A Review and Summary of “Malolactic Fermentation: Importance of Wine Lactic Acid Bacteria in Winemaking”

A Review and Summary of “Malolactic Fermentation: Importance of Wine Lactic Acid Bacteria in Winemaking” By Joy Ting, Michael Shaps Wineworks Chardonnay can be made in several different styles (tank vs. barrel fermentation, no malolactic to full malolactic). We make a variety of these styles, and try to match the fruit source with the style in which it will be made. Last year, one of our lots intended for full malolactic fermentation struggled to progress and we ended up stopping it early. In an effort to better understand why some Chardonnays progress through malolactic fermentation more easily and consistently than others, and to know if there were some changes I needed to make in my winemaking this harvest to encourage malolactic fermentation when it is intended, I did a little research. A friend leant me her copy of “Malolactic Fermentation – Importance of Wine Lactic Acid Bacteria in Winemaking”, published by Lallemand (1). It is written by a group of academics that represent a mixture of company scientists as well as consultants and those working in university settings. Unlike many trade publications, this resource does not primarily sell products, but rather provides good scientific background into what is known of this microbial process. (It does have a “modern” view of winemaking, assuming intervention is better than letting the ambient microbes work it out.) Following is a summary of information I found helpful when considering aspects I need to think about at harvest that will help the Chardonnays progress well through malolactic fermentation. The publication also discusses the role of malolactic fermentation in red wine making, and has a fairly extensive section on the timing of malolactic inoculation (co-inoculation vs. sequential inoculation), but I will let you read it on your own for those insights. An Introduction to Oenococcus oeni There are many strains of lactic acid producing bacteria on grapes. The population dynamics of these microbes are largely driven by pH. At pH<3.5 the dominant strain is Oenococcus oeni, however, wines with pH>3.5 are able to support many different strains of LAB (including Pediococcus). Oenococcus is considered the most desired lactic acid bacteria due to the flavor characteristics it produces as well as its ability to complete malolactic fermentation in high alcohol and acidic environments. Others, such as Pediococcus, are often seen as spoilage organisms, not completing the conversion and potentially producing high amounts of VA or mouse taint. This may be why some wineries will inoculate for malolactic fermentation in red wines but not in Chardonnays, as red wines generally have higher pH than white wines. There are several strains of Oenococcus oeni that are well separated by DNA, physiology and geographic origins, indicating this bacterial line has been evolving in separate regions of the world for some time. Several strains have been isolated and are available for commercial purchase. Due to their physiological differences, sometimes a mixture of strains is inoculated to produce complexity in the wine and avoid stalling MLF if bacteriophage is encountered (more on bacteriophage later). Physiologically, Oenococcus has “complex nutrient needs”. This means that any technique or circumstance that increase overall nutrient need may limit needed nutrients. Examples include ambient ferments and highly clarified juice (removing pulp that might provide micronutrients). These fermentations may require nutrient supplementation formulated specifically for Oenococcis oeni. Oenococcus can be slow growing, requiring 5-7 days to grow visible colonies on lab plating tests. In this winery, this means it may need some time to build up to metabolically active population levels in your wine. Lactic Acid Bacterial Nutrition Nutritionally, lactic acid bacteria need a carbon source to build their cell bodies, amino acids to build proteins, and a source of energy to fuel this construction. Oenococcus mostly use hexose sugars like glucose and fructose as a carbon source. Even when wines test “dry” for sugar, there are usually sufficient number of glucose and fructose molecules remaining to provide carbon backbones. In addition, Oenococcus will cleave larger carbohydrate molecules in the wine to release simple sugars. Lactic acid bacteria do not generate amino acids on their own, and therefore need an extraneous source. These can be provided by the breakdown of yeast cells in the lees. Lees stirring makes these products more available to LAB. Bacterial cells pick up peptides (small protein chains) better than amino acids due to the kinetics of the membrane and the charge distribution of amino acids. Nutrient supplements specially formulated for malolactic fermentation contain peptides and can help Oenococcus overcome inhibiting factors (such as higher SO2 levels and lower pH) through synthesis of cellular factors that invoke tolerance. Unlike YAN, there is not a straightforward winery lab test to determine if nutrient supplementation is needed. A good rule of thumb is that if the alcoholic fermentation was stressful for yeast, then it is likely that the environmental conditions and nutrient environment will also be stressful for Oenococcus . This is especially true if juice was highly clarified, since this means it will have lower nutrients. Energy is provided by the conversion of malic acid to lactic acid. The consumption of one proton during this reaction and subsequent export of lactate out of the cell sets up a proton gradient that can be used by the cell to harness energy in the form of ATP. Juice also contains small amounts of citric