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.     

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.     

Purification and characterization of carbon monoxide dehydrogenase from the aerobic hyperthermophilic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis>

The aerobic hyperthermophilic archaeon Aeropyrum pernix expresses carbon monoxide (CO) oxidation activity under heterotrophic growth conditions. Using activity stain gel analysis, CO oxidation activity was detected in a protein with a molecular mass of 210 kDa. The 210 kDa CODH protein was purified to homogeneity from A. pernix. Aeropyrum Mo-CODH catalyzed the oxidation of CO with a specific activity of 2.1 μmol CO min⁻¹ mg⁻¹ at 95°C, pH 8.0 using methyl viologen as the electron acceptor. The CODH protein showed high oxygen and thermo stability. The protein contains three subunits: L (86.6 kDa), M (34.5 kDa), and S (12.6 kDa), which form the LM₂S complex. The molecular mass of the complex was calculated by gel filtration and found to be 163.7 kDa. N-terminal amino acid sequencing and peptide mass fingerprinting analysis of the subunits indicated that they corresponded to NP_148462.1, NP_148464.2, and NP_148465.1, and their genes annotated the molybdo iron-sulfur flavoprotein carbon monoxide dehydrogenase S, L, and M subunits, respectively. Phylogenetic analysis revealed that CODH belongs to a novel clade of diverse CODHs.     

Antioxidant and anti‐inflammatory activities of Pinus radiata bark extract in salmonid cell lines

A fish meal supply shortage is limiting aquaculture development. Currently, plant‐based proteins, such as soya bean meal, are being used as an alternative protein source, despite that such a diet can adversely affect fish, such as by inducing an inflammatory response. A possible solution is to include dietary additives in farm diets to counteract negative effects. One such solution originates from pine bark extracts, which present bioactive properties. In this study, the antioxidant and anti‐inflammatory properties of Pinus radiata bark extracts were evaluated for the first time in a salmonid cell line. This extract chemically demonstrated antioxidant activity through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH = 58.4 ± 1.1%) and ferric ion reducing antioxidant power (FRAP = 575 ± 17 mgEqFe(II)·g extract^?1) assays. Additionally, the extract showed high flavonoid and phenolic compound contents. Up to 100 mg mL^?1, the P. radiata extract showed no cytotoxicity in the CHSE‐214 salmonid embryo cell line. Moreover, the antioxidant activity of the extract (50 μg mL^?1) was evaluated by a dichlorofluorescein (DCFH) assay in the SHK‐1 salmon cell line challenged with an oxidant stimulus (H₂O₂), showing 58.9% activity. The extract also protected DNA from oxidative damage, as observed through a comet assay. When assessing anti‐inflammatory properties in an in vitro inflammation model, the extract significantly reduced the relative expression of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) and of the inducible cyclooxygenase‐2 (COX‐2) enzyme. These results suggest a potential application of P. radiata bark extract in functional foods in aquaculture.     

Protective effect of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> polysaccharide against carbon tetrachloride-induced hepatic damage in precision-cut carp liver slices

The aim of the present study was to investigate the protective effects of Ganoderma lucidum polysaccharide (GLPS) against carbon tetrachloride (CCl₄)-induced hepatotoxicity in vitro in common carp. Precision-cut liver slices (PCLSs), which closely resemble the organ from which they are derived, were employed as an in vitro model system. GLPS (0.1, 0.3, and 0.6 mg/ml) was added to PCLS culture system before the exposure to 12 mM CCl₄. The supernatants and slices were collected to detect molecular and biochemical responses to CCl₄ and PCLS treatments. The levels of CYP1A, CYP3A, and CYP2E1 were measured by ELISA; the mRNA expressions of TNF-α, IL-1β, IL-6, and iNOS were determined by RT-PCR; and the relative protein expressions of c-Rel and p65 were analyzed by western blotting. Results showed that GLPS inhibited the elevations of the marker enzymes (GOT, GPT, LDH) and MDA induced by CCl₄; it also enhanced the suppressed activity of antioxidant enzymes (SOD, CAT, GSH-Px, T-AOC). The treatment with GLPS resulted in significant downregulation of NF-κB and inflammatory cytokine mRNA levels and significant decreases in the hepatic protein levels of CYP1A, CYP3A, and CYP2E1. These results suggest that GLPS can protect CCl₄-induced PCLS injury through inhibiting lipid peroxidation, elevating antioxidant enzyme activity, and suppressing immune inflammatory response.     

