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

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

Effect of marinating ingredients on temperature-induced denaturation of hemoglobin and its relation to red blood spot formation in cooked chicken breast

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 (T_d) 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 T_d 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.     

Effect of microbial transglutaminase on autolysis and gelation of lizardfish surimi

In the absence of microbial transglutaminase (MTGase), the textural properties of lizardfish surimi (Saurida spp) improved when pre‐incubated at 4 and 25 °C for 24 and 4 h, respectively. MTGase optimally catalyzed incorporation of monodansylcadaverine (MDC) into surimi at 40 °C. Addition of MTGase appeared to reduce autolytic activity at 25 and 40 °C, but had no effect on autolytic activity at 65 °C. Breaking force and deformation of lizardfish surimi significantly improved when 0.1 unit MTGase g^?1 surimi (1.8 g kg^?1) was added and pre‐incubated at either 25 or 40 °C. Textural properties improved concomitant with cross‐linked polymers of myosin heavy chain and tropomyosin, but not actin. Addition of MTGase also improved the storage modulus (G′). The gel network of surimi mixed with MTGase and pre‐incubated at 40 °C readily formed during the pre‐incubation period, while formation of the gel network began at 48.1 °C in the absence of MTGase. Copyright © 2005 Society of Chemical Industry     

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

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

In vitro comparative hemostatic studies of chitin,chitosan, and their derivatives

The effects of chitin, chitosan, and their derivatives on in vitro human blood coagulation and platelet activation were comparatively studied. The coagulation was assessed by the measure of the whole blood clotting time (WHBCT) and plasma recalcification time (PRT). The tested materials were chitin, chitosan, partially N‐acetylated chitosan (PNAC), N,O‐carboxymethylchitosan (NOCC), N‐sulfated chitosan, N‐(2‐hydroxy)propyl‐3‐trimethylammonium chitosan chloride, and SPONGOSTAN® standard (a positive control). The results revealed that the WHBCTs of whole blood mixed with chitin, chitosan, NOCC, or SPONGOSTAN® standard were significantly decreased with respect to that of the pure whole blood (a blank control) (P < 0.05), while the WHBCT value of whole blood mixed with PNAC was not significantly reduced. However, the presence of PNAC significantly lowered the PRT value, similar to the addition of chitin, NOCC, or SPONGOSTAN® standard. Chitosan was found to reduce PRT, but not significantly. In the platelet adhesion and activation studies, the morphology of platelets adherent to the film surfaces of tested materials was examined using a scanning electron microscopic technique. Because of their effective coagulation activites, chitosan, PNAC, and NOCC were further evaluated to determine how platelets behaved when in contact with these film samples for given periods. It was found that NOCC activated platelets most effectively. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 445–451, 2006     

Antioxidant activity of protein hydrolysates derived from threadfin bream surimi byproducts

Antioxidant activities of protein hydrolysates from threadfin bream surimi wastes, including frame, bone and skin (FBS) and refiner discharge (RD), were investigated. FBS and RD were rich in Lys, Glu, Gly, Pro, Asp, Leu, His, Tyr and Phe. FBS was hydrolysed to a greater extent than RD regardless of proteinases tested (Virgibacillus sp. SK33 proteinase, Alcalase, pepsin and trypsin). Pepsin-hydrolysed FBS, at a 5% degree of hydrolysis (DH), showed the highest antioxidant activity based on 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) radical (0.455 ± 0.054 mg Trolox equivalents/mg leucine equivalents), ferric reducing antioxidant power (FRAP) (0.221 ± 0.005 mM Trolox equivalents) and inhibition of β-carotene bleaching assays. FBS hydrolysates showed higher antioxidant activity based on chemical assays than their RD counterparts. However, FBS and RD hydrolysates protected HepG2 cells against tert-butyl hydroperoxide-induced oxidative damage to a similar extent. Therefore, FBS and RD hydrolysates have a potential as antioxidative neutraceutical ingredients.     

pH-dependent characteristics of gel-like emulsion stabilized by threadfin bream sarcoplasmic proteins

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.     

Comparative Study of Washing Treatments and Alkali Extraction on Gelation Characteristics of Striped Catfish (Pangasius hypophthalmus) Muscle Protein

ABSTRACT:  The effect of washing treatments (water and alkali washing) and alkali extraction/acid precipitation on the physicochemical and textural properties of striped catfish ( Pangasius hypophthalmus ) muscle protein was compared. Alkali extraction/acid precipitation process resulted in the highest protein recovery of 98.77% (dry weight basis) and lowest fat content of 0.98% (dry weight basis). The extractable proteins of fish protein isolates (FPI) at various ionic strengths (0 to 0.6 M NaCl) exhibited majority of protein at 38 kDa with trace amount of others. Unsaturated fatty acids, namely, oleic, linoleic, and docosahexaenoic acid, were greatly reduced in FPI. Water-washed mince (W) exhibited higher autolytic activity than mince (M), suggesting the presence of myofibril-bound proteinase(s). Autolytic activity was lowest in the FPI. Breaking force of striped catfish gels was greatly improved when set at 40 °C for 30 min, regardless of preparation treatment. The typical water washing process was effective to improve gel-forming ability of striped catfish. FPI gels contained the lower total sulfhydryl content, indicating the greater extent of disulfide bond formation as compared to that of washed mince. The FPI gels showed higher breaking force but lower deformation values than W and alkali-washed (AW) gels. Addition of NaCl improved deformation, but adversely affected breaking force of FPI gel. Whiteness of FPI gel increased with NaCl addition.     

Structural Changes and Functional Properties of Threadfin Bream Sarcoplasmic Proteins Subjected to pH-Shifting Treatments and Lyophilization

ABSTRACT: 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 α-helical and β-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.