Research Journal of Biotechnology
8 (7) July (2013)
Chemical and Laccase catalysed oxidation of gallic acid:
Determination of kinetic parameters
Zinnai A., Venturi F.,* Sanmartin C., Quartacci M.F. and Andrich G.
Department of Agriculture, Food and Environment (DAFE), University of Pisa, Via del Borghetto 80, 56124 Pisa, ITALY
*fventuri@agr.unipi.it
Abstract
During grape crushing, the release in the liquid phase
of oxidizing enzymes can reduce the concentration of
phenols which are natural antioxidants important for
human health. On the contrary, in the following
phases, related to the aging of wine, it is the slower
chemical oxidation that causes the unwished
degradation of the phenolic compounds. In spite of the
wide literature on the field, kinetic evaluations which
allow to quantify the extent of the phenomenon with
temperature (30 and 45°C) are not yet available. To
evaluate the phenol degradation kinetics, a model
solution containing gallic acid which was subjected to
both chemical and enzymatic oxidation was used.
After determination of the kinetic equation able to
describe the time reduction of the gallic acid
concentration in the solution, the kinetic parameters
related to the degradation curves and the oxidative
process involved were calculated. In both the
chemical and enzymatic oxidation experiments, an
increase in the temperature of 15°C induced a
reduction of the gallic acid oxidation rate, evidencing
an influence of temperature on the oxygen amount
dissolved in the liquid phase. In the presence of
laccase, the oxidative kinetics was three orders of
magnitude higher than that recorded without the
enzyme.
Keywords: Gallic acid, kinetic models, laccase, oxidation,
phenols.
Introduction
The browning of wine, primarily due to the oxidation
(enzymatic and not) of the phenolic compounds12,18,20,
represents one of the most feared processes which may
arise during winemaking7 especially with regard to white
and rosé wines10.
During grape crushing, the release in solution of laccase
from grapes affected by Botrytis cinerea1 may cause a
significant reduction of the content of the phenolic
compounds9. As a matter of fact, this enzyme catalyzes
with low specificity, the conversion of o- and p-diphenols
into the corresponding quinones, thus oxidizing a large
number of phenolics12. Furthermore, the oxidation of
phenolic compounds adversely affects the sensory
properties of wine (loss of colour, flavour, aroma) even if
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present in low concentration (3 U/mL) and results in the
loss of nutritional value of wine7. Moreover laccase activity
persists even during the winemaking process as this
enzyme is stable at pH values.11,18
Differently from what happens in the phases immediately
following grape crushing, during the aging of wine, the
reduction of the phenol concentration is mainly due to the
development of chemical oxidative processes. O-diphenols,
in particular caffeic acid and its esters, catechin and
epicatechin as well as gallic acid, are considered among the
most susceptible compounds to the oxidation caused by the
non enzymatic browning process8,12.
As the phenol concentration depends on the difference
between the amount of compounds transferred from grape
solid parts into wine and that removed by degradation
processes (oxidation of the end product or adsorption on
the walls of yeast cells)3, the different winemaking
protocols should include the use of operating conditions
(temperatures, gas concentrations, enzymatic coadjutants)
able to optimize the extraction yields and preserve at the
same time the integrity of the extracted compounds.
However, maceration also promotes the dissolution in the
must of many compounds derived from the skin which may
have negative effects on the quality of wine. Among these,
the oxidizing enzymes (responsible of the oxidation and
browning of must) and ions such as potassium, calcium and
magnesium (responsible of the pH increase due to organic
acid salification) are particularly important. The browning
of the phenolic compounds extracted during maceration
involves a chromatic evolution of wine towards
golden/amber tonalities with a consequent alteration of the
sensory properties and an acceleration of the product
evolution leading to a reduction of the wine storability15,16.
Furthermore, in spite of the fact that the content in white
wines is significantly lower than in grapes, increased
research in recent years concerning the phenol composition
of white wines and their effect on human health has been
developed. It was observed that the in vitro oxidation of a
human low density lipoprotein is delayed in the presence of
a phenol extract obtained from a white wine made in a
traditional way. This delay is even longer compared to the
one induced by a red wine extract if the white grapes were
subjected to cuticle maceration before fermentation6.
Researchers19 at Reading University found that champagne
has the same health benefits as previously found in red
wine because of polyphenol antioxidants which are
Research Journal of Biotechnology Vol. 8 (7) July (2013)
Res. J. Biotech

believed to reduce the effects of cell-damaging free radicals temperature.

in the body. In particular, these antioxidants slow down the
removal of nitric oxide from the blood, lowering blood
pressure and therefore reducing the risk of heart problems
and strokes.

