Journal of Food Engineering 241 (2019) 97–104

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Journal of Food Engineering

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

Maximisation of the polyphenols extraction yield from green tea leaves and T
sequential clarification
Marlon Menezes Maciel Bindesa, Vicelma Luiz Cardosoa, Miria Hespanhol Miranda Reisa,
Daria Camilla Boffitob,∗
a
Faculdade de Engenharia Química, Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121, 38400-902, Uberlândia, Minas Gerais, Brazil
b
Department of Chemical Engineering, Polytechnique Montréal, C.P., 6079, Succ. CV Montréal, H3C 3A7, Québec, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: We designed a parametrical study to optimise the polyphenol liquid-solid extraction in green-tea by clarification
Polyphenols by centrifugation followed by microfiltration through a 0.22 μm cellulose acetate membrane. Grounding, sieving
Extraction and selecting the optimal granulometry (0.15–0.74 mm) increased the total polyphenol concentration by 14%
Clarification compared to unground leaves, while using 61% of the initial tea leaves mass. The optimal polyphenol extraction
Microfiltration
conditions were water-to-tea ratio of 20:1 mL g−1, temperature of 80 °C, and extraction time of 60 min. The
Green tea
sequential clarification process decreased the turbidity of the extract (from 1197 to 13 NTU), while only re-
ducing the polyphenol concentration by 13%. The permeate of the microfiltration process formed 82% less tea
cream than the centrifuged extract after 30 days under refrigeration at 5 °C. This sequential clarification is
suitable to produce a green tea extract with low turbidity and reduced tea cream.

1. Introduction 2013). Extraction time is a significant factor influencing the extraction

The consumption of functional foods and beverages has recently
risen across the world mainly due to the concern about prevention and
treatment of certain diseases (Corbo et al., 2014). In this scenario, tea is
the most widely consumed drink after water. Green tea (Camellia si-
nensis) has received great attention due to its high content of poly-
phenols. Polyphenols are a large group of biocompounds from plant
materials (Sasikala et al., 2018), which include phenolic acids and
flavonoids (Carloni et al., 2013; Komes et al., 2010). The most im-
portant flavonoids are catechins (Komes et al., 2010; Yamamoto et al.,
1997). Several studies confirmed the pharmacological properties of
catechins as anti-oxidant compounds for free radical induced disorders
(Hong et al., 2002; Manna et al., 2014). The main catechins present in
green tea are (−)-epicatechin (EC), (−)-epigallocatechin (EGC),
(−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG)
(Banerjee and Chatterjee, 2015).
Extraction is the key step to produce tea as a beverage. The pro-
duction of natural extracts demands proper processing steps to max-
imise the conservation of the bioactive substances (Bucić-Kojić et al.,
2007; Pinela et al., 2016; Şahin et al., 2015). Therefore, the optimisa-
tion of the variables that influence the biomolecule extraction from
plant materials is of great importance for the industry (Azmir et al.,
yield of bioactive compounds from plants (Vuong et al., 2011b). This
parameter mostly depends on the extraction method used, the plant
matrix and the production standards required (Domínguez-Rodríguez
et al., 2017; Plaza and Rodríguez-Meizoso, 2013). Particle size is an-
other variable to consider in the extraction of biomolecules. Literature
data are available on the impact of particle size on the bioactive com-
pounds extraction yield (Hu et al., 2016; Kuhn et al., 2017; Sari and
Velioglu, 2011; Vuong et al., 2011a). However, literature lacks re-
porting the percentage of the raw material obtained after sieving. An-
other misconception about solid-liquid extraction concerns the way of
expressing the solvent-to-material ratio, which influences the extraction
of biomolecules from fruits and plant materials (Jovanović et al., 2017;
Rezende et al., 2017; Stamatopoulos et al., 2013). Some authors such as
Sousa et al. (2016) and Kumar et al. (2012) expressed the concentration
of polyphenols in mg L−1. Others like Rao et al. (2011) and Zhang et al.
(2012) expressed it in mg g−1. However, the scientific community
seems to neglect at a practical level the difference of expressing it in mg
g−1 or in mg L−1. Both data are indeed very important to design se-
quential extraction/filtration modules in industry and should be always
reported in tandem.
Ready-to-drink teas are vulnerable to the tea cream formation,
which is an apparent precipitate that spontaneously forms upon tea

