Accepted Manuscript Separation and purification of phenolic compounds from pomegranate juice by ultrafiltration and nanofiltration membranes
Carmela Conidi, Alfredo Cassano, Francesca Caiazzo, Enrico Drioli PII:
To appear in:
Journal of Food Engineering
27 July 2016
16 September 2016
19 September 2016
Please cite this article as: Carmela Conidi, Alfredo Cassano, Francesca Caiazzo, Enrico Drioli, Separation and purification of phenolic compounds from pomegranate juice by ultrafiltration and nanofiltration membranes, Journal of Food Engineering (2016), doi: 10.1016/j.jfoodeng. 2016.09.017
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ACCEPTED MANUSCRIPT Research highlights
Flat-sheet membranes were studied to purify phenolic compounds from clarified pomegranate juice Productivity and separation capability of selected membranes were investigated
Desal GK membrane displayed high productivity, low fouling index and a good separation
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The retentate fraction displayed high antioxidant activity
Glucose and fructose were mainly recovered in permeate and diafiltrate streams
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Separation and purification of phenolic compounds from pomegranate juice by ultrafiltration and nanofiltration membranes
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Carmela Conidi, Alfredo Cassano*, Francesca Caiazzo, Enrico Drioli
Institute on Membrane Technology, National Research Council, ITM-CNR, via P. Bucci, 17/C
University of Calabria, I-87036 Rende, Cosenza, Italy
11 12 13
21 22 23 24
* Corresponding Author. Tel.: +39 0984 492067; Fax: +39 0984 402103 E-mail address: [email protected]
Pomegranate juice is well recognized for its nutritional and health benefits due to the presence of
phenolic compounds, including anthocyanins, ellagic acid, phytoestrogenic flavonoids and
tannins. Therefore, the demand for the production of functional foods containing bioactive
compounds isolated from the juice has remarkably increased in the last decade.
In this study ultrafiltration (UF) and nanofiltration (NF) flat-sheet membranes, with nominal
molecular weight cut-off (MWCO) ranging from 1,000 to 4,000 Da, were tested to purify
biologically active compounds from clarified pomegranate juice. The filtration process was
evaluated in a crossflow pilot unit equipped with a Sepa CFII Membrane Cell System featuring
an effective membrane area of 0.014 m2.
A first screening was made in order to evaluate the performance of selected membranes in terms
of productivity, fouling index and retention towards sugars, phenolic compounds and total
antioxidant activity. Among these membranes the Desal GK membrane, with a MWCO of 2,000
Da, displayed higher permeate fluxes, lower fouling index and a good separation efficiency of
sugars from phenolic compounds in comparison with the other tested membranes. Therefore
further experiments were addressed to evaluate the separation capability and the productivity of
this membrane at different transmembrane pressure (TMP) values. Concentration/diafiltration
experiments were also performed in order to obtain a retentate fraction enriched in phenolic
compounds and a permeate stream mainly containing glucose and fructose.
According to the proposed process the yields of polyphenols and anthocyanins in the retentate
stream were of the order of 84.8% and 90.7%, respectively. The diafiltration step allowed to
obtain a recovery efficiency in the permeate side for glucose and fructose up to 90% and 93%,
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Keywords: Pomegranate juice; ultrafiltration; nanofiltration; anthocyanins; membrane processing.
Pomegranate is one of the first five cultivated foods in the world widely grown in many countries
including Iran, India, Turkey, Tunisia, Pakistan, China, USA and Spain. Popular in Eastern as
well as Western parts of the world, pomegranate thrives well in regions with semi-arid and sub-
tropical climatic conditions but is also naturally adapted in regions with cold winters and hot
summers (Ozgen et al., 2008). The total world production is estimated currently at 2 million
tons/year (Erkan, 2011). In recent years, the interest for pomegranate fruit and its derivatives has
increased remarkably as evidenced by hundreds of publications on their chemical composition,
potential uses and proven health-promoting effects (Gumienna et al., 2016; Jurenka, 2008;
Lansky and Newman, 2007). Several studies have focused on the ability of different components
of the fruit, including the juice, seed oil, peel, flower extracts or their derivatives to protect
against several diseases such as cancer (Dai and Mumper, 2010), type 2 diabetes (Banihani et al.,
2013), atherosclerosis (Al-Jarallah et al., 2013) and cardiovascular diseases (Aviram et al., 2008)
providing the scientific basis for some use of pomegranate in traditional medicine. In addition,
these products have been shown to posses antimicrobial, anti-hepatoxic and antiviral properties
(Faria and Calhau, 2011). These health benefits have been attributed to the high antioxidant
capacity that is strongly correlated with the high concentration and chemical composition of
phenolic anthocyanins and hydrolysable tannins such as punicalagins, punicalin, peduncalagin,
and ellagic acid (Vegara et al., 2013). Different studies have also shown that the antioxidant
activity of pomegranate juice is much higher than most other fruit juices and beverages (Gil et al.,
2000; Seeram et al., 2008).
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Given the wide spectrum of health promoting activities exerted by pomegranate and the
enormous interest that bioactive compounds isolated from this fruit have raised in the scientific
community, the interest of researchers has been addressed in recent years to the optimization of
the extraction and purification procedures of these compounds for the development of functional
foods meeting the consumer requirements.
