EFFECT OF PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON

APPLE JUICE TURBIDITY

V. SORRIVAS
1
, D.B. GENOVESE
2
and J.E. LOZANO
2,3
1
CRIBABB
2
PLAPIQUI (UNS-CONICET)
Camino La Carrindanga Km 7, Baha Blanca (8000), Argentina
Accepted for Publication November 17, 2005
ABSTRACT
The mechanisms governing the enzymatic clarication of apple juice
were studied by electron microscopy techniques. Full ripe and unripe apple
juice samples (Granny Smith) were treated with commercial pectinase (Solvay
5XLHA) and amylase (Rhalase HT) enzymes, respectively. Scanning electron
microscopy studies revealed that commercial amylolytic enzymes quickly
reduced starch content in unripe apple juice to undetectable values. It was also
observed that after pasteurization of this juice (90C, 5 min) all starch granules
gelatinized. Using transmission electron microscopy, it was possible to
observe pectin bonded to ripe apple juice particles. This protective colloid is
known to be responsible for cloudy juice stability. The effect of pectic enzyme
to destroy the protective pectin colloid was also detected with this technique.
As a result of the enzymatic treatment, average particle size initially increased
from 1000 to 1500 nm and decreased thereafter to 1100 nm, and Z-potential
increased in absolute values from -9.6 to -11.4 mV. It was speculated that the
destruction of the weak pectin net by the action of the specic enzyme caused
particle aggregation, followed by the collapse of aggregates, increasing the
number of particles 500 nm.
INTRODUCTION
The main purpose of the clarication treatment employed in industrial
apple juice processing is to eliminate constituents responsible for the turbidity
and cloudiness in freshly produced juice. Conventional clarication process
3
Corresponding author. TEL: +54-291-4861700; FAX: +54-291-4861600; EMAIL: jlozano@
plapiqui.edu.ar
Journal of Food Processing and Preservation 30 (2006) 118133. All Rights Reserved.
2006, The Author(s)
Journal compilation 2006, Blackwell Publishing
118
includes hydrolysis of pectin and starch with specic enzymes, occulation of
turbidity with clarifying agents (bentonite, gelatin and/or silica-sol) (Grampp
1977) and ltration through plate and frame or vacuum Oliver-type lters.
Application of ultraltration (UF) as an alternative to conventional processes
for the clarication of fruit juices is gaining acceptance in the industry (Lozano
et al. 2000). However, these membrane techniques partially substituted the use
of specic enzymes (Mondor and Brodeur 2002), and the adequacy of UF as
a technique for preventing haze formation still requires further studies. The
other important purpose of clarication is to remove substances that may cause
haze and sediment formation during storage, at reconstitution of the concen-
trate or after bottling of the juice (Tajchakavit et al. 2001).
Apple juice is one of the juices that may contain considerable amounts of
starch, particularly at the beginning of the harvest season. Unripe apples
contain as much as 15% starch (Reed 1975), and up to 1% starch may be
present in juice after milling and pressing (Carrn et al. 2004). Starch is a
common problem for apple juice processors, complicating ltration and
causing postprocess cloudiness.
The process of depectinization involves the use of commercial enzymes,
generally a blend of pectinases (e.g., pectinase, polygalacturonase, cellulase,
pectin lyase) to degrade pectic substances. Starch-degrading enzymes such as
amylase and amyloglucosidase are also commonly added during the depecti-
nization stage. These enzymes degrade starch into smaller units and may
contribute to postbottling haze formation by aggregating among themselves or
through the formation of proteinstarch complexes. Fining of apple juice also
involves the use of gelatin and bentonite as ning agents (Stock 1998).
Differences in the nature of ionic charges of protein, polyphenols and the
ning agents induce occulation and sedimentation and result in the removal
of these potential haze precursors from juices.
Turbidity of opalescent apple juice is provided by particulate material that
remains in suspension (Genovese et al. 1997). The cell wall of fruit comprises
a complex mixture of cellulose, hemicelluloses, pectins and proteins, which
incorporate to pressed juice in different ways, providing cloudiness.
Cloud particles have been modeled to consist of a negatively charged
pectin wrapped around a positively charged proteincarbohydrate core
(Yamasaki et al. 1964; Endo 1965). Pectin slows aggregation by coating the
particles, preventing the close-enough approach of one to another needed for
the formation of hydrogen bonds or effective dipolar interactions (Van Buren
1989). In addition, they may give particles enough charge such that signicant
electrostatic repulsion takes place between particles. Particle sizes above
0.5 mm are unstable and may easily settle out (Genovese et al. 1997). Below
this range, particles are retained in suspension because of their small size,
mutual charge repulsion and protective effect of pectin.
