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Article history: The major interest in pulsed electric eld treatment of biological tissues is derived from
Received 15 December 2015 its non-thermal application: increasing cell permeability. This application has an important
Received in revised form 2 March implication in extraction of complex organic molecules. In this work, pulsed electric eld
2016 treatment is investigated as a mild (non-thermal) processing method for opening the cell
Accepted 5 March 2016 structure in fresh tea leaves. Pulsed electric eld utilizes short-duration high-voltage pulses
Available online 12 March 2016 for opening the cell structure by the process called electroporation. Upon the treatment,
subsequent extraction of complex organic molecules, particularly, polyphenols, occurs. The
Keywords: amount of extracted polyphenols (in this case, the extraction yield) has been determined
Pulsed electric eld as a function of electric eld strength, duration and number of applied pulses, as well as
Polyphenols energy input per unit of mass of the sample. The results indicate that the used conditions
Fresh tea leaves during the treatment increase in temperature did not exceed 10 C. This limited temperature
Non-thermal method rise provides a valid evidence that pulsed electric eld processing is a non-thermal method
applied under used conditions.
2016 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Corresponding author. Tel.: +49 421 218 63301; fax: +49 421 218 63302.
E-mail address: edwin.zondervan@uni-bremen.de (E. Zondervan).
http://dx.doi.org/10.1016/j.cherd.2016.03.010
0263-8762/ 2016 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
chemical engineering research and design 1 0 9 ( 2 0 1 6 ) 586592 587
2.2. Moisture content analysis number of pulses (N) from 10 to 50. The total treatment time
was dened as the product of the number of pulses and the
Fresh tea leaves were plucked 1 day before shipment to Europe pulse width applied to the system (Fig. 1b). Apart from the
(The Netherlands) and they were stored at 5 C until required. treatment time, the pause between two pulses (PBP) is the
Before treatment, leaves were allowed to reach the ambient interval between two pulses and represents the relaxation
temperature of approximately 20 C. To determine the mois- time.
ture content, fresh leaves were freeze-dried and subsequently The temperature was measured both at the inlet and outlet
subjected to a hot-air oven at 105 C. Moisture content was of the treatment chamber. In all experiments, the increase in
7580 wt%. Samples that were used for the moisture content the temperature due to the treatment never exceeded 10 C.
analysis were separated from the samples used for PEF treat- All experiments were duplicated.
ment. For all experiments, thin slices of fresh tea leaves were
used (approximately 1 cm width). 2.5. Specic energy input
2.3. Natural extraction without electrical treatment: The pulse shape (square wave bipolar) was monitored on-line
control experiments with an oscilloscope (Model RTM 2000) during the treatment.
For a square-wave pulse, the specic energy input for each
For the control experiments without electrical treatment, PEF-treated sample was calculated on the basis of Eq. (3):
slices of fresh tea leaves (untreated, m = 20 g) were placed into
a cylindrical glass beaker. Distilled water (200 g) was added
n
ttreatment
n
at 20 C, and then diffusion was studied. A careful agita- Wspec = U(t) I(t) dt (eq.3)
m
tion at 250 rpm was provided. The concentrations of the total 0
0
polyphenols in water ware measured after 20 min. It is possi-
ble to determine the length of experiments, using the Fourier The total specic energy input was calculated by multiply-
number to calculate how much time is needed to reach steady ing the energy delivered per pulse with the number of pulses
state. At that point in time, the extracted amount of polyphe- applied into the system.
nols is in equilibrium with the rest of polyphenols in the spent Wspec is the specic energy input (in kJ/kg). The energy per
leaves: pulse (in kJ) is calculated from the power of the pulse (Ppulse )
multiplied by pulse duration (s). Ppulse is the result of out-
Dd t
F0 = (eq. 1) put voltage U(t) and the total electric current I(t) supplied to
R2
the sample on the basis of Ohms law. n is the pulse number
where D is the diffusion coefcient (m2 /s), t the time (s) and applied to the system (dimensionless) and m is the total weight
R the radius of the particle (m). of sample (kg) charged to the treatment chamber for the PEF
When the Fourier number reaches Eq. (1), steady state treatment applied.
is achieved. The time necessary to reach equilibrium is the
time needed for our experiments. The equilibrium can be 2.6. Determination of extraction yieldmeasurement
represented with the following mass balance. Here d in the of total polyphenols content
subscript represents the dispersed PPs (in spent leaves) and c
the continuous phase (water) at time t = 0 and t = . The disintegration of the cellular membrane was detected
indirectly by a total polyphenols (PPs) content measurement.
