The use of commercial pectinase in fruit juice industry.

Part 3:
Immobilized pectinase for mash treatment
Nilay Demir
*
, Jale Acar, Kemal Saroglu, Mehmet Mutlu
Department of Food Engineering, Hacettepe University, 06532 Ankara, Turkey
Received 24 March 2000; accepted 24 July 2000
Abstract
Enzymatic mash treatment is a well-known modern process for gaining more juice from fruits and vegetables. According to the
technique, cell wall and middle-lamina pectin of the fruit are degraded by pectinase activities. Besides increasing press capacity and
the yield of juice up to 20%, it has also a positive eect to achieve high carotene and dry matter content of the product. The aim of
the research was to investigate the activity and reusability of immobilized commercial pectinase named as ``Pectinex Ultra SP-L'' on
carrot puree. Immobilization process was carried out by using ion exchange resin particles washed with 0.05 M phosphate buer at
pH 4.5. Pectinase activity of immobilized enzyme was determined by the measurement of viscosity reduction of pectin solution
model system at pH 4.5 and 35C and found to be 1.252-pectin (w/v, %)/s ml. Activity loss was only 20% after nine batches run
model system studies. The optimum initial enzyme concentration was detected by measuring the highest pectinase activity in pectin
solution and found to be 6% (v/v). Enzyme immobilized particles were added to the carrot puree with an amount of 1.5 g particle/
100 g puree at pH 4.5 and 35C to degrade soluble and insoluble pectin and haze-provoking polysaccharides. The activity of im-
mobilized enzyme was determined by measuring pH, dry matter content and viscosity of the puree. Immobilized enzyme preparation
reduced the viscosity of the carrot puree from 90 to 6.5 Poise, after 60 min of incubation. While the viscosity and pH of the puree
were decreased, dry matter content and total yield were found to be increased because of the polysaccharide degradation. An
average yield increment was 30.23% with respect to the yield obtained from non-enzymic processed carrot juice. Immobilized en-
zyme was used 5 times in carrot puree medium at the above described conditions and the activity loss was found to be only 6.5%.
Activity of the immobilized enzyme was quite stable. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Carrot juice; Immobilization; Pectinase; Pectin; Enzymic treatment
1. Introduction
Pectolytic enzymes degrade pectic substances of sev-
eral kinds. Aspergillus niger or Aspergillus aculeatus is
used for industrial production of pectolytic enzymes
(Naidu & Panda, 1999). Pectolytic enzymes are used for
the fruit-processing industry to increase yields, improve
liquefaction, clarication and lterability of juices,
maceration, and extraction of plant tissues (D orrich,
1996; van den Broek, den Aantrekker, Voragen, Beld-
man, & Vincken, 1997).
Enzymatic mash treatment leads to an extensive de-
gradation of the middle-lamina and cell wall pectin by
polygalacturonase, pectinmethylesterase and pectinlyase
activities (Stutz, 1996). The synergetic eect of the
combination of pectinases and cellulases is the crucial
part of the process of enzyme treatment of pulp to an
almost complete liquefaction of pulped fruit and vege-
tables (D orreich, 1996). Enzymatic hydrolysis of the cell
walls increases the extraction yield, reducing sugars,
soluble dry matter content and galacturonic acid content
and titrable acidity of the products (Joshi, Chauhan, &
Lal, 1991; Drilleau, 1994). The resultant pulp has a
lower viscosity and the quantity of waste pomace is
reduced (D orreich, 1996; Doco, Williams, Vidal, &
Pellerin, 1997).
Although immobilization of the pectic enzymes oers
several advantages (Bernath & Venkatasubramanian,
1986; Fiedurek, Ilczuk, Lobarzewski, & Pleszczynska,
1992; Bayindirli, 1995), relatively little research has been
carried out on their immobilization. Immobilized pectic
enzymes have been suggested as clarifying agents for
various fruit juices. Lozano et al. (1987) developed a
Journal of Food Engineering 47 (2001) 275280
www.elsevier.com/locate/jfoodeng
*
Corresponding author. Tel.: +90-312-297-7100; fax: +90-312-299-
2123.
E-mail addresses: nilay@eti.cc.hun.edu.tr, nilay@hacettepe.edu.tr
(N. Demir).