∗
Corresponding author.
E-mail address: daria-camilla.boffito@polymtl.ca (D.C. Boffito).

https://doi.org/10.1016/j.jfoodeng.2018.08.006
Received 25 May 2018; Received in revised form 1 August 2018; Accepted 7 August 2018
Available online 10 August 2018
0260-8774/ © 2018 Elsevier Ltd. All rights reserved.
M.M.M. Bindes et al.
Fig. 1. (a) Sequence of the clarification processes and (b) Schematic diagram of the microfiltration system.
cooling (Dickinson, 1994; Lin et al., 2015) and confers it a creamy
appearance. The formation of tea cream consists of a change in the
molecular weight and the solubility of phenolic polymers upon com-
plexation with other tea constituents such as metal cations, proteins,
lipids and polysaccharides (Monsanto et al., 2014a; Tolstoguzov, 2002).
Therefore, clarification consists in the removal of these major compo-
nents responsible for this complexation and consequently for tea cream
formation. Centrifugation and microfiltration are two different pro-
cesses to clarify fruit juices and extracts (Laorko et al., 2013; Sagu et al.,
2014; Sentandreu et al., 2011). These two operations are safe for the
food and beverage industry, especially because they avoid chemicals
(Chhaya et al., 2013). Microfiltration systems have been used for
clarification of tea extracts. However, there is a lack of data reporting
centrifugation as a pre-treatment of tea extracts before microfiltration.
A primary clarification of the extract by centrifugation may remove the
major suspended solids and increase the efficiency of the sequential
membrane filtration. Aroma enhancement and reduction of tea cream
are examples of important procedures to guarantee the sensory quality
of tea as a beverage (Banerjee and Chatterjee, 2015). In this regard, it is
key to find a technique to increase the efficiency of the clarification of
tea beverages while keeping bioactive compounds intact at the same
time.
This work is therefore original for several reasons. The following
aspects have not been reported in literature, yet: i) we design a para-
metrical study to produce a green tea extract, ii) we clarify the extract
in sequence by centrifugation and microfiltration through a cellulose
acetate membrane of 0.22 μm, iii) we obtain an unprecedented poly-
phenols extraction yield of 21% (9501 mgGAE L−1) with a reduced
water volume. Here, the extraction optimisation aimed at evaluating
the entire sequential extraction and clarification process. iv) Stability
tests were also conducted to evaluate the variation in the polyphenols
content, solid concentration and turbidity of the centrifuged extract and
of the permeate of the microfiltration during 30 days of storage under
Fig. 1. (continued)
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Journal of Food Engineering 241 (2019) 97–104
refrigeration.
2. Material and methods
2.1. Materials
Dried green tea leaves were purchased from a local market (Minas
Gerais state, Brazil) in July 2016. Standards for caffeine, Catechin (C),
(−)-Epigallocatechin gallate (EGCG), (−)-Epigallocatechin (EGC),
(−)-Epicatechin (EC) and gallic acid (GA) were purchased from Sigma-
Aldrich. All reagents were of analytical grade and used without further
purification.
2.2. Processing and characterization of green tea leaves
The dried green tea leaves were grounded in a commercial blender
(50 Hz, 500 W) for 5 min. The ground leaves were then separated in
eight different particle size by a stainless steel sieving system
(Granouest™). Sieves of 9, 16, 24, 35, 48, 100 and 270 mesh were used
and the retained fraction of tea leaves was determined by percentage.
We adopted Equation (1) to calculate the mean Sauter diameter.
1
D = Δsi
∑i Di (1)
D is the mean Sauter diameter, Δsi is the particle mass fraction and Di is
the average band diameter.
2.3. Polyphenols extraction from green tea leaves
