Antioxidant properties of peptide fractions from silver carp (Hypophthalmichthys molitrix) processing by-product protein hydrolysates evaluated by electron spin resonance spectrometry

Silver carp processing by-product protein is usually discarded as an industrial solid waste. In this study the protein was recovered using a pH-shift method, after which seven commercial proteases were separately employed to prepare antioxidative hydrolysates. Among the hydrolysates, pepsin hydrolysates, which had the highest free radical-scavenging activity, were further separated into five peptide fractions, SCPH-I (>10 kDa), SCPH-II (5–10 kDa), SCPH-III (3–5 kDa), SCPH-IV (1–3 kDa), and SCPH-V (<1 kDa), by using ultrafiltration. The antioxidative properties of the peptide fractions were investigated, using a free radical-scavenging assay, by electron spin resonance. The results show that SCPH-V had the highest scavenging effects on DPPH (1,1-diphenyl-2-picrylhydrazyl), hydroxyl and superoxide anion radicals. SCPH-V had potent antioxidant activity in the prevention of the peroxidation of linoleic acid and alleviation of H₂O₂-induced oxidative stress in human intestinal epithelial caco-2 cells. The results indicated that the antioxidant capacity of silver carp by-product hydrolysates could be enhanced by ultrafiltration.     

Acid-induced aggregation of actomyosin from silver carp (Hypophthalmichthys molitrix)

Acid-induced denaturation and aggregation properties of silver carp actomyosin added with d-gluconic acid-δ-lactone (GDL) during incubation at chilled temperature (4 °C) were studied. Actomyosin underwent aggregation with decreasing pH, as evidenced by increased turbidity and decreased protein solubility in 0.6 M NaCl solution. Ca²⁺-ATPase activity decreased continually with increasing incubation time in the presence of GDL, accompanied by an initial increase in surface reactive sulphydryl (SH) content, indicating the conformation changes of actomyosin during acidification. Protein solubility in selected solvents and electrophoresis analysis showed that the major myofibrillar proteins, particularly, myosin heavy chain and actin were involved in the formation of protein aggregates mainly through noncovalent bonds including hydrophobic interactions and hydrogen bonds during acidification. The slight decrease in total SH content during acidification suggested that disulphide bonds were also involved in acid-induced aggregation of silver carp actomyosin to a lesser extent.     

Compositions,functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi)

Composition, functional properties and antioxidative activity of a protein hydrolysate prepared from defatted round scad (Decapterus maruadsi) mince, using Flavourzyme, with a degree of hydrolysis (DH) of 60%, were determined. The protein hydrolysate had a high protein content (48.0%) and a high ash content (24.56%). It was brownish yellow in colour (L^∗ = 58.00, a^∗ = 8.38, b^∗ = 28.32). The protein hydrolysate contained a high amount of essential amino acids (48.04%) and had arginine and lysine as the dominant amino acids. Na⁺ was the predominant mineral in the hydrolysate. The protein hydrolysate had an excellent solubility (99%) and possessed interfacial properties, which were governed by their concentrations. The emulsifying activity index of the protein hydrolysate decreased with increasing concentration (p < 0.05). Conversely, the foaming abilities increased as the hydrolysate concentrations increased (p < 0.05). During storage at 25 °C and 4 °C for 6 weeks, the antioxidative activities and the solubility of round scad protein hydrolysate slightly decreased (p < 0.05). Yellowness (b^∗-value) of the protein hydrolysate became more intense as the storage time increased but the rate of increase was more pronounced at 25 °C than at 4 °C.     

Characterisation of acid-soluble collagen from skin of silver carp (Hypophthalmichthys molitrix)

Acid-soluble collagen (ASC) from the skin of silver carp (Hypophthalmichthys molitrix) was isolated and some properties of ASC were investigated. SDS–PAGE patterns showed ASC from silver carp skin was type ? collagen. Sulfopropyl-Toyopearl 650(M) column chromatography indicated that ASC from silver carp skin was composed of three kinds of α chains, α₁, α₂ and α₃. Hydroxyproline and proline content of ASC from silver carp skin was 192 residues/1000 residues, which was similar to that of ASC from carp skin. Denaturation temperature (T_d) of ASC from silver carp skin was around 29 °C. The results showed that some properties of ASC from silver carp skin were similar to those of ASC from carp skin. However, the peptide map of ASC from silver carp skin digested by pepsin was distinguished with that of ASC from carp skin.     

