栽培措施和灰霉菌病害对葡萄与葡萄酒香气成分的影响
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摘要
香气成分是影响葡萄酒香气质量的重要化合物。在葡萄酒生产中,可以通过控制水分、光照、病虫害等栽培措施来改变葡萄生长状况,调节葡萄与葡萄酒中的成分,从而达到提高葡萄酒质量的目的。本研究以搅拌子萃取法结合气相色谱质谱法(SBSE-GC-MS)为主要分析方法,研究了葡萄亏缺灌溉、日光照射处理、植株生长势区域差异及葡萄果实灰霉菌感染对葡萄或葡萄酒香气成分的影响,旨在为改进葡萄栽培管理措施和控制葡萄病害,生产高品质的葡萄酒提供依据和参考。主要研究结果如下:
     1.建立了葡萄果实中游离态和结合态香气成分的SBSE-GC-MS分析方法,并对这种方法进行了评价。该方法利用柠檬酸缓冲液对葡萄果实中的香气成分进行提取,然后对提取液进行离心和过滤处理。澄清提取液中的游离态香气成分直接利用搅拌子以1000r/min的转速提取3h后进行GC-MS分析。结合态香气成分经过C18柱分离和酶水解(45°C,20h)后进行SBSE-GC-MS分析。该方法所检测葡萄果实中香气物质标准曲线的线性关系良好,精密度和准确度较高。对黑比诺和梅尔诺葡萄果实的分析结果表明,这两种葡萄果实中游离态香气成分主要是脂肪酸衍生物、萜烯类、C_(13)-降异戊二烯类化合物,而结合态香气成分主要是醇类、萜烯类、C_(13)-降异戊二烯类物质和苯衍生物。结合态香气成分的含量比游离态香气成分的含量略高。
     2.对葡萄酒中香气成分的SBSE-GC-MS分析方法进行了评价,并利用该方法分析了4个黑比诺品系(292、MV6、114和G5V15)葡萄酒中的香气成分。这种方法是利用10mL饱和食盐水将10mL葡萄酒稀释,用搅拌子以1000r/min的转速提取3h后进行GC-MS分析。结果表明,这种方法可以快速、准确地分析葡萄酒中的香气成分。4个黑比诺品系葡萄酒中的香气成分主要是醇类、酯类、脂肪酸类、萜烯类和C_(13)-降异戊二烯类等。不同黑比诺品系葡萄酒中一些香气成分的含量存在显著差异(p <0.05)。
     3.研究了亏缺灌溉和高岭土处理对梅尔诺葡萄果实游离态和结合态香气成分的影响。本试验于2007年和2008年对梅尔诺葡萄植株进行不同水平的亏缺灌溉(需水量的35%、70%、100%和35-70%)结合叶面喷施或未喷施高岭土的处理。结果表明,亏缺灌溉降低葡萄果实中游离态C6化合物的含量,增加结合态萜烯类和C_(13)-降异戊二烯类物质的含量(如橙花醇、香叶醇、β-大马士酮、3-羟基-β-大马士酮、1,1,6-三甲基-1,2-二氢萘和3-酮基-α-紫罗兰醇)。高岭土处理对葡萄果实中多数香气成分的影响较小,且与亏缺灌溉没有交互作用。葡萄果实中结合态橙花醇、香叶醇和β-大马士酮的含量与其葡萄酒中相应游离态橙花醇、香叶醇和β-大马士酮的含量呈正相关,证明亏缺灌溉可以通过提高品种香气物质的含量来增强葡萄酒的花香和果香。
     4.研究了黑比诺果实日光暴露(摘叶处理)对其葡萄酒香气成分的影响,并通过紫外光阻隔试验探讨了紫外光的作用。结果表明,果实日光暴露处理对葡萄酒中多数酯类物质的含量没有显著影响,但是显著降低脂肪酸的含量,显著增加葡萄酒中一些萜烯类物质(香茅醇、橙花醇和香叶醇)的含量,且显著增加β-紫罗兰酮的含量。果实紫外光照射处理对葡萄酒中脂肪酸含量的降低和萜烯类物质的增加起着非常重要的作用。紫外光照射处理还显著降低葡萄酒中己酸乙酯、辛酸乙酯、壬酸乙酯和癸酸乙酯等酯类物质的含量,显著增加苯乙醇的含量,但是对C_(13)-降异戊二烯类物质没有显著影响。
     5.研究了黑比诺葡萄植株生长势的区域差异对其葡萄酒香气成分的影响。利用遥感图像技术将黑比诺葡萄园分为4个不同生长势区域(极低生长势、低生长势、中生长势和高生长势区域)。对不同生长势区域葡萄酒中香气成分的研究结果表明,随着葡萄生长势的降低,葡萄酒中一些酯类和醇类物质的含量呈增加的趋势。极低生长势区域葡萄酒中萜烯类如里哪醇和橙花醇的含量显著高于其它生长势区域葡萄酒中,但是低生长势和极低生长势区域葡萄酒中香茅醇的含量比中生长势和高生长势区域葡萄酒中低。另外,随着葡萄生长势的降低,葡萄酒中6,9-环氧基-3,5(13)-巨豆二烯和β-紫罗兰酮的含量呈增加的趋势,而β-大马士酮的含量无明显的变化规律。极低生长势区域葡萄酒中黑色浆果的香气最浓郁,而高生长势区域葡萄酒中这种香气的强度最低。高生长势区域葡萄酒中青草味和泥土味最强。利用判别分析法可以将不同生长势区域的葡萄酒进行很好的区分。遥感图像技术作为一种快捷的葡萄生长势测定方法可以为优质葡萄酒的生产提供了有力保障。
     6.比较了灰霉菌不同感染率(1%、2.5%、5%、10%、50%,w/w)的黑比诺葡萄果实所酿葡萄酒中香气成分含量的差异。与未感染葡萄相比,灰霉菌低感染率(<10%)的葡萄果实所酿葡萄酒中绝大多数香气成分的含量变化不显著,但50%灰霉菌感染显著增加葡萄酒中庚酸乙酯、苯乙酸乙酯、琥珀酸二乙酯的含量,而显著降低异丁酸乙酯、2-甲基丁酸乙酯和异戊酸乙酯的含量。与其它灰霉菌感染葡萄相比,50%灰霉菌感染葡萄所酿葡萄酒中庚醇和1-辛烯-3-醇含量最高,而异戊醇的含量最低。葡萄果实灰霉菌感染率较低时(<10%),其葡萄酒中的萜烯类物质如里哪醇和香茅醇的含量略有增加,但是随着灰霉菌感染率的进一步增大,葡萄酒中里哪醇、香茅醇、橙花醇的含量呈下降趋势。另外,随着果实灰霉菌感染程度的增加,其葡萄酒中壬醛、苯甲醛和1-辛烯-3-醇的含量呈增加趋势。果实灰霉菌感染对葡萄酒中C_(13)-降异戊二烯类物质的含量没有显著影响。
