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Introduction

Fermentation is the transformative action of microorganisms, which results in


something delicious. Humans didnt invent or discover fermentationit happens
spontaneously. Our accomplishment is creating vessels to make these processes
happen on our terms. Bread, miso, sauerkraut, wine these foods are produced by
invisible life forces, microorganisms, which adds a sort of alchemy to the whole
thing. -

A. Background of the Study

Wine has been with us since the dawn of civilization and has followed humans and
agriculture along diverse migration paths .Serendipity presumably played a part in
its genesis more than 7,000 years ago: damaged grapes spontaneously fermented
in harvesting vessels; curious farmers tasted the resultant alcoholic beverage; the
curious farmers liked what they tasted and enjoyed its effects; said farmers
preferred fermented grape juice to the unfermented fruit. The fate of the grape was
sealed.

The natural occurrence of fermentation means it was probably first observed long
ago by humans. The earliest uses of the word "fermentation" in relation to
winemaking was in reference to the apparent "boiling" within the must that came
from the anaerobic reaction of the yeast to the sugars in the grape juice and the
release of carbon dioxide. The Latin fervere means, literally, to boil. In the mid-19th
century, Louis Pasteur noted the connection between yeast and the process of the
fermentation in which the yeast act as catalyst and mediator through a series of a
reaction that convert sugar into alcohol. The discovery of the EmbdenMeyerhof
Parnas pathway by Gustav Embden, Otto Fritz Meyerhof and Jakub Karol Parnas in
the early 20th century contributed more to the understanding of the complex
chemical processes involved in the conversion of sugar to alcohol. [

One might argue that the most important test tube in the birth and growth of the
modern life sciences is the fermenter

Figure 1
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A generalized scheme of the spread of Vitis vinifera noble varieties of grapevine and
winemaking from their centre of origin in Asia Minor to other parts of the world.

One might argue that the seeds of science and technology, particularly
biotechnology, were also sown at this time. Empirical observations of natural events
and processes were harnessed in repeat experiments'which is to say, vintages
and improvements were made by trialling modifications to practices, retaining those
that were beneficial and discarding failures, with the results communicated down
through the generations. At that time, there was no EMBO reports or alternative
means by which to facilitate horizontal dissemination of information, but the
principle of developmentsans peer reviewis clear: experimentation and
invention lead to progresstechnological and otherwiseand new knowledge is
shared and built upon.

Of course, early inventions and innovations in grape and wine production were
based on little or no knowledge of the biology of grapevines or the microbes that
drive fermentation. In fact, it would be several thousand years before it was even
known that microscopic organisms exist: using a primitive microscope, Antonie van
Leeuwenhoek observed cells for the first time in 1680 (Fig 2).

Figure 2
Selected milestones that mark the path of research in microbiology and yeast
biology that have affected, directly or indirectly, wine science and winemaking.

Scientific knowledge grows at an exponential rate, and nowhere is this more evident
than in the historical milestones of chemistry and biology that have shaped our
understanding of the biology of the microorganisms that drive fermentation (Fig 2).
This progress has been adorned with some of the most significant names in the
chemical and biological sciences, including van Leeuwenhoek, Lavoisier, Gay-
Lussac, Pasteur, Buchner and Koch. One might argue that the most important test
tube in the birth and growth of the modern life sciences is the fermenter, and the
most important model organism has been the yeast Saccharomyces cerevisiae
commonly known as baking, brewing or wine yeast. As readers might know, this is
exemplified in the origin of the word enzymeen' meaning within and zyme'
meaning leaven. Yeast has been integral to pioneering work in microbiology and
biochemistry, particularly in the fields of metabolism and enzymology (Barnett,
1998, 2000; Barnett & Lichtenthaler, 2001).

Throughout the early decades of the twentienth century the place for S. cerevisiae
in fundamental research was affirmed, and there are several good reasons for this.
Our close relationship with this yeast in food and beverage production over
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millennia tells us that it is safe to work with; as confirmed by its Generally


Recognised as Safe' designation by the US Food and Drug Administration. In
addition, it is inexpensive, easy to grow and can be stored for long periods in
suspended animation. Perhaps the most important thing is that it has accessible
genetics that can be followed through sexual and asexual cycles (Barnett, 2007).

The 1970s set the stage for another explosion of knowledge, sparked by the advent
of gene technology and driven by a convergence of genetics, biochemistry, cell
biology, microbiology, physical and analytical chemistry, as well as computing
brought together under the banner of molecular biology (Fig 3). Yeast molecular
biology was established when Gerald Fink's group in the USA demonstrated that
yeast could be transformed with foreign DNA (Hinnen et al, 1978). In the same year,
Jean Beggs in the UK developed a shuttle vector between Escherichia coli and S.
cerevisiae that enabled cloning in yeast (Beggs, 1978). The research community
now had a eukaryotic host that was amenable to genetic engineering, benefiting
both fundamental research and offering the potential of precise engineering of
novel strains for industrial applications. It was the first host cell for industrial-scale
production of a recombinant vaccine against hepatitis B and a recombinant food-
grade enzyme, chymosin, which is used in cheese processing (Pretorius et al, 2003).

Figure 3
Selected milestones that mark the path of research in genetics and molecular
biology that have affected, directly or indirectly, wine science and winemaking.

Ever since, S. cerevisiae has been one of the most important model organisms in
molecular biology and emerging fields; breakthroughs and technological advances
in molecular, systems, and now synthetic biology rarely happen without S.
cerevisiae figuring somewhere prominently in the story (Fig 3). The international
yeast science community has been particularly progressive and proactive in
establishing large collaborative projects and building resources that are available to
the scientific community. S. cerevisiae was the first eukaryote to have its genome
sequenced (Goffeau et al, 1996), a feat that was achieved through an international
effort that involved 600 scientists, which paved the way for the first chip-based
gene array experiments (Schena et al, 1995). It was the first organism to be used to
build a systematic collection of bar-coded gene deletion mutants (Winzeler et al,
1999; Giaever et al, 2002), in which there are deletion strains for most of the open-
reading frames in the S. cerevisiae genome. This has enabled high-throughput
functional-genomic experiments, and anyone seeking information on just about any
aspect of S. cerevisiae biology has access to the amazing community resource: the
Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/).
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All of this is important to wine research; our winemaking workhorse is centre stage
in thousands of research projects worldwide, so we know more about this humble
eukaryote than any other organism on the planet. It is therefore unsurprising that
wine research has benefited enormously from the privileged place that S. cerevisiae
occupies in life sciences research. This is particularly evident in the impact that
advances in molecular biology and related fields have had on winemaking.

In the hands of molecular biologists, S. cerevisiae is the most tractable of


organisms; it is amenable to almost any modification that modern biology can throw
at a cell. This makes it an ideal host for generating variants with improved and even
exotic phenotypes that will benefit winemaking. The following gives some examples
of current research and directions in this field.

In modern winemaking, fermentations are driven largely by single-strain


inoculations; pure cultures of selected strains of S. cerevisiae are added to grape
must as soon as possible after crushing. This ensures greater control of vinification,
leads to more predictable outcomes and decreases the risk of spoilage by other
microorganisms. There are manyprobably hundreds ofdifferent yeast strains
available, and the winemaker's choice can substantially effect the quality of the
wine (Lambrechts & Pretorius, 2000; Swiegers et al, 2005).

One of the reasons for the yeast-induced variation in wine quality is that, during
fermentation, S. cerevisiae produces an abundance of aroma-active secondary
metabolites and releases many aroma compounds from inactive precursors in grape
juice, which greatly affect the sensory properties of the wine (Swiegers & Pretorius,
2007). Thus, any genetic variation in wine yeast that affects the production or
release of sensorially important molecules will affect wine quality. In this context it
has been demonstrated, for example, that different commercial yeast strains
generate wines with very different profiles of volatile thiols (Swiegers et al, 2009).
These thiolswhich are present in grape juice as non-volatile cysteinylated
precursors (Tominaga et al, 1998)are often described as passionfruit', tropical
fruits' and citrus' by tasters, flavours that are particularly important in wine
varieties such as Sauvignon Blanc (Dubourdieu et al, 2006).

Molecular biology and its tools are crucial to our understanding of the genetic and
molecular bases of yeast-driven volatile thiol release from non-volatile precursors in
grape juice. Howell et al (2005) have used bioinformatic tools and the SGD to
identify candidate S. cerevisiae carbonsulphur lyase genes that might be involved
in the release of volatile thiols from cysteinylated precursors during fermentation.
The researchers used targeted gene deletion to remove these candidate carbon
sulphur lyases from the wine and laboratory yeast strains, and they identified four
genes that potentially contribute to the release of these important aroma
molecules.

Swiegers et al (2007) then engineered a wine yeast, VIN13, to constitutively express


a carbonsulphur lyase gene, tnaA, from E. coli. Sensory analysis revealed that,
compared with its non-engineered relative, this transgenic yeast, VIN13 (CSL1), had
a positive impact on the release of volatile thiols from a Sauvignon Blanc grape
juice. The authors commented that wine assessors preferred the VIN13 (CSL1)-
derived experimental wines to the relatively neutral VIN13-derived wines.
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A similar approach has been used to engineer yeasts for the enhanced production of
fruity esters (Lilly et al, 2006a) and to increase the production of higher, fusel
alcohols (Lilly et al, 2006b)all of which contribute to the flavour profiles of wines.
Although this work is in the early stages of development, it shows the value of yeast
molecular biology, and the amazing resources that come with it.

