Grape, Juice and Must Processing Impacts on Flora

The crushing of grapes brings the microbes of the berry surface in contact with the nutrients of the fruit. Ample carbon, nitrogen, sulfur and phosphate sources exist in the typical berry to promote the growth of a variety of bacteria and yeasts. Micronutrients are also present and only the most nutritionally fastidious organisms will be unable to proliferate. How grapes are processed can create conditions that select for the growth of a subset of the organisms originally present. This can be used to the advantage of the winemaker to enrich for desired populations. However more often the grape processing steps selected were chosen to manipulate extraction of flavor components, not to manage microbial populations. The populations will respond to any selections imposed, even if this is unintended.

Processing also introduces winery flora to the juice. All winery equipment will build up biofilms of yeast and bacteria and the presence of nutrients and flow of liquid will lead to proliferation of these microbes as well. The levels introduced by equipment will depend upon cleaning and sanitation practices. With sound fruit the level of Saccharomyces coming in from the vineyard is generally less than 1 colony forming unit per mL. The contribution from unsanitized equipment is on the order of 103 to 104 colony forming units/mL. Thus the resident winery flora can have a significant impact on the fermentation,

Skin Contact Factors: Grape berry flora are largely associated with the surface of the fruit. These organisms exist as biofilms, meaning they are firmly attached to the surface of the fruit. When transferred to batch fermentation conditions, the biofilm will produce free-living, or planktonic, cells during division. Thus, the grape skins are a source of inoculation for the juice and must. The greater the mixing of the must, the greater the dispersion of the planktonic cells. The presence of skins can impact the microorganism present during the fermentation.  

Pre-fermentation Processing: Several aspects of grape, must and juice processing will influence the numbers and types of organisms present as fermentation is initiated. In many cases, the impact of processes depends upon the bioload present. The effects of various treatments can be interactive, serving to increase or decrease the presence of organisms present (skin contact at a warm temperature) or can counterbalance each other (skin contact or cold soaks in the presence of an antimicrobial agent such as sulfite).  Again, what is desired depends upon the wine style, its impact compounds and innate character, and the nature of the organisms present in the first place. An inexpensive microscope with phase contrast capability is a sound winery investment. Although this will not allow identification to the genus and species level in many cases, examination of the juice prior to and during fermentation can often indicate early on that the population is atypical so that corrective action can be taken.

Clarification: The level of juice solids can have a strong impact on microbial processes. Grape solids, if derived from the surface of the fruit, will carry a high bioload of organisms. If skins are efficiently removed and solids largely generated from pulp material, they can form a surface of adherence for microbial growth, also fostering cell division. Solids are a source of nutrition and may provide growth requirements early on in the fermentation. Solids also entrap oxygen and this may be important to sustain aerobic populations and to facilitate adaptation of fermentative organisms to the juice or must following crushing. Mixing is generally facilitated by the presence of solids and mixed populations tend to grow more rapidly than static ones. Overly clarified juices may lack nutrients and not be able to sustain air pockets in the juice during fermentation or assist mixing and loss of carbon dioxide. Depending upon the varietal, a solids content from 2 to 5% may be desirable. Higher solids contents have been associated with off-odors and lower solids content with arrested fermentation. A graduated test tube can be used for a rough estimate of solids content. A specific amount of juice can be added to the test tube and allowed to settle in the refrigerator overnight. The next day if the solids have settled the wet solids percentage can be obtained. Solids can also be estimated via centrifugation of a sample or via filtration of a specific weighed amount of juice followed by weighing of the pellet and supernatant fractions. Solids content can also be estimated using turbidity if a turbidity meter is available. However, this technique needs to be applied carefully since solids may settle during the reading process, giving a false low percentage. Filtration can also be used on a sample of known mass to collect a solids with the solids then weighed to determine the percentage of the weight that was solid material.  These methods may yield slightly different percentages depending upon the presence of non-solids materials that is collected. Generally all that is needed is a rough estimate of the solids content.

Temperature Treatments: there are two general kinds of temperature treatments of juices and musts. High temperature short time treatments are used to enhance skin extraction, inhibit fungal oxidase activity, block microbial growth and accelerate chemical hydrolytic reactions and loss of volatile compounds. Generally the HTST treatments used are not long enough to lead to significant loss of microbial viability with the exception of thermovinification treatments that steam the surface of the fruit. The temperature shock does make the organisms more sensitive to other types of inhibition, ethanol, carbon dioxide, acidity and sulfite. So, if used in combination with other practices, a significant loss of microbial viability can result. Modest temperature increases serve to encourage microbial growth and may shift the relative ratios of organisms present. Warmer temperatures favor the bacteria and later in fermentation will favor Saccharomyces as ethanol accumulates. To inhibit wild yeasts, the temperature must exceed 35 to 40°C and to inhibit Saccharomyces it must exceed 42-45°C. Below these limits, the warmer the temperature, the faster the rate of growth.

