| The Clitocybe squamulosa has a unique flavor,balanced nutrition,rich in a variety of nutrients required by the human body,and has high edible and medicinal value.In this experiment,Clitocybe squamulosa fruiting body powder was used as the material.The response surface method was used to optimize the extraction process of the Clitocybe squamulosa protein and its structure and rheological properties were analyzed.Further,the antioxidant peptide was prepared by enzymatic hydrolysis of Clitocybe squamulosa protein and a variety of isolation and purification methods were used to obtain a higher purity antioxidant peptide.The test results are presented as follows:Extracting Clitocybe squamulosa protein under the condition of slightly alkaline,the best extraction process was optimized using Box-Behnken test design and response surface method: liquid-material ratio31:1(m L/g),extraction liquid p H11.0,extraction time 60 min,extraction temperature 51?,the protein extraction rate reached 33.65±0.17%.Under these preparation conditions,the molecular weight of the protein isolated from the Clitocybe squamulosa is mainly distributed in the range of 31~66.2 k Da?14.4~22k Da;The secondary structure of the protein analyzed by the fitted infrared spectrum is mainly composed of?-helix,?-sheet,and ?-turn,and the relative contents which are 36.53%,27.66%,and 24.08%,respectively,and the proportion of random curl is at least 11.73%.The denaturation temperature and denaturation enthalpy of the sample protein analyzed by thermal differential scanning are 82.80?and 57.09 J/g.Exploring the steady-state rheological properties of Clitocybe squamulosa isolated protein under different processing conditions: Under steady-state shearing conditions,the rheological characteristic curve of the protein solution with mass concentration of 0.5%~5%,conforms to Herschel Bulkley model,presenting as shear thinning pseudoplastic fluid.When the shear rate is close to 100 S-1,the viscosity of the solution system tends to be stable,and the viscosity increases with the increase of protein concentration.When the heat treatment temperature is 75?,the apparent viscosity of the protein solution with a mass concentration of 3% reaches the maximum.High temperature treatment,slightly alkaline conditions and the amount of sodium chloride added will increase the apparent viscosity of the Clitocybe squamulosa protein solution.Steady state thixotropic properties: The concentration of 3% protein solution can form a clockwise thixotropic ring system.Dynamic rheological properties: Under dynamic oscillating shear conditions,Clitocybe squamulosa protein solution of 0.5%,1%,2% and 4% concentration exhibits a characteristic of viscoelasticity,as the mass concentration increased,the gelatinity of the protein solution system increased.Five commercial proteases were screened under the optimal conditions for enzymatic hydrolysis of Clitocybe squamulosa isolated protein.The degree of hydrolysis and the DPPH free radical scavenging rate of the hydrolyzed product were the measurement indicators.The test results showed that the hydrolysis effect of alkaline protease was the best,with a hydrolysis degree of 22.16% and a DPPH radical scavenging rate of the enzymatic hydrolysis product(5.0 mg/m L)of 64.52%.Then,the combined methods of ultrafiltration separation,DEAE-52 anion exchange chromatography and Sephadex G-15 glucan gel chromatography were used to hydrolyze Clitocybe squamulosa protein from alkaline protease.The hydrolyze product was separated and purified to obtain the highly active component CP2-2.After analysis by MALDI-TOF-MS laser analysis ionization flight mass spectrometry,its molecular weight was 305 Da.Analysis of the stability of CP2-2,an antioxidant peptide,under the processing conditions of lower than55?,p H4~8,has a good antioxidant stability;The strong acidity,alkalinity and high temperature and the environment of metal ion Fe3+and Cu2+ have obvious influence on the oxidation stability. |
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