Microbial Ecology Changes with ph Thomas Henick-Kling Director, Viticulture & Enology Program Professor of Enology
Winemaking Involves Different Population of Microorganisms Kloeckera / Hanseniaspora Schizosaccharomyces Zygosaccharomyces Brettanomyces/Dekkera Hansenula Candida Saccharomyces cerevisiae Oenococcus oeni Acetobacter Gluconobacter Pediococcus sp Lactobacillus sp. Wine Yeast Wine Bacteria Spoilage Microbes
Good winemaking practice is guiding microbial populations and closing the spoilage windows
Spoilage windows Damaged fruit Cold soak Start of alcoholic fermentation Sluggish and stuck fermentation High ph and delay of malolactic fermentation Growth of microbes after completion of fermentation
Succession of Bacteria During Wine Production ph below 3.5 O. oeni ph 3.5-4.0 Lactobacillus Pediococcus O. oeni Wibowo et. al., Occurrence and Growth of Lactic Acid Bacteria in Wine: A Review, Am J. Enol. Vitic., Vol. 36
High Must and Wine ph Increases Fermentation Rate and Growth of Spoilage Yeast and Bacteria ph between 3.5 and 4.0 increases fermentation rates and results in larger overall yeast and bacteria populations can produce biogenic amines (e.g. histamine) by decarboxylation of amino acids Oughi, C.S. (1996). FERMENTATION RATES OF GRAPE JUICE II effect of initial Brix, ph and fermentationa temperature. Am. J. Enol. Vitic. 17 Pediococcus Lactobacillus Hanseniaspora uvarum/ Kloeckera apiculata
ph 3.5 is a critical dividing line for the growth of LAB At ph above 3.5: Potential spoilage bacteria grow much faster than O. oeni (e.g. Lactobacillus sp. and Pediococcus sp.) Results in spoilage flavors and stuck fermentation Affect wine metabolite profiles To suppress growth of unwanted bacteria, the wine ph should be below 3.5 before start of MLF Or, a ML starter culture can be added
ph and L-malic Acid of Washington Red Grapes Typical grape must and wine ph in Washington red wines is above 3.5 In a typical year, red grape varieties in WA have low malic acid content (can be as low as 1 g/l) - Affect the growth and metabolic activity of LAB - Important energy source for LAB in wine during MLF
High Must and Wine ph Leads to Increased Production of Biogenic Amines From Landete. Biogenic amines in wine from three Spanish regions. J Agric. Food Chem. 2005 After MLF, sulfur dioxide additions may not completely halt all bacterial amino acid conversions especially in high ph wines
Effect of High ph On the Wine Metabolite Profile Different and distinct metabolite profile when MLF was conducted at low vs. high ph Higher total red fruit esters at the lower ph At higher ph, these esters were either absent or present in much lower amounts Malolactic Fermentation-importance of Wine Lactic Acid Bacteria in Winemaking, Published LALLEMAND
The Effect of Malolactic Fermentation On the Overall Wine Composition and Quality Malolactic Fermentation (MLF): NAD + Mn 2+ Cox, D.J. and Henick-Kling, T. (1989) Chemiosmotic energy from malolactic fermentation. J Bacteriol. 3H + Malic acid Malolactic Enzyme (malate: NAD + Carboxylase) Lactic acid ooh 3H + During MLF, L-malic acid is decarboxylated to L-lactic acid Increases wine ph and decreases acidity MLF affects wine characteristics Starter culture should contain at least 2 g/l of L-malic acid Figure. Model of the ATP-generating mechanism for the malolactic conversion. In this model the electrogenic transport of L-malate with electroneutral export of L-lactate provides energy through the proton gradient for transport processes and it can be converted by the membrane ATPase into ATP.
