| This study is supported by two projects: the Jilin modern agriculture key research project-key technologies of mussel products processing and comprehensive product development(20150204034NY) and Changchun modern agricultural technology development support project-key technology of mussel products processing and comprehensive utilization of mussel resources(13NK13).China is rich in wild mussel resources with low prices. Mussel meat is tough, result in its low degree of processing and utilization. Biological active peptides have many functional properties which are beneficial to human body and are easy to absorb. By now, biological active peptides have become one of research hotspots. Therefore, the extraction of antioxidant peptides from wild mussel meat has a wide development prospects. In this study, ultrasound-assisted enzymolysis method is applied to obtain antioxidant peptides from wild mussel meat. The antioxidant peptides are purified by tangential flow filtration system and gel chromatography. Then the composition of amino acid, antioxidant activity, physicochemical properties and stability of the peptides are analyzed. Finally, mussel antioxidant peptides are encapsulated by liposome technology. This research aims to provide a theoretical basis for the comprehensive utilization of mussel and efficient extraction of antioxidant peptides. The main contents and conclusions are as follows:1. The effects of 6 proteases on the reducing power and hydrolysis degree of mussel hydrolysates were analyzed. Both singer-factor test and Box-Behnken design were used to optimize the experiment conditions. The results indicated that neutral protease was better and the optimum conditions were as follows: dosage of protease of 4141 U/g, temperature of 51℃, p H of 6.0 and enzymolysis time of 3.5 h. The effects of the factors on the reducing power of the enzymatic hydrolysates were in the following order: enzymolysis time > dosage of protease >p H> temperature.2. Based on the ordinary digestion process, ultrasonic technology was used to assist the preparation of mussel antioxidant peptides. Single-factor experiment and orthogonal test were carried out to obtain the optimal conditions. Results indicated that adding protease before ultrasonic treatment(dosage of protease 4141 U/g, p H 6.0) was better and the mixture was treated by ultrasonic under the condition of 40℃ and 150 W for 30 min. Then, the mixture was carried out at 51℃ with stirring for 180 min. Comparing with the common enzymatic hydrolysis method, the antioxidant activities of the product obtained by ultrasonic-assisted enzymatic hydrolysis were higher and more effective.3. Ultrafiltration separation of UAH(ultrasonic-assisted hydrolysates) has been studied. The UAH-Ⅳ(the molecular weight <3ku) showed a stronger antioxidant capacity among the four peptide fractions(with molecular weight >10 ku, 6~10 ku, 3~6 ku and <3 ku). Then the Sephadex G-25 was used to separate UAH-Ⅳand the separation conditions were studied. The results showed that the concentration of sample was 25mg/m L(1 m L), and de-ionized water was used to elute the peptides at a flow rate of 0.7m L/min. Four separate parts(F1, F2, F3, F4) were obtained under the condition, and F2 has higher antioxidant capacity.4. The composition of amino acid of the mussel meat, UAH-Ⅳand F2 were analyzed. The results showed that the content of hydrophobic amino acids of mussel antioxidant peptides after purified increased. Mussel antioxidant peptides are high in nutritional value. This product can be used for developing functional health food or advanced seasoning with the antioxidant activity.5. The antioxidant activities of UAH-Ⅳand F2 were evaluated by 6 kinds of tests in vitro. After purified by Sephadex G-25, the reducing power, radical scavenging activities and the ability to inhibit the oxidation of linoleic acid of mussel antioxidant peptides were increased, especially the hydroxyl radical scavenging capacity which increased by nearly 2 times. Both UAH-Ⅳand F2 showed a strong ability of chelating Fe2+. Mussel antioxidant peptides also showed a strong ability to inhibit linoleic acid oxidation, which was better than VC and GSH at the same concentration.6. The physicochemical properties and stability of the antioxidant peptides from mussel were analyzed and the conclusions were as follows: p H had a great influence on the solubility of UAH, but had little effect on UAH-Ⅳ. The emulsifying properties and foaming properties of UAH were higher than that of UAH-Ⅳ. The antioxidant activity of the mussel peptides was almost the same at the temperature of 4~50℃, however, its high temperature resistance ability was poor but can tolerate the low temperature pasteurization processing; p H had little effect on the reducing power of the sample, but had significant effect on the DPPH radical scavenging activity. Zn2+ had a strong inhibitory effect on the antioxidant activity of UAH and UAH-Ⅳ, while the effect of K+ was relatively weak. There were a strong inhibitory effect on the reducing power of the sample when treated with 6 kinds of food additives, but the impact on the DPPH free radical scavenging rate is relatively weak. Both UAH and UAH-Ⅳ had a strong ability of resisting gastrointestinal digestion.7. The preparation process of mussel antioxidant peptide conventional liposome was studied by singer-factor test and quadratic general rotary unitized design. The optimum conditions were as follows: ultrasonic time of 21 min, the ratio of lecithin to cholesterol of 3.3:1 and the ratio of lecithin to antioxidant peptides of 7.1:1.8. Mussel antioxidant peptide long circulation liposome was prepared by the modification of polyethylene glycol(PEG). And the freeze dried liposome was also prepared with sucrose/mannitol as protective agents. The results indicated that the activity retention rate was more than 90% after the preparation of liposome. And liposome can play a role in slow release. The storage stability of the freeze dried liposome was strong, and no change was found in organization status after 30 days.Innovation: 1. Ultrasonic assisted enzymatic method is applied to obtain antioxidant peptides from wild mussel, and activity, physicochemical properties, stability and amino acid composition were also studied to provide a theoretical basis for mussel high value utilization. 2. The rotary-film evaporation method and the freeze-drying method are applied to obtain mussel antioxidant peptide liposome, and to provide reference for the production of antioxidant peptide products. |
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