Technique

The Biology of Malolactic “Bugs”

In this excerpt from the technical binder “Malolactic Fermentation in Wine” (Lallemand, 2005) written for commercial winemakers, researchers at Lallemand, Inc. offer an insider’s view of the more technical aspects of malolactic fermentation (MLF).

Lactic acid bacteria (LAB) are natural inhabitants of vineyards and wineries. These bacteria can transform malic acid into lactic acid. Wild LAB may not completely degrade all the malic acid in wine must and may additionally produce off aromas or flavors.

However, specially selected strains of LAB, such as certain strains Oenococcus Oeni, are the desired microorganisms for performing a successful malolactic fermentation.

Lactic acid bacteria (LAB) are found naturally on grapes, leaves, soil and equipment surfaces and have the ability to grow on a variety of sources, including grape juice. The most common LAB belong to the genera Lactobacillus, Pediococcus, Leuconostoc and Oenococcus. These bacteria are generally microaerophillic (they respire anaerobically, but are stimulated by small amounts of oxygen), require carbohydrates and must be supplied with amino acids and vitamins in order to proliferate. Wild or cultured LAB may cause a malolactic fermentation in wine. Knowing the factors that affect the viability and health of LAB will enable winemakers to stimulate or inhibit their action, according to their winemaking plans.

Typically, LAB identified in grape musts are present at approximately 104 cells per mL. The majority of these bacteria are not tolerant towards the changing environmental conditions associated with winemaking and disappear during alcoholic fermentation. However, many species are able to survive, in particular Oenococcus oeni, which is often found in wines with a pH below 3.5. Wines exhibiting a pH greater than 3.5 are capable of supporting a broader range of species. Regardless of the species of LAB, the main significance of these organisms in wine production is their ability to conduct malolactic fermentation (MLF). MLF is characterized as the degradation of L-malic acid to L-lactic acid and CO2, a process which decreases the amount of acidity in the wine. However, MLF not only represents a biological deacidification process, it also exerts a significant impact on the organoleptic aspects of wine. These sensory effects can be positive or negative, depending on the bacterial species or the strain of LAB employed to conduct the MLF. LAB strains that produce particularly favorable characteristics in wine, and hence are more desirable to perform the MLF, are often termed “malolactic bacteria” (MLB). LAB strains that negatively influence the final product may cause a range of undesirable changes to wine sensory properties, altered wine color and may even lead to the generation of biogenic amines.

Microorganisms, MLF and Wine

Given the important role of the organism employed for MLF, it is an increasingly common practice to inoculate a fermentation with a known malolactic bacterial strain or a mixture of strains, rather than depend on the naturally occurring flora. The advantage of inoculating is that the time and the extent to which MLF occurs can be controlled and the quality of the final product can be predicted. Species of Lactobacillus or Pediococcus may conduct MLF, especially in wine exhibiting a pH higher than 3.5, but usually result in non-acceptable wines. These genera are poorly tolerant to low pH and produce undesirable flavors as well as high levels of acetic acid.

Enter Oenococcus oeni

Despite the potential of many LAB for use in wine production, Oenococcus oeni remains the organism of choice for many wine producers. Oenococcus oeni, the bacteria formerly known as Leuconostoc oenos, is a facultative anaerobe (It can respire anaerobically or aerobically). It’s nutritional requirements are complex.

A source of carbon (derived from sugars), nitrogen (derived from free amino acids or short peptides), vitamins (nicotinic acid, thiamine, biotin and pantothenic acid), mineral ions (Mn2+, Mg2+, K+ and Na+) and purine derivatives (guanine, adenine, xanthine and uracil) are all required for optimum growth. Oenococcus oeni cells are spherical and occur in chains when grown on solid media. Growth is generally slow and can take from 5 to 7 days to form visible colonies at incubation temperatures between 68–86 °F (20–30 °C).

Although previously grouped with the Leuconostoc species, DNA analysis of Oenococcus oeni strains has placed them in a group that is clearly distinguishable from the Leuconostoc species. It is widely believed that Oenococcus oeni represents the best candidate to conduct MLF because of its resistance to a variety of environmental stresses, in particular the acidic conditions and the high alcohol levels which are typical of wine. Inoculating wine with carefully selected strains of Oenococcus oeni has the advantage of enabling the winemaker to have more control over MLF. In addition, employing a specific strain of Oenococcus oeni allows the winemaker to ensure that particular characteristics are produced in the final product, thus creating wines that are more distinctive and characteristic.

