Separation and Purification Technology 168 (2016) 188198

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Purification of polyphenols from green tea leaves by ultrasound assisted

ultrafiltration process
Larissa dos Santos Sousa a, Bruna Vieira Cabral a, Grasiele Scaramal Madrona b, Vicelma Luiz Cardoso a,
Miria Hespanhol Miranda Reis a,
a
Faculdade de Engenharia Qumica, Universidade Federal de Uberlndia, Av. Joo Naves de vila, 2121, 38400-902 Uberlndia, Minas Gerais, Brazil
b
Departamento de Engenharia de Alimentos, Universidade Estadual de Maring, Av. Colombo, 5790, 87020-900 Maring, Paran, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Application of ultrafiltration membranes is a promising alternative to recovery target components from
Received 5 April 2016 natural substrates, but some drawbacks should be considered such as the membrane fouling. Here we
Received in revised form 23 May 2016 propose the application of the ultrasound assisted ultrafiltration process for the purification of phenolic
Accepted 26 May 2016
compounds from green tea extract. Comparison between the processes with and without ultrasound
Available online 27 May 2016
showed that the state steady flux through the membrane of 5 kDa was 4 times greater in the ultrasound
assisted process than in the process without ultrasound. This flux improvement is associated with the
Keywords:
decrease in cake formation and in the total resistance (from 17.8 to 10.2
1013 m1). The flux decay pro-
Green tea leaves
Phenolic compounds
files were better described by pore blockage models than by the cake formation model, since the cake was
Membrane filtration process formed in the first minutes of filtration and, after that, the particles of relative small size entered in the
Ultrasound assisted process membrane pores. Moreover, the ultrasound facilitated the permeation of phenolic compounds through
Fouling the membrane. Comparison between ultrafiltration membranes of different MWCO suggests that the
Stability membrane of 20 kDa presented high purity of catechin components in the permeate (49% of EGCG related
to the total polyphenol content). Turbidity values of ultrafiltration permeates remained lower than 5 NTU
after 30 days of storage under refrigeration and any tea cream formation was observed, which suggests
great permeate stability. Thus, the ultrasound assisted ultrafiltration process with the membrane of
20 kDa is suggested for the purification of phenolic compounds from green tea extract in order to ensure
great permeate flux, purity of phenolic compounds and extract stability.
2016 Elsevier B.V. All rights reserved.

1. Introduction 3. Extraction, 4. Isolation and purification and 5. Product formation

Tea is the most consumed beverage worldwide mainly due to
its enjoyable flavour and medicinal properties. Pharmacological
properties of green tea leaves (Camellia sinensis) are highlighted
due to the presence of large amount of alkaloids (caffeine) and cat-
echins [1,2]. The main catechins of green tea leaves are: ()-
epicatechin (EC), ()-epigallocatechin (EGC), ()-epicatechin gal-
late (ECG) and ()-epigallocatechine gallate (EGCG), and, cumula-
tively, all catechins are related to the tea total polyphenol
content [3]. Extraction and purification of these target components
are of great interest to the pharmaceutical industry and the scien-
tific community.
Five stages can be identified for extraction, fractionation and
isolation of high added-value compounds from foods: 1. Macro-
scopic pre-treatment, 2. Macro- and micro-molecules separation,
Corresponding author.
E-mail address: miria@feq.ufu.br (M.H.M. Reis).
[4]. In the extraction step, studies are mainly focused on the appli-
cation of different solvents and process conditions [5]. Rosello-Soto
et al. [6,7] highlighted the application of ultrasound and electrical
devices for greater extractions of phenolic compounds from food.
Isolation and purification of catechins can be achieved in chro-
matographic columns at small scale and with high cost [8]. Mem-
brane separation processes have been used to separate micro and
macromolecules from several substrates [9], also including con-
ventional beverages such as juice, beer and wine [1014]. Torun
et al. [15] obtained great polyphenol recovery from sage extract
using a sequential process of osmotic distillation and reverse
osmosis. Zagklis and Paraskeva [12] proposed the purification of
phenolic compounds from grape marc by a sequential membrane
filtration process (ultrafiltration and nanofiltration) for a better
isolation of these components. Similarly, Diaz-Reinoso et al. [16]
proposed the concentration of phenolic compounds from Castanea
sativa leaves in a series of two ultrafiltration membranes (5 and
10 kDa) in order to minimize the fouling occurrences. In these

http://dx.doi.org/10.1016/j.seppur.2016.05.029
1383-5866/ 2016 Elsevier B.V. All rights reserved.
 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198 189

