Document [original]

Impact of Pulsed Electric Fields (PEF) on post-

permeabilization processes in plant cells

vorgelegt von

Diplom-Ingenieurin

Anna Winter

von der Fakultät III – Prozesswissenschaften

der Technischen Universität Berlin

zur Erlangung des akademischen Grades

Doktorin der Ingenieurwissenschaften

- Dr.-Ing. -

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. Dipl.-Ing. Frank-Jürgen Methner

1. Berichter: Prof. Dr. Dipl.-Ing. Dietrich Knorr

2. Berichter: Prof. Dr. Sc. agr. Monika Schreiner

Tag der wissenschaftlichen Aussprache: 06.06.2011

Berlin 2011

D 83

Für Oliver, Mae und Milla

Abstract I

Abstract

The exposure of biological cell material to Pulsed Electric Fields (PEF) leads to a spectrum of

biophysical and biochemical responses. The most important effect, the electrical breakdown

of cellular membranes, realizes the temporary or permanent pore formation in cell

membranes, which induces an increase in membrane permeability. The loss of

semipermeability enables the transport of non-permeating molecules across the cell

membrane. The disintegration of the cell membrane as well as the alteration of structural

properties offers numerous options to apply this novel, non-thermal and short-time technique

in food- and bioengineering. In this thesis the impact of PEF on plant single cells as well as on

vegetable tissues was investigated. In order to understand underlying mechanisms at cellular

level and to clarify the influence of cell wall on the degree of cell membrane disintegration,

protoplasts from cultured tobacco cells (Nicotiana tabacum b.y.-2) and cells with cell wall

were compared during and after reversible as well as irreversible PEF treatment. Results

showed higher sensitivity of protoplasts to electric fields related to native cells. Protoplasts

sizes were measured before and after different treatment intensities and protoplasts shrinkage

was used as an indicator for cell rupture. It could be demonstrated that cell volume decrease is

influenced by PEF intensity, initial cell size, cell orientation in the electric field and nucleus

position. Focus was also put on the potential of PEF to gentle disintegrate plant tissue and

thus to apply this technique in food industry. Hence, the enhancement of mass transfer after

irreversible membrane permeabilization from potato and asparagus tissue was examined.

Results showed the enhanced release of intracellular molecules from permeabilized tissue as

well as improved uptake of low molecular substances into the sample. Sugar, one substrate for

the Maillard reaction, was decreased in PEF treated potatoes due to membrane

permeabilization and the subsequent release of cell vacuole sugar, while conductivity

increased after electroporation and soaking in sodium chloride solution, indicating the

improved diffusion of salt caused by PEF. Higher release of cell liquid during drying was

noticed additionally. This effect increased with the treatment intensity. Furthermore, it was

revealed that PEF application leads to a significant reduction of fat content after deep fat

frying of potato stripes and thus provides a potential for the production of low-fat French

fries. It was noticed additionally that PEF treatment decreases the content of the biopolymer

lignin in white asparagus in order to improve macroscopic characteristics and gain softer

texture of the spears. It can be presumed that PEF is a capable assistance to thermal treatments

in the processing of potato snack products or in the preserving of asparagus for the

achievement of structural modifications and the improvement of process conditions.

Zusammenfassung II

Zusammenfassung

Der hochspannungsimpulsinduzierte Aufschluss der Zellmembran und die daraus folgende

Änderung der strukturellen Eigenschaften bergen großes Potential für die Anwendung dieses

nicht-thermischen und zeiteffektiven Verfahrens in der Bio- und Lebensmitteltechnologie.

Ziel dieser Arbeit war es, den Einfluss von Hochspannungsimpulsen (HSI) auf einzelne

Pflanzenzellen als auch auf pflanzliches Zellgewebe zu untersuchen. Zur Erforschung

grundlegender Mechanismen auf zellulärer Ebene und zur Klärung des Einflusses der

Zellwand auf den Grad der Zellmembranpermeabilisierung wurden Protoplasten kultivierter

Tabakzellen (Nicotiana tabacum b.y.-2) und Zellen mit Zellwand bezüglich ihres Verhaltens

während und nach reversibler als auch irreversibler HSI-Behandlung untersucht. Es konnte

gezeigt werden, dass Protoplasten sich sensibler gegenüber dem elektrischen Feld verhalten

als native Zellen. Die Zellgröße der Protoplasten wurde vor und nach verschiedenen HSI-

Behandlungsintensitäten gemessen. Die Verringerung der Zellgröße diente als Indikator für

den Grad des Zellaufschlusses. Es zeigte sich, dass die Reduktion des Zellvolumens von der

HSI-Behandlungsintensität, der Ausgangszellgröße, der Zellorientierung im elektrischen Feld

und der Position des Zellkerns abhängt. Zudem sollte das Potential elektrischer Felder zum

milden Zellaufschluss von pflanzlichem Gewebe für einen möglichen Einsatz in der Spargel-

und Kartoffelindustrie untersucht werden. Verbesserte Stofftransportvorgänge HSI-

behandelter Kartoffeln führten sowohl zu einer erleichterten Freigabe von intrazellulären

Molekülen als auch zu einer verbesserten Aufnahme von niedermolekularen Substanzen in

das Gewebe. HSI-behandeltes Kartoffelgewebe zeigte einen geringeren Gehalt an

reduzierendem Zucker, ein Substrat für die Maillard-Reaktion, was sich auf die erleichterte

Freigabe des Vakuoleninhalts durch die permeabilisierte Zellmembran zurückführen lässt. Im

Hinblick auf die verbesserte Molekülaufnahme in das aufgeschlossene Gewebe wurde eine

erleichterte Diffusion von Salzionen in HSI-behandelte Kartoffelscheiben beobachtet.

Zusätzlich erhöhte sich der Trocknungsgrad permeabilisierter Kartoffelscheiben mit

steigender HSI-Behandlungsintensität. Eine Fettextraktion frittierter Kartoffelstäbchen zeigte,

dass eine HSI-Vorbehandlung der Fettaufnahme während des Frittierens entgegenwirkt. Der

Einsatz von HSI bei der Herstellung fettreduzierter Pommes frites ist daher denkbar. Bei HSI-

behandeltem Spargel war eine Reduzierung des Biopolymers Lignin nachweisbar. Dies

könnte die ligninbedingte Verholzung der Spargelstangen bei der Verarbeitung vermindern.

HSI-induzierte strukturelle Modifikationen und die dadurch verbesserten Prozessbedingungen

lassen den Einsatz von HSI in der Kartoffel- und Spargelverarbeitungsindustrie als viel

versprechend erscheinen.

Danksagung III

Danksagung

Ich möchte mich an dieser Stelle bei all denen bedanken, ohne deren Hilfe diese

Arbeit nicht möglich gewesen wäre.

Ich danke Herrn Prof. Dr. Dietrich Knorr sowohl für Überlassung des Themas und die

fachliche Betreuung als auch für seine unkomplizierte und motivierende Art, die mich bei

meiner Arbeit sehr unterstützte.

Ich danke Frau Prof. Dr. Monika Schreiner für ihre sofortige Bereitschaft als Gutachterin tätig

zu sein und für ihre professionelle Hilfe beim Projektanträge schreiben.

Danke auch an meine Institutskollegen für ihre Hilfsbereitschaft und das freundliche

Arbeitsklima. Liebe Ana, danke für die schöne und turbulente Zeit, die wir beim Teilen

unserer Büros und beim Bearbeiten unserer Projekte hatten. Bok do Zagreb!

Ganz besonders danken möchte ich Paul und Suse Janositz, nicht nur für uneingeschränkte

Unterstützung, sondern auch für ihre grenzenlose und liebevolle Bereitschaft für ihre

Enkelinnen zu sorgen, ohne die ich meine Promotion nie hätte fertig stellen können.

Danke, danke, danke an meinen Mann Oliver und die zwei kleinen Mäuse für alles…

Table of Content IV

Table of Conten t

Abstract .......................................................................................................................................I

Zusammenfassung.................................................................................................................... II

Danksagung............................................................................................................................ III

Table of Content ......................................................................................................................IV

List of Figures ...........................................................................................................................V

List of Abbreviations ............................................................................................................. VII

List of original Articles.........................................................................................................VIII

1. Introduction........................................................................................................................... 1

2. Background ........................................................................................................................... 3

2.1 Biological cell material................................................................................................................ 3

2.1.1 Plant tissue............................................................................................................................................ 3

2.1.2 Plant cell culture................................................................................................................................... 4

2.2 Pulsed Electric Fields (PEF) Technology.................................................................................. 6

2.2.1 Mechanisms of action........................................................................................................................... 6

2.2.2 Applications.......................................................................................................................................... 8

3. Summary of research methodology .................................................................................... 10

3.1 Biological raw material............................................................................................................. 10

3.2 Experimental set-up and electric field pulses protocol.......................................................... 11

3.3 Examination of cell vitality through impedance measurement ............................................ 12

3.4 Determination of sugar content (D- Glucose, D- Fructose)................................................... 13

3.5 Analysis of fat content............................................................................................................... 13

3.6 Analysis of lignin content ......................................................................................................... 14

4. Main findings ...................................................................................................................... 16

4.1 Basic principles of PEF on cellular level: Microscopic visualization of cell structure

changes............................................................................................................................................. 16

4.1.1 Comparison of protoplasts (digested cell wall) and native cells (intact cell wall).............................. 16

4.1.2 Protoplasts as a model system to visualize influence factors on PEF induced membrane rupture -

determinant factors: PEF treatment intensity and cell size.......................................................................... 18

4.2 Applications of PEF on plant tissue: Enhanced mass transfer of low molecular substances

.......................................................................................................................................................... 20

4.2.1 PEF-induced release of intracellular substances Î sugar.................................................................. 21

4.2.2 PEF-induced release of intracellular substances Î cell liquid Î lowering of French fries fat content

..................................................................................................................................................................... 23

4.2.3 PEF-induced uptake of extracellular substances Î sodium chloride................................................. 25

4.3 PEF-induced changes on food ingredients

lignin.............................................................. 25

5. Conclusions ......................................................................................................................... 28

5.1 Outlook and Future work......................................................................................................... 30

References................................................................................................................................ 34

Curriculum Vitae..................................................................................................................... 40

List of Figures V

List of Figures

Figure 1: Plant cell structure (http://de.wikipedia.org/). ......................................................................... 4

Figure 2: Isolated protoplasts of seven-day-old Nicotiana Tabacum cell suspension after enzymatic

… cell wall degradation (Janositz & Knorr, 2010)....................................................................... 5

Figure 3: Electroporation of a cell membrane (Tsong, 1991). ................................................................ 7

Figure 4: Schematic depiction of food and process improvement due to pulsed electric fields (Janositz,

…...unpublished results)................................................................................................................. 8

Figure 5: Protoplasts (Nicotiana Tabacum L. cv Bright Yellow-2) untreated and after PEF treatment

…...(E= 0. 5 kV/cm, n= 10, f= 2 Hz) (Janositz & Knorr, 2010)................................................... 17

Figure 6: Tobacco cells with cell wall (Nicotiana Tabacum L. cv Bright Yellow-2) in vital dye

…....solution (Phenosafranine) untreated and after PEF treatment (E= 2. 5 kV/cm, n= 20, f= 2

……Hz) (Janositz & Knorr, 2010)............................................................................................... 17

Figure 7: Cell disintegration index of untreated and PEF treated protoplasts and native cells after

…...different PEF treatment conditions (Janositz & Knorr, 2010)............................................... 18

Figure 8: Cell area of untreated and PEF treated protoplasts before and after PEF processing with

…...different treatment conditions. Statistical significance (* P<0.05, ** P<0.01, *** P<0.001)

…...(Janositz & Knorr, 2010)....................................................................................................... 19

Figure 9: PEF treated (E= 5 kV/cm, n= 20, 5 min. after treatment) and untreated potato tissue stained

…...with ruthenium red. Light microscope (Nikon Eclipse TS 100, Japan) (Janositz, Noack &

…...Knorr, 2011). ......................................................................................................................... 20

Figure 10: Sugar content in potato slices after PEF treatment (E= 1.5 kV/cm, n= 20) in comparison to

…….untreated samples. □ = PEF treated potato samples, ■ = untreated potato samples.

