摘要
本论文以挥发性嗅感物质分析鉴定为主要技术手段，开展啤酒麦芽生产链嗅感物质的构成及变化规律的研究。对制麦过程中影响麦芽风味的微生物进行生态学分析，确定不同制麦工艺阶段的主要微生物，同时考察不同外源微生物自身嗅感物质的构成及其对制麦过程绿麦芽嗅感物质的影响，探究制麦过程中外源微生物对麦芽风味的影响机制。
论文主要研究结果如下：
（1）利用固相微萃取与气质联用技术可以快速准确地分析鉴定酿造大麦中嗅感物质的组成。从10个酿造大麦品种中共分析鉴定出41种挥发性嗅感风味化合物，这些嗅感物质由醛类、醇类、酮类、有机酸类以及少量的芳香族和呋喃类化合物所构成。各供试大麦品种的嗅感物质组成基本相同，只是在个别化合物上存在一定的差别。
（2）通过计算嗅感风味化合物的气味活度值（OAV），确定了构成酿造大麦的16种关键性嗅感物质：其中醛类8种（乙醛、异丁醛、异戊醛、2-甲基丁醛、正己醛、正庚醛、正辛醛、壬醛）；醇类2种（异戊醇、环戊醇）；酮类2种（2,3-丁二酮、2-庚酮）；有机酸类一种（乙酸）；其他类化合物3种（乙酸乙酯、2-戊基呋喃、苯乙醛）。
（3）应用主成分分析法（PCA）对不同产地的酿造大麦进行了分析。国产大麦品种与进口大麦品种的风味构成存在一定的差异，乙醛、异丁醛、异戊醛、2-甲基丁醛、正己醛、正庚醛、正辛醛、异丁醇、正戊醇、2,3-戊二酮、2-庚酮、乙酸、庚酸、乙酸乙酯、正己醇和2-戊基呋喃对进口大麦品种的风味贡献较大；而壬醛、环戊醇、异戊醇、2,3-丁二酮、己酸及苯乙醛则对国产大麦品种的风味贡献较大。
（4）在工业化制麦过程中共分析鉴定出48种挥发性嗅感物质，它们同样由醛、醇、酮、有机酸、呋喃和一些芳香族化合物所组成，通过将这些化合物分成六大类可以直观地反映出各化合物在制麦过程中的出现时间及其变化规律。同时，通过考察不同嗅感物质在制麦过程中含量变化的趋势发现，麦芽干燥、焙焦过程是整个制麦过程中对挥发性嗅感物质影响较大的主要工艺流程。
（5）应用变性梯度凝胶电泳技术（PCR-DGGE）对工业制麦过程中的微生物多样性进行了研究。研究结果发现，制麦过程不同工艺阶段的菌群构成差异性较大，而同一工艺阶段、不同时期的微生物群落结构相似度较高。原料大麦的优势微生物菌种为单胞菌属和肠杆菌属两种；浸麦阶段优势菌种同样为单胞菌属和肠杆菌属；发芽阶段菌群构成快速增加，此时的优势菌种为肠杆菌属、奈瑟菌属、欧文氏菌属、克雷伯氏杆菌属；成品麦芽菌群数量最少，此时的优势菌种为肠杆菌属。从整个制麦过程来看，肠杆菌属和欧文氏菌属是制麦过程中的两个最重要菌属，而单胞菌属、肠杆菌属、Sandarakinorhabdus和欧文氏菌属是原料大麦固有的表面微生物，其余菌种均来自于制麦过程中通入的空气。
（6）应用固相微萃取-气质联用技术（SPME/GC-MS）分析鉴定了制麦过程中主要外源微生物梨孢镰刀菌、草酸青霉菌、大肠杆菌及克雷伯氏杆菌在不同培养时期所产生的主要挥发性物质。结果发现，3-甲基-1-丁醇、1-辛烯-3-醇、3-辛酮和3-辛醇等8个碳的酮类和醇类物质为上述四种微生物的主要挥发性物质。另外，不同培养阶段、各菌体所产生的挥发性物质不同。培养前期主要以碳链较短的醇酮类化合物为主，而培养后期则以脂肪类及烯烃类等长链分子为主。梨孢镰刀菌对整个麦芽生产链条有重要影响，其自身产生的挥发性物质可以基本覆盖大部分大麦品种所能够鉴定出的挥发性成分，对于原料大麦的贮藏、麦芽生产、以及成品阶段的风味影响很大。
（7）制麦过程中外源微生物对麦芽风味的影响主要体现在对麦芽嗅感风味物质含量的影响上，但没有产生新的风味代谢物质。另外，主要微生物均对各嗅感物质的生成产生一定的抑制作用，该抑制作用的产生机制在于：大麦发芽过程中，醛、酮、醇类及部分有机酸类等嗅感物质主要来源于麦芽中酯类的氧化和水解作用，当外源微生物侵入时，制麦过程中各嗅感风味物质的含量是外源微生物与绿麦芽两个生理代谢系统共同作用的结果。在此过程中，一方面微生物分泌出的蛋白酶会对大麦内部脂类相关水解氧化酶系产生一定的水解作用，从而降低了其酶活力；另一方面微生物利用了相关嗅感物质的前体物质进行自身的生长和代谢。
In this dissertation, the mechanism of microbe effection on the flavors of malt, thecompostions and changing patterns of the flavors during malting process were investigated,with the aids of volatile compounds analysis techniques. Ecological analysis wasimplemented to identifiy the microbes which affected the flavors in malting process and todetermine the major microbes during different stage in malting process. In addition, thecompositions of aroma-active compounds originated from different microbes and theirinfluences on green-malt based flavors in malting process were also studied. The major resultsof this dissertation were summarized as below:
(1) The techniques of SPME combined with GC-MS were used to fastly and accurately toidentify the compositions of volatile compounds in barley. A total of41volatile compoundswere identified from10barley cultivars. The volatile compounds were constituted byaldehydes, alcohols, ketones, organic acids, and tiny amounts of aromatics and funancompounds. The compositions of the flavors in various barley with different origins weresimilar, with some discrepancies on certain individual compound.