cases, membranes of lower molecular weight cut-off (MWCO) can
retain polyphenols with high molecular weight, while membranes
of greater MWCO can concentrate simple polyphenols. Few studies
are reported in the literature about the application of membrane
processes for green tea processing. Kumar et al. [17] showed that
an ultrafiltration membrane of 30 kDa was able to produce a puri-
fied permeate with about 80% of EGCG related to the total polyphe-
nol content. The separation of EGCG from other polyphenols in
crude tea was also obtained by Zhu and Qin [18] with a polymeric
asymmetric membrane. Manna et al. [19] successfully recovered
catechins from green tea leaves extract using a hollow fibre sup-
ported liquid membrane module.
Other studies are focused on the stabilization and the clarifica-
tion of tea extract due to tea cream formation. Tea cream is the
precipitation of solids from the tea solution that are formed upon
cooling as a result of polyphenolprotein interaction, and this phe-
nomenon is a great concerning in the storage of ready-to-drink
teas. According to Todisco et al. [20], an ultrafiltration membrane
of 40 kDa is able to maintain the polyphenol content in final pro-
duct for up to 2 months, besides the haze reduction in black tea.
Chandini et al. [21] showed that microfiltration membranes were
able to stabilize black tea extracts with low feed concentrations.
Argyle and Bird [22] also suggested the application of microfiltra-
tion membranes for haze removal from black tea. Evans and Bird
[23] concluded that the ultrafiltration process produces a clarified
black ready to drink tea beverage with an increased stability and a
significantly reduced haze. On the other hand, Rao et al. [24] con-
cluded that the ultrafiltration process was not able to clarify green
tea infusion, since the infusion became cloudy when stored in the
refrigerator for just one week.
Thus, a proper application of the membrane filtration process
for green tea extract treatment should evaluate the performance
of micro and ultrafiltration membranes for the recovery and purifi-
cation of target components and the stabilization of the produced
permeate. Moreover, fouling occurrences and low permeate fluxes
are major concerns in membrane filtration processes. The better
understanding of fouling occurrences during the filtration is
important to propose strategies to maximize the permeate flux
and the membrane reuse [25]. Application of ultrasound during
the filtration is a promising alternate to reduce fouling occurrences
[26,27] and, although being successfully applied in the filtration of
several fluids, the ultrasound effect was not yet reported for filtra-
tions of green tea extract.
The main objective of this study was to evaluate the application
of the ultrasound assisted ultrafiltration process for the recovery
and purification of polyphenols from green tea extract. The influ-
ence of time and temperature on the polyphenol extraction from
green tea leaves was evaluated. Microfiltration membranes of dif-
ferent pore sizes were used for the clarification of green tea extract.
Ultrafiltration processes were carried out with and without ultra-
sound and with membranes of different MWCO. Stabilities of the
permeate from micro and ultrafiltration processes were verified.
Fouling occurrences were investigated according to flux decay
and the resistance-in-series models. This comprehensive analysis
of membrane filtration process focused on alternative processes,
membranes of different pore sizes and MWCOs and flux modelling
will enable the proposal a suitable process for the obtainment of
phenolic compounds from green tea extract.
for gallic acid, caffeine, ()Epigallocatechin gallate (EGCG), Cate-
chin (C), ()Epicatechin (EC) and ()Epigallocatechin (EGC)
were purchased from Sigma-Aldrich. All other reagents were of
analytical grade.
2.2. Aqueous extraction
Although other organic solvents could result in a better
polyphenol extraction [5], we have decided to perform the aqueous
extraction in order to guarantee a better food product quality, as
also proposed by Diaz-Reinoso et al. [16].
Kumar et al. [17] suggested ratios of leaves to water from 1:10
to 1:25. Green tea extract was prepared by mixing green tea leaves
into distilled water at a mass ratio of 1:10. This mixture was kept
under mechanical agitation at 400 rpm for 120 min at different
temperatures (from 30 to 80 C). The beaker containing the green
tea leaves with water was inserted in a water bath for temperature
control. For each evaluated temperature, samples were collected at
every 10 min in order to evaluate the time influence on the extrac-
tion of polyphenolic compounds. After the infusion process, leaves
were separated from the extract by filtration through a filter paper.
2.3. Membrane filtrations
Flat membranes of different materials and pore sizes or MWCO
were applied for micro and ultrafiltrations of green tea extract. The
membrane characteristics are presented in Table 1. Water fluxes
were measured at several pressures in order to determine the
water permeability of each membrane.
Dead-end microfiltration processes were carried out using a cell
of 6.4
103 m2 of filtration area, which was coupled to a nitrogen
cylinder for pressurization, as presented in Fig. 1a. Membranes of
0.22, 0.3 and 0.8 lm were used for microfiltrations of green tea
extract at transmembrane pressure of 0.7 bar for 60 min.
The microfiltered extract was fed to the ultrafiltration system.
Cross-flow ultrafiltrations were carried out in a Convergence
Inspector Minos System with a membrane module of
3.8
103 m2 and using membranes of 30, 20, 10 and 5 kDa at
6 bar for 180 min with and without ultrasound, as presented in
Fig. 1b. Filtration times were chosen to guarantee flux stabilization.
For ultrafiltrations with ultrasound, the filtration module was
inserted in an ultrasound bath of 40 Hz (Ultracleaner model
1650A). The temperature increase during the 180 min of ultrafil-
tration was controlled by re-circulating water from a water bath
with temperature control in the ultrasound equipment.
All filtrations were carried out at room temperature (approxi-
mately 25 C). The initial feed volume was of 300 mL.
2.4. Physico-chemical analyses
Permeate samples were analyzed for total phenolic and protein
contents, turbidity, total solids, colour intensity and concentrations
of gallic acid, caffeine, ()Epigallocatechin gallate (EGCG), Cate-
Table 1
Membrane characteristics.
Pore size or Material Manufacturer Water permeability
MWCO (105 kg h1 m2 Pa1)

