Sunday, June 1, 2014

Lactic acid bacteria: Pre-, intra-, and post-malolactic-fermentation development

Either concurrent with, or post-alcoholic-fermentation, a number of wines are subjected to a process --malolactic fermentation (MLF) -- wherein the harder malic acid is converted to lactic acid by lactic acid bacteria (LAB). The perceived benefits of this process to the final wine are:
  • Deacidification, with a resultant increase in pH
  • Increased microbial stability through removal of malic acid as a possible carbon substrate; and
  • Modification of the wine's aroma profile.
I described the origin and evolution of malic acid in my most recent post and provide a similar treatment of LAB in the current.

The genera from which the LABs are drawn are shown below. The species associated with wine are (Wibowo et al., AJEV, 36 (4) 1988):
  • Oenococcus.oeni -- mainly during MLF
  • Pediococcus cerevisiae -- mostly after MLF; predominantly in wines with high pH
  • Pediococcus pentosaceous -- mostly after MLF; predominantly in wines with high pH
  • Lactobacillus spp. -- mainly after MLF.

Phylogenetic trees of Lactobacillales constructed on the
basis of concatenated alignments of ribosomal proteins

Doctor Murli Dahrmadikari (Lactic Acid Bacteria and Wine Spoilage, describes LAB thusly:
These organisms are gram positive, catalase negative, nonsporing cocci, coccobacilli or rods. They are microaerophilic (sic) that means that they grow well under conditions of low oxygen content. Since they can grow under low oxygen conditions, they can grow throughout the wine (as opposed to on the surface of the wine) even though the container is kept full. The bacteria can metabolize sugars, acids and other constituents in wine and produce several compounds. Some of these are undesirable and constitute spoilage.
LAB utilize two pathways for the metabolism of glucose and a third for the metabolism of pentose (Lerm et al., Malolactic Fermentation: The ABCs of MLF, S. Afr. J. Enol. Vitic. 31 (2), 2010). One of the glucose pathways (EMP) converts glucose into pyruvate over a number of steps and then into lactic acid. In this process, 1 mole of glucose yields 2 moles of lactic acid plus 2 ATPs. This process is called homolactic fermentation and all Pediococcus species utilize this mechanism. The second glucose pathway (6-PG/PK) yields lactic acid, carbon dioxide, ethanol, acetate, and 1 ATP. The bacteria utilizing this pathway are called heterolactic fermenters and this includes all strains of Leuconstoc, some Lactobacillus strains and Oenococcus.oeni. The pentose pathway combines pentose with a phosphate derivative before coalescing with the later portions of the 6-PG/Pk pathway for completion. The outputs of the pentose pathway are lactic acid, acetic acid, and carbon dioxide.

The LAB in wine originate from grape and grape leaves and are brought into the winery at harvest (Dharmadhikari; Lerm et al.; Wibowo et al.). The LAB diversity and population density are limited by  grape maturity and sanitary conditions and levels are generally on the order of 100 cells/gm (Wibowo et al.) The species that are present at this time include Pediococcus and Leuconstoc (Lerm et al.).

Once the grapes are brought into the winery, the LAB population increases dramatically, implicating the winery environment in this proliferation. According to Lerm et al., the population rises to 103 - 104 cells per ml shortly after crushing and prior to alcoholic fermentation (AF). In a study of the evolution of LAB, Lafon-Lafourcade et al. (Occurrence of Lactic Acid Bacteria During the Different Stages of Vinification and Conservation of Wines, Appl. Environ. Microbiol. 1983, 46 (4)) show populations of 104 cells/ml at 14℃ and 19℃ if (i) unsulfited and (ii) sulfited at 50 mg/l. At 100 mg/l of SO2, however, the LAB population declines 10-fold. The LAB species present at this stage are L. plantarium, L. casei, Leuconstoc mesenteries, P. damnosus, and, to a lesser extent, O. oeni (Lerm et al.).

During the course of AF, the LAB population levels fall precipitously with only 200 cells/ml surviving the process (Wibowo et al.; Lafon-Lafourcade et al.). Wibowo et al., attribute this decline to ethanol sensitivity. The only LAB strain that survives AF is O. Oeni.

AF is followed by a lag phase which is, in turn, followed by rapid proliferation of LAB with levels rising to between 106 - 1010 prior to MLF. Lafon-Lafourcade et al., posit that this growth originates from winery equipment which serve as incubators for the LAB that would perform a natural inoculation of MLF. O. oeni is the main species that develops here but Lactobacillus and Pediococcus spp. may proliferate and conduct the MLF if the wine pH approaches 4.0.

There are a number of factors that affect the development of LAB and, as a result, the activation and effectiveness of MLF. For example, wine pH affects (Wibowo et al.):
  • LAB growth rate
  • The LAB species that proliferate
  • The metabolic behavior of the species that grow
  • The survival of LAB.

A listing of the factors beyond pH that affect LAB growth and development are provided in Tables 1 and 2 below.

TABLE 1. The influence of different winemaking practices on LAB growth
Degree of must clarification
Significant impact on bacterial growth. Yeast produce more medium chain fatty acids in highly clarified must
Skin contact prior to AF
Direct effect on extraction of nitrogenous and other macromolecules stimulate LAB growth and malolactic activity
Choice of yeast strain
Inhibitory and stimulatory effects differ between strains
Aging of wine on yeast lees
Yeast autolysis release nutrients that stimulate LAB growth and malolactic activity
Source: Lerm et al., TABLE 2

TABLE 2. Yeast activity inhibiting LAB via the production of yeast metabolites
Yeast Metabolite
Effect on LAB and/or MLF
Affects growth ability
AF with SOproducing yeast strain results in wine inhibitory to MLF
Medium chain fatty acids
Affect LAB growth and reduce ability to metabolise malic acid. Combination of fatty acids (hexanoic, octanoic and decanoic acid) cause greater inhibition than individual compounds.
Metabolites of protein nature
Peptide produced by S. cerevisiae during AF: inhibit O. oeni by disruption of cell membrane; inhibition dependent on SO2
Source: Lerm et al., TABLE 3

According to Wibowo et al., additions of sulfur dioxide, and storing the wine at higher temperatures, leads to the progressive loss of viability of any LAB surviving MLF. According to Lafon-Lafourcade et al., under standard conditions, the surviving LAB remained viable post-MLF, exhibiting, initially, a tendency for further growth and then showing a slow, progressive decline over a 200-day storage period. At temperatures over 20℃, a rapid decline in viability was recorded and at 26℃, no LAB activity was recorded after 80 days. Lafon-Lafourcade et al. found that the decline in LAB viability was accelerated by increasing temperature, lowering the wine pH, and increasing the alcohol concentration. These actions, it seems, combine to provide a toxic environment for the bacteria. Addition of sulfur dioxide, they agree, does result in a rapid loss in cell viability but growth recommences at a later date.

At the end of barrel aging, the wine microbial population is stabilized but its population prior to bottling is 103 - 104 cells/ml, if unfiltered, with microbes such as Acetobacter, S. cerevisiae, and O. oeni predominant (Renouf et al., Survival of Wine Microorganisms in the Bottle during Storage, AJEV 58(3), 2007). Filtering with a 0.4 micron filter sheet will eliminate all bacteria from the wine.

Now that the two most important players in the MLF drama have been described, I will cover the MLF process itself when I revisit this topic.

©Wine -- Mise en abyme

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