| Bioactive protein or peptide is an important active substance which has the functions of enhancing immunity,resisting oxidation,resisting fatigue,assisting in lowering blood pressure and so on.It was found that the bioactive peptides obtained by different proteases hydrolysis of the same protein source often have different biological activities,and the peptides obtained by pepsin hydrolysis often have better immune enhancement effect.In addition,polypeptides with different molecular weight sizes after enzymatic hydrolysis of the same protease have different biological activity effects.Chlorella protein content up to 50%of the dry weight,has antioxidant,antitumor,radiation resistance,disease-resistant microorganisms,reducing blood lipid,and enhance immunity and other biological activities,these functions must be closely related to the Chlorella protein and polypeptide.However,there are no reports on the function of Chlorella polypeptide in enhancing immunity,including which protease hydrolyzed Chlorella polypeptide has better activity of enhancing immunity,and which molecular weight range of peptides has the best effect of enhancing immunity.Therefore,Chlorella protein was extracted by high pressure homogenization combined with cellulase and pectinase.Chlorella protein was enzymatically hydrolyzed by pepsin,and the reaction conditions were optimized by response surface methodology.Chlorella polypeptides with different molecular weight intervals were prepared by multi-stage membrane separation technique.In vitro,RAW264.7 cells were used as cell model,and the best Chlorella polypeptide interval was screened by animal experiments.The results are as follows:(1)The cell wall of Chlorella was broken by high-pressure homogenization method.The optimum parameters of high-pressure homogenization were determined as follows:concentration of algae solution was 15%,homogenization pressure was 40MPa,homogenization times were 2 times.The cell wall-breaking rate of Chlorella was above 98%,and the protein concentration was 16.55mg/mL,accounting for 11.03%of the dry weight of Chlorella:(2)The optimum parameters of cellulase and pectinase were determined by orthogonal experiment:adding 10000U:8000U cellulase and pectinase per g Chlorella powder,pH 4.0,enzymolysis temperature 55℃,enzymolysis time 5h.At this time,the protein concentration was 21.85mg/mL,accounting for 14.57%of the dry weight of Chlorella,and the extraction efficiency was improved by 24.3%compared with 11.03%of the dry weight of Chlorella after homogenization.Subsequently,Chlorella protein powder was prepared by centrifugation,concentration,low temperature freeze drying and other experimental techniques.(3)The optimum parameters were determined by response surface methodology:substrate concentration 50g/L,enzyme dosage 1778 U/g,pH2.2,enzymolysis temperature 44.4℃ enzymolysis time 3.4 h,and the degree of hydrolysis of Chlorella protein was 54.94%.(4)Combining a multi-stage membrane separation technology,namely preparing the small ball polypeptides with different molecular weight intervals through ultrafiltration membrane separation with different pore diameters,and obtaining the Chlorella polypeptides with different molecular weight intervals through low-temperature freeze drying.(5)RAW264.7 cells were used as cell model to detect the effects of Chlorella polypeptide on proliferation,phagocytosis and cell morphology.The results showed that Chlorella polypeptide with molecular weight range of O～1kDa had the most significant effect on the proliferation and phagocytosis of RAW264.7 cells.At the same time,when RAW264.7 cells were stimulated by Chlorella polypeptide with different molecular weight range,the smooth and round cells showed irregular shape and extended pseudopodia.(6)The results of DNFB induced delayed allergic reaction in mice showed that the weight difference between the left and right ears of mice with 0～1 kDa polypeptides was significantly different from that of the negative control group(P<0.01),indicating that Chlorella polypeptides with 0～1kDa polypeptides could promote delayed allergic reaction in mice.Through ConA-induced lymphocyte transformation experiment in mice,it was found that the lymphocyte transformation ability of mice given peptides with molecular weight range of 0～1 kDa and 3.5-5kDa was significantly different from that of the negative control group(P<0.01),indicating that Chlorella polypeptides with molecular weight range had the effect of promoting lymphocyte proliferation and transformation in mice.In conclusion,Chlorella polypeptide with molecular weight ranging from 0～1kDa was found to have the best activity of enhancing immunity. |
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