Application of Pulsed Electric Field (PEF)

Techniques in Food Processing

Francesca De Vito

Unione Europea UNIVERSITÀ DEGLI

STUDI DI SALERNO

FONDO SOCIALE EUROPEO Programma Operativo Nazionale 2000/2006

“Ricerca Scientifica, Sviluppo Tecnologico, Alta Formazione” Regioni dell’Obiettivo 1 – Misura III.4

“Formazione superiore ed universitaria”

Department of Chemical and Food Engineering

Ph.D. Course in Chemical Engineering (IV Cycle-New Series)

APPLICATION OF PULSED ELECTRIC FIELD (PEF)

TECHNIQUES IN FOOD PROCESSING

Supervisor Ph.D. student Prof. Giovanna Ferrari Francesca De Vito Scientific Referees Prof. Giorgio Donsì Prof. Vincenzo Tucci Prof. Petr Dejmek Ph.D. Course Coordinator Prof. Ernesto Reverchon

v

Acknowledgments

I wish to thank the scientific advisors of this PhD thesis i.e. Prof. Giorgio Donsì, Prof. Giovanna Ferrari and Prof. Vincenzo Tucci, for their professional support and scientific advices.

A special acknowledgment is for Prof. Petr Dejmek, who introduced me in this research field at Lund University and gave me precious suggestions and encouragements.

I’m very grateful to Dr. Maria Rosaria Vincensi for her cooperation and precious help in the experimental work..

I’d like to thank Eng. Raffaele Raimo and Eng. Biagio De Vivo for their technical support.

At the end my best thanks to all the members of my research group i.e. Dr. Maria Carmela Bruno, Eng. Luigi Esposito, Eng Paola Maresca, Eng. Gianpiero Cataro and Raffaele Taddeo for their friendship.

To my beloved grandfather

Publications

Fincan, M., De Vito, F., and Dejmek, P. Pulsed electric field treatment for solid-liquid extraction of red beetroot pigment, Journal of Food Engineering, 64, 381-388, 2004.

De Vito, F., Donsì G, and Ferrari, G. Effects of Pulsed Electric Field on permeabilization of cell membranes of fruits and vegetables. Proceedings. of INTRADFOOD, Innovations in Traditional Foods, Valencia (Spain), 2, 1515-1518, 25th-28th October 2005.

Contents

INDEX OF FIGURES V INDEX OF TABLES IX ABSTRACT 1 CHAPTER I INTRODUCTION 3 I.1 METHODS OF CELL MEMBRANE DISINTEGRATION IN FOOD

PROCESSING 3 I.2 THE PRINCIPLES OF PULSED ELECTRIC FIELD 5 I.3 PULSED ELECTRIC FIELD TECHNOLOGY 11

I.3.1 MAIN ELECTRICAL PARAMETERS OF PEF TREATMENTS 11 I.3.2 PEF PROCESSING SYSTEM 12

I.3.3 ENERGY CONSUMPTION AND INCREASE OF FOOD

TEMPERATURE DURING PEF TREATMENTS 21 CHAPTER II STATE OF THE ART 25 II.1 PLANT CELL AND TISSUE STRUCTURE 25

II.1.1 THE PROTOPLAST 25 II.1.2 THE CELL WALL 28

II.2 ELECTROPLASMOLYSIS IN PLANT TISSUE 28 II.3 DETECTION AND CHARACTERIZATION OF CELL

DISINTEGRATION IN VEGETABLE TISSUE 29 II.4 THE INFLUENCE OF THE ELECTRICAL PARAMETERS ON

PLANT MEMBRANE PERMEABILIZATION 32 II.5 USE OF PEF IN VEGETABLE PROCESSING 34

II.5.1 DRYING 34

II

II.5.1.1 EFFECT OF PEF ON MASS TRANSFER DURING AIR DRYING 34 II.5.1.2 EFFECT OF PEF ON OSMOTIC DEHYDRATION 35 II.5.2. EXTRACTION OF FRUIT AND VEGETABLE JUICES BY PEF AND MECHANICAL PRESSING 37 II.5.3 POTENTIAL APPLICATION OF PEF IN SUGAR BEET AND COCONUT PROCESSING 39

II. 6 PEF AND RECOVERY OF HIGH VALUABLE INTRACELLULAR

METABOLITES 41 II.7 TEXTURE AND STRUCTURE MODIFICATIONS INDUCED BY

PEF 43 II.8 PEF AND SOLID-LIQUID DIFFUSION 44 II.9 CONTINUOUS PEF TREATMENT IN SUGAR BEET PROCESSING

45 CHAPTER III PEF PERMEABILIZATION STUDIES 47 III.1 INTRODUCTION 47 III.2 THE EXPERIMENTAL APPARATUS 47

III.2.1 THE HIGH VOLTAGE PULSE GENERATOR 48 III.2.2 THE BATCH TREATMENT CHAMBER 52 III.2.3 THE SAFETY ENCLOSURE 53 III.2.4 THE OSCILLOSCOPE 53

III. 3 THE SYSTEM FOR IMPEDANCE MEASUREMENT 54 III.3.1 THE IMPEDANCE ANALYZER 54 III.3.2 THE SAMPLE HOLDER 55

III.4 MATERIALS AND SAMPLING 56 III.5 EXPERIMENTAL PROCEDURES 57

III.5.1 PULSED ELECTRIC FIELD TREATMENT 57 III.5.2 ELECTRICAL MEASUREMENT AND DATA ANALYSIS 57

III.6 RESULTS AND DISCUSSION 59 III.6.1 EFFECTS OF ELECTRIC FIELD STRENGTH ON TISSUE PERMEABILIZATION59 III.6.1.1 APPLE TISSUE 59 III.6.1.2 POTATO TISSUE 62 III.6.2 EFFECTS OF PULSE NUMBER ON TISSUE PERMEABILIZATION 64

CHAPTER IV PEF AS A PRE-TREATMENT STAGE IN DEHYDRATION OF PLANT

FOOD 59 IV.1 INTRODUCTION 59

Contents

III

IV.2 SAMPLE PREPARATION 59 IV.3 EXPERIMENTAL PROCEDURES 60

IV.3.1 PULSED ELECTRIC FIELD TREATMENT 60 IV.3.2 DRYING PROTOCOL 60 IV.3.3 CHARACTERIZATION OF SAMPLES AFTER DRYING 61 IV.3.3.1 MEASUREMENT OF BULK DENSITY 61 IV.3.3.2 MEASUREMENT OF PARTICLE DENSITY 61 IV.3.3.3 CALCULATION OF OPEN PORES POROSITY 62 IV. 3.3.4 REHYDRATION RATE 63 IV.3.3.5 TEXTURE ANALYSIS 63

IV.4 RESULTS AND DISCUSSION 63 IV.4.1 THE EFFECT OF PEF ON DRYING RATE 63 IV.4.2 THE EFFECT OF PEF ON SAMPLE POROSITY 65 IV.4.3 THE EFFECT OF PEF ON REHYDRATION RATE 66 IV.4.4 TEXTURE ANALYSIS 68

CHAPTER V PEF AS A PRE-TREATMENT STAGE IN EXTRACTION OF

ANTHOCYANINS 69 V.1 INTRODUCTION 69

V.1.1 ANTHOCYANINS: CHEMICAL COMPOSITION 69 V.1.2 ANTHOCYANINS : HEALTH EFFECTS AND PHYSICAL PROPERTIES 71 V.1.3 ANTHOCYANINS IN GRAPES 73 V.1.4 COMMERCIAL PREPARATION OF GRAPE SKIN EXTRACT: ENOCYANIN 74

V.2 MATERIALS AND METHODS 75 V.2.1. SAMPLE PREPARATIONS AND CHEMICALS 75 V.2.2 EXPERIMENTAL PROCEDURES 75 V.2.2.1 PEF TREATMENT 75 V2.2.2 ELECTRICAL IMPEDANCE MEASUREMENT 78 V.2.2.3 EXTRACTION 78 V.2.2.4 ANALYSIS 78

V.3 RESULTS AND DISCUSSION 79 V.3.1 COMPOSITION OF GRAPE EXTRACTS 79 V.3.2 THE EFFECT OF PEF ON TISSUE PERMEABILIZATION AND EXTRACTION OF ANTHOCYANINS 82

CHAPTER VI DESIGN OF A PEF CONTINUOUS SYSTEM 89 VI.1 PEF CONTINUOUS SYSTEM 89 VI.2 DESIGN OF A CONTINUOUS PEF TREATMENT CHAMBER 90

VI.2.1. PARALLEL PLATE CONTINUOUS TREATMENT CHAMBER 91

IV

VI.2.2 CO-FIELD TREATMENT CHAMBER 97 VI.3 FLUID-HANDLING SYSTEM 99 CHAPTER VII CONCLUSIONS 101 REFERENCES 105 SYMBOLS 113 APPENDIX 117

Index of Figures

Figure 1 Schematic diagram of a cell exposed to electric field (Zimmermann,1986). 8 Figure 2 Schematic diagram of reversible and irreversible membrane breakdown (Zimmerman, 1986). 10 Figure 3 Scheme of a pulsed electric field system for food processing. 12 Figure 4 Exponential decaying pulse generating circuit (Qin et al., 1994). 14 Figure 5 Exponential decaying pulse (Gongóra-Nieto et al., 2002). 15 Figure 6 Square wave pulse generating circuit (Qin et al, 1994). 16 Figure 7 a) Square pulse. b) Pseudosquare pulse (Gongóra-Nieto et al., 2002). 17 Figure 8 Bipolar pulse generating circuit (Qin et al., 1994). 18 Figure 9 Effective energy of pseudosquare and exponential decaying pulse useful for PEF processing purposes (Gongóra-Nieto et al., 2002). 22 Figure 10 Anatomy of a plant cell (Heldt, 1996). 26 Figure 11 Cell membrane (Wolfe,1985). 27 Figure 12 Equivalent circuit model of: a) intact and b) permeabilized plant cell. R1, R2 plasma membrane and tonoplast resistance, C1, C2, plasma membrane and tonoplast capacitance;R3 cytoplasmatic resistance surrounding the vacuole in the direction of the current ;R4 cytoplasmatic resistance in vacuole direction; R5 resistance of the vacuole interior; R6 resistance of the extracellular compartment (Angersbach et al., 1999.). 32 Figure 13 PEF laboratory scale apparatus. 48 Figure 14 Electrical parameters of pulses. 49 Figure 15 Electrical circuit of the pulse generator. 50 Figure 16 Panel control of the software HPVEE v. 5.0. 51 Figure 17 Plastic connection case. 53 Figure 18 Impedance analyzer (1260, Solartron). 55 Figure 19 Sample holder (1296 2A, Solartron). 56 Figure 20 Schematic representation of electrical impedance measurement. 58 Figure 21 Relative complex conductivity of apple tissue at E=0 and E=0,5 kV/cm for 10 pulses. 60 Figure 22 Relative complex conductivity of apple tissue at E=0,5 and E= 1 kV/cm for 10 pulses. 61

VI

Figure 23 Relative complex conductivity of apple tissue at E=1 and E= 1,5 kV/cm for 10 pulses. 61 Figure 24 Relative complex conductivity of apple tissue at E=1,5 and E= 2 kV/cm for 10 pulses. 62 Figure 25 Relative complex conductivity spectra of potato tissue as a function of the electric field strength at n=10 pulses. 63 Figure 26 Relative complex conductivity spectra of apple tissue as a function of the number of pulses at E= 1,5 kV/cm. 64 Figure 27 Relative complex conductivity spectra of potato tissue as a function of the number of pulses at E= 1kV/cm. 65 Figure 28 Electrical impedance of intact and PEF-treated apple tissue as a function of the frequency. 65 Figure 29 Electrical impedance of intact and PEF-treated potato tissue as a function of the frequency. 66 Figure 30 Complex conductivity spectra of intact apple and potato tissue. 67 Figure 31 Sampling region in apple tissue. 60 Figure 32 Dehydration curves of intact and PEF-treated apple discs aE=1,5 kV/cm and 10 pulses. 64 Figure 33 Dehydration curves of intact and PEF-treated apple discs at E=1,5 kV/cm and 100 pulses. 65 Figure 34 Dehydration curves of intact and PEF-treated apple discs at E=1,5 kV/cm and 500 pulses. 65 Figure 35 Rehydration curves of intact and PEF-treated apple discs at 10 pulses. 66 Figure 36 Rehydration curves of intact and PEF-treated apple discs at 100 pulses. 67 Figure 37 Rehydration curves of intact and PEF-treated apple discs at 500 pulses. 67 Figure 38 Six major anthocyanidins in foodstuff (Nyman and Kumpulainen, 2001). 70 Figure 39 Structural changes in malvidin-3-glucoside with pH (Timberlake, 1980). 72 Figure 40 Effects of pH on anthocyanin structures of malvidin-3-glucoside. A, B, C and AH+ refer to forms in Figure 39 (Timberlake, 1980). 73 Figure 41 Draw of the batch treatment chamber set-up. 77 Figure 42 Chromatogram of anthocyanin extract obtained from untreated grape berry samples at 520 nm. 79 Figure 43 Comparison in the anthocyanin composition of extract obtained from untreated grape berry and standard malvidin-3-glucoside. 80 Figure 44 Comparison in the anthocyanin composition of extract obtained from untreated grape berry and standard cyanidin-3-glucoside. 80 Figure 45 Comparison in the anthocyanin composition of extract obtained from untreated grape skin and standard malvidin-3-glucoside. 81 Figure 46 Comparison in the anthocyanin composition of extract obtained

Index of Figures

VII

from untreated grape skin and standard cyanidin-3-glucoside. 81 Figure 47 Comparison in the anthocyanin composition of extract obtained from untreated and PEF-treated grape berry. 82 Figure 48 Voltage and current traces during PEF treatment at 1 kV/cm at a) 1 pulse and b) 100 pulses. 83 Figure 49 Impedance spectrum of control and PEF-treated grape samples.

84 Figure 50 Comparison in the anthocyanin composition of extract obtained from untreated and PEF-treated grape skin at 1 kV/cm. 85 Figure 51 Comparison in the anthocyanin composition of extract obtained from untreated and PEF-treated grape skin at 3 kV/cm. 86 Figure 52 Scheme of a PEF continuous system to process food slurries. 89 Figure 53 Draw of the parallel plate treatment chamber. 96 Figure 54 Distribution of the electric field vector in the parallel plate treatment chamber. 97 Figure 55 Draw of the co-field treatment chamber. 98

VIII

Index of Tables

Table 1 Electrical pulses characteristics. 60 Table 2 Porosity of dry control and PEF-treated apple discs at E=1,5 kV/cm with different number of pulses. 66 Table 3 Maximum compressive force and energy in rupture test on control and PEF-treated apple discs at E=1,5 kV/cm with different number of pulses. 68 Table 4 Maximum compressive force energy in puncture test on control and PEF-treated apple discs at E= 1,5 kV/cm with different number of pulses. 68 Table 5 Anthocyanins in some fruits. 71 Table 6 Anthocyanins concentrations in grape berries extracts obtained before and after PEF at 1 kV/cm. 83 Table 7 Anthocyanins concentration in grape skin extracts obtained before and after PEF at 3kV/cm 86 Table 8 Electrical conductivities of food slurries. 93 Table 9 Characteristics of the parallel plate treatment chamber. 93 Table 10 Length of entrance and related variables. Fmin is the minimum flow rate. 94 Table 11 Simulation parameters. 96 Table 12 Characteristics of the co-field treatment chamber. 98

X

Abstract

Pulsed electric field (PEF) treatment is an innovative and promising method for non-thermal processing of foodstuff. It is a good alternative to conventional cell membrane permeabilization methods such as thermal treatments and the addition of chemicals as well as of enzymes.

In this thesis the effect of some process parameters on the cellular membrane permeabilization of different plant foods by means of Pulsed Electric Fields was studied. PEF treatment was carried out in a laboratory batch equipment set-up ad hoc. Plant tissues were exposed to rectangular pulses varying the intensity of the treatment.

The measurement of the electrical impedance of untreated and PEF-treated tissues as a function of the frequency was used to detect cell membrane permeabilization. The relative complex conductivity was also introduced to characterize the degree of permeabilization as a function of the applied electrical variables.

Permeabilization studies carried out on apple and potato tissue have demonstrated that the electric field strength and the number of pulses are the main process parameters determining the effectiveness of the treatment. The degree of permeabilization increased with increasing the electric field strength and the pulse number.

At a fixed number of pulses, a threshold level of tissue permeabilization is achieved. At 10 pulses, for potato tissue the electric field strength necessary for permeabilization was 1 kV/cm while a higher value was found for apple i.e. 1,5 kV/cm.

By means of the developed methodology, total permeabilization conditions were determined.

By applying critical electric field strength to potato and apple tissue, total permeabilization was achieved at 500 pulses.

The applicability of PEF as pre-treatment stage of conventional thermal drying was also investigated. Whatever the intensity of the treatment, the application of PEF determined the initial loss of water as a consequence of the rupture of the cell membrane. At 1,5 kV/cm with increasing the number of pulses from 10 to 100 a higher dehydration rate was detected, while no significant enhancement was observed at higher number of pulses. However,

2

the electrical treatment improved the dehydration rate only in the initial stage. The final water content was the same for untreated and PEF-treated samples.

Characterization tests were also carried out to investigate the effect of PEF on the quality of the final product. After PEF at 1,5kV/cm and 500 pulses a better rehydration of samples was achieved.

Texture measurements of the rehydrated samples revealed that PEF determined the softening of the tissue due to the loss of turgor caused by the permeabilization.

The use of PEF as a pre-treatment stage in the extraction of anthocyanins from grape tissue was also tested. The complete process was developed and the use of distilled water as solvent was proposed. The malvidine-3-glucoside, the major anthocyanin in the extract composition was identified. PEF allowed to preserve the integrity of pigments and to increase the extraction yield. For instance, treatment of grape skins after PEF at 3 kV/cm and 100 pulses the increase of 15% in the malvidine content was observed.

Chapter I Introduction

I.1 Methods of cell membrane disintegration in food processing

Cell membrane disintegration is defined as the irreversible breakdown of the cell membrane in order to increase its permeability and/or cause cell death (lysis).

The disintegration of the cell membrane is used in many fields such as biotechnology, cell biology, medicine and food industry.

Mass transfer processes such as solid-liquid extraction and drying as well as food preservation are important unit operations of the food industry requiring the disintegration of the cell membrane.

When the cell membrane of a microorganism is broken, cell lysis takes place and the inactivation of the microorganism occurs. This is the way to achieve food preservation and extending the shelf life of food products inactivating pathogens responsible of undesired food degradation.

On the other hand, cell membrane acts as a physical barrier in removing the intracellular substances (water, juices and solutes) from plant food tissues in solid-liquid extraction and drying. Thus, the disintegration or permeabilization of the cell membrane in a plant food tissue causes the release of intracellular water and solutes (secondary metabolites) to migrate in an external medium. This can be seen as an effective tool to promote mass transport processes, so that higher yields and shorter contact time can e achieved in the above mentioned unit operations.

According to these applications, the degree of the cell membrane disintegration is very important determining the efficiency of the process.

Presently, the rupture of the cell membrane can be obtained by means of several methods according to the desired degree of disintegration and to the particular application. It is possible to identify: thermal and non-thermal methods.

High temperature is used in food preservation (pasteurization/sterilization) and in pre-treatment and/or complementary stages before extraction processes (sugar beet processing) and drying (hot

Chapter I

4

water blanching). In this way it is possible to achieve a high degree of cell membrane breakdown, but due to the thermal denaturation of the cell membrane induced by heating, this treatment damages sensory properties (flavour, taste, smell and texture) and nutrients (vitamins), thus compromising the quality of the processed food.

The denaturation of the cell membrane can be avoided by means of non-thermal techniques including mechanical pressing, addition of chemicals and use of enzymes. Mechanical pressing is commonly used in many food extraction processes (juice, oil and wine production). In this case the breakdown of the cell membrane is achieved by means of the mechanical action of pressure. However, normal moderate pressure doesn’t assure a high degree of disintegration, thus in most cases a low extraction yield is obtained. Therefore, a combination with thermal method is required.

The addition of enzymes is largely used in juice processing to increase the extraction yield. Due to the reactions induced by biological catalysts, this method reduces the quality of juices and requires additional operating costs of post-processing stages to remove enzymes and by-processing products.

The use of chemicals such as alkaline (NaOH) or acid (HCl) solutions is suitable for pre-treatment of vegetable and fruit tissues in drying. As for the use of enzymes, also in this case additional operation costs of post-processing stage are necessary to remove residuals. In alternative to the above mentioned methods, non-thermal cell membrane modification is possible also by means of High Intensity Pulsed Electric Field (HIPEF) i.e. the application of high intensity electric field (kV/cm) in form of repetitive very short pulses (pulse duration ranges between few µs up to ms).

HIPEF is an innovative technology in food processing. In fact, the application of HIPEF can be proposed for the inactivation of vegetative bacteria and yeasts at ambient or even low temperatures (≥4°C) and non-thermal disintegration (permeabilization) of cell membranes in plant food (fruit and vegetable) tissue. In both cases, minimum changes of the food take place at low energy consumption. Even if a high electric field strength is applied, this technique involves a very short treatment time (usually less that 1s) resulting in a negligible ohmic heating of the processed food.

Due to these advantages, in recent decades the use of HIPEF in food processing has gained attention from universities, research units as well as food private companies. Some groups identify HIPEF as HELPs (High Intensity Electric Field Pulses) or shortly PEF (Pulsed Electric Field).

The use of PEF for inactivation of vegetative microrganisms has been extensively studied. Successful results were obtained in processing of several liquid foodstuff including apple juice, milk, liquid eggs, and pea soup (Barbosa-Cánovas et al., 1993; Zhang et al., 1994a; Barbosa-Cánovas et al., 1995; Qin et al., 1995).

Introduction

5

Only in recent years, this technique has been proposed not only for non-thermal pasteurization of liquid foodstuff but also as mild technology for more careful and controlled modification of plant based food tissue.

The employment of PEF has been investigated and reported to be very promising in the recovery and production of commercially interesting and high value metabolites (natural pigments, sugar) from food (Brodelius et al., 1988; Dörnerburg and Knorr, 1993; Fincan et al., 2004; Eshtiaghi and Knorr, 2002), the improvement of fruit an vegetable juices yield in solid-liquid extraction (McLellan et al., 1991; Knorr, 1994; Bouzrara and Vorobiev, 2000; Ade-Omowaye et al., 2001d; Rastogi, 2003), intensifying diffusion (Jemai and Vorobiev, 2002; El-Belghiti and Vorobiev, 2005) and acceleration of mass transport in drying processes (Angersbach and Knorr, 1997; Rastogi et al., 2000; Ade-Omowaye et al., 2001a; Ade-Omowaye et al., 2001c; Teijo et al., 2002; Taiwo et al., 2001).