2. Material and methods 0.8 lm Cellulose acetate MilliporeTM 810.70

0.3 lm Cellulose acetate MilliporeTM 376.81
0.22 lm Cellulose acetate MilliporeTM 28.88
2.1. Material 30 kDa Polyethersulfone Nadir 42.99
20 kDa Polyethersulfone Nadir 28.66
Dried ground green tea leaves (C. sinensis) were bought from a 10 kDa Polyethersulfone Nadir 23.97
5 kDa Polyethersulfone Nadir 16.18
local market (Minas Gerais, Brazil) in June 2015. The standards
190 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198
Fig. 1. Membrane systems for micro (a) and ultrafiltrations (b).
chin (C), ()Epicatechin (EC) and ()Epigallocatechin (EGC). All
analyses were carried out in triplicate. The Tukey HDS test was
applied to identify significant differences in mean values regarding
to the membrane pore size or MWCO and to the process (with or
without ultrasound) with a confidence level of 5%.
Total phenolic content was determined by the Folin-Ciocalteu
method using gallic acid as standard and with absorbance measure-
ments at 760 nm (UVmini 1240 Shimadzu spectrophotometer)
[17]. Individual catechins (EGCG, C, EC and EGC) and gallic acid
and caffeine were determined by liquid chromatography according
to the methodology suggested by Wang et al. [28] in a Shimadzu
HPLC (model LC-20A Prominence) equipped with a Discovery HS
C18 column. The purity of each evaluated catechin was calculated
related to the total phenolic concentration, as suggested by Kumar
et al. [17].
Total solid concentrations were measured by weighting a sam-
ple of 5.0 mL before and after drying at 103 C for 24 h. Turbidity
was measured with a Nova Organica HD 114 turbidimeter. Colour
was determined as a measure of absorbance at 400 nm in a Shi-
madzu UV mini 1240 spectrometer. Todisco et al. [20] and Evans
et al. [29] suggested the colour analysis in the visible spectrum
range between 380 and 770 nm. The wavelength of 400 nm was
chosen to obtain results of absorbance between 0.5 and 0.8. Total
protein content was determined by the Lowry method.
2.5. Flux modelling
The flux decay in micro and ultrafiltrations were analyzed
according to the differential equations proposed by Hermia [30]
and Field et al. [31], respectively. The Hermia model, which was
written in terms of flux for constant pressure filtrations (Eq. (1)),
was applied to describe the flux decay in the dead-end microfiltra-
tions of green tea extract.
dJ 3n
 kJ
dt
where J is the mass flux (kg h1 m2), t is the filtration time (min), k
is the Eq. constant and n is the number indicating fouling
mechanism.
Field et al. [31] included the concept of critical flux in the Her-
mia model in order to describe cross-flow filtrations, as presented
in Eq. (2). Eq. (2) was used to model the flux decay in the cross-flow
ultrafiltrations of green tea extract.
dJ
 kJ  J  J 2n
dt
where J is the critical flux defined as the constant flux with time
[31].
The superscript n in Eqs. (1) and (2) was fixed at 0, 1, 1.5 or 2
to represent each fouling mechanism, as described in Table 2.
For each n value, Eqs. (1) and (2) were numerically solved
using the LevenbergMarquardt method with an integration step
of 103 and precision of 108. The LevenbergMarquardt method
was programmed in Fortran language, combining the Gauss and
the Steepest Descent methods. Calculated flux profiles were com-
pared with the experimental flux data.
2.6. Membrane resistances
Membrane resistances were determined according to the
resistance-in-series model, in which the total resistance (RT) was
understood as the sum of the membrane hydraulic resistance
(RM) and the resistances due to pore blockage and cake formation
(RP and RC, respectively).
The membrane hydraulic resistance was determined after filtra-
tion of distilled water through a clean membrane. The sum of the
resistances due to pore blockage and cake formation was deter-
mined by filtering distilled water through the membrane that
was previously used for green tea extract filtration. Sequentially,
the membrane surface was physically cleaned with a sponge and
the water flux was measured in order to determine the resistance
due to pore blockage.
2.7. Stability tests
Green tea extract and permeate samples were stored at 5 C for
30 days in order to evaluate their stabilities according to the fol-
lowing parameters: turbidity, colour intensity, total phenolic con-
Table 2
1 Characteristics of each fouling mechanism.
n Fouling Phenomenological background Representation
mechanism
2 Complete Particle sizes are greater than the
pore membrane pore size and totally block
blocking the membrane pore
1.5 Internal Particle sizes are smaller than the
pore membrane pore size and are retained
blocking in the internal surface of the
membrane pore
2 1 Partial pore Particles connect each other to bridge
blocking a pore or to stick it
0 Cake Particles form a cluster on the
filtration membrane surface
 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198
tent and tea cream formation. These parameters were evaluated at
every 7 days.
Tea cream formation was measured according to the procedure
suggested by Chandini et al. [21] as the amount of insoluble parti-
cles that are formed when the sample is kept under refrigeration.
After at least 16 h of refrigeration, the sample was centrifuged
for 20 min at 5600 rpm in order to remove the insoluble particles.
Tea cream (%) was calculated as the amount of total solids in the
supernatant related to the amount of solids in the initial sample
[21].
3. Results and discussion
3.1. Polyphenol extraction from green tea leaves
