MALOLACTIC FERMENTATION

Management

Gordon Specht February 25, 2010

Grand Rapids

Lallemand produces both wine yeast & wine bacteria products

St. Simon, France

Bacteria Production Facility Virtual Tour:

http://stsimon.lallemand.com/tour_coeno.htm

Malolactic Fermentation Topics:

• Background

• Monitoring

• Selecting MLB Starter Cultures

• Yeast & Bacteria Interaction

• Timing of MLB Inoculation

Bacteria in juice and/or in wineLACTIC BACTERIA – gram positive rods and cocci• Oenococcus oeni (ex Leuc. oenos)• Leuconostoc mesenteroides• Lactobacillus plantarum • Lactobacillus casei• Lactobacillus brevis• Pediococcus damnosus• Pediococcus pentosaceus• Lactobacillus hilgardi

ACETIC BACTERIA – gram negative cocci• Gluconobacter oxydans• Acetobacter aceti• Acetobacter pasteurianus

Oenococcus oeni(formerly Leuconostoc oenos) cells are coccoid to rod-shaped, dividing into long chains.

Oenococcus oeni cells are among the smallest that grow in wine, with 1 µm diameters; lengths typically run 1-3 µm, but may be much longer.

Malolactic Fermentation (MLF)

Lactic bacteria

wine type

inoculation rate

fermentation time

O. oeni strain

SO2

aeration

temperature

contact withyeast lees

Diacetyl concn

variable

white - lowerred - higher

104 - higher

106 - lower

longer MLF - higher

Diacetyl concn

18°C - higher25°C - lower

binds to diacetyl- sensorially inactive

air - higheranaerobic - lower

long contact- lower

lower pH may favourpH

Diacetyl - management during winemaking

From: Dr. Eveline Bartowski (AWRI) Trier (D) April 2008

Environmental Factors that Impact Malolactic Fermentation

Good Moderate Difficult Extreme

Alcohol (% vol) <13 13 - 15 15 - 17 >17

pH >3.4 3.1 - 3.4 2.9 - 3.1 <2.9

Free SO2(mg/L) <8 8 - 12 12 - 15 >15

Total SO2(mg/L) <30 30 - 40 40 - 60 >60

More Environmental Factors that Impact Malolactic Fermentation

Good Moderate Difficult Extreme

Temperature (°C) 18 - 22

14 - 18 or

18 - 24

10 -or

24 -

14

29or

>29

<10

Yeast's nutritional

needsLow Medium High Very high

Ease of Alcoholic

Fermentation

No problems

Transient yeast stress

Sluggish / stuck

AF

Prolonged yeast

contact

More Environmental Factors that Impact Malolactic Fermentation

Good Moderate Difficult Extreme

Initial level of malic

acid (g/L)2 - 4

4 - 5 or

1 - 2

5 -or

0.5 -

7 1

>7 or

<0.5

Maximum AF rate

(maximum loss of

brix/day)

<2 2 - 4 4 - 6 >6

Other, currently less well-known factors that may impact Malolactic

Fermentation and are not considered in this scorecard may include the level of

dissolved oxygen, polyphenoliccontent, lees compacting, pesticide

residues, etc.

Malolactic Fermentation Topics:

• Background

• Monitoring

• Selecting MLB Starter Cultures

• Yeast & Bacteria Interaction

• Timing of MLB Inoculation

Monitoring MLF• Most accepted methods for

following malic acid degradation used by Winemakers…– Paper Chromatography– Enzymatic

• Most accepted method for following MLB used by winemakers…

–MLF has not started yet if you see double cells or very short chains rather than long chains. –You should see about 5 short chains per view and the cells should give contrast. If they look shiny they are most probably dead.

The Microscope…

The Microscope…

Pure cultures of Oenococcus such as in these ML starter cultures ready for buildup often have long chains of cells; on the other hand, some cultures don't make very long chains under winery conditions.

