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‫תהליכים כימיים המתרחשים במהלך התסיסה הכוהלית‬

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‫מבוא‬
C6H12O6  2CH3-CH2OH + 2CO2
Mw = 180 g/mol Mw = 46 g/mol

2x46 = 92  (46/92)*100 = 51%

Actually ~5% of the sugar is consumed to produce many by-products such as glycerol, succinic acid, lactic
acid, acetic acid, 2,3-butandiol etc. Also, ~2.5% is consumed by the yeast as carbon source, and ~0.5% is
left over as unfermented residual sugars. Thus, ~8% of the Bx is not converted into ethanol. This makes the
total weight yield to be 51x0.92 = 47% which comes to 180x0.47 = 84.7g of ethanol.

d = 0.789 g/ml  V = 84.6/0.789 = 107.2 ml

Dilution with water to 1L cause contraction of 0.7% in volume (e.g. for 100ml ethanol one should add 907 ml
water to yield 1 L with 10% alcohol v/v).

From 180g/L sugar  107.2 x 1.007 = 108 ml alcohol in 1L of wine which is 10.8% (v/v). In Brix units (where
~95% of the total solids content in must is sugar), in order to produce 1% (v/v) of alcohol, the following brix
will be needed:
(180/0.95/108 = 1.75), namely, each 1.75 Bx  1% alcohol (v/v); Must’s Brix ratio 1.0/1.75 = 0.57.

Adapted from: Margalit, A. 2004: “Concepts in Wine Chemistry"

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The meaning of this number is that the potential alcohol content in a given must can be
predicted by the quite good approximation:

% potential alcohol (v/v) = 0.57 Brix

The experimental value of this number was checked in various studies and was found
to be in the range of (0.55-0.60), namely, it can be written as:

% potential alcohol (v/v) = (0.57 ± 0.03) Brix

The formula has the following limitations:

1) The non-sugar solids which are part of the must`s brix, depend on the grape variety, growing
region, and state of ripeness. Grapes which are less ripe have higher non-sugar solids content,
and hence, lower alcohol/brix ratio.
2) Ethanol yield depends on the fermentation temperature: the higher the temperature, the lower the
yield, partly because of other by-products such as glycerol, and partly by ethanol evaporation at
higher fermentation temperature.
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...‫תזכורת‬
Adenosine triphosphate (ATP)

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Microbiology of Wine and Vinifications”
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Nicotinamide adenine dinucleotide (NAD+)

(a) Structure of nicotinamide adenine dinucleotide in the oxidized form (NAD +).
(b) Equilibrium reaction between the oxidized (NAD+) and reduced (NADH) forms

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Microbiology of Wine and Vinifications”
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Glycolysis
 Glycolysis refers to the metabolism of six‐carbon hexoses (e.g., fructose,
glucose) to pyruvate.

 Yeast will utilize glycolysis for two reasons:

1. To generate energy, in the form of adenosine triphosphate (ATP)

2. To produce compounds necessary for growth and other physiological functions,

for example, lipids, polysaccharides, proteins, nucleic acids.

 Glycolysis generates an excess of reducing compounds, particularly the reduced

formed of nicotinamide adenine dinucleotide (NADH) and related metabolites.

 Under aerobic conditions, many eukaryotes will undergo respiration, in which

NADH is converted back to its oxidized form (NAD+) by an external electron
acceptor, typically O2.

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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Glycolysis
 This metabolic process can generate a large amount of energy, approximately 36
ATP per hexose sugar.

 A related redox couple, NADPH and its oxidized form (NADP+) can be produced
through hexose sugar oxidation (pentose phosphate pathway).

 In addition to energy production, these compounds have important roles as

reductants, with NADH primarily involved in catabolism and NADPH in anabolism.

 In fermentation, no external electron acceptor is involved. In humans and other

mammals, pyruvate is reduced to regenerate NAD+ during fermentation, yielding
lactic acid.

 In yeast, pyruvate is decarboxylated to acetaldehyde, which is subsequently

reduced to ethanol.

 The net equation for this reaction is:

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry" (Crabtree effect)
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Glycolysis Pathway

Glycolysis Pathway. This sequence of 10 reactions oxidizes glucose to pyruvate. Each reaction is catalyzed
by a different enzyme to produce two net ATP (4 ATP are produced, but 2 are invested), two molecules of NADH, and two
molecules of pyruvate. In step 4, fructose-1,6-bisphosphate is divided into two products that both proceed through steps 6–
10. The amounts for “What goes in” and “What goes out” are the combined totals for both molecules.

