葡萄酒抗氧化功能与结合态香气研究

葡萄酒抗氧化功能与结合态香气研究

STUDY ON WINE’S ANTIOXIDANT CAPACITY AND BOUND VOLATILES

作者：田　玲　　导师：王　华

西北农林科技大学　　食品科学2011届博士

摘　要

目前葡萄酒研究领域的两大热点为葡萄酒的抗氧化功能与葡萄酒中结合态香气化合物，二者是葡萄酒消费者与爱好者最为关心的问题，本论文分别对葡萄酒抑制由自由基引发的胆固醇氧化的抑制能力与葡萄酒中结合态香气化合物以及影响其固相萃取回收率的因子进行了研究。

适量饮用葡萄酒对心脏病、动脉粥样硬化、血小板凝聚等心血管疾病以及中风、某些癌症都具有一定的预防与抑制的作用，主要是因为葡萄酒中所含具有抗氧化、清除自由基功能的各类多酚类物质；同时也有研究证明，胆固醇与胆固醇的氧化产物在人体中含量与心血管疾病紧密相关，故抑制由自由基引发的胆固醇氧化对降低上述疾病的发病率以及死亡率都具有重要意义。本研究采用了与健康人体中胆固醇含量基本一致的体外由自由基引发的胆固醇氧化模型调查了圆叶(Vitis Muscadinia rotundifolia）红葡萄酒，欧亚种( Vitis Vihifera )红，白葡萄酒抑制胆固醇氧化的能力。将供试样品葡萄酒与胆固醇悬浮液按照不同的体积比例混合，在该反应体系中加入2,2'-偶氮(2-甲基丙基脒)二盐酸盐（AAPH）作为自由基引发剂，加速胆固醇氧化，保持37℃的反应温度（与健康人体温度一致），利用搅拌子使其反应体系维持在均匀混合的状态，反应0 h、24 h、48 h、72 h后，利用高效液相色谱HPLC 测试胆固醇氧化的主要终端产物7-酮基胆固醇（7-ketocholesterol）的含量。对欧亚种红葡萄酒与白葡萄酒进行研究，结果表明欧亚种梅尔诺（Merlot）与赤霞珠（Cabernet Sauvignon）红葡萄酒二者拥有类似的抑制由自由基引发的胆固醇氧化的能力；欧亚种白诗南（Chenin Blanc）与缩味浓（Sauvignon Blanc）白葡萄酒二者也具有类似的抑制由自由基引发的胆固醇氧化的能力，但是欧亚种红葡萄酒抑制胆固醇氧化能力远远大于欧亚种白葡萄酒，红葡萄酒抑制胆固醇氧化的能力是白葡萄酒抑制胆固醇氧化能力的50倍；同时发现梅尔诺与赤霞珠红葡萄酒中儿茶素含量分别为140.6mg/L,132.5mg/L;白藜芦醇的含量分别为0.7 mg/L,1.0mg/L;而欧亚种白诗南与缩味浓白葡萄酒中儿茶素含量分别为10.6mg/L,11.0mg/L;白藜芦醇的含量均为0.3mg/L。同时本部分研究还证明了儿茶素抑制胆固醇的氧化的能力强于白藜芦醇。对圆叶红葡萄酒与欧亚种红葡萄酒进行研究，结果表明圆叶（Muscadine）与欧亚种梅尔诺红葡萄酒对由自由基引发的胆固醇氧化都具有很强的抑制作用，其中梅尔诺红葡萄酒抑制由自由基引发的胆固醇氧化的能力强于圆叶红葡萄酒。二者的主要抗氧化物质为儿茶素与花青素，在本部分实验中，样品酒中没有检测到白藜芦醇，圆叶与梅尔诺红葡萄酒花青素含量分别为150.1mg/L和150.3mg/L；儿茶素含量分别为15.6mg/L与140.6mg/L。相比较花青素而言，儿茶素与白藜芦醇在人体中消化后不易发生降解，同时更容易进入到人体血液系统中，而葡萄酒中白藜芦醇的含量又很低，由此可以推断出，在红葡萄酒抑制由自由基引发的胆固醇氧化儿茶素比白藜芦醇过程中发挥了更大的作用。依照实验测定结果，普通的佐餐红葡萄酒中儿茶素含量为150 mg/L左右，那么健康人每天饮用60-70mL红葡萄酒就能有效抑制人体中低密度脂蛋白胆固醇的氧化。