We first investigated the effect of tea particle size on the extraction
efficiency of polyphenolic compounds. For each sample of different
particle size, extraction conditions were fixed at 80 °C for 30 min at a
water-to-tea ratio of 20:1 (mg L−1), as suggested by Vuong et al.
M.M.M. Bindes et al.
(2011b). All extractions were carried out under magnetic stirring at
120 rpm.
Further polyphenols extractions were carried out with the green tea
leaves of optimal particle size. We designed a parametric study to
evaluate the effect of water-to-tea ratio, temperature and time on the
extraction of polyphenolic compounds from green tea leaves. The
water-to-tea ratio varied from 10:1 to 100:1 (mg L−1) for extractions at
80 °C for 30 min. Aqueous extractions at 40, 60, 80 and 90 °C were
carried out for a water-to-tea ratio fixed at 20:1 (mg L−1) for up to
120 min with sample collection at different time intervals. The extracts
were then analysed to quantify total phenolic content, solid con-
centration, turbidity, concentration of gallic acid, caffeine, and in-
dividual catechins (EGCG, C, EC and EGC).
2.4. Green tea extract clarification
We retained the green tea extract obtained at the optimal conditions
(tea particle size 0.15–0.74 mm, water-to-tea ratio 20:1 mL g−1, 80 °C,
and 60 min) following the sequential process as presented in Fig. 1 (a).
The mixture passed through a 11 μm filter paper (Whatman) to remove
rough particles, and the filtrate was then centrifuged (Beckman Coul-
terTM centrifuge) at 8000 rpm for 20 min, as suggested by Rao et al.
(2011). The supernatant was collected for analyses and further mem-
brane filtration. The centrifuged green tea extract was sequentially
filtered through a cellulose acetate flat membrane of 0.22 μm pore size
(Millipore™) with a filtration area of 3.9 x10−3 m2. A membrane fil-
tration module was adopted for the cross-flow microfiltration of green
tea extract (Fig. 1 (b)). The conditions were 0.8 bar of transmembrane
pressure, room temperature, and a concentration factor of 1.5.
Permeate stream was then collected for further analyses.
2.5. Physico-chemical analyses
Feed and permeate streams were analysed for total phenolic con-
tent, turbidity, solid concentration and concentration of gallic acid,
caffeine and individual catechins (EGCG, C, EC and EGC). The total
phenolic content was measured by the Folin-Ciocalteu method, as
suggested by Kumar et al. (2012) but with slight modifications. In-
itially, the tea extract was diluted in distilled water at a 1:10 (v:v) ratio.
Then, 0.25 mL of the Folin–Ciocalteu reagent were added to 0.05 mL of
this diluted extract. After 2 min, 2 mL of sodium carbonate (10 wt%)
were added and the final volume was adjusted to 4 mL with distilled
water. The mixture was kept in dark for 1 h. The absorbance was
measured at 760 nm in a Shimadzu 1240 spectrophotometer (Japan). A
calibration curve of the gallic acid standard solution was adopted to
determine the total polyphenols concentration (mg L−1). Total solid
concentration was determined by weighing a 2.0 mL sample before and
after drying for 24 h at 105 °C. Turbidity was measured with a Nova
Organica HD 114 turbidimeter. Ash and moisture content were de-
termined using the methodology of AOAC International (AOAC). Con-
centrations of gallic acid, caffeine and individual catechins (EGCG, C,
EC and EGC) were determined by liquid chromatography in a Shimadzu
HPLC model LC-20AT Prominence equipped with a Controller CBM-
20A system, an automatic liquid sampler (ALS), an UV-VIS Detector
(model SPD-20AV) and a VP-ODS C18 (4.6 × 150 mm) column. The
mobile phase consisted of a solution of methanol, water and ortho-
phosphoric acid in the ratio of 20, 79.9 and 0.1 by volume respectively.
The flow rate of the mobile phase was 1.0 mL min−1, the injection
