Advances in Bioscience and Biotechnology, 2011, 2, 89-96 ABB
doi:10.4236/abb.2011.22014 Published Online April 2011 (
Published Online April 2011 in SciRes.
Pectinases yeast production using grape skin as carbon source
María Arévalo-V illena1*, Mercedes Fernández2, Jesús López2, Ana Briones1
1Tecnología de Alimentos, Facultad de Ciencia Químicas, Universidad de Castilla la Mancha, Ciudad Real, Spain;
2Escuela de Ingeniería Técnica Industrial, Universidad de Castilla la Mancha, Ciudad Real, Spain.
Received 17 January 2011; revised 9 March 2011; accepted 11 March 2011.
Pectinases are used in Enology for some different
utilities. Enzymatic preparations from moulds are a
mixed of different enzymes with strong and unspe-
cific activities. Some Saccharomyces cerevisiae pro-
duce pectinases which can be used instead of com-
mercial preparations. The objectives of this work
were to study the enzyme secretion by one Saccharo-
myces cerevisiae (CECT 11783) for growing on grape
skin (industry oenological by-product) as carbon
source. Preliminary experiments showed that the
strain produced pectinases for growing on grape skin
without any other carbon source. Statistical treat-
ment (factorial design 25) was applied to evaluate the
influences of related factors (agitation, temperature,
presence of peptone and detergent in the medium and
time of growth) Variables with the most significant
interactions for pectinase production were agitation
and nitrogen source concentration. Response surface
methodology showed that a first order model was not
adequate for results. Nevertheless, the built of a sec-
ond order model offered a polynomial equation
which surface predicted a maximum of activity (52.68
enzymatic units) for specific values of the studied
variables (147.8 rpm of agitation and 15.9 g of pep-
tone/L culture medium).
Keywords: Pectinase Enzyme from Yeast; Enology;
Grape Skin; Statistical Treatment; Response Surface
Pectinolytic enzymes are found mainly in moulds and
bacteria, but they also occur in some yeasts [1-3]. Given
the role played by yeasts, especially of the genus Sac-
charomyces, in fermented products, further research into
their pectinolytic enzymes would be useful for two pur-
poses: one, so that yeast can be used to synthesize then
purify the enzymes for addition to fruit juices as clarifi-
cation and extraction enhancers; and two, in the case of
fermented products, so that the enzyme can be produced
by the yeast as part of the process rather than having to
be added to the medium.
Most commercial pectinase preparations used in the
food industry are derived from Aspergillus niger, a
GRAS microorganism producing large quantities of
these enzymes. However, this mould secretes other en-
zymes which may trigger collateral reactions, such as th e
release of volatile phenols less desirable for the produc-
tion of wine or fruit juices, for instance arabinofuranosi-
dase, which can cause turbidity [4].
Pectinases are used in winemaking to enhance must
extraction by degrading structural polysaccharides which
interfere with the extraction process [5], thus increasing
the release of colour and aroma compounds in musts
both before and during fermentation. At the same time,
the addition of pectinases improves maceration, clarifi-
cation and filtration during the winemaking process
Pectinolytic enzymes derived from Saccharomyces
cerevisiae would provide a useful alternative to mould-
derived pectinases, since a genuine product can only be
obtaine d f rom yeasts .
Certain strains of S. cerevisiae have been found to
break down polygalacturonic acid, which could be im-
portant for the fermentation of plant-derived substrates
It has been demonstrated that when the enzyme ex-
tract from Saccharomyces bayanus is added to fresh
must, the effects on turbidity are the same as when a
commercial enzyme preparation is added [10].
A study reported that when PG+ strains of S. cere-
visiae were used in winemaking, in some cases the fil-
tration time was reduced by half without any appreciable
changes in viscosity [11]. Moreover, a transformed strain
with good winemaking qualities has more recently been
engineered using the PGU1 gene from another strain,
transcriptionally bonded to the PGK1 gene promoter, in
order to enhance its expression during growth [12].
M. Arévalo-V illena et al. / Advances in Bioscience and Biotechnology 2 (2011) 89-96
Copyright © 2011 SciRes. ABB
In some countries, current legislation prohibits the u se
of genetically-modified organisms, though not of GMO-
derived enzymes, in winemaking. The first step towards
achieving this goal is to develop appropriate enzyme-
production technology.
