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MALOLACTIC FERMENTATION QUESTIONS AND
ANSWERS SESSION
ML SCHOOL September 2016
University Stellenbosch
QUESTIONS • Why should I care about specific wine lactic acid bacteria? • Why should I pay if MLF comes spontaneously in my
wines? • How can I avoid a spontaneous MLF and rather go for a
controlled MLF? • What is the importance of pH management in managing
MLF? • Does the choice of a specific yeast strain impact on the
subsequent process of MLF? • When do I add malic acid in order to support my MLF? • How much malic acid can I add? • What type of malic acid can I use?
MORE QUESTIONS???
QUESTIONS • Why should I care about specific wine lactic acid bacteria? • Why should I pay if MLF comes spontaneously in my
wines? • How can I avoid a spontaneous MLF and rather go for a
controlled MLF? • What is the importance of pH management in managing
MLF? • Does the choice of a specific yeast strain impact on the
subsequent process of MLF? • When do I add malic acid in order to support my MLF? • How much malic acid can I add? • What type of malic acid can I use?
Microbial metabolism - flavor-active compounds
From Swiegers, Bartowsky, Henschke & Pretorius, 2005 Eveline Bartowski, AWRI, 2009
ML strain influence - ester production
Adelaide Hills, 2008
80
100
120
140
Rela
tive
% to
no
MLF
Eveline Bartowsky 22nd Entretiens Scientifique Lallemand, Dubrovnic
Wine bacteria – Biological tools
Security of MLF under difficult conditions
QUESTIONS • Why should I care about specific wine lactic acid bacteria? • Why should I pay if MLF comes spontaneously in my
wines? • How can I avoid a spontaneous MLF and rather go for a
controlled MLF? • What is the importance of pH management in managing
MLF? • Does the choice of a specific yeast strain impact on the
subsequent process of MLF? • When do I add malic acid in order to support my MLF? • How much malic acid can I add? • What type of malic acid can I use?
Days to complete MLF and Brett. population
From V. Renouf et al., 2005, J. Int. Sc. Vigne Vin., 39, 179-190
Origin of the problem: development and metabolism of Brettanomyces
Vinyl phenols: 4 vinyl phenol 4 vinyl guaiacol 4 vinyl catechol
Cinamic acid esters: P-coumaril tartaric acid FeriLyl tartaric acid
Cinamil-Esterasa
Cinamato decarboxilasa
Vinil-fenol reductasa
Cinamic acids: P-cumaric acid Ferulic acid Cafeic acid
Volatile phenols: 4-ethyl- phenol (4EF) 4-ethyl-guaiacol (4EG) 4-ethyl-catecol (4EC)
Before MLF After MLF Coutaric
acid (mg/L)
p-coumaric acid (mg/L)
Coutaric acid (mg/L)
p-coumaric acid (mg/L)
Control 9.7 1.3 9.6 1.3 VFO 9.9 1.2 3.1 6.3 Alpha 9.7 1.2 9.8 1.3 VP41 9.8 1.2 9.6 1.3
Bound cinnamic acids degraded by VFO resulted in increased p-coumaric acid
Impact on volatile phenol production by Brettanomyces?
James Osborn Int. ML School 2016 Toulouse
0
200
400
600
800
1000
1200
1400
1600
1800
Control VFO Alpha VP41
Vola
tile
phen
ols
(ug/
L)
4-EP 4-EG
Significantly higher 4-EP and 4-EG produced by Brettanomyces in wine that underwent MLF with VFO
Reported sensory
thresholds
4-EP
4-EG
Cheschier et al 2015, AJEV 66: doi:10.5344
O. oeni and Brettanomyces • 14 commercial strains evaluated to date VFO, CH-35, VP41, MBR31, Inobacter, Elios 1, Alpha, Beta, 350
PreAC, 450 PreAC • No strains could degrade p-coumaric or ferulic acid • To date only VFO degraded coutaric and fetaric acid • After MLF higher p-coumaric and ferulic acid concentration in
wine Increased production of volatile phenols by Brettanomyces
• O. oeni VFO not cause of volatile phenols or spoilage Increased precursor compounds may lead to increased volatile
phenols if Brettanomyces infection occurs
17
James Osborn Int. ML School 2016 Toulouse
Inoculation with selected wine bacteria to avoid contamination
QUESTIONS • Why should I care about specific wine lactic acid bacteria? • Why should I pay if MLF comes spontaneously in my
wines? • How can I avoid a spontaneous MLF and rather go for a
controlled MLF? • What is the importance of pH management in managing
MLF? • Does the choice of a specific yeast strain impact on the
subsequent process of MLF? • When do I add malic acid in order to support my MLF? • How much malic acid can I add? • What type of malic acid can I use?
How can I avoid a spontaneous MLF and rather go for a controlled MLF?
