Off-Character Formation during Fermentation Anita Oberholster Introduction Aroma Compounds • Grape-derived – provide varietal distinction • Yeast and fermentation-derived • If known – WHAT – HOW – WHY – Prevention and treatment – next talk Introduction Aroma Compounds • Grape-derived – provide varietal distinction – Methoxypyrazines (vegetative, herbacious, bell pepper or earthy aroma) – 2- isobutylmethoxypyrazine, 3-butylmethoxypyrazine, 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 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 Grape derived thiols 4MMP Threshold 3 ng/L Boxtree 3MHA Threshold 4 ng/L Passionfruit, grapefruit, gooseberry guava 3MH Threshold 60 ng/L) Grape-derived Aroma Compounds • Isoprenoids – Monoterpenes (fruity, floral) • Muscat, Gewürtztraminer – C-13 Norisoprenoids • -Damascenone (apple, rose, honey) • Vitispirane (green odor of chrysanthemum, flowery-fruity note) • Present in many wines • In Riesling – Riesling acetal (fruity, ionone-like) – TDN (kerosene-like) citronellol Introduction Aroma Compounds • Yeast and fermentation – volatile metabolites: – Esters – Higher alcohols – Carbonyls – Volatile acids – Sulfur compounds Yeast and Fermentation Produced Aroma Compounds • Esters (fruity flavors) Compound Wine (mg/L) Threshold (mg/L) Aroma descriptor Ethyl acetate 22.5-63.5 7.5* Fruity, VA, nail polish Isoamyl acetate 0.1-3.4 0.03* Banana, pear Isobutyl acetate 0.01-1.6 1.6*** Banana, fruity 2-Phenylethyl acetate 0-18.5 0.25* Flowery, rose, fruity, Hexyl acetate 0-4.8 0.07** Sweet, perfume Ethyl butanoate 0.01-1.8 0.02* Floral, fruity Ethyl hexanoate 0.03-3.4 0.05* Green apple Ethyl octanoate 0.05-3.8 0.02* Sweet soap Ethyl decanoate 0-2.1 0.2**** Floral, soap *10% ethanol, **wine, ***beer, ****synthetic wine Swiegers et al., 2005 Austr. J. Grape Wine Res. 11: 139-173 Esters – 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 • 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 ethyl hexanoate ethyl 2-hydroxypropanoate ethyl octanoate Esters • 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, SO2 levels, duration of MLF – Biggest influence is air, increased production under aerobic conditions Yeast and Fermentation Produced Aroma Compounds • Higher alcohols (fusel alcohols) – Secondary yeast metabolites and can have both positive and negative impacts on aroma Compound Wine (mg/L) Threshold (mg/L) Aroma descriptor Propanol 9-68 500** Fruity, sweet, pungent, harsh 2-methylpropanol 25.8-110 4 Fruity, wine-like Butanol .5-8.5 150* Fusel, spiritous Isobutanol 9-174 40* Fusel, spiritous Isoamyl alcohol 6-490 30* Harsh, nail polish Hexanol 0.3-12 4* Green, grass 10* Floral, rose 2-Phenylethyl alcohol 4-197 *10% ethanol, **wine 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 Fusel alcohols • Conc fusel alcohols produced: – Amount of precursor - amino acids Ehrlich Pathway – EtOH conc, fermentation temp, pH, must composition, amount of solids, skin contact time etc. influence conc of higher alcohols From Linda Bisson: The Fusel Family Yeast and Fermentation Produced Aroma Compounds • 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 SO2 can cause accumulation of acetaldehyde • Acetaldehyde in white wine is indication of oxidation Carbonyl compounds • 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 • Diacetyl (butter or butterscotch, low conc nutty or toasty) – Variety of factors influence production – Fermentation temp, SO2 levels, duration of MLF – Biggest influence is air, increase production under aerobic conditions 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 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 Yeast and Fermentation Derived Volatile Compounds • Volatile phenols (produced from hydroxycinnamic acid precursors in the grape must) -CO2 p-coumaric acid ferulic acid 4-vinylphenol 4-vinyl-guaiacol Reduced 4-ethylphenol 4-ethyl-guaiacol 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) Main contributor Present below threshold values Volatile Phenols – Brettanomyces/Dekkera spp. • Produce high conc of 4EP, 4EG, 4EC, regarded as spoilage organisms • Band-aid, medicinal, pharmaceutical, barnyardlike, 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 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 Sulfur compounds • Sulfides – Hydrogen sulfide (H2S) – rotten egg • Aroma threshold (10-80 g/L) – Produced by yeast from: • Inorganic sulfur compounds, sulfate (SO42-) and sulfite (SO32-) • Organic sulfur compounds, cysteine and glutathione Glutathione Hydrogen sulfide – Amount produced varies with: • • • • Amount of sulfur compounds available Yeast strain Fermentation conditions Nutrient status of environment – H2S 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 N2 and glutathione present after fermentation Hydrogen sulfide – Grape must typically deficient in organic sulfur – Yeast synthesize org sulfur from inorganic sources – H2S is metabolic intermediate in reduction of sulfate or sulfite needed for synthesis – If enough N2 present, formed H2S used by Oacetyl serine and O-acetyl homoserine, derived from N2 metabolism, to form org sulfur compounds – Otherwise build-up of H2S in cells Thiols (mercaptans) • Formation of sulfides such as DMS (dimethylsulfide, asparagus, corn, molasses) not clear • Mercaptans such as ethanethiol can be formed by reaction of H2S with EtOH or CH3CHO • Yeast can reduce disulfides to thiols such as ethane- and methanethiol 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 methanethiol ethanethiol Sulfur compounds Swiegers et al., (2005) Austr. J. Grape Wine Res. 11: 139-173 Mousy off-flavor • 3 known compounds causes mousy aroma • Lactic acid bacteria (LAB) can produce all 3 compounds • Dekkera/Brettanomyces can produce 2 ETPY 2-ethyltetrahydropyridine ACTPY ACPY 2-acetylpyrroline 2-acetyltetrahydropyridine 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 2-acetylpyrroline 2-ethyltetrahydropyridine 2-acetyltetrahydropyridine 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 2-acetylpyrroline 2-ethyltetrahydropyridine 2-acetyltetrahydropyridine Snowdown et al. (2006) J. Agric. Food Chem. 54: 6465-6474 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, Fe2+ pos environment for mousy off-flavor production Snowdown et al. (2006) J. Agric. Food Chem. 54: 6465-6474 Concluding remarks • Main off-flavors • VA, ethyl acetate, H2S 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 SO2 use Contact details • Anita Oberholster – – – – – – – RMI North, room 3146 aoberholster@ucdavis.edu Tel: (530) 754-4866 Mobile: (530) 400-0137 http://wineserver.ucdavis.edu http://enologyaccess.org http://www.facebook.com/aoberholster