Seasonal differences in the properties of gelatins extracted from skin of silver carp (Hypophthalmichthys molitrix)

The gelatins were extracted from skins of silver carp (Hypophthalmichthys molitrix) caught in winter and summer, respectively. The physicochemical (molecular weight distribution and melting point) and rheological characteristics (viscosity property), as well as functional properties (emulsifying capacity and stability) of the gelatin from winter silver carp skin were compared with those of the summer equivalent. The results showed the properties of the summer gelatin were superior to those of the winter one, showing higher viscosity, emulsion stability, melting point and lower concentration for gelling. The summer gelatin has slightly denser strands of the gel microstructure which was observed by scanning electron microscopy (SEM). Different properties of gelatins from skin of silver carp may be attributed to the big discrepancy of the environmental temperature in the two seasons.     

Effect of microwave heating on the low-salt gel from silver carp (Hypophthalmichthys molitrix) surimi

Gels from silver carp (Hypophthalmichthys molitrix) surimi were obtained using microwave (MW) heating (15 W/g power intensity for 20–80 s) at different levels of salt (0 g/100 g, 1 g/100 g, or 2 g/100 g). And the gel heated by MW was compared with the gel obtained by conventional water-bath heating (85 °C for 30 min). The gel strength increased when the salt level was increased. The mechanical and functional properties of non-salted, low-salt and regular-salt products were improved by MW heating for 60 s and 80 s, significantly (p < 0.05), except for the cook loss. The content of TCA-soluble peptides indicated that the MW heating inhibited the autolysis of proteins significantly (p < 0.05) during gelling. The SDS-PAGE and total content of –SH group proved that MW enhanced the cross-linking of proteins effectively through disulphide bonds and non-disulphide covalent bonds. The microstructure of the samples revealed that a fine compact network, with particles of protein aggregates, was formed in the low-salt gels (1 g/100 g) heated by MW for 60 s. All of these properties might be responsible for the formation of a superior textural low-salt gel induced by MW.     

银鲳酶解物抗氧化活性研究

选用胃蛋白酶、胰蛋白酶、碱性蛋白酶和中性蛋白酶对银鲳蛋白进行酶解以制备蛋白酶解物,以羟基自由基清除活性为指标确定银鲳最佳水解酶。结果显示,碱性蛋白酶的水解物抗氧化活性最强。实验对碱性蛋白酶水解银鲳的酶解条件(时间、温度、pH、酶添加量和固液比)进行正交实验设计,并对最佳水解条件下所获得的酶解物进行抗氧化活性测试。结果表明,银鲳蛋白碱性蛋白酶水解物对DPPH自由基和羟基自由基具有清除作用,其自由基清除效果呈现剂量依赖性,而且银鲳蛋白水解物还具有明显还原能力。所有这些体外抗氧化数据说明,银鲳蛋白水解物有明显的抗氧化效力。     

Gel properties of surimi from silver carp (Hypophthalmichthys molitrix) as affected by heat treatment and soy protein isolate

The effects of setting conditions and soy protein isolate (SPI) on textural properties of surimi produced from silver carp were investigated. Effects of setting temperature, setting time and protein concentration on the gel strength were evaluated and compared utilizing response surface methodology. Models for breaking force and breaking distance of silver carp surimi were established. The total protein content was 13.4% in all experimental samples. Setting temperature and protein concentration were the major factors affecting the gel strength. In the range of the additive SPI protein (10–40%), breaking force and distance of silver carp surimi gels decreased when the protein ratio of SPI was increased in the total protein at 30 and 40 °C for 60 min setting and heating at 85 °C for 30 min, but the breaking force obtained for 90% surimi protein plus 10% SPI protein was higher than surimi alone at 50 °C for 60 min incubation and heating at 85 °C for 30 min.     

Antioxidant activities and functional properties of grass carp (Ctenopharyngodon idellus) protein hydrolysates

BACKGROUND: Grass carp, with an annual production exceeding 4 × 10⁶ t in China in 2009, has not been developed into a high‐value product. In this study the antioxidant activities and functional properties of grass carp protein hydrolysates prepared with Alcalase 2.4L (HA) and papain (HP) were investigated. The hydrolysate with strongest radical‐scavenging activity and reducing power was assessed further for changes in its antioxidant activity during simulated gastrointestinal digestion. RESULTS: As the degree of hydrolysis (DH) increased, the metal‐chelating activity of both HA and HP increased while their reducing power and 1,1‐diphenyl‐2‐picrylhydrazyl radical (DPPH^?)‐scavenging activity decreased (P < 0.05). At the same DH, HP possessed higher DPPH^?‐scavenging activity and reducing power than HA (P < 0.05). The metal‐chelating activity of HP with 10% DH was significantly increased after in vitro gastrointestinal metabolism (P < 0.05). Regarding their functional properties, all hydrolysates were more than 81% soluble over a wide range of pH (3–8). At the same DH, HP showed higher emulsion activity index but lower solubility and foaming capacity than HA. CONCLUSION: Grass carp protein hydrolysates showed high solubility over a wide pH range and could be used as natural antioxidants in food systems. Copyright © 2011 Society of Chemical Industry     