119 PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY
However, the mechanism of particle stabilization in cloudy juices is not
completely clear yet. For colloidal stability, the repulsive forces must be
dominant. There are different mechanisms that affect dispersion stability in a
colloid, including Van der Walls attractive and electrostatic repulsive forces
and some others interactions such as steric repulsion, depletion, hydrophobic
and hydrophilic interactions (McClements 1999).
Z-potential (x) measurements are used to assess the stability of colloidal
systems because it is a very good index of the magnitude of electrostatic
repulsive forces between colloidal particles. On the one hand, when all the
particles have a large negative or positive x, they will repel each other and
increase dispersion stability. On the other hand, when the particles have low x
values, then the net repulsive force to prevent the particles coming together is
weak or null. x is a function of the nature of particles surface, and the
composition of the dispersed phase (pH, ionic strength and concentration of
specic ions and polyelectrolyte) (McClements 1999). Genovese and Lozano
(2001) worked with the addition of hydrocolloids to cloudy apple juice. They
found that x was satisfactory to predict the haze stability in cloudy fruit juices.
Scanning electron microscopy (SEM) is a microscopic technique used to
visualize the aggregation and occulation process (Ferworn and Svrcek 1998).
In addition to the generalized application of commercial enzymes in the
juice industry, there is also a lack of information about the phenomena that
govern the formation of stable colloidal particles on turbid juices and how
specic enzymes contribute to make this colloid unstable. The objective of this
study was to examine the effect of commercial pectinase and amylase on
turbid apple juice using different microscopic techniques, supported by par-
ticle size and x determinations.
MATERIALS AND METHODS
Figure 1 schematically represents the methodology followed in this study.
On the one hand, juice made from unripe apples was used to: (1) isolate starch
granules; (2) study the effect of pasteurization on starch; and (3) verify the
action of amylolytic enzymes. On the other hand, juice made from ripe apples
was used to study the presence of pectin as a protective colloid (nontreated
juice, NT) and effect of pectinolytic enzymes on cloud structure and stability
(enzyme-treated juice, ET). Particle size and x were also determined in both
NT and ET samples.
Cloudy apple juice of 10Brix was obtained from full ripe and unripe
Granny Smith apples at the laboratory. The unripe apples were picked two
weeks before normal harvest date and showed a rmness of 88.3 N, as
determined with an 11-mm-wide probe Effegi FT 327 Penetrometer (Effegi,
120 V. SORRIVAS, D.B. GENOVESE and J.E. LOZANO
Milano, Italy). The generic starchiodine index chart for apples was used to
determine fruit maturity (Blanpied and Silsby 1992). Manufacturing included
crushing individual apples in a laboratory mill, steam-heating the mash at
6570C for 1520 s to inactivate native polyphenol oxidase and pressing it in
a hydraulic press (100-mesh lter) with the same type of equipment and
procedures as previously described (Genovese et al. 1997). Aliquots of juices
were pasteurized (90C, 5 min). Soluble solids were determined with a digital
Abbe-type refractometer according to 932.12 AOAC (2000) method. The
pectin used was apple pectin from Sigma-Aldrich S.A. (Buenos Aires, Argen-
tina) (Anhydro-galacturonic acid = 77.5). Apple juice turbidity was measured
Ripe
apples
Starch
granules
separation
SEM observations
TEM observations
Unripe
apples
Amylolytic
enzyme treatment
Unripe apple juice
pasteurization
Juice manufacturing
(crushing and pressing)
Sample preparation for
TEM study
Particle size
and Z-
potential
determinations
Sample preparation for
SEM study
Ripe apple juice
pasteurization
(NT)
Pectic enzyme
treatment
(ET)
Iodine
test
FIG. 1. SCHEME OF THE EXPERIMENTAL METHOD FOLLOWED DURING THIS STUDY
SEM, scanning electron microscopy; TEM, transmission electron microscopy;
NT, nontreated; ET, enzyme treated.
121 PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY
as nephelometric turbidity unit (NTU) with an Aqualytic PC-COMPACT
(Dortmund, Germany) turbidimeter. Samples of juices (30 mL) were placed in
a 30-mL cell, capped and gently inverted twice to ensure even mixing. Pres-
ence of starch in juice was determined by the standard iodine test (IFFJP
1984). All the calibration assays and analytical determinations were made in
triplicates.
