Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
Malolactic fermentation Malolactic fermentation 20052005
RR--ee--ss--pp--ee--cc--ttthe bacteria in your wines the bacteria in your wines and and managemanage your MLF your MLF
Geneva on the Lake, February, 2005Geneva on the Lake, February, 2005
BACTERIA IN MUST & WINE
Only a few bacteria are adapted to survive in wine
alcoholacidityLow pH???SO2Nutrient deficiency
Microflora in must and wine
LactobacillusLactobacillusBrettanomycesBrettanomyces PediococcusPediococcus
SaccharomycesSaccharomyces
Acetobacter e OenococcusAcetobacter e Oenococcus
THE CHEMISTRY...
CH3 . CHOH . COOH
90
lactic acid
CO2
44carbon dioxide
HOOC . CH2. CHOH . COOH
134
malic acid
+
LALLEMAND
THE CHEMISTRY
L (-) malic acid converted into L(+) lactic acid (commercial additions of D(+) will remain untouched).Not really a ‘fermentation’ as no energy is producedReduction of acidity by 1-3 g/LAddition of diacetyl: good or bad?
Metabolism in heterofermentative Lactic Acid Bacteria
GLUCOSEFRUCTOSE
acetyl-P
ACETATE
Pyruvate
CITRATE
D-LACTATE
Acetyl-CoA
Fatty acidsLIPIDS
Acetaldehyde-TPP DIACETYL
acetoin
MALATE
L-LACTATEATP
ATP
ATP
pH, temp
100-700 ppm
300-1000 ppm1000-4000 ppm
100-200 ppm
100-200 ppm
2-8 ppm
670-2680 ppm
Bacteria found in must and in wine
LACTIC ACID BACTERIA
fermentation:Oenococcus oeni (ex Leuc. oenos) heteroLeuconostoc mesenteroides heteroLactobacillus plantarum homoLactobacillus casei homoLactobacillus brevis heteroPediococcus damnosus homoPediococcus pentosaceus homo
ACETIC BACTERIAGluconobacter oxydans sugarsAcetobacter aceti ethanolAcetobacter pasteurianus ethanol
BACTERIA EVOLUTIONUNDER FAVOURABLE CONDITIONS
107
106
105
104
103
102
108yeasts Oenococcus
LactobacillusPediococcus
Gluconobacter
Acetobacter
10Cells/
mlHARVESTDELIVERY
ALCOHOLICFERMENTATION
MALOLACTICFERMENTATION
STORAGE
BACTERIA EVOLUTION UNDER DIFFICULT CONDITIONS
107
106
105
104
103
102
108
yeasts Oenococcus
LactobacillusPediococcus
Gluconobacter
Acetobacter
10Cells/
mlALCOHOLIC
FERMENTATIONMALOLACTIC
FERMENTATIONSTORAGEHARVEST
DELIVERY
Most important parameters
SO2 > pH > temperature > alcohol
(achtung! O. Oeni cannot use ammonia)
Influence of the wine yeast from AF
CHEMICAL/PHYSICAL
NUTRIENTS
MICROBIOLOGICAL
POSSIBLE INTERACTIONSBETWEEN YEAST
& ML BACTERIA
YEAST
ML BAC
TERI
A
INHIBITION OF OENOCOCCUS OENI BY THE YEAST
STIMULATION OF OENOCOCCUS OENI BY THE YEAST
YEAST
ML BACTERIA
NO INFLUENCE = INDIFFERENT
IN SOME CASES INHIBITION OF SACCHAROMYCES CEREVISAE BY WILD LACTIC ACID BACTERIA WAS REPORTED
LB. KUNKEEISACCHAROMYCES
IN SOM
E CA
SES TH
EY
EVEN
CAN
HAV
E MU
CH
FUN
ITV 99 - TastingPinot noir – Qualitative differences
020406080
Blackcurrant
Cherry
Pepper
Caramel
Hay
EarthyControl
EQ 54
Lalvin 31
Bacteria X
ML Properties based on organoleptic properties
RED WINES
-tropical fruit, -vanilla in barrel fermentation-peach and melon characterisitcs
Maintain fruit characterisitcs
WHITE WINES
Reduction of vegetal aromas
Heightened
MOUTHFEEL: volume and balance in mouth
Mature fruit and jam
Increase
Uncontrolled Malolactic Fermentation...THE MASKS...
(MLF Sensory Defects Kit) presented by...Dr. Sibylle KriegerDidier Theodore
Dr. Antonio Palacios
Uncontrolled Malolactic Fermentation...THE MASKS...
(MLF (MLF SensorySensory DefectsDefects KitKit) ) presentedpresented by...by...Dr. Sibylle Dr. Sibylle KriegerKriegerDidierDidier TheodoreTheodore
Dr. Antonio PalaciosDr. Antonio Palacios
LALL
EMA
ND
WinemakersWinemakers are are concernedconcerned withwith::•Limiting chemical inputs (optimizing theSO2 dosage).•Limiting health risk concerns and spoilage(low biogenic amines).•Avoiding heavy curative treatments ofclarification, filtration y stabilization.•Developing and stabilizing positive aromas tannin perception.•Maintaining quality through thewinemaking process
Wine bacteria:The drawbacks...Wine bacteria:The drawbacks...
