Yeast strains available from the AWRI Wine Microorganism Culture Collection are provided as live yeast growing on agar slopes. These slopes should be stored sealed at 2–4°C and not frozen. If the slope is more than three months old, please discard it and obtain a replacement slope, or revitalize it in Yeast Media (YM) or on yeast extract peptone dextrose (YPD) agar.

Starter culture preparation

Prepare 2 L sterile filtered diluted juice

• Prepare 2 L of a juice or juice concentrate/chlorine-free water mixture at approximately 15°Brix/8 Baume, in a 2 L bottle. Note this will require approximately 1 L of juice and 1 L of water.
• Use mineral water/rainwater/clean tap water (free of inhibitory substances such as chlorine). Chlorine can be removed by sparging/boiling.
• Measure the concentration of sulfur dioxide (SO₂). The concentration of SO₂ (free/total) in the diluted juice should not exceed 5/50 mg/L. Oxidative techniques, including addition of hydrogen peroxide, can be used to lower excessive residual SO₂ (see Removal from and addition of sulfur dioxide to must, juice and wine and How to determine a hydrogen peroxide addition calculator).
• Sterile filter the diluted juice into a 2 L sterilised vessel, such as a conical flask or Schott bottle, fitted with a cotton wool plug. Options for sterilisation of the vessel include heat (autoclave), rinsing with 70% alcohol or dipping the vessel in a 2% sulfur dioxide (SO₂) solution and then draining it. An alternative approach is to sterile filter the juice and then add the required volume of sterilised water into the 2 L sterilised vessel.
• Add 2 g/L diammonium phosphate (DAP).

Inoculation

Transfer the yeast strain from the agar slope into 2 L diluted filtered juice

• Wash the contents from the agar slope with around 5 mL of the sterile juice using a sterile Pasteur pipette (or similar) and pour this into the juice mixture. Aerate the culture with sterile-filtered air using a compressed air cylinder or aquarium aerator. The aeration should be approximately 2.5 – 5% v/v/min (50 – 100 mL air per min) for the 2 L culture and continued until yeast cell numbers reach the desired concentration.
• Maintain the temperature at 20 – 25°C. Note that a warmer temperature will increase the rate of growth.

Inoculation of barrels

• Check the yeast count every 8 hours. When the cell number in the starter culture reaches approximately 2 x 10⁸ per mL (which could be the same day or overnight) or if the cell number ceases to increase any further, inoculate one 200 L barrel with the culture (2 L inoculum will result in an inoculation rate of 2 x 10⁶ cells per mL in 200 L juice if the desired density of 2 x 10⁸ cells/mL is achieved in the starter culture).
• Prior to inoculation, acclimatise the culture slowly (not more than 2°C change per hour) to the temperature of the juice/must to which it will be added. The temperature difference at the time of inoculation should not exceed 5°C. Large or rapid temperature changes induce physiological stress that could increase the fermentation lag phase.

Propagation – scale-up the starter culture for larger juice volumes

Propagation of yeast to required volume for inoculation into grape juice/must of 1,000L or less

2 L of starter culture is required for each 200 L of juice/must to be inoculated. For volumes up to 1,000 L of juice/must, scale-up the prepared culture further as follows:

• Prepare a further volume of a juice or juice concentrate/chlorine-free water mixture at approximately 15°Brix/8 Baume in an appropriately sized container. Add DAP as required to adjust the YAN to >180 mg/L and adjust the pH to 3.1-3.4 to support good yeast growth and minimise bacterial growth.
• Add the 2 L yeast culture prepared above to sufficient juice detailed below for the desired volume so that 2 L of starter culture is available for each 200 L juice/must to be inoculated. For example:
  • Add 2 L yeast culture to 2 L juice to inoculate 400 L juice/must
  • Add 2 L yeast culture to 4 L juice to inoculate 600 L juice/must
  • Add 2 L yeast culture to 6 L juice to inoculate 800 L juice/must
  • Add 2 L yeast culture to 8 L juice to inoculate 1,000 L juice/must
• Aerate the culture/juice mixture with filtered air (approx. 2.5 –5% v/v.min; or 250 – 500 mL air per min) using a compressed air cylinder or aquarium aerator and maintain the temperature at 20 – 25°C. Note that a warmer temperature will increase the rate of growth.
• Check the yeast count every 8 hours. When the cell number reaches approximately 2 x 10⁸ per mL, or if the cell number ceases to increase, inoculate each 200 L juice/must with 2 L of the starter culture (a 2 L inoculum will result in an inoculation rate of 2 x 10⁶ cells per mL in 200 L juice if the desired density of 2 x 10⁸/mL is achieved in the starter culture).

Propagation of yeast to required volume for inoculation into grape juice/must of more than 1,000 L

This step will normally require the use of a dedicated yeast propagator (Doddridge 1991) such that a starter culture volume of 2 – 2.5% of the juice/must volume can be produced. The inoculation volume of 2 – 2.5% assumes that the cell number in the starter culture can reach a minimum of 2 x 10⁸ cells/mL.

Expansion of the starter culture volume is normally carried out in 10-fold (v/v) increments. For example, the yeast cells contained on an agar slope are transferred to 2 L sterile juice, then to 20 L, followed by 200 L, 2 kL, 20 kL etc., until the required volume has been produced. Since each incremental step can take 1–2 days, sufficient time must be allocated for the expansion phase. If necessary, the starter culture can be chilled to no less than 5°C for short periods (for example, to hold the culture overnight) on the condition that the concentration of residual sugar (3–5°Be) and yeast assimilable nitrogen (> 100 mg N/L) is not permitted to become limiting. When using colder temperatures to delay the progress of a starter culture, maintain the air supply to reduce yeast sedimentation, which can have a severe negative impact on the culture’s vitality.

Reference

Doddridge, P.N. Propagation of yeast on a large scale. Aust. N.Z. Wine Ind. J. 6: 297-298; 1991.

Need help?

For more information, please contact AWRI Wine Microorganism Culture Collection team on 08 8313 6600 or culture@awri.com.au.