Although a single bacterial strain is generally employed, in some instances a mixture of strains may be used in the inoculum. This procedure can not only produce certain preferred characteristics in the wine, but is also capable of maximizing the chances of bacterial survival if a bacteriophage (a virus that targets bacteria) is encountered in the wine.

Interactions with Yeast

Although some strains of yeast are capable of contributing to MLF, the role of yeast is generally negative, given its main function of converting nutrients to ethanol. The inhibitory effect of certain yeast strains on MLF has been reported and is generally caused by the production of yeast metabolites that can have a negative influence on bacteria as well as competition for nutrients. Interestingly, studies of the growth patterns of yeast and bacteria on agar plates performed in the Lallemand laboratories and elsewhere have indicated that some yeast strains may actively inhibit the growth of Oenococcus oeni by the production of an “antimicrobial” substance (or substances). Such data supports the belief that the relationship between yeast and bacteria is complex and matching the appropriate strains of both is the key to successful MLF. The autolytic activity of wine yeast during aging on lees can greatly affect the concentrations of nitrogenous compounds available to malolactic bacteria, including amino acids, peptides. It has been suggested that leaving wine on yeast lees specifically to maintain a higher level of carbon dioxide (CO2) may further encourage MLF.

In wine, sulfur dioxide (SO2) exists in a pH-dependent equilibrium between bound SO2, molecular or free SO2 and bisulfite and sulfite ions. Low levels of SO2 can inhibit the growth of LAB in wine, resulting in stuck malolactic fermentation, and high levels of SO2 can kill bacterial cells. Molecular SO2 is considered to be the most toxic form for LAB and it has been reported that a molecular SO2 concentration as low as 0.1–.15 mg/L may be inhibitory to the growth of some strains.

Although SO2 concentrations are dependent on the chemistry of the wine, they may also be influenced by the yeast strain used to conduct the alcoholic fermentation. Some yeast strains are capable of producing rather large amounts of SO2. If MLF is required, it is important to use a yeast strain that produces little, if any, SO2.

Factors Affecting LAB

The composition of the wine, the method of vinification and the interrelationships between LAB and other microorganisms present can affect the survival and growth of LAB in wine and therefore influence MLF. Environmental conditions such as pH, temperature, alcohol level, nutritional status and levels of sulfur dioxide (SO2) may also play a significant role.

A pH Around 3.4

A critical parameter for successful MLF is pH, and the minimum pH at which bacterial growth can occur in wine is approximately 2.9–3.0. Bacterial growth is faster and MLF is completed earlier as the pH increases above 3.0. Although a pH of 6.3 is optimum for the activity of the malolactic enzyme, degradation of malic acid by non-growing cells of Oenococcus oeni is most rapid at lower pH values due to an increase in intracellular pH to 4.0. It is widely accepted that in terms of initiation and completion of MLF, a pH of approximately 3.4 is the most desirable.

Under 15% Alcohol

Alcohol tolerance is an important characteristic of many LAB, and resistance to alcohol varies among them. Most strains are not capable of proliferating in wines with an ethanol concentration greater than 15%, but some have been observed to grow in the presence of 20% ethanol.

Some Like It Hot

The optimum growth temperature for LAB is between 77 and 95 °F (25–35 °C) and the rate of malate degradation by non-growing cells is highest at approximately the same temperatures. The rate of growth of malolactic bacteria and the speed of the MLF are inhibited by low temperatures. This can be problematic, particularly during the production of white wines, which tend to be fermented at lower temperatures.

Carbohydrates Required

Growth conditions during the malolactic fermentation (MLF) in wine are very difficult for LAB. During the malolactic fermentation, 0.4–0.8 g/L of sugar is degraded, the bulk of which is represented by glucose and fructose, which are the most important sources of energy for bacterial growth. Nearly all wines contain adequate amounts of these sugars to sustain sufficient bacterial growth to ensure a complete MLF, but it has been shown that the MLF may be inhibited in wines in which the sum of the concentrations of glucose and fructose is less than 0.2 g/L. Oenococcus oeni is heterofermentative (utilizes more than one fermentation pathway) and converts glucose to L-lactic acid, CO2 and acetic acid (or ethanol). Almost all strains of malolactic bacteria ferment glucose and fructose, with most prefering fructose.

