| Letinous edodes is a kind of medicinal and edible fungus,not only succulent crisp and delicious taste,but also has a high nutritional value and medicinal value.Letinous edodes handle is a by-product of letinous edodes products in the process,letinous edodes accounted for about 20%-30%of dry weight,because the mushroom stem showed fibrosis,thick and tough,most have been abandoned,a small amount of feed every year,tens of thousands of tons of discarded letinous edodes handle,causing a great waste of resources.In fact,letinous edodes handle and mushroom cover is also rich in protein,amino acids,vitamins,minerals and other nutrients,so letinous edodes handle has a certain value of development and utilization.The effects of different extraction methods on the extraction rate of letinous edodes stem protein were studied.The extraction rate of letinous edodes handle protein by alkali extraction,cellulase and ultrasonic assisted extraction were 13.45%,18.57%and 22.44%,respectively.The ultrasonic extraction method was the best extraction method.The results of single factor experiments showed that,with the increase of the ratio of material to liquid,pH value,ultrasonic power and ultrasonic time,the extraction rate of protein showed a trend of rising first and then decreasing.The results of orthogonal experiments showed that the effects of various factors on the extraction rate of the protein were as follows:pH value>the ratio of material to liquid>ultrasonic time>ultrasonic power.The best combination of ultrasonic assisted extraction of letinous edodes stem protein was:the ratio of material to liquid 1:35,pH12,ultrasonic power 400 W,ultrasonic time 20 min,the extraction rate of letinous edodes handle protein was 25.971%.The results of variance analysis showed that pH had a significant effect on the extraction rate of protein(P<0.05),but the effect of the ratio of material to liquid,ultrasonic time and power on the extraction rate of protein was not significant.The functional properties of letinous edodes handle protein extracted by alkali extraction,cellulase and ultrasonic assisted extraction were compared.The solubility,emulsifying stability,foaming and foam stability of the letinous edodes handle protein obtained by ultrasonic assisted extraction were higher,which were 81.92%,86.56%,263.16%and 65%,respectively.However,the oil absorption and emulsifying activity of the letinous edodes stalk protein extracted by cellulase were higher,which were 6.60 mL/g and 1.54%.,respectively.In contrast,the letinous edodes stalk protein derived from the traditional alkali extraction method has a higher water holding capacity than other methods,and other functional properties are lower than those obtained by ultrasonic assisted or cellulase extraction.The letinous edodes stem protein was hydrolyzed by papain,neutral protease,trypsin,alkaline protease and compound protease,using the degree of hydrolysis and DPPH-scavenging rate as the index to determine the best enzyme source was alkaline protease.The hydrolysis of single factor experimental results showed that,with the increase of substrate concentration,the hydrolysis degree of letinous edodes handle enzymatic hydrolysate decreased gradually.And the DPPH· radical scavenging rate increased first and then decreased,which reached the highest while the substrate concentration was 3%.With the increase of the amount of enzyme,the hydrolysis degree of the hydrolysate of the letinous edodes stalk increased first and then remained stable.When the amount of the enzyme was 5000 U/g,the free radical scavenging rate of DPPH· reached the maximum value of 36.10%,and then showed a downward trend.The hydrolysis degree and DPPH· free radical scavenging rate of the enzyme hydrolysate of letinous edodes stalk increased firstly and then decreased with the increase of temperature.At 55? the degree of hydrolysis and DPPH· free radical scavenging rate reached the maximum.With the increase of pH,the degree of hydrolysis and DPPH· radical scavenging rate increased first and then decreased.When pH was 8.5,the degree of hydrolysis reached the highest,while pH was 9,the highest rate of DPPH· radical scavenging reached the highest.The degree of hydrolysis increased with the increase of time,and reached the maximum value at 3 h,and then remained unchanged,while the DPPH· radical scavenging rate showed a trend of increasing first and then decreasing with the extension of time.Using the DPPH· radical scavenging rate as the response value,the response surface experiment of four factors and three levels was carried out by using the concentration of substrate,the amount of enzyme,pH and enzymolysis temperature as the influencing factors.The results showed that the optimal parameters for the preparation of the letinous edodes handle polypeptide as follows:substrate concentration 2%,protease dosage 4700 U/g,pH 8.3 and hydrolysis temperature 55?.Under these conditions,to prepare letinous edodes handle antioxidant peptide when the hydrolysis time was 2.5 h,DPPH· radical scavenging rate can reach the theoretical value 63.20%.To do experiments under the same experimental conditions,DPPH· radical scavenging rate can get the actual value 62.02%,and there was little difference with the theoretical value.The letinous edodes handle peptide was separated by ultrafiltration to get three components:LP1(>10 kDa),LP2(3 kDa?10 kDa),LP3(<3 kDa).The determination results of antioxidant activity showed that LP3 has the highest antioxidant activity,the ·OH and O2-· scavenging rate and reducing power were 57.33%,51.74%and 1.1224.The component LP3 was separated by gel chromatography to get four components:LP3-1,LP3-2,LP3-3 and LP3-4.The different components was collected to determine the antioxidant activity.The results showed that the free radical scavenging rate and reducing power of LP3-1 and LP3-2 were lower,while the antioxidant capacity of LP3-3 and LP3-4 were higher.The ·OH and O2-· scavenging rate and reducing power of component LP3-3 were 50.75%,68.75%and 1.1913.While the ·OH and O2-·scavenging rate and reducing power of component LP3-4 were 60.44%,57.73%and 1.1630.According to the gel chromatography standard curve regression equation and different components retention time,the molecular weight of four components LP3-1,LP3-2,LP3-3,LP3-4 from letinous edodes handle polypeptide were 4150 Da,3264 Da,1587 Da and 1248 Da.Namely,the molecular weight of letinous edodes handle polypeptide range from 1248 Da to 4150 Da.Due to the large area of the components LP3-3 and LP3-4,so it could be concluded that the molecular weight of letinous edodes handle polypeptide was concentrated in 1248?1587 Da.The antioxidant activity and molecular weight of each component of the polypeptide from letinous edodes stem were determined.It was concluded that the antioxidant capacity of antioxidant peptide was closely related to its molecular weight. |
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