Preparation Of Antifreeze Polypeptide From Pigskin Gelatin And Study On The Cryoprotective Activity

In this study, food-grade pigskin gelatin was hydrolyzed with Alcalase. Setting the cryoprotective activity of catalase as an index, the conditions for preparing antifreeze peptides were studied. The cryoprotective effect of gelatin peptides on catalase, surimi and Bulgaria lactobacillus were investigated. The detailed contents were as follows.(1) Research on the method of detecting cryoprotective effect by enzyme activity:a-amylase and catalase were adopted to study the cryoprotective activity of antifreeze peptides. With the same addition of antifreeze peptides, their residual activity were compared after the same freezing-thawing progress. The result showed that catalase was suitable to be chosen as the carrier to study the cryoprotection of gelatin peptides.(2) The optimization of conditions for producing antifreeze peptides from pigskin gelatin:Setting the cryoprotection of antifreeze peptides on catalase as an index, pigskin gelatin was hydrolyzed with Alcalase at various temperatures, times, pHs. and enzyme-to-gelatin ratios. After investigating the conditions that infect the cryoprotective effect by single factor experiment, the optimized conditions hydrolysis temperature at 37℃, hydrolysis time in 3 h, pH 9, and the enzyme-to-gelatin ratio in 1:50 were obtained by the orthogonal experiment.(3) The characterization of physicochemical property of antifreeze peptides:The hydrolysis degree of natural antifreeze peptides was 7.2%, the molecular mass range of pigskin gelatin hydrolysis was mostly between 150-2000 Da, their surface hydrophobicity and isoelectric point were respectively 363.0 and 3.93. The THA of antifreeze peptides was 0.14 ℃, and they show a certain effect of inhibiting of ice recrystallisation.(4) The investigation of cryoprotective effect on catalase:By comparing the cryoprotective activity of BSA, conventional cryoprotectant(4%sugar and 4%sorbitol)on catalase, the result showed that antifreeze peptides were 8.1%more effective than conventional cryoprotectant. Their cryoprotection on catalase and concentration had positive correlation.(5) The investigation of cryoprotective effect on Bulgaria lactobacillus:Methods of OD600 and flat plate counting were used to detect the cryoprotection of antifreeze peptides on Bulgaria lactobacillus. The result showed that with the addition of 0.25 mg/mL,the survival rate of Bulgaria lactobacilluswas 10% more than the control using the flat plate counting method. When using the method of OD600, with the addition of 5mg/mL, the survival rate was 34% more than the control.(6) By testing the changes of disulfide bonds, surface hydrophobicity, salt extractable protein content, Ca2+-ATPase activity during extend storage at-18℃, the cryoprotection of antifreeze peptides at levels of 2%,4%,8% in silver carp surimi was studied in comparison with adding 8% conventional cryoprotectant and blank group. The results showed that antifreeze peptides had cryoprotective effect on the silver carp surimi as shown by the effectiveness in retarding the increase of disulfide bonds and the surface hydrophobicity and maintaining protein solubility and Ca2+-ATPase activity. The cryoprotection of antifreeze peptides at level of 8% in surimi was the best, followed by 4%, conventional cryoprotectant,2% antifreeze peptides, and the cryoprotective effect of antifreeze peptides on surimi was superior to commercial cryoprotectant.