Solvent organic extractions (SOEs) are the most commonly used procedures to extract bioactive
compounds from pomegranate fruits (Sood and Gupa, 2015). It is generally known that the yield
of chemical extraction depends on type of solvent (polarity), extraction time and temperature
(Singh et al., 2014). However, solvents commonly employed such as methanol, ethanol, acetone
and ethyl acetate are not always “food friendly” and not suitable or safe for their utilization in the
food industry (Amyrgialaki et al., 2015). In addition, long extraction times and high temperatures
may produce an oxidation of phenolics leading to a decreased yield of phenolics in the extracts. It
has been also shown that high temperatures (>70 °C) cause a rapid degradation of anthocyanins
(Havlíková and Miková, 1985). Alternative methods, such as microwave extraction (Zengh et al.,
2011), ultrasound-assisted extractions (Tabaraki et al., 2012) and supercritical fluid extractions
(He et al., 2012), have been also applied in the extraction of phenolic compounds from
pomegranate peels and seeds. However, low extraction efficiency, partial oxidation and
degradation of compounds of interest, high requirements of istrumentations and costs on
industrial scale are typical drawbacks which often outweigh the technical benefits. Therefore, it is
of critical importance to select efficient extraction procedures in order to maintain the stability of
In this contest, membrane separation processes (MSPs) represent a valid alternative to traditional
technologies due to their low operating and maintenance costs, mild operating conditions of
temperature and pressure, easy control and scale-up and highly selective separations. They do not
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require any extraction mass agents or chemical additives, avoiding product contaminations and
preserving the biological activity of the compounds of interest (Drioli and Romano, 2001).
Pressure-driven membrane operations, such as microfiltration (MF), ultrafiltration (UF),
nanofiltration (NF) and reverse osmosis (RO) are today well-established technologies in food and
beverage industries for the treatment of several products and by-products (Daufin et al., 2001;
Patsioura et al., 2011; Tylkowski and Tsibranska, 2015). Other membrane processes, such as
osmotic distillation (OD), membrane distillation (MD) and pervaporation (PV) have been also
investigated in recent years for selected applications in the same area. Moreover, the development
of hybrid processes based on the combination of different membrane unit operations and
conventional separation technologies offers new and much more opportunities in terms of
competitiveness, improvement of quality, process or product novelty and environmental
friendliness (Cassano, 2016; Conidi et al., 2014). The use of membrane technology in the
treatment of pomegranate juice has been recently investigated. In particular, MF and UF
processes have been studied to clarify pomegranate juice as alternative technologies to the
traditional use of fining agents (gelatin, bentonite, diatomaceous earth, silica sol, etc.) and other
techniques including centrifugation, decantation, depectinization and filtration (Baklouti et al.,
2012; Cassano et al., 2015; Mirsaeedghazi et al., 2010a; Mirsaeedghazi et al., 2010b); MD and
OD have been evaluated for their potential in the concentration of the juice as alternative to the
thermal evaporation (Cassano et al., 2011; Onsekizoglu, 2013).
In recent years UF and NF operations have gained a great interest for the separation and
concentration of bioactive compounds from plant extracts and by-products of agro-food
industries (Cassano et al., 2014; Cissé et al., 2011; Diaz-Reinoso et al., 2009; Galanakis et al.,
2013; Giacobbo et al., 2013; Li and Chase, 2010; Mello et al., 2010; Murakami et al., 2013;
Tsibranska and Tylkowski, 2013; Tylkowski et al., 2010). According to the so-called "5-Stages
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Universal Recovery Processing" approach, the production of target compounds from food wastes
includes the following steps: (i) macroscopic pre-treatment, (ii) separation of macro- from micro-
molecules, (iii) extraction, (iv) purification and (v) product formation (Galanakis, 2012). UF and
NF are considered key physicochemical and non-destructive techniques applied in the second,
third and fourth step of the above downstream processing (Galanakis, 2015).
NF membranes have a nominal pore size in the range of 0.5-1 nm; the typical range of MWCO
levels is between 200 and 1,000 Dalton. UF involves the use of membranes with a MWCO in the
range of 1-300 kDa and a pore size of about 0.01 m (Baker, 2004). The separation capabilities
of UF and NF membranes are mainly related to size exclusion but interactions between solutes
and membrane like charge interactions, bridging and hydrophobic interactions may play an
important role in the formation of fouling layers at the membrane surface (or within the
membrane pores) which will exert some influence on passage of solutes through the membrane.
The formation of fouling layers due to macromolecules like proteins and dietary fibres has been
also reported in literature (Galanakis et al., 2014; Patsioura et al., 2011). Moreover, diafiltration
conditions can be employed in order to remove contaminants with low molecular weight (MW)
from valuable products with higher molecular weight in order to increase the product yield of the
process (Aspelund and Glatz, 2010; Teixeira et al., 2014).
No literature is readily available on the performance of UF and NF membranes for the separation
and purification of phenolic compounds from sugars in pomegranate juice. In the light of these
considerations, this work investigated the performance of flat-sheet UF and NF membranes, with
different membrane material and molecular weight cut-off (MWCO), for separating and
concentrating phenolic compounds from clarified pomegranate juice. The performance of the
selected membranes was compared in terms of permeate fluxes, retention towards sugars, total
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antioxidant activity and biologically active compounds (mainly total polyphenols and
anthocyanins). To fulfil the final aim to purify the selected bioactive compounds from sugars, the
membrane process was also studied in a diafiltration mode.
