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VEN 124 Section IV

VEN 124 Section IV. The Malolactic Fermentation. Lecture 12: The Biology of the Lactic Acid Bacteria. Reading Assignment: Chapter 6, pages 244-250; 262-278.

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VEN 124 Section IV

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  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 CH2 CH3 + CO2 CHOH CHOH COOH COOH Dicarboxylic Monocarboxylic Can give up 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-CH3 Pyruvate HO-C-CH-CH3 Lactate O O NADH2 NAD+ O OH

  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 ATP ADP NADP+ NADPH CO2 Ribulose 5-phosphate NADP+ NADPH Xylulose 5-phosphate Acetyl-phosphate Glyceraldehyde 3- phosphate NADH NAD+ ADP ATP ADP ATP (glycolysis) Acetate Acetaldehyde Pyruate NADH NAD+ Ethanol Lactate

  13. Lactic Acid Bacteria: Genera • Oenococcus • Pediococcus • Lactobacillus • Leuconostoc

  14. Oenococcus • O. oeni

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

  16. 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 Lactobacillus

  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. Deacidification •  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 CO2 • 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 OH H3C-C=O

  28. Flavor Changes Associated with Malolactic Fermentation • Acetic acid • Diacetyl

  29. Diacetyl • 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 Acetate Oxaloacetate Pyruvate Pyruvate CO2 Pyruvate Acetaldehyde* Acetaldehyde* Acetyl CoA Acetaldehyde* Pyruvate -Acetolactate O O H3 C-C-C-CH3 Diacetyl CO2

  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. Acetoin • 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 Acetate Oxaloacetate Diacetyl CO2 Pyruvate NAD+ NADH Acetaldehyde* Pyruvate Acetoin -Acetolactate O OH H3 C-C-CH-CH3 CO2

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

  36. 2,3-Butanediol • 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 O OH Acetoin H3C-C-CH-CH3 NADH NAD+ HO OH 2,3 Butanediol H3C-CH-CH-CH3

  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 O OH H3C-C-O-C-C-CH3 H H

  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 O H H O H H3C-C-O-C-C-C-C-O-C-CH3 H H H H

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

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

  44. Acrolein Glycerol -hydroxypropionaldehyde H H Acrolein H2C=C-C=O

  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.

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