Nutrients Are Nice

If yeast with high nutrient demands conduct the alcoholic fermentation, the juice is rapidly depleted of factors necessary to support the growth of LAB. Under this condition, a bacterial nutrient must be added. Similarly, the addition of a bacterial nutrient is critical in juices with naturally low nutrient levels because some yeast may produce excessive levels of SO2, which will strongly inhibit the MLB. Suffice it to say that proper nutrition of both yeast and MLB is always essential. Under the difficult pH, SO2, alcohol and nitrogen conditions found in wine, the use of supplemental nutrients will make it possible for Oenococcus oeni to survive and multiply. Careful preparation of malolactic (ML) starter cultures and proper use of the nutrient preparations designed for those cultures will ensure the rapid start of the MLF. Although malic acid is the most important acid metabolized by LAB in wine, other organic acids are also metabolized.

Tartaric acid Small decreases in the concentration of tartaric acid are sometimes observed during the MLF. These changes are most likely due to changes in the solubility of tartaric acid rather than to actual microbial degradation. The degradation of tartaric acid is always associated with wine spoilage.

Malic acid Tartaric and malic acids are the two major organic acids in wine, especially in wines from cool climates. Malic acid is naturally present in the L- form. D-malic acid is not naturally present in grape juice and is not metabolized by wine LAB. Several studies have shown that L-malic acid stimulates growth and biomass production of Oenococcus oeni. During growth at low pH, MLB degrade malic acid at a high rate, whereas carbohydrate is degraded at a low rate. This phenomenon results in an overall increase in pH, which, in itself, allows for an increase in carbohydrate utilization, thus explaining the observation of malic acid induced growth.

Citric acid Citric acid is a major compound in grape must and wine and can be found in concentrations ranging from 0.1 to 0.7 g/L. Citric acid metabolism by Oenococcus oeni has been correlated with the synthesis of acetic acid, diacetyl and acetoin. Oenococcus oeni is not able to grow on citric acid as a sole carbon and energy source, but in the presence of an energy source such as glucose, the growth rate of Oenococcus oeni is enhanced. Citric acid is completely metabolized in some wines, but to a lesser extent in others. Production of diacetyl and acetoin by Oenococcus oeni is stimulated by increased citric acid concentrations and the maximum concentration of diacetyl is found upon completion of malic acid degradation. During the MLF, degradation of citric acid is delayed as compared to the degradation of malic acid.

Winery Practices

Clarification of juice and wine not only can physically remove a large portion of LAB, it can reduce the amount of bacterial growth obtainable, thus impacting wine quality. In addition, clarification will remove nutrients and suspended particles stimulatory to bacterial growth, further impacting the MLF.

Timing of the inoculation of MLB will also influence the kinetics of MLF. The availability of nutrients will be affected by interactions among wine microorganisms. It is common to expect that mixed cultures of microorganisms will introduce the possibility of antagonistic and synergistic relationships but, in some minor cases, they exert little or no influence over each other. In winemaking, there is always the possibility of interactions occurring among LAB and yeast, fungi, acetic acid bacteria and even bacteriophage. Moreover, there also may be interactions among different species and strains of LAB. The antagonistic effect of yeast on MLB has been explained through competition for nutrients and the production of substances that inhibit bacterial growth, such as SO2 or medium-chain length fatty acids. Conversely, yeast may support the growth of LAB in wine and stimulate the progress of MLF. During the process of yeast autolysis, vitamins and amino acids are released into wine, and the associated extended lees contact enriches the wine with micronutrients that stimulate MLF.