In addition, other applications of PEF have been also explored, including biosynthesis of microbial metabolites of commercial interest (Knorr et al., 2001), infusion of solutes in foods (Barsotti et al., 1999), decontamination of wastewater (Castro et al., 1993), pre-treatment of milk for cheese-making (Sepulveda-Ahumada et al., 2000) and biofouling prevention in cooling water (Abou-Ghazala and Schoenbach, 2000). Moreover in the food processing area the inhibition of enzymes activity by means of PEF has been also studied. Whereas some researchers reported a very high efficiency of the technology in the inactivation of some enzymes, others (Hamilton and Sale, 1967) report no significant effect of PEF on the enzymatic activity. However, due to the significant differences existing in the enzymes structure the results suggest that some enzymes are more sensitive to PEF than other.

I.2 The principles of Pulsed Electric Field

Exposing a biological cell (plant, animal and microbial) to a high intensity electric field (kV/cm) in form of very short pulses (µs to ms) induces the formation of temporary or permanent pores on the cell membrane.

This phenomenon, named electroporation, causes the permeabilization of cell membrane i.e. an increase of its permeability and if the intensity of the treatment is sufficiently high, cell membrane disintegration occurs.

The mechanism of electroporation is not yet fully understood. Several models have been suggested to explain this complex phenomenon based on: increase in the treansmembrane potential, electromechanical compression of the cell membrane and conformational changes in the lipids or protein molecules.

Zimmermann et al. (1974) proposed the so called “dielectric breakdown theory”.

Chapter I

6

The model considers the cell membrane as a capacitor filled with a dielectric material of low permittivity 2=mε (i.e. lower than the corresponding values existing inside and outside the cell).

The main cause of cell membrane destabilization is attributed to the increase of the “so called” transmembrane potential.

Cell membrane maintains an electrical and chemical gradient, the electrochemical gradient, between the outside and the inside of the cell. The electrical gradient is due to the excess of negative ions accumulating immediately at the inner surface of the cell membrane and to an equal number of positive ions accumulated immediately outside the cell. This establishes across the cell membrane a transmembrane potential, typically of 0,1 V, called the resting potential (Guyton, 1986; Weaver 1993).

When the external electric field E is applied, the ions inside the cell move along the field up to the membrane surface. As a result, free charges accumulate on both sides of the membrane (Zimmermann et al., 1976).

The accumulation of these surface charges results in an increase of the potential difference across the membrane. The electric field induces an additional transmembrane potential ∆ϕ, larger than the cell’s natural potential rϕ∆

The induced transmembrane potential (defined as the difference between intra and extra cellular potentials) is unevenly distributed over the surface of cell membrane, that is the potential difference is high on the membrane sites close to the electrodes and the low as the distance between the membrane sites and the electrodes increases:

( ) ( )⎥⎥

⎦

⎤

⎢⎢

⎣

⎡−−=

⎟⎟⎠

⎞⎜⎜⎝

⎛−

m

t

cm MCosErFMEt τϑεϕ exp1,,∆ (I.1)

where ϕ∆ is the transmembrane potential, M is the point of interest on

the membrane, F is the shape factor, E is the applied electric field strength, cr is the cell radius,ϑ is the angle between the direction of E and M , t is the time after the field is turned on; mε is the relative permittivity of the membrane evaluated from the following Equation:

( )( ) ( )iemimme

iem r σσσ

δσσσσ

σσε

+⎟⎠⎞

⎜⎝⎛+++

=222

2 (I.2)

where eσ , iσ and mσ are the electrical conductivities of the external medium, the cytoplasm and the cell membrane, respectively.

Introduction

7

mτ is the characteristic time constant, called relaxation time of membrane, defined by the Equation:

⎟⎟⎠

⎞⎜⎜⎝

⎛+=

eimcm Cr

σστ

211

(I.3)

where mC is the membrane capacitance per unit area (Teissiè and Rols,

1999). Assuming that the membrane charging time is much smaller than the

pulse duration (i.e.τm<<t) Equation (I.1) can be reduced to a simple, time-independent form:

ϑεϕ coscm ErF=∆ (I.4)

If the membrane is considered as a pure dielectric (i.e. 0=mσ ), 1=mε

and Equation (I.3) becomes:

ϑϕ coscFEr=∆ (I.5) For a spherical cell the shape factor F is equal to 1.5:

ϑϕ cos5.1 cEr=∆ (I.6) At the poles of cell membrane (i.e. ϑcos =1), the transmembrane

potential (TMP) assumes its maximum value maxϕ∆ :

cEr5.1max =ϕ∆ (I.7) Breakdown of the cell membrane occurs when the overall transmembrane

potential (sum of the induced potential and the resting potential) reaches a threshold value, that is the critical transmembrane potential cϕ∆ :

ccc rE5.1=ϕ∆ (I.8)

where cE is the critical electric field strength (Fig.1).

Chapter I

8

Figure 1 Schematic diagram of a cell exposed to electric field (Zimmermann,1986).

For an elongated cell, F =0.5:

El5.0max =ϕ∆ (I.9) where l is the length of the cell and the electric field is parallel to the cell

(Grahl and Märkl, 1996). Studies on the TMP and on the environment surrounding the cell

membrane have shown that the charges generated on the membrane surfaces are of opposite sign and attract each other. This attraction gives rise to a compression pressure that causes the decrease of the membrane thickness. (Zimmermann et al., 1976).

The electro-compressive force, Pe, per unit area of the membrane is given by:

dxEddP me

20

0 21

εεδ

δ

∫−= (I.10)

where δ is the thickness of the membrane, E is the electric field, and 0ε is the dielectric constant of the empty space. If the compressive force is independent on the position x, Eq. (I.9) becomes:

2

20

2δϕεε ∆m

eP = (I.11)

The compression of the membrane creates elastic strain forces. Assuming that the cell membrane is an ideal elastic material, the mechanical restoring force ( mP ) per unit area is given by:

Introduction

9

0

lnδδYPm = (I.12)

where Y is the elastic modulus of the membrane and 0δ is the initial unstrained membrane thickness. When the electrocompressive force exceeds the membrane elastic strength membrane breakdown or pore formation occurs. The critical potential difference for electromechanical breakdown of the membrane cϕ∆ is given by:

0

203679,0

εεδ

ϕm

cY

=∆ (I.13)

With much greater field strengths, larger and more areas of the membrane are subjected to breakdown. If the size and number of pores become large in relation to the membrane surface, irreversible breakdown associated with mechanical destruction of the cell occurs (Figure 2).

The breakdown voltage was found to be approximately 1 V for many types of cells (Ho and Mittal, 1996).

Electroporation determines the increase of the cell membrane permeability because new channels on the cell membranes (i.e. pores) are created.

Depending on the electric field strength, duration and number of pulses applied the induced membrane breakdown and subsequent permeabilization can be reversible or irreversible (Benz and Zimmermann, 1980).

In reversible breakdown, small size and transient pores are formed that reseal when the electric field is turned off. Electroporation is considered a dynamic process with three stages: pore formation, pore expansion and pore reasiling. Reversible permeabilization of cell membrane is studied and used in biotechnology for the transfer of genetic materials (DNA) inside bacterial cells as well to improve fusion of cells (Chang et al., 1992). Electroporation have been used also in biomedicine to allow the permeation of cytotoxin through the membranes of cancerous cells.

In 1987, Okino and Mohri for the first time showed that electropermeabilization could be used in vivo to increase the concentration of the anticancer agent in solid tumours.

In vivo, electroporation used to increase the chemotherapy efficiency in cancer treatment has been termed electrochemotherapy (ECT) (Miklavcic et al., 1998). The first clinical trial using ECT on head and neck tumours are reported by Belehhradek et al. (1993).

Irreversible permeabilization is investigated in food science.

Chapter I

10

Figure 2 Schematic diagram of reversible and irreversible membrane breakdown (Zimmerman, 1986).

Introduction

11

I.3 Pulsed Electric Field Technology

I.3.1 Main electrical parameters of PEF treatments

During a PEF treatment, the food material is placed between two electrodes forming a treatment chamber and high voltage repetitive pulses are applied across the system in order to achieve membrane breakdown. The processing time or total duration of the treatment is defined as:

pnt τ= (I.14)

where τp is the pulse duration and n is the number of the pulses applied to

the food. Equation (I.14) shows that the treatment time increases either with the

number of pulses or with the pulse duration. It is important to observe that increasing the number of pulses, the total energy consumption increases, while with increasing the pulse duration the temperature of the food arises (see section. I.3.3).

During treatments, the food material is in close contact with the electrodes and, therefore, is subject to an electric field. Assuming the sample homogeneous (i.e. permittivity constant across the sample), the average electric field strength E is as follows:

dVE = (I.15)

where V is the voltage across the food sample and d the distance between

the electrodes. From Equation (I.15) it can be seen that with increasing the gap a higher

voltage is required to obtain the desired electric field (i.e. critical electric field corresponding to the membrane breakdown).

The design of the treatment chamber (i.e. the geometry and the dimension of the electrodes) determines a more or less uniform distribution of the field inside the food. The more uniform is the electric field the more homogeneous will be the treatment.

Besides the number of pulses and pulse duration, (i.e. total treatment time), a PEF treatment is defined by other electrical parameters such as: the pulse shape and the pulse repetition rate i.e. the number of pulses in 1s.

Chapter I

12

I.3.2 PEF processing system

A PEF system for food processing in general consists of three basic components: a high voltage pulse generator, a treatment chamber and a control system for monitoring the process parameters.

Figure 3 Scheme of a pulsed electric field system for food processing.

I.3.2.1 High voltage pulse generator

The high voltage pulse generator provides electrical pulses of the desired voltage, shape and duration by using a more or less complex pulse forming network (PFN). More in detail, a PFN is an electrical circuit consisting of several components: one or more DC power supplies, a charging resistor, a capacitor bank formed by two or more units connected in parallel, one or more switches, and pulse-shaping inductors and resistors.

The DC power supply charges the capacitors bank to the desired voltage. Using this device, the ac power from the utility line (50-60Hz) is converted in high voltage alternating current (AC) power and then rectified to high-voltage dc power (Zhang et al., 1995). Depending on the required voltage, the generation of the high dc voltage can be achieved with different devices most commonly using transformers ad rectifiers. The energy provided by the power supply is temporarily stored in the capacitor(s) and then delivered very quickly, i.e. in form of pulses, to the food to generate the necessary electric field strength. The total power rating of the power supply limits the maximum number of times that the capacitor can be charged and discharged

Treatment chamber

Intactproduct

Treatedproduct

High Voltage pulsegenerator

Control system

Treatment chamber

Intactproduct

Treatedproduct

High Voltage pulsegenerator

Control system

Introduction

13

in a given interval time. Moreover the power required to the power supply for charging the capacitor bank depends on the electrical resistance of the charging resistor and on the size and number of capacitors charged: a larger capacitor will take more time and/or more power to be charged than a smaller one. Similarly, a smaller charging resistor will speed up the charging process but increases the power requirement. Power supply of the order of 1.5kW of average power is commonly used for PEF laboratory scale equipment.

The energy stored in the capacitor bank cW depends on its capacitance C0

and the charge voltage U :

202

1 UCWc = (I.16)

According to Eq. (I.12), a low capacitance capacitor can store less energy

than a larger one when both are charged at the same level. Due to the losses only an aliquot of this energy is transferred to the food

samples. The discharge of the energy stored in the capacitor(s) is accomplished

through the switch closed by the applying a high voltage trigger signal. The switch is the most important and delicate component of the pulse generator.

It must be able to operate reliably at high power and repetition rate. Many devices may be used as discharge switch. They can be classified in

two main groups: ON switches and ON/OFF switches. ON switches are devices able to connect the discharging capacitor system and the PFN, that show a lack of ability to interrupt the connection as the voltage level remains high. If these devices are turned on, it is not possible to turn them OFF until the voltage drops below a certain level. This kind of devices is useful when complete discharge of a capacitor is desired. Generally speaking, ON devices can handle higher currents at a higher voltages (~100KV up to 1 MA) at relatively low cost. However, low pulsing frequencies, short life span and the difficulty of turning ON and OFF frequently limit the utilization of this kind of devices. Most of these devices work by ionizing gas or vapour confined between two electrodes to promote conduction of the main current. Some examples are the Ignitron, Gas Spark Gap, Trigatron and Thyratron.

ON/OFF switches, on the other hand, can be easily turned ON and OFF that improves the control of the pulses generation. This type of devices allows the direct generation of square pulses with a power supply, and can also be used to partially or completely discharge capacitors through pulse forming networks. ON/OFF switches were considerably developed in the last few years, thanks to the development in the field of solid-state pulsed power. Semiconductor solid-state switches are considered the most

Chapter I

14

convenient option for future applications of the PEF technology. Solid-state switches have a very large operation life span when compared to other type of switches, have better performance, are easier to handle, do not require mechanical components (electrodes or gases), allow higher pulsing frequencies and have low switching and conducting losses; their price also tends to drop, which is common with semiconductor operated equipment. A drawback of this type of switches is that they can usually handle only very limited amount of current at relatively low voltages (~1,2 kV, 1 kA). Thus, it is necessary to use several units connected in series and parallel to increase the switch capacity, causing a significant increase in the price of the unit. Examples of solid-state switches are the gate turn-off (GTO) thyristor, the insulated gate bipolar transistor (IGBT) and the symmetrical gate commutated thyristor (SGCT).

Due to the connections of the single components, the voltage across the electrodes doesn’t increase instantaneously. The electrical wiring as well as the closing rate of the switch determine the voltage rise across the electrodes. It may take less than a microsecond.

As the switch discharge the voltage from the capacitor(s), the PFN is used to modify the shape and the length of the pulses.

Electrical pulses can be generated with different wave shapes including exponentially decaying, square waves and oscillating pulses. Moreover, they can be of constant polarity (monopolar pulses) or alternate polarity (bipolar pulses).Monopolar exponential decaying and monopolar and bipolar square wave pulses are the most common in the application of PEF in food technology.

The simplest PFN is a RC (resistance/capacitance) circuit producing an exponential decaying pulse (Fig.4).

Figure 4 Exponential decaying pulse generating circuit (Qin et al., 1994).

Introduction

15

In a monopolar exponential decaying pulse, the voltage across the treatment chamber rises rapidly to a set point 0V and then decays slowly with time t according to the following Equation:

)/(

0 exp)( τtVtV −= (I.17) where 0V is the initial charging voltage of the capacitor bank and τ is the

time constant defined as: RC=τ (I.18)

where R and C is the load resistance and C is the total circuit

capacitance, respectively.

Figure 5 Exponential decaying pulse (Gongóra-Nieto et al., 2002). The resistance of the electrodes is negligible with respect to that of the

food sample. Similarly the capacitance of the food sample is negligible compared to that of the capacitor(s), therefore it can be assumed that:

0CR f=τ (I.19)

Chapter I

16

where fR is the food resistance and 0C is the capacitance of the capacitor bank.

In a RC circuit, i.e. an exponential pulse, the pulse duration τp is approximately equal to τ5 (Cogdell, 1999).

Since the time constant represents the time during which the voltage across the electrodes decreases from the initial value V0 to V0/e or to 37% V0, it can be practically assumed that all the energy of the pulse is delivered during the first constant time. Therefore, several researchers considered the time constant τ as the real pulse duration (Zhang et al., 1995; Grahl and Märkl, 1996; Barsotti et al., 1999). Other researchers, instead, use the total pulse duration (Knorr and Angersbach, 1998).

In theory, the generation of a square wave pulse requires a transmission line. The use of a transmission line to generate square pulses has two different problems. First of all, it is difficult and inconvenient to match the impedance of the food material with the characteristic impedance of the transmission line. In addition, the real transmission line (i.e. a coaxial cable) is not suitable for short pulses (i.e. pulse duration is in the microseconds range).

These problems can be overcome using the PFN consisting of arrays of capacitors and inductors, in which the inductors-capacitors sections emulate the transmission line (Fig.6).

Figure 6 Square wave pulse generating circuit (Qin et al, 1994).

In a square pulse the voltage across the electrodes increases to a peak

voltage remaining constant during all the pulse duration. Thus, a constant

Introduction

17

a ba b

electric field is applied to the food. In practice, this condition is guaranteed only when the impedance of the pulse forming network matches the impedance of the food to be processed. In practice, this is very difficult especially for chambers with low resistance (<50 Ω). Therefore the wave shape is distorted and a pseudo square pulse is produced.

The real pulse shape approximates the ideal one when the impedance of the treatment chamber and of the PFN are equal.

Figure 7 a) Square pulse. b) Pseudosquare pulse (Gongóra-Nieto et al., 2002).

Due to its simplicity and flexibility (for example, pulse duration can be

varied changing the discharging capacitors of the PFN), several exponential pulse generators have been designed and used to study inactivation of bacteria and enzymes as well as plant tissue permeabilization (Brodelius et al., 1988; Dörnerburg and Knorr, 1993; Zhang et al., 1994b; Pothakamury et al., 1996; Angersbach and Knorr, 1997; Rastogi et al., 1999; Angersbach et al., 2000; Ade-Omowaye et al., 2001a; Ade-Omowaye et al., 2001b; Angersbach et al., 2002; Esthiaghi and Knorr, 2002; Taiwo et al., 2002).

Although the PFN producing square pulses is more complex and expensive that the exponential pulses circuit, square wave pulses are more efficient in cellular permeabilization. For this reason, square pulse generators have also been used in PEF applications (Sale and Hamilton, 1967; Zhang et al., 1994b; Bazhal and Vorobiev, 2000; Bouzhara and Vorobiev, 2000; Lebovka et al., 2000; Lebovka et al., 2001; Jemai and Vorobiev, 2002; Lebovka et al., 2002).

Using multiple switches in the same basic circuit configurations, it is possible to generate bipolar pulses (Figure 8). Due to their high cost, bipolar

Chapter I

18

pulses has not been extensively studied. In a study of Qin et al. (1994) on the inactivation of microorganisms in liquid foodstuffs by different waveforms, bipolar pulses were demonstrated to be more efficient than monopolar ones offering the advantage of minimum energy utilization, reduced deposition of solid on electrodes surfaces and reduced food electrolysis.

In the design of pulse generators is necessary to include a control system to protect the device from unexpected faults during the treatment. In fact, electrical components, and in particular the switch, can be seriously and irreversibly damaged when processing food samples with too high or too low resistivity, with respect to the resistivity limits permitted by the electrical circuit. For example, if the resistivity of the food sample is too low, there will be an increase of the current intensity crossing the sample and this may cause an overheating of the switch, instead of the food, unless a current limiting device is employed in the circuit. To overcome this problem, generally a resistor is added to the circuit in series with the treatment chamber.

A food with high resistivity keeps a high voltage across the switch. Thus, to protect the device a shunting resistance is added to the circuit in parallel with the treatment chamber.

Figure 8 Bipolar pulse generating circuit (Qin et al., 1994).

I.3.2.2 Treatment chamber

The other major component of a PEF unit is the treatment chamber, used to transfer electrical voltage pulses to food. It basically consists of two

Introduction

19

electrodes. Generally, for safety reasons is better to connect one electrode to the voltage source (i.e. the pulse generator) and the other to the ground. However, systems with floating electrodes (i.e. both connected to the pulse generator) are also used.

An insulating material holds the electrodes in fixed positions and forms the chamber containing the food to be processed. The insulator, in fact, acts as a spacer between the electrodes, protects the food from the undesired effects of electrodes and even more important is its function of minimizing ohmic losses.

Treatment chambers can be static (batch) or continuous. Static chambers are mostly used in laboratory scale studies for PEF treatments of both liquid and solid products. Continuous chambers, instead, are more suitable for pilot plant and industrial scale applications.

The proper design of the treatment chamber is important in order to assure the effective cellular permeabilization during the process. Many aspects have to be considered such as geometry, size and construction materials.

In order to be able to carry out the treatment, the material used as insulator of the treatment chamber should have dielectric strength exceeding the applied electric field intensity.

A part from this the principal criterion to be followed in the design of the treatment chamber is that the dielectric breakdown of the processed food should be avoided. Dielectric breakdown consists of the generation of a spark inside the chamber (Barbosa-Cánovas et al., 1995).

A local enhancement of the electric field intensity as well as the presence of air bubbles can cause dielectric breakdown of foods. Therefore, this drawback, should be prevented:

by choosing a geometry of the treatment chamber that can

achieve as better as possible a uniform distribution of electric field,

introducing in the PEF apparatus prior to treatment a food degassing system to prevent or eliminate air bubble formation.

Moreover, materials selected to build up the treatment chamber have to

be washable (cleaning in place) or autoclavable and chemically inert with respect to foods.

According to the different choices, several static treatment chambers have been proposed for processing both liquid (Sale and Hamilton, 1967; Dunn and Pearlman, 1987; Mizuno and Hori, 1988; Grahl et al., 1992; Zheng-Ying and Yan, 1993; Zhang et al., 1995) and solid foodstuff (Bouzhara and Vorobiev, 2000; Angersbach et al., 2000; Bazhal and Vorobiev, 2000; Angersbach and Knorr, 1997; Fincan and Dejmek, 2002).

Chapter I

20

Stainless steel represents the most suitable choice for the electrodes and has been extensively used (Dunn and Pearlmann, 1987; Zhang et al., 1995; Bouzrara and Vorobiev 2000; Angersbach et al., 2000; Bazhal and Vorobiev, 2000; Esthiaghi and Knorr, 2002; Fincan et al., 2004).

Anyway, the use of other electrochemical inert material including carbon, gold, platinum and metal oxides has been reported for the electrodes or electrode surfaces (Bushnell et al., 1993; Angersbach and Knorr, 1997; Jemay and Vorobiev, 2002). The use of carbon-brass electrodes has also been reported (Sale and Hamilton, 1967; Grahl et al., 1992).

Insulator materials used are: polythene (Sale and Hamilton, 1967), polypropylene (Bouzrara and Vorobiev, 2000); nylon (Dunn and Pearlman, 1987); polysulfone (Zhang et al., 1995), Plexiglas (Grahl et al., 1992; Zheng-Ying and Yan, 1993; Angersbach and Knorr, 1997; Esthiaghi and Knorr, 2002); PVC (Mizuno and Hori, 1988; Angersbach et al., 2002).

Several geometries of the electrodes have been proposed. Parallel plate electrodes with a small gap between them provide a

uniform electric field and represent the most practical choice of batch chambers (Dunn and Pearlmann, 1987; Mizuno and Hori, 1988; Angersbach and Knorr, 1997; Rastogi et al., 1999; Angersbach et al., 2002; Fincan et al.,2004). Disk-shaped and round edged electrodes can minimize the electric field enhancement reducing also the possibility of dielectric breakdown (Zhang et al., 1995). Polished electrode surfaces reduce the risk of dielectric breakdown of the food to be processed (Angersbach et al., 2000).

