Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.     

Evaluation of the Antioxidant Activity In Vitro and in Hippocampal HT-22 Cells System of Protein Hydrolysates of Common Carp (Cyprinus carpio) By-Product

Fish processing by-products may become more than 50% of the starting material. If mismanaged, these large quantities of discarded fish can create serious pollution problems and can also generate cost associated with their disposal. Enzymatic hydrolysis is one of the techniques that is currently being developed in order to recover and add value to these biomolecules. There is an increasing interest in natural antioxidants that are safer for consumers compared with synthetic antioxidants. In this study, common carp by-product was hydrolyzed using the enzymes Alcalase (A) and Protamex (P) to reach degrees of hydrolysis (DH) of 10 and 15%, respectively. Antioxidant capacity against peroxyl radicals (ACAP); 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging; and the measurement of intracellular reactive oxygen species (ROS) concentration after exposure to common carp protein hydrolysates were investigated. The results revealed that the hydrolysate A15 exhibited significantly (p < 0.05) higher antioxidant activity against the DPPH radical. A15 showed the highest in vitro antioxidant competence against peroxyl radicals, whereas P15 showed the lowest activity against peroxyl radicals (p < 0.05). Hydrolysates having the highest and the lowest in vitro antioxidant activity (A15 and P15, respectively) were selected for the determination of antioxidant activity in the HT-22 cells system. Measurement of intracellular ROS concentration revealed that P15 at the concentration of 1.25 mg/mL significantly (p < 0.05) reduced the intracellular ROS concentration. These results showed that common carp by-product protein hydrolysates are a source of antioxidant peptides with a high potential for food and pharmaceutical industries to develop new nutraceuticals and functional foods.     

Protein Hydrolysate Fractions from Sea Cucumber (Isostichopus badionotus) Inhibit Angiotensin-Converting Enzyme

Angiotensin-converting enzyme (ACE) inhibitory capacity was evaluated in hydrolysates from sea cucumber Isostichopus badionotus. The pepsin-pancreatin and Alcalase®-Flavorzyme® sequential systems were used. Hydrolysates were characterized in terms of degree of hydrolysis (DH), electrophoretic profile, and inhibitory activity. The Alcalase®-Flavorzyme® system produced hydrolysates with the highest DH, with that produced at 90 min exhibiting the highest ACE inhibitory action (86%). This hydrolysate was chosen for purification by ultrafiltration with five molecular weight cut-offs (< 1, 1–3, 3–5, 5–10, and > 10). The > 10-kDa fraction exhibited the highest ACE inhibitory action (80.7%) and was fractionated by gel fractionation chromatography. Of the 11 resulting fractions, IX had the highest ACE inhibitory action (73.4%) and lowest IC₅₀ value (2-μg prot/mL). Hydrolysis of sea cucumber I. badionotus with the Alcalase®-Flavorzyme® system produced hydrolysates with ACE inhibitory action. These contained a peptide with an IC₅₀ value comparable to those of synthetic ACE inhibitors.     

Structural Changes,Volatile Compounds and Antioxidant Activities of Maillard Reaction Products Derived from Scallop (Patinopecten yessoensis) Female Gonad Hydrolysates

In order to improve the utilization of scallop byproducts, scallop female gonad hydrolysates (SFGHs) were prepared using alcalase and ribose at a mass ratio of 1:2 and pH 7.0 (95°C, up to 12 h) to develop heterogeneous Maillard-type antioxidant and flavor compounds. The formation of Schiff’s base as well as modification of the amide bands in Maillard reaction products (MRPs) were investigated using ultraviolet–visible, fluorescence, and Fourier transform infrared spectroscopy. The significant decrease of amino acid residues explained the strong Maillard reactivity of SFGHs. MRPs with enhanced ABTS⁺ radical scavenging capacity over SFGHs promoted the survival of HepG2 cells treated with hydrogen peroxide. Moreover, 19 new volatile compounds were produced by the reaction of SFGHs with ribose. These results suggest that SFGHs–ribose MRPs can be potentially used as antioxidants with flavor properties.

Abbreviations: SFGs: scallop female gonads; SFGHs: scallop female gonad hydrolysates; ABTS: 2?2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); BHT: butylated hydroxytoluene; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; MRPs: Maillard reaction product; H₂O₂: hydrogen peroxide; HS-SPME/GC/MS: Headspace solid phase micro-extraction/gas chromatography/mass spectrometry.     