Antioxidant Potential of Water Hyacinth (Eichornia crassipes): In Vitro Antioxidant Activity and Phenolic Composition

The aims of the present study were (a) to extract and quantify the main phenolic acids and tocopherols from the petiole, leaves, and flowers of Eichornia crassipes; (b) to evaluate the antioxidant capacity of the extracts in four in vitro systems (1,1-diphenyl-2-pycryl-hydrazyl [DPPH] radical scavenging ability, iron chelating activity, reducing power, and prevention of oxidation in a liposome model system); and (c) its effectiveness in retarding lipid peroxidation in fish oil by accelerated stability test. Significant differences were observed in total and individual phenolic contents and in the antioxidant activities of extracts from the various parts of E. crassipes. Out of the 11 phenolic acids analyzed, ethanolic extracts contained high amounts of gallic, protocatechuic, gentisic, and p-hydroxybenzoic acid, whereas, water extracts contained less amounts of a varied number of phenolic acids. Ethanolic extracts of flower, which contained the highest total phenolic content, were found to have high DPPH radical scavenging activity and reducing power. However, ethanolic extracts of leaf exerted a high Fe²⁺ chelating activity and also inhibited lipid peroxidation process both in liposomes and fish oil. Our results demonstrate that E. crassipes, an underutilized aquatic weed, could be a potential natural antioxidant source for food, feed, and pharmaceutical applications.     

Improvement of Antioxidant Activity of Grass Carp (Ctenopharyngodon idella) Protein Hydrolysate by Washing and Membrane Removal Pretreatments and Ultrasonic Treatment

To improve the antioxidant activity of grass carp hydrolysate, washing and membrane removal pretreatments and ultrasonic treatment were applied in this study. Various pretreatment methods (washing methods and membrane removal methods) for removing the prooxidants and phospholipid in minced fish were evaluated. The effects of ultrasonic treatment prior to enzyme hydrolysis on the antioxidant activities of minced carp hydrolysate were investigated. The minced carp subjected to washing with 0.4% NaCl solution and subsequent membrane removal with CaCl₂ and citric acid solution had the lowest content of prooxidants and phospholipid remaining in the minced fish. The hydrolysate produced from washing/membrane removal pretreated carp with ultrasonic treatment for 20 min had the highest ferric reducing antioxidant power and scavenging activities against 1,1-diphenyl-2-picrylhydrazyl and 2,2?-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radicals. This suggested that the combination approach of washing/membrane removal pretreatment and ultrasonic treatment could improve the antioxidant activity of carp hydrolysate, and this hydrolysate could be a potential antioxidant ingredient for functional foods.     

Lipid peroxidation,protein oxidant and antioxidant status of muscle and serum for juvenile Jian carp (Cyprinus carpio var. Jian) fed grade levels of methionine hydroxy analogue

Lipid peroxidation, protein oxidation and antioxidant status of serum and muscle in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of methionine hydroxy analogue (MHA: 0, 5.1, 7.6, 10.2, 12.7, 15.3 g kg^?1 diet) for 60 days were investigated. Both malondialdehyde and protein carbonyl content in serum and muscle decreased with increasing dietary MHA level up to 5.1–10.2 g kg^?1 diet (P < 0.05). Anti‐hydroxyl radical and activities of catalase, glutathione peroxidase and glutathione reductase in muscle and serum, as well as anti‐superoxide anion, superoxide dismutase activity and glutathione content in serum, increased with optimal MHA supplement (P < 0.05). Meanwhile, glutathione‐S‐transferase activity in serum showed a downward trend with dietary MHA up to 7.6 g kg^?1 diet (P < 0.05). These results indicated that MHA improved antioxidant status and depressed lipid and protein oxidation in serum and muscle.     

