Co-Inoculation as a risk-mitigation strategy in malolactic fermentation

I have previously discussed the malolactic fermentation (MLF) of wine both as a 'natural" occurrence and being induced through inoculation. In that treatment I discussed MLF as occurring after alcoholic fermentation (AF) in a sequential process. The gap between the conclusion of AF and the initiation of MLF is a risky period as no sulfur can be applied to the wine due to its toxic effect on the bacteria that facilitate the latter. During this time, the wine is exposed to the potential of oxidation and attack by spoilage organisms. While inoculation reduces the duration of the gap, it does not completely eliminate the risk.

Under the "right" circumstances, the lactic acid bacteria (LAB) can themselves pose a risk to wine quality. In a normal AF, all of the fermented sugars are consumed by the yeasts, with residual sugars coming in at < 2g/l. Also, the fermented sugars will have been fully consumed prior to the full depletion of the malic acid. In cases of stuck or sluggish fermentations, however, the malic acid may be fully depleted while fermentable sugars are still available. LABs are heterofermentative and while they preferentially consume malic and citric acids, they "possess the metabolic machinery to utilize glucose and fructose for the production of energy." The by-product of such energy production is acetic acid, an undesirable characteristic in wine.

Co-inocualtion, an emerging approach,  has been employed as a strategy to minimize the identified risks. The figure below shows MLF considerations and the options in the inoculation space.

Co-inoculation involves introducing selected LAB into the wine within 12 to 48 hours of its inoculation with AF yeasts. If the LAB is introduced earlier than 12 hours post yeast inoculation, 99% of the viable cells would be killed off by free sulfites. In the early stages of AF, however, the yeasts produce compounds which detoxify the free sulfites, rendering the environment more hospitable for the LAB. 

Advantages of Co-inoculation

Following are some of the benefits associated with co-inoculation:

• Better control over the winemaking process in terms of time management
• Shorter AF-MLF cycle
• Bacteria are introduced into a low-ethanol, high-nutrient environment with the lowest pH the wine will probably experience
• Bacterial activity is suppressed during active fermentation but cells acclimatize their membranes to the rising alcohol during this time
• Microbiological activity of both the yeasts and the bacteria work to limit  development of contaminating organisms 
• As yeasts begin to die at the end of AF, they release nutrients into the wine just as malic bacterial production begins to transition from lag to log phase
• When MLF occurs under the reductive conditions of AF, lower levels of diacetyl and higher levels of fruity esters manifest in the wine. 

©Wine -- Mise en abyme

I will have some butter with my Chardonnay, please

If you are tempted to address the title statement to your host after tasting the un-oaked Chardonnay served at the party, then you are enamored with diacetyl (butanedione, butane-2,3-dione), an organic compound which donates buttery, butterscotch, nutty, and toasty aromas to wine and, at the appropriate levels, a desirable complexity.

Diacetyl is produced at low levels (< 5 mg/L) during alcoholic fermentation (especially at high fermentation temperatures) but is absorbed by the yeast and reduced to acetoin and 2,3-butanediol (Jackson). Its production, however, is most strongly associated with malolactic fermentation (MLF) and the metabolism of citric acid by lactic acid bacteria (LAB), a process wherein its maximum concentration occurs simultaneously with the exhaustion of L-malate. This chemically unstable compound is reduced to acetoin and 2,3-butanediol (sensorially insignificant compounds) by the actions of the LAB Oenoccus oeni and yeasts. The accumulation of diacetyl is a function of the MLF rate, with lower levels produced at higher rates and vice versa (Lern, et al.). 

Bauer and Dicks describe diacetyl as one of the most important flavors produced during MLF. The threshold levels for its detection varies by variety and wine type. The threshold for detection in Chardonnay is 0.2 mg/L, is 0.9 mg/L in Pinot Noir, and is 2.8 mg/L in Cabernet Sauvignon. In general, diacetyl levels in young red wines range between 0.2 and 1.84 mg/L while aged reds have levels that range between 1.25 and 3.39 mg/L (Lern, et al.). At concentrations between 1 and 4 mg/L, the compound contributes the aforementioned aromas and complexity to the wine but at levels above 4 mg/L the aromas are overpowering and undesirable (Lern, et al.). Jackson refers to the resulting aroma as lactic off odor and notes that it is generally associated with spoilage induced by certain strains of LAB.

