The goal of fermentation management is to develop a robust population of microorganisms that will complete the fermentation and resist any biotic or abiotic stress that may occur along the way in addition to the production of positive aromatic characters contributing to complexity. Grape juice is rich in nutrients, particularly in sugars from which energy will be derived. The end products of sugar catabolism, aldehydes and alcohols, are inhibitory to growth so the cell must be able to successfully adapt both growth and continued metabolism to the presence of these biological stressors. Sufficient macronutrients (N, S, P) and micronutrients (vitamins, minerals and growth factors) must be present to support cell growth and proliferation. Nutrient supplementation can be used to augment the grape content. Excessive use of nutrients may detract from wine quality by preventing the natural competition that keeps certain populations in check and may block production of desired aroma compounds derived from catabolism of less preferred nutritional sources. Fermentation management must also consider establishing conditions that will allow completion of the fermentation, conversion of all available sugar to either biomass, ethanol or carbon dioxide. Specific nutrients and growth conditions are needed for optimal ethanol and aldehyde tolerance and these condition should be established during fermentation to prevent fermentation arrest.
Successful fermentation management strategies also consider the impacts of temperature, oxygen, pH and mixing on microbial growth and metabolism. If bioloads of non-Saccharomyces organisms are high, then the use of antimicrobial agents to assure rapid domination of the fermentation may be optimal. Inoculation with specific cultures can be used to assure metabolically hearty strains are present.
Inoculated versus Uninoculated Fermentations: There are two basic options for the initiation of fermentation: inoculated and uninoculated. Uninoculated fermentations are conducted by the flora present on the surface of the grapes and winery. True uninoculated fermentations use no yeast additions at the onset of fermentation. Inoculated fermentations give a boost to the existing Saccharomyces population by introducing large numbers of this organism to the juice following processing. There are two basic types of inoculations: commercial strains and amplified indigenous flora. In commercial strain inoculations an active dry strain with desired fermentation properties is rehydrated and added to the juice or must. In amplified indigenous strain inoculated fermentations an initial “starter” uninoculated fermentation is allowed to develop a dominant population of winery/vineyard Saccharomyces and that starter is used to subsequently inoculate other fermentations. This is a “best of both worlds” concept – allowing native or regional yeasts to conduct the fermentation but growing up a healthy population of those yeasts so as to effect a more controlled fermentation.
The addition of high populations of Saccharomyces will obviously impact the microbial flora of the fermentation. The inoculant often but not always is the strain that can be found as the dominant strain at the end of fermentation. However in native inoculated fermentations there may be a progression of different strains present, each blooming as the specific conditions under which it is metabolically optimized arise during the fermentation.
For commercial inocula, it is important to follow manufacturer’s instructions for the rehydration step. If not rehydrated properly the yeast will lose viability. Some yeasts are more tolerant of drying and rehydration than others. They are not all the same so a practice that works well with one strain may not with another. Following the directions is always a best practice. Open packets of yeast will lose viability during storage. Manufacturerers have monitored loss of viability of their products and the “use by” dates assure that the inoculant will be healthy.
Some strains benefit from the presence of nutrients during the rehydration process. It is a well known phenomenon in laboratory yeast that the presence of sugar even under non-growing conditions provides the energy needed to sequester other nutrients from the environment. Thus if glucose is present the strains will accumulate N, S or P as well as micronutrients. If N is omitted from the rehydration mix, the yeast will accumulate the other nutrients present and they will be available for use later on in the fermentation. The nutrient-charged cells will be better able to adapt to and dominate the fermentation. Since the inoculum rather than the entire fermentation is being fed, the inoculant gets the full and sole benefit of the addition.
Indigenous Flora versus Commercial Preparations: Grape juice following harvest and processing at the winery will have some level of Saccharomyces yeasts present. If the fruit is sound and harvested below 22 Brix, the levels of Saccharomyces will be low coming from the vineyard, but still present. Saccharomyces is a minor resident of grape fruit surfaces. Rot, seepage or the establishment of anaerobic conditions in the clusters will increase the level of Saccharomyces present. Saccharomyces is often, but not always, the dominant organism of the winery flora. In some rare cases the strains of the related genus Candida were found on winery surfaces at levels higher than Saccharomyces. Even at these low levels, the establishment of anerobic conditions will favor the growth and metabolism of Saccharomyces. Uninoculated fermentations will eventually be dominated by Saccharomyces. Several studies have shown that indigenous fermentations, particularly inoculated amplified indigenous flora fermentations are largely conducted by indigenous winery not vineyard flora unless significant rot has occurred in the vineyard and the rotted fruit is used in the fermentation. If commercial strains have been used in the winery, the commercial strains often become indigenous winery flora. Even the most stringent sanitation practices reduce surface bioloads but cannot eliminate the microbes entirely.
