH2-tagged peptides were selectively purified by avidin-affinity chromatography. Amino acid sequences of BPNH2-tagged peptides were identified by comparing their MALDI-TOF mass spectra with the theoretical mass profiles from the MASCOT database. The BPNH2-modification sites catalysed by MTG were glutamine (Q)13, Q68, Q15 or Q20, Q155 or Q159, whilst FTG only incorporated BPNH2 into BLG at Q68. The different reactivities between FTG and MTG might be due to the different accessibilities of these TGases to the Q residues as well as to differences in substrate specificities.