2.1. Pomegranate juice extraction and clarification
Pomegranates, of Calabria origin, were purchased from a local open market (Cosenza, Italy).
Fruits were washed in cold tap water and drained. They were manually cut-up into two halves
and then squeezed by using an electric juicer (Aristalco S.r.l., Treviso, Italy). The obtained juice,
having a deep-red color, was pre-filtered with a cotton fabric filter. The extracting procedure
gave an average juice yield of 40% (w/w).
The raw juice was clarified by using a laboratory unit supplied by Verind SpA (Milan, Italy)
equipped with a cellulose triacetate UF membrane module (FUC 1582, Microdyn Nadir,
Wiesbaden, Germany) in hollow fiber configuration with a nominal MWCO of 150 kDa ad a
membrane surface area of 0.26 m2. The juice filtration was conducted according to the batch
concentration mode (the retentate stream was flowed back to the feed tank while the permeate
stream was collected separately) up to a weight reduction factor (WRF) of 4.8. The WRF is
defined as the ratio between the initial feed weight and the weight of the resulting retentate
according to the following equation:
𝑊𝑅𝐹 = 𝑊 = 1 + 𝑊
where Wf, Wp and Wr are the weight of feed, permeate and retentate, respectively.
Material and methods
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The UF system was operated at a transmembrane pressure (TMP) of 0.6 bar, an axial feed
flowrate (Qf) of 400 L/h and a temperature (T) of 25±2 °C. The produced clarified juice was
stored at -18 °C and defrosted before use
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2.2. Treatment of clarified juices with UF and NF membranes
Membrane filtration experiments were performed by using a lab crossflow membrane filtration
unit (Sepa CF II, GE Water and Process Technologies, Canada, USA) equipped with a stainless
steel rectangular cross-flow cell. This cell is designed to simulate flow dynamics of commercially
available spiral membrane elements, by using a combination of stainless steel shims, feed spacers
and permeate carriers. The dimensions of the cell are 14.6 cm, 9.5 cm and 0.86 mm for channel
length, width, and height, respectively. These channel dimensions provide an effective membrane
area of 140 cm2 and a cross-sectional flow area of 0.82 cm2. The test cell of the unit is rated for
operating pressures up to 69 bar.
The assembled cell body was inserted into a cell holder and compressed against the cell holder
top through a piston. The feed stream (clarified pomegranate juice) was pumped from a stainless
steel vessel with a capacity of 2 L to the feed inlet located on the cell body bottom through a high
pressure pump (SSE1507 - Interpump 63SS Series Pump). The permeate flowing to the center of
the cell body top was collected in a manifold and through a permeate outlet connection into a
permeate vessel. Two manometers placed before and after the cell were used to measure the inlet
and the outlet pressure and, consequently, the applied TMP. The feed flow rate (Qf) and the TMP
values were regulated by a pressure control valve on the retentate side. The temperature of the
juice was controlled by using a cooling coil placed into the feed vessel fed with tap water. The
permeate flux was periodically monitored by acquisition of its weight, using an electronic
balance (with an accuracy of 0.1 g) placed under the permeate vessel.
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Four commercial flat-sheet membranes from different manufacturers were studied in this work,
namely Etna 01PP from Alfa-Laval (Lund, Sweden), PES 004H from Mycrodin-Nadir
(Wiesbaden, Germany), SelRO MPF-36 from Koch Membrane Systems (Wilmington, USA) and
Desal GK from GE Water & Process Technologies (Trevose, USA). Their typical characteristics
according to the manufacturers’ data sheet are reported in Table 1.
2.2.3. Batch concentration experiments
A first set of experiments was performed according to the batch concentration configuration in
which the permeate stream was collected separately while the retentate was recycled bach to the
feed reservoir up to a WRF of 1.5.
All experiments were performed at a TMP of 10 bar and an operating temperature of 25±1 °C.
The membrane performance was evaluated in terms of productivity (permeate flux), solute
rejection and fouling index.
The permeate flux (Jp) was determined by measuring the collected permeate weight in a given
time through the membrane surface area by using the following equation:
𝐽𝑝 = 𝐴 ∙ 𝑡
where Jp is the permeate flux (kg/m2h), Wp the permeate weight (kg) at time t (h) and A the
membrane surface area (m2).
The retention (R) of selected membranes towards specific compounds was determined as:
R = 1 ‒ C ∙ 100
where Cp and Cf are the concentration of a specific component in the permeate and feed,
The fouling index (FI) was calculated by comparing the pure water permeability before and after
the juice filtration according to the following equation:
where WP1 is the pure water permeability after the pomegranate juice filtration and WP0 the
water permeability of the virgin membrane.
The water permeability of each membrane was determined by the slope of the straight line
obtained by plotting the water flux values, measured in fixed conditions of temperature (25±1
°C), versus the applied TMP. After the treatment with the clarified juice, membranes were rinsed
with tap water for 30 min and their pure water permeability was measured; then, the fouled
membranes were submitted to a cleaning procedure by using a 0.125 M NaOH solution, at 40±1
°C for 60 min. At the end of the chemical cleaning procedure, the pure water permeability was
The cleaning efficiency (CE) was evaluated according to the following equation:
where WP3 is the water permeability after the chemical cleaning and WP0 is the water
permeability of the virgin membrane.