Wine alcohol content is of growing importance to the wine industry. In some wine
regions, it has been increasing during recent decades (Godden & Muhlack, 2010).
The main reason for this increase is that grapegrowers tend to leave fruit on the
vine as long as possible to increase fruity characterswhich develop as berries
matureand reduce undesirable green' characters. This practice, however,
produces fruit with a higher sugar content, which translates to higher ethanol
concentrations in the wine.

A recent review by Kutyna et al (2010) discusses several metabolic engineering


strategies that have been explored to generate wine yeasts that can divert some
carbon metabolism away from ethanol production, with the aim of decreasing
ethanol yields during vinification. Understanding the central metabolism of yeast
and the genes that drive it has been crucial to this work. Candidate genes that are
likely to influence ethanol yields can be identified from a range of sources, including
the SGD, and then manipulated and cloned as required. Several laboratories have
targeted the glycerol-3-phosphate dehydrogenase isozymes GPD1 and GPD2, which
divert carbon from glycolysis to glycerol production (Michnick et al, 1997; Remize et
al, 1999; de Barros Lopes et al, 2000).

Increased expression of either of the GPD paralogues increased glycerol and


decreased ethanol yields. However, increased Gpd activity also led to increased
amounts of acetic acid in the fermentation product. This was probably owing to
rectificationby one or more of the five aldehyde dehydrogenase isozymesof a
redox imbalance that resulted from excessive Gpd-driven oxidation of NADH.
Aldehyde dehydrogenase isozymes drive the oxidation of acetaldehyde to acetic
acid with concomitant reduction of coenzymes NAD + or NADP, depending on which
isozyme is involved (Navarro-Avio et al, 1999). This might be good for a yeast cell
struggling with an imposed redox imbalance, but an increase in acetic acid
production is not good news for winemakers; excessive vinegar is not desirable in
wine. This problem was alleviated by knocking out one of the five aldehyde
dehydrogenase isozymes, ALD6 (Eglinton et al, 2002; Cambon et al, 2006).

Similar approaches have targeted S. cerevisiae pyruvate decarboxylase isozymes,


alcohol dehydrogenase isozymes and glycerol transporters, leading to increased
glycerol yields and reduced ethanol production (Kutyna et al, 2010). However, while
there are probably several good candidate low-ethanol' wine yeast strains sitting in
various labs around the world, none have been tested in commercial-scale,
industrial fermentations. This is largely because consumers are generally
unaccepting of genetically modified organisms (GMOs) in foods and beverages.

Another area of ongoing research in wine yeast molecular biology is the


development of strains that flocculatethat is, form clumpsat the end of
fermentation. This facilitates the process of settling them out of suspension and
separating them from the wine, thereby reducing the need for clarification. The
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timing of flocculation is crucial; it must not happen too early, as yeast in large flocs
are inefficient at sugar utilization and can generate suboptimalstuck or sluggish
fermentations (Pretorius, 2000).

Generally, wine yeasts are not good at flocculation; they do not form large clumps
that settle out of suspension. Many years of research using laboratory strains of S.
cerevisiae led to the identification and characterization of genes that encode cell-
surface glycoproteinsincluding lectin-like flocculinsthat cause, among other
things, flocculation and subsequent settling to the bottom of the fermentation
vessel (Pretorius, 2000).

Recent findings have identified a problem with extrapolating basic research on


laboratory strains to those used in industry; yeasts domesticated for different
purposes have different phenotypes. Work by Govender et al (2008) on the
flocculation genes FLO1, FLO5 and FLO11, for example, demonstrated the potential
ability of engineered ADH2- or HSP30-promoter/FLO gene combinations to switch on
flocculation at the end of fermentation; ADH2 and HSP30 are both upregulated in
stationary-phase cells, so their promoters are suitable candidates to drive the
expression of genes in later stages of wine fermentation.

The results of this work were promising, but, when they were carried over to wine
yeast, the findings were rather different. There were even substantial differences
between wine yeast strains, leading the authors to caution that optimisation of the
flocculation pattern of individual commercial strains will have to be based on a
strain-by-strain approach (Govender et al, 2010). Nonetheless, controlled
expression of FLO genes at the end of fermentation remains a plausible technique
for improving the performance of wine yeast, but the strategies required to achieve
a desirable outcome might be more complex than was originally thought.

While the complexity of biological systems is a cause for excitement and wonder to
most biologists, it can make engineering novel strains for industrial applications
trickier than molecular biology and biotechnology textbooks might suggest. For
those of us working on industrial yeast strains, it might be pertinent to directly
tackle the issue of complexity and use systems biology approaches to better
understand the workings of yeast metabolism. This should lead to more accurate
modelling of metabolic processes for better-informed manipulations, to achieve
targeted, predictable outcomes.

However, molecular biologists face one important obstacle to this progress: near
worldwide refusal to permit the use of GMOs in the production of foods and
beverages

S. cerevisiae has been at the forefront of -omics' research. This provides us with
enormous opportunities to improve understanding of wine yeast complexity, which,
in turn, will inform the design of new strains for industrial applications. Increased
and improved knowledge from a huge number of studies investigating strains of S.
cerevisiae at the various -omic levels gives wine yeast scientists a head start in this
field (Borneman et al, 2007; Petranovic & Vemuri, 2009).
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One of the most interesting developments has come from the sequencing of a wine
yeast genome, and its comparison with the genomes of a laboratory strain and an
opportunistic pathogenic S. cerevisiae (Borneman et al, 2008). The authors found a
difference of about 0.6% in sequence information between the wine yeast and the
other strains. They also found, perhaps more importantly, 100 kb of additional
genome sequence in the former; enough to carry at least 27 genes. Open reading
frames (ORFs) in the additional sequences do not resemble anything found in other
strains of S. cerevisiae, but seem to be similar to genes found in distant fungal
relatives. BLAST searches have indicated that some of the genes that are specific to
wine yeast are similar to those encoding cell-wall proteins. This might contribute to
the greater robustness of wine yeast, compared with laboratory strains. Other genes
might encode proteins associated with amino acid uptake, which is significant in the
context of wine sensory attributes; amino acid metabolism is central to the
production of many sensorially important volatile aroma compounds.

Novo et al (2009) published similar findings from a different wine yeast strain
(EC1118) and suggested that the extra sequence was probably the result of
horiziontal gene transfer. Further work using functional geneticsto determine the
effects of knocking out and overexpressing the ORFsshould enable
characterization of the phenotypes of these ORFs, determine their relevance in the
context of winemaking and might also reveal their origins.

There have also been numerous studies describing transcriptomic, proteomic and
metabolomic analyses of wine-yeast fermentations. This work is beginning to
provide insights into wine-yeast fermentations, but it is still early days. It should
also be noted that much of the -omics work on wine yeast has used resources and
databases that are based on laboratory strains. It is now clear that there are
genomic differences between wine and lab strains of S. cerevisiae, and these might
affect -omics data acquisition and analysis. For example, gene-array chips based on
the reference laboratory strain S288c will not include the additional ORFs found in
wine strains. This does not suggest that earlier work is invalid, but that there are
likely to be gaps in it.

As the various -omics fields progress, it should be possible to build systems-based


mathematical models of metabolism that will facilitate the in silico design of new
wine yeast strains (Borneman et al, 2007). In parallel, we see the emergence of
synthetic biology where, yet again, S. cerevisiae is a key player. It should not be too
long before we have customised S. cerevisiae genomic componentsregulatory
elements to control the expression of targeted genes, or cassettes carrying genes
encoding metabolic pathways to shape wine-relevant traits, for exampleavailable
off the shelf' for designing, building and refining metabolic processes in our wine
yeast. But are consumers ready for this brave and exciting new world?

The engineered wine yeast strains described in this paper show the potential of
novel yeast strain development to improve wine quality. But molecular biologists
face a major obstacle to this progress: near world-wide refusal to permit the use of
GMOs in the production of foods and beverages, at least in developed' countries
(Gross, 2009; Pretorius & Hj, 2005). Wine industries in most parts of the world have
eschewed the use of GMOs in commercial winemaking, leaving most new-
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generation wine yeasts on the laboratory shelf, where they await more enlightened
times.

Two genetically modified wine yeast strains have been released to market in a
limited number of countries including the USA, Canada and Moldova: ML01 and
522EC. ML01, a transgenic wine yeast, has genes that enable it to perform
malolactic fermentation (MLF), a deacidifying secondary fermentation in which malic
acidpresent in grape juiceis decarboxylated to lactic acid. MLF is usually
performed by the lactic acid bacterium Oenococcus oeni after alcoholic
fermentation. However, this bacterium is rather fastidious, being inhibited by a
range of conditions that are typical of fermented grape juicelow pH, high alcohol
content, poor nutrient availability and the presence of sulphur dioxideand can
become stuck' or take considerable time to complete fermentation (Davis et al,
1985). In addition, lacitic acid bacteria can produce a range of biogenic amines,
which are associated with health risks (Lonvaud-Funel, 2001).

A wine yeast that completes both primary and secondary fermentations should
therefore have great potential in the wine industry. The genetically modified wine
yeast ML01 carries two foreign genesthe Schizosaccharomyces pombe malate
transporter gene (mae1) and the O. oeni malolactic enzyme gene (mleA)which are
both chromosomally integrated and regulated by the S. cerevisiae PGK1 promoter
and terminator (Husnik et al, 2006). This enables the host wine yeast to perform
MLF, in parallel with alcoholic fermentation.