Spanish grape-skin production, as a by-product of the
winemaking process, is estimated at around 750 000 ton-
nes per year. At present, it is used mostly as animal feed.
Polygalacturonase activity in grape musts has been
shown to increase markedly one day after the addition of
yeast, whereas no enzyme activity was detected through-
out fermentation in must made from juice al one [13].
The incorporation of grape skin in the formulation of
culture media for use in industrial enzyme production
would bring both economic and environmental benefits
for winemaking areas, by enabling commercial exploita-
tion of this by-product. The composition of grape skin
may well enhance yeast growth as well as inducing pecti-
nase synthesis.
The aim of this research was to optimize the culture
medium using grape skin as substrate for the growth and
synthesis of pectinases derived from a genetically-
modified yeast strain.
Statistical optimization was preferred because it en-
abled evaluation of interactions between parameters and
involved a specific experimental design [14,15].
A genetically-modified Saccharomyces cerevisiae strain
(CECT 11783) (12) was used, containing the gene PGU1
from a spontaneous winemaking yeast, which conferred
the ability to hydrolyze polygalacturonic acid and there-
fore pectins.
For all assays, cells were precultured in YPD broth to
enable inoculation of a final population of 107 cells/mL
onto each tested growth medium.
2.1. Enzyme Method for Determining
Pectinolytic Activity
Pectinolytic activity was evaluated by quantifying the
amount of galacturonic acid released from apple pectin
(Fluka) using the DNSA (dinitrosalicylic acid) reaction.
The method was optimized by adjusting reagent concen-
trations and incubation times. A commercial pectinase
was used as positive control, and a commercial Sac-
charomyces strain (UCLM S325) not possessing pecti-
nolytic activity served as negative control.
Results were plotted on a galacturonic-acid calibration
curve covering the appropriate range of concentrations.
2.2. Preliminary Tests. Relationship between
Yeast Growth and Pectinase Production
2.2.1. First Experiment
A number of prior experiments were performed to con-
firm the ability of yeast to grow and to synthesize poly-
galacturonase in the presence of grape skin. The yeast
was grown on the following media:
7 g/L grape skin
7 g/L grape skin + 5 g/L gl ucose
7 g/L grape ski n + 10 g/ L pep t o ne
7 g/L grape skin + 5 g/L gl ucose + 10 g/L pepto ne
A set of 100-mL flasks containing 20 mL of each me-
dium were inoculated and incubated at 28ºC in a ther-
mostatically-controlled water bath shaker (150 rpm).
Polygalacturonase activity was measured at 24, 46 and
96 hours of yeast growth.
All assays were performed in triplicate, and results
were expressed in enzyme units (i.e. the amount of en-
zyme required to liberate 10 ug of galacturonic acid
from apple pectin in the conditions outlined above).
2.2.2. Second Experiment
In view of the results obtained, a second experiment was
performed to ascertain whether grape skin and/or glu-
cose concentrations significantly influenced enzyme
synthesis. For this purpose, peptone concentration and
growth time (determinant variables) were fixed at 10 g/L
and 24 h, respectively. Yeast was grown in a r efriger ated
orbital shaker which was used until the end of th e work.
The new media formulations were as follows:
0 g/L grape skin + 5 g/L gl ucose + 10 g/L peptone
7 g/L grape skin + 5 g/L gl ucose + 10 g/L peptone
21 g/L grape skin + 5 g/L glucose + 10 g/L peptone
7 g/L grape ski n + 10 g/ L pep t o ne
21 g/L grape skin + 10 g/L peptone
2.3. Optimization of the Culture Medium for
Pectinase Production. Statistical Analysis
In the light of the preliminary study results, the grape
skin concentration was set at 21 g/L and glucose was
omitted from the culture broth; a study was therefore
made of other variables potentially influencing enzyme
A two-stage statistical analysis was performed: the
first stage identified significant factors, while in the
second stage response surface methodology (RSM) was
used to maximize enzyme activity.
2.3.1. Fi r st Stage
The conditioning variables studied were: agitation
(shaking speed), temperature, presence of detergent
(Tween 80), cell harvest time and presence of a nitrogen
source (peptone); these variables were selected in view
of their marked influence on enzyme synthesis by yeasts
Each variable was studied at two levels, so that the
combination of five variables (factorial design 25 with
two replications) gave a total of 64 runs.
The experimental design used is shown in Table 1.