If indigenous inoculants are to be used, it is important to make sure that the starter culture contains viable yeasts. If the starter is too old the yeast present may have shut down metabolically and are likely not to regrow when placed into fresh juice. It is also possible that a spoilage organism has taken hold in the inoculants, particularly if it was created in a small volume at high temperature, and use of this starter then boosts the level of the contaminating organism. Visual inspection of the starter under the microscope can determine if bacteria are present and will show that yeast are present but many yeast look similar and it may be difficult to know that the desired strain is present. It is important to smell the starter and not ignore any off characters or attribute them to the starter juice in the belief that they will not appear in fruit harvested at a later time.
The desirable yeast traits are:
- Fermentation to dryness
- Reasonable rate of fermentation
- Predictable fermentation characteristics
- Good ethanol tolerance
- Good temperature tolerance
- Sulfur dioxide tolerance
- Simple nutrient requirements
- Little to no off-character production
- Sulfur volatiles
- Acetic acid
- Ethyl carbamate
- Little to no inhibition of other desirable microbes
- Killer factor resistant
- Production of desired aroma characters
The undesired traits are:
- Off-character production
- Fastidious nutrient requirements
- Inhibition of desired microbes
- High production of sulfites
- Erratic fermentation properties
- Lack of ethanol and temperature tolerance
Yeast strains vary in biological properties. These differences arise due to selective pressure in the environment that leads to genetic modification of the strains. Commercial strains can be strains ofSaccharomyces cerevisiae var. cerevisiae, Saccharomyces cerevisiae var. bayanus, Saccharomyces bayanus, hybrid strains generated from forced crosses of different species, native isolates, and native isolates following mutagenesis and selection for improved traits. Genetically modified commercial strains containing non-Saccharomyces DNA have also been generated to reduce malate levels in wine. Commercial strains may be in pure or mixed culture with the latter thought to provide greater complexity and insurance of a completed fermentation. Non-Saccharomyces strains are also available as inocula again as pure or mixed cultures with Saccharomyces.
There are some key factors to consider when choosing a strain. First the ethanol tolerance of the strain should exceed the projected final ethanol level of the fermentation. Second the nitrogen requirements should match the nutritional conditions of the juice. The temperature tolerance should also be considered if uniform temperature control is an issue. The compatibility of the yeast strain with the malolactic fermentation is also important to consider if the MLF is also desired. The production of specific aroma compounds is also a consideration but the ability to produce a spectrum of volatile characters is dependent upon the composition of the juice. The aromas produced will vary depending upon the levels of precursors present.
Timing of Inoculation: The time following harvest and fruit processing at which an inoculant is added can impact the flora present during fermentation. At crush, the juice or must will contain large populations of bacteria and non-Saccharomyces yeasts. These microbes will be able to initiate growth under anaerobic conditions and were it not for the competition from Saccharomyces and production of toxic end products would be able to persist in the fermentation. The non-Saccharomyces yeasts generate a broad spectrum of aromatic compounds and produce hydrolytic enzymes that may release bound aromatic components of the grape. The do not have the ethanol and stress tolerance of Saccharomyces so rarely complete a fermentation, depending upon the organism present. If non-Saccharomyces characters are desired in the wine there are two options available. Commercial preparations of non-Saccharomyces yeasts exist and can be used to boost numbers of desired organisms prior to domination by Saccharomyces. However this is not the same as an indigenous or native fermentation as this practice is equivalent to the use of commercialSaccharomyces preparations.