The profiles of total polyphenol concentration according to the
extraction time at several temperatures are presented in Fig. 2.
According to Fig. 2, polyphenol extraction increases with the
increase in the process time up to approximately 60 min. The time
influence is more pronounced at low (45 C) and high (75 C) tem-
peratures. Todisco et al. [20] analyzed the time influence of
polyphenol extraction from black tea and observed that the
polyphenol concentration remained constant for extraction times
greater than approximately 80 min. Vuong et al. [32] analyzed
the time influence on the extraction of individual catechins from
green tea and observed the catechin concentration reached a pla-
teau when the extraction time was increased from 30 min.
The increase in the process temperature from 45 to 75 C
increased the polyphenol extraction. Vuong et al. [32] analyzed
the temperature influence on the water extraction of individual
catechins from green tea leaves. According to the results of Vuong
et al. [32], low concentrations of EGCG (the major catechin compo-
nent in green tea) were obtained at temperatures bellow 50 C
(lower than 20% of the maximum concentration of this compo-
nent). In our study, total polyphenol concentrations were not
detected at temperatures below 45 C probably because the
applied methodology analysis for total polyphenols is less sensitive
than the HPLC analysis for individual cathechins.
However, the polyphenol extraction decreased when the tem-
perature was increased from 75 to 80 C probably due to the degra-
dation of polyphenol compounds at temperatures higher than
75 C [33]. Vuong et al. [32] also indicated that excessive extraction
temperatures above 80 C could lead to an increased epimerization
Fig. 2. Influence of time and temperature on polyphenol extraction from green tea
leaves.
191
of the epistructured catechins to non-epistructured catechins.
Although Vuong et al. [32] and Perva-Uzunalic et al. [33] have sug-
gested that the polyphenol degradation starts at 80 C, we have
observed this degradation for temperatures higher than 75 C so
that this limit of 5 C should be considered for security purposes.
The literature presents several optimal conditions for the
extraction of active components from green tea leaves depending
on the applied process, solvent and temperature [3]. Analyzing
the time influence for extraction with water, Perva-Uzunalic
et al. [33] suggested the application of lower extraction times if
the temperature is higher than 80 C in order to avoid catechin
degradations. Manna et al. [34] prepared the aqueous extract of
green tea leaves at 60 C for 10 h. The greater required time may
be due to the low extract concentration (7.7 g L1) used by Manna
et al. [34]. Kumar et al. [17] produced an aqueous green extract at
50 g L1 and carried out the extraction at 80 C for 1 h.
According to results presented in Fig. 2, the best conditions for
total polyphenol extraction from a solution at 100 g L1 are tem-
perature of 75 C and extraction time of 80 min. These conditions
were applied to prepare the extracts for further microfiltration
and stability experiments. The characteristics of the prepared
green tea extract are presented in Table 3. The polyphenol concen-
tration is in agreement with the value reported by Kumar et al. [17]
for extraction with water at similar temperature. Component con-
centrations in the green tea extract measured by HPLC analyses
were, in mg L1: 2395.1 44.1 of EGCG, 1240.5 36.2 of EC,
204.1 25.0 of EGC, 201.3 6.2 of C, 122.9 2.3 of gallic acid and
1621.0 49.1 of caffeine. Concentrations of EC, EGCG and caffeine
are in agreement with the values reported by Hu et al. [5] for
extraction of green tea leaves with water. EGCG accounts for 59%
of total catechins and it was confirmed to be the predominant cat-
echin compound [33,35].
3.2. Microfiltrations of green tea extract
The obtained extract was microfiltered through membranes of
different pore sizes and the properties of permeates are presented
in Table 3.
Comparing the extract and the permeate, the membranes of 0.3
and 0.22 lm decreased all the evaluated parameters. The mem-
brane of 0.8 lm decreased the extract turbidity and solid concen-
tration, while colour intensity and total polyphenol concentration
remained constant. According to the statistical analysis, mem-
branes of different pore sizes produced permeates with different
characteristics, except the colour intensity of the permeate
obtained with the membranes of 0.3 and 0.22 lm. The retentions
of solids and polyphenols increased with the decrease in the mem-
brane pore size. Membranes of 0.8 and 0.22 lm retained 8 and 20%
of solids and 4 and 30% of polyphenols, respectively. Chandini et al.
[21] observed similar retentions of solids and polyphenols for the
microfiltration of black tea with membranes of 0.2 and 0.45 lm.
Colour reductions were of 2 and 22% with the membranes of 0.8
Table 3
Properties of microfiltered green tea extract.
Membrane Turbidity Colour Solid Total
pore size (NTU) intensity (ABS) concentration polyphenol
(lm) (mg L1) concentration
(mg L1)
Extract 458a 0.5 0.632a 0.0015 61,080a 85 6402a 81
0.8 29b 0.5 0.621a 0.013 56,440b 74 6160a 33
0.3 25c 0.5 0.522b 0.018 50,736c 69 5059b 48
0.22 21d 1.0 0.495b 0.012 48,882d 62 4451c 37
Values are expressed as mean standard deviation of triplicate analyses. Mean
values denoted by a different letter along a column are significantly different at
p 6 0.05 with respect to the increase in membrane pore size.
192 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198