Knowing the qualities imparted to the wine by diacetyl, the enterprising winemaker can take action to influence the levels in his/her final product. The table below identifies the factors which can impact on the final diacetyl levels in the finished wine.

Influencing Factors	Effect on diacetyl concentration/sensory perception
LAB strain	Varying production levels
Wine type	Higher levels of diacetyl in red wine production than in white
MLF inoculation rate	Lower inoculation rate favors dactyl production
Yeast and lees contact	Reduces diacetyl content
Aeration during MLF	Increases dactyl production
Sulfur dioxide content	Sulfur binds with diacetyl, rendering it sensorially inactive but this is reversible.  Sulfur dioxide addition inhibits yeast/LAB activity, stabilizing diacetyl content at time of addition
Citric acid concentration	Favors diacetyl production but acetic acid is also produced
MLF temperature	Diacetyl production enhanced between 18 and 25℃
Wine pH at MLF	Lower pH favors diacetyl production
Wine stabilization	Immediate stabilization post malic and citric acid metabolism will increase dactyl content

Source: Lern, et al.

References:
Bauer and Dicks, Control of Malolactic Fermentation in Wine: A Review, S. Afr. J. Enol. Vitic., 25(2), 2004.
Jackson, Wine Science.
Lern, Engelbrecht, and du Toit, Malolactic Fermentation: The ABCs of MLF, S. Afr. J. Enol. Vitic., 31(2), 2010.

©Wine -- Mise en abyme

Monitoring the malolactic fermentation of wine

I have recently described the malolactic fermentation process. In general, a winemaker needs to be able to tell if/when MLF has commenced, how it is proceeding, if any problems are in the offing, and when it has concluded. Key to addressing these concerns are the establishment of benchmarks for the beginning and end of MLF and a regime for tracking its progress. 

According to Boulton et al. (Principles and Practices of Winemaking, Chapman and Hall, 1996), there is a difference in perception as to what constitutes the MLF duration depending on whether you are a microbiologist or a winemaker. The microbiologist sees MLF as commencing with the “introduction of viable bacteria into the wine or must” and ending “when the bacteria have gone through the growth phase and have re-entered their final resting or stationary phase.” The winemaker, on the other hand, will equate the start of fermentation with a noticeable drop in malic acid levels (grape juice contains between 1 and 8 g/l malic acid) and as completed when the malic acid has finally disappeared (vintessential.com.au (Malolactic Fermentation Monitoring -- Resources for Winemakers) recommends a figure of < 0.05 g/l as safe for declaring the end of MLF while Katherine Mansfield (Monitoring Malolactic Fermentation 3 Ways, Cornell University College of Agriculture and Life Sciences, Cornell Cooperative Extension) cites a number of 30 mg/l).

Even though acknowledging the microbiologists' view of MLF duration, Boulton et al. accept the winemakers perspective in that they see the “measurement of the disappearance of malic acid as the accepted means for determining whether the malolactic fermentation has occurred.” Lerm et al., place this measurement of the presence of malic acid within a broader context: “The continuous monitoring of MLF is essential and often neglected by winemakers.” Continuous monitoring “allows the winemaker to follow the progress of malic acid degradation as well as the bacteria responsible for the fermentation. This is also a way for the winemaker to identify any difficulties before they can affect the quality of the wine.”

There are a number of tools that are available for the monitoring of MLF and they are summarized in the table below. 