volume for all samples was 20 μl, wavelength of 270 nm and an oven
temperature of 30 °C.
2.6. Stability tests
Stability tests evaluated the effect of the microfiltration on the
product stability. Parameters such as turbidity, total polyphenolic
concentration and amount of tea cream were analysed. For this
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Journal of Food Engineering 241 (2019) 97–104
purpose, feed and permeate samples were stored at 5 °C for 30 days. Tea
cream was determined as the amount of insoluble particles that are
formed in the sample after at least 16 h of refrigeration (Chandini et al.,
2012). For this determination, samples were centrifuged at 5600 rpm
for 20 min and tea cream (%) was calculated as the amount of total
solids in the supernatant related to the amount of solids in the initial
sample (Sousa et al., 2016).
2.7. Statistical analyses
All physicochemical analyses were done in triplicate and data are
expressed as mean ± standard deviation. The statistical Tukey test was
applied for the analyses of variance (ANOVA) and a significance level of
5% was adopted for rejection of the null hypothesis (p ≤ 0.05).
3. Results and discussion
3.1. Tea leaves characterization and polyphenols extraction maximisation
Particle size characterization of the matrix is key to design the
polyphenols extraction process from tea leaves (Zderic and Zondervan,
2017). The calculated Sauter mean diameter of the unground tea was of
0.54 ± 0.02 mm. Table 1 presents the values of moisture and ash
content for different particle size ranges of green tea leaves. The
moisture content varied from 6.12 to 7.45%, while the ash content
varied from 4.65 to 4.93%. Although the range of variation of the
moisture and ash content was very small, the Tukey's test (p ≤ 0.05)
indicates significant differences for these parameters among the ma-
jority of the studied particle size fractions. This is explained by the fact
that each particle size range corresponds to different parts of the plant
(Fig. 2). In other words, the non-homogeneity among the different size
fractions of tea leaves had a significant impact (p ≤ 0.05) on the ash
and moisture content. Adnan et al. (2013) evaluated the chemical
composition of five green tea samples and observed that ash and
moisture content were 4.57 and 6.46%, respectively, similarly to the
values reported in Table 1.
3.1.1. Effect of the tea particle size on the extraction yield of total
polyphenols
Fig. 2 highlights the influence of particle size on the yield of total
polyphenols extraction from green tea leaves. The yield of total poly-
phenols was statistically higher (p ≤ 0.05) when tea particle ranged
from 0.15 to 0.74 mm, providing a total polyphenols concentration of
approximately 9497 mgGAE L−1. However, particle size lower than
0.15 mm and from 0.74 to 2.83 mm resulted in a lower concentration of
polyphenols (mgGAE L−1). For particles smaller than 0.15 mm, the
polyphenols concentration was lower mainly because of the tendency of
fine particles to agglomerates. Agglomerates, even under agitation, may
deposit at the bottom of the vessel and reduce diffusional transport
between the tea leaves and the solvent. This is in agreement with
Table 1
Moisture and ash contents in green tea samples. The mean values indicated by a
different letter along a column are significantly different for the Tukey test
(p ≤ 0.05).
Tea particle size (mm) Moisture (%) Ash (%)
< 0.05 6.12 a ± 0.04 4.93 a ± 0.04
0.05–0.15 6.50 b ± 0.06 4.81 a, b ± 0.01
0.15–0.32 6.94 c ± 0.03 4.69 b, c ± 0.00
0.32–0.5 7.22 d ± 0.03 4.66 c ± 0.03
0.5–0.74 7.18 d ± 0.03 4.67 c ± 0.04
0.74–1.19 7.42 e ± 0.04 4.70 b, c ± 0.08
1.19–2.19 7.36 d, e ± 0.05 4.68 b, c ± 0.01
2.19–2.83 7.45 e ± 0.03 4.65 c ± 0.01
Unground tea 7.31 d, e ± 0.05 4.69 b, c ± 0.02
M.M.M. Bindes et al. Journal of Food Engineering 241 (2019) 97–104

