Download Scientific Validation

Wellness for Wine
H2S-PREVENTING
WINE YEAST
PHYTERRA YEAST
Now from
Pacific Rim Oenology Services
Why use Phyterra Yeast?
Scientific slides by Dr. John Husnik, Phyterra
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H2S prevention
“Preventative” instead of remedial
Same features as yeasts already being used
Improved wine quality
Lower overall cost of winemaking
NO GMO technology is involved: Organic and Kosher
Phyterra: a Unique Approach
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Other yeast producers use mutagens (agents causing mutations) on
commercial yeast strains, to create low-H2S strains.
These mutagens have a “scattershot” effect, causing a number of
different mutations. The resultant mutants are screened for low- or
no-H2S producing isolates, but other mutations are present as well.
Many low-H2S strains (regardless how made) may also produce
larger-than-normal levels of Total SO2, significantly more than the
parent strain.
By contrast, Phyterra yeasts have a single, natural mutation that
prevents the cell from excreting H2S from the metabolic pathway
mainly responsible for H2S in yeast and have been further selected
for normal SO2 production.
How Sulphides Form
Sulphate Reductase Pathway
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sulphates
MET5, MET10
H2S
other nutrients,
amino acids
(nitrogen pool)
sulphides
Terminology
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Elemental S: the sulphur atom with no other atoms attached
Sulphur-containing amino acids: methionine and cysteine are
needed by yeasts so they are usually synthesised by the yeast
Sulphates: SO4=, found naturally in grape juice.
Sulphites: exist in several forms in wine, including molecular
SO2, sulphite ion (SO3=) and bisulphite ion (HSO3-)
Sulphides: H2S, plus more complex sulphides such as
mercaptans and disulphides, all produced by yeasts
Sulphate Reductase Pathway
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Grape musts generally contain low levels of the amino acids
methionine and cysteine, so yeasts need to synthesise them.
Sulphur is required to make those amino acids, so sulphate is
transported into the cell and is reduced until being
incorporated into methionine and cysteine.
Some sulphide is “leaked” out of the pathway and becomes
converted to H2S and causes smelly fermentations.
Research at U.C. Davis
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At U. C. Davis, Dr Linda Bisson has studied yeast metabolism and
H2S, for at least 25 years.
Her lab checked many U.C. Davis library strains for H2S production
and found UCD932, a Saccharomyces cerevisiae wine strain with a
unique, natural mutation in the MET10 gene.
The MET10 gene encodes for part of the enzyme responsible for the
conversion of sulphite to sulphide, and the mutation in UCD 932
does not allow “leakage” of H2S out of the cell from the sulphate
reductase pathway.
UCD 932 does not make very good wine, but Dr Bisson realised that
this natural mutation could be bred into more desirable strains, by
crossing and backcrossing.
H2S-Preventing Technology
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A Single Amino Acid
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The difference between UCD 932 and
other S. cerevisieae strains is a single
amino acid change: threonine to lysine.
Threonine to Lysine
Threonine is a neutral amino acid, while
lysine is charged. The charge apparently
prevents H2S from exiting the cell.
Phyterra Yeast Breeding
Programme
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To develop Phyterra yeasts commercially, UCD932 and a
commercial, desirable Saccharomyces cerevisiae strain (called
the “Parent Strain”) are both induced to sporulate, and the
spores are dissected out.
A number of matings of the spores of UCD932 and the Parent
Strain are set up. Once they have mated, successful zygotes
(the product of mating) are checked to make sure that they
are progeny of UCD932 and the Parent Strain.
After the first hybrids are created, they are screened for the
presence of the UCD932 MET10 gene.
Yeast Breeding
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Saccharomyces yeasts mating
Can any Saccharomyces yeast strain
be used as the Parent Strain?
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Theoretically, yes; however…
Requirements :
1) The Parent Strain with desirable fermentation characteristics
must be able to be induced to sporulate.
2) The spores must mate successfully with UCD932 spores and
produce viable zygotes.
If a yeast strain fails either of these requirements, it is unable
to be used. Several strains have failed, though most are
successful.
Phyterra Programme, continued
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Around 50 trial fermentations, using the first progeny
containing the UCD932 MET10 gene, are selected for positive
fermentation characteristics, including sensory qualities and
low to normal total SO2 production.
The best are then backcrossed to the Parent Strain. Trial
fermentations are repeated and again the best are selected.
THIS BACKCROSSING PROCESS IS REPEATED AT LEAST 6
TIMES AFTER THE INITIAL HYBRID IS MADE.
Backcrosses are eventually selected that bring the UCD932
MET10 gene together with all the desired fermentation
characteristics.
Selection for H2S Production
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Selection for the UCD932
MET10 allele can be completed
on BiGGY agar, where white
colonies indicate strains not
producing H2S and tan, brown
and black colonies indicate
various levels of H2S production.