Commercial pectinase and amylase used were a liquid fungal pectinase,
Solvay 5XLHA (Genencor Enzymes, Arroyito, Argentina), and a fungal amy-
loglucosidase, Rhalase HT(RO) (ABEnzymes GmbH, Darmstadt, Germany),
respectively. Ceci and Lozano (1998) and Carrn et al. (2002) reported both
methods for enzymatic activity determination and optimal conditions for the
application of these enzymes to the clarication of apple juice.
Indicated relative polygalacturonase and pectinesterase activities, as per-
centage of maximum pectinase activity, were 75 and 33%, respectively, at
pH = 3.5 and T = 50C. Amylase (RO) activity was determined (Carrn et al.
2002) to be 1.6 U at pH = 3.6 (1 U of enzymatic activity was taken as the
enzyme required to release 1 mg of maltose in 1 min).
The pectic enzyme treatment (80 mg/L) was carried out after the hot
technique (Toribio and Lozano 1984). To study the effects of the commercial
amylase on the apple starch, pasteurized unripe apple juice was treated with
enzyme solution (RO, 100 mg/L) at 50C for 150 min. Enzymatic reactions
were inhibited by cooling in ice; the mixture was centrifuged (9000 g,
1 min), and the precipitate was washed three times with 50% (v/v) ethanol/
water.
Starch granules were isolated by repetitive washing and centrifugation of
cloudy juice made from unripe apples. The cloudy juice was centrifuged
(9000 g, 1 min) in a Beckman Microfuge centrifuge (Beckman Instruments,
Inc., Irvine, CA). Supernatant was removed and replaced with water/ethanol
solution (50% v/v). Suspension was treated in an ultrasonic bath (25C, 1 min)
and centrifuged (9000 g, 1 min). This operation was repeated three times.
The supernatants were collected and pasteurized, and the sediments were
suspended in 2 M NaOH, dissolved as previously mentioned and diluted in
0.1 M acetate buffer (pH = 4.6). Insoluble starch was determined in these
dissolved and diluted sediments by the iodometric method, as described by
Carrn et al. (2004).
SEM of Apple Starch and Unripe Juice
Drops of both cloudy juice of unripe apples and isolated starch granules
were put on glass slides, dried in oven under vacuum at 40C (30 min), gold-
covered in a Pelco model 3 Sputter Coater 91000 metal evaporator (Ted Pella,
Inc., Redding, CA) and observed with a Japan Electron Optics Laboratory
(JEOL) model 35CF SEM (JEOL Ltd, Tokyo, Japan) at 5 kV.
122 V. SORRIVAS, D.B. GENOVESE and J.E. LOZANO
Transmission Electron Microscopy (TEM) of Pectin
To verify the role of pectin and pectic enzymes in turbidity, cloudy juice
of ripe apples was centrifuged (9000 g, 1 min). The supernatant was dis-
carded, and the sediment was diluted (1:100) with distilled water. One drop of
each dispersion, made with both ET and NT juices, was xed with 2.5%
glutaraldehyde solution in 0.1 M sodium cacodylate buffer (pH: 7.47.6) with
0.3% ruthenium red, dehydrated with 25, 50, 70, 90% and three times 100%
acetone (30 min each step), post-xed with 2% osmium tetroxide with 0.3%
ruthenium red for 30 min at room temperature, embedded with Spurrs resin
and cured at 70C overnight. Thin sections were cut with a diamond knife in an
LKB Ultramicrotome, mounted in 200-mesh grids and examined and photo-
graphed in a JEOL 100 CXII transmission electronic microscope (JEOL Ltd).
Selected cuts were stained with 1% aqueous uranyl acetate for 30 min, washed
with water, stained with lead citrate for 3 min, washed with a 0.01 N NaOH
solution and placed on a lter paper in a Petri dish to dry. ET and NT apple
juice samples were examined with a JEOL 100 CXII transmission electronic
microscope at 80 kV.
Particle Size and x Determinations
Ripe apple juice particle size was determined by photon correlation
spectroscopy. The x was determined by laser Doppler electrophoresis applying
the Smoluchowski equation. Both particle size and electrophoretic mobility
measurements were conducted with a Malvern Zetasizer 3000 (Malvern
Instrument, Inc., London, England) as in a previous work (Genovese and
Lozano 2000). The juice samples were centrifuged (4200 g 5 min) in
advance, and no dilution was required. The results given are the average values
of ve samples for each treatment.