……Statistical significance (* P<0.05, ** P<0.01, *** P<0.001) (Janositz, Noack & Knorr,

……2011)..................................................................................................................................... 21

Figure 11: Glucose (a) and fructose (b) content of PEF treated (E= 5 kV/cm, n= 20) and untreated

…….asparagus after 0 and 4 days of storage. Statistical significance (* P<0.05, ** P<0.01, ***

…….P<0.001) (Janositz, Semrau & Knorr, 2011)....................................................................... 22

Figure 12: Cell disintegration index of PEF treated asparagus (E= 5 kV/cm, n= 20) with electrode

…….orientation in longitudinal or diagonal path direction. Statistical significance (* P<0.05, **

…….P<0.01, *** P<0.001) (Janositz, Semrau & Knorr, 2011)................................................... 23

Figure 13: Comparison of blanching (T= 80 °C, t= 2 min.) and PEF (E= 1.8 kV/cm, n= 40) pre-

…….treatment with untreated potato stripes concerning fat uptake during frying. □ = PEF treated

…….potato samples, ▒ = blanched potato samples, ■ = untreated potato samples. Statistical

…….significance (* P<0.05, ** P<0.01, *** P<0.001) (Janositz, Noack & Knorr, 2011).......... 24

Figure 14: Conductivity of PEF treated (E= 1.5 kV/cm, n= 20) and untreated potato samples without

…….NaCl immersion and after soaking in 1 g/100g NaCl solution for 15 or 30 minutes. □ = PEF

List of Figures VI

……. treated potato samples, ■ = untreated potato samples. Statistical significance (* P<0.05, **

… P<0.01, *** P<0.001) (Janositz, Noack & Knorr, 2011).................................................... 25

Figure 15: Cross section of asparagus spear (a) and longitudinal cut of asparagus pod (b) performed

…….after reaction with phloroglucin to visualize lignin (Janositz, Semrau & Knorr, 2011)...... 26

Figure 16: Amount of Acid Detergent Lignin (= raw lignin) of PEF treated (E= 5 kV/cm, n= 20) and

…….untreated asparagus. Statistical significance (* P<0.05, ** P<0.01, *** P<0.001) (Janositz,

…… Semrau & Knorr, 2011)....................................................................................................... 27

List of Abbreviations VII

List of Abbreviations

ADF Acid Detergent Fibre

ADL Acid Detergent Lignin

AOAC Association of Official Analytical Chemists

a* value sample position between red and green

b* value sample position between yellow and blue

C capacity (F)

CDI

cell disintegration index

Cl Cellulases

d electrode gap

DW dry weight (g)

electric field strength (kV/cm)

ƒ Frequency (Hz)

Fc form factor for cells with spherical shape

(= 1.5)

IARC International Agency for Research on Cancer

Kl ; Kl’ electrical conductivity of untreated and

treated cell material in a low- frequency field

(1-5 kHz)

Kh ; Kh` electrical conductivity of untreated and

treated material in a high- frequency field

(3-50 MHz)

L* value Lightness of a sample

sample mass (g)

pulse number

PEF pulsed electric fields

PL pectin lyases

PPO polyphenol oxidase

Τ pulse width (µs)

TUB

Berlin University of Technology

voltage (V)

V m transmembrane potential (V)

energy input (kJ/kg)

Wpulse

energy per pulse (J)

WC Water content (g)

κ (T)

electric conductivity (mS/cm)

List of original Articles VIII

List of original Articles

This PhD thesis is based on the following publications, which are referred to by their Roman

numerals in the text:

Article I Janositz, A. & Knorr, D. (2010). Microscopic visualization of Pulsed Electric

…………….Field induced changes on plant cellular level. Innovative Food Science and

………………Emerging Technologies, 11, 592–597.

Article II Janositz, A., Noack, A.-K. & Knorr, D. (2011). Pulsed Electric Fields and their

……………..impact on the diffusion characteristics of potato slices. LWT-Food Science and

……………….Technology, 9, 1939-1945.

Article III Janositz, A., Semrau, J. & Knorr, D. (2011). Impact of PEF treatment on quality

…… parameters of white asparagus (Asparagus officinalis L.). Innovative Food

……………...Science and Emerging Technologies, 12, 269-274.

Introduction

1. Introduction

The effect of Pulsed Electric Fields (PEF) on cellular material has been one of the most

interesting scientific research topics in food- and biotechnology since 1960. First efforts were

made concerning the increase of plant tissue permeability (Doevenspeck, 1960) as well as the

inactivation of microorganisms due to electroporation (Sale & Hamilton, 1967). The

application of PEF involves the subject of biological cell material to a pulsed high voltage

field for a very short time, inducing pore formation and subsequent permeabilization of the

cell membrane. Electroporation can be reversible or permanent, dependent on the applied

treatment intensity. Transient membrane permeabilization maintains cell viability and can be

adopted in biotechnology and medicine for the delivery of drugs and genes into living cells

(Gehl, 2003; Neumann et al., 1982). Irreversible cell disintegration results in the loss of cell

vitality and presents an effective tool for mild pasteurization of liquid foods (Alvarez et al.,

2006; Heinz et al., 1999; Jaeger et al., 2009) as well as for the enhancement of mass transfer

effectiveness of intracellular substances (Ade-Omowaye et al., 2001a; Bazhal & Vorobiev,

2000; Chalermchat et al., 2004). Based on the various applications of PEF, the emerged non

thermal processing method owns a great potential to assist or replace common thermal food

manufacturing by producing fresh-like foods with less determinations on nutritional value and

thus with a high standard of quality.

Many PEF-assisted operations as extraction, pressing or drying of cellular solid food are

based on the irreversible electrical breakdown resulting in pore formation of the semi

permeable cell membrane. Thus, mass transfer is positively affected during subsequent

processing of food.

The effectiveness of PEF technology depends on several factors which can be classified in

technical and chemical process conditions as well as in biological product characteristics.

Besides technical factors, including PEF process parameters such as electric field intensity,

treatment time, pulse shape and applied energy (Hülsheger et al., 1981; Tatebe et al., 1995;

Zhang et al., 1994a) as well as chemical and physical characteristics of treated products, the

biological aspects like species, cell size, shape or physiological state influence the degree of

membrane permeabilization additionally. Small microorganisms cells were found to be less

sensitive against the external electric field, whereas membrane disintegration of larger plant

cells occurs in markedly higher percentage by applying same PEF treatment conditions (Sale

& Hamilton, 1967).

Introduction

Although, applications of PEF to improve and modify operations in commercial plant

processing have been largely discussed in literature, knowledge about the influence factors of

membrane permeabilization and the impact of the cell wall on the degree of cell rupture is still

expandable. In order to tap the full potential of PEF technology in food- and bioengineering, it

is required to obtain better insight of the PEF-induced changes in the structure of plant tissue

at the basic cellular level.

The aim of this thesis is to gain better understanding of the PEF-induced changes in the

structure of plant tissue with focus on (i) the permeabilization at cellular level during and after

reversible as well as irreversible PEF treatment; (ii) the enhancement of mass transfer as a

post-permeabilization process after irreversible cell membrane disintegration.

Background 3

2. Background

2.1 Biological cell material

2.1.1 Plant tissue

The cell is the structural unit of living organisms (Virchow, 1858). All plant cells are

surrounded and structured by a rigid cell wall, providing shape and strength to cell and

protecting the plasma from external damage. The firm structure is based on a network of

cellulose and hemicelluloses, being associated with pectic material. Each cell has usually one

nucleus which is surrounded by cytoplasm. In higher plants the nucleus is enclosed by nuclear

membrane. Up to 80 % of the entire plant cell compartment constitutes to parenchyma tissue.

In ground tissue systems, large parenchyma plant cells are embedded in a matrix with

intercellular spaces between cells and confined by cell walls which are in contact with

neighbouring cell walls. Parenchyma cells consist of cytoplasm with plastids and large central

fluid-filled vacuoles storing near high amounts of cell sap also ions, sugars, organic and

amino acids and other substances. Plant vacuoles are enclosed by a membrane termed

tonoplast, which controls the inner vacuole composition due to its highly selectivity in

transporting only small molecules through the membrane phospholipid bilayer. Based on this

separation the vacuole sap consistence can vary markedly from the cytoplasm content. The

solutes in the vacuoles cause an influx of water resulting in the formation of a large internal

pressure, the turgor pressure, in plant cells. The maintenance of turgor pressure leads to the

rigidity and stability of plant tissue since the pressure is exerts from cell to cell, leading to a

large tissue tension.

Background 4

Figure 1: Plant cell structure (http://de.wikipedia.org/).

2.1.2 Plant cell culture

Plant cells, removed from tissues, are able to grow in-vitro if they are supplied with

appropriate nutrients and conditions. Plant cell cultures are generally initiated from sterile

parts of a whole plant and can be bred as cell suspension cultures in liquid medium or as

callus cultures on solid medium. After initial cell division the cells volume increases and, in a

batch culture, further expand until limited by some culture variable such as nutrient depletion.

Main applications of plant cell cultures are the manufacturing of high-value secondary

metabolites (Mewis et al., 2011; Krumbein et al., 2010; Endress, 1994; Knorr, 1994) as well

as the production of pharmaceuticals or chemicals from root cultures (Schreiner et al., 2011;

Flores et al., 1999; Norton & Towers, 1986) as cost-effective alternatives to classical

approaches, using the whole crop as a source. Numerous food additives including flavours,

pigments, essential oils, sweeteners and antioxidants have been produced in culture

(Dörnenburg & Knorr, 1996; Chung et al., 1994; Swanson et al., 1992; Berlin et al., 1986).

Furthermore, the initiation of plant cell culture can be used to realize metabolite extraction

from rare and threatened plants contemporary and economically. Plant cells act as

independent units and are biosynthetically totipotent, which means that each cell in culture

has the ability to retain the full genome and hence can produce the same range of chemicals as

Background 5

its precursor (Schleiden, 1838; Schwann, 1839). The major benefit in the use of cell culture is

the assurance of the uniformity and reproducibility of results. However, the instability of cell

lines, insufficient yields and slow growth can be mentioned as long-term problems. In the

field of scientific research plant cells can be used as model system in order to understand

plant metabolism basics as well as to study the effects of unit operations on plant foods.

Relevant non-thermal applications for the food industry as pulsed electric fields, high pressure

and ultrasound, which cause the disintegration of biological cell material can be applied to

gentle release desired cell metabolites (Cai et al., 2011; Ye et al., 2004). Dörnenburg and

Knorr (1993) studied the impact of pulsed electric field and high pressure treatment,

respectively on the plant cell cultures Chenopodium rubrum and Morinda citrifolia and their

intracellular pigments, amaranthin and anthraquinones. They found an increased release of 85

% from amaranthin and 5.7 % release of the anthraquinones after PEF application, whereas

treatment at pressure level of 350 MPa caused a pigment release of 99 % and 9.4 %.

Moreover, novel technologies can be optimized through the use of plant cells as model

systems. Studies of process-induced changes of single cells cause better understanding of

basic underlying principles and help to tap the full potential of these technologies. The

preparation of protoplasts (cells with removed cell wall) from plant tissue or cell suspensions

is also of scientific interest. The isolation of cell wall is often performed enzymatically with

the cell wall degrading enzymes cellulases and pectinases. The obtained spherical cells must

be cultured carefully in an isotonic medium. Near the use of DNA transformation and plant

breeding by electrofusion, protoplasts are ideal targets to study membrane biology (Costa et

al., 2003; Morse et al., 2004).

Figure 2: Isolated protoplasts of seven-day-old Nicotiana Tabacum cell suspension after enzymatic cell

wall degradation (Janositz & Knorr, 2010).

Background 6

2.2 Pulsed Electric Fields (PEF) Technology

2.2.1 Mechanisms of action

The mechanism of PEF-induced pore formation in cell membrane is not yet fully elucidated.

One of the most accepted theories about cell membrane permeabilization caused by an

external electric field is related to electrocompression of the cell membrane. The

electromechanical model developed by Zimmermann et al. (1974) considers the cell

membrane to be a capacitor that separates ionic species and free charges on inner and outer

side of the membrane. The different charges on both sides of the membrane cause a natural

transmembrane potential in cell. When subjecting biological cell material to an electric field,

accumulation and attraction of oppositely charged ions on both sites of the non conductive

cell membrane occur. These reactions cause the reduction of membrane thickness. With

further increase in the transmembrane potential, as a consequence of the increased electric

field, and by reaching a critical value of 1 V, membrane compression intensifies, which lead

to the formation of either temporary or permanent pores and the loss of semi-permeability in

the cell membrane.

Unlike the theory of membrane compression, other theses are based on molecular realignment

within the lipid bilayer and protein channels which cause pore formation in cell membrane

when subjecting a cell to an electric field. Based on studies with protoplasts as model systems,

it has been suggested that PEF treatment could cause alteration of membrane composition by

reorientation of bipolar phospholipids and subsequent membrane permeabilization. These

conformational changes could cause destabilization with the loss of membrane semi-

permeability and thus the loss of cell vitality (Sale & Hamilton, 1968, Tsong, 1991). Tsong

(1991) described the presence of hydrophobic and hydrophilic pores in lipid matrix induced

by the electric field and assumed that hydrophilic pores conduct electricity which causes Joule

heating. Thus, increase of temperature might cause changes in membrane structure and affect

its function as a barrier. Membrane disintegration is believed to be caused by osmotic

imbalances and swelling of permeabilized cells. Which means it can be seen as a result of the

difference in the permeabilities of ions and macromolecules inside the cell, building up an

osmotic pressure that press water into the cells and leads to cell elongation (Fig.3) (Kinosita

& Tsong, 1977; Tsong, 1991).

Background 7

Figure 3: Electroporation of a cell membrane (Tsong, 1991).

Additionally, membrane permeabilization might be a consequence of denaturated protein

channels in the lipid layer, since their functionality depends on the natural transmembrane

potential. Protein channels getting activated about 50 mV, considerably lower than the critical

transmembrane potential. Thus, by exposing cells to PEF, many voltage-sensitive channel

proteins might open which induces electrical injury. However, it has to be in mind that protein

channel opening may not effectual enough to inhibit an increase in transmembrane potential

to equal the breakdown potential of the lipid bilayer. Due to the high current Joule heating or

electric modification of the protein channels with subsequent denaturation may occur,

identifying that electroporation can take place in protein channel as well as in lipid fraction of

the membrane.