Effect of Enzyme Type on the Antioxidant Activities and Functional Properties of Enzymatic Hydrolysates from Sea Cucumber (Cucumaria frondosa) Viscera

ABSTRACT

Effects of various proteases were obvious on the amino acid composition, antioxidant activities, and functional properties of enzymatic hydrolysates prepared from sea cucumber viscera (SCV). Alcalase, trypsin, and flavourzyme conferred better antioxidant activities on hydrolysates and endowed them with a higher degree of hydrolysis, coming to 19.08, 32.38, and 15.94%, respectively. For functional properties, neutrase- and flavourzyme-derived hydrolysates showed better foaming properties. Amino acid composition suggested that papain, neutrase, alcalase, and bromelain granted the hydrolysate’s high nutritional quality. The results indicated that SCV hydrolysates could be used in food systems to impart desirable characteristics to products.     

Antioxidant and ACE Inhibitory Activities of Kawakawa (Euthynnus affinis) Protein Hydrolysate Produced by Skipjack Tuna Pepsin

ABSTRACT

Pepsin enzyme from skipjack tuna was extracted for the production of kawakawa (Euthynnus affinis) fish protein hydrolysate. Using ultra-fractionation, Kawakawa protein hydrolysates were separated into four different fractions, including fractioned protein hydrolysate I (FPH I) (< 1 kDa), FPH-II (1–3 kDa), FPH-III (3–10 kDa), and FPH-IV (> 10 kDa). The antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, inhibition of linoleic acid oxidation, reducing power tests, and chelating activity of metal ions. Results indicated that FPH II fraction peptides had higher antioxidant activity in comparison with the other fractions, followed by FPH I. Further, the fractions were evaluated for angiotensin converting enzyme (ACE) inhibition, and IC₅₀ value ranged from 0.45 to 1.86 mg/ml with higher activity in FPH I (IC₅₀ 0.45). Finally, the amino acid profile of different fractions was analyzed. The fractions exhibited significant amounts of hydrophobic amino acids, which could perform as hydrogen donors, frustrate the free radicals, and inhibit the ACE. The recovered pepsin from the viscera was used to produce hydrolysates with good biological activities. Peptides lower than 3 KDa had antioxidant activity as positive controls and significant ACE activity. These are very important findings that could be used to conduct further research in a preclinical study of these peptides.     

Optimization of Hydrolysis Conditions for the Production of the Angiotensin-I Converting Enzyme Inhibitory Peptides from Sea Cucumber Collagen Hydrolysates

The effects of different hydrolysis conditions on the angiotensin-I converting enzyme (ACE) inhibitory properties of giant red sea cucumber (Parastichopus californicus) collagen hydrolysates were investigated. Optimal conditions predicted by central composite rotatable design (CCDR) modeling for producing ACE inhibitory peptides were found to be 54.9°C, 1.76 h and an enzyme to substrate (E/S) ratio of 0.064. Compared with experiments performed under these optimal conditions, the predicted degree of hydrolysis (DH) and ACE inhibitory activities had error rates of 3 to 6%. Under optimum conditions, the molecular weights of collagen hydrolysates were less than 6.5 kDa. Lineweaver-Burk plots suggest that the collagen hydrolysates acted as competitive inhibitors with an inhibition constant (Ki) value of 0.706 mg/mL.     

Setting Ability of Threadfin Bream (Nemipterus japonicus) Meat as Affected by Freezing and Frozen Storage with Special Reference to Transglutaminase Enzyme Activity and Rheological Properties

The effect of freezing and frozen storage of threadfin bream fish (Nemipterus japonicus) meat on the setting and gel-forming ability has been evaluated. The dynamic viscoelastic properties of fish meat during setting and thermal gelation process were evaluated using Controlled Stress Rheometer under oscillatory mode. A sharp decrease in setting ability was recorded immediately after freezing as revealed by storage modulus (G?) values. The transglutaminase (TGase) enzyme activity of fish meat decreased from the initial value of 81.09 to 51.46 U/g meat/min at the end of 200 days of frozen storage. A decrease in setting ability of fish meat beyond 160 days of frozen storage is probably related to lower TGase enzyme activity. The gel-forming ability was related to setting ability during the frozen storage period. Although the protein solubility showed a decreasing trend during 200 days of frozen storage, the decrease was not significant. The effect of freezing and frozen storage on calcium-activated adenosine triphosphatase (Ca²⁺-ATPase) enzyme activity of fish mince was significant (P < 0.05). A reduction in protein solubility and Ca²⁺-ATPase enzyme activity is an indication of aggregation/denaturation of myofibrillar proteins.     

