Diagnostic Key Inoculated with Active Dry Yeast

Inoculated with Active Dry Yeast

1.      How was strain hydrated? Water? Juice? Diluted Juice? Temperature?

Solution: if hydration conditions were not optimal the cells in the inoculant may have lost viability and a new inoculant should be made. The temperature of hydration should remain at or below 104°F/40°C. Above this temperature the yeast will receive a heat shock impacting viability. Dropping below 30°C/86°F will impact the hydration step as well. The dried cells will not rehydrate properly at lower temperatures. Often if the water solution is heated or hot tap water is used without benefit of a thermometer the hydration may be done at too high of a temperature. The hydration water should be slightly warm to the touch but if it is too hot to touch it is too hot for the yeast. Pouring boiling water from a microwave into the bucket with the yeast preparation is an excellent way to kill off the entire batch of yeast.

It would be prudent to have a standard operating procedure for yeast rehydration that could be easily followed by all employees. This SOP should also include tips on best practices to follow if there is a problem such as yeast not used in a timely manner, sulfite not mixed in to the tank prior to inoculation, insufficient rehydration time, all errors that are likely to happen during a hectic crush. Check manufacturer’s suggestions for rehydration and base the SOP on their recommendations.

2.      How long was hydrated preparation held before inoculation?

Solution: if the hydrating suspension was held for longer than recommended, the yeast could have lost viability due to the lack of nutrients and inability to repair any cellular damage incurred during the drying and packaging process. Some yeast strains are more sensitive to the rehydration process than others and benefit from the addition of nutrients or sugar during rehydration. Manufacturer’s recommendations for the strain being used should be reviewed. If there is an incompatibility with what was done and the actual recommendations, a new inoculant should be made.

3.      Was strain past its expiration date?

Solution: the expiration dates should always be trusted. Dehydrated cells will slowly leak nutrients or accumulate damage that cannot be repaired upon rehydration. Inocula past the use date may contain enough yeast to start a fermentation but this will be a subpopulation of survivors and not necessarily have the same properties as the major strain present. If viable cell counts are too low then the indigenous yeast of the juice will initiate the fermentation. Strain variation arises normally during growth and does not indicate contamination. This natural population variation allows domination of more marginal environmental conditions by a subgroup of the strains present. The subgroup may not retain the same biological properties of the original mixed population so the fermentation performance of the survivors may differ than that of the originally packaged strain. Although some cells may still be viable past the expiration date, many of the cells in the packet will have lost viability upon continued storage particularly if the packets were not stored under ideal conditions. A new inoculant should be made.

4.      Was packet opened previously and not refrigerated?

Solution: yeast in open packets lose viability more quickly than in sealed packets. Water from the atmosphere becomes absorbed by the yeast and they can partially hydrate and form clumps that are difficult to resuspend. Partial hydration puts the cells at greater risk of loss of viability. Cells can be leakier under these conditions and not retain enough survival and growth factors to be able to grow in the juice. If the packet was not refrigerated or held under proper conditions to maintain viability, a new inoculant should be made.

5.      What were the sulfite addition practices? Could there have been a mistake in sulfite addition?

Solution: the sulfite level should be checked if there is a sluggish initiation. An all too common mistake is adding sulfite to the same tank multiple times if records were not kept. Also errors in calculations can be made that result in very high addition levels. If the level is high but below 200 ppm, the yeast will eventually start the fermentation. At higher sulfite levels trying to eliminate the sulfite by inert gas sparging or addition of hydrogen peroxide can be risky and ineffective depending upon the pH and presence of compounds that bind sulfite. The juice can be blended with other juices and serve as a source of sulfite addition for those juices, if possible.

It can be very difficult to remove excess sulfite so it is important that practical sulfite addition practices be in place in the winery – calculations should be simple and quick and easy methods employed to tag when tanks have had a sulfite addition to avoid multiple additions to the same tank. Overly tired employees tend to make mistakes so sulfite additions should not be relegated to the end of a shift.