Vd (cd,0 cd, ) = Vc (cc, cc,0 ) (eq. 2) Based on the fact that the cellular membrane is ruptured,
transport of the cell material together with the polyphenols
where V is the volume (m3 ) and c the concentration (kg/m3 ). occurred and the extraction yield is dened as a fraction
of extracted PPs. After the treatment, samples were stored
2.4. Pulsed electric eld treatments at ambient temperature and left in an aqueous solution for
20 min (extraction time). Upon extraction, the leaves were sep-
Tea leave samples were treated using the pulsed electric eld arated from the aqueous solution. The amount of extracted
(PEF) equipment with a batch treatment conguration of the PPs was measured in the solution. The total amount of phe-
Nutri-Pulse NP110-60 System (IXL Netherlands B.V.) which nols was determined by directly reading the absorbance at
consists of a PEF treatment chamber and a high-voltage gen- 725 nm (SpectraMax 190 Absorbance Microplate Reader, USA)
erator. The batch treatment chamber (100 mm length 70 mm of diluted samples 1/10 (v/v). The total amount of PPs was
width 50 mm height, 350 mL capacity) consists of two par- expressed as gallic acid equivalents (GAE) by means of a cor-
allel stainless steel electrodes of 5 mm thickness separated responding calibration curve with standard gallic acid. The
by a distance of 20 mm (see Fig. 1a). A high-voltage genera- extraction yield of PPs was calculated according to Eq. (4):
tor provided rectangular pulses in the range of 0.1 103 0.1 s
with a maximum voltage of 2.2 kV and a maximum number extraction yield of PPs
of pulses 50. Samples were placed in the treatment chamber
polyphenols content (aquoues solution)
between the two stainless steel electrodes lled with a ster- = 100% (eq.4)
polyphenols content (fresh tea leaves)
ile salt solution (Fig. 1a). The conductivity of the sterile salt
solution was adjusted to correspond with the conductivity of
the sample ( = 3.5 mS/cm). NaCl as a salt was used for the
preparation of the salt solution. After cutting, the leaves were 2.7. Statistical analysis
subjected to various PEF treatments. All experiments were
carried out using an electric eld strength (E) ranging from All experiments and measurements of characteristics were
0.1 to 1.1 kV/cm, pulse duration (PD) from 0.1 103 0.1 s and repeated at least twice. A one-way analysis of variance was
chemical engineering research and design 1 0 9 ( 2 0 1 6 ) 586592 589
Fig. 1 Experimental set up (lab scale). Scheme of PEF treatment chamber (a) and PEF pulse protocol (b).
used for statistical analysis of the data using the Statgraphics membrane. Therefore, when PEF-treated leaves were in con-
Centurion XVI. For each analysis, a signicance level of 5% tact with an aqueous solution after 20 min of extraction time,
was assumed. The error bars presented in the gures corre- their color changed in yellowish brown. This phenomenon
spond to the standard deviations. occurs due to the transport of cellular material into the sur-
rounding aqueous solution and the reaction of PPs catalyzed
3. Results and discussion by the enzyme polyphenol oxidase (PPO). PPO is located in the
cytoplasm of the plant cells and PPs are in organelles called
There are several techniques for extraction of PPs from tea vacuoles. When PPO and PPs are brought into contact, oxi-
leaves such as microwave-assisted extraction (Nkhilli et al., dized PPs are produced as result of enzymatic oxidation. The
2009; Pan et al., 2003) solvent-based extraction (Meterc et al., formation of oxidized PPs was identied by visual observa-
2007) and (Dong et al., 2011), molecular distillation (Tang tion: a change in color of the solution during the oxidation
et al., 2011) and ultrasonication (Koiwai and Masuzawa, 2006). reaction occurs. Furthermore, UV spectrophotometer analy-
Microwave-assisted extraction uses hot/boiling water and in sis was used to determine the total amount of PPs by reading
addition, irradiation power of 600 W (Nkhilli et al., 2009). PPs, the absorbance at 725 nm.
proteins and other valuable components from fresh tea leaves
are sensitive to high temperatures. Usually, ethanol is used 3.1. Characterization of pulsed electric eld process
as solvent for extraction of PPs from tea. In a later stage, and electric measurements
ethanol is removed from the process. However, there could
still be some residue of ethanol in the nal product. In the In the experiments performed, electrical measurements, i.e.,
proposed process, no additional solvent is used and due to voltage and current, were recorded for each PEF experiment at
complete absence of ethanol, the proposed process is more regular time intervals. Fig. 2 shows an example of a recording
effective compared to the solvent-based extraction method. of voltage (V) and current (mA) using an oscilloscope.
