【作者】 张良；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学与工程， 2015， 博士

【摘要】 蓝莓是一种营养丰富、富含花青素和各种微量元素的浆果,其食用价值与药用价值都非常高。目前已经被大量种植,但是经采摘后的蓝莓难以长久保存,除部分鲜食外,大部分需要通过冷冻得以保存,然后再进一步加工。蓝莓发酵酿酒是一种重要的深加工方法。由于其口感特殊、功能强大、营养丰富,蓝莓酒也越来越受到大众的青睐。为了更好的开发蓝莓资源,本研究以辽宁的蓝丰、伯克利、北陆、圣云、北青五个品种的蓝莓为材料,系统的研究蓝莓酒自然发酵特性、蓝莓酒发酵动力学、蓝莓酒发酵工艺优化、发酵过程中有机酸的变化及其对发酵的影响以及利用基因工程方法改良蓝莓酒酵母,以解决果汁中柠檬酸含量高抑制产酒的问题,研究结果如下：1.对五种蓝莓进行自然发酵,结果表明,“蓝丰”品种在试验中CO2减重最大达到0.74g。通过对生长曲线、降糖特性、产酒能力测定,以及比较发酵过程中的电导率变化、发酵液中溶氧变化,发现五个供试蓝莓品种中,“蓝丰”酵母生长最快,且降糖、产酒迅速。进一步对各个品种的发酵动力学研究显示,发酵过程的动态变化可以很好的被预测。五种供试蓝莓酒在发酵过程种菌种生长速率相差不大(除北青酵母菌生长比较慢外)；酒精生成速率从快到慢依次是蓝丰、伯克利、北陆、圣云、北青；底物消耗速率由快到慢依次是蓝丰、伯克利、圣云、北青、北陆。由此确定蓝莓酒自然发酵酒的最佳酵母,并拟合蓝莓果酒发酵动力学曲线,为蓝莓酒自然发酵生产提供理论依据。2.通过对酒精发酵过程的动力学分析和研究,建立了发酵过程中菌体生长、酒精生成以及糖消耗的动力学模型。方差分析结果显示,三组模型的决定系数分别为R12=0.991,R22=0.985,R32=0.979,因此可以很好的预测发酵过程的动态变化。进一步对发酵动力学方程求一阶导数得到蓝莓酒发酵过程中,菌种增长速率、酒精生成速率、底物消耗速率,因而更加清楚地认识蓝莓酒发酵过程。3.通过中心组合响应面设计试验确定了蓝莓酒发酵的最佳工艺参数是：加酶量0.13%o,温度22.0℃,接种量1.63%o,在此条件下酒精量为11.8%vol。对影响酒精量的3个因素(加酶量、发酵温度、接种量)的最佳工艺条件进行研究,结果显示3个因素对酒精量的影响是显著的,影响的主次顺序是：温度>接种量>加酶量。验证试验所得样品的酒精量为11.823%vol,与预测值相差无几,从而验证了模型的可靠性。4.发酵有机酸变化及其对发酵的影响总酸含量随着蔗糖和果糖添加量的增加不断增加,随着葡萄糖添加量的增加不断降低。pH值随蔗糖、果糖和葡萄糖添加量的变化趋势与总酸的变化趋势相反。随着蔗糖含量逐渐增加,草酸和乙酸含量先增加后减少,柠檬酸含量逐渐降低；随着果糖含量逐渐增加,草酸含量先增加之后趋于平缓；随着葡萄糖含量逐渐增加,草酸含量先降低后增加,柠檬酸逐渐降低,说明葡萄糖的加入有助于蓝莓酒中柠檬酸的转化。随着有机酸浓度的增加,PDC酶活不断降低,柠檬酸含量在14.5mg/mL时PDC酶活接近0,而草酸和乙酸对PDC的影响相对较小。随着有机酸含量的增加酶活缓慢降低。随着有机酸浓度的增加,AHD酶活不断降低,柠檬酸含量在25mg/mL时AHD酶活接近0,而草酸和乙酸对AHD的影响相对较小。随着有机酸含量的增加酶活缓慢降低。5.利用基因工程方法将烟曲霉中的柠檬酸裂解酶基因导入到酿酒酵母细胞中,经过表达后,分离纯化柠檬酸裂解酶,并测定该酶的生物学性质,进一步通过发酵试验,验证改良后的菌株发酵蓝莓酒的特性,通过比较得知,新构建的菌株,降糖快,产酒速率提高,发酵周期短。更多还原

【Abstract】 Blueberry is a kind of berries rich in nutrition, anthocyanin, and all kinds of trace elements, and has higher edible and medicinal value. Blueberry has been planted a lot currently, but it’s difficult to save the blueverry for a long time after being picked. Few portion of blueberries is eated directly as fresh fruits, while most of them are freezed to preserve for further process. Blueberry wine fermentation is one kind of important further processing methods, and blueberry wine is becoming more and more favorable by the public for its special taste, powerful function and rich nutrition. For better development of blueberry resources, this study researched systematically the characteristics of blueberry wine natural fermentation, the blueberry wine fermentation kinetics, blueberry wine fermentation process optimization, and the change of organic acids in the fermentation process and its effect on fermentation, improved the blueberry wine yeast by using genetic engineering methods, soloved the problem of the inhibition of the wine production from high citric acid content in fruit juice. The tested blueberries-Lan feng, Berkeley, Sheng yun, Bei qing, Bei lu, were sampled in Liaoning province.The results were as follows:1. Fermented naturally five kinds of blueberries, the results showed that the biggest weight reduction of CO2 of "Lan feng " is 0.74g in the experiment condition. Moreover, it was proved that " Lan feng " yeast grew most fast comparing with the other four kinds of blueberries, reduced sugar and produced wine quickly through the growth curve, hypoglycemic properties, determination of wine production capacity, as well as conductivity and dissolved oxygen changes during the fermentation. Further studies dynamics of the varieties of fermentation showed that the dynamic changes of the fermentation process can be predicted well. During fermentation, the strain growth rate was similar (except the yeast growth of "Bei qing" was slow). Alcohol generation, Lan feng>Berkeley>Bei lu>Sheng yun>Bei qing. The substrate consumption rate, Lan feng>Berkeley>Sheng