Control of malolactic fermentation

Why do we conduct MLF?

• de-acidification - especially in cool climates as malic acid levels are higher in cool regions, and especially in varieties that are naturally high malic producers

• reduce risk of bacterial growth in bottle - removal of nutrients

• sensory effects - change in pH

What are the sensory effects of conducting MLF?

• change in perception of varietal aromas (reduction of vegetative/herbaceous notes)

• increased mouthfeel (contribute to fullness & softness, related to pH/acid changes)

• production of flavour compounds (buttery, butterscotch, malty characters - diacetyl (0-5 mg/L - any more and becomes too much)

• increase in overall complexity/integration

• decrease in colour intensity

What varieties are naturally high in malic acid?

Pinot Noir, Malbec, Chenin Blanc

Why is MLF generally not done in white wines?

As we like the acid, pH is generally too low for bacteria, whites generally not fermented on skins so not a lot of nutrients available for bacteria to use

What is the main bacteria used for MLF called?

Oenococcus oeni

What culture options do you have for MLF (uninoculated)?

• uninoculated/indigenous LAB

  • 10² - 10^4 cpu/mL at crushing, from vineyard & winery equipment

  • high pH favours pediococcus and lactobacillus (>3.7)

  • low pH favours ornococcus oeni (<3.6)

What culture options do you have for MLF (inoculated)?

• Inoculated/selected LAB

  • agar slant, liquid & freeze dried (concentrates) of cultures of lactiplantibacillus plantatum or oenococcus oeni that require propagation prior to inoculation

  • highly concentrated freeze dried, or frozen pre-adapted lactiplantibacillus plantatum or oenococcus oeni for direct inoculation

What are strain selections based on?

• previous experience

• timing of inoculation

• chemistry of wine

• financial constrains (between $100-120 for 25 hL pack)

a large number of strains are commercially available from different manufacturers

What other factors can be used in strain selection?

• direct inoculation, no or limited reactivation

• strains selected on basis of physiochemical tolerance

• preparation of cultures via progressive adaptation

What are chemical limits in terms of MLF options?

Chemistry of wine critical

• energy sources

• growth factors

• pH

• SO2 (total)

• ethanol

• temp

In terms of energy sources for LAB, how are organic substances (hexoses & pentoses) oxidised to produce ATP?

Hexoses (flu & fru) and pentoses (ribose, xylose) are metabolised by LAB in two main pathways:

1. Homofermentative

2. Heterofermentative

These pathways ensure that LAB have a steady supply of ATP and maintain the redox balance by regenerating NAD+ through locate production

What is involved in the homofermentative pathway and what is an example of a LAB that is characterised as homofermentative?

• producing only lactic acid

• converts glucose -> pyruvate -> lactate

• Produces 2 ATP per glucose

• NADH is re-oxidised to NAD+ by lactate dehydrogenase (LDH), converting pyruvate into lactic acid

Pediococcus is an example of homofermentative LAB

What is involved in the heterofermentative pathway and what is an example of a LAB that is characterised as heterofermentative?

• producing lactic acid, acetic acid and CO2

• This type of metabolism is characterised by gas production from glucose

• Generates 1 ATP per glucose

• Re-oxidation of NADH occurs through various fermentation steps

Leuconostoc is an example of heterofermmentative LAB

In terms of energy sources for LAB, how are organic substances (citrate) oxidised to produce ATP?

Some LAB can metabolise citrate into diacetyl (butter character), acetoin (butter character) and CO2

In terms of energy sources for LAB, how are organic substances (arginine) oxidised to produce ATP?

Some LAB can break down arginine into ornithine, ammonia and ATP

This provides an additional energy source in environments with limited sugar, I.e., MLF after alcoholic fermentation where the wine is RS dry

How does the use of proton motive force work to generate ATP?

• The PMF drives the proton back into the cell through F0F1-ATPase synthase converting ADP + pyruvate to ATP

• This allows LAB to generate additional ATP even in energy limited conditions like wine

List the steps in the generation of a proton motive force (ATPase)

1. influx of negatively charged malate

2. efflux of neutral lactate + 2 protons

3. alkalisation of cytoplasm and increased pH gradient

What happens in the generation of a proton motive force (ATPase)?

LAB use a PMF to generate ATP through membrane bound F0F1-ATPase. PMF is created by pH and electrochemical gradients across the cell membrane.

Outline what occurs during the influx of negatively charged malate in the generation of PMF

• malate (negatively charged) enters the cell - often bringing a proton

• this reduces intracellular acidity, helping LAB survive in wine’s low pH

Outline what occurs during the efflux of neutral lactate + 2 protons in the generation of PMF

• once inside, malate is decarboxylated into lactate + CO2

• lactate exits the cell along with 2 protons

• this further increases the pH difference strengthening the PMF

Outline what occurs during the alkalisation of cytoplasm and increased pH gradient in the generation of PMF

• since malate uptake brings in protons but lactate removes it, inside the cell becomes less acidic

• this reinforces the pH gradient, making the PMF stronger

What are the growth factors required by LAB?

• carbon: sugars & organic acids

• nitrogen: amino acids, peptides, purines & pyrimidines

• vitamins: B-group required by LAB

• minerals: enzyme co-factors

What carbon sources are required by LAB (growth factors)?

Glucose, fructose (preferred by oenococcus oeni), arabinose, xylose, ribose (trace)

What nitrogen sources are required by LAB (growth factors)?

• strain dependent - possibly Ile, leu, val essential for oenococcus

• deficiency typically not occur, yeast breakdown and release

• nucleic acids act as growth activators

What vitamins are required by LAB (growth factors)?

Nicotinic, thiamin, biotin & pantothenic acid

What minerals are required by LAB (growth factors)?

