Yeast & fermentation management

What are the most ESSENTIAL things a winemaker wants from a yeast strain?

They must be able to SURVIVE, ADAPT & GROW under conditions such as:

• Anaerobic

• High sugar (10-15 Bé)

• pH below 4.0

• Temp range 8-35*C

What are some other things a winemaker wants from a yeast?

• Nutritional requirements that allow predictable behaviour and convenient fermentation management

• Alcohol tolerance for complete fermentation

• Consistent end-product formation under fermentation conditions

In the yeast’s perspective; what must it be able to do?

• Increase population from 1-5 × 10^6 cells/mL to 1-2 × 10^8 cells/mL

• Accommodate ‘batch’ system design

  • decline in nutrients

  • accumulation of toxic end-products

What is the highlighted phase called?

Inoculation

What is the highlighted phase called?

Stationary phase

What is the highlighted phase called?

Decline/death phase

What is the highlighted phase called?

Lag phase

What is the highlighted phase called?

Exponential phase

What is the oenological consideration at the lag phase and decline phase?

To AVOID temperature shocks

What does the lag phase in yeast growth/death involve?

Healthy yeast and gradual environmental transition

Temperature control is difficult in which phase of yeast growth/death?

The exponential phase

What does the exponential phase in yeast growth/death involve?

Heat evolution and sugar transport rate limiting

During the stationary phase it is important to what?

Avoid nutrient limitation

What does the stationary phase in yeast growth/death involve?

Nutrient decrease and end-product build up

What does the decline phase in yeast growth/death involve?

Nutrient exhaustion, ageing/dying population

What can you expect occurs during alcoholic fermentation in terms of yeast populations?

You can expect the yeast population to increase to 1-2 × 10^8 cells/mL

For sugar accumulation in alcoholic fermentation, distinction occurs between two phases. What are the phases and what is the distinction?

Exponential phase: approximately 50% of sugar is directed to biomass and energy production

Stationary phase: near 50% of sugar is directed to energy production and by-product formation without a significant change in cell biomass

What is the exponential phase characterised by and what should you do to support yeast cells/the fermentation?

Characterised by high viability and high budding

• do haemocyctometer counts, flow cycometry

• do viable/vital staining

What is the stationary phase characterised by and what should you do to support yeast cells/the fermentation?

Characterised by declining viability and budding

• do carefully monitor progress of fermentation

• do accurately determine residual sugar towards end of fermentation

During the stationary phase, in addition to ethanol and CO2 what are some other metabolic end-products that are formed?

• organic acids

• higher alcohols

• esters

• carbonyl compounds

• polyols (glycerol)

The theoretical ethanol yield is never realised

200g of glucose makes 102.2g of ethanol and 97.8g of CO2

What are the range and concentration of yeast by-products determined by?

• Characteristics of the strain

• Wine production steps

• Nutritional and physiochemical conditions of fermentation

What sort of style impacts does yeast choice have on resulting wine?

• commercially available (sacch & non-sacch) - selection based upon documented features and experience

• In-house strains - special features, on-site propagation

• Indigenous, uninoculated strains (a succession of non-sacch & sac species) - additional complexity, associated risks

What features and risks do a indigenous ferment bring?

Add complexity as there will be more than 1 strain working

Risks: extended lag phase - low population to start with

What are some other things you can do to ensure you have selected the correct yeast for your ferment?

Continuing to research:

• extending knowledge of yeast behaviour

• Yeast performance can be value adding (alternative genera & species)

However:

• still experience sluggish/stuck fermentations

How can yeast influence on quality of wine?

Can influence wine in different ways:

• be consistent & reliable with minimal impact plus:

  • can influence grape varietal expression (extracellular enzyme activity) & mouthfeel perception (polysaccharide production)

  • can contribute via addition of yeast derived flavours (esters etc.)

What are the basic features of a saccharomyces selection?

