【作者】 姜旭；

【导师】 李艳丽；

【作者基本信息】 吉林农业大学 ， 生物与医药（专业学位）， 2023， 硕士

【摘要】 赤松茸作为一种高端优质的食用菌,其具有长速快、产量高、适应力强等优点,近年来在我国的种植规模不断扩张。但草腐菌水分含量较多,容易开伞、腐败,失去商品价值。赤松茸蛋白含量在25%-35%之间,含有人体必需的8种氨基酸,营养价值评价与FAO/WHO提出的参考蛋白质模式相接近。近年来赤松茸的研究主要集中在多糖、黄酮和酚类等方面的开发利用,对赤松茸蛋白的开发鲜见报道。随着赤松茸种植产业的发展,需大力开发精深加工产业,提高赤松茸的利用度和附加值。鉴于此,本实验主要对赤松茸蛋白进行开发,制备赤松茸小分子生物活性肽,并探究赤松茸肽的抗氧化活性和免疫活性。1.采用超声辅助碱提法对赤松茸蛋白提取工艺进行研究,结果表明:碱提法提取赤松茸蛋白的最佳工艺参数是:料液比1:30,p H值为11,碱提温度为65℃,碱提时间为90 min,离心取上清,继续对废料进行超声辅助碱提,在料液比1:30,超声时间20 min,超声温度50℃,超声p H 10,超声功率200 W的条件下,蛋白提取率为51.09%,较碱提提高了5.11%。最后通过等电点沉淀(p I为3.8)和盐析(硫酸铵饱和度为50%)分离出赤松茸蛋白,冷冻干燥得蛋白质冻干粉。2.以赤松茸蛋白酶解物的水解度为参考指标,研究双酶同步酶解对赤松茸蛋白酶解液水解度的影响。通过单因素和响应面优化获得赤松茸蛋白酶解的最佳工艺参数为:p H值为7.8,温度为48℃,底物浓度为3.8%,加酶量为11000 U,酶解时间为6 h,最终赤松茸酶解液的水解度达到67.3%。3.通过超滤法和凝胶层析法对赤松茸酶解液进行分离纯化和冷冻干燥,并对各组分进行体外抗氧化活性和免疫活性的初步评价。结果表明:(1)酶解产物及各超滤组分均具有抗氧化活性,其中,酶解产物展现了最强的DPPH自由基清除率,3-10 k Da表现出最强的羟自由基清除率;<3 k Da表现出了最强的ABTS自由基清除率和金属螯合能力;酶解产物及<3 k Da均表现出最强的总还原力;凝胶过滤各组分中F1表现出最强的DPPH自由基清除率及总还原力;F2组分表现出最强的羟自由基清除率和金属螯合能力;F2及F1均表现出最强的ABTS自由基清除率。(2)赤松茸酶解产物及各组分均能促进RAW 264.7细胞的增殖,其中<3 k Da组分相对增殖率最高,还能促进RAW 264.7细胞对中性红的吞噬能力和NO的分泌。凝胶过滤后F2组分相对增殖率最高;F1组分促进细胞吞噬中性红的能力最强;F2组分促进细胞分泌NO的能力最强。更多还原

【Abstract】 Stropharia rugoso-annulata,as a high-end and high-quality edible mushroom,has advantages such as fast growth rate,high yield,and strong adaptability.In recent years,its planting scale in China has been continuously expanding.However,grass rot fungi have a high moisture content and are prone to opening umbrellas,spoilage,and loss of commercial value.The protein content of Stropharia rugoso-annulata is between 25%-35%,containing 8 essential amino acids for the human body.The nutritional value evaluation is similar to the reference protein model proposed by FAO/WHO.With the development of the Stropharia rugoso-annulata planting industry,it is necessary to vigorously develop the deep processing industry to improve the utilization and added value of Stropharia rugoso-annulata.In view of this,this experiment mainly develops the protein of Stropharia rugoso-annulata,prepares small molecule bioactive peptides of Stropharia rugoso-annulata,and explores the antioxidant and immune activities of Stropharia rugoso-annulata peptides.1.The ultrasonic assisted alkaline extraction method was used to study the extraction process of Stropharia rugoso-annulata protein.The results showed that the optimal process parameters for alkaline extraction of Stropharia rugoso-annulata protein were: material liquid ratio 1:30,p H value 11,alkaline extraction temperature 65℃,alkaline extraction time 90 minutes,centrifugation to extract the supernatant,and continued ultrasonic assisted alkaline extraction of the waste.Under the conditions of material liquid ratio 1:30,ultrasonic time 20 minutes,ultrasonic temperature 50 ℃,ultrasonic p H 10,and ultrasonic power 200 W,The protein extraction rate is 51.09%,which is 5.11% higher than alkaline extraction.Finally,Stropharia rugoso-annulata protein was separated by isoelectric precipitation(with a p I of 3.8)and salting out(with an ammonium sulfate saturation of 50%),and freeze-dried to obtain protein freeze-dried powder.2.Using the hydrolysis degree of Stropharia rugoso-annulata protease hydrolysate as a reference index,study the effect of dual enzyme synchronous enzymatic hydrolysis on the hydrolysis degree of Stropharia rugoso-annulata protease hydrolysate.The optimal process parameters for enzymatic hydrolysis of Stropharia rugoso-annulata were obtained through single factor and response surface optimization: p H value of 7.8,temperature of 48 ℃,substrate concentration of 3.8%,enzyme dosage of 11000 U,enzymatic hydrolysis time of 6 hours.Finally,the hydrolysis degree of Stropharia rugoso-annulata enzymatic hydrolysis solution reached 67.3%.3.The enzymatic hydrolysate of Stropharia rugoso-annulata was separated,purified and freeze-dried by ultrafiltration and gel chromatography,and the antioxidant activity and immune activity of each component were preliminarily evaluated in vitro.The results indicate that:(1)The enzymatic hydrolysates and various ultrafiltration components all exhibit antioxidant activity,among which the enzymatic hydrolysates exhibit the strongest DPPH free radical scavenging rate and 3-10 k Da exhibit the strongest hydroxyl free radical scavenging rate;< 3 k Da exhibited the strongest ABTS radical scavenging rate and metal chelating ability;The enzymatic hydrolysis products and<3 k Da showed the strongest total reducing power;F1 in each component of gel filtration showed the strongest DPPH radical scavenging rate and total reducing power;The F2 component exhibits the strongest hydroxyl radical scavenging rate and metal chelating ability;Both F2 and F1 showed the strongest ABTS radical scavenging rate.(2)The enzymatic digestion products and fractions of Stropharia rugoso-annulata promoted the proliferation of RAW 264.7 cells,with the <3 k Da fraction showing the highest relative proliferation rate,and also promoted the phagocytosis of neutral red and the secretion of NO in RAW 264.7 cells.The F2 fraction had the highest relative proliferation rate after gel filtration;the F1 fraction had the strongest ability to promote phagocytosis of neutral red;the F2 fraction had the strongest ability to promote NO secretion by the cells.更多还原