The researchers went to great lengths to ensure the safety of ML01. The transgenes
came from microorganisms found in wine, there were no antibiotic resistance genes
or vector sequences carried by the yeast and transcriptome and proteome analysis
showed no important differences in gene expression profiles between the
genetically modified strain and its parent. The FDA granted Generally Regarded As
Safe' status to ML01, but it has not been widely adopted, even in countries where it
is approved for use. This is largely owing to concerns about export markets that do
not tolerate GMOs. In fact, wine industries in many countries have banned the use
of GMOs in wine production, in order to avoid jeopardizing their exports.exports.

The genetically modified wine yeast 522EC was engineered to reduce the risk of
ethyl carbamate production during fermentation. Ethyl carbamate, a potential
carcinogen, is the product of yeast-derived urea reacting with ethanol. It is usually
produced at such low levelsif at allthat it is not a cause for concern, but it
sometimes can make an appearance in some wine-producing regions.

S. cerevisiae is able to degrade urea before it is secreted and release ammonia


instead, thereby reducing the risk of generating ethyl carbamate. This is achieved
by the action of an enzyme encoded by DUR1,2, but this gene is repressed by
nitrogen and therefore downregulated throughout much of wine fermentation.
Coulon et al (2006) placed a copy of DUR1,2 behind a constitutive (PGK1) S.
cerevisiae promoter, which led to a reduction in ethyl carbamate yields.
Interestingly, this genetically modified yeast is self or cis cloned; it carries no
foreign DNA and therefore is not transgenic. Nonetheless, because it was generated
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by using techniques that involved the manipulation of DNA in vitro, the regulations
of many countries classify it as a GMO. Again, to the best of our knowledge, this
yeast is not being used in the industry. This might be because ethyl carbamate
production is not a widespread problem, but it probably also reflects the influence of
GMO bans and the reluctance of winemakers to risk losing market share in countries
that harbour strong anti-GMO sentiment.

Who knows what bottled masterpieces await us as we sculpt novel yeast strains in
the laboratory using molecular, systems and synthetic biology

Winemaking, science and technology have interwoven histories and have grown
together over the millennia, benefiting from each other. Although science is an
important part of an oenologist's training and scientific methods and equipment are
routinely employed in the winery, winemakers are not scientists per se. They are,
perhaps more appropriately regarded as artisans, with the emphasis on the art'. As
for many human endeavours, the Arts progress with developments in technology;
think of the use of acrylic paint in the fine arts since its introduction in the 1950s, or
David Hockney's use of a Polaroid camera to create photocollages. In the way that
acrylic paint and photography have provided more options to artists, enabling them
to broaden their horizons, yeast science and technology is adding to the
winemaker's palette. Who knows what bottled masterpieces await us as we sculpt
novel yeast strains in the laboratory using molecular, systems and synthetic biology.
The only real obstacle that we face is consumer acceptance of GMOs; we can only
hope that rationality will eventually prevail.

An important topic of discussion regarding natural wine is native, spontaneous,


or indigenous yeast fermentation. Perhaps youve heard these terms mentioned
at your local wine shop, or seen them in marketing materials from a winery you like.
Like most topics wine-related, its subject to lots of opinions and debate.

Yeasts are what ferment the sugar in grape juice into alcohol, transforming it into
wine. Yeasts exist everywhere in our environment, and certain strains are
indigenous to certain places. This is why San Francisco sourdough is so unique we
have our own strain of wild yeast that lives in the foggy San Francisco air, giving our
bread its own unique flavor.

Many wineries use commercially-sold yeasts to ferment their wines. These yeasts
are selectively bred or genetically altered by laboratories to enhance favorable
flavor profiles in a wine (such as spiciness or fruit flavor), to tolerate heat extremes
(cold and hot), to limit malodor in fermentations, and to produce wines with
consistent flavor profiles year after year. These yeasts are also resistant to SO2, the
chemical additive used in wine to prevent unfavorable microbal growth and
oxidation. There are many labs out there making hundreds of yeast strains available
in freeze-dried or liquid form for wineries, and each strain comes with its own name
and marketing materials explaining its virtues. These commercially-sold yeasts are
strong and hearty; pitch some into your grape must and they will quickly overpower
any wild yeasts living in it.
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Commercial yeast strains are a relatively new development in the business of


winemaking. People have been making wine for thousands of years, where
commercial yeasts have only existed in modern times. Grapes dont always need
help from commercial labs to turn into wine the vineyard is full of flora that will
happily do the job for free.

Native or indigenous yeast fermentations are started by wild yeasts occurring


naturally on the grapes, in the winery, and in the vineyard. Pick the grapes, crush or
press them, and the fermentation should just start on its own. These wild yeasts
are usually capable of fully fermenting the wine, but at times the fermentation may
seem to slow down or get stuck, requiring the winemaker to wait it out and see if
it restarts later in the season, or to pitch a strong commercial yeast to finish the job
quickly. The other issues with indigenous yeasts are that the wines may taste
different from year to year, and they are more prone to producing malodor during
fermentation. Its a risk for any winemaker to choose a spontaneous fermentation
over inoculating with commercial yeasts, but those who know how to do it well
create (in my opinion) MUCH more interesting, complex, and soulful wines.

Some winemakers believe that theres no such thing as a truly native or


indigenous yeast fermentation, since many wineries have been using commercial
yeasts for years. Those commercial yeasts get into the air and equipment in the
winemaking facility, and can take up permanent residence. Its possible that any
future spontaneous yeast fermentation happening in that facility would be
because of the commercial yeasts used in the past.

Whos to say which yeast fermented a wine? You would have to be a scientist to
figure it out. My belief is that less is more, and I would rather sell a wine
fermented spontaneously, even if that means the fermentation started with a
commercial yeast that just happens to be part of the landscape. So many new
winemakers are making wine in shared custom crush facilities and co-ops, its
hard to say anymore. Unless your wine came from a centuries-old chateau in France
that has never seen a commercial yeast, its hard to say for sure whether or not
your wine is truly indigenous or native yeast fermented.

It would be nave for us to be dogmatic about this topic, insisting that we only sell
native yeast wine, so instead we only offer wines that are fermented
spontaneously, and never through direct inoculation with commercial yeasts. We
simply feel that these wines are better they are more interesting, delicious, and
unique.

The fermenting of grapes and grape juice into wine, is a natural process that has
been enjoyed by mankind for thousands of years. The choices made about soil and
particular grapes in the vineyard and winery, are of relevance to the wine
enthusiast as they directly effect the final product. Wine derives its alcohol by the
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process of fermentation. Grapes on the vine are covered with yeast, mold and
bacteria. By putting grape juice into a container at the right temperature, yeast will
turn the sugar in the juice into alcohol and carbon dioxide. The grape juice will have
fermented and become wine. But fermentation is not the only step in the process of
making wine. The next several pages will discuss in basic terms the total process of
wine making. Links to individual steps in the process are listed to the left if you
prefer to skip around. To start at the beginning of the delicate process of making
one of the world's oldest drinks click on harvesting, where the grapes take their first
step to reaching the goblet.

B. Significance of the Study


In very general terms a wine fermentation occurs when yeast consumes sugar
and converts it into approximately half alcohol and half CO2 gas (carbonation)
by weight.

For example, if you had five gallons of juice that had 10 pounds worth of sugar
in it, and you fermented all of that sugar with yeast, you would end up with 5
gallons of juice that has roughly 5 pounds of alcohol in it.

The other five pounds of sugar would dissipate into the air as CO2 (carbonic)
gas. So in fact the five gallon batch would become five pounds lighter than it
was before the fermentation started.

Realize that the breakdown of alcohol verses gas would not be exactly half and
half, but usually it would be very close. Some variances do occur depending on
external factors such as the amount of available air, nutrients as well as the
type of yeast used. But, rest assured that it would be within 46% one way or
another.

It is important to note here that the 10 pounds of sugar that was in the five
gallon batch may not have come all from sugar you added, but partially from
the fruit as well. And in some cases, such as when making a wine from grapes,
there may be no sugar required at all. In these cases enough sugar is already in
the fruit itself to produce a wine with 11 or 12 percent alcohol.

Alcoholic Fermentation
The important bit converting the sugars to alcohol. Fermentation requires the
action of yeasts to convert the sugars to alcohol. These yeasts can be the natural
yeasts from the vineyard, or specially selected, cultured yeasts. Cultured yeasts are
much easier to control and ensure a more consistent fermentation. Natural yeasts,
on the other hard, ensure a truer manifestation of the vineyards terroir, but are less
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reliable. Fermentations can be more challenging and can sometime be a bit


sluggish. Each has its own set of advantages and disadvantages.

Fermentation vessel is a big decision oak, stainless steel or other inert vessel.
Because of its affinity with oak, Chardonnay is often fermented in small oak barrels.
In contrast aromatic grapes such as Riesling or Sauvignon Blanc are typically
fermented in stainless steel or other inert vessels to preserve their vibrant aromas
and flavors.

Fermentation temperature also impacts the wine. Overall, white wines are typically
fermented at cooler temperatures than red. The cooler the temperature the more
well-preserved the primary fruit aromas and flavors. Warmer temperatures make for
a more structured wine.

