Vol.4, No.9B, 45-50 (2013) Agricultural Sciences
http://dx.doi.org/10.4236/as.2013.49B008
Pectinmethylesterase extraction from orange solid
wastes: Optimization and comparison between
conventional and ultrasound-assisted treatments
Antonio D. Rodriguez-Lopez1*, Luis Mayor2, María M. Galfarsoro2, Jorge Martinez-Otalo2,
Esperanza M. Ga rcia-Castello2*
1Department of Chemical and Nuclear Engi neering, Universitat Politècnica de València, Cam ino de Ve r a , s/n. CP 46022, Valencia ,
Spain; *Corresponding Author: [e-mail]
2Institute of Food Engineering for Development, Universitat Politècnica de València, Camino de Vera, s/n. CP 46022, Valencia,
Spain; *Corresponding Author: [e-mail]
Received August 2013
AB STRACT
During orange juice production, a half of fresh
oranges weight is considered as production
waste (peels, pulp, seeds, orange leaves and
damaged orange fruits). An alternative for the
management of these wastes is their treatment
by addition of lime and a latter pressing, ob-
taining a press cake and a press liquor rich in
sugars (10˚ Brix) and citric acid, protein, pectin
and ethanol. For non-thermal concentration of
press liq u o r to obtain citruss molasses (65˚ - 70˚
Brix), the removal of pectin is necessary. Tradi-
tionally, depectinization of juices has been done
by using pectinmethylesterase (PME) enzymes
from ext ernal source s. In this w ork it performed
the extraction of PME enzymes from orange
peels to obtain the optimum extraction condi-
tions. Two different methods of solvent extrac-
tion were compared (conventional and ultra-
sound-assisted methods). For the conventional
extraction experiments, a central composite de-
sign with three variables ([NaCl], pH and time)
and five replicates of the center point was used.
For ultrasound-assisted extraction, exp eriments
were done at pH = 5.5 and [NaCl] = 1.25 M), va-
rying extraction time (1 - 30 min). Response va-
riables were PME activity, protein content and a
ratio between them, named PME effectiveness
(ηPME). At the same experimental conditions (pH
=5 .5, [NaCl] = 1.25 M, t = 15 min) it was found
that conventional extractions led to slightly bet-
ter results in terms of ηPME than ultrasound-as-
sisted extraction method.
Keywords: Orange Peel; Solid-Liqu id Extr actio n;
Ultrasound-Assisted Extraction;
Pectinmethylesterase; Press Liquor
1. INTRODUCTION
During orange juice production o nly approxi mately the
half o f fresh or anges weight is tra nsformed into juic e [1]
while the other half is considered as production waste
(peels, pulp, seeds, orange leaves and damaged orange
fruits) [2]. The United States Department of Agriculture
(USDA) forecasted a world production of orange juice of
2.2 × 106 metric tons (MT) [3] in 2010, which would
lead to around 1.1 × 106 MT of solid wastes.
These wastes are, in most cases, spread on soil areas
adjacent to the production locations, for a final use as
raw material for cattle feed, or burned [4]. This way of
waste handling produces highly polluted wastewater in
terms of chemical and biological oxygen values (COD
and BO D) [1] t ha t c an ne gat ive l y a ffect t he s oi l t he ground
and superficial water.
The most desirable procedure under bo th environ men-
tal and ec o no mica l poin ts o f vi e w is t he selec tion of waste
treatment alternatives directed to their integrated valori-
zation. An alternative to improve the management of
orange solid wastes is their tr eatment by addition o f lime
and a latter pressing, obtaining a press cake and press
liquo r rich in su gars (sucr ose, glucose and fructo se) with
a total concentration of around 10˚ Brix. Other compo-
nents present in the press liquor are citric acid, protein,
pec tin and ethanol [1].
Currently, the orange press liquor is concentrated up to
65˚ - 70˚ Brix by multiple effect evaporation to obtain
molasses that are used in the production of beverage al-
cohol [5], and as cattle feed [6]. The evaporative concen-
tration implies very high energy consumptions when com-
par ed wit h non-thermal membrane operatio ns, as reported

A. D. Rodriguez-Lopez et al. / Agricultural Sciences 4 (2013) 45-50
46
in the preconcentration of sucrose solutions by reverse
osmosis (RO) [7].
