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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 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...

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.

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.

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.

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

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.

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.

Virgibacillus sp. SK1-3-7 exhibited the highest fibrinolytic activity among 25 bacterial isolates obtained from fish sauce fermentation. Results of 16S rRNA gene sequence analysis showed 99% homology to Virgibacillus halodenitrificans ATCC 49067. It was, therefore, identified as V. halodenitrificans SK1-3-7. Fibrinolytic enzymes from V. halodenitrificans SK1-3-7 were partially purified using ammonium sulfate fractionation, hydrophobic and ion-exchange chromatographies. The enzymes with molecular weight of 20- and 36-kDa showed fibrinolytic activity on a fibrin zymogram. The enzymes were stable between pH 4 and 10 and below 60 °C. The enzymes were activated by 20 mM CaCl 2 and 0.15 M NaCl. The activity increased with CaCl 2 up to 100 mM and increased with NaCl concentration up to 2 M. In addition, the residual fibrinolytic activity of 61% was found at 4 M NaCl. The enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA, suggesting a subtilisin-like serine proteinase. V. halodenitrificans SK1-3-7 enzymes hydrolyzed fibrin to a greater extent than did plasmin. In addition, the enzymes were resistant to pepsin and trypsin digestion. The de novo peptide homology analysis of a 20- and 36-kDa proteinase revealed no matches to bacilli serine proteinases, suggesting that both were novel fibrinolytic enzymes.

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.

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.

Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated.

Aims: Virgibacillus sp. SK37 isolated from Thai fish sauce produced numerous NaCl-activated subtilisin-like proteinases. Our objectives were to purify, characterize and identify these extracellular proteinases. Methods and Results: Three major subtilisin-like enzymes including 19, 34 and 44 kDa were partially purified and showed maximum activity at pH 8, 55– 60� C, 25–30% NaCl and 70–100 mmol l )1 CaCl2. Enzymes showed stability at 0–30% NaCl and

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.

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.

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.

Cell-bound proteinase from Virgibacillus sp. SK37 isolated from the first month of fish sauce fermentation was characterized. The enzyme showed the maximum activity at 65 °C, pH 7.0 and 9.5, using azocasein as a substrate. The enzyme required at least 10 mmol/l Ca 2+ to effectively hydrolyze casein and the extent of casein degradation increased with Ca 2+ concentration. Ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF) largely inhibited the activity, indicating a characteristic of Ca 2+ -activated serine proteinase. Among six synthetic substrates tested, the enzyme preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, indicating a subtilisin-like proteinase. Although activity towards actomyosin at 20 g/100 ml NaCl decreased to 63% compared to at 5 g/100 ml, the enzyme showed high stability at 25 g/100 ml NaCl, 30 °C. This was the first study to report biochemical characteristics of cell-bound proteinase from a moderately halophilic bacterium isolated from fish sauce.

Transglutaminase (TGase) from the threadfin bream ([TB]Nemipterus sp.) liver was partially purified using ion exchange, size exclusion and affinity chromatography. Three protein bands with molecular weight (Mw) of 95, 63 and 43 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were observed. Only one distinct fluorescent band appeared on the TGase activity staining of the native-PAGE. When the protein band was eluted and analyzed on the SDS-PAGE, it showed a single band with an Mw of 95 kDa. The enzyme required Ca2+up to 1 mM for full activation. TGase was also activated by 10 mM Sr2+. Dithiothreitol had no effect on activity. TGase activity was unaffected by NaCl up to 0.6 M and reduced to 75% at 1.2 M NaCl. Optimum pH and temperature was 8.5–9.0 and 50C, respectively. TGase activity was markedly inhibited by the sulfhydryl reagents. The enzyme catalyzed the cross-linking of the TB myosin heavy chains. PRACTICAL APPLICATIONS Threadfin bream (TB) liver transglutaminase (TGase) exhibited Ca2+-dependent characteristic similar to muscle TGase. The enzyme catalyzed the cross-linking of myosin, and therefore, could be applied to improve the textural properties of muscle proteins. The enzyme remained its high activity up to 1.2 M NaCl (≈7% w/v), indicating its potential application in foods containing a relatively high NaCl. The biochemical characteristics of the TB liver TGase are critical information leading to more understanding in the nature of the enzyme as well as determining the optimum conditions for food applications. Moreover, the presented purification scheme could be adopted to recover TGase from the TB liver, which is currently a solid waste of the surimi industry.