Aroma compounds play an important role in the quality of wine. Viticultural practicesby controlling water supply, sunlight exposure or disease were used to improve the quality ofgrape, and subsequently the wine. In this study, the effect of deficit irrigation, grape berrysunlight exposure, grapevine vigor variation, and the influence of grape berry Botrytis cinereainfection on volatile compounds of grape and wine were evaluated. Aroma compounds wereanalyzed by stir bar sorptive extraction-gas chromatography-mass spectrometry(SBSE-GC-MS). The major results were summarized as follows:
     1A SBSE-GC-MS detection method was developed to quantify free and bound aromacompounds in grape berries. Aroma compounds were extracted with citric acid buffer fromgrape berries, blended under liquid nitrogen, and the extracts were centrifuged and filtered.Free aroma compounds were determined directly by SBSE-GC-MS. Bound aromacompouonds were isolated with C18cartridges, and the resulting glycoside extracts wereenzymatically hydrolyzed. The released aglycons were determined by SBSE-GC-MS. Thevalidation results (correlation coefficient, recovery and repeatability) showed that this methodwas simple, fast, and could be used to accurately analyze most of aroma compounds in grapeberries. Free aroma compounds in Merlot and Pinot Noir grape berries, including fatty acidderivatives, terpenes and C_(13)-norisoprenoids were detected, while bound aroma compounds,classified into alcohols, terpenoids, C_(13)-norisoprenoids, benzene derivatives, were determined.In general, the concentrations of free aroma compounds were lower than those of theircorresponding bound aroma compounds.
     2The SBSE-GC-MS detection technique for analyzing aroma compounds in wine wasvalidated. A10mL wine sample was diluted with10mL salt water, and volatile compoundswere extracted by stir bar at1000r/min for3h. The validation results indicated that thismethod was fast, sensitive and accurate. The aroma compounds including esters, alcohols,fatty acids, terpenoids and C_(13)-norisoprenoids were determined in wines from Pinot Noirclones and concentration differences of aroma compounds in the Pinot Noir wines wereobserved.