Some attempts o f press liquor pr econcentration b y RO
and forward osmosis (FO) have been done. In the RO
preconcentration it was found that the presence of pectin
in synthetic press liquo r made its treatment very difficult
mainly due to the high viscosity of the solution whilst
when the press liquor was prepared without pectin, the
solution was satisfactorily preconcentrated for all tested
conditions [8]. In the case of the FO treatment, it was
found that synthetic press liquor solution prepared with-
out pectin could be concentrated up to 2.5 folds the press
liquor prepared with pectin [9]. Hence, a depectinization
step is strongly recommended for membrane concentra-
tion of press liquor.
Pectin is a heteropolysaccharide that consists of long
sequences of partially methyl-esterified d-galacturonate
residues interrupted by other sugars such as d-xylose, d-
glucose, l-rhamnos e, l-arabinose and d-galactose [10].
There are many studies of juice depectinization to ob-
tain clarified products b y using commercial enz ymes such
as pectinmethylesterase (PME) (EC 3.1.1.11) or polyga-
lacturonase (EC 3.2.1.15) [11]. PME enzymes are natu-
rally present in the peel of several fruits such as oranges
and apples [1,10,12]. Based on results obtained by Gar-
cia-Castello et al. [8] and Garcia-Castello and McCut-
cheon [9] with synthetic press liquor solutions it would
be expected a considerable improvement in the precon-
centration yield of press liquor by membra ne tec hnology.
In this work it performed the extraction of PME en-
zymes from orange peels to obtain the optimum extrac-
tion conditions. Two different methods of solvent extrac-
tion were compared (conventional and ultrasound-assisted
extraction).
2. MATERIALS A ND METHODS
2.1. Raw Materials
“Valencia Late” (Citru s sinensis L.) oranges were pur-
chased from a local market in Valencia, Spain. Oranges
were carefully washed with tap water and stored at 5˚C
until use. Fruits were cut into halves and the juice was
extracted using a domestic squeezer (Philips, Royal Phil-
ips Electronics, Inc., The Netherlands). The remaining
peels were used for the conventional and ultrasound-
assisted extraction of PME.
2.2. Extraction of Pectinmethylesterase
Enzymes. Experimental Design
Two different methods of solvent extraction were com-
pared: conventional and ultrasound-assiste d extraction.
For the conventional extraction experiments, a central
composite design (CCD) with three variables (NaCl con-
centration, pH and time) and five replicates of the center
point was used. Real and coded values for each variable
are listed in Table 1. For ultrasound-assisted extraction,
so me of the e xp er i me nts s hown in Table 1 were selected,
namely those of the center point (pH = 5.5, [NaCl] =
1.25 M), but in this case tested times were 1, 5, 15, 25
and 30 min. Response variables in every experiment for
both extraction methods were PME activity and protein
content in extracts.
Independently of the method used the extraction was
done as follows: a 25 g sample of orange peels (natural
or pressed) was suspended into 100 mL of extracting
aqueous solution at different [NaCl]. The mixture was
homogenized in an electrical blender (Turbomix plus 300,
Moulinex, France). The pH of the homogenate was ad-
justed to different values according to the experimental
design using small volumes of concentrated NaOH and
HCl solutio ns. I n the con vent io nal e xtra c tio n exp eri ments,
homogenates were stirred in an orbital shaker at 175 rpm
and at constant temperature of 4˚C for the time estab-
lished in Table 1. In the ultrasound-assisted extraction
experiments, homogenates at pH 5.5 and with a [NaCl]
of 1.25 M were introduced in flasks and then in an ultra-
sound device (ATM-7,0LCD, Labbox; frequency, 40 ± 2
kHz) at a constant temperature of 4˚C for the times
commented previously in this section.
Afterwards, samples were vacuum filtered. The crude
extract was centrifuged at 13,000 rpm for 30 minutes at
4˚C (Medifriger BL-S, JP Selecta, S.A., Spain). The su-
pernatants (enzymatic extract) were kept at −20˚C until
analysis.