High ph of Must and Wine Increases Growth of Bacteria Bacteria Population Changes in Grape Must within 3 days 1.00E+05 4.0 Bacteria Colony Counts (CFU/ml) 9.00E+04 8.00E+04 7.00E+04 6.00E+04 5.00E+04 4.00E+04 3.00E+04 2.00E+04 1.00E+04 E+00 3.8 3.8 3.8 3.75 3.8 3.5 3.8 3.5 3.5 AW=3.75 TA=3.5 AW=3.5 MA=3.5 Piao et al. 2017. Unpublished
Alcoholic Fermentation Rate Sugar content (g/l) Sugar content (g/l) Sugar content (g/l) 30 25 20 15 10 5 30 25 20 15 10 5 30 25 20 15 10 5 Add Yeast No MLB Inoculation AW_3.75 TA_3.5 AW_3.5 MA_3.5 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Add Yeast MLB Co-Inoculation AW_3.75 TA_3.5 AW_3.5 MA_3.5 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 Add Yeast Sequential MLB Inoculation AW_3.75 TA_3.5 AW_3.5 MA_3.5 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Rapid alcoholic fermentation was observed between day 4 and day 6 (Correlated with fast yeast population increase) Alcoholic fermentation was not been affected by ph adjustment or by MLB inoculation methods
Malolactic Fermentation Rate L-malic Acid (g/l) L-malic Acid (g/l) L-malic Acid (g/l) 3.50 3.00 2.50 2.00 1.50 1.00 0.50 3.50 3.00 2.50 2.00 1.50 1.00 0.50 3.50 3.00 2.50 2.00 1.50 1.00 0.50 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 Add MLB MLB Co-Inoculation AW_3.75 TA_3.5 AW_3.5 MA_3.5 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 Add MLB No MLB Inoculation Sequential MLB Inoculation Add MLB End MLF (All) End MLF AW_3.75 TA_3.5 AW_3.5 MA_3.5 End MLF End MLF AW_3.75 TA_3.5 AW_3.5 MA_3.5 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 Without MLB starter culture addition, MLF took up to 10 weeks to complete With MLB co-inoculation, MLF completed within 5 weeks; MLF is faster in the acid wine adjusted lower ph wine while it goes slower in the tartaric acid added wine With sequential inoculation, MLF completed in 3-4 weeks after MLB inoculation (two treatments were not added MLB right after AF)
Phylogenetic Profiles of the Bacterial Communities during Wine Fermentation Relative Abundance 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Malic Acid Tar + Mal Malic Acid Tar + Mal Malic Acid Tar + Mal Malic Acid Tar + Mal Malic Acid Tar + Mal Acidobacteria Actinobacteria Bacteroidetes Chlamydiae Chloroflexi Firmicutes Tenericutes Gemmatimonadetes Planctomycetes Proteobacteria Verrucomicrobia Unclassified Day 2 Day 4 Day 8 Day 15 Day 25 Phylogeny was assigned in the phylum level based on SILVA reference database after quality-filtered reads were clustered using 97% sequence identity cut-off.
ph Acid addition Tartaric acid DL-malic acid Tartaric + DLmalic acid Day 2 4.51 + 0.13 3.36 + 0.07 3.54 + 0.06 3.64 + 0.01 Day 4 3.94 + 0.02 3.56 + 0.03 3.78 + 0.01 3.80 + Day 8 4.16 + 3.74 + 0.02 3.98 + 0.03 3.94 + 0.04 Day 15 4.19 + 0.02 3.78 + 0.01 4.01 + 0.01 4.00 + 0.04 Day 25 4.14 + 0.01 3.81 + 0.04 4.02 + 0.02 4.00 + 0.02 Titratable Acidity (TA) (g/l) Acid addition Tartaric acid DL-malic acid Tartaric + DLmalic acid Initial TA 3.31 + 0.03 3.27 + 0.08 3.23 + 0.04 3.12 + 0.08 Addition + 3.13 + 0.11 + 0.54 + 0.11 DL-Malic Acid Addition + + 3.18 + 0.06 2.80 + Target TA 3.31 + 0.03 6.50 + 6.50 + 6.50 + (g/l) Acid addition Tartaric acid DL-malic acid Tartaric + DLmalic acid Day 2 3.72 + 0.05 6.02 + 0.33 3.31 + 0.17 4.33 + 0.03 Day 4 4.03 + 0.01 5.09 + 0.54 4.05 + 0.10 4.13 + 0.02 Day 8 2.00 + 0.03 2.42 + 0.18 1.99 + 0.04 2.16 + 0.05 Day 15 1.57 + 1.93 + 1.51 + 1.67 + Day 25 1.54 + 1.74 + 1.45 + 1.62 +
A. B. Glucose + Fructose (g/l) 30 25 20 15 10 5 Glucose/Fructose DL-malic Acid Tartaric & DL-Malic 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Glucose (g/l) 14 12 10 8 6 4 2 Glucose DL-malic Acid Tartaric & DL-Malic 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 C. D. Fructose (g/l) 14 12 10 8 6 4 2 Fructose DL-malic Acid Tartaric & DL-Malic 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Alcohol (%) 2 18.00 16.00 14.00 12.00 1 8.00 6.00 4.00 2.00 Alcohol DL-malic Acid Tartaric & DL-Malic Acid 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
E. F. (g/l) 7.00 6.00 5.00 DL-Malic Acid 4.00 Tartaric & DL-Malic Acid 3.00 2.00 1.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 L-Malic Acid (g/l) 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 L-Malic Acid DL-Malic Acid Tartaric & DL-Malic Aicd 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 G. H. L-Maic Acid (g/l) 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 L-Lactic Acid DL-Malic Acid Tartaric & DL-Malic Acid 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 L-Acetic Acid 0.60 0.50 0.40 0.30 0.20 0.10 L-Acetic Acid DL-Malic Acid Tartaric & DL-Malic Acid 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Thank You Collaborators: Dr Hailan Piao, Dr Tom Collins, Dr Jim Harbertson Tyler Williams - graduate student Mitchell Williamson, Dru Seed undergrad students Research assistants: Rosemary Veghte, Maria Mireless, Caroline Merrell Richard Larsen and Cary Wilton research winemakers