Study On The Separation Of Anticancer Peptides From Chlorella Protein And Its Microencapsulation

Chlorella, a kind of single-celled algae bloom, has many active ingredients and remarkablepharmacology and nutrition health care function. As very good health food and drug source, ithas extremely broad development prospects. This article optimized extraction of protein fromchlorella by the response surface, prepared peptides by enzymic method, at the same time toachieve protection of antitumor activity and the purpose of the slow release throughmicroencapsulation technology. The main contents and results were as follows:(1)Effects of ratio of liquid-solid, extraction pressure and broken numbers on total proteinsextraction were determined individually. By response surface design, the results showed thatoptimum conditions: ratio of liquid-solid21:1, extraction pressure, broken numbers4, theextraction yield of proteins was45.78%.(2) Three kinds of enzyme (Papain, Trypsin, Alcalase2.4L) were chose for hydrolysis. Basedon single-factor tests analysis and orthogonal experimental design, the conditions of eachenzymatic reaction were determined and optimized. Optimum enzymatic conditions of Papainwere E/S2%, temperature70°C, pH6.0, time4h, the degree of hydrolysis was14.33%.Optimum enzymatic conditions of Trypsin were E/S4%, temperature47°C, pH7.0, time8h,the degree of hydrolysis was8.47%. Optimum enzymatic conditions of Alcalase2.4L wereE/S2%(V/V), temperature50°C, pH8.0, time3h, the degree of hydrolysis was18.31%.(3)Each enzyme respectively hydrolyzed chlorella proteins under each optimum enzymeaticconditions. Three kinds of hydrolysate after wultrafiltration were obtained nine products andthe result showed that the3KD-5KD of hydrolysate by Papain had the strongest inhibitoryability on HepG2cell. Chlorella peptide was finally got after the ion exchangechromatography and gel chromatography separation technology on the basis of the inhibitoryability on HepG2cell.(4) The MTT assay results showed that, chlorella peptide and fluorouracil couldinhibit HepG2cell in vitro and proliferate of HepG2cell in a dose-dependentmanner. Its IC50were426μg/mL and314μg/mL. After treated with differentconcentrations of Chlorella peptide (0.1-0.5mg/mL) respectively, HepG2cells whichwere observed by inverted microscope manifested typical Characteristic of apoptosis.(5) The activity of antitumor Chlorella peptide that was gained and its activity wasprotected by microencapsulation. Microballoons of Chlorella antitumor peptide wereprepared by complex coacervation and ionotropic gelation in this experiment. The embedding rate were74.5%and30.1%, dosage were12.7%and12.3%. The vitro testcertified: the release rate of two kinds of microballoons was both low in the gastricfluid, but continued to release in the intestinal juice for the purpose of delayed release.Comparing the activity of antitumor embedment with not embedment, activity ofEmbedment had a little loss. The test showed that the activity of antitumor peptidewas protected by microencapsulation..