0260-8774/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 2 6 0 - 8 7 7 4 ( 0 0 ) 0 0 1 2 7 - 8
reactor conguration in which, a cross-ow microl-
tration system and an immobilized pectolytic enzyme
system on a derivative nylon membrane have been
combined (Bayindirli, 1995). Similarly, Szaniawski and
Spencer (1997) examined the eect of immobilized pec-
tinase on the microltration of dilute pectin solutions by
macroporous titanium membranes and immobilized
enzyme was found to be very eective for the degrada-
tion of the pectin solution. Endopectin-lyase immobili-
zation onto tailor-made core-shell microspheres is
another research on the immobilization of the pectolytic
enzymes (Dinnella, Doria, Laus, & Lanzarini, 1996).
In this research commercial pectolytic enzyme prep-
aration was immobilized onto porous anion exchange
resins with electrostatic attraction. Anion exchange St-
DVB macroporous base resins (Dowex Marathon
WBA) were used for immobilization because of the ease
of the additional enzyme loading on to the matrix sur-
face to increase activity during the operation and the
possibility of regeneration (Bailey & Ollis, 1986; Bernath
& Venkatasubramanian, 1986; Shuler & Kargi, 1992).
Since, pH and ionic strength are important parameters
associated with immobilization by electrostatic adsorp-
tion (Bailey & Ollis, 1986), during the trials and im-
mobilization processes pH was kept constant at 4.5.
2. Materials and methods
2.1. Materials
Enzyme source. Pectinex Ultra SP-L from A. aculea-
tus was obtained from Novo Nordisk Ferment (Swit-
zerland) and stored at 4C.
Reagents. Pectin (galacturonic acid content approxi-
mately 79.1% and methoxy content approximately 8.2%
(Sigma, USA)) and commercial anion-exchange beads
with the particle size of 525 lm (Dowex WBA Mara-
thon, USA) were used during the treatments.
2.2. Methods
2.2.1. Immobilization pectinase on to anion exchange
resin particles
Coupling of enzyme. Initially, anion exchange resin
particles were washed with distilled water and 0.05 M
Mc-Ilvane citrate buer at pH 4.5. Coupling procedure
was carried out at +4C and at pH 4.5. Dierent initial
enzyme concentrations (2, 6, 10, 14, and 18%, v/v) were
used to determine the optimal conditions on the im-
mobilization of Pectinex Ultra SP-L. In a typical pro-
cedure, enzyme solutions were prepared in citrate buer
at pH 4.5, then 1 g of the particles were poured over 20
ml enzyme solution and immobilization was carried out
by continuous stirring at +4C for 24 h. To remove free
enzymes, particles were washed with water and citrate
buer and then stored in 0.05 M citrate buer at +4C
until use.
Enzyme activity. Immobilized and free enzyme ac-
tivities were determined by using viscosimetric method
as described elsewhere (Mutlu, Sarioglu, Demir, Ercan,
& Acar, 1999). Briey, pectolytic activities of immobi-
lized and free pectinases were determined by monitoring
the viscosity reduction of the pectin solution at 35C by
using Brookeld Viscometer (Model DV-I) at 100 rpm.
4.0% (w/v) of pectin solution was used as a model system
including 40% of sucrose solution and 0.05 M phosphate
buer solution at pH 4.5.
Operational stability of the immobilized pectinase. The
operational stability of the immobilized pectinase was
determined by quantifying its activity in consecutive
cycles of repeated use of the enzyme. After each cycle,
immobilized particles were washed with deionized water
and 0.05 M citrate buer.
Storage stability of the immobilized pectinase. Pectin-
ase immobilized anion exchange particles were stored in
0.05 M citrate buer at 4.5 pH and +4C for 7 weeks.
Periodically every week, samples were taken from the
buer solution and the enzyme activities were deter-
mined by the viscosimetric method (Mutlu et al., 1999).
Carrot puree. Carrot puree was prepared from fresh
carrots. Carrots (Daucus carota) were blended and ho-
mogenized with a kitchen type of blender. After the heat
treatment at 85C for 15 min, the pH of the puree was
adjusted to 4.5 by using 50% of citric acid.