Low temperature treatments of grapes, musts and juices may also occur. Cold soaks incubate skins and seeds with the expressed juice of red grapes for a defined period of time with the goals of enhanced extraction and driving chemical reactions that occur more readily at low temperature. The temperature used is below that which will support fermentation (generally less than 12°C) but microbes may still proliferate to a limited extent under these conditions. The non-Saccharomyces yeasts, particularly Hanseniaspora uvarum, are the most cold-tolerant organisms found on grape surfaces; so cold soaks favor a bloom of these organisms. They can be prolific producers of acids and ethyl acetate, so impacts on the aroma profile of the wine may not be positive. Other lower-temperature tolerant wild strains produce a more diverse ester profile, but may become over-dominated by Hanseniaspora.  Since Hanseniaspora is an apiculate yeast, with experience the population size of this organism may be determined by microscopic examination. The pointed or apiculate nature of the cells only develops after multiple rounds of budding, so there may be non-apiculate members also present. Skin contact in white must processing is similar to a cold soak in reds, in that the desired outcome is the extraction of skin components into the juice. If cryo-processing methods are used, the juice is exposed to very low temperatures so that water may be removed as crystals of ice, leaving a more concentrated juice. In general, the bacteria, fungi, and yeasts of grape surfaces are prevented from growing at these low temperatures but do not lose viability. As soon as the juice is warm enough, they are able to initiate growth. Water removal also serves to concentrate the organisms remaining.

Enzymatic Treatments: Enzymatic treatments are often used to degrade pectins and complex polysaccharides to facilitate extraction and settling of the juice. Such treatments, to the extent that they release a variety of nutrients, may impact fermentation flora. Generally, there is such a high content of hexoses already present that the availability of more complex sugars has little to no impact on the major yeast species. However, the degradation of cell wall material may lead to greater extraction of nitrogen, sulfate and phosphate compounds that meet microbial nutritional needs. Micronutrients may also be released at this time, potentially providing requirements for more fastidious organisms. Again, with supplemented musts and juices, the additions from enzyme treatments are minor.

Oxygen Exposure

The organisms present on the surface of the grape, regardless of whether they are aerobic (requiring molecular oxygen for growth) or anaerobic (capable of fermentation), will be exposed to oxygen. Organisms requiring oxygen are able to proliferate, but organisms that can grow in the absence of oxygen will often be adapted to the presence of oxygen in the environment. Cells cycle between oxidation states during growth, with some oxidative activities occurring prior to and post- DNA replication. During DNA replication, oxidative activity is minimized and the cytoplasm is operating in a reduced chemical environment. This cycling is important to degradation and biosynthesis of macromolecules and is strongly influenced by the presence of molecular oxygen in the environment. The range of metabolic options available to cells will define the redox conditions that are permissive for growth.

Oxygen exposure of the juice or must allows aerobic organisms to continue to grow, but it also enables anaerobic organisms to more rapidly adapt to the impending loss of oxygen. Oxygen is required for the synthesis of certain cellular components that are required to tolerate increasing ethanol concentrations, so it is important for cells to be able to make and sequester these compounds when oxygen is present. The oxidative enzymes of grapes (polyphenol oxidase) and fungi (laccase) compete with microbes for available oxygen. Chemical reactions also consume oxygen, but these are generally much slower and require catalysts. Catalyst-driven oxygen consumption, microbial or enzymatic, will block chemical consumption of molecular oxygen.

Role of pH and Acidity

An excess or shortage of protons can be inhibitory to growth. Microorganisms have a window of pH that is permissive for growth. Optimal pH values provide the needed hydrogen ion concentration. At extremes of pH, the impact of low or high hydrogen ion content is mitigated by other factors, such as the use of proton pumps to excrete hydrogen ions or the substitution of hydrogen ions in biological processes by other charged molecules, such as potassium. If the hydrogen ion concentration of the cytoplasm is too high, protein denaturation will occur and cellular metabolism will be inhibited. If hydrogen (positive) ion concentration is too low, protonated species will lose protons, likewise inhibiting or preventing activity. Similarly to redox conditions, the cells have the capacity to buffer hydrogen ion concentrations.

Grape juice is traditionally low in pH, generally below pH 4.0. This low pH is inhibitory to many organisms. Yeast are very low-pH-tolerant, with some species such as Brettanomyces able to grow at pH 2.5 and below.Saccharomyces is able to grow across a wide pH range, from pH 3.0 to 8.0. At lower pH values, growth is dependent upon the presence of hydrogen ions to facilitate transport of substrates from the environment. At higher pH values, potassium plays this role in yeast. Many bacterial species in wine are inhibited at pH values below 3.5. Low pH juices, therefore, favor the growth of yeasts rather than bacteria. The pH value also impacts the amount of sulfite that is present in a free, or inhibitory, form. Sulfite additions should be corrected by pH.

