【作者】 刘鑫；

【导师】 谢彦海； 胡小平；

【作者基本信息】 南昌大学 ， 生物与医药硕士（专业学位）， 2025， 硕士

【摘要】 石螺,学名方形环棱螺(Sinotaia quadrata),是我国淡水中常见的软体动物,富含蛋白质、维生素和矿物质。其肉质鲜美,且肌肉组织富含高质量蛋白质,是制备生物活性肽的优质原料。食源性抗炎肽通常由2至20个氨基酸构成,来源于可食用蛋白质,经酶解、发酵或合成后展现生物活性,相较于蛋白质和游离氨基酸更易被机体吸收,具有开发功能性食品和保健品的潜力。因此,本课题以石螺为研究对象,通过酶解超滤等手段从石螺肌肉组织分提取具有抗炎活性的多肽,利用动物实验探究其对炎症性肠病(Inflammatory Bowel Disease,IBD)的影响,并完成石螺肽相关的工业化产品设计,旨在提高石螺附加值,开发高生物活性产品。本论文主要研究方法、结果及结论如下:1.本实验成功筛选出碱性蛋白酶作为最佳酶解酶,并对石螺肌肉组织粗蛋白的酶解工艺进行优化。实验结果显示,碱性蛋白酶酶解产物无细胞毒性,并能显著抑制RAW264.7细胞NO的释放,具有较高的体外抗炎活性。最佳酶解条件为:碱性蛋白酶添加量1270.7 U/g,初始pH值8.9,酶解温度46.6℃,酶解6 h,料液比1:6。在此条件下,测得NO抑制率为43.17%±0.59%,验证了模型预测的有效性。此外,响应面实验表明加酶量和酶解温度对NO抑制率有显著影响,其中酶解温度影响最大。2.采用超滤法将石螺肽分离为三个次级组分:SQP(<3 kDa)、SQPⅠ(3-10kDa)和SQPⅡ(>10 kDa),通过RAW 264.7细胞模型评价各次级组分的抗炎活性。实验结果表明,SQP(<3 kDa)在抑制NO释放、TNF-α、IL-6和IL-1β表达水平方面表现出最优效果。为进一步验证SQP的抗炎活性,采用LC/MS-MS技术鉴定其中的肽段。共鉴定出133个肽段,其中84.96%的肽段长度为7-14个氨基酸。使用Peptide Ranker、AIPpred、PreAIP和PreTP-Stack四个工具预测肽段的抗炎活性,结果显示大多数肽段具有潜在抗炎活性。综合得分最高的四个肽段分别为GKFLWPGFGDNIR、FLWPGFGDNIR、APDWPPGMNFFPR和GSKPWISLPK。3.通过构建DSS诱导的IBD小鼠模型,研究SQP的抗炎活性及其对疾病症状的缓解效果。实验选用5周龄雌性BALB/c小鼠,分为5组:Control组、DSS组、DSS-Low SQP组、DSS-High SQP组和High SQP组。实验过程中,通过监测小鼠的体重变化、疾病活动指数(DAI)、脾脏指数、结肠长度、结肠组织学变化以及结肠组织中细胞因子(IL-6、IL-1β、TNF-α)的表达水平,评估SQP的干预效果。结果显示,SQP能够显著减轻DSS诱导的体重下降、DAI评分增加、脾脏肿大和结肠缩短等症状,并有效抑制促炎细胞因子(IL-6、IL-1β、TNF-α)的过度表达,尤其在高剂量(200 mg/kg)SQP干预下效果更为显著。组织学评价显示,SQP干预组的小鼠结肠组织损伤明显减轻,表现为炎性细胞浸润减少、杯状细胞和隐窝结构的保存。实验结果表明,SQP具有良好的抗炎活性,能够有效缓解DSS诱导的溃疡性结肠炎症状。4.利用16S rDNA测序技术,分析了Control组、DSS组、DSS-High SQP组和High SQP组小鼠盲肠内容物的肠道菌群结构和丰度变化。结果显示,DSS处理显著降低了小鼠肠道菌群的Alpha多样性,表明肠道微生物的丰富度和均匀度下降。然而,SQP干预显著缓解了这一现象,尤其是DSS-High SQP组的Shannon指数显著高于DSS组,表明SQP有助于维持肠道菌群的多样性。在物种组成方面,SQP显著抑制了DSS诱导的变形菌门丰度增加,并在一定程度上缓解了厚壁菌门丰度的下降。特别是在属水平上,SQP显著减少了有害菌如志贺氏菌属的丰度,同时增加了有益菌如拟杆菌属的丰度。这些结果表明,SQP可以通过调节肠道菌群结构,减轻DSS诱导的肠道炎症,进而改善溃疡性结肠炎的症状。5.以石螺肽为主要原料进行工业化产品设计。确定了石螺活性肽固体饮料的生产工艺、主要设备选型、物料衡算等,并进行了经济技术分析,同时绘制了厂区平面图、车间平面图及工艺设备图,最终完成年产15吨石螺活性肽固体饮料的工厂设计。总之,本研究成功得到石螺肽的最佳酶解工艺参数,利用细胞和动物模型对超滤后次级组分的抗炎活性进行评价,证明SQP具有良好的抗炎活性,并以此进行了年产15吨石螺活性肽固体饮料的工厂设计。更多还原

