Process Biochemistry 44 (2009) 353–356
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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Short communication
Application of electrospun poly(ethylene terephthalate) nanofiber mat to apple
juice clarification
Beatriz Veleirinho, J.A. Lopes-da-Silva *
Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 8 March 2008
Received in revised form 25 October 2008
Accepted 5 November 2008
Electrospinning was used to produce self-supporting nanofibrous poly(ethylene terephthalate)
membranes with good mechanical properties and straightforward handling. The application of this
type of membranes in apple juice clarification process was investigated. Processing characteristics and
quality parameters of apple juice were analyzed in order to compare the proposed method to traditional
clarification techniques. In general, the apple juice obtained from electrospun nanofiber membrane
filtration revealed physico-chemical characteristics comparable to those from juice clarified by
ultrafiltration or by conventional clarification using filtering aids. Nevertheless, the new process showed
a high flux performance and revealed to be much faster, simple and more economical than the traditional
processes. This work demonstrated the filtration potential of an electrospun PET membrane thus
introducing a new concept of clarification and opening new approaches for the juice processing industry
or even for other food industry fields.
ß 2008 Elsevier Ltd. All rights reserved.
Keywords:
Nanofiber mat
Electrospinning
Juice processing
Apple juice
Clarification
Membrane filtration
1. Introduction
Juice and fruit juice products represent a very important
segment of the total processed fruit industry and their consumption significantly increased during the last years. The major
amount of apple juice is consumed as a brightly clear product
obtained through the clarification of the raw apple juice [1].
Traditionally, apple juice clarification is achieved by addition of
filtering aids, such as gelatin and bentonite, promoting the
adsorption and/or coagulation of a wide range of compounds.
The particles in suspension are then removed by centrifugation or
common filtration, improving juice’s stability and appearance and
thus increasing the consumer’s acceptability [2]. However, this
process is not only expensive and laborious but it can also causes
modifications on sensory and nutritional properties of the juice
[3,4]. In addition, in recent years there has been an increasing
demand for natural, free-additive products, motivating the juice
processing industry to develop and employ free-additive clarification techniques. Ultrafiltration is the mostly widespread freeadditive clarification method due to many advantages, including
higher juice yield, cost reduction and high quality products [2,5].
Unfortunately, a disadvantage of this process is the rapid reduction
of permeate flux by fouling of the membrane which declines the
system performance [6].
* Corresponding author. Tel.: +351 234370360; fax: +351 234370084.
E-mail address: [email protected] (J.A. Lopes-da-Silva).
1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.11.008
Electrospinning technique has gained much attention due to its
ability to produce nanofibrous mats with distinct characteristics,
such as high porosity, small pore sizes with an interconnected
structure and a large surface area per unit volume [7,8]. These
characteristics make them attractive in a variety of applications,
including both air and fluid filtration [9–12]. In fact, in liquid
filtration applications, the nanofibrous membranes produced by
electrospinning are expected to overcome some of the limitations
associated to the porous polymeric membranes manufactured by
conventional methods, e.g. low-flux and high-fouling performance,
due to their highly interconnected pore structure and large surface
area to volume ratio.
In despite of their potential as filtration membranes, to the best
of our knowledge the use of electrospun membranes in juice
processing has not yet been reported. Therefore, we have studied
the preparation and application of a nanofibrous electrospun
filtration membrane for apple juice clarification. Poly(ethylene
terephthalate) (PET) was selected as the polymer to prepare the
nanofibrous mats, due to its low cost, attractive structural and
mechanical characteristics, and good electrospinning properties
[13,14].
2. Experimental
2.1. Materials
The apples used in this study (Golden Delicious variety) were purchased from a
local market and washed with tap water to remove any adhering substances. PET
pellets used to produce the filtration membranes were kindly supplied by Flexitex
(Portugal). All solvents were analytical grade from Sigma–Aldrich Chemical
Company and were used without further purification.
B. Veleirinho, J.A. Lopes-da-Silva / Process Biochemistry 44 (2009) 353–356
354
2.2. Preparation and characterization of the electrospun membrane
The nanofibrous membranes were prepared by electrospinning 30% (w/v) PET
solutions in a mixture of trifluoracetic acid (TFA) and dichloromethane (DCM)
(80:20 v/v), following the conditions previously reported [14].
