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)

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0,1

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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

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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