Besides parallel plate other examples of continuous configurations are concentric cylinder, concentric cone, converged electric field and co-field treatment chamber (Bushnell et al., 1993; Qin et al., 1996; Yin et al. 1997; Matsumoto et al., 1991).

I.3.2.3 Control system

Electrical and process parameters can be monitored by the control system consisting of two major devices: an oscilloscope and a temperature probe.

The oscilloscope measures the voltage across the treatment chamber and shows the output voltage shape. A current probe connected with the treatment chamber interfaced with the oscilloscope measures, also, the current flowing trough the food. In order to minimize electromagnetic interference, the oscilloscope should be placed in a shielded area.

Samples temperature is measured with a thermal probe placed inside the treatment chamber.

Introduction

21

I.3.3 Energy consumption and increase of food temperature

during PEF treatments

The energy consumption of a PEF treatment is an important parameter in maintaining the non-thermal characteristics of the treatment. This parameter depends on the number of pulses n and on the energy delivered in each pulses pW :

nWW p= (I.20)

The energy pW dissipated in each pulse is:

∫τ

=p

dttpW0

)( (I.21)

where p(t) is the instantaneous power and time t = 0 corresponds to the

time when the circuit switch is closed. The instantaneous power p(t) is defined as:

)()()( titutp = (I.22) where )(tu is the instantaneous voltage across the electrodes and i(t) is

the instantaneous current crossing the food sample in the treatment chamber. Thus Equation (I.21) becomes:

∫=p

dttitvWp

τ

0)()( (I.23)

As can be seen from Eq. (I.23), the energy in a PEF treatment is

determined by sample properties (i.e. resistivity/conductivity, size) and pulse characteristics ( i.e. wave shape, width, peak voltage).

Compared to exponential pulses, square pulses allow the optimization of energy consumption, due to the fact that irreversible permeabilization and/or breakdown of cells occurs only at a threshold value of the electric field.

In fact, exponential decay pulses show a long tail with a low electric field, during which only heat is generated in the food without bactericidal and/or permeabilization effects. On the other hand, square pulse maintains a

peak voltage for a longer time than the exponential decay pulse. Since both waveforms cause the breakdown of cells, square wave pulses

allow energy saving (Fig.9).

Chapter I

22

Figure 9 Effective energy of pseudosquare and exponential decaying pulse useful for PEF processing purposes (Gongóra-Nieto et al., 2002).

Introduction

23

More often the specific energy or energy density Q is considered in literature, defined as follows:

vW

Q p= (I.24)

where v is the volume of the treatment chamber. Other researchers refer to the energy per unit mass of sample mQ :

ff

p

f

pm

WmW

Qνρ

== (I.25)

where m, fρ and fν are mass, density and volume of the food sample,

respectively (Angersbach and Knorr, 1997; Rastogi et al., 1999; Bazhal and Vorobiev, 2000; Ade-Omowaye et al., 2001b).

The total electrical energy input W is discharged in the food in the form of Joule heating causing an increase of the sample temperature. According to this, when the pulse duration is high, the treatment chamber should be equipped with a cooling system in order to keep the sample temperature below an acceptable value according to the non-thermal features of PEF.

The maximum temperature increase ∆T after each pulse can be calculated by an energy balance under the condition that all the energy is provided to the sample in the absence of external losses (i.e. no cooling is provided):

TCmW pfp ∆= (I.26)

where pC is the specific heat of the food in the treatment chamber. Measurement of ∆T should be used to determine the energy dissipated in

the food sample, but the measurement of this parameter is not always possible and often is not accurate. In practice, Equation (I.26) is used to calculate the temperature increase of the processed sample.

Introducing the energy density discharged in each pulse Q and the number of pulses delivered to the food n, the temperature increase is given by:

pCnQT =∆ (I.27)

(Barsotti et al., 1999).

Chapter I

24

Chapter II State of the art

II.1 Plant cell and tissue structure

Plant food tissue is a biological material with complex structure. Tissue consists of layers of an array of horizontal and vertical units i.e. plant cells, separated by the extracellular fluid. Plant food tissue contain different cells among which the most common is the parenchyma. Parenchyma cells are polyhedral or spherical in shape. Their size can vary from 10 up to 100 µm. Depending on the type, spatial arrangement and relative shapes of constituent cells, plant tissue shows a variable amount of intercellular air space (1-25%) (Jackman and Stanley, 1995). While parenchyma cells of different fruit and vegetable differ in size, they all have essentially the same fundamental anatomy. A plant cell mainly consists of a protoplast and a cell wall. A detailed description is presented in the following sections.

II.1.1 The protoplast

Electron microscopy techniques allowed the identification of structural details of the protoplast and revealed the architecture of the cell wall.

The protoplast represents the protoplasm i.e. the living portion of the cell. It includes a complex mixture of proteins, lipids and other substances in colloidal suspension as well as some compounds dissolved in solution. The protoplast consists mainly of the cytoplasm and the nucleus. The cytoplasm consists of a crystalline, fluid-viscous material in which cellular organelles as well as membrane systems float. These include the mitochondria, golgi bodies, endoplasmic reticulum, ribosomes and plastids. The water content of cytoplasm is high (60-90%). The dry solid content (40-50%) consists of proteins. A second class of substances forming the cytoplasm are lipids (2-3% of the dry solid matter). The nucleus is the more evident structure in the protoplast. It is a spherical or ovoidal body surrounded by the nuclear membrane holding the genetic material (DNA). The cytoplasm is separated

Chapter II

26

from the cell wall by a thin cell membrane also called plasmalemma.(Fig.10).

Figure 10 Anatomy of a plant cell (Heldt, 1996).

The main function of the cell membrane is to regulate the flow of

substances from the outside to the interior of the cell. The cell membrane surrounding each cell is a very thin, only 7.5-10 nm thickness, elastic structure almost entirely composed of lipids and proteins. The approximate membrane composition is 55% proteins, 25% phospholipids, 13% cholesterol and 7% other lipids and carbohydrates (Guyton, 1986; Tortora and Grabowski, 1996). The lipid bilayer is an oil-like film, only 2 molecules thickness and consists almost entirely of cholesterol and phospholipids. The outer part of the bilayer is hydrophilic (water soluble) and the inner part is hydrophobic, i.e. only soluble in fats. Because the hydrophobic (fatty) portions are repelled by water but are mutually attracted to each other, they tend to line up occupying the centre of the bilayer while the hydrophilic portions face the water surrounding the cell. The membrane lipid bilayer is a major barrier impermeable to common water-soluble molecules such as ions, glucose, urea and others (Guyton, 1986). The “so-called” integral proteins floating in the lipid bilayer protrude through the membrane, creating channels through which water-soluble substances, especially ions, can diffuse. Cell membrane structure is shown in Fig.11.

State of the art

27

Figure 11 Cell membrane (Wolfe,1985). On the other hand, fat-soluble substances, such as oxygen and alcohols,

can penetrate the lipids portion easily. Of course, substances essential for the function and metabolism of the cell are able to enter the cell through the membrane, and waste products or harmful substances must be able to leave the cell. The mechanisms of removing undesired materials through the membrane without expending energy (by ATP splitting) are called passive processes (e.g. diffusion and facilitated diffusion by integral proteins) and include transportation of water, O2, CO2, N2, K+, Ca2+, Cl- and urea. Substances, crossing the membrane against the concentration gradient, require active processes. These are ATP-consuming processes and are mainly mediated by ATP-driven pump proteins that "push" ions e.g. Na+, K+, Ca2+, I- and Cl- across the membrane.

Plant cells also present one or more vacuoles. The vacuoles are cavities containing an aqueous liquid named vacuole sap in which many substances are dissolved in high concentration. These substances are salts and organic molecules, such as sugar and amino acids. Often soluble pigments such as anthocyanins are located in the vacuoles. The vacuole is surrounded by another membrane, the tonoplast structurally equivalent to the cell membrane with a higher lipids content. As the cell membrane, the tonoplast plays an important role in the active transport and retention of ions in the

Chapter II

28

vacuole. In this way, a higher concentrations of ions, compared to the cytoplasm are dissolved into the vacuole sap. Young cells contains several small vacuoles and as the cells grow the size of the vacuoles increases due to coalescence of small vacuoles to form a big unit. The vacuole often constitutes the 90% of the total cell protoplast and the protoplasm is reduced to a thin layer pressed against the cell wall.

II.1.2 The cell wall

The cell wall makes the difference between plant and animal cells. It determines the shape of the cell and prevents also its rupture due to the increase of vacuole’s volume. The wall of parenchyma cells is thin (0,1-10 µm) and is formed by two layers: the primary wall and the middle lamella. In most of the cells a secondary layer, named secondary wall, is present. The primary wall is an elastic thin layer formed before and during cells growing. After cell growing has finished, the secondary wall is formed inside the primary wall. The walls of two adjacent cells are joined together by means of the middle lamella. The major component of the cell wall is cellulose. Long chains of cellulose are assembled in form of microfibrils. The network of microfibrils (i.e. alternating layers of microfibrils) is dispersed in a aqueous matrix consisting of hemicellulose and pectines, chemically similar to hemicellulose. Sometimes also lignin can be present. The primary and secondary wall differ for composition. A part of cellulose, hemicellulose and pectines, primary lamella contains glico-proteins. The latter substances don’t appear in the secondary lamella, which also exhibits a much higher cellulose content. Due to the highly ordered arrangement of cellulose, the cell wall has crystalline properties and its architecture determines the texture of the plant cell.

II.2 Electroplasmolysis in plant tissue

During PEF treatment of a plant tissue the voltage is applied to the whole tissue. Since conductivity of the intracellular juice is significantly higher than the conductivity of the plasmatic membranes surrounding the cells, up to 95% of the voltage applied across the tissue samples drop at the cellular membranes.

The electric field in the cell membrane at the poles mE can be expressed as the ratio between the induced transmembrane potential ϕ∆ and the thickness of the cell membrane δ according to:

δϕ∆

=mE (II.1)

State of the art

29

The mean size of a plant cell is between 10-100 µm and the cell membrane thickness is about 10 nm. Therefore, from the definition of the transmembrane potential, the electric field strength in plant cell membranes is 103 -104 times higher than the electric field strength applied to the whole tissue. This can explain the irreversible plant membrane permeabilization (i.e. electroplasmolysis).

From Equation (I.8), the critical transmembrane potential is attained with external electric field decreasing with the cell radius. Due to the size of a vegetable cell, usually bigger than that of microorganism, i.e. mean size between 10nm and 1 µm (Rogers et al., 1980), the electric field strength to achieve the electroplasmolysis of vegetable material is lower than that necessary for inactivating microorganisms. The values of the electric field strength required for electroplasmolysis in plant cells and tissues range from 0,5 kV/cm up 5 kV/cm (Knorr, 1999) whereas electropermeabilization of microbial cells is reported to occur between 10 and 50 kV/cm (Barbosa-Cánovas et al., 2000).

II.3 Detection and characterization of cell disintegration in

vegetable tissue

The early attempts in studying the degree of cell permeabilization was based on quantifying the release of intracellular metabolites (i.e. pigments) from plant cultured cells after electroporation induced by the application of Pulsed Electric Field (Brodelius et al., 1988; Dörnerburg and Knorr ,1993).

The irreversible permeabilization of cells in vegetable tissues has been demonstrated, for the first time on potato tissue (exposed to PEF treatment), determining the release of the intracellular liquid from the treated tissue using a centrifugal method. From PEF–treated samples a liquid leakage from the tissue was detected while no-release occurred from control samples. Thus, this leakage has been interpreted as a consequence of the cellular damage induced by the electrical pulses inside the cells of the tissue (Angersbach and Knorr, 1997).

Due to fact that electrical complex conductivity of a biological tissue, such as a food material, is sensitive to the mobility of ionic species in the tissue, the determination of the frequency spectra of this variable before and after PEF treatment has been proposed as another and more reliable method to detect cell permeabilization in food tissue (Anghersbach et al., 1997). Electrical impedance of the tissue was measured before and after PEF. More in detail, the electrical conductivity )(ωk for intact and processed samples was obtained from impedance spectra, according to the following equation:

Chapter II

30

)()(

ωω

jZAl

ks

s= (II.2)

where sl is the length of the sample, As is the area perpendicular to the

electric field, j is the complex number and Z(jω) is the total system impedance.

The angular frequency ω is related to the circuit frequency f by:

f2πω = (II.3)

The two impedance or conductivity frequency-spectra were determined with frequency between 1 kHz and 100 MHz i.e. in the so called β-dispersion range. The presence of two non-conductive membranes (the plasma membrane and the tonoplast) of a vegetable cell in the intact tissue, determines the dependence of the whole vegetable tissue impedance on the frequency. When exposed to an alternate voltage (i.e. electric field), the process of repeated charging of these two membranes, produces a typical AC frequency trend of the electrical conductivity and this change is visible in a frequency range between 1 kHz and 100 MHz.

The impedance-frequency spectra were determined with an impedance measurement equipment according to the following experimental procedure: the sample was placed between two stainless steel electrodes and a square wave voltage signal of alternate polarity with fixed amplitude (1 V peak to peak ) and variable frequency (3 kHz up to 50 MHz) was applied. Thus, impedance was determined by the indirect measurement of the electrical current flowing into the sample.

By comparison of the two spectra, an increase of the electrical conductivity of the tissue after PEF treatment was observed. In particular a drastic change at low frequency range was evident, while in the high frequency range the conductivity value of intact and PEF-treated tissue were practically the same. The significant increase observed at low frequency ranges has been explained by the authors as the evidence of the irreversible induced permeabilization of the plant membrane. In an intact tissue, in fact, the value of electrical conductivity is related to the concentration of ionic species in the external medium between the cells, being the other ions kept inside the cell by the cell membrane. After irreversible permeabilization of cell membranes, ions are free to migrate from the cells to the extracellular region determining an increase of the current flowing in the sample and, therefore, of the electrical conductivity.

The coincidence of the values of the electrical conductivity if treated and untreated samples at high frequency has been explained according to the fact that the cell membrane do show any resistance to current flow at high frequency.

State of the art

31

To quantify the cellular degree of permeabilization, a coefficient Zp, the cell disintegration index, has been defined on the basis of the measurement of the electrical complex conductivity of intact and permeabilized tissue in the low and high frequency range:

( )( )lh

'l

'h

p KKKK

b1Z−−

−= (II.4)

'h

h

KK

b = (II.5)

10 ≤≤ pZ (II.6) where lK and '

lK are the electrical conductivities of untreated and treated materials in the low frequency range (1-5 kHz), respectively;

hK and 'hK are the electrical conductivity of untreated and treated materials

in the high frequency range (3-50 MHz), respectively. The disintegration index characterizes the proportion of damaged

(permeabilized) cells in the plant tissue. For intact cells 0=pZ and for total

cell disintegration 1=pZ . The definition of the cell disintegration index was developed on the basis of an electrophysiological model of intact and processed tissue suggested by Angersbach et al. (1999).

According to this model, the tissue consists of m elementary layers put in series and each layer contains intact and permeabilized cells, regularly distributed, as well as extracellular compartments.

The single intact cell present in a layer, is electrically equivalent to a combination of resistors (cytoplasm and extracellular medium) and capacitors (the plasma membrane and tonoplast). A permeabilized cell, instead, is represented by a different circuit in which, due to the breakdown of the membranes, the capacitors are replaced by additional resistors. (Fig.12).

Furthermore, based on the electrical conductivity measurement and the above mentioned model, the damage percentage area was defined as follows:

⎟⎟⎠

⎞⎜⎜⎝

⎛−

⎟⎟⎠

⎞⎜⎜⎝

⎛−

=treatS

ltreatSl

IShIS

h

treatSltreatS

h

ISh

m

p

IF

,,

,,

,,

,

2

σσσ

σ

σσσ

(II.7)

Chapter II

32

where ISl

,σ , ISh

,σ , treatSl

,σ treatSh

,σ are the conductivities of intact and treated sample at low and high frequencies( Angersbach et al., 2002).

Figure 12 Equivalent circuit model of: a) intact and b) permeabilized plant cell. R1, R2 plasma membrane and tonoplast resistance, C1, C2, plasma membrane and tonoplast capacitance;R3 cytoplasmatic resistance surrounding the vacuole in the direction of the current ;R4 cytoplasmatic resistance in vacuole direction; R5 resistance of the vacuole interior; R6 resistance of the extracellular compartment (Angersbach et al., 1999.).

Another cell disintegration index was proposed by Lebovka et al. (2002)

according to the definition of Rogov and Gorbatov (1974): ( )

( )id

iZ p σσ

σσ

−

−= (II.8)

where σ is the measured electrical conductivity value at low frequency

(1-5KHz) and the subscripts “i” and “d” refer to the conductivities of intact ant totally destroyed tissue, respectively. As in the previous case 0=pZ for

intact tissue and 1=pZ for totally disintegrated material.

II.4 The influence of the electrical parameters on plant membrane

permeabilization

Electroplasmolysis induced by pulsed electric field depends on the electrical parameters of the processing system. In PEF treatments, in fact, electric field strength, number, duration and shape of pulses can be varied and this affects the treatment intensity and, consequently, the degree of permeabilization achieved.

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Electric field strength and number of pulses are the main parameters determining permeabilization induced by PEF.

Permeabilization induced by PEF was studied in terms of different parameters, among which the total energy density Q. The estimation of this parameter was carried out in relation to the pulse shape.

For exponential pulses Q was approximated according to:

fRnV

Q2

20 τ= (II.9)

where fR is the resistance of the food sample (Geulen et al, 1994; Esthiaghi and Knorr, 2002).

The method to determine fR was reported by Knorr and Angersbach

(1998). A ballast resistance of known value bR was placed in parallel to the treatment chamber and the total resistance of the system sR (parallel of bR and fR ) was determined by the time constant τ and the capacitance of the

system’s capacitors 0C :

00 5CCR p

s

ττ== (II.10)

where the pulse duration pτ was obtained from the pulse shape recorded by the oscilloscope.

The resistance fR was, then, determined as a function of bR and sR :

( )RRRR

Rb

bf −= (II.11)

For rectangular pulses, the total energy density was evaluated in a

different way:

∑=n

ipjUIQ τ (II.12)

where n is the total number of pulses, U is the applied voltage, Ij is the

value of electric current after each pulse (Bazhal and Vorobiev, 2000). There is an energy saturation level, beyond which further energy increase

has no influence on PEF application, thus reducing the treatment efficiency. Maximum electroplasmolysis rate was reported at an energy input of 3-5kJ/kg for apple tissue (Bhazhal and Vorobiev, 2000), 14-16 kJ/kg for potato

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34

samples (Knorr and Angersbach, 1998) and 60-70 kJ/kg for sugar beet samples (Bhazhal, 1998).

II.5 Use of PEF in vegetable processing

The potential application of PEF in solid food processing has been investigated in several studies. In these investigations PEF has been associated with extraction and drying processes of fruit and vegetables as a non-thermal pre-treatment alternative. However, the degree of efficiency obtained using PEF as well as the optimization of the electrical variables were different in each application.

II.5.1 Drying

II.5.1.1 Effect of PEF on mass transfer during air drying

High intensity pulsed electric field pre-treatment is reported to improve mass transfer during drying of several vegetable and fruit tissues.

Angersbach and Knorr (1997) showed that HELP pre-treatment of potato tissue improved mass transfer during air drying in a fluidized bed drier (air temperature 70°C, air velocity 2 m/s). The treatment chamber consisted of a Plexiglas cuvette in which two parallel plate carbon electrodes (surface area 140 cm2) have been placed. The sample was placed between the electrodes and air in the chamber was eliminated by filling the cuvette with tap water covering the surface of the electrodes and, thus, exponential pulses were applied.

The effect of electric field strengths in the range 0,35 to 3,0 kV/cm and number of pulses between 1-70 on the degree of permeabilization of potato cubes (1×1×1 cm) was evaluated detecting the release of the intracellular liquid after centrifugation (700×g for 10 min).

Optimum conditions could be achieved at field strengths between 1,5 and 3,0 kV/cm and number of pulses between 15 and 30.

At low specific energy (6,4 up to 16,2 kJ/kg) the maximum liquid release was observed (29%). The temperature increase of treated product was only of 1,8-4,5 °C in this case. Electrical treatment with optimal parameters (number of pulses between 5-30 pulses and electric field strength 0.9-2 kV/cm) accelerated drying of potato samples. At E = 1.1 kV/cm and 25 pulses the drying time of potato cubes was reduced to 1/3 with respect to the control. However, total energy input higher than 10 kJ/kg doesn’t improve drying rate.

Ade-Omowaye et al (2001b) studied the influence of PEF on red paprika dehydration in a fluidized bed dryer (at 60 °C for 6 h and air velocity of 1m/s). Red paprika slices (length 1 cm) were pre-treated with different methods including water blanching (boiling water for 3 min), skin treatments

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(5% w/v NaOH solution at 25 °C or 35 °C for 20 min, and 5% v/v HCl solution at 25 or 35°C for 20 min ), high hydrostatic pressure (400 MPa for 10 min at 25 °C) and pulsed electric field (exponential decay pulses, peak field strength = 2,4 kV/cm, pulse duration = 300 µs, number of pulses = 10). After each pre-treatment mass and heat transfer coefficients during the constant rate period of drying were evaluated. The transfer coefficients obtained after PEF treated samples were comparable with the those corresponding to the maximum value, measured after blanching. The cell disintegration index (Equation I.27) was evaluated for all pre-treatments. Among non-thermal processes, PEF induced the highest index of 0,61, while the absolute maximum was 0,88 achieved by hot water blanching, due to thermal disintegration effects. The permeabilization induced by PEF determined the enhancement of mass transport during drying. In fact, PEF pre-treated samples showed a reduction of approximately 25 % of drying time compared with control samples. The total specific energy consumption was 3.0 kJ/kg and the temperature increase due to PEF treatment was less than 1°C.

II.5.1.2 Effect of PEF on osmotic dehydration

Pulsed Electric Field has also been suggested as a method of pre-treatment to accelerate mass transfer during osmotic dehydration.

Rastogi et al. (1999) investigated the effect of PEF on the osmotic dehydration of carrot. Carrot discs (2 cm of diameter, 1 cm of thickness) were pre-treated by PEF at different level with exponential pulses, 5 pulses and a total specific energy input in the range of 0,04 to 2,25 kJ/kg. The electric field strength was in the range between 0,22 to 1,6 kV/cm and pulse duration between 378 up and 405 µs. The temperature rise in all the experimental conditions was <1 °C. The cell disintegration index Zp detected was 0,09 with increasing the total energy up to 0,84-0,86 kJ/kg. Above this value, no further increase of cell membrane disintegration was detected. Both PEF-treated and untreated samples were osmotically dehydrated (immersion in 50° B sucrose solution at 40 °C for 5 h). PEF pre-treated samples showed a decreased moisture content as well as an increased solid content during osmotic dehydration. Water and solute diffusion rates increased with increasing the applied electric field strength. The effective diffusion coefficients of water and solute, determined using Fickian diffusion model, increased exponentially with the applied electric field strength E with two coefficients A and B according to:

⎟⎠⎞

⎜⎝⎛−

= EB

expAD (II.13) where D is the diffusion coefficient.