M. Arévalo-V illena et al. / Advances in Bioscience and Biotechnology 2 (2011) 89-96
Copyright © 2011 SciRes. ABB
Table 1. Experimental design for identification of significant factors in enzyme production. (Agitation, temperature, presence of
detergent (Tween 80), cell harvest time and presence of a nitrogen source (peptone)).
Experiment A Experiment B Experiment C Experiment D Tween 80 Time (h) Peptone
A1 B1 C1 D1 – 12 –
A2 B2 C2 D2 + 12 –
A3 B5 C5 D5 + 48 –
A4 B6 C6 D6 + 12 +
A5 B3 C3 D3 – 48 –
A6 B4 C4 D4 – 12 +
A7 B8 C8 D8 + 48 +
A8 B7 C7 D7 – 48 +
- Experiment A: Agitation 50 rpm, temperatu re 18ºC
- Experiment B: Agitation 150 rpm, temperature 18ºC
- Experiment C: Agitation 5 0 rp m, temperature 28ºC
- Experiment D: Agitation 150 rpm, temperature 28ºC
The four possible combinations of shaking speed (50 and
150 rpm) and temperature (18 and 28ºC) were fixed; 8
experiments were performed in duplicate for each com-
bination to quantify the amount of enzyme produced,
expressed as enzyme units.
The replicated 25 factorial model was constructed us-
ing the SPSS statistical software package. The univariate
GLM procedure was used to examine the magnitude and
direction of fac t o r effe ct s.
The design model was based on the equation:
 
()( )
ijklmhij kl mij ik
illm ijkklm
ijkl ijklm
 
 
 
  
 
where i, j, k, l, m took the values 1 and 2, and h varied
between 1 and the number of replicates (in this case, 2).
In the first instance, a full factorial model was con-
structed using the five main factors, ten second-order
interactions and ten third-order interactions. Factor ef-
fects and significant interactions were then estimated by
the UNIANOVA procedure using pairwise comparisons
and profile plots.
2.3.2. Second Sta ge
The results from the first stage indicated that neither
detergent (Tween 80) nor cell harvest time were signifi-
cant variables, and that the optimum temperature was
28ºC. For technical and economic reasons, therefore, the
following variables were fixed: temperature (28ºC),
grape skin concentration (21 g/L) and cell harvest time
(24 h); thus only shaking speed and peptone concentra-
tion were studied at the seco nd stage.
First, a linear approach to optimal cond itions was car-
ried out using first-order strategies and a 22 factorial
design with three replications of the centre-point (values
of the central conditions of each assay): agitation = 150
rpm, peptone concentration = 10 g/L. Model suitability
was assessed by analysing fit and curvature, and esti-
mating experimental error. Shaking speed was set at 150
rpm, and peptone concentrations were tested at 2 g/L
intervals up to 20 g/L, giving a total of 7 assays.
Since results were not determinant (data not shown),
the test was repeated with a new centre-point (shaking
speed = 150 rpm, peptone = 14 g/L), which involved
performing a further 7 experiments.
3.1. Enzyme Method for Determining
Pectinolytic Activity
The enzyme reaction providing the best results was a
mixture of 500 µL of supernatant (enzyme) with 500 µL
of 0.25% apple pectin, incubated for 30 minutes at 37ºC.
Once the reaction was complete, 500 µL of the mixture
was reacted with 500 µL of DNSA, and incubated for 10
minutes at 100ºC.
The cooled reaction mixture was diluted with 1.2 mL
of water, and data were plotted on a galacturonic acid
calibration curve ranging from 0.1 to 1 mg/mL.
3.2. Preliminary Tests. Relationship between
Yeast Growth and Pectinase Production
3.2.1. First Experiment
Extracellular pectinase activity at various harvest times
using different growth media is shown in Figure 1. Ac-
tivity was influenced by medium composition and the
presence of a nitrogen source (peptone) stimulating
pectinase synthesis (growth media C and D).