Background 8

2.2.2 Applications

Figure 4: Schematic depiction of food and process improvement due to pulsed electric fields

(Janositz, unpublished results).

Most potential applications of PEF in food industry can be referred to the disintegration of the

cell membrane. As a mild alternative preservation method to heat pasteurization, PEF can

extend shelf-life at sub-lethal temperatures while maintaining physical, chemical and sensory

properties of food. Liquid and semi-solid products such as fruit and vegetable juices

(Barbosa-Cánovas et al., 1995, Heinz et al., 2003, Molinari et al, 2004), milk (Zhang et al.,

1994b, Sampedro et al., 2005), liquid eggs (Amiali et al., 2004, Hermawan et al., 2004) and

soups (Vega-Mercado et al., 1996) exposed to PEF in continuous systems showed significant

reduction of most pathogenic bacteria. However, studies about PEF-induced pasteurization

concerning spores showed only limited inactivation effects (Raso et al., 1998). Especially in

recent years, PEF technology research has been investigated not only in microbial safety of

food products but also for gentle and controlled modification of plant cell tissue. Very

promising results have been achieved concerning the release and production of cell

metabolites (Eshtiaghi & Knorr, 2002; Fincan et al., 2004; Guderjan et al., 2005; Puertolas et

al., 2010). Due to their function as health related ingredients and/or their use as colouring and

flavouring substances in food the recovery of intracellular molecules in its natural state are of

Background 9

high commercial interest. The improvement of juice yield and rates with simultaneous

retaining of fresh-like characteristics in solid-liquid extraction of fruit and vegetables (Knorr

et al., 1994; Bouzrara & Vorobiev, 2000; Schilling et al, 2008) as well as the acceleration of

mass transport in drying processes (Rastogi et al., 1999, Ade-Omowaye et al., 2001b,

Lebovka et al., 2007) are counted among the benefits of PEF employed in food processing.

Besides this, PEF treatment offers a potential involving decontamination of waste water

(Koners et al., 2004; Kopplow et al., 2004), improvement of textural and sensory properties of

cheese made from PEF treated milk (Sepulveda-Ahumada et al., 2000) as well as the

prevention of biofouling in cooling water (Abou-Ghazala & Schoenbach, 2000). However,

only limited report exists concerning the effect of PEF on enzyme activity. Different

conclusions have been drawn varying from significant reduction of some enzymes after PEF

application (Schuten et al., 2004) to no effect of PEF on enzyme activity (Van Loey et al.,

2001). This contradiction could be referred to difference treatment conditions as well as to the

differences in enzyme molecular structure, causing higher sensitivity of some enzymes to PEF

than other. Jaeger et al. (2010) found only 5 % reduction of Lactoperoxidase activity due to

PEF without thermal effects, but marked that the benefit in maintaining LPO-activity lies in

the retention of antimicrobial effect, which can be referred to the presence of LPO.

Summary of research methology 10

3. Summary of research methodology

This section summarizes main methodologies that were conducted in the research work.

3.1 Biological raw material

Article I (Janositz & Knorr, 2010) Plant cell studies were carried out with cultured tobacco

cells (Nicotiana tabacum L. cv Bright Yellow-2), grown in MS medium (Murashige, 1962)

for 7 days at 25 °C in the dark with reciprocatory shaking at 120 rpm.

For protoplast preparation, tobacco cells were vacuum filtered and 2 g fresh weight cells were

resuspended in 10 ml solution of isotonic buffer W5 (154 mM NaCl, 125 mM CaCl2, 5 mM

KCl, 5 mM Glucose, pH 5.7) combined with a mixture of cellulolytic and pectolytic enzymes

(0.01 g Rohament Cl, 0.1 g Rohament PL) (AB Enzymes, Darmstadt, Germany) for the

residence time of 4 hours. After digestion of cell wall components, the obtained spherical

protoplasts were washed twice with 0.6 M mannitol. Isolated protoplasts were finally

resuspended in 6 ml unbuffered isotonic mannitol solution to perform pulsed electric field

treatment (Fig.1). Buffer was excluded in order to render a low conductivity medium for PEF

operation.

Pre-treatment of tobacco cells with cell wall was carried out with vacuum filtration and

resuspension of 2 g cells in 6 ml mannitol solution before PEF processing.

Article II (Janositz, Noack & Knorr, 2011), Article III (Janositz, Semrau & Knorr,

2011) Plant tissue experiments were performed with potatoes (Solanum tuberosum) and white

asparagus (Asparagus officinalis). Potatoes were obtained from the potato processing

company Lorenz Snack-World GmbH & Co KG (Neu-Isenburg, Germany) and stored in the

dark at 8-10 °C. Asparagus spears were bought in a local store in Germany and stored at 4 °C

in a refrigerator.

Summary of research methology 11

3.2 Experimental set-up and electric field pulses protocol

PEF treatment was performed on plant suspension culture (Article I) and plant tissue (Article

II, III).

In Article I, exponential electric field pulses were applied with the PEF microscope,

constructed in the Department of Food Biotechnology and Food Process Engineering (TU

Berlin). The microscope (Zeiss Optik, Jena, Germany) enabled the study of direct cell

structure changes during the treatment. Main components were a camera (Nikon E 8700,

Japan), which was fixed to the microscope, 3 objectives, with a maximum magnification of

400 fold, and a glass slide with two copper foil electrodes (gap 2 mm, length 3 mm, thickness

0.2 mm, area 0.6 mm²). The treatment chamber was connected to the micro pulse modulator,

consisting of a power supply FUG HCK, 800 M- 20.000, 20 kV, 80 mA (FUG, Rosenheim,

Germany) to a capacitor bank of three capacitors with 6.8 nF each. The pulse parameters were

examined by a high voltage and a current probe, coupled to a TDS220 (Sony Tektronix,

Beaverton, US) oscilloscope. A PC computer was used to control PEF treatment intensities,

namely electric field strength E: 0.25 – 7.5 kV/cm; pulse number n: 10, 20; specific energy

input W: 2,206 – 1985 J/g, pulse width τ: 2 -8 µs and frequency ƒ: 2 Hz. The images obtained

with the microscope from the samples were recorded with the camera and single pictures of

untreated and PEF treated were selected to analyze PEF induced cell disintegration. Camera

was activated manually before treatment. For microscopic analysis, each process condition

was performed approximately 10 times. Recorded cells per experiment/ picture varied

between 1 and 8. Cell area was measured by the program AnalySis 2.11 (Muenster, Germany)

from pictures taken from the recorded movie before and after (after the last pulse) PEF

treatment. T-tests were used for the analysis of statistical significance. Cell area reduction was

calculated by the formula:

(1-(cell size of PEF treated protoplasts/cell size of untreated protoplasts))*100. (1)

In Article II, III exponential electric field pulses were applied to a parallel plate treatment

chamber for batch-wise operations, which was connected to a capacitor bank of four DP

30560 (GA, San Diego, USA), 15 kV, 2 μF in series. Thus, a total capacity of 0.5 μF was

achieved. Capacitors were charged using an ALE802 (Lambda-Emi, Neptune, USA), 40 kV

power supply. The applied PEF treatment intensities for potatoes (Article II) and asparagus

(Article III) are listed in table 1:

Summary of research methology 12

Table 1: PEF treatment parameter

Output

voltage

Electrode

gap

Electric

field

strength

Pulse

number

Pulse

duration

Pulse

frequency

Article II

Section

4.2

U= 1000 V d= 0.2 cm

E= 5 kV/cm

n= 20

τ = 100 µs ƒ= 2 Hz

Section

4.2.1

U= 12000 V

U= 20000 V

d= 8 cm

E= 1.5 kV/cm

E= 2.5 kV/cm

n= 20

τ = 400 µs ƒ= 2 Hz

Section

4.2.2

U= 9000 V

d= 5 cm

E= 1.8 kV/cm

n= 40 τ = 400 µs ƒ= 2 Hz

Article III

Section

4.3

U= 15000 V

d= 3 cm

E= 5 kV/cm

n= 20

τ = 400 µs

ƒ= 2 Hz

3.3 Examination of cell vitality through impedance measurement

Electrical properties of biological tissue define the impact of PEF on the degree of

permeabilization. Thus, the determination of Cell Disintegration Index (CDI) is basically

necessary. CDI was analyzed after Angersbach et al. (1999). The method based on the

frequency depending conductivity of intact and permeabilized tissue. The cell disintegration

index CDI analysis was carried out via impedance measurement equipment (Biotronix GmbH,

A. Angersbach, Hennigsdorf, Germany).

CDI was calculated by following equation:

b= 0≤CDI≤1 (2)

where Kl and Kl’ indicate the electrical conductivity of untreated and treated cell material in a

low- frequency field (1-5 kHz), respectively; and Kh and Kh` indicate the electrical

conductivity of untreated and treated material in a high- frequency field (3-50 MHz).

bCDI −

−

−= )''(

Summary of research methology 13

The CDI varies between 0 for intact cells and 1 for total disintegration. Cylindrical pieces

were cut out of the tissue and placed into a plastic test tube. The electrode area of the

measuring cell was 2 cm². The gap was adjusted to 1.0 cm.

3.4 Determination of sugar content (D- Glucose, D- Fructose)

In Article II and III sugar content was analysed before and after PEF treatment to examine

the influence of PEF on the release of low molecular substances.

For the analysis, samples were washed after treatment in tap water (500 ml), cut, 50 g were

mixed with 50 ml distilled water and homogenised with an Ultra- Turax (T 25 digital Ultra-

turrax, IKA laboratory technology, Germany) at 15000 rpm for three minutes at room

temperature. 5 ml Carrez I solution (3.60 g K4 [Fe(CN)6] x 3H2O (potassium

hexacyanoferrate/ 100 ml) and 5 ml Carrez II solution (7.20 g ZnSO4 x 7 H2O (zinc sulfate

hepta hydrate/ 100 ml) were added to sample mash (pH= 7.0-7.5). In a volumetric flask 0.3 ml

n- Octanol were added to the sample and shook till foam was dissolved. Filtration was

performed after addition of distilled water to the mark of 250 ml. Sugar content was analysed

spectrophotometrically (Kontron 25/Germany) at 334 nm wavelength.

3.5 Analysis of fat content

In Article II the oil uptake of PEF treated potato stripes during frying was analyzed and

compared to the fat content of blanched and untreated samples in order to study effect of PEF

on the drying behavior during deep fat frying.

Blanching and PEF processing were performed for the comparison of different pre-treatments

to reduce fat content during frying. Warm water blanching was accomplished for 2 minutes at

80 °C. Blanched potatoes were cooled in tab water for 10 minutes and dripped of water. After

cutting 100 g potato stripes were fried in two liter rapeseed oil for 13 minutes at 190 °C. The

frying sieve was shaken to release the surface oil and cooling of the fries was performed for

10 minutes at room temperature. Oil content of potato stripes was determined by 3 h Soxhlet

extraction using petroleum ether as a solvent (AOAC, 1995).

Summary of research methology 14

3.6 Analysis of lignin content

In Article III amount of lignin was measured to analyse impact of PEF on delignification and

thus on softer texture.

-Qualitative-

Phloroglucin, a benzentriol (1,3,5- trihydroxybenzene, Merck, Darmstadt/Germany), was

solubilized in a mixture of ethanol/water (1:1) (w= 5%). For the qualitative detection of

lignin, phloroglucin solution was applied to asparagus sample and one drop of hydrochloric

acid was added to turn the contained lignin red.

Pictures were recorded by using a light microscope (Nikon Eclipse E400) equipped with a

digital camera (JVC, TK -10070E).

-Quantitative-

Lignin content determination is based on Association of Official Analytical Chemists

[AOAC] methods (1984) according to the procedures of Goering & Van Soest (1970). The

analysis includes the detection of ADF (Acid Detergent Fiber) and ADL (Acid Detergent

Lignin).

The freeze dried samples were homogenized in an Ultra- Turrax (T 25 digital ultra-turrax,

IKA laboratory technology, Germany) at 24000 rpm for 5 minutes at room temperature.

Detection of ADF content: 100 ml of acid detergent dissolution (20 g N-trimethyl-ammonium

bromide) were solute in sulphuric acid (c: ½ H2 SO4 = 1 mol / l) and added with 0.5 ml

Octanol. 1 gram of grounded sample was weighted out and mixed with the solution and boiled

for 60 minutes. After boiling, content of the glass beaker was vacuum-filtrated through a filter

crucible and washed afterwards with 250 ml hot water and acetone. Filter crucible was dried

over night in a drying oven at 100 °C and weighted out after cooling in a dehydrator. The

content of ADF was determined by the formula:

ADF 100*)12( −

= (3)

where 1m indicates the mass of the filter crucible [g], 2m indicates the mass of the filter

crucible and ADF [g] and

notifies the initial weight [g].

The filter residue can be used for the detection of raw lignin = ADL (Acid Detergent Lignin).

Summary of research methology 15

Determination of ADF content:

Filter crucible with residue of ADF analysis was weight out and placed in a beaker glass.

