【作者】 陈双；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 黄酒香气特征是黄酒品质的重要组成部分，其在很大程度上决定了消费者的消费选择。黄酒中的微量挥发性组分是构成我国黄酒独特香气特征的物质基础。黄酒中特征香气组分的解析和研究是明晰我国黄酒香气化学本质，实现黄酒香气特征调控的前提，对于提升我国黄酒产品品质具有重要意义。但是目前对我国黄酒中微量香气组分的研究还十分肤浅，构成黄酒香气特征的化学本质尚不清晰。本课题以我国民族特色酒精饮料——黄酒为研究对象，采用现代风味化学研究的研究方法，针对目前风味化学研究领域存在的技术挑战，通过新的风味化学研究技术手段及策略的开发，试图解析构成我国黄酒独特香气特征的风味化学本质及其变化规律。主要研究内容如下：（1）首次采用全二维气相色谱-飞行时间质谱联用技术（GC×GC-TOFMS）对我国典型黄酒样品挥发性组分特征进行了深入研究。建立并优化了适于黄酒挥发性组分鉴定分析的GC×GC-TOFMS方法。本研究联合使用固相萃取技术和固相微萃取技术结合GC×GC-TOFMS分析将我国黄酒中挥发性组分检测能力从百种化合物水平提升至千种化合物水平。初步分析鉴定出古越龙山黄酒中挥发性组分975种，其中有机酸类93种、酯类149种、醇类61种、醛类52种、缩醛类55种、呋喃及内酯类98种、硫化物42种、含氮化合物154种、酮类73种、酚类46种、其他类化合物152种。大量对黄酒香气特征可能具有贡献的香气化合物通过GC×GC-TOFMS分析得以检出。黄酒挥发性组分的GC×GC-TOFMS分析结果充分体现了我国黄酒挥发性组分特征的复杂性和多样性特征。（2）针对酒精饮料复杂基质中挥发性香气组分提取分离存在的挑战，研究创新了酒精饮料中挥发性香气组分提取及预分离技术。基于特殊吸附材料LiChrolut EN树脂建立的黄酒香气组分直接吸附萃取技术具有劳动强度小、有机溶剂用量少、操作简单等优点，能够有效的替代传统液液萃取的提取方法。基于快速蛋白纯化系统（FPLC）改造而成的正相硅胶色谱分离系统克服了传统硅胶柱色谱操作复杂、无法实现自动化、分离效率低的缺点，实现了黄酒复杂挥发性组分自动化、梯度化的高效预分离，极大的简化了后期香气化合物分离鉴定的难度。（3）采用气相色谱-闻香法（GC-O）结合气相色谱-质谱联用（GC-MS）分析技术在古越龙山传统型黄酒样品中共鉴定出香气化合物90种，其中近30种香气化合物在黄酒中首次检出。在鉴定出的香气化合物中，除了酒精饮料中常见的醇、酸、酯类香气物质外，苯甲醛（苦杏仁香）、香兰素（香草香）、反-1,10-二甲基-反-9-癸醇（泥土/发霉气味）、γ-壬内酯（椰子香）、愈创木酚（药香）、2,6-二甲基吡嗪（坚果香）、二甲基三硫（烂白菜气味）等香气物质具有较高的香气强度。这些香气化合物往往具有较为独特的香气描述，可能对构成我国黄酒独特香气特征具有重要贡献。采用香气稀释分析法（AEDA）比较了传统麦曲黄酒和无麦曲黄酒香气组分特征的差异，发现麦曲黄酒中苯甲醛、反-1,10-二甲基-反-9-癸醇、糠醛及大部分酚类香气物质的香气稀释因子（FD值）明显高于非麦曲黄酒样品。这些香气化合物可能是形成两种风格黄酒香气特征差异的主要香气物质。AEDA研究结果同时显示了黄酒酿造原料麦曲对黄酒香气特征的重要影响。（4）针对黄酒中香气组分物理化学性质差异大、含量跨度大的特点，提出了联合使用多种方法对黄酒香气组分进行全面定量分析的策略。利用不同分析方法各自的优点，建立了一套全面、精确测定黄酒中香气组分的方法体系。实现了对黄酒中从易挥发性组分（如乙醛）到难挥发性组分（如香兰素），从常量组分（如乙酸乙酯、3-甲基丁醇）到痕量组分（如反-1,10-二甲基-反-9-癸醇）的精确定量分析。该方法体系具有针对性强、灵敏度高的特点，是目前黄酒中香气组分研究最全面的定量分析方法体系。（5）采用本研究中提出的分析策略对我国典型黄酒样品的香气组分含量进行了全面定量分析。通过对古越龙山传统型黄酒77种香气组分含量的准确测定及香气活力值（OAV）的计算确定了对黄酒香气特征贡献较大的化合物有：香兰素、3-甲基丁醛、二甲基三硫、反-1,10-二甲基-反-9-癸醇、苯甲醛、γ-壬内酯等。通过比较传统型黄酒和清爽型黄酒香气物质香气活力值的差异明确了形成两种风格黄酒香气特征差异的主要香气化合物。进一步通过香气重构试验对前期研究结果进行了验证分析。试验发现使用香气化合物标准物质能够配制出与黄酒样品香气特征较为接近的黄酒模拟溶液，从而验证了前期定性、定量分析结果的有效性。（6）采用AEDA结合大体积进样GC-MS分析在甜型黄酒中鉴定出对黄酒“焦糖香”香气特征具有重要贡献的香气物质葫芦巴内酯（sotolon）。基于SPE和大体积进样GC-MS分析技术建立了黄酒中微量葫芦巴内酯含量的精确测定方法。该方法标准曲线线性范围为3.5-916.67μg L-1，定量限低至1.77μg L-1，远低于该物质在酒中的香气阈值（9μg L-1）。通过对不同类型黄酒中葫芦巴内酯含量分析发现不同类型黄酒样品中葫芦巴内酯含量范围在35.93-526.17μg L-1之间，明显高于其香气阈值，说明葫芦巴内酯对不同类型黄酒香气特征均有重要贡献。分析葫芦巴内酯在黄酒中的分布规律发现甜型和半甜型黄酒中葫芦巴内酯含量明显高于半干型和干型黄酒，说明黄酒中葫芦巴内酯的含量可能与黄酒中糖含量具有紧密的联系。（7）探索了黄酒酿造过程中酵母菌株及麦曲的使用对黄酒发酵过程及香气物质生成的影响。结果显示不同黄酒酵母对黄酒酿造常规理化指标并无显著影响，但对黄酒风味物质的形成具有显著影响。不同区域黄酒酵母的香气特征具有典型的差异。本论文研究结果表明黄酒酵母生产菌株的选用是调控黄酒香气品质的有效措施。研究了麦曲对黄酒发酵及香气物质形成的影响。结果发现麦曲在黄酒生产过程中不仅起到糖化剂的作用，还发挥着为酵母提供营养和“生香”的重要作用。麦曲通过对酵母代谢活性的影响及香气化合物前体物质的供给，能够显著影响黄酒香气特征的形成。麦曲的使用是我国黄酒酿造技艺的精华部分，这一结论早期体现在麦曲中复合酶系在黄酒独特边糖化边发酵过程中发挥的重要作用，如今更多的体现在麦曲对我国黄酒独特风味特征形成的重要作用。更多还原

【Abstract】 Aroma characteristic is one of the most important parameters for the quality of Chineserice wine, which greatly affects the choices of consumers. The aroma characteristic ofChinese rice wine is the result of the mixture and interaction of different aroma compounds.Characterization of the aroma profiles of Chinese rice wine is the precondition to modulatethe aroma characteristic of Chinese rice wine. It is very important for the improvement of thequality of Chinese rice wine. However, there are only a few researches have been carried outon the aroma composition of Chinese rice wine. The number and nature of the odorantsresponsible for the aroma characteristic of Chinese rice wine are still not known.The thesis is concerned with the volatile and aroma characteristics of Chinese rice wine.Modern flavor chemistry research approaches were employed to qualitatively andquantitatively measure the volatile and odor-active compounds in Chinese rice wine. We