葡萄酒中香气主要是源自于酿酒葡萄（Vitis. vinifera）果实,在葡萄酒的酿造与陈酿过程中结合态香气前体物质对香气的释放，其主要是糖结合的半萜类化合物。在葡萄酒的生产过程中，葡萄果实中的一部分结合态香气化合物在其葡萄果实自身的酶，以及酵母释放的酶类物质的作用下，会被水解一部分，构成最初的葡萄酒香气，例如里拉醇；同时，另一类结合态香气化合物在酸作用下水解，发生化合物结构重排，释放出来游离态，阈值很低的葡萄酒香气主要贡献物，例如大马酮、TDN。由于葡萄酒中的结合态香气化合物始终影响到葡萄酒协调及优雅的香气的形成，故准确定量定性葡萄酒中结合态香气化合物至关重要。本研究采用 Lichrolut EN 商业包装固相萃取柱，利用固相萃取（SPE）提取方法提取葡萄酒中结合态香气化合物，然后在100℃温度下酸水解 1h提取到的结合态葡萄酒香气化合物，再利用GC-MS和标准样品鉴定葡萄酒中的结合态香气化合物。在本部分研究中，雷司令（Riesling）白葡萄酒与赤霞珠红葡萄酒中37与36种结合态香气化合物被鉴定，按照葡萄酒中香气化合物的不同生物来源前体物质，将其分为脂肪类香气化合物、异戊二烯类香气化合物、萜类香气化合物以及莽草酸类香气化合物。同时对可能影响葡萄酒结合态香气化合物固相萃取回收率的因子进行了研究，采用统计分析软件MODDE 6.0，利用响应面与全因子实验设计方法对实验进行了设计。研究结果表明葡萄酒参数乙醇含量、葡萄糖浓度、pH值以及单宁含量等对不同类型的结合态香气化合物固相萃取回收率影响不一样。总体上来讲，在雷司令白葡萄酒中，当乙醇浓度为3.75 mg /ml到　.75 mg /ml时，乙醇是影响其固相萃取结合态香气化合物回收率最主要的因素，第二是葡萄糖含量，第三是葡萄糖含量的平方；当乙醇含量降为3.75 mg /ml到63.75 mg /ml时，乙醇仍然是最重要的影响葡萄酒中固相萃取结合态香气化合物回收率最主要的因素，第二是pH值与乙醇含量的交互作用，第三是pH值。在赤霞珠红葡萄酒中，当乙醇浓度为3.75 mg /ml到　.75 mg /ml时，乙醇是影响其固相萃取结合态香气化合物回收率最主要的因素，第二是乙醇与单宁的交互作用，第三是pH值与单宁的交互作用；当乙醇含量降为3.75 mg /ml到63.75 mg /ml时，乙醇仍然是最重要的影响葡萄酒中固相萃取结合态香气化合物回收率最主要的因素，第二是乙醇的平方的作用，第三是pH值。当供试红白葡萄样酒中的乙醇浓度降为23.75 mg/ ml后，其葡萄酒中结合态香气化合物固相萃取的回收率不受上述因子的影响；通过本研究，弄清楚了影响葡萄酒结合态香气固相萃取（SPE）回收率的因子；同时也建立了一种简单、准确、快速定性定量检测葡萄酒中结合态香气化合物的方法。

关键词　葡萄酒　胆固醇氧化　结合态香气化合物　HPLC　GC-MS

Abstract

At present,wine antioxidant function and bound aroma attracted many studies. In this research,we focused on the capability of red and white wine to prevent cholesterol oxidation induced by free radical,the bound aroma of wine and the wine parameters which affect the wine bound volatiles SPE extraction recovery.