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The authors attributed the rise of the effective diffusion coefficient to the increase of cell permeability. The permeabilization induced by PEF also resulted in the softening of the tissue. The compressive strength required for samples rupture was measured using a texture analyzer of 25 Kg load cell. The compressive strength decreased exponentially with increasing the applied electric field strength. However, above 1,09 kV/cm further softening was very limited.

The influence of HIPEF and other pre-treatment methods on the osmotic dehydration of apple slices was investigated by Taiwo et al. (2001). HIPEF treatment (exponential decay pulses, 20 pulses, duration of 800µs and electric field strength of 1,4 kV/cm) was carried out on apple discs (diameter 38 mm, thickness 8mm) placed between two parallel stainless steel electrodes in tap water. The specific energy per pulse in the tissue was 0,15 kJ/kg. Osmotic dehydration was performed by the immersion of samples in 50 °B sugar solution at 40 °C for 6 h.

The other pre-treatments were blanching (direct immersion of samples in hot water at 60°C in a water bath for 5 min), freezing/thawing (freezing at -28°C overnight of samples sealed in polyethylene bags followed by thawing at room temperature for 1 h and 30 min) and High Pressure (400MPa for 20 min at 25°C).

For all treatments studied, water loss increased with OD time. PEF-treated samples showed a higher water loss compared to untreated tissue. Water loss after PEF was comparable to that obtained with other pre-treatment studied such as high pressure and blanching. The enhanced water loss was attributed to the increased permeability of the cell membrane after PEF treatment. The effect of HIPEF treatment on solid gain was minimal. Similar values were obtained for both PEF-treated and untreated samples being lower than those of high pressure, freezing and blanching treated samples. In a later report, Taiwo et al., 2002 investigated the effect of PEF in combination with osmotic dehydration (OD) on the rehydration characteristics of apple discs at different temperatures (24-90°C). In this study, apple discs (diameter 38 mm, thickness 8 mm) were treated by PEF (exponential pulses, 20 pulses, duration of 400 µs and electric field strength of 1 kV/cm) and osmotic dehydration (immersion in 50 °B sugar solution at 40 °C for 4 h). The specific energy consumption during PEF treatment was 4,8 kJ/kg. Different treated samples (control, control + OD, HELP but no OD, HELP + OD) were dried in air drier at 80 °C for 27 h. Rehydration was carried out with distilled water at the following temperature and time combinations: room temperature 24° C, (1-6 and 24h); 45°C (1-6 h, and 16h); 60°C (0,5-5 h) and 90°C (10-60 min.). The authors reported that the combination of PEF and osmotic dehydration improved the rehydration capacity (RC) of apple samples. PEF-treated samples without OD showed the lowest rehydration in the whole temperature range. The low rehydration capacity of PEF-treated without OD samples was attributed to their

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shrinkage due to faster water loss during air drying, as a result of the increased membrane permeabilization. The enhanced rehydration capacity for PEF+OD treated samples was attributed to reduced compactness of the structure due to the absorbed sugar.

II.5.2. Extraction of fruit and vegetable juices by PEF and

mechanical pressing

The increase of the yield of juice extraction from fruit and vegetables by cellular permeabilization using high intensity pulsed electric fields was studied by many authors. The use of PEF as a pre-treatment stage before pressing and in combination with the mechanical operations was investigated. Reports shown also that the application of pulsed electric fields affected positively sensory and quality characteristics of the extracted juices.

The pre-treatment of finely or coarsely ground carrots with HEFP followed by expression (10 MPa, room temperature, 5min) enhanced the extraction of juice. PEF treatment of fine particles (average size of 1,5 mm) with electric field strength of 2,6 kV/cm resulted in a maximum juice yield of 76,1% compared with 51,3 % for the control. For coarse particles (average size of 3.0mm) the juice yield after PEF was 70,3% and 30% for the control samples. Variation of the number of pulses at a given electric field strength (1,6 kV/cm) indicated that an increase above 50 pulses did not alter juice yield substantially. Evaluation of selected quality indexes showed that HEFP treated carrot mash resulted in lighter colored, less oxized products with higher β-carotene values than samples pre-treated with commercial pectinase (Rohament P, 80 min at 45°C and pH 4,5). Comparison of dry matter, pH values and total acidity of the resultant juice revealed almost identical spectra for the control samples and HEFP-treated ones, while enzyme-treatment resulted in higher levels of dry matter, lower pH values and higher total acidity than control. Maximum juice yields for the enzyme-treated juices were 68,8% for finely ground samples and 61,8% for coarsely ground samples (Knorr et al., 1994).

Bazhal and Vorobiev (2000) investigated the effect of pulsed electric field treatment in combination with pressing on apple juice extraction yield. The authors proposed a new process of solid-liquid expression assisted by PEF in which PEF was applied as an intermediate treatment after mechanical precompression of the samples. In this study apple cossettes (obtained by grinding of uniform sub-lots of apples with a 6 mm grater) were treated in a laboratory filter-press cell with a monopolar rectangular pulse generator. A layer of cossettes of 2 cm was placed in the pressure cell and compressed between two electrodes: a steel plate used as stationary electrode and a mobile wire gauze electrode. The following experimental procedure was applied: a first stage of cossettes pressing, followed by PEF treatment in

Chapter II

38

combination with another pressing step, and a final pressing of the PEF-treated cake. Parameters adopted for PEF treatment were: 1000 pulses with a voltage of 1000 V, a pulse duration of 100 µs and a period of 10 ms. The authors studied the effect of pressure on total juice yield (at the end of the cake pressuring step), and pressure was varied between 0 bar up to 30 bars, before and after PEF treatment. The same pressure value was used in each different pressurizing step. PEF treatment of cossettes leaded to an increase in total juice yield depending on the value of the compressive pressure. At a pressure of 30 bar, PEF treatment resulted increased the juice yield referred to the first step pressing juice of about 12-13 % . The total amount of juice obtained at this pressure was very close to that obtained at 2-3 bar, where the PEF treatment increased the yield by about 40%. The maximum juice yield corresponded to a minimum energy input of 3-5 kJ/kg.

Similar results on the increase of the yield were obtained, in this study, applying pressing in combination to electro osmotic treatment. The pressure cell was equipped with a DC electric field generator. The experiments were performed in this way: a first stage of pressing followed by the application of a direct current (successively 50, 100 and 200 mA for 30 min each) in combination with a second pressing stage. For both the pressing steps, the same previous conditions were applied as described above. Calculation of the total energy consumption (i.e. sum of the energy consumption for pressing and that for the electrical treatment) for both the electrical treatments, revealed that the energy consumption was 50-100 times lower in case of PEF treatment.

The application of pulsed electric field also resulted in an improved quality of the extracted juice. Absorbance measurements of the juice, revealed that the juice obtained from the second pressing and PEF at 3 bar was clearer even than the juice obtained from pressing at 30 bars, while electro osmosis (EO) produced a darkest juice (as a result of happened ohmic heating and thermal denaturation). A further comparison of pH and electrical conductivity of the juice, showed that EO induced a considerable increase in both these parameters, while with PEF only a negligible effect was observed. Thus, on the basis of the results obtained, the authors suggested that the use of PEF in combination of pressing steps at 3 bar, could be a suitable method of increasing apple juice yield, affecting positively the quality of the extracted juice.

A similar test was conducted by Bouzrara and Vorobiev (2000) using sugar beet cossettes, to investigate the effect of PEF in combination with pressing on juice yield. The same set-up, consisting of a laboratory filter–press cell equipped with a monopolar rectangular pulse generator, was used. The protocol consisted of: initial pressing for 20 min. (pressure between 0.5 up to 30 bars), followed by PEF treatment (1000 pulses, 1000 V, 100 µs and a pulse period of 10 ms) and final pressing at the same pressure of the previous stage for 30 min. Application of PEF increased the juice yield at all

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studied pressures. Increasing pressure between 0.5 up to 3 bars resulted in the maximum yield increase of about 54%. The total juice yield was 78%. PEF treatment allows to obtain a juice of a good quality. Juice exhibited a higher sugar content, the absence of pectic substances (galacturonic acid) and less colour than the product obtained after pressing stage.

II.5.3 Potential application of PEF in sugar beet and coconut

processing

Eshtiaghi and Knorr (2002) investigated the possibility of using high

intensity electric field pulses (HELPs) in sugar beet processing as an alternative to conventional thermal treatments. Sugar beet slices (diameter 2.0 cm and thickness 1.20 cm) were treated by exponential pulses with variable electric field strength and number of pulses and a repetition frequency of 1 Hz. The electrical treatment was performed with stainless steel electrodes (total area of 140 cm2) spaced of 3.8 cm placed in a Plexiglas cuvette with tap water (conductivity 0.75 mS/cm).

The dependence of the cell disintegration index either on the electric field strength from 1,2 kV/cm up to3,6 kV/cm or on the number of pulses in the range 1-200 has been studied. The cell disintegration index increased rapidly with increasing field strength in the early stage i.e. from 1,2 kV/cm up to 2,4 kV/cm and then more slowly from 2,4- 3,6 kV/cm.

Increasing the pulse number from 1 to 5 an increase of the cell disintegration index was detected. Further increase from 20 pulses up to the upper limit of 200 pulses had no marked effect on the induced cell disintegration.

The permeabilization effect induced in PEF-treated samples was compared to that induced by a thermal treatment. Heating was performed as follows: samples were vacuum-packed in polyethylene bags and immersed in warm water ( 20-95°C) for 15 min and immediately cooled in tap water to room temperature.

The cell disintegration index was measured as a function of the heating temperature. Disintegration did not occur up to 55°C, and above this temperature the cell disintegration increased.

The comparison between the cell disintegration index of PEF and thermal treated samples, showed that the degree of permeabilization achieved by PEF treatment at 2,4 kV/cm and 20 pulses, was equal to that obtained with thermal treatment at 72°C for 15 min. At this temperature, however, denaturation normally occurs.

To extract sugar, samples after PEF were mechanically pressed in hydraulic press either to 2 and 5 MPa for 5 min or to 30 MPa for 15 min and the obtained pulp suspended in distilled water. After repetition this cycle,

Chapter II

40

pressed pulp and raw juice were analysed and the results compared with the product of the processing cycle without PEF pre-treatment. PEF parameters were the following: exponential pulses, electric field strength of 2,4 kV/cm, 20 pulses and frequency repetition of 1 Hz). The three cycle protocol yielded 97% sugar extraction and the speed of extraction was 2-3 times faster than that using conventional thermal process (70-90 min.). Moreover the pulp obtained from HELP treated samples contained more dry matter (dm 30%) than that obtained with the conventional thermal process (dm 15%).

The impact of high intensity electric field pulses (HELPs) on permeabilization of coconut was studied by Ade-Omowaye et al. (2001a). Cylinders of coconut pulp (10 mm diameter and thickness 10 mm) have been treated with exponential pulses, a pulse width of 575 µs and a repetition frequency of 1 Hz. The authors used the treatment chamber reported by Angersbach and Knorr (1998) replacing carbon with stainless steel electrodes. The electric field strength was ranging from 0.1 to 2.5 kV/cm and number of pulses from 0-200 pulses. The effect of the electrical variables on the cell disintegration index was investigated. Increasing of the electric field and the number of pulses resulted in an initial sharp arise of the disintegration index (i.e.1 kV/cm and 50 pulses, respectively). Further increase of the index was only marginal with increasing the processing variables. Cell permeabilization however was more affected by electric field strength than by number of pulses.

The highest value of the cell disintegration index was 0.91 achieved at 2,5 kV/cm and 200 pulses. At 2,5 kV/cm, and 20 pulses corresponded the minimum energy consumption (25,4 kJ/kg the product with a satisfactory degree of permeabilization. The effect of HELPs treatment carried out at optimal conditions was compared with the results obtained with other disintegration methods, determining the disintegration index.

The disintegration methods included mechanical rupture (samples were finely and coarsely grated into stripes with average cross-sectional areas of 6.5 mm×1 mm and 19 mm×2.4 mm, respectively), thermal treatment (samples were vacuum packed in polyethylene bags, heated in water bath a 70 °C for 15 min and cooled rapidly in tap water to room temperature), and freezing (samples were vacuum packed in polyethylene bags, frozen to –20°C for 5 hr and thawed rapidly in tap water to room temperature). Due to the formation of ice crystals freeze-thawing resulted in total disintegration ( pZ = 1); 90% of the cells were disintegrated by mechanical rupture (finely grated samples). The level of cell disintegration obtained by HELPs was comparable to that corresponding to thermal treatment and mechanical rupture (control, coarsely grated). However, at temperature higher than 60° C thermal denaturation occurred.

Extraction of milk coconut from treated samples was performed and yield, fat and protein contents of the milk obtained were compared. Strips of an average cross sectional area of 6.5 mm×1 mm were manually expressed

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in a laboratory hydraulic press at a pressure of 15 MPa for 10 min. HELP treatment induced an increase of 20% of coconut milk yield with respect to the control samples. The yield obtained with HELP process was of the same order than that from freeze-thaw samples (highest yield). Protein and fat contents analysis showed that HELP did not affect the quality of the extracted milk. The fat content of the extracted milk from electrically treated and untreated samples was respectively 58% and 61.2 %, and the corresponding proteins content was 50% and 51,6%. In this work the effect of HELP on coconut dehydration has also been investigated.

HELP-treated samples(E=2,5 kV/cm, n=20 pulses, t=575 µs and frequency 1 Hz) were centrifuged (10000×g for 10 min.) prior air dehydration in a fluidized bed drier (air temperature of 60 °C, air velocity 1 m/s). The combination of the these two treatments enhanced the drying rate resulting in a reduction of about 22% of the drying time (to achieve 4 % moisture content) if compared to that of untreated samples, therefore showing potential advantages in production of copra.

II. 6 PEF and recovery of high valuable intracellular metabolites

The first attempt to use PEF in the recovery of high valuable components such as pigments was made by Brodelius et al. (1988) on plant cell suspension cultures treated with exponential pulses with electric field strength between 0.3 to 1.6 kV/cm and 3 impulses. The time interval between pulses was 10 s. During the treatment also the pulse duration was varied. Changing the value of the capacitance of the discharging system capacitor between 10 and 40 nF, exponential decay constant for pulse between 6 and 23 µs was obtained in this way .

Two cell cultures have been used i.e. Thalictrum rugosum and Chenopodium rubrum. The release of berberine from T. rugosum. and betacianins from C. rubrum was studied as a function of the applied electric field strength and pulse duration. In addition, cells viability was investigated. The pigment release was expressed as the ratio between the pigment amount obtained after electroporation and the total amount stored in the cells. The latter quantity was evaluated by solvent extraction with methanol. For complete permeabilization (i.e. release of all the intracellular product) T. rugosum required an electric field strength of 6 kV/cm, while in case of C. rubrum a strength of 10 kV/cm was necessary. The pulse duration didn’t affect significantly the pigment release but the cell viability was strongly influenced by this electrical variable. A marked decrease of the viability of T. rugosum cells was observed with increasing the pulse duration. The amount of product released was constant. Thus, long pulses electroporation induced the cell death without release of significant amount of intracellular compounds from the vacuoles. The authors concluded that it is not possible

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42

to release secondary product efficiently without inducing irreversible damage on cells.

Further attempts to release secondary metabolites from electroporated cultured plant cells suspensions keeping cells viability were carried out by Dörnerburg and Knorr (1993). The authors studied the effect of pulsed electric fields on the release of two intracellular pigments i.e. amaranthin and antrachinone from Chenopodium Rubrum and Morinda Citrifolia cells (in exponential phase). Cellular suspensions were placed in a dialysis tube permeable to electrical pulses and here exposed to exponential pulses. 85% of the total amaranthin content and 5.7% of the total amount of antrachinone were released in the culture medium after PEF treatment with 1.6 kV/cm, 10 pulses and 0,75 kV/cm, 20 pulses, respectively. The effect of electric field strength between 0,5 up to 1,6 kV/cm and number of pulses from 1 up to 30 pulses on the intracellular pigments release and viability was investigated. With increasing the value of both parameters, an increase of the release of amaranthin was observed, but the effect of the field strength was more significant than that of the number of pulses. Cells viability decreased strongly with the release of pigment. At 0,75 kV/cm and low number of pulses (i.e. up to 9 pulses) viability of Chenopodium Rubrum was still satisfactory. Further increase of the electric field strength and the number of pulses determined the loss of cells viability. For M. Citrifolia at 0,5 kV/cm and 3 pulses, the viability of cells was lost.

Cell viability was lost at release rates higher 16% for C. Rubrum and 2% for M. Citrifolia. Experimental results showed that in both cases it is not possible to obtain a significant amount of pigments without loosing cell viability, confirming in this way the conclusions of Brodelius et al. (1988).

The authors attributed the differences in extraction rates to a different location of the unit of pigments in the plant cell, (cytoplasm or vacuole). They also postulated that the breakdown of vacuoles and the subsequent changes of pH of cytoplasm should be responsible of cells death.

(Fincan et al. 2004) investigated the PEF-induced extractability of betanine from red beet tissue in solid-liquid extraction process. Discs of red beet (diameter 4 mm, thickness 1mm) were placed between two stainless steel parallel plate electrodes and exposed to rectangular positive pulses. PEF was performed using the following electrical parameters: field strength of 1 kV/cm, pulse duration of 1µs, interpulse delay of 20 ms. Three different values of the number of pulses have been selected: 27, 54 and 270 pulses. PEF-induced permeabilization of tissues has been detected as a function of treatment intensity, determining the frequency-dependence spectra of the electrical complex conductivity before and after the electrical treatment. With increasing the number of pulses, the conductivity of the tissue increased, as a result of permeabilization. In addition, the release of the red pigment and ionic species from the PEF-treated samples in an isotonic solution (0,25M of Mannitol) as a function of time was monitored until

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saturation using a spectrophotometer and a conductivity meter, respectively. Since freezing/thawing is a well-establish method leading to full cellular permeabilization, after 1 h, the time necessary to reach mass transfer equilibrium in the solution, PEF-treated samples have been frozen/thawed. Since concentration of the pigment and ions in the extraction medium are proportional to the absorbance and conductivity of the solution respectively, the induced releases of pigment and ions were expressed as the relative solution absorbance RSA and conductivity, RSC respectively defined as follows

RSA=AF

t

ABSABS

(II.14)

RSC= AF

t

KK

(II.15)

where tABS and AFABS are the absorbance at time t and after freezing/thawing, respectively; tK and AFK are the conductivities (mS/cm) at time t and after freezing /thawing, respectively.

The result showed that the release of ions was faster than the release of pigment as expected, pigment molecules being bigger than ions. After 1 h of extraction, from PEF-treated samples at maximum intensity, i.e. at 270 pulses resulted an extraction yield of almost 90% of the red pigment was detected.

II.7 Texture and structure modifications induced by PEF

The changes of textural properties caused by PEF have been reported by different authors. The electro-permeabilization determined the decrease of compressive strength in carrot tissue (Rastogi et al., 1999). Moreover, compressibility and failure stress of plant tissues also increase.

The effect of PEF on the porous structure of apple tissue have been investigated by Bazhal et al.(2003). Apple discs (57 mm of diameter and 15 mm of thickness) were treated in laboratory scale treatment cell with rectangular pulses, an electric field strength of 1 kV/cm, pulse width of 300 µs, pulse frequency of 1 Hz and 60 pulses. Porosity and pore size distribution of dry control and PEF-treated samples were measured by means of a mercury porosimeter. After PEF, the porosity of the tissue increased from 63% to 69,4 %. Moreover, increase of pores volume in the tissue was observed. The electrical treatment caused the formation of additional pores smaller than that of untreated samples. Mean pore diameter decreased from 6,64 to 5,32 µm. Finally, since the size of pores was comparable with the cell wall thickness, the authors suggested that electroplasmolys affected not only the cell membrane but also the integrity of the cell wall. The effect of the electric field strength and number of pulses on the permeabilization of apple

Chapter II

44

tissue was investigated by Lebovka et al. (2000) measuring the conductivity of PEF-treated samples as a function of the electrical variables. Electrical treatment was performed in the following conditions: field strength between 0.2-2 kV/cm, number of pulses from 1 to 10000, pulse length of 100 µs and interpulse delay 10 ms. The results obtained suggested that the combination of small number of pulses (up to 200) and high electric field strength produced the most significant permeabilization effects inducing a rapid and continuous increase in the relative conductivity. High number of pulses (up to 10000) and field strength of 0.2-0.4 kV/cm did not lead to a continuous increase of conductivity that remains constant before reaching the final value.

II.8 PEF and solid-liquid diffusion

Jemai and Vorobiev (2002) investigated the effect of moderate electric field pulses (MEFP) on the permeability of apple tissue determining the diffusion coefficient of soluble substances from apple slices. Apple discs (52 mm of diameter and 3,8 mm of thickness) were exposed to electrical treatments with monopolar rectangular pulses in an electroplasmolysis chamber consisting of two platinum electrodes at different MEFP levels followed by diffusion in a constantly agitated water bath at constant temperature of 20°C for 60 min. An interpulse delay of 10 ms and 1000 pulses were used while the electric field strength and the pulse duration were varied. The diffusion coefficient of soluble substances from treated slices was evaluated with a Fickian model. With increasing electric field strength and pulse duration, the diffusion coefficient increased. For each value of pulse duration values (50, 100 e 200 µs)and with an electric field strength of 100 V/cm, a significant increase of the diffusion coefficient as a consequence of permeabilization was detected. A further increase of field intensity and pulse duration, intensified the membrane permeabilization process, leading to an enhancement of the diffusion coefficient. Although a considerable increase of diffusion was obtained after 100 µs, the coefficient decreased slowly if the pulse duration was increased from 100 to 200 µs. In addition, for treatment above 500 V/cm, pulses of 100 and 200 µs determined comparable diffusion coefficients.

