Modified Yeast for Beer Fermentation Flashcards
What are the main steps in beer manufacturing? [4]
- Malting (Germination; Kilning)
- Mashing (Milling; Mash-In; Mash-Out; Lautering [Recirculation; Sparging])
- Boiling (Wort Kettle Boil; Addition of Hops)
- Cooling and Clarification (Whirlpool Centrifugation; Aeration)
- Pitching
- Fermentation (Dry Hopping - Optional)
- Post-Fermentation (Maturation and Clarification; packaging)
Why is barley converted to malt? [2]
So that (1) the enzyme synthesis is maximized and (2) the enzyme activity is stable and well preserved
During malting, barley grains are germinated so that enzymes needed to break down starch are expressed, followed by drying in a kiln to preserve enzyme activity.
Why are barley grains germinated during malting?
- So that enzymes needed to break down starch are expressed, followed by drying in a kiln to preserve enzyme activity
What occurs during kilning? [2]
Malting
- Non enzymatic browning
- Heat generated flavour reactions
Compare dark beers and paler beers, re: malting.
- Dark beers (stouts, porters) use a darker, more flavourful malt
- Paler beers (pale ale) use lightly-coloured, less roasted malt
- Since less ethanol can be generated from dark malt, dark beers are usually made with a combination of dark and lighter based malts
- The darker the malt, the less sugar is generated from the starch because the less amylolytic activity present.
Generally speaking, the darker the malt, the […] because […].
Generally speaking, the darker the malt, the less amylolytic activity because less sugar is generated from the starch.
Since less ethanol can be generated from dark malt, dark beers are usually made with a combination of
dark and lighter based malts
Why is mashing necessary?
The starches, proteins and enzymes necessary for beer production need to be extracted from the malted and kilned barley
The malt must be milled to […].
Break the kernal and expose the interior portion to an aqueous environment
The finer the malt, the more easily the enzymes and other materials are extracted.
True or False?
True.
However, the insoluble residue can be difficult to separate later on after the mashing step
The coarser the malt, the more easily the enzymes and other materials are extracted.
True or False?
False.
The finer the malt, the more easily the enzymes and other materials are extracted.
However, the insoluble residue can be difficult to separate later on after the mashing step
The finer the malt, the less easily the enzymes and other materials are extracted.
True or False?
False.
The finer the malt, the more easily the enzymes and other materials are extracted.
However, the insoluble residue can be difficult to separate later on after the mashing step
The coarser the malt, the less easily the enzymes and other materials are extracted.
True or False?
True.
However, the insoluble residue can be difficult to separate later on after the mashing step
It is easier to separate coarsely ground malt with minimum husk damage.
True or False?
True.
It is harder to separate coarsely ground malt with minimum husk damage.
True or False?
False.
It is easier to separate coarsely ground malt with minimum husk damage.
It is easier to separate finely ground malt with minimum husk damage.
True or False?
False.
It is easier to separate coarsely ground malt with minimum husk damage.
It is harder to separate finely ground malt with minimum husk damage.
True or False?
True.
What is the purpose of mashing?
- To extract the enzymes, starches, and proteins and solubilize them in a hot aqueous solution
- It also results in the reaction of the enzymes with the starches and proteins to form nutrients that the yeast can use for growth during fermentation.
When does mashing begin?
- When the malt (20-30%) is mixed with brewing water at ambient temperature in a specialized tank called a ‘mash tun’.
What is the malt-water mixture called?
The mash.
The mash is […] from 20 to 60-65C.
Gradually heated
By gradually increasing the heat,
different amylase enzymes will
get activated
Why is the mash gradually heated and not quickly heated?
By gradually increasing the heat, different amylase enzymes will get activated
Describe the carbohydrate fraction of the malt.
It is mostly in the form of starch.
What are the main starch-degrading enzymes released during malting?
Alpha and beta amylase
What will yeast use as the sugar source? [3]
- Maltotriose
- Maltose
- Glucose
What are the products of alpha-amylase?
