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…
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