Selected MLF Monitoring Techniques and their Attributes

Monitoring Technique	Advantages	Disadvantages
Paper Chromatography (PC) separate compounds based on their polarity visually follow disappearance of malic acid commonly used in winery	easy to use simple, affordable and indicative of MLF progress	strictly qualitative so still need quantitative values to verify MLF completion not precise not specific for L-malic acid
Thin Layer Chromatography (TLC) - similar to PC but uses TLC plates instead of paper	easy to use simple and affordable results in one hour,; much faster than PC	not precise not specific for L-malic acid strictly qualitative so still need quantitative values to verify MLF completion
Reflectance Reflectoquant® based on reflectance photometry use reactive test strips to analyze for various wine components	a fraction of the cost of a spectrophotometer half the cost of an enzymatic kit measure multiple wine parameters fastest method currently available (5 min/sample) relative accuracy of 10%	measure relative malic acid levels so still need to qualify absolute levels operating range 1 to 60 mg/L, so some samples need to be diluted or decolorised need to be calibrated with reference method
Enzymatic Analysis uses enzyme that specifically react with L-malic acid then use UV-visible spectrometer to monitor enzymatic reaction most commonly used method MLF complete if malic acid is less than 200 to 300 mg/l	quantitative excellent precision kits readily available quantify very low levels of malic acid results in 30 minutes	more complex more expensive short shelf life of reagents after activation require use of accurate micro-pipettes turbid samples need to be centrifuged
Capillary Electrophoresis (CE)	highly accurate short analysis time; fast results	extremely expensive not recommended for everyday use in winery
Fourier-transform Infrared (FT-IR) Spectroscopy - use infrared spectra to quantify wine parameters	accurate small sample volume short analysis time, fast results	expensive equipment accuracy dependent on reference values and calibration curve
High Performance Liquid Chromatography (HPLC) - separation of compounds based on polarity and interaction with stationary or solid phase	- highly accurate	extremely expensive not recommended for everyday use in winery

Source: Lerm et al., Table 7.

Of the mechanisms listed above, the one that is most commonly used in wineries today is paper chromatography. Paper chromatography detects the presence of malic acid but does not tell its concentration. As such, it should never be used to make a decision regarding the end of MLF. According to Mansfield, the lower level of malic acid detection for paper chromatography is 100 mg/L, way above the 30 mg/L considered by her lab to be the “safe number” for the end of MLF. The risk associated with prematurely calling the end of MLF is residual malic acid which can be metabolized by latent LAB in the bottle with a host of resultant problems (change in color, aroma, flavor; CO₂ production; clouding (Lisa Van de Water, Monitoring microbes during cellaring/bottling, practicalwinery.com, January/February 2010)). In a table accompanying the article, the author shows that Oenococcus would be found at levels of 500+/ml if residual malic acid were present. Ms. Van de Water suggests checking wines during MLF for the presence of spoilage bacteria and doing so by microscope as well as by PCR.

A robust MLF monitoring protocol which incorporates some of the tools listed in the table above is as follows:

1. Take representative samples of cellar barrels for testing. Care should be taken to ensure that ease of access does not determine the barrels utilized for sampling.
2. Test consistently.  I propose that the sampling be conducted every two weeks or at topping.
3. Position paper chromatography as a tool in the MLF monitoring toolbox for measuring progress. Paper chromatography detects the presence of malic acid but does not tell the concentration. The level of malic acid in the wine needs to be below 30 mg/L in order for the microbial stability benefits of the MLF process to accrue. 
4. Utilize enzymatic analysis in the MLF monitoring protocol as a means of certifying the end of MLF. As the cost of the equipment for enzymatic tests are very high, I recommend the services of an outside lab for this purpose. As recommended by vintessential.com.au, the costs of these outside tests can be minimized by submitting barrel composites. If no malic acid is detected, then all of the barrels will be assumed to have completed MLF. If the test comes back positive, then the barrels would be tested individually in order to identify the offending barrel(s).
5. Include the human factors (nose, palate, and skills; Dr Wann, Power Point presentation) in the MLF monitoring process. Dr. Wann recommends: (i) checking for CO₂ evolution; (ii) smelling and tasting the barrel at every topping (heads up on potential spoilage activity); (iii) maintaining an appropriate temperature (between 18 and 22℃); and (iv) to be aware of increasing pH levels (potential foothold for spoilage organisms). In that CO₂ is one of the outputs of the MLF process, its presence is indicative of ongoing malate degradation.
6. Institute a number of post-MLF processes to ensure the microbial stability of the wines through to the blending and bottling processes. According to Wibowo et al., additions of SO₂ and storing the wine at higher temperatures leads to the progressive loss of viability of any bacteria surviving the MLF. The wines themselves may be sterilized by filtering with membranes having pore sizes of 0.22 to 0.45µm. Lafon-Lafourcade et al., sees the decline in LAB accelerated by increasing temperatures, lowering the wine pH, and increasing the alcohol concentration, actions which, it seems, combine to provide a toxic environment for the bacteria. The addition of SO₂ does result in a rapid loss in cell viability, they agree, but growth recommences at a later date. The specific post-MLF operations that should be undertaken, according to Boulton et al. are as follows:

