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 Sigrid@lallemand.com

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