Diagnostic Key Inoculated with an Active Tank Fermentation
Inoculated with an Active Tank Fermentation
1. What was the ethanol level of the inoculant fermentation?
Solution: there are three main factors that impact plasma membrane functionality: water availability, ethanol displacement of water of hydration and temperature. The yeast cell changes membrane composition to adapt to these different environmental conditions. If the inoculant tank exceeds 7-9% ethanol (v/v, or roughly greater than 10 to 12 Brix has been fermented) then the cells will have adapted their membranes to ethanol. Using ethanol-adapted yeast as a juice starter requires that the yeast reverse that adaptive process and recreate water-dependent membranes. If juice conditions are permissive for growth the yeast will re-adapt their membranes, but this will lead to a lag in onset of fermentation. If a microscope is available, the vitality of the yeast population can be evaluated. If there is evidence of budding cells then there is a good chance the population is lagging in order to adapt to the new conditions. If cell death is evident in the starter culture, then inoculation with an active dry preparation should be considered. Also if the cells go from an active fermentation with a low sulfite level to juice with a high sulfite level they will need to adapt to the new sulfite level as well.
2. What was the Brix level of the target tank?
Solution: high sugar in the environment can lead to a removal of water from the cells during fermentation. If the water drops below a critical value cell growth will be inhibited. Most commercial strains are osmotolerant, but osmotolerance is impacted by growth conditions so a growing population may not show the same osmotolerance as a newly rehydrated population. High ethanol inoculant to a high Brix target juice is particularly challenging for the yeast. The ethanol has lead to a displacement of water of hydration which will be further exacerbated by the high osmolarity of the juice. Again the yeast will likely be able to adapt to this situation but it will lead to a longer lag prior to initiation of fermentation.
3. Were there temperature differences between the inoculant and target tank?
Solution: temperature also impacts plasma membrane functionality. The yeast must simultaneously produce a membrane that is adapted to the temperature and ethanol conditions of their environment. Moving from the presence of ethanol to a greatly reduced level of ethanol will force changes in the membrane composition. If there is a temperature difference on top of the ethanol difference, the adaptation of the membrane will be more complex and if the differences are large enough the population may not be able to re-adapt. Re-adaptation generally requires that the cells be metabolically able to generate energy in the form of ATP and have sufficient reserves of nitrogen and phosphate to make the components necessary for the adaptation process. The greater the number of differences in two environments the more difficult the adaptation process will be for the yeast. Long lags caused by challenging adaptation conditions will not necessarily lead to slow or incomplete fermentations as once the cells have adapted they are able to grow and ferment. If the lag is persisting longer than expected, inoculation with an active dry starter may be prudent.
4. Were nutrients added?
Solution: the passaging of yeast through grape juice can lead to depletion of micronutrients. Commercial strains are grown under conditions that lead to accumulation of micronutrients and survival factors and as such are fairly resistant to limiting micronutrient conditions. Grape juice typically contains ample micronutrients for yeast but if there is any bacterial or wild yeast metabolic activity prior to inoculation and fermentation these organisms can deplete the fermentation of micronutrients. Over time the yeast internal stores of micronutrients will become depleted. If a micronutrient-depleted culture is used as an inoculum it will struggle to dominate the fermentation and a long pre-fermentation lag will ensue. Addition of micronutrients to a depleted culture may be ineffective if they are not competitive with other organisms present for acquiring those nutrients. Under these conditions it is important to examine the fermentation microscopically to see if there is evidence of bacteria and wild (apiculate) yeasts. If there is a high bioload then use of a pre-fed rehydrated active dry culture is warranted.
5. Was the inoculant tank evaluated under the microscope?
Solution: it is important when using an in-house grown inoculum to evaluate the inoculum under the microscope. Often the criterion used for an active inoculant is to take active bubbling as evidence of yeast fermentation. Most organisms will produce some level of carbon dioxide from the substrates present in grape juice. If the pH and temperature are high enough and sulfite was not used it is possible to grow up a healthy population of lactic acid bacteria. We have seen this situation numerous times – that the inoculant population was not in fact Saccharomyces. This can lead to very high bioloads in the target tank that may inhibit the indigenous Saccharomyces strains from growing and dominating the fermentation. This is worse than an uninoculated fermentation as there has been a deliberate increase in the numbers of juice-adapted bacteria. If this has occurred then it will be necessary to inoculate with a highly tolerant strain ofSaccharomyces.