• Transfer the wines off the lees and a rough filtration
• Adjustments of temperature and pH and addition of SO₂. The recommendation here is for 0.8 mg/L
• Fining operations could also be performed at this time

Following this set of procedures will minimize the potential for microbes indicating their presence in the bottle.


©Wine -- Mise en abyme

The malolactic fermentation of wine

In recent posts I have discussed malic acid and lactic acid bacteria (LAB), two of the major actors in the malolactic fermentation (MLF) drama. In this post I will elaborate on the arena within which this drama unfolds and how the interaction of malic acid and other minor players with LAB results in lactic acid and other metabolites. Let us begin by defining MLF.

According to Sauvageot and Vivier (Effects of Malolactic Fermentation on Sensory Properties of Four Burgundy Wines, AJEV 48(2), 1997), MLF is a bacterial conversion -- most commonly performed by Leuconostoc strains due to their tolerance of the high acid and alcohol content associated with wine -- of L-malic acid to L-lactic acid and CO₂. The MLF process can be represented thusly (Lerm et al., Malolactic Fermentation: The ABCs of MLF, S. Afr. J. Enol. Vitic. 31(2), 2010):

L-malic acid  + LAB  →    L-lactic acid          + CO₂
(dicarboxylic)                  (monocarboxylic)

wherein a carboxyl group (C(O)OH) is removed from the dicarboxylic L-malic acid. The reaction is catalyzed by the LAB along one of three pathways (Lerm et al., Bauer and Dicks, Control of Malolactic Fermentation in Wine: A Review, S. Afr. J. Enol. Vitic. 25(2), 2004):

1. Direct conversion of malic acid to lactic acid via malate decarboxylase (the preferred pathway for wine LAB)
2. L.casei and Enterococcus faecales possess a malic enzyme that converts L-malic to pyruvic acid which is in turn reduced to lactic acid by L-lactate dehydrogenase
3. Via L. fermentum,  malate is reduced by malate dehydrogenase to oxaloacetate, followed by decarboxylation to pyruvate which is further reduced to lactic acid.

The main effects of MLF on wine are (i) a reduction in titratable acidity (by 0.1 to 0.3%) and an increase in pH (0.15 to 0.30). In addition, dramatic organoleptic changes to the wine are evidenced (Lonvaud-Funel, Microbiology of the Malolactic Fermentation: Molecular Aspects, FEMS Microbiology Letters):

• The specific taste of malic acid disappears
• Sugars are catabolized to produce mainly lactic and acetic acid
• Citric acid is transformed into acetic acid and carbonyl compounds, notably the butter-flavored diacetyl
• Wine taste and color are modified due to the metabolic activity of bacteria on phenolic compounds (tannins, anthocyannins).

By synthesizing anti-bacterial compounds and depriving the wine of nutrients, MLF also contributes to its microbial stability (Lonvaud-Funel).

In practice, most red wines, and selected white wines, undergo MLF. The process is encouraged (Bauer and Dicks, Control of Malolactic Fermentation in Wine, S. Afr. J. Enol. Vitic. 25(2), 2004): in cooler areas where grapes have high malic acid content; in cases where the wine is aged in oak barrels; and when the wine style calls for long-term aging in bottle. The practice is sometimes forsworn in warmer, lower-acid areas and in the cases where undesirable organoleptic changes or the production of biogenic amines result.

