Preparation of a malolactic fermentation (MLF) starter culture using freeze dried bacteria


The protocol sets out the steps required to adapt a freeze dried bacteria culture for growth in the relatively harsh conditions of wine. The procedure is recommended when either direct inoculation has failed or when the winemaker has determined by measurement of critical wine parameters (temperature, pH, alcohol, sulfur dioxide (SO2) and compatibility of yeast strain) the wine conditions are particularly unfavourable. The protocol is based on the collective experience of bacteria suppliers, winemakers and AWRI staff.

There is no guarantee that the protocol will resolve a specific problem wine, however, many winemakers have reported success when applying this procedure to their failed or stuck MLFs.

Critical wine parameters

Before proceeding with initiation of MLF, confirm (measurement/assay) the critical wine parameters and make adjustments where possible this step can greatly increase the chances of success.

Critical parameters to consider:

  • temperature should be within 15 – 25°C (preferably 18 – 22°C, when other parameters are unfavourable);
  • pH should exceed 3.0 (or higher when alcohol and SO2 concentration are high);
  • alcohol should be less than 15% v/v (<16% v/v when using most alcohol tolerant strains); and
  • total SO2 concentration should be < 40mg /L (try to keep total SO2 concentration less than 50 – 70 mg/L immediately before alcoholic fermentation).

In addition to the above parameters, possible yeast-bacteria interactions should also be considered consult your supplier on choosing a malolactic bacteria strain that is most compatible with the fermentation yeast used.

Procedure (for induction of MLF in 1,000 L of problem wine)

Step 1. Preparation of activation medium

5 litre grape juice1(preservative/SO2free)
5 litre water (chlorine free)
nutrient mix (use as per label instructions)

  1. Mix to dissolve and to achieve a homogeneous preparation.
  2. Adjust pH to 4.0 (using calcium carbonate).

Step 2. Rehydration of freeze dried Oenococcus oeni bacteria

  1. Dissolve bacteria in lukewarm (25 – 30°C) water (as per label instructions).
  2. Allow bacterial suspension to stand for 30 minutes.
  3. Mix reactivation medium (from step 1) with the fully rehydrated malolactic bacteria and maintain temperature between 18- 22°C with as little ullage as possible.
  4. Monitor the L-malic acid concentration by enzymatic analysis and when approximately 50% (approximately 48 – 72 hours) of the malic acid has been utilised, proceed to step 3. At this stage there should be carbon dioxide (CO2) evolution from the culture (and foam islands), and a slight lactic smell should be detectable.

Note that care must be taken to ensure that the bacteria do not consume all of the L-malic acid (do not allow the L-malic acid concentration to fall below 1 g/L). If the culture runs out of L-malic acid a rapid loss of bacteria activity can be expected.

Step 3. Acclimatization of malolactic bacteria to difficult wine conditions

  1. Add 10 L of the problem wine to be inoculated to the 10 L of starter culture and maintain at 18 – 22C. Monitor the L-malic acid concentration and do not allow the L-malic acid concentration to fall below 1 g/L.
  2. After approximately 24 hours (or when the L-malic acid concentration drops to approximately 1 g/L), proceed to step 4. Alternatively, you can conduct another acclimatisation step, (but again, do not allow the L-malic acid to fall much below 1 g/L).

Step 4. Inoculation of the starter culture into the bulk wine

With gentle stirring, transfer the acclimatised, active malolactic culture into 1,000 L of the problem wine.


Costello, P.J. 1993. Induction procedures for malolactic fermentation Aust. N.Z. Wine Ind. J. 8(1): 51-56.

1 Australian grape juices usually contain 2 g/L or more of malic acid.