• tolerance to sugar, alcohol, SO2

• predictable & controllable (some can be FAST fermenters - research & decide what’s best)

• desirable fermentation by-product profile

• nitrogen demand (generally need more nutrition than suggested by tech reports)

Desirable characteristics of saccharomyces selection

• basic growth factor requirements

• high osmotolerance

• ability to tolerate SO2

• tolerate alcohol produced

• minimal production of undesirable by-products

• low foaming, ability to flocculate & settle

Yeast performance is dependent on environmental conditions:

• physio-chemical

• influence of winemaker

Special features of saccharomyces selection

• low temp fermentation

• beta-glucosidase fermentation aromatics

• mouthfeel & phenolic extraction

• colour enhancement

• compatibility with LAB

Other features of saccharomyces selection

• regional & style contribution (e.g., France regions where you are bound to a particular style)

• low ethanol yield

• killer activity (not sensitive to other yeasts killer factors/toxins)

Why is yeast strain a critical step?

• take into account strain characteristics related to wine style

• predictable and controllable fermentation at desired temp (fit with refrigeration/control capacity)

• predictable nutritional demands

• other physio chem limitations

What are some characteristics of non-saccharomyces strains?

• typically not able to complete AF

  • need to over-inoculation with sacch

• range of different impacts

  • bioprotection (grape transport, cold soak, stabulation)

  • mimicking uninoculated fermentation (early to mid stages of AF

Why might one use non sacch yeasts?

To get a range of different compounds and added complexity - higher costs as you need to buy more than one yeast strain though

What are some important things to note when rehydrating dried yeast?

• follow instructions of yeast packet

• if you inoculate below 15 degrees - more problems

• don’t inoculate with a temp difference greater than 5 degrees

What is ergosterol?

ergosterol is a sterol, a type of lipid, that yeast cells synthesize under aerobic (oxygen present) conditions, essential for maintaining cell membrane integrity and functionality

Why is ergosterol important?

It enhances yeast growth, metabolism and viability during fermentation and improves resistance to stress

What is an example of an easily assimilable form of nutrients for yeast?

Yeast ghosts or hulls

What are some critical factors for inoculation?

• temp of water/juice at rehydration can be detrimental to viability

• yeast physiological restrictions and nutritional requirements must be respected

• addition of complex nutrients

• always follow manufacturers recommendations

• winery hygiene, microbial/chemical control

• forward planning essential

Lower temperature rehydration

• need to respect manufacturers instruction

  • some non sacch need lower temp e.g., 25 degrees

  • lower temp rehydration protection products can be used (e.g., Lallemand Go Ferm Sterol Flash)

What is critical to the success of a ferment?

Initial nutrient concentration and aeration

fermentation conditions need to support 4-6 yeast generations

completion of fermentation requires yeast numbers of 1 × 10^8 cells/mL

What needs to be done in terms of inoculation if you need to run a quick ferment?

Higher inoculation rate is important

What is assimilable nitrogen in terms of yeast nutrients? And in what form can be added?

It is the second most important macronutrient after carbon

required for amino acid synthesis - proteins

DAP or DIAMMONIUM PHOSPHATE is used for YAN (22% N)

What is DAP addition dependent on?

• strain

• oxygen availability (i.e., next limiting nutrient)

• initial sugar content (i.e., how much work the yeast must do to finish fermentation)

What is the max amount of DAP you can add to a ferment?

Max DAP you can add before exceeding limit is 1700 mg/L

What is minimum N VS maximum N?

Minimum N to complete or maintain fermentation rate

or

Maximum N or Total N used when present in excess

what is the Minimum YAN requirement for low risk fermentation?

• Whites – approx. 150 mg/L

• Reds – approx. 100 mg/L

What is the YAN requirement for clean/fruity flavour?

• Whites – approx. 250 to 350 mg/L

• Reds – unknown (research in progress)

What is the Maximum YAN demand?

• Mean = 400 mg/L

• Range = 330 to 470 mg/L

What are the consequences of nitrogen limitation?