Isolation of cathepsin B from the muscle of silver carp (Hypophthalmichthys molitrix) and comparison of cathepsins B and L actions on surimi gel softening

Cathepsin B from silver carp muscle was purified to 263-fold by acid treatment, ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 29 kDa as determined by SDS-PAGE and immunoblotting. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64, suggesting the purified enzyme belongs to the cysteine proteinase family. Optimal pH and temperature were 5.5 and 35 °C, respectively. The enzyme catalyzed the hydrolysis of Z-Arg-Arg-MCA with a parameter of K_m (90 μM) and K_cat (20.3 s⁻¹), but hardly hydrolyzed Arg-MCA. Analysis of surimi gel strength and microstructure showed that cathepsins B and L were capable of destroying the network structure of silver carp surimi gels, consequently causing gel softening. Cathepsin L might play an important role in the modori effect.     

Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates

Porcine haemoglobin hydrolysates were prepared through hydrolysis by Alcalase followed by Flavourzyme, and their protein compositions were analyzed using Sephadex G-50 gel filtration chromatography. The antioxidant activities, including reducing power, ferrous ion chelating ability, and DPPH radical scavenging activity, of the hydrolysates were evaluated. The results showed that the hydrolysates of haemoglobin exhibited low reducing powers, but high ferrous ion chelating abilities and DPPH radical scavenging activities. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 h and followed by 1% Flavourzyme for 6 h, had the highest ferrous ion chelating ability of 63.54% at a concentration of 5.0 mg/mL. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 hrs, had the highest DPPH radical scavenging activity of 41.94% at a concentration of 5.0 mg/mL. According to the results of protein composition analysis, we divided the hydrolysates into three groups, including high molecular weight (MW) group (Group I), medium MW group (Group II), and low MW group (Group III). The reducing power and ferrous ion chelating ability of the hydrolysates were significantly and positively correlated to the relative amount of Group I, and negatively correlated to the relative amount of Group III. This study revealed that the antioxidant activities of porcine haemoglobin hydrolysates were dependent on their protein compositions. The high MW protein fraction (Group I) was responsible for the high reducing power and ferrous ion chelating ability of the hydrolysate.     

Autolysis-assisted production of fish protein hydrolysates with antioxidant properties from Pacific hake (Merluccius productus)

Fish protein hydrolysates (FPH) with antioxidative properties were prepared using Pacific hake fish with high endogenous proteolytic activity from Kudoa paniformis parasitic infection. Infection level of ∼10⁷K. paniformis spores/g fish mince or higher yielded FPH with high antioxidant potential by autolysis and/or Validase^® BNP or Flavourzyme^® 500L. Autolyzing fish mince containing 30 × 10⁶ spores/g for 1 h at 52 °C and pH 5.50 produced FPH (named E-1h) with Trolox equivalent antioxidant capacity (TEAC) in the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical assay of 262 ± 2 μmol/g and oxygen radical absorbing capacity (ORAC) of 225 ± 17 μmol Trolox equivalents/g freeze-dried sample. E-1h FPH also exhibited a marked concentration-dependent scavenging activity on 1,1-diphenyl-2-picrylhydrazyl radical. Antioxidant activity of E-1h FPH was higher (p < 0.05) than BHA and α-tocopherol in a linoleic acid peroxidation system over prolonged storage (∼162 h). Antioxidative FPH from Pacific hake may be useful ingredients in food and nutraceutical applications.     

The effect of enzymatic hydrolysis time and temperature on the properties of protein hydrolysates from Persian sturgeon (Acipenser persicus) viscera

Protein hydrolysate was prepared from the viscera of Persian sturgeon (Acipenser persicus), a major sturgeon species in the Caspian Sea. Hydrolysis was performed at three different temperatures (35, 45 and 55 °C), pH 8.5, using commercially available Alcalase^® and an enzyme to substrate ratio of 0.1 AU/g viscera protein over a 205 min incubation period. Protein and lipid content of the hydrolysate were 65.82%, and 0.18%, respectively. Protein recovery and degree of hydrolysis ranged from 34.97% to 61.96% and 13.32% to 46.13%, respectively. The highest degree of hydrolysis was observed at 55 °C after 205 min (p < 0.05). The amino acid score of the hydrolysates was similar to that of the FAO/WHO reference protein. It is revealed that Persian sturgeon visceral protein hydrolysate amino acid fulfils adult human requirements. Based on National Research Council guidelines, phenylalanine is the first limiting amino acid in the hydrolysate. However, the hydrolysate has the potential for application as an ingredient in formulated diets.     

