| Large yellow croaker (Pseudosciaena crocea) belongs to the Perciformes, Sciaenidae family, which are rich in protein and is an important economic fish in China. As the breeding areas of large yellow croaker, Fujian province enjoys natural advantage. However, the majority of large yellow croakers in Fujian province were sold fresh or frozen with low value added. Due to the improvement and expanded scale of large yellow croakers, the supply exceeds the demand of people. In response, enzymatic hydrolysis that produces peptides would be one of the most efficient ways to promote deep processing of large yellow croakers. In the present study, antioxidant peptides were prepared obtained from protein of large yellow croaker by controlled enzymatic hydrolysis assisted with ultrasonic pretreatment. The large yellow croaker peptides (LYCP) were fractionated by a series of ultrafiltration separation technology, and their antioxidant capacity was evaluated by various in vitro tests and animal experiments. To provide a technical basis for its application, the basic physical properties and oxidative stability of large yellow croaker peptides were examined. The metabonomics technology was employed to study the pathway and mechanism of peptides. On this basis, the peptides were further isolated and purified by ion chromatography, gel chromatography and reversed-phase high performance liquid chromatography. Consequently, the sequence of the active peptide was identified by mass spectrometry.First, the basic composition of large yellow croakers was analyzed, and amino acid composition was used to evaluate the nutritive value. The content of moisture, crude protein, fat and ash of large yellow croaker were 69.1%,17.5%,11.1% and 1.1%, respectively. Its amino acid composition was within the reasonable range and the proportion of essential amino acids was 40.84%, most of which were higher than ideal protein pattern except for valine. Among them, the proton affinity amino acids, aromatic amino acids and hydrophobic amino acids accounted for 40.96%,9.49% and 36.79% respectively, which exhibited a good protein resource for antioxidant peptides preparation.Alkaline protease, neutral protease, papain, trypsin, pepsin and compound protease were used in hydrolysis in large yellow croaker protein. In addition, through the comparison of hydrolysis degree of protein,O2-· scavenging ability and reducing power of hydrolysate, the neutral protease was chosen to be the best hydrolysis enzyme. With regards to single factor experiment and response surface experimental design, the optimal process conditions of neutral protease were determined as follows:hydrolysis temperature is 46℃; substrate concentration is 26.55%; hydrolysis time is 7.2 h; hydrolysis pH is 7.0 and enzyme dosage is 2400U·g-1. Under these experimental conditions, the hydrolysis degree of protein was 36.51%, and O2-·scavenging ability and reducing power reached 87.46% and 0.963. The yield of antioxidant peptides was 34.8%. Subsequently, ultrasonic pretreatment conditions were examined by means of single factor experiment and response surface optimization. The results indicated that ultrasonic pretreatment could significantly improve the antioxidant activity of peptides, for which the optimal ultrasonic pretreatment conditions were:ultrasonic power is 700 W; ultrasonic time is 24 min; and ultrasonic temperature is 59.3℃. In addition, the yield of peptides reached 40.1%.Subsequently, the LYCP was isolated by a series of ultrafiltration membrane with molecular weight cut-off of 10 kDa,5 kDa and 3 kDa and four different molecular weight ranges of peptides were obtained as followed:LYCP-Ⅰ (10 kDa), LYCP-Ⅱ (5～10 kDa), LYCP-Ⅲ(3～5 kDa) and LYCP-Ⅳ (< 3 kDa). On the basis of several in vitro chemical test, the antioxidant activity of different ultrafiltration fractions were compared. It turned out that LYCP-Ⅳ boasted the strongest in vitro antioxidant activity, the IC50 values of O2-· scavenging ability, DPPH-scavenging and Cu2+ chelating ability were 7.610,7.681,7.842 mg/mL respectively, and the reducing power was 0.721 at the concentration of 15 mg/mL. The effects of LYCP-Ⅳ on the cytotoxicity and H2O2-induced oxidative stress of HepG2 cells were further investigated. Consequently, LYCP-Ⅳ treatment was found to exhibit no significant toxic in HepG2 cells. To sum up, LYCP-Ⅳ significantly reduced H2O2 induced oxidative damage in HepG2 cells, increased the cell viability, and significantly increased the levels of SOD, CAT, GSH-Px and GSH in the cells.The in vivo protective effect of LYCP-Ⅳ on oxidative stress was investigated by virtue of D-galactose induced aging mice model. Results showed that low, medium and high-dose LYCP-Ⅳ and antagonistic group could ameliorate apathy, lethargy, unresponsiveness, hair removal and loss of appetite of mice