Moreover grape skin extracts (GSE) were tested and both

had in vitro yeast and mammalian α-glucosidase inhibitory
activity. The GSE was 32-times more potent at inhibiting
yeast α-glucosidase than acarbose, a commercial oral
hypoglycemic agent. From HPLC and LC-MS analysis,
three phenolics from the GSE (resveratrol, ellagic acid and
catechin) were identified as active inhibitory compounds.
The acute administration of GPE (400 mg/kg bw) to mice
reduced postprandial blood glucose level by 35% following
an oral glucose tolerance test compared to the control. The
daily supplementation (250 mg/kg bw) of GSE for 12-
Fig. 1: Batch biofermentator (V = 5 L) used for the
weeks to mice affected fasting blood glucose levels,
chemical and enzymatic oxidation trials
oxidative stress and inflammatory biomarkers associated
with obesity and type 2 diabetes5.
Results and Discussion
The chemical oxidation of phenols as a function of time
In addition, it was observed that tyrosol and
was described according to a first order kinetics (eq. 1)
hydroxytyrosol, non-flavonoid phenols present in white
using the following differential equation:
wines but not in the must before fermentation, cause a
higher expression of genes codifying for anti-aging
v = -d[S]t=t /dt = k · [O2] · [S]t=t (1)
proteins4, 17. Tyrosol and hydroxytyrosol have an effect
analogous to resveratrol and could have antioxidant
For each temperature the dissolved oxygen amount was
abilities similar of the latter in white wines. As all non-
kept constant:
flavonoid phenols are particularly subject to oxidation
processes, they are greatly affected by laccase activity and
their concentration deeply decreases in presence of that k · [O2]  constant = kox
enzyme12.
and then:
Notwithstanding the information available in the literature,
the values of the kinetic parameters related to both the v = -d[S]t=t /dt = kox · [S]t=t
oxidation of each phenol compound and the total phenols
are still not known. The availability of kinetics constant where v is the degradation rate due to gallic acid oxidation
would allow tocontrast the evolution of the oxidative (g L-1 s-1), [S]t=t is the phenol concentration (g L-1) at a
reactions in white wines and in particular in those generic t time (s) and kox is the oxidation kinetic constant of
characterised by high phenol content and then easily the oxidable substrates (s-1). By integrating the equation it
subjected to oxidative processes. follows that:

On the other hand, because of laccase from Trametes ln [S]t=t = -kox · t + ln [S]t=0 (2)
versicolor can be potentially utilised for phenolic removal
in must for white wine stabilization10 , the kinetic In the absence of the enzyme, the relation describing the
characterization of the enzymatic activity in a reaction course of phenol oxidation as a function of time (Figure 2)
medium simulating a wine can be useful to define the best can be represented by the following exponential equation:
working conditions to be adopted in induced degradation of
phenols by controlled addition of the laccase to wine. [S]t=t = [S]t=0 · e- kox  t (3)

In this study an evaluation of the kinetic constants related
to the course of both enzymatic and chemical oxidations of
phenols was carried out. Gallic acid was used as reference
compound for the non-flavonoids to verify the suitability of
this kinetic approach. The experimental runs were carried
out using a model solution at two different temperatures
(30 and 45°C), maintaining constant the percentage of the
oxygen dissolved in the reaction medium at a fixed
The calculated values of the functional parameters (kox and
[S]t=0), together with the squares of the correlation
coefficients which attest the statistical reliability, are shown
in table 1. The validity of the theoretical approach is
demonstrated by the good degree of overlapping between
the experimental and calculated data. In the trials at 45°C, a
significant decrease of the rate of gallic acid chemical
oxidation was observed (Figure 2) probably due to the