( ) ∙ 100
( ) ∙ 100
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2.2.4. Experiments with the membrane Desal GK
After the first set of experiments, according to the obtained results, the Desal GK membrane was
selected to perform experimental runs in total recycle configuration (both permeate and retentate
were continuously recycled to the feed reservoir) in order to study the effect of TMP on the
permeate flux and selectivity towards the compounds of interest. The TMP value was varied in
the range 5-25 bar maintaining the operating temperature at 25±1 °C.
In order to improve the removal of glucose and fructose from the UF retentate constant volume
diafiltration experiments were performed in a discontinuos way. In particular, the clarified juice
was firstly concentrated in batch concentration mode in selected operating conditions (TMP, 15
bar; T, 25±1 °C) until a minimum retentate hold-up of 500 mL was reached in the filtration unit,
corresponding to a WRF of 5. Then the retentate was diluted with the same amount of purified
water and the permeate was removed separately. The filtration and dilution procedure was
repeated three times, thus approaching diafiltration conditions. The last filtration run was
operated until the minimum retentate hold-up was reached.
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2.3. Analytical evaluations
Permeate and retentate samples coming from different experiments were immediately frozen and
kept at -18 °C until analysed. Samples were analysed for total phenols, total soluble solids (TSS),
anthocyanins, sugars and total antioxidant activity (TAA).
2.3.1. Total phenols
Total phenols were estimated colorimetrically by using the Folin-Ciocalteu method (Singleton et
al., 1999). The method is based on the reduction of tungstate and/or molybdate in the Folin-
Ciocalteu reagent by phenols in alkaline medium resulting in a blue colored product. A sample
aliquot (0.2 mL) was mixed with 1 mL of a 10 fold diluted Folin-Ciocalteu reagent and 0.8 mL of
7.5% sodium carbonate. Then the mixture was allowed to stand for 30 min at room
temperature. The absorbance was measured at 760 nm by using an UV-visible spectrophotometer
(Shimadzu UV-160A, Japan). Gallic acid solutions with concentrations ranging from 10 to 100
mg/L were used for calibration. A dose response linear regression was generated by using the
gallic acid standard absorbance and results were expressed as mg/L gallic acid equivalent (GAE)
All measurements were performed in triplicate in triplicate and the results were averaged.
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Anthocyanins were assessed by high-performance liquid chromatography (HPLC) using an
Agilent 1100 series system (Agilent Technologies, Waldbronn, Germany) equipped with a
vacuum degasser, a quaternary pump, an autosampler and an UV-Vis diode array detector.
Chromatographic separation was performed by using a Luna C18(2) column (250 mm x 4.60
mm, 5m, Phenomenex, Torrance, CA, USA) according to the following conditions: V = 1
mL/min; T = 25 °C; = 518 nm. The mobile phase was a mixture of H2O/HCOOH (9:1) as
solvent A and H2O/HCOOH/CH3CN (4/1/5) as solvent B. Anthocyanins separation was achieved
by using the following linear gradient: starting condition, 88% A, 12% B; 26 min, 70% A, 30%
B; 35 min, 100% B; 43 min, 88% A, 12% B; 46 min 88% A, 12% B. Anthocyanins were
identified by matching the retention time and their spectral characteristics against those of
standards (cyanidin 3,5-diglucoside, delphinidin 3-glucoside, cyanidin 3-glucoside, pelargonidin
3-glucoside). Quantification was made according to the linear calibration curves of standard
compounds (Mondello et al., 2000).
The quantitative determination of glucose and sucrose was carried out by an HPLC system
(Thermo Scientific Accella 600, USA) equipped with a binary pump, an autosampler, a
thermostated column compartment and refractometer index detector. Separation was achieved
with a Luna NH2 100A column (250 mm x 4.60 mm, 5 Phenomenex, Torrance, CA, USA).
Samples were eluted in isocratic mode by using a mixture of acetonitrile/water (80:20). Operating
conditions were as follows: V = 1 mL/min, T = 40 °C, pressure = 85 bar (Ruiz-Rodríguez et al.,
Prior to HPLC analysis all samples were diluted with acetonitrile (9:1) and filtered by using 0.45
m nylon filters. A sample volume of 20 L was used. The peak areas in the chromatograms
were plotted against calibration curves obtained from standard solutions (external standard
method), in a concentration range of 0.5-2 mg/mL for each compound.
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2.3.4. Total antioxidant activity (TAA)
ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical decolourisation assay in which the
radical cation is generated by reaction with potassium persulfate before the addition of the
antioxidant (Re et al., 1999). This method gives a measure of the antioxidant activity of pure
substances and of mixtures by monitoring the reduction of the radical cation as the percentage
inhibition of absorbance at 734 nm. Spectrophotometric measurements were performed by using
an UV–Visible recording spectrophotometer (UV-160 A, Shimadzu Scientific Instruments, Inc.,
Japan) at 30 °C. ABTS was dissolved in water at 2 mM concentration: ABTS radical cation was
produced by reacting 10 mL of ABTS stock solution with 100 L of 70 mM potassium persulfate
solution (ABTS:K2S2O8 = 1:0.35 molar ratio) and allowing the mixture to stand in the dark at
room temperature for 12–16 h before use. The work solution was prepared diluting 1 mL of the
ABTS radical cation solution to 25 mL with PBS buffer (5 mM Na2HPO4, 5 mM NaH2PO4, NaCl
9 g/L, pH= 6.8) to a final UV absorbance of 0.70 ± 0.02 at 734 nm. After addition of 10 L of
sample to 10 mL of ABTS work solution, the absorbance at 734 nm was recorded every min for a
total of 6 min. The value at 5 min was used to calculate the results reported as TAA, expressed in
terms of mM Trolox equivalent. Each determination was performed in triplicate. Results were
expressed as mean ± SD of three samples.