The second way to increase the contribution of the indigenous flora is to delay the time of inoculation withSaccharomyces. Fermentations may be started by warming the tank and not inoculated for one to three days post temperature increase or the inoculation may occur after the indigenous Saccharomyces strains have become established to assure a robust strain will complete the fermentation. This strategy allows the winemaker to monitor the fermentation and if it appears to be going to be able to complete on its own, no inoculation is used. If it becomes sluggish then an inoculation can occur. A good strategy is to inoculate 10% of the production which can then serve as an inoculation for the remaining 90% if difficulties appear to be arising with those fermentations, such as the appearance of an off-character. This allows the progression of the fermentation to dictate timing of addition of an inoculant rather than following a recipe, but both methods lead to an enhanced contribution of the native flora.
Inoculation Level: The typical fermentation will reach 108 cells/mL at the end of active yeast growth. This level may vary 2-3 fold during fermentation or hold steady at this level. The recommended inoculation level is 100- fold lower than the expected final population level, generally 106 cells/mL or a 1-2% of an amplified starter. This is not a randomly selected number of cells. A dilution of 100 fold will mean that roughly 7 generations are needed to recreate the final population level. This allows strains to grow and adapt to the conditions of the fermentation. Since wild yeasts can be present in the must or juice at levels of 104 to 106cells/mL, depending upon the level of rot and seepage, use of an equivalent level of Saccharomyces levels the playing field and assures a more rapid domination by Saccharomyces. Seven generations will not lead to depletion of micronutrients of the starter population. Thus commercial inocula that are well fed with micronutrients can grow and complete fermentations in micronutrient-deficient juices and supplementation is not needed.
Lower and higher levels can be used. Lower inoculation levels will mean more generations need to occur and more growth nutrients need to be present in the juice to support production of the cells. It might seem that the bulk of the nutrients would be needed in the final one or two cell divisions so that starting at a 105cells/mL would only require a miniscule increase in nutritional needs, but the yeast assess nutritional status prior to each cell division and if nutrients are low will stop dividing and not increase the cell population. Lower level inoculations allow greater contributions of the indigenous flora.
In contrast higher levels of inoculation allow more rapid domination by Saccharomyces. Fewer generations will occur before maximal cell density and arrest of growth is attained. If the fruit has a high bioload, use of a higher inoculation level may be warranted. However, high inoculation levels are often associated with high yeast ester formation which may not be desired.
Sulfur dioxide addition has multiple impacts on yeast fermentation. SO2 inhibits sensitive yeast and bacteria thereby reducing competition for Saccharomyces. However this effect is generally not that important if the grapes are sound and rot is minimal. If a healthy inoculum is used the yeast will dominate the fermentation without the need for sulfite inhibition. If there is a high bioload of bacteria in the juice use of SO2 as an antimicrobial agent may be important to prevent formation of inhibitory acids that would negatively impact fermentation progression. SO2 also inhibits polyphenol oxidase activity. This inhibition prevents consumption of molecular oxygen by this protein making oxygen available to the yeast for metabolic activities. SO2 stimulation of fermentation may be due to enhancing oxygen availability.
The winery flora, particularly of any surface that comes in contact with the grapes, juice or must can be a source of microbes that can impact the fermentation. Winery flora are generally well-adapted to fermentative conditions so if transferred to the juice or must will be able to grow and impact fermentation. The bioload of winery surfaces is impacted by cleaning and sanitation practices. The rigorous and frequent sanitation of all winery surfaces will minimize the transfer of microbes to the juice. Yeast as well as bacteria can form biofilms on surfaces, and often mixed biofilms are present. A mixed biofilm is more resistant to inhibitory conditions and more versatile metabolically. If sanitizers are used, instructions should be followed. Short-cuts in either timing or concentration of application will generally result in higher survival rates of the microbes. Sound sanitation practices also include insect control. Fruit flies in particular are attracted to oxidized and spoiled wine will serve as a source of inoculation for sound wines and juices.
One of the most important tools of fermentation management is nutrient addition. Depending upon the growing region, juice may be limited in nitrogen, phosphate or micronutrient levels. A deficiency of potassium may also exist. Nutrient limitation has two important effects: reduced size of the cell population and reduced ethanol tolerance. In general yeast populations reach 5 x 107 to 1 x 108 cells/mL during fermentation. The more cells the faster the fermentation as there are more “bioreactors” present. Sufficient cellular building blocks need to be available in the medium to support the production of new cells. The fermentation rate per cell is also influenced by the level of nutrients. High nitrogen in particular leads to higher concentrations of metabolic enzymes resulting in a faster rate of fermentation per cell than lower N concentrations will support. Overfeeding of fermentations can be as problematic as underfeeding as very rapid fermentation rates may ensue leading to greater heating of the fermentation and enhanced loss of volatile aroma compounds.
