1. Lecture 13:
Managing the Malolactic Fermentation

2. Reading Assignment:
Chapter 6, pages

3. The Malolactic Fermentation
Requires NAD+, Mn++
Occurs after exponential growth phase
Used to generate energy

4. 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.

5. Factors Affecting the Malolactic FermentationpH

6. pH Affects which strains/species will grow Affects rate of growth
Affects survival of organism
Affects metabolic behavior of strains that are growing

7. Factors Affecting the Malolactic FermentationpH
SO2

8. 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

9. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition

10. 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

11. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen

12. Oxygen Stimulatory to growth Affects spectrum of end products
Can produce more energy (and acetic acid) in presence of oxygen

13. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen
CO2

14. Carbon dioxide
Stimulatory to malolatic fermentation
Mechanism unknown

15. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen
CO2
Alcohol

16. Alcohol High alcohol slows malolactic fermentation
Affects bacterial viability
Affects which species/strains are present

17. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen
CO2
Alcohol
Temperature

18. Temperature
Growth of malolactic bacteria better at higher temperatures
Malolactic fermentation faster at higher temperatures

19. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen
CO2
Alcohol
Temperature
Organic acids

20. 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

21. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen
CO2
Alcohol
Temperature
Organic acids
Phenolic compounds

22. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen
CO2
Alcohol
Temperature
Organic acids
Phenolic acids
Presence of other lactic acid bacteria

23. 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

24. Factors Affecting the Malolactic FermentationpH
SO2
Nutrient composition
Oxygen
CO2
Alcohol
Temperature
Organic acids
Phenolic acids
Presence of other lactic acid bacteria
Bacteriophage

25. 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

26. First Decision:
Do you want the MLF?

27. Reasons MLF Is Desirable
Acidity reduction
Addition of flavors
Bacterial stability of product

28. Reasons MLF Is Undesirable
Acidity reduction
Addition of flavors

29. 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

30. MLF Inhibited By: Use of SO2 Early racking Downward pH adjustment
Low temperature
Filtration/Fining
Addition of fumaric acid
Bacteriocin (lysozyme) addition

31. Second Decision:
Inoculated versus Spontaneous Malolactic Fermentation

32. 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

33. 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

34. Spontaneous MLF Uncontrolled timing of process
Risk of unwanted species/strains
Off-characters can be produced if MLF occurs when undesired

35. Third Decision:
Timing of Malolactic Fermentation

36. Timing of MLF: Options Prior to yeast fermentation
Simultaneous with yeast fermentation
Mid-way through yeast fermentation
After yeast fermentation

37. 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

38. Timing of MLF: Options Prior to yeast fermentation
Simultaneous with yeast fermentation
Mid-way through yeast fermentation
After yeast fermentation

39. 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

40. Timing of MLF: Options Prior to yeast fermentation
Simultaneous with yeast fermentation
Mid-way through yeast fermentation
After yeast fermentation

41. 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

42. Timing of MLF: Options Prior to yeast fermentation
Simultaneous with yeast fermentation
Mid-way through yeast fermentation
After yeast fermentation

43. 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

44. Fourth Decision:
Choice of Strain

45. MLF: Choice of Strain Compatible with yeast
Production of desirable characters
Ability to complete ML fermentation
Vigor
Availability as freeze-dried inoculum

46. Fifth Decision:
Method of Monitoring MLF

47. 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

48. Sixth Decision:
Alternative Method of Acid Reduction

49. 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

50. Overall Goal:
To have all microbial activity finished prior to bottling.

51. This concludes the section on the Malolactic Fermentation