alsocompared the aroma profiles of different type Chinese rice wines and investigated theformation aroma compounds during Chinese rice wine manufacture. During the researchprocess new techniques and strategies were developed to deal with the technical challenges inflavor chemistry research. The main results were shown as follows:(1) A sensitive method for characterizing the volatile profile of Chinese rice wine usingcomprehensive two-dimensional gas chromatography coupled with time-of-flight massspectrometry (GC×GC-TOFMS) was developed for the first time. Comprehensivetwo-dimensional gas chromatography parameters were optimized for Chinese rice winevolatile compounds analysis. By combination of two sample preparation techniques andGC×GC-TOFMS, we investigated the detail of volatile profile of Chinese rice wine. A total of975volatile compounds were tentatively identified in Chinese rice wine, including93kindsof acids,149kinds of esters,61kinds of alcohols,52kinds of aldehydes,55kinds of acetals,98kinds of furans and lactones,42kinds of sulphur compounds,154kinds of nitrogencompounds,73kinds of ketones,46kinds of phenolics and152kinds of other compounds.The huge number of volatile compounds identified in Chinese rice wine showed the highlycomplex and diverse characteristics of Chinese rice wine.(2) New aroma extraction and pre-separation techniques for aroma composition researchin Chinese rice wine were developed in this study. LiChrolut-EN solid-phase extraction wasused to isolate the aroma components in Chinese rice wine. The results showed thatLiChrolut-EN had limited absorption of sugars, pigments, and other non-volatile compounds,while extracting a vast majority of polar and non-polar aroma compounds. This approach iseasy to use, labor-saving, solvent-saving and can be used to instead of traditional liquid-liquidextraction approach. A high-efficiency silica gel normal phase chromatography system wasdeveloped base on a fast protein liquid chromatography (FPLC) system. The newly developedsilica gel normal phase chromatography system overcame the disadvantage of traditionalsilica gel normal phase chromatography system, and could work automatically andgradiently.This system was very suitable for the pre-separation of complex aroma componentsin Chinese rice wine. (3) A total of90aroma compounds were identified in Guyuelongshan Chinese rice winesample by GC-O and GC-MS analysis, among them, nearly30kinds of aroma compoundswere identified in Chinese rice wine for the first time. In addition to acids, esters and alcohols,which commonly found in alcoholic beverages, benzaldehyde, vanillin, geosmin,γ-nonalactone, guaiacol,2,6-dimethylpyrazine and dimethyl trisulfide were identified to bepotentially important to Chinse rice wine. The aroma differences of Chinese rice winemanufactured with and without wheat Qu were investigated by aroma extract dilution analysis(AEDA). The results showed the aroma intensities of benzaldehyde, vanillin, geosmin, andphenolic compounds were much higher in Chinese rice wine manufactured with wheat Quthan that without wheat Qu. These aroma compounds might be the key aroma compoundswhich contribute to the characteristic aroma different of these two type Chinese rice wine.(4) A new strategy by combination of different quantitative methods was suggested forthe quantification of different aroma compounds in Chinese rice wine. By the combination ofHS-GC-FID, HS-SPME, SPE-GC-MS, and HS-SPME-GC-PFPD, a total of77aromacompounds were accurately quantified in Chinese rice wine.