【作者】 俞志敏；

【导师】 赵谋明；

【作者基本信息】 华南理工大学 ， 食品科学， 2011， 博士

【摘要】 本文以一株啤酒工业酿造酵母(Saccharomyces pastorianus)FBY0095为研究对象,并与其诱变得到的优良超高浓(VHG)酿造酵母(S. pastorianus)L6对比,以阐明VHG酿造对酵母生理代谢的影响为目标,在充分了解酿酒酵母胞内碳源代谢途径、辅因子代谢过程和代谢环境的基础上,运用代谢工程和微生物生理学的理论和方法,在代谢水平对影响VHG酿造酵母发酵速率和生理活性的关键因素进行分析和调控。主要研究结果如下:(1)以酿酒酵母(S. pastorianus)FBY0095为初始菌株对其进行UV照射和甲基磺酸乙酯复合诱变,然后在含有高浓度麦芽糖和乙醇的培养基上进行驯化培养。突变菌株L3和L6在含有15%(w/v)麦芽糖和15%(w/v)乙醇培养基上表现较好的生长状态。突变株L3和L6的VHG发酵速率比初始菌株FBY0095显著提高,使发酵周期缩短了21.43%。VHG发酵结束时(28d),相对初始菌株FBY0095,突变株L3和L6的残糖较低,乙醇浓度较高,细胞活力较高,其生产的啤酒乙酸乙酯含量显著下降,风味明显改善。(2)以不同浓度的葡萄糖和麦芽糖为底物,模拟啤酒酿造过程(厌氧和低温发酵),研究了糖浓度对酿酒酵母(S. pastorianus)FBY0095和优良VHG酿酒酵母(S. pastorianus)L6发酵特性的影响。运用拟稳态方法定量分析了酵母胞内的代谢通量,并分析了酵母胞内糖酵解途径关键酶(己糖激酶、磷酸果糖激酶和丙酮酸激酶)活性及其对代谢流的定量调控作用。结果发现,随着糖浓度升高,酵母FBY0095糖酵解途径的代谢通量和关键酶活逐渐降低。在VHG条件下,酵母L6糖酵解途径的代谢通量和关键酶活均比酵母FBY0095的高。酵母胞内磷酸果糖激酶和丙酮酸激酶对代谢通量控制作用较强,可认为这两种酶的催化反应是糖酵解流的关键调控步骤。(3)研究了不同浓度麦汁对酿酒酵母(S. pastorianus)FBY0095和优良VHG酿酒酵母(S. pastorianus)L6胞内辅因子(磷酸腺苷和辅酶I)代谢的影响,并且分析了辅因子对发酵速率的调控作用。随着麦汁浓度升高酵母FBY0095胞内ATP含量和能荷(EC)水平逐渐升高,对磷酸果糖激酶和丙酮酸激酶起到抑制作用,从而导致糖利用速率降低。麦汁浓度升高导致酵母FBY0095胞内NADH/NAD~+值升高,使乙醇生成速率降低,胞内代谢流分布也发生了变化。另外,在VHG条件下,酵母L6胞内ATP含量、EC水平和NADH/NAD~+值比酵母FBY0095的低,因此酵母L6的代谢活性,糖利用速率和乙醇生成速率均比FBY0095高。(4)研究了不同浓度麦汁对酿酒酵母(S. pastorianus)FBY0095和优良VHG酿酒酵母(S. pastorianus)L6胞内代谢环境的影响,主要是分析了胞内H+动态变化过程,并阐述了乙醇压力对细胞膜的影响。随着麦汁浓度升高,酵母FBY0095细胞膜上H+-ATPase活性逐渐降低,胞内H+输出能力降低,从而导致胞内pH降低,胞内代谢环境酸化,不利于代谢反应进行,并且胞内外pH差异也相应降低,导致酵母细胞对底物的利用能力下降。当乙醇浓度达到4%(w/v)时酵母代谢活性受到显著的抑制。另外,相对酵母FBY0095,酵母L6的细胞膜不饱和度较高,使其耐乙醇性能升高,代谢环境得到改善。(5)研究了不同分子量大豆肽对酿酒酵母(S. pastorianus)FBY0095生长和耐乙醇性能的影响,结果发现大豆肽分子量小于1kDa时有利于酵母细胞生长,分子量为3～5kDa的大豆肽可提高酵母的耐乙醇性能。将0.2%大豆肽添加到含有12%(w/v)乙醇的YNB培养基中,发现大豆肽吸附于细胞表面,使细胞膜透性降低,细胞活性升高。添加大豆肽使酵母的耐乙醇和代谢相关基因(OLE1、ELO1、TPS1、PMA1和MAL1)的表达水平升高,特别是OLE1基因提高最显著。在VHG酿造过程中添加0.2%大豆肽(3kDa < Mw < 5kDa)使酵母发酵性能显著提高,并且使酵母细胞膜不饱和度增加,提高了细胞的耐乙醇性能,从而使H+-ATPase活性增加,改善胞内代谢环境。更多还原

