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.     

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.     

Simultaneous Extraction and Fractionation of Fish Oil from Tuna By-Product Using Supercritical Carbon Dioxide (SC-CO2)

ABSTRACT

Fish oil was extracted and simultaneously collected into six fractions based on molecular weight and the chain length of triglycerides in terms of fatty acid constituents without splitting of the triglycerides, using supercritical carbon dioxide (SC-CO₂) at optimized conditions of 40 MPa, 65°C, and a flow rate 3 mL min^?1. In each type of fractionation, the first fraction (F1) was rich in saturated fatty acids (SFA; 52.57 to 61.26%), followed by monounsaturated fatty acids (MUFA; 22.17 to 23.22%) and polyunsaturated fatty acids (PUFA; (0.54 to 20.37%); the sixth fraction (F6) was rich in PUFA (48.93%), followed by MUFA (33.59%) and SFA (13.61%). It was obvious that short-chain fatty acids were extracted at an earlier fraction; therefore, the latter fractions were dominant in long-chain fatty acids, especially MUFA and PUFA. Thus, omega-3 fish oil (last three fractions) was successfully separated to be used as a value-added health product.     

The Feasibility of Increasing Lipid Extraction in Tilapia (Oreochromis niloticus) Waste by Proteolysis

ABSTRACT

The aim of the present study was to evaluate the feasibility of increasing lipid extraction in tilapia (Oreochromis niloticus) waste using different enzymes and to minimize the periods of hydrolysis to evaluate the performance of the lipid fractions. When processing waste tilapia, commercial enzymes were added that were composed of neutrase and alcalase acting at different hydrolysis times to produce the best yields. The treatment proved feasible for obtaining the protein hydrolysates. The type of enzyme and the hydrolysis time determined the degree of fractionation. The enzymes performed well, and the highest efficiency occurred within 2 h of hydrolysis. By fractionating, the obtained by-products can be applied in the preparation of feed, as the lipid fraction treatments yielded significant amounts of polyunsaturated fats and suitable n-6/n-3 ratios.     

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.     

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.     

Antioxidant Activity of Hydrolysates and Peptide Fractions of Nemipterus japonicus and Exocoetus volitans Muscle

The focus of the study was to investigate the antioxidant activity of hydrolyzed muscle protein of Nemipterus japonicus and Exocoetus volitans. The trypsin protein hydrolysates of both fish showed maximum free radical scavenging potential and lipid peroxidation inhibition. Furthermore, it was purified by chromatographic methods followed by the lipid peroxidation inhibition; free radical scavenging assay was performed before and after purification. The purified peptide fractions of N. japonicus and E. volitans exhibited higher activity against polyunsaturated fatty acids (PUFA) peroxidation which was similar to natural antioxidants like α-tocopherol. Free radical scavenging potencies were measured by electron spin resonance technique (ESR). The purified peptide of E. volitans quenched free radicals (DPPH, hydroxyl, and superoxide) slightly more than N. japonicus. The amino acid composition of both fish protein hydrolysates showed variations in their ratio. The purified peptides were tested for cell cytotoxicity for Vero (kidney epithelial cells of the African Green Monkey) and Hep G₂ (human hepatocellular liver carcinoma) cell lines. It was found that peptides did not show any cytotoxic effect for Vero cell lines and exerted a significant antiproliferative effect on Hep G₂ cell lines.     

Estradiol-17β-induced calcium uptake and resorption in juvenile rainbow trout,Oncorhynchus mykiss

Juvenile rainbow trout, Oncorhynchus mykiss, were injected with estradiol-17β (E₂) in order to study the source of extra calcium needed during vitellogenesis. E₂-treatment increased the calcium uptake from the external medium as well as calcium mobilization from muscle and scale. Judged by the increase in plasma protein-bound calcium levels, the E₂-induced increase in calcium uptake is an apparent over-mobilization of calcium, i.e., the calcium uptake of the fish is in excess of what is found bound to plasma proteins. As the calcium excretion and calcium space (calculated from free plasma calcium levels) were unaffected, the excess calcium is suggested to be incorporated into internal calcium stores. This implies that the systems regulating vitellogenesis and calcium balance are integrated on the mechanistic or endocrine level, and that E₂ causes calcium mobilization of a magnitude geared to the needs of the sexually maturing female.     

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.     