and 0.22 lm, respectively. The reductions in turbidity were greater

than 90% with all membranes. Thus, the microfiltration process
was able to clarify the tea extract with low retention of polyphenol
compounds.
Fig. 3 presents calculated and experimental flux profile with all
microfiltration membranes. Calculated data were obtained by the
adjustment of the experimental data to the model proposed by
Hermia [30] according to n values that represent the fouling
mechanisms (n = 2 for complete pore blocking, n = 1.5 for internal
pore blocking, n = 1 for partial pore blocking and n = 0 for cake
filtration).
Fig. 3 shows that the more abrupt flux decline in the first 10 min
of filtration can be attributed to all the fouling mechanisms. For the
membranes of 0.8 and 0.22 lm, the steady state flux profile was
better described by the internal pore blocking mechanism
(n = 1.5) while the partial pore blocking mechanism (n = 1.0) better
described the steady state flux profile with the membrane of
0.3 lm. In addition to the adjustment of the experimental flux data
to Eq. (1) with the characteristic n values, Eq. (1) was also solved
in order to find the best n value for each microfiltration. Table S1
presents the calculated equation constants and the root-mean-
square deviations (RMSD) for the adjustment to each n value.
In fact, the best n value did not significantly differ from those
that is characteristic for the fouling mechanism for the membranes
of 0.8 and 0.22 lm, by means the internal pore blocking (n = 1.5)
was the predominant fouling mechanism for the microfiltrations
of green tea extract through these both membranes. A slight
greater difference was observed for the membrane of 0.3 lm, since
the best found n value was equal to 1.16, which suggests the pre-
dominant fouling mechanism was something between internal and
partial pore blockings (n = 1 and 1.5, respectively). For all the
membranes, the steady state flux profile was not well described
by the cake formation model probably because the pore size of
the components of green tea extract are smaller than the pore size
of these membranes and, thus, the flux behaviour was better
described by the pore blockage models. Moreover, given such a
high solids concentration in the extract, the cake is formed just
in the first minutes of filtration and after that the particles are
deposited in the membrane pores due to the constant pressuriza-
tion of the vase. A cross-flow operation is suggested to minimize
the cake formation.
Argyle and Bird [22] analyzed the flux decay in microfiltrations
of black tea extracts and observed that the predominant fouling
mechanism is dependent on the extract concentration. According
to Argyle and Bird [22] an instantaneous cake formation occurs
for extracts at compositions higher than 10.0 wt%. Domingues
et al. [36] applied different pre-treatments for microfiltration of
passion fruit juice and observed that the best fouling mechanism
adjustment depends on the applied pre-treatment. The internal
pore blocking was the best adjustment reported by Domingues
et al. [36] for the pre-treatment that enabled the highest solid
reduction, while cake formation was the main fouling mechanism
for all the other pre-treatments.
The steady state fluxes through the membranes of 0.8, 0.3 and
0.22 lm were of 10.3, 7.4 and 4.0 kg h1 m2, respectively. Since
the flux in the membrane of 0.8 lm was greater, the phenolic
Fig. 3. Calculated and experimental flux data for microfiltrations of green tea
retention was lower and the decrease in the other parameters
extract in membranes with pore sizes of (a) 0.8, (b) 0.3 and (c) 0.22 lm.
was satisfactory (Table 3), this membrane was chosen as a pre-
treatment for further ultrafiltrations.
fed to the ultrafiltration systems. As presented in Table 3, the
3.3. Permeate characterizations for ultrafiltrations of green tea extract physico-chemical characteristics of this stream are: Total polyphe-
with and without ultrasound nol concentration of 6160 33 mg L1, Protein concentration of
2067 13 mg L1, Solid concentration of 56,440 74 mg L1 and
Ultrafiltrations with and without ultrasound of green tea Turbidity of 29 0.5 NTU. Fig. 4 presents the permeate characteris-
extract were carried out with membranes of different MWCO. tics regarding to reductions of total polyphenols, protein, total
The extract microfiltered through the membrane of 0.8 lm was solids and turbidity.
 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198
The statistical analysis showed that most results are signifi-
cantly different (p 6 0.05) in respect to the increase in membrane
MWCO. Greater rejections are obtained with lower MWCO mem-
branes of lower MWCO. The differences are not significant for
polyphenol, protein and total solid permeations in the membranes
of lower MWCO (5 and 10 kDa) probably because these mem-
branes are closed enough to retain the same amount of these com-
ponents. The statistic equivalence for turbidity reduction was
verified for the membranes of 20 and 30 kDa in the process with-
out ultrasound and for the membranes of 10 and 20 kDa in the pro-
cess with ultrasound. However, reductions in green tea extract
turbidity were greater than 90% with all membranes.
The membranes of 10 and 5 kDa retained almost 100% of
polyphenols from the feed stream, while the retention of the mem-
branes of 20 and 30 kDa were lower than 70%. Galanakis et al. [37]
evaluated the polyphenol retention from winery sludge by an
ultrafiltration membrane of 20 kDa and reported a similar reten-
tion of approximately 70%. Galanakis et al. [38] also observed great
polyphenol retentions (up to 91%) from Cypriot wines by an ultra-
filtration membrane of 1 kDa. Polyphenol retentions of 71 and 91%
by membranes of 10 and 2 kDa, respectively, were reported by
Galanakis et al. [39] for the ultrafiltration of olive mill wastewater.
These great polyphenol retentions cannot be associated with only
the size exclusion, since catechins, which are the major polypheno-
lic compounds in green tea, have molecular sizes ranging from 290
to 458 Da approximately. In fact, the polyphenol retention by
ultrafiltration membranes may be attributed to the dynamic layer
formation [21] and/or to interactions between polyphenols and
proteins [24]. Galanakis et al. [37] compared the solute retentions
from winery sludge by ultrafiltration membranes of different
MWCOs (100, 20 and 1 kDa) and also reported that the retention
was affected mainly by severe fouling phenomena instead of size
exclusion.
Protein rejections were around 80% for the membranes of 20
and 30 kDa and around 90% for the membranes of 10 and 5 kDa.
Total solids and turbidity reductions were in the same range than
protein rejection. Chandini et al. [21] used a membrane of 25 kDa
for black tea clarification and achieved rejections of 77% of solids,
83% of polyphenols and 80% of proteins. Thus, based only in the
physico-chemical characteristics of permeate, the membrane of
Fig. 4. Influence of membrane MWCO and process condition (with and without ultrasound) on the reduction of physico-chemical parameters of green tea extract. (Bars
denoted by different lower-case letters and by different upper-case letters are significantly different at p 6 0.05 with respect to the membrane MWCO and to the process
(with and without ultrasound), respectively.)
193
30 kDa should be chosen for green tea treatment, since it enabled
high total solids, protein and turbidity reductions and the lowest
polyphenol rejection. However, other parameters (purity of perme-
ate, membrane flux and permeate stability) should be considered
for a proper choice.
The comparison between both proposed processes (with and
without ultrasound) showed that the polyphenol permeation was
up to 1.5 times higher in the ultrasound assisted process than in
the process without ultrasound. With the membrane of 5 kDa,
polyphenol permeations were statically equivalent in both pro-
cesses, since the ultrasound action was negligible due to the low
MWCO of this membrane. Protein retentions were statistically
equivalent in both processes probably due to the large size of these
molecules compared to the membrane MWCOs. Reductions in total
solids were around 1.2 times greater in the ultrasound assisted
process than in the process without ultrasound. The membranes
of 20 and 30 kDa enabled similar total solid permeations in both
processes. The ultrasound should act by removing the cake formed
on the membrane surface so that the total solid permeation was
lower in the ultrasound assisted process than in the process with-
out ultrasound especially in the membranes of lower MWCO.
Thus, the ultrasound assisted process enabled higher perme-
ations of polyphenols, similar permeations of proteins and lower
permeations of total solids than the process without ultrasound.
Polyphenol compounds have smaller molecule sizes than the other
components of green tea extract (proteins and carbohydrates) and
the ultrasound facilitated polyphenol permeation. The lower tur-
bidity reduction in the ultrasound assisted process compared to
the process without ultrasound is probably due to the higher per-
meation of polyphenols and proteins.
Table 4 presents the concentrations of individual catechins, gal-
lic acid and caffeine in the permeate with membranes of different
MWCOs and in both processes (with and without ultrasound).
The permeate is mainly composed by EGCG, EC and caffeine, as
well as the feed extract. The increase in the membrane MWCO
increased the permeation of most components. Only concentra-
tions of catechin (C), ()-epigallocatechin (EGC) and gallic acid
with membranes of 30 and 20 kDa in the process without ultra-
sound and of ()-epigallocatechin (EGC) and gallic acid with the
same membranes but in the process with ultrasound are not statis-
194 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198