Oxidative stability of emulsion sausages prepared from African walking catfish (AF: Clarias gariepinus) and rohu (RH: Labeo rohita) fortified with three levels of the refined tuna oil (2%, 6%, and 10%) with 150 μg KI/100 g sample and without KI was investigated. The control was prepared using 20% soybean oil and without KI. Samples were vacuum-packed and stored at 4 °C. Iodine content decreased approximately 14% after cooking and remained constant throughout storage. Sausages fortified with tuna oil showed higher level of n-3 (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), but lower level of n-6 fatty acids (P

Virgibacillus sp. SK33 newly isolated from one-month-old Thai fish sauce was studied for its proteinase production. Neopeptone broth (NEO), halobacterium broth (HL) and HL without either yeast extract (HL-Y), peptone (HL-P) or casamino acid (HL-C) were found to be suitable for proteinase production, whereas fish broth (FB) and skim milk salts broth (SKS) appeared to suppress proteinase production. Moreover, yeast extract, peptone and casamino acid equally stimulated proteinase production. The optimal enzyme production for Virgibacillus sp. SK33 was at 5% NaCl, 40 °C. Maximum proteinase production was achieved at 36 h and maximum cell growth was obtained at 72 h in the modified HL supplemented with only yeast extract (Ym). Extracellular proteinase of Virgibacillus sp. SK33 exhibited optimum activity at 50 °C and pH 8, 10 and 11. Crude proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating the presence of serine alkaline proteinase. The enzymes preferentially cleaved Suc-Ala-Ala-Pro-Phe-AMC, a substrate for subtilisin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) activity staining showed molecular weights of 56, 46, 42, 32, 25 and 19 kDa. All proteinases exhibited caseinolytic activity at 25% NaCl. Proteolytic activity towards synthetic substrate increased about 4 times in the presence of 25% NaCl, indicating the characteristic of NaCl-activated proteinase. In addition, crude proteinase from Virgibacillus sp. SK33 showed higher proteolytic activity towards anchovy than did commercial proteinases at 25% NaCl, demonstrating its potential for protein hydrolysis at high salt content.

Maximum autolytic activity of Indian anchovy ( Stolephorus indicus ) was found at 60 °C. Autolytic activity decreased with increased NaCl concentration. Remaining autolytic activity at 25% NaCl (w/w) was 52%. Crude proteinase extracts exhibited the highest activity at 60 °C, using either casein or acid-denatured hemoglobin (dHb) as a substrate. Optimal pH of crude extracts was found at 8.5 for casein and 9.5 for dHb. Activity of crude extract decreased >50% when NaCl concentration was greater than 0.1 M. Crude extract was stable for up to 8 h at 4, 30, and 60 °C. Crude proteinase hydrolyzed several synthetic substrates of trypsin, including Boc-Asp(oBzl)-Pro-Arg-MCA, Boc-Val-Leu-Lys-MCA, and Boc-Gln-Ala-Arg-MCA. Soybean trypsin inhibitor (SBTI), leupeptin, phenylmethanesulfonyl fluoride (PMSF), and N -tosyl- l -lysine chloromethyl ketone (TLCK) inhibited activities of proteinase, indicating trypsin-like characteristics. Molecular weight of proteinases exhibiting caseinolytic activity at 4.0 M NaCl were estimated to be 63, 53, 46, 40, 35, and 31 kDa, using electrophoresis activity staining.

The protein solubility and molecular-weight distribution of freeze-driedsarcoplasmic proteins (SPs) from rockfish treated under low and high pH aswell as various NaCl concentrations were elucidated. The solubility of SPswas significantly suppressed at an acidic pH (2.0–4.0) and in the presence ofhigh salt concentration (0.5 M NaCl). The least amount of protein was lostwhen SPs were treated at pH 2.0 or 3.0 followed by precipitation at pH 5.5.The interaction of SPs with Alaska pollock surimi (myofibrillar proteins) wasalso investigated. The addition of SPs appeared to delay the thermal denatur-ation of myosin and actin. The SPs positively contributed to the gelation ofmyofibrillar proteins as judged by breaking force. INTRODUCTION A new method for preparing fish protein isolate using pH shift hassignificant advantages over conventional surimi processing. This revolutionaryapproach can reduce water usage and subsequently minimize wastewater prob-lems. The new method can also provide extremely high yields (35–40%)because it solubilizes nearly all myofibrillar and sarcoplasmic proteins (SPs)through homogenization and pH adjustment before centrifugal recovery. Incontrast, the conventional process removes SPs through washing and dewater-ing, which results in significant protein loss and excessive water usage, andcontributes to wastewater problems (Hultin and Kelleher 1999; Hultin

Optimum autolytic activities of lizardfish ( Saurida tumbil ) mince and surimi were at pH 6 and 7, respectively, with optimum temperature at 65 °C. Autolysis of surimi was mainly inhibited by phenylmethanesulfonyl fluoride and p -tosyl- l -phenylalanyl-chloromethylketone, indicating the involvement of myofibrillar-associated serine proteinase. Myosin heavy chain (MHC) and tropomyosin were preferentially hydrolyzed, resulting in poor textural properties. Based on TCA-soluble oligopeptide assay, egg white powder (EW) and whey protein concentrate (WPC) showed 77% and 96% inhibition, respectively. However, a significant loss of MHC was found. At any pre-incubation condition (25 °C/4 h, 40 °C/1 h and 65 °C/1 h), EW improved gel-forming ability of lizardfish surimi to a greater extent than WPC. Addition of 1% EW and pre-incubation at 25 °C resulted in an increase of higher molecular weight cross-linked proteins, corresponding to a twofold increase in the breaking force.