【Abstract】 This dissertation chose the metabolism of carbon sources, cofactors and environment in an industry brewer’s yeast (Saccharomyces pastorianus) FBY0095 and its variant (S. pastorianus) L6 with improved very high gravity (VHG) fermentation performances as the model system to study the principal physiological and metabolic characteristics. The effect of VHG condition on fermentation rate and physiological activity of yeast at the metabolic level were investigated in detail. The main results were described as follows:(1) The brewer’s yeast FBY0095 (S. pastorianus) was used as starting strain to select variants for efficient VHG brewing by UV irradiation and ethyl methanesulfonate mutagenization combined with the domestication of ultra-high concentration of maltose and ethanol. Two variants (L3 and L6) screened displayed the better growth profile and the improvedα-glucosides transport and alcohol dehydrogenase activity on the plates with 15% (w/v) maltose and 15% (w/v) ethanol. Moreover, compared with FBY0095, both L3 and L6 variants showed faster fermentation rates, and their fermentation times were decreased in 21.43%. Furthermore, there were increases in apparent attenuation and ethanol concentration and improvement on flavor of resulting beer by L3 and L6 at the end of VHG fermentation (28 d).(2) The effect of concentration of glucose and maltriose on fermentation performances of S. pastorianus FBY0095 and S. pastorianus L6 with improved VHG fermentation performances under low temperature and anaerobic conditions were investigated. Furthermore, the metabolic flux and its control by glycolytic key enzymes (hexokinase, phosphofructokinase and pyruvate kinase) were quantitatively analyzed. The metabolic flux and key enzymes activities in S. pastorianus FBY0095 were decreased with the increase in wort gravity. The metabolic flux and key enzymes activities in S. pastorianus L6 were higher than those in S. pastorianus FBY0095 under VHG condition. Furthermore, the metabolic flux in S. pastorianus cells was intensely controlled by phosphofructokinase and pyruvate kinase, meaning that the catalytic reactions of the two key enzymes were regulation steps of glycolysis flux.(3) The effect of wort gravity on cofactors (adenosine phosphate and coenzyme I) in S. pastorianus FBY0095 and S. pastorianus L6 with improved VHG fermentation performances and the regulation of intracellular cofactors to fermentation rate were investigated. The ATP content and EC level in S. pastorianus FBY0095 were increased with the increase in wort gravity, which resulted in the decrease in wort sugars consumption. The increase in NADH/NAD~+ value in S. pastorianus FBY0095 companied with the increase in wort gravity, which led to the decrease in ethanol production rate and the changes of metabolic flux distribution in S. pastorianus FBY0095. In addition, the ATP content, EC level and NADH/NAD~+ value in S. pastorianus L6 were lower than those in S. pastorianus FBY0095, which resulted in the improved metabolic activity of S. pastorianus L6 and the increases in wort sugars consumption rate and ethanol production rate.(4) The effect of wort gravity on metabolic environment, including of the dynamic change process of intracellular H+ and the influence of ethanol stress on membrane, in S. pastorianus FBY0095 and S. pastorianus L6 with improved VHG fermentation performances were investigated. The activities of H+-ATPase on membrane of S. pastorianus FBY0095 decreased with the increase in wort gravity, which resulted in the decrease of H+ efflux rate, intracellular pH, difference of intra- and extracellular pH, and the acidification of intracellular metabolism environment and inactivation of metabolic reactions and carbon sources utilization. The metabolic activity of S. pastorianus cells was obviously inhibited by ethanol of 4% (w/v). In addition, compared to S. pastorianus FBY0095, S. pastorianus L6 displayed that higher unsaturation index of membrane, better ability of ethanol tolerance and improved metabolism environment.(5) The effect of soy peptides with different molecular weight on the growth and vitility S. pastorianus FBY0095. The results indicated that the soy peptide (Mw < 1kDa) could increase growth of yeast cells and the soy peptide (3kDa < Mw < 5kDa) could increase ethanol tolerance of yeast cells. The soy peptide (3kDa < Mw < 5kDa) adhere on membrane of S. pastorianus FBY0095 in YNB medium containing ethanol 12% (w/v), which resulted that decrease of permeability of membrane and increase of cells vitality. The effect of soy peptide (3kDa < Mw < 5kDa) on ability of ethanol tolerance of S. pastorianus FBY0095 and the reaction mechanism between soy peptide and yeast cells were investigated. Furthermore, The gene expression level on ethanol tolerance and metabolism (OLE1、ELO1、TPS1、PMA1 and MAL1) of S. pastorianus FBY0095 were increased by addition of 0.2% soy peptide, especially OLE1. S. pastorianus FBY0095 displayed that better fermentation performances, increased membrane unsaturation index, ethanol tolerance ability and H+-ATPase activity and improved metabolism environment by addition of 0.2% soy peptide during VHG brewing.更多还原