Malolactic fermentation (also known as malolactic
conversion or MLF) is a process in winemaking
in which tart-tasting malic acid, naturally
present in grape must, is converted to softer-tasting
lactic acid.
Malolactic fermentation is most often performed
as a secondary fermentation shortly after
the end of the primary fermentation, but can
sometimes run concurrently with it.
The process is standard for most red wine
production and common for some white grape
varieties such as Chardonnay, where it can
impart a "buttery" flavor from diacetyl, a
byproduct of the reaction.The fermentation
reaction is undertaken by the family of lactic
acid bacteria (LAB); Oenococcus oeni, and
various species of Lactobacillus and Pediococcus.
Chemically, malolactic fermentation is a decarboxylation,
which means carbon dioxide is liberated in
the process.The primary function of all these
bacteria is to convert one of the two major
grape acids found in wine called L-malic acid,
to another type of acid, L+ lactic acid.
This can occur naturally.
However, in commercial winemaking, malolactic
conversion typically is initiated by an inoculation
of desirable bacteria, usually O. oeni.
This prevents undesirable bacterial strains
from producing "off" flavors.
Conversely, commercial winemakers actively
prevent malolactic conversion when it is not
desired, such as with fruity and floral white
grape varieties such as Riesling and Gewürztraminer,
to maintain a more tart or acidic profile
in the finished wine.Malolactic fermentation
tends to create a rounder, fuller mouthfeel.
Malic acid is typically associated with the
taste of green apples, while lactic acid is
richer and more buttery tasting.
Grapes produced in cool regions tend to be
high in acidity, much of which comes from
the contribution of malic acid.
Malolactic fermentation generally enhances
the body and flavor persistence of wine, producing
wines of greater palate softness.
Many winemakers also feel that better integration
of fruit and oak character can be achieved
if malolactic fermentation occurs during the
time the wine is in barrel.A wine undergoing
malolactic conversion will be cloudy because
of the presence of bacteria, and may have
the smell of buttered popcorn, the result
of the production of diacetyl.
The onset of malolactic fermentation in the
bottle is usually considered a wine fault,
as the wine will appear to the consumer to
still be fermenting (as a result of CO2 being
produced).
However, for early Vinho Verde production,
this slight effervesce was considered a distinguishing
trait, though Portuguese wine producers had
to market the wine in opaque bottles because
of the increase in turbidity and sediment
that the "in-bottle MLF" produced.
Today, most Vinho Verde producers no longer
follow this practice and instead complete
malolactic fermentation prior to bottling
with the slight sparkle being added by artificial
carbonation.
== History ==
Malolactic fermentation is possibly as old
as the history of wine, but scientific understanding
of the positive benefits of MLF and control
of the process is a relatively recent development.
For many centuries, winemakers noticed an
"activity" that would happen in their wines
stored in barrel during the warm spring months
following harvest.
Like primary alcoholic fermentation, this
phenomenon would release carbon dioxide gas
and seem to have a profound change on the
wine that was not always welcomed.
It was described as a "second fermentation"
in 1837 by the German enologist Freiherr von
Babo and the cause for increased turbidity
in the wine.
Von Babo encouraged winemakers to quickly
respond at the first sight of this activity
by racking the wine into a new barrel, adding
sulfur dioxide, and then following up with
another set of racking and sulfuring to stabilize
the wine.In 1866, Louis Pasteur, one of the
pioneers of modern microbiology, isolated
the first bacteria from wine and determined
that all bacteria in wine were a cause for
wine spoilage.
While Pasteur did notice an acid reduction
in wine with the lactic bacteria, he did not
link that process to a consumption of malic
acid by the bacteria, but rather assumed it
was just tartrate precipitation.
In 1891, the Swiss enologist Hermann Müller
theorized that bacteria may be the cause of
this reduction.
With the aid of peers, Müller explained his
theory of "biological deacidication" in 1913
to be caused by wine bacterium Bacterium gracile.In
the 1930s, the French enologist Jean Ribéreau-Gayon
published papers stating the benefits of this
bacterial transformation in wine.
During the 1950s, advances in enzymatic analysis
allowed enologists to better understand the
chemical processes behind malolactic fermentation.
Émile Peynaud furthered enology understanding
of the process and soon cultured stock of
beneficial lactic acid bacteria was available
for winemakers to use.
== Role in winemaking ==
The primary role of malolactic fermentation
is to deacidify wine.
It can also affect the sensory aspects of
a wine, making the mouthfeel seem smoother
and adding potential complexity in the flavor
and aroma of the wine.
