science behind wine fermentation sara belchik 28 june 2014
TRANSCRIPT
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SCIENCE BEHIND WINE FERMENTATIONSara Belchik
28 June 2014
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Wine fermentation
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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Wine predates written records
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But we did not understand it• 6000 BCE
• Earliest evidence of wine production
• 1680 AD• Description of small spheres during fermentation
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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
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Pasteur experiment
Sterilized Not sterilized
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Pasteur experiment
Sterilized Not sterilized
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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
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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What are yeast?
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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
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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
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Many yeast species
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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
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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Early earth had no oxygen
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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
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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
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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
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NADH and NAD+
Need to regenerate this to continue using glucose
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Regenerate NAD+ to keep using glucose
This pathway uses oxygen and generates lots of ATP
Our muscles sometimes do this
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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Yeast use ethanol fermentation
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From glucose to ethanol
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Glucose comes from the grape
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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But…wine isn’t just ethanol
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Glycerol in wine
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Glycerol production by yeast
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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
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Oenococcus oeni• Certain bacteria are preferred over others based on
byproducts of fermentation
• Products of malolactic acid fermentation• Lactic acid• Acetoin and diacetyl• Acetic acid• Various esters• Higher alcohols
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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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
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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
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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Yeast added to control end product
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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
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Overview• History• Yeast• Sugar metabolism• Ethanol fermentation• Other products• Spontaneous fermentation• Inoculated fermentation• How to get the perfect yeast
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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
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Not a perfect process
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Target specific genes in yeast
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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
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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!
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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!
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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
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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
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Yeast GMOs result in more wine varieties
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Wine fermentation
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Questions?