Table 4
Concentration of individual catechins (()-epigallocatechine gallate (EGCG), ()-epicatechin (EC), catechin(C) and ()-epigallocatechin (EGC)), gallic acid and caffeine in
permeates with membranes of different MWCO for processes with and without ultrasound.

Process Membrane MWCO (kDa) Component concentration (mg L1)

EGCG EC C EGC Gallic acid Caffeine
Without ultrasound 5 151d;B 1 114d;B 1 21c;B 1 24c;B 0 10c;A 1 98d;B 4
10 245c;B 5 200c;B 5 48b;A 1 52b;B 2 25b;B 3 307c;B 10
20 1115b;B 9 647b;B 8 100a;A 3 100a;A 5 59a;B 3 642b;B 10
30 1205a;B 8 845a;B 8 111a;A 7 109a;A 1 61a;B 3 714a;B 5
With ultrasound 5 223d;A 7 143d;A 5 31d;A 1 40c;A 0 18c;A 2 112d;A 2
10 555c;A 6 309c;A 9 56c;A 3 102b;A 5 64b;A 2 409c;A 5
20 1448b;A 15 804b;A 10 98b;A 4 112a,b;A 7 77a;A 1 802b;A 10
30 1419a;A 10 904a;A 11 112a;A 3 123a;A 5 86a;A 5 904a;A 10

Mean values denoted by different lower-case letters and by different upper-case letters are significantly different at p 6 0.05 with respect to the membrane MWCO and to the
process (with and without ultrasound), respectively.

tically different (p 6 0.05), as well as concentration of ()- membrane of 20 kDa in processes with and without ultrasound.
epigallocatechin (EGC) with membranes of 20 and 10 kDa in the Similar behaviours were observed for membranes of different
process with ultrasound. Membranes of different MWCO may have MWCO. Calculated data were obtained according to the model pro-
permeated the same amount of these molecules (C, EGC and gallic posed by Field et al. [31] for cross-flow filtrations.
acid) due to the low concentration of them, so that no statistical The modelling of the experimental flux data showed that all the
differences were observed. fouling mechanisms are responsible for the flux decay in the first
The comparison of the processes with and without ultrasound 10 min of filtration. The transition of accentuate flux decay to
showed that the ultrasound assisted process enabled higher per- steady state flux is described by the cake filtration model (n = 0).
meation of the evaluated components. Permeation of these com- For the ultrafiltration without ultrasound, the cake formation is
pounds was at least 1.4 times higher in the ultrasound assisted the main fouling mechanism until almost 100 min of filtration,
process than in the process without ultrasound. Thus, based on
the requirement of having high concentration of catechin compo-
nents in the permeate, the ultrasound assisted process with the
membrane of 30 kDa should be chosen.
The purity of each catechin in the permeate is also an important
parameter to be evaluated, as presented in Table 5 for the ultra-
sound assisted process.
Table 5 shows that the ultrafiltration membranes where able to
increase the purity of each individual catechin compared to the
purity of the feed stream, and that the decrease in the membrane
MWCO increased the component purity. However, C and EGC puri-
ties of the feed stream and of the permeate with membranes of 20
and 30 kDa and EGCG purities of the feed stream and of the perme-
ate with the membrane of 30 kDa are statistically equivalents.
Thus, if the main objective is to increase the purity of EGCG (the
major catechin component in green tea), a membrane of at least
20 kDa is suggested.