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.     

Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein⁻¹ min⁻¹) followed by silver carp (1.084 ± 0.061 U mg  protein⁻¹ min⁻¹), and catla (0.193 ± 0.006 U mg  protein⁻¹ min⁻¹). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.     

Oxidative stress and antioxidant responses in juvenile Brazilian flounder <Emphasis Type="Italic">Paralichthys orbignyanus</Emphasis> exposed to sublethal levels of nitrite

This study evaluated the effects of short-term exposure to sublethal levels of nitrite on oxidative stress parameters and histology of juvenile Brazilian flounder Paralichthys orbignyanus. An assessment of fish recovery was also performed. Fish were exposed to 0.08 (control), 5.72, 10.43, and 15.27 NO₂-N mg L^?1 for 10 days followed by the same recovery time. Gill, liver, and muscle samples were collected after 1, 5, and 10 days of exposure and after recovery for the measurement of antioxidant capacity against peroxyl radicals (ACAP), glutathione-S-transferase (GST) activity, content of non-protein (NPSH) and protein thiols (PSH), and lipid peroxidation levels by thiobarbituric acid-reactive substances (TBARS) content. Nitrite exposure induced alterations which compromised the overall antioxidant system (reduced ACAP and GST activity) and enhanced oxidative damage in lipids and proteins. Increases in GST activity and NPSH and PSH contents were also demonstrated. The recovery period allowed for resumption of basal levels for all (treatment 5.72 NO₂-N mg L^?1) or some of the evaluated parameters (other treatments). In conclusion, exposure to nitrite concentrations from 5.72 to 15.27 NO₂-N mg L^?1 induced oxidative stress and antioxidant responses in juvenile Brazilian flounder. The 10-day recovery period was sufficient for a complete resumption of basal physiological condition of fish exposed to concentrations of up to 5.72 NO₂-N mg L^?1.     

Identification and characterization of maturation-promoting factor from catfish,Clarias batrachus

Maturation-promoting factor (MPF) extracted from maturing oocytes of catfishes (Clarias batrachus andHeteropneustes fossilis) and carp (Labeo rohita) induces 100% germinal vesicle breakdown (GVBD) when microinjected intoClarias immature unstimulated oocytes. The presence of a similar MPF activity has also been demonstrated in the active fractions collected after superose 12. SDS-PAGE analyses of cytosolic extracts (CE) prepared from immature and mature oocytes revealed the presence of 34- and 46 kDa proteins apart from a few others. Antibody against the PSTAIR sequence of p34^cdc2 recognized 32- and 34 kDa proteins of immature as well as mature oocytes while, 46 kDa protein of mature oocytes was recognized by anti-cyclin B1 antibody. Moreover, labelling of [³⁵S]methionine was observed mainly in the region of 46 kDa protein band indicatingde novo synthesis of this particular protein. Anti-cyclin A antibody did not recognize any proteins of immature or mature oocytes. Cyclin B1 was absent in immature oocytes and ovulated eggs. These findings indicate the presence of p34^cdc2 homologs and cyclin B in the MPF of the catfishes and carp oocytes.     

Evaluation of the effects induced by dietary diphenyl diselenide on common carp Cyprinus carpio

Several diets employed in aquaculture are enriched with selenium (Se), as it is a fundamental element to aquatic vertebrates. Diphenyl diselenide [(PhSe)₂], which is a synthetic organoselenium compound, has been considered a potential antioxidant agent in different experimental models. Thus, the aim of this study was to evaluate the effects of dietary diphenyl diselenide at concentrations of 1.5, 3.0, and 5.0 mg/kg for 60 days and to determine its optimal supplemental level for carp, Cyprinus carpio. Neither growth retardation nor hepatoxicity was induced by the inclusion of diphenyl diselenide at concentrations ranging from 1.5 to 5.0 mg/kg. In addition, the inclusion of 3.0 mg/kg of diphenyl diselenide stimulated the weight and length of the carp. The supplementation with 1.5 and 3.0 mg/kg of diphenyl diselenide did not produce oxidative damage in the tissues, verified by peroxidation lipid and protein carbonyl assays. However, at 5.0 mg/kg, it caused an increase of the lipid peroxidation in the liver, brain, and muscle, and inhibited the cerebral acetylcholinesterase activity. An increase of the hepatic superoxide dismutase activity and non-protein thiols content in all tissues and ascorbic acid in the liver, gills, and brain was verified in carp fed with the diet containing 3.0 mg/kg of diphenyl diselenide. This diet had advantageous effects for the fish used in experiments. Therefore, this compound could be considered a beneficial dietary supplement for carp nutrition.     

Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2′,5′-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K _m values for G6-P and NADP⁺ were determined to be 0.026, 0.0068 mM, respectively. The V _max values for G6-P and NADP⁺ were 2.20 and 2.27 μM min^?1 mg protein^?1, respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10^?6 min^?1 mg protein^?1, respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC₅₀ values of Zn⁺², Mn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn⁺², Al⁺³, and Cd⁺² showed competitive inhibition, while Cu⁺² inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.     

Effects of temperature and dietary protein on gene expression of Hsp70 and Wap65 and immunity of juvenile mirror carp (Cyprinus carpio)

Juvenile mirror carp were fed diets containing 303.4, 321.7, 341.2, 361.0 and 379.1 g kg^?1 proteins, respectively, and reared at different water temperatures (18, 23 and 28°C) for 60 days. Gene expression of heat shock protein gene (Hsp70) and the warm temperature acclimation‐related 65 kDa protein gene (Wap65), immunity and antioxidant status in the carp were investigated. Results indicated that the contents of serum complement 3 (C3), complement 4 (C4) and immunoglobulin M (IgM), as well as activities of liver superoxide dismutase (SOD) and lysozyme (LSZ) were significantly enhanced with increasing dietary protein (P < 0.05), while content of malondiadehyde (MDA) decreased. Gene expression level of Wap65 in the liver significantly increased with dietary protein, while gene expression of Hsp70 decreased. The contents of C3, C4 and IgM, the activities of SOD and LSZ and gene expression level of Wap65 in the liver significantly increased with temperature. These results suggest that: Serum immune parameter, antioxidant enzymes and Hsp70 and Wap65 expression interact in fish to improve ability to adapt to the environment; and the optimal conditions for the immunity of carp are 348.1?354.5 g kg^?1 protein at 18°C, 352.3?364.9 g kg^?1 at 23°C and 360.2?364.3 g kg^?1 at 28°C, and the optimum temperature for carp is 23°C.     

Investigation into the possible role of androgens in the induction of hepatic vitellogenesis in the European eel:in vivo andin vitro studies

Changes in the levels of plasma vitellogenin (Vg), estradiol (E₂) and testosterone (T) were examined following gonadal development induced by carp gonadotropin treatment (cGTH) of freshwater female yellow and silver eels (Anguilla anguilla L.). The animals received injections of cGTH (250 μg kg⁻¹ body weight) or saline vehicle three times a week, for 6 to 8 weeks. No effect of vehicle was observed. Steroidogenic activity of the ovary was stimulated by cGTH treatment as shown by the increase in circulating steroid levels in both stages. However, the responses of T, E₂ and Vg differed according to the stage of development of eels. At the yellow stage, the initial steroid plasma levels were undetectable (< 0.01 ng ml⁻¹) before treatment and ovarian steroidogenic activity was slightly stimulated following cGTH treatment; steroid levels reached their highest values after 3 weeks and 6 weeks of treatment for E₂ (0.62 ± 0.13 ng ml⁻¹ and T (0.33 ± 0.30 ng ml⁻¹), respectively. The cGTH treatment slightly increased plasma Vg levels (0.2–0.7 μg ml⁻¹ during the experiment compared with the initial values of the group. At the silver stage, the initial steroid levels were detectable (0.7 ng ml⁻¹ for E₂ and 0.1 ng ml⁻¹ for T); cGTH treatment did not significantly increase plasma E₂ level which remained at initial levels. Nevertheless, plasma T levels dramatically increased from 0.1 to 3 ng ml⁻¹ and peaked after 1 or 2 weeks of cGTH treatment; a rapid increase in plasma Vg levels occurred, reaching its highest value at 5 mg ml⁻¹ after 3 weeks of treatment. Thus, the steroid kinetic profiles in relation to the appearance of Vg in the plasma following cGTH treatment was closely related to androgen levels and there was a strong vitellogenic response induced by chronic cGTH treatment. In order to test if androgens could be implicated in the vitellogenic response, we evaluated the potencies of various androgens (testosterone and 5α-androstane-3β,17β-diol)in vivo andin vitro, associated with E₂ to induce the production of Vg.In vitro experiments showed that Vg synthesis was induced by high doses (10⁻⁶ to 10⁻⁵ M) of androgen in the eel. Tamoxifen totally inhibited the action of androgens suggesting that androgens were acting through binding to the E₂ receptor.In vivo, androgens given alone at 50 μg kg⁻¹ 3 times a week for 1 months had no significant effect on plasma Vg levels. In addition, E₂-androgen cotreatment showed that the presence of androgen did not modify the vitellogenic response induced by E₂.     

