Transcript 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
But we did not understand it
• 6000 BCE
• Earliest evidence of wine production
• 1680 AD
• Description of small spheres during fermentation
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
Pasteur experiment
Sterilized
Not sterilized
Pasteur experiment
Sterilized
Not sterilized
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
Overview
• History
• Yeast
• Sugar metabolism
• Ethanol fermentation
• Other products
• Spontaneous fermentation
• Inoculated fermentation
• How to get the perfect yeast
What are yeast?
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
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
Many yeast species
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
Overview
• History
• Yeast
• Sugar metabolism
• Ethanol fermentation
• Other products
• Spontaneous fermentation
• Inoculated fermentation
• How to get the perfect yeast
Early earth had no oxygen
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
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
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
NADH and NAD+
Need to regenerate this to
continue using glucose
Regenerate NAD+ to keep using glucose
Our muscles
sometimes do this
This pathway uses oxygen
and generates lots of ATP
Overview
• History
• Yeast
• Sugar metabolism
• Ethanol fermentation
• Other products
• Spontaneous fermentation
• Inoculated fermentation
• How to get the perfect yeast
Yeast use ethanol fermentation
From glucose to ethanol
Glucose comes from the grape
Overview
• History
• Yeast
• Sugar metabolism
• Ethanol fermentation
• Other products
• Spontaneous fermentation
• Inoculated fermentation
• How to get the perfect yeast
But…wine isn’t just ethanol
Glycerol in wine
Glycerol production by yeast
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
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
Overview
• History
• Yeast
• Sugar metabolism
• Ethanol fermentation
• Other products
• Spontaneous fermentation
• Inoculated fermentation
• How to get the perfect yeast
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
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
Overview
• History
• Yeast
• Sugar metabolism
• Ethanol fermentation
• Other products
• Spontaneous fermentation
• Inoculated fermentation
• How to get the perfect yeast
Yeast added to control end product
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
Overview
• History
• Yeast
• Sugar metabolism
• Ethanol fermentation
• Other products
• Spontaneous fermentation
• Inoculated fermentation
• How to get the perfect yeast
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
Not a perfect process
Target specific genes in yeast
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
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!
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!
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
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
Yeast GMOs result in more wine varieties
Wine fermentation
Questions?