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Essential Knowledge to Manage Malolactic Fermentation

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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 풄푵풄풄풄 풑푵푵 푵풄 풘풘풘푵 ퟕퟐퟕퟕퟕퟕퟕ 푵풄 = >2.8x106 CFU/ml 푵푵푵푵푵푵 풐풐 풄푵풄풄풄 풑푵푵 푵풄 풘풘풘푵 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 = ퟕ >ퟕퟐ 푪푪푪 푵푵푵푵푵푵 풐풐 풄푵풄풄풄 풑푵푵 푵풄 풘풘풘푵 ퟕퟐퟕퟕퟕퟕퟕ 푵풄 = >1.0x106 CFU/ml Some products have added a carrier that 푵푵푵푵푵푵 풐풐 풄푵풄풄풄 풑푵푵 푵풄 풘풘풘푵 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.
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  • Genomic Analysis of Oenococcus Oeni PSU-1 and Its Relevance to Winemaking

    Genomic Analysis of Oenococcus Oeni PSU-1 and Its Relevance to Winemaking

    FEMS Microbiology Reviews 29 (2005) 465–475 www.fems-microbiology.org Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking David A. Mills a,*, Helen Rawsthorne a,1, Courtney Parker a,2, Dafna Tamir a,3, Kira Makarova b a Department of Viticulture and Enology, University of California, Davis, CA 95616, United States b National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, United States Received 24 February 2005; accepted 23 April 2005 First published online 28 August 2005 Abstract Oenococcus oeni is an acidophilic member of the Leuconostoc branch of lactic acid bacteria indigenous to wine and similar envi- ronments. O. oeni is commonly responsible for the malolactic fermentation in wine and due to its positive contribution is frequently used as a starter culture to promote malolactic fermentation. In collaboration with the Lactic Acid Bacteria Genome Consortium the genome sequence of O. oeni PSU-1 has been determined. The complete genome is 1,780,517 nt with a GC content of 38%. 1701 ORFs could be predicted from the sequence of which 75% were functionally classified. Consistent with its classification as an obli- gately heterofermentative lactic acid bacterium the PSU-1 genome encodes all the enzymes for the phosphoketolase pathway. More- over, genes related to flavor modification in wine, such as malolactic fermentation capacity and citrate utilization were readily identified. The completion of the O. oeni genome marks a significant new phase for wine-related research on lactic acid bacteria in which the physiology, genetic diversity and performance of O.