Preparation Of Oligo-peptide With High Fisher Ratio By Immobilized Enzymes From Squid

Squid minced meat, which is rich in protein and essential amino acids as well as squid whose essential amino acid composition is close to the whole egg protein, is produced in the squid processing. Although different physiological active proteins and peptides can be prepared by commercial proteolytic ways at present, higher cost and difficult large-scale applications limited this direct adding method. Immobilized enzyme is relative to the free enzyme. The immobilized enzymes have been widely used in many fields for their advantages especially for their stability, usability, low cost and easy separation from reaction system.Therefore, the preparation of High Fischer Ratio Oligo-peptide from the immobilized enzyme hydrolysis of squid minced meat by optimizing the enzyme immobilization process, controlling immobilized enzyme reaction conditions was studied. The realization of enzymolysis target products have provided technical basis for this technological method.The main aspects of this paper are as follows:The preparation of High Fischer Ratio Oligo-peptide from squid minced meat being hydrolyzed by immobilized pepsin and flavourzyme and adsorbed by multistage tandem activated carbon was studied. 10 g squid minced meat was taken as sample. Firstly, the optimal immobilization condition of pepsin by Box-Behnken response surface was studied. The results were: concentration of chitosan 2.50%, immobilized on the carrier for 3.40 h, immobilized temperature 35 ℃. The recovery of the immobilized pepsin can reach 74.38% ± 0.13% at the optimum conditions. And then the optimum hydrolysis method by immobilized pepsin was studied by orthogonal optimization. The results were: ratio of squid minced meat to water 1:5(g/m L), amount of immobilize pepsin to substrate 9 g, time 5 h, temperature 45~50 ℃. The degree of hydrolysis can reach 32.09% ± 0.58% at the optimum conditions. Secondly, the optimal immobilization condition of flavor enzyme by Box-Behnken response surface was studied. The results were: the concentration of sodium alginate 2.50%, ratio of the enzyme solution volume to sodium alginate volume 1:2, immobilization time of 3.00 h. Then the recovery of the immobilized flavor enzyme can reach 80.05% ± 1.33 %. And then the optimum hydrolysis method by immobilized flavor enzyme was studied by orthogonal optimization. The results were: amount of enzyme to substrate 8 g, p H 7.0, temperature 55 ℃. Then the degree of hydrolysis can reach 32.09%±0.58% and OD280 can reach 0.238±0.001. Thirdly, hydrolysates derived from squid minced meat were dynamically adsorbed by activated carbon in order to remove liberated aromatic amino acids(AAA). And then the optimum adsorption technology was studied by orthogonal optimization. The results were: volume of hydrolysates 150 m L, flow rate of hydrolysates 1.0 BV/h, p H 3.0, temperature 50 ℃, height of carbon layer 36 cm. The OD220/OD280 of hydrolysates can reach 24.61 ± 0.1044 after enzymatic hydrolysate being adsorbed by multistage tandem activated carbon. Lastly, amino acids compositions of hydrolysates absorbed by activated carbon were measured to calculate the F value. The determination results showed that the F value is much greater than 20. So the sample prepared by above technologies have already met the requirements of High Fischer Ratio Oligo-peptide.