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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– [email protected]– Tel: (530) 754-4866– Mobile: (530) 400-0137– http://wineserver.ucdavis.edu– http://enologyaccess.org– http://www.facebook.com/aoberholster