Selected Strains: No H2S
Production
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Lead acetate strips (black
in the presence of H2S) and
quantitative analysis
confirm no H2S production
by a Phyterra strain.
Selection: Total SO2 Production
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Mini wine fermentations are
completed on about 50 individual
yeast clones from each breeding
step. Interestingly, each yeast
colony produces different levels of
Total SO2. By selecting low Total
SO2 producers, we can also insure
that not only are these strains H2Snegative, but also retain similar
Total SO2 levels as the Parent Strain.
Trials: Total SO2 Levels
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PS
Total SO2 Graph
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By constantly selecting for low Total SO2 (and low H2S) we
push the selection to make potentially desirable strains with
low to normal Total SO2 levels.
Across a number of juices, the Total SO2 levels of the selected
strains (called “P1Y0-6X” in the graph) fall in a normal range,
as does the Parent Strain (called “PS”).
However, without the selection process, they can quickly
become strains that produce high levels of Total SO2 (in the
graph, this is shown by a strain called P1Y0-SC, which was not
selected for production because of undesirable attributes).
Trials: Complete Fermentations
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Complete Fermentation Graph
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The same fermentations were evaluated for residual
fermentable sugar.
The three selected strains (P1Y0-6X) left very low levels of
fermentable sugar, as did the Parent Strain (called “wt” in the
graph).
The strain P1Y0-SC left 1.4 g/L of fructose, so it is not suitable
for production on this basis, as well as Total SO2 production.
Trials: Total SO2 – Low YAN
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Total SO2 – Low YAN Graph
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The strain P1Y0-B6X, which became Vivace, produced low
Total SO2 even in nitrogen-deficient juice (low yeast-available
nitrogen, or YAN), as did the Parent Strain.
The strain P1Y0-SC again produced high levels of Total SO2.
Aerobic Growth
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Aerobic growth, by means of which yeast is produced
commercially, is normal for all the crosses.
H2S Review
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•H2S (hydrogen sulphide) is a natural by-product of wine yeast fermentation
•Noxious odor – smells of rotten egg , coffee; wine lacks fruitiness
•Removal can be time-consuming, expensive and degrades the wine
Common Treatment and Control Methods for H2S
Phyterra Progress 2010-2012
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2010-2011:
• Two years of successful no-H2S fermentations with
Phyterra Yeast in the US and EU
2012:
• Another 30% growth in Phyterra usage by US
winemakers
• NO reports of elevated Total SO2
• Numerous reports of varietal fruit aromas not usually
perceived
Dominant vs. Ambient Strains
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 Phyterra prevents the emission of Hydrogen Sulphide from
metabolic pathways during fermentation when it is the dominant
strain.
 Ambient yeast from vineyard or cellar may produce hydrogen
sulfide if allowed to grow.
 Ambient strains especially tend to grow in non-sulphited grape
must, must soaked at a cool but not cold temperature, or in must as
it warms up from cold soak before inoculation.
 “Cold soaking” is defined at 10 degrees C or < . Warmer
temperatures will allow growth of other species/strains.
Making Phyterra the Dominant
Strain
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 Best Winemaking Practices to encourage dominance of the
inoculated strain require careful evaluation of the fruit and
the following actions:
1) Addition of sulphites to the grapes/must
2) Temperature control especially during cold-soak (not
over 10° C)
3) Timely inoculation of must when warmed after soaking
4) Nitrogen supplementation if necessary; winemakers
using Phyterra cannot use production of H2S to indicate
the need for nitrogen supplementation
Real-Life Example:
 This 2011 must was kept at ambient temperature for
4 days to soak on skins, then inoculated with a
Phyterra strain. Different species/strains took over
during the soaking and made large amounts of H2S.
Some of these yeasts have the classic
round or “egg” shape of Saccharomyces
and thus may be Phyterra yeasts, but
many have elongated or other odd
shapes, so they are different species of
yeasts from grapes or cellar sources.
These “volunteers” produced H2S.
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Sensory Effects of H2S
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The Enology Department at UCD can now ferment wines
without any hydrogen sulphide.
These wines can be evaluated by the standing sensory panel.
H2S can be dosed into these same wines at sub-threshold
levels and the wines can be evaluated sensorily again, to
quantify the effects.
Curent Progress
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22 strains in Breeding Program
 Organic Vivace (P1Y0), Allegro (P2Y3) and Andante are available in
2013
 Normal levels of Total SO2 for all strains
 Investigation of genetic component of Total SO2 and Juice
composition
 Collaboration with UC Davis
 Conventional yeast production for fall 2013
 Work in Progress
Current Proprietary Products
Organic Wine Yeast
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Products available for 2013 HARVEST
Vivace (P1Y0): for whites, sparkling, and high-Brix wines
Allegro (P2Y3): for fruit-forward whites, especially Sauvignon
Blanc and Chardonnay
Andante: for reds, especially Pinot Noir
See Page 9 in 2013 PROS catalogue for more details
Organic and
Kosher
Certifications