【作者】 李景明；

【导师】 蔡同一；

【作者基本信息】 中国农业大学 ， 农产品加工及贮藏工程， 2003， 博士

【摘要】 白藜芦醇是葡萄在抵御真菌侵染、机械和紫外线等伤害时产生的植保素类次生代谢产物，通过发酵进入葡萄酒中。近年来研究发现它对人类健康特别是在预防心血管疾病等方面具有积极意义。 本论文采用食品检测分析方法和分子生物学研究手段，分别在产物水平和RNA转录水平上研究了紫外线(UV)照射、水杨酸(SA)喷施处理对采后葡萄中白藜芦醇合成的诱导作用，同时以葡萄酒企业生产为研究对象，通过对葡萄酒酿造过程中各工艺环节的监测及调控实验，发现了白藜芦醇在葡萄酒酿造过程中变化，提出了初步的调控意见。 论文建立了葡萄及葡萄酒中白藜芦醇的提取和检测方法。对比了常温提取、冷冻提取和热烫提取等三种方式从葡萄果皮中提取白藜芦醇的效果，发现采用热烫提取方法白藜芦醇含量明显高于常温提取。 实验确定了UV照射和SA喷施处理对玫瑰香和赤霞珠葡萄合成白藜芦醇的诱导作用。确定了UV诱导适宜剂量赤霞珠为450秒(1.35KJ／m~2)、玫瑰香为300秒(0.9KJ／m~2)，SA喷施的适宜浓度玫瑰香为100mg／L、赤霞珠为50mg／L。实验发现低温(10℃)贮藏不利于白藜芦醇的合成；光照(日光和弱光)减弱了玫瑰香葡萄UV诱导后白藜芦醇的合成，而对SA诱导后白藜芦醇的合成影响较小。UV和SA诱导后96小时的贮藏期内，玫瑰香葡萄中白藜芦醇含量和持续时间都高于赤霞珠。有关SA对白藜芦醇的合成诱导作用以及贮藏条件的影响均未见相关报道。 从UV和SA不同诱导处理后的赤霞珠葡萄果皮中提取到RNA，以sts和pal基因的保守性片段为探针进行斑点杂交，发现UV和SA处理引起了pal和sts基因的表达，其中sts表现出比pal更高的敏感性，受到诱导后容易产生表达，从而在转录水平上确认了UV和SA对赤霞珠葡萄采后白藜芦醇合成的诱导作用。 对葡萄酒生产的全过程监测发现，白藜芦醇总量在发酵结束后达到最高，澄清工艺和高温贮藏是造成白藜芦醇损失的主要因素，损失率分别达到15.7％和24.5％，氧化作用使葡萄在开启后4和8小时后，白藜芦醇总量减少了18％和38％。 原料品种和部分酿造工艺影响葡萄酒中白藜芦醇含量。赤霞珠葡萄酒中白藜芦醇含量高于梅露辄葡萄酒，采用UV诱导后赤霞珠葡萄酿造的葡萄酒，白藜芦醇含量提高了30％；采用贝酵母发酵，葡萄酒中白藜芦醇含量比D254酵母提高了11.8％；发酵温度提高(28℃)，白藜芦醇总量提高了32％。微孔错流膜分离技术(CMF)和速冷处理等澄清工艺对葡萄酒主要理化品质影响小，对白藜芦醇有较完整的保留。 本文的创新之处在于，以葡萄及葡萄酒品质为前提，通过UV和SA等无毒、无有害残留的方法对采后葡萄进行人工诱导处理，使在自然条件下只有遭受病虫害、机械伤时才能大量产生的白藜芦醇，在健康、品质良好的鲜食和酿酒葡萄中的含量得到显著提高，同时结合葡萄酒酿造、贮运等环节的调控，合理、安全地提高葡萄酒中白藜芦醇的含量，以此增加正常膳食中白藜芦醇的摄入。此项研究在葡萄产地加工、葡萄酒工业化生产中具有较大的推广应用前景。更多还原

【Abstract】 Resveratrol (trans-3,4’,5-trihydoystilbene) , as a phytoalexin, occurs in the berry skins of most grape cultivars and its synthesis is stimulated by the UV light, fungal infection and injury. It presents in red wine due to the fermentation with grape skin and seed. More and more researches prove that resveratrol has many beneficial effects on people health, such as reducing the risk of coronary heart disease (CHD), inhibiting platelets aggregation and low-density lipoproteins oxidation. In this research, the inducement of resveratrol with Ultraviolet(UV) and Salicylic acid (SA) in grape was studied and the change of resveratrol concentration during winemaking was analyzed. Base on the results obtained, some enological practices are suggested to be regulated in order to enhance the concentration of resveratrol in wine, so that the people can get more benefit from resveratrol via normal diet safely.Firstly, based on the published reports, a modified HPLC method for detection of four resveratrol isomers, including trans- and cis- resveratrol, trans- and cis- resveratrol glucosides, was established by using a PDA detecter. Three methods for extraction of resveratrol isomers from Muscat grape (after UV induce) skin were compared. The concentrations of fnms-resveratrol in the samples obtained by hot extraction and by RT extraction (Room Temperature) were 7.36 ug/g and 0.72 ug/g, respectively.The effects of UV and SA on the induction of resveratrol in Muscat and Cabemert Sauvignon were determined. According to concentration of resveratrol and the quality of grape (including sensory characters), the optimum time of UV exposure for the maximum resveratrol production was 300s (0.9KJ/m2) for Muscat (concentration of resveratrol rise to 11.13g/g) and 450s (1.35KJ/m2) for Cabemert Sauvignon (50.02g/g); the optimum concentration of SA treatment was 100mg/L for Muscat (65.42g/g) and 50mg/L for Cabemert Sauvignon (23g/g). Storage at low temperature (10 C) or under the sunlight after UV treatment restrained the synthesis of resveratrol in grape. However, the ray only has minor effect on resveratrol synthesis after SA inducement. In Muscat, the maximum concentrations of resveratrol is higher and last longer time than its in Cabemert Sauvignon. As we known, it is the first time to report the inducement of resveratrol by SA in grape.The total RNA was extracted from grapes skin (Cabemert Sauvignon) that had been treated by UV and SA, respectively. The Northern dot blot was performed using DNA fragments of sts and pal genes from grape as probes. Our results showed that the expression of sts and pal in grape skin was stimulated by UV and SA treatment. The expression of Pal gene strongly increased at 4-6h after the treatment with SA and at 6-24h after the treatment with UV. Sts expression increased at 1-2h after SA treatment and 1-6h after UV treatment It might indicate that the expression of sts gene was more sensitive than pal in grape skin upon the UV and SA application under our experimental conditions (Cabemert Sauvignon).Total Resveratrol, including four isomers, in red wine (Cabemert Sauvignon) get highest in the end of fermentation and decreased 15.7% in clarification subsequently. Under unsuitable storage condition, the resveratrol in wine might decrease greatly. The total content of resveratrol in wine could decrease 24.5% at high temperature(37 C) in darkness within 30 days. Opening for 4h, the loss of totalresveratrol in wine could be 18%, but it grew up to 38% after 8h.The wine made of Cabernert Sauvignon had more 30% total resveratrol than that made of Merlot. Using S.bayanus yeast was more efficient in total resveratrol accumulation than D254. Fermentation at 28 C took 32% more resveratrol than that at 25 C. Some new technology in winemaking, such as cross micro fluid membrane filtration (CMF) and rapid cold stabilization dynamic continous contact process were believed to fit wine filtration since they had only minor effect on the wine qualities and no effect on the total resveratrol.By UV and SA更多还原