1. Anita Oberholster Off-Character Formation during Fermentation

2. Introduction Aroma Compounds Grape-derived – provide varietal distinction Yeast and fermentation-derived If known – WHAT – HOW – WHY – Prevention and treatment – next talk

3. Introduction Aroma Compounds Grape-derived – provide varietal distinction – Methoxypyrazines (vegetative, herbacious, bell pepper or earthy aroma) – 2- isobutylmethoxypyrazine, 3-butylmethoxy- pyrazine, 3-isopropylmethoxypyraxine – Threshold 2 ng/L, in wine 9-42 ng/L Sauv. blanc, Semillon, Sauv. Cab. Recognized 4-8 ng/L white wine Recognized 7-15 ng/L red wine Undesirable  25 ng/L IBMP

4. Introduction Aroma Compounds Grape-derived aroma compounds – provide varietal distinction – Thiols very low thresholds (box tree, broom, passion fruit, grapefruit) – Formed during fermentation from odorless precursor (S-cysteine conjugate) Sauv. Blanc,Sauv. Cab., Merlot Seen as positive aroma contributor More important in white then red wine

5. Grape derived thiols 4MMP 3MHA Threshold 3 ng/L 3MH Passionfruit, grapefruit, gooseberry guava Threshold 60 ng/L) Boxtree Threshold 4 ng/L

6. Grape-derived Aroma Compounds Isoprenoids – Monoterpenes (fruity, floral) Muscat, Gewürtztraminer – C-13 Norisoprenoids  -Damascenone (apple, rose, honey) Vitispirane (green odor of chrysan- themum, flowery-fruity note) Present in many wines In Riesling – Riesling acetal (fruity, ionone-like) – TDN (kerosene-like)

7. Introduction Aroma Compounds Yeast and fermentation – volatile metabolites: – Esters – Higher alcohols – Carbonyls – Volatile acids – Sulfur compounds

8. Esters (fruity flavors) Yeast and Fermentation Produced Aroma Compounds Swiegers et al., 2005 Austr. J. Grape Wine Res. 11: 139-173 CompoundWine (mg/L)Threshold (mg/L) Aroma descriptor Ethyl acetate22.5-63.57.5*Fruity, VA, nail polish Isoamyl acetate0.1-3.40.03*Banana, pear Isobutyl acetate0.01-1.61.6***Banana, fruity 2-Phenylethyl acetate 0-18.50.25*Flowery, rose, fruity, Hexyl acetate0-4.80.07**Sweet, perfume Ethyl butanoate0.01-1.80.02*Floral, fruity Ethyl hexanoate0.03-3.40.05*Green apple Ethyl octanoate0.05-3.80.02*Sweet soap Ethyl decanoate0-2.10.2****Floral, soap *10% ethanol, **wine, ***beer, ****synthetic wine

9. – Produced mainly by yeast (through lipid and acetyl-CoA metabolism) Variable amounts, mixed strains higher levels of esters compared to fermentations with Saccharomyces cerevisiae Also variety depended Some esters produced by yeast from specific grape precursors Esters

10. Lactic acid bacteria show esterase activity – Esters such as ethyl acetate (nail polish), ethyl hexanoate (apple), ethyl lactate (creamy, fruity, coconut) and ethyl octanoate (sweet soap) increase with MLF and some others decrease – Suggest that esterases is both involved in the synthesis and hydrolysis of esters – This may increase or decrease wine quality Esters

11. Ethyl acetate (nail polish, solvent, glue) – Aroma threshold 7.5 mg/L – Wine normal 22.5-63.5 mg/L, spoiled  150 mg/L – Fermentation temp, SO 2 levels, duration of MLF – Biggest influence is air, increased production under aerobic conditions Esters

