Lecture 13: Managing the Malolactic Fermentation.
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Transcript of Lecture 13: Managing the Malolactic Fermentation.
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Lecture 13:
Managing the Malolactic Fermentation
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Reading Assignment:
Chapter 6, pages 251-261
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The Malolactic Fermentation
• Requires NAD+, Mn++
• Occurs after exponential growth phase
• Used to generate energy
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Energy Generation from the Malolactic Conversion
ATP ADP Lactate Malate
Lactate Malate
Proton Motive Force
The conversion of malate to lactate and accompanying “fixing” of a proton decreases the proton content of the cytoplasm upon efflux of lactate thereby creating a “proton motive force” across the membrane; the energy of the proton movement can then be captured in ATP.
H+
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Factors Affecting the Malolactic Fermentation
• pH
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pH
• Affects which strains/species will grow
• Affects rate of growth
• Affects survival of organism
• Affects metabolic behavior of strains that are growing
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Factors Affecting the Malolactic Fermentation
• pH
• SO2
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SO2
• Sulfur dioxide is inhibitory
• All genera/species/strains appear to be equally sensitive
• Even if SO2 is not added, it may be produced by yeast at an inhibitory concentration
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Factors Affecting the Malolactic Fermentation
• pH
• SO2
• Nutrient composition
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Nutrient Composition
• Lactic acid bacteria are fastidious: numerous growth requirements
• Aging on yeast lees increases micronutrient content via autolysis
• Extended skin contact enhances lactic acid bacteria
• Higher solids/less clarification enhances lactic acid bacteria
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Factors Affecting the Malolactic Fermentation
• pH
• SO2
• Nutrient composition
• Oxygen
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Oxygen
• Stimulatory to growth
• Affects spectrum of end products
• Can produce more energy (and acetic acid) in presence of oxygen
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Factors Affecting the Malolactic Fermentation
• pH
• SO2
• Nutrient composition
• Oxygen
• CO2
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Carbon dioxide
• Stimulatory to malolatic fermentation
• Mechanism unknown
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Factors Affecting the Malolactic Fermentation
• pH
• SO2
• Nutrient composition
• Oxygen
• CO2
• Alcohol
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Alcohol
• High alcohol slows malolactic fermentation
• Affects bacterial viability
• Affects which species/strains are present
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Factors Affecting the Malolactic Fermentation
• pH
• SO2
• Nutrient composition
• Oxygen
• CO2
• Alcohol
• Temperature
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Temperature
• Growth of malolactic bacteria better at higher temperatures
• Malolactic fermentation faster at higher temperatures
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Factors Affecting the Malolactic Fermentation
• pH
• SO2
• Nutrient composition• Oxygen
• CO2
• Alcohol• Temperature• Organic acids
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Organic Acids
• Fumarate inhibitory at low concentrations
• Can be produced by yeast
• Fatty acids can also be inhibitory
• Malate stimulates growth prior to malolactic fermentation
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Factors Affecting the Malolactic Fermentation• pH• SO2
• Nutrient composition• Oxygen• CO2
• Alcohol• Temperature• Organic acids• Phenolic compounds
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Factors Affecting the Malolactic Fermentation• pH• SO2
• Nutrient composition• Oxygen• CO2
• Alcohol• Temperature• Organic acids• Phenolic acids• Presence of other lactic acid bacteria
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Presence of Other Lactic Acid Bacteria
• Mixed cultures may yield “better” complexity
• Can be stimulatory– Increase in pH
• Can be inhibitory– Bacteriocin production– Competition for nutrients
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Factors Affecting the Malolactic Fermentation• pH• SO2
• Nutrient composition• Oxygen• CO2
• Alcohol• Temperature• Organic acids• Phenolic acids• Presence of other lactic acid bacteria• Bacteriophage
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Bacteriophage
• Bacterial “viruses” that can be spread from one bacterium to another and that cause cell death
• Not known if this is a problem in wine production or not; it is a problem in other lactic acid bacteria fermentations
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Do you want the MLF?
