Pectinmethylesterase extraction from orange solid wastes: Optimization and comparison between conventional and ultrasound-assisted treatments

doi:10.4236/as.2013.49B008

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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: [email protected]

2Institute of Food Engineering for Development, Universitat Politècnica de València, Camino de Vera, s/n. CP 46022, Valencia,

Spain; *Corresponding Author: [email protected]

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

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

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).

011223341 52

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

A. D. Rodriguez-Lopez et al. / Agricultural Sciences 4 (2013) 45-50

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

(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

=−+ +−

−− +−

+−

(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

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 ).

REFERENCE S

[1] Braddock, R.J. (1999) Handbook of citrus by-products

and processing technology, John Wiley & Sons, Inc.,

Hoboken.

[2] García, E., Gozálvez, J.M. and Lora, J. (2002) Use of

reverse osmosis as a preconcentration system of waste

leaching liquid from the citric juice production industry.

Desalination, 148, 137-142.

http://dx.doi.org/10.1016/S0011-9164(02)00667-7

[3] United States Department of Agriculture, USDA

(2010) http://usda.mannlib.cornell.edu/MannUsda/viewSt

atic-

Page.do?.url=http://usda.mannlib.cornell.edu/usda/ers/./8

9022/2006/index.html.

[4] Martín, M.A., Siles, J.A., C hica, A.F. and Martín, A. (201 0)

Biomethanization of orange peel waste. Bioresource

Technology, 101, 8993-8999.

http://dx.doi.org/10.1016/j.biortech.2010.06.133

[5] Bampidis, V.A. and Robinson, P.H. (2006). Citrus by-

products as ruminant feeds: A review. Animal Feed

Scien ce and Technology, 128, 175-217.

http://dx.doi.org/10.1016/j.anifeedsci.2005.12.002

[6] Wing, J.M., Van Horn, H.H., Sklare, S.D. and Harris Jr.,

B. (1988) Effects of citrus molasses distillers solubles and

molasses on rumen parameters and lactation. Journal of

Dairy Science, 71, 414-420.

http://dx.doi.org/10.3168/jds.S0022-0302(88)79571-5

[7] Garci a-Castello, E., Lora-Garcia, J., Garcia-Garrido, J.

and Rodriguez-Lopez, A.D. (2006). Energetic comparison

for leaching waste liquid from citric juice production us-

ing both reverse osmosis and multiple-effect evapo ration.

Desalination, 191, 178-185.

http://dx.doi.org/10.1016/j.desal.2005.09.012

[8] Garci a-Castello, E.M., Mayor, L., Chorques, S., Argüelles,

A., Vidal-Brotóns, D. and Gras, M.L. (2011) Reverse

osmosis concentration of press liquid from orange juice

solid wastes: Flux decline mechanisms. Journal of Food

Engineering, 106, 199 -205.

http://dx.doi.org/10.1016/j.jfoodeng.2011.05.005

[9] Garci a-Castello, E.M. and McCutcheon, J.R. (2011) De-

watering press liquor derived from orange production by

forward osmosis. Journal of Membrane Science, 372, 97-

101. http://dx.doi.org/10.1016/j.memsci.2011.01.048

[10] Pagan, J., Ibarz, A., Llorca, M., Pagan, A. and Barbosa-

Canovas, G.V. (2001) Extraction and characterization of

pectin from stored peach pomace. Food Research Inter-

national, 34, 605-612.

http://dx.doi.org/10.1016/S0963-9969(01)00078-3

[11] Cassano, A., Conidi, C. and Drioli, E. (2010) Phys i co-

chemical p ara meters of ca ctu s pear (Opuntia ficus-indica)

juice clarified by microfiltration and ultrafiltration pro-

cesses. Desalination, 250, 1101-1104.

http://dx.doi.org/10.1016/j.desal.2009.09.117

[12] Liu, Y.Z., Dong, T., Lei, Y., Deng, X.-X. and Gu, Q.-Q.

(2011) Isolation of polygalacturonase gene from Citrus

sinensis fruit and its expression relative to fruit mastica-

tion trait, fruit development, and calcium and boron

treatments. Plant Molecular Biology Reporter, 29, 51-59.

http://dx.doi.org/10.1007/s11105-010-0206-0

[13] Arbaisah, S.M., Asbi, B.A., Junainah, A.H. and Jamilah,

B. (1996) Determination of optimum conditions for pec-

tinest erase extraction from soursop fruit (Anona muricata)

using response surface methodology. Food Chemistry, 55,

289-292.

http://dx.doi.org/10.1016/0308-8146(95)00105-0

[14] Con treras-Esquivel, J.C., Correa-Robles, C., Aguilar, C.N.,

Rodríguez, J., Romero, J. and Hours, R. (1999) Pectines-

terase extraction from Mexican lime (Citrus aurantifolia

Swingle) an d prickly pear (Opuntia ficus indica L.) peels.

Food Chemistry, 65, 153-156.

http://dx.doi.org/10.1016/S0308-8146(98)00012-0

[15] Hou, W.N., Jeong, Y., Walker, B.L., Wei, C.I. and Mar-

shall, M.R. (1997) Isolation and characterization of pec-

tinesterase from Valencia orange. Journal of Food Bio-

chemistry, 21, 309-333.

http://dx.doi.org/10.1111/j.1745-4514.1997.tb00211.x

[16] Vivar-Vera, M.A., Salazar-Montoya, J.A., Calva-Calva, G.

and Ramos-Ramirez, E.G. (2007). Extraction, termal sta-

A. D. Rodriguez-Lopez et al. / Agricultural Sciences 4 (2013) 45-50

bility and kinetc behavior of pectinmethylesterase from

hawthorn (Crataegus pubescens) fruit. LWT-Food Science

and Technology, 40, 278-284.

http://dx.doi.org/10.1016/j.lwt.2005.10.005

[17] Zhou, H.-W., Ben-Arie, R. and Lurie, S. (2000) Pectin

esterase, polygalacturonase and gel formation in peach

pectin fractions. Ph yt ochemistry, 55, 191-195.

http://dx.doi.org/10.1016/S0031-9422(00)00271-5

[18] Kertesz, Z.I. (1955) Pectic enzymes. Methods in Enzi-

mology, 1, 158-166.

[19] Bradford, M.M. (1976) A rapid and sensitive method for

quantitation of microgram quantities of protein utilizing

the principle of protein-dye binding. Analytical Bioche-

mistry, 72, 248-254.

http://dx.doi.org/10.1016/0003-2697(76)90527-3