One-way analysis of variance (ANOVA, 95% CI) re-
vealed that enzyme production differed significantly in
all tested media at 46 h. Maximum enzyme production
was observed with the grape skin + glucose + peptone
M. Arévalo-V illena et al. / Advances in Bioscience and Biotechnology 2 (2011) 89-96
Copyright © 2011 SciRes. ABB
20 30 4050 60 70 8090100
Tim e (h)
Enzym e units
Figure 1. Evolution of pectinolitic activity in some culture
media at different harvest time. A. 7 g/L skin; B. 7 g/L skin + 5
g/L glucose; C. 7 g/L skin + 10 g/L peptone; D. 7 g/L skin + 5
g/L glucose + 10 g/L peptone.
combination (medium D) at 46 hours; the maximum
value of 78.25 ± 0.2 enzyme units was significantly
higher than that obtained with all the other combin ations.
This higlights the importance of peptone for yeast
growth and thus for enzyme synthesis; indeed, the sec-
ond-highest value (60.3 ± 1.4) was obtained with pep-
tone-containing medium C.
The influence of cell harvest time on enzyme produc-
tion in each medium varied as a function of medium
composition: variations in harvest time had no signifi-
cant effect on production in the medium containing only
grape skin, but prompted significant differences in the
other three media. For the grape skin + glucose + pep-
tone combination (medium D), enzyme production
peaked at 46 h. When glucose was removed (medium C),
production peaked earlier (24 h); however, the difference
between the two harv est times, though significant, was
not marked. Finally, in the medium containing glucose
but not peptone (medium B), enzyme synthesis was sig-
nificantly inhibited in the early stages; although some
activity was detected later, values never approached
those obtained using a nitrogen source.
3.2.2. Second Experiment
Yeast was grown in media I, II, III, IV and V (detailed
under Material and Methods) to determine the influence
of glucose and/or grape skin on pectinolytic activity. No
significant differences (95% CI) in enzyme production
were noted, values of around 40 enzyme units being re-
corded in all cases (Tabl e 2). The positive effect of glu-
cose in medium II was matched by the increased
grape-skin concentration in medium V.
In the absence of statistically-significant differences,
the decision to use a peptone-containing medium in
which the carbon source (glucose) was replaced by an
increased grape skin concentration (21 g/L) was prompt-
ed by the fact that this medium proved cheaper and also
made profitabl e use of a wi nemaking by-pro duct .
3.3. Optimization of the Culture Medium for
Pectinase Production. Statistical Analysis
3.3.1. Fi r st Stage
Enzyme activity for the 64 runs detailed under Material
and Methods is shown in Table 3. Enzyme production
was significantly affected by all tested variables except
presence of detergent. In experiment A (agitation 50 rpm,
temperature 18ºC), activity was n eg ligible or no nexisten t
due to minimal yeast growth, attributable to stress condi-
With these results, statistical analysis was repeated,
Table 2. Production of pectinolitic enzyme in media with different skin and glucose concentration.
Culture medium Enzyme units
I. 0 g/L skin + 5 g/L glucose + 10 g/L peptone 43.3 ± 3.1
II. 7 g/L skin + 5 g/L glucose + 10 g/L peptone 48.5 ± 2.2
III. 21 g/L skin + 5 g/L glucose + 10 g/L peptone 39.8 ± 7.9
IV. 7 g/L skin + 10 g/L peptone 42.4 ± 4.5
V. 21 g/L skin + 10 g/L peptone 47.5 ± 1.5
Table 3. Pectinolitic activity obtained by Table 1 design.
Experiment A (50 rpm/18ºC) Tw een 80 Time (h) Peptone Enzyme units
1 – 12 – 3.2 ± 0.6
2 + 12 – 3.6 ± 2 .6
3 + 48 –
4 + 12 +
5 – 48 –
6 – 12 +
7 + 48 +
8 – 48 +
M. Arévalo-V illena et al. / Advances in Bioscience and Biotechnology 2 (2011) 89-96
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Experiment B (150 rpm/18ºC) Tween 80 Time (h) Peptone Enzyme units
1 – 12 – 20.3 ± 0.5
2 + 12 – 26.7 ± 8.8
3 – 48 – 18.3 ± 0.9
4 – 12 + 21.6 ± 0.5
5 + 48 – 30.8 ± 0.5
6 + 12 + 23.6 ± 6.7
7 – 48 + 25.1 ± 1.7
8 + 48 + 25.6 ± 1.2
Experiment C (50 rpm/28ºC) Tween 80 Time (h) Peptone Enzyme units
1 – 12 – 15.7 ± 0.3
2 + 12 – 15.0 ± 3.9
3 – 48 – 12.7 ± 0.2
4 – 12 + 34.6 ± 1.3
5 + 48 – 13.6 ± 0.2
6 + 12 + 37.0 ± 0.8
7 – 48 + 35.1 ± 1.6
8 + 48 + 35.3 ± 0.9
Experiment D (150 rpm/28ºC) Tween 80 Time (h) Peptona Enzyme units
1 – 12 – 23,8 ± 0,2
2 + 12 – 27,8 ± 1,1
3 – 48 – 11,3 ± 0,5
4 – 12 + 41,1 ± 0,2
5 + 48 – 12,4 ± 0,4
6 + 12 + 44,1 ± 0,2
7 – 48 + 25,5 ± 1,8
8 + 48 + 30,9 ± 0,6
* Nd. No detected
retaining those main variables and second-order interac-
tions significantly influencing polygalacturonase synthe-
sis. The shaking speed*temperature combination was not
significant, and was therefore omitted from the statistical
analysis. The time factor, though also non-significant,
was retained since it was contained in two significant
interactions. This gave rise to a custom model 2:
  
  
where µ was the overall mean of runs,
, β,
, and
parameters due to the effects of agitation, temperature,
time and presence of peptone, respectively; and (
and β
) the parameters corresponding to the respec-
tive interactions.
