Preparation Of Enzymatic Peptides From Chinese Giant Salamander(Andrias Davidiauns) Meat And Its Antioxidant And Immunomodulatory Activity Activity

Immunomodulating peptide is one of bioactive peptides that can improve the body's immune function."Compendium of Materia Medica"records that the Chinese giant salamander?Andrias davidiauns?has the effect of improving immunity,but there is no detailed evidence.Through the enzymolysis of the Chinese giant salamander meat,the immunity of the enzymatic peptide was screened by the cell immunity in vivo and in vitro,demonstrating that the giant salamander could be digested by commercial enzymes,and enzymatic peptides possessed good immune-enhancing activity,which can provide a theoretical basis for development of the Chinese giant salamander meat health-promoting products.The main findings were as follows:1.Preparation of enzymatic peptides derived from Chinese giant salamander meat.Flavourzyme,alkaline protease and trypsin were used to enzymatically hydrolyze the salamander meat.The enzymolysis conditions were pH 7.0,8.0,7.0,temperature 50°C,55°C,50°C,and the enzymolysis time was 3 h.The prepared three kinds of giant salamander enzymolysis peptides with hydrolysis degrees was 10.36%,28.55%,and 15.58%,and yields was 16.59%,18.27%,and 10.10%,respectively.Gel permeation chromatography?GPC?combined with internal standard method found that the molecular weight distribution of the three enzymatic peptides concentrated below 5 kDa,which were 99.73%,97.65%and 99.64%,respectively.Tricine-SDS-PAGE electrophoresis analysis found that the separation gel had two bands on 4.1 kDa and 14.4 kDa.The results of in vitro immunity experiments showed that the proliferation rates of the three enzymatic peptides on RAW 264.7 macrophages at the concentration of 400?g/mL were 35.97±1.56%,17.96±0.49%and 16.57±0.78%,respectively,used flavourzyme to prepare enzymatic peptide.The enzymatic peptides were separated by Sephadex G-25 gel filtration chromatography,and 4 components were observed and collected.The proliferation rate of RAW 264.7 macrophages for the four peaks at50?g/mL was 9.73±6.97%,23.23±7.23%,2.81±4.39%and 39.2±0.91%,the fourth component has strong cell proliferation ability.2.Enzymatic peptides by Flavourzyme possessed antioxidant activity.The results of the in vitro antioxidant assay showed that the scavenging rate of DPPH radical at 10 mg/mL was65.01±0.24%,and the IC₅₀ was 3.54 mg/mL;the scavenging rate of superoxide anion radicals at 2.5 mg/mL was 50.98±0.41%,its IC₅₀ was 2.47 mg/mL;at 2 mg/mL,the chelating capacity of ferrous ion was 74.11±0.42%,and its IC₅₀ was 1.14 mg/mL.The results of in vivo anti-oxidation experiments showed that enzymatic peptides can significantly increase the activity of GSH-Px in serum,heart,liver and kidney?p<0.01?;It can significantly improve T-AOC ability in the liver?p<0.01?;flavor proteolytic peptides at a concentration of 100 mg/mL can significantly increase SOD activity in the blood and heart?p<0.01?.Enzymatic peptides can significantly decrease the MDA level in serum and heart?p<0.01?.At 100 mg/mL,it can also significantly reduce the MDA level in liver?p<0.01?,and there is only a slight decrease in kidney?p>0.05?.3.Enzymatic peptides by Flavourzyme possessed immune activity.Cyclophosphamide is used to establish a low-immunity mice model by intragastric administration,and different doses of proteolytic peptides from salamander flesh protease,100 mg/mL of hydrolysate peptides from salamander meat can significantly improve the splenic lymphocyte proliferation capacity of normal mice.The phagocytic activity of mouse macrophages and the secretion of IL-6?p<0.01?,slightly increase the secretion of IFN-?and TNF-??p>0.05?.