2.3. Estimations
Yield. Carrot pulp was pressed to obtain juice till the
dryness. The juice yield was estimated as the percentage
of the juice obtained from the carrot pulp.
Viscosity. The viscosities of the samples were deter-
mined using spindle no. 4 at 100 rpm of a Brookeeld
viscometer model DV-I (Brookeld Engineering, USA).
3. Results and discussion
3.1. Kinetic parameters of the free and immobilized
pectinases
The kinetic parameters of free pectinase were re-
ported in the rst part of this study by Mutlu et al.
(1999). The pectolytic activity of Pectinex Ultra SP-L
was found to be 0.025 pectin (w/v, %)/s/enzyme %(v/v).
The MichaelisMenten constant (K
m
) and activation
energy (E
a
) were found to be 1.137-pectin (w/v, %) and
9.316 kcal mol
1
, respectively.
The kinetic parameters of immobilized pectinase were
reported in the second part of this study. These pa-
rameters were found to be V
max
1.252 pectin (w/v, %)/s,
276 N. Demir et al. / Journal of Food Engineering 47 (2001) 275280
K
m
2.172-pectin (w/v, %) and E
a
11.98 kcal mol
1
(Saroglu, Demir, Acar, & Mutlu, 2000).
3.2. Quantication of immobilized Pectinex Ultra SP-L
on the surface
Specic activity of the immobilized enzyme prepara-
tion is a characteristic of the immobilization technique
and is aected by the amount of enzyme loading. In the
selection of optimum enzyme loading to the particles, a
set of activity runs was performed in a batch reactor by
using pectin model solution with a series of commercial
pectinase immobilized anion exchange resins prepared
by changing the initial enzyme concentrations between
2% and 18% (v/v) prepared in a 0.05 M citrate buer. All
runs were performed at 35C within 20 ml of reaction
volume. The results in Table 1 indicated that, it was
possible to load from 8.10% to 81.96% of enzyme to the
anion exchange resins by changing the initial enzyme
concentration. The maximum activity based on per ml
of immobilized enzyme was found to be 3.28 pectin (w/v,
%)/ml enzyme s with an initial enzyme concentration of
6% (v/v). With the initial enzyme concentration of 2%
(v/v), immobilization yield had reached a maximum
value of 81.96%, but the apparent enzyme activity was
not related to the yield and showed 2.52 pectin (w/v, %)/
ml enzyme s. This may be due to the steric hindrance of
resin inhibiting maximal immobilized enzyme activity
and conformational changes in enzyme structure (Bailey
& Ollis, 1986). In addition, the way in which the enzyme
is attached to the support renders an orientation that is
important in the magnitude of the steric hindrances
(Shuler & Kargi, 1992; Alkorta, Garbisu, Llama, &
Serra, 1996).
3.3. Immobilized enzyme activity
Operational stability of the immobilized enzyme
preparation aects enzyme stability, the amount of en-
zyme leaching and the resistance of the support to at-
trition (Alkorta et al., 1996). Pectinase activity of
immobilized enzyme was determined by the measure-
ment of viscosity reduction of the pectin solution model
system at pH 4.5 and 35C (Fig. 1). As seen here, after
the second use, no signicant decrease in the stability of
immobilized resin particle was observed. Activity loss
was only 20% after running nine batches through the
model system. For that reason, pectinase activity was
taken as the second run activity and found as 1.252
pectin (w/v,%)/s ml.
3.4. Storage stability of the immobilized pectinase
Dinnella et al. (1996) stated that the stability of the
activity of the enzyme is greatly improved once the en-
zyme is adsorbed on to the microspheres. To evaluate
the eect of storage on the activity of immobilized
pectinase, immobilized weak base anion exchange resin
particles (Dowex WBA Marathon, USA) were stored in
pH 4.5 citrate buer at +4C for 7 weeks. Immobilized
enzyme particles were taken from the buer medium
periodically every week and the activities were deter-
mined by using pectin solution at pH 4.5. The related
activities were found to be 3.280 pectin (w/v, %) /s ml for
the rst and 3.024 pectin (w/v, %) /s ml for the last run,
respectively. The immobilized enzyme was found to be
stable and retained its initial activity for at least 7 weeks
of storage at +4C and pH 4.5. The activity loss was
found to be only 7.80% (Fig. 2). In a similar manner,
Alkorta et al. (1996), showed that pectinlyase immobi-
lized on nylon retained all its initial activity for 80 days
of storage at +4C and pH 8.0.
