Anita Oberholster Off-Character Formation during Fermentation

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-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

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 3MHA Threshold 3 ng/L 3MH Passionfruit, grapefruit, gooseberry guava Threshold 60 ng/L) Boxtree Threshold 4 ng/L

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)

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

Esters (fruity flavors) Yeast and Fermentation Produced Aroma Compounds Swiegers et al., 2005 Austr. J. Grape Wine Res. 11: CompoundWine (mg/L)Threshold (mg/L) Aroma descriptor Ethyl acetate *Fruity, VA, nail polish Isoamyl acetate *Banana, pear Isobutyl acetate ***Banana, fruity 2-Phenylethyl acetate *Flowery, rose, fruity, Hexyl acetate **Sweet, perfume Ethyl butanoate *Floral, fruity Ethyl hexanoate *Green apple Ethyl octanoate *Sweet soap Ethyl decanoate ****Floral, soap *10% ethanol, **wine, ***beer, ****synthetic wine

– 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 Esters

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

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 Propanol **Fruity, sweet, pungent, harsh 2-methylpropanol Fruity, wine-like Butanol *Fusel, spiritous Isobutanol *Fusel, spiritous Isoamyl alcohol *Harsh, nail polish Hexanol *Green, grass 2-Phenylethyl alcohol *Floral, rose *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 – 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

Carbonyl compounds – Acetaldehyde (bruised apple, nutty) Sensory threshold of 100 mg/L, typical conc. in wine 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

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, SO 2 levels, duration of MLF – Biggest influence is air, increase production under aerobic conditions Carbonyl compounds

Yeast and Fermentation Produced Volatile Compounds Volatile acids ( 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  mg/L depending on wine style Production by Saccharomyces cerevisiae strains varies widely 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)

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

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

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 (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

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

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

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

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

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

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

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  g/L – 2-acetylpyrroline (ACPY) Threshold 0.1  g/L, detected in wine in trace – 7.8  g/L amounts

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:

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:

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

Contact details Anita Oberholster – RMI North, room 3146 – – Tel: (530) – Mobile: (530) – – –