     3The effect of deficit irrigation and kaolin particle film on aroma compounds inMerlot grape berries was investigated. Merlot grapevines were provided with100,70, or35%of estimated crop evapotranspiration (ETc) or35%ETcuntil veraison followed by70%ETcuntil harvest throughout berry development, and particle film (PF) was applied to half vinesin each irrigation main plot in2007and2008. The results indicated that the concentrations of free C6compounds (hexanal, trans-2-hexenal, and1-hexanol) in Merlot grapes decreased,and bound terpene alcohols (nerol and geraniol) and C_(13)-norisoprenoids (β-damascenone,3-hydroxy-β-damascenone,1,1,6-trimethyl-1,2-dihydronaphthalene, and3-oxo-α-ionol)increased in the berries each year in response to severity of vine water stress. Particle filmapplication had minimum effect on free and bound volatile composition in the grapes, andthere was no interactive effect between particle film and deficit irrigation. The concentrationsof bound nerol, geraniol and β-damascenone in grapes were positively correlated with theirconcentrations in wines, suggesting that water deficit could potentially contribute to Merlotwine aroma through influencing grape varietal aroma.
     4The effect of Pinot Noir grape berry sunlight exposure on volatile compounds of theresulting wine was studied, as well as the role of ultraviolet radiation. Pinot Noir grapevineswere subjected to sunlight exposure treatment conducted by basal leaf removal at the earlydays of veraison with cluster-shading treatment as control. UV radiation treatment wasconducted using filter to transmit natural UV radiation with UV exclusion treatment ascontrol. The results showed that most of esters were not affected by sunlight exposuretreatment, while sunlight exposure treatment resulted in significantly higher concentrations ofterpenoids including citronellol, nerol and geraniol, caused a decrease in the contents of fattyacids (octanoic acid, nonanoic acid and decanoic acid) and an increase in the level ofβ-ionone, as compared to bunch shading treatment. UV radiation exclusion treatmentsindicated that UV radiaton during grape ripening plays an important role in modifying thelevels of some volatile compounds including fatty acids, terpenes and esters in Pinot Noirwines. However, UV radiation had no effect on the levels of C_(13)-norisoprenoids, whichcontradicted the previous hypothesis.
     5The influence of spatial variation of vine vigor on aroma compounds of the resultingwines was investigated. Four vine vigor zones including ultra low vigor, low vigor, moderatevigor and high vigor zone, were determined by plant cell density (PCD) obtained from aerialphotography in Pinot Noir vineyard. In general, with the increase of vine vigor, theconcentrations of some esters and alcohols were decreased. The levels of terpenoids includinglinalool and nerol in wines from ultra low vigor were higher than those from other vine vigorzones with the exception of citronellol. Meanwhile, with the decrease of vine vigor, theconcentrations of vitispirane and β-ionone were increased. Based on the volatile composition,discriminant analysis illustrated the differences between wines from the four vine vigor zones.In addition, wines from ultra low vigor zones scored highest dark fruit, while those from highvigor zones scored the lowest. Wines from high vigor zones had the highest level ofherbaceous character, while ultra low vigor had the lowest. The results demonstrate thepotential of remote sensing as a rapid method to delineate vine vigor zones, producing wineswith high quality.
     6The effect of grape berry B. cinerea infection on volatile compounds of the resulting wines was investigated. Different levels of B. cinerea infected grapes (1%,2.5%,5%,10%,50%, w/w) harvested from Pinot Noir grapevines were fermented separately with healthygrapes as control. No significant difference was observed for the content of most of aromacompounds in wines from grapes with low B. cinerea infection rate (<10%). Compared withhealthy wines, wines fermented from50%B. cinerea infected grapes had higher contents ofethyl heptanoate, ethyl2-phenylacetate, diethyl butanedioate, while lower levels of ethyl2-methylpropanoate, ethyl2-methyl butanoate, ethyl3-methylbutanoate. The concentrationsof heptan-1-ol and1-octen-3-ol in wines fermented from50%B. cinerea infected grapes weresignificantly higher than those from healthy grapes, while the content of3-methylbutan-1-olwas lower. In addition, the concentrations of linalool and citronellol in wines from grapeswith low B. cinerea infection rate (<10%) were slightly higher than those in healthy wines,although, with the increase of B. cinerea infection rate, the concentrations of terpenes such aslinalool, citronellol, nerol in the resulting wine were decreased, while the contents of nonanal,benzaldehyde and1-octen-3-ol were increased. The content of C_(13)-norisoprenoids was notaffected by B. cinerea infection.
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