MLF is initiated either naturally or through inoculation of the wine with an LAB strain. In the case of indigenous initiation, upon the completion of alcoholic fermentation, and following a lag phase, the surviving LAB begin to multiply rapidly. MLF begins when their numbers approach 10⁶ cells/ml (Savageot and Vivier; Wibowo et al.; Lonvaud-Funel; Lerm et al.). Lafon-Lafourcade et al., posit that this growth originates from winery equipment which serve as incubators for the LAB. Oenococcus. 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. If MLF is not desired, clarification of must or newly fermented wine will remove the majority of the LAB and, if excessive, its potential nutrient sources, and reduce the possibility of indigenous inoculation (Wibowo et al.). In addition, wines that have undergone thermovinification (rapid heating of the must to near boiling point in order to extract anthocyanins and tannins) are less susceptible to MLF (Wibowo et al.).

During the time between the end of AF and the initiation of MLF, no SO₂ can be applied to the wine because of its toxic effect on the LAB. During this time, then, the wine is exposed to the potential of oxidation and attack by spoilage organisms. The use of starter cultures reduces this risk by shortening the time between the end of AF and the initiation of MLF and by ensuring a rapid onset of MLF with a very high population of viable bacteria (on the order of 10¹¹ cells/g, according to Lerm et al.). Given the environment within which the LAB has to operate, a starter culture should have the following characteristics (Lerm et al.):

• Tolerance to low pH, high ethanol, and SO₂
• Good growth characteristics under winemaking conditions
• Compatibility with the S. cerevisiae strain(s) being used for alcoholic fermentation
• Ability to survive the production environment
• Does not produce biogenic amines
• Does not produce off-flavors or off-odors
• Production of aroma compounds that will favorably impact the wine’s aroma profile.

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.

Temperature is synergistic with ethanol levels as it relates to inhibiting LAB (Lerm et al.):

• The optimal growth temperature of LAB decreases at high ethanol concentrations
• Elevated temperatures lower the ability of LAB to withstand increased ethanol concentration
• Temperatures of 25℃ and above, combined with ethanol levels of 10 -14%, almost completely inhibits LAB growth.

A listing of the factors beyond pH and temperature that affect LAB growth and development are provided in the tables below.

TABLE 1. The influence of different winemaking practices on LAB growth

Practice	Influence
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
Ethanol	Affects growth ability
SO2	AF with SO2 producing 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 Boulton et al. (Principles and Practices of Winemaking, Chapman and Hall, 1996), there is a difference in perception as to what constitutes the MLF period, depending on whether you are a microbiologist or a winemaker. The microbiologist measures the MLF from the “introduction of viable bacteria into the wine or must” and it ends “when the bacteria have gone through the growth phase and have re-entered their final resting or stationary phase.” The winemaker, on the other hand, will equate the start of fermentation with a noticeable drop in malic acid levels (grape juice contains between 1 and 8 g/l malic acid) and as completed when the malic acid has finally disappeared (vintessential.com.au (Malolactic Fermentation Monitoring -- Resources for Winemakers) recommends a figure of < 0.05 g/l as safe for declaring the end of MLF while Katherine Mansfield (Monitoring Malolactic Fermentation 3 Ways, Cornell Cooperative Extension) cites the number 30 g/ml. That discrepancy in the metric notwithstanding, Boulton et al., see the “measurement of the disappearance of malic acid as the accepted means for determining whether the malolactic fermentation has occurred.”