饲料中添加两种蛋白水解物对大菱鲆(Scophthalmus maximus L.)幼鱼生长性能及肠道组织学结构的影响

本研究共配制了3种等氮等脂的实验饲料,其中,对照组饲料使用15％鱼粉提供部分饲料蛋白,2个实验组饲料分别用鱼水解蛋白和鸡水解蛋白替代了饲料中10％的鱼粉蛋白.对初始体重为4.16g左右的大菱鲆(Scophthalmus maximus L.)幼鱼进行了为期12周的投喂,研究了其对大菱鲆生长性能及肠道组织学结构的影响.结果显示,鱼水解蛋白组(PHf)的特定生长率、饲料效率、蛋白效率比和蛋白质沉积率较对照组(FM)无显著差异(P＞0.05),但显著高于鸡水解蛋白组(PHc)(P＜0.05);3组实验鱼的摄食率无显著差异(P＞0.05);PHf和PHc组的鱼体蛋白含量显著高于FM组(P＜0.05),3组实验鱼的脂肪含量无显著差异(P＞0.05).PHf和PHc组的鱼体肌肉必需氨基酸含量显著高于FM组(P＜0.05).PHf和PHc组的前肠和中肠黏膜皱襞高度大于FM组,且PHf组较FM组差异显著(P＜0.05);3组实验鱼的前肠和中肠上皮细胞的高度无显著差异(P＞0.05);FM组前肠和中肠的肠壁厚度大于其他2组,且显著大于PHf组(P＜0.05).大菱鲆饲料中使用鱼水解蛋白替代部分鱼粉蛋白在生长性能及肠道组织学结构方面要优于使用鸡水解蛋白.     

Optimization of Hydrolysis Conditions for Production of Angiotensin-Converting Enzyme Inhibitory Peptides from Basa Fish Skin Using Response Surface Methodology

ABSTRACT

Basa fish skin free from fat and noncollagenous proteins was hydrolyzed by different proteinases to produce angiotensin-converting enzyme (ACE) inhibitory peptides. Protamex hydrolysates were found to have the highest ACE inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions of Protamex—including enzyme-to-substrate ratio, hydrolysis time, and solid (fish skin)-to-liquid (water for hydrolysis)—at a temperature of 50°C and a pH of 7.0. The mathematical model demonstrated a good fit with the experimental data. An enzyme-to-substrate ratio of 6,108 U/g, a hydrolysis time of 1.24 h, and a solid-to-liquid ratio of 1:8.05 g/mL were found to be the optimal conditions to achieve the highest value of ACE inhibitory activity. The IC₅₀ of the hydrolysates for ACE inhibitory activity was 1.417 mg/mL under these conditions.     

Characterization of Collagen Enzymatic Hydrolysates Derived from Lizardfish (Synodus fuscus) Scales

Acid soluble collagen was extracted from the scales of lizardfish and characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis. After 8 h of collagen hydrolysis, hydrolysates had an estimated degree of hydrolysis (DH) from 4 ± 0.05 to 25 ± 0.63% (p < 0.05). All hydrolysates had angiotensin converting enzyme inhibitory and antioxidant activity. These activity levels showed little change after treatment with gastrointestinal proteases. Results indicated that the lizardfish by-products may be improved by enzymatic treatment with acid-soluble collagen from lizardfish scales.     

Optimization of Hydrolysis Conditions for Production of Gelatin Hydrolysates from Shark Skin Byproduct and Evaluation of Their Antioxidant Activities

ABSTRACT

The effect of Alcalase hydrolysis conditions on antioxidant activities of shark skin gelatin hydrolysate (SSGH) byproduct was studied. Optimal hydrolysis condition for SSGH at 60°C, pH 7.5, and 2.70 AU kg^?1 gelatin protein was used for varied hydrolysis times (10–90 min). The IC₅₀ against DPPH radicals of SSGH hydrolyzed for 90 min (SSGH-90) was 27.39 mg ml^?1, which is greater than ascorbic acid. SSGH-90 was predominantly composed of 15–20 kDa protein fragments that regulated increases in peroxide value and thiobarbituric acid reactive substances values and suppressed decrease in DHA of a fish oil-in-water emulsion during storage at 30°C for 7 days.     

