Diagnostic Key Uninoculated Fermentation
1. Was there rot in the vineyard?
Solution: Vineyard Saccharomyces populations are low compared to both total microorganisms and to total yeasts. Saccharomyces has evolved to dominate anaerobic fermentation of high sugar substrates so will eventually take over a grape juice fermentation. The presence of rot has two impacts. It is true that rot gives a boost to the numbers of Saccharomyces cells present in the cluster, but other organisms also increase in number. If these other organisms are competitive with Saccharomyces during the fermentation the fermentation may have a sluggish start. If there is rot in the vineyard addition of sulfite to the juice will give the wild strains of Saccharomyces more of an advantage. Also, avoid very cool (below 12°C/53°F) as this will favor the growth of the non-Saccharomyces yeasts (unless such growth is desired) and very warm (above 28°C/83°F) as this will encourage the growth of bacteria. If rot is extensive then the juice is likely not a good candidate for a native fermentation and an inoculation should be used.
2. What are the winery sanitation practices?
Solution: Although Saccharomyces may be a minor resident of the grape flora, it is often the major resident of the winery flora. Stringent sanitation practices serve to reduce winery resident flora. If sanitation is lax then biofilms of Saccharomyces will build up on equipment and hoses and serve as a point of inoculation of the fermentation. Such fermentations would still be considered uninoculated but the indigenous strain may be indigenous to the winery but not the vineyard. If a true vineyard indigenous strain is desired as the organism conducting the fermentation then great pains should be taken to minimize any possible role of the yeasts indigenous to the winery. Most often though it is not that important if the yeast is indigenous to the vineyard or the winery. Also if an inoculant is used in some tanks this strain will become part of the indigenous winery flora. The more stringent the sanitation practices the lower the bioload from the winery flora and the greater the reliance will be on vineyard Saccharomyces flora. the vineyard flora are often not as adapted to fermentation conditions as are the winery flora so extended pre-fermentation lags may occur. The longer the lag the greater the opportunity for a spoilage organism to become established. Inert gas blanketing can be used to reduce the potential for growth. But it is important to keep in mind that inert gas blanketing will also inhibit the growth of the yeast population. If it is taking an inordinate amount of time for the fermentation to initiate (longer than 2 weeks) it may be wise to inoculate the fermentation.
3. What is the starting non-Saccharomyces microbial bioload?
Solution: uninoculated fermentations are generally slower than inoculated ones simply because there is a lower initial population level of Saccharomyces and longer time is needed to build up the cells to the same density as is immediately present upon inoculation. Inoculation levels are typically 106 cells/mL. Vineyard population levels of Saccharomyces are generally 10-3 to 100 cells/mL. If winery biofilms are allowed to build up then inoculation levels from winery equipment are 103 to 104 cells/mL. Thus starting cell populations may vary over 109 fold. There will be similar variation in the populations of non-Saccharomyces yeasts and bacteria. The higher the non-Saccharomyces to Saccharomyces bioload ratio, the more difficulty the yeast will have in dominating the fermentation. This does not mean that they will not dominate, as many studies have shown that they will eventually take over the fermentation even when outnumbered millions to one, but the initiation phase may be quite sluggish until populations build. Again, if the fermentation is taking a long time to initiate, inoculation should be considered.
4. What is the starting Brix level?
Solution: high Brix juices may have difficulty starting due to their high osmolarity and low water availability. Native yeasts are no more tolerant or resistant to high osmolarity than are commercial yeasts. In fact some wine strains may be more osmo-sensitive than commercial strains. Also, extended hang time in the vineyard can lead to a bloom of the lactic acid bacteria on the surface of the fruit, adding to the competition for nutrients. If the indigenous strain is not highly osmotolerant, a long lag may result as the strains attempt to adapt. A larger problem with uninoculated fermentations of high Brix substrates is in ethanol tolerance. Native strains may not be as tolerant to ethanol as are some of the commercial strains and may arrest growth it the ethanol content exceeds their tolerance level.