In this work, we will focus on the pulsed electric eld method. In addition, in the experiments carried out in this study
Fresh tea leaves were subjected to pulsed electric eld next to the electrical measurements (voltage and current),
(PEF) treatments. Extraction of polyphenols occurred as a temperature and resistance were also monitored and recorded
result of pore formation and disintegration of the cellular at regular intervals during each experiment. Fig. 3 shows a
2500 600
500
2000
400
Current, mA
1500
Voltage, V
300
200
1000
100
500
0
0 -100
-2.00E-04 -1.00E-04 0.00E+00 1.00E-04 2.00E-04 3.00E-04 4.00E-04 5.00E-04 6.00E-04
Time, s
Fig. 2 A recording of voltage (blue dotted line) and current (red dotted line) during a PEF experiment.
590 chemical engineering research and design 1 0 9 ( 2 0 1 6 ) 586592
10.00 21.90
8.00
21.70
Temperature, C
Resistance,
6.00
21.50
4.00
21.30
2.00
0.00 21.10
-1 1 3 5 7 9 11 13 15
Time, s
Table 1 Summary of PEF treatment conditions for each PEF experiment and the treatment impact on the changes in
electrical parameters and temperature.
Pulse duration, (103 , s) Number of pulses () Specic energy input (kJ/kg) Change in temperature ( C)
The total treatment time is equal to the product of the num- Koiwai, H., Masuzawa, N., 2006. Extraction of catechins using
ber of pulses and pulse width (duration). Experimental results ultrasound. Proc. Symp. Ultrason. Electron. 27, 483484.
showed that longer pulses which means a longer treatment Lebovka, N., 2009. Electrotechnologies for Extraction from Food
Plants and Biomaterials. Springer, New York.
time of 2.5 s were more effective for moderate electric eld
Lebovka, N.I., Bazhal, M.I., Vorobiev, E., 2000. Simulation and
(E = 0.4 kV/cm). Moreover, to achieve the same extraction yield experimental investigation of food material breakage using
of 27% but with a shorter total treatment time of 1.5 s, a higher pulsed electric eld treatment. J. Food Eng. 44 (4), 213223.
electric eld (E = 0.9 kV/cm) is required. Also a total treatment Lebovka, N.I., Shynkaryk, M.V., El-Belghiti, K., Benjelloun, H.,
time of 5 s (for both electric eld strengths), the experiment Vorobiev, E., 2007. Plasmolysis of sugarbeet: pulsed electric
was unfortunately not possible due to limitation of the PEF elds and thermal treatment. J. Food Eng. 80 (2), 639644.
unit. Meterc, D., Petermann, M., Weidner, E., 2007. Extraction of green
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ACKNOWLEDGEMENTS Monfort, S., Gayn, E., Saldana, G., Purtolas, E., Condn, S., Raso,
J., lvarez, I., 2010. Inactivation of Salmonella typhimurium and
The work was nancially supported by the Institute for Staphylococcus aureus by pulsed electric elds in liquid whole
Sustainable Process Technology under the project FO-10-06 egg. Innov. Food Sci. Emerg. Technol. 11 (2), 306313.
Nkhilli, E., Tomao, V., El Hajji, H., El Boustani, S., Chemat, F.,
Selective Opening and Fractionation. The authors would like
Dangles, O., 2009. Microwave-assisted water extraction of
to acknowledge Biobased Research Wageningen University, green tea polyphenols. Phytochem. Anal. 20, 408415.
especially to Dr. Hennie Mastwijk and ir. Ariette Matser for Pan, X., Niu, G., Liu, H., 2003. Microwave-assisted extraction of tea
valuable input and feedback. polyphenols and tea caffeine from green tea leaves. Chem.
Eng. Process Intensication 42 (2), 129133.
Parsija, D., Anandharamakrishnan, C., 2015. Techniques for
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