Nutrients are also required for tolerance to inhibitory conditions. Sufficient nutrients need to be available to allow adaptation to the inhibitory conditions. The higher the starting sugar concentration the higher the nitrogen level will need to be to complete the fermentation. The nitrogen level of the must should be evaluated. There are several techniques that can be used: yeast available nitrogen (YAN), free amino nitrogen (FAN), nitrogen via o-Phthaldialdehyde/N-Acetyl-L-Cysteine (NOPA). Any of these methods will provide an assessment of juice nitrogen. If a deficiency is identified, nitrogen supplements can be used to increase the level of nitrogen. There are two types of N-containing nutrients that can be added: ammonia and organic amino acids. Diammonium phosphate (DAP) or ammonium sulfate can be used. Ammonium ions are incorporated into organic amino acids by the cell. Alternately amino acids can be added directly. In this case the yeast is saved the trouble of synthesizing the amino acid and does not need to divert sugar carbon to amino acid biosynthesis. Mixtures of amino acids and ammonia support faster growth rates than individual compounds. Commercial nutrients differ in nitrogen content. DAP also provides phosphate to the cells. In some growing regions phosphate may be limiting. In these cases nitrogen is also limiting and addressing the nitrogen limitation through addition of amino acids may not restore fermentation rates as phosphate then becomes limiting. Sulfate has rarely been reported as a limiting nutrient. Less sulfur is required for cell proliferation and generally juice contains sufficient levels of this compound.
Juice deficiencies of micronutrients may also impact ethanol tolerance. Vitamins and minerals are important catalysts and if not available critical biological activities may not be able to occur. Saccharomyces only requires a single vitamin, biotin, but the presence of other vitamins stimulates both growth and ethanol tolerance. A deficiency of potassium will also lead to increased sensitivity to ethanol. Sterols and unsaturated fatty acids also enhance ethanol tolerance. These compounds may be incorporated into commercial nutrient preparations. There are no convenient analytical methods that allow ready quantification of micronutrients. Commercial strains are often grown under conditions that result in high internal micronutrient levels, sufficient to complete a fermentation without additional supplementation. However if not rehydrated properly micronutrients may be lost from the cells requiring supplementation.
Microorganisms require the same types of micronutrients. A nutrient addition will feed the existing populations so it is important to time the addition to benefit the desired population. For example, addition of nutrients at the onset of a cold soak will feed the wild yeasts and lead to their proliferation rather than aideSaccharomyces. Yeast nutrients will also supply bacterial requirements. The most effective time of addition of nutrients is once the Saccharomyces population has become dominant, generally 24 to 48 hours after inoculation if commercial strains are added, or once visible carbon dioxide is being produced if native inoculated or uninoculated fermentations are being conducted.
A second factor to consider with nutrient additions is the impact of high levels of ethanol on absorption. Ethanol is inhibitory to substrate transport and if components are added after this inhibition has occurred the substrates will not be taken into the cell. Also some nutrients required for ethanol tolerance need to be present while new cells are being made during the rapid phase of cell growth. Addition after cell division and net growth has been inhibited will not allow incorporation of the compounds into cellular structures.
Prudent fermentation management cannot exist if the progression of the fermentation is not followed. Soluble solids can be followed using hydrometry (Brix), refractometry, tank weight loss or rates of CO2evolution. It is also important to maintain historical records so that problems with progression of the fermentation can be recognized early as failure to treat a problematic fermentation often results in arrest or the appearance of off-characters (see section on problem fermentations).
It is also important to monitor fermentation volatiles. Off-character production early during active growth can often, but not always, be removed by entrainment in the carbon dioxide stream produced during the vigorous phase of fermentation. Off-characters produced after this stage or as fermentation is becoming sluggish will not be driven off. The presence of a spoilage organism can also be detected by aroma of the fermentation.
If a microscope is available the presence and nature of the microbes present can be monitored during fermentation. This is particularly important if indigenous flora fermentations are conducted in order to determine if Saccharomyces has successfully dominated the fermentation.
