1. VEN 124 Section IV The Malolactic Fermentation

2. Lecture 12: The Biology of the Lactic Acid Bacteria

3. Reading Assignment: Chapter 6, pages 244-250; 262-278

4. The “malolactic fermentation” refers to the conversion of the grape acid malate to lactate conducted by members of the lactic acid bacteria

5. Malolactic Fermentation COOH CH 2 CH 3 +CO 2CHOHCOOH DicarboxylicMonocarboxylic Can give up 2 protons 1 proton

6. Lactic Acid Bacteria: Characteristics Prokaryotes: no membrane around nucleus Gram positive –Peptidoglycan –Teichoic acid Divide by binary fission

7. Binary Division DNA

8. Lactic Acid Bacteria: Divisions Group I: Strict homofermenters Group II: Facultative heterofermenters Group III: Strict heterofermenters

9. Homofermentative Metabolism 85% of Glucose Lactic acid Glucose Pyruvate via glycolytic pathway HO-C-C-CH 3 Pyruvate HO-C-CH-CH 3 Lactate O O OH NADH 2 NAD +

10. Heterofermentative Metabolism Organisms metabolize glucose via the pentose phosphate pathway. End products can vary depending upon level of aeration and presence of other proton and electron acceptors. Acetyl- phosphate can be converted to acetate and ATP or reduced to ethanol without ATP production.

11. Pentose Phosphate Pathway Lactic Acid Bacteria can also metabolize pentoses such as ribose, arabinose and xylose, via the pentose phosphate pathway. Acetyl-phosphate leads to the generation of acetate and ATP exclusively in pentose metabolism.

12. Pentose Phosphate Pathway Glucose Phospho-6-gluconate Ribulose 5-phosphate Xylulose 5-phosphate CO 2 Glyceraldehyde 3- phosphate Pyruate Lactate Acetyl-phosphate Acetate Acetaldehyde Ethanol (glycolysis) NADP + NADPH NADP + NADPH NADH NAD + NADH NAD + ADP ATP ADP ATP ATP ADP

13. Lactic Acid Bacteria: Genera Oenococcus Pediococcus Lactobacillus Leuconostoc

14. Oenococcus O. oeni

15. Pediococcus P. damnosus P. parvulus P. pentosaceus P. acidilactici

16. Lactobacillus Homolactic on hexoses L. bavaricus L. casei L. homohoichii L. curvatus L. saki L. plantarum Heterolactic on hexoses L. fermentum L. brevis L. buchneri L. fructovorans L. hilgardii

17. Leuconostoc Wine species of Leuconostoc have been reclassified as Oenococcus.

18. Lactic Acid Bacteria: Prevalence in Wine Only O. oeni is found at low (  3.5) pH Pediococcus and Lactobacillus grow at pH values above 3.5.

19. Effects of Malolactic Fermentation Deacidification

20.  Titratable acidity by 0.01 to 0.03 g/L because of H + fixation  pH by 0.1 to 0.3 units Important for high acid wines May be undesirable in low acid situations

21. Effects of Malolactic Fermentation Deacidification Bacterial stability

22. Bacterial Stability Consume nutrients that would otherwise be available for other organisms Produce toxins (bacteriocins) that may inhibit growth of other bacteria Prevent malolactic fermentation from occurring in bottle

23. Malolactic Fermentation in Bottle: Increases turbidity due to cell growth Produces noticeable gas as CO 2 May produce polysaccharide material –Haze –Ropiness May raise pH allowing growth of spoilage organisms Does not allow for control of flavor/aroma profile of wine

24. Effects of Malolactic Fermentation Deacidification Bacterial stability Flavor changes

25. Flavor Changes Associated with Malolactic Fermentation Acetic acid

26. Acetic Acid From sugar metabolism Amount formed versus ethanol depends upon aeration and presence of other electron acceptors Level produced can be significant Can also be produced from citrate metabolism Low levels can be made by Saccharomyces

27. Acetic Acid H 3 C-C=O OH

28. Flavor Changes Associated with Malolactic Fermentation Acetic acid Diacetyl

29. Made from pyruvate Multiple pathways to pyruvate 1-4 mg/L adds complexity “buttery” Over 4 mg/L dominates “movie popcorn butter/rancid” Low amounts can be produced by yeast Dicarbonyl compounds important in the chemical generation of wine grape characters

30. Pathways to Diacetyl Citrate Oxaloacetate Pyruvate Acetate CO 2 Acetaldehyde *  -Acetolactate Diacetyl Acetaldehyde * Pyruvate CO 2 Acetyl CoA H 3 C-C-C-CH 3 O

31. Acetaldehyde * refers to “active acetaldehyde” which indicates the enzymatically bound form of acetaldehyde with the coenzyme thiamine pyrophosphate.

32. Flavor Changes Associated with Malolactic Fermentation Acetic acid Diacetyl Acetoin

33. Also produced from pyruvate Can be derived from diacetyl Generally present below threshold of detection May be important in other chemical reactions in wine

34. Pathways to Acetoin Citrate Oxaloacetate Pyruvate Acetate CO 2  -Acetolactate Acetoin Acetaldehyde * Pyruvate CO 2 H 3 C-C-CH-CH 3 O OH Diacetyl NAD + NADH

35. Flavor Changes Associated with Malolactic Fermentation Acetic acid Diacetyl Acetoin 2,3-Butanediol

36. Derived from acetoin Generally present below threshold of detection Mild “sweet alcohol” flavor that borders on bitterness Can be produced by yeast

37. 2,3 Butanediol Acetoin 2,3 Butanediol H 3 C-C-CH-CH 3 H 3 C-CH-CH-CH 3 O OH HO OH NADH NAD +

38. Flavor Changes Associated with Malolactic Fermentation Acetic acid Diacetyl Acetoin 2,3 Butantediol Ethyl lactate

39. Ethyl Lactate “Generic fruit” character Ester of lactate, a monocarboxylic acid H 3 C-C-O-C-C-CH 3 H H HO OH

40. Flavor Changes Associated with Malolactic Fermentation Acetic acid Diacetyl Acetoin 2,3 Butanediol Ethyl lactate Diethyl succinate

41. Diethyl Succinate “Generic fruit” similar to ethyl lactate Ester of succinate, a dicarboxylic acid H 3 C-C-O-C-C-C-C-O-C-CH 3 H H OH O H H

42. Flavor Changes Associated with Malolactic Fermentation Acetic acid Diacetyl Acetoin 2,3 Butanediol Ethyl lactate Diethyl succinate Acrolein

43. Made from glycerol Creates an intensely bitter taste when combined with phenolic compounds

44. Acrolein Glycerol  -hydroxypropionaldehyde Acrolein H 2 C=C-C=O H

45. Flavor Changes Associated with Malolactic Fermentation Acetic acid Diacetyl Acetoin 2,3 Butanediol Ethyl lactate Diethyl succinate Acrolein Other compounds

46. Other Compounds The Lactic Acid Bacteria are capable of producing numerous other aroma compounds, especially from the degradation of amino acids. It is likely that some of these compounds are also being produced during growth in wine.

47. Tartrate Some strains of L. plantarum and L. brevis are capable of metabolizing tartrate to acetic acid, referred to as “tourne disease” by Pasteur. This is always undesirable.

48. Flavor Changes Associated with the Malolactic Fermentation Amounts of specific compounds produced are strain dependent and dependent upon the composition of the juice and level of aeration.

49. In the next lecture we will learn how to manage the malolactic fermentation in the winery.