Typically when the yeasts have converted all the sugars to alcohol the fermentation
is over and you have a dry wine. However, if the intended style off dry or medium
sweet, the winemaker will stop the fermentation before all the sugars have been
converted, leaving the desired amount of residual sugar.

Malolactic Fermentation (MLF)


After the alcoholic fermentation, some white wines go through a process called
malolactic fermentation. Technically this is not a true fermentation but rather a
conversion of any remaining tart malic acid (think green apples) in the wine to the
softer lactic acid (think milk). Once again, Chardonnay is one of the main wines to
undergo full or partial malolactic fermentation. Ever notice that many Chardonnay
wines have a buttery note? Well, in part due to this process.

C. Scope and Limitations


A wine fermentation has two distinct stages: primary and secondary--also
sometimes described as aerobic and anaerobic fermentations.

* The Primary Fermentation will typically last for the first three to five days. On
average, 70 percent of the fermentation activity will occur during these first few
days. And in most cases, you will notice considerable foaming during this time
of rapid fermentation.

The primary fermentation is also called an aerobic fermentation because the


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fermentation vessel is allowed to be opened to the air. This air plays an


important role in the multiplication of the yeast cells.

Here's how important. The little packets of yeast that is generally called for in a
five gallon wine recipe will typically be multiplied up to 100 to 200 times during
the few days of primary/aerobic fermentation. Without air this multiplying stage
is hindered. That is why it is important that you do not use an air-lock during
the first few days of fermentation and allow the fermentation to be open to air.

Alcohol is being produced during the primary fermentation as well, but a


significant portion of the yeast's energy is being devoted to reproducing itself.

* The Secondary Fermentation is when the remaining 30 percent of of


fermentation activity will occur. Unlike the typical four to seven days the
primary fermentation takes, the secondary fermentation will usually last
anywhere from one to two weeks depending on the amount of nutrient and
sugars still available.

So as you can start to see, the secondary fermentation is much slower with less
activity at any given time. You will also notice the activity becoming slower and
slower with each passing day.

The secondary fermentation is an anaerobic fermentation which means that air


exposure is to be kept to a minimum. This can easily be done by attaching an
air-lock to the fermentation vessel.

It is this reduction in air exposure during the secondary fermentation that


entices the yeast to forget about multiplying and start giving its energy
completely to making alcohol.

* Temperature plays an extremely vital role in the fermentation process. If the


fermentation temperature is too cool, the yeast may not be invigorated enough
to ferment. It will simply remain in the juice, dormant.

If the fermentation temperature is too warm, the yeast may ferment fine, but
the flavor of the wine will usually suffer. This is because of the increased
production of unwanted enzymes by the yeast and the possible growth of
micro-organisms that thrive in warmer temperatures.

The optimum temperature for a fermentation is 72 degrees, but anywhere


between 70 and 75 will do fine.
Page 14 of 36

* Throughout the fermentation process you will need to transfer the wine off the
sediment into a clean container. This is a process that is referred to as "racking"
in most wine making books.

This should be done at the end of the primary fermentation or when the
Specific Gravity reading on your hydrometer reaches approximately 1.030. It
should also be racked after the secondary fermentation as well as right before
bottling the wine.

* It is also important to understand that once the wine's fermentation activity


has stopped that it also needs to be given time to clear as well before bottling.
Yeast is a silty substance that can take up to 2-4 additional weeks to clear up
once the fermentation has stopped.

Once you feel comfortable with the wine making process, your next step is to
purchase the necessary equipment that goes along with each step. You can find
everything you need to start your wine making career at EC Kraus, including a
Wine Making Kit.

Concerns with Stuck Fermentation


One of the greatest concerns in low temperature fermentations is the risk of an
increased lag phase and a decreased growth rate of yeast (Blateryon and
Sablayrolles 2000). This is sometimes referred to as a sluggish or stuck
fermentation. In these situations, yeast are minimally consuming sugar to convert
it to ethanol (Bisson 1999). However, sluggish and stuck fermentations differ from
slow fermentations. In stuck or sluggish fermentations, the rate of sugar
consumption decreases dramatically toward the end of primary fermentation,
while slow fermentations have a decreased sugar consumption rate throughout
the entire duration of primary fermentation (Blateyron and Sablayrolles 2000).
Stuck and sluggish fermentations do not directly result from the low temperature,
but rather from other yeast stressors including, but not limited to: nitrogen
deficiency, lack of oxygen, excessive clarification, microbial spoilage growth, pH,
and fatty acid development (Van de Water 2000). However, the use of
temperature control should be regularly monitored as cooler temperature can
inflict some of these properties on the wine during fermentation.
When a stuck or sluggish fermentation occurs, winemakers are encouraged to test
the wine to determine the cause and develop options for treatment. Delle Units
are one way to evaluate a sluggish wine. With this method, it is first necessary to
test alcohol and reducing sugar separately. Delle Units can be calculated using
the equation [(4.5 x % alcohol)
+ % reducing sugar]. If the Delle Unit is at or above 65, restarting the
fermentation is possible but will be difficult (Van de Water 2000). At or above 80,
the chances of restarting fermentation are slim to none (Delfini 2001). When
Page 15 of 36

fermentation cannot be restarted, several measures can be taken to get the


fermentation out of its sluggish state, such as introducing oxygen or re-inoculating
with a hardy yeast strain (Sablayrolles et al. 1996).
Testing for Lactobacilli is another way to provide insight on a sluggish wine. A
presumptive confirmation of Lactobacilli can be made using a phase contrast
microscope to look for
rod-shaped bacteria (Figure 2). Some species of Lactobacilli can produce acetic
acid, and growth can occur during a time when S. cerevisae growth is stuck and
wine is generally unprotected by sulfur dioxide (Edwards et al. 1999). The
presence and growth of Lactobacilli can make restarting fermentation challenging.
If no Lactobacilli or other spoilage bacteria are present, it is possible to take
measures and get the fermentation progressing by slightly increasing wine
temperature to more optimal ranges for yeast growth. If temperature increase
alone does not restart the fermentation, re-inoculation of a hardy wine yeast is
typically recommended (Van de Water 2000). Winemakers may need to ensure
that all other conditions, including oxygen content, are optimal for their selected
yeast to finish the fermentation to dryness. Many suppliers offer stuck or sluggish
fermentation protocols to restart fermentation.

Going beyond ideal fermentation temperatures can cause problems. Ferment too
hot or too cold and your wine will suffer.

Fermentations that get too hot not only ferment too fast but it could lead to
cooked flavors. Your wine will taste like it was boiled on the stove. Additionally,
yeast can only tolerate fermentation temperatures that are so high. Go beyond their
maximum temperature tolerance and theyll die.

Keep in mind that the fermentation process is exothermic which means that heat is
produced as the yeast are doing their work. So even if your wine is stored where the
room temperature is within the ideal temperature range your wine could still over
get over heated.

At the other extreme if your wine gets too cold your yeast will go dormant. The good
news here is that when your fermentation temperature rises again the yeast will
likely wake up again and continue fermenting. Even if they dont come back you
can pitch more yeast and continue where you left off. Your wine wont get damaged
by excessively cold temperatures like it will with excessively warm temperatures.

The simplest way to monitor your fermentation temperature is to use a sanitized


kitchen thermometer. Just open up your fermentation vessel and take a
measurement. Be sure to work as quickly as you can to limit the amount of time
your wine is exposed to oxygen.

Another option is to get a self adhesive temperature strip. It sticks right to the side
of your carboy or fermenter and displays the internal temperature. While these
Page 16 of 36

arent accurate to the tenth of a degree or anything they will at least give you a
good idea of where you are in the temperature range.

There are several inexpensive products to help you warm up your wine when
fermenting in a cool environment. The least expensive option is using an insulative
wrap on your carboy. These are passive and only trap heat produced by
fermentation.

Another option is to use a heating belt or a ferm wrap. These are electric heaters
that wrap around your carboy or fermenter. Once plugged in they will raise the
temperature of your wine by approximately 10 degrees F.

To get finer control of how warm your wine is you can pick up a thermostat which
allows you to dial in the temperature to within a degree of what you set it
at. Heating bands usually run around $20 and the thermostat is another $80.

Another similar device is a heating panel. These products are just a flat pad that you
place your carboy on top of. When plugged in they heat up to a pre-determined
temperature. Again, you can pick up thermostats for these too.

The easiest way to bring down a high fermentation temperature is to place the
fermenter or carboy in an ice bath. Simply place your container in a plastic tub, add
cool water, and then add ice. Be very careful when moving your carboys especially
if theyre wet. Remember, if you go too far your fermentation can stop.

Wine cooling equipment can be quite expensive. There are a few products on the
market to help you with this task, however, most solutions cost well over $1000.

Beyond using ice water you could opt for a glycol chiller to use on the wine itself or
you can cool the entire room. The latter may not be practical if you dont have a
thermally isolated room to ferment your wine in. Glycol chillers start at around
$1600 and go up from there. Theyre designed to work on larger fermentation tanks.

One intermediate option is to pick up a stainless steel tubing coil or heat exchanger
used in glycol chiller system and run cold water through it. This works a little more
slowly and youll have to figure out your own system of recirculating the cold water
or find a garden to pump it into. Coils start at around $300.