3.4. Flux analyses for ultrafiltrations of green tea extract with and
without ultrasound

More than evaluating the physico-chemical characteristics of

the permeate, the fluxes through the different membranes should
be considered. Fig. 5 presents experimental and calculated flux val-
ues according to the filtration time for ultrafiltrations with the

Table 5
Purity of permeates regarding to individual catechins in the ultrasound assisted
process with membranes of different MWCO.

Membrane Component purity (%)

EGCG EC C EGC
Feed 41.32d 0.54 22.57c 0.53 4.08c 0.29 4.13c 0.29
30 kDa 42.99d 0.24 25.75b 0.06 3.20c 0.03 3.52c 0.06
20 kDa 48.83c 0.19 27.08b 0.05 3.34c 0.06 3.78c 0.12
10 kDa 58.93b 0.54 32.75b 0.12 5.94b 0.12 7.70b 1.55
5 kDa 71.94a 0.33 45.80a 1.78 10.09a 0.52 12.94a 0.82

Values are expressed as mean standard deviation of triplicate analyses. Mean Fig. 5. Calculated and experimental flux data for ultrafiltrations of green tea extract
values denoted by a different letter along a column are significantly different at in the membrane of 20 kDa for processes (a) without ultrasound and (b) with
p 6 0.05 with respect to the sample. ultrasound.
 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198
Table 6
Stead steady fluxes in ultrafiltrations with membranes of different MWCO and for
processes with and without ultrasound.
Membrane MWCO Stead steady flux (kg h1 m2)
(kDa)
Process without Process with
ultrasound ultrasound
30 9.89 11.10
20 6.20 8.74
10 3.49 5.16
5 0.40 1.47
while for the ultrafiltration with ultrasound this time is decreased
to 60 min.
The less extended cake formation in the ultrasound assisted fil-
tration process is expected since there was a constant clean of the
membrane surface due to the vibration caused by the ultrasound
frequency. For the both processes, the steady state flux profiles
are described by all the pore blockage models. The cake formation
model was the worst model to describe the steady state flux pro-
files probably because the molecules blocked the membrane pores
after the cake compacting on the membrane surface.
Table S2 presents the calculated equation constants and the
root-mean-square deviations (RMSD) for the adjustment to each
n value. Considering the whole process, the best adjust of the
experimental flux data to the flux decay equation was for n
equals to 1.0 by means partial pore blocking is the predominant
fouling mechanism. In fact, the optimization of the n value did
not significantly decrease the RMSD values for both processes
(with and without ultrasound). However, the best found n value
slight differs from 1.0 for the ultrasound assisted process. This dif-
ference is probably an attempt to better describe the flux data of
the transition stage from the accentuated flux decline to the steady
state flux, since this transition stage was controlled by the cake for-
mation model (n = 0).
Table 6 presents the steady state fluxes in ultrafiltrations with
membranes of different MWCO and for processes with and without
ultrasound.
The steady state flux was in average 1.3 times greater in the
ultrasound assisted process than in the process without ultrasound
with the membranes of 30, 20 and 10 kDa. For the membrane of
5 kDa, the ultrasound effect was more accentuated for the flux
increase and resulted in a steady state flux 3.7 times greater than
Fig. 6. Membrane resistances (membrane hydraulic resistance (RM), resistance due to pore blockage (RP) and resistance due to cake formation (RC)) for ultrafiltrations with
(w/) and without (wo/) ultrasound.
195
in the process without ultrasound. The ultrasound effect is not
reported in the literature for filtrations of green tea extract, but
the flux increase due to ultrasound application during filtrations
were already observed for other fluids. Liu et al. [40] observed an
increase of approximately 50% in the permeate flux in the ultra-
sound assisted process compared to the conventional dead-end
microfiltration of grape pomace extracts. Aghdam et al. [41] related
the increase in the permeate flux of pomegranate juice in the ultra-
sound assisted process to the decrease in the membrane resistance.
3.5. Resistance analyses for ultrafiltrations of green tea extract with
and without ultrasound
Fig. 6 presents the calculated resistances for each membrane in
processes with (w/) and without (wo/) ultrasound.
The membrane resistance (RM) is increased with the decrease in
the membrane MWCO. The resistance due to cake formation (RC)
accounts for approximately 70% of the total resistance and it is
the controlling resistance during the filtration process. Evans and
Bird [23] also concluded that cake formation was the main fouling
mechanism during ultrafiltrations of black tea liquor. According to
the Field model, the cake formation was responsible for the accen-
tuated flux decline and to the transition to steady state fluxes
(Fig. 5). The resistance-in-series model considers the whole process
and, in this case, the cake formation was the controlling fouling
mechanism.
Application of ultrasound during the filtration of green tea
extract decreased the total resistance of the process. The reduction
in the total resistance (44%, in average) was more pronounced for
the membranes of intermediated pore sizes (10 and 20 kDa). For
the membranes of 30 and 5 kDa, reductions on total resistances
were of 23 and 24%, respectively. For the membrane of 30 kDa,
cake and pore resistances decreased in 20 and 48%, respectively,
in the ultrasound assisted process compared to the process with-
out ultrasound. However, for the membranes of 20, 10 and 5 kDa,
the ultrasound effect was more pronounced in the reduction of
cake formation than of pore blockage. Thus, for more opened mem-
branes, in which the pore blockage is more accentuated, the ultra-
sound acted more in preventing the deposition of the molecules
inside the pores of the membranes. If the membrane is closer, a