Measurement of the degradation of malic acid is one of the key aspects of monitoring MLF, the topic of my next post

©Wine -- Mise en abyme

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
Source: pnas.org

Doctor Murli Dahrmadikari (Lactic Acid Bacteria and Wine Spoilage, extension.iastate.edu) 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

Practice	Influence
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
Ethanol	Affects growth ability
SO2	AF with SO2 producing 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

Malic acid: Preparation for malolactic fermentation

Malolactic fermentation (MLF) has been practiced by winemakers for many a year but the benefits have not always been widely acclaimed. But, as Jackson (Wine Science: Principles and Applications, 3rd ed., Elsevier, 2008) notes, the controversy seems to be over and it is now generally accepted that the process can impact wines as follows (Lerm et al., Malolactic Fermentation: The ABCs of MLF, S. Afr. J. Enol. Vitic., 31 (2), 2010):

• Deacidification, with a resultant increase in pH
• Contribute to the microbial stability through removal of malic acid as a possible carbon substrate; and
• Modification of the wine's aroma profile.

In its simplest terms, MLF is the conversion of the "hard" malic acids into the softer lactic acid by lactic acid bacteria (LAB). But what is the origin of the malic acid so unceremoniously neutered? And what was the path that it trod to get here? I explore these questions in this post.

As shown in the figure below, malate makes its first appearance in the grape berry in Stage I of its development. Malic acid is produced in the berry by one of two processes: (i) Fixation of carbon dioxide by PEP carboxylase (an enzyme that catalyzes the addition of bicarbonate to PEP to form oxaloacetate and inorganic phosphate) and (ii) synthesis from sugars via glycolysis and the Tricarboxylic acid cycle (TCA). The oxaloacetic acid produced in the PEPC process is reduced to malate by the enzyme malate dehydrogenase (MDH)

Grape berry development (Source:www.extension.org)

It is estimated that tartaric and malic acids constitute between 70% and 90% of total berry acid content, with malic acid's contribution on the order of 23% - 40%. Malic acid concentration increases constantly in the first stage of development, increases sharply in Stage II, and then declines rapidly in Stage III. Prior to véraison, malic  acid levels are the highest of any of the organic acids in the berry, reaching levels of up to 25 g/L, with a resultant pH of 2.5. Pre- and post-véraison occurrences, however, contribute to a significant reduction in malic acid levels (Straus et al., Malic Acid in Wine, S. Afr. J. Enol. Vitic., 27 (2), 2006):

• Malic acid in the berry vacuoles are diluted by water influx in Stage II
• There is a significant decrease in L-malic acid biosynthesis post-véraison
• The slowing of glycolytic carbon flow during véraison results in increased glucose and fructose in the berry vacuole and a decrease in malic acid synthesis via pyruvic acid in the TCA cycle
• The biosynthesis of malic acid via the PEPC cycle is reduced due to véraison-induced lack of PEPC transcription 
• During véraison, chlorophyll is degraded so the berry shifts its metabolism from sucrose respiration to malic acid respiration.

As a result of the pre- and post-véraison retrenchments, malic acid concentration will decline to between 4 and 6.5 g/l; in some cases, getting down to 1 g/l.

A point of note is the role that malic acid plays in the development of flavor and color compounds in the berry. While the berry turns its attention to its reproductive phase -- and directs the attention of its sugars to plumping and sweetening the berry --  it fails to utilize those energy sources for the development of the compounds that are so important for wine quality. But, as I have mentioned previously, the berry is not interested in producing great wines; it is interested in genetic survival. Malic acid, then, is respired to meet the berry's energy needs for development of the flavor and color compounds.

Of the three reasons listed for malolactic fermentation, two would seem to be intrinsically related to malic acid: de-acidification and substrate for microbial instability. And de-acidification would only seem to be appropriate in the cooler-climate regions. In the warmer regions, where low acidity and high pH are already an issue, the downsides associated with the pursuit of malolactic fermentation has to be significantly outweighed by the aroma-enrichment benefits.

In his study of solutes in grape berries, Coombe (Distribution of Solutes within the Developing Grape Berry in Relation to its Morphology, AJEV, 38 (2), 1987) showed the evolution of malate levels during grape development by measuring its presence at four stages identified thusly:

1. Close to véraison, berries hard and free; 6.2ºBrix
2. Berries ripening; 10.2ºBrix
3. Early ripe stage; 17.4ºBrix
4. Overripe; 26.4ºBrix

Some of his key findings, re malate, were as follows:

• Malate was the most abundant solute in the flesh of unripe berries but declined during development by proportions comparable with the degree of increase that occurred in glucose and fructose concentrations
• The decline in malate was most notable between 10ºBrix and 17ºBrix
• The levels of malate in the skin and brush showed smaller changes as the berries developed (they were smaller to begin with and declined less over the stages of berry development)
• The malate decline in the skin occurred earlier in the regions closest to the pedicel
• Within the flesh, the lowest malate concentrations were found where vascular bundles occur.

