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)

citronellol

O H

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: 139-173

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

– 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 hexanoate

O

O

ethyl 2-hydroxypropanoate

OO

O

ethyl octanoate

O

O

• 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

Esters

Yeast and Fermentation Produced Aroma Compounds

• Higher alcohols (fusel alcohols)– Secondary yeast metabolites and can have

both positive and negative impacts on aromaCompound 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

2-Phenylethyl alcohol

4-197 10* 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 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

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, SO2 levels, duration of

MLF– Biggest influence is air, increase production

under aerobic conditions

Carbonyl compounds

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 Reduced4-ethylphenol4-vinylphenol

4-ethyl-guaiacol4-vinyl-guaiacolferulic acid

p-coumaric acidO H

OOH

O H

O

OH O

O H

O

O H O H

O H

O

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 (H2S) – rotten egg• Aroma threshold (10-80 g/L)

– Produced by yeast from:• Inorganic sulfur compounds, sulfate (SO4

2-) and sulfite (SO3

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

– 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 O-

acetyl 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

S

ethanethiol

S

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

2-acetylpyrroline

NO

2-ethyltetrahydropyridine

N

2-acetyltetrahydropyridine

O

N

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

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

NO

2-ethyltetrahydropyridine

N

2-acetyltetrahydropyridine

O

N

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

NO

2-ethyltetrahydropyridine

N

2-acetyltetrahydropyridine

O

N

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

• 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

Concluding remarks

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