Moderate consumption of wine can reduce the incident of Cardiovascular diseases,platelet aggregation,stroke and some cancers,cause wine contain resveratrol and different kinds of phenols,and their antioxidant and capability. So it is very necessary to prevent cholesterol oxidation inducing by free radicals,which will decrease the incidence of above disease and mortality. In this study,capabilities of Muscadine (MCD),Merlot (MLT),Cabernet Sauvignon red wine,Chenin Blanc and Sauvignon white wine in inhibiting cholesterol oxidation were evaluated using cholesterol emulsion (CE) system,a free radical generator,2,2’-Azobis(2-methylpropionamidine) dihydrochloride (AAPH),was used to accelerate cholesterol oxidation at 37℃,the production of 7-ketocholesterol was detected after 0h,24h,48h and 72 h oxidation. In the research about white wine and red wine,the results show that Merlot and Cabernet Sauvignon red wines have the similar capability in reducing cholesterol oxidation reducing by free radical;and the Chenin Blanc and Sauvignon Blanc white wines have the similar capability of anti-cholesterol-oxidation,too;however,the red wines’ activity are almost equal to 50 times of white wines’ activity. The level of catechin in Merlot and Cabernet red wines are 140.6mg/L and 132.5mg/L,respectively;the resveratrol are 0.7 mg/L,1.0mg/L,respectively;　at the same time,the level of catechin in Chenin Blanc and Sauvignon Blanc are 10.6mg/L,11.0mg/L,respectively;and the resveratrol level of the two white sample wines are 0.3 mg/L. also,the results demonstrated that the antioxidant capability of catechin are much stronger than the resveratrol’s capability. In the research about Muscadine and Merlot red wine,the results demonstrated that the both of sample red wines can strongly prevent cholesterol oxidation inducing by free radicals,meanwhile,the activity anti-cholesterol-oxidation of　Merlot red wines is stronger than Muscadine red wines’;The mainly antioxidant compounds are catechin and anthocyanins,meanwhile,resveratrol was not determined,the content of anthocycanin in Muscadine and Merlot red wine are 150.1mg/L and 150.3 mg/L,respectively;and the content of catechin are 15.6 mg/L and 140.6mg/L,respectively. Compared with anthocyanins,catechin and resveratrol are more readily absorbed in the bloodstream without degradation after ingestion,it can be known that catechin rather than resveratrol may significantly contribute to the antioxidant capability of red wine in retarding cholesterol oxidation. Based on the results of that study,catechin level of normal table red wine is about 150 mg/L,consuming 60-70mL of the red wine would be helpful in inhibiting the oxidation of LDL cholesterol of a healthy person for 24 hours.

The aromas of a finished wine are absent in many wine-grape cultivars (Vitis. L. vinifera),but are instead generated during fermentation and storage from non-volatile precursors such as the glycoconjugates. Glycoconjugates (glycosides) posses an aglycone covalently bound to one or four sugar moieties via an ether linkage.　During wine production,glycosides may be hydrolyzed enzymatically by yeasts and exogenous glycosidases or non-enzymatically by acid-catalyzed hydrolysis.　Following hydrolysis,the liberated aglycone can have immediate odor impact (e.g. linalool) or else may rearrange under acid conditions to form odor-active compounds (e.g. β-damascenone,1,1,6-trimethyldihydronapHthalene). Because the bound volatiles always effect on the wine’s desirable aroma,it is very important to determine the bound volatiles. In this research,the bound volatiles of wine was extracted by　solid-phase extraction (SPE),with LiChrolut EN polymeric packing resin,then those bound compound were hydrolyzed in water bath at 100℃. In Riesling white wine and Cabernet Sauvignon red wine,there are 37 and 36 different bound volatiles were identified. Those aroma compounds are grouped according to the biological origin of the precursors which presumably generated the volatiles,using this criterion,four large groups can be differentiated: lipid derivatives,norisoprenoid,terpenes and shikimic acid derivates. In generally,the different wine parameters: ethanol level,glucose,pH and tannin have the different response to those aroma compounds of SPE extraction recovery. In white wine ‘Riesling’,when then ethanol level is between 3.75 mg/ml and.75mg/ml,ethanol is the most important parameter which affects SPE of bound volatiles recovery ,the following is glucose and Glucose×Glucose;when the ethanol level decreased,between 3.75mg/ml and 63.75mg/ml,the ethanol is still the most important factor,the following is pH×Eth and pH;In Cabernet Sauvignon red wine,when the ethanol level is between 3.75mg/ml and.75mg/ml,ethanol also is the most important parameter which affects SPE of bound volatiles recovery,the following is ethanol × tannin and pH ×tannin;when the ethanol decreased,between 3.75mg/ml and 63.75mg/ml,the ethanol is still the most important parameters,the following is Eth×Eth and pH. When the ethanol level is under 23.75mg/ml,there are no bound volatiles compounds will be affected by wine parameters. According to our study,we figured out the wine parameters affect the bound aroma’s SPE recovery;Meanwhile,a quickly,precise method was developed to determine those wine bound volatiles.

Key words　Wine　Cholesterol oxidation　Bound Volatiles　HPLC　GC-MS