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VEN124 Section III

VEN124 Section III. The Alcoholic Fermentation. Lecture 8: Yeast Biology. Reading Assignment: Text, Chapter 4, pages123-168. In this lecture we will cover the basic biology of yeast and the topics of yeast nutrition and selection for wine production. The Alcoholic Fermentation.

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VEN124 Section III

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  1. VEN124 Section III The Alcoholic Fermentation

  2. Lecture 8: Yeast Biology

  3. Reading Assignment: Text, Chapter 4, pages123-168

  4. In this lecture we will cover the basic biology of yeast and the topics of yeast nutrition and selection for wine production.

  5. The Alcoholic Fermentation The microbiological conversion of the grape sugars, glucose and fructose, to the end product, ethanol, is called the alcoholic fermentation. Fermentation means that an organic compound serves as terminal electron acceptor.

  6. The Alcoholic Fermentation Is conducted by the yeast Saccharomyces cerevisiae or Saccharomyces bayanus

  7. Characteristics of Saccharomyces • Eukaryote: possesses a membrane bound nucleus Endoplasmic reticulum Nucleus Nucleus is surrounded by a double membrane layer with the outer membrane contiguous with the endoplasmic reticulum

  8. Characteristics of Saccharomyces • Eukaryote: possesses a membrane bound nucleus • Reproduces by budding

  9. Reproduction by Budding Daughter Cell Mother Cell

  10. Characteristics of Saccharomyces • Eukaryote: possesses a membrane bound nucleus • Reproduces by budding • Grows vegetatively as haploid (1N) or diploid (2N)

  11. Yeast Life Cycle New daughters must grow before initiating their first cell cycle

  12. Characteristics of Saccharomyces • Eukaryote: possesses a membrane bound nucleus • Reproduces by budding • Grows vegetatively as haploid (1N) or diploid (2N) • Capable of conjugation (1N ⃗ 2N) and sporulation (2N ⃗ 1N)

  13. Yeast Life Cycles: Conjugation Mating Pair  a a  a/ Budding Zygote Diploid Cell Haploid Cells

  14. Yeast Life Cycles: Sporulation Ascus Spore Vegetative Cell 2N Tetrad 4 x 1N

  15. Characteristics of Saccharomyces • Eukaryote: possesses a membrane bound nucleus • Reproduces by budding • Grows vegetatively as haploid (1N) or diploid (2N) • Capable of conjugation (1N ⃗ 2N) and sporulation (2N ⃗ 1N) • Non-motile

  16. Characteristics of Saccharomyces: Sub-Cellular Organization • Plant-like cell wall: comprised of carbohydrate (glucan, mannan) and glycosylated protein (phosphomanno-protein) • Mitochondria: site of oxidative reactions • Vacuoles: site of storage and hydrolysis • Secretory pathway • Nucleus

  17. Saccharomyces Nucleus Mitochondrion Secretory Pathway Golgi Vacuole Endoplasmic reticulum

  18. Glycolysis The set of biochemical reactions converting hexose (6 carbon) sugars to two 3 carbon pyruvate molecules, during which energy is released and recaptured in the form of ATP.

  19. Glycolysis Glucose + 2 ATP + 2 NAD+ + 2 ADP + 2 Pi 2 Pyruvate + 4 ATP + 2 NADH + heat

  20. Glycolysis glucose ATP fructose glucose-6-phosphate fructose-6-phosphate ATP fructose 1,6-diphosphate dihydroxyacetone phosphate glyceraldehyde 3-phosphate NAD+ NADH 1,3 -diphosphoglycerate ATP 3-phosphoglycerate 2-phosphoglycerate phosphoenol pyruvate ATP pyruvate

  21. “Upper Glycolysis”: consumes two molecules of ATP “Lower Glycolysis”: produces four molecules of ATP NET PRODUCTION: TWO MOLECULES OF ATP

  22. Where does ethanol come from? The end products of glycolysis are pyruvate and 2 molecules of the reduced co-factor NADH. Yeast cells regenerate NAD+ by transferring the hydrogen molecule (electron) to an organic molecule: acetaldehyde