12. Yeast and Fermentation Produced Aroma Compounds Higher alcohols (fusel alcohols) – Secondary yeast metabolites and can have both positive and negative impacts on aroma CompoundWine (mg/L)Threshold (mg/L) Aroma descriptor Propanol9-68500**Fruity, sweet, pungent, harsh 2-methylpropanol25.8-1104Fruity, wine-like Butanol.5-8.5150*Fusel, spiritous Isobutanol9-17440*Fusel, spiritous Isoamyl alcohol6-49030*Harsh, nail polish Hexanol0.3-124*Green, grass 2-Phenylethyl alcohol4-19710*Floral, rose *10% ethanol, **wine

13. Fusel alcohols  300 mg/L add complexity (fruity characteristics)  400 mg/L (strong, pungent smell and taste) Different yeast strains contribute variable amount of fusel alcohols – Non-Saccharomyces yeast – higher levels of fusel alcohols

14. Fusel alcohols Conc fusel alcohols produced: – Amount of precursor - amino acids – EtOH conc, fermentation temp, pH, must composition, amount of solids, skin contact time etc. influence conc of higher alcohols Ehrlich Pathway From Linda Bisson: The Fusel Family

15. Carbonyl compounds – Acetaldehyde (bruised apple, nutty) Sensory threshold of 100 mg/L, typical conc. in wine 10-75 mg/L Major intermediate in yeast fermentation Increase over time due to oxidation of EtOH - due to aeration Use of high conc of SO 2 can cause accumulation of acetaldehyde Acetaldehyde in white wine is indication of oxidation Yeast and Fermentation Produced Aroma Compounds

16. Diacetyl (butter or butterscotch, low conc nutty or toasty) – Aroma thresholds 0.2 mg/L in white, 2.8 mg/L in red wine –  1-4 mg/L buttery or butterscotch –  5 mg/L undesirable – rancid butter – Significant production during MLF by lactic acid bacteria (LAB) – Intermediate in reductive decarboxylation of pyruvic acid to 2,3-butanediol Carbonyl compounds

17. Diacetyl (butter or butterscotch, low conc nutty or toasty) – Variety of factors influence production – Fermentation temp, SO 2 levels, duration of MLF – Biggest influence is air, increase production under aerobic conditions Carbonyl compounds

18. Yeast and Fermentation Produced Volatile Compounds Volatile acids (500-1000 mg/L) – Volatile fatty acids (propionic and hexanoic acid) Produced by fatty acid metabolism of yeast and bacteria – Acetic acid (90%) High conc. vinegar-like aroma Fault  0.7-1.1 mg/L depending on wine style Production by Saccharomyces cerevisiae strains varies widely 0.1-2 mg/L However, commercially used strains produce less than native strains

19. Volatile acids (VA) Acetic acid – Excess conc. largely the result of metabolism of EtOH by aerobic acetic acid bacteria – Small increase in VA with MLF 2 possible pathways Produced from res. sugar through heterolactic metabolism First step in citric acid metabolism

20. Yeast and Fermentation Derived Volatile Compounds Volatile phenols (produced from hydroxycinnamic acid precursors in the grape must)

21. Volatile Phenols Trace amounts present in grapes Mostly produced during fermentation from precursors during fermentation – Saccharomyces cerevisiae 4-ethylphenol (medicinal, barnyard) 4-ethylguaiacol (phenolic, sweet) 4-vinyl phenol (phamaceutical) 4-vinylguaiacol (clove-like phenolic) Present below threshold values Main contributor

22. Volatile Phenols – Brettanomyces/Dekkera spp. Produce high conc of 4EP, 4EG, 4EC, regarded as spoilage organisms Band-aid, medicinal, pharmaceutical, barnyard- like, horsey, sweaty, leathery, mouse urine, wet dog, smoky, spicy, cheesy, rancid, metallic – Brett is not an fermentation problem but sanitation problem in cellar/air and barrel

23. Yeast and Fermentation Derived Volatile Compounds Sulfur compounds – Sulfides, polysulfides, heterocyclic compounds – Thiols, thioesters Produced by yeast – Degradation of sulfur-containing amino acids – Degradation of sulfur-containing pesticides – Release and/or metabolism of grape-derived sulfur-containing precursors