(2)16volatile compounds were identified as the key aroma compounds of brewing barley, bycalculating the odor active valves (OAV) of each compound. Among these compounds,8aldehydes (acetaldehyde,2-methyl-propanal,3-methyl-butanal,2-methyl-butanal, hexanal,heptanal, octanal and nonanal),2alcohols (3-methyl-1-butanol and cyclopentanol),2ketones(2,3-butanedione and2-heptanone),1organic acid (acitic acid) and3other compounds (ethylacetate,2-pentyl-furan, benzeneacetaldehyde) were found.
(3) Principal component analysis technique showed that the aroma compositions of domesticbarley cultivars were different from those of the imported ones. The major volatilecompounds contributing the flavors of imported barley cultivars were acetaldehyde,2-methyl-propanal,3-methyl-butanal,2-methyl-butanal, hexanal, heptanal, octanal,2-methyl-1-propanol,1-pentanol,2,3-pentanedione,2-heptanone, hexanoic acid, heptanoicacid, ethyl acetate,1-hexanol, and2-pentyl-furan, while those of domestic barley cultivarswere nonanal, cyclopentanol,3-methyl-1-butanol,2,3-butanedione, hexanoic acid andbenzeneacetaldehyde.
(4) A total of48volatile compounds were identified in an industrial malting process, and theywere also constituted by the substances of aldehydes, alcohols, ketones, organic acids, funanand aromatic compounds. By classifying these compounds into six catagories, the appearancetime and changing patterns of each volatile compound during malting process could be clearlyindicated. These results also showed that, among the entire malting process, the kilning androasting process had a significant effection on volatile compounds concentration.
(5) The microbe diversity for industrial malting process was investigated using PCR-DGGE technique. The results indicated that the microbe group constitutions differed largely indifferent malting stage, but they kept higher similarity at different instant within one particularmalting stage. The dominant stains were Sphingomonas spp. and Enterobacter spp. in rawbarley and barley subject to steeping operation. In germinating stage, the microbe floraincreased rapidly, and the dominant species were Enterobacter spp., Neisseria spp., Erwiniaspp. and Klebsiella spp. The mibrobe flora in malt was the least and the dominant strain wasEnterobacter spp. During malting process, Enterobacter spp. and Erwinia spp. were the majorflora, while Sphingomonas spp, Enterobacter spp. Sandarakinorhabdus spp. and Erwinia spp.were microbes existed at the surface of barley, and all the other mibrobes were from theaerated air during operation.
(6) The volatile compounds at different instant during cultivations of Fusarium poae,Penicillium oxalicum, Escherichia coli and Klebsiella peneunomiae was collected andanalyzed using the techniques of SPME combined with GC-MS. The results showed that3-methyl-1-butanol,1-octen-3-ol,3-octanone and3-octanol were the major volatilemetabolites produced by the four microbes. It was also found that the volatile compoundsproduced were different at different cultivation stage: during the early stages, the majorvolatile metabolites were alcohols and ketones with short-chains; while in the later stage, themain volatile compounds turned to be lipids and terpeness. Fusarium poae was veryimportant for the entire malting process, as its volatile compounds almost covered most ofthose identified in each individual barley. Fusarium poae greatly affected barley storage,malting, and malt flavors.
(7) During malting process, microbes could affect the flavors by varying volatile compoundscontents or concentrations, but they could not produce new volatile metabolites. The majormicrobes had certain inhibitory effects on the formation of volatile compounds, and theinhibitory mechanism lied on the follwing facts: the volatile metabolites were originated fromlipid oxidation and hydrolysis reactions in malting. When microbe infection occurred, volatilecompounds contents were determined by the coordinate actions of microbes and green-maltmetabolisms. During malting process, proteases secreted from microbes deterioated lipidoxidation and hydrolysis enzymatic system in green-malt, leading to a lower enzymaticactivity; while at the same time, the infected microbes utilized the precurosors of volatilecompounds for their growth and metabolism.
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