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Science behind wine fermentation

Science behind wine fermentation. Sara Belchik 28 June 2014. Wine fermentation. Overview. History Yeast Sugar metabolism Ethanol fermentation Other products Spontaneous fermentation Inoculated fermentation How to get the perfect yeast. Wine predates written records.

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Science behind wine fermentation

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  1. Science behind wine fermentation Sara Belchik 28 June 2014

  2. Wine fermentation

  3. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  4. Wine predates written records

  5. But we did not understand it • 6000 BCE • Earliest evidence of wine production • 1680 AD • Description of small spheres during fermentation

  6. But we did not understand it • 6000 BCE • Earliest evidence of wine production • 1680 AD • Description of small spheres during fermentation • 1830 AD • Small spheres identified as living organisms

  7. Pasteur experiment Sterilized Not sterilized

  8. Pasteur experiment Sterilized Not sterilized

  9. Yeast are responsible agents • 6000 BCE • Earliest evidence of wine production • 1680 AD • Description of small spheres during fermentation • 1830 AD • Small spheres identified as living organisms • 1863 AD • Yeast identified as microbe responsible for fermentation of grapes into wine • 1890 AD • Addition of pure yeast culture to help with fermentation • 1965 AD • Commercially available dehydrated yeast for sale

  10. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  11. What are yeast?

  12. Yeast are eukaryotic cells • Unicellular organisms • Kingdom Fungi • Complex interior allows for complex reactions to occur • Yeast is umbrella term • Almost 2000 species • Earliest domesticated organism

  13. Semi-permeable membrane • Water-loving parts on exterior • Water-hating portions on interior • Difficult for many molecules to get through • Require transport proteins to help get across membrane

  14. Many yeast species

  15. Wine yeast genera • Predominant species on surface of grape berries • Kloeckera • Also found on grapes but to lesser extent • Brettanomyces • Candida • Crytococcus • Kluyveromyces • Metschnikowia • Pichia • Rhodotorula • Found in vineyards or on winemaking equipment • Debaryomyces • Saccharomyces • Saccharomycodes • Schizosaccharomyces • Zygosaccharomyces

  16. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  17. Early earth had no oxygen

  18. How did early microbes generate energy? • Need to generate energy in absence of oxygen • Life evolved to utilize glucose • Glucose molecule has energy within its bonds • Multiple enzymes adapted to produce maximum energy out of these bonds • First part of glucose metabolism is glycolysis • All life on earth uses glycolysis ENERGY

  19. Glycolysis generates pyruvate and NADH • Glucose  2 pyruvate • 2 ATP generated • Energy currency of the cell • Needed for most cellular processes • 2 NADH generated • Electron carrier of the cell • Needs to be oxidized to NAD+ for glycolysis to continue • Fate of NADH determined by environment

  20. Glycolysis generates pyruvate and NADH • Glucose  2 pyruvate • 2 ATP generated • Energy currency of the cell • Needed for most cellular processes • 2 NADH generated • Electron carrier of the cell • Needs to be oxidized to NAD+ for glycolysis to continue • Fate of NADH determined by environment

  21. NADH and NAD+ Need to regenerate this to continue using glucose

  22. Regenerate NAD+ to keep using glucose Our muscles sometimes do this This pathway uses oxygen and generates lots of ATP

  23. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  24. Yeast use ethanol fermentation

  25. From glucose to ethanol

  26. Glucose comes from the grape

  27. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  28. But…wine isn’t just ethanol

  29. Glycerol in wine

  30. Glycerol production by yeast

  31. Organic acids • Tartaric, malic, and citric acid present in grapes • Tartaric and citric acid mostly stable throughout fermentation • Malic acid is tart and considered undesirable in a red wine • Luckily, bacteria (not yeast) can convert malic acid to more palatable lactic acid

  32. Oenococcusoeni • Certain bacteria are preferred over others based on byproducts of fermentation • Products of malolactic acid fermentation • Lactic acid • Acetoinand diacetyl • Acetic acid • Various esters • Higher alcohols

  33. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  34. What about before 1863? • Microorganisms are ubiquitous and live in various environments • Grapes are just one example of a habitat • Yeast and certain bacteria live on grapes • When grapes are crushed, the natural microbes mix with grape juice • Called spontaneous fermentation

  35. Spontaneous fermentation • Low concentrations of microbes on grapes • Fermentation takes longer to start • Entire process requires more time • Wild yeast may not have favorable traits • Low alcohol tolerance • Results in high sugar content • Other microbes present • Uncertainty of final product • Off-aromas and weird esters

  36. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  37. Yeast added to control end product

  38. Inoculated fermentation • Added at high concentrations to grape must • Inoculum outcompetes other microorganisms present • Preferred yeast is Saccharomyces cerevisiae • Multiple strains exist • Different strains for different wines • Other yeast can also be used • Kloeckera • Zygosaccharomyces

  39. Overview • History • Yeast • Sugar metabolism • Ethanol fermentation • Other products • Spontaneous fermentation • Inoculated fermentation • How to get the perfect yeast

  40. Breeding to generate perfect yeast • Yeast can be haploid (one copy of chromosomes) or diploid (two copies) • Take two yeast with desirable traits and breed them • Result is diploid cell containing genetic information from both parents

  41. Not a perfect process

  42. Target specific genes in yeast

  43. Let’s target glycolysis • Process involves a series of enzymes • We can increase number of enzymes in the process • Increased flux through glycolysis results in more pyruvate • More pyruvate results in more ethanol • Not much success • Rate limiting step is getting glucose into the cell

  44. Let’s target sugar transport • Increase amount of glucose getting into cell • Increased glucose in cell ultimately results in more ethanol • Target glucose transporters • Success!

  45. Let’s target sugar transport • Increase amount of glucose getting into cell • Increased glucose in cell ultimately results in more ethanol • Target glucose transporters • Success!

  46. Let’s improve ethanol tolerance • Different yeast strains have different ethanol tolerance limits • Ethanol destabilizes the membrane • Target membrane fluidity or detoxification enzymes • Many GMOs with increased tolerance

  47. Other modifications • Sulfur dioxide resistance • SO2 used to decrease unwanted microbes • Higher SO2 resistance in yeast means we can increase concentration and kill off even more unwanted microbes • Nitrogen assimilation • Yeast require carbon and nitrogen • Nitrogen not abundant in grapes • Increase nitrogen uptake or utilization to increase robustness of yeast

  48. Yeast GMOs result in more wine varieties

  49. Wine fermentation

  50. Questions?

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