– pH adjustment to pH 3.5 or lower (in combination with moderate SO2 (≤ 50 ppm)
– Co-inoculation with O.oeni for the early dominace of a selected strain
– ML-PRIME – inoculation with a high biomass and import enzymatic pool
– (Bactiless – polymer composed of Chitosan and Chitin-glucan with activity against LAB and AAB)
QUESTIONS • Why should I care about specific wine lactic acid
bacteria? • Why should I pay if MLF comes spontaneously in my
wines? • How can I avoid a spontaneous MLF and rather go for
a controlled MLF? • What is the importance of pH management in
managing MLF? • Does the choice of a specific yeast strain impact on
the subsequent process of MLF? • When do I add malic acid in order to support my MLF? • How much malic acid can I add? • What type of malic acid can I use?
Potential risk of spontaneous MLF - The influence of the pH
wine pH
4.0 3.5 3.3 3.7 3.0
Risk, that MLF does not
occur
Difficulties to induce a
spontaneous MLF
Oenococcus oeni
Lactobacillus sp. / Pediococcus sp.
Increasing risk of spontaneous MLF induced by Lactobacillus & Pediococcus sp.
OFF-FLAVORS
Potential risk of sugar
degradation after MLF by O. oeni
at high pH
Evolution of bacteria under difficult conditions
107
105
104
103
102
106
10
Viab
le Ce
lls (c
fu/m
L)
108 Yeast Oenococcus
Lactobacillus Pediococcus
Gluconobacter
Acetobacter
3 - 24 weeks
HARVEST TRASPORT
ALCOHOLIC FERMENTATION
MALOLACTIC FERMENTATION
AGING
Bacterial grwoth in must and wine under easy conditions
107
105
104
103
102
106
10 cfu/ ml HARVEST
TRANSPORT ALCOHOLIC
FERMENTATION MALOLACTIC
FERMENTATION MACERATION/
STORAGE
108
3 - 24 days
yeast
Lactobacillus Pediococcus
Oenococcus
Gluconobacter
Acetobacter
8
8
8
8
2
34
SO2 and MLF
• The molecular SO2 molecular is the most active form of the sulfuric acid with the
highest antimicrobial activity. • Molecular SO2 = free SO2 / ( 10(pH - 1.81) + 1 )
)
• The molecular SO2 increases with a decrease in pH and increase of temperature
and/or alcohol • The lethal level for wine bacteria: 0.3 mg/L- 0.5 mg/L.
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
3.0 3.1 3.2 3.3 3.4
SO2
mol
écul
aire
(mg/
l)
pH
SO2 libre : 10mg/l - 18°C
0 % v/v 13 % v/v
added free pH 3.0 pH 3.1 pH 3.2 pH 3.3 pH 3.4
20 6 0.36 0.29 0.23 0.19 0.18
30 9 0.55 0.44 0.35 0.28 0.23
40 12 0.73 0.59 0.47 0.38 0.30
50 15 0.91 0.73 0.59 0.47 0.38
60 18 1.09 0.88 0.70 0.56 0.45
SO2 (mg/l) molecular SO2 (mg/l) at 18⁰C
QUESTIONS • Why should I care about specific wine lactic acid
bacteria? • Why should I pay if MLF comes spontaneously in my
wines? • How can I avoid a spontaneous MLF and rather go for
a controlled MLF? • What is the importance of pH management in
managing MLF? • Does the choice of a specific yeast strain impact on
the subsequent process of MLF? • When do I add malic acid in order to support my MLF? • How much malic acid can I add? • What type of malic acid can I use?
How yeast can impact malolactic fermentation ?
• Production of ethanol and SO2 • Modification of wine nutrients content • Production of medium chain fatty acids • Production of antibacterial peptides or proteins • Malic acid consumption • Impact of other yeast metabolites?
5 groups of yeast depending on MLF compatibility
1- Yeast inhibitory for MLF (very high production of SO2 or other toxic compounds for bacteria) 2- Yeast unfavourable to MLF (high production of SO2 or other toxic compounds) 3- Yeast that could be positive or negative for MLF: - high nutrient needs and early autolysis - low to medium nutrient needs and medium production of SO2 or other toxic compounds 4- Yeast favourable to MLF: low nutrient needs and low production of inhibitory compounds 5- Yeast very favourable to MLF: very low nutrient needs and no production of inhibitory
compounds
1 2 3 4 5
QUESTIONS • Why should I care about specific wine lactic acid
bacteria? • Why should I pay if MLF comes spontaneously in my
wines? • How can I avoid a spontaneous MLF and rather go for
a controlled MLF? • What is the importance of pH management in
managing MLF? • Does the choice of a specific yeast strain impact on
the subsequent process of MLF? • When do I add malic acid in order to support my MLF? • How much malic acid can I add? • What type of malic acid can I use?
What about the addition of malic acid to wine? – How much can I add? Within legal limits –(OIV: 1.5 g/L tartaric acid to must (1.34 g/L malic acid) + 3 g/L tartaric acid to wine ( 2.68 g/L malic acid)
Tartaric Malic acid acid
Tartaric acid Malic acid
8
8
8
8
2
34
What about the addition of malic acid to juice/wine?
– How much can I add?
– What should be the lowest dosage of natural
malic acid before I can add more to
stimulate/get MLF going?