Fig. 2. Effect of the tea particle size on the yield of total polyphenols of green tea. The extraction conditions were 80 °C, 30 min and 20:1 mL g-1. Values with different
letters are statistically different (p ≤ 0.05) according to the Tukey's test.

literature data (Spiro and Jago (1982), Gujar et al. (2010) and Hu et al.
(2016)). In the case of larger particles, in the range from 0.74 to
2.83 mm, the dominant presence of harder parts of the leaves, such as
the veins and the petioles, explains the reduction in the polyphenols
concentration. These parts of the leaves were more difficult to grind and
presented lower concentration of polyphenols than other regions like
the lamina (blade). Ochanda et al. (2015) observed that the content of
phenolic compounds varies in different parts of the plant Camellia si-
nensis and suggested to use a mixture of tea leaves retained in the sieves
of 35, 48 and 100 mesh (particles between 0.15 and 0.74 mm). This
range of particles, besides providing the highest concentration of total
polyphenols mentioned above, corresponds to approximately 61% of
the total mass of retained tea after sieving. The range of tea leaves
particle size in this work complies with this optimal range, thus
granting a efficient use of the plant material.
Vuong et al. (2011a) observed that the yield of catechins increased
when tea leaves particle size reduced to 1 mm or less. However, smaller
particles were more difficult to separate from the solution after brewing
Fig. 3. Effect of tea-to-water ratio (g L−1) on the yield of polyphenols in mg g−1
either through filtration or centrifugation. Vuong et al. (2011b) ana-
and mg L−1. Extraction conditions: 80 °C, 30 min, water-to-tea from 8:1 to
lysed the effect of granulometry in the extraction of theanine from
100:1 mL g−1. The uncertainty corresponds to the symbol size.
green tea and observed that extraction yield was higher and sig-
nificantly different (p ≤ 0.05) for tea leaves particle size between 0.5
and 1 mm. This size range is close to our data (0.15–0.74 mm). Sari and extraction for values ranging from 8 to 100 mL g−1. The ratio of sol-
Velioglu (2011) evaluated the effect of tea particle size on the extrac- vent-to-material affects the extraction of biocompouds from vegetable
tion of theanine from Camellia sinensis leaves and obtained a greater matrices (Jovanović et al., 2017; Stamatopoulos et al., 2013). Previous
extraction yield for the tea particle 0.15–0.3 mm. Hu et al. (2016) found studies report the impact of the material-to-solvent ratio in the ex-
that the extraction of total catechins and caffeine increased significantly traction yield of bioactive compounds, but few of them focus on the
while reducing the particle size. With a mesh of 45 (0.354 mm), close to difference between expressing the concentration of the bioactive com-
the 24–48 mesh range (0.15–0.74 mm) used in our work, Hu et al. pound in mg L−1, rather than in mg g−1. From the point of view of the
(2016) obtained intermediate values for caffeine and total catechins scalability of the extraction of active molecules from raw materials, it is
extraction. Previous studies reported that smaller particle size favours preferable to include both data. Expressing the biocompouds con-
the extraction of several compounds, except when agglomeration oc- centration in mg L−1 is necessary since the volume of required solvent
curs (Sari and Velioglu, 2011). Hu et al. (2016) suggested the use of determines the size of the extraction unit. Water is a clean and low-cost
aqueous ethanol as an alternative to water for the extraction of ca- solvent, but it may require a greater reactor volume compared to other
techins from green tea leaves. They reported that the extraction effi- solvents and more energy i to heat it up. If a dry extract is desired,
ciency with absolute ethanol mainly depends on the particle size of the evaporating water is often more energy consuming than evaporating
sample, and rarely on solvent penetration. lower boiling point solvents. In this study, polyphenols purification is
the next step after the extraction procedure. Therefore, the best water-
to-tea ratio favours both extraction and purification steps. After ana-
3.1.2. Effect of the water-to-tea ratio on the extraction yield of total
lysing the water-to-tea profiles (Fig. 3), the ratio 20:1 mL g−1 was se-
polyphenols
lected as the optimal condition. Our findings are in accordance with
Fig. 3 shows the impact of water-to-tea ratio on the polyphenol

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M.M.M. Bindes et al. Journal of Food Engineering 241 (2019) 97–104

previous data. Vuong et al. (2011a) optimised the extraction of ca-

techins from green tea leaves and concluded that a water to tea ratio of
20:1 mL g−1 provided the best results in terms of water efficiency and
cost-effectiveness. Kumar et al. (2012) studied four different water-to-
tea ratio (10:1, 15:1, 20:1 and 25:1 mL g−1) and found that 20:1 was
the best choice in terms of polyphenols extraction from green tea.

