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Lecture 13: Managing the Malolactic Fermentation. Reading Assignment: Chapter 6, pages 251-261. The Malolactic Fermentation. Requires NAD + , Mn ++ Occurs after exponential growth phase Used to generate energy. Energy Generation from the Malolactic Conversion. Lactate Malate.

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slide1

Lecture 13:

Managing the Malolactic Fermentation

slide2

Reading Assignment:

Chapter 6, pages 251-261

the malolactic fermentation
The Malolactic Fermentation
  • Requires NAD+, Mn++
  • Occurs after exponential growth phase
  • Used to generate energy
energy generation from the malolactic conversion
Energy Generation from the Malolactic Conversion

Lactate Malate

H+

ATP ADP

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.

slide6
pH
  • Affects which strains/species will grow
  • Affects rate of growth
  • Affects survival of organism
  • Affects metabolic behavior of strains that are growing
slide8
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
nutrient composition
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
factors affecting the malolactic fermentation11
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
oxygen
Oxygen
  • Stimulatory to growth
  • Affects spectrum of end products
  • Can produce more energy (and acetic acid) in presence of oxygen
factors affecting the malolactic fermentation13
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
  • CO2
carbon dioxide
Carbon dioxide
  • Stimulatory to malolatic fermentation
  • Mechanism unknown
factors affecting the malolactic fermentation15
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
  • CO2
  • Alcohol
alcohol
Alcohol
  • High alcohol slows malolactic fermentation
  • Affects bacterial viability
  • Affects which species/strains are present
factors affecting the malolactic fermentation17
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
  • CO2
  • Alcohol
  • Temperature
temperature
Temperature
  • Growth of malolactic bacteria better at higher temperatures
  • Malolactic fermentation faster at higher temperatures
factors affecting the malolactic fermentation19
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
  • CO2
  • Alcohol
  • Temperature
  • Organic acids
organic acids
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
factors affecting the malolactic fermentation21
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
  • CO2
  • Alcohol
  • Temperature
  • Organic acids
  • Phenolic compounds
factors affecting the malolactic fermentation22
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
  • CO2
  • Alcohol
  • Temperature
  • Organic acids
  • Phenolic acids
  • Presence of other lactic acid bacteria
presence of other lactic acid bacteria
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
factors affecting the malolactic fermentation24
Factors Affecting the Malolactic Fermentation
  • pH
  • SO2
  • Nutrient composition
  • Oxygen
  • CO2
  • Alcohol
  • Temperature
  • Organic acids
  • Phenolic acids
  • Presence of other lactic acid bacteria
  • Bacteriophage
bacteriophage
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
slide26

First Decision:

Do you want the MLF?

reasons mlf is desirable
Reasons MLF Is Desirable
  • Acidity reduction
  • Addition of flavors
  • Bacterial stability of product
reasons mlf is undesirable
Reasons MLF Is Undesirable
  • Acidity reduction
  • Addition of flavors
mlf stimulated by
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
mlf inhibited by
MLF Inhibited By:
  • Use of SO2
  • Early racking
  • Downward pH adjustment
  • Low temperature
  • Filtration/Fining
  • Addition of fumaric acid
  • Bacteriocin (lysozyme) addition
slide31

Second Decision:

Inoculated versus Spontaneous Malolactic Fermentation

inoculated mlf
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
inoculum preparation
Inoculum Preparation
  • Start culture from slant in medium supporting good growth of organism
  • Inoculate “diluted” juice (with water) from starter with addition of nutrients
  • Use #2 to inoculate full strength wine or juice with addition of nutrients
  • Use #3 to inoculate rest of wine
spontaneous mlf
Spontaneous MLF
  • Uncontrolled timing of process
  • Risk of unwanted species/strains
  • Off-characters can be produced if MLF occurs when undesired
slide35

Third Decision:

Timing of Malolactic Fermentation

timing of mlf options
Timing of MLF: Options
  • Prior to yeast fermentation
  • Simultaneous with yeast fermentation
  • Mid-way through yeast fermentation
  • After yeast fermentation
timing of mlf pre fermentation inoculation
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
timing of mlf options38
Timing of MLF: Options
  • Prior to yeast fermentation
  • Simultaneous with yeast fermentation
  • Mid-way through yeast fermentation
  • After yeast fermentation
timing of mlf simultaneous with yeast inoculation
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
timing of mlf options40
Timing of MLF: Options
  • Prior to yeast fermentation
  • Simultaneous with yeast fermentation
  • Mid-way through yeast fermentation
  • After yeast fermentation
timing of mlf mid fermentation
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
timing of mlf options42
Timing of MLF: Options
  • Prior to yeast fermentation
  • Simultaneous with yeast fermentation
  • Mid-way through yeast fermentation
  • After yeast fermentation
timing of mlf post fermentation
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
slide44

Fourth Decision:

Choice of Strain

mlf choice of strain
MLF: Choice of Strain
  • Compatible with yeast
  • Production of desirable characters
  • Ability to complete ML fermentation
  • Vigor
  • Availability as freeze-dried inoculum
slide46

Fifth Decision:

Method of Monitoring MLF

monitoring the mlf
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

slide48

Sixth Decision:

Alternative Method of Acid Reduction

alternative methods of acid reduction
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
slide50

Overall Goal:

To have all microbial activity finished prior to bottling.