The effect of PEF parameters on the rate of sugar extraction from sugar beet tissue was studied by El-Belghiti et al. (2005). Sugar slices of 1,5 mm thickness were used, treated with rectangular pulses inside a cylindrical device between two wire gauze electrodes. Pulse width was 100 µs and pulse frequency was 1000 Hz. Different values of electric field strength (270, 400, 540, 670 and 800 V/cm) and number of pulses (50, 250, 500 and 1000) were selected. The release of solute during extraction in distilled water at room temperature was monitored using a digital refractometer. Solute concentration was measured until equilibrium (2 h) and yield was expressed

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as °Brix in water solution at time t with respect the theoretical equilibrium solute concentration. In absence of PEF, the quantity of solute obtained after 2 hr of extraction is nearly 40%. With PEF treatment at the maximum number of pulses selected i.e. 1000, the yield of solute increased significantly with increasing the applied voltage reaching the value of 93% at 670 V/cm. A further increase of the field strength to 800 V/cm didn’t produce any further increase of the extraction efficiency. At a fixed electric field strength of 670 V/cm, with increasing the number of pulses from 50 to 250, the yield of solute was increased from 60% to nearly 93%. However, an increase of the number of pulses to 1000 did not increase the solute yield.

El-Belghiti and Vorobiev (2005) investigated the effect of PEF energy and process conditions on the kinetics of solid-liquid extraction of solutes from carrot coarse slices (1.5 mm and 6 mm). Pulse width was 100 µs and pulse period was 10 ms. Different values of electric field strength (200, 270, 550 and 650 V/cm) and number of pulses (200, 300, 700 and 1000) were selected to study the effect of PEF in terms of the energy provided. The release of solute during extraction in distilled water at room temperature was monitored using a digital refractometer. Solute concentration was measured until equilibrium (8 h) and yield was expressed as the ratio between the initial solute concentration 0c and the total equilibrium solute concentration

∞c .PEF increased the solute yield with increasing the electrical energy provided. In absence of PEF the solute yield was only 45%. With 9 kJ/kg about 95% of the solute yield was achieved. Further increase of the energy provided didn’t produce any further enhancement of the solute yield.

II.9 Continuous PEF treatment in sugar beet processing

More recently the use of pulsed electric fields as pre-treatment stage in sugar beet processing was tested using a continuous PEF system (Schlutheiss et al., 2002).

Entire sugar beets were processed in a cylindrical treatment chamber consisting of a PP-tube (18 cm of inner diameter) equipped with four pairs of twisted disk shaped stainless steel electrodes. High voltage pulses up to 300 kV with 1 µs of duration and repetition rate of 1 Hz were applied. Specific input energy ranged between 1 kJ/kg to 174 kJ/kg. The electrodes were located on the axial and azimuth to assure that the cells of sugar beet samples passing through the treatment chamber were exposed to at least the minimum electric field strength at the centre between the two electrodes (i.e. 12 kV/cm). The set-up allowed the processing of the maximum mass rate of 300 kg/h. The treated beets leaving the reaction tube were transported with a screw conveyor. PEF-treated samples and control samples were cut in pieces and subjected to two different extraction procedures including: mechanical pressing ( 32 bar or 15 min) and thermal diffusion at different temperature

Chapter II

46

values up to 72°C. For PEF pre-treated samples the juice yield obtained after pressing was higher than for untreated samples. Both PEF and thermal extraction at72°C allowed the same value of juice yield, but PEF ensured energy saving. The specific energy required by thermal treatment was 174 kJ/kg much higher compared to 12 kJ/kg as for PEF pre-treatment. The application of PEF before thermal extraction resulted in the same juice yield as for thermal extraction but a lower temperature in the extraction process could be used, enhancing sugar juice purity and attaining a low raw-juice draft. The authors concluded that the introduction of PEF in a sugar production line presented the advantage of an extraction step at low temperatures with a low juice draft.

Chapter III PEF permeabilization studies

III.1 Introduction

PEF permeabilization studies were carried out in order to investigate the effect of pulse parameters on tissue permeabilization. Among all the possibilities, two parameters have been chosen: the electric field strength and the number of pulses. A method to detect cell membrane permeabilization induced by PEF was developed, based on the measurement of the electrical conductivity of the tissue before and after pulsed electric field treatment. Two different vegetables were used as test materials: apple and potato tissue, to evaluate the effect of the structure of the tissue on PEF permeabilization.

III.2 The experimental apparatus

The experimental apparatus was designed to study the cellular permeabilization of food tissues obtained with the application of pulsed electric field (PEF) technology and to investigate modifications induced into the tissue by the electrical treatment. It is formed by two main units:

a PEF laboratory scale equipment

a diagnostic system.

The PEF apparatus was set-up in the first part of the research activity and

consists of: a high- voltage pulse generator, a batch treatment chamber, a safety enclosure and an oscilloscope. Figure 13 shows a scheme of the experimental set-up.

The diagnostic system includes an impedance analyzer and a sample holder. A detailed description of each component is provided in the following sections.

Chapter III

48

Figure 13 PEF laboratory scale apparatus.

III.2.1 The high voltage pulse generator

The high voltage pulse generator was designed by Services Electronique (University of Technology of Compiègne, France). The equipment provides bursts of monopolar (i.e. negative polarity), rectangular shape, high voltage pulses. The electrical pulses rising time is 1 µs and the unit is able to generate a maximum voltage output of 1500 V. The maximum current output is 20A. It is possible to change the voltage in the range 1-1500 V (step 1 V), the number of burst in the range of 1-10, the number of pulses for each burst in the range of 1-10000, the pulse width of the single pulse in the range of 10-100µs (accuracy +/- 2µs) and the repetition period between 1ms and 0.1s (step 0.1ms). Electrical characteristics of pulses are shown in Figure 14.

The generator acts by means of a complex electrical circuit shown in Figure 15. A high voltage adjustable power supply, able to generate a current of 400mA under a voltage of 1500V (HEINZINGER PCN 1500V-400 mA), allows the charging of a capacitor bank of four capacitors of 4700µF 500V assembled in series. The voltage pulses are, then, obtained by an IGBT transistor operating as an electronic switch. The unit is managed by a

COMPUTER SAFETY ENCLOSURE

OSCILLOSCOPE

HV

LV

COMPUTER SAFETY ENCLOSURE

OSCILLOSCOPE

TREATMENT CHAMBER

COMPUTER SAFETY ENCLOSURE

PULSE GENERATOR

OSCILLOSCOPE

HV

LV

HV

LV

PEF permeabilization studies

49

Pulse width(10µs à 1ms)

Period (1ms à 0,1s)

Rest time (1s à 3600s)Number of

pulses by burst

(1 à 10000)

Number of bursts(1 à 1000)

Pulse width(10µs à 1ms)

Period (1ms à 0,1s)

Rest time (1s à 3600s)Number of

pulses by burst

(1 à 10000)

Number of bursts(1 à 1000)

microcontroller electronic board which communicates with the PC computer through a serial port. The digital signal to turn on the IGBT transistor is transmitted by a galvanic insulation consisting of a high- voltage opto-coupler.

Figure 14 Electrical parameters of pulses.

The high voltage power supply is programmable in voltage and current

thanks to two analog inputs (0-10V). The control voltage is generated by two digital/analog 12 bits converters. Monitoring of the voltage and current are carried out by two analog output (0-10V). The microcontroller board is equipped with a 12 bits Analog to Digital converter allowing the control, in addition of the two power supply monitoring signals of the charging voltage of the capacitors, peak values of the voltages and currents of generated pulses and load resistance.

The pulse current is measured with a restive shunt of 0,1Ω. The voltage of the pulses as well as the charging voltage of the capacitors are transmitted to the electronic board after attenuation by two resistive dividing bridges.

The two rectangular signals, proportional to the values of voltage and current of the pulses, feed a peak detector, whose resetting is controlled by the microcontroller.

The generation of pulses is actuated and controlled with a dedicated software developed with the graphic programming language HPVEE v. 5.0 (Hewlett-Packard), adapted by Service Electronique. From the front panel of the software it is possible to set the pulse parameters such as voltage amplitude, number of burst, number of pulses per burst, pulse width and

Chapter III

50

repetition period. After setting the treatment parameters, the generation of the pulses can be activated (Figure 16). The software allows also recording, in a txt format file, of both voltage and current peak values of the generated pulses.

Figure 15 Electrical circuit of the pulse generator.

H.V

. pow

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+ -

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PEF permeabilization studies

51

Figure 16 Panel control of the software HPVEE v. 5.0.

Settings and chancingof the electrical

parameters

Optional manualttiggering of resistance

measurement

Activation of Pulses generation

Settings and chancingof the electrical

parameters

Optional manualttiggering of resistance

measurement

Activation of Pulses generation

Chapter III

52

Moreoever, in order to preserve the high valuable electric components, several safety devices are provided with the pulser. Before generating the pulses, the value of the inter-electrodes resistance is checked by generating rectangular current pulses of 1mA or 10mA, according to the voltage range (positive pulse of 10ms followed by a negative pulse of 10ms).

Via software the current corresponding to the desired voltage is evaluated the safety circuit not authorize the generation of pulses if the estimated current is higher than 20A.

The generator is also equipped with a hardware control of the pulse current. If the current exceeds 25A during pulse generation, the gate voltage of the IGBT is forced to zero. If this over-current detection occurs more than 10 times during a burst, the software inhibits the generation of the following pulses. This system makes possible to preserve the power transistor in the case of electric arcs formation due to a too weak inter-electrodes distance. Finally, the generator output is automatically disconnected in case of main supply breakdown.

III.2.2 The batch treatment chamber

A first batch type treatment chamber has been designed to process plant tissues by pulsed electric fields.

The treatment chamber consists of a plastic holder containing a cylindrical frame of Teflon closed on both sides with two stainless steel disc shaped electrodes. The cylindrical compartment has a thickness of 20 mm and a height of 50 mm. The electrodes diameter is 30 mm. The lower electrode is fixed on the base of the Teflon cylinder and a guard ring is placed on it to guide the current flow through the sample during treatments avoiding undesired current paths. The upper electrode is fixed on a metallic plate. The contact with the sample takes place by sliding the plate along two small bars mounted on the plastic holder.

During the treatment, a constant pressure is applied to each sample corresponding to the weight of the upper system, assuring the uniformity of treatment conditions. Moreover, polished surfaces of electrodes are used in order to reduce the risk of dielectric breakdown of the food during pulses generation.

The electrodes are connected to the pulser by means of a plastic connection case and two banana plugs(Fig. 17).

PEF permeabilization studies

53

Figure 17 Plastic connection case. III.2.3 The safety enclosure

A safety enclosure containing the treatment chamber and the plastic connection case was build-up. The aim of this system is to ensure the protection of the user by preventing the access, and thus avoiding any possibility of accidental contact with high voltage elements, when the generator is under operation. The protection consists of a parallelepiped structure (70×70×30 mm) made with tubes of square section (25 mm side) and five sheets (50 mm thickness) fixed on it. The structure is made in aluminium and the walls are of transparent polycarbonate, that permits the visualization of the inside of the shell. All the walls are fixed except the front panel that is removable for the internal access to the electrodes. The handling of the door is facilitated by two small handles. This door is fixed with two banana plug connectors, ensuring also the control of the envelope lock. The terminals of these two connectors are connected to a safety switch. In the event of opening the protection enclosure, the switch is opened acting to a relay that disconnect the output of the high voltage generator. In this way the generation of pulses is inhibited.

III.2.4 The oscilloscope

A digital phosphor four-channel oscilloscope (3014B, Tektronix, USA) connected to the pulse generator, allows to monitor on-line the electrical pulses during treatments. The acquisition of both voltage and current waveforms is possible thanks to two probes integrated the generator to which the oscilloscope is connected by means of two BNC cables.

Chapter III

54

The oscilloscope has an acquisition rate of 1,25 G/sample and allows the recording of the waveforms in different format files including excel and bitmap.

III. 3 The system for impedance measurement

Measurement of the electrical impedance of the tissue before and after the treatment as a function of the frequency is carried out by an experimental system including: an impedance analyzer and a sample holder.

III.3.1 The impedance analyzer

The impedance analyzer (1260, Solartron, UK) performs the measurement of the electrical properties of the plant food tissue (Fig.18). This device acts by means of two main units: a generator and an analyzer.

The generator provides the input signal to the sample under test. It is possible to produce a voltage as well as a current signal. In both cases, the signal is sinusoidal and frequency can be changed in the range 10 µHz-32 MHz. The maximum output voltage and current amplitudes (rms values) depends on the frequency range adopted. For frequency ≤ 10MHz they are 3V and 60mA and for frequency > 10MHz, 1V and 20mA. The generator uses a feedback control system to hold the input signal (voltage/current) applied to the sample at a constant level. The generator output is varied within defined limits to maintain the selected input at the desired value. An error message advices the user in case of failure of the control system.

The analyzer correlates the input signal to the drive signal frequency to obtain the frequency response and the impedance of the sample under test. It measures the phase angle and computes the electrical impedance as the ratio of the voltage drop across the sample and the current crossing it during the test. Three different quantities are provided: the magnitude of the impedance, the real part and the imaginary part of the impedance.

The impedance analyzer communicates with a PC computer by means of a GPIB communication board and a dedicated software allows the control of the device. Two packages are integrated in the software: Zplot and Zview.

With Zplot the set-up and the control of the experiments as well as the acquisition of the measured data are made. It is possible to perform the measurement at a fixed frequency or, in alternative, to acquire data increasing or decreasing the frequency in a selected range. This experiment type is called the frequency sweep. Sweep can be linear or logarithmic and in both cases the number of points for the sweep can be selected. Moreover, Zplot displays the status of the experiment including the estimated time remaining. At the end of the test, the measured data are saved in txt format compatible files.

PEF permeabilization studies

55

With Zview the graphical visualization of the data is possible. It can display either live data from Zplot or the data saved as files. Up to 10 data files can be displayed simultaneously. Three different graphs are possible: the impedance as a function of the frequency, the phase angle as a function of frequency and the real part of the impedance as a function of its imaginary part.

Figure 18 Impedance analyzer (1260, Solartron).

III.3.2 The sample holder

The sample holder (1296 2A, Solartron, UK) to measure electrical impedance of solid materials at room temperature consists of two disk shaped parallel electrodes. The lower has a fixed position and the upper can be moved in contact with the sample by adjustment of a micrometer (Fig. 19Figure 19). The electrodes have a diameter of 20 mm. The thickness of the sample can be read from a micrometer digital display, either in mm or in inches. Sample thickness can range from a minimum of 0.2 mm to a maximum of 25.4 mm. A guard ring on the fixed electrode reduces the effect of stray field lines at the edge of sample which would otherwise lead to measurement errors. The guard ring ensures that electric field lines are parallel to the axis of the sample undergoing electrical impedance measurements. In fact, there is no potential difference between the lower electrode and the guard ring, therefore, the field lines at the edges of the lower electrode are kept parallel to each other. In this way the impedance of the sample is calculated taking into account only the current which flows in the central part of the sample where the field lines are parallel. The current which goes through the edge of the samples and the air surrounding the

Chapter III

56

sample is not considered (it goes directly to earth) ad therefore does not contribute to the measurement.

.

Figure 19 Sample holder (1296 2A, Solartron).

III.4 Materials and sampling

Apples (Granny Smith), potatoes (Agraria) were purchased from a local market and stored in a refrigerator at 4°C before use. After removing from the fridge, the samples were allowed to reach the room temperature, before use. Slices of about 2 ± 0,1 mm were prepared using an industrial slicer. Then discs of fresh tissue with a diameter of 16 mm were sampled by means of a coark borer. Due to the heterogeneity of the tissue, in order to assure a good reproducibility of data samples were taken from the same parenchimatic region. The thickness of the sample was checked with a micrometer and discs with the same thickness were used in the experiments.

PEF permeabilization studies

57

III.5 Experimental procedures

III.5.1 Pulsed electric field treatment

Pulsed electric field treatments were performed in the experimental apparatus shown in Fig.13.

A disc of fresh tissue was introduced in the treatment chamber in contact with the two electrodes. After closing the safety enclosure and setting the treatment parameters, the electric pulses were activated and oscilloscope’s waveform acquisition was launched. The samples were treated at different

intensities varying the voltage amplitude as well as the pulse number. In all the experiments, pulse width and interpulse duration were kept constant.

In order to investigate the effect of the electric field strength and pulse number on tissue permeabilization, two series of PEF treatment were carried out for both apple and potato tissues. In the first experiments, the electric field strength corresponding to the maximum degree of cellular permeabilization at a fixed number of pulses was determined. To this purpose, keeping constant the number of pulses, the applied electric field strength was increased until the required value.

In the second, a value of the electric field strengths was set and the pulse number was varied. In this way, it was possible to determine the optimal values of electrical parameters able to permeabilize completely the food tissues.

In each test one burst of 10 negative rectangular pulses was applied to the tissue. Pulse duration of 100 µs was used and pulse repetition period was set at 10 ms. Electrical voltage was varied in the range 100-400V and three different values of the number of pulses were selected: 10,100 and 500. In order to assure a good reproducibility of the experimental data, a number of four replicates was used in each treatment condition.

III.5.2 Electrical measurement and data analysis

The electrical impedance of the tissue and the phase angle were measured as a function of frequency before and after PEF treatment. A disc of the tissue was introduced in the sample holder, between the two parallel electrodes, the desired voltage rms value and the frequency range were set and the sweep was launched (Fig. 20).

The sinusoidal voltage had a rms value of 1 V and the frequency changed in the range from 1 KHz up 10 MHz. For data acquisition, logarithmic sweep have been used and 100 measurement points have been chosen.

The rms value and the frequency range used for the measurements were determined by means of preliminary tests carried out on the intact tissues. The optimal values were selected to minimize polarization effects, significant in the low frequency range (<1KHz), and to avoid the occurrence

Chapter III

58

of resonance phenomena visible in the high frequency range (>10MHz) (data not shown).

The tissue electrical complex conductivity ( )ωσ * was calculated from the impedance measurement, according to the Equation reported below:

( )ωσjZs

h=* (III.1)

being d the sample thickness, s the contact surface area between the sample and electrodes, ( )ωjZ the electrical impedance of the tissue and ω the angular frequency of the applied signal.

Finally, the degree of permeabilization was evaluated introducing relative complex conductivity *

RELσ defined as follows:

*

**

UNTR

TRREL σ

σσ = (III.2)

being *

UNTRσ and *TRσ the complex conductivities before and after the

PEF treatment, respectively.

Figure 20 Schematic representation of electrical impedance measurement.

V=Vmsenωt

I

Food tissueFood tissue hV=Vmsenωt

I

Food tissueFood tissue h

PEF permeabilization studies

59

III.6 Results and discussion

III.6.1 Effects of electric field strength on tissue

permeabilization

The effects of the electric field strength was investigated by carrying out PEF treatments of different voltage magnitude utilizing 10 pulses. The electric field strength was gradually increased from the minimum of 0,5 kV/cm to the maximum of 2 kV/cm by a constant step of 0,5 kV/cm (corresponding to 100V).

III.6.1.1 Apple tissue

Figure 21 shows the graph of the average relative complex conductivity of apple tissue as a function of the measurement frequency for intact tissue and after PEF at the electric field strength of 0,5 kV/cm.

Pulsed electric field affects the electrical properties of the treated tissue. After PEF, an increase of the complex conductivity of the tissue occurs as a consequence of the achieved permeabilization of the cell membrane. As clearly shown by the graph, the increase of tissue conductivity is more significant in the low frequency range (1-10 kHz) than at high frequency field (>105 Hz) where there is almost no difference between the conductivity values of non-treated and treated samples. This can be explained with the different electrical behaviour of the cell membranes in plant cells shown in the low frequency and high frequency range.

The presence of the cell membranes determines the frequency dependence of the whole plant tissue electrical impedance (i.e. conductivity). In the low frequency field, the cell membrane acts as a capacitor preventing the flow of the electrical current in the intracellular medium. The cell is poorly conductive if compared with the surrounding electrolyte (i.e. the extra cellular medium). Thus, in this frequency range, only the extra cellular fluid is available to current flow and the complex conductivity represents only the contribution of the extra cellular medium i.e. is related to the ionic species concentration in the extra cellular medium. With increasing the frequency, the cell membranes become less and less resistant to the current flow in the intracellular liquid. At very high frequency values, the membranes are totally shorted out and complex conductivity is representative of the contribution of both extra cellular and intracellular medium.

Thus, the tissue permeabilization induced by PEF is detectable in the low frequency range while, due to the electrical transparency of the membranes in the high frequency range, even if the permeabilization occurs, it is not possible to account for the effects of the electrical treatment. Therefore, the observed increase of the conductivity can be correctly attributed to an

Chapter III

60

increase of the concentration of ionic species in the extra cellular space resulting from the permeabilization of the membranes induced by the electrical treatment.

With increasing the electric field strength up to 1 kV/cm it is not detected a considerable enhancement of the permeabilization. The conductivity spectrum shows the same trend described above. Only a sligth increase of the tissue conductivity can be observed (Fig. 22).

0,0

1,0

2,0

3,0

4,0

5,0

6,0

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07Frequency (Hz)

Rel

ativ

e co

mpl

ex c

ondu

ctiv

ity

E= 0,5 kV/cmno PEF

Figure 21 Relative complex conductivity of apple tissue at E=0 and E=0,5 kV/cm for 10 pulses.

PEF permeabilization studies

61

0,0

1,0

2,0

3,0

4,0

5,0

6,0

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07Frequency (Hz)

Rel

ativ

e co

mpl

ex c

ondu

ctiv

ity

E= 0,5 kV/cmE= 1 kV/cm

Figure 22 Relative complex conductivity of apple tissue at E=0,5 and E= 1 kV/cm for 10 pulses.

On the contrary, the electric field strength of 1,5 kV/cm causes a

significant degree of permeabilization.(Fig. 23).

0,0

1,0

2,0

3,0

4,0

5,0

6,0

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07Frequency (Hz)

Rel

ativ

e co

mpl

ex c

ondu

ctiv

ity

E= 1 kV/cmE=1,5 kV/cm

Figure 23 Relative complex conductivity of apple tissue at E=1 and E= 1,5 kV/cm for 10 pulses.

After treatment, the relative complex conductivity is four times higher

than that corresponding to the untreated tissue

Chapter III

62

A further increase of the applied electric field strength from 1,5 kV/cm up to 2 kV/cm doesn’t determine any additional increase of the electrical conductivity (Fig.24Figure 24).

0,0

2,0

4,0

6,0

8,0

10,0

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07Frequency (Hz)

Rel

ativ

e co

mpl

ex c

ondu

ctiv

ity

E=1,5 kV/cmE= 2 kV/cm

Figure 24 Relative complex conductivity of apple tissue at E=1,5 and E=2 kV/cm for 10 pulses.

Due to the overlapping of the two conductivity spectra at 1,5 kV/cm and

2 kV/cm shows, the existence, at the assumed treatment time, of a threshold value of the electric field strength necessary for permeabilization can be hypothesized. Above this electric field intensity, PEF treatment doesn’t contribute to intensify cell membrane breakdown process thus not improving the achieved degree of permeabilization.