One of the most critical parameters of fermentation is temperature. As with pH and oxygen status, microbes have a specific window of permissive temperatures for growth and metabolism. But aside from impacts on microbial growth and metabolism, temperature impacts grape component extraction, loss of aromatic compounds via volatilization and rates of chemical reactions. Low temperatures favor retention of existing volatiles but reduce growth rates and rates of chemical hydrolysis with some notable exceptions. In red wine production localized cap temperatures are very important for the extraction of skin components such as anthocyanins. Often the temperature requirements of the yeast are secondary to these other factors.
Some organisms are adapted to grow optimally at low temperatures, psychrophilic (cryophilic), and some to high temperatures, thermophilic. Others are mesophilic growing optimally at moderate temperature, 15 to 40°C. Suboptimal growth may occur at the extremes of this temperature range. Within the mesophilic range there may be subspecialization with some strains more tolerant of lower or higher temperatures than others. The organisms found in wine production are generally classified as mesophilic. The optimum temperature for Saccharomyces strains is generally in the range of 25 to 30°C. Other non-Saccharomycesyeasts have lower temperature optima, in the range of 15 to 20°C with permissive temperatures as low as 10°C for some organisms. Temperature impacts membrane integrity and functionality. The cell must maintain sufficient membrane fluidity to allow protein movement and function and must be resilient enough to prevent leakage into or out of the cell. At higher temperatures lipids are more fluid than at lower temperatures. Cells can adapt to differences in temperature by changing membrane composition. However if the temperature change is large and fast enough the cells may become non-functional before they have time to adapt. At lower temperatures membranes are less fluid and the membrane “freezes up” blocking protein function but retaining cellular components. Some strains tolerate swings of temperature more so than others.
Ethanol impacts temperature tolerance. In general the higher the ethanol concentration the narrower the permissive temperature range of growth. The converse is also true, the greater the temperature the lower the maximal tolerable level of ethanol. This is because ethanol like temperature impacts membrane functionality and integrity. The specific membrane changes that allow ethanol tolerance are different than those allowing temperature tolerance. Thus the cell cannot become tolerant to both conditions. Generally the first condition imposed is the one they will become tolerant of. Since temperature shocks generally occur early in the fermentation the impact on ethanol tolerance may not be immediately apparent and the culture may appear healthy. The temperature shock has lead to a re-tooling of the plasma membrane. Continued fermentation may raise the ethanol or other metabolite concentration to a level that is now inhibitory to continued growth or fermentation. Heat is released as an end product of fermentation along with ethanol and CO2. The simultaneous production of heat and ethanol is doubly inhibitory to sensitive microbes.
In addition to being driven off by temperature, volatile compounds can also be removed by a vigorous fermentation. Temperature is an excellent way to control yeast metabolic rates. Lower temperature fermentations will be slower and retain more volatile compounds than warmer temperatures. Higher temperatures lead to greater extraction from skins and seeds.
Tank temperature control systems generally involve jackets or submerged heating or cooling units. In most cases the temperature will not be uniform across the tank. In tanks that are improperly sized for their refrigeration units or that are not mixed thereby allowing local temperature spiking to occur will develop different flora in the different temperature zones. Distinct zones of bacteria in the warmer sections of the tank and yeast near the cooling apparatus have been reported. These fermentations may be more prone to arrest if they bacteria are producing inhibitory substances for the yeast.
Yeast are tolerant of the pH range typical of grape juice. Their optimum pH range for growth is around pH 6.0 but they are able to grow well below and above this pH value. Both fermentation and growth rates will be higher at optimal pH values, but the pH optimum curve is quite broad. In contrast most bacteria are inhibited at pH values below 3.5. Low pH juices are at minimal risk of bacterial spoilage. One of the impacts of high ethanol is the enhanced leakiness of the membranes to protons. The higher the proton content in the environment the higher the concentration of protons that can leak into the cell. The pH tolerance ofSaccharomyces is not as impacted by ethanol as it is for other yeasts.
pH also impacts rates of oxidative and other chemical reactions. Very high pH wines are more prone to oxidative damage than low pH wines. As with temperature ethanol impacts the permissive pH range. SO2 is less effective at high pH values which adds to the higher occurrence of oxidation of the juice and wine. “Early oxidation disorder” is a term that has been used to describe wines that appear to have oxidized characters normally associated with barrel aged wines immediately post-fermentation. These are generally high pH wines that have not had effective SO2 additions and that have been exposed to air or oxygen. These wines also seem to be predisposed towards oxidative damage suggesting they contain metal ion catalysts required for the efficient formation of hydrogen peroxide from molecular oxygen. In this case reduction of the pH may be of benefit.