Malolactic Fermentation Topics:

• Background

• Monitoring

• Selecting MLB Starter Cultures

• Yeast & Bacteria Interaction

• Timing of MLB Inoculation

MBR® Bacteria PROCESS: PLANT SCALE FERMENTATION - PHYSIOLOGICAL ADAPTATION

STRESSES

Multiplication stage Adaptation stage

5 to 7 days

STRESS CSTRESS A

STRESS B

COMPARISON SUMMARY &

Controlled MLF 1-Step

Inoculation Direct After acclimatization

MLF Kinetics Medium-fast Medium-fast

Acclimatization/Handling

Lallemand does the

acclimatization

Winemaker does the acclimatization

COST $$$ $$

Winemaker selection criteria for ML Bacteria

1. MLF Reliability

Alc.(%) pH

Total SO2

(mg/L)Temp(oC)

Typical

fermentation Kinetics

Nutrient Demand

Lalvin 31 < 14 > 3.1 < 45 > 13 Slow start,

fast finish Medium

PN4< 15.5 > 3.0 < 60 > 16

Moderate start & fast

finishMedium

1. - MLF Reliability…

How to use 1-STEP…

Winemaker selection criteria for ML Bacteria

MLF Reliability for extreme Low pH

Champagne base wine

CIVC protocol

Extreme Low pH Champagne base wine

CIVC protocol Inobacter STANDARD freeze-dried culture

• Acclimate in Juice• No pH adjustment needed• Shorter reactivation step (3 days

instead of 7), duration of Pied de Cuve is maximum 12 days

Winemaker selection criteria for ML Bacteria

• MLB Sensory Effect

Impact on Mouthfeel

Impact on Fruitiness

Lalvin 31 * ****PN4 ** ***

AWRI / Lallemand Research Project Impact of ML Bacteria on Aroma

(Bartowski/Costello) 2006-2009

To demonstrate the role of MLF in affecting the varietal aroma (red berry fruit)

Parameters and details of the Cabernet Sauvignon fruit and wine for the 2008 MLF trial

Batch No. I II III IV Fruit Source Clare

Valley Langhor

ne Creek

Padthaway

Adelaide Hills

FINISHED WINE Alcohol† (% v/v) 13.9 14.7 14.4 14.0 pH* 3.42 3.44 3.44 3.46 TA* (pH

8.2;g/L) 8.3 8.0 8.0 7.9

Specific gravity 0.9949 0.9936 0.9937 0.9938 Glu+Fru† (g/L) 0.4 0.3 0.5 0.6 Free SO2

†† (mg/L) nd** nd nd nd

Total SO2†† (mg/L) 11 8 10 10

L-Malic acid††† (g/L) 1.60 1.76 1.94 2.18 VA†‡ (g/L) 0.2 0.37 0.2 0.25 † analysis performed by NIR (Winescan) †† analysis performed by FIA ††† analysis performed by enzymatic procedure

AUSTRLIA: AWRI

Malic acid metabolism during MLF of 2008 Cab. Sauvignon wines I, II, III and IV. Oenococcus oeni strains R1106 (BETA)

Un-inoculated wines treated with 60mg/L total SO2 served as non-MLF controls. - AWRI

1105

1106

No MLF

Dark berries

Red berries

Jammy/Cooked fruit

Mint/Eucalyptus

Chocolate

SpiceMusty

Boiled Egg

Overall fruit flavour

Dark berries flavour

Red berries flavour

Mint/Eucalyptus flavour

Fresh green flavour

Chocolate flavour

Astringent

Bitter

Fruit after taste

PC1 (68%)

PC2 (32%)

1105

1106

1118

No MLF

Dark berries

Red berries

Jammy/Cooked fruit

Mint/Eucalyptus

Chocolate

Spice

MustyBoiled Egg

Overall fruit flavour

Dark berries flavour

Red berries flavour

Mint/Eucalyptus flavour

Fresh green

flavour

Chocolate flavour

Astringent

Bitter

Fruit after taste

PC1 (48%)

PC2 (32%)

1105

1106

1118

No MLFDark berries

Red berries

Jammy/Cooked fruit

Mint/Eucalyptus

Chocolate

Spice

Musty

Boiled Egg

Overall fruit flavour

Dark berries flavour

Red berries flavour

Mint/Eucalyptus flavour

Fresh green

flavour

Chocolate flavour

Astringent

Bitter

Fruit after taste

PC1 (63%)