RESULTS AND DISCUSSION
Starch
The juice from unripe apples showed a large quantity of insoluble starch
granules. Apple starch granules could be considered practically spherical
(Fig. 2). Mean diameter (f) was determined to be f = 9 (3) mm, which is
similar to what is reported in previous work (Carrn et al. 2004). Nonpasteur-
ized juice showed ne particulates, supposed to contain considerable protein
and carbohydrate as basic constituents (Endo 1965; Beveridge 1997) over
which the starch granules are easily observed (Fig. 3a). Although a few of
them collapsed to some degree, most of the granules retained their integrity.
123 PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY
Figure 3b shows an SEM micrograph of the pasteurized sample (90C, 5 min).
After heat treatment, mainly gelatinized starch was detected. This micrograph
(Fig. 3b) shows that apple starch granules collapsed after heat treatment and
only gel-like starch fragments dispersed among the other components of cloud
may be observed. Similar behavior was found when wheat starch was gelati-
nized by heat in an excess of water (Lineback and Wongsrikasem 1980).
SEM studies revealed that amylase normally used in fruit juices clari-
cation quickly reduced starch contents in pasteurized juices to an undetectable
level (micrographs not shown). Starch was not detected by iodometry either.
Pectin
Ruthenium red binding to free carboxyl groups of pectin with different
degrees of esterication was investigated by Hou et al. (1999). Ruthenium
red with glutaraldehyde for staining mucopolysaccharides, and with osmium
tetroxide for enhancing contrast, is a well-known staining reagent in histology.
In Fig. 4, TEM micrographs of NT apple juice at different magnications are
presented. Typical particle shapes, including vesicles and agglomerations
FIG. 2. SCANNING ELECTRON MICROGRAPH OF ISOLATED APPLE STARCH GRANULES
(5 kV 3200)
124 V. SORRIVAS, D.B. GENOVESE and J.E. LOZANO
already described in the literature (McKenzie and Beveridge 1988), were
identied (Fig. 4a). Presence of pectin bers in a network structure, speci-
cally stained with ruthenium red, was observed at a higher magnication
(Fig. 4b). Presence of brous material in apple haze, attributable to pectin, was
also observed by Beveridge et al. (1996). Ruthenium red is a dye, which
selectively binds to the intramolecular spaces between carboxyl groups of
pectin. The dye group links the carboxyl oxygen of one galacturonide moiety
to a hydroxyl oxygen of an adjacent neighbor galacturonide in the pectate
chain. The number of staining sites for ruthenium red depends on the number

b
a
FIG. 3. SCANNING ELECTRON MICROGRAPHS OF PRECIPITATES FROM
UNRIPE APPLE JUICE
(a) Nonpasteurized; (b) Pasteurized (5 min, 90C).
125 PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY
of monomers of anhydrogalacturonide units in the pectin polysaccharide
(Sterling 1970).
A TEM micrograph of a commercial pectin gel (0.017 g pectin/mL) is
also presented for comparison (Fig. 4c). As the gure shows, size and charac-

c
a
v
v
g
g

2 2. .2 2 m m
v
p p
b
0 0. .2 2 m m 0 0. .2 2 m m
FIG. 4. TRANSMISSION ELECTRON MICROGRAPHS OF RIPE APPLE JUICE PARTICLES
WITHOUT ENZYMATIC TREATMENT
(a) Low magnication (6700); (b) High magnication (40,000); (c) Commercial pectin gel
(0.017 g/mL).
g, particle agglomerates; p, pectin bers; v, vesicles.
126 V. SORRIVAS, D.B. GENOVESE and J.E. LOZANO
teristics of commercial and native pectins are very similar, taking into consid-
eration the difference in concentration. High methoxyl apple pectin is
composed of open networks, with the strands arranged in bundles or loose
aggregates. Lofgren and Hermansson (2002) also found pectin gels that
showed aggregated networks with large pores around 500 nm.
Apple juice particles after the treatment with Solvay 5XLHA enzyme
may be observed in Fig. 5a. The effect was a reduction in the size of vesicles
and an increase in the number of particles smaller than 0.5 mm. In Fig. 5b, ET
particles can be observed at a higher magnication. It was determined, after
analyzing nearly 40 micrographs, that ber-like pectin practically disappears
after the action of pectinase, as expected.
b
0 0. .2 25 5 m m
a
g
g
v v
0 0. .7 7 m m
FIG. 5. TRANSMISSION ELECTRON MICROGRAPHS OF RIPE APPLE JUICE PARTICLES
AFTER PECTINOLYTIC ENZYME TREATMENT AT DIFFERENT MAGNIFICATIONS
(a) 12,000; (b) 40,000.
g; particle agglomerates; v; vesicles.
