Acetobacter in wine
Non-Saccharomyces yeasts
from grapes
There are yeasts on grapes, though
very few are Saccharomyces. Kloeckera
apiculata (teleomorph Hanseniaspora
uvarum) is the major species, and
there are quite a few other species
also. Some are sensitive to alcohol, or
are aerobic so they cannot grow during
fermentation, but they may grow
before or during the start of fermentation.
Other species may survive
until middle to late in the fermentation
process.
If odors of ethyl acetate (airplane
glue/nail polish remover), amyl
acetate (banana skin), or other offodors
appear in juice or must, examine
immediately under a phase-contrast
microscope for yeasts that are
not the round/ovoid shape of
Saccharomyces. Non-Saccharomyces
yeasts are sometimes
round, but more
often have other appearances: apiculate
(pointy), elongated, or bizarrely
shaped, and they may be unusually
small or large.
Brettanomyces in stuck wine.
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Kloeckera is especially problematic
for several reasons. Their SO2 sensitivity
is comparable to that of Saccharomyces,
and they are naturally coldtolerant
so they grow readily in cool
must (10 to 15°C). They can survive
much farther into the fermentation
than most other non-Saccharomyces
species, sometimes all the way to the
end.
Sometimes a thick, slimy scum
forms, or stringy clumps that fall out
later as fluffy lees. Kloeckera can make
prodigious amounts of ethyl acetate
and amyl acetate, and is quite efficient
in quickly depleting nutrients, particularly
thiamine and other vitamins.
Microscopically, it resembles bowling
pins or thin lemons with a knob on
each end.
Kloeckera is easy to culture because it
is cycloheximide-resistant and grows
in one to two days, unlike Brettanomyces
(three to seven days), which
Kloeckera resembles. There are also
PCR primers for Kloeckera, and for
Pichia membranifaciens, which could be
used at this stage, though not for other
non-Saccharomyces vineyard species, so
those will not be detected by PCR.
However, the question is notwhether
there are Kloeckera and other vineyard
yeasts in themust (there are), but howto
monitor their population, and how to
handle themust so that they do not grow
too extensively. For this purpose, following
the must microscopically, estimating
percentages of non- Saccharomyces
species, is more useful. Before fermentation
begins, if more than 5 or 10 non-Saccharomyces yeasts are seen in a 40x
field, a problem is developing.
Examine some musts microscopically
every day, especially any must
over 10°C that are soaking on skins or
warming up from soaking/settling, or
musts that start fermenting spontaneously.
Also examine microscopically
if acetate or amyl acetate odor is noted.
YEAST FERMENTATION
Microbes to watch out for: Non-
Saccharomyces yeasts, lactic acid bacteria
Danger signals: Spontaneous fermentation,
ethyl acetate or other off-odor,
sluggish fermentation, spontaneous
malolactic fermentation (MLF), low
viability of yeast inoculum, VAincrease
Monitoring methods: Microscopic
exam, sensory cues, chemical tests,
PCR-based genetic testing
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Yeast and Lactobacilli in stuck wine
(by Patricia Roca)
Saccharomyces
When preparing dry yeast, check
viability with methylene blue stain to
make sure that enough survived transport,
storage, and rehydration in the
winery. Approximately half of the
rehydrated yeasts should be viable, as
evidenced by lack of blue staining.
Counting yeasts with a hemocytometer
also helps to monitor the population
to be sure that it reaches 108
cells/ml, or a stuck ferment is likely.
Non-Saccharomyces
Some winemakers encourage the
growth of some non-Saccharomyces
vineyard species to contribute to aromatics,
complexity, andmouthfeel before
Saccharomyces, which must become
dominant for the fermentation to go to
completion, takes over. Potential problems
with this approach include depletion
of vitamins and nutrients (natural
and added) that Saccharomyces will
need, production of off-characters such
as H2S, ethyl acetate, and acetic acid,
and possibly inhibition of Saccharomyces.
It is very important for winemakers
encouraging participation by vineyard
yeast species to guard vigilantly
against their extensive growth. Many
stuck fermentations have their origins
early on, as growth of competing
species did not allow Saccharomyces to
achieve a large population of healthy
yeasts. The winemaker may look for a
cause late in fermentation, but what
happens at the beginning can set the
process in motion.
Spontaneous fermentations, whether
intentional or not, should be checked
microscopically until Saccharomyces
becomes the dominant yeast. If Saccharomyces
does not take over within the
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