III.6.1.2 Potato tissue

The cellular permeabilization induced by PEF in potato tissue has also been studied. In the electrical treatment, the same number of pulses i.e. 10 has been used. In Figure 25 the average relative complex conductivity of potato tissue for untreated samples and after PEF at the different applied electric field strengths ranging from 0,5-1,5 kV/cm is plotted.

PEF permeabilization studies

63

0,00

2,00

4,00

6,00

8,00

10,00

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07Frequency (Hz)

Rel

ativ

e co

mpl

ex c

ondu

ctiv

ity

E= 0,5 kV/cmE= 1 kV/cmE=1,5 kV/cm

Figure 25 Relative complex conductivity spectra of potato tissue as a function of the electric field strength at n=10 pulses.

As for apple tissues, the increase of the electrical conductivity in the low

frequency range after PEF occurs. This electrical variable increases with the applied electric field strength. Thus, the degree of permeabilization shows the same trend found for apple tissues. Also in this case at 0,5 kV/cm a slight degree of permeabilization is observed, while the application of 1 kV/cm determines a significant improvement in cell membrane breakdown.

From 1 kV/cm to 1,5kV/cm, the overlapping of the two conductivity spectra can be observed. This behaviour confirms the existence of the threshold value of the electric field strength required for tissue permeabilization. For potato tissue the threshold value is 1 kV/cm. This result is in agreement with that of Angersbach and Knorr (1997). The authors reported that cells disintegration increased with the field strength ranging from 0,5-1,1 kV/cm, while above 1,1 kV/cm no significant difference was observed. The existence of the threshold value of the electric field strength in other plant food tissue permeabilization experiments, has been showed by Rastogi et al. (1999). Studying carrot tissues, the authors found a sharp increase of the permeabilization index with increasing the field strength up to a certain value, after which further increase is only marginal. In following investigations on coconut and paprika tissue, The same trend was also observed by other authors (Ade-Omowaye et al., 2001 and Ade-Omowaye et al. ,2002).

Chapter III

64

III.6.2 Effects of pulse number on tissue permeabilization

The effect of the number of pulses on cellular permeabilization induced by PEF in both apple and potato tissue has been also investigated. In this case PEF experiments were carried out, for both food materials, at the threshold value of the electric field strength. Three different values of the pulse number were selected: 10, 100 and 500 pulses.

Figures 26 and 27 show the average relative complex conductivity spectra at different number of the pulses of PEF-treated apple and potato discs.

As for the parameters previously studied, the higher the number of pulses the higher the degree of cell membrane breakdown achieved by the electrical treatment, reaching the maximum value at 500 pulses in both cases.

0,00

2,00

4,00

6,00

8,00

10,00

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07Frequency (Hz)

Rel

ativ

e co

mpl

ex c

ondu

ctiv

ity

n=10n=100n=500

Figure 26 Relative complex conductivity spectra of apple tissue as a function of the number of pulses at E= 1,5 kV/cm.

PEF permeabilization studies

65

0,00

2,00

4,00

6,00

8,00

10,00

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07Frequency (Hz)

Rel

ativ

e co

mpl

ex c

ondu

ctiv

ity

n=10n=100n=500

Figure 27 Relative complex conductivity spectra of potato tissue as a function of the number of pulses at E= 1kV/cm.

Figures 28 and 29 reported the electrical impedance of untreated and

PEF-treated apple and potato samples as a function of frequency. As can be clearly observed, total permeabilization in both cases occurs. In fact, after PEF the impedance is independent on frequency.

.

Figure 28 Electrical impedance of intact and PEF-treated apple tissue as a function of the frequency.

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+03 1,00E+04 1,00E+05 1,00E+06

Frequency (Hz)

|Z| O

hm

PEF, E=1,5Kv/cm, 500pulses

Intact

Chapter III

66

Cell membranes are totally permeabilized and, thus, are shorted out even in low frequency field. Therefore, as already stated, not only the electric field strength but also the number of pulses is a key process parameter determining the cell membrane breakdown. The electrical impedance measurement confirmed to be a reliable method to individuate the optimal values of processing parameters to achieve total permeabilization.

Experimental results show that even if the total permeabilization of both samples is obtained with the same pulses number, i.e. 500, the electric field necessary to permeabilize potato samples of 1 kV/cm is lower than that necessary for apple tissue, 1,5 kV/cm

Figure 29 Electrical impedance of intact and PEF-treated potato tissue as a function of the frequency.

A possible explanation of this behaviour leads back to the electrical

conductivity that is higher for potato tissues than for apple (Fig. 30). In fact, a higher conductivity corresponds to a lower resistance to the flow of current in the tissue during the treatment, thus allowing permeabilization at lower electric field strength.

The difference of the electrical conductivity of potato and apple tissue observed is not surprising since structure of these materials is different. The presence of a porous structure characterized by air (i.e. dielectric) spaces in apple tissue, in fact, can clearly explain the lower value of the complex conductivity shown.

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+03 1,00E+04 1,00E+05 1,00E+06

Frequency (Hz)

|Z| O

hm

Intact

PEF, E=1,0Kv/cm, 500pulses

PEF permeabilization studies

67

0,0

2,0

4,0

6,0

1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07

Frequency (Hz)

Com

plex

con

duct

ivity

(mS/

cm)

potato

apple

Figure 30 Complex conductivity spectra of intact apple and potato tissue. Moreover at total permeabilization conditions, the complex conductivity

a 1kHz becomes 8-9 times higher than that of non-treated tissue in apple and potato tissue. These results are in agreement with the experimental findings for the same food materials of Knorr and Angersbach (1998) and Lebovka et al. (2001) reporting an increase of 8-10 times of the tissue complex conductivity.

Chapter IV PEF as a pre-treatment stage in

dehydration of plant food

IV.1 Introduction

The application of PEF as a pre-treatment stage of dehydration of plant food materials has been tested. The effect of PEF process parameters on the drying rate and quality properties of rehydrated samples has been also evaluated.

IV.2 Sample preparation

Discs of apple (Granny Smith) 2 mm thickness and 16 mm diameter were prepared as described in Section III.2. Due to the heterogeneity of the tissue, in order to assure a good reproducibility of experimental data samples were taken from the same parenchimatic region shown in Fig. 31.

Immediately after sampling, apple discs were dipped in 25 ml of a solution at 1% of citric and ascorbic acid for about 1 min to prevent enzymic browning. Before PEF treatment, the excess of solution on the sample surface was removed by blotting the samples with adsorbent paper.

After anti browning treatment, one set of four samples was dried while the other was exposed to electrical pulses before undergoing drying. Initial content of water of apple was ranging from 86-88%.

Chapter IV

60

Apple tissue 2 mm

16 mm

Figure 31 Sampling region in apple tissue.

IV.3 Experimental procedures

IV.3.1 Pulsed electric field treatment

PEF experiments were carried out in the laboratory scale apparatus described in Chapter III. The combination of the electrical parameters adopted in PEF treatment have been selected on the basis of the results of permeabilization studies reported in Section III.6. The electric field strength was kept constant at the threshold value of 1,5 kV/cm while the pulse number was varied. Electrical pulses characteristics have been summarized in the Table reported below.

Table 1 Electrical pulses characteristics.

Pulse duration (µs)

Electric field strength

(kV/cm)

Pulse number

Interpulse duration (ms)

100

1.5

10, 100, 500

10

IV.3.2 Drying protocol

Dehydration of control and PEF treated samples was performed in an air natural convection oven (HERAUS 6060 T 5042 E). Samples were dried at 50°C and atmospheric pressure for 4 h. The temperature was chosen in order

PEF as a pre-treatment stage in dehydration of plant food

61

to prevent thermal denaturation of cell membranes in the food tissue, as revealed by preliminary studies (data not shown). At regular intervals (30 min for the first two hours and 1 hour for the remaining drying time) samples were taken from the oven, weighted on an analytical balance (Mettler Toledo) and weight losses recorded. Drying was performed until no further changes of weight was observed. At different drying time, sample moisture content, X, was determined as follows:

( ) ( )( ) S

S

WPWPtP

X*0

*0−=

⎥⎥⎦

⎤

⎢⎢⎣

⎡

soliddry

OH

kgkg

.

2 (IV.1)

being P(t) the sample weight at time t, P(0) the initial sample weight and

Ws the sample dry solid content. Samples weight before and after PEF treatments was also recorded. The dry solid content was determined after drying at 105°C for 24 h. Dehydration curves were, then, obtained plotting the moisture content X as a function of the drying time t.

IV.3.3 Characterization of samples after drying

After drying, characterization tests were performed on the samples to evaluate the modifications induced by Pulsed Electric Field to the physical properties of the tissue as well as to evaluate the quality of the final product.

IV.3.3.1 Measurement of bulk density

The bulk volume of both untreated and PEF-treated dry samples was determined with the liquid displacement method based on Archimedes principle. Each sample was immersed in glycerol by means of an anchored pivot and the force (the weight) necessary to submerge the sample in the liquid completely was determined by means of an analytical balance (Mettler Toledo). Before immersion, each sample was coated with a thin film of metacrilic glue to prevent liquid absorption on the surface. Thus, the bulk volume was obtained as the ratio between the weight, defined above, and the density of glycerol (1,26 g/m3). The bulk density was then evaluated as the ratio between the weight of the sample M and its overall (bulk) volume Vb.

IV.3.3.2 Measurement of particle density

A helium electronic picnometer, with a maximum operative pressure of 20 psi (Ultrapicnometer 1000, Quantacrome Instruments), was used to determine the particle density ρP of intact and PEF treated dry samples, according to the technique developed by Donsì et al. (1995). The equipment performed the determination of the volume of the solid trough the

Chapter IV

62

comparison, by the means of the ideal gas equations, of the pressure values recorded before and after the expansion of an inert gas in the measuring cell.

IV.3.3.3 Calculation of open pores porosity

The overall (bulk) volume Vb can be expressed as the sum of three terms:

aldb VVVV ++= (IV.2) being, Vd, the volume of solid matter, Vl the volume of the liquid matter

and Va the volume of air. Considering Equation (IV.2 ) the bulk density can be expressed according

to :

( )aldb VVV

M++

=ρ (IV.3)

The total volume occupied by air Va can be considered as the sum of two

contributions acpV and aopV related to the pores closed and open to the outside, respectively:

aopacpa VVV += (IV.4)

Particle density is defined as the ratio between the current weight of the

sample and its overall volume diminished by the volume of the pores open to the outside according to:

( ) ( )acpldaopbp VVV

MVV

M++

=−

=ρ (IV.5)

The open pores porosity ε is the ratio between the volume of the pores

filled with air connected to the outside and the overall volume and is defined as follows:

b

aop

VV

=ε (IV.6)

Combining Equations (IV.3), (IV.5) and (IV.6), open pores porosity is

then expressed as follows:

PEF as a pre-treatment stage in dehydration of plant food

63

p

b

ρρ

ε −= 1 (IV.7)

being bρ and pρ the bulk and the particle density of the sample,

respectively. IV. 3.3.4 Rehydration rate

Dehydrated samples were weighted and placed in glass beakers containing distilled water immersed in a thermostatically controlled water bath at 50 °C. At defined time intervals, samples were taken out from the rehydration solution, gently blotted with tissue paper to remove the adhering water and weighted with an analytical balance. Rehydration experiments were carried out until a constant weight of the samples was reached.

Rehydration capacity of the samples RC was then determined as:

0RRRC = ⎥

⎦

⎤⎢⎣

⎡kgkg

(IV.8)

being R the sample weight at time t and 0R the sample weight before

rehydration, respectively. Finally, rehydration curves were obtained plotting the RC parameter as a function of the rehydration time.

IV.3.3.5 Texture analysis

In order to evaluate the firmness of fully rehydrated samples, rupture and puncture tests were carried out using a computerized texture analyzer ( TA-XT2, Stable Microsystems, United Kingdom) consisting of a flat platform and a 25 kg load cell. Each sample was placed on the platform and the maximum force and the energy required to cause either rupture or puncture were measured. Rupture test was carried out using a 10 mm OD cylindrical probe, while in puncture test a 2mm OD punch was used. In both cases the cross-head speed was 2 mm/s.

IV.4 Results and discussion

IV.4.1 The effect of PEF on drying rate

Figures 32, 33 and 34 show the comparison between the dehydration curves of untreated and PEF-treated samples at the electric field strength of 1,5 kV/cm and 10, 100 and 500 pulses, respectively.

Chapter IV

64

In all cases, after the electrical treatment the samples exhibit an initial loss of water content due to the induced cellular permeabilization. Moreover, this effect becomes more significant with increasing the applied number of pulses. At 10 pulses there is no difference in initial moisture content between control and PEF treated samples. This result validate the electrical impedance measurement showing that a very low degree of permeabilization is reached at this process conditions. Increasing the number of pulses from 10 to 100, PEF affects positively drying process improving dehydration rate during the first stage as shown by the fact that the electrical treated samples hold a lower moisture content than the control. Further increase of the pulse number doesn’t produce any enhancement of the drying rate. The final water content of dry samples is the same, irrespective of the pre-treatment stage utilised. The same behaviour was found on red bell pepper by Knorr et al. (2003). The authors showed that PEF increased initially the rate of drying increasing the number of pulses from 10 to 30, while no significant difference was observed for higher number of pulses. However, at the end of the process the moisture content of untreated and treated sample was practically the same.

A possible explanation of this behaviour might be that the initial losses as well as the faster rate might have caused a deposition of solid layer around the sample thus creating a resistance to further water outflow.

Figure 32 Dehydration curves of intact and PEF-treated apple discs at E=1,5 kV/cm and 10 pulses.

0

1

2

3

4

5

6

7

8

0 30 60 90 120 150 180 210 240

t (min)

x (K

g.H 2

O/K

g s.

s.)

controlPEF

PEF as a pre-treatment stage in dehydration of plant food

65

Figure 33 Dehydration curves of intact and PEF-treated apple discs at E=1,5 kV/cm and 100 pulses.

Figure 34 Dehydration curves of intact and PEF-treated apple discs at E=1,5 kV/cm and 500 pulses.

IV.4.2 The effect of PEF on sample porosity

The porosity of dried untreated and PEF pre-treated apple is shown in Table 2Table 2. The electrical treatment induces an increase of the porosity as a result of the induced cellular permeabilization, the higher the number of pulses utilised, the higher the final porosity. This result validate the results achieved by means of the electrical impedance measurement. After PEF, when total permeabilization of samples occurs, the porosity increases from

0

1

2

3

4

5

6

7

8

0 30 60 90 120 150 180 210 240

t (min)

x (K

g.H

2O/K

g s.

s.)

controlPEF

0

1

2

3

4

5

6

7

8

0 30 60 90 120 150 180 210 240

t (min)

x (K

g.H

2O/K

g s.

s.)

controlPEF

Chapter IV

66

0

1

2

3

4

5

6

0 50 100 150 200

t (min)

R/R

0

PEFControl

0,767 to 0,854 for PEF treated samples. These results are in agreement with the experimental findings of Arevalo et al. (2004), reporting an increase of the porosity of 12% in MacIntosh apple slices after PEF.

Table 2 Porosity of dry control and PEF-treated apple discs at E=1,5 kV/cm with different number of pulses.

IV.4.3 The effect of PEF on rehydration rate

Figures 35, 36 and 37 reported the rehydration rate of control and PEF-treated samples at 10, 100 e 500 pulses, respectively. There isn’t a significant difference between the control and PEF-treated samples at 10 and 100 pulses, while at 500 pulses a better rehydration of PEF-treated sample is recognizable. Thus, rehydration tests confirm the previous porosity measurement. The same results have been found by Knorr et al. (1998) on potato tissue, after convective forced air drying.

Figure 35 Rehydration curves of intact and PEF-treated apple discs at 10 pulses.

Sample ε Control 0,767

PEF, 10 pulses 0,801 PEF, 100 pulses 0,823 PEF, 500 pulses 0,854

PEF as a pre-treatment stage in dehydration of plant food

67

0

1

2

3

4

5

6

0 50 100 150 200t(min)

R/R

0

PEFControl

0

1

2

3

4

5

6

7

0 50 100 150 200

t(min)

R/R

0 PEFControl

Figure 36 Rehydration curves of intact and PEF-treated apple discs at 100 pulses.

Figure 37 Rehydration curves of intact and PEF-treated apple discs at 500 pulses.

Chapter IV

68

IV.4.4 Texture analysis

Tables 3 and 4 show the maximum force and the energy in rupture and puncture test, respectively. In both cases pulsed electric field results in the reduction of the maximum compressive force as well as the energy, thus determining a decrease of the firmness of the samples.

The softening of the tissue can be attributed to the cellular damage induced by the electrical treatment and the subsequent loss of the pressure turgor into the cell.

Moreover, texture studies show that the softening of the tissue increases with increasing the applied number of pulses.

Table 3 Maximum compressive force and energy in rupture test on control and PEF-treated apple discs at E=1,5 kV/cm with different number of pulses.

Table 4 Maximum compressive force energy in puncture test on control and PEF-treated apple discs at E= 1,5 kV/cm with different number of pulses.

Therefore, this trend supports the results of the electrical impedance

measurement and validates the previous permeabilization studies

Sample F (N) E (N*mm) Control 29,8497 11,2350

PEF, 10 pulses 28,3675 10,0015 PEF, 100 pulses 14,7020 5,9065 PEF, 500 pulses 5,5352 2,8235

Sample F (N) E (N*mm) Control 29,8497 11,2350

PEF, 10 pulses 28,3675 10,0015 PEF, 100 pulses 14,7020 5,9065 PEF, 500 pulses 5,5352 2,8235

Chapter V PEF as a pre-treatment stage in

extraction of anthocyanins

V.1 Introduction

These investigations aim to evaluate the impact of pulsed electric field on the permeabilization of berry tissue. Moreover, the use of PEF as a pre-treatment stage in solid/liquid extraction processes was tested for the recovery of anthocyanins from grapes.

V.1.1 Anthocyanins: chemical composition

The anthocyanins are probably the best known natural pigments. The world anthocyanin is derived from Greek terms, anthos, meaning flower and kyanos meaning blue. These pigments are widely spread in the plant kingdom and are responsable for the most red, blue and purple colors in fruits, berries and flowers. The use of anthocyanins as food colorants, dates back to ancient Romans. They used highly coloured berries for colouring wine. Due, to their visibility, many efforts in the research world have been made in the past 75 years to improve the recovery tecniques.

Anthocyanins are phenolic plant metabolites belonging to the flavonoid family. Over 250 different anthocyanins, part of about 5000 flavonoid compounds, have been isolated from plants of similar chemical structure (Wang at al., 1997). An anthocyanin molecule constitutes the aglycone or anthocyanidin body, the chromophore group, that in its natural stage is glycosylated by one or more different sugars. Infact, anthocyanins are glycosylated derivatives of the 3,5,7,3'-tetrahydroxy-flavylium cation. Free aglycones are formed very rarely in plants. One reason for this is that the electron deficiency of the flavylium cation makes the free aglycones highly reactive and thus the molecule is very unstable. Since sugar stabilizes the molecule, glycosides are then more stable than the aglycones.

Chapter V

70

The differences between the aglycon bodies are due to the number of hydroxyl groups and their degree of methylation. Twenty two aglicones are known eighteen of which are natural. However, the most important anthocyanidins in foodstuff are only six including: cyanidin, delphinidin, peonidin, petunidin, pelargonidin, and malvidin (Nyman and Kumpulainen, 2001; Francis, 1999). The structure of these compounds is shown in Figure 38.

Figure 38 Six major anthocyanidins in foodstuff (Nyman and Kumpulainen, 2001).

The glycosyl moiety is usually located at carbons 3, 5, 7, 3', and 5'. The sugars linket to the aglycones, listed in relation to their relative

amount, are: D-glucose, L-rhamnose, D-galactose, D-xylose, Arabinose and Glucuronic acid. Anthocyanins may also be acylated an this accounts for the third component of the molecule. One or more molecules of the acyl acids (p-Coumaric, Ferulic, and Caffeic) or the aliphatic acids (Malonic and Acetic) maybe esterified to the sugar molecule (Francis, 1989).

The anthocyanins in some fruits are listed in Table 5. Most of them contains 3-glycosides.

PEF as a pre-treatment stage in extraction of anthocyanins

71

Table 5 Anthocyanins in some fruits.

Apple Cy 3-galactoside, 3-glucoside, 3-

arabinoside, 3-xyloside, acilated derivates

Blackberry Cy 3-glucoside and 3-rutinoside

Blackcurrant Cy e Dp 3-glucosides and 3-rutinosides

Cherry (sour) Cy 3-glucoside, 3-rutinoside, 3-

sophoroside and 3-glucorutinoside

Cherry (sweet) Cy e Pn 3-glucosides and 3-rutinosides

Cranberry Cy, Pn, Dp, Pt e Mv 3-galactosides, 3-

arabinosides, 3-glucosides

Elderberry Cy 3-sambubioside, 3-glucoside, 3-

sambubioside-5-glycoside

Grapes

(V.vinifera)

Mv, Dp, Pt, Pn, Cy 3-glucosides and

acetilated with p-coumaric and caffeic acids

Plum Cy and Pn 3-rutinosides, 3-glucosides

Raspberry Cy and Pg 3-glucosides, 3-rutinosides, 3-

sophorosides and 3-glucorutinosides

Redcurrant Cy 3-glucosides, 3- rutinosides and others

Strawberry Pg and Cy 3-glucosides

Pg = pelargonidin; Cy = cyanidin; Pn = peonidin; Dp = delphinidin;

Pt = petunidin; Mv = malvidin

V.1.2 Anthocyanins : health effects and physical properties

Several factors affect the stability of anthocyanins. Their colour is strongly dependent on the pH. This characteristics is utilized when

Chapter V

72

anthocyanins are used as taxonomic markers. Anthocyanins are more stable at low pH values. They gradually loose colour as the pH is increased. Above pH 4, most anthocyanins appear colourless (Brouillard, 1982; Mazza and Miniati, 1993). This colour loss is reversible and the red colour return by means of acidification. In aqueous solutions, common anthocyanins exist as a mixture of four structures: AH+ flavylium cation (red), A quinoidal anydrobase (blue), B carbinol pseudobase (colour-less) and C chalcone (colourless or light yellow). These structures are shown in Fig. 39.

Figure 39 Structural changes in malvidin-3-glucoside with pH (Timberlake, 1980).

The relative amount of these structural forms varies with pH, thus

determining colour changing. In very acid solution (pH<5), only the red AH+

cation exists. With increasing pH, its concentration decreases due to the hydration to the colourless carbinol base. Between pH 2 and 3 small amounts of the colourless chalcone and the blue chinoidal are also present. With increasing pH, together with these forms, the concentration of the carbinol base continues to increase against the red cationic form. Above pH 4 no red cationic compound appears and only the colourless form is present. (Timberlake, 1980). Figure 40 shows the variation of the pigment structures with the pH.

