【作者】 杨晶；

【导师】 高冷；

【作者基本信息】 长春工业大学 ， 生物学， 2023， 硕士

【摘要】 本文以黑果腺肋花楸为原料,通过酿造工艺得到黑果腺肋花楸酒,应用大孔吸附树脂对酒中成分进行分离纯化;通过高效液相色谱-质谱联用技术（HPLC-MS/MS）确定果酒的功能性成分;利用网络药理学及分子对接技术分析黑果腺肋花楸酒中多酚类物质抗氧化及抗炎机制,并对其活性进行考察。研究内容如下:以单因素为基础实验,利用响应面法优化黑果腺肋花楸酒酿造工艺,最佳工艺为:发酵液含糖量17%、发酵温度26℃、发酵时间7天、酵母接种量0.8 g/L。在此工艺条件制得的黑果腺肋花楸酒,果酒酒精度为8.5±0.2%vol,呈红宝石色,澄清透明,口感醇厚,果香味与酒香味协调,感官评价达85分。检测酒中多酚含量为3.580±0.05 mg/mL,通过HPLC-MS/MS对其组成进行分析,得到黑果腺肋腺肋花楸酒中多酚类物质共7种。通过物质的保留时间及物质的特征碎裂方式分析,得知物质分别为矢车菊-3-O-半乳糖苷、矢车菊-3-O-葡萄糖苷、矢车菊-3-O-阿拉伯糖苷、矢车菊-3-O-木糖苷、槲皮素-3-O-芸香糖苷、金丝桃苷和槲皮素-3-O-葡萄糖苷。利用网络药理学对黑果腺肋花楸酒中多酚类物质进行研究,构建黑果腺肋花楸酒中7种活性物质与抗氧化“成分-靶点-通路”网络图及核心靶点互作图,分析其抗氧化作用机制。结果显示:黑果腺肋花楸酒中活性成分靶点与抗氧化疾病靶点相交119个,筛选出核心靶点有14个,有效通路10个,得知黑果腺肋花楸酒中活性成分以多靶点、多通路进行抗氧化调节。基于分子对接技术对黑果腺肋花楸酒中多酚类物质与抗氧化结合程度,明确黑果腺肋花楸酒中多酚类物质具有抗氧化作用机理。通过自由基清除实验研究黑果腺肋花楸酒中多酚类物质的抗氧化能力,结果表明酒中多酚类物质对DPPH自由基、·OH自由基和ABTS⁺自由基的IC₅₀分别为:0.476mg/m L、1.098 mg/m L和0.506 mg/m L,其抗氧化能力与多酚类物质浓度呈正比;通过脂多糖（LPS）诱导小鼠巨噬细胞（RAW264.7）设计体外炎症模型,MTT法检测多酚类物质对细胞生存率的影响,考察了黑果腺肋花楸酒中多酚类物质对RAW264.7中一氧化氮（NO）、前列腺素E₂（PGE₂）释放量的影响,证明黑果腺肋花楸酒中多酚类物质具有抗炎活性。更多还原

【Abstract】 Aronia melanocarpa is a new type of small berry with high nutritional and economic value.In this paper,the constituents of aronia melanocarpa wine were analyzed by brewing technology with aronia melanocarpa as raw material,and the constituents were separated and purified by macroporous resin.The functional components of fruit wine were determined by high performance liquid chromatography-mass spectrometry（HPLC-MS/MS）.The antioxidant and anti-inflammatory mechanisms of polyphenols were analyzed by network pharmacology and molecular docking,and their activities were investigated.The research contents are as follows:Response surface method was used to optimize the brewing process of aronia melanocarpa wine based on single factor experiment.The optimal process was as follows:the sugar content of fermentation liquid was 17%,fermentation temperature was 25.5℃,fermentation time was 7 d and yeast inoculum amount was 0.8 g/L,and the polyphenol content reached 3.580 mg/m L.Under the best technological conditions,the average alcohol content of the fruit wine was 8.5±0.2%vol,ruby color,clear and transparent,mellow taste,fruit flavor and wine flavor coordination,sensory evaluation up to 85 points.The polyphenol content was 3.580±0.05 mg/m L.HPLC-MS/MS was used to an alyze its composition,and 7 kinds of polyphenolic compounds were obtained.Accor ding to the retention time of the compounds and the characteristic splintering metho d of the compounds,we know that the compounds are cyanidin-3-O-galactoside,cy anidin-3-O-glucoside,cyanidin-3-O-arabinoside,cy-anidin-3-O-xyloside,quercetin-3-O-rutinose,hypericin,isoquercetin,among which cya-nidin-3-O-galactoside content was the highest.Based on network pharmacology,the"compose-target-pathway"network diagram and core target interaction diagram of 7 active substances and antioxidant of aronia melanocarpa were constructed to analyze the mechanism of antioxidant action.The results showed that 119 targets of active constituents and antioxidant disease targets intersected,14 core targets and 40 effective pathways were screened out,and it was concluded that the regulation of L.Aronia melanocarpa was multi-target and multi-pathway.The mechanism of antioxidant activity of polyphenols of aronia melanocarpa was analyzed based on molecular docking.The antioxidant capacity of polyphenols in the aronia melanocarpa wine was studied by free radical scavenging experiments.The results showed that the IC50 values of DPPH radical,·OH radical and ABTS⁺radical of polyphenols in wine were 0.476 mg/m L,1.098mg/m L and 0.506 mg/m L,respectively.The antioxidant capacity of polyphenols was directly proportional to the concentration of polyphenols.The cell inflammation model of mouse mononuclear macrophage（RAW264.7）cells was constructed by LPS induction.The cell survival rate was detected by MTT assay,and the release of nitric oxide（NO）and prostaglandin E₂（PGE₂）in RAW264.7 cells at different doses of polyphenols was investigated,which proved that polyphenols of aronia melanocarpa wine had anti-inflammatory activities.更多还原