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lower solubility of oxygen in the reaction medium.
Fig. 2: Time evolution of the concentration of gallic acid
(chemical oxidation) in the model solution.
The enzymatic oxidation of gallic acid was evaluated in the
same conditions used for the chemical oxidation with the
addition of a commercial laccase product in a concentration
similar to that of a must obtained from grapes affected by
Botrytis cinerea. The time course of the enzymatic
oxidation of phenols at both temperatures shows a
progressive inactivation of the enzyme (Figure 3) which
accounts for the dramatic reduction of the oxidation rate
and above all, for the tendency to reach almost asymptotic
values of minimum. Assuming that the enzymatic
inactivation follows a first order kinetics, it follows that the
enzyme concentration at a generic t time ([E]t=t ) will be
represented by:
[E]t=t = [E]t=0 · e- kE  t
and then:
v = -d[S]t=t/dt = k2 · [O2] · [E]t=t · [S]t=t = k2 · [O2] · [E]t=0 · e-
kE  t
· [S]t=t
where k2 is the kinetic constant related to the enzymatic
catalysis, [S]t=t the phenol concentration (g L-1) at a generic
t time (s) and kE is the kinetic constant of inactivation of
the enzyme (s-1). If the concentration of oxygen dissolved
in the reaction medium does not vary with time, the
following relation can be written:
kox = k2 · [O2] · [E]t=0
and then:
v = -d[S]t=t/dt = kox · e- kE  t · [S]t=t
The integration of the above relation gives the equation
which describes the time course of gallic acid
concentration:
[S]t=t
 d[S]t=t/[S]t=t = - kox ·  e- kE  t · dt
[S]t=0
Vol. 8 (7) July (2013)
Res. J. Biotech
ln [S]t=t/[S]t=0 = kox/kE · (e- kE  t – 1)
- kE  t – 1)
[S]t=t = [S]t=0 · e kox/kE · (e (9)
lim [S]t=t = [S]t=0 · e -kox/kE = [S]t=0 / e kox/kE = cost.
t →∞
lim [S]t=t = [S]t=0
t →0
where [S]t=t is the phenol concentration (g L-1) at a generic t
time (s), [S]t=0 is the phenol concentration (g L-1) at t=0 (s),
kox is the kinetic constant related to the oxidation of the
oxidable substrates (s-1) and kE is the kinetic constant
related to enzyme inactivation (s-1).
Fig. 3: Time evolution of the concentration of gallic acid
(enzymatic oxidation) in the model solution.
(4)
In table 2, the squares of the correlation coefficients as well
as the calculated values of the functional parameters (kox,
kE and [S]t=0) are obtained using the Burenl statistical
program. Also in this case the validity of the theoretical
approach is demonstrated by the good degree of
(5)
overlapping between the experimental and calculated data.
In the presence of laccase the oxidation kinetics exceeds of
about three orders of magnitude, the one detected without
the enzyme. Also in the enzymatic degradative
experiments, the enhancement of the temperature by 15°C
induced a reduction of the gallic acid oxidation rate (Table
2), stressing the importance on the amount of oxygen
dissolved in the reaction medium.
(6) Further experimental runs carried out at temperatures
different from those already used are needed to identify the
(7) optimal thermal conditions which can maximize the
catalytic activity of laccase in model solution. An
interesting development for reaching a better understanding
of the oxidative degradation of phenols will be the
identification of the kinetic constants related to the
oxidation of other phenolic compounds either singly or in
combination, in order to evaluate their sensitivity to oxygen
(8) and verify the existence or not of synergistic/antagonistic
behaviour.
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Research Journal of Biotechnology
Table 2
Values assumed by the kinetic constants kox and kE and [S]t=0 parameter (mean ± confidence interval) and
corresponding square values of the correlation coefficient following the enzymatic
oxidation of gallic acid in model solution
Temperature kox (s-1)·104 kE (s-1)·104
(°C)
30 3.07  0.17 2.47  0.17
45 1.15  0.08 0.71  0.11
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(Received 27th February 2013, accepted 20th April 2013)