127 PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY
In Fig. 6a, pectin bers connecting particles in an NT apple juice, was
identied at a higher magnication (40,000). Figure 6b is a micrograph of an
ET sample (40,000) to have better view on the effect of pectin hydrolysis on
particle structure. It can be also veried (Fig. 6b) that pectin bers disappeared
in apple juice particles after the enzymatic treatment.
Turbidity, x and Particle Size Determinations
Figure 7 shows the variation in turbidity of an apple juice sample treated
with pectic enzyme. After reduction in the rst 10 min, the juice turbidity

a
v
v

p p
b
0 0. .5 5 m m
0 0. .5 5 m m
FIG. 6. NONTREATED RIPE APPLE JUICE (a), AND TREATED WITH PECTINOLYTIC
ENZYME (b), AT THE SAME MAGNIFICATION
p, pectin bers; v, vesicles.
128 V. SORRIVAS, D.B. GENOVESE and J.E. LOZANO
recovered back to the initial range (1300-1400 NTU). On the contrary,
average particle size increased right after the enzyme addition, reached a
maximum in about 10 min, decreased sharply afterwards and nally remained
stable around 1100 nm. This behavior may be explained by considering that
some particles aggregated immediately after addition of the pectolytic
enzyme. During aggregation, particles increased in size and decreased in
amount, while their total mass remained constant. When the total aggregate
mass remains constant, above a critical f of 0.3 mm for an aggregate, the
800
900
1000
1100
1200
1300
1400
1500
1600
0 20 40 60 80 100 120
Time (min)
N
T
U
/
n
m
NTU
Size (nm)
FIG. 7. CHANGE IN TURBIDITY (NEPHELOMETRIC TURBIDITY UNIT, NTU) AND MEAN
PARTICLE SIZE (nm) OF RIPE APPLE JUICE DURING PECTINASE TREATMENT
Vertical bars represent SDs.
129 PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY
scattered light intensity declines in proportion to 1/f because the particle count
decreases in proportion to the cube root, although the f increases in relation to
the square (Anon 2004). This may explain the observed decrease in the
measured turbidity during initial aggregation.
Moreover, as a result of the enzymatic treatment, x increased in absolute
values from -9.6 to -11.4 mV, after 2 h.
We propose that enzyme depectinization had two effects:
(1) It destroyed the weak soluble pectin net, and reduced juice viscosity. The
role of pectin in increasing cloudy apple juice viscosity was discussed
previously by Beveridge (2002);
(2) It caused the aggregation of cloud particles. Insoluble pectin forms a
protective coat around proteins in suspension (Lozano 2003). In acidic
environment (apple juice typically has a pH of 3.5), pectin molecules carry
a negative charge. This causes them to repel one another. Pectinase
degrades the pectin molecule and exposes part of the positively charged
protein beneath. The electrostatic repulsion between cloud particles is
thereby reduced so that they clump together.
However, it seems that after most of the native pectin was destroyed,
aggregation structures collapsed, increasing the number of small particles,
resulting in the decrease in size and increase in turbidity as observed in Fig. 7
after the rst 10 min. This fact, which is associated to the changes observed in
x, may explain the need of other clarifying agents (bentonite and gelatin) for
occulation.
CONCLUSIONS
This study has used electron microscopy to show the distribution of
starch and pectin in cloudy apple juice. SEM studies and specic starch test
revealed that amylases normally used in fruit juices clarication quickly
reduced starch contents to undetectable levels. By using TEM, it was possible
to identify: (1) a weak net of soluble pectin suspended in the NT cloudy juice;
and (2) insoluble pectin bonded to apple juice particles. The effect of pecti-
nolytic enzyme, destroying both the pectin protective colloid and the pectin
net, came also to light with this technique. It is known from industrial practice
that viscosity reduction is one of the controlling steps during apple juice
clarication. This study revealed that the role of pectinase during clarication
was more associated with an increase in particle mobility (because of the
pectin net disintegration) than a reduction of electrostatic repulsion between
cloud particles.
130 V. SORRIVAS, D.B. GENOVESE and J.E. LOZANO
ACKNOWLEDGMENT
This research was supported by a grant (BID 1201/OC-AR) from the
Agencia Nacional de Promocin Cientca y Tecnolgica of Argentina.
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