Essential Knowledge to Manage Malolactic Fermentation
Dr. Peter Sommer
May 2017
EXECUTIVE SUMMARY
Fact box
>1,000,000 cells of Oenococcus oeni per ml wine to start a malolactic fermentation (MLF)
24,983,700,000,000.00 cells in a 6,660 Gal tank – (3,334 times the earth human population)
No! Not all commercial products are the same in adaptation quality and cell numbers
No! Adding too low cell amount will not make your MLF go fast - if at all - if at all with the strain you inoculated!
Choose the right MLF strain for the right Gary Larsson wine condition
Presentation outline
• The genus of Oenococcus oeni
• The metabolism of Oenococcus oeni • Carbohydrate • Malic acid • Citric acid
• Oenococcus oeni strain characteristics
• Preparation of Oenococcus oeni for inoculation
• Magic Number (of Cells) for successful malolactic fermentation
The genus of Oenococcus oeni
Gram positive bacteria Heterofermentative Coccoid shape Grows optimally in pairs (CH35 in chains of 5 cells) Growth between pH 2.9-5.0 Growth between 57.2-77°F Makarova K. 2006 Inhibited by Total SO2 >30-45 PPM (pH dependent)
The metabolism of Oenococcus oeni
Sugar metabolism Malic acid metabolism
Bisson & Walker 2015
Cibrario et al. 2016 Lonvaud-Funel, A. 1999.
Citrate metabolism in Oenococcus oeni
Citrate lyase
Decarboxylation
Aerobic chemical decarboxylation at low pH
Acetoin reductase (irreversible)
Acetoin reductase (reversible – acetoin preferred substrate)
Olguı´n et al., 2009
Citrate metabolism in Oenococcus oeni The impact of oxygen and diacetyl production
• Anaerobic conditions (0.2 mg/L O2) 2 mg/L diacetyl
• Semi anaerobic conditions
(2-4 mg/L O2) 13 mg/L diacetyl
Nielsen & Richelieu 1999
Citrate metabolism in Oenococcus oeni The impact of oxygen and diacetyl production 1 g/L citrate added
• Anaerobic conditions (0.2 mg/L O2) 5 mg/L diacetyl
• Semi anaerobic conditions
(2-4 mg/L O2) 30 mg/L diacetyl
Nielsen & Richelieu 1999 Citrate metabolism in Oenococcus oeni
C6H8O7 CH3CHOHCOOH + C4H6O2 + 2 CH3COOH + 4 CO2 Citric acid Lactic acid Diacetyl Acetic acid Carbon dioxide
1 mole of citric acid is converted to 2 moles of acetic acid.
Molecular formula and weight of citric acid is C6H8O7 and 192.12 g/mole. Molecular formula and weight of acetic acid is CH3COOH and 60.05 g/mole.
1 mole of citric acid weighs 192.12 g and is converted to 2 moles of acetic acid weighing 120.10 g (2 x 60.05 g)
This means that addition of 1 g of citric acid is converted to 0.63 g of acetic acids (in theory). This is significant.
O. oeni use the citric acid pathway for more than just getting Aerobic chemical decarboxylation at low pH more ATP (energy), but also a way to bio synthetize amino acids when needed. The study also shows that the amount of acetic acid produced from citric acid can be lowered due to these activities.
Olguı´n et al., 2009
Citrate metabolism in Oenococcus oeni
SO2 interaction with diacetyl
Reversible Exothermic (releases energy) Reaction shift left with increasing temperature
Reaction of SO2 with a carbonyl compound. Nielsen & Richelieu, 1999
The metabolism of Oenococcus oeni
In summary
O. oeni metabolize sugars to ethanol, acetic acid, D-lactic acid, and CO2 at pH>3.5
O. Oeni metabolize L-malic acid into L-lactic acid and CO2 at pH<3.5
O. Oeni metabolize citrate into diacetyl, acetoin, and 2,3-butanediol
O. Oeni produce significantly more diacetyl under semi-aerobic conditions (up to 13 mg/L compared to 2 mg/L under anaerobic conditions) (30 mg/L by adding 1 g/L of citric acid)
O. Oeni metabolize L-malic acid before citric acid
Strain characteristics of Oenococcus oeni
Different strains isolated from various wines globally
Strains adapt to a specific wine
condition (pH, temperature, [SO2])
Commercial strains do not have equally amount of cells in the pouch
Commercial strains cope differently to direct inoculation and will die if not pre-adapted for wine conditions
Some commercial strains standardize the number of cells with a carrier (e.g. Maltodextrin)
Preparation of Oenococcus oeni for inoculation
Build up cultures Add culture to prepared juice (heated, cooled, pH adjusted etc.) Add 10% inoculum to wine at each stage, i.e. 0.53 gal into 5.28 gal into 52.83 gal. Risk of contaminations during process Very low inoculation level, but cells well adapted to wine Can take several weeks
Pre activation Dissolve activator and bacteria in 26.4 gal water at 64.4-77°F Wait 20 minutes Add this mixture to 26.4 gal wine (pH>3.5), temp 62.6-77°F Wait 18-24 hours Transfer to 26,417 gal wine Time consuming Problems with administering temperature etc. Potential contamination risk
Quick reactivation Dissolve contents in sterile filtered water at 70°F Wait 5-15 minutes, add to wine
Direct inoculation Open pouch and add to wine Magic number (of cells) for successful malolactic fermentation
>1,000,000 viable/adapted cells of O. oeni per ml of wine is needed for starting the malolactic fermentation
Direct inoculation cultures should be adapted for wine conditions Low pH High ethanol
SO2 Temperature
Ask your vendor for documentation on how they adapt the O. oeni cells for wine conditions Magic number (of cells) for successful malolactic fermentation
Scenarios
22 days 34 days 48 days
22 days 12 days 14 days Magic number (of cells) for successful malolactic fermentation
Direct inoculation
Magic number (of cells) for successful malolactic fermentation
Stretching cultures
Magic number (of cells) for successful malolactic fermentation
Spontaneous
Magic number (of cells) for successful malolactic fermentation
How can you calculate the number of cells you buy?