First Decision:
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Reasons MLF Is Desirable
• Acidity reduction
• Addition of flavors
• Bacterial stability of product
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Reasons MLF Is Undesirable
• Acidity reduction
• Addition of flavors
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MLF Stimulated By:
• Low to no use of SO2
• Warm temperatures
• Addition of nutrients
• Use of inocula
• Low ethanol (avoid late harvest wines)
• Delay racking off yeast lees
• Acid/pH adjustment
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MLF Inhibited By:
• Use of SO2
• Early racking
• Downward pH adjustment
• Low temperature
• Filtration/Fining
• Addition of fumaric acid
• Bacteriocin (lysozyme) addition
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Inoculated versus Spontaneous Malolactic Fermentation
Second Decision:
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Inoculated MLF
• Better control over both timing and organisms present
• Difficult to maintain inocula
• Starter culture must be “pure”
• Percent inoculation: 1-50% depending upon vigor of culture
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Inoculum Preparation
1. Start culture from slant in medium supporting good growth of organism
2. Inoculate “diluted” juice (with water) from starter with addition of nutrients
3. Use #2 to inoculate full strength wine or juice with addition of nutrients
4. Use #3 to inoculate rest of wine
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Spontaneous MLF
• Uncontrolled timing of process
• Risk of unwanted species/strains
• Off-characters can be produced if MLF occurs when undesired
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Third Decision:
Timing of Malolactic Fermentation
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Timing of MLF: Options
• Prior to yeast fermentation
• Simultaneous with yeast fermentation
• Mid-way through yeast fermentation
• After yeast fermentation
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Timing of MLF: Pre-Fermentation Inoculation
• Decreases yeast nutrients– Stuck/sluggish fermentation– Production of off-characters
• May lead to production of inhibitory compounds (acetic acid) due to presence of oxygen
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Timing of MLF: Options
• Prior to yeast fermentation
• Simultaneous with yeast fermentation
• Mid-way through yeast fermentation
• After yeast fermentation
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Timing of MLF: Simultaneous with Yeast Inoculation
• See increase in acetic acid
• See a decrease in viability of both yeast and bacteria
• Yeast “rebound” better than bacteria
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Timing of MLF: Options
• Prior to yeast fermentation
• Simultaneous with yeast fermentation
• Mid-way through yeast fermentation
• After yeast fermentation
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Timing of MLF: Mid-Fermentation
• Nutrients left for bacteria
• Ethanol low and not inhibitory
• Yeast-produced SO2 may be inhibitory
• May lead to arrest of yeast fermentation
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Timing of MLF: Options
• Prior to yeast fermentation
• Simultaneous with yeast fermentation
• Mid-way through yeast fermentation
• After yeast fermentation
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Timing of MLF: Post-Fermentation
• Nutrients have been depleted– Add nutrients– Encourage yeast autolysis
• Ethanol concentration high
• Concentration of other yeast inhibitory compounds also high
• Better temperature control
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Fourth Decision:
Choice of Strain
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MLF: Choice of Strain
• Compatible with yeast
• Production of desirable characters
• Ability to complete ML fermentation
• Vigor
• Availability as freeze-dried inoculum
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Fifth Decision:
Method of Monitoring MLF
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Monitoring the MLF
• By conversion of malate to lactate– Loss of malate not appearance of lactate*
– HPLC, Enzymatic, Paper chromatography
• By flavor changes– Tells you bacteria are active– Does not tell you when they are done
* Lactate can be produced from other sources
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Sixth Decision:
Alternative Method of Acid Reduction
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Alternative Methods of Acid Reduction• Immobilized enzyme• Immobilized cells• Yeast mediated conversion of malate to
ethanol– Conducted by S. pombe– S. cerevisiae has been genetically engineered to
perform this conversion
• Expression of ML enzyme in Saccharomyces• Chemical precipitation
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Overall Goal:
To have all microbial activity finished prior to bottling.
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This concludes the section on the Malolactic Fermentation