Table 1. Natu ral and co ded (in brackets) variab les for th e expe-
rimental fact ors pH, NaC l concentratio n and time in the cent ral
composite design for conventional extraction of PME enzyme
from orange peel wastes.
Run pH (X1) [NaCl] (M) (X2) Time (min) (X3)
1 7.0 (+1.0) 0.5 (−1.0) 40 (−1.0)
2 5.5 (0.0) 1.25 (0.0) 20 (−1.682)
3 8.0 (+1.682) 1.25 (0.0) 70 (0.0)
4 7.0 (+1.0) 0.5 (−1.0) 100 (+1.0)
5 4.0 (−1.0) 0.5 (−1.0) 40 (−1.0)
6 5.5 (0.0) 1.25 (0.0) 70 (0.0)
7 5.5 (0.0) 1.25 (0.0) 70 (0.0)
8 7.0 (+1.0) 2.0 (+1.0) 40 (−1.0)
9 7.0 (+1.0) 2.0 (+1.0) 100 (+1.0)
10 5.5 (0.0) 1.25 (0.0) 121 (+1.682)
11 5.5 (0.0) 1.25 (0.0) 70 (0.0)
12 5.5 (0.0) 2.5 (+1.682) 70 (0.0)
13 5.5 (0.0) 1.25 (0.0) 70 (0.0)
14 4.0 (−1.0) 0.5 (−1.0) 100 (+1.0)
15 5.5 (0.0) 1.25 (0.0) 70 (0.0)
16 4.0 (−1.0) 2.0 (+1.0) 100 (+1.0)
17 5.5 (0.0) 0.0 (−1.682) 70 (0.0)
18 4.0 (−1.0) 2.0 (+1.0) 40 (−1.0)
19 3.0 (−1.682) 1.25 (0.0) 70 (0.0)

A. D. Rodriguez-Lopez et al. / Agricultural Sciences 4 (2013) 45-50
47
2.3. Analytical Determinations
2.3.1. Pectinmethylesterase Activity
The PME activity of the extracts was determined as
described by several authors [13-18]. This method meas-
ures the releasing rate of carboxylic groups from a 1%
(w/v) standar d citrus pecti n (Sig ma) solution at 30 ˚C and
pH 7 when the enzymatic extract is added. A volume of
20 mL of the 1% pectin solution was adjusted to pH 7
keep ing the temperature constant at 30˚C. 4.75 mL of the
enzymatic extract were added and as soon as the pH de-
creased due to the release of carboxylic groups, the pH
was re-adjusted to a value of 7 using a solution of 0.01 M
NaOH. This pH re-adjustment was made as often as ne-
cessary during 10 minutes. One unit of PME activity was
defined as 1 μequivalent of carboxyl groups released per
minute and millilitre of enzymatic extract (U/mL). PME
activity units were given in U per mg of orange peel in
dry basis (U/mg (d.b.).
2.3.2. Protein Content
The protein content determination was done according
to the Bradford method [19]. This method is based on a
colorimetric determination of the presence of proteins at
a wavelength of 595 nm. The protocol was the following:
3 mL of the Bradford reagent (Sigma Aldrich Co., St.
Louis, MO, USA) were added to 300 μL of sample, and
after 30 min. the absorbance was measured in a UV/
Visible spectrophotometer (Ultrospec 3300pro; Amer-
sham Bioscience, Piscataway, NJ, USA). A calibration
curve was done using standard bovine serum albumin
(Sigma Aldrich Co., St. Louis, MO, USA) solutions.
Protein content was given in mg of protein per mg of
orange peel in dry basis (mg prot/mg (d.b.).
2.3.3. Moisture Content in Orang e Pee ls
Five orange fruits were cut and squeezed as described
before. Peels were cut by hand in pieces of similar size
and then were weighted. Afterwards, samples were in-
troduced in a vacuum oven at 60˚C and −0.60 bar (Va-
cioterm, JP Selecta, S.A., Spain) until constant weight.
Moisture content was determined from weight difference
before and after sample drying.