3.1.3. Effect of temperature and time on the extraction yield of total

polyphenols
Fig. 4 shows the profiles of polyphenols concentration at different
times and temperatures. The polyphenols concentration increases with
the time up to around 60 min for temperatures of 40 °C, 60 °C and 80 °C,
while at 90 °C and after 40 min the polyphenols concentration started to
decrease. Perva-Uzunalić et al. (2006) and Vuong et al. (2011a) re-
ported that polyphenols degradation begins at 80 °C, in accordance with
the results presented in Fig. 4. Kumar et al. (2012) reported that higher
polyphenols concentration was obtained at 80 °C when compared to
60 °C. Banerjee and Chatterjee (2015) authored an exhaustive review
Fig. 4. Influence of temperature and time on polyphenols extraction from green on extraction methods from Camellia sinensis and reported that most
tea leaves. When not visible, the uncertainty corresponds to or is lower than the literature data are collected at temperatures around 80 °C. Additionally,
symbol size.
Perva-Uzunalic et al. (2006) suggested either high temperature (95 °C)
and short extraction time (5–10 min), or lower temperature (60 or
Table 2 80 °C) and longer extraction time (20 min) to avoid catechin degrada-
Percentage of total polyphenols after infusion of green tea leaves for different tion. Thus, our results about the temperature influence on the extrac-
studies. tion of polyphenolic compounds from green tea leaves are in the same
Author Water-to- Temperature (°C) Time Percentage of range as consolidated literature data.
tea ratio (min) total In our case, a tea-to-water ratio of 20:1 mL g−1 (50 g L−1), 80 °C and
(mL g−1) polyphenols (%) 60 min provided the greatest polyphenols content (10686 mg L−1) and
This work 20 80 60 21
these conditions were set as operational. We report an unprecedented
Sousa et al. (2016) 10 75 60 7 polyphenols extraction yield compared to previous studies (Table 2),
Zhang et al. (2012) 16 96 40 13 confirming a successful optimisation of green tea extraction conditions.
Kumar et al. (2012) 20 80 100 8
Bharadwaz and 20 60 – 19
Bhattacharjee,
2012 3.2. Green tea extract clarification
Rao et al. (2011) 100 60 20 11
Perva-Uzunalić et al. 40 80 120 19 Table 3 presents the concentrations of total polyphenols and solids,
(2006) as well as the values of turbidity for the feed and the centrifuged ex-
tracts and for the permeate of the microfiltration process. After cen-
trifugation, polyphenols reduced by 9%, total solids by 7%, and the
Table 3
turbidity by 91%. Pretreatments such as centrifugation play an im-
Properties of the feed and centrifuged extracts and of the permeate of the mi-
crofiltration. Values are expressed as mean ± standard deviation of triplicate
portant role to remove suspended solids, avoiding the rapid flux decay
analyses. Mean values denoted by a different letter along a column are sig- during the sequential filtration processes.
nificantly different at p ≤ 0.05 with respect to the sample analysed. Concentrations of individual catechins, gallic acid and caffeine are
statistically different (p ≤ 0.05) in the feed stream and clarified sam-
Sample Total polyphenols Solids Turbidity
concentration concentration (mg (NTU)
ples (Table 4). The EGCG was the most abundant catechin present in the
(mgGAE L−1) L−1) feed and clarified extracts, with a recovery of 82% after the sequential
clarification (from 1615 to 1330 mg L−1).
Feed extract 10686 a ± 46 20644 a ± 97 1197 a ± 5 Fig. 5 shows the samples of the feed and centrifuged extracts and the
Centrifuged extract 9771 b ± 48 19306 b ± 106 108 b ± 8
permeate of the microfiltration. Chandini et al. (2012) reported that
Permeate of the 9304 c ± 45 18933 c ± 104 13 c ± 1
microfiltration clear packaged tea beverages are those with turbidity lower that 50
NTU. Our sequential process of centrifugation/microfiltration provided
a clear green tea extract, with low turbidity (13 NTU), and can be po-
tentially applied to produce ready-to-drink teas, especially with high
recovery of total polyphenols.