•Volatile Acidity•Too much Diacetyl•Undesirable Aromas & Flavors
•Varietal character loss•Color loss•Ethyl Carbamate•Biogenic Amines •Geranium Aromas
The Usual Suspects:Some OenococcusMany LactobacillusMany Pediococcus
Wine Bacteria:The positive side…Wine Bacteria:The positive side…
•Lowering acidity•Ethyl Lactate / Diacetyl•Varietal aroma enhancement•Reducing Vegetative notes•Rounding the mouthfeel•Lowering astringency•Lowering bitterness•Increasing complexity•Lowering overall SO2
The Usual Suspects:Some Oenococcus
LALL
EMA
ND
LALL
EMA
ND
Ethyl Lactate AromaEthyl Lactate Aroma
Formed mainly during MLF.Low levels contribute mouthfeel volume.High concentrations add milk and yogurt aromas.Above 15 mg/L is usually considered negative in wines.
LALL
EMA
ND
AACETALDEHYDECETALDEHYDEC
CH3H
O
Threshold detection ~ 100 mg/L.Odor: overripe apple, fish in vinegar, “oxidation”.Sometimes formed during MLF, and other times its levels are reduced.Normaly the levels are reduced by binding with free SO2.
LALL
EMA
ND
GGERANIUM ODORERANIUM ODOR
Derived from the metabolism of sorbic acid, which hydrogenates to SORBINOL, which isomerizes to 3,5-Hexadien-2-ol, then reacting with the ethanol part of 2-ethoxy-hexa-3,5-dien, resulting in the spoilage odor.Perception threshold is 0,1 µg/L.O. oeni has low a production.Not a problem in beverages without ethanol.
LALL
EMA
ND
0
1
2
3
4
5
6
7
8
9
10
NoMLF
A B C D E
Diacetyl in wine (mg/l)
ChardonnayCab. Sauv.
Diacetyl ImpactDiacetyl ImpactDiacetyl Impact
- 5-14 mg/LButter- 2- 4 mg/Lnuttycaramelyeastyhoney(threshold > reds)
The final concentration depends on the bacteria strainused for the MLF & on its citric acid metabolism
Bacteria Strain Diacetyl Production
Metabolism of citrate by OeMetabolism of citrate by Oenococcus oeninococcus oeni and the and the transformation of diacetyl by yeaststransformation of diacetyl by yeasts
CitrateAcetate
2-ButandiolAcetoinDiacetyl
Oxalacetate
Pyruvate
The transformation of diacetyl to butandiol lowers the buttery aromas and transforms them into mouthfeel volume and fattness.
LALL
EMA
ND
Biogenic Amines Decarboxylation Amino Acids
Proteins
Decarboxylation of amino acids,Ex.: histidine decarboxylase for
histamine
MECHANISM OF BIOGENIC AMINE MECHANISM OF BIOGENIC AMINE FORMATION FORMATION
Biogenic amines are unhealthy (histamine) and alsocontribute negative sensory compounds (putrescine & cadaverine)
BIOGENIC AMINE FORMATION: BIOGENIC AMINE FORMATION: examplesexamples
Amino acids – Biogenic AminesHistidine
Tyrosine
Lysine
Arginine
Arginine
Arginine
Histamine
Tyramine
Cadaverine
Putrescine
Espermine
Epermidine
Ethanolamine
Phenylethylamine
Isopentylamine
0
5
10
15
20
25
Strain Y Strain A Spontaneous Strain X
mg/l
CadaverineIsoamylaminePutrescinePhenyl ethylamineTyramineEthylamineMethylamineHistamine
LacticLactic AcidAcid Bacteria Bacteria StrainStrain InfluenceInfluence on on ConcentrationConcentration ofof BiogenicBiogenic Amines Amines
afterafter MLFMLF
Putrescine, a putrid “dirty sponge” aroma, is the highest contributor to Biogenic Amines in this trial.
LALL
EMA
ND
LactobacillusLactobacillus can produce can produce volatile phenolsvolatile phenols
R1R2
H2C-CH3
R1R2
HC=CH-COOH R1R2
HC=CH2
44--ETHYL DERIVATIVESETHYL DERIVATIVES44--VINYL DERIVATIVESVINYL DERIVATIVES
CINNAMIC ACIDCINNAMIC ACID
Activity ofLactobacillus plantarum
Volatile phenols are formed from the grape precursors by contaminating microorganisms...
BrettanomycesBrettanomyces ContaminationContamination......
Contaminant yeast, responsible for the formation in wine of volatile phenols resulting in very negative aromas
Ethylphenols 4-ethyl phenol4-ethyl guaiacol
Vinylphenols 4-vinyl phenol4-vinyl guaiacol
4- ethyl phenol results in the descriptors (“poorly cured leather”, “horse sweat”, “used socks”, “horse stables”).
