H 2 S-Preventing Wine Yeast

1. Wellness for Wine H2S-Preventing Wine Yeast PHYTERRA YEAST Now from Pacific Rim Oenology Services

2. Why use Phyterra Yeast?Scientific slides by Dr. John Husnik, Phyterra • 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

3. Phyterra: a Unique Approach • 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.

4. sulphates How SulphidesFormSulphateReductasePathway MET5, MET10 H2S other nutrients, amino acids sulphides (nitrogen pool)

5. Terminology • 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

6. SulphateReductase Pathway • 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.

7. Research at U.C. Davis • 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 sulphatereductase pathway. • UCD 932 does not make very good wine, but DrBissonrealised that this natural mutation could be bred into more desirable strains, by crossing and backcrossing.

8. H2S-Preventing Technology

9. A Single Amino Acid The difference between UCD 932 and other S. cerevisieaestrains 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.

10. Phyterra Yeast Breeding Programme • 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.

11. Yeast Breeding Saccharomyces yeasts mating

12. Can any Saccharomyces yeast strain be used as the Parent Strain? • 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.

13. PhyterraProgramme, continued • 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.

14. Selection for H2S Production • Selection for the UCD932 MET10 allele can be completed on BiGGYagar, where white colonies indicate strains not producing H2S and tan, brown and black colonies indicate various levels of H2S production.

15. Selected Strains: No H2S Production Lead acetate strips (black in the presence of H2S) and quantitative analysis confirm no H2S production by a Phyterra strain.

16. Selection: Total SO2 Production • 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 H2S- negative, but also retain similar Total SO2levels as the Parent Strain.

17. Trials: Total SO2 Levels PS

18. Total SO2 Graph • By constantly selecting for low Total SO2 (and low H2S) we push the selection to make potentially desirable strains with low to normal Total SO2levels. • 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).

19. Trials: Complete Fermentations

20. Complete Fermentation Graph • 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.

21. Trials: Total SO2 – Low YAN

22. Total SO2 – Low YAN Graph • 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.

23. Aerobic Growth Aerobic growth, by means of which yeast is produced commercially, is normal for all the crosses.

24. H2S Review • 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

25. Phyterra Progress 2010-2012 • 2010-2011: • Two years of successful no-H2S fermentations with PhyterraYeast 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

26. Dominant vs. Ambient Strains • Phyterra prevents the emission of Hydrogen Sulphidefrom 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.

27. Making Phyterra the Dominant Strain • Best Winemaking Practices to encourage dominance of the inoculated strain require careful evaluation of the fruit and the following actions: 1) Addition of sulphitesto 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

28. 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.

29. Sensory Effects of H2S • 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.

30. Curent Progress • 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

31. Current Proprietary ProductsOrganic Wine Yeast 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

32. Organic and Kosher Certifications