- Dextrins
- Limit dextrins
What are the main products of beta-amylase? [4]
- maltose
- maltotriose
- glucose
- small branched dextrins
Define: mashout
When the temperature of the mash is raised to 75 degrees C which inactivates enzymatic
activity.
The insoluble material in the mash liquid, (which still contains grain solids, insoluble proteins, carbohydrates) needs to be separated from the liquid
What needs to happen after mashout?
- The insoluble material in the mash liquid, (which still contains grain solids, insoluble proteins, carbohydrates) needs to be separated from the liquid
- Some mash tuns have filtration capacity
- Mash can also be pumped into a separate tank called a lauter tun.
What are the 3 steps of lautering?
- Mashout
- Recirculation
- Sparging
Define: wort
- The liquid material, or filtrate, that is collected at the end of the mash separation
When are hops added to beer?
After mashing and heating, before kettle boil and mashout
Hops were not in the ‘original’ beer recipes but were added during the middle ages
Why are hops added to beer?
Hops are derived from the plant Homulus lupulus
Hops were not in the ‘original’ beer recipes but were
added during the middle ages
- The flowers of the hop plants provide flavour and aroma to beer
- A large variety of hop cultivars or varieties are commercially available and are distinguished based on their aroma and flavour properties
- The predominant flavour that hops provide is bitterness due to the presence of alpha-acids and beta-acids
Brewers select hops based on […]
- Alpha-acid content (compared to whole hop weight)
- Alpha-acid content can range from 5% (low bitterness) to 14% (very bitter).
How is the bitterness contributed by hops expressed?
- In terms of International Bitterness Units (IBU)
- 1 IBU = 1mg of iso-alpha-acids per litre of beer
- U.S. lager = bitterness less than 15 IBU
- U.K. ale = bitterness of 50 IBU
One predominant flavour that hops provide is bitterness due to the presence of alpha-acids and beta-acids.
What is the role of alpha-acids? [2]
- Alpha-acids serve as precursors for thermal isomerization reactions that result in iso-alpha-acids formation during wort boiling (iso-humolone and iso-cohumulone)
- The iso-alpha-acids increase the shelf-life of the beer due to antimicrobial activity.
Why does adding hops improve beer shelf-life?
- The iso-alpha-acids formed during wort boiling increase the shelf-life of the beer due to antimicrobial activity.
What is responsible for the aroma or bouquet property of hops?
- The essential oil fraction of hops
Terpenoids, esters, ketones, and other volatiles
What comprises the essential oil fraction of hops?
- Terpenoids
- Esters
- Ketones
- Other volatiles
Define: dry hopping
adding hops into the fermentation vessel close to the end of fermentation
If hops are added during the beginning of the kettle boil step:
Most volatiles will be lost, but iso-alpha-acids will form (=bitterness)
When high-oil hops are used, they are usually added […]
Near the end of the kettle boil step to preserve the volatiles (less iso-alpha-acid formation; less bitterness)
What are the 3 categories of hops?
- Aroma
- Bitter
- Dual-purpose
Describe aroma hops. [2]
- Have a lower alpha-acid percentage and an oil profile associated with good aroma
- Generally used for finishing or dry hopping
Dry Hopping: adding hops into the fermentation vessel close to the end of fermentation
Describe bitter hops. [2]
- Have a higher alpha-acid percentage
- Generally used in the boiling process to extract bitterness.
Describe dual-purpose hops. [2]
- Have qualities of both the aroma and bitter hops
- Are both aromatic and bittering
Describe the characteristics of amarillo hops.
- Flowery
- Citrus-like aroma
Describe the characteristics of chinook hops.
- Slightly spicy
- Very piney
- Robust grapefruit
What is the purpose of boiling the wort? [5]
The wort is pumped into a special heating tank called the brew kettle; hops are added (or not); mixture is boiled for 1-1.5 hours.
- Kills microbes; making the wort sterile
- Extracts oils and resins from the hops
- Accelerates isomerization of the hop alpha-acids
- Enhances colour development by catalyzing Maillard reaction products
- Remove excess water
What is the 2nd round of precipitate that needs to be removed during beer production?