Crucible content was covered with 72 % sulphuric acid, which was cooled to 15 °C. Over a

period of three hours, sulphuric acid was refilled and mixture was stirred hourly at a

temperature of 20-23 °C. Suction, hot water washing, drying and weighting were performed

subsequently. After incineration of organic substances the specimen was weighted again. The

annealing loss equates the amount of raw lignin. The experiments were performed in

duplicates and replicated five times for statistical purposes.

Main findings 16

4. Main findings

This section summarizes the most important results of the three publications.

4.1 Basic principles of PEF on cellular level: Microscopic visualization of cell structure

changes

In Article I (Janositz & Knorr, 2010) cultured tobacco cells (Nicotiana tabacum b.y.-2)

were used as model systems and microscopic images were recorded during the PEF treatment

to visualize the PEF- induced changes on cell structure. Protoplasts were prepared

enzymatically and compared with native cell behaviour in the electric field to identify the

influence of cell wall on the degree of cell disintegration. Cell shrinkage was observed for

protoplasts after PEF exposure. Thus, cell area was measured before and after PEF treatment

and different cell sizes were compared with different treatment intensities. The reduction of

cell area served as an indicator for cell membrane permeabilization.

4.1.1 Comparison of protoplasts (digested cell wall) and native cells (intact cell wall)

Visual observation showed higher sensitivity of protoplasts to electric fields than cells with a

cell wall. The elimination of cell walls leads to a loss of structural support. Therefore

irreversible membrane pore formation after PEF processing of protoplasts was indicated by

the reduction of cell size whereas membrane disintegration of cell wall cells could only be

noticed with vital dye (phenosafranine) diffusion. Noticeable decrease of protoplast cell area

was already shown after the first pulses at quite low treatment conditions (≥ E= 0.5 kV/cm, n=

10, ≥ W= 8.824 J/g; Fig.5).

Main findings 17

Figure 5: Protoplasts (Nicotiana Tabacum L. cv Bright Yellow-2) untreated and after PEF treatment (E=

0. 5 kV/cm, n= 10, f= 2 Hz) (Janositz & Knorr, 2010).

In contrast to cells with cell wall, where the phenosafranine uptake, which indicates

irreversible pore formation, was only registered at higher PEF intensities (≥ E= 1.2 kV/cm, n=

20, ≥ W= 2541 J/g; Fig.6).

Figure 6: Tobacco cells with cell wall (Nicotiana Tabacum L. cv Bright Yellow-2) in vital dye solution

(Phenosafranine) untreated and after PEF treatment (E= 2. 5 kV/cm, n= 20, f= 2 Hz) (Janositz & Knorr,

2010).

The cell disintegration index correlated with microscopic observations and demonstrated the

intensified effect of PEF on protoplasts (Fig.7). It could be shown that the presence of cell

wall highly influence the degree of membrane permeabilization. Both cell types showed

higher degree of cell disintegration with the application of higher PEF intensities. The extent

of protoplast cell rupture was nearly twice as high compared to the cells with cell wall with

same treatment conditions, demonstrating the protective effect of plant cell walls.

Main findings 18

Figure 7: Cell disintegration index of untreated and PEF treated protoplasts and native cells after

different PEF treatment conditions (Janositz & Knorr, 2010).

4.1.2 Protoplasts as a model system to visualize influence factors on PEF induced

membrane rupture - determinant factors: PEF treatment intensity and cell size

Cell shrinking after irreversible PEF treatment could only be observed in protoplasts. Hence,

cell volume of untreated and PEF treated protoplasts can serve to detect the degree of

membrane permeabilization. Microscopic analysis during the treatment could visualize the

fact that higher PEF intensities cause higher degree of cell rupture, indicated by major cell

area reduction at stronger PEF energy inputs (Fig. 8).

Main findings 19

Figure 8: Cell area of untreated and PEF treated protoplasts before and after PEF processing with

different treatment conditions. Statistical significance (* P<0.05, ** P<0.01, *** P<0.001) (Janositz &

Knorr, 2010).

For cells of 250-350 μm² cell area, application with field strength of 0.5 kV/cm and 10 pulses

resulted in cell area reduction of 12.5 % whereas for E= 5 kV/cm and n= 10 the protoplast

shrinking reached 34 %. Furthermore, it could be shown that the cell size determines the

required external electric field intensity which causes membrane disruption. Larger cells were

more affected by the electric field than cells with smaller size. For cells with less than 250

μm² cell area, lower size reduction was noticed after PEF treatment and the different

intensities caused minor differences in cell area as it could be monitored for larger cells. The

impact of cell size on the effectiveness of PEF treatments is clearly shown in the reduction of

the cell size after PEF processing with the highest applied treatment intensity (E= 5 kV/cm,

n= 10, W= 883.353 J/g), where cell area differed from 11.8 % for smallest cells (< 250 μm²)

to 39.8 % for cell size in the range over 350 μm². The observed effect of cell size on the

degree of cell area reduction corresponds with other experimental studies (Sale & Hamilton,

1967; Hülsheger et al., 1983; Zhang et al., 1994b) and is based on the required electric field

intensity to induce a given transmembrane potential into a cell.

Another focus of our study was the microscopy of reversible pore formation through

following the resealing processes. Furthermore, it was not only possible to visualize

irreversible cell disintegration by the reduction of cell size but also to image temporary pore

formation in cell membrane after the application of PEF with low energy inputs. The PEF

induced stress reaction which causes reversible pores in plasmalemma could be indicated by

Main findings 20

cell swelling after the exchange of intra- and extracellular fluids due to slight osmotic

imbalance in the medium (Fig.8). Temporary formed pores leads to a break in the osmotic

barrier. Subsequently the gradient for osmotic pressure between intra- and extracellular

liquids drops to zero. For draining permeabilized cells, a hyperosmotic medium is used. Vice

versa, liquid uptake occurs in a hypoosmotic medium.

In figure 8 the differences in protoplast cell area before and after PEF treatment are

represented. Whereas treatment conditions higher than E= 0.5 kV/cm and n= 10 led to a

reduction of cell area, the utilization of low process parameter (E= 0.25 kV/cm, n= 10, W=

2.206 J/g) resulted in an increase of cell size, which could indicate the resealing of temporary

formed pores in membranes after PEF implementation.

4.2 Applications of PEF on plant tissue: Enhanced mass transfer of low molecular

substances

In Article II (Janositz, Noack & Knorr, 2011), Article III (Janositz, Semrau & Knorr,

2011) PEF were applied on plant tissue with the main aim of irreversible permeabilization of

the cell membrane and subsequent improved diffusion of intra- and extracellular molecules.

Figure 9: PEF treated (E= 5 kV/cm, n= 20, 5 min. after treatment) and untreated potato tissue stained

with ruthenium red. Light microscope (Nikon Eclipse TS 100, Japan) (Janositz, Noack & Knorr, 2011).

In Fig. 9 untreated and PEF treated (E= 5 kV/cm, n= 20) potato tissues with stained cell wall

pectin are shown. The dye ruthenium red binds to deesterified carboxyl groups and stains

pectin in cell wall and middle lamellae. It is seen that the tissue compartment is slightly

changed. Still, it is not clear whether cell wall components are changed directly due to the

PEF treatment or due to cell membrane disintegration and the release of cytoplasm. However,

Main findings 21

it was shown in Article III (Janositz, Semrau & Knorr, 2011) that the content of the cell wall

biopolymer lignin reduces after PEF application (see 4.3).

4.2.1 PEF-induced release of intracellular substances Î sugar

As demonstrated in Article II PEF application on potatoes slices improves the removal of

reducing sugars from the tissue (Fig. 10). A significant increase in the release of glucose and

fructose was observed after PEF application of potatoes with the field strength E= 1.5 kV/cm

and 20 pulses. The enhanced diffusion characteristics after PEF induced electroporation

resulted in a one third reduction of fructose content and a nearly bisection of glucose rate.

Figure 10: Sugar content in potato slices after PEF treatment (E= 1.5 kV/cm, n= 20) in comparison to

untreated samples. □ = PEF treated potato samples, ■ = untreated potato samples. Statistical significance

(* P<0.05, ** P<0.01, *** P<0.001) (Janositz, Noack & Knorr, 2011).

In contrast to the PEF-induced sugar release in potatoes, PEF processing on asparagus

(Article III) did not result in pronounced differences of glucose and fructose content. As

represented in figure 11a no alteration of glucose level was found for untreated and PEF

treated asparagus directly after treatment. However, on the fourth day of storage, both

samples showed significant reduction of glucose content. PEF treated samples amounted 3

g/100g less glucose than the reference.

Main findings 22

Figure 11: Glucose (a) and fructose (b) content of PEF treated (E= 5 kV/cm, n= 20) and untreated

asparagus after 0 and 4 days of storage. Statistical significance (* P<0.05, ** P<0.01, *** P<0.001)

(Janositz, Semrau & Knorr, 2011).

The lowering in glucose content during storage could be attributed to respiratory processes,

microbial load and the enhanced release of endogenous enzymes due to PEF-induced cell

disintegration causing degradation of glucose (Bisson et al., 1926). As demonstrated in figure

11b, only minimal changes in fructose content due to PEF treatment were observed, but no

further decrease of fructose in untreated as well as in PEF treated asparagus was noticed.

These observations mark the different influence of PEF on different food matrices. The

electric field direction can be mentioned as another impact factor, which affects the extent of

cell membrane permeabilization. In Article III, Cell Disintegration Index (CDI)

measurements were performed of PEF processed (E= 5 kV/cm, n= 20) asparagus, treated and

measured in longitudinal and diagonal direction.

Main findings 23

Figure 12: Cell disintegration index of PEF treated asparagus (E= 5 kV/cm, n= 20) with electrode

orientation in longitudinal or diagonal path direction. Statistical significance (* P<0.05, ** P<0.01, ***

P<0.001) (Janositz, Semrau & Knorr, 2011).

Figure 12 shows that CDI increased by 9.06 % when orientating the electrodes longitudinally

relative to the major axis of the tissue. This observation demonstrates that the direction of the

electric field has a significant influence on the effectiveness of PEF treatment. Asparagus

tissue can be seen as distinctly anisotropic, since the cells have a diameter of approximately

20 µm, but a length of up to 100 µm (Gassner et al., 1989). Electrical conduction along the

length of cell, filled with rich ionic intracellular liquid, is thus easier than conduction between

the cells in the less conductive extracellular matrix and the non-conductive cell membrane.

4.2.2 PEF-induced release of intracellular substances Î cell liquid Î lowering of

French fries fat content

Several studies reported about the enhancement of drying processes after PEF treatment of

plant tissue (Ade-Omowaye et al., 2001b; Taiwo et al., 2002; Lebovka et al., 2007). In Article

II (Janositz, Noack & Knorr, 2011) higher water loss of PEF treated potato slices after baking

in drying oven was found (data not shown). In the present investigation, the effect of PEF pre-

treatment on the fat uptake of potato strips during frying was examined. As presented in

figure 13 markable fat reduction of 38.66 % was observed for PEF pre-treated samples

compared to untreated fried strips. This distinct decrease of fat content could not be found for

blanched samples, which showed no significant fat reduction regarding to the reference

samples. The blanching-induced layer of gelatinized starch (Moreira et al., 1999) was shown

Main findings 24

to be less efficient concerning limitation of oil absorption in comparison to PEF pre-

treatment. This finding was attributed to the modified frying characteristics of the PEF treated

potato strips. Frying is mainly a drying process that involves heat and mass transfer. After

initial heating of the food through the surrounding oil, surface boiling begins including water

vaporizing and the formation of bubbles. Moisture is transferred from the surface to the oil

and later by diffusion of inner cellular liquid to the surface. The water vapour layer on the

potato surface acts as a barrier against the oil and depends on the vapour pressure difference

between food moisture and oil, which influence the rate of drying (Jason, 1958). Due to the

permeabilized cell membranes of PEF treated tissue cell liquid diffusion from the core to the

surface is enhanced, which result in higher vapour pressure difference and thus thicker water

vapour layer, reducing dehydration and fat uptake. As revealed visually and haptically the

surface of PEF treated potato strips is smooth and flat, which assist additionally the decreased

oil uptake during frying and post-frying (Thanatuksorn et al., 2005). Due to the even cut, oil

absorption during frying can be reduced in contrast to the more distinct roughness of non PEF

treated tissue. During the cooling period PEF treated samples were less susceptible to oil

absorption of the adverse crust oil because of the smooth and even outer surface, causing

better oil draining (Bouchon & Pyle, 2006).

Figure 13: Comparison of blanching (T= 80 °C, t= 2 min.) and PEF (E= 1.8 kV/cm, n= 40) pre-treatment

with untreated potato stripes concerning fat uptake during frying. □ = PEF treated potato samples, ▒ =

blanched potato samples, ■ = untreated potato samples. Statistical significance (* P<0.05, ** P<0.01, ***

P<0.001) (Janositz, Noack & Knorr, 2011).

Main findings 25

4.2.3 PEF-induced uptake of extracellular substances Î sodium chloride

The uptake of sodium chloride after PEF implementation of potatoes was analyzed in order to

examine the potential of PEF to assist the infusion of flavor carrier or pigments in the tissue.