Interaction of Factors

The best understood factors governing successful MLF include SO2, pH, alcohol and temperature. For the MLF to be successful, the values of these chemical parameters must correspond to those which allow the bacterial cultures to function successfully. It is important to remember that these factors function synergistically, i.e., their actions together have a greater total effect than the sum of their individual actions. Similarly, a favorable level of one component may compensate for an unfavorable level of one or several of the other components. Although assigning exact values to each component is difficult, abiding by the parameters as defined by the different producers of commercial cultures is imperative. Adherence to this rule is perhaps the most important consideration to ensure successful MLF. In some cases, it can be very difficult to produce a wine whose analyses conform to these general parameters. Red wines from the New World that are harvested at very high maturity and with subsequent high alcohol levels are a typical example. In these cases it is important to select the proper yeast strain to produce the wine, as well as the correct bacterial strain to conduct the MLF. Even when all chemical factors fall within the desired parameters, the course of MLF will occasionally be problematic. Possible causes of these anomalies will be discussed below.

Lesser-Known Factors

A number of lesser-known factors can influence the course of MLF. The fact that they are lesser known does not mean that their impact is less significant. These factors include the following:

Tannins Recent research has shown that certain grape tannins can have a negative influence on malolactic bacteria, and consequently on the course of MLF. In fact, some research has indicated that certain red cultivars, such as Merlot, can have great difficulty undergoing a successful MLF. Latest results at Lallemand indicate that phenolic acids influence the growth of certain bacterial strains in laboratory growth media. The effect on growth stimulation can be either positive or negative, depending on the bacterial species, the specific phenolic acid used and its concentration. A nutrient to support the course of MLF under these limiting circumstances might be considered.

Lees compaction As a result of hydrostatic pressure, the lees found at the bottom of a tank can be compacted to such an extent that yeast, bacteria and nutrients are “captured” and cannot function properly. It has recently been observed that larger tank sizes may correlate with increasing delays in the initiation of the MLF. The inhibition of the start of the MLF in larger tanks can be overcome by pumping over either on the day of inoculation or on the second day after inoculation with the bacteria. A general recommendation would be to stir the lees regularly (at least weekly) to ensure that bacteria and nutrients are kept in suspension. Of course, these observations were made at commercial wineries. At home, the size of your fermenter is not likely to be large enough to begin to inhibit the action of LAB.

Residual lysozyme activity If lysozyme is used during the production of wine, residual levels of this enzyme may impact the time required for the onset of MLF. Care must be taken to follow the supplier’s recommendations with regard to the required time between the addition of lysozyme and the inoculation of the commercial MLF culture. In most cases, racking the wine off of the gross lees is recommended.

Excessive amounts of oxygen Malolactic bacteria have been shown to be sensitive to excessive amounts of oxygen. This means that exposure of the bacteria to undue amounts of oxygen after the completion of alcoholic fermentation should be avoided. Although it has been noted that even low concentrations of oxygen may detrimentally influence MLF, micro-oxygenation may have a positive effect on MLF due to the gentle stirring action associated with the micro-oxygenation process itself.

Fungicide residues Certain fungicide and pesticide residues, especially the former, may have a detrimental effect on the functioning of malolactic bacteria. Most effective, in a negative sense, are residues of the systemic compounds often used in humid years to control the Botrytis fungus. Careful precautions should be taken in years with high incidence of Botrytis contamination. Wine producers must be familiar with the spraying programs and products used, and they must adhere to the prescribed withholding periods required for the various antifungal products.

Initial malic acid concentration Malic acid concentrations differ among grape musts. As such, the duration of an MLF may differ from one year to the next. It is especially difficult to induce an MLF in wines with malic acid levels below 0.8 g/L. In this case, using ML starter cultures with a high malate permease activity is recommended.

Fatty acids Sufficient levels of oleic acid are necessary for malolacic bacteria to reach and maintain high cell viability in the wine. In fact, the success of MLF is influenced by the ability of the bacterial strain to assimilate oleic acid. Certain practices, such as must clarification, can lead to a wine deficient in oleic acid. If the MLF was induced using very high cell numbers, this phenomenon may not be observed. Medium-chain length fatty acids can have a negative impact on the course of MLF. The antagonism between yeast and lactic acid bacteria could be explained by the production of certain medium-chain length fatty acids.

This knowledge of the biology of malolactic bacteria should allow you to encourage or inhibit MLF, as your winemaking plan dictates.

The authors of this binder excerpt are yeast researchers at Lallemand, Inc.