Study On Preparation Of Antioxidant Peptide From Morchella And Its Antifatigue

Morchella spp.is a kind of precious edible mushroom with the same origin as medicine and food.It grows widely all over the world and is popular with people because of its delicious taste and anti-oxidation,anti-fatigue,liver and stomach protection and other physiological activities.The various functions of Morchella come from its rich bioactive substances,which are especially rich in protein.However,the current research on the physiological activity of Morchella is focused on the mixtures such as water extract and fermentation products,and the research on Morchella protein and peptide is very few,lack of sufficient development and utilization.Therefore,this paper used M.importuna as raw material,the structure and antioxidant activity of Morchella protein prepared by alkali extraction and acid precipitation,salt-soluble and salt-out,water extraction were compared.M.importuna peptide G3 component was prepared by sequential enzymatic hydrolysis and purification.The amino acid sequence of G3 components was identified by HPLC-MS/MS,and the fatigue model of mice was established to explore the potential mechanism of G3 components on fatigue,in order to provide a theoretical basis for the development of Morchella functional food.The main results are as follows:（1）M.importuna protein was prepared by alkali extraction and acid precipitation method,salt-soluble and salt-out method and water extraction method.It was found that the highest protein extraction rate was alkali extraction and acid precipitation method（AAMP）,and the highest purity was salt-soluble and salt-out method（SSMP）,The extraction rate and purity of protein prepared by water extraction method（MP）were the lowest.The amino acids of the three kinds of M.importuna proteins are rich,the essential amino acids of AAMP,SSMP and MP are 398.5 mg/g Pro,395.5 mg/g Pro and 311.0mg/g Pro,respectively,but the contents of hydrophobic amino acids and aromatic amino acids of SSMP are the highest,all of these amino acids contribute to the functional activity of proteins and peptides.SSMPS also have the loosest structure.The number of disulfide bonds was 2.57μmol/L/g,and the surface hydrophobicity was 116583.Infrared spectroscopy showed that the proportion ofαhelix,βfolding,βAngle and random curling in SSMP was 23.47%,22.87%,17.88%and 35.78%,respectively.The results indicated that SSMP was more inclined to react with protease during enzymolysis.The antioxidant activities of AAMP,SSMP and MP were all higher enough.It was found that MP had the highest scavenging ability of DPPH free radical,scavenging ability of ABTS free radical and reducing ability of iron ion,followed by AAMP and SSMP,there was no significant difference of bioactivity between the latter two.（2）Six proteases including alkaline protease,neutral protease,trypsin,pepsin,papain and flavourzyme were selected.The degree of hydrolysis,peptide yield,protein recovery and antioxidant activity were used as indexes to evaluate the enzymolysis of M.importuna protein prepared by salt-soluble and salt-out method,and the alkaline protease and neutral protease were selected to optimize the complex enzymolysis.Finally,it was decided to prepare M.importuna peptides by the sequential enzymolysis method of firstly adding alkaline protease,and then adding neutral protease.After purification by DEAE-32 and Sephadex G-25,the highest antioxidant activity of G3 was selected.The IC₅₀ value of scavenging DPPH free radicals was 0.122±0.003 mg/m L,the IC₅₀ value of scavenging ABTS free radicals was 0.652±0.025 mg/m L,and the reducing capacity of iron ion was 0.355±0.010 when the protein concentration was 1 mg/m L（Expressed as the absorbance of the sample reaction solution at 700 nm）.The active peptide sequences in G3 were identified as RVDWEF,VDWEFT,VDWE and DWEFT by HPLC-MS/MS.（3）Explore the fatigue-relieving effect and the enhancing-locomotor-ability effect on mice of M.importuna peptide G3 component by the exhaustion swimming test and the grip strength test.The results showed that the mice from medium-dose and high-dose group can swim longer and climb longer significantly than control group,moreover,the mice from high-dose group were recover better from liver and skeletal muscle damage.The treatment of G3 component significantly increased the amoumt of energy substances such as liver glycogen and blood glucose in mice,significantly decreased the metabolites such as lactate and urea nitrogen in serum,activated the endogenous substances such as creatine kinase and lactate dehydrogenase,and increased the activities of SOD,CAT and GSH-Px and decreased the MDA content.Besides,the G3 component also regulated the inflammatory response in the test mice,reducing the levels of anti-inflammatory factors IL-6,IL-1βand TNG-αand increasing the level of pro-inflammatory factor IL-10.（4）In order to further explore the potential mechanism of anti-fatigue of M.importuna peptide G3 component,the skeletal muscle protein of mice was analyzed by western blotting.The results showed that G3 component could activate Nrf2/ARE signaling pathway,reduce the expression of Keap-1 protein,and increase the expression of Nrf2 and HO-1 protein,so as to relieve oxidative stress and inflammation in the body.G3 component can also activate AMPK/PGC-1αsignaling pathway,promote AMPK phosphorylation,increase the expression of PGC-1αand TFAM protein,and enhance mitochondrial synthesis.In conclusion,M.importuna peptide G3 component may relieve fatigue and improve exercise ability by reducing the damage and inflammation caused by oxidative stress in tired mice and improving the energy material in the body.