Inhibitory activities of the edible brown alga <Emphasis Type="Italic">Laminaria japonica</Emphasis> on glucose-mediated protein damage and rat lens aldose reductase

Increased aldose reductase (AR)-related polyol pathway activity and the subsequent formation of advanced glycation end-products (AGEs) have been implicated in the onset of diabetic complications. We have evaluated the inhibitory effects of the methanolic extract and its fractions of Laminaria japonica on rat lens AR (RLAR) and AGE formation. The CH₂Cl₂ fraction was separated by high-performance liquid chromatography to yield active porphyrin derivatives, including pheophorbide a (1) and pheophytin a (2), which were assayed for their anti-diabetic complications and yield. Compound 1 exhibited potent inhibitory activities against both AGE formation and RLAR, with respective half-maximal inhibitory concentration (IC₅₀) values of 49.4 and 12.3 μM. Conversely, compound 2 was found to be active against AGE formation, with an IC₅₀ of 228.7 μM. For further elucidation of the structure–inhibitory activity relationship of the porphyrin derivatives, the inhibitory activities of four commercially available porphyrin derivatives on AGE formation and RLAR were measured. The presence of a carboxyl group and the absence of a phytyl group at the C-17² position of the porphyrin were found to contribute to the inhibitory effects towards both AGE formation and RLAR. These results suggest that the L. japonica and its porphyrin derivatives may represent a potential functional food resource for further prevention of diabetic complications.     

Effect of environmental calcium levels on calcium uptake in tilapia larvae Oreochromis mossambicus

Effects of environmental calcium concentrations on the survival, growth, body calcium content and calcium uptake kinetics in developing tilapia (Oreochromis mossambicus) larvae were studied. Fertilized eggs were incubated in high- and low-calcium artificial freshwater (0.88–0.96 mmol l^–1 vs. 0.02–0.03 mmol l^–1 CaCl₂ or CaSO₄) until 3 days after hatching. Tilapia larvae showed similar hatching rates and wet weights in either high- or low-calcium medium, indicating neither the development nor the growth in tilapia larvae was affected by the environmental calcium levels. The body calcium content in low-calcium groups was about 90–95% that of high-calcium groups, No matter what calcium source was used (CaCl₂ or CaSO₄), acclimation to low calcium medium caused a stimulation of calcium uptake (measured in 0.2 mmol l^–1 calcium),i.e., 1.2–1.3 fold higher than that of high calcium groups. This enhanced calcium uptake capacity was characterized by a 50% decrease in K_m and a 25% increase in J_max. Effect of different calcium salts on calcium influx was significant only in low calcium level,i.e., calcium influx in low-CaCl₂ group higher than that in low-CaSO₄ group. These results suggest that tilapia larvae are able to modulate their calcium uptake mechanism to maintain normal body calcium content and growth in environments with different levels of calcium.     

Replacement of fishmeal by two types of fish protein hydrolysate in feed for postlarval shrimp Litopenaeus vannamei

After filleting of tilapia, the material remaining is discarded and this waste represents about 700 g/kg of fish body volume, corresponding to carcass and viscera. These leftovers are important sources of proteins that can be used as feed in aquaculture industry by producing protein hydrolysates. In this study, two protein hydrolysates of tilapia were produced, with one (FPH1) and two (FPH2) hours of hydrolysis. The nutritional composition of the hydrolysates showed desirable levels of crude protein and essential amino acids. Electrophoresis revealed peptides ranging from 10 to 250 kDa. In addition, caseinolytic activity was recorded by zymogram. The hydrolysates were incorporated separately in experimental diets to replace fishmeal at distinct levels: 0, 40, 80 and 120 g/kg, totalizing seven diets named 0 (control), 40H₁, 80H₁, 120H₁, 40H₂, 80H₂ and 120H₂. A 45‐day feeding trial was carried out to evaluate the zootechnical performance of postlarvae fed these diets. In conclusion, the use of FPH2 as a substitute for fishmeal promotes better shrimp growth than FPH1 and allows higher levels of substitution. In addition, it is recommended a 60 g/kg fishmeal replacement by FPH2 to improve growth.     

Amino Acid Composition,Volatile Compounds and Bioavailability of Biocalcium Powders from Salmon Frame as Affected by Pretreatment

Biocalcium powders, Bio-cal-H, and Bio-cal-A, obtained from non-alkaline treated and alkaline treated salmon frame were characterized. Glycine was the dominant amino acid (216.45–321.80 mg/g), followed by glutamic acid (102.52–110.38 mg/g), proline (72.96–96.10 mg/g), and hydroxyproline (72.98–83.75 mg/g) in both biocalcium powders. Bio-cal-H possessed a high abundance of volatile compounds such as 2-propanone, acetonitrile, and 2-butanone than Bio-cal-A. Bio-cal-A had a higher solubility in in vitro simulated gastrointestinal tract than Bio-cal-H and CaCO₃ (P < .05). However, transportation of calcium across Caco-2 cell monolayer of Bio-cal-H was higher than Bio-cal-A and CaCO₃ (P < .05). Overall, biocalcium could serve as a promising source of calcium supplementation.     