For these other reasons, most red wines throughout
the world (as well as many sparkling wines
and nearly 20% of the world's white wines)
today go through malolactic fermentation.Malolactic
fermentation deacidifies the wine by converting
the "harsher" diprotic malic acid to the softer
monoprotic lactic acid.
The different structures of malic and lactic
acids leads to a reduction of titratable acidity
(TA) in the wine by 1 to 3 g/l and an increase
in pH by 0.3 units.
Malic acid is present in the grape throughout
the growing season, reaching its peak at veraison
and gradually decreasing throughout the ripening
process.
Grapes harvested from cooler climates usually
have the highest malic content and have the
most dramatic changes in TA and pH levels
after malolactic fermentation.
Malolactic fermentation can aid in making
a wine "microbiologically stable" in that
the lactic acid bacteria consume many of the
leftover nutrients that other spoilage microbes
could use to develop wine faults.
However, it can also make the wine slightly
"unstable" due to the rise in pH, especially
if the wine already was at the high end of
wine pH.
It is not unusual for wines to be "deacidified"
by malolactic fermentation only to have the
winemaker later add acidity (usually in the
form of tartaric acid) to lower the pH to
more stable levels.
=== Conversion of malic into lactic ===
Lactic acid bacteria convert malic acid into
lactic acid as an indirect means of creating
energy for the bacteria by chemiosmosis which
uses the difference in pH gradient between
inside the cell and outside in the wine to
produce ATP.
One model on how this is accomplished notes
that the form of L-malate most present at
the low pH of wine is its negatively charged
monoanionic form.
When the bacteria move this anion from the
wine into higher pH level of its cellular
plasma membrane, it causes a net-negative
charge that creates electrical potential.
The decarboxylation of malate into L-lactic
acid releases not only carbon dioxide, but
also a proton that generates the pH gradient
which can produce ATP.Lactic acid bacteria
convert L-malic acid found naturally in wine
grapes.
Most commercial malic acid additives are a
mixture of the enantiomers D+ and L-malic
acid.
=== Sensory influences ===
Many different studies have been conducted
on the sensory changes that occur in wines
that have gone through malolactic fermentation.
The most common descriptor is that acidity
in the wine feels "softer" due to the change
of the "harsher" malic acid to the softer
lactic acid.
The perception of sourness comes from the
titratable acidity in the wine, so the reduction
in TA that follows MLF leads to a reduction
in perceived sour or "tartness" in the wine.The
change in mouthfeel is related to the increase
in pH, but may also be due to the production
of polyols, particularly the sugar alcohols
erythritol and glycerol.
Another factor that may enhance the mouthfeel
of wines that have gone through malolactic
fermentation is the presence of ethyl lactate
which can be as high as 110 mg/l after MLF.The
potential influence on the aroma of the wine
is more complex and difficult to predict with
different strains of Oenococcus oeni (the
bacterium most commonly used in MLF) having
the potential to create different aroma compounds.
In Chardonnay, wines that have gone through
MLF are often described as having "hazelnut"
and "dried fruit" notes, as well as the aroma
of freshly baked bread.
In red wines, some strains metabolize the
amino acid methionine into a derivative of
propionic acid that tends to produce roasted
aroma and chocolate notes.
Red wines that go through malolactic fermentation
in the barrel can have enhanced spice or smoke
aromas.However, some studies have also shown
that malolactic fermentation may diminish
primary fruit aromas such as Pinot noir, often
losing raspberry and strawberry notes after
MLF.
Additionally, red wines may endure a loss
of color after MLF due to pH changes that
causes a shift in the equilibrium of the anthocyanins
which contribute to the stability of color
in wine.
== Lactic acid bacteria ==
All lactic acid bacteria (LAB) involved in
winemaking, whether as a positive contributor
or as a source for potential faults, have
the ability to produce lactic acid through
the metabolism of a sugar source, as well
as the metabolism of L-malic acid.
Species differ in how they metabolise the
available sugars in wine (both glucose and
fructose, as well as the unfermentable pentoses
that wine yeasts do not consume).
Some bacteria species use the sugars through
a homofermentative pathway, meaning only one
main end product (usually lactate) is produced,
while others use heterofermentative pathways
that can create multiple end products such
as carbon dioxide, ethanol, and acetate.
While only the L-isomer of lactate is produced
by LAB in the conversion of malic acid, both
hetero- and homofermenters can produce D-,
L- and DL-isomers of lactic from glucose which
may contribute to slightly different sensory
properties in the wine.While O. oeni is often
the LAB most desired by winemakers to complete
malolactic fermentation, the process is most
often carried out by a variety of LAB species
that dominate the must at different points
during fermentations.
