Factors Impacting the Fermentation

Factors Impacting the Malolactic Fermentation

Several factors impact the initiation and progression of the malolactic fermentation. Temperature, pH, acidity, ethanol, sulfite and availability of nutrients are all important for the growth and metabolic activities of the lactic acid bacteria. The lactic acid bacteria are more fastidious in their growth requirements than the yeast. It can be challenging to get the malolactic conversion to occur at the desired time in the wine.

The lactic acid bacteria generally require higher temperatures than the yeast in order to grow. The temperature should be above 18°C (64°F) to allow growth of the bacteria. The bacteria will grow more rapidly at higher temperatures but this can lead to deterioration of the wine. Successful malolactic conversions have been reported to occur at lower temperatures and this may indicate some strains can develop temperature tolerance but this is not a general phenomenon.

The pH of the wine is also an important factor. The pH will affect which strains and species can grow in the wine or juice and will impact growth rate and metabolic activities of the organisms. In general Oenococcus is more tolerant of low pH than the lactobacilli or pediococci. If the pH is 3.8 or higher, then strains of Lactobacillus and Pediococcus can readily grow. The process of the malolactic conversion and fixation of protons increases the pH of the wine. The metabolic activity of Oenococcus may therefore make conditions more conducive for the growth of other lactic acid bacteria.

Lactic acid bacteria can also be inhibited by ethanol. Most wine lactic acid bacteria are tolerant to ethanol levels of up to 14% (vol/vol), and there are some highly tolerant strains that have been identified. Ethanol tolerance is strongly influenced by the pH. As with yeast, ethanol increases the passive proton flux into bacterial cells and can tax the cells normal mechanisms that maintain pH homeostasis. The higher the hydrogen ion concentration (the lower the pH) the more difficult maintaining hydrogen ion gradients becomes.

The lactic acid bacteria evolved in nutrient-rich environments and therefore have more growth requirements than they yeast. The lactic acid bacteria generally all require supplementation with amino acids and vitamins and have lost the ability to make these compounds de novo. At the same time they are more diverse than yeast in the compounds that they can use to meet their growth requirements. Peptides can be taken up and degraded to amino acid constituents and a broader spectrum of nitrogen-containing compounds can be used. The lactic acid bacteria display even more complex growth requirements. The compound 4’-O-(β-D-glucopyranosyl)-D-pantothenic acid found in tomato and other fruit juices is a growth factor required by the lactic acid bacteria. Cultivation media for lactic acid bacteria generally contain tomato or apple juice as components. The yeast may deplete the wine of essential nutrients needed by the bacteria. If lysis of the yeast cells occurs at the end of fermentation some nutrients may be returned to the wine that can then be used by the bacteria but if the wine is racked off of the yeast lees, the nutritional content may be too low to allow growth of the lactic acid bacteria.  Growth of the lactic acid bacteria is stimulated by extended skin contact and high juice solids content as this provides a greater nutritional content for the organisms.

Lactic acid bacteria have diverse mechanisms for creating the energy needed to support and sustain biological activities. These organisms can degrade carbon substrates and capture energy from bond rearrangements as seen with yeast. However they can also generate energy anaerobically from creation of a proton motive force across their plasma membranes. This process can be accomplished using movements of acids, derived from organic acids or from amino acids. Thus the availability of organic acids can be important in allowing growth and metabolism of the lactic acid bacteria. Obviously if the grape has consumed the malate such that wine malate levels are low the malolactic conversion will not occur. Oxygen is also stimulatory to the growth of the malolactic bacteria. In the presence of oxygen more energy can be obtained from catabolism and higher levels of acetic acid can be produced as oxygen serves as terminal electron acceptor. The presence of carbon dioxide can also stimulate the malolactic conversion.

The malolactic bacteria are more sensitive to the inhibitory effects of sulfite than are yeast. Free sulfite is inhibitory but in contrast to the yeast some bound forms of sulfite will also be taken up by the bacteria releasing the bound sulfite inside of the cell post-transport.  Sulfite is a reactive molecule and can inhibit biological activities occurring within the cells. Bacteria can also be inhibited by organic acids made by the yeast, such as fumaric acid, and can be inhibited by the presence of yeast fatty acids.

The presence of other lactic acid bacteria can also impact the malolactic conversion. Lactic acid bacteria make bacteriocins, compounds that inhibit the growth of other bacteria. Therefore an early bloom of wild lactic acid bacteria on the surface of the grape or under conditions of cluster rot can result in the presence of inhibitory bacteriocins that may block the progression of subsequent growth of other members of the lactic acid bacteria and the malolactic conversion. Alternately, the presence of a mixed culture of bacteria may speed the malolactic conversion due to the increase in pH.

Finally the presence of bacteriophage may impact the progression of the malolactic conversion. Bacteriophage are virus-like particles that can infect a bacterial cell hijacking the metabolism of the cell to produce more bacteria and ultimately lysing the cell and releasing more infective particles. Bacteriophage can cause havoc in dairy fermentations and in vegetable fermentations. The malolactic bacteria have been shown to be susceptible to bacteriophages but it is not clear if this is a problem in wine production or not.