【作者】 郭安鹊；

【导师】 李华；

【作者基本信息】 西北农林科技大学 ， 果树学， 2007， 博士

【摘要】 葡萄酒的氧化褐变严重影响了葡萄酒的质量,是葡萄酒生产中长期存在的一个重要问题。传统抗氧化剂二氧化硫（SO₂）因存在诸多问题尤其是对人体健康的不利影响而日益引起人们的忧虑,急需减少其使用量或寻找其替代物。然而,SO₂可能的抗氧化替代物-抗坏血酸（Vc）在葡萄酒中的应用也并不成功。因此,寻找有效防止葡萄酒氧化褐变的新型抗氧化剂,对降低SO₂用量,稳定和改善葡萄酒的质量,促进葡萄酒的发展具有重要的现实意义。论文基于对葡萄酒氧化褐变的机制和葡萄酒抗氧化研究现状的全面的、综合的分析,筛选出可能代替SO₂的抗氧化剂EDTA（乙二胺四乙酸）,通过模拟系统和葡萄酒中的抗氧化实验与SO₂的抗氧化作用进行了比较,并探讨了它们间的抗氧化协同作用,主要获得了以下研究结果:A.理论研究结果:铜铁离子在葡萄酒非酶氧化褐变过程中可能起着关键的作用。有关葡萄酒氧化褐变机制的大量文献综合分析表明:葡萄酒的氧化主要表现为酚类物质的氧化,它们的非氧化性褐变反应和它们在偶合氧化反应中的再生能力是导致葡萄酒褐变的根本原因;多酚氧化酶是酶促氧化链式反应的引发剂,而邻醌是能够产生多酚链式反应的活化粒子,以褐色素为最终产物,但主要限于在葡萄汁中发生;而在葡萄酒中,主要表现为非酶氧化褐变,虽然非酶氧化过程中也有邻醌的形成,且醌随后发生的反应与酶促氧化十分相似,但形成醌的速率十分缓慢,需要铜铁离子的参与,更多地表现为依赖铜铁离子的氧化褐变:铜铁离子在启动和延伸葡萄酒非酶氧化褐变反应中起着关键的催化作用。这对有目的地寻找葡萄酒中SO₂的抗氧化替代物有重要的指导意义。B.实验研究结果1.模拟酒系统中的实验结果:证实了铜铁离子（尤其是亚铁离子）在葡萄酒氧化褐变过程中的关键作用,并初步验证了EDTA的抗氧化效果（与SO₂相比）。参照目前典型葡萄酒中铜铁离子的平均浓度范围,实验首先研究了铜铁离子对模拟酒氧化褐变的影响,结果显示:随着离子浓度的增加,Fe²⁺促氧化作用增强,在实验的42天内,从第7天起酒样的褐变均表现为0级动力学,褐变速率常数（k）的变化范围是10～40×10-5/天,42天后,Fe²⁺浓度与酒样的褐变程度极显著正相关（R2=0.9838）;而在实验浓度条件下,Cu²⁺几乎没有催化氧化作用,一定程度上甚至抑制了酒样的褐变,与Fe²⁺配合使用也没有表现出协同催化氧化作用。实验42天后,多元线性回归分析结果显示:溶解氧消耗量（?DO）和氧化还原电位降低量（?Eh）与酒样褐变增加量（?A420）间极显著正相关（R2=0.8241 p<0.01）。随后,过氧化氢（H2O2）对模拟酒氧化褐变影响的实验结果表明:单独添加一定量的H2O2对酒样褐变的影响很小,但当与铜铁离子一起添加时,酒样中溶解氧（DO）含量,氧化还原电位（Eh）均显著下降,而褐变程度则急剧上升,现象十分明显。这说明:?OH在葡萄酒氧化褐变的过程中可能比H2O2发挥着更为重要的作用,也即Fenton反应对深入理解葡萄酒的氧化褐变十分重要。EDTA（25mg/L）和SO₂（50mg/L）抗氧化的初步实验结果显示,虽然EDTA效果不及SO₂,但低浓度的EDTA就能阻止铜铁离子的催化氧化作用,有效地抑制模拟酒的氧化褐变。2.葡萄酒中的抗氧化实验结果:在实验条件下,EDTA和SO₂均表现出了一定的抗氧化作用;虽然它们在白葡萄酒和红葡萄酒中的作用效果不同,但是两者配合使用后都表现出了很强的协同抗氧化作用。实验以EDTA和SO₂为抗氧化剂,通过饱和氧的方法,研究了它们在赤霞珠干红葡萄酒和霞多丽干白葡萄酒中的抗氧化作用。结果表明:在白葡萄酒中,在低浓度条件下（≤30mg/L）,EDTA的抗氧化效果优于SO₂,在红葡萄酒中,低浓度条件下（≤30mg/L）,SO₂的抗氧化效果则优于EDTA;两者抗氧化的机制也不同,EDTA主要是抑制了酒样对DO的消耗,但很难降低酒样的Eh,而SO₂抑制酒样消耗DO的作用很有限（且依赖于浓度）,主要是降低了酒样的Eh;两者配合使用后,低浓度下就表现出了很好的协同抗氧化作用,不仅有效地抑制了酒样对DO的吸收,而且稳定了葡萄酒的Eh,有效地降低了酒样的褐变程度。低浓度条件下,EDTA和SO₂均有效地防止了酚类物质的降解,但对主要酚类物质组成的影响在白葡萄酒中不同,在红葡萄酒中却差异不大;与应用同浓度的单一抗氧化剂相比,两者配合使用后更有效地抑制了葡萄酒中主要酚类物质的氧化降解,起到了很好的保护作用。3. EDTA和SO₂配合使用对葡萄酒质量影响的实验结果:两者配合使用后,EDTA都很好地增强了SO₂的抗氧化作用,对葡萄酒的质量也没有产生不利的影响。低浓度条件下（≤30mg/L）,EDTA和SO₂配合使用,处理酒样在室温下放置三个月后,评价了它们对葡萄酒理化指标和感官质量的影响,结果显示:无论是在红葡萄酒还是在白葡萄酒中,低浓度条件下,两者配合使用后,EDTA都显著增强了SO₂的抗氧化作用,有效地降低了葡萄酒的褐变程度,抑制了酒样中酚类物质的氧化,而且没有对酒样的理化性质和感官质量产生不利的影响。感官质量综合评价的结果表明:在实验条件下,白葡萄酒中以（20mg/L EDTA+30mg/L SO₂）处理的酒样最好,红葡萄酒中以（20mg/L EDTA+15mg/L SO₂）处理的酒样最好。综上表明,EDTA配合SO₂使用在酿酒学上具有重要的现实意义:不仅有效防止了葡萄酒的氧化褐变,没有对葡萄酒的质量产生不利的影响,而且显著降低了SO₂在葡萄酒中的使用量,在葡萄酒生产中有很好的应用前景。更多还原

【Abstract】 Oxidative browning badly affects the quality of wine and is a long-standing problem in winemaking. Sulfur dioxide （SO₂） is the generally used antioxidant to control wine browning. However, concerns over its ability to induce severe allergic reactions have created a great need for its reduction or replacement in recent years. However, ascorbic acid seems unsuccessful as an alternative antioxidant in winemaking, especially in combination with SO₂. So it is critically important to study new antioxidant（s） other than SO₂ to stabilize and improve wine quality. Based on the systematically comprehensive review of numerous literatures on wine oxidative browning and the prevention of wine browning, EDTA （ethylenediaminetetraacetate） was chosen as a possible alternative antioxidant, and then antioxidant experiments in model systems and wines were done to evaluate the antioxidant activities of EDTA compared with those of SO₂, as well as the synergistic effect between