2.4. Statistical Analysis
All the analyses were conducted in triplicate. Data was
expressed as the average of these values. The results of
the CCD were analyzed using the software “Statgraphics”
versio n Ce ntur ion X VI, fro m Stat Po int Technolo gies, Inc,
USA. Linear and quadratic effects of the three variables
considered, as well as their interactions on the response
variables were calculated. Their significance was eva-
luated by analysis of variance (ANOVA).
Moreover, experimental data were fitted to a second-
order polynomial model (Eq.1) and regression coeffi-
cients were obtained (R2).
22
011223341 52
2
63712 813 923
Y XXXXX
XXX XX XX
ββ ββββ
βββ β
=+++ ++
+++ +
(1)
where Y is the studied response, β0 is the independent
coefficient, β1, β2, β3 are the lineal coefficients, β4, β5, β6
are the quadratic coefficients and β7, β8, β9 are the inte-
raction coe fficients.
3. RESULTS A ND DISCUSSION
3.1. Conventional Pectinmethylesterase
Extraction
For the pectinmethylesterase activity, it was ob tained a
wide range of values between 0.01 (run 17) and 1.23 (run
3) U/mg (d.b.). The lowest PME activity was obtained at
the lowest NaCl concentration and at center point condi-
tions for pH and time. On the other hand, highest PME
activity was obtained at highest pH (pH = 8.0) and at
center point co nd itions for [NaCl] a nd time.
Moreover, the ANOVA analysis showed that the sig-
nificant effects (p < 0.05) were linear terms of NaCl
concentratio n and pH wit h a p ositive e ffect in both cases.
On the other hand, the extracting time, at least in the
range 20 - 120 minutes was not significant for the PME
activity.
Experimental data were fitted to a second-order poly-
nomial model for PME activity (Table 2).
Table 2. Second-order model equations for the response surface
fitted to the experimental data points obtained in the conven-
tional extraction as a function of pH (1), NaCl concentration (2)
and time (3).
Coeff icient Pectinmethylesterase activity Protein c ontent
U/mg (d.b.) mg∙prot/mg (d.b.)
Independent
β0 −1.46069 −0.17714
Li n e ar
β1 0.43937 0.16595
β2 1.39288 0.15610
β3 −0.01002 −0.00200
Quadratic
β4 −0.01799 −0.00899
β5 −0.23622 −0.02864
β6 0.00003 −0.00001
Crossproduct
β7 −0.11444 −0.03726
β8 0.00058 0.00038
β9 0.00150 0.00096
Regression
R2 (%) 78.9 88. 1

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48
For the protein content results, the ANOVA analysis
showed that linear pH term was highly significant with
positive effect. Linear [NaCl] and crossproduct pH and
[NaCl] terms, were also significant with a negative effect.
As occurred with PME activ ity, time did not s how signi-
ficance. Protein content data were fitted to a second-
order poly n om i a l model (Table 2).
Worst results in protein content (0.001 mg∙prot/mg
(d.b.)) were obtained with run 3 what is in agreement
with results obtained for the PME activity. The PME ac-
tivit y dep end s in so me wa y o n the p ro tein c onte nt; he nce,
if experi mental conditio ns in run 3 led to an extract with
very limited protein content, the PME activity must be
extremel y lo w.
On the other hand highest protein content (0.52
mg∙prot/mg (d.b.)) was obtained at run 4 (pH = 7.0,
[NaCl] = 0.5 M and t = 100 min). These conditions differ
from experimental conditions leading to highest PME
activity. It was evaluated the relation between PME ac-
tivity and protein content and no correlation was found.
This fact proves that not all extracted proteins show cat-
alytic function. Thus, in order to evaluate the catalytic
power of the extracted protein it is possible to define a
factor of PME effectiveness, ηPME (U/mg prot) (Eq.2).
activity
PME content
PME
Protein
η =
(2)
The ANOVA analysis was done for the ηPME too. It
was found that the only significant effect was the NaCl
concentration (positive effect) what was corroborated by
the RSM plot (Figure 1).
The second-order polynomial equation for the ηPME
(Eq.3) showed a R2 of 82.9%.
PME1 2 3
222
123 12
13 23
3.1295 0.95153.61440.0253
0.0403 0.34420.0002 0.2876
0.0006 0.0007
XXX
XXXXX
XX XX
η =−+ +−
−− +−
+−
(3)
Figure 1. Response surface pl ot for the PME effectivenes s as a
function of the NaCl concentration and time. The pH was fixed
at 5.5.
