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