Jackson noted that the latter point may result from "malate metabolism initiating around the axial vascular bundles and progressing outwards." Regardless, at maturity, the malic acid levels in the skin may be higher than the levels in the flesh.

The overall regional climate will determine the final berry malate levels. In colder climates, the rate of berry respiration is low and this results in immature grapes at harvest with high total acidity (TA) and low pH (Jackson, Straus et al.). In this situation, malic acid can be as much as 50% of the TA in the grape berry, resulting in a sour tasting wine. In warmer climates, conversely, the rate of malic acid respiration is higher with a resultant low (insufficient) TA and high pH at harvest. Wines made from these grapes can have a flat taste and may be susceptible to microbial spoilage (Jackson). Further, these wines may not age well. Malic acid content is thus a key factor in the determination of optimal harvest date.

According to Straus et al., mature grapes have between 2 and 6.5 g/l at maturity with levels above that only present in grapes harvested after cold summers in the cool-climate viticultural regions of the world. In those cases, the levels could rise to as much as 15 - 16 g/l. And the levels at harvest are, for the most part, the levels in the wine when the decision regarding malolactic fermentation is made. The type of crushing equipment used may have some impact on malic acid levels but that is minimal (Jackson).  Also, according to Jackson, during alcoholic fermentation, the yeasts may increase the wine pH by converting some of the malic acid to lactic acid but this is highly variable by yeast strain and has not been established to any reliable degree.

So, after its many contributions along the way; its sacrifices to keep the berry alive during its most vulnerable time, this is malic acid's reward. A neutering; a sex change operation by some egotistic, self-serving winemaker who wants to make his/her wine softer. Where are your cojones man? Well that is not a question that I can answer. When I next visit this topic I will look at the other player in this drama: the lactic acid bacteria.


©Wine -- Mise en abyme

The role of acidity in wine balance

Now that I have provided a contextual framework for wine balance, I can return to the promised discussion of acidity, one of its constituent elements.  As a refresher on acid's relationship to balance, Dr. Bruce Zoecklein's (Virginia Tech oenologist) Palate Balance Equation is reproduced below. The arrow on the figure following the equation illustrates where balance and acidity fall within the wine quality assessment framework.

              Sweet ⇄ Acid + Phenolics (Astringency and Bitterness).

Acids play an important role in the cellular and metabolic functions of the grape berry and in the color and texture of the fermented wine.  The precursors of acid are formed in the leaves of the grape plant and are transported to the berries where they are synthesized to acids.  Acid accumulation begins at the start of berry development and continues unabated until the beginning of the ripening process.  Acid levels tend to vary acording to the controlling temperatures of the growing region; in warmer regions acid is used up during respiration, resulting in lower acidity levels in the fruit at harvest.  Conversely, acid levels are higher and sugar levels lower in cooler-climate growing regions.

The primary acids found in grapes and fermented wine are tartaric, malic, and citric acids as well as the tartaric and malic derivatives potassium hydrogen tartrate (cream of tartar) and potassium hydrogen malate.  Tartaric acid -- which occurs in nature in fruits such as grapes, bananas, and tamarinds -- represents between 50% and 66% of the acid content in a ripe berry and, as such, controls the acid content in the finished wine.  The tartaric acid level falls off as the grape ripens but not as much as in the case of malic acid.  Crystallized tartaric acid precipitates out of the wine during fermentation and can form crystals on the underside of the wine bottle cork if the wine is stored below 50ºF.  Tartaric acid is resistant to attack by wine microbes (and thus lends ageability with lower spoilage risk to the finished product) and is the winemaker's material of choice if/when a decision is made to add acid to a wine.