High pH grapes generally contain a richer bioload of microbes on the cell surface, particularly if any rot is present. The higher bioload and the higher pH can lead to higher numbers of bacteria and non-Saccharomyces yeast early in the fermentation. Addition of sulfite may not be effective under these conditions and reduction of the pH is necessary.
Oxygen in juice is a substrate for polyphenol oxidases (tyrosinases) as well as for chemical reactions yielding hydrogen peroxide (H2O2). Some oxidative reactions such as those leading to the formation of a desired tannin structure and stabilized pigment are desired while those leading to sherry-like aldehydic characters are not. In white wines the presence of oxygen may lead to browning or other off-color formation. Oxygen is required by aerobes in order to persist in the grape juice environment and can stimulate the growth of acetic acid bacteria and formation of acetic acid by both the acetic and lactic acid bacterial populations. Oxygen also serves as a yeast nutrient, allowing the cells to synthesize unsaturated fatty acids and sterols needed to construct ethanol tolerant membranes. Thus oxygen has multiple positive and negative impacts on juice chemistry and microbial populations.
The trick with oxygen treatments of juices and musts is to encourage desired effects while minimizing those that are not wanted. Oxygen stimulation of yeasts can be achieved after the yeasts have dominated the fermentation. Once yeast populations are high enough, 107 cells/mL, ten orders of magnitude higher than the bacterial or non-Saccharomyces yeast populations, they will be highly competitive for introduced oxygen and capture it for metabolic purposes. Therefore the timing of oxygen exposure can be used to the advantage of the resident yeast populations.
Oxygen exposure to drive chemical reactions can also be attempted. Hyper-oxidation of some white juices leads to rapid and complete browning with subsequent precipitation of the accumulated pigmented polymers. In this case the hyper-oxidation results in a wine stable to further oxidative pigment damage, although the wines are still susceptible to aldehyde formation and microbial growth. In other cases, hyper-oxidation leads to formation of brown polymers, but not their precipitation, as this is dependent upon the presence of other phenolic compounds. If bacteria are present in high concentrations and the pH of the juice is high at the time of hyper-oxidation, hyper-production of acetic acid can also occur.
The variables available for starting juice composition, inoculation and supplementation of fermentation, and desired wine composition preclude a “one recipe fits all” approach to fermentation management. The most critical variables are the nature of the bioload of the juice or must at the end of grape processing, the type of inoculation practices and the specific conditions of the fermentation.
- The bioload at the onset of fermentation is a function of the bioload from the vineyard, the additions to that bioload from winery equipment, and any pre-fermentation manipulations that may have enriched for sub-populations of that bioload. High bioloads are not necessarily bad, as the nature of the organisms present is as important as their relative numbers. Spoilage lactic and acetic acid bacteria can inhibit subsequent yeast metabolic activities by producing inhibitory organic and short chain fatty acids. This inhibition may not be immediately observe but only become apparent once the ethanol content of the juice has increased. Bioloads are higher with the presence of rot in the vineyard, lack of sanitation in the winery, higher pH values and greater oxygen exposure of the grapes during processing.
- Inoculation practices: successful indigenous and commercial flora fermentations are equally possible depending upon the nature of the organisms present and winemaking practices. Indigenous fermentations are not necessarily unpredictable or more prone to spoilage or arrest, if appropriate management strategies are employed. It is important to monitor these fermentations carefully so that the winemaker can intervene if a problem arises. If the grapes are high in percentage of rot or damage during harvesting, the use of a commercial starter may be a better option than indigenous fermentation. Sulfite can also be used in indigenous fermentations to inhibit berry flora and enrich for winery flora.
- Fermentation conditions: successful fermentation management requires careful attention to fermentation progression. Wild swings of temperature should be avoided. Juice nutrient concentrations should be determined and supplemented when needed and provision of oxygen and nutrients should be timed to feed the desired yeast populations.