PEF as a pre-treatment stage in extraction of anthocyanins

73

Figure 40 Effects of pH on anthocyanin structures of malvidin-3-glucoside. A, B, C and AH+ refer to forms in Figure 39 (Timberlake, 1980).

In addition to pH sensitivity, anthocyanins degenerate in contact with

light, heat, oxygen, metals (iron, copper and tin), ascorbic acid and sulphur dioxide (Francis, 1999; Kearsley and Rodriguez, 1981). Besides the high colour attributes, the interest towards anthocyanins increased due to their beneficial pharmacological activities and possible health benefits. Their benefits to human health have been reported in several studies and include: antioxidant activity (Tamura and Yamagami, 1994; Wang et al., 1997), anticancer properties (Kamei et al., 1995), visual acuity promotion (Timberlake and Henry, 1988) and probable defence against coronary heart disease (Waterhouse, 1995)

V.1.3 Anthocyanins in grapes

Many food plants have been suggested as commercial sources of natural colorants. In his review, Francis (1999), reported pigment profiles and extraction methods for over 40 plants considered as potential sources and listed 49 patents on anthocyanin sources. Among the food plants available in nature, grapes are the major source of anthocyanins. Pigments content in grapes ranges between 30-75 mg/100g and varies greatly according to cultivar, season and environmental factors (Bridle and Timberlake, 1997). Anthocyanins are based on five aglycones: cyanidin, peonidin, malvidin, petunidin and delphidin and the related organic acids are acetic, coumaric and caffeic, The only sugar present is glucose. Grapes, usually, show a very complex anthocyanins profile depending mainly on their cultivar.

For instance, the Concord variety has 31 anthocyanins, the greatest number in any single cultivar. In European grape (Vitis Vinifera) the only anthocyanins are the 3-glucosides derivates (Wulf and Nagel, 1978), while

Chapter V

74

the 3,5-diglucosides compounds also appear in American species (V. Riparia, V. rupestris) and hybrid varieties (Hrazdina, 1975; Baublis et al., 1995).

V.1.4 Commercial preparation of grape skin extract: Enocyanin

Despite of the large number of potential sources, only grapes are used in commercial application of anthocyanins extraction.

Colorants from grapes have been suitable for nearly 120 years, primarily from press cake as a by-product of the wine industry. Grapes are the world’s largest fruit crop for processing. In 1995, the annual production of grapes was estimated to be 60 million metric tons, of which about 80% was used to make wine. This ensures an limited-less source of inexpensive raw material for colorant production.

Enocianin is the regulatory approved natural colorant made from grape skins. This product is classified as a E 163 by the European community and as 21 CFR 73 by the American Food and Drug Administration and is widely used in food industry as dye of soft drinks, jams, snacks, dairy products and confectionery and even in pharmaceutical products in many countries like Canada, United States, Japan and European Union.

The most common commercial method of producing grape skin extract involves treatment of the skins with water solution of sulphur dioxide up to 3.000 p.p.m., or its equivalent in bisulphite or metabisulfite. After 48-72 h, the liquid is removed from the skins, filtered, desulfured, and concentrated. Sometimes, a fermentation step is allowed before the extraction and in this case the alcohol is removed in the next steps of the process. The final product is a particle free liquid of high colorant power, which may also be dried to produce a water-soluble dry powder. The presence of sulphur dioxide in the extracting medium gives rise to an increased extraction yield of the pigments as well as increased stability of the final product. The sulphite-anthocyanin complex is colourless, thus the sulphite must be removed before the final concentration and filtration step. The addition of acids makes the extraction step more efficient, but mineral acids require a neutralization step at some point. If tartaric acid is used, it can be removed by addition of potassium hydroxide since the resulting precipitate of potassium hydrogen tartrate can be easily removed by filtration.

Purification of the crude extract can be accomplished in a bed of ion- exchange resins. The eluate can be fractionated. The first portion is red while the tail portion is blue due to the higher molecular weight compounds of the latter compounds crossing the bed of resins at a lower rate. Treatment with resins produces a more pure products but the cost of the colorant extract is higher. The FDA- approved procedures for extraction are specified in 21 CFR 73.170.

PEF as a pre-treatment stage in extraction of anthocyanins

75

In the last two decades investigations on anthocyanins were concerned with the extraction techniques. In particular, the use of different solventsis discussed including methanol, acetone, ethanol and water, all of which are generally acidified with HCl or with SO2 (Dikmen and Yildiz, 1988; Yokotsuka and Nishino, 1990; Bocevska and Stevcesvska, 1997; Garcia-Viquera, et al., 1998; Perez-Munuera et al., 1998; Revilla et al., 1998)

Extraction of anthocyanins is much more efficient with acidified methanol or ethanol. The use of these solvents in the enocyanin production process would require an additional step to remove alcohol. While wineries are usually familiar with handling alcohol, other producers of colorants seem to be reluctant to use an alcohol recovery unit in the processing plants. As a result, alcohol extraction has no commercial appeal.

V.2 Materials and methods

V.2.1. Sample preparations and chemicals

Black Grape of Palieri variety were used for the experiments. The grape skin has a dark colour while the pulp is clear. Grapes, bought from a local market, were stored in refrigerated conditions at 4 °C until used. Before each test, the samples were removed from the refrigerator in order to reach the room temperature i.e. 25-30°C. Grape diameter was in the range 15 to 20 mm. Two different preparations steps of the samples were used. Whole berries with skin were cut in pieces, deseeded and 10 g have been used in the experiments. Moreover, whole berries were manually peeled to remove skins. 1g of this material was used in the experiments.

Standards of anthocyanins (Cyanidin 3-glucoside, Malvidine 3-glucoside, Pelargonidin 3-glucoside, Peonidin Chloride), acid formic and acetonitrile were purchased from Sigma Aldrich Company.

V.2.2 Experimental procedures

V.2.2.1 PEF treatment

Pulsed electric field treatment was carried out with the high voltage pulse generator described previously in Section III.2.1, equipped with the new treatment chamber set up ad hoc for these investigations.

The treatment chamber consists of two polished surface stainless steel disc shaped electrodes with polished surfaces and a cylindrical spacer acting as insulator. The electrodes diameter is 30 mm. The spacer is made of polycarbonate and its height is 70 mm. The cylindrical frame holds the electrodes in fixed position and contains the material undergoing PEF treatment. The lower electrode is fixed to the base of the cylinder, while the upper one can be moved as a piston inside the plastic frame.

Chapter V

76

Due to its configuration, the system is flexible since it allows to treat different volumes of the food sample. Moreover, the container was specifically made of transparent material to measure the distance i.e. the gap between the electrodes as well as for inspection of the sample before the treatment. In this way, it is possible to detect the presence of air bubbles accidentally entrapped in the food material, before the generation of pulses. All the system lies on a plastic base. Electrical connections between treatment pulse generator is guaranteed by the side hole on the base wall. A detailed draw of the described treatment chamber is reported in Fig. 41.

Grape samples were placed in the treatment chamber and, due to their irregular shape, care was taken to guarantee a good contact with the electrodes. To reach this purpose, it was necessary to exert a light pressure on the samples and, due to the softening of the tissue, the leakage of a small amount of juice was observed. This juice was added to the treated material in the next extraction process. The same experimental procedure was applied to control samples except for the generation of the electrical pulses. In fact, the samples were kept in the treatment chamber for all the time required for PEF treatment. In this way it was possible to induce the same stress to the tissue as for PEF-treated samples to evaluate only the effect of PEF on the extraction yield. The gap between the electrodes was measured by means of a digital calliper (327.15, LTF) in order to guarantee the same electric field strength during each PEF experiment. At a given electric field strength and gap, the voltage to be applied was then determined. In spite of gap variations, in each experiment, the desired electric field strength was guaranteed.

Rectangular pulses with the following characteristics were used:

• Pulse duration, τp= 100 µs; • Total number of pulses, n=100; • Interpulse duration, ∆t=10 ms; • Electric field strength, E = 1 and 3 kV/cm.

Optimal process parameters were selected from specific preliminary

investigations (data not shown). For in stance, the maximum electric field strength depends on the electrical properties of the sample and is limited by the current output sustainable by the pulse generator. According to this consideration, the electrical filed strength of 3 kV/cm couldn’t be used for berries. Moreover, to limit ohmic heating effect, the pulses were not applied in a single burst. 10 bursts of 10 pulses were used. Finally, during the application of pulses, voltage ad current waveforms were recorded by means of an oscilloscope (3014B, Tektronix, USA).

PEF as a pre-treatment stage in extraction of anthocyanins

77

Figure 41 Draw of the batch treatment chamber set-up.

Chapter V

78

V2.2.2 Electrical impedance measurement

Before and after PEF treatment, the electrical impedance of the sample was measured using the methodology described previously in Section III.5.2 Moreover, the experimental procedure was optimized ad hoc by avoiding to use of the sample holder carrying out the measurements in the PEF treatment chamber.

V.2.2.3 Extraction

Extracts from both untreated and PEF-treated samples were obtained prepared using distilled water as solvent. The samples were removed from the treatment chamber and suspended in the solvent at room temperature with a fruit/solvent ratio g/ml of 1:2, for 1 min in agitated conditions. Samples were centrifuged (PK 121 R, ALC centrifuge) at 5000 rpm for 5 min at a temperature of 10 °C. The supernatant was filtered with acetate membrane 0,45 µm syringe filters (Schleicher & Schuell, Whatman). Untreated and PEF-treated samples were prepared in three replicates.

V.2.2.4 Analysis

Anthocyanins extracts were analyzed by means of high performance liquid chromatography (HPLC). Compared to spectrophotometric techniques, this method makes possible the identification of individual pigments in complex mixtures. HPLC system consists of a UV detector (2487, Waters, USA) equipped with a binary pump (1525, Waters). Samples of 20 µl were injected in a reversed-phase C18 column (Spherisorb 4,26 *250 mm, Waters). Chromatograms were observed at 520 m, the maximum absorbance wavelength of this class of compounds (Lapornik et al, 2005).

Elution solvent A was a solution of formic acid/water 1:9. Elution solvent B was a solution of acid formic/water/acetonitrile 1:4:5.

The extraction yield was expressed in terms of the malvidine-3-glucoside and cyanidine-3-glucoside contents. The concentration of these anthocyanins in the extracts was determined using peak areas from standard curves. Standard were dissolved in the solvent A. Calibration curves of malvidine 3-glucoside and cyanidine-3-glucoside are reported in Appendix.

PEF as a pre-treatment stage in extraction of anthocyanins

79

1

2

1

2

V.3 Results and discussion

V.3.1 Composition of grape extracts

The chromatogram of the extract obtained from an untreated berry sample is shown in Figure 42. Two major peaks are evident (peak 1) and (peak 2), and other small peaks can be observed.

Figure 42 Chromatogram of anthocyanin extract obtained from untreated grape berry samples at 520 nm.

The main anthocyanin, corresponding to peak 2, is malvidine-3-glucoside (Figure 43). In the extract also cyanidine-3-glucoside was identified (Figure 44). Grape skin extracts showed the same chromatographic profile (Figures 45 and 46) confirming that anthocyanins are stored in the skin of the fruit. It can be observed that extract composition is similar to that reported by Lapornik et al. (2005). The authors found that malvidine-3-glucoside is the major anthocyanin in water extract from grapes. In the same way, the

Chapter V

80

Control

Cyanidin-3-glucoside

Control

Cyanidin-3-glucoside

Control

Malvidin-3-glucoside

Control

Malvidin-3-glucoside

presence of small amounts of cyanidine-3-glucoside was shown by Goiffon et al. (1999).

Figure 43 Comparison in the anthocyanin composition of extract obtained from untreated grape berry and standard malvidin-3-glucoside.

Figure 44 Comparison in the anthocyanin composition of extract obtained from untreated grape berry and standard cyanidin-3-glucoside.

PEF as a pre-treatment stage in extraction of anthocyanins

81

Control

Malvidin-3-glucoside

Control

Malvidin-3-glucoside

Figure 45 Comparison in the anthocyanin composition of extract obtained from untreated grape skin and standard malvidin-3-glucoside.

Figure 46 Comparison in the anthocyanin composition of extract obtained from untreated grape skin and standard cyanidin-3-glucoside.

Control

Cyanidin-3-glucoside

Control

Cyanidin-3-glucoside

Chapter V

82

Control

PEF

Control

PEF

V.3.2 The effect of PEF on tissue permeabilization and extraction

of anthocyanins

In Figure 47 the chromatograms of extracts obtained from untreated and PEF treated berry samples are compared. It can be observed that Pulsed Electric Field doesn’t determine the change of the composition in anthocyanins of the extract. Moreover, results indicate that the electrical treatment induces an increase of the anthocyanins concentration, Since these pigments are stored in the vacuoles of the cells, this increase is a direct consequence of cell membrane permeabilization induced by PEF pre-treatment. The relative increase of the extraction yield produced by PEF is 3% for malvidine-3-glucoside and 4% for cyanidine-3-glucoside (Table 6). After PEF, the final concentration of pigments achieved is higher . Therefore, it can be concluded that PEF-treatment increases the anthocyanins extraction yield without causing the alteration of the pigments.

Figure 47 Comparison in the anthocyanin composition of extract obtained from untreated and PEF-treated grape berry.

PEF as a pre-treatment stage in extraction of anthocyanins

83

Table 6 Anthocyanins concentrations in grape berries extracts obtained before and after PEF at 1 kV/cm.

Membrane permeabilization effects can be also observed monitoring the

current flowing through the sample during treatments. In intact tissues the current reaches a peak due to the polarization of cell membranes, and after which decreases with exponential decay.

Current shape after the application of the first pulse is reported in Figure. 48.

After the first pulse, cell membrane breakdown can be observed. In fact, the current shows the characteristics curve of permeabilized tissues as reported by Angersbach et al., (2000).

Figure 48 Voltage and current traces during PEF treatment at 1 kV/cm at a) 1 pulse and b) 100 pulses.

Sample

Cyanidin-3-glucoside (mg/g)

Malvidin-3-glucoside

(mg/g) Control

3,61*10-5 3,70*10-3

PEF 1,68*10-4 1,48*10-2

a) 1 pulse b) 100 pulses

membranebreakdown

voltage voltage

currentcurrent

a) 1 pulse b) 100 pulses

membranebreakdownmembranebreakdown

voltage voltage

currentcurrent

Chapter V

84

With increasing the number of pulses and the degree of permeabilization achieved, the current increases. At 100 pulses the current shape validates the impedance measurement revealing a pure resistive behaviour and thus the occurrence of total permeabilization (Figure 48).

In Figure 49 rate of the electrical impedance measurement is shown. Before the electrical treatment, the impedance is a function of frequency. This is due to the capacitive contribution of cell membranes. After PEF at 100 pulses, almost total permeabilization of cell membranes occurs, thus the electrical impedance is independent on frequency and the curve reduces to a straight line. Results obtained are in agreement with the current shape reported in Fig. 48.

0,00E+00

5,00E+02

1,00E+03

1,50E+03

2,00E+03

2,50E+03

1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06

Freq (Hz)

|Z| O

hm Control

PEF Treated

Figure 49 Impedance spectrum of control and PEF-treated grape samples.

The electrical energy applied during the PEF treatment and the

corresponding increase of temperature were also evaluated. The energy density Q was determined according to the following Equation:

f

n

p

m m

VIQ

∑= 1

τ (V.1)

PEF as a pre-treatment stage in extraction of anthocyanins

85

Control

PEF

Control

PEF

Control

PEF

Control

PEF

where m is the mass of the product (kg), τ is the pulse duration (µs), V is the applied voltage (kV) and I is the current peak value (A). Voltage and current peak values were recorded by means of the software HPVEE of the pulse generator.

Finally temperature increase was evaluated as follows:

p

m

CQ

T =∆ (V.2)

where Cp is the specific heat of the sample, assuming for the latter parameter the value of 3,53kJ/kg°C (Rao and Rizvi, 1995), the temperature increase ranged between 3-4°C.

Figures 50 and 51 report the chromatograms of the extracts obtained from untreated and PEF-treated grape skins at 1 kV/cm and 3 kV/cm. PEF treatment doesn’t produce any enhancement of the extraction yield at 1 kV/cm. On the contrary, at 3 kV/cm the degree of permeabilization achieved induces a high increase in the extraction yield. The increase of anthocyanins concentration after PEF is 11% for cyanidin-3-glucoside and 15% for malvidin -3-glucoside (Table 7)

Figure 50 Comparison in the anthocyanin composition of extract obtained from untreated and PEF-treated grape skin at 1 kV/cm.

Chapter V

86

PEF

Control

PEF

Control

Figure 51 Comparison in the anthocyanin composition of extract obtained from untreated and PEF-treated grape skin at 3 kV/cm.

Table 7 Anthocyanins concentration in grape skin extracts obtained before and after PEF at 3kV/cm

Sample

Cyanidin-3-glucoside (mg/g)

Malvidin-3-glucoside

(mg/g) Control

1,99*10-5 0,102

PEF 2,44*10-4 0,163

PEF as a pre-treatment stage in extraction of anthocyanins

87

Results show that for skins an electric field strength of 1 kV/cm is ineffective to achieve a good degree of permeabilization. This difference can be attributed to the low conductivity of the skins with respect to the whole berry due to the absence of pulp. Therefore even if the energy provided by the electrical treatment is significant, the local resistance offered by the skin to the current flow is very high. Results of these investigations confirm that the electrical conductivity of samples is a key parameter in determining the permeabilization of cells membranes. Finally, grape marcs, i.e. the by-products of wine processing were used in these investigation. Fermented marcs were provided by Mastro Berardino Wine Company, Avellino. The results revealed that even at 3 kV/cm there is no enhancement in the extraction yield (data not shown). This should be attributed to the effects of the fermentation process, that induces cell membranes disintegration, thus limiting the effectiveness of PEF treatments.

Chapter VI Design of a PEF continuous

system

VI.1 PEF continuous system

A laboratory scale continuous PEF system has been designed to apply pulsed electric field membrane permeabilization to food slurries. In the design of the PEF system many aspects have been taken into account including the electrical as well as fluid dynamic characteristics. PEF continuous system consists of: a PEF treatment section, a pump, and a cooling device as reported in Figure 52.

Figure 52 Scheme of a PEF continuous system to process food slurries.

Initial product Treatment chamberTreated product

Pulse generator

Coolingdevice

PumpInitial product Treatment chamberTreated product

Pulse generator

Coolingdevice

Pump

Chapter VI

90

The food material, previously comminuted, is feed with a pump in the PEF treatment section where is exposed to electrical pulses. The continuous PEF treatment section includes the high voltage pulse generator (Service Electronique, 1500V-20 A) and the continuous treatment chamber designed ad hoc. Before entering the PEF treatment section, the food material cross a cooling section. In fact, in very high intensity treatments the refrigeration of the fluid is necessary to counteract the increase of temperature occurring to the sample during pulses generation. PEF system elements and the design choices are described in the following sections.

VI.2 Design of a continuous PEF treatment chamber

To design the treatment chamber properly the electrical properties of the slurries to be treated and fluid dynamics aspects related to the flow of the viscous material have to be taken into account. In fact, the treatment chamber must guarantee the homogenous and continuous PEF treatment of the food material. Moreover, it should be operated wide range of treatment conditions with different food materials. Several continuous treatment chambers have been proposed. Examples of these chambers are parallel plate, concentric cylinder and co-field treatment chambers. However, these chambers have been designed for inactivation purposes treating almost liquid foodstuff and thus homogenous material. In this case, an additional parameter i.e. the presence of solid particles in the food material has been considered in determining the chamber configuration.

As mentioned in Section I.3.1 the principal requirement to be guaranteed in designing a treatment chamber is the homogeneity of the generated electric field. Homogeneity of the electric field throughout the gap between the electrodes, in fact, ensures homogeneous treatments. When different field intensities are present between the electrodes some of the product maybe sub-treated, while other portion of the product maybe over-treated. Parallel plate configuration allows a constant electric field throughout the chamber volume.

Concentric cylinder electrode treatment chambers on the other hand, show a radial distribution of the electric field with a decreased electric field vector density from the inner high-voltage electrode to the outer low-voltage electrode. The intensity of the electric field (E) at any point r between the electrodes in a concentric electrode treatment chamber is defined according to the following Equation:

⎟⎟⎠

⎞⎜⎜⎝

⎛=

1

2lnRR

r

VE (VI.1)

Design of a PEF continuous system

91

where R2 is the radius of the low-voltage electrode and R1 is the radius of the high-voltage electrode (Zhang et al, 1995).

Also in a co-field treatment chamber, the electric field is not constant. The distribution of the electric field in this type of chamber is variable depending on the exact configuration of the electrodes and to their relative position with respect to the insulating elements. A proper relationship to define the electric field intensity in the area between the electrodes has not been developed yet, thus Equation (I.15) is commonly employed.

Even though coaxial and co-field treatment chamber show an heterogeneous distribution of the electric field strength, they are more suitable for continuous operations than parallel plate treatment chambers. However, co-field electric chambers provide better fluid dynamics characteristics. On the other hand, although coaxial treatment chambers are more simple, they show a smaller cross section available for the flow, that can be easily blocked by particulate products. The co-field treatment chamber (for the same electric field strength) allows to treat large particle and is not blocked by particulate products.

Considering the energy consumption and low product heating effects, treatment chambers with high electrical resistance are the most suitable. Co-field treatment chambers have higher resistance than parallel plate and coaxial treatment chambers. In fact, depending on design and foodstuff characteristics, coaxial and parallel plate treatment chamber have intrinsic resistances in the range of 3 to 30 Ω, whereas the resistance of co-field treatment chamber is of the order of 50 to 300Ω (Gòngora and Nieto, 2002). Due to the high intrinsic resistance of Co-field treatment chambers, they can be used in the configuration of several treatment chambers in series, thus increasing the volume of the treated material (Yeom et al, 1999; Evrendilek et al., 2004).

On the basis of the above considerations, two treatment chambers have been designed: a parallel plate and a co-field chamber. The parallel plate configuration has been chosen for the uniformity of the electric field. It is suitable to study the permeabilization induced by PEF in a continuous configuration. On the contrary, the co-field treatment chamber represents a more advanced and engineered solution view of industrial application of PEF technique.

VI.2.1. Parallel plate continuous treatment chamber

The parallel plate treatment chamber consists of two stainless steel plate electrodes and a Teflon insulator. The insulator is cylindrical and presents internal square cross section cavities in which the two electrodes are placed. Two side holes allows the electrical connection of the electrodes with the pulser. The chamber consists of two symmetric halves, easily opened for cleaning.