PC2 (21%)PadthawayLanghorne Creek

Adelaide Hills Clare Valley

1105

1118

No MLF

Dark berries

Red berries

Jammy/Cooked fruit

Mint/Eucalyptus

Chocolate

Spice

Musty

Boiled Egg

Overall fruit flavour

Dark berries flavour

Red berries flavour

Mint/Eucalyptus flavour

Fresh greenflavour

Chocolate flavourAstringent

Bitter

Fruit after taste

PC1 (51%)

PC2 (33%)

1106

Malolactic Fermentation Topics:

• Background

• Monitoring

• Selecting MLB Starter Cultures

• Yeast & Bacteria Interaction

• Timing of MLB Inoculation

Competition or availability of nutrients

Negative effects of the yeast on the bacteria (inhibition) could be

caused by:

Yeast Strain Nitrogen DemandJ.M. Sablayrolles, A. Julien INRA-IPV Montpellier, France

0

0.5

1

1.5

2

2.5

mg

N2/

g C

O2

rele

ased

71B

EC

1118

K-1

V11

16

L205

6

RC

212

S6U

CY3

079

BM

45 VL1

Relative Nitrogen Demand

or... Production of inhibitory metabolites :

- SO2 (variable from one strain to another)

- Medium chain fatty acids- Anti-bacterial compounds

Negative effects of the yeast on the bacteria (inhibition) could be

caused by:

0.0

2.0

4.0

6.0OSU

Bac

teria

(log

CFU

/mL)

end of alc.ferm.

0 10 20 30 40 Days

SO2

(mg/

L)

20

30

40

50

60

70

80

Wädenswil

71-B

EC-1118

K-1yeast strains:

Chardonnay juice:20°Brix, pH 3.3, SO2 17/35 mg/L at 20°C

Production of SO2 by yeast & effect on Oenoccocus

growth

Eno

logy

Gro

up

CornellUniversity

YEAST / BACTERIA COMPATIBILITY MLF

compatibilityNitrogenNeeds yeast

POOR MEDIUM GOOD

LOWDV10

EC1118PM

ICV D21 / QA23 Rhone 4600

AMH / ICV D47VRB / 71B

MEDIUM SIMI WHITE*

RHSTBRL 97 / L2056

RC212 / R2 / S6UBDX*

ICV D254*CEG*

HIGH**BM45*

ICV K1*

T306*RA17*/ R2*

L2226*

CY3079*ICV D80*

* Rehydration in GOFERM / ** GOFERM + FERMAID after 1/3 of AF

The result of many years of studying the interaction between yeast and bacteria …the perfect couple

Malolactic Fermentation Topics:

• Background

• Monitoring

• Selecting MLB Starter Cultures

• Yeast & Bacteria Interaction

• Timing of MLB Inoculation

107

105

104

103

102

106

10

Popu

latio

n (c

fu/m

l)

HARVESTTRANSPORT

AF MLF AGEING

108

SelectedML Bacteria

LYSOZYMESelectedYeast

Model Microbial Populations for Post AF inoculation of ML Bacteria…

107

105

104

103

102

106

10

Popu

latio

n (c

fu/m

l)

HARVESTTRANSPORT

AF & MLF AGEING

108

SelectedML culture

LYSOZYMESelectedYeast

Co-inoculation

When to Add Malolactic Bacteria (MLB)?

• Co-inoculation – Yeast addition followed 24-72 hours later by Malolactic bacteria addition

• Why co-inoculate?

– Want positive mouthfeel impact of MLF

– Want to avoid MLF aromas masking fruit character

– Limited tank space faster turn over of fermentors

– Helps the MLB adapt to more difficult wine conditions

When to Add Malolactic Bacteria (MLB)?

• Co-inoculation – Yeast addition followed 24 hours later by Malolactic bacteria addition

– Safe to practice if…• pH < 3.5

• Reliable Alcohol Fermentation

• Post Alcohol MLB addition – Less risky

– Stylistic choice - more classical MLF sensory character impact

Sensory descritors for a 2003 Riesling - MLF induced simultaneously, after alcoholic fermentation and no MLF

0

1

2

3

4

5buttery

lactic

acid

reducedbalanced

body

varietal fruitsimultaneousEC1118

post AFEC1118

no MLFEC 1118

Lallemand MLF available resources…

THANK YOU!gspecht@lallemand.com