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2.3.5.Total soluble solids
Total soluble solids, expressed as °Brix, were measured by using a hand refractometer (Atago
Co., Tokyo, Japan) with a scale range of 0-32 °Brix.
3. Results and discussions
3.1. Pomegranate juice clarification and juice composition
Figure 1 shows the dependence of permeate flux and WRF on time observed in the clarification
of the raw juice in the selected operating conditions. The initial permeate flux of about 37 kg/m2h
decreased gradually with operating times due to concentration polarization and fouling
phenomena to reach a steady-state value of about 7 kg/m2h. As reported in Table 2, the UF
treatment produced a complete removal of suspended solids with the production of a clear juice
with a red brilliant colour.
The raw juice was characterized by a total soluble solids (TSS) content of 17.03±0.04 °Brix
which is in agreement with data reported by other Authors (Dafny-Yalin et al., 2010; Ferrara et
al., 2011). This value appeared to be higher in comparison to the clarified juice: this phenomenon
can be attributed to the presence of suspended solids in the raw juice that can interfere with the
measurement of the refractive index.
As previously reported, polyphenols from pomegranate juice have taken great attention in the last
years for their health-promoting properties (Viuda-Martos et al., 2010; Sreeja et al., 2014). The
content of polyphenols in the clarified juice (2457.50±15.30 mg/L) was well preserved in
comparison to the raw juice and resulted higher than that reported for typical cultivars grown in
southern Turkey, Greece and Chile (Ferrara et al., 2011). The predominant polyphenols detected
in both raw and clarified juices were anthocyanins; in the clarified juice cyanidin 3,5-O-
diglucoside resulted the most representative compound (136.10±5.30 mg/L), followed by
cyanidin 3-O-glucoside (53.71±2.06 mg/L), delphinidin-3-O-glucoside (14.61±0.48 mg/L) and
pelargolidin 3,5-O-diglucoside (4.77±0.43 mg/L).
The amount of phenolic compounds is directly correlated with the antioxidant activity of the
clarified juice (Mousavinejad et al., 2009) which, according to the ABTS method, was of
26.0±2.8 mM Trolox.
According to Tezcan et al. (2009) glucose and fructose were the only sugar types detected in the
juice. The fructose content (19.45±0.62 g/L) resulted higher in comparison with the glucose one
(12.53±0.48 g/L). The absence of sucrose in the fruit can be explained by the enzymatic
hydrolysis of this sugar into glucose and fructose during the ripening process (Zarei et al., 2011).
In the clarified juice the content of glucose and fructose resulted 2.8% and 8.3% lower than that
measured in the raw juice, respectively.
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3.2. Performance of selected membranes
The pure water permeability (WP0) values measured for each membrane are summarized in Table
3. According to the results the WP values decreased in the following order: 15
PES 004H > Etna 01PP> MPF-36 > Desal GK.
These values are not correlated with the MWCO of the selected membranes: indeed, the PES004
H membrane, with the highest MWCO (4,000 Da) showed the highest permeability value
(WP0=47.81 kg/m2hbar); Etna 01PP and MPF-36 membranes with the same MWCO (1,000 Da),
but different membrane material (thin-film composite and fluoro polymer, respectively) showed
WP0 values of 22.16 kg/m2hbar and 14.54 kg/m2hbar, respectively. The lowest WP0 value (10.5
kg/m2hbar) was found for the Desal GK membrane with a MWCO of 2,000 Da. These results can
be attributed to the internal structure of the selected membranes which is strongly related to their
composition, morphology and hydrophilicity/hydrophobicity (Benítez et al., 2009).
A different behavior was observed in the treatment of the clarified pomegranate juice. In the
selected operating conditions the permeate flux decreased gradually up to reach a steady-state
value. The initial decrease of permeate flux is generally explained by the effect of concentration
polarization phenomena whereas the second phase of decrease is due to the accumulation of
molecules and particles on the membrane surface or inside the pores of the membrane as far as
the concentration of the feed solution increases (Conidi et al., 2015).
The Desal GK membrane presented the highest initial and steady-state permeate flux values (22.6
kg/m2h and 11.3 kg/m2h, respectively) when compared with the other tested membranes (Figure
2). On the other hand, the PES 004H membrane presented the lowest permeate flux values (2.4
kg/m2h and 0.7 kg/m2h, respectively). These results confirm that the permeate flux is affected not
only by TMP but also by membrane material and structure as well as by interactions between
solute and membranes.
The obtained results in term of permeate fluxes, are in agreement with experimental data reported
by other Authors. Cissé et al. (2007) tested different UF and NF membranes to separate and
concentrate anthocyanins compounds from Hibiscus sabdariffa L. extracts. They found that
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Desal thin film composite membranes, with a MWCO in the range 500-1,000 Da, presented
higher permeate fluxes when compared with polyethersulphone membranes with higher MWCO
(in the range 5-50 kDa) working in the same operating conditions (operating pressure 20 bar).