Several factors influence which species will
be dominant, including fermentation temperature,
nutritional resources, the presence of sulfur
dioxide, interaction with yeast and other
bacteria, pH, and alcohol levels (Lactobacillus
species, for example, tend to prefer higher
pH and can tolerate higher alcohol levels
than O. oeni), as well as initial inoculation
(such as "wild" ferments versus an inoculation
of cultured O. oeni).
=== Oenococcus ===
The genus Oenococcus has one main member involved
in winemaking, O. oeni, once known as Leuconostoc
oeni.
Despite having the name Oenococcus, under
the microscope, the bacterium has a bacillus
(shape) rod shape.
The bacteria is a Gram-positive, facultative
anaerobe that can utilize some oxygen for
aerobic respiration but usually produces cellular
energy through fermentation.
O. oeni is a heterofermenter that create multiple
end products from the use of glucose with
D-lactic acid and carbon dioxide being produced
in roughly equal amounts to either ethanol
or acetate.
In reductive conditions (such as near the
end of alcoholic fermentation), the third
end product is usually ethanol while in slightly
oxidative (such as early in alcohol fermentation
or in an untopped barrel), the bacteria are
more likely to produce acetate.Some O. oeni
strains can use fructose to create mannitol
(which can lead to wine fault known as mannitol
taint), while many other strains can break
down the amino acid arginine (which can be
present in the wine that is resting on the
lees after fermentation from the autolysis
of dead yeast cells) into ammonia.In addition
to the hexose glucose and fructose sugars,
most strains of O. oeni can use the residual
pentose sugars left behind from yeast fermentation
including L-arabinose and ribose.
Only around 45% of O. oeni strains can ferment
sucrose (the form of sugar usually added for
chaptalization that gets converted by yeast
into glucose and fructose).Winemakers tend
to prefer O. oeni for several reasons.
First, the species is compatible with the
main wine yeast Saccharomyces cerevisiae,
though in cases where both MLF and alcoholic
fermentation are started together, the yeast
most often outcompetes the bacterium for nutritional
resources which may cause a delay in the onset
of malolactic fermentation.
Second, most strains of O. oeni are tolerant
to the low pH levels of wine and can usually
deal with the standard alcohol levels that
most wines reach by the end of fermentation.
Additionally, while sulfur dioxide levels
above 0.8 molecular SO2 (pH dependent but
roughly 35-50 ppm) will inhibit the bacteria,
O. oeni is relatively resistant compared to
other LAB.
Finally, O. oeni tends to produce the least
amount of biogenic amines (and most lactic
acid) among the lactic acid bacteria encountered
in winemaking.
=== Lactobacillus ===
Within the genus Lactobacillus are both heterofermentative
and homofermentative species.
All lactobacilli involved in winemaking are
Gram-positive and microaerophilic, with most
species lacking the enzyme catalase needed
to protect themselves from oxidative stress.Species
of Lactobacillus that have been isolated from
wine and grape must samples across the globe
include L. brevis, L. buchneri, L. casei,
L. curvatus, L. delbrueckii subsp.
lactis, L. diolivorans, L. fermentum, L. fructivorans,
L. hilgardii, L. jensenii, L. kunkeei, L.
leichmannii, L. nagelii, L. paracasei, L.
plantarum, and L. yamanashiensis.Most Lactobacillus
species are undesirable in winemaking with
the potential of producing high levels of
volatile acidity, off odors, wine haze, gassiness,
and sediment that can be deposited in the
bottle, especially if the wine had not been
filtered.
These bacteria also have the potential to
create excessive amounts of lactic acid which
can further influence the flavor and sensory
perception of the wine.
Some species, such as the so-called "ferocious
Lactobacillus", have been implicated in causing
sluggish or stuck fermentations, while other
species, such as L. fructivorans, have been
known to create a cottony mycelium-like growth
on the surface of wines, nicknamed "Fresno
mold" after the wine region where it was discovered.
=== Pediococcus ===
So far, four species from the genus Pediococcus
have been isolated in wines and grape must,
P. inopinatus, P. pentosaceus, P. parvulus,
and P. damnosus, with the last two being the
species most commonly found in wine.
All Pediococcus species are Gram-positive
with some species being micro-aerophilic while
others utilizing mostly aerobic respiration.
Under the microscope, Pediococcus often appear
in pairs of pairs or tetrads which can make
them identifiable.
Pediococci are homofermenters, metabolizing
glucose into a racemic mixture of both L-
and D-lactate by glycolysis.
However, in the absence of glucose, some species,
such as P. pentosaceus, begin using glycerol,
degrading it into pyruvate which later can
be converted to diacetyl, acetate, 2,3-butanediol
and other compounds that can impart unfavorable
characteristics to the wine.Most Pediococcus
species are undesirable in winemaking due
to the high levels of diacetyl that can be
produced, as well as increased production
of biogenic amines that has been implicated
as one potential cause for red wine headaches.