Thus, Article II focused not only on the enhanced release of molecules out of the cell but also

by the increased infusion of substances into the sample. In figure 14 conductivity of untreated

and PEF treated potatoes after soaking in sodium chloride solution is presented. It was

observed that conductivity of PEF treated samples was higher and increased with residence

time, indicating the higher uptake of sodium chloride in the tissue. Two mass processes

occurred, water release out of the cells as well as salt diffusion into the tissue dependent on

the applied concentration gradient.

Figure 14: Conductivity of PEF treated (E= 1.5 kV/cm, n= 20) and untreated potato samples without NaCl

immersion and after soaking in 1 g/100g NaCl solution for 15 or 30 minutes. □ = PEF treated potato

samples, ■ = untreated potato samples. Statistical significance (* P<0.05, ** P<0.01, *** P<0.001)

(Janositz, Noack & Knorr, 2011).

4.3 PEF-induced changes on food ingredients Î lignin

In Article III (Janositz, Semrau & Knorr, 2011) lignin content of PEF treated asparagus was

analyzed to clarify impact of PEF on the biopolymer lignin, gaining improved macroscopic

characteristics of the spears.

In figure 15 asparagus tissue with red stained lignin is shown. The chemical reaction with

phloroglucin and sulphuric acid was performed to visualize the distribution of lignin in the

Main findings 26

spear. It is seen that lignin is particularly abundant in the pod (a) and located in longitudinal

direction of the spear. This was clarified by viewing cross-sectional imaging (b). Lignin

deposition was noticed as compact and bundled grown in asparagus tissue.

Figure 15: Cross section of asparagus spear (a) and longitudinal cut of asparagus pod (b) performed after

reaction with phloroglucin to visualize lignin (Janositz, Semrau & Knorr, 2011).

PEF application was found to have an influence on lignin content in asparagus. As

represented in figure 16, the amount of raw lignin decreased from 12.6 % (± 0.08) in

untreated asparagus sample to 10.2 % (± 0.34) in the PEF treated asparagus base section. The

behaviour of macromolecules exposed to an intense electric field is not well understood

(Neumann, 1986). Lignin, a complex phenolic polymer, is seen as highly resistant to

biodegradation (Crawford, 1981). Its chemical structure is branched and the macromolecule is

bonded with various lignin cross-links and also linkaged between lignin and polysaccharides

as cellulose and hemicellulose (Eriksson et al., 1980). Application of PEF may be able to

enhance separation of cellulosic material from lignin. High voltage pulses may be effective to

break intermolecular and intramolecular bonds within or between the cellulose, hemicellulose,

and lignin (Navapanich & Giorgi, 2008). Explanation for the breakage could be that cellulose

microfibrills contain large number of hydroxyl groups on the surface causing interactive force

attraction with the hydroxyl and methoxyl groups of coumaryl, coniferyl, and sinapyl alcohols

from lignin (Houtman & Atalla, 1995). These findings indicate that the dominant force

connecting lignin and cellulose is caused by electrostatic dipole-dipole interactions.

Subsequent delignification can occur when the bonds are cleaved resulting in solubilization of

polymer fragments (Goring, 1971).

Main findings 27

Figure 16: Amount of Acid Detergent Lignin (= raw lignin) of PEF treated (E= 5 kV/cm, n= 20) and

untreated asparagus. Statistical significance (* P<0.05, ** P<0.01, *** P<0.001) (Janositz, Semrau &

Knorr, 2011).

Conclusions and Outlook 28

5. Conclusions

This PhD thesis is focused on the basic principles underlying PEF technology and the

applications of PEF particularly in the enhancement of mass transfer processes.

The following conclusions can be drawn:

Basic research

• A novel microscopic technique allows the in situ analysis of plant cell material under

PEF treatment.

Microscopic analysis of cell structure changes during PEF treatment is a useful tool to

gain a better insight in the permeabilization mechanism of plant cell material. The

microscope connected with a pulse modulator aims to achieve further information

concerning the influence factors of PEF-induced cell membrane rupture.

• Protoplasts as model systems are adequate facilities for PEF basic research.

Plant cells with removed cell wall can help to understand the basic effects of PEF on

plant cell components and to study the impact of cell wall on cell protection in the

electric field.

• Plant cell walls have a protective effect against the electric field.

Protoplasts show higher sensibility to the electric field than suspension cells with intact

cell wall. Thus, the presence of cell wall highly influences the degree of cell membrane

permeabilization.

• Changes of cell size can serve as an indicator for cell vitality.

Protoplast cell size is reduced after irreversible cell disruption and slightly increased

after reversible membrane permeabilization. Determination of cell viability can help to

evaluate the effectiveness of applied technology on biological material and to assay

different process conditions during process development.

Conclusions and Outlook 29

• The direction of the electric field influences degree of cell disintegration.

Because of the anisotropy of asparagus tissue the electric field orientation has a significant

influence on the degree of electroporation of asparagus. These findings confirm the

relevance of electrode orientation in order to ensure the efficiency of tissue

permeabilization.

Applications

• PEF improve the removal of reducing sugars

PEF treatment on potato slices causes an increase in reduction of glucose and fructose.

It can be considered that PEF pre-treatment is a capable assistance or alternative to

conventional thermal processing for the removal of reducing sugars, which represents

relevant substrates for the Maillard reaction and acryl amide formation. However, the

effectiveness to release low molecular substances depends on food matrix. No

pronounced sugar reduction was noticed after PEF processing of asparagus.

• PEF improve drying rates and thus reduce fat uptake of potato strips during deep fat

frying

PEF application enhances diffusion coefficients within potato tissue and causes higher

release of cell liquid during oven drying of potato slices. Improved drying

characteristics of the disintegrated food matrix may the reason for the reduction of fat

content in PEF pre-treated French fries, as well. Thus, PEF treatment provides a

potential to be implemented in potato processing in order to apply a non-thermal

method for the production of low-fat French fries, energy and water saving and with

only minimal losses on the basic product.

• PEF enhance infusion of sodium chloride

PEF assist the infusion of common salt into potato tissue. In agreement with the

observations of Toepfl and Heinz (2007), who reported about improved diffusion of

salt and nitrite into pork haunches after PEF treatment, PEF application is considered

to be a method able to target insert pigments or flavour carrier not only in animal but

also in plant tissue.

Conclusions and Outlook 30

• PEF reduce lignin content in white asparagus

PEF application decreases amount of lignin in white asparagus spears. Thus, PEF may

be applied as a pre-treatment before preserving to minimize lignification in order to

improve macroscopic characteristics and gain softer texture of the spears.

5.1 Outlook and Future work

Basic research

• PEF-microscopy enables in situ analysis of how the electric field influences cell wall

substances

The application of pulsed electric fields in combination with simultaneous

microscopic visualization provides a promising tool to observe cell structure changes

instantaneously during treatment. This technique offers new ways to study the

immediate effects of PEF on cellular level and to identify influencing factors on the

degree of cell membrane disintegration. The development of innovative methods for

the examination of cell vitality shall help to convert the basic knowledge into effective

processes. Based on the different characteristics of protoplasts and native cells in the

electric field it is of great interest if PEF application influences biopolymers in cell

wall. With regard to the reduction of lignin content in PEF treated asparagus spears

and the possible separation of cellulosic material from lignin due to PEF, it should be

tested if the external electric field has an influence on other macromolecules like

celluloses, hemicelluloses and pectin. It can be supposed that PEF affect glycosidic

bonds, polar hydroxyl groups and/or the charged carboxyl groups on the molecule

pectin.

Conclusions and Outlook 31

Applications

• The enhanced mass transfer in potato and asparagus tissue due to PEF treatment

needs to be examined at industrial scale processes

PEF treatment was shown to be effective for the enhancement of mass transfer in

potato slices and asparagus spears. To apply PEF in food industry, more studies in

technical scale need to be performed. Thus, PEF equipment design and treatment

conditions should be optimized. This includes PEF treatments with continuous PEF

treatment chambers that are high in diameters in order to achieve flow-rates up to 5

t/h.

• Consumer acceptance of PEF treated potato products with lower fat content and

hardened crust texture needs to be evaluated

Another aspect of great importance is the consumer acceptance of PEF pre-treated

French fries. Altered drying characteristics of the PEF- treated potato stripes leads to

lower fat content but also to a harder crust structure. Due to extensive sensory

evaluations, it can be clarified to which extent the cross texture is noticed and accepted

by the human taste.

• PEF application improves the removal of reducing sugars. Future studies shall clarify

whether PEF treatment improves the diffusion of amino acid as well and to which

extent this reflects reduced acrylamide formation.

As far as PEF enhance the release of low molecular substances it should be tested

whether PEF treatment increases the removal of amino acids (asparagine, glutamine)

from potato tissue likewise. This is of great interest, because reducing sugars and

amino acids represent relevant substrates for the Maillard reaction. Finally, the

acrylamide formation after processing of PEF pre-treated French fries and potato

crisps should be determined.

Conclusions and Outlook 32

• The effect of electrode positions on the level of mass transfer in PEF- treated

asparagus should be examined

It was shown that PEF treatment with longitudinal electrode orientation causes higher

cell disintegration degrees in asparagus than placing the electrodes in longitudinal

direction. Thus, it is of relevance to analyze effects of PEF on asparagus

characteristics additionally by orientating the electrodes in longitudinal direction

relative to the major axis of the spear. This would help to optimize PEF processing

and achieve more effective process conditions.

Acknowledgements 33

Acknowledgements

This work has been supported by an EU-integrated Project NovelQ “Novel Processing

Methods for the Production and Distribution of High-Quality and Safe Food”, FP6-CT-2006-

015710, Priority 5 `Food Quality and Safety`.

References 34

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Curriculum Vitae and List of Publications 40

Curriculum Vitae

Anna Winter née Janositz

PhD Student, Dipl.-Ing. (M.Eng) Food Engineering

Biography:

Since 06/2005 Ph.D. student at Berlin University of Technology, Prof. D. Knorr,

Thesis: Impact of Pulsed Electric Fields on post-permeabilization processes of plant cells.

04/2005 Dipl.-Ing. (M.Eng.) Food Technology, Berlin University of Technology

Thesis: Auswirkungen von Hochspannungsimpulsen auf das Schnittverhalten von Kartoffeln

(Solanum tuberosum).

1998-2005 Studies of Food Technology at Berlin University of Technology

Date of birth May, 20th, 1977

Children two daughters (*29/01/07, *18/06/09)

List of Publications:

Janositz, A., Semrau, J. & Knorr, D. (2011). Impact of PEF treatment on quality parameters

of white asparagus (Asparagus officinalis L.). Innovative Food Science and Emerging

Technologies, 12, 269-274.

Janositz, A., Noack, A.-K. & Knorr, D. (2011). Pulsed Electric Fields and their impact on the

diffusion characteristics of potato slices. LWT-Food Science and Technology, 9, 1939-1945.

Janositz, A. & Knorr, D. (2010). Microscopic visualization of Pulsed Electric Field induced

changes on plant cellular level. Innovative Food Science and Emerging Technologies, 11,

592–597.

Jaeger, H., Janositz, A. & Knorr, D. (2010) The Maillard reaction and its control during food

processing. The potential of emerging technologies. Pathologie Biologie (Paris), 58, 3, 207-

213.

Balasa, A., Janositz, A. & Knorr, D. (2010) Electric Field Stress on Plant Systems.

Encyclopedia of Biotechnology in Agriculture and Food (EBAF), Taylor and Francis Group

LLC.

Curriculum Vitae and List of Publications 41

Knorr, D., Balasa, A., Guderjan, M., Janositz, A., Volkert, M. (2006) Keine

Qualitätsverluste- Schonende Aufbereitung und Verarbeitung von bioaktiven Inhaltsstoffen.

dei, dei- die Ernährungsindustrie, 1, 1, 10-13.

Oral and poster presentations:

Balasa, A., Janositz, A. & Knorr, D. (2009) Pulsed electric field treatment of plant tissue:

an overview , EuroFoodChemXV 2009, Copenhagen/Denmark.

Janositz, A., Toepfl, S. & Knorr, D. (2006) The impact of Pulsed Electric Field treatment on

the reduction of potato cutting energy. Food is life, IUFoST, Nantes/Frances.

Janositz, A. & Knorr, D. (2006) Factors of influence in plant cell membrane

permeabilization. COST meeting 928-300606, Cost, Reykjavik/Iceland.

Janositz, A., Toepfl, S. & Knorr, D. (2006) Impact of pulsed electric fields on membranes on

a cellular level. Food factory of the future, SIK - The Swedish Institute of Food and

Biotechnology, Gothenburg/Sweden.

Janositz, A. & Knorr, D. (2006) Die Reduzierung der Zellfläche als Indikator der HSI-

induzierten Permeabilisierung. BioPerspectives im Fokus: Nahrung für die Zukunft, Industrie

und Handelskammer (IHK) Potsdam, Potsdam/Germany.

Janositz, A., Smetanska, I. & Knorr, D. (2006) Factors of influence in plant cell membrane

permeabilization. Workshop Molecular Interactions, Max-Planck-Insitut für Molekulare

Genetik, Berlin/Germany.

Janositz, A., Toepfl, S. & Knorr, D. (2005) Microscopic visualization of plant tissue during

pulsed electric field treatment. Nonthermal Processing Division, IFT –institute of food

technology, Philadelphia/USA.