Utilization of Chum Salmon (Oncorhynchus keta) Skin Gelatin Hydrolysates to Attenuate Hydrogen Peroxide-Induced Oxidative Injury in Rat Hepatocyte BRL Cell Model

Six chum salmon skin gelatin hydrolysates with degree of hydrolysis of 4.7–13.5% were generated by Alcalase and papain and showed scavenging activities to 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion radicals. Potential protection of these hydrolysates against H₂O₂-induced oxidative injury in rat hepatocyte line (BRL cells) was assessed in vitro, based on cell viability, leakage of lactate dehydrogenase, thiobarbituric acid reactive substances (TBARS), glutathione content, glutathione reductase, and catalase of the cells. The hydrolysates exhibited protection on the hepatocytes, reflected by an enhanced cell viability (about 6.2–87.9%), glutathione reductase (about 28.2–85.9%) or catalase (about 43.3–116.9%) activity, and decreased lactate dehydrogenase leakage (about 2.7–34.7%) or TBARS content (about 4.1–39.3%). On the contrary, cellular glutathione content gave an unnoticeable difference among the investigated groups. These hydrolysates also showed protection against DNA damage in the cells. Cytoprotection of these hydrolysates possessed a dosage-dependence manner. More importantly, there was a high correlation (0.7 < r < 0.9) between DPPH or superoxide anion radical scavenging activity of these hydrolysates and three cellular antioxidant indices (glutathione content, glutathione reductase, and catalase activity).     

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.     

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus,a rhabdovirus from salmonid fish

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon‐farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk‐derived proteins as bovine caseins or casein‐derived peptides at different stages during the course of IHNV infection. The results indicate that the 3‐h fraction of casein and α_S2‐casein hydrolysates reduced the yield of infectious IHNV in a dose‐dependent manner and impaired the production of IHNV‐specific antigens. Hydrolysates of total casein and α_S2‐casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein‐treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV‐inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.     

Antioxidant,ACE-Inhibitory,and Antimicrobial Activities of Peptide Fractions Obtained From Dried Giant Squid Tunics

ABSTRACT

Squid tunics were divided in two batches: freeze-dried and air-dried. Dried squid tunics were directly hydrolyzed with pepsin, Alcalase, and Esperase. Freeze-dried tunics showed better aptitude for hydrolysis than air-dried tunics. Pepsin showed the lowest efficiency in protein breakdown and gave hydrolysates with low antioxidant activity, whereas Alcalase and Esperase peptide fractions showed strong radical scavenging ability, ACE-inhibitory activity, and noticeable antimicrobial properties. B. cereus, B. coagulans, and D. hansenii were found to be the most sensitive bacteria. Dried squid tunics proved to be an alternative to a previously extracted gelatin for obtaining bioactive peptides.     

Influence of Different Hydrolysis Processes by Trypsin on the Physicochemical,Antioxidant, and Functional Properties of Collagen Hydrolysates from Sphyrna lewini,Dasyatis akjei,and Raja porosa

ABSTRACT

Acid soluble collagen hydrolysates from the cartilages of Sphyrna lewini, Dasyatis akjei, and Raja porosa on different hydrolysis conditions by trypsin were prepared and named as S, D, and R, respectively. The hydrolysates (S1, D1, and R1) obtained from hydrolysis in pH 2.5, 3 h, 37°C presented wonderful foaming and emulsifying capacities, caused by their high average molecular weights (AMWs), high degree of Pro and Lys hydroxylation, and imino acid contents (17.1, 15.3, and 14.1%). At the concentration of 0.1% (w/v), the foaming capacities of S1, D1, and R1 were 104.75 ± 2.57, 63.87 ± 2.73, and 76.87 ± 2.02%; and the emulsifying activity indexes of them were 116.07 ± 1.89, 91.04 ± 1.79, and 123.85 ± 2.14 m²/g, respectively. Conversely, hydrolysates (S2, D2, and R2) obtained from hydrolysis in pH 7.8, 3 min, 37°C exhibited strong scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC₅₀ 0.568, 0.680, and 1.634 mg/mL), hydroxyl radical (EC₅₀ 0.253, 0.376, and 0.438 mg/mL), and moderate reducing power (0.0465–0.4702 at the concentration of 5 mg/mL), due to their low AMWs. The results indicated that S1, D1, and R1 could be used as emulsifiers and foaming agents, and S2, D2, and R2 could be used as natural antioxidants in food systems.     

Purification and Identification of an Antioxidative Peptide from Digestive Enzyme Hydrolysis of Cutlassfish Muscle

ABSTRACT

The major objective of this study was to investigate the pepsin digests of cutlassfish muscle for their antioxidant activity. The peptide isolated after 3 h of pepsin hydrolysis demonstrated the highest scavenging activity for both 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and alkyl radicals. It also had the highest yield of the isolates with the highest protein content, which was likely related to its higher degree of hydrolysis than the other hydrolysates. The antioxidant peptide purified from the 3 h pepsin hydrolysate indicated the amino acid sequence, Phe-Ser-Gly-Gly-Glu. This study suggests that cutlassfish muscle is an excellent antioxidant source and could be effectively used as a food ingredient and in pharmaceuticals.