Malic acid is the second most important contributor to grape acid levels with amounts ranging between 23% and 40% of the total acid content.  The grape utilizes malic acid during respiration at a rate higher than for tartaric acid, leading to a higher ratio of tartaric-to-malic acid at harvest than at the earlier stages of fruiting.  Unlike tartaric acid, malic acid can be metabolized by a number of organisms and winemakers take advantage of this fact to to reduce wine acidity through malolactic fermentation, a process wherein the bacteria convert the hard malic acid to the softer lactic acid and carbon dioxide.  Malolactic fermentation increases the wines aging potential as the bacteria that metabolize the malic acid also scavenge remaining nutrients and, in so doing, reduce the potential for future microbial spoilage.  Malolactic fermentation occurs post-alcoholic-fermentation and is automatic for most red wines and selected whites.

Acetic acid is produced during fermentation by the conversion of ethanol to acetic acid by a species of  Acetobacter or from the actions on glucose by selected anaerobic bacteria.  The acid is present in most wines at levels of approximately 0.5 g/L and is detectable by humans as a pungent odor at levels of 1.0 g/L and above.  The legal limit for acetic acid in wine is 1.2 g/L in California and 1.4 g/L elsewhere in the U.S.  Acetic acid boils off when heated and as such is referred to as volatile acidity.

The winemaker needs to know the acid content of the grape and must in order to: decide when to harvest; determine pre-fermentation must treatment; monitor wine stability; and comply with TTB requirements of 0.5% minimum acid levels.  Total acidity is the sum of the hydrogen ions of both fixed and volatile acids that are present in the wine and, as such, is the most accurate representation of acid concentration.  Total acidity is difficult to measure accurately, however, and so the more easily measurable titratable acidity (TA) is used as its proxy.  Acids and bases neutralize each other to water so the acidity of a liquid can be approximated by determining the amount of an alkaline solution that is required to neutralize it to water.  The acidity level revealed in this manner is called the substance's titratable acidity.  Red table wines generally range between 0.6% and 0.7% TA as levels below 0.4% render the wine susceptible to infection and spoilage.

A second method for measuring the acidity of a wine is through observation of its pH (potential of hydrogen) level.  The higher the number of hydrogen ions (H+) in a liquid, the more acidic it is while the higher the number of hydroxide ions (formed when an oxygen ion bonds to a hydrogen ion and represented as OH-) in the liquid, the higher its alkalinity. The pH scale (illustrated below) runs from 0-14 with acidic solutions falling below 7,

Source: epa.gov

7 as a point of neutrality, and alkaline solutions falling between 7 and 14.  A change of 1 unit on the scale represents a 10-fold change in pH.

The pH level of a wine affects the way it is perceived by the wine drinker as well as its reaction to micro-organisms.  Low-pH wines are generally viewed as sour and render tannins more astringent but they also  limit micro-organism growth.  Higher pH provides a more favorable environment for micro-organism growth and reduces the functionality of sulfur application.  White wine pH ranges between 3.0 and 3.3 while red wine pH falls between 3.3 and 3.5.  Low pH values are often correlated with high TAs and vice versa.

To summarize, acid gives wine a tartness and freshness while countering the effect of sweetness and magnifying the astringency of tannins.  If a wine has too much acid it will be puckery and sour; too little and it will be flat, flabby, and dull.  As stated previously, wine balance is viewed as a key indicator of wine quality.  If a wine has insufficient sugar in relation to its acids and phenols, it will present as harsh and acidic and will retard the evolution of flavors in the mouth.  In such a case the winemaker may choose to de-acidify using potassium bicarbonate or calcium carbonate or dilute the wine with water or a low-acid wine.  If the wine has too much sugar, it will be flabby and cloying and will not refresh the palate.  In such a case the winemaker may choose to acidify the wine by adding tartaric, malic, or citric acid.

Whether naturally obtained or engineered, appropriate acidity is a key element of wine balance.

© Wine -- The View From Orlando