Many species of Pediococcus also have the
potential to introduce off odors or other
wine faults to the wine such as the bitter-tasting
"acrolein taint" that comes from degradation
of glycerol into acrolein which then reacts
with phenolic compounds in the wine to produce
a bitter-tasting compound.One species, P.
parvulus, has been found in wines that have
not gone through MLF (meaning malic acid is
still present in the wine), but has still
had its bouquet altered in a way that enologist
have described as "not spoiled" or flaw.
Other studies have isolated P. parvulus from
wines that have gone through malolactic fermentation
without the development of off odors or wine
faults.
=== Nutritional requirements ===
Lactic acid bacteria are fastidious organisms
that cannot synthesize on their own all of
their complex nutritional requirements.
For LAB to grow and complete malolactic fermentation,
the constitution of the wine medium must provide
for their nutritional needs.
Like wine yeast, LAB require a carbon source
for energy metabolism (usually sugar and malic
acid), nitrogen source (such as amino acids
and purines) for protein synthesis, and various
vitamins (such as niacin, riboflavin, and
thiamine) and minerals to assist in the synthesis
of enzymes and other cellular components.The
source for these nutrients is often found
in the grape must itself, though MLF inoculations
that run concurrent with alcoholic fermentation
risk the yeast outcompeting the bacteria for
these nutrients.
Towards the end of fermentation, while most
of the original grape must resources have
been consumed, the lysis of dead yeast cells
(the "lees") can be a source for some nutrients,
particularly amino acids.
Plus, even "dry" wines that have been fermented
to dryness still have unfermentable pentose
sugars (such as arabinose, ribose and xylose)
left behind that can be used by both positive
and spoilage bacteria.
As with wine yeast, manufacturers of cultured
LAB inoculum usually offer specially prepared
nutritional additives that be used as a supplement.
However, unlike wine yeast, lactic acid bacteria
can not use the supplement diammonium phosphate
as a nitrogen source.Before the introduction
of complex nutritional supplements and advances
in freeze-dried cultures of LAB, winemakers
would cultivate their inoculum of lactic acid
bacteria from culture slants provided by laboratories.
In the 1960s, these winemakers found it easier
to create starter cultures in media that contained
apple or tomato juice.
This "tomato juice factor" was discovered
to be a derivative of pantothenic acid, an
important growth factor for the bacteria.As
with yeast, oxygen can be considered a nutrient
for LAB, but only in very small amount and
only for microaerophilic species such as O.
oeni.
However, no evidence exists currently to suggest
that malolactic fermentation runs more smoothly
in aerobic conditions than in complete anaerobic
conditions, and in fact, excessive amounts
of oxygen can retard growth of LAB by favoring
conditions of competing microbes (such as
Acetobacter).
=== Native LAB species in the vineyard and
the winery ===
Oenococcus oeni, the LAB species most often
desired by winemakers to carry out malolactic
fermentation, can be found in the vineyard,
but often at very low levels.
While moldy, damaged fruit has the potential
to carry a diverse flora of microbes, the
LAB most often found on clean, healthy grapes
after harvest are species from the Lactobacillus
and Pediococcus genera.
After crushing, microbiologists usually find
populations under 103 colony forming units/ml
containing a mix of P. damnosus, L. casei,
L. hilgardii, and L. plantarum, as well as
O. oeni.
For musts that do not receive an early dose
of sulfur dioxide to "knock back" these wild
populations of LAB, this flora of bacteria
compete with each other (and the wine yeasts)
for nutrients early in fermentation.In the
winery, multiple contact points can be home
to native population of LAB including oak
barrels, pumps, hoses, and bottling lines.
For wines where malolactic fermentation is
undesirable (such as fruity white wines),
the lack of proper sanitation of wine equipment
can lead to the development of unwanted MLF
and result in wine faults.
In cases of oak barrels where full and complete
sanitation is almost impossible, wineries
often mark barrels that have contained wines
going through MLF and keep them isolated from
"clean" or brand new barrels that they can
use for wines that are not destined to go
through MLF.
=== Schizosaccharomyces yeast ===
Several species in the genus Schizosaccharomyces
use L-malic acid, and enologists have been
exploring the potential of using this wine
yeast for deacidifying wines instead of the
traditional route of malolactic fermentation
with bacteria.
However, early results with Schizosaccharomyces
pombe have shown a tendency of the yeast to
produce off odors and unpleasant sensory characteristics
in the wine.
In recent years, enologists have been experimenting
with a mutant strain of Schizosaccharomyces
malidevorans that has so far been shown to
produce less potential wine flaws and off
odors.