II. Body of the Research Paper

A. Related Literature/ Studies( Local and Foreign)

Temperature control in the winery involves manipulating temperature to slow down


or accelerate winemaking processes or control chemical changes in the wine. Since
wine is easily influenced by temperature, this technique is frequently employed for
processes such as fermentation, cold settling, aging, and storage.
Temperature is a vital part of fermentation in winemaking. Fermentation occurs
when yeast convert sugar to alcohol and carbon dioxide (Figure 1). Due to the
Page 17 of 36

exothermic nature of fermentation, temperature increases as sugars are


metabolized. Heat production can be managed with the usage of external
temperature control. This typically involves controlling tank or cellar temperature
to keep the wine at a relatively lower temperature than it would reach naturally.
Temperature manipulation changes the dynamics of the fermentation system and
can be a beneficial source of control in winemaking.

Figure 1. Chemical equation of alcoholic fermentation (Pavia 2015).

Desirable fermentation temperatures vary for red and white wines. Red wine
fermentation temperatures are optimally between 68-86F (20-30C), while white
wine fermentation temperatures are recommended at or below 59F (15C)
(Reynolds et al. 2001). Higher temperatures are favorable in red winemaking to
enhance extraction of color, phenolics, and tannins from skins (Reynolds et al.
2001). The goal of white wine fermentation temperature control is to preserve
compounds that contribute to aroma and flavor (Molina et al. 2007).
versus 28C. These findings exemplify that temperature creates differences in
flavor and aroma.
Temperature control has also been shown to prevent the development of off-
flavors, specifically volatile
sulfur-containing compounds like hydrogen sulfide. Higher (above 30C/86F)
fermentation temperatures increase the rate of fermentation, which may enhance
production of undesirable byproducts produced by the yeast (Reynolds et al.
2001). The most common off-flavors associated with high temperature and rapid
fermentation are sulfur-derived compounds including hydrogen sulfide,
mercaptans (thiols) and disulphides (Gladish 1999). These compounds have been
associated with aromas or flavors reminiscent of rotten eggs, cooked cabbage,
skunk, and landfill, amongst other descriptors. While some of these off-aromas
may be remediated with copper additions, use of clean lees, or other product
additions, these techniques can be challenging to achieve a clean wine and time
consuming on cellar personnel. Furthermore, additional flavor stripping of
desirable aromatics may occur (Pickering et al. 2005).
Cooler fermentations have been shown to improve the clarity of wine (Comfort
2012). Clarity is typically achieved through racking, cold settling, cold
stabilization, and fining, but low temperature fermentation may be an alternative
option for enhanced clarity. At lower temperatures, yeast cells are less likely to
give off colloids, thus improving clearness (Jackson 2014). Colloids derive from
yeast and pectin, which form polysaccharides and aggregates after a
physiochemical change (i.e. temperature), diminishing clarity (Drioli et al. 2010).
Therefore, the use of lower temperature fermentation may minimize potential
for cloudy or hazy wines by the end of primary fermentation.
Lowering fermentation temperature can also better control microbial growth and
Page 18 of 36

minimize potential spoilage (Jackson 2014). For example, at higher fermentation


temperatures (above 21C), some bacteria can produce undesirable amounts of
diacetyl, creating a buttery flavor that may be unwanted (Bartowsky 2009). Also,
spoilage yeasts such as Pichia membranifaciens, Pichia anomala, and Candida
species can develop in the absence of proper temperatures, contaminating wine
surfaces and producing unwanted acetic acid or ethyl acetate volatiles (Loureiro
and Malfeito-Ferreira 2003). The growth of spoilage organisms is dependent on
various factors, such as oxygen exposure. But, the use of temperature control can
be an effective hurdle in minimizing their presence. This can potentially reduce
the need for higher doses of sulfur dioxide through production. As consumer
perception encourages limited sulfur dioxide use (Pretorius and Bauer 2002), such
practices can be advantageous to wineries brands.
White wines, especially aromatic varieties (e.g. Reisling, Gewrztraminer,
Traminette, Vidal Blanc), ross, and fruit wines benefit from the effects of lower
temperature fermentations (below 15C or 59F) to help retain their delicate
aroma and flavor described previously. Red wines traditionally require higher
temperature fermentation (~28C,
82.4 F) to achieve color and tannin extraction from the skins (Molina et al. 2007).
Color extraction is particularly important

The harvest season marks a monumental time where it all comes together for grape
growers and winemakers. The lack of rainfall during the last few weeks of the
growing season usually transpire into a relatively ideal harvest. Warm, dry days and
cool, dry nights allow grapes to mature optimally.

In the eastern U.S., however, each growing season is unpredictable. Preparation for
incoming, diseased fruit can help winemakers make well-informed decisions during
processing to minimize the influence of disease on wine quality. Botrytis, which
typically flourishes in more humid or higher precipitated years, is one disease that
winemakers should be prepared to handle with incoming fruit. While management
and prevention in the vineyard is obviously the best strategy for dealing with
Botrytis, some years it's presence is unpreventable.

In Loinger, C. et al. (1977), Semillon grapes were fermented to assess the effects of
Botrytis rot on chemical composition of grape must, the wine qualitys sensory
attributes, and the chemical composition of wine. This research showed several
changes in the must and wine chemical composition chemically, associated several
sensory generalizations (i.e., color, aroma, flavor) in wines produced with rot.
Additionally, Bruce Zoecklein has noted in his Enology Notes that Botrytis routinely
causes a loss of fruitiness in wine, and may produce off-flavors that are phenolic
and iodine-like in their descriptions. Loinger et al.s guidelines on cluster rot
infestation can be summed up as follows:

5 10% rot on clusters: noticeable reduction in wine quality; wine quality is


still good (as opposed to very good with 0% rot on clusters)
Page 19 of 36

20 40% rot on clusters: marked reduction in wine quality; wine quality is


low

>80% rot on clusters: wine is commercially unacceptable

The paper concluded, although not tested, that greater than 40% rot on clusters
would create a wine in the range of low quality to commercially unacceptable. It is
reasonable for winemakers and cellar assistants to evaluate incoming fruit,
regardless of disease or rot infestation, and decide if it is worth the time and
finances to ferment the received crop.

What is a vintner to do?

Fermenting with Rot: Emphasis Botrytis (Gray Mold)

Create a sorting table and sort through diseased fruit


Investing in a sorting table may be an integral part in many winery's standard
operating procedures during harvest operations. However, if the winery is not set
up with a sorting table, taking the time at harvest to carefully pull out diseased fruit
manually can save a lot of time and effort during and after fermentation. Remember
that the rot organisms will impart characteristics on the wine as it ferments, causing
changes in the aroma and flavor composition and depleting the varietal characters
associated with properly ripened grapes. The goal in removing diseased fruit prior to
fermentation is to minimize the influence of disease-associated attributes on the
wine by reducing the amount of contact the product has with diseased component.

Do not contaminate clean fruit with rot infested fruit


Simple cleaning and sanitizing protocols need to be utilized when dealing with
rotted fruit. Washing off crusher/destemmers with hot water is not enough to
decontaminate the processing equipment from the rot infestation. There are a few
easy steps wineries can integrate into harvesting operations to minimize the
potential for disease contamination:

If you are processing more than 1 variety in 1 day, process the cleanest fruit
first. You should end with the dirtiest fruit.

Wash and sanitize all equipment prior to use. Theres no point in allowing
dead bugs, old mildews, and dirt in your product. This practice decreases the
potential risk that can be detrimental to wine quality as soon as grapes get to
the processing area.

Physically wash off equipment in between lots of grapes. This is just good
practice. Use water to initially clean leftover fruit and debris from the
processing equipment, and manually scrub off any debris before properly
sanitizing equipment. In our research winery, we use a citric acid/SO 2 rinse,
but you can also use commercial cleaners and sanitizers. If the sanitizer
Page 20 of 36

requires a neutralizing agent or water rinse, make sure you do this before
putting in the next lot of fruit. Always follow supplier safety instructions.

Make sure you take the time to properly wash and sanitize processing
equipment at the end of the day. This keeps your equipment in good
condition, reduces biofilm formation, and minimizes chances for any external
contamination.

With whites and reds, limit contact time with the skins
Botrytis resides on the skins with the grapes, and minimizing skin contact with the
pulp will inhibit extraction of Botrytis-related components into your wine. Remember
that red wines require skin contact in order to extract the red pigments
(anthocyanins) from the skins. However, there are several available options for
winemakers. Pre-fermentation flash pasteurization techniques that will quickly
increase red pigmentation and inhibit Botrytis. It should be noted that this
technique does impart its own flavor characteristics on the wine, but it will likely be
more beneficial than the disease-related flavors and aromas. If flash pasteurization
is not an option, consider removing

Whole cluster press your whites, and separate press runs


Fugelsang and Edwards (2007) recommend separating out the first 10+ gallons that
are rich in Botrytis metabolites. Separation of press fractions is key here, as it allows
more control over the phenolic content, solids, and potential off-flavors associated
with Botrytis.

During cold settling of whites, it may be necessary to do a slight bentonite fining


A light bentonite fining in the juice should reduce your laccase enzyme content.
Laccase, unlike polyphenol oxidase, is not inhibited by the alcohol content produced
during fermentation. Therefore, laccase can cause premature browning of young
wines. Grapes high in Botrytis infection are often rich in laccase concentrations.
Refer to your suppliers product recommendations for this step if you think it is
essential.

Press lightly this is not the time where you want to over-extract
With high Botrytis-infected fruit, pressing for increased yield is not recommended.
The increased pressure will not only over-extract tannins, but also extract more rot-
associated compounds and flavors that are detrimental to wine quality.