Antioxidant Efficacy of Protein Hydrolysates from Large Yellow Croaker (Pseudosciaena crocea) in D-galactose-Induced Aging Mice

The antioxidant efficacy of protein hydrolysates prepared from large yellow croaker (Pseudosciaena crocea) by enzymatic hydrolysis using neutral protease was evaluated in D-galactose-induced aging mice. Animals were divided into normal and model control groups, and low-, middle-, and high-dose groups (50, 100, and 300 mg/kg; LYCHs), as well as tocopherol (VE) and antagonistic (LYCHs + VE) groups. The study was carried out for 30 days. Administration of D-galactose induced oxidative damage with a significant rise (p < 0.01) in malondialdehyde and a reduction in several endogenous antioxidant enzymes and glutathione (GSH). Treatment with large yellow croaker protein hydrolysates stimulated an increase in superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities, increased levels of reduced glutathione, and decreased malondialdehyde, compared with the model control group. Additionally, hepatocellular damage was evaluated by measuring aspartate aminotransferase and alanine aminotransferase levels. These results demonstrate the protective role of large yellow croaker protein hydrolysates in D-galactose-induced oxidative stress, as well as their potential as nutraceuticals or adjuvant agents for disease prevention.     

Characterization and Cryoprotection of Invasive Silver Carp (Hypophthalmicthys molitrix) Protein Hydrolysates

ABSTRACT

Silver carp (Hypophthalmicthys molitrix) is regarded as an invasive, underutilized freshwater fish causing environmental and economic complications along the U.S. Mississippi River System. In this study, silver carp protein hydrolysates (FPH) were obtained from 30, 60, 90, 120, and 240 min hydrolysis with Protamex®. Amino acid composition showed polar amino acids, including Asp and Glu, and freed hydrophobic residues in FPH-90 and FPH-240. Protein surface hydrophobicity of FPH increased with increasing degree of hydrolysis. Cryoprotection was evaluated using a muscle (mince) food system with 6% FPH (w/w) from each hydrolysis time condition, an untreated mince (CTRL), and 8% (w/w) 1:1 sucrose-sorbitol (SUSO), respectively. After six freeze-thaw cycles (D7), all FPH treatments had lower (p < 0.05) expressible moisture, indicating strong water-holding capacity by the FPH. FPH formulations also imparted antifreeze activity equal to or better than SUSO, with the FPH-90 exhibiting higher (p < 0.05) proportion (43.9%) of unfrozen water at D7. Results from this study provide preliminary evidence for development of effective cryoprotectants using an underutilized, invasive fish species that can be exploited for environmental and economic gain in the form of value-added ingredients and further provide opportunities to understand the fundamental physicochemical properties governing cryoprotection of FPH in a frozen mince system.     

Angiotensin Converting Enzyme and Dipeptidyl Peptidase-IV Inhibitory,and Antioxidant Activities of a Blue Mussel (Mytilus edulis) Meat Protein Extract and Its Hydrolysates

ABSTRACT

Protein extracted from mussel processing coproducts was hydrolyzed with four different food-grade enzyme preparations and assessed for angiotensin converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory and oxygen radical antioxidant capacity (ORAC) activities. All hydrolysates tested showed higher activity than the intact protein. ACE and DPP-IV IC₅₀ values in the range 1.13–3.34 and 0.33–2.43 mg mL^?1, respectively, and ORAC values in the range 66.26–121.56 µmol trolox equivalents g^–1 were obtained. These results suggest that some of the mussel meat protein hydrolysates may have potential as functional food ingredients for the management of diseases such as type II diabetes and hypertension.     

Evaluation of Plasticizing and Antioxidant Properties of Silver Carp Protein Hydrolysates in Fish Gelatin Film

Physico-mechanical and antioxidant properties of glycerol plasticized fish gelatin films were investigated at different levels of fish protein hydrolysates (FPH) from silver carp muscle (5, 10, 15, and 20 g FPH/100 g gelatin). The films supplemented with FPH, especially at higher concentration showed significant increase in 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 20-azinobis-(3-ethylbenzothiazoline-6-sulfnic acid) (ABTS) radical scavenging, and ferric reducing antioxidant power (FRAP), elongation at break, difference of color, water vapor permeability and opacity, while tensile strength, elastic modulus, L*, b*, and contact angle decreased markedly (p < 0.05). However, no significant differences were observed for a* and solubility of all samples (p > 0.05). Films prepared using 20% FPH had lower glass transition temperature and more homogeneous structure, compared with control film. Also, Fourier transform infrared (FTIR) spectroscopy confirmed the increase of free groups of hydrolysates and lower interaction between chains of film incorporated with the highest content of FPH. Thus, these results indicate that FPH enhanced the antioxidant activities and affected some characteristics due to less interaction between gelatin and FPH.     