Effects of pyridoxine on antioxidative parameters in juvenile Jian carp (Cyprinus carpio var. Jian)

To investigate the effects of dietary pyridoxine (PN) on antioxidant status of fish in serum, intestine and trunk kidney, 1050 juvenile Jian carp (11.7 ± 0.1 g) were used for the experiment. The carp were divided into seven groups and fed diets containing graded levels of PN (0.20, 1.71, 3.23, 4.96, 6.32, 8.58 and 12.39 mg kg^?1 of diet) for 80 days. Results of the study showed that content of malondialdehyde in serum, intestine and kidney tissues was the highest when fed the diet containing 1.71 mg PN kg^?1 diet (P < 0.05). Meanwhile, the protein carbonyl content of intestine and kidney tissue showed a downward trend to a point (P < 0.05). Conversely, activities of superoxide dismutase, catalase, glutathione peroxidase (GSH‐Px), glutathione S‐transferase, glutathione reductase and glutathione (GSH) content in serum, intestine and kidney tissue were generally higher in PN‐supplemented diets than unsupplemented diet (P < 0.05). The present results indicated that the PN decreased lipid peroxidation and protein oxidation in fish, and partly because of its improved antioxidant enzymes activities and levels of GSH.     

鲢鱼鱼皮蛋白肽的制备与抗氧化活性评价

为制备抗氧化活性良好的鲢鱼鱼皮蛋白肽,采用胰蛋白酶、碱性蛋白酶、菠萝蛋白酶和木瓜蛋白酶等4种常见的商业酶对鲢鱼鱼皮进行酶解,测定酶解物的ABTS自由基清除力和Fe2+螯合力来评价其抗氧化活性,并用超滤及凝胶层析对酶解物进行分离,以期得到活性更好的酶解物分离组分。酶解后产物的抗氧化活性均有所提高,其中碱性蛋白酶酶解2 h产物活性较强。对此酶解物用截留分子量为10 k Da、5 k Da和3 k Da的中空纤维超滤膜进行超滤,得到的4个组分中,分子量越小的组分抗氧化活性越强。分子量小于3 k Da的组分经Sephadex G-15凝胶层析得到3个组分,其中分子量最大的组分活性较好,在0.51 mg/m L质量浓度下测定其ABTS自由基清除率和Fe~(2+)螯合力分别为(79.65±0.87)%和(93.40±0.20)%。该研究成果对鲢鱼鱼皮抗氧化肽的开发具有较好的指导作用。     

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACT

The serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl₂ (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as K_m (0.104 mM), k_cat (44.25 s^?1), and catalytic efficiency (427.54 s^?1 mM^?1). Monovalent, bivalent, and trivalent ions (Li⁺, K⁺, Hg²⁺, Al³⁺, Mg^2+, Cd^2+, Co^2+, Zn²⁺, and Al³⁺) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-_L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.     

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.