5. Was there an addition of SO2?
Solution: sulfite addition can be either beneficial or detrimental to an uninoculated fermentation. SO2 at low levels (20-25 ppm), depending upon the pH, can inhibit juice bacteria preferentially over wild strains ofSaccharomyces. However as discussed under Question 3 #5, if too high of an addition of SO2 is made the tolerances of even the most robust of the wild strains of Saccharomyces may have been exceeded. If a mistake in SO2 addition occurred, it will be necessary to reduce that level and to inoculate as the native yeasts may not recover.
6. What is the pH?
Solution: low pH musts and juices favor the growth of wild yeast strains and high pH musts and juices favor the growth of bacteria. It is important to determine the pH of the native flora fermentation to determine if yeast or bacteria will be favored and if low sulfite additions will likely be effective. The pH can be adjusted if it is too high to favor the desired microbial populations. If the pH is below 3.0, Saccharomyces may be inhibited. In rare cases Brettanomyces has been found to dominate very low pH juices as it is the most pH tolerant of the vineyard or winery flora. Microscopic examination will be helpful here in determining what species are dominating the fermentation and if growth has occurred. If other organisms have dominated the juice or must it may be necessary to reduce their numbers via filtration or centrifugation and then inoculate with Saccharomyces.
7. What is the temperature of the juice/must?
Solution: low temperatures favor the bloom of non-Saccharomyces yeasts such as the apiculate yeasts. The apiculate yeasts are capable of scavenging micronutrients from the juice and may lead to a depleted situation. If the limitation is severe enough a long lag may ensue and Saccharomyces have difficulty increasing in biomass. This is often evidenced by high populations of non-Saccharomyces yeasts early in the fermentation. High temperatures favor the bacteria, particularly the lactic acid bacteria. Some species of lactic acid bacteria produce inhibitory substances that will block growth of the yeast. Again this is easily detected by looking under the microscope and seeing large populations of bacteria in the fermentation. If there are toxins present then starting these fermentations may require a mild fining agent treatment or use of yeast ghosts to strip the juice of the inhibitory compounds. In this case an inoculation should be used and the native yeasts should not be relied upon to rebound and conduct the fermentation.
8. Were nutrients added? What and when?
Solution: it is just as important to know the nutrient level of uninoculated fermentations as it is for inoculated ones. In the case of an inoculated fermentation unless the nutrient limitation is severe, the fermentation will initiate normally. However in the case of a native flora fermentation the cells will not have the benefit of having been pre-grown in nutrient sufficient conditions so a limitation may be manifest by a sluggish start to the fermentation. Nitrogen and micronutrient levels are the two most critical factors in native fermentations. Indigenous yeast can have more complex nutrient requirements than the commercial strains as the commercial strains have largely been pre-selected to not be fastidious in requirements. Wild strains can display vitamin requirements not typical of Saccharomyces. They may also have higher nitrogen requirements than commercial strains. It is important if adding nutrients to a native flora fermentation to add those nutrients once the Saccharomyces population has become established or after ethanol has started to accumulate. If nutrients have been added and the fermentation is still displaying a lag in onset then use of an inoculant may be desirable.
9. Are there any treatment residues from the vineyard?
Solution: use of antimicrobial agents in the vineyard, fungicides and pesticides, incorrectly or too close to harvest can lead to the presence of inhibitory residues in the juice at the onset of fermentation. SinceSaccharomyces is a fungus, growth and metabolism may be inhibited as it detoxifies the environment. This is a very rare problem in wine production and such wines generally are defective as the presence of the inhibitory condition generally leads to off-character production. It is important to make sure vineyards are properly managed. This is not a problem in countries such as the United States that require training for agricultural application of any potentially environmentally toxic substances but may be a problem elsewhere where such training is not required.
10. Are there any residues from sanitation treatments?
Solution: occasionally sanitation of a tank or piece of winery equipment may not have been completed prior to use for grape processing. This can lead to high and inhibitory residues of cleaning or sanitizing components in the juice or can lead to drastic alterations of pH. Fermentation will be inhibited, which is a good thing since the wine made from such contaminated juices cannot be used. It is important to make sure sanitation practices are completed prior to use of equipment and that good communication practices among workers are in use. Such errors are almost non-existent in wineries especially if trained personnel are used for sanitation. Here again having clearly defined protocols will prevent such errors from occurring.