As a side note, sulfite solutions should always be made with proper respiratory protection by individuals who have been trained to work with sulfite properly. A standard operating procedure for both sulfite additions and making of the sulfite solution to be added to the tank should be made. Employees with asthma should not be engaged in sulfite additions as they are more sensitive to this compound than non-asthmatics.

6.      Was sulfite mixed in prior to yeast addition?

Solution: sometimes an inexperienced cellar worker will not understand the rationale behind making and mixing in two separate components: yeast and sulfite, and shortcuts will be taken to get done faster. These can be mixing yeast in the sulfite solution or adding a high dose of sulfite to the yeast so that only one addition needs to be made. Alternately two additions could have been made but not mixed in until after the second addition again because the purpose of each addition is not well understood.  These practices lead to the yeast being exposed to a toxic dose of sulfite. If this has happened, a new inoculant should be prepared. This can be prevented by making sure crew members understand the reason for not taking shortcuts at this step.

7.      Was yeast addition mixed in well?

Solution: if a vigorous properly rehydrated yeast suspension was added but not mixed in the fermentation will start once the yeast is distributed. This is important to keep in mind if the yeast were simply added to the top of the fermentor but the sample for Brix readings is taken from a bottom valve – the yeast will not be active in the area of sampling but could be quite active elsewhere in the tank. Mixing will disperse the yeast, but once fermentation gets started the bubbling action from the carbon dioxide will serve to mix the yeast in. Occasionally a mixing problem arises from the yeast not having been hydrated and just poured in the top of the tank. Some of the cells in the inoculant will likely survive this process and become hydrated and grow but the majority may die off due to the osmotic shock experienced in the juice. Again patience may be rewarded and the yeast biomass builds back up to a level that will initiate fermentation. The danger here is that the wild yeasts and bacteria will have an advantage, especially if nutrients were added at the time of inoculation, and compounds could be produced that are inhibitory to subsequent growth and metabolism ofSaccharomyces.

8.      What was the juice/must temperature versus the inoculant temperature?

Solution: if there was a significant temperature difference between the inoculant and the must/juice, in either direction (juice cooler or warmer than the inoculants), the yeast will receive a temperature shock upon addition to the tank. Shocks of 10 degrees or more (F, 5 degrees C), can impact plasma membrane functionality. Generally cells can recover from a moderate temperature shock, but this will add to the lag time preceding onset of fermentation. If the shock has been too great, then a new inoculum should be prepared that is adapted to the temperature conditions of the juice/must.

9.      Is the Brix value high? (above 30 Brix)

Solution: Yeast are sensitive to osmotic shock. If the solute concentration outside of the cell is high then the cells can lose water. Adaptation to the high solute (which in the case of juice is sugar) can occur but this will add to the pre-fermentation onset lag time. Some strains are more tolerant of high osmolarity and one of these should be used as the inoculant. Alternately, the inoculant can be more gradually adapted to the juice sugar content by mixing the well-hydrated inoculant with an equal amount of juice and letting that mixture get started (generally overnight or 24 hours would be sufficient) and adding that mixture as the inoculant.

10. Is the pH low (below 3) or high (above 4)?

Solution: if the pH of the juice is below 3.0 the yeast may have difficulty adapting to the juice and initiating fermentation. The low pH means high hydrogen ion concentrations and the cells will have to adjust the plasma membrane to minimize passive influx of protons to the cytoplasm. Growth conditions will need to be permissive in order for this to happen and any other addition that also increases ATP demand, such as sulfite addition, may make it quite difficult for the cells to adapt.  A subpopulation should be able to adapt and proliferate. In contrast high pH values favor the growth of bacteria and if the bacteria present produce organic and fatty acids or other substances inhibitory to yeast the inoculant may again have difficulty adapting to the juice conditions and initiating fermentation. In both cases it may be prudent to adjust the pH or remove acids (such as acetic acid) if the acid content is too high. Use of an inoculant that is more tolerant of bacterial end products or less sensitive to low pH may be advisable.