EDTA and SO₂. The major results achieved were as follows:A. Results achieved in theory: iron and copper may play central roles in the process of wine non-enzymic browning.Advanced research on mechanisms of oxidative browning of wine shows that, phenols′（especially vicinal diphenols′） regenerative capacity in coupled oxidation with other compounds and their non-oxidative browning reactions constitute the major reason for wine browning; PPO is the initiator of wine enzymic oxidation, which converts some phenols into unstable active o-quinones quickly in the presence of oxygen, consequently forming brown pigments, but it happens almost in grape must; although the formation of quinones and subsequent reactions induced by them are similar to those in enzymic oxidation, non-enzymic browning predominantly prevails in wine and mainly depends on iron and copper because the formation of quinones is too slow to compare with that occurring in enzymic browning. It indicates that iron and copper may play central roles in the initiation and propagation of wine non-enzymic browning, which has important implications in studying antioxidant substitute for SO₂ in wine.B. Results achieved in experiments 1. The results obtained in model systems verified the central roles played by iron and copper in the process of wine non-enzymic browning and the preliminary efficient antioxidant properties of EDTA and SO₂ in model wines were also investigated.Considering the concentrations of the present average range found for iron and copper in typical wines, we studied their roles involved in the browning in a wine-like system treated with saturated oxygen. Results showed that Fe²⁺ markedly accelerated the browning of samples and there were significantly positive correlations between browning degrees and Fe²⁺ levels in treated wines after 42 days of incubation （R2 = 0.9838）. Over a period of 42 days, browning development was shown to obey zero-order kinetics from the seventh day of the treatment, and the browning rate constants （k） varied from 10 to 40×10-5 day-1. However, with the levels used in the experiment, Cu²⁺ hardly influenced the browning of model wines and in turn had a negative effect on the browning as time went on. In addition, no synergistic effect of enhancing browning was observed between Cu²⁺ and Fe²⁺. At the end of the experiment （day 42）, the results of multivariate regression analysis indicated that, a significantly positive correlation existed between consumption of dissolved oxygen （?DO）, reduction of redox potential （?Eh） and enhancement of browning （?A420） （R2 = 0.8241 p < 0.01）. On the other hand, the addition of hydrogen peroxide （H2O2） alone hardly appeared to oxidize （+）-catechin in model solutions, but once in combination with iron and copper, the oxidative browning of samples was significantly increased, which might be explained by the Fenton reaction. It means the production of hydroxyl radical （·OH） other than H2O2 may be the major reason for wine browning.The primary results obtained in the antioxidant experiments with EDTA （25 mg/L） and SO₂ （50 mg/L） showed that EDTA had great potentials in preventing the samples from browning.2. The results attained in wines showed that, under the experimental conditions, both EDTA and SO₂ displayed good antioxidant activities, and a powerful