Adapted from: Chapter 9 Cellular Respiration and Fermentation , MasteringBiology

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Adapted from: Chapter 9 Cellular Respiration and Fermentation , MasteringBiology

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The Reactions of Glycolysis

Adapted from: Chapter 9 Cellular Respiration and Fermentation , MasteringBiology

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Glycolysis and alcoholic fermentation pathway
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7 steps:
1. Phosphorylation
2. Hexose breaking
3. Phosphorylation
4. Dephosphorylation I
5. Dephosphorylation II
6. Decarboxylation
7. Reduction

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Microbiology of Wine and Vinifications”
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Fermentation
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Cellular Respiration and Fermentation Are Alternative Pathways for Producing ATP. When oxygen
or another final electron acceptor used by the ETC is present in a cell, the pyruvate produced by glycolysis enters the citric
acid cycle and the electron transport system is active. But if no electron acceptor is available to keep the ETC running, then
pyruvate undergoes reactions known as fermentation.

Fermentation Regenerates NAD∙ So That Glycolysis Can Continue. These are just two examples of the many
types of fermentation that occur in prokaryotes and eukaryotes.

Adapted from: Chapter 9 Cellular Respiration and Fermentation , MasteringBiology

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Fermentation

Change in concentration of sugars (hexose + glucose), live yeast cells, and key glycolysis metabolites during fermentation of a
Chardonnay juice. Live cells increase during the growth phase, plateau during the stationary phase, and decrease during the decline
phase. Note that an initial delay (lag phase) can also occur during which yeast adapt to must conditions.

 The major outcome of glycolysis is production of ethanol, but a portion of glycolysis products are diverted to
biomass formation, yielding glycerol and acetic acid. Beyond this, several other minor metabolites important
to flavor can be formed, and changes to the fermenting grape must will result from the reducing environment
and entrainment of volatiles in CO2 gas.

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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Alcoholic Fermentation

Overview of glycolysis (EMP pathway) and subsequent ethanol formation to restore the
NADH: NAD+ redox balance. Utilization of hexose sugars requires active transport into the
cell via hexose transporters

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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‫ סיכום מקוצר‬- ‫תסיסה כוהלית‬

Taken from: http://resizeimage.club/openphoto.php?img=http://classconnection.s3.amazonaws.com/583/flashcards/751135/jpg/alcohol-fermentation.jpg

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Glyceropyruvic fermentation
 Experimentally, the ethanol produced by commercial yeast strains doesn’t reach
the theoretical maximum.

 Although ethanol and CO2 represent the major products of fermentation, yeast
cells must divert some carbon to form biomass compounds such as lipids,
proteins, and DNA to grow, and will form other metabolites like glycerol and
acetic acid as part of this process.

Molar yield of key fermentation products from glucose (mol C of product/mol C

of glucose) during exponential growth phase of an alcoholic fermentation at 28 °C and two
different glucose concentrations (240 and 280 g/L)

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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Glyceropyruvic fermentation pathway

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Microbiology of Wine and Vinifications”
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Glyceropyruvic fermentation pathway

Overview of the glyceropyruvic pathway, showing the formation of glycerol and acetyl ‐CoA for
utilization in biomass synthesis

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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Glyceropyruvic fermentation
 Total biomass production will account for about 1% of sugar substrate that does not form CO 2.

 Many of the building blocks of these compounds will be formed from pyruvate.

 …creates an excess of NADH as compared to NAD +. The yeast’s solution is to divert a portion of
DHAP away from pyruvate formation, where it is reduced to glycerol in a two ‐step process.

 The coupled glycerol/pyruvate formation is referred to as glyceropyruvic fermentation.

Glyceropyruvic fermentation generates only 1 ATP per hexose, but allows for the diversion of
pyruvate to fates other than ethanol while still maintaining NADH:NAD+ balance. For example,
pyruvate can form Acetyl-CoA and fatty acids, with concurrent production of ethanol to retain
NADH:NAD+ balance.

Balance of ATP energy and NADH:NAD+ ratio during alcoholic and glyceropyruvic fermentation

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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Succinic acid and other Krebs cycle intermediates

The Krebs cycle, aka TCA cycle or citric acid cycle. Red arrows show normal clockwise direction of the Krebs cycle under aerobic
conditions, in which acetyl‐CoA is incorporated into citrate as part of a cycle. Under anaerobic conditions, certain enzymes in the
oxidative branch are inhibited (succinic dehydrogenase, citrate lyase), as indicated by “x” marks, and the cycle splits into two
separate branches, with succinic acid accumulation primarily occurring through the reductive branch (black arrows).
NAD(P)+/NAD(P)H indicates that either NAD+/NADH or NADP+/NADPH may participate

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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Consequences of glycolysis on wine chemistry

 Loss of sugars and formation of fermentation metabolites.

 Enzymatic reduction of unsaturated compounds.

 Entrainment of volatile in CO2.

 Heat formation.
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