• H2S liberated - no action, mercaptan (off aroma/flavour)

• sugar transport slows or ceases

  • hexose permeases irreversibly inactivated

  • delayed additions may be slow to help

• biomass formation limited

  • fermentation rate is a function of biomass and vitality

• FERMENTATION STOPS

What are the key factors in H2S production?

1. yeast strain genetic propensity (sulphate reduction sequence (SRS) metabolism)

2. Yeast assimilable nitrogen (YAN) concentration

3. SO2 (exogenous or derived from the reduction of sulphate) import in yeast SRS metabolism

4. vitamins )thiamine, pyridoxine, biotin pantothenic acid) are important in yeast SRS metabolism

Generally speak, lower initial N concentration, higher H2S response

Nitrogen additions: how much?

• varies according to strain

• varies according to composition/conditions

• need to know how much is in the juice

• measure YAN - YAN = free amino nitrogen + ammonia

• routine analysis difficult in terms of:

  • methodologies and constraints of vintage

  • easier methods = approximation

  • historic data helpful

Make an educated guess

Nitrogen additions: timing - earlier additions?

Earlier additions can lead to:

• fermentation duration reduced

• greater stimulation of fermentation rate

• increased cell numbers

Nitrogen additions: timing - later additions?

Later addition leads to:

• reduced cell numbers

• reduced range in fermentation rates

• reduced fermentation duration BUT to less extent

Conclusions of nitrogen additions

• additions helpful

• need a guide - YAN vs yeast requirements, analysis, experience, history

• consider timing - moderate initial dose, more later, signals of depletion - H2S, slowing of ferment

What is the average N demand of wine yeast strains?

350 mg YAN/L

What are the recommendations for YAN?

• yeast strain plays a critical role

• Measure Yan

• understand strain N demand and H2S potential

• consider timing of DAP additions

• DAP additions can also effect flavour outcomes

What are the major grape sugars?

Glucose and fructose

sucrose in low concentration

glu and fru are the major carbon sources utilised by yeast during fermentation

What are importation consideration when it comes to sugars and fermentation?

• very high sugar concentrations (>250 g/L) cause osmotic stress - consider this impact for ferment

• glucose and fructose are utilised at different rates - can be important in stuck ferments

What happens in sugar consumption during fermentation?

• initially grape juice sugar composition ~ 50/50 glu/fru

• during fermentation ration glu:fru decreases

• if the fermentation sticks, residual sugar is mainly fru

What are the important steps needed to determine the termination of fermentation?

• towards end of ferment MUST be diligent in determining end point

• necessity for accurate sugar analysis, not just hydrometry

  Need residual sugar analysis: enzyme G&F, rebelein etc

• once completed (sugar dry <2 g/L), post fermentation processing occurs immediately

further consideration must be given to microbial stability (e.g., SO2 additions for whites, or MLF inoculation in reds)

What are some factors for sluggish or stuck ferments?

• excessive clarification of juices/low solids

• nutritional deficiencies in juice

• strain selection

• unsuccessful inoculation

• initial temp too low

• temp too high

• temp shocks, causing cell damage/death

• presence of inhibitors (e.g., ethanol, medium chain fatty acids, yeast killer toxins, bacterial contamination and pesticide residues)

During the monitoring of fermentation; what can we measure?

• sugar (g & f)

• YAN

• temp, pH, TA, SO2 - free & total

• microscopic observations, cell counts

• turbidity

How can one monitor and manage yeast during fermentation? (1)

• at inoculation, must/juice temps >15 degrees to minimise yeast viability losses

• ferm monitored by measuring sugar content (hydrometer) 1-2x daily, and then determining rate of change in sugar concentration

Need to plot a fermentation curve - helps to manage any problems that arise

How can one monitor and manage yeast during fermentation? (2)

• for whites - average 0.5-1 bé/day expected under controlled fermentation conditions

• reds ferment much faster due to the presence of skins with reduction on average 1-2 bé/day preferred

• cooling should be delayed until about 1 bé of sugar is fermented and avoided once ferment drops to about 2 bé

• agitation of the ferment is generally required when cell vitality declines