Morphology and diameter of the nanofibers were analyzed by scanning electron
microscopy (Philips XL 30 ESEM) at an accelerating voltage of 10 kV. The average
fiber diameter was calculated from 300 measurements of the sample fibers, using
the SEM images of magnification 1500 and appropriate software (Image J 1.37c,
Wayne Rasband, National Institute of Health, USA). The thickness of the electrospun
membranes was measured using a digital micrometer (model MDC-25S, Mitutoya
Corp., Japan). The apparent density and porosity of the fibrous membrane was
determined as previously described [13,14]. Mechanical properties were evaluated
by uniaxial tensile tests performed in parallel with the principal axis of fiber partial
alignment using a texture analyser equipment (model TA.Hdi, SMS, England).
2.3. Preparation of apple juice
Peeled apples were introduced in a centrifugal juice extractor (Molynex
EazyClean) in order to obtain the raw apple juice. The juice was depectinized
with 0.75 mL/L Pectinex Ultra SPL (Sigma pectinase from Aspergillus aculeatus) and
0.15 g/L a-amylase (Fluka amylase from Bacillus subtilis, 49 U/mg), for 2 h at 50 8C.
The absence of detectable pectin and starch in the clarified juice was confirmed by
ethanol and iodine method separately. The enzymes were inactivated by heat (90 8C
for 5 min) and the juice was filtered through a ‘‘cheese cloth’’ in order to remove the
remaining pulp before the clarification step.
2.4. Clarification of apple juice
Clarification of apple juice is normally performed by addition of clarification
agents (conventional clarification) or by ultrafiltration. For this reason, we have
used these two clarification methods as a reference to the filtration method using
the electrospun nanofibrous mats proposed in this work. The unclarified juice was
used as a control for comparison among clarification treatments.
The conventional clarification method was based on a that previously described
by Gokmen et al. [2]. Accordingly, clarification was achieved by adding 0.5 g/L of
gelatin and 2.5 g/L of bentonite to the enzymatically treated juice, at 50 8C for 2 h.
The precipitate was removed by centrifugation (4000 rpm, 10 min) and the clear
juice was stored at 4 8C. The ultrafiltration technique was performed using a stirred
ultrafiltration cell (Millipore, model 8200). In order to produce the clear juice,
150 mL of enzymatically treated juice were ultrafiltered through a regenerated
cellulose membrane (Millipore, cut off 100 kDa), under N2 pressure. The clarified
juice was stored until analysis at 4 8C.
Apple juice clarified by electrospun nanofibrous membrane (ENM) filtration was
obtained using also the stirred ultrafiltration cell (Millipore, model 8200). The ENM
was cut into a 63.4 mm diameter circle and introduced into the ultrafiltration cell.
The unclarified juice was forced to pass through the membrane using nitrogen
pressure. The clarified juice was stored until analysis at 4 8C.
2.5. Juice characterization
Colour and turbidity were measured by UV–vis spectrophotometry. Colour was
expressed as the extinction coefficient at 440 nm and turbidity as the percentage
transmission at 650 nm [2]. Soluble solids of samples were determined with a
manual refractometer (Atago, Tokyo), at 20 8C, and the results were expressed in
8Brix. The pH was measured using a pH meter (WTW InoLab). For titratable acidity
measurement, 25 mL of juice was titrated with 0.25 M NaOH to pH 8.1, and the
suitable volume was converted into malic acid equivalent. The total phenolic
content of samples was determined according to the Folin-Ciocalteau method [15].
Total protein was measured using a commercial protein kit (Micro Lowry,
Peterson’s Modification—Sigma).
Free sugars (glucose, fructose and sucrose) where measured by HPLC using a
Beckman system with RI detector and an YMC-Pack polyamine II S-5um column
(250 mm 4.6 mm). Isocratic elution was carried out at room temperature using
acetonitrile/water (75:25) at 1.5 mL/min as the mobile phase. Organic acids were
quantified by HPLC using a Beckman System Gold, a diode array detector (210 nm)
and a Aminex Ion Exclusion HPX-87H column (300 mm 47.8 mm). Isocratic
elution was carried out at 40 8C using 5 mmol/L sulphuric acid at 0.6 mL/min as the
mobile phase.