Janositz, A., Toepfl, S. & Knorr, D. (2005) Mikroskopische Visualisierung von pflanzlichem

Gewebe während der Behandlung mit Hochspannungsimpulsen. FEI Diskussionstagung, FEI,

Berlin/Germany.

Ich erkläre an Eides statt, dass die vorliegende Dissertation in allen Teilen von mir

selbständig angefertigt wurde und die benutzten Hilfsmittel vollständig angegeben

worden sind.

Anna Winter

Microscopic visualization of Pulsed Electric Field induced changes on plant

cellular level

A. Janositz ⁎, D. Knorr

Technical University of Berlin, Institute of Food Technology and Food Chemistry, Department of Food Biotechnology and Food Process Engineering, Germany

abstractarticle info

Article history:

Received 22 April 2009

Accepted 23 July 2010

Keywords:

Pulsed Electric Fields (PEF)

Plant cell culture

Protoplast

Microscope

Cell size

Stress induction

The effects of Pulsed Electric Fields (PEF) on protoplasts from cultured tobacco cells (Nicotiana tabacum b.y.-2)

in comparison to the changes on cultured plant cells with cell walls were visualised in order to study the direct

impact of PEF on cell components and to clarify the inﬂuence of the cell wall on electroporation. Optical

microscopic analyses were carried out and images were recorded during PEFtreatment. Results showed higher

sensitivity of protoplasts to electric ﬁelds related to cells with a cell wall. Protoplasts sizes were measured

before and after different treatment intensities and protoplasts shrinkage was used as an indicator for cell

rupture. It could be demonstrated that cell volume decrease is inﬂuenced by PEF intensity, initial cell size, cell

orientation in the electric ﬁeld and nucleus position.

Industrial relevance: Since the beginning of the 20th century the relevance of Pulsed Electric Fields (PEF)

technology in food- and biotechnology has increased substantially. However, the mechanism of membrane

permeabilization and the PEF induced changes in cell structure remain poorly understood, diminishing the

optimal use in food industry. In this study the direct effects of PEF on cultured plant cell material and

inﬂuencing factors of the degree of membrane disintegration were visualized and identiﬁed. The development

of new methods to examine cell vitality shall help to convert the basic knowledge into effective processes.

1. Introduction

During the 1990's, industrial interest in developing gentle food

technologies, to replace the currently commonthermal processing, has

increased substantially. The non-thermal application of Pulsed Electric

Fields (PEF) is counted among these emerging processes and received

considerable relevance in bio- and food technology. It involves the

exposure of biological cell material to short repeated pulses of a high

voltage with the result of pore formation in cell membrane leading to

membrane permeabilization and cell rupture. The beneﬁt of this

approach is an important aspect of process and product development

because it is aimed to protect quality food attributes, such as sensory

quality and nutrition value, as well as to control the microbial safety

with minimal or no changes during processing. Besides the use of non

thermal technologies to inactivate microorganisms through mechan-

ical destruction of cellular structure (Ho & Mittal, 2000; Wouters,

Alvarez, Angersbach & Knorr, 2001; Heinz, Alvarez, Angersbach &

Knorr, 2001) the main ﬁeld of interest is the treatment of plant cell

material, for cell membrane disruption leading to increased membrane

permeability and to improved mass transfer of inner liquid and cell

components (e.g. health related plant metabolites, pigments) from the

intracellular vacuoles. Processes such as drying, osmotic dehydration

and extraction are facilitated by PEF treatment (Angersbach, Heinz &

Knorr, 1998; Ade-Omowaye, Angersbach, Taiwo & Knorr, 2001). The

application of osmotic dehydration can be used as a pre-treatment to

conventional drying, or freezing for the enhancement of diffusion

characteristics with simultaneous maintenance of fruit product

attributes and the reduction of energy consumption. Prior PEF

treatment intensiﬁes the desired effect due to the improvement of

water and solution mass transfer into and out of the tissue. Rastogi,

Eshtiaghi and Knorr (1999) investigated the impact of PEF on the

dehydration characteristics of carrots and found out that PEF

processing of carrot cubes caused a lowering of moisture content

during osmotic dehydration. Further successful results have been

gained concerning apple slices (Taiwo, Angersbach & Knorr, 2002),

mango (Tedjo, Taiwo, Eshtiaghi and Knorr, 2002), and bell peppers

(Ade-Omowaye, Rastogi, Angersbach & Knorr, 2002).

Several studies reported on the gentle recovery of sensitive

vacuole components such as ﬂavours and dyestuffs in different

plant food and on the increase of extraction yield after PEF processing

(Eshtiaghi & Knorr, 1999; Bouzrara & Vorobiev, 2000; Bazhal, Lebovka

& Vorobiev, 2001; Guderjan, Toepﬂ, Angersbach & Knorr, 2005). These

low energetic cost and short treatment time applications offer

alternative possibilities to thermal processes and therefore to

optimize process control on food sector. Additionally, the employ-

ment of mild heat can be used to intensify the desired target and

Innovative Food Science and Emerging Technologies 11 (2010) 592–597

⁎Corresponding author.TU Berlin, Department of Food Biotechnology and Food

Process Engineering,Koenigin-Luise-Str. 22,D-14195 Berlin, Germany.Tel.: +49 30

314 71411.

E-mail address: [email protected] (A. Janositz).

doi:10.1016/j.ifset.2010.07.004

Contents lists available at ScienceDirect

Innovative Food Science and Emerging Technologies

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

therefore to obtain synergistic effects with the combination of both

treatments (Schilling et al., 2008). Another beneﬁt of PEF processing

lies in the treatment with mild conditions to induce the production of

secondary metabolites with the maintenance of cell viability. The

stimulation caused by PEF can be used to target inﬂuence the plant for

the generation of health related compounds as a stress response of the

electric ﬁeld (Guderjan et al., 2005).

The most accepted theory about the permeabilization mechanism

is conceived to be related to electrocompression of the cell membrane

(Zimmermann, 1986). Due to the electric ﬁeld, accumulation and

attraction of oppositely charged ions on both sites of the non

conductive cell membrane occur, causing membrane thickness

reduction. With further increase in the transmembrane potential, as

a consequence of the increased electric ﬁeld, a critical value is reached

and membrane compression is intensiﬁed leading to the formation of

pores and the loss of semi-permeability in the cell membrane. This

pore formation can be temporary (reversible) or irreversible

(permanent), depending on treatment intensity and sample compo-

sition (Zimmermann, 1986). Reversible pore formation takes place

when the external electric ﬁeld is removed while the critical

membrane potential is reached and the generated pores are small

related to the membrane surface. Characteristic for the processing

with mild PEF conditions is that the cell retains its viability in contrast

to high energy input treatment (for plant cells, E≥1 kV/cm), which

results in the loss of cell vitality. The effectiveness of PEF technology to

permeable cell membranes depends on several factors which can be

classiﬁed in technical and chemical process conditions as well as in

biological product characteristics. The technical factors include PEF

process parameters such as electric ﬁeld intensity, treatment time,

pulse shape and applied energy, whereas the electric ﬁeld intensity

has been described as the most relevant factor deﬁning membrane

rupture by pulsed electric ﬁelds (Hamilton & Sale, 1967; Hülsheger,

Potel & Niemann, 1981; Schoenbach, Joshi, Stark, Dobbs & Beebe,

2000; Zhang, Monsalve-González, Barbosa-Cánovas & Swanson, 1994;

Tatebe, Muraji, Fujii & Berg, 1995). The chemical and physical

characteristics of treated products have also an important impact on

the efﬁciency of PEF. Further product parameters are the composition

of treated media, including pH, temperature and especially ionic

strength, which is responsible for the conductivity of treated media

(Jayaram, Castle & Margaritis, 1993; Vega-Mercado, Pothakamury,

Chang, Barbosa-Cánovas & Swanson, 1996). Biological characteristics

such as species, size, shape or physiological state inﬂuence the degree

of membrane permeabilization additionally. Therefore, small micro-

organism cells were found to be less sensitive against the external

electric ﬁeld, whereas membrane disintegration of larger plant cells

occurs in markedly higher percentage by applying same PEF

treatment conditions (Sale & Hamilton, 1967; Zhang, Monsalve-

González et al., 1994).

Although background knowledge and theories about the PEF

induced plasmolysis increased (Cruzeiro-Hanson, 1988; Dimitrov,

1984; Sugar & Neumann, 1984), the mechanism of membrane

permeabilization and the changes on cell level after PEF processing

remain poorly understood, degrading the full potential use of this

technology. One problem in the study of biological cells is that the

dynamic process of electroporation is extremely rapid. Pore building

occurs within 10 ns at sites where the membrane potential reaches/

obtain 1 V (Dimitrov & Jain, 1984). This celerity causes difﬁculties in the

visualization of pore formation and the subsequent exchange of intra-

and extracellular compounds. Membrane recovery can be happen in a

broader range, from 0.1 ms to 2.8 h (Ho & Mittal, 1996). Furthermore,

research not only about effects of PEF on cell membranes but also on

other cell materials as cell walls is still scarce. Bazhal, Lebovka and

Vorobiev (2003) analysedtextural parameters of PEFtreatedapples and

reported that the electric ﬁeld affects not only plasmalemma mem-

branes but also the cell wall integrity. Moreover, the function of the cell

wall as a possible protection for the cell against the electric ﬁeld as well

as the interaction between cytoplasm and cell wall during post

permeabilization are largely unknown.

In the study undertaken, enzymatic protoplasts (cells without cell

wall) have been prepared from a tobacco plant cell culture to create a

model system for the analysis of PEF induced membrane changes on

cell level. The cells were exposed to a microscope with an integrated

PEF unit in order to visualize membrane disintegration not only

afterwards but also during the period of PEF processing. To gain better

inside of the permeabilization mechanism, speciﬁc methods for the

indication of cell vitality of plant cell cultures after PEF treatment were

developed. Additionally, protoplasts were compared with cell wall

cells to analyse the impact of PEF on different cell types and to explain

the role of cell wall in electric cell rupture.

2. Materials and methods

2.1. Plant cells and protoplasts

Cultured tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2)

(Takebe, Otsuki & Aoki, 1968; Mathur & Koncz, 1998; Nagata &

Kumagai, 1999) were grown in MS medium (Murashige, 1962) for

7 days at 25 °C in the dark with reciprocatory shaking at 120 rpm.

For protoplast preparation, tobacco cells were vacuum ﬁltered and 2 g

freshweightcellswereresuspendedin10mlsolutionofisotonicbuffer

W5 (154 mM NaCl, 125 mM CaCl

5 mM KCl, 5 mM Glucose, pH 5.7)

combined with a mixture of cellulolytic and pectolytic enzymes (0.01 g

Rohament Cl, 0.1 g Rohament PL) (AB Enzymes, Darmstadt, Germany)

for the residence time of 4 h. After digestion of cell wall components, the

obtained spherical protoplasts were washed twice with 0.6 M mannitol.

Isolated protoplasts were ﬁnally resuspended in 6 ml unbuffered isotonic

mannitol solution to perform pulsed electric ﬁeld treatment (Fig. 1).

Buffer was excluded in order to render a low conductivity medium for

PEF operation.

Pre-treatment of tobacco cells with cell wall was carried out with

vacuum ﬁltration and resuspension of 2 g cells in 6 ml mannitol

solution before PEF processing. The measured conductivity of both cell

types (protoplasts and cells with cell wall) mixed with mannitol was

3.3 mS/cm.

2.2. Tobacco cells

2.2.1. Staining

For the visualization of cell rupture from cells with cell wall after

PEF processing, vital dyes were needed, to penetrate into permeabi-

lized cells and indicate irreversible membrane disintegration. There-

fore, freshly prepared solution (0.1%) of the vital dye Phenosafranine

(dry content 80%, Sigma-Aldrich, USA) was added to cell suspension

in a ratio 1:2 directly before treatment.

Fig. 1. Isolated protoplasts of seven-day-old Nicotiana tabacum cell suspension after

enzymatic cell wall degradation.

593A. Janositz, D. Knorr / Innovative Food Science and Emerging Technologies 11 (2010) 592–597

2.2.2. Experimental set-up and electric ﬁeld pulses protocol

The exponential electric ﬁeld pulses were applied with the PEF

microscope, schematic drawing shown in Fig. 2, constructed in the

Department of Food Biotechnology and Food Process Engineering (TU

Berlin). The microscope (Zeiss Optik, Jena, Germany) enabled the study

of direct cell structure changes during the treatment. Main components

were a camera (Nikon E 8700, Japan), which was ﬁxedtothemicroscope,

3 objectives, with a maximum magniﬁcation of 400 fold, and a glass slide

with two copper foil electrodes (gap 2 mm, length 3 mm, thickness

0.2 mm, area 0.6 mm²). The treatment chamber was connected to the

micro pulse modulator, consisting of a power supply FUG HCK, 800 M–

20.000, 20 kV, 80 mA (FUG, Rosenheim, Germany) to a capacitor bank of

three capacitors with 6.8 nF each. The pulse parameters were examined

by a high voltage and a current probe, coupled to a TDS220 (Sony

Tektronix, Beaverton, US) oscilloscope. A PC computer was used to

control PEF treatment intensities, namely electric ﬁeld strength E:0.25–

7.5 kV/cm; pulse number n:10,20;speciﬁc energy input W: 2206–

1985 J/g, pulse width τ:2–8μsandfrequencyƒ:2Hz.Theimages

obtained with the microscope from the samples were recorded with the

camera and single picturesof untreated and PEF treated were selectedto

analyze PEF induced cell disintegration. Camera was activated manually

before treatment. For microscopic analysis, each process condition was

performed approximately 10 times. Recorded cells per experiment/

picture varied between 1 and 8.

