Preparation Of High Fisher Ratio Peptide From Pinus Koraiensis Sieb.et Zucc. And Its Anti-fatigue And Antioxidant Activity

With the fast-paced life and work,people in high-pressure state feel fatigued,manifesting as mental inactivity,decreasing work efficiency,mental and cognitive dysfunction,which seriously affect physical health.In recent years,there have been many reports that oxidative damage is an important factor in causing fatigue.Because its unique structural features,physicochemical properties and biological functions,high Fisher ratio peptide has become important research topics for research in recent years.In this paper,Pinus koraiensis Sieb.et Zucc.was used as raw material to optimize the enzymatic hydrolysis process of high Fisher ratio peptide.The target active peptides were separated and purified by ultrafiltration,ion exchange chromatography and gel filtration chromatography.The molecular weight distribution and amino acid composition of the target active peptides were initially investigated using Gel Permeation Chromatography and an amino acid automatic analyzer.The amino acid sequence was identified by matrix assisted laser analytical ionization time-of-flight mass spectrometry combined with amino acid analysis.An animal model was established to analyze the anti-fatigue and antioxidation effects in vivo.The optimum enzymatic hydrolysis conditions of the high Fisher ratio peptide of Pinus koraiensis Sieb.et Zucc.were determined.High Fisher ratio peptide from Pinus koraiensis Sieb.et Zucc.was prepared by pepsin and trypsin.The enzymatic hydrolysis conditions of trypsin were optimized mainly.The response surface analysis was carried out on the basis of the degree of hydrolysis and OD220/OD280.The optimum enzymatic hydrolysis conditions of trypsin were obtained as follows,p H was 7.98,the hydrolysis temperature was 44.68,the amount of enzyme was 5886.11 U/g,the time of enzymatic hydrolysis was 90 min,and the concentration of substrate was 6%.Fisher ratio was20.75(F>20)in pine nut high Fisher ratio peptide by automatic amino acid analyzer.,which accorded with the high Fisher ratio.The separation and purification of high Fisher ratio peptide from Pinus koraiensis Sieb.et Zucc.The high Fisher ratio peptide of pine nut were separated by ultrafiltration,ion exchange chromatography and gel filtration chromatograph.After separation,the component PPH1-D-? with the strongest antioxidant activity was obtained.GPC is used to determine the molecular weight distribution.The results show that the molecular weight of PPH1-D-? is mainly concentrated in 500~1500 Da,and the amino acidcomposition and content in the PPH1-D-? component are determined by the amino acid automatic analyzer.The results show that the Fisher ratio of PPH1-D-? is 29.58.Two peptides were identified by MALDI-TOF-MS combined with amino acid analysis.Their sequence structures were Asn-Gln-Lys-Pro-Asp-Phe-Glu-Asn-Asp-Asn-Gly-Arg-Phe and Arg-Gly-Glu-Glu-Glu-Glu-Glu-Ala-Glu-Glu-Arg,with molecular weights of1587.745 Da and 1362.598 Da,respectively.The anti-fatigue and anti-oxidation effects of PPH1-D-? were evaluated.The results showed that PPH1-D-? did not affect the body weight change and normal growth of mice.It also increased the organ index of mice,prolonged the time of heavy swimming in mice,increased the level of blood glucose and liver glycogen in mice,and improved the antioxidant enzyme system SOD and CAT.And the activity of GSH-PX decreased the content of lactic acid,serum urea nitrogen and malondialdehyde.The results showed that PPH1-D-? had a certain anti fatigue effect,and the correlation coefficient between SOD activity and liver glycogen,serum urea nitrogen and lactic acid in each dose of PPH1-D-? was 0.8688,0.9838 and 0.9312 respectively.The activity of GSH-PX in each dose and the liver were the same as the liver.The correlation coefficient of glycogen,serum urea nitrogen and lactic acid were 0.9216,0.9547 and 0.9685 respectively,which showed that PPH1-D-? had both anti fatigue and antioxidant activity in vivo,and there was a good correlation between them.The existence of fatigue and oxidative stress will cause the body's antioxidant defense system and immune system to be out of balance.Therefore,this subject further demonstrates the protective effect of PPH1-D-? on the body's fatigue damage system.The study found that PPH1-D-? can improve the immune ability of mice,reduce the body's energy consumption and accumulation of harmful substances in the fatigue state,improve the body's antioxidant enzyme activity,reduce the production of large amounts of free radicals.This study provides theoretical support for explaining the relationship between anti-fatigue and anti-oxidation,and has application value and guiding significance for further development of natural products as anti-fatigue and antioxidant substances.