Table 4
Concentration (mg L−1) of individual catechins ((−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), catechin(C) and (−)-epigallocatechin (EGC)), (GA)
gallic acid and caffeine in feed extract and centrifuged extract and in the permeate of the microfiltration. 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 ≤ 0.05 with respect to the sample analysed.
Sample EGCG (mg L−1) EC (mg L−1) C (mg L−1) EGC (mg L−1) GA (mg L−1) Caffeine (mg L−1)

Feed extract 1615 a ± 5 639 a ± 4 106 a ± 1 1403 a ± 9 75 a ± 0 1035a ± 1

Centrifuged extract 1500 b ± 18 625 b ± 1 101 b ± 2 1332 b ± 10 72 b ± 1 981 b ± 8
Permeate of the microfiltration 1330 c ± 9 585 c ± 3 96 c ± 2 1269 c ± 19 68 b ± 3 930 c ± 4

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Fig. 5. Green tea samples ((S1) feed extract, (S2) centrifuged extract and (S3) permeate of the microfiltration).

Fig. 6. Stability of green tea extract and permeate samples: (a) total poly-
Fig. 6. (continued)
phenols content, (b) turbidity and (c) tea cream. When not visible, the un-
certainty corresponds to or is lower than the symbol size.
content decreased by 12 and 9% and turbidity increased to values
greater than 290 and 25 NTU causing a tea cream formation of 41% and
3.3. Stability tests
23%, respectively. In this sense, microfiltration through the membrane
of 0.22 μm reduced tea cream formation by 82% after 30 days of
Fig. 6 shows the stability of the obtained clarified green tea samples
cooling. Monsanto et al. (2014b) reported that the formation of tea
in terms of polyphenols concentration, turbidity and tea cream forma-
cream caused a removal of up to 25% of the polyphenols present in the
tion during 30 days of storage.
black tea extract. Sousa et al. (2016) evaluated the stability of green tea
For the centrifuged green tea extract and for the permeate of the
and observed a drop of approximately 27% in the polyphenols con-
microfiltration, during 30 days of refrigeration, the total polyphenols
centration in the 0.22 μm flat membrane permeate after storage for 30

102
M.M.M. Bindes et al.
Fig. 6. (continued)
days at 5 °C. Chandini et al. (2012) observed stability of black tea
permeates of the microfiltration through membranes of 0.2 and 0.45 μm
during 30 days under cooling. Chandini et al. (2012) considered stable
permeates with turbidity values less than or equal to 4 NTU.
4. Conclusions
The impact of extraction conditions on the content of total poly-
phenols from green tea leaves applying water as a solvent was ex-
amined. Tea particles smaller than 0.15 mm and from 0.74 to 2.83 mm
presented lower polyphenols concentration upon extraction. Smaller
agglomerated leaves reduce the diffusional transport. An ash content of
almost 5% in green tea leaves indicates a considerable concentration of
inorganic impurities. Regarding the water-to-tea ratio influence on
polyphenol extraction, a water-to-tea ratio of 20:1 mL g−1 maximised
the yield of polyphenols (9501 mgGAE L−1), which is unprecedented in
literature. An extraction time of 60 min and a temperature of 80 °C were
the best conditions for polyphenols extraction from green tea leaves. In
this study, although the centrifuged and filtrated samples were not
stable after 30 days of storage at 5 °C, the permeate of the micro-
filtration presented a tea cream formation 82% lower than the cen-
trifuged extract. These findings confirm that the sequential clarification
produced a clear green tea extract with high recovery of polyphenols
and low tea cream formation under refrigeration.
Acknowledgements
We acknowledge financial support from FAPEMIG, CAPES and
CNPq. This research was financed, in part, thanks to funding from the
Canada Research Chairs program.
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