acid, which can be used in a separate conversion to produce energy, resulting in acetic acid (when higher amounts of oxygen are present) or diacetyl (when little to no oxygen is present). Most Oenococcus have a higher affinity for malic acid than citric acid, and therefore citric acid consumption (and diacetyl production) may only occur as malic acid becomes scarce, mid-way or later in MLF. Different strains of Oenococcus have different affinities for citric acid, and therefore different timing and probability of producing diacetyl. In the highly reducing conditions of alcoholic fermentation, diacetyl is quickly reduced to acetoin or 2,3 butanediol, which have much higher thresholds for sensory perception. If full malolactic conversion is desired without perceptible diacetyl (buttery flavor), co-inoculation during fermentation should be considered. Why yeast strain matters: Successful MLF can be inhibited by fermentative yeast for several reasons. Microorganisms are in competition for resources, and therefore produce compounds to inhibit each other’s growth. Yeast produce several compounds that effectively limit competition. Alcohol produced during fermentation disrupts the cell membranes of microbes that don’t have specific mechanisms to tolerate it. Oenococcus can have a tolerance limit of up to 15% alcohol, but can show signs of inhibition at lower levels, depending on environmental conditions and bacterial strain. Yeast also produce SO2, which can inhibit malolactic fermentation at low levels and kill lactic acid bacteria at higher levels. SO2 bound to acetaldehyde is especially inhibitory because lactic acid bacteria will metabolize acetaldehyde and release SO2, thus increasing the free SO2 levels in the course of malolactic fermentation. Molecular SO2 is most toxic, showing inhibition at 0.1-0.15 mg/L and toxicity between 0.3 – 0.5 mg/L depending on strain. Generally, free SO2 should be less than 10ppm and total SO2 <90ppm for successful MLF. One study showed that 75% of the inhibitory effects of yeast on malolactic bacteria are due to alcohol, but 25% is due to other inhibitory compounds produced by yeast. An as-yet-unidentified 5-10 kDa protein produced by yeast caused significant reduction in consumption of malic acid when present. Yeast also produce medium chain fatty acids that can inhibit malolactic fermentation. The amount of inhibition is dependent on strain, grape cultivar, and other environmental conditions. Suppliers consider these and other factors when they rate yeast for malolactic fermentation potential. General inhibitors of growth: There are several biotic and abiotic factors that limit growth and/or activity of lactic acid bacteria: • pH less than 2.9-3.0 inhibits malolactic fermentation while a pH = 3.4 is considered most desirable. • Alcohol above 15% is inhibiting to most strains. • Temperatures that are too high (above 25 degrees C) increase the toxicity of alcohol while those that are too low (less than 16 degrees C) limit the multiplication and activity of bacteria. For wines with 12.5 – 14 % alcohol, 18-22 degrees C is optimal. • Lysozyme is active for about 2 weeks, so this treatment may temporarily inhibit malolactic bacteria. • Bacteriophages are viruses that infect bacterial cells. They are usually strain specific, and are most active in growing cells. This may be the cause if a malolactic fermentation suddenly stops. Bacteriophages take time to build up (just like any infection), so the best approach is to help the malolactic fermentation outpace the phage. If a bacteriophage infection is suspected, try a different strain of Oenococcus, which may be immune to the infecting phage, and a higher inoculation rate, to allow the bacteria to multiply faster than phage can kill the cells. • Yeast lees seem to be stimulatory because they release amino acids, detoxify polysaccharides, and absorb inhibitory compounds. • An initial malic acid range of 2-4 g/L malic acid is most favorable. As the conversion progresses, increasing lactic acid levels become inhibitory. • Initial lactic acid levels >0.3 g/L are inhibitory to starter cultures. Some yeast produce lactic acid, so this confounds yeast strain decisions. This is also why re-inoculation after a stalled malolactic fermentation may be difficult. • Chitosan may have an inhibitory effect . This effect was being tested at the time of publication (2015), so more updated information may now be available. • Excess oxygen is inhibiting. • Residual fungicidal sprays (for botrytis) are inhibiting. • Deficiency of oleic acid due to over clarifying can inhibit cell growth. Cumulative Effects When considering any of the above factors affecting the growth of lactic acid bacteria, it is important to realize that they work synergistically. This means a favorable level of one may offset the stressful effects of another. This also means that a specific strain of Oenococcus reported to be tolerant to 14% alcohol, may be less tolerant if temperature, nutritional environment, or SO2 are at marginal levels. Stress in one area diminishes tolerance in another area. As part of their online resources, Scottlabs provides a scorecard for determining the ease of malolactic fermentation (2). This website also provides guidelines for each of the malolactic bacterial inoculants they sell. Other suppliers likely have this info as well. The choice of a LAB strain requires understanding of both sensory impacts and compatibility with environmental conditions. References: Costello, P. et al (2015). Malolactic Fermentation – Importance of Wine Lactic Acid Bacteria in Winemaking. Quebec, Canada: Lallemand. (2) Malolactic Review. (n.d.). Retrieved August 30, 2017, from http://www.scottlab.com/uploads/documents/Malolactic%20Overview.pdf