Download 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
The “malolactic fermentation” refers
to the conversion of the grape acid
malate to lactate conducted by
members of the lactic acid bacteria
Malolactic Fermentation
COOH
CH2
CH3
CHOH
CHOH
COOH
COOH
+
CO2
Dicarboxylic
Monocarboxylic
Can give up
Can give up
2 protons
1 proton
Lactic Acid Bacteria: Characteristics
• Prokaryotes: no membrane around
nucleus
• Gram positive
– Peptidoglycan
– Teichoic acid
• Divide by binary fission
Binary Division
DNA
Lactic Acid Bacteria: Divisions
• Group I: Strict homofermenters
• Group II: Facultative heterofermenters
• Group III: Strict heterofermenters
Homofermentative Metabolism
85% of Glucose
Glucose
Lactic acid
Pyruvate via glycolytic pathway
OO
HO-C-C-CH3
Pyruvate
NADH2
NAD+
O OH
HO-C-CH-CH3
Lactate
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. Acetylphosphate can be converted to acetate and
ATP or reduced to ethanol without ATP
production.
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.
Pentose Phosphate Pathway
Glucose
Phospho-6-gluconate
ATP ADP
NADP+
NADPH
CO2
Ribulose 5-phosphate
NADP+
NADPH
Xylulose 5-phosphate
Acetyl-phosphate
ADP
Acetate
ATP
NADH
NAD+
Acetaldehyde
NADH
Glyceraldehyde 3- phosphate
(glycolysis)
ATP
Pyruate
NAD+
Ethanol
ADP
Lactate
Lactic Acid Bacteria: Genera
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Oenococcus
Pediococcus
Lactobacillus
Leuconostoc
Oenococcus
• O. oeni
Pediococcus
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•
P. damnosus
P. parvulus
P. pentosaceus
P. acidilactici
Lactobacillus
Homolactic on hexoses
Heterolactic on hexoses
•
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L. bavaricus
L. casei
L. homohoichii
L. curvatus
L. saki
L. plantarum
L. fermentum
L. brevis
L. buchneri
L. fructovorans
L. hilgardii
Leuconostoc
Wine species of Leuconostoc have been
reclassified as Oenococcus.
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.
Effects of Malolactic Fermentation
• Deacidification
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
Effects of Malolactic Fermentation
• Deacidification
• Bacterial stability
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
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
Effects of Malolactic Fermentation
• Deacidification
• Bacterial stability
• Flavor changes
Flavor Changes Associated with
Malolactic Fermentation
• Acetic acid
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
Acetic Acid
OH
H3C-C=O
Flavor Changes Associated with
Malolactic Fermentation
• Acetic acid
• Diacetyl
Diacetyl
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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
Pathways to Diacetyl
Citrate
Acetate
Oxaloacetate
CO2
Pyruvate
Pyruvate
Acetaldehyde*
Acetaldehyde*
Acetaldehyde*
Acetyl CoA
Pyruvate
-Acetolactate
CO2
Pyruvate
Diacetyl
OO
H3 C-C-C-CH3
Acetaldehyde* refers to “active
acetaldehyde” which indicates the
enzymatically bound form of acetaldehyde
with the coenzyme thiamine
pyrophosphate.
Flavor Changes Associated with
Malolactic Fermentation
• Acetic acid
• Diacetyl
• Acetoin
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
Pathways to Acetoin
Citrate
Acetate
Oxaloacetate
Diacetyl
CO2
Pyruvate
NAD+
NADH
Acetaldehyde*
Pyruvate
-Acetolactate
CO2
Acetoin
O OH
H3 C-C-CH-CH3
Flavor Changes Associated with
Malolactic Fermentation
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Acetic acid
Diacetyl
Acetoin
2,3-Butanediol
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
2,3 Butanediol
O OH
H3C-C-CH-CH3
Acetoin
NADH
NAD+
2,3 Butanediol
HO OH
H3C-CH-CH-CH3
Flavor Changes Associated with
Malolactic Fermentation
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Acetic acid
Diacetyl
Acetoin
2,3 Butantediol
Ethyl lactate
Ethyl Lactate
• “Generic fruit” character
• Ester of lactate, a monocarboxylic acid
H
O OH
H3C-C-O-C-C-CH3
H
H
Flavor Changes Associated with
Malolactic Fermentation
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Acetic acid
Diacetyl
Acetoin
2,3 Butanediol
Ethyl lactate
Diethyl succinate
Diethyl Succinate
• “Generic fruit” similar to ethyl lactate
• Ester of succinate, a dicarboxylic acid
H
OHH O
H
H3C-C-O-C-C-C-C-O-C-CH3
H
HH
H
Flavor Changes Associated with
Malolactic Fermentation
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Acetic acid
Diacetyl
Acetoin
2,3 Butanediol
Ethyl lactate
Diethyl succinate
Acrolein
Acrolein
• Made from glycerol
• Creates an intensely bitter taste when
combined with phenolic compounds
Acrolein
Glycerol
-hydroxypropionaldehyde
Acrolein
HH
H2C=C-C=O
Flavor Changes Associated with
Malolactic Fermentation
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Acetic acid
Diacetyl
Acetoin
2,3 Butanediol
Ethyl lactate
Diethyl succinate
Acrolein
Other compounds
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.
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.
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.
In the next lecture we will
learn how to manage the
malolactic fermentation in
the winery.