Cell area was measured by the program AnalySis 2.11 (Muenster,

Germany) from pictures taken from the recorded movie before and

after (after the last pulse) PEF treatment.

T-tests were used for the analysis of statistical signiﬁcance.

Cell area reduction was calculated by the formula:

ð1−ðcell size of PEF treated protoplasts=cell size of untreated protoplastsÞÞ*100:

2.2.3. Examination of cell vitality through impedance measurement

The extent of cell membrane permeabilization was determined

using the cell disintegration index (CDI) (Angersbach, Heinz & Knorr,

1999). The method is based on the frequency dependence of

conductivity of intact and permeabilized tissue.

The cell disintegration index Z

was calculated by:

Zp=1−bK‵h−K‵l

Kh−Kl

b=Kh

K‵h

0≤Zp≤1

where K

and K

'indicate the electrical conductivity of untreated and

treated cell material in a low-frequency ﬁeld (1–5 kHz), respectively;

and K

` indicate the electrical conductivity of untreated and

treated material in a high-frequency ﬁeld (3–50 MHz).

The CDI values between 0 for intact cells and 1 for total

disintegration.

Impedance measurement of protoplasts and cell with cell wall at

low and high frequencies within the frequency range of 3 kHz and

100 MHz was carried out via impedance measurement equipment

(Biotronix GmbH, A. Angersbach, Hennigsdorf, Germany). The

electrode area of the measuring cell was 2 cm². The gap was adjusted

to 1.3 cm.

3. Results and discussion

3.1. Effect of PEF on protoplasts (digested cell wall) and native cells

(intact cell wall) —a comparison

Visual observation showed higher sensibility of protoplasts to the

electric ﬁeld compared to cells with cell walls (Figs. 3 and 4). The

elimination of cell walls leads to a loss of structural support. Therefore

irreversible membrane pore formation after PEF processing of

protoplasts was indicated by the reduction of cell size whereas

membrane disintegration of cell wall cells could only be noticed with

vital dye (phenosafranine) diffusion. Noticeable decrease of protoplast

cell area was already shown after the ﬁrst pulses at quite low

treatment conditions (≥E=0.5 kV/cm, n=10,≥W=8824 J/g; Fig. 3).

In contrast to cells with cell wall, where the phenosafranine uptake,

which indicates irreversible pore formation, was only registered at

higher PEF intensities (≥E=1.2 kV/cm, n=20,≥W=2541 J/g; Fig. 4).

The cell disintegration index correlated with microscopic observations

and demonstrated the intensiﬁed effect of PEF on protoplasts (Fig. 5). It

could be shown that the presence of cell walls highly inﬂuence the

degree of membrane permeabilization. Both cell types showed higher

degree of cell disintegration with the application of higher PEF

intensities. The extent of protoplast cell rupture was nearly twice as

high compared to the cells with cell wall with same treatment

conditions, demonstrating the protective effect of plant cell walls.

3.2. Protoplasts as a model system to visualize inﬂuence factors on PEF

induced membrane rupture

Cell shrinking after irreversible PEF treatment could only be

observed in protoplasts. Hence, cell volume of untreated and PEF

Fig. 2. Schematic set-up of pulsed electric ﬁeld treatment chamber combined with a

microscope.

Fig. 3. Protoplasts (Nicotiana tabacum L. cv Bright Yellow-2) untreated and after PEF treatment (E=0. 5 kV/cm, n=10, f=2 Hz).

594 A. Janositz, D. Knorr / Innovative Food Science and Emerging Technologies 11 (2010) 592–597

treated protoplasts can serve to detect the degree of membrane

permeabilization.

3.2.1. Determinant factors: PEF treatment intensity and cell size

Microscopic analysis during the treatment could visualize the fact

that higher PEF intensities cause higher degree of cell rupture,

indicated by major cell area reduction at stronger PEF energy inputs

(Fig. 6). For cells of 250–350 μm² cell area, application with ﬁeld

strength of 0. 5 kV/cm and 10 pulses resulted in cell area reduction of

12.5% whereas for E=5 kV/cm and n=10 the protoplast shrinking

reached 34%. Furthermore, it could be shown that the cell size

determines the required external electric ﬁeld intensity which causes

membrane disruption. Larger cells were more affected by the electric

ﬁeld than cells with smaller size. For cells with less than 250 μm² cell

area, lower size reduction was noticed after PEF treatment and the

different intensities caused minor differences in cell area as it could be

monitored for larger cells. The impact of cell size on the effectiveness of

PEF treatments is clearly shown in the reduction of the cell size after

PEF processing with the highest applied treatment intensity (E=5 kV/

cm, n=10, W=883,353 J/g), where cell area differed from 11.8% for

smallest cells (b250 μm²) to 39.8% for cell size in the range over

350 μm². The observed effect of cell size on the degree of cell area

reduction corresponds with other experimental studies (Sale &

Hamilton, 1967; Hülsheger et al., 1983; Zhang, Chang & Barbosa-

Cánovas, 1994), and is based on the required electric ﬁeld intensity to

induce a given transmembrane potential into a cell, which can be

calculated by the equation

Vm=f*a*Ec

(Schwan, 1957) (1).

Where V

is the transmembrane potential induced by an external

ﬁeld of the strengthE

[kV/cm], a[μm] is the cell radius and fis the

form factor for spherical shape (=1.5). This formula enables the

calculation of the induced potential for tobacco protoplasts of

different cell sizes. The calculated relation between transmembrane

potential to cause disruption of cell membrane and cell diameter is

represented in Fig. 7. The graph shows cells of different diameters

sizes evaluated from the ﬁeld strength E=2.5 kV/cm. It is shown that

external electrical ﬁelds induce higher transmembrane potentials in

larger cells and that the potential rises proportional to the increase in

cell size. Thus, the average cell diameter of the smallest protoplast

group was 14.5 μm and the associated transmembrane potential

2.72 V, whereas for larger cells (N18 μm) the mean cell diameter was

20.73 μm with a transmembrane potential of 3.89 V.

Another focus of our study was the microscopy of reversible pore

formation through following the resealing processes. Furthermore, it

was not only possible to visualize irreversible cell disintegration by

the reduction of cell size but also to image temporary pore formation

in cell membrane after the application of PEF with low energy inputs.

The PEF induced stress reaction which causes reversible pores in

plasmalemma could be indicated by cell swelling after the exchange

of intra- and extracellular ﬂuids due to slight osmotic imbalance in the

medium (Fig. 8). Temporary formed pores leads to a break in the

osmotic barrier. Subsequently the gradient for osmotic pressure

between intra- and extracellular liquids drops to zero. For draining

permeabilized cells, a hyperosmotic medium is used. Vice versa, liquid

uptake occurs in a hypoosmotic medium.

In Fig. 8 the differences in protoplast cell area before and after PEF

treatment are represented. Whereas treatment conditions higher than

E=0.5 kV/cm and n=10 led to a reduction of cell area, the utilization

of low process parameter (E=0.25 kV/cm, n=10, W=2206 J/g)

resulted in an increase of cell size, which could indicate the resealing

of temporary formed pores in membranes after PEF implementation.

Fig. 4. Tobacco cells with cell wall (Nicotiana tabacum L. cv Bright Yellow-2) in vital dye solution (Phenosafranine) untreated and after PEF treatment (E=2. 5 kV/cm, n=20,

f=2 Hz).

Fig. 5. Cell disintegration index of untreated and PEF treated protoplasts and cells with

cell wall after different PEF treatment conditions.

Fig. 6. The effect of different PEF treatment conditions on cell size reduction of

protoplasts.

595A. Janositz, D. Knorr / Innovative Food Science and Emerging Technologies 11 (2010) 592–597

3.2.2. Determinant factors: electric ﬁeld direction and cell nucleus

The in situ study of plant cell permeabilization aimed to achieve

some further information concerning the inﬂuence factors of PEF

induced membrane rupture. On the basis of microscopic analyses it

could be frequently noticed that cell shrinking started at the cell poles,

which were nearest to the electrodes (Fig. 9). An observation, which

was described by some research groups (e.g. Kinosita et al., 1988;

Hibino, Itoh & Kinosita, Jr, 1993). Electroporation does not occur

uniformly across the cell membrane. The areas nearest the electrodes

are easily electroporated, whereas others still remain intact, based on

different build transmembrane potentials.

The beginning of cell size decrease at these areas can be seen as an

indicator for the initiation of pore formation on the membrane sites

facing to the electrodes.

Furthermore, it was assumed that membrane disruption depends

on cell nucleus position (Fig. 10). The nucleus stabilizes the cell which

results in higher membrane damage probability at the opposite side.

In contrast to the described increase of transmembrane potential due

to the position of the cell in the electric ﬁeld, it cannot be concluded

that pore formation starts at the obverse membrane side were the cell

core is located, but only to nucleus induced cell ﬁrmness. Further

investigations have to be performed to intensify the possibility of

nucleus induced cell stability.

4. Conclusions

Microscopic analysis before, during and after PEF treatment is a

useful tool to gain a better insight in the permeabilization mechanism

of plant cell material. The comparison between protoplasts and native

plant cells concerning their PEF induced cell structure changes helps

to determine the effect of PEF on the cell wall. Visual observation

showed higher sensibility of protoplasts to the electric ﬁeld in

contrast to cells with cell wall. The cell disintegration index correlated

with microscopic analysis and indicated higher degree of disinte-

grated cells for protoplasts.

It was detected that protoplast cell size is reduced after irreversible

cell disruption and slightly increased after reversible permeabiliza-

tion. Hence, it was concluded that measurement of cell size before and

after treatment can serve as an indicator for cell vitality. It could be

demonstrated through the measurement of cell size and the visual

analysis that cell area decrease is inﬂuenced by PEF intensity, cell size,

electrode situation and nucleus position.

Fig. 9. Recorded images of untreated and PEF treated (E=4 kV/cm, n=10, f=2 Hz) Tobacco protoplasts at different treatment times.

Fig. 7. Exemplary example for the relationship between cell size and transmembrane

potential, induced at the ﬁeld strength of E=2.5 kV/cm and 10 pulses.

Fig. 8. Cell area of untreated and PEF treated protoplasts before and after PEF processing

with different treatment conditions. Statistical signiﬁcance (* Pb0.05, ** Pb0.01, ***

Pb0.001).

Fig. 10. Recorded images of untreated and PEF treated (E=2 kV/cm, n=10, f=2 Hz) Tobacco protoplasts at different treatment times.

596 A. Janositz, D. Knorr / Innovative Food Science and Emerging Technologies 11 (2010) 592–597

Acknowledgments

This work has been supported by an EU-founded Integrated

Project NovelQ “Novel Processing Methods for the Production and

Distribution of High-Quality and Safe Food”, FP6-CT-2006-015710,

Priority 5 `Food Quality and Safety`.

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597A. Janositz, D. Knorr / Innovative Food Science and Emerging Technologies 11 (2010) 592–597

Pulsed electric ﬁelds and their impact on the diffusion characteristics

of potato slices

A. Janositz

, A.-K. Noack, D. Knorr

Department of Food Biotechnology and Food Process Engineering, Institute of Food Technology and Food Chemistry, Technical University of Berlin, Germany

article info

Article history:

Received 6 October 2010

Received in revised form

13 April 2011

Accepted 18 April 2011

Keywords:

Pulsed electric ﬁelds (PEF)

Potato

Diffusion

Reducing sugars

Drying

abstract

Mass transfer in potato slices and strips after Pulsed Electric Fields (PEF) treatment was examined to

evaluate potential application of PEF in potato processing. PEF treatment on cell material leads to pore

formation in cell membrane and thus modiﬁes diffusion of intra- and extracellular media. Results

showed enhanced release of intracellular molecules from permeabilized tissue as well as improved

uptake of low molecular substances into the sample. Sugar, one substrate for the Maillard reaction, was

decreased in PEF treated potatoes, while conductivity increased after electroporation and soaking in

sodium chloride solution, indicating the improved diffusion of salt caused by PEF. Higher release of cell

liquid during drying of PEF treated potatoes was noticed in comparison to untreated potato slices. This

effect increased with the treatment intensity. Furthermore, it was revealed that PEF application leads to

a distinct reduction of fat content after deep fat frying and thus provides a potential for the production of

low-fat French fries. It can be presumed that PEF is a capable assistance to thermal treatments in the

processing of potato chips or French fries for the achievement of structural modiﬁcations and improved

process conditions.

1. Introduction

Mass transfer processes are important unit operations in food

industry requiring the disintegration of biological material. Espe-

cially the processing of plant cells is of great commercial interest

because of the high amount of health related ingredients, pigments

and cell liquid in the vacuoles but also due to the diversiﬁed

potential to be further manufactured. Cell membranes can be seen

as a barrier in diffusion processes, which are therefore strongly

inﬂuenced by the degree cell membrane permeabilization. To

soften the tissue, disrupt cell membranes and to release intracel-

lular content out of the vacuoles, diffusion processes are often

applied with thermal, enzymatic or mechanical exposures.