Similary, the Desal GK membrane exhibited permeate fluxes values of about 40 kg/m2h at 30 bar
in the concentration of ellagitannins from blackberry juice which were higher than those
measured with a polyethersulphone membrane of 5000 Da (UP005, Microdyn Nadir) at the same
operating pressure (Acosta et al., 2014).
The tested membranes were also compared in terms of fouling index (FI) and cleaning efficiency
(CE) (Table 3). The lowest FI value was measured for the Desal GK membrane (51%), followed
by MPF-36 (77%) and PES 004H (88%) membranes; on the other hand the highest FI value was
detected for the Etna 01PP membrane (95%). The CE resulted higher than 90% for all
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3.3. Retention of UF and NF membranes towards analyzed compounds
The analyses of anthocyanin compounds in samples coming from batch concentration
experiments revealed that the rejection of the selected membranes towards these compounds were
higher than 80%, with the exception of the ETNA 01PP membrane (Figure 3a). The MPF-36
membrane exhibited the highest rejection towards the more representative anthocyanins of the
juice; the observed retention for the PES 004H membrane was in the range 60-99%, while the
Etna 01PP membrane showed a lower retention (in the range 60-80%). These results were
confirmed by analyses of phenolic compounds and TAA. In particular, the rejection of all
selected membranes towards phenolic compounds was
membrane exhibiting the highest retention values (Figure 3b). As expected, with the exception of
the Etna 01PP membrane, a strict correlation was observed between the rejection towards
higher than 80% with the MPF-36
phenolic compounds and TAA, since phenolic compounds mainly contribute to the TAA of the
juice (Tezcan et al., 2009). In particular, the observed retention of Desal GK membrane towards
TAA and phenolic compounds was of 78% and 88%, respectively; similar results were obtained
with the PES 004H membrane (retention values were of 86% and 95%), while the retention of the
MPF-36 membrane was higher than 95% for both components. The lowest retention value
towards TAA was measured with the Etna 01PP membrane (retention of about 57%); adversely,
the retention towards polyphenols was of about 85%. As reported by Galanakis et al. (2015) the
high concentration of phenols in samples obtained from the filtration of Cypriot wine with the
Etna 01PP membrane did not reflect higher antioxidant capacity, probably due to the observed
antagonistic effect between different phenolic compounds and anthocyanins.
All selected membranes exhibited a low rejection towards sugar compounds. In particular, with
the exception of the MPF-36 membrane, the rejection towards soluble solids was of the order of
30%. For glucose and fructose the observed rejections were lower than 10%, with the Desal GK
exhibiting the lowest retention towards both compounds (in the range 1-4%) (Figure 3c).
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3.4. Experiments with Desal GK membrane
On the basis of the preliminary results related to membrane productivity, membrane fouling and
membrane selectivity the Desal GK membrane was selected to perform other experiments both in
total recycle configuration (in order to evaluate the effect of TMP on the rejection of compounds
of interest and productivity) and in batch concentration configuration at higher WRF, in order to
increase the concentration of phenolic compounds in the retentate stream. Diafiltration was also
used to improve the removal of sugar compounds from phenolics.
Figure 4 shows the effect of TMP on the steady-state permeate flux and retention towards TSS,
TAA and total polyphenols for the Desal GK membrane. A linear increase of the permeate flux,
from 7 to 40 kg/m2h, was observed by increasing the operating pressure in the range of
investigated values (5-25 bar): the absence of a limiting flux can be attributed to the preliminary
treatment of the raw juice by UF which removed suspended solids that, as well known are the
main responsible compounds in the formation of fouling layers. On the other hand, the retention
of phenolic compounds was not affected by TMP: indeed, the retention of phenolic compounds
was higher than 90%, independently by the applied pressure. Accordingly, the TAA retention
showed a similar trend. Adversely, an increasing of TMP induced higher retention of TSS with a
significant retention (higher than 40%) at 25 bar, suggesting that this pressure should be avoided
in order to reach high separation factors between sugars and phenolic compounds. The increased
retention levels can be explained not only by steric considerations but also through the
interactions between solutes and membrane material and the association of the solutes with
Data of steady-state permeate fluxes at different TMP were in agreement with those related to the
fouling index. In particular, in the range of investigated pressures, a little increase of the fouling
index (from 30 to 35%) was observed when TMP was increased at 25 bar. Data of water
permeability before and after cleaning procedures, confirmed a greater contribution of the fouling
resistance to the total resistance if compared to the cake layer resistance (Table 4).
The effect of TMP on the anthocyanins rejection for the Desal GK membrane is illustrated in
Figure 5. For all the investigated compounds the retention was higher than 90% independently by
the operating pressure. In addition, a strict correlation between the retention value and the
molecular weight of each compound was observed, with cyanidin-3,5-O-diglucoside, exhibiting
the highest rejection.