3.5. Stability and the activity of immobilized anion
exchange resins in carrot puree
Enzyme immobilized particles were added to the
carrot puree at a concentration of 1.5 g particle/100 g
puree at pH 4.5 and at 35C to degrade soluble and
insoluble pectins and haze-provoking polysaccharides.
As it was shown by several researches, enzymatic
treatment results in higher yields of fruit and vegetable
products (Sreenath, Frey, Radola, & Scherz, 1984; Acar
Table 1
The activities of commercial pectinase immobilized anion exchange
resins prepared with dierent enzyme loadings
Initial enzyme
concentration
(%, v/v)
a
Loaded enzyme
(%, v/w)
b
Activity (pectin
(w/v, %) /s ml)
2 81.96 2.52
6 61.72 3.28
10 14.28 2.82
14 23.08 2.11
18 8.10 2.11
a
The free enzyme concentration in 4.5 pH citrate buer.
b
The percentage loaded enzyme per gram of particle.
Fig. 1. The variation of the initial activity rates of immobilized
enzymes with the run in the model system studies.
N. Demir et al. / Journal of Food Engineering 47 (2001) 275280 277
& Alper, 1996; Czukor & Nyarady, 1999). From this
point, the activity of immobilized enzyme was deter-
mined by measuring pH, dry matter content and vis-
cosity of the puree. Table 2 shows the eect of
immobilized enzyme on some analytical properties of
carrot puree. During the enzymatic mash treatment of
carrots, mash was continuously stirred at 4060 rpm for
60 min at 35C in order to obtain higher yields as de-
scribed by Stutz and Felix (1988). During the mash
treatment, the juice acidity increased and also dry matter
content value (% w/w, at 20C) changed from 9.2 to
10.8; this is mainly due to the degradation of the pectin
and partially due to the cellulose hydrolysis (Fau-
quembergue & Grassin, 1996). The immobilized en-
zymes were tested by running ve batches each of 1 h.
After completion of each run, immobilized particles
were easily removed from the pomace by washing with
water.
Immobilized enzyme preparation reduced the viscos-
ity of the carrot puree from 90 to 6.5 Poise, after 60 min
of incubation (Fig. 3). The enzymic activity was calcu-
lated from the slope of the linear part of the viscosity
reduction versus time curves and found to be 3.9 pectin
(w/v,%) /min ml and 1.392 pectin (w/v,%) /min ml for the
free and immobilized enzymes, respectively. As stated by
Sreenath et al. (1984), the enzymatic degradation rate of
the carrot mash was found to depend on the time of
incubation (Fig. 3). The most conspicuous eect was the
very rapid degradation in the rst 20 min. After 20 min
incubation, a maximum was reached and remained un-
changed on further incubation. This eect can be ex-
plained by accumulation of an enzymatically resistant
core and limitation of the substrate (Sreenath et al.,
1984). The use of liquefying enzymes was found to be
induced by an enrichment of the derived products of
Rhamnogalacturonan II whereas other pectic polysac-
charides like homogalacturonan or rhamnogalacturo-
nan rich regions on the cell wall were heavily degraded
(Doco et al., 1997).
With enzymatic mash treatment, pectins, other
polysaccharides and cell-substances were partially or
completely disintegrated (D orreich, 1996). Thus, the
enzymatic activity was followed by the Brix (soluble dry
matter, %), total yield and at the same time by the re-
duction in viscosity and pH. An average yield increment
was 17.7% with respect to the yield obtained from non-
enzymic processed carrot juice with 86.63% and 68.93%,
respectively.