Purification of antioxidative peptides prepared from enzymatic hydrolysates of tuna dark muscle by-product

To produce and identify bioactive peptides, two commercial enzymes, orientase (OR) and protease XXIII (PR) were used to hydrolyze tuna dark muscle by-product for up to 6 h, and the hydrolysates were evaluated for antioxidative properties. The results showed that, 60-min OR and 120-min PR hydrolysates possessed the highest antioxidative activity. Then, the protein hydrolysates were subjected to a Sephadex G-25 gel filtration chromatography, and the molecular weight of the peptide fractions which showed the highest antioxidative activity ranged from 390 to 1400 Da. The peptide fractions were further isolated using the two-step high-performance liquid chromatography (HPLC-1 and HPLC-2), and the amino acid sequences of the two antioxidative peptides from OR and PR hydrolysates were Leu–Pro–Thr–Ser–Glu–Ala–Ala–Lys–Tyr (978 Da) and Pro–Met–Asp–Tyr–Met–Val–Thr (756 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna dark muscle by-product may be useful ingredients in food and nutraceutical applications.     

Functional properties of proteins recovered from silver carp (Hypophthalmichthys molitrix) by isoelectric solubilization/precipitation

Isoelectric solubilization/precipitation at acidic and basic pH ranges was applied to whole gutted silver carp (Hypophthalmichthys molitrix) in order to recover muscle proteins. Thermal denaturation (T_onset, T_max, and ΔH), viscoelasticity (G′), and texture properties (shear stress) of proteins recovered from carp as affected by functional additives (beef plasma protein, potato starch, exogenous transglutaminase, polyphosphate, and titanium dioxide) were determined and compared to Alaska pollock surimi. Proteins recovered from carp showed typical endothermic transitions only when functional additives were used. Similar to endothermic transitions, viscoelasticity in carp proteins increased only when the additives were used. Typical endothermic peaks and viscoelasticity increase were recorded for Alaska pollock surimi. Carp protein-based gels with functional additives had lower (P < 0.05) shear stress than their surimi counterparts, but greater (P < 0.05) or similar (P > 0.05) when compared to surimi gels without functional additives. In addition, generally higher shear stress was measured for carp protein-based gels developed from basic pH treatments than the acidic counterparts. The present study indicates that proteins can be recovered from whole gutted carp using isoelectric solubilization/precipitation. However, if the recovered proteins are used for subsequent development of restructured food products, functional additives should be used.     

Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type

Antioxidative activity and functional properties of protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) meat, hydrolyzed by Alcalase 2.4L (HA) and Flavourzyme 500L (HF) with different degrees of hydrolysis (DH) were investigated. As the DH increased, DPPH radical-scavenging activity and reducing power of HA decreased (p < 0.05) but no differences were observed for HF (p > 0.05). Metal chelating activity of both HA and HF increased with increasing DH (p < 0.05). HF generally had a higher (p < 0.05) chelating activity than had HA at the same DH tested. At low DH (5%), HA exhibited a better DPPH radical-scavenging activity while, at high DH (25%), HF had a higher (p < 0.05) reducing power. For the functional properties, hydrolysis by both enzymes increased protein solubility to above 85% over a wide pH range (2–12). When the DH increased, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity, foam stability) of hydrolysates decreased (p < 0.05), possibly caused by the shorter peptide chain length. At the same DH, the functionalities of protein hydrolysate depended on the enzyme used. The results reveal that antioxidative activity and functionalities of protein hydrolysates from yellow stripe trevally meat were determined by the DH and by the enzyme type employed.     

Angiotensin converting enzyme (ACE) inhibitory,antihypertensive and antihyperlipidaemic activities of protein hydrolysates from Rhopilema esculentum

Angiotensin-converting enzyme (ACE) inhibitory, antihypertensive and antihyperlipidaemic activities of protein hydrolysates (RPH) from the jellyfish Rhopilema esculentum were investigated. R. esculentum was hydrolysed sequentially with pepsin and papain, and then the hydrolysate was ultrafiltered with a 2000 Da cut-off membrane. It was found that RPH contained high levels of Gly, Glu, Pro, Asp and Ala, having potential ACE inhibitory activity in vitro with an IC₅₀ of 1.28 mg/ml. It was also found that systolic blood pressure was reduced markedly in spontaneously hypertensive rats after single and chronic oral administration of RPH, indicating that RPH had an antihypertensive effect. In addition, oral administration of RPH decreased total serum cholesterol and triglyceride, and increased high-density lipoprotein cholesterol in rats fed with high-fat diet. These results indicate that RPH may prove to be a promising functional food for the prevention and treatment of hypertension and hyperlipidaemia.     