compared with the model group, as well as significantly reduced the liver index. Besides, the liver index of the high-dose group and the antagonist group exhibited no significant difference (p>0.05) from the normal group. However, the weight gain of low, middle, and high-dose group, VE group and the antagonist group were significantly increased compared with the model group. Among them, the antagonist group exhibited the biggest weight gain. It turned out that LYCP-IV could reduce MDA content of D-galactose aging mice, enhance SOD, CAT, GSH-Px and GR activity, and increase GSH level. Additionally, the serum ALT and AST activity decreased significantly. And LYCP-IV exhibited satisfactory in vivo antioxidant activity.Subsequently, the protective effect of LYCP-Ⅳ on oxidative stress injury was examined from the perspective of metabonomics by means of established CCl4 oxidative damage model. Compared with the normal group, oxidative damage caused metabolism disturbance in glycolysis, amino acid catabolism, tryptophan cycle, tricarboxylic acid cycle and arachidonic acid metabolism. The metabolites of pyruvate, acetoacetate, methionine, cysteine,16 (R)-hete and arachidonic acid were reported to decrease significantly in the model group, while threonine, proline, tryptophan, serine, succinic acid, fumaric acid and linolenic acid increased significantly. By contrast, LYCP-IV treatment could adjust pyruvate, acetoacetate, threonine. proline, methionine, cysteine, succinic acid, fumaric acid, linolenic acid,16 (R)-hete and arachidonic acid content to the opposite trend or alleviate the disorder, suggesting that LYCP-IV could adjust the disordered metabolismof oxidative damage to a certain extent.The physicochemical and functional properties of LYCP-IV under different conditions and different concentrations were examined, including solubility, water absorption capacity, water holding capacity, oil absorption capacity, apparent viscosity, foaming properties and emulsifying properties. The results showed that the solubility of LYCP-IV was more than 80% at the range from pH 3 to 10. LYCP-IV at 40℃ boasted the maximum oil absorption capacity, which was significantly reduced when the temperature was higher than 70℃. LYCP-Ⅳ boasted preferable water absorption capacity but poor water-holding capacity. In addition, the lower apparent viscosity may be correlated with good fluidity of LYCP-IV. The pH4 may act as pI due to the lowest solubility, at which LYCP-IV possessed maximum emulsion stability, forming and forming stability that decreased with the increase of pH. On the contrary, emulsifying capacity decreased firstly with the increase of pH and then increased, which attained its minimum while pH reached 4. To sum up, the emulsifying capacity, foaming capacity and foam stability increased with the increase of concentration, while the emulsion stability showed a decline.The effects of NaCl, temperature, pH, ultraviolet radiation, sugars, metal ions, and simulated gastrointestinal digestion were assessed based on the antioxidant activity of LYCP-Ⅳ. LYCP-IV exhibited good stability when they were mixed with sugar at 30℃ or a low amount of NaCl, K+, or Ca2+. Temperature lower than 80℃ and short exposure to ultraviolet irradiation proved beneficial to the stability. Exposure to ultraviolet irradiation for longer than 24 h, or adding Fe2+, Fe3+, and Zn2+caused significant damage to LYCP-IV. Under the acidic and neutral pH conditions, pH value had little effect on the DPPH-scavenging activity of LYCP-Ⅳ, however, under pH>8, the DPPH-scavenging activity was significantly decreased. However, the Cu2+ chelating ability showed an opposite trend. The DPPH-scavenging activity of LYCP-IV was increased when peptides were mixed with reduced amount of sugar at 70℃. In addition, the antioxidant activity was increased when exposure to pepsin was less than 30 min, but declined with extended gastric digestion or further intestinal digestion. In addition, the change of antioxidant activity of LYCP-IV was consistent with the trend of hydrophobicity.According to the difference of charged, molecular weight size and hydrophobicity of LYCP-IV, ion exchange chromatography, gel chromatography and reversed-phase high performance liquid chromatography separation technology were employed for further separation and purification, and then the antioxidant activity of purified peptide fractions were evaluated. Finally, ESI-MS/MS were used for identification of amino acid sequence, and peptides with strong antioxidant activity were identified as Ser-Arg-Cys-His-Val and Pro-Glu-His-Trp. After being synthesized, antioxidant activity of the two peptides were verified by O2-·scavenging ability and DPPH-scavenging ability, which exhibited effective antioxidant capacity. |
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