2.6. Statistical analysis
A one-way ANOVA was used to test for any significant difference between
treatments and control on each independent variable under study. A t-Student test
was performed to test for specific statistical significance among data means.
3. Results and discussion
3.1. Membrane characterization
The electrospinning process was performed until the membrane reaches an average thickness of 0.20 mm (similar to the
ultrafiltration membrane used). The PET electrospun nanofibrous
membrane (ENM) was typically characterized by a uniform
smooth surface and a good resistance to rupture despite of its
exceptional lightness. Fig. 1 shows the general aspect of the ENM
before and after apple juice filtration.
The electrospun membranes revealed good mechanical proprieties, such as a high tensile strength (2.7 0.2 MPa) and relatively
high elongation (35 8%) which are also important features for a
membrane filtration in order to avoid damage during handling. The
strength of the PET nanofibers web was high enough to use for filter
without the need of any supporting matrix like meltblown or
spunbonded nonwovens, thus avoiding one of the common problems
of handling electrospun nanofiber mats, related to their usually low
mechanical strength.
The average density of the membrane (0.33 g cm 3) was 4.2
orders of magnitude lower than the original PET density which
reveals the presence of large amounts of pores among the fibers. In
fact, the porosity of the ENM was about 80%.
Fig. 2 shows representative SEM images of the PET ENM,
revealing the detailed picture of the membrane morphology. This
analysis revealed a membrane with many attractive characteristics
for the required end-use as a filtration device: a random fibrous
arrangement, free of beads and with a tridimensional porous
Fig. 1. Images showing the general aspect of the PET ENM membrane (A) before filtration and (B) after apple juice filtration.
B. Veleirinho, J.A. Lopes-da-Silva / Process Biochemistry 44 (2009) 353–356
355
Fig. 2. SEM images showing the morphology of the PET electrospun membrane (A) magnification 500; (B) magnification 1500.
structure, high porosity and small pore diameters. The average
fiber diameter was 420 nm.
3.2. Clarification process
3.2.1. Processing characteristics
The most remarkable difference between the studied clarification processes was the total clarification time (Table 1). ENM
filtration was clearly the fastest clarification technique, being more
than 20 times faster than the conventional one. Additionally, heat
or clarification agents are not required by the ENM filtration
method, allowing a cost reduction, as well as avoiding off-flavours
in the juice.
Comparing to the ultrafiltration technique the new proposed
method allowed for a faster juice flux during the process, thus also
providing less time consumption. An additional economical
advantage of the PET ENM filtration refers to a much lower work
pressure compared to the ultrafiltration process.
3.2.2. Changes on apple juice quality
The influence of the different treatments on the juice quality is
shown in Table 2. All the treatments significantly decreased the
apple juice colour (p < 0.05). The final colour of the juice samples
was very similar for all treatments and visibly higher than the
control (unclarified juice). The preliminary removal of pectins and
starch, common to all the clarification methods, is the main
responsible for the improved colour and turbidity of the clarified
juice samples. However, ENM filtration and ultrafiltration promoted a significant decrease in the turbidity of the juice (p < 0.05)
and the best clarification efficiency. No significant effects of the
treatments were found for the total solids content, soluble solids,
pH and acidity (p < 0.05).
Table 3 shows the concentration of total phenolic compounds,
proteins, selected free sugars and organic acids. As expected, the
protein content of the apple juice is very low and relatively high
errors are associated to the results, which should be regarded as
indicative. Nevertheless, in general, a decrease in the protein
concentration was observed after the clarification treatments,
being more evident for the conventional clarification and PET ENM
filtration (p < 0.05). Since the protein content of the juice is very
low, removing this kind of compounds will not reduce the
nutritional value of this product, but can be useful to reduce
turbidity and improve juice stability.
In general, it was observed a more pronounced reduction of free
sugars when the clarification method was performed using the PET
ENM filtration. Under the analytical conditions used, no significant
Table 1
Operational parameters for conventional clarification, ultrafiltration and PET ENM filtration, related to the treatment of 150 mL juice volume.
