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The Metabolic Analysis and Regulation of Brewer’s Yeast under Very High Gravity Brewing

Author: YuZhiMin
Tutor: ZhaoMouMing
School: South China University of Technology
Course: Of Food Science
Keywords: Saccharomyces cerevisiae Beer ultra high gravity brewing Metabolic Analysis Metabolic regulation Resistance to ethanol Soybean peptide
CLC: TS262.5
Type: PhD thesis
Year: 2011
Downloads: 136
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Abstract


This article to a beer industry the brewing yeast (Saccharomyces pastorianus,) FBY0095 research object, and its mutagenic get excellent ultra-high concentrated (VHG) brewers yeast (S. pastorianus) L6 contrast, in order to clarify the VHG brewing yeast physiological metabolism objective, fully understand the carbon source metabolic pathway in Saccharomyces cerevisiae intracellular cofactor metabolic processes and metabolic environment based on the theories and methods of use of metabolic engineering and microbial physiology at the metabolic level impact VHG brewing yeast fermentation rate and physiological activity The key factor in the analysis and regulation. Main results are as follows: (1) Saccharomyces cerevisiae (S. pastorianus), FBY0095 original strain to UV irradiation and ethyl methane sulfonate compound mutagenesis, and then in a medium containing a high concentration of maltose and ethanol domesticated. Mutant strain L3 and L6 containing 15% (w / v) maltose and 15% (w / v) ethanol medium, the better the performance state of growth. The mutant strain L3 and L6 the VHG fermentation rate than the initial strain FBY0095 increased significantly, 21.43% of the fermentation period is shortened. VHG fermentation end (28d), relative to the initial strains FBY0095, the lower the residual sugar of the L3 and L6 of the mutant, the ethanol concentration is higher, the higher cell viability, a significant decrease in the production of beer ethyl acetate content, significantly improve flavor. (2) with different concentrations of glucose and maltose as substrate the analog brewing process (anaerobic fermentation at low temperature), a the sugar concentration FBY0095 yeast (S. pastorianus), and excellent VHG brewing yeast (S. pastorianus) L6 fermentation characteristics. Use to be steady-state method for quantitative analysis of yeast intracellular metabolic flux analysis of yeast intracellular key enzyme in the glycolytic pathway (hexokinase, phosphofructokinase and pyruvate kinase) activity and its quantitative regulation of metabolic flux role. The results found that the metabolic flux and activities of key enzymes of the glycolytic pathway in yeast FBY0095 gradually decreased with increasing sugar concentration. In the VHG conditions of the metabolic flux and activities of key enzymes of the glycolytic pathway of yeast L6 than the yeast FBY0095 the high. Yeast intracellular phosphofructokinase and pyruvate kinase stronger role of metabolic flux control, these two enzymes catalyzed reaction is the key regulatory step of glycolysis stream. (3) research of different concentrations wort of Saccharomyces cerevisiae (S. pastorianus) FBY0095 and excellent VHG brewing yeast the (S. pastorianus) L6 intracellular metabolism of cofactors (ATP and coenzyme I), and analysis of the cofactor regulation of the rate of fermentation. With the the wort concentration increased yeast FBY0095 intracellular ATP content and energy charge (EC) level gradually increased, phosphofructokinase and pyruvate kinase play an inhibitory effect, resulting in reduced glucose utilization rate. Wort concentrations cause the yeast FBY0095 intracellular NADH / NAD ~ value increased ethanol formation rate decreased intracellular metabolic flux distribution also changed. Further, in VHG under conditions, the yeast L6 intracellular ATP content, EC level and the NADH / NAD ~ value than yeast FBY0095 low, therefore, the metabolic activity of the yeast L6 of glucose utilization rate and ethanol formation rate is high than FBY0095. (4) to study the impact of different concentration of wort the brewing yeast (S. pastorianus), FBY0095 and excellent the VHG brewers yeast (S. pastorianus) L6 intracellular metabolic environment, is to analyze the dynamic changes of intracellular H and elaborated of ethanol pressure is on the cell membrane. H-ATPase activity with increasing wort, yeast FBY0095 membrane gradually reduced reduced intracellular H output capability, resulting in intracellular pH decreased the acidification of intracellular metabolic environment is not conducive to the metabolic reaction was conducted, and the intracellular and extracellular pH differences also reduced accordingly, resulting in the utilization decreased ability of yeast cells to substrates. Be significantly suppressed when the ethanol concentration of 4% (w / v) yeast metabolic activity. Further, relative yeast FBY0095, the cell membrane of the yeast L6 unsaturated higher degree, so the performance of resistance to ethanol increases, the metabolic environment to be improved. (5) of different molecular weight soybean peptide of Saccharomyces cerevisiae (S. pastorianus), FBY0095 growth resistance and ethanol performance and found that soy peptide molecular weight less than 1kDa conducive to the growth of yeast cells, the molecular weight of 3 ~ 5kDa soybean peptides can improve yeast resistance to ethanol performance. 0.2% soy peptides added to a solution containing 12% ethanol (w / v) YNB medium and found that soy peptides adsorbed on the cell surface, so that the cell membrane permeability reduction, cell activity increased. The soy peptide of yeast resistance to ethanol and metabolism-related genes (OLE1, ELO1, TPS1, PMA1 and MAL1) expression levels increased, especially OLE1 gene to improve the most significant. VHG brewing process to add 0.2% soybean peptide (3kDa lt; Mw lt; 5kDa) the yeast fermented performance improved significantly, and make the yeast cell membrane unsaturated degree increase, improve the cells resistant to ethanol performance, so as to make H-ATPase activity increased improve intracellular metabolic environment.

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CLC: > Industrial Technology > Light industry,handicrafts > Food Industry > Brewing industry > A variety of wine and its manufacturing > Beer
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