Enzyme Hydrolysis Of Tuna Meat For Preparation And Isolation Of Active Peptide

Tuna is one of the important fishing species in the world ocean fishing and it is often used for producing sashimi, fish fillets, all kinds of canned tuna and other products. During the process, a lot of tuna waste, about 50%⁷0% of the total weigh, will be left. The waste is mostly produced into feed and has low economic value. We intend to extract peptides with antioxidant activity from tuna meat with protease digestion and ultrafiltration, which will be a reference for the comprehensive utilization of tuna meat.In this paper, four kinds of enzymes, trypsin, papain, neutral protease and alkaline protease were used for hydrolysis. Trypsin and papains were with good hydrolysis effects and selected for two-enzyme cooperative hydrolysis system. In order to maximize the efficiency of hydrolysis, the raw materials were first pretreated: the homogenated meat were first mixed with equal volume of isopropanol for degreasing at 50℃for 1.5h and then the defatted meat were under the microwave for 10min at microwave power of 500W and temperature of 50℃and the hydrolysis rate is about double increased. The main factors that influence hydrolysis rate were the addition order of two enzymes, enzyme dosage, reaction temperature, and reaction pH and hydrolysis time. Ater the single factor experiments and orthogonal tests, the results showed that: when the two enzymes were added together, the hydrolysis rate was the best and the optimum conditions for hydrolysis were: reaction temperature 45℃, reaction time 6h, trypsin and papain dosage were 15000U/g and 45000U/g, system pH7.0. Under these conditions, the degree of hydrolysis can reach 15.83%, about 2 times of the single enzyme hydrolysis.Enzyme solution usually contains many impurities, such as unhydrolysis meat and degenerated protease. Ultrafiltration is a commonly used separation method in enzyme solution. The enzyme solution was first filtered through 0.45μm membrane and then with membrane flux as an index, the solution was separated by membrane with molecular weight retention of 10kDa and 5kDa. A higher permeability of the membrane could be got under operating pressure of 30psi, ultrafiltration time of 10min, enzyme concentration of 10%, stirrer speed of 90r/min. Three sub-components were collected: componentâ… (molecular weight>10kDa), componentâ…¡(5kDa ¹0kDa), componentâ…¢(<5kDa) and the yields were 7.69%, 30.71% and 19.23%, respectively. The peptide concentrations of the three components were 2.3223 mg / mL (componentâ… ), 4.5858 mg/mL (componentâ…¡), and 3.3929 mg/mL (componentâ…¢) , in which peptide concentrations of componentsâ…¡andâ…¢were higher than sample (3.0024 mg/mL) and the enrichment effect is obvious. Antioxidant activity of peptide is measured through the minimum concentration required of eliminating 50% of free radicals (IC₅₀). With Vc as a positive control, antioxidant activity of three components were measured: (1) concentration of each component was diluted to 2.0000mg/mL while Vc was diluted to 0.0500mg/mL and Oyaizu method was used to measure the reduce power. The order from strong to weak was Vc> componentâ…¢> componentâ…¡> componentâ… . (2) Three components were diluted into a series of concentration gradient and regression curve about scavenging rate for peptide concentration was made. The results showed that the IC₅₀ of Vc and the three components for cleaning hydroxyl radical (?OH) was 1.0034mg / mL, 1.8923mg/mL, 1.7135mg/mL and 1.6419mg/mL, and scavenging ability order was Vc> componentâ…¢> componentâ…¡> componentâ… ; IC₅₀ of Vc and the three components for the superoxide radical ( ) were 0.7361 mg / mL, 2.1078mg/mL, 2.0327mg/mL, 1.7697mg/mL, and the scavenging order was Vc> componentâ…¢> componentâ…¡> componentâ… ; IC₅₀ for for diphenyl generation of bitter acyl radical (DPPH?) were 0.0793 mg / mL, 0.7662mg/mL, 0.6470mg/mLand 0.6541mg/mL and the order was Vc> componentâ…¡> componentâ…¢> componentâ… . This indicates that peptide has strong antioxidant activity in enzyme solution was concentrated in molecular weight below 10kDa and peptide molecular weight below 5kDa has the best antioxidant activity, but there is still a gap compared with Vc. If the purity can further be enhanced, there should be a good prospect.In this study, the nutritional characteristics of peptide were analyzed by amino acid score (AAS). The results showed that the amino acid composition of the sample is different with the reference amino acid pattern; the sampleâ…¡is rich in lysine and other amino acid compositions are similar to the reference amino acid pattern, and it can be used for the preparation of products with high lysine content; the sampleâ…¢is with methionine and phenylalanine Alanine deficiency and it can be added a certain amount of methionine and phenylalanine for a balanced amino acid diet made of protein products.