Acidity is also an important factor. Total acid levels are not directly influenced by pH, but the molecular form of the acid is. At low pH, with high concentrations of protons, acid species will tend to be protonated or neutral in charge. The cellular surface membrane is very permeable to protonated acids, so they readily gain entry into the cell. Once inside of the cell, the relatively high pH of the cell causes the acids to lose a proton. If  the rate of uptake of protonated acids exceeds the cells capacity to fix or excrete the protons, the pH of the cytoplasm will decrease and metabolic activities will be arrested. Thus, both the pH and total acidity will impact the numbers and types of microorganisms that can be present in an environment.

Impact of Antimicrobial Additions

Microbial populations can be manipulated by the judicious use of antimicrobial agents. There are two agents commonly used in grape juice or must to control microbial growth: sulfur dioxide (sulfite) and lysozyme. Other inhibitory compounds, sorbate, fumaric acid and dimethyldicarbonate (DMDC; Velcorin), are approved additions for wines prior to bottling, but not used in actual juices pre-fermentation.

Sulfur dioxide is a reactive molecule. The free or gaseous form of sulfur dioxide (SO2) can be formed from burning inorganic sulfur in the presence of oxygen (air) or it can be liberated from sulfite salts (potassium metabisulfite; K2S2O5) under acidic conditions. Sulfur dioxide inhibits and kills cells by interfering with essential oxidation/reduction reactions. It can bind to proteins or to metabolites blocking further metabolism. Sulfite exists in different forms as a function of pH. The free form is the most reactive and therefore the most inhibitory:  it results in a general loss of metabolic activity and ATP (energy) production. Some organisms are more tolerant of sulfite than others. Tolerance is generally achieved by complexing the sulfite with a reactant and excreting the complex from the cell. In the case of Saccharomyces, the compound filling this role is acetaldehyde. The sulfite reacts with the abundant aldehyde, thereby preventing damage to sensitive metabolic processes. Different species of yeast and bacteria show differing sensitivities to sulfite. The critical form of sulfite is the molecular form, not the total amount of all species present. During active growth and, therefore, at the height of metabolic activity, a molecular SO2 concentration of 6 mg/L is needed to inhibit the growth of Saccharomyces. In contrast, in finished wine where sugar has been catabolized, only 0.825 mg/L of molecular SO2 is needed, as tolerance is decreased in the absence of the ability to create adjuncts with aldehydes.

In contrast to yeast, the bacteria in wine are inhibited, also, by sulfite bound to aldehydes, as the bound version is transported into the bacterial cells, where it dissociates within the cytoplasm. Again, the level of sensitivity is dependent upon the cell’s capacity to continue to make ATP and to grow. Thus, sulfite can be used at concentrations below the yeast inhibitory threshold, but above the inhibitory threshold for bacteria.

Lysozyme is an enzyme that can damage bacterial cell walls. It has no inhibitory impact on yeast, as their walls are of a different composition. Lysozyme is particularly effective in the inhibition of gram positive organisms, such as the lactic acid bacteria. However, use of this compound may inhibit desired bacteria as well. It will be inactivated over time, so it is possible to use lysozyme at the point of inoculation with yeast and still obtain an ML fermentation during wine aging and storage. If an excessive amount of lysozyme is used, then the ML may not occur. Lysozyme can be used to control bacterial populations without fear of impact on yeast.

Summary and Recommendations

With respect to grape, must and juice processing, the most important variables are the pH of the juice, sulfite addition practices, and establishment of selective conditions.

  1. pH: the lower the pH, the lower the number of different species that can be present; but, also the slower the metabolic activities of those that are present. In general, wines below pH 3.5 will support rapid development of large yeast populations, while higher pH values favor establishment by bacteria.
  2. Sulfite addition: the earlier the addition of sulfite, the greater the inhibition of the non-Saccharomycesflora. Free levels of sulfite, not merely total added, should be monitored to target inhibition to unwanted species.
  3. Establishment of selective conditions: microbes will commence growth immediately upon damage to the fruit, and the concurrent release of sugar and other growth substrates. The ability to utilize glucose and fructose is fairly ubiquitous in the microbial world. Which species dominates depends upon the establishment of other types of selective conditions: temperature, oxygen availability, solids content, presence of inhibitors. Warmer temperatures and high oxygen generally favor the bacteria over the yeast. Bacterial cells are less complex than yeast cells and they can divide more quickly. The optimal processing conditions are going to vary by the situation: the existing bioload at harvest, the bioload contributions from winery equipment, the nature of the organisms present and the presence of any stressful or selective conditions.