Use PVPP prior to fermentation to minimize color oxidation


PVPP will help strip out potential browning compounds or their precursor forms
before fermentation begins.

Do not undergo a native/natural fermentation: use a commercial yeast strain


With disease-heavy fruit, the microflora associated with the disease are a part of the
potential microorganisms that can contribute off-flavors to your final product. It is
Page 21 of 36

recommended that winemakers inoculate with a commercial yeast strain that is


recommended for Botrysized fruit. Most suppliers will have a recommendation for
red, white, and ros wines under Botrytis conditions. Generically, you want a yeast
strain that is robust and can ferment under disease pressure and its associated
fermentation inhibitors.

Limit your oxygen exposure to crushed fruit: use Nitrogen and Argon gas blanketing
Although this will slightly inhibit your commercialized yeast strain, the lack of
oxygen will completely knock out invasive yeasts and microorganisms, including
Acetobacter, which are usually more prevalent in rotted grapes. Ask your supplier if
the commercial yeast can handle this treatment.

If you have more than 10% rot on clusters, consider getting a Laccase Test from a
wine lab
Laccase is an oxidative enzyme that is produced in higher concentrations with
Botrytis infection. Laccase generally progresses the rate of oxidation (i.e., turning
wines brown or making them taste like sherry) even in very young wines. The
results of the Laccase Test will tell you whether or not you need to go extreme
treatments (e.g., heat treatment, early bentonite fining) to avoid rapid oxidation.

Consider the use of high temperature, short time (HTST) treatments


Some people also call this Flash Vinification or Pasteurization in which the must
is heated very quickly to a high temperature and then quickly chilled back down to
a reasonable fermentation temperature. The quick cooling time is essential. Too
much heat for too much time will not only kill the bacteria and denature harmful
enzymes, but it will also denature proteins and other compounds in the fruit. It can
also cause cooked flavors in the final wine, which are not preferred by consumers.

Add SO2 to inhibit the natural microflora on the clusters


There are several recommendations on how much SO 2 to add, which is dependent
on the variety (white, ros, or red) and the extent of Botyris infection. In general, for
lower Botrytis infections, a 30 ppm SO2 addition in the harvest bins followed by an
additional 20-30 ppm after crushing and destemming should suffice. For larger
Botrytis infections, a 40 ppm SO2 addition in the harvest bins followed by an
additional 30-40 ppm addition after crushing and destemming is recommended.

Decrease the pH of your red wines


Decreasing the pH will help inhibit natural microflora from growing and proliferating
during primary fermentation. However, it is important for winemakers to choose a
commercial yeast strain suitable for low pH conditions. Additionally, lowering the pH
can have potential sensory implications.
Page 22 of 36

Treat the must with Lysozyme


A Lysozyme addition will not do anything to the Botrytis infection directly, but it will
decrease populations of potential lactic acid bacteria spoilage organisms. This may
be necessary if the fermentation has a hard time getting started. (It is
recommended that winemakers evaluate the lactic acid bacteria levels in the must
prior to treating it with Lysozyme. You can do this by simply evaluating the must
under a microscope or sending a sample to a wine lab.)

Add tannins prior to red wine fermentations


The use of pre-fermentation tannins will do several things for your fermentation
when dealing with Botrytis-infected fruit:

Binds with some of the active enzymes that may be destructive towards the
fermentation (e.g., laccase).

Will offer binding materials for anthocyanins to enhance for better color
stability throughout and after fermentation.

Enhance the mouthfeel of the red wine. Botrytis (and disease in general)
tends to thin the mouthfeel of many wines, which extenuates the off-flavors
associated with Botrytis. Building mouthfeel will off-set this phenomena.

Use a gluconase enzyme product, especially in white wines during cold settling
and before fermentation
This enzyme will help break down some of the solids associated with Botrytis
infections. Additionally, this enzyme should help make clarification and filtration
easier on the wine after fermentation. Both processes are often challenging
for Botrysized white wines.

Manage your free SO2 after primary fermentation and malolactic fermentation (MLF)
Checking the free SO2 concentration every other week or once every month can
help inhibit spoilage microorganisms from proliferating during times when the wine
is not actively worked on. Remember that the molecular SO 2 concentration is always
changing and heavily associated with pH. Every time the wine is moved, the
free SO2 should be confirmed to ensure that the proper molecular level is being
obtained in the wine.

Manage your numbers


Good quality control programs are essential for winemakers even when making
wines without disease pressure. However, having analytical records from the start of
processing through bottling of the wine will assist winemakers in making proper
processing decisions. Get a juice panel (Brix, pH, TA, malic acid, and YAN) prior to
fermentation, with, perhaps, some of the additional analyses for heavily-
infested Botrysized fruit (i.e., Laccase Test). At the completion of primary
fermentation, check your glucose/fructose levels (reducing sugar to ensure that
Page 23 of 36

your fermentation is dry to avoid any microorganisms from utilizing left over sugar),
volatile acidity (VA) to ensure that it is not high and to know the starting level post-
primary fermentation, and malic acid concentration. Make sure that your MLF is
complete by getting the malic acid concentration checked enzymatically after a
paper chromatography result shows that it has completed. Regularly
monitoring SO2 is also an important step.

Trust your palate dont just add sugar


Some people will add sugar to sweeten wines when less than optimal grapes were
received at harvest. If the wine successfully ferments to dryness (<1 g/L RS) and
completes MLF, it is recommended that winemakers stabilize the problem wine and
taste it periodically over time before making sugar additions. If it has no residual off-
flavors from the Botrytis infection, then do a bench trial and determine adequate
sugar additions. However, do not underestimate the possibility of blending out the
wine into blended wines. Depending on the starting level of Botrytis, there may be
some off-flavors that developed during primary fermentation that are not well
complemented with sugar additions. However, they may be minimized, or masked,
through blending with clean wines.

These are all suggestions that will depend on the extent of Botrytis infection on the
incoming fruit, and what you are capable of accomplishing at your facility. If you can
estimate that less than 10% of the fruit is infected, minor treatments are usually
needed to ensure a successful fermentation and quality product. Above 15%
infected fruit is where processing operations can get a bit tricky to maintain wine
quality.

Remember that clarification and filtering is a challenge with wines produced


from Botrysized fruit. Taking some of these suggestions above, should help
minimize filtration problems at later stages in wine processing.

The knowledge and the understanding of the microbial terroir how the microbiome
contributes to the natural environment of grapes and to the identity of wine, is a
process that starts at the vineyards, at the harvest of grapes, and then evolves
along the different stages of fermentation (Van Leeuwen and Seguin, 2006; Bokulich
et al., 2013). Indeed, it is known that grapes harbor a complex microbiome,
including a high range of filamentous fungi, yeasts and bacteria with different
physiological and metabolic characteristics (Pretorius, 2000; Fleet, 2003; Barata et
al., 2012). The microflora of the grapes is highly variable, mostly due to the
influence of external factors as environmental parameters, geographical location,
grape cultivars and application of phytochemicals on the vineyards (Pretorius, 2000;
Cadez et al., 2010; Pinto et al., 2014). These microbial communities play an
Page 24 of 36

important role during the winemaking process, as they metabolize the sugars from
the grapes and produce a whole set of secondary metabolites that influence the
wine aromatic quality (Fleet, 2003). In fact, the natural diversity of those metabolic
pathways, and the contribution of the different microorganisms involved on the
fermentation process, is well documented (Setati et al., 2012). Therefore, unveiling
the microbial biodiversity of grapes and during their fermentation will expand our
understanding on fermentation dynamics, on its control (Bisson, 1999; Bisson and
Butzke, 2000) and may also contribute to the identification of novel starter cultures
(Fleet, 2008; Ciani et al., 2010).
The spontaneous wine fermentation is carried out by indigenous microbiota (Heard,
1999; Pretorius, 2000; Ciani et al., 2006; Renouf et al., 2007). Species of
Metschnikowia, Candida, Hanseniaspora, Pichia, Lachancea (Kluyveromyces), and
Saccharomyces are often present at the initial stages of wine fermentations and
form the dominant consortium (Cocolin et al., 2000; Mills et al., 2002; Fleet, 2008).
However, during the wine fermentation, the ethanol content increases and
Saccharomyces cerevisiae strains dominate the alcoholic fermentation (AF; Fleet,
2008). Additionally, a deacidification may occur, by conversion of malic acid into
lactic acid. This process is known as malolactic fermentation (MLF) and is due to the
activity of lactic acid bacteria (LAB; Lonvaud-Funel, 1999; Lerm et al., 2011). The
LAB species associated with MLF generally belong to the Oenococcus, Pediococcus,
Lactobacillus, and Leuconostoc genera (Lonvaud-Funel, 1999). Indeed, MLF mainly
influences the organoleptic characteristics and the aging of wines (Lonvaud-Funel,
1999). On the other hand, acetic acid bacteria (AAB) may cause a negative impact
on the winemaking process, due to the production of undesirable metabolites, as
acetic acid, thus affect negatively the quality of wine and so are considered spoilage
microorganisms (Zoecklein et al., 2000).
The majority of the wine microbiology studies focus on the characterization of S.
cerevisiae strains (Pretorius, 2000; Fleet, 2008; Nisiotou et al., 2011). Nevertheless,
recent studies based on culture-independent methods, started to explore the
microbial communities associated with wine grapes (Bokulich et al., 2013; Taylor et
al., 2014). It is widely accepted that unveiling the indigenous microbial community
associated with particular grape varieties, from specific locations, could represent
an important source of distinctive metabolites and introduce an authenticity terroir
to the region (Heard, 1999; Jolly et al., 2006; Fleet, 2008). The biogeographical
distribution of the wine associated microorganisms has been recently investigated
in vineyards from different regions of California (Bokulich et al., 2013), New Zealand
Page 25 of 36

(Taylor et al., 2014), and in conventional, biodynamic, and integrated vineyards of


South Africa (Setati et al., 2012). These studies allowed for a better spatial and
temporal characterization of the wine grapes microbiome and brought new insights
of its dynamics and biodiversity. Also, other biogeography wine studies have been
previously published focusing on S. cerevisiae (Schuller et al., 2012). Nevertheless,
there is still a lack of knowledge on the diversity and the dynamics of microbial
communities as a whole from the wine grapes until the wine fermentation, which
can now be obtained using high-throughput sequencing technologies and
metagenomics approaches that allow for the identification of both non-cultivable
microorganisms, and of less represented species.