According to this ANOVA analysis, the experimental
conditions that maximize the ηPME are: pH = 5.9, NaCl
concentration = 2.5 M and time = 20 min, obtaining a
maximum value of ηPME = 3.33 U/mg∙prot.
3.2. Ultrasound-Assisted
Pectinmethylesterase Extraction
In the ultrasound-assisted extraction, experiments were
done at pH = 5.5 and [ Na Cl] = 1.2 5 M va r yin g ext ra ct ion
time between 0 - 30 min.
It was found that both PME activity and extracted
protein showed a similar hyperbolic behavior with time
course during extraction. Asymptotic values for PME
activity and protein content were 0.85 U/mg (d.b.) and
0.41 mg∙prot/mg (d.b.), respectively. The stabilization
time was around 15 min in both cases.
Regarding the PME effectiveness evolution with the
extraction time course (Figure 2), a hyperbolic trend was
found with an asymptotic value of about 2.0 U/mg∙prot.
In order to compare conventional and ultrasound- as-
sisted methods, second order model equations in Table 2
were used to find the expected values of PME activity
and protein content at the stabilization conditions for the
ultrasound-assisted extra ction (pH = 5.5; NaCl = 1.25 M;
t = 15 min). The obtained values were 0.93 U/mg (d.b.)
and 0.38 mg∙prot/mg (d.b.) for PME activity and protein
content, respectively.
As observed, expected PME activity for the conven-
tional extraction (0.93 U/mg (d.b.)) was higher than for
the ultrasound-assisted method (0.85 U/mg (d.b.)). The
contrary occurs with protein content, 0.38 mg∙prot/mg
(d.b.) with conventional extraction vs. 0.41 mg∙prot/mg
(d.b.).
Comparing the PME effectiveness for both extraction
methods at the same extracting conditions (pH = 5.5;
NaCl = 1.25 M; t = 15 min), the expected value for the
conventional extraction reached 2.5 U/mg∙prot. vs. the
Figure 2. Evolution of the PME effectiveness as a function of
the extraction time course. Experimental conditions: pH = 5.5
and NaC l concentrati on = 1.25 M.
0.0
0.5
1.0
1.5
2.0
2.5
010 2030 40
PME efectiveness (U/mg prot)
Time (min)

A. D. Rodriguez-Lopez et al. / Agricultural Sciences 4 (2013) 45-50
49
2.0 U/mg∙prot. obtained for the ultrasound-assisted me-
thod. Hence, conventional extraction led to higher PME
effectiveness in the extracts than the ultrasound-assisted
extraction.
4. CONCLUSIONS
Variables studied (pH, [NaCl] and t), had different ef-
fect on the PME activity, protein content and PME effec-
tive ness ( ηPME) in extracts for the co nventional extra ction:
For PME activity, [NaCl] and pH were significant and
had positive effect; for protein content, pH was highly
significant with positive effect while, [NaCl] was less
significant with a negative effect; for the ηPME, [NaCl]
was found significant with positive effect. Model equa-
tions describing the PME activity, protein content and
ηPME of the extracts presented adequate regression coef-
ficients. Experimental conditions that maximize ηPME
were: pH = 5.9, [NaCl] = 2.5 M and time = 20 min.
For ultrasound-assisted extraction, PME activity, pro-
tein content and ηPME showed a similar hyperbolic beha-
vior with ti me course d uring e xtraction. T he stabilization
time was around 15 min in both cases.
Comparing results for both extraction methods at the
same extracting conditions (pH = 5.5; NaCl = 1.25 M; t
= 15 min), conventional extraction led to slightly higher
PME effectiveness than the ultrasound-assisted extrac-
tion, what is interesting in future ind ustrial a pplications.
5. ACKNOWLEDGEMENTS
Authors acknowledge the Vicerrectorado de Investigación of the
Universidad Politecnica de Valencia for the financial support (project
1965) from the call Proyectos de Nuevas Líneas de Investigación Mul-
tidiscipli nares (PAID05-11 ).
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