yun>Bei qing>Bei lu. Then the best yeast of the natural fermented blueberry wine was identified, and blueberry wine fermentation kinetics was fited which provided the theoretical basis for blueberry wine natural fermentation production.2. The dynamic models of bacteria growth, alcohol production and sugar consumption were established through analysis and study on the dynamics of alcohol fermentation process. The decision coefficients of three models were 0.991,0.985,0.979, respectively according to the results of variance analysis, so the dynamic change of fermentation process can be well predicted by the models. The strain growth rate, alcohol generating rate and the substrate consumption rate during the blueberry wine fermentation process were calculated through solving the first derivative of the fermentation kinetics equation to know more clearly about the blueberry wine fermentation process.3. The optimum parameters of blueberry wine fermentation were tested through the center combination of response surface design, such as enzyme addition quantity of 0.13 ‰, temperature of 22.0℃, the inoculum amount of 1.63‰. The wine production was 11.8% vol in this condition. The best process condition of three factors of affecting the amount including enzyme addition quantity, fermentation temperature, inoculation amount (enzymatic) was studied, and the results showed that three factors influenced the amount of alcohol significantly, and the order of their importance is, temperature>inoculation quantity> amount of enzyme. The alcohol content of validation test samples was 11.823% vol, similar to that of forecast, which can verify the reliability of the model.4. Organic acids change of fermentation and its effect on fermentationThe total acid amount increased with the increase of sucrose and fructose addition amount, while decreased with the increase of the glucose addition amount. The trend of pH value changing with the sucrose, fructose,and glucose was opposite to that of the total acid.As sucrose content increased gradually, oxalate and acetic content reduced firstly and then increased, citric acid content gradually reduced; With fructose content increased, the oxalic acid content increased firstly and then smoothed. As the glucose content increased gradually, oxalic acid content decreased firstly and then increased, citric acid reduced gradually, which indicated that addition of glucose to the transformation was helpful for the transformation of citric acid in the blueberry wine.With the increase of organic acid concentration, PDC enzyme activity decreased gradually, and approached zero when the content of citric acid was 14.5 mg/mL. While the effects of oxalic acid and acetic acid on PDC was relatively small. With the increase of organic acid, enzyme activity decreased slowly.With the increase of organic acid concentration, AHD enzyme activity decreased gradually, approached zero when the content of citric acid was 25 mg/mL.While the e effects of oxalic acid and acetic acid on AHD was relatively small. With the increase of organic acid enzyme activity decreased slowly.5.The citrate lyase gene of aspergillus fumigatus was imported into saccharomyces cerevisiae by using genetic engeering methods. Then the citrate lyase was separated and its biological characteristics was detected after expression. Also improved strain fermentation characteristic of blueberry wine was verified through further fermentation test. Finally, by compairing the datas, we can get the result that the new improved strain can degradate sugar and producte alcohol faster, with shorter fermentation period更多还原