Presentation Transcript

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?