Experimental runs in selected operating conditions (15 bar and 26 °C) were performed according
to the batch concentration configuration up to WRF 5. In these conditions the initial permeate
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flux of 30 kg/m2h was reduced up to 10 kg/m2h when the final WRF was reached. The analyses
of permeate and retentate streams revealed that, according to the high retention of the Desal GK
membrane towards phenolic compounds, phenolic compounds and anthocyanins were
concentrated in the retentate stream. In particular, the content of single anthocyanins, total
phenols and TAA in the retentate stream was in agreement with the reached WRF. On the other
hand, the content of soluble solids, glucose and fructose in the permeate stream was of the same
order of the clarified juice due to the low rejection of the membrane towards these compounds
In Table 6 the mass balance of the process at WRF 5 for total anthocyanins, polyphenols and
sugars, is reported. This balance is referred to an experimental run in which starting from 3.3 kg
of clarified pomegranate juice, 2.64 kg of the permeate and 0.65 kg of retentate, were obtained
(final WRF 5, recovery factor 80.3%). The recovery factor of glucose and fructose in the
permeate stream was of the same order of the recovery factor of the process (about 80%); 86% of
phenolic compounds and more than 89% of anthocyanins were recovered in the retentate stream.
In order to improve the removal of glucose and fructose from the retentate a diafiltration step was
applied. The process was operated until to WRF 5; then the retentate was filled up with purified
water to its original volume. The filtration and dilution procedure was repeated thus approaching
diafiltration conditions. The last filtration run was operated until the minimum retentate hold-up
was reached. The redilution of the retentate had a positive impact on the transmembrane flux. As
soon as the retentate was filled up with water a significant increase in the flux, up to 20 kg/m2h
was observed (Figure 6). The addition of water reduces the osmotic pressure of the retentate and
a higher driving force filtration is obtained (Schütte et al., 2015).
The composition of the collected diafiltrate in terms of glucose and fructose revealed a similar
content of the initial retentate solution indicating that these compounds are still removed in the
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permeate side. On the other hand, the observed rejection for all investigated anthocyanin
compounds was higher than 97%. High retentions of phenolic compounds and TAA were also
detected (Table 7).
Consequently, the efficiency of the Desal GK membrane in the purification of bioactive
compounds from clarified pomegranate juice is enhanced by combining the concentration step
with diafiltration due to the different selectivity of the membrane towards these compounds: the
water addition increases the quantity of sugars in the diafiltrate increasing at the same time the
purification factor of biologically active compounds in the retentate. The water addition may also
reduce the amount of membrane fouling and improve the solute separation by reducing the
solute-solute interactions in the retentate (Almanasrah et al., 2015; Nguyen et al., 2016).
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3.5. Mass balance of the process
The growing interest of health promoting activities exerted by pomegranate juice has addressed
the researchers towards the optimization of new extraction and separation techniques in order to
produce extracts enriched in biologically active compounds for their use in other markets such as
those of functional ingredients, nutraceuticals, cosmeceuticals and food colourings. On the basis
of the obtained results, a mass balance of the membrane fractionation process was carried out in
order to quantify the amount of biologically active compounds and sugars recovered in the
different permeate and retentate fractions. The mass balance, illustrated in Figure 7, was
estimated for an initial volume of clarified juice of 1000 L. According to the final volume
reduction factor of the process, about 200 L of concentrated solution are obtained. In these
conditions the yields of polyphenols and anthocyanins in the retentate stream are of the order of
84.8% and 90.7%, respectively. By applying the diafiltration step the efficiency of glucose and
fructose recovery can be increased up to 90% and 93%, respectively.
The final retentate of the process exhibits very high antioxidant activity: it can be reused for the
formulation of nutraceutical products or as a natural colorant in alternative to the use of synthetic
ones; the residual permeate and diafiltrate streams, with high content of sugars, can be reused as
food additives or as bases for soft drinks.
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The potential of commercial flat-sheet membranes in the separation and purification of bioactive
compounds from sugars in clarified pomegranate juice was evaluated. Preliminary experiments
were carried out to assess the performance of the membranes in terms of productivity, fouling
index, cleaning efficiency and rejection coefficients. All the tested membranes showed high
retention towards biologically active compounds and low retention towards sugars. Among the
investigated membranes the Desal GK exhibited higher productivity, lower fouling index and
good cleaning efficiency. Permeate fluxes and rejection coefficients of phenolic compounds were
not affected by the operating pressure in the range of investigated values. The phenolic content,
the content of single anthocyanins and the antioxidant activity of the retentate stream was in
agreement with the reached WRF when the clarified juice is treated according to the batch
concentration configuration, confirming the suitability of the selected membrane for
concentration purposes. At the same time the low retention of the Desal GK membrane towards
fructose and glucose allows the recovery of these compounds in the permeate stream. The sugar
yield can be improved significantly if diafiltration is applied.
The retentate stream, enriched in phenolic compounds, exhibits very high antioxidant activity
suggesting its reuse for the formulation of nutraceutical products. It can be also reused as natural
colorant as alternative to the use of synthetic ones due to the presence of anthocyanins.
Permeate and diafiltrate fractions enriched in sugar compounds can be reused as food additives or
as bases for soft drinks.
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ACCEPTED MANUSCRIPT Figure captions
Figure 1 - Clarification of pomegranate juice by UF. Time course of permeate flux and WRF.
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(Operating conditions: TMP = 0.6 bar; Qf = 400 L/h; T = 25±1 °C).