Significant effect estimations showed that an agitation
of 150 rpm increased enzyme activity by 4.76 units, i.e.
an increase of 25.2% compared to 50 rpm. Similarly,
enzyme production at 28ºC was 5.76 units (33%) higher
than at 18ºC. Production in peptone-containing media
was 11.49 units (65.5%) higher than in peptone-free
Significant interaction estimations were obtained us-
ing profile plots, and the results were used as the basis
for the next experiment. An example of a profile plot is
provided in Figure 2. No interaction was recorded be-
tween presence of peptone and either agitation (B) or
temperature (D); by contrast, both these latter variables
interacted with time (A and C, respectively).
To summarize, the highest enzyme activity was found
at 28ºC, 150 rpm and in media containing peptone as
nitrogen source.
3.3.2. Second Sta ge
On the basis of these results, temperature was fixed at
28ºC, harvest time at 24 h and grape skin concentration
at 21 g/L. The results indicated that the response func-
M. Arévalo-V illena et al. / Advances in Bioscience and Biotechnology 2 (2011) 89-96
Copyright © 2011 SciRes. ABB
Figure 2. Profile graphics of some interactions between studied variables on pect inolitic enzymes formation. (a) Agitation a nd time
interaction; (b) Agitation and peptone presence interaction; (c) Temperature an d time interacti on; (d) Temperature and peptone pres-
ence interaction.
tion curve was not suitable for the first-order model. A
second-order model was therefore constructed, involving
a central composite design containing a 22 factorial de-
sign with three centre points and a star design with a
further three centre points. The polynomial equation
representing the second-order model used to account for
enzyme activity was:
'51'987 0'0522'107
3'557 1'6450'71
 
The fitted response surface and contour plot for pect-
inase production are shown in Figure 3(a) and 3(b),
respectively. The three-dimensional umbrella-shaped
curve represented the main effect of the tested variables
(presence of peptone and agitation) and their interaction
with maximum pectinase production by Saccharomyces
cerevisia strain CECT 11783. As Figure 3 shows, a
maximum point was located at roughly 16 g/L of peptone
and around 150 rpm of agitation. According to the model,
(150 + 30 X1) and (14 + 3 X2), predicted maximum
Agitation (rpm) 50150
me units
Time (hours)
Agitation (rpm) 50 150
With peptone
Without peptone
Enzyme units
Time (hours)
Temperature 18 28
With peptone
Without peptone
Peptone (peptona)
(c) (d)
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Copyright © 2011 SciRes. ABB
Figure 3. Response surface (a) and contour plot for second order model in nature variables (agitation and
peptone concentration) for pectinases production.
pectinase produ ction u sing grap e sk in was 52.68 enzyme
units, using an agitation of 147.8 rpm and a grape skin
concentration of 15.9 g/L.
The present study offers the possibility of obtaining
pectinases from yeast using a byproduct from the same
industry: grape skin. The statistical studies guarantied
the maximum enzyme production under specific culture
conditions: growth of CECT 11783 using 16 g/L of pep-
tone, 21 g/L of dried grape skin as carbon source and
inductor of the pectinases synthesis with an agitation of
150 rpm. These concentrations could be adjusted getting
a compromised between quantity of produced enzyme
Eenzyme units
Agitation (rpm)
Peptone (g/L)
Agitation (rpm)
Peptone (g/L)
146 148 150 152 154
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Copyright © 2011 SciRes. ABB
and economic factors.