== Influence of inoculation timing ==
Winemakers differ in when they choose to inoculate
their must with LAB, with some winemakers
pitching the bacteria at the same time as
the yeast, allowing both alcoholic and malolactic
fermentations to run concurrently, while some
wait till the end of fermentation when the
wine is racked off its lees and into barrel,
and others doing it somewhere between.
For practitioners of minimalist or "natural
winemaking" who choose not to inoculate with
cultured LAB, malolactic fermentation can
happen at any time depending on several factors
such as the microbiological flora of the winery
and the competing influences of these other
microbes.
All options have potential benefits and disadvantages.The
benefits of inoculating for MLF during alcoholic
fermentation include:
More potential nutrients from the grape must
(though the bacteria will be competing with
the yeast for these)
Lower sulfur dioxide and ethanol levels which
can otherwise inhibit the LAB
Higher fermentation temperatures which are
more conducive to LAB growth and an earlier
completion of MLF: The optimal temperatures
for malolactic fermentation are between 20
and 37 °C (68 and 98.6 °F), while the process
is significantly inhibited at temperatures
below 15 °C (59 °F).
Wine stored in the barrels in the cellar during
the winter following fermentation will often
have a very prolonged malolactic fermentation
due to the cool cellar temperatures.
Early completion of malolactic fermentation
means the winemaker can make a postfermentation
SO2 earlier to protect the wine from oxidation
and spoilage microbes (such as Acetobacter).
Since sulfur dioxide can inhibit MLF, delaying
LAB inoculation till after alcoholic fermentation
may mean a delay in sulfur addition till early
spring when cellar temperatures warm up enough
to encourage the completion of MLF.
Less diacetyl productionThe disadvantages
for early inoculation include:
Wine yeast and LAB competing for resources
(including glucose) and potential antagonism
between the microbes
Heterofermenters such as O. oeni metabolizing
the glucose still present in the must and
potentially creating undesirable byproducts
such as acetic acidMany of the advantages
for postalcoholic fermentation answer the
disadvantages of early inoculation (namely
less antagonism and potential for undesirable
byproducts).
Also, the advantage is seen of the lees being
a nutrient source through the autolysis of
the dead yeast cells, though that nutrient
source may not always be enough to ensure
MLF runs successfully to completion.
Conversely, many of the disadvantages of late
inoculation are the absence of the advantages
that come from early inoculation (higher temperatures,
potentially quicker completion, etc.).
== Preventing MLF ==
For some wine styles, such as light, fruity
wines or for low-acid wines from warm climates,
malolactic fermentation is not desired.
Winemakers can take several steps to prevent
MLF from taking place, including:
Limited maceration, early pressing, and early
racking to limit contact time of the LAB with
potential nutrient sources
Maintain sulfur dioxide levels to at least
25 ppm of "free" (unbound) SO2, depending
on the pH of the wine, this may mean an addition
of 50–100 mg/l of SO2
Maintain pH levels below 3.3
Keep the wine cool at temperatures between
10 and 14 °C (50.
0 to 57.2 °F)
Filter the wine at bottling with at least
a 0.45-micron membrane filter to prevent any
bacteria from making it into the bottleIn
addition, winemakers can use chemical and
biological inhibitors such as lysozyme, nisin,
dimethyl dicarbonate (Velcorin), and fumaric
acid, though some (like Verlcorin) are restricted
in winemaking countries outside the United
States.
Fining agents, such as bentonite, and putting
the wine through cold stabilization will also
remove potential nutrients for LAB, thus inhibiting
malolactic fermentation.
Some experimentation with the use of bacteriophages
(viruses that infect bacteria) has been conducted
to limit malolactic fermentations, but disappointing
results in the cheesemaking industry have
led to skepticism about the practical use
of bacteriophages in winemaking.
== Measuring malic content ==
Winemakers can track the progression of malolactic
fermentation by paper chromatography or with
a spectrophotometer.
The paper chromatography method involves using
capillary tubes to add small samples of the
wine to chromatograph paper.
The paper is then rolled and placed in a jar
filled with a butanol solution containing
bromocresol green indicator dye for several
hours.
After the paper is pulled out and dried, the
distance of yellow-colored "splotches" from
the base line denotes the presence of various
acids, with tartaric being closest to the
baseline followed by citric, malic, and finally
lactic acids near the top of the paper.A significant
limitation to paper chromatography is that
it will not show exactly how much malic is
still remaining in the wine, with the size
of the "splotch" on the paper having no correlation
to a quantitative figure.