TheThe ProblemProblem
4-ethyl-guaiacol
4-ethyl-phenol
Threshold perception:Ethyl phenol: 600µg/LSum of ethyl phenols: 430 µg/L
LALL
EMA
ND
LALL
EMA
ND
Causal compounds of Mousy offCausal compounds of Mousy off--flavourflavourNN--heterocyclic bases: 2heterocyclic bases: 2--ethyltetrahydropyridineethyltetrahydropyridine,,22--acetyltetrahydropyridineacetyltetrahydropyridine & 2& 2--acetylacetyl--11--pyrrolinepyrroline
2-ethyltetrahydropyridine (ETPY)
N
Taste threshold (wine): 150 µg/L (Craig & Heresztyn 1984)Conc’n reported in wines exhibiting mousy off-flavour: 2.7-18.7 µg/L
2-acetyltetrahydropyridine (ACTPY)
NO
Odour threshold (water): 1.6 µg/L (Teranishi et al. 1975)Conc’n reported in wines exhibiting mousy off-flavour: 4.8-106 µg/L
2-acetyl-1-pyrroline (ACPY)
NO
Odour threshold (water): 0.1 µg/L (Buttery et al. 1983)Conc’n reported in wines exhibiting mousy off-flavour: Tr-7.8 µg/L
Grbin, P.R.; Costello, P.J.; Herderich. M.; Markides, A.J.; Henschke, P.A.; Lee, T.H. (1996) Proceedings 9th
Aust. Wine Industry Technical Conference, Adelaide, Australia, (Winetitles: Adelaide) pp.57-61.
Possible formation pathways of ACTPY Possible formation pathways of ACTPY & ACPY from & ACPY from L. L. hilgardiihilgardii DSM 20176DSM 20176
Heterolactic
Acetyl-CoA
Acetaldehyde
Ethanol
ACTPY
Lysine
CO2Ac. Lactic
Acylation
Ornithine
ACPY
Acylation
NH2
COOH
NH2
N
NO
D-Glucose / D-Fructose
Acetyl-phosphate Acetate
NH2
COOH
NH2
N
NO
Costello, P.J.; Henschke, P.A. (2002) J. Agric.Food Chem. 50: 7079-7087.
To avoid these “Masking” To avoid these “Masking” components…components…
Limit the duration of lactic bacteria in the wine.Limit the duration of lactic bacteria in the wine.
Control the winery cleanliness to limit spoilage organisms Control the winery cleanliness to limit spoilage organisms ((PediococcusPediococcus, Lactobacillus, Lactobacillus…) …)
Use selected ML bacteria to control the MLF and avoid Use selected ML bacteria to control the MLF and avoid wine spoilage including biogenic amines. wine spoilage including biogenic amines.
Thank you for your
attention !
Thank you for your
attention !
For more info please contact [email protected]
Nitrogen requirements of yeastsNitrogen requirements of yeasts
Different demand depending on the strains fermentation temperature and pHJiranek et al., 1991, Manginot et al., 1998, Julien et al., 2000
71B / QA23 / 71B / QA23 / DV10 / BC / DV10 / BC / EC8 / D47 / EC8 / D47 / EC1118 EC1118
BDX / CSM / BDX / CSM / CY3079 / BM45 / CY3079 / BM45 / K1 marque / K1 marque / L2226 / L2323 / L2226 / L2323 / VL1 / CEG VL1 / CEG
EC7 / K1 / D254 EC7 / K1 / D254 / CAW / L2056 / / CAW / L2056 / R2 / RC212 / R2 / RC212 / S6US6U
Nutrient requirements for O.oeni
Oenococcus oeni (Leuconostoc oenos)does not grow on malic acid only
it needs complex nutrients
Improved nutrient formulationsfor Oenococcus oeni
Negative effects of the yeast on the bacteria (inhibition)
could be caused by:
Competition on nutrient levelProduction of inhibitory metabolites
- SO2- CO2- Medium chain fatty acids- Antibacterial compounds
LALVIN MBR in difficult wine
0
20
40
60
80
100
120
140
160
days
to c
ompl
ete
MLF
CH '98, pH 3.13, SO2T 30, Alc 14.0 Spont. MLF
MBR 1
MBR 31
MBR 2
MBR 3
MBR EQ54
STRAIN X
MBR 4
OSU
EQ54 1 STEP
MT01 STD
LALLEMAND
LALVIN MBR pH tolerance:ITV Beaune, SOFRALAB ‘98
0
5
10
15
20
25
30
35
40
45
50
days
to c
ompl
ete
MLF
Pinot pH 3.7; Alc 14.5 Syrah pH 2.97; Alc 13.0
Spont. MLF
MBR 1
MBR 31
MBR 2
MBR 3
MBR 4
MBR EQ 54
MBR®: Tolerance at low temperatures
0
20
40
60
80
100
120
140
days
to c
ompl
ete
MLF
18°C 13°C
ITV Beaune 1998, Pinot noir pH 3.35, alc. 13.20 %
spon.FMLALPHALalvin 31mbr2mbr2mbr4EQ54mbr5