The formation of biogenic amines in fish sauce made from fresh and temperature-abused (left at 35°C for 8 and 16 h) Indian anchovy (Stolephorus indicus) was investigated. Histamine, cadaverine, putrescine, and tyramine were the predominant biogenic amines found in anchovy left at 35°C for 16 h and its fish sauce product. Changes of biogenic amines were subtle during the course of fermentation at room temperature (RT) and at 40°C, suggesting that the main source of biogenic amines was associated with raw material, rather than with the fermentation process. Soluble peptide and total nitrogen of fish sauce prepared from temperature-abused anchovy were higher at the initial stage of fermentation at RT and 40°C and became comparable to those prepared from fresh anchovy at the end of fermentation. Total free amino acid contents of samples fermented at RT for 52 wk (7208.3 to 8473.6 mg/100 mL) were higher than those fermented at 40°C for 13 wk (4560.9 to 5730.9 mg/100 mL). Fish sauce prepared from temperature-abused anchovy contained less free histidine and arginine. Fish sauce of a good quality was obtained using fresh anchovy fermented at RT. Besides total nitrogen content, biogenic amines should be considered as quality indicators of fish sauce.

Transglutaminase(TGase) activity of threadfin bream mince was 99.6 units/g of dry weight. After washing and screw-pressed dewatering, 44% residual activity was retained. Covalent cross-linking of myosin heavy chain (MHC) was observed at both 25 and 40°C and supported by increased gel strength. When pre-incubation at 40°C was prolonged to 4 h, breaking force and MHC decreased due to endogenous proteinase(s). TGase activity towards MHC and synthetic substrates was effectively inhibited by iodoacetic acid (IAA). Autolytic activity and degradation of MHC was inhibited by phenylmethanesulfonyl fluoride (PMSF). Addition of 0.2% Ca(2+) significantly improved breaking force and increased MHC cross-linking of surimi gels pre-incubated at 40°C for 2 h. Keywords: transglutaminase, myosin heavy chain, cross-linking, threadfin bream.

The NaCl-activated and detergent-stable proteinases from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation were purified and characterized. The enzymes with molecular masses of 20 and 36 kDa showed caseinolytic activity on a zymogram. Optimum azocaseinolytic activity was at 60 °C and pH 9. The proteolytic activity increased in the presence of 10 mM CaCl2 and 0.5 M NaCl and showed high stability at 0–2 M NaCl. The enzymes were stable at pH 4–10 and 10–50 °C. The enzymes preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA and were completely inhibited by phenylmethanesulfonyl fluoride (PMSF), showing subtilisin-like characteristics. Activity and stability remained high in the presence of H2O2 and various surfactants. The enzymes exhibited high stability (>95 %) in various organic solvents (DMSO, butanol, ethanol, 2-propanol, and acetonitrile) at concentration of 50 %. The V. halodenitrificans SK1-3–7 proteinases showed potential as a biocatalyst in aqueous-organic solvent systems and as an additive in laundry detergent.

Proteolytic degradation of tropical tilapia surimi was biochemically and rheologically characterized to identify a group of proteinase(s) responsible for its textural degradation. Proteolysis of tilapia surimi occurred as the temperature increased and attained the highest activity at 65 °C. Smaller proteins with molecular weight of 116-97 kDa were noted as a result of myosin heavy chain (MHC) degradation. MHC completely disappeared when incu- bated at 65 °C for 4 h. Proteolysis was significantly inhibited by soybean trypsin inhibitor (SB) and leupeptin (LE). Storage modulus (G) of surimi gels mixed with either SB or LE was higher than other inhibitors indicating that serine type proteinase(s) were involved in proteolysis of tropical tilapia surimi.

Gelation behavior and potential cross-linking of Pacific whiting (Merluccius productus) surimi were affected by setting temperatures and an enzyme inhibitor. Gels of Pacific whiting surimi with salt and beef plasma protein were compared with those containing guanidine hydrochloride, sodium dodecyl sulfate, and β-mercaptoethanol. The strongest gels were formed at 25 o C setting followed by 90 o C heating. Hydrogen and hydrophobic bonds appeared to strongly influence gel formation, while the influence of disulfide bonds was moderate. Viscosity scanning during setting at different temperatures was also useful to estimate effects of enzymes and inhibitors

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

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.

Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.

Background Microbial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications. Results A gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX). Conclusions The stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.