Method
Conventional clarification
Ultrafiltration
PET ENM filtration
Average processing
time (min)
Required
temperature (8C)
Required
pressure (psi)
Clarification
agents added
Average flux
(mL cm 2 min
160
35
6
50
N.A.
N.A.
N.A.
50.8
0.7
Bentonite and gelatin
N.A.
N.A.
N.A.
0.17
3.5
1
)
N.A. = not applicable.
Table 2
Physico-chemical properties (mean standard deviation, n = 3) of apple juice samples obtained from different clarification treatments.
Treatment
Coloura Abs440 nm
Turbiditya% T650 nm
Total solids (%)b
Soluble solids (8Brix)b
Acidityb (%w/w)c
Unclarified (control)
Conventional clarification
Ultrafiltration
PET ENM filtration
0.53 0.07
0.41 0.04
0.40 0.03
0.40 0.03
75 4
82 4
87 2
88 1
13.1 0.6
13.2 0.6
13.0 0.2
13.1 0.7
14.5 0.4
14.5 0.5
14.2 0.2
14.4 0.5
0.33 0.03
0.32 0.03
0.35 0.01
0.32 0.02
a
a
b
b
b
a
b
c
c
Means with different letters within a column indicate significant differences (p < 0.05).
All scores within each column were not significantly different to p < 0.05 level.
c
As malic acid equivalent.
b
356
B. Veleirinho, J.A. Lopes-da-Silva / Process Biochemistry 44 (2009) 353–356
Table 3
Composition in protein, phenolic compounds, sugars and organic acids (mean standard deviation, n = 3) for apple juice samples obtained from different clarification
treatmentsa.
Unclarified (control)
Conventional clarification
Ultrafiltration
PET ENM filtration
Total protein content (mg L 1)
Total phenolic content (g L 1)b
194 16 a
0.15 0.01
83 16 b
0.13 0.01
145 14 c
0.14 0.02
98 3 b
0.15 0.02
Sugars (g/L)
Fructose
Glucose
Sucrose
95.4 0.8 a
37.2 0.3 a
13.6 0.5 a
91.3 0.5 b
35.2 0.5 b
12.3 0.3 b
94.8 0.9 a
33.7 0.8 c
13.1 0.4 a
89.9 0.6 b
33.6 0.4 c
10.9 0.4 c
Organic acids (g/L)b
Malic
Oxalic
Lactic
Citric
9.7 0.2
0.14 0.03
n.d.c
n.d.
9.9 0.3
0.10 0.02
n.d.
n.d.
9.8 0.1
0.18 0.05
n.d.
n.d.
9.8 0.2
0.13 0.03
n.d.
n.d.
a
Means with different letters within a row indicate significant differences (p < 0.05).
Not significantly different at p < 0.05 level.
c
n.d.—not detected.
b
differences could be detected for the total phenolics and detectable
organic acids present in the different apple juice samples.
4. Conclusions
In this work, we have demonstrated that fibrous mats
composed of randomly oriented submicron-size fibers can be
prepared by electrospinning and successful used, for the first time,
as nonwoven filters in fruit juice clarification. Filtration using the
ENM allowed for a less time consuming and more economical
process, still originating a clarified juice with characteristics
similar to that obtained by ultrafiltration.
It is important to point out that the investigated electrospun
membranes have not been fully optimized for further improvements. One relevant aspect, taking into account that the findings
described came out from a lab-scale process, is the need for
further scale-up studies in order to understand how the process
would run on a larger scale. In addition, retention capability, flux
rate and the fouling problems can be further improved and
optimized by adequate control of the electrospinning process, in
order to manipulate fiber diameter, porosity and membrane
thickness.
In addition, the possibility to incorporate bioactive compounds
or additional fiber components into the nanofibers may allow
preparing affinity membranes for the selective removal of certain
components, rather than operating merely by sieving mechanisms.
Acknowledgments
Authors gratefully acknowledge Fundação para a Ciência e
Tecnologia (FCT, Lisboa, Portugal) for funding through the
programmes POCI 2010 and FEDER (project POCI/CTM/58312/
04). The authors thank Dr. Manuel Rei (CITEVE, Portugal) for the
help with the SEM analysis. Authors also thank Mrs. Cristina Santos
(College of Biotechnology, Porto, Portugal) for helping with the
HPLC analysis.
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