Many notable wine regions have been recognized with an integrated use of
temperature control, as this processing aid has been shown to improve wine
quality dramatically.
Temperature control is an essential tool when crafting
high-quality wines, but winemakers are encouraged to monitor its use. White,
ros, fruit, and aromatically delicate wines have a greater need for temperature
control during fermentation to enhance aroma and flavor retention. Red wines
require higher temperature fermentations for color and tannin extraction, but can
still benefit from temperature control throughout wine production.
There are a number of solutions for temperature control, based upon winery size
and financial flexibility. These options provide incremental improvement steps
directly related to wine quality, overall. Over time, wineries can slowly enhance
temperature control steps to impact wine quality as economic means are
retained.

B.Discussion

During the primary fermentation of wine, the two grape sugars, glucose and
fructose are converted to alcohol (ethanol) by the action yeast. The by-products of
primary fermentation are aromas and flavours, the gas carbon dioxide, and heat.
The production of heat during fermentation (i.e. it is an exothermic process) means
that during fermentation the temperature of the fermentation vessel will rise, and
will require action on the part of the winemaker to cool it down. White fermentation
is usually conducted in the range of 8-19OC, and red wine fermentions typically are
allowed to run at between 25 and 32oC. At temperatures higher than this, there can
be a loss of desirable aroma and flavour compounds, and unattractive aroma
characters in the spectrum of caramel, burnt or cooked characters can be produced.
There are many types of yeast, but two closely related types known as
Saccharomyces cerevisiae and Saccharomyces bayanus are the ones that are
responsible for fermentation.

These species of yeast are encouraged to conduct the fermentation because they:
Page 26 of 36

Are alcohol tolerant. That is, they can continue to ferment sugars to alcohol
even during the latter stages of fermentation when the sugar is low but the
alcohol content is high.

Can establish a viable population in an environment of high sugar (190-270


grams per litre) and high acidity.

Are strong and consistent fermenters even at cold temperatures.

They ferment quickly and only stop when all the grape sugars have been
utilised. Otherwise we would be buying sweet low alcohol wines.

Are more tolerant to sulfur dioxide than other yeasts and bacteria.

They produce wine like aroma and flavour characters. A free list of wine
fermentation characters can be found here, or can be purchased as a beaut
full colour laminated wine tasting wheel here.

In white winemaking the juice is usually inoculated with yeast following its
clarification. The most common type of inoculation is that using an active dried
yeast culture. Yeasts are freeze dried and stored in vacuum packed tins by yeast
supply companies ready for use. The winemaker then re-hydrates the yeast in warm
water bringing them out of their state of suspended animation. The hydrated yeast
is then added to the juice.

Another less interventionist approach is to let nature take its course. The grapes
have a bloom that contains active cells of Saccharomyces cerevisiae. After juicing
the grapes these grape yeast cells and other yeast cells picked up from the winery
equipment then start to convert the grape sugars to alcohol. This type of
fermentation is variously called natural, indigenous or spontaneous fermentation.

The former approach is common in New World winemaking whilst the latter is more
often practiced in Europe. So what are the advantages and disadvantages of each
approach to inoculation?

The advantages of winemaker inoculation are:

Choice. Many different strains of Saccharomyces cerevisiae have been


isolated and have different properties. Some produce very fruity (estery)
aromas, others produce more neutral characters. Some ferment better at
colder temperatures than others.

Consistency. All strains are selected for their ability to meet the general
necessary criteria given above. This maximises the chances of clean and
complete fermentations occurring.

The advantages of spontaneous inoculation are:


Page 27 of 36

Better Mouthfeel. Many commentators' report that they feel that spontaneous
fermentation promotes better mouthfeel in wines. That is, the wines are
thought to be softer and creamier than those made using single strain starter
cultures. This suggestion however has not been conclusively demonstrated
scientifically. The rational for the suggestion is that the natural yeast flora on
the grape is genetically heterogeneous, i.e. consists of multiple strains and
that this is the reason for the improved mouthfeel.

Cost. Spontaneous fermentation costs nothing to initiate.

The disadvantages of spontaneous fermentation are:

Lag time. Yeast cells increase in number by division. One becomes two, two
become four, four become eight etc. The initial numbers of yeast cells in an
un-inoculated juice are by nature low. Dividing from a low initial base of cells
means that it takes longer for fermentation to become active. A wait of 3-4
days is typical. In large commercial wineries where the vintage is planned to
work like clockwork, with tanks becoming free just when purchased fruit is to
arrive at the weighbridge, the uncertainties of spontaneous fermentation
present excessive risk. When inoculating, the winemaker adds about 5 billion
cells per litre. Quite a head start over spontaneous fermentation.

Higher probability of spoilage. In theory this should be a problem but in


practice it rarely is. Sometimes other spoilage yeasts and bacteria take
advantage of the ecological void caused by the shortage of active yeast cells
in the early stages of spontaneous ferments. In practice this can be avoided
by ensuring that the acidity of the juice is sufficiently high. Saccharamyces
cerevisiae is more tolerant to acidity and sulfur dioxide than most other
organisms, and winemakers use these facts and engineer the juice
environment to favour its growth over the undesirable microbes.

The white juice may either be entirely fermented in barrel, or in tank, and when
partially completed, transferred to barrel. With red wines, practicalities demand that
they be fermented in tank for most of duration of the ferment (so as to ensure
appropriate extraction of colour and tannin from the skins), before the coloured
partly fermented juice is drained into barrel to complete the fermentation.
Temperature control in barrel ferments is pretty basic. The barrels are placed in a
cool room and the wine is left to ferment. The smaller volume and relatively high
surface area of the barrel aids in temperature control. Barrel fermentation is
generally carried out for premium full bodied styles and used with varieties such as
Chardonnay, Semillon and occasionally Sauvignon Blanc. Any full bodied red wine
can benefit from partial barrel fermentation.

White wines are often left in contact with the dead yeast cells (lees) that fall to the
bottom of the barrel following the completion of fermentation. This contact is
usually for a period of six to nine months, and the lees can either be left or stirred
(battonage) at regular intervals. Barrel fermentation and lees contact increases the
Page 28 of 36

aroma and flavour complexity, imparting smokey, toasty and cheesy flavours to
white wines. Some winemakers also feel that barrel ferment results in better
integration of fruit and oak, and that imparts a creamy texture to the mouth-feel.
However if it is overdone, the wine can take on overt doughy and vegemite
characters.

Fermentation is completed when all the fermentable sugars have been converted to
alcohol. This end point is measured chemically. Once the wine is deemed free of
fermentable sugar, i.e. dry it is cooled to 4oC and the dead yeast cells (known as
gross lees) are allowed to settle. The relatively clear wine is then racked to remove
the gross lees before it it is stabilised and filtered ready for bottling.

Even a very complex wine is only alcoholic grape juice. The alcohol is produced by a
process called fermentation.

Fermentation requires two things : sugars and yeasts.

A ripe organic grape is full of natural sugars and there are wild yeasts living on its
skin. As soon as the skin of the grape is broken, fermentation can begin.

To make wine, all the winemaker has to do is collect his grapes and gently crush
them, releasing the sugary juice and exposing it to the yeasts.

Fermentation will continue until all the sugar has been turned into alcohol or the
level of alcohol in the juice reaches around fifteen percent, whichever is sooner.

At around fifteen percent alcohol, the yeasts will die naturally and any left over
sugars will remain in the wine.

Yeasts
A natural wine is fermented only with the wild yeasts native to its terroir.

Yeast strains vary widely from place to place and contribute significantly to the
odour of the finished wine. The yeasts indigenous to a particular area are an
important part of what gives its wines their character.

Conventionally grown grapes have little or no wild yeast living on their skin.

The winemaker will kill whatever yeast remains with sulphur dioxide, and reseed the
grapes with a single strain of commercially produced yeast.
Page 29 of 36

Wines fermented in this way have less personality, all using the same few
commercial yeast strains, and are less an expression of their terroir. This is one
reason they taste so similar.

They are also less complex, as each of the many wild yeasts present on an organic
grape will contribute something to the finished wine.

Sugars
The level of alcohol in the finished wine is determined by the level of sugar in the
grapes from which it is made.

More sugar means there is more for the yeast to convert into alcohol.

Grapes grown further north see less sun and therefore contain less stored sugar
than those grown in the south. Traditionally, therefore, northern wines contain a
lower level of alcohol.