  23. Ethanol Formation Pyruvate CH3-CO-COOH Acetaldehyde CO2 + CH3-CHO NADH NAD+ H+ Ethanol CH3-CH2OH

  24. Other organisms use different strategies to regenerate NAD+ Their presence in wine leads to a diversity of end products of sugar catabolism

  25. Carbon Distribution at End of Fermentation • 95% = ethanol + carbon dioxide • 1% = new cells • 4% = other end products • Pyruvate • Acetate • Acetaldehyde • Glycerol • Lactate

  26. Ethanol Yield 1M glucose (fructose) 2 M CO2 + 2 M ethanol Theoretical Maximum: 180 g 2(46g) = 92/180 = 51.1% w/w = 63.9% v/w = 0.6 original Brix value

  27. Yeast will ferment even in the presence of oxygen. Why?

  28. Fermentation vs. Respiration Fermentation: 2 ATP/glucose(fructose) Respiration: 36-38 ATP/ glucose Efficiency of ATP yield is only an issue if sugar is limiting

  29. In Saccharomyces, glucose concentration regulates the switch between fermentation and respiration.

  30. Regulation of Glycolysis • Transport: site of global rate control • Allosteric enzymatic steps: localized rate control • Hexokinase • Phosphofructokinase • Pyruvate kinase • Effectors of regulation: ATP, ADP, AMP fructose 2,6 bisphosphate, citrate, glucose

  31. Yeast Choice and Nutrition

  32. Yeast Choice: Desirable Traits • Fermentation to dryness • Reasonable rate of fermentation • Predictable fermentation characteristics • Good ethanol tolerance • Good temperature tolerance • Sulfur dioxide tolerance

  33. Yeast Choice: Desirable Traits • Little to no off-character production • Sulfur volatiles • Acetic acid • Ethyl carbamate • Little to no inhibition of other desirable microbes • Killer factor resistant • Production of desired aroma characters

  34. Yeast Nutrition • Macronutrients: Building blocks needed for new cell material • Micronutrients: Catalysts needed to facilitate biochemical reactions

  35. Macronutrients • Carbon/Energy Sources: glucose, fructose, sucrose • Nitrogen Sources: amino acids, ammonia, nucleotide bases, peptides • Phosphate Sources: inorganic phosphate, organic phosphate compounds • Sulfur Sources: inorganic sulfate, organic sulfur compounds

  36. Macronutrient Energy Sources • Monosaccharides: glucose, fructose, galactose, mannose • Disaccharides: sucrose, maltose, melibiose • Trisaccharides: raffinose • Pentoses: None • Oxidative substrates: pyruvate, acetate, lactate, glycerol, ethanol

  37. Categories of Yeast Nitrogen Sources • Compound may be used as that compound for biosynthesis • Compound may be converted to related compounds for biosynthesis • Compound may be degraded with release of nitrogen

  38. Yeast Nitrogen Sources • Degradation may depend upon availability of other components: vitamins and oxygen • Utilization impacted by other environmental factors such as pH

  39. Micronutrients • Minerals and Trace Elements: Mg, Ca, Mn, K, Zn, Fe, Cu • Vitamins: biotin is the only required vitamin, but others are stimulatory

  40. Nutritional Requirements of Different Phases of Fermentation • Growth Phase: Building blocks and catalysts • Stationary Phase: Survival factors

  41. Yeast Nutritional Phases stationary death Cell # log Brix lag Time

  42. Most of the fermentation is conducted by stationary phase cells Stationary phase: 1. rate of growth = rate of death 2. quiescent, no growth, no death

  43. Role of Survival Factors • Maintain viability of cells • Increase ethanol tolerance • Maintain energy generation

  44. Survival Factors • Oxygen • Fatty Acids • Sterols • Nutritional Factors

  45. How Does Ethanol Inhibit Yeast? • Displaces water of hydration changing the properties of protein-lipid interactions • Denatures proteins • Disrupts protein active sites • Allows increased passage of protons from the medium into the cell leading to acidification of the cytoplasm • Removal of protons requires expenditure of energy

  46. Survival Factors Needed to alter composition of the plasma membrane (sterols, fatty acids and proteins) so that it can withstand the perturbing effects of ethanol Both phospholipid and protein content must be adjusted

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