24. Sulfur compounds Sulfides – Hydrogen sulfide (H 2 S) – rotten egg Aroma threshold (10-80  g/L) – Produced by yeast from: Inorganic sulfur compounds, sulfate (SO 4 2- ) and sulfite (SO 3 2- ) Organic sulfur compounds, cysteine and glutathione Glutathione

25. Hydrogen sulfide – Amount produced varies with: Amount of sulfur compounds available Yeast strain Fermentation conditions Nutrient status of environment – H 2 S produced during early – middle stages of fermentation Associated with yeast growth and respond to nutrient addition Mechanism not well known In white wine inversely correlated with initial amount of N 2 and glutathione present after fermentation

26. Hydrogen sulfide – Grape must typically deficient in organic sulfur – Yeast synthesize org sulfur from inorganic sources – H 2 S is metabolic intermediate in reduction of sulfate or sulfite needed for synthesis – If enough N 2 present, formed H 2 S used by O- acetyl serine and O-acetyl homoserine, derived from N 2 metabolism, to form org sulfur compounds – Otherwise build-up of H 2 S in cells

27. Thiols (mercaptans) Formation of sulfides such as DMS (dimethylsulfide, asparagus, corn, molasses) not clear Mercaptans such as ethanethiol can be formed by reaction of H 2 S with EtOH or CH 3 CHO Yeast can reduce disulfides to thiols such as ethane- and methanethiol

28. Thiols (mercaptans) – Low aroma thresholds  1.1  g/L – Ethanethiol (onion, rubber, natural gas) – Methanethiol (cooked cabbage, onion, putrefaction (rot), rubber) Their presence during fermentation suggest that they are by-product of yeast metabolism

29. Sulfur compounds Swiegers et al., (2005) Austr. J. Grape Wine Res. 11: 139-173

30. Mousy off-flavor 3 known compounds causes mousy aroma Lactic acid bacteria (LAB) can produce all 3 compounds Dekkera/Brettanomyces can produce 2 ACPY ETPY ACTPY

31. Mousy off-flavor – 2-ethyltetrahydropyridine (ETPY) Threshold 150  g/L, up to 162  g/L can be produced by LAB – 2-acetyltetrahydropyridine (ACTPY) Threshold 1.6  g/L, isolated in wine at levels of 4.8-106  g/L – 2-acetylpyrroline (ACPY) Threshold 0.1  g/L, detected in wine in trace – 7.8  g/L amounts

32. Mousy off-flavor Following needed for mousy-flavor production – L-Lysine, L-ornithine Responsible for ring formations of 3 mousy heterocycles – EtOH and acetaldehyde Responsible for the acetyl side chain Snowdown et al. (2006) J. Agric. Food Chem. 54: 6465-6474

33. Mousy off-flavor Formation restricted to heterofementative bacteria, general order of magnitued for LAB – Lactobacillus (heterofermentative)  Oenococcus  Pediococcus and Lactobacillus (homofermentative) – Oxygen, high redox potential, high pH, Fe 2+ - pos environment for mousy off-flavor production Snowdown et al. (2006) J. Agric. Food Chem. 54: 6465-6474

34. Main off-flavors VA, ethyl acetate, H 2 S and ethanethiol, acetaldehyde, volatile phenols, mousy Most off-flavors can be minimized or prevented by Using clean fruit Sufficient nutrient and temperature control during fermentation Good winery sanitation and adequate SO 2 use Concluding remarks

35. Contact details Anita Oberholster – RMI North, room 3146 – aoberholster@ucdavis.edu aoberholster@ucdavis.edu – Tel: (530) 754-4866 – Mobile: (530) 400-0137 – http://wineserver.ucdavis.edu http://wineserver.ucdavis.edu – http://enologyaccess.org – http://www.facebook.com/aoberholster http://www.facebook.com/aoberholster