Protocol for Difficult Malo-lactic fermentation
Overview
The success of a smooth Malo-lactic fermentation depends on several physio-chemical factors where alcohol, pH, temperature and the sulfur dioxide content of the wine all act as a factor in the dynamic of the fermentation. It is essential to monitor these parameters as each individual parameter has a range over which MLF is favourable. As more of these factors become unfavourable, the environment for fermentation will become increasingly harsh and therefore, causing difficulties for the bacteria to complete the fermentation smoothly.
The favourable and unfavourable wine condition for MLF conductions are as below:
| Parameter | Favourable | Unfavourable |
| Temperature (Celsius) | 18-22 | Below 16 or over 25 |
| pH | 3.3-3.5 | Below 3.1 |
| Free SO2 (ppm) | Below 8 | Above 10 |
| Total SO2 (ppm) | Below 30 | Over 40 |
| Alcohol (%) | Below 13 | Over 14 |
As temperature increases, the toxic effects of ethanol on bacteria also raises, along with increased risk of bacterial spoilage and volatile acidity. It is especially important to keep an ideal temperature range during inoculation or preparation of the bacteria culture as bacteria is in its most sensitive stage during growth phase.
Both free SO2 and total SO2 have an inhibitory effect on bacterial growth, and it is essential to keep the concentration of SO2 low for MLF fermentation. Yeast selection for primary fermentation should also be taken into consideration as specific yeast strains have high SO2 producing property, and although the SO2 formed will be in bound form (with acetaldehyde), bacteria is able to metabolise the bound fraction and convert them into inhibitory free SO2.
As the molecular SO2 concentration increases when pH decreases, low pH is also unfavourable for bacterial growth, especially when pH is below 3.1. However, higher pH (over 3.5) could also favour the growth of other spoilage microorganism, therefore it is essential to maintain the pH of the wine between 3.3-3.5, preferably.
Alcohol concentration ideally should be kept under 13%, and a strong alcohol tolerant bacterial strain should be selected when alcohol is over 14%. In the case of a grape juice with very high sugar concentration which can potentially yield over 15% alcohol, co-inoculation can be another strategy to prevent sluggish MLF fermentation.
Difficult MLF Conditions
Under harsh MLF condition, the availability of certain nutritional components can pose a significant impact on a smooth completion of fermentation. Oenococccus Oeni., the most common species of LAB used for MLF, has a diverse requirement of nutrients, including organic source of nitrogen (amino acids and peptides), vitamins and trace elements (minerals). Inorganic form of nitrogen such as DAP can not be utilised by LAB bacteria.
Furthermore, although commercially available frozen bacteria packs are widely adopted in wineries due to their ease of application, especially for MLF in barrels, they are not the best strategy to adopt for wines with difficult MLF condition. Under such circumstances, it is advised to prepare a MLF starter culture and inoculate the wine in tank prior to barrelling down.
Difficult MLF inoculation protocol
This protocol is recommended for MLF inoculation in difficult wines (when two or more of the parameters are unfavourable), and is based on example of 25hL (2500L) wine:
| Inoculation Protocol |
|---|
| 1. Rehydrate 1 dose for 25 hL of bacteria in 0.5L of still mineral water, kept at 20-25 Celsius. |
| 2. Let it stand for 15 minutes, monitor temperature and make sure it is within range. |
| 3. Once rehydration is completed, dissolve 50g of Nutriferm Osmobatic directly in the same water suspension and mix. |
| 4. Wait for 2-4 hours and keep the suspension at 18-20 Celsius. |
| 5. Meanwhile, prepare a starter blend with 25L of water, 25L of wine and 0.5 kg of Nutriferm Energy or Nutriferm ML. |
| 6. Adjust the starter to pH >3.3 if necessary. |
| 7. Inoculate the starter blend with the bacteria suspension (refer to step 3). Keep the culture at 18-20 Celsius. |
| 8. Monitor malic acid depletion by enzymatic kits. At half of 2/3 malic acid depletion, add 100L of wine into the starter culture and keep at 18-20 Celsius. |
| 9. At another half of 2/3 malic acid deletion, add the starter culture to the remaining wine. Keep at 18-20 Celsius. |
| 10. Once fermentation is ticking away, transfer wine from tank to barrel. Keep at 18-20 Celsius. |
Difficult MLF spontaneous fermentation strategy
In the case of a spontaneous MLF fermentation, it is recommended to add Nutriferm ML at the beginning of fermentation whilst keeping the wine at optimal temperature range (18-22 Celsius) and adjust pH to > 3.3 if necessary. The recommended dosage is 20-30 g/hL, which corresponds to 45-67 g/barrel (assuming 225L per barrel). Simply dissolve nutrient in a small amount of water or wine, then add to the tank/barrel at the very beginning of fermentation (for a spontaneous fermentation, add after completion of AF/once wine is transferred to barrel).
*If Nutriferm ML is unavailable, Nutriferm Energy can also be used at 10-20 g/hL.
Nutriferm Osmobacti is a nutrient and osmotic pressure controller, consists of yeast hulls, cellulose, malic acid and bi-ammonium phosphate. It is used towards the end of rehydration phase which helps improve bacterial resistance towards osmotic shock and increases the rate of surviving cells at inoculation.
Nutriferm Energy is a nutrient blend consists of autolyzed yeast rich in amino acid and survival factors as well as 0.1% vitamin B1. It stimulates the growth of bacteria and improves multiplication.
Nutriferm ML is a biological fermentation energiser containing inactivated yeast rich in amino acids, polysaccharides, cell wall polypeptides, co-factors and vitamins, which stimulates the growth of bacteria and improves their multiplication. Cell wall polypeptides and cellulose acts as essential support for the bacteria cells and can also absorb toxins both from the wine and bacteria (as by-products of metabolism).
