MALOLACTIC FERMENTATION QUESTIONS AND ANSWERS SESSION

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???

Microbial metabolism - flavor-active compounds From Swiegers, Bartowsky, Henschke & Pretorius, 2005 Eveline Bartowski, AWRI, 2009

ML strain influence - ester production 140 Relative % to no MLF 120 100 80 Adelaide Hills, 2008 Eveline Bartowsky 22 nd Entretiens Scientifique Lallemand, Dubrovnic

Wine bacteria Biological tools

Security of MLF under difficult conditions

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 Cinamic acid esters: P-coumaril tartaric acid FeriLyl tartaric acid Cinamil- Esterasa Cinamic acids: P-cumaric acid Ferulic acid Cafeic acid Cinamato decarboxilasa Vinyl phenols: 4 vinyl phenol 4 vinyl guaiacol 4 vinyl catechol Vinil-fenol reductasa Volatile phenols: 4-ethyl- phenol (4EF) 4-ethyl-guaiacol (4EG) 4-ethyl-catecol (4EC)

Bound cinnamic acids degraded by VFO resulted in increased p-coumaric acid Coutaric acid (mg/l) Before MLF After MLF p-coumaric acid (mg/l) Coutaric 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 p-coumaric acid (mg/l) Impact on volatile phenol production by Brettanomyces? James Osborn Int. ML School 2016 Toulouse

Significantly higher 4-EP and 4-EG produced by Brettanomyces in wine that underwent MLF with VFO 1800 Volatile phenols (ug/l) 1600 1400 1200 1000 800 600 400 200 0 4-EP 4-EG Control VFO Alpha VP41 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

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 SO 2 ( 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)

Potential risk of spontaneous MLF - The influence of the ph Risk, that MLF does not occur Difficulties to induce a spontaneous MLF Increasing risk of spontaneous MLF induced by Lactobacillus & Pediococcus sp. OFF-FLAVORS Lactobacillus sp. / Pediococcus sp. Oenococcus oeni 3.0 3.3 3.5 3.7 wine ph 4.0 Potential risk of sugar degradation after MLF by O. oeni at high ph

Evolution of bacteria under difficult conditions Viable Cells (cfu/ml) 10 8 10 7 10 6 10 5 10 4 10 3 Yeast Lactobacillus Pediococcus Oenococcus Acetobacter 10 2 10 Gluconobacter 3-24 weeks HARVEST TRASPORT ALCOHOLIC FERMENTATION MALOLACTIC FERMENTATION AGING

Bacterial grwoth in must and wine under easy conditions 10 8 10 7 10 6 10 5 10 4 yeast Lactobacillus Pediococcus Oenococcus Acetobacter 10 3 10 2 Gluconobacter 10 cfu/ ml HARVEST ALCOHOLIC TRANSPORT FERMENTATION 3-24 days MALOLACTIC FERMENTATION MACERATION/ STORAGE

8 8 8 8 2 34

SO 2 and MLF SO2 moléculaire (mg/l) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 The molecular SO 2 molecular is the most active form of the sulfuric acid with the highest antimicrobial activity. Molecular SO 2 = free SO 2 / ( 10 (ph - 1.81) + 1 ) SO2 libre : 10mg/l - 18 C 3.0 3.1 3.2 3.3 3.4 ph ) 0 % v/v 13 % v/v SO2 (mg/l) The molecular SO 2 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. molecular SO2 (mg/l) at 18⁰C 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

How yeast can impact malolactic fermentation? Production of ethanol and SO 2 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 2 3 4 5 1- Yeast inhibitory for MLF (very high production of SO 2 or other toxic compounds for bacteria) 2- Yeast unfavourable to MLF (high production of SO 2 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 SO 2 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

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 acid Malic 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)

Portugal (lab results) Merlot/Cabernet Sauvignon Alcohol=14% ph=3.3 Malic acid=0.6 g/l Total SO 2 =50ppm Temperature=20 C Progress of MLF Merlot/Cabernet Sauvignon (Portugal) Malic acid (g/l) 0,7 0,6 0,5 0,4 0,3 0,2 0,1 Non-inoculated SAB 1-Step Alpha Lalvin VP41 0 0 10 20 30 40 Time (days) Failure for direct inoculation / Success for Alpha 1-Step Initial low malic acid level can explain the difficulty to induce MLFwith direct inoculation

2007 - Italy (lab results) Sangiovese Alcohol=14.1% ph=3.12 Malic acid=0.7 g/l Total SO 2 =36 ppm Free SO 2 =6ppm Temperature=20 C Malic acid (g/l) 1,00 0,90 0,80 0,70 0,60 0,50 0,40 0,30 2nd trial - Malic acid degradation 1-Step preculture only for 6 hours 1-Step Alpha Double 1-Step Alpha Control 0,20 0,10 0,00 0 5 10 15 20 25 30 35 40 Time (days) 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

Malic acid tolerance / Lactic acid sensitivity 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 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.

Impact of initial malic acid content Chardonnay 12.5%v/v ph=3.25 Malic acid=2.6g/l Temp.= 16 C Time to achieve 90% of MLF Duration of MLF of course increases with malic acid content. 35 28 21 14 7 Time (days) malate : 0.75 malate : 1.35 malate : 2,6 malate: 5,2 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. 0 Expertise S Lalvin 31 Alpha 49A1 Addition of D-malic acid (D,L malic acid) has no noticeable effect on MLF

Impact of addition of L-lactic acid before MLF Chardonnay 12.5%v/v ph=3.25 Malic acid=2.6g/l Temp.= 16 C Time to achieve 50% of MLF Initial L-malic acid=3g/l 1,0E+07 Bacteria population - Alpha Time (days) 110 100 90 80 70 60 50 40 30 20 10 0 Expertise S Lalvin 31 Alpha 49A1 Lactate : 0 Lactate : 1.5 g/l Lactate: 3.0 g/l log cfu/ml 1,0E+06 1,0E+05 Lactic acid 0 Lactic acid 1.5 Lactic acid 3.0 1,0E+04 0 1 2 3 4 5 6 7 Time (days) 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.

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