thick layer of cake is formed and the ultrasound effect was more
pronounce in cake removal.
196 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198
Fig. 7. Stability of green tea extract and permeate samples related to (a) total polyphenol concentration, (b) tea cream, (c) colour intensity and (d) turbidity.
3.6. Stabilities of permeates from micro and ultrafiltrations
Fig. 7 presents the variation of turbidity, colour, total phenolic
content and tea cream formation according to the storage days of
green tea extract and permeates of micro and ultrafiltration mem-
branes. The stability of permeate samples of ultrafiltrations with
and without ultrasound are similar and results presented in
Fig. 7 are regarding to ultrafiltration with ultrasound.
For all samples, there was an accentuated variation in all the
evaluated parameters during the first 7 days of storage and, in gen-
eral, the values remained almost constant until the thirty day of
storage. During the first 7 days of storage, total phenolic concentra-
tion (Fig. 7a) of the green tea extract decreased in approximately
7%, while total phenolic concentration of permeates of micro and
ultrafiltrations decreased in at least 9 and 60%, respectively. The
decrease in total phenolic concentration probably occurs due to
polyphenolprotein interaction, which may cause tea cream for-
mation [20]. However, although the permeate samples from ultra-
filtration had presented a greater polyphenol concentration
decrease, initial and final values are lower compared to the perme-
ate samples of microfiltration and to the green tea extract sample.
Fig. 7b presents the tea cream formation in extract and microfil-
tered samples. Any tea cream formation was detected in the per-
meate samples of ultrafiltration during 30 days. Fig. 7b shows
that almost 90% of total solids of green tea extract are precipitated
when the sample is stored for 16 h at 5 C (day 0 of stored). For the
permeate of microfiltration, the tea cream formation increased
from 66 to 75%, 43 to 54% and 41 to 43% for the membranes of
0.8, 0.3 and 0.22 lm, respectively. Colour and turbidity increases
(Fig. 7c and d, respectively) were more accentuated for the perme-
ate samples of micro than of ultrafiltrations. Turbidity values of
green tea extract varied from 489 to 519 NTU and are not pre-
sented in Fig. 7d due to the scale range. According to Chandini
et al. [21], ready to drink tea beverage is considered clear if the tur-
bidity is below 50 NTU. Turbidity values of permeates of microfil-
trations were greater than 50 NTU after 30 days of storage, while
the turbidity of permeates of ultrafiltrations were lower than
5 NTU during the 30 days of storage. Thus, based on the criteria
of low turbidity and none tea cream formation, the membrane of
30 kDa is enough to guarantee a stabilized green tea permeate.
4. Conclusions
The best conditions for aqueous extraction of polyphenol com-
pounds from green tea leaves were at 75 C and 80 min. Tempera-
tures up to 75 C presented a positive effect on polyphenol
extraction, but polyphenol concentrations decreased when the
temperature was increased from 75 to 80 C due to the degradation
of polyphenol compounds. Total polyphenol concentration
remained constant for extraction times greater than 80 min.
The microfiltration membrane of 0.8 lm produced a permeate
with low turbidity and low total phenolic concentration reduction
(from 458 to 29 NTU and from 6402 to 6160 mg L1, respectively).
 L.S. Sousa et al. / Separation and Purification Technology 168 (2016) 188198
Due to the highest permeate flux through the membrane of 0.8 lm
compared to the membranes of 0.3 and 0.22 lm, this membrane
was chosen to clarify green tea extract. Mathematical analysis of
flux decay during microfiltrations showed that pore blockage and
cake formation models were responsible for the initial flux decay
and that the steady state flux profiles were better adjusted by pore
blockage models than by the cake formation model. These results
are due to the high solid concentration which resulted in the for-
mation of cake on the membrane surface in the first minutes of
operation. Moreover, the extract particle sizes are smaller than
the membrane pore sizes and entered in the membrane pores after
the cake formation.
In the ultrafiltration process, the comparison of membranes of
different MWCO showed that the membrane of 20 kDa should be
chosen in order to ensure higher catechin purities in the permeate.
The ultrasound effect was positive for ultrafiltrations of green tea
extract. With ultrasound, the steady state permeate flux and the
total polyphenolic compound permeation increased both in 1.4
times for the membrane of 20 kDa. The modelling of flux decay
during ultrafiltrations showed that the extension of cake formation
was lower in the ultrasound assisted process than in the process
without ultrasound. Moreover, the application of ultrasound
reduced in 44% the total resistance during ultrafiltration of the
membrane of 20 kDa.
Permeate samples of microfiltration cannot be considered
stable due to tea cream formation and high turbidity values. On
the other hand, turbidity values of permeate samples of ultrafiltra-
tion remained lower than 5 NTU after 30 days of storage under
refrigeration and any tea cream formation was observed.
In conclusion, ultrasound assisted ultrafiltration process with a
membrane of 20 kDa is suggested to purify catechins from green
tea extract with a relative high permeation flux and great catechin
components purity and stability.
Acknowledgement
We acknowledge financial support from FAPEMIG (Fundao de
Amparo Pesquisa do Estado de Minas Gerais), CAPES (Coor-
denao de Aperfeioamento de Pessoal de Nvel Superior) and
CNPq (Conselho Nacional de Desenvolvimento Cientfico e
Tecnolgico).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.seppur.2016.05.
029.
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