synergistic effect was found between them when used together, although they presented different antioxidant activities in white wines and red wines.The antioxidant properties of EDTA and SO₂ in wines were examined, employing a dry red wine （Cabernet Sauvignon） and a dry white wine （Chardonnay） and the treatments saturated with oxygen. The results showed that at low concentration （equal or lower 30 mg/L） in white wine, the antioxidant activity of EDTA was better than that of SO₂, and however, under the same conditions in red wines, SO₂ was better than EDTA. The antioxidant mechanisms were different between them: EDTA mostly seemed to inhibit dissolved oxygen （DO） consumption by wine samples while it could hardly reduce the redox potential （Eh） of them; on the other hand, SO₂ could inhibit dissolved oxygen consumption by samples to some extent, especially depending on its concentrations, whereas it greatly appeared to decrease the Eh of wine samples. However, when EDTA and SO₂ were used together at low concentration, a strong synergistic effect was observed: not only inhibiting the consumption of DO by samples, but also stabilizing the Eh, and effectively decreasing the browning degrees of wines.The data on the effects of antioxidants on individual phenolics in wine indicated that EDTA and SO₂ both protected the individual phenols in wine with a low concentration, and had few different effects on the compostion of individual phenols in red wine, but the compostion of individual phenols in white wine influenced by them were different. Compared with a single antioxidant at the same concentration, EDTA in combination with SO₂ seemed more effective to prevent the oxidation of main individual phenols in wine.3. The effect of EDTA in combination with SO₂ on the quality of wine revealed that EDTA greatly increased the antioxidant activity of SO₂ both in white wine and red wine, not only effectively decreasing the browning degrees of wine, but also having no adverse effect on wine quality.The application of EDTA in combination with SO₂ at a low concentration to a dry white wine （Chardonnay） and a dry red wine （Cabernet Sauvignon） was examined, and the physical and chemical properties and the organoleptic qualities of the treated samples were investgated after they were stored at common temperature and common pressure for three months. The results showed that EDTA significantly increased the antioxidant activity of SO₂ both in white wine and red wine, effectively decreasing the browning degrees of wine and preventing the phenols from oxidation, and furthermore, not adversely affecting the physical and chemical properties and sensory qualities of wine. A full organoleptic evaluation of the treated wine samples showed that, under the conditions used in the experiment, samples added with 20 mg/L EDTA in combination with 30 mg/L SO₂ were the best in white wines, and samples added with 20 mg/L EDTA in combination with 15 mg/L SO₂ were the best in red wines.In conclusion, it suggests that the application of EDTA in combination with SO₂ has important implications in winemaking: it can effectively prevent wine from browning without adversely affecting the quality of wine, and most of important, significantly decrease the amount of SO₂ applied in winemaking, which seems very promising in the future.更多还原