Figure 2 - Time course of permeate flux for clarified pomegranate juice processed with: (a) Etna 01PP, MPF-36 and PES 004H membranes and (b) Desal GK membrane (operating conditions: TMP, 10 bar; T, 25±1 °C; WRF, 1.5).
Figure 3 - Retention of selected membranes towards: (a) anthocyanins; (b) phenolic
compounds and TAA; (c) sugars.
Figure 4 - Ultrafiltration of clarified pomegranate juice with Desal GK membrane. Effect of TMP on steady-state permeate flux and retention towards polyphenols, total antioxidant
activity (TAA) and total soluble solids (TSS).
Figure 5 - Ultrafiltration of clarified pomegranate juice with Desal GK membrane. Effect of
TMP on anthocyanins retention.
Figure 6 - Ultrafiltration of pomegranate juice. Time course of permeate flux and WRF
(Operating mode: batch concentration/diafiltration; operating conditions: TMP, 15 bar; T, 25±1 °C)
Figure 7 – Mass balance of the fractionation process of the pomegranate clarified juice with Desal GK membrane (GLU, glucose; FRU, fructose; POL, polyphenols; ANT, anthocyanins).
Operating time (min)
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Jp (kg/m h)
8 ETNA 01PP MPF 36 PES 004H
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Jp (kg/m h)
(a) 0 0
Operating time (min)
Jp (kg/m h)
Operating time (min)
100 Cyanidin 3,5 diglucoside Cyanin 3, glucoside Pelargolidin 3-glucoside Delphinidin-3-glucoside
(a) 0 PES 004
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Total polyphenols TAA
TSS glucose fructose
0 PEES 004
Polyphenols TAA TSS Jp
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cyanidin 3,5-diglucoside cyanidin 3-glucoside pelargolidin 3-glucoside delphinidin-3-glucoside
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Operating time (min)
Vfeed = 1000 L GLU = 11.09 kg FRU = 19.18 kg POL = 2.704 kg ANT = 0.183 kg
UF, 150 kDa
UF, 2kDa VRF = 5
Vret = 200 L
GLU = 1.855 kg FRU = 3.560 kg POL = 2.295 kg ANT = 0.166 kg
Vperm = 300 L
GLU = 8.815 kg FRU = 15.86 kg POL = 0.301 kg ANT = 0.018 kg
GLU = 1.117 kg FRU = 2.14 kg POL = 0.071 kg ANT = 0.004 kg
UF diafiltrate Food additives
Water (300 L)
Vperm = 800 L
V = 200 L
GLU = 1.15 kg FRU = 1.17 kg POL = 2.22 kg ANT = 0.16 kg
Table 1- Characteristics of selected flat-sheet membranes Etna 01PP
pH operating range
Max. operating temperature (°C)
Max. operating pressure (bar)
pH range in cleaning conditions
Composite fluoro polymer
ACCEPTED MANUSCRIPT Table 2 - Chemical composition of raw and clarified pomegranate juice Raw juice Clarified juice
Suspended solids (%w/w)
Total soluble solids (TSS) (°Brix)
Fructose (g/L) Cyanidin 3,5-O-diglucoside (mg/L) Cyanidin 3-O-glucoside (mg/L) Delphinidin 3-O-glucoside (mg/L) Pelargolidin 3,5-O-diglucoside (mg/L) TAA (mM Trolox)
Total polyphenols (mg GAE/L)
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ACCEPTED MANUSCRIPT Table 3 – Water permeabilities, fouling index and productivity of selected membranes in the treatment of clarified pomegranate juice
Membrane type PES 004H
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Legend: WP0 =water permeability before the treatment with pomegranate juice; WP1 =water permeability after the treatment with pomegranate juice; WP3 =water permeability after chemical cleaning; FI = fouling index; CE = cleaning efficiency; Jo = initial permeate flux; Jstaz = steady-state permeate flux
ACCEPTED MANUSCRIPT Table 4 – Water permeabilities and fouling index of Desal GK membrane for experimental runs performed at different TMP values WP0
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Legend: WP0 =water permeability before the treatment with pomegranate juice; WP1 =water permeability after the treatment with pomegranate juice; WP2 =water permeability after cleaning with water; WP3 =water permeability after chemical cleaning; FI = fouling index
ACCEPTED MANUSCRIPT Table 5 - Analyses of TSS, sugars, total polyphenols, anthocyanins and TAA in juice samples from UF with Desal GK membrane (WRF 5) Feed
Total polyphenols (mg/L)
TAA (mM Trolox)
Cyanidin 3,5-O-diglucoside (mg/L)
Cyanidin 3-O-glucoside (mg/L) Delphinidin 3-O-glucoside (mg/L)
Pelargolidin 3,5-O-diglucoside (mg/L)
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ACCEPTED MANUSCRIPT Table 6 - Mass balance of the UF process with Desal GK membrane (WRF 5) Feed
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ACCEPTED MANUSCRIPT Table 7 - Analyses of TSS, sugars, total polyphenols, anthocyanins and TAA in samples of pomegranate juice coming from the diafiltration process Rejection
Total polyphenols (mg/L)
TAA (mM Trolox)
Cyanidin 3,5-O-diglucoside (mg/L)
Cyanidin 3-O-glucoside (mg/L)
Delphinidin 3-O-glucoside (mg/L)
Pelargolidin 3,5-O-diglucoside (mg/L)
MA ED PT CE AC
NU SC RI PT