This study was funded by INIA pr o j ec t (RM 2007 00004-00-00).
[1] Fernández-González, M., Ubeda, J.F., Vasudevan, T.G.,
Otero and Briones, R.A. (2004) Evaluation of polygalac-
turonase activity in Saccharomyces cerevisiae wine
strains. FEMS Microbiology Letters, 237, 261-266.
[2] Jayani, R. Singh, Saxena, S. and Gupta, R. (2005) Mi-
crobial pectinolitic enzymes: A review. Proccess Bio-
chemistry, 40, 2931-2944.
[3] Oliveira, K.F., Malavolta, L., Souza, C.S., Vicente, E.J.
and Laluce, C. (2006) Pectinoytic activity secreted by
yeasts isolated from fermented citrus molasses. Journal
of Applied Microbiology, 100, 633-640.
do i:10 .1111/j. 1365 -2672.2006.02823.x
[4] Whitaker, J.R. (1990) Microbial pectinolitic enzymes. In:
Fogarty, W.M. and Kelly, C.T., Eds., Microbial Enzymes
and Biotechnology. 2nd Edition, Elsevier Science Ltd.,
London, 133-176.
[5] Roldán, A., Palacios, V., Peñatez, X., Benitez, T. and
Pérez, L. (2006) Use of Trichoderma enzymatic extracts
on vinification of Palomino fino grapes in the Sherry re-
gion. Journal of Food Engineering, 75, 375-382.
[6] Villetaz, J.C. (1990) Les colloides colmatant et la
filtration des vins. Reveu Francaise d’oenologie, 122,
[7] Villetaz, J.C. (1996) Utilisitain les enzymes en oenologie
pour l’extraction de la couleur et por l’extration et la
revelation des aromes. Bulletin de l'OIV, 787, 843-869.
[8] Servili, M., Begliomini, A.L. and Montedor, G. (1992)
Utilization of a yeast pectinase in olive oil extraction and
red wine making processes. Journal of Science and Food
Agriculture, 58, 253-260. doi:10.1002/jsfa.2740580214
[9] McKay, A.M. (1990) Degradation of polygalacturonic
acid by Saccharomyces cerevisiae. Letter in Applied Mi-
crobiology, 11, 41-44.
[10] Gainvors, A., Frézier, V., Lemarasquier, H., Lequart, C.,
Aigle, M. and Belarbi, A. (1994) Detection of polygalac-
turonase, pectin-lyase and pectin-esterase activities in a
Saccharomyces cerevisiae strain. Yeast, 10, 1311-1319.
[11] Blanco, P., Sieiro, C., Díaz, A., Reboredo, M. and Villa,
T.G. (1997) Grape juice biodegradation by polygalactu-
ronases from Saccharomyces cerevisiae. International
Biodeterioration and Biodegradation, 40, 115-118.
[12] Fernández-González, M., Úbeda, J.F., Cordero-Otero,
R.R., Thanvanthri Gururajan, V. and Briones, A.I. (2005)
Engineering of an oenological Saccharomyces cerevisiae
strain with pectinolytic activity and its effect on wine.
International Journal of Food Microbiology, 102, 173-
183. doi:10.1016/j.ijfoodmicro.2004.12.012
[13] Takayanagi, T., Uchibori, T. and Yokotsuka, K. (2001)
Characteristics of yeast polygalacturonases induced dur-
ing fermentation on grape skins. American Journal of
Enology and Viticulture, 52, 41-44.
[14] Fernández Valdivia, D.G., Espinola Lozano, F. and Moya
Vilar, M. (2008) Influencia de diferentes coadyuvantes
tecnológicos en la calidad y rendimiento del aceite de
oliva virgen utilizando la metodología de superficies de
respuesta. Grasas y Aceites, 59, 39-44.
[15] Vasconcelos, A.F., Barbosa, A., Dekker, R., Scarminio, I.
and Rezende, M.I. (2000) Optimization of lacase produc-
tion by Botriosphaeria sp., in the presence of veratryl
alcohol by the response-surface method. Process in Bio-
chemistry, 35, 1131-1138.