Preparation And Immunological Study Of Active Peptides From Nibea Japonica Skin

In recent years,marine bioactive peptides have emerged in the field of food and health products because of their safety,non-toxicity and excellent characteristics.Nibea japonica is rich in nutrition and delicious,and its by-products,such as skin and scales,which produced in its food production and refining process,have high added value.In order to provide a basis for further research on its application,the preparation technology of bioactive peptide from Japanese croaker skin was optimized and its immune activity in vivo and in vitro was studied.In this study,protease hydrolysis method was used to determined the effect of enzymatic hydrolysate on the relative proliferation rate of RAW 264.7 cells.The enzymatic hydrolysis preparation process of active peptides from Nibea japonica skin was optimized by screening protease experiment,single factor experiment and response surface experiment.The immune activity of NJSAP on RAW 264.7 cells in vitro was investigated by MTT cell proliferation test,cell morphological changes,neutral red phagocytosis test,determination of nitric oxide?NO?secretion,determination of TNF-?,interleukin-6?IL-6?,interleukin-12?IL-12?secretion;the mice model of immunosuppression induced by cyclophosphamide was established by intraperitoneal injection.After the model has been established successfully,the mice was administered orally of NJSAP with 100,200 and400 mg/kg for 25 consecutive days.The changes of body weight and organ index were recorded.ConA-induced splenic lymphocyte transformation test,delayed allergy test,median hemolytic dose(HC ₅₀),phagocytic capacity of peritoneal macrophages,serum levels of tumor necrosis factor-??TNF-??,immunoglobulin?IgG,IgA,IgM?,total antioxidant capacity?T-AOC?,malondialdehyde?MDA?,superoxide dismutase?SOD?,catalase?CAT?and HE staining of spleen of mice were measured to investigate immune activity of NJSAP in vivo.The results showed that the optimum protease for enzymatic hydrolysis of Nibea japonica skin was neutral protease.The optimal enzymatic hydrolysis conditions were pH7,temperature 45.5°C,time 5.5 h,enzyme dosage 1500 U/g,liquid to material ratio 10:1.?mL/g?,the relative proliferation rate of the obtained enzymatic hydrolysis product on RAW264.7 was 57.47%,and the activity of the<3kDa segment was the highest after ultrafiltration;NJSAP in the range of 12.5-500?g/mL could increase the relative proliferation rate of RAW 264.7 cells.NJSAP can also differentiate the extended pseudopods of RAW 264.7 cells and promote the phagocytosis and secretion of NO,TNF-?,IL-6 and IL-12 of RAW 264.7 cells.Different doses of NJSAP could significantly increase the body weight and organ index of immunocompromised mice,and the difference of plantar thickness and spleen lymphocyte of mice were significantly enhanced,which indicated that NJSAP could promote cellular immunity of mice.Median hemolytic dose increased significantly,which indicated that NJSAP could promote the humoral immunity of mice,and the phagocytosis ability of peritoneal macrophages increased significantly,which indicated that NJSAP could promote the specific immunity of mice.The contents of TNF-?,IgG,IgA,IgM,T-AOC,SOD and CAT in serum increased significantly,while the contents of MDA in serum decreased.HE staining showed that the spleen of mice recovered.In conclusion,NJSAP has certain immune activity and can improve the immunity of mice,which provides a theoretical basis for further study of NJSAP.