The variation in stability with the run number is given
in Fig. 4. Here, the stability was dened as the ratio of
initial activity rate in any run to that observed in the rst
run (as relative activity). Pectinase activity rates were
calculated from the viscosity reduction of carrot puree
Table 2
The eect of immobilized commercial pectinase on some analytical
properties of carrot puree
Run number pH Dry matter
content (% w/w,
at 20C)
Viscosity
(Poise)
Run 1 Initial 4.493 9.2 90
Final 3.965 10.8 6.5
Run 2 Initial 4.428 9.4 90
Final 4.042 10.6 10
Run 3 Initial 4.526 9.2 90
Final 4.168 10.8 10
Run 4 Initial 4.521 9.0 95
Final 4.161 10.8 11
Run 5 Initial 4.561 9.2 90
Final 4.237 11 12
Fig. 3. Eect of immobilized pectinase on the carrot puree viscosity.
Fig. 2. Storage stability of the immobilized pectinase in model system
studies.
Fig. 4. The variation of the immobilized resin activity and stability
with the run number for carrot puree media.
278 N. Demir et al. / Journal of Food Engineering 47 (2001) 275280
for each run. As seen here, after the second use, no
signicant decrease in the stability of immobilized resin
particle was observed over ve batch runs performed
within one week. The lower activity in the second run
relative to the rst one may be explained by the leakage
of enzyme molecules weakly bound on the surface of
resin particles during the rst run (Bayhan & Tuncel,
1988). Pectinase activities were 1.392-pectin (w/v, %)/
min ml for the rst run and 1.300 pectin (w/v, %)/min ml
for the last run, respectively. The total activity decrease
was found to be approximately 6.5%. Signicant de-
creases were not observed in the activities for both the
model system and carrot puree medium after more than
5-h studies.
4. Conclusion
In this study, commercial pectinase was immobilized
onto 525 lm sized, uniformly spherical anion exchange
resin beads with electrostatic adsorption. The pectolytic
activities of free and immobilized Pectinex Ultra SP-L
were measured from the viscosity reduction of pectin
solution at pH 4, 5 and 35C. The kinetic parameters of
free and immobilized pectinases were studied in a batch
reactor in previous studies of this research. In this part
of the study, the behavior of the immobilized mash
treatment on the pectolytic enzyme in carrot puree me-
dium was investigated. The activity of immobilized en-
zyme was determined by measuring pH, dry matter
content and viscosity of the carrot puree. Immobilized
enzyme preparation reduced the viscosity of the carrot
puree from 90 to 6.5 Poise, after 60 min of incubation.
After 20 min incubation, a maximum was reached and
remained unchanged on further incubation by the ac-
cumulation of the enzymatically resistant core and the
limitation of the substrate. The enzymic activity was
found to be 3.900 pectin (w/v, %)/min ml and 1.392
pectin (w/v, %)/min ml for the free and immobilized
enzymes, respectively. Besides good stabilization, the
17.7% increase in carrot juice yield caused by the im-
mobilized enzyme preparation, can be concluded that
the immobilization of commercial pectinase appears to
be of potential interest for carrot mash treatment to
obtain high yields of carrot juice.
Acknowledgements
This research is supported by Turkish State Planning
Organisation (DPT) project [Project No: 96 K 120890].
The authors wish to thank Prof. Dr. S.A. Tuncer
(Chemical Engineering Department, Hacettepe Univer-
sity) for his contribution to the immobilization part of
the study.
References
Acar, J., & Alper, N. (1996). Production of fermented carrot juices
with enzymatic liquefaction. In XII international congress of fruit
juice report of congress (pp. 183197). IFU, Interlaken, 2024 May.
Alkorta, I., Garbisu, C., Llama, M. J., & Serra, J. L. (1996).
Immobilization of pectin lyase from Penicillium italicum by
covalent binding to nylon. Enzyme and Microbial Technology, 18,
141146.
Bailey J. E., & Ollis F. D. (1986). Biochemical engineering fundamentals
(2nd ed.). New York: McGraw-Hill, 984 pp.
Bayhan, M., & Tuncel, A. (1988). Uniform poly(isopropylacrylamide)
gel beads for immobilization of a-chymotrypsin. Journal of Applied
Polymer Science, 67, 11271139.
Bayindirli, A. (1995). Immobilization of enzymes and potential
applications in food industry. GIDA, 20/2, 113116.
Bernath, F. R., & Venkatasubramanian, K. (1986). Methods of
enzyme immobilization. In A. Demain, & N. A. Solomon (Eds.),
Manual of industrial microbiology and biotechnology (pp. 230247).