- The precipitate formed during the kettle boil needs to be removed
How is the precipitate formed during the kettle boil removed?
- The hot wort is centrifuged using continuous centrifuges called a whirlpool separator
- The precipitate collects in the center of the tank which has solids and hop remains
- After the precipitate is removed, the wort is cooled in plate-type heat exchangers
Why after the precipitate is removed, is the wort is cooled in plate-type heat exchangers?
Because high temperature will kill the yeast.
What must be done before yeast is added to the wort? [2]
- The wort temperature is reduced to 10C to 15C
- The wort will be aerated or sparged with sterile air
Why is wort aerated?
Even though beer fermentation turns
into an anaerobic environment,
starting the process with an aerobic
environment helps the yeast rapidly
enter logarithmic growth
What is pitching?
After the wort is cooled and aerated,
the yeast is added in a step called
‘pitching’
Why is the distinction of top and bottom fermenting yeast less relevant now?
Due to the modern use of enclosed fermentation vessels, the distinction between top and bottom fermenting yeast is less relevant as ale yeasts can even drop to the bottom of the fermentor
Describe top-fermenting strains.
- Ales are fermented with top-fermenting strains
- Saccharomyces cerevisae
Ales are fermented at […]
High temperatures from 18-27C
Lagers are fermented at […]
- Temperatures below 15C.
Describe bottom fermenting strains.
- Lagers are fermented with bottom fermenting strains
- Saccharomyces pastorianus
90% of the beer worldwide is produced by […]
Lager yeast
How much yeast is added during pitching?
- 100 billion yeast per 5 gallons of wort
Describe the growth of yeast during fermentation.
- After lag phase, the yeast cells enter logarithmic growth and alcoholic fermentation begins
- The log phase growth of the yeast lasts ~2-3 days for ales and up to 6-7 days for lager (due to lower temperature)
- After log phase, all of the mono and di-saccharides will have been fermented and most of the maltotriose
How do brewers monitor the sugar fermentation of beer?
- By measuring specific gravity of the beer
- The specific gravity of a liquid is the liquid’s density compared to water
- As the fermentation progresses, the specific gravity will decrease
- e.g. specific gravity of 1.030 is 1.03 times the density of water
- Once the specific gravity stops decreasing, the fermentable sugars are depleted and the beer is “fully attenuated”
The specific gravity of beer decreases during fermentation because:
- Sugars are consumed, reducing the density.
- Ethanol is produced, which has a lower density than water.
- CO₂ escapes the liquid, further reducing the mass of dissolved components.
When is beer considered “fully attenuated”?
Once the specific gravity stops decreasing, the fermentable sugars are depleted and the beer is “fully attenuated”
What is the problem with hops to justify this type of GMO?
- Hops are an expensive ingredient for breweries to source
- ~100 billion L of water are required for annual irrigation of domestic hops (US)
- Flowers of the hop plant provide bitterness and ‘hoppy’ flavour to beer
- Essential hops oils are rich in terpenes which impart a hoppy flavour to beer
- Two monoterpene molecules, linalool and geraniol are important for the floral aroma of Cascade hops which is widely used in American craft brewing
Hops are pricey and thirsty!
How could we get around the requirement to add flavour (dry hopping) in fermentation of beer?
- Created a brewer’s yeast (Saccharomyces cerevisiae) strain that can biosynthesize monoterpenes
Outline the method for creating a GMO brewer’s yeast that can synthesize monoterpenes.
- The yeast Saccharomyces cerevisiae naturally produces FPP (farnesyl pyrophosphate) through the ergosterol (yeast version of cholesterol) biosynthesis pathway
- Blue arrow represents reaction carried out by hydroxymethyl-glutaryl-CoA reductase (HMG-CoA reductase)
- Removal of the HMGR inhibitory regulatory domain results in increased flux towards end products
- Orange arrow represents reaction carried out by FPP synthase
- A mutant form of FPPS reduces conversion rate and increases amount of GPP
- Green and purple arrows that are new non yeast enzymes engineered into the pathway.