Use of viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus) for the production of protein hydrolysate from toothed ponyfish (Gazza minuta) muscle

Proteolytic activity of viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was studied. The optimal pH and temperature were 9.0 and 50 °C, respectively, when toothed ponyfish (Gazza minuta) muscle was used as a substrate. When viscera extract from hybrid catfish was used for the production of protein hydrolysate from toothed ponyfish muscle, extract concentration, reaction time, and fish muscle/buffer ratio affected the hydrolysis and nitrogen recovery (NR) (p < 0.05). Optimum conditions for toothed ponyfish muscle hydrolysis were 3.5% hybrid catfish viscera extract, 15 min reaction time and fish muscle/buffer ratio of 1:3 (w/v). High correlation between the degree of hydrolysis (DH) and NR (R² = 0.974) was observed. Freeze-dried hydrolysate had a high protein content (89.02%, dry weight basis) and it was brownish yellow in colour (L^∗ = 63.67, a^∗ = 6.33, b^∗ = 22.41). The protein hydrolysate contained a high amount of essential amino acids (48.22%) and had arginine and lysine as the dominant amino acids.     

Compositions and antioxidant properties of protein hydrolysates from the skins of four carp species

Compositions and antioxidant properties of protein hydrolysates prepared from four carp skins: black carp, grass carp, silver carp and bighead carp, using pepsin, with a degree of hydrolysis (DH) of 6–15%, were investigated. The yield of freeze‐dried hydrolysates was in the range of 54–62 g/100 g (dry skin). The content of protein and ash in four freeze‐dried hydrolysates was 72–81% and 8–17%, respectively. All hydrolysates contained high amount of hydrophobic amino acid residues (389–480 residues/1000 residues). Meanwhile, their antioxidant properties were evaluated by in vitro assays. The results revealed that all hydrolysates possessed potent antioxidant activities and showed dose dependency as the activity increased with sample concentration, capable of scavenging 72–88% of DPPH and 61–69% of hydroxyl radicals, respectively, at the highest tested concentration. The hydrolysates exhibited high reducing power and β‐carotene–linoleic acid oxidation inhibition. Among the four hydrolysates, the hydrolysate derived from bighead carp skin was superior to others in terms of yield, DH and antioxidant activities.     

The effects of pretreatments on antioxidative activities of protein hydrolysate from the muscle of brownstripe red snapper (Lutjanus vitta)

Different pretreatments of mince from brownstripe red snapper (Lutjanus vitta) including 1) washing; 2) membrane separation; 3) washing followed by membrane separation and 4) membrane separation followed by washing were conducted prior to hydrolysis. Among the resulting minces, that subjected to membrane separation with subsequent washing (MS/W) contained the lowest remaining myoglobin content, phospholipid content, heme iron and non-heme iron contents (p < 0.05) and showed the lowest TBARS values throughout 9 days of storage at 4 °C in the presence and absence of 0.15 mol L⁻¹ cupric acetate (p < 0.05). When hydrolysates from 1) mince, 2) MS/W and 3) protein isolate from MS/W (PI) with different degree of hydrolysis (DH) (20, 30 and 40%) were prepared using proteases from pyloric caeca of brownstripe red snapper, antioxidative activities determined by DPPH, ABTS radical scavenging activities, ferric reducing antioxidant power and metal chelating activity varied with hydrolysates and DH. Antioxidative activities increased with increasing DH up to 40% (p < 0.05). At all DH tested, hydrolysate prepared from MS/W exhibited the highest antioxidative activities determined by all assays, compared to those from mince and PI (p < 0.05). Hydrolysate from MS/W with 40% DH had the molecular weight lower than 6.5 kDa as determined by SDS-PAGE. In liposome oxidation system, the addition of hydrolysate from MS/W resulted in the lower TBARS, compared with the control throughout the incubation period of 48 h at room temperature (25-28 °C). Therefore, fish mince with membrane separation followed by washing was the most appropriate source for production of hydrolysate possessing antioxidative activity with the lowered amount of lipids and pro-oxidants.