Inhibitory Activities of Protein Hydrolysates from Spotted Babylon Snails on Tyrosinase and Melanogenesis

The optimal conditions for protein hydrolysates preparation from spotted babylon Babylonia areolata exhibiting tyrosinase inhibitory activity and antioxidant activity using alkaline protease (Protease G6) were undiluted Protease G6 (5.8 × 10⁵ DU/g) at 60 min of hydrolysis time and eightfold diluted Protease G6 (7.25 × 10⁴ DU/g) at 240 min of hydrolysis time. These two conditions were fractioned using molecular weight (MW) cutoff values of 10, 5, and 3 kDa membranes, and their anti-melanogenic and antioxidant properties were further analyzed. Among the fractions, the MW < 3 kDa fraction exhibited high levels of inhibitory activity toward the mono- and diphenolase activities of tyrosinase, with IC₅₀ values of 1.758 and 8.995 μg/mL, respectively. Kinetic studies revealed that this fraction behaved as an uncompetitive inhibitor. The results demonstrated that the MW < 3 kDa fraction suppressed melanin synthesis and decreased cellular tyrosinase activity with no cytotoxicity to B16F10 melanoma cells.     

Effect of Degree of Hydrolysis and Protease Type on the Antioxidant Activity of Protein Hydrolysates From Cuttlefish (Sepia officinalis) By-Products

We have investigated the antioxidant activities of eight hydrolysates from cuttlefish by-products obtained by treatment with various gastrointestinal proteases (chymotrypsin, trypsin, and crude alkaline enzyme extracts from cuttlefish and sardinelle) and bacterial proteases (Alcalase and crude enzymes from Bacillus pumilus A1, Bacillus mojavensis A21, and Bacillus cereus BG1). The antioxidant activities of the cuttlefish by-products protein hydrolysates (CPHs) were evaluated using various in vitro antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power, and total antioxidant capacity. All hydrolysates showed different degrees of hydrolysis (DH) and varying degrees of antioxidant activity. Among the different hydrolysates, cuttlefish crude enzyme hydrolysate exhibited the highest antioxidant activity, followed by sardinelle crude enzyme and Alcalase hydrolysates. Further, CPHs with different degrees of hydrolysis were prepared by treatment with proteolytic enzymes from cuttlefish, sardinelle, and B. mojavensis A21. All hydrolysates showed a greater antioxidative activity as indicated by all the methods considered. In addition, antioxidant activity in hydrolysates was positively correlated with the increase of DH. The results of this study indicated that CPHs might be a good candidate for further investigation in developing new antioxidants.     

Autolysis of Anchovy (Engraulis japonicus) Protein: Evaluation of Antioxidant Properties and Nutritional Quality of Resulting Hydrolysates

Protein hydrolysis of underutilized marine organisms is a method to improve added value. In the present article, anchovy protein was autolyzed at pH 8.5 and 50°C. After autolysis processing for 24 h, total nitrogen recovery reached 92.1%. The protein hydrolysates had antioxidant properties on reducing power, 1, 1-diphenyl-2-picryhydrazyl radical, and hydroxyl radical scavenging activity. More than 79% of the peptides in the hydrolysates had a molecular weight of lower than 3 kDa. Protein hydrolysates had balanced amino acid compositions, and total content of essential amino acids had no significant difference after hydrolysis. Protein hydrolysates from anchovy autolysis have potential in nutritional and functional foods.     

Purification and Characterization of an Antioxidant Protein from Pearl Oyster (Pinctada fucata martensii)

Protein from pearl oyster (Pinctada fucata martensii) was purified and characterized. Results showed that the pearl muscle protein (PMP) was composed of a single polypeptide with a molecular weight of approximate 11.6 kDa and contained 95.1% of the protein and 4.92% of the carbohydrate of the oyster. The polypeptide appears to be a glycoprotein, since the Fourier transform infrared (FTIR) spectroscopy of the PMP shows the typical characteristics of protein and polysaccharides. The denaturation temperature of the PMP was 81.3°C by differential scanning calorimetry. Circular dichroism spectroscopy showed that the PMP has a highly ordered structure. The PMP (200 μg/mL) showed a high oxygen radical absorbance activity (ORAC) of 6.57 μmol Trolox/μmol protein. Its antioxidant activity was stable at temperatures from 30–80°C and pH 2–10. The antioxidant activity was significantly inhibited by Zn²⁺, Ca²⁺, and EDTA and enhanced by Mn²⁺. The results suggest that the PMP could be used as a potential natural antioxidant.