Describe the changes made to the monoterpene biosynthesis pathway in the creation of a GMO brewer’s yeast that can synthesize monoterpenes.
- Black arrows are the enzymes naturally present in S. cerevisiae that were not changed.
- Blue arrow represents reaction carried out by hydroxymethyl-glutaryl-CoA reductase (HMG-CoA reductase)
- Orange arrow represents reaction carried out by FPP synthase
- Green and purple arrows that are new non yeast enzymes engineered into the pathway.
What is the key rate-limiting step of the monoterpene biosynthesis pathway?
HMG-CoA Reductase
Controlled by an inhibitory regulatory domain.
Removal of the HMGR inhibitory regulatory domain results in increased flux towards end products (truncated HMGR = tHMR)
What is isopentyl pyrophosphate converted to in the monoterpene biosynthesis pathway?
Geranyl pyrophosphate
What limits the amount of GPP available in the monoterpene biosynthesis pathway?
FPPS has a high conversion rate of GPP to FPP.
A mutant form of FPPS (FPPS*) reduces the conversion rate and increases the amount of GPP
What is geranyl pyrophosphate converted into?
Farnesyl phosphate
By FPP synthase
What does the mutant form of FPPS do?
- Reduces the conversion rate of GPP to FPP to icnrease the amount of GPP available
Which monoterpene synthases that catalyze GPP (geranyl pyrophosphate) to linalool and geraniol in hops were identified for creating a GMO brewer’s yeast to synthesize monoterpenes?
- Linalool synthase (LIS) gene in mint
- Geraniol synthase (GES) from basil
Both can be expressed and function in yeast.
How were genes for LIS and GES introduced to the monoterpene biosynthesis?
- Used a beer brewing strain with deletion of the ADE2 gene (ADE2-delta) which is red
- The repair (donor DNA) template has homology to upstream and downstream regions of the ADE2 gene
- Designed a synthetic guide RNA (sgRNA) to allow Cas9 to cut at the location indicated by a red line
- The ~10kb repair template contains the ADE2, tHMR, LIS, GES and FPPS* genes with promoters
- If the repair template is integrated successfully into the yeast genome, colonies will be white
Describe testing of GMO beer yeast strains.
- Many combinations of promoters and genes were tested
- Micro-fermentation conditions
In the creation of a GMO yeast strain that can synthesize monoterpenes, what happened after the first iteration?
- After first iteration they realized that GES abundance was not correlated with geraniol production
- Since GES and LIS compete for GPP supply, they found that the fraction of geraniol correlated with the ratio of GES and LIS
- Second iteration design had strong promoters driving tHMGR, GES and FPPS* expression with a range of promoter strengths for LIS expression
Describe pilot-scale beer fermentations with engineered strains.
- JBE strains produce more consistent linalool and geraniol than fermenting with parent strain and dry hopping
- The JBE strains produce different amounts.
Describe sensory analysis of pilot-scale beer fermentations with engineered strains.
- Figure 5c
- American ale produced in pilot scale
- 3 different GMO strains (JME)
- Parent strain - WPL001 with no dry hopping
- Figure 5d
- JBE fermented beer with no dry hops
- Compared to WL001 with dry hopping and two dry hopped beers (WA and ID dry hop)
- JBE strain generates more flavour than dry hopping.
How long is log phase for ale fermentation vs. lager fermentation?
The log phase growth of the yeast lasts ~2-3 days for ales and up to 6-7 days for lager (due to lower temperature)
What is the incubation temperature for ales vs. lagers?
Ales: 18-27 degrees C
Lagers: Below 15 degrees C
Which yeast strains are used to ferment ales vs. lagers?
Ales: Saccharomyces cerevisae (top fermenting)
Lagers: Saccharomyces pastorianus (bottom fermenting)
Think: Lager - lower - bottom - pastorianus