Chapter VI

92

The size of the parallel plate treatment chamber was defined on the basis of the value of its electrical resistance cR according to:

fc S

dRσ

= (VI.2)

being d, the gap between the electrodes, fσ the electrical conductivity of

the food material and S the contact superficial area of the electrodes. After PEF permeabilization during the generation of pulses the electrical

current flowing in the foodstuff, thus in the treatment chamber, can significantly increase becoming 10 or more times higher than its initial value.

Food slurries are very high conductive materials, thus electrical current can assume very high values easily exceeding eventually the output limit sustainable by the pulse generator to which the treatment chamber is connected.

In the design of the treatment chamber this undesired drawback has to be taken into account. As already stated in Section III.2.1, in fact, if during the generation of the pulses the current exceeds 20A the generation of the pulses is inhibited and PEF treatment can’t be completed. Thus, it is necessary to fix the minimum resistance value allowing the correct operability of the unit. While the limiting current of 20A should not be exceeded during operation whatever the value of the operating voltage, the critical condition occurs by applying the pulser maximum voltage output of 1500 V. Thus, to operate in “safety” conditions, the minimum value of the resistance should be equal to 400 Ω. The design of the treatment chamber was made according to this value.

The first variable to be considered in the design of the treatment chamber has been the gap between the electrodes. As stated by Equation. (I.15), the electric field strength depends on the gap, the lower the gap, the higher the electric field strength.

However the gap should not be too small, to void its blockage due to the food slurry flowing in the same treatment chamber. Based on this considerations, a gap of 75 mm has been chosen. To this value maximum electric field strength of 2 kV/cm can be applied.

In order to be able to treat food slurries with different electrical conductivities, the maximum electrical conductivity was determined. To this purpose, electrical impedance measurements were carried out on two different food slurries: strawberry and grape mashes. Electrical conductivities were determined from the impedance spectrum at 100 Hz. At this frequency, in fact, the food can be assumed as only resistive. Table 8 shows the calculated electrical conductivities of food slurries.

Design of a PEF continuous system

93

Table 8 Electrical conductivities of food slurries.

Food Electrical conductivity (S/mm)

Strawberry mash (1÷2) 10-4

Grape mash (0.5÷1) 10-4

As can be observed strawberry slurry exhibits the highest value of the electrical conductivity and thus this value was selected for design purposes. On the basis of the latter value of the electrical conductivity and at a fixed value of the gap, the electrode section has been determined according to Equation (VI.2) in which it is assumed that the minimum resistance value is that corresponding to the maximum value of the electrical conductivity of the food material. Thus, the selected electrodes surface area is 56,25 mm2 and the volume of the treatment zone is 1,4 ml. Moreover, to allow a simple connection of the treatment chamber with the flow system, a square flowing section has been chosen with the width of the electrodes equal to the gap size i.e. 75 mm. Finally, the length of the electrodes has been calculated as the ratio between the surface area and the width i.e. 25 mm. Table 9 summarizes the geometry characteristics of the designed treatment chamber.

Table 9 Characteristics of the parallel plate treatment chamber. Gap between the electrodes, d 7,5 mm

Electrode width, EW 7,5 mm

Electrode surface area, S

fcRdSσ

= = 56,25 mm2

Electrode length, L LSH = =25 mm

Volume of the treatment zone, tv tv = SH =1,4 ml

Chapter VI

94

1A reduction of the flow section occurs from the connection piping to the inlet of the treatment chamber. To avoid the formation of a stagnant zone at the inlet of the treatment chamber, the spacer is longer than the electrodes. In this way, after the entering section the reaches the treatment zone with a flow field parallel parallel to the axis of the treatment chamber.

The length of the entrance zone LE has been evaluated according to:

hE DL Re02875,0= (VI.3)

(Reileigh, 1975) Re is the Reynolds number and hD is the hydraulic diameter defined as:

µρ hff DF

=Re (VI.4)

p

rh w

AD

4= (VI.5)

where fF is the flow rate, fρ is the food sample density, µ is the fluid

viscosity, rA is the square section and pw is the wetted perimeter. The values of the entrance length and of related variables are reported in the Table 10.

Table 10 Length of entrance and related variables. Fmin is the minimum flow rate.

Flow rate , fF == min10FFf 85 ml/min

Hydraulic diameter, Dh 7,5 mm

Reynolds number , Re

µρ hff DF

=Re = 129

Length of entrance, EL hE DL Re02875,0= =28 mm

Figure 53 shows the draw of the parallel plate treatment chamber.

Design of a PEF continuous system

95

95ø4

ø7.5

28

10

ø10 25

28

ø4

7.5

Chapter VI

96

Figure 53 Draw of the parallel plate treatment chamber. The electric field distribution in the designed treatment chamber have

been evaluated using the simulation software “Maxwell 2D”. The software generates the bidimensional solution of the Maxwell equation. In the simulations the voltage of 1500 V and the gap of 7,5 mm have been assumed. The Table 11 summarizes the parameters used in the simulation.

Table 11 Simulation parameters.

Material Relative permittivity

Electrical conductivity (S/mm)

Electrodes Stainless steel 1 1.1 *106 Insulator Teflon 2,08 0,0051

Fluid Water 81 0,01 Assuming an uniform distribution of the electric field, its the value can be

calculated as the ratio between the applied voltage and the gap between the electrodes, i.e. according to Equation (I.15). Therefore, assuming for the applied voltage its maximum value of 1500 V and for the gap the design value, the theoretical value of the electric field strength is 2 kV/cm.

The electric field lines in the treatment chamber are shown in Figure 54. The electric field intensity between the electrodes is almost constant. As

expected, the electric field vectors are parallel and perpendicular to the electrodes and parallel with presenting the same density throughout the region between the electrodes. Moreover, the electric field strength evaluated via software is equal to the expected theoretical value. As can be observed, the electric field intensity decreases near the exit of the treatment chamber and in this region the field lines become curves. The non-uniformity of the electric field is negligible since it occurs in the areas external to the PEF treatment section.

Design of a PEF continuous system

97

Figure 54 Distribution of the electric field vector in the parallel plate treatment chamber.

VI.2.2 Co-field treatment chamber

The co-field treatment chamber includes two electrodes to supply the electric field to the food product. Each electrode includes an electrode flow chamber, allowing the flow of the food to be processed and the electrical contact with the food products. The electrodes are made in stainless steel with a cylindrical geometry. Each electrode includes an inlet and outlet opening to introduce the food material through the same electrodes in the axial direction. Thus, in this configuration the field as well as the electric field lines are parallel. This is the reason why the chamber is called “co-field”. A Teflon spacer between the first and the second electrode ensures

Chapter VI

98

20.0

ø10.0

ø20.0

15.0

45.0

ø20.0

ø10.0

ø30.0

the electrical insulation of the two conductive elements. The insulator and of the electrodes are designed to include a single tube flow chamber. The insulator flow chamber inlet aperture and the first electrode flow chamber outlet aperture are adjacent to each other and show the same geometry of the cross-section. The treatment zone, between the two electrodes, has a length of 15 mm and a diameter of 10 mm equal to the diameter of the apertures in the electrodes. The diameter was chosen to connect the treatment chamber with the pump, while the length of the electrodes allows to obtain an adequate volume of the treated sample. Figure 55 shows the draw of the co-field treatment chamber.

Figure 55 Draw of the co-field treatment chamber. The volume of the treatment zone is about 1,2 ml. Assuming the

electrical conductivity of 2* 10-4 S/mm, the electrical resistance of the treatment chamber is about 1 kΩ above the critical value of the high voltage pulse generator. Table 12 summarizes the geometric characteristics of the co-field treatment chamber

Table 12 Characteristics of the co-field treatment chamber.

Gap between the electrodes, d 15 mm

Electrode diameter, Φ 10 mm

Flow Section, Σ

4

2ΦΣ π= = 78.5 mm2

Design of a PEF continuous system

99

Volume of the treatment zone, tv dvt Σ= =1.17 ml

To provide the electrical insulation between high voltage elements and

other components of the apparatus that can be damaged by high voltage, i.e. the pump, the co-field treatment chamber is introduced in a insulating cylindrical holder made of Teflon. This holder also realizes the matching of the single components of the treatment chamber (electrodes ed insulator).

VI.3 Fluid-handling system

The continuous PEF system is equipped with the piping and the pump necessary to feed the food material in the treatment chamber. The pump should ensure a continuous and uniform flow of the material treatment homogeneity. The viscosity of the food material is an important parameter determining the choice of the feeding pump. On this basis, a single screw pump has been chosen. Single screw pumps are rotary positive displacement pump able to move materials of different viscosity, such as fluid, media with suspended solids or slurries, and are commonly used in food processing applications. Moreover this kind of pump has the advantage of moving viscous media at low velocities. The pump (MN 15 10/7, C.M.O. Pompe, Italy) operates by means of a rotating assembly consisting of a stainless steel screw, i.e. rotor, and a stator. The stator is formed by a stainless steel container covered by a layer made in natural rubber. Rotation of the screw inside the stator creates a series of cavities that are moved along the suction-delivery axis, thereby creating the pumping action. The pump is also equipped with a temperature probe on the stator monitoring the temperature and preventing the damage of the device caused by excessive heat.

The food material is fed at the top through a stainless steel hopper. The flow rate can be manually regulated in a wide range between 0-0,4 m3/h according to the rotation speed (0-1000 rpm).

In a PEF continuous process, the flow rate F is defined as:

T

tf t

vF = (VI.6)

being Tt the total treatment time and tv the volume of the treatment zone

i.e. the volume between the two electrodes. The minimum flow rate required to the pump has been chosen on the

basis of the maximum value of the treatment time allowed by the technical features (pulse number and pulsing frequency) of the pulser, that is 10 s.

Chapter VI

100

For parallel plate treatment chamber, the minimum flow rate is 8,5 ml/min, while for the co-field treatment chamber is 7,1 ml/min.

The continuous processing system is completed by a circulating water bath (F25-ED, Julabo, Italy). If necessary, before entering the treatment chamber the food material, flowing through a stainless steel coil, can be cooled.

The refrigeration fluid is ethylene glycol. The bath allows the temperature variation in a wide range (-28-100°C). The choice of this device has been based on the maximum temperature increase occurring during PEF treatment and, thus, on the required cooling power. Assuming no heat losses during treatments, the increase of sample temperature during PEF treatment can be estimated from the energy delivered by the electrical treatment to the food for given sample properties i.e. the density and specific heat. The energy depends on the applied voltage, the food resistance and the total treatment time. To evaluate the maximum temperature increase, the electrical energy calculation was made assuming the maximum applied voltage (1500 V), the maximum treatment time(10 s) and the minimum food resistance (400 Ω). The maximum increase of temperature is 26°C, assuming for density and heat capacity the values of 3,6 kJ/kg °C and 1062 kg/m3, respectively. Assuming the maximum flow rate Fmax (100 ml/min), the maximum cooling power required P can be evaluated according to:

maxQFP = (VI.7)

Thus, the cooling power required is 160 W.

Chapter VII Conclusions

The starting point of the work performed in this thesis was the question of how wide could be the utilization of Pulsed Electric Field techniques in processing food materials. In fact, while the microbial stabilization effect of PEF on foods was recognized, collateral effects on permeabilization of vegetable cells as a potential enhancement of extraction or dehydration processes were considered matter of concern. Literature results were not exhaustive about and the attitude of potential users of this kind of treatment was, at least, doubtful.

As a consequence, most of this work was devoted to explore PEF applications as a pre-treatment for promoting permeabilization of foodstuff and to set up methods for a quantitative evaluation of the effectiveness of the process. From this point of view, some interesting results were obtained. Of course, not all the fundamental questions were answered properly; however, some useful conclusions could be drawn also in order to give a more precise direction to further work.

The following final statements can be formulated on the basis of the work performed:

- Pulsed Electric Field treatment can be considered an effective non-

thermal permeabilization technique in food processing, provided that a careful analysis of process conditions for the specific foodstuff is carried out. It was demonstrated that an effective permeabilization can be obtained, in general, for a number of vegetables, but optimal operative conditions must be determined previously through laboratory tests.

- A new experimental methodology was proposed and set up to measure

the degree of cell membrane permeabilization. The method is based on the direct measurement of the electrical impedance of the tissue as a function of the frequency in the beta dispersion range characteristics for biological systems, before and after the electrical treatment.

Chapter VII

102

This method, easy and fast to be performed in laboratory on small samples through pure electrical instruments, was proved to be useful in studying the effect of the investigated parameters on the cell membrane permeabilization. It is suitable for different foodstuff and is a valid help to determine optimal operating conditions.

- According to the experimental analysis performed, it was demonstrated

that among the process variables influencing cell permeabilization, the electric field strength and the number of pulses are the most significant.

Experimental results confirmed, as expected, that the effectiveness of PEF is mainly influenced by the intensity of the treatment. Higher degree of permeabilization can be achieved with increasing both the electric field strength and the number of pulses. However, the system reaches a saturation level, above which further increase of the treatment intensity doesn’t contributes to improve the degree of permeabilization. The level of saturation depends on the structure of the vegetable tissue. Thus, it is a property of the foodstuff, that can be determined by means of preliminary laboratory tests on electrical impedance.

- Besides the external electrical variables, cell membrane

permeabilization is influenced by the original conductivity of the tissue to be treated. A high conductivity of the sample requires a lower electric field strength to produce effective permeabilization.

Differently to what reported by relevant literature, which considers conductivity as a minor factor in permeabilization treatment, this suggests that conductivity is a key factor and thus has to be taken into account in the design of a PEF process. To compare the latter effect to that of other electrical variables, further investigations are needed.

- As far as the application of pulsed electric fields as a pre-treatment stage

before conventional thermal drying is concerned, experimental results demonstrated that cellular permeabilization induced by the electrical treatment enhances the first stage of the process determining, in this phase, an increase of the drying rate. However, the moisture content attained at the end of the drying process is almost the same for untreated and PEF-treated samples and, moreover, PEF doesn’t significantly enhance the quality of the product with respect to rehydration properties. These results suggest that PEF could be used fruitfully for drying mostly when an intermediate moisture content is required. However, this application seems to be not promising.

- As far as the application of pulsed electric fields as a pre-treatment stage

before the extraction of natural pigments from vegetables, experimental

Conclusions

103

results demonstrated that in this case PEF technique is a valuable tool in specific processes.

In particular, an assisted-extraction process for the recovery of high added-value components (anthocyanins) from grape was set-up and tested. To this purpose, a new batch treatment chamber was designed and a specific extraction procedure, based on the use of distilled water as solvent, was developed Experimental results showed that PEF is efficient also in achieving irreversible permeabilization in the part of the tissue, the skin, which offers the higher resistance to the mass transport process involved. In this case, permeabilization phenomena determine also a measurable increase of the extraction yield.

- As a secondary, but very interesting achievement, the new extraction

process set up, due to its non-thermal characteristic, fully preserves the integrity of the pigments. This could become, with time, the main key for the success of the application of PEF techniques to the extraction of natural biologically active components from vegetables.

Finally, we can mention that the promising results obtained so far

allowed to design a pre-pilot scale PEF apparatus operating in continuous. This apparatus will facilitate the performing of further work oriented to process development necessary to answer the basic question of how useful will be PEF technique in food industry and under what conditions.

Chapter VII

104

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Ade-Omoyawe B.I.O., Angersbach A., Taiwo K.A.and Knorr D. (2001c) Use of pulsed electric field pre-treatment to improve dehydration characteristics of plant based foods Trends in Food Science & Technology 12: 285-295.

Ade-Omoyawe B.I.O., Angersbach A., Taiwo K.A.and Knorr D. (2001d) Te use of pulsed electric fields in producing juice from paprika (Capsicum Annum L.) Journal of Food Processing Preservation 25: 353-365.

Ade-Omoyawe, B.I.O., Angersbach, A., Eshtiaghi, M. N. and Knorr, D. (2001a) Impact of high intensity electric field pulses on cell permeabilization and as pre-processing step in coconut processing Innovative Food Science and Emerging Technologies 1: 203-209.

Ade-Omoyawe, B.I.O., Angersbach, A., Rastogi, N. K. and Knorr, D. (2001b) Effects of high hydrostatic pressure or high intensity electric field pulse pre-treatment on dehydration characteristics of red paprika Innovative Food Science and Emerging Technologies 2: 1-7.

Angersbach, A. and Knorr, D. (1997) Anwendung elektrischer Hochspannungsimpulse als Vorbehandlungsverfahren zur Beeinflussung der Trocknungscharakteristika und Rehydratationseigenschaften von Kartoffelwürfeln Nahrung 41 (4): 194-200.

Angersbach, A., Heinz, V. and Knorr, D. (1999) Electrophysiological model of intact and processed plant tissues: cell disintegration criteria Biotechnology Progress 15: 753-762.

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106

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Symbols

A coefficient in the equation of the coefficient diffusion [ ]−

rA square section in parallel plate chamber [ ]2m

sA food sample area perpendicular to the electric field [ ]2m

tABS absorbance of the solution at time t [ ]−

AFABS absorbance of the solution after freezing/thawing [ ]− b coefficient in the equation of the cell disintegration index [ ]− B coefficient in the equation of the coefficient diffusion [ ]−

0c initial solute concentration [ ]lg /

∞c total solute concentration [ ]lg /

C capacitance [ ]F

oC capacitance of the capacitor bank [ ]F

mC capacitance per unit area of the cell membrane [ ]2/ mF

pC specific heat of the food sample [ ]CkgJ °/

D diffusion coefficient [ ]sm /2

hD hydraulic diameter in Eq. (VI.5) [ ]m d distance between the electrodes [ ]m E electric field strength [ ]mV /

cE critical electric field [ ]mV /

mE electric field strength at the cell membrane [ ]mV / F shape factor [ ]−

fF food sample flow rate [ ]sl /

f frequency [ ]Hz h thickness of the food sample [ ]m

)(ti instantaneous current crossing the food sample [ ]A K electrical conductivity of food tissue [ ]mS /

114

hK electrical conductivity of untreated tissue at 3-50 MHz [ ]mS /

lK electrical conductivity of untreated tissue at 1-5kHz [ ]mS /

tK electrical conductivity of the solution at time t [ ]mS /

AFK electrical conductivity of the solution after freezing/thawing [ ]mS /

hK ' complex conductivity of treated tissue at 3-50 MHz [ ]mS /

lK ' electrical complex conductivity treated tissue at 1-5 kHz [ ]mS / L electrode length [ ]m

EL length of the entrance zone in the parallel plate chamber [ ]m l cell length [ ]m

sl length of the food sample [ ]m

fm is the mass of the food sample [ ]kg

n number of pulses [ ]− )0(P initial weight of the food sample [ ]kg )(tP weight of the sample at time t [ ]kg

eP electro-compressive force per unit area [ ]2/ mN )(tp instantaneous power [ ]W

Q energy density per pulse [ ]3/ mJ

mQ energy per pulse per unit mass [ ]kgJ / R electrical resistance [ ]Ω

21 , RR radius of the inner and outer electrode surface [ ]m

bR ballast resistance placed in parallel to the treatment chamber [ ]Ω Re Reynolds number [ ]−

fR electrical resistance of the food sample [ ]Ω

RSA relative solution absorbance [ ]− RSC relative solution conductivity [ ]− r current radius [ ]m

cr cell radius [ ]m

S electrode surface area [ ]2m t time after the application of the electric field [ ]s

Tt treatment time [ ]s )(tu instantaneous voltage across the electrodes [ ]V

U charging voltage of the capacitor bank [ ]V

Symbols

115

tv volume of the treatment chamber [ ]3m

fv volume of the food sample [ ]3m

V voltage between the electrodes [ ]V

bV bulk volume of the food sample [ ]3/ mkg

0V peak voltage [ ]V W electrical energy dissipated in the food sample [ ]J

cW energy stored in a capacitor [ ]J

EW electrode width [ ]m

pW electrical energy per pulse dissipated in the food sample [ ]J

sW dry solid content of the food sample [ ]− X moisture content of the food sample [ ]− Y elastic modulus of the cell membrane [ ]2/ mN Z electrical impedance of the food sample [ ]Ω

pZ cell disintegration index [ ]− Greek symbols ϕ∆ transmembrane potential [ ]V

maxϕ∆ maximum transmembrane potential [ ]V

c,ϕ∆ critical transmembrane potential [ ]V

rϕ∆ resting potential [ ]V t∆ interpulse duration [ ]s T∆ temperature increase of the food sample [ ]C°

δ thickness of the cell membrane [ ]m

0δ unstrained thickness of the cell membrane [ ]m Φ electrode diameter [ ]m

mε relative permittivity of the cell membrane [ ]− ϑ angle between the electric field direction and the normal [ ]rad

bρ bulk or apparent density of the food sample [ ]3/ mkg

fρ density of the food sample [ ]3/ mkg

pρ particle or true density of the food sample [ ]3/ mkg

116

Σ Flow section in co-field treatment chamber [ ]2m

eσ electrical conductivity of the extracellular medium [ ]mS /

iσ electrical conductivity of the cytoplasm [ ]mS /

mσ electrical conductivity of the cell membrane [ ]mS / *σ complex conductivity of the food cylinder [ ]mS /

REL*σ relative complex conductivity of food cylinder [ ]mS /

UNTR*σ complex conductivity of untreated food cylinder [ ]mS /

TR*σ complex conductivity of the PEF-treated food cylinder [ ]mS /

τ constant time [ ]s

mτ relaxation time of the cell membrane [ ]s

pτ pulse duration [ ]s

ω angular frequency [ ]srad /

Appendix

Tarature curve of anthocyanin standards

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

A (u

Vsec

)

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

Abs

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

A (u

Vsec

)

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

Are

a

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

A (u

Vsec

)

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

Abs

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

A (u

Vsec

)

tarature Mv-3-O-glu

y = 3E+07x + 51197R

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

A (u

Vsec

)

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

Abs

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

A (u

Vsec

)

tarature Mv-3-O-glu

y = 3E+07x + 51197R2= 0.9996

y = 3E+07x + 51197R2= 0.9996

0

500000

1000000

1500000

2000000

2500000

3000000

0 0.02 0.04 0.06 0.08 0.1

c (mg/ml)

Are

a

tarature Cy-3-O-glu

y = 7E+07x + 86789R2= 1

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 0.02 0.04 0.06 0.08 0.1 0.12

c (mg/ml)

Abs

tarature Cy-3-O-glu

y = 7E+07x + 86789R2= 1

y = 7E+07x + 86789R2= 1

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 0.02 0.04 0.06 0.08 0.1 0.12

c (mg/ml)

Are

a

tarature Cy-3-O-glu

y = 7E+07x + 86789R2= 1

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 0.02 0.04 0.06 0.08 0.1 0.12

c (mg/ml)

Abs

tarature Cy-3-O-glu

y = 7E+07x + 86789R2= 1

y = 7E+07x + 86789R2= 1

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 0.02 0.04 0.06 0.08 0.1 0.12

c (mg/ml)

Are

a