Chaptalization is a way of boosting the level of alcohol in the finished wine by


adding sugar to the juice during fermentation.The technique is named after Jean
Antoine Chaptal, Napoleon's minister for agriculture, who is said to have invented it.

A natural wine is fermented only with its own sugars.

Malolactic fermentation
Malolactic fermentation is a secondary process of bacterial conversion, which may
follow or overlap with primary fermentation.

Harsher tasting malic acid is converted into softer, and less acidic, lactic acid.
Carbon dioxide is also produced.

In practical terms this means a reduction in the acidity of the wine and an increase
in its complexity. The level of alcohol is unaffected.

Like primary fermentation, malolactic fermentation can be induced by the


introduction of cultured bacteria, or suppressed with sulphur dioxide.

If a wine is bottled quickly, it may take place inside the bottle. One reason SO2 is
used at bottling is to prevent this.

A natural winemaker has to wait for the malo to finish naturally before he can bottle
his wine.
Page 30 of 36

III. Summary and Recommendation

In winemaking, there are distinctions made between ambient yeasts which are
naturally present in wine cellars, vineyards and on the grapes themselves
(sometimes known as a grape's "bloom" or "blush") and cultured yeast which are
specifically isolated and inoculated for use in winemaking. The most common
genera of wild yeasts found in winemaking include Candida,
Klckera/Hanseniaspora, Metschnikowiaceae, Pichia and Zygosaccharomyces. Wild
yeasts can produce high-quality, unique-flavored wines; however, they are often
unpredictable and may introduce less desirable traits to the wine, and can even
contribute to spoilage. It should be noted that few yeast, and lactic and acetic acid
bacterial colonies naturally live on the surface of grapes, but traditional wine
makers, particularly in Europe, advocate use of ambient yeast as a characteristic of
the region's terroir; nevertheless, many winemakers prefer to control fermentation
with predictable cultured yeast. The cultured yeasts most commonly used in
winemaking belong to the Saccharomyces cerevisiae (also known as "sugar yeast")
species. Within this species are several hundred different strains of yeast that can
be used during fermentation to affect the heat or vigor of the process and enhance
or suppress certain flavor characteristics of the varietal. The use of different strains
of yeasts is a major contributor to the diversity of wine, even among the same
grape variety. Alternative, non-Saccharomyces cerevisiae, yeasts are being used
more prevalently in the industry to add greater complexity to wine. After a winery
has been in operation for a number of years, few yeast strains are actively involved
in the fermentation process. The use of active dry yeasts reduces the variety of
strains that appear in spontaneous fermentation by outcompeting those strains that
are naturally present.

The addition of cultured yeast normally occurs with the yeast first in a dried or
"inactive" state and is reactivated in warm water or diluted grape juice prior to
being added to the must. To thrive and be active in fermentation, the yeast needs
access to a continuous supply of carbon, nitrogen, sulfur, phosphorus as well as
access to various vitamins and minerals. These components are naturally present in
the grape must but their amount may be corrected by adding nutrients to the wine,
in order to foster a more encouraging environment for the yeast. Newly formulated
time-release nutrients, specifically manufactured for wine fermentations, offer the
most advantageous conditions for yeast. Oxygen is needed as well, but in wine
making, the risk of oxidation and the lack of alcohol production from oxygenated
yeast requires the exposure of oxygen to be kept at a minimum.
Page 31 of 36

Dry winemaking yeast (left) and yeast nutrients used in the rehydration process to
stimulate yeast cells.

Upon the introduction of active yeasts to the grape must, phosphates are attached
to the sugar and the six-carbon sugar molecules begin to be split into three-carbon
pieces and go through a series of rearrangement reactions. During this process, the
carboxylic carbon atom is released in the form of carbon dioxide with the remaining
components becoming acetaldehyde. The absence of oxygen in this anaerobic
process allows the acetaldehyde to be eventually converted, by reduction, to
ethanol. During the conversion of acetaldehyde, a small amount is converted, by
oxidation, to acetic acid which, in excess, can contribute to the wine fault known as
volatile acidity (vinegar taint). After the yeast has exhausted its life cycle, they fall
to the bottom of the fermentation tank as sediment known as lees. Yeast ceases its
activity whenever all of the sugar in must has been converted into other chemicals
or whenever the alcohol content has reached 15% alcohol per unit volume; a
concentration strong enough to halt the enzymatic activity of almost all strains of
yeast.

Other compounds involved


The metabolism of amino acids and breakdown of sugars by yeasts has the effect of
creating other biochemical compounds that can contribute to the flavor and aroma
of wine. These compounds can be considered "volatile" like aldehydes, ethyl
acetate, ester, fatty acids, fusel oils, hydrogen sulfide, ketones and mercaptans) or
"non-volatile" like glycerol, acetic acid and succinic acid. Yeast also has the effect
during fermentation of releasing glycoside hydrolase which can hydrolyse the flavor
precursors of aliphatics (a flavor component that reacts with oak), benzene
derivatives, monoterpenes (responsible for floral aromas from grapes like Muscat
and Traminer), norisoprenoids (responsible for some of the spice notes in
Chardonnay), and phenols.

Some strains of yeasts can generate volatile thiols which contribute to the fruity
aromas in many wines such as the gooseberry scent commonly associated with
Sauvignon blanc.
Brettanomyces yeasts are responsible for the "barnyard aroma" characteristic in
some red wines like Burgundy and Pinot noir.[11]
Page 32 of 36

Methanol is not a major constituent of wine. The usual concentration range is


between 0.1 g/liter and 0.2 g/liter. These small traces have no adverse effect on
people and no direct effect on the sens Winemaking considerations

Carbon dioxide is visible during the fermentation process in the form of bubbles in
the must.

During fermentation, there are several factors that winemakers take into
consideration, with the most influential to ethanol production being sugar content in
the must, the yeast strain used, and the fermentation temperature. The biochemical
process of fermentation itself creates a lot of residual heat which can take the must
out of the ideal temperature range for the wine. Typically, white wine is fermented
between 18-20C (64-68F) though a wine maker may choose to use a higher
temperature to bring out some of the complexity of the wine. Red wine is typically
fermented at higher temperatures up to 29C (85F). Fermentation at higher
temperatures may have adverse effect on the wine in stunning the yeast to
inactivity and even "boiling off" some of the flavors of the wines. Some winemakers
may ferment their red wines at cooler temperatures, more typical of white wines, in
order to bring out more fruit flavors.

To control the heat generated during fermentation, the winemaker must choose a
suitable vessel size or else use a cooling device. Various kinds of cooling devices are
available, ranging from the ancient Bordeaux practice of placing the fermentation
vat atop blocks of ice to sophisticated fermentation tanks that have built-in cooling
rings.

A risk factor involved with fermentation is the development of chemical residue and
spoilage which can be corrected with the addition of sulfur dioxide (SO2), although
excess SO2 can lead to a wine fault. A winemaker who wishes to make a wine with
high levels of residual sugar (like a dessert wine) may stop fermentation early either
by dropping the temperature of the must to stun the yeast or by adding a high level
of alcohol (like brandy) to the must to kill off the yeast and create a fortified wine.

The ethanol produced through fermentation acts as an important co-solvent to the


non-polar compound that water cannot dissolve, such as pigments from grape skins,
giving wine varieties their distinct color, and other aromatics. Ethanol and the
Page 33 of 36

acidity of wine act as an inhibitor to bacterial growth, allowing wine to be safely


kept for years in the absence of air.

Other types of fermentation

A California Chardonnay that shows it has been barrel fermented.

In winemaking, there are different processes that fall under the title of
"Fermentation" but might not follow the same procedure commonly associated with
wine fermentation.

Bottle fermentation
Bottle fermentation is a method of sparkling wine production, originating in the
Champagne region where after the cuvee has gone through a primary yeast
fermentation the wine is then bottled and goes through a secondary fermentation
where sugar and additional yeast known as liqueur de tirage is added to the wine.
This secondary fermentation is what creates the carbon dioxide bubbles that
sparkling wine is known for.

Carbonic maceration
The process of carbonic maceration is also known as whole grape fermentation
where instead of yeast being added, the grapes fermentation is encouraged to take
place inside the individual grape berries. This method is common in the creation of
Beaujolais wine and involves whole clusters of grapes being stored in a closed
container with the oxygen in the container being replaced with carbon dioxide. ]
Unlike normal fermentation where yeast converts sugar into alcohol, carbonic
maceration works by enzymes within the grape breaking down the cellular matter to
form ethanol and other chemical properties. The resulting wines are typically soft
and fruity.
Page 34 of 36

Malolactic fermentation
Instead of yeast, bacteria play a fundamental role in malolactic fermentation which
is essentially the conversion of malic acid into lactic acid. This has the benefit of
reducing some of the tartness and making the resulting wine taste softer.
Depending on the style of wine that the winemaker is trying to produce, malolactic
fermentation may take place at the very same time as the yeast fermentation.
Alternatively, some strains of yeast may be developed that can convert L-malate to
L-lactate during alcohol fermentation. For example, Saccharomyces cerevisiae strain
ML01 (S. cerevisiae strain ML01), which carries a gene encoding malolactic enzyme
from Oenococcus oeni and a gene encoding malate permease from
Schizosaccharomyces pombe. S. cerevisiae strain ML01 has received regulatory
approval in both Canada and the United States

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