The sensitivity of the paper is also limited
to a detection threshold of 100–200 mg/L
while most measurements of "MLF stability"
target a malic level of less than 0.03 g/l
(30 mg/L).The enzymatic method allows for
a quantitative measurement of both malic and
lactic acids, but requires the expense of
reagent kits and a spectrophotometer that
can measure absorbance values at 334, 340,
or 365 nm.
== Other products produced ==
The main products of malolactic fermentation
are lactic acid, diacetyl, acetic acid, acetoin,
and various esters.
The amount and exact nature of these products
depends on the species/strain of LAB conducting
the malolactic fermentation and the condition
influencing that wine (pH, available nutrients,
oxygen levels, etc.).Some strains of O. oeni
can synthesize higher alcohols which can contribute
to fruity notes in the aroma of the wine.
Additionally, some strains of the bacterium
have beta-glucosidase enzymes that can break
down monoglucosides which are aroma compounds
attached to a sugar molecule.
When the sugar component is cleaved, the rest
of the compound becomes volatilized, meaning
it can potentially be detected in the aroma
bouquet of the wine.In the early 21st century,
some strains of O. oeni were shown to use
acetaldehyde by breaking it down into ethanol
or acetic acid.
While this may help for wines with excessive
levels of acetaldehyde, for red wines, it
can also destabilize the color of the wine
by interfering with acetaldehyde's reaction
with anthocyanins to create polymeric pigments
that help create a wine's color.
=== Diacetyl ===
Diacetyl (or 2,3-butanedione) is the compound
associated with the "buttery" aromas of Chardonnays,
but it can affect any wine that has gone through
malolactic fermentation.
At an odor detection threshold of 0.2 mg/l
in white wines and 2.8 mg/l in red wines,
it can be perceived as slightly buttery or
"nutty" while at concentrations greater than
5 to 7 mg/l (5-7 ppm) can overwhelm other
aroma notes in the wine.Diacetyl can be produced
by the LAB through metabolism of sugar or
of citric acid.
While citric acid is naturally present in
grapes, it is in a very small amount with
most of it coming from deliberate addition
by the winemaker to acidify the wine.
In the presence of both malic and citric acids,
the LAB use both, but use the malic much more
quickly, with the rate of citric use/diacetyl
formation influenced by the particular bacterial
strain (with most strains of O. oeni producing
less diacetyl than Lactobacillus and Pediococcis
species), as well as the redox potential of
the wine.
In wine conditions that have a low redox potential
(meaning it is more oxidative such as in a
barrel that is not fully topped up), more
citric acid will be consumed and diacetyl
formed.
In more reductive conditions, such as in alcoholic
fermentations where yeast populations are
at their peak and the wine is heavily saturated
with carbon dioxide, the formation of diacetyl
is much slower.
The yeasts also help keep levels low by consuming
diacetyl and reducing it to acetoin and butylene
glycol.Diacetyl production is favored in fermentations
that run warm with temperatures between 18
°C (64.4 °F) and 25 °C (77 °F).
It also tends to be produced at higher levels
in wines with lower pH levels (under 3.5),
though at levels below 3.2, most strains of
LAB desirable for MLF tend to be inhibited.
"Wild" (as in uninoculated) malolactic ferments
have the potential to produce more diacetyl
than inoculated ferments due to the lower
initial populations during the lag phase with
inoculated ferments usually having an initial
inoculum of 106 CFU/ml.
Late MLF inoculations, after alcoholic fermentation,
also tend to produce higher levels of diacetyl.
Chardonnay producers desiring to make the
high-diacetyl "buttery style" will often do
late or "wild" inoculation in the barrel after
primary fermentation, allowing the wine to
spend several weeks or even months sur lie
in reductive conditions that promote diacetyl
production.
Some sources point out that diacetyl is actually
decreased by sur lie, due to surviving yeast
metabolizing diacetyl, and therefore malolactic
fermentation is best performed apart from
lees.With wines that have excessive levels
of diacetyl, some winemakers use sulfur dioxide
to bind with the compound and reduce the perception
of diacetyl by 30 to 60%.
This binding is a reversible process and after
only a few weeks aging in the bottle or tank,
the high levels of diacetyl return.
However, sulfur dioxide added earlier in the
malolactic fermentation process limits diacetyl
production by inhibiting the bacteria and
limiting their activity in its entirety, including
the conversion of malic to lactic acid.
== Wine faults ==
The most common fault associated with malolactic
fermentation is its occurrence when it is
not desired.
This could be for a wine that is meant to
be acidic and fruity (such as Riesling) or
it could be a wine that was previously thought
to have gone through MLF and bottled only
to have malolactic fermentation commence in
the bottle.
The outcome of this "in-bottle" fermentation
is often gassy, hazy wine that can be unpalatable
to consumers.