(5) The quantification and odor active values (OAVs) of Guyuelongshan Chinese ricewine showed that vanillin,3-methylbutanal,2-phenylacetaldehyde, dimethyl trisulfide,geosmin, guaiacol, benzaldehyde, and γ-nonalactone had important contribution to thecharacteristic aroma of Guyuelongshan Chinese rice wine. Comparison of the OAVs of aromacompounds between traditional and Qingshuang types of Chinese rice wines showed thatbenzaldehyde, γ-nonalactone, geosmin, guaiacol, vanillin, ethyl pentanoate, ethyl butanoate,ethyl3-methylbutanoate, ethyl hexanoate,3-methylbutanoic acid,3-methylbutanal, phenol,γ-nonalactone, and ethyl cinnamate were the most important aroma compounds responsiblefor the aroma profile differences between those two types of Chinese rice wine. Aromareconstitution model prepared by mixing aroma compounds quantified in Chinese rice wine inan odorless Chinese yellow rice wine matrix showed a good similarity to the aroma of theoriginal Chinese yellow rice wine.(6) The aroma characteristics of sweet-type Chinese rice wine were studied by sensoryanalysis, aroma extract dilution analysis (AEDA), and quantitative analysis. Sotolon wasidentified as key aroma compounds in Chinese rice wine for the first time. AEDA resultsindicated that sotolon (caramel-like) was the potentially key odorant responsible for thetypical caramel-like descriptor of sweet-type Chinese rice wine. The concentration of sotolonin Chinese rice wine was further quantified by Lichrolut-EN solid phase extraction coupledwith microvial insert large volume injection method. The content of sotolon that wasquantified ranged from35.93to526.17μg/L, was above its threshold (9μg/L) for all Chineserice wine samples. The highest concentration of sotolon was found in the sweet-type Chineserice wine, which highlighted the key aroma role of sotolon for this particular type of Chineserice wine.(7) The influence of yeast strains and wheat Qu on the Chinese rice wine fermentationand aroma compounds formation were investigated in this study. The volatile flavor profilesof the different Chinese rice wines showed statistically significant differences depending onthe yeast strains used. Yeast strains from the Shaoxing region showed a higher capacity to produce the esters, β-phenylethanol and3-methylthiopropanol, while yeast strains from theShanghai region stood out for their production of branched-chain higher alcohols. Chineserice wine fermented with a yeast strain from the Jiangsu region had the highest levels oforganic acids. The data obtained in this work shows that the yeast strains contributedsignificantly to the flavor differences of the Chinese rice wines from the different regions.Small-scale Chinese rice wine brewing showed that wheat Qu not only act as saccharifyingagent, but also can increase the yeast activity and the formation of aroma compounds. Higheryeast growth and fermentation rate, and lower yeast dead rate were observed when wheat Quwas used as saccharifying agent. Global analysis of the aromatic profiles revealed that the useof wheat Qu can greatly promote the aroma compounds formation in Chinese rice winefermentation. The total concentration of aroma compounds was1.40folds higher in Chineserice wine fermented with16%wheat Qu than the Chinese rice wine fermented with mixedenzymes. The concentrations of volatile phenolic compounds and higher alcohols wereobviously higher in Chinese rice wine samples fermented with wheat Qu. The role of wheatQu cannot be simply replaced by commercial enzymes, but the Chinese rice wine aromaticprofiles can be modulated depending on the amount of wheat Qu used in Chinese rice winefermentation.更多还原