Mg2+, Mn2+, K+, Na+, phosphorus

What four parameters directly determine the growth rate of LAB in wine?

1. pH

2. temperature

3. alcohol concentration

4. sulphur dioxide concentration

factors are not indecent of each other

a favourable level of one can compensate an unfavourable value of another (i.e., cells able to tolerate higher alcohol content&/or SO2 concentrations in wines with more favourable pH)

difficult to give exact limits

What do these colours represent in this diagram?

The different conditions and levels of physiochemical factors and their relationship together

What is not good for LAB?

Free SO2

Why is it difficult to get sparkling wines through MLF?

Due to super low pH - is possible, need to be careful & use proper adaptation methods

What are the two implications of pH on LAB growth?

1. influences growth rate

2. influences malolactic activity

In terms of pH, how does it influence growth rate?

• related to intracellular pH limit

• growth stops below this limit

• oenococcus oeni can maintain a higher pH (hence greater PMF) at low extracellular pH, relative to other LAB

In terms of pH, how does it influence malolactic activity?

• optimum pH

• relates to growth, therefore typically faster at higher pH

• threshold pH for malic vs sugar breakdown could be important in relation to acetic acid production

What is the impact of SO2 on MLF?

• molecular SO2 is antimicrobial

• concentration of molecular SO2 is pH dependent

• inhibits both growth and& malolactic enzyme

• bound SO2 is toxic

  • LAB can metabolise acetaldehyde bound to SO2

  • bound is 5-10x less active than free

What levels of ethanoll inhibit the growth of LAB? And what species are more sensitive than others?

8-10% v/v

Cocci are more sensitive than lactobacilli

What percentage of ethanol stimulates the growth of O.oeni?

5-6% v/v

What happens when bacteria grow in wine? LAB wise

They adapt to ethanol and the harsh conditions of a wine environment

Is ethanol strain dependent?

Yes

Lactobacillus (heterofermentors) grow in fortified wines

What are the optimum wine conditions in terms of temperature for LAB/MLF?

• optimum temp range 20-23 degrees

• as EtOH concentrations increase, temp optimum decreases

• growth limited below 14 degrees

• above 25 degrees MLF slows - growth reduced

• below 15-18 degrees MLF initiation delayed

How do sacch yeast and LAB compete or coexist during winemaking?

LAB growth is influenced by yeast-derived compounds. Some yeast strains produce metabolites that promote LAB activity, while others inhibit it. Factors such as pH, ethanol levels, and residual nutrients determine whether LAB can successfully perform MLF

MLF options: timing of inoculation (before alcoholic fermentation)

• inoculation with non-proliferating homofermentative

  • high biomass inoculated

  • rapid degradation of malic acid - good option if you have heaps of malic acid

  • advantage in carrying out MLF before ethanol production by yeast without acetic acid production

Risks

• not ethanol tolerant (<6%) & sensitive to SO2

• uncontrolled alcohol fermentation & partial MLF

MLF options: timing of inoculation (simultaneous)

• inoculation with O.oeni concurrently with yeast, allow adaptation to envionemtal conditions

• get population decline, then growth at end of alc fermentation, MLF initiates, rapid completion

• positive flavour effects (white wine)

• more research required

Risks

• get population decline, and then death - no advantage

• get yeast/bacterial antagonism, stuck ferment, production of acetic acid

Why is simultaneous inoculation with Saccharomyces yeast more suitable in high-Baume wines than sequential inoculation of lactic acid bacteria (LAB) after alcoholic fermentation?

When starting with a high sugar concentration, yeast fermentation produces more ethanol, which can be inhibitory to LAB if they are added sequentially after alcoholic fermentation. By inoculating LAB at the beginning, they are exposed to a gradual increase in ethanol rather than a sudden shock, improving their survival and ensuring a smoother malolactic fermentation (MLF). Additionally, early inoculation allows LAB access to nutrients before yeast deplete them, reducing the risk of sluggish or stuck MLF

MLF options: post alcoholic fermentation (sequential)

• most common as it involves a “dry” wine and is considered to be the safest

• minimal risk of sugar catabolism by LAB to produce acetic acid (particularly at <3.5pH)

• yeast breakdown may provide nutrients

• minimise any antagonism between yeast & LAB

Risks

• LAB may struggle to establish an active population due to a number of nutritional and physiochemical factors

• extended MLF period

MLF options: just prior to end of alcoholic fermentation

• inoculate 1-2 days before or after press

• similar to SIM inoculation - some of the advantages of acclimitisation

• limited scientific research some anecdotal evidence

  • loss of bacteria biomass post pressing, racking off gross lees

  • strategy for high alcohol wines?

When monitoring MLF what should you do during?

monitor once a week

• look & listen, smell & taste

• check & maintain temp 15-20 degrees

• paper or thin later chromatography, test strips, enzyme analysis

• L-malic acid analysis

• microscopic observations - chain formation in o.oeni

• qualitative not quantitative testing

What is the ideal rate of decrease in L-malic acid analysis?

0.1-0.2 g/L/day

How long should you wait between adding LAB after inoculating with sacch yeast?

24-48hr period after yeast added to add bacteria, you need to get rid of the SO2 or LAB will die

When monitoring MLF what should you do at the completion?

• malic acid conc less than 0.1 g/L

• indigenous MLF approximately 4-12 weeks

• inoculated MLF approx 2-8weeks

• citric acid metabolism may occur following the completion of MLF

• inactivation of LAB following MLF by lowering temp, addition of SO2 and acid adjustment if needed

What does citric acid metabolism create?

Acetic acid and diacetyl

What do you need other than know before inoculation?

• timing

• purpose: risk management/sensory effects

• physiochemical parameters (temp, pH, SO2, EtOH)

with this info: strain and inoculation selection possible