=> Preferably addition in the juice stage, but also
in wine if
⇒ ≤ 0.8 g/L L-malic acid
⇒ Wine LAB can’t degrade D-malic acid. Better,
but more expensive to add only L-malic acid
Induction of MLF in wines with low malic acid content
Malic Levels < 0.8 g/L:
1. Co-inoculation (24 h after yeast inoculation) in combination
with a yeast strain with low capacity to degrade malic acid
2. Sequential inoculation: We achieved best results applying a
1-Step starter culture and shortening the acclimatization to
6 – 8 hours (instead of 24 hours)
Failure for direct inoculation / Success for Alpha 1-Step ® Initial low malic acid level can explain the difficulty to induce MLFwith direct inoculation
Progress of MLFMerlot/Cabernet Sauvignon (Portugal)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0 10 20 30 40
Time (days)
Mal
ic a
cid
(g/L
)
Non-inoculated SAB1-Step AlphaLalvin VP41
Portugal (lab results) Merlot/Cabernet Sauvignon Alcohol=14% pH=3.3 Malic acid=0.6 g/L
Total SO2=50ppm Temperature=20°C
2nd trial - Malic acid degradation
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
0,90
1,00
0 5 10 15 20 25 30 35 40
Time (days)
Mal
ic a
cid
(g/L
)
1-Step Alpha
Double 1-StepAlphaControl
• Success for Alpha 1-Step ® with a reduced acclimation phase.
• Initial low malic acid level and low pH can explain the difficulty to induce MLF with direct inoculation
2007 - Italy (lab results) Sangiovese Alcohol=14.1% pH=3.12 Malic acid=0.7 g/L Total SO2=36 ppm Free SO2=6ppm Temperature=20°C
1-Step® preculture only for 6 hours
Malic acid tolerance / Lactic acid sensitivity
Organic Acids • From practical experience:
- wines with L-malic acid levels below 1 g/L not as conducive to MLF as are wines with L-malic acid concentrations between 2 and 4 g/L.
- wines with levels of L-malic acid above 5 g/L start L-malic acid degradation, but do not always go to completion.
Inhibition of the bacteria by increasing concentrations of L-lactic acid derived from the MLF itself ?
• Acidification with the organic acids lactic acid, L(-) or DL malic acid, L(+) tartaric acid and citric acid is authorized in many wine regions.
=> In collaboration with Lallemand, IFV (V. Gerbaux) in France has studied the influence of organic acid additions on MLF.
Factors that impact MLF Easy Moderate Difficult Extreme
Initial level of malic acid (g/L) 2 - 4
4 - 5 or
1 - 2
5 - 7 or
0.5 - 1 >7 or <0.5
Impact of initial malic acid content
• Duration of MLF of course increases with malic acid content.
• However: - Same latency phase
duration for all conditions. - Speed of malic acid
degradation increases with malic acid content.
- Some strains little affected by an increase of malic acid content.
• Addition of D-malic acid (D,L
malic acid) has no noticeable effect on MLF
Chardonnay – 12.5%v/v pH=3.25 Malic acid=2.6g/L Temp.= 16°C
0
7
14
21
28
35
Expertise S Lalvin 31 Alpha 49A1
Tim
e (d
ays)
Time to achieve 90% of MLF
malate : 0.75
malate : 1.35
malate : 2,6
malate: 5,2
Impact of addition of L-lactic acid before MLF
Strong effect of lactic acid addition on bacteria population and MLF duration. Addition of 1.5g/L highly increases the time to achieve MLF. Addition of 3g/L induces a high loss of viability which leads to stuck MLF. Alpha seems to be highly sensitive. Full screening of Lallemand strains regarding lactic acid sensitivity on-going.
0
10
20
30
40
50
60
70
80
90
100
110
Lactate : 0 Lactate : 1.5 g/L Lactate: 3.0 g/L
Tim
e (d
ays)
Time to achieve 50% of MLFInitial L-malic acid=3g/L
Expertise S
Lalvin 31
Alpha
49A1
Chardonnay – 12.5%v/v pH=3.25 Malic acid=2.6g/L Temp.= 16°C
1,0E+04
1,0E+05
1,0E+06
1,0E+07
0 1 2 3 4 5 6 7
log
cfu/
mL
Time (days)
Bacteria population - Alpha
Lactic acid 0
Lactic acid 1.5
Lactic acid 3.0
Malic acid tolerance/ L-lactic acid sensitivity Malic acid: • Increasing concentration of malic acid increases the speed of malic acid
degradation, but of course also increase the duration of MLF.
• Some strains more suitable than others for high malic acid content (characterization of the full range on-going)
Lactic acid: • The presence of L-lactic acid in the wine inhibits the implantation and growth of
the inoculated wine LAB resulting in an inhibition of MLF. • An initial content of L-lactic acid in the range of 1.5 g/L strongly slows MLF, but a
content of 3.0 g/L fully inhibits MLF. • Problems inducing MLF by inoculation with selected wine bacteria may be
encountered when L-lactic acid was added to must or wine or in wines with a partial MLF.
THE MORE WE KNOW THE MORE WE STILL
NEED TO KNOW
THANK YOU FOR ALL YOUR QUESTIONS