摘要
微生物在葡萄酒酿造和储藏过程中起到重要作用，如何运用对葡萄酒品质影响最小的方法，对其中微生物进行有效合理的控制，在很大程度上决定了葡萄酒产品的价值。作为一种无污染，绿色节能易操作的技术，超声波技术越来越多的应用于食品工业领域。本研究为大功率超声波对葡萄酒酿造过程中常见微生物的灭菌效果提供详细数据；针对超声水浴对酿酒酵母发酵性能的影响进行研究，提供超声水浴对酿酒酵母发酵促进作用的详实数据；并选取13个目标基因进行超声波水浴影响下酵母基因表达量变化研究。旨在为超声波在葡萄酒酿造及储藏过程中的应用提供参考。
主要研究结果如下：
1.在间歇性大功率超声波辐射下，本文所研究的6株酵母菌及5株细菌在红葡萄酒，白葡萄汁及生理盐水中存活率均有不同程度的下降。受到超声辐射影响较大的酵母菌有Saccharomyces cerevisiae及Schizosaccharomyces pombe，经过20min超声辐射后，两者在生理盐水中最终存活率仅为20%；乙酸菌Acetobacte pasteurianus存活率仅为12.8%。
2.超声波在不同介质中传播时，受到介质蒸汽压，粘度及表面张力等因素的影响，对介质中细胞的杀灭效果不同。在本论文所用地生理盐水，白葡萄汁及红葡萄酒等三种悬浮介质中，20℃条件下，超声波在生理盐水中对细胞的杀灭作用最为明显。除Pediococcus sp外，其他菌体的存活率在生理盐水中最低。菌体在葡萄汁中有较高的存活率。
3.微生物细胞的不同结构及形态使其对超声辐射的抵御能力不同。Oenococcusoeni，Pediococcus sp.及Lactobacillus plantarum为G+细菌，具有较厚的细胞壁及胞壁肽葡聚糖层，比Acetobacter aceti和Acetobacter pasteurianus等G—菌更好的抵御来自超声波的影响，在实验过程中存活率相对于高。而从形态上比较，球菌比杆菌在超声波中更易受到超声影响。细胞团聚性也会影响超声辐射效果。在对超声过程中所取Zygosaccharomyces bailii一系列菌液样品显微观察可以看到，初始阶段，在超声辐射下菌团被打散，然而存活率并未下降。随着超声辐射的继续进行，细胞受到伤害，变得不完整，活菌数减少。
4.实验中所选用的酿酒酵母S.erevisiae AWRI796，在高频低功率超声波水浴作用下，发酵进程加快，与其他组比，至少提前72h完成发酵。并且发酵后甘油产量显著增高，乙酸产量显著降低，乙醇含量并无差异。
5.在发酵初期接受了48h超声水浴辐射的酿酒酵母细胞，停止超声水浴辐射后，与摇床培养的酿酒酵母细胞在相同条件下的发酵比较中，仍比后者提前24h完成发酵。此时，该组酵母细胞在前期超声辐射下已经改变了发酵活性，并能保持这种活性，但超声辐射并未改变其繁殖特性。两组发酵液最终甘油产量无差异，乙酸产量有显著性差异。在酿酒酵母发酵过程中，甘油产生于超声波对细胞的持续性刺激。而在酵母发酵前期对酵母进行超声辐射，会对乙酸产生有较大影响。
6.本实验在选取目标基因时，借鉴临床医学中所得到的鼠及人类对超声波敏感的基因在酵母中的同源基因，再结合以往研究酵母胁迫实验中，出现频率最高的基因，两者综合选取交集，在其中共确定13个目标基因。其中SSE2，TDH1，SIS2在超声辐射影响下，初期表达量升高，后期回落。SPI1019在后期表达量变化一直处于被激发状态。其余占多数的9个目标基因ADE13，HSP12，HSP42，HSP78，HSP104，HXT5，SSA4，TDH2，TDH3在超声水浴中表达量均有不同程度受到抑制，并且在后期有缓慢回升的趋势。
Microorganism is important for wine making and storage. Wine quality depended on theway which was used to control the microbe. As a green and low power consumptiontechnology, ultrasound has been used increasly in food industry. In this study, effect onviability of high power ultrasound of common microbe, fermentation of Saccharomycescerevisiae in ultrasound water bath, and13related S. cerevisiae genes had been evaluated.The major results were summarized as follows:
1. After high power ultrasound (HPU) irradiation, the ability of yeast and bacteria insaline, juice and wine were decreased differently. S. cerevisiae and S. pombe. It’s were greatlyaffected by HPU. The viability of the cells for S. cerevisiae and S. pombe was20%, and12.8%for A. pasteurianus in saline.
2. The medium that the cells were suspended in also modulated the effect of HPU.Cavitation efficiency was affected by the vapour pressure, viscosity coefficient and surfacetension of thesuspended liquid. The viscosity coefficient was lowest, the vapour pressure andthe surface tension were highest in saline at20℃, so the cavitation effeiciency was storagestin saline, beside Pediococcus sp., the other organisms were more sensitive than in the othersmediums. The cavitation efficiency was stronger in wine than in the juice. The nonlinearproperty of12%ethanol had also influenced the cavitation efficiency.
3. The different structure of microbe made the difference viability after HPUirradiation. The gram-positive organisms with a thicker and more tightly adherent layer ofpeptidoglycans, was more resistant to HPU than the gram-negative organisms. As for theshape, the rod one was more sensitive than the long one in this paper. During the irradiation,the ultrasound had ruptured the cluster of Z.baillii, and broken the cells to death.
4. The cavitation of ultrasound enhanced the permeability of the membrane that wasimportant to accelerate enzymes releasing from the cell, and induced substance exchangebetween extra and intracellular. The fermentation witch irradiated had finished72h faster thanthe shaking group.
5. After48h ultrasound iraadistion the cells were reinocuulated in the new medium, theultrasound cell group was more activity than the shaking cell group, the fermentation was24hfaster than the other. The two groups had the almost same vital yeast cells, the Ultrasound irradiation changed the cells’ activity but not the multiplication characterisitics.
6. Compared the shaking group, the ultrasound group had the higher glycerol and theacitic acid contend, and the same alcohol contend after fermentation. But after stop ulatrsoundirradiation after48h, there was no difference between the two groups of glycerol, but the acticacid was still different.
7. Ultrasound irradiation degassed the dissolved CO2from the medim, thus mightenhance the fermentation but not the main reason.
8. There were few paper about ultrasound affection on yeast and the investigation onthe genes, thus the genes chosen for this paper were from the genes that appeared frequentlyin some papers about yeast stress tolerance, and clinical paper in which yeast homology genesexpression different after ultrasound irradiation. Gene expression difference had beenobserved. SSE2, SIS2, TDH1showed increasing in the beginning, the fold change of SPI1decreased a little in the beginning and increasing till3.467in the end. ADE13, HSP12, HSP42,HSP78, HSP104, HXT5, SSA4, TDH2, TDH3decreased from the beginning and down to thelowest at time2h or1d, and increasing later.
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