Production and characterization of chicken blood hydrolysate with antihypertensive properties.

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.

Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37.

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) CaCl(2) . Enzymes showed stability at 0-30% NaCl and <20 mmol l(-1) CaCl(2) and were completely inhibited by phenylmethanesulphonyl fluoride but not by ethylenediaminetetraacetic acid. The isoelectric points of 19-, 34- and 44-kDa proteinases were at 3·6, 5·2 and 3·8, respectively, based on 2D electrophoresis. Peptide mass fingerprint and de novo peptide homology analysis of tryptic peptides using MALDI-TOF and LC-MS/MS, respectively, suggested that all three enzymes were novel and homologous to bacillopeptidase F. CONCLUSIONS: The three major proteinases are a member of bacillopeptidase F-like enzymes exhibiting thermophilic and halotolerant characteristics with high stability at 30% NaCl. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on bacillopeptidase F-like proteinases in genus Virgibacillus with a distinct halotolerant feature. They showed potential to be a processing aid for food and biotechnological applications, particularly in high salt condition.

Comparative study of washing treatments and alkali extraction on gelation characteristics of striped catfish (Pangasius hypophthalmus) muscle protein.

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 degrees 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.

Biochemical and conformational changes of myosin purified from Pacific sardine at various pHs.

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

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

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.

Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures.

A means to accelerate fish sauce fermentation without adversely affecting fish sauce quality was investigated. Starter cultures prepared from Virgibacillus sp. SK33, Virgibacillus sp. SK37, and Staphylococcus sp. SK1-1-5 were added separately to anchovy that was hydrolyzed by 0.25% Alcalase at 60 degrees C for 2 h followed by 0.5% Flavourzyme at 50 degrees C for 4 h. The mixtures were then adjusted to contain 25% solar salt and incubated at 35 degrees C for 4 mo. alpha-Amino contents of all inoculated samples were higher than the control (without the addition of starter culture) during the course of fermentation. After 4-mo fermentation, the samples inoculated with Staphylococcus sp. SK1-1-5 contained the highest alpha-amino content of 733.37 +/- 13.89 mM while that of the control was 682.67 +/- 3.33 mM. Amino acid profiles of inoculated samples showed similar patterns to that of commercial product fermented for 12 mo, with glutamic, aspartic, and lysine being predominant amino acids. Virgibacillus sp. SK33 appeared to decrease histamine content of fish sauce by 50% when compared to the control. Volatile compounds analyzed by GC-MS of all inoculated samples fermented for 4 mo exhibited a similar pattern to those of the 12-mo-old commercial product. Samples inoculated with Staphylococcus sp. SK1-1-5 produced higher levels of volatile fatty acids and showed similar sensory characteristics to the commercial fish sauce fermented for 12 mo. Staphylococcus sp. SK1-1-5 is a potential strain that can be applied to produce fish sauce with overall sensory characteristics of traditional fish sauce in shorter time.

Proteolysis and gelation of fish proteins under ohmic heating.

Pacific whiting surimi gels heated slowly in a water bath exhibited poor gel quality, while the ohmically heated gels without holding at 55 degrees C showed more than a twofold increase in shear stress and shear strain over conventionally heated gels. Degradation of myosin and actin was minimized by ohmic heating, resulting in a continuous network structure. Ohmic heating with a rapid heating rate was an effective method for maximizing gel functionality of Pacific whiting surimi without enzyme inhibitors. In linear heating, slow heating rates increased proteolysis in Pacific whiting surimi as shown by degradation of myosin heavy chain and low shear stress and shear strain. Proteolysis of whiting surimi was decreased by the presence of beef plasma protein (BPP) to a greater extent at rapid heating rates (20 and 30 degrees C/min) than at slow heating rates (1 and 5 degrees C/min). Shear stress of Alaska pollock surimi gels with or without BPP increased as heating rate decreased, but shear strain was unaffected. An increase in shear stress was accompanied by an increase of cross-linked myosin heavy chain.