Washington, DC: American Society for Microbiology.
Czukor, B., & Nyarady, Z. F. (1999). Production of high-ber
products by enzymatic treatment. In Euro food chem (pp. 335342)
10, 2224 September, Budapest, Hungary, FECS-Event No. 234.
Dinnella, C., Doria, M., Laus, M., & Lanzarini, G. (1996). Reversible
adsorption of endopectin-lyase to tailor-made core-shell micro-
spheres prepared by dispersion polymerization. Biotechology and
Applied Biochemistry, 23, 133140.
Doco, T., Williams, P., Vidal, S., & Pellerin, P. (1997). Rhamnoga-
lacturonan II, a dominant polysaccharide in juices produced by
enzymic liquefaction of fruits and vegetables. Carbohydrate
Research, 297, 181186.
D orreich, K. (1996). Investigations on production of apple juice
without the utilisation of presses. In XII International congress of
fruit juice report of congress (pp. 183197). IFU, Interlaken, 2024
May.
Drilleau, J. F. (1994). Biochemical characteristics of apple juices and
fermented products from musts obtained enzymatically. Fruit
Processing, 4, 108113.
Fauquembergue, P., & Grassin, C. (1996). Apple pomace liquefaction:
a new technology. Fruit Processing, 12, 490494.
Fiedurek, J., Ilczuk, Z., Lobarzewski, J., & Pleszczynska, M. (1992).
Optimization of pectinolytic enzymes biosynthesis by immobilized
mycelium of Aspergillus niger 71. Zentralbl. Mikrobiol., 147, 1521.
Joshi, V. K., Chauhan, S. K., & Lal, B. B. (1991). Extraction of juices
from peaches, plums and apricots by pectinolytic treatment.
Journal of Food Science Technology, 28(1), 6465.
Lozano, P., Manjon, A., Romojaro, F., Canovas, M., & Iborra, J. L.
(1987). A cross ow reactor with immobilized pectolytic enzymes
for juice clarication. Biotechnol. Lett., 9, 875880.
Mutlu, M., Sarioglu, K., Demir, N., Ercan, M. T., & Acar, J. (1999).
The use of commercial pectinase in fruit juice industry. Part 1:
Viscosimetric determination of enzyme activity. Journal of Food
Engineering, 41, 147150.
Naidu, N. G. S., & Panda, T. (1999). Performance of pectolytic
enzymes during hydrolysis of pectic substances under assay
conditions: a statistical approach. Enzyme and Microbial Technol-
ogy, 25, 116124.
Sarioglu, K., Demir, N., Acar, J., & Mutlu, M. The use of commercial
pectinase in fruit juice industry/Part 2: Determination of the kinetic
behavior of immobilized commercial pectinase. Journal of Food
Engineering, 47, 271274.
Shuler, M. L., & Kargi, F. (1992). Enzymes bioprocess engineering.
Englewood clis, NJ: Prentice-Hall.
Sreenath, H. K., Frey, M. D., Radola, B. J., & Scherz, H. (1984).
Degradation of a washed carrot preparation by cellulases and
pectinases. Biotechnology and Bioengineering, 26, 788796.
N. Demir et al. / Journal of Food Engineering 47 (2001) 275280 279
Stutz, C., & Felix, R. (1988). Application and eect of mash enzymes
in fruit juice plants. Fruit Processing, 83(5), 160168.
Stutz, C. (1996). Enzymatic liquefaction dream or reality. Fruit
Processing, 9, 358362.
Szaniawski, A. R., & Spencer, H. G. (1997). Eects of immobilized
pectinase on the microltratin of dilute pectin solutions by
macroporous titania membranes: resistance model interpretation.
Journal of Membrane Science, 127, 6976.
Van den Broek, L. A. M., den Aantrekker, E. D., Voragen, A. G. J.,
Beldman, G., & Vincken, J. P. (1997). Pectin Lyase is a key enzyme
in the maceration of potato tuber. Journal of the Science of Food
and Agriculture, 75, 167172.
280 N. Demir et al. / Journal of Food Engineering 47 (2001) 275280