Improvement in sanitation and control of lactic
acid bacteria in the winery can limit the
occurrence of these faults.For early Vinho
Verde producers, the slight effervesce that
came from in-bottle malolactic fermentation
was considered a distinguishing trait that
consumers enjoyed in the wine.
However, wineries had to market the wine in
opaque bottles to mask the turbidity and sediment
that the "in-bottle MLF" produced.
Today, most Vinho Verde producers no longer
follow this practice and instead complete
malolactic fermentation prior to bottle with
the slight sparkle being added by artificial
carbonation.While not necessarily a fault,
malolactic fermentation does have the potential
of making a wine "protein unstable" due to
the resulting change in pH which affects the
solubility of proteins in wine.
For this reason, protein fining and heat stability
tests on wine usually take place after malolactic
fermentation has run to completion.
=== Volatile acidity ===
While volatile acidity (VA) is usually measured
in terms of acetic acid content, its sensory
perception is a combination of acetic (vinegary
aromas) and ethyl acetate (nail polish remover
and model airplane glue aromas).
High levels of VA can inhibit wine yeast and
may lead to a sluggish or stuck fermentation.
Several microbes can be a source for VA, including
Acetobacter, Brettanomyces, and film yeast
such as Candida, as well as LAB.
However, while LAB usually only produce acetic
acid, these other microbes often produce ethyl
acetate, as well as acetic acid.Most wine-producing
countries have laws regulating the amount
volatile acidity permitted for wine available
for sale and consumption.
In the United States, the legal limit is 0.9
g/l for foreign wine exported to the United
States, 1.2 g/l for white table wine, 1.4
g/l for red wine, 1.5 g/l for white dessert
wine, and 1.7 g/l for red dessert wine.
European Union wine regulations limit VA to
1.08 g/l for white table wines and 1.20 g/l
for red table wines.Heterofermenting species
of Oenococcus and Lactobacillus have the potential
to produce high levels of acetic acid through
the metabolism of glucose, though with most
strains of O. oeni, the amount is usually
only 0.1 to 0.2 g/l.
Several species of Pediococcus can also produce
acetic acid through other pathways.
Wines starting out with a high pH levels (above
3.5) stand the greatest risk of excessive
acetic acid production due to the more favorable
conditions for Lactobacillus and Pediococcus
species.
L. Kunkeei, one of the so-called "ferocious
Lactobacillus" species, has been known to
produce 3 to 5 g/l of acetic acid in wines—levels
which can easily lead to stuck fermentations.
=== "Ferocious" Lactobacillus ===
In the late 20th century, among American winemakers,
seemingly healthy fermentation were reported
becoming rapidly inundated with high levels
of acetic acid that overcame wine yeasts and
led to stuck fermentations.
While a novel species of Acetobacter or wine
spoilage yeast was initially thought to be
the culprit, it was eventually discovered
to be several species of Lactobacillus, L.
kunkeei, L. nagelii, and L. hilgardii, collectively
nicknamed "ferocious" Lactobacillus for their
aggressive acetic acid production, how quickly
they multiply, and their high tolerance to
sulfur dioxides and other microbiological
controls.Ferments of high-pH wines (greater
than 3.5) that spent time cold soaking prior
to yeast inoculations and received little
to no sulfur dioxide during crushing seem
to be at the most risk for "ferocious" Lactobacillus.
While infection seems to be vineyard-specific,
currently, none of any of the implicated lactobacilli
has been reported as being found on the surface
of freshly harvested wine grapes.
=== Acrolein and mannitol taint ===
The degradation of glycerol by some strains
of LAB can yield the compound acrolein.
Glycerol is a sweet-tasting polyol present
in all wines, but at higher levels in wines
that have been infected with Botrytis cinerea.
An "active-aldehyde", acrolein can interact
with some phenolic compounds in wine to create
highly bitter-tasting wines, described as
amertume by Pasteur.
While at least one strain of O. oeni has been
shown to produce acrolein, it is more commonly
found in wines that have been infected by
strains of Lactobacillus and Pediococcus species
such as L. brevis, L. buchneri, and P. parvulus.
Acrolein taint has also shown to be more common
in wines that have been fermented at high
temperatures and/or made from grapes that
have been harvested at high Brix levels.Heterofermenting
species from the genus Lactobacillus, as well
as some wild strains of O. oeni, have the
potential to metabolize fructose (one of the
main sugars in wine) into the sugar alcohols
mannitol and (less commonly) erythritol.
These are sweet-tasting compounds can add
sweetness to a wine where it is not desired
(such as Cabernet Sauvignon).