Look at the product info sheet and find number of cells per g or per pouch.
Find info on dosage. E.g. 1 pouch for 660 gal or 25 g per 2500 L/660 gal
= 660 gal = 2,500,000 ml
= >ퟕퟕퟕퟕퟕퟕ 푪푪푪
푵푵푵푵푵푵 풐풐 풄�풄풄풄 풑𝒑 𝒎 풘풘풘� �ퟐ𝟐𝟐� 𝒎
= >2.8x106 CFU/ml
푵푵푵푵푵푵 풐풐 풄�풄풄풄 풑𝒑 𝒎 풘풘풘� Magic number (of cells) for successful malolactic fermentation
How can you calculate the number of cells you buy?
Look at the product info sheet and find number of cells per g or per pouch.
Find info on dosage.
= 660 gal = 2,500,000 ml
= � >�ퟐ𝒙𝒙 푪푪푪
푵푵푵푵푵푵 풐풐 풄�풄풄풄 풑𝒑 𝒎 풘풘풘� �ퟐ𝟐𝟐� 𝒎 = >1.0x106 CFU/ml Some products have added a carrier that 푵푵푵푵푵푵 풐풐 풄�풄풄풄 풑𝒑 𝒎 풘풘풘� dilute the cell number by half
Strain characteristics of Oenococcus oeni
Malolactic fermentation Colony Forming Unit/ml wine
1.00E+07 +MLF
) 1
- 1.00E+06 g mL
1.00E+05
1.00E+04 ÷MLF
Colony Colony Forming Units ( 1.00E+03
1.00E+02 Commercial strains
Chr. Hansen A B C
Strain characteristics of Oenococcus oeni Cells inoculated per ml of wine compared to VINIFLORA OENOS (%) 100.0 100.00 90.00 80.00
70.00
60.00
50.00
40.00
30.00
20.00 14.3 14.3 14.3 14.3 14.3 14.3 14.3 14.3
10.00 0.4 0.4 0.4 0.4 1.1 0.0 1.1 1.1 0.7 0.00
A B C Chr. Hansen Chr. Magic number (of cells) for successful malolactic fermentation
Td td [h] [days]
86.6 3.6
69.3 2.9
57.8 2.4
49.5 2.1
49.5 2.1
(Ln2) Td (h)= µmax
Zhang & Lovitt, 2005
Strain characteristics of Oenococcus oeni
Generations to reach same cell numbers as VINIFLORA OENOS 49 days
14 13.5 Red line indicated generations to 12 reach the magic number
29 days 10 Doubling time of O. oeni under optimal lab conditions 3.6 days 24 days 8 8 8 8 8 7.2 12.6 days 6.6 6.6 6.6 6
4 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 Number of generationsNumber
2
0 0
C B C A VINIFLORA OENOS Magic number (of cells) for successful malolactic fermentation
Scenarios
22 days 34 days 48 days
22 days 12 days 14 days EXECUTIVE SUMMARY
Fact box ASK YOUR VENDOR
>1,000,000 cells of Oenococcus oeni per ml How many O. oeni cells is in the product wine to start a malolactic fermentation (MLF) per g or per pouch?
No! Not all commercial products are the How many O. oeni cells is inoculated per ml same in adaptation quality and cell numbers of wine?
No! Adding too low cell amount will not How many O. oeni cells do I get per $? make your MLF go fast - if at all - if at all with the strain you inoculated! How adapted are the O. oeni cells for wine? Choose the right MLF strain for the right wine condition How is that adaptation tested?
Literature cited
Cibrario, A., Peanne, C., Lailheugue, M., Campbell-Sills, H., Dols-Lafargue M. 2016. Carbohydrate metabolism in Oenococcus oeni: a genomic insight. BMC Genomics Vol.17:984
Grbin, P.R., Herderich, M. 2007.The Role of Lysine Amino Nitrogen in the Biosynthesis of Mousy Off-Flavor Compounds by Dekkera anomala. J. Agric. Food Chem., Vol. 55, No. 26
Lonvaud-Funel, A. 1999. Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie van Leeuwenhoek Vol. 76(1-4):317-31
Maicas, S., P. Gonzalez-Cabo, S. Ferrer, I. Pardo. 1999. Production of Oenococcus oeni biomass to induce malolactic fermentation in wine by control of pH and substrate addition. Biotechnology Letters Vol. 21: 349–353
Makarova, K. Comparative genomics of the lactic acid bacteria. 2006. Proceedings of the National Academy of Sciences of the United States of America Vol. 103 (42): 15611–15616
Nielsen, J.C., M. Richelieu. 1999. Control of flavor development in wine during and after malolactic fermentation by Oenococcus oeni. Appl. Environ. Microbiol. Vol. 65:740-745.
Olguı´n, N., A. Bordons, C. Reguant. 2009. Influence of ethanol and pH on the gene expression of the citrate pathway in Oenococcus oeni. Food Microbiology Vol. 26:197–203
Zhang, D.-S., Lovitt, R.W. 2005. Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures. J. Appl. Biol. Vol. 99, (3): 565–572
Zhang, D.-S, Lovitt, R.W. 2006. Performance assessment of malolactic fermenting bacteria Oenococcus oeni and Lactobacillus brevis in continuous culture. Applied Microbiology and Biotechnology. Vol. 69 (6): 658–664 .