【Abstract】 Stone Snail(Sinotaia quadrata),a common freshwater mollusk in China,is rich in protein,vitamins,and minerals.Its tender muscle tissue,abundant in high-quality protein,serves as an excellent raw material for preparing bioactive peptides.Food-derived anti-inflammatory peptides,typically composed of 2–20 amino acids,are derived from edible proteins through enzymatic hydrolysis,fermentation,or synthesis.Compared to intact proteins or free amino acids,these peptides are more easily absorbed and hold potential for developing functional foods and health products.This study focuses on extracting anti-inflammatory peptides from stone snail muscle tissue via enzymatic hydrolysis and ultrafiltration,investigating their effects on inflammatory bowel disease(IBD)through animal experiments,and designing industrial products to enhance the added value of stone snails.The main methods,results,and conclusions are as follows:1.Alkaline protease was identified as the optimal enzyme for hydrolyzing stone snail muscle protein.Under optimized conditions(enzyme dosage:1270.7 U/g,pH8.9,temperature:46.6℃,hydrolysis time:6 h,solid-to-liquid ratio:1:6),the hydrolysate exhibited no cytotoxicity and significantly inhibited nitric oxide(NO)release in RAW264.7 cells,achieving a NO inhibition rate of 43.17%±0.59%.Response surface analysis revealed that enzyme dosage and temperature had the most significant impacts on NO inhibition.2.Ultrafiltration separated stone snail peptides(SQPs)into three fractions:SQP(<3 kDa),SQP I(3–10 kDa),and SQP II(>10 kDa).SQP(<3 kDa)demonstrated the strongest anti-inflammatory activity by suppressing NO,TNF-α,IL-6,and IL-1βin RAW264.7 cells.LC-MS/MS identified 133 peptides,with 84.96%containing 7–14amino acids.Four peptides—GKFLWPGFGDNIR,FLWPGFGDNIR,APDWPPGMNFFPR,and GSKPWISLPK—were predicted as top candidates for anti-inflammatory activity using Peptide Ranker,AIPpred,PreAIP,and PreTP-Stack tools.3.In a DSS-induced IBD mouse model,high-dose SQP(200 mg/kg)alleviated symptoms including weight loss,elevated disease activity index(DAI),splenomegaly,and colon shortening.SQP suppressed pro-inflammatory cytokines(IL-6,IL-1β,TNF-α)and reduced histopathological damage(e.g.,inflammatory cell infiltration,crypt structure preservation).4.16S rDNA sequencing revealed that SQP restored gut microbiota diversity(increased Shannon index),reduced harmful Proteobacteria(e.g.,Shigella),and enriched beneficial Bacteroidetes,demonstrating its role in mitigating DSS-induced dysbiosis.5.An industrial design for producing 15 tons/year of SQP-based solid beverages was completed,including process flow,equipment selection,material balance,economic analysis,and factory layout diagrams.In conclusion,this study optimized enzymatic hydrolysis for SQP extraction,validated its anti-inflammatory efficacy in vitro and in vivo,and proposed an industrial production design,offering a foundation for high-value functional products derived from stone snails.更多还原