Mannitol taint, described as mannite by Pasteur,
in wines is often accompanied by other wine
faults, including the presence of excessive
levels of acetic acid, diacetyl, lactic acid,
and 2-butanol, which can contribute to a "vinegary-estery"
aroma.
The wine may also have a slimy sheen on the
surface.
=== Fresno mold and ropiness ===
In the mid-20th century, a cottony mycelium-like
growth began appearing in the bottles of some
sweet fortified wines produced in California's
Central Valley.
Being fortified, these wines often had alcohol
levels in excess of 20% which is usually a
level that discourages growth of most spoilage
organisms associated with winemaking.
Nicknamed "Fresno mold" due to where it was
first discovered, the culprit of this growth
was determined to be L. fructivorans, a species
which can be controlled by sanitation and
maintaining adequate sulfur dioxide levels.Some
Lactobacillus and Pediococcus species (particularly
P. damnosus and P. pentosaceus) have the potential
to synthesize polysaccharides that add an
oily viscosity to the wine.
In the case of Lactobacillus, some of these
saccharides may be glucans that can be synthesized
from glucose present in the wine as low as
50–100 mg/l (0.005 to 0.01% residual sugar)
and afflict seemingly "dry" wines.
While "ropiness" can occur in the barrel or
tank, it is often observed in the wines several
months after they are bottled.
Wines with pH levels above 3.5 and low sulfur
dioxide levels are at most risk for developing
this fault.Called graisse (or "grease") by
the French and les vins filant by Pasteur,
this fault has been observed in apple wines
and cider.
It can also be potentially be caused by other
spoilage microbes such as Streptococcus mucilaginous,
Candida krusei, and Acetobacter rancens.
=== Mousiness and geranium taint ===
Wines infected with L. brevis, L. hilgardii,
and L. fermentum have been known to occasionally
develop an aroma reminiscent of rodent droppings.
The aroma becomes more pronounced when the
wine is rubbed between the fingers and, if
consumed, can leave a long, unpleasant finish.
The aroma can be very potent, detectable at
a sensory threshold as low as 1.6 parts per
billion (µg/l).
The exact compound behind this is derivatives
of the amino acid lysine created through an
oxidation reaction with ethanol.
While undesirable LAB species have been most
commonly associated with this fault, wine
infected by Brettanomyces yeast in the presence
of ammonium phosphate and lysine have also
been known to exhibit this fault.Sorbate is
often used as a yeast-inhibitor by home winemakers
to stop alcoholic fermentation in the production
of sweet wines.
Most species of lactic acid bacteria can synthesize
sorbate to produce 2-ethoxyhexa-3,5-diene
which has the aroma of crushed geranium leaves.
=== Tourne ===
Compared to malic and citric acids, tartaric
acid is usually considered microbiologically
stable.
However, some species of Lactobacillus (particularly
L. brevis and L. plantarum) have the potential
to degrade tartaric acid in wine, reducing
a wine's total acidity by 3-50%.
French winemakers had long observed this phenomenon
and called it tourne (meaning "turn to brown")
in reference to the color change that can
occur in the wine at the same time likely
due to other processes at work in addition
to the tartaric loss.
While Lactobacillus is the most common culprit
of tourne, some species of the spoilage film
yeast Candida can also metabolize tartaric
acid.
=== Health-related faults ===
While the presence of ethyl carbamate is not
a sensory wine fault, the compound is a suspected
carcinogen which is subjected to regulation
in many countries.
The compound is produced from the degradation
of the amino acid arginine which is present
in both grape must and released in the wine
through the autolysis of dead yeast cells.
While the use of urea as a source of yeast
assimilable nitrogen (no longer legal in most
countries) was the most common cause of ethyl
carbamate in wine, both O. oeni and L. buchneri
have been known to produce carbamyl phosphate
and citrulline which can be precursors to
ethyl carbamate formation.
L. hilgardii, one of the "ferocious Lactobacillus"
species, has also been suspected of contributing
to ethyl carbamate production.
In the United States, the Alcohol and Tobacco
Tax and Trade Bureau has established a voluntary
target limit of ethyl carbamate in wine to
less than 15 µg/l for table wines and less
than 60 µg/l for dessert wines.Biogenic amines
have been implicated as a potential cause
of red wine headaches.
In wine, histamine, cadaverine, phenylethylamine,
putrescine, and tyramine have all been detected.
These amines are created by the degradation
of amino acids found in grape must and left
over from the breakdown of dead yeast cells
after fermentation.
Most LAB have the potential to create biogenic
amines, even some strains of O. oeni, but
high levels of biogenic amines are most often
associated with species from the Lactobacillus
and Pediococcus genera.
In the European Union, the concentration of
biogenic amines in wine is beginning to be
monitored, while the United States currently
does not have any regulations