However, these pre-treatments are connected with some draw-

backs because processes such as extraction, osmotic dehydration or

drying are leading to high energy consumption, long holding times

and determination on nutritional value (Carlsson-Kanyama & Faist,

2000; Santos, Veggi, & Meireles, 2010; Tangka, 2003).

1.1. PEF processing

The newly emerged application of Pulsed Electric Fields (PEF)

constitutes an alternative to conventional processing of plant cell

material, with the main aim of mass transport enhancement

through permeabilization of the cell membrane. PEF processing is

a non-thermal technology to treat plant cell material with less

degradation of nutritional compounds. The application of PEF

includes the implementation of short repeated high voltage pulses

to biological cell material resulting in pore formation of cell

membranes. Two different kinds of pore structure are leading to

different application ﬁelds of PEF. Mild reversible PEF treatment

(E¼0.5e1 kV/cm) maintains the viability of cells and can be used to

target stimulate plant cells to produce secondary metabolites

(Guderjan, Toepﬂ, Angersbach, & Knorr, 2005). Higher energy

pulsing (E>1.0 kV/cm) causes permanent membrane disintegra-

tion and therefore the mechanical destruction of the cell. Irre-

versible pore formation offers several ﬁelds of application in the

non-thermal treatment of plant cell material. Mass transfer

processes are enhanced by PEF treatment due to the facilitated

release of intracellular liquid out of the cell after membrane

disruption. Improvement of juice rates and intracellular metabolite

extraction (Bazhal & Vorobiev, 2000; Bazhal, Lebovka, & Vorobiev,

2001; Knorr, Geulen, Grahl, & Sitzmann, 1994; Sensoy & Sastry,

2004) or the enhancement of drying efﬁciency (Ade-Omowaye,

*Corresponding author. TU Berlin, Department of Food Biotechnology, and Food

Process Engineering, Koenigin-Luise-Str. 22, D-14195 Berlin, Germany. Tel.: þ49 30

314 71411.

E-mail address: annajanositz@yahoo.de (A. Janositz).

Contents lists available at ScienceDirect

LWT - Food Science and Technology

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

doi:10.1016/j.lwt.2011.04.006

LWT - Food Science and Technology 44 (2011) 1939e1945

Angersbach, Esthiahgi, & Knorr, 2001) can be mentioned besides

the use of PEF as a pasteurization method to decontaminate solid

and liquid food (Alvarez, Condon, & Raso, 2006; Heinz, Alvarez,

Angersbach, & Knorr, 2002; Wouters & Smelt, 1997).The critical

external ﬁeld strength is highly dependent on cell size as well as on

cell orientation in the ﬁeld (Heinz et al., 2002). Compared to small

bacterial cells (1e10

m), which require high treatment conditions

to disrupt (critical electric ﬁeld strength of 10e14 kV/cm), lower

energy input is needed for the PEF induced membrane per-

meabilization (critical electric ﬁeld strength of 1e2 kV/cm) of plant

cells, due to their larger cell size, which is in the range of

10e100

m(Janositz & Knorr, 2010; Knorr, Angersbach, Eshtiaghi,

Heinz, & Lee, 2001). The advantage of using mild process condi-

tions for plant cells is based on the easier generation of trans-

membrane potential in large cells, which is required to form pores

in the phospholipid layer.

1.2. PEF assisted mass transfer

The employment of PEF in the treatment of plant tissue can be

used to increase extraction yield in the production of fruit and

vegetable juice. PEF applied as a pre-treatment before pressing

retains product quality and results in the production of fresh taste

juices with a high concentration of heat-sensitive ingredients. It was

demonstrated by Knorr et al. (1994) that PEF assisted extraction of

carrot juice increased not only extractions rate but also preserved

natural composition of functional compounds as

-carotene. Higher

availability of

-carotenewas attained as well as an improvement in

extraction efﬁciency from 51% to 67% by using PEF. PEF processing

can be employed to enhance wine extraction and the amount of

phenolic compounds (Puértolas, López, Condón,Álvarez & Raso,

2010). An increased wine yield after pressing of PEF treated white

grapes was found by Praporscic, Lebovka, Vorobiev, and Mietton-

Peuchot (2007). They showed that high-quality wine can be

produced after PEF application due to rapid expression of low

oxidized must with smallest possible rate ofmire formation. Besides

the use of PEF to enhance extraction yield and composition in the

production of fruit and vegetable liquids, the implementation of PEF

in sugar processing has also attracted attention. The disaccharide

sucrose, located in the intracellular structure of the sugar beet, is

traditionally extracted by thermal treatment for 10e20 min at high

temperatures, about 75e80



C to achieve denaturation of cellular

membranes (Van der Poel, Schiweck, & Schwarz,1998). This thermal

treatment leads near high water consumption and energy costs to

some undesirable effects on the extracted product, as it reduces the

juice purity after thermal destruction of cell wall components and

transformation of high-molecular-weight substances. Several

studies demonstrated the improved sugar extraction of PEF treated

sugar beets (Lebovka, Shynkaryk, El-Belghiti, Benjelloun, & Vor-

obiev, 2007). Eshtiaghi and Knorr (2002) reported after PEF treat-

ment of sugar beets about a 97% sugar yield and a 2e3 times faster

extraction rate as that achieved byconventional thermalprocessing.

Also López, Puértolas, Condón, Raso, and Álvarez (2009) investi-

gated in PEF assisted extraction of sucrose from sugar beets and

found out that temperature of thermal treatment could be reduced

from 70



Cto40



C after PEF pre-treatment. Loginova, Vorobiev,

Bals, and Lebovka (2011) demonstrated that previous PEF treat-

ment enhances the exhaust of sugar beet pulp and increases pulp

dryness. Another point of view that contributes to the great

importance of thefacilitated sugarextraction after PEFapplicationis

the potential cancer-causing agent acrylamide. Acrylamide can be

formedinthermallyprocessedfoodattemperaturesabove120



Cas

a result of the Maillard reaction, which occurs between amino acids

as asparagine or methionine and reducing sugars as fructose or

glucose (Ledl & Schleicher, 1990; Rosén & Hellenäs, 2002; Stadler

et al., 2002). Maillard reactions are important in food processes

such as baking or frying. The formed Maillard products are partly

responsible for color and aroma of many food products. In processes

such as drying, pasteurization and sterilization Maillard reactions

are undesirable and cause major food spoilage due to the darkening

of color, the reduced nutritional availability of certain amino acids

and the formation of unfavored reaction products (Zhang & Zhang,

2007). The formation and reduction of acrylamide in the Maillard

reaction mainly depends on the variables: temperature, processing

time, pH, water activity, product composition and the availability of

reactants, which were regulated by the processing of food material

(Ruﬁa’n-Henares, Delgado-Andrade, & Morales, 2009). Since

temperature and time of food process count as the most important

factors affecting Maillard reaction, the application of PEF provides

a new concept to reduce thermal energy and treatment time with

the combination of target control of food composition. Jaeger,

Janositz, and Knorr (2010) demonstrated the advantageous use of

PEF in the processing of potato chips. A higher release of sucrose,

glucose and fructose was observed after PEF lab scale application

based on the enhanced diffusion of cell water and low molecular

substances out of the cells. A 50% increase in reduction of glucose

content could be shown after PEF treatment in comparison to

untreated potato slices. Better diffusion after PEF implementation

was not only indicated by the enhanced sugar release out of the cell

but also by the improved enzyme infusion of glucoseoxidase in the

potato. It was possible to increase the enzymatic decrease of glucose

content from 52% to 65% and therefore to present an effective

method to improve enzyme diffusion as well as enzyme substrate

accessibility (Jaeger et al., 2010).

The enhanced diffusion properties after PEF induced electro-

poration can also be used to accelerate the drying process efﬁciency

of plant cell material. A shorter drying time of PEF treated potato

cubes during ﬂuidized bed drying was reported by Angersbach and

Knorr (1997).Ade-Omowaye,Rastogi, Angersbach & Knorr (2003)

studied the effect of PEF on red bell pepper slices and compared

the dehydration characteristics with other pre-treatments. They

reported about signiﬁcant effects on drying time after PEF treat-

ment. Drying process time decreased from 360 min without pre-

treatment to 220 min with PEF pre-treatment and thus could be

reduced to one third. Lebovka, Praporscic, and Vorobiev (2003) and

Lebovka, Shynkaryk, and Vorobiev (2007) demonstrated an

essential inﬂuence of PEF treatment at moderate electric ﬁeld

strengths on potato drying rates and showed that PEF application

allows performing thermal pre-treatment at milder temperatures.

For the process of osmotic dehydration several studies reported

about PEF improved water and solution diffusion. Rastogi,

Eshtiaghi, and Knorr (1999) investigated the impact of PEF on

dehydration of carrot cubes and described a decrease in moisture

content during osmotic treatment. Similar observations were made

for mango (Tedjo, Taiwo, Eshtiaghi, & Knorr, 2002) and apple slices

(Taiwo, Angersbach, & Knorr, 2002).

In the study undertaken, PEF were applied to show the potential

of this technology as a disintegration method to improve mass

transfer processes of potato slices. Potato food matrix was used to

study the different effects of facilitated diffusion after PEF pre-

treatment with regard to potential application of PEF in potato

chips processing. Drying of potato slices was investigated as well as

the enhanced release of monosaccharides, which represent rele-

vant substrates for the Maillard reaction. Furthermore, oil content

of fried potato strips was examined to analyze the inﬂuence of PEF

pre-treatment on fat uptake during frying. The study focused not

only on mass transfer from but also to the tissue. The uptake of

sodium chloride after PEF implementation of potatoes was

analyzed in order to examine the potential of PEF to assist the

infusion of ﬂavor carriers or pigments into the tissue.

A. Janositz et al. / LWT - Food Science and Technology 44 (2011) 1939e19451940

2. Material and methods

2.1. Sampling

Potato tubers, Solanum tuberosum, varieties `Karlena’(mid-early

maturing Dutch-German variety, BSA 1993) and `Saturna’(mid-late

maturing Dutch-German variety, BSA 1993) were obtained from the

potato processing company Lorenz Snack-World GmbH & Co KG

(Neu-Isenburg, Germany) and stored in the dark at 8e10



C. The

analyses of sugar content and the uptake of sodium chloride were

performed with the variety `Karlena’, `Saturna’potatoes were

investigated additionally in dryingexperiments.Thesecultivarshave

similar starch content (ca. 18 g/100 g, Kita, 2002) and are capable

productsfortheprocessingofpotatochips.Fatanalyseswererealized

with potato tubers, S. tuberosum, of the variety `Russet Burbank’(late

maturing US variety) which is often used in French fries production.

Samples were obtained from a potato cropping farm in Sachsen-

Anhalt/Germany. Before treatment, unpeeled potato tissue slices,

1.5e2mminthicknessapprox. 5 cm in diameter, were sampled

withastainlesssteelvegetableslicerandwashedinadeﬁnedvolume

(500ml)oftapwaterfor5s.Experimentsinlabscalewereperformed

with ca. 200 g specimen (15 potato slices ¼ﬁve potatoes with three

slices of each potato) larger scale studies were carried out with 1 kg

potatoes per test cycle (75 potato slices ¼25 potatoes with three

slices of each potato). For the analysis of fat content (see 2.8), PEF

treatment was performed on whole tubers (ca. 200 g each) and

blanchingwascarriedoutonpotatohalves.Aftertreatmentssamples

were subsequentlycutinto stripes (10 10 40 mm) bya French fry

cutter with a stainless steel cutting grid.

2.2. Experimental set-up and electric ﬁeld pulses protocol

PEF treatment was applied with exponential pulses. The treat-

ment chamber was connected to a capacitor bank of four DP 30560

(GA, San Diego, USA),15 kV, 2

F in series was used to achieve a total

capacity of 0.5

F and was charged using an ALE802 (Lambda-Emi,

Neptune, USA), 40 kV power supply. The parallel plate treatment

chamber for laboratory batch-wise operation was built with an

electrode size of 20 7 cm. Larger scale batch-wise PEF applications

were performed in a parallel plate treatment chamber which was

built with an electrode size of 49.5 32 cm and 45 29.5 cm for the

upper and lower electrode, respectively. Samples were treated at

room temperature in tab water. The applied PEF treatment parame-

tersarelistedinTable1.TheﬁeldstrengthsofE¼1.5e2.5kV/cmwere

selected in order to achieve irreversible cell disintegration at a low

energy consumption. Zimmermann (1986) reported that ﬁeld

strengths E>1 kV/cm are sufﬁcient to result in permanent pore

formation of plant cell membranes. The microscopic study (see 2.3)

wasperformedwiththehigherﬁeldstrengthof E¼5 kV/cmJanositz,

Semrau,andKnorr(2011)demonstratedthatthecellwallcomponent

lignin was reduced in white asparagus after PEF treatment with the

same treatment intensities. Thus, parameter were chosen to have

conditions that enable the breakingof covalent bonds within the cell

wall and allow the investigation of PEF induced effects in this cell

compartment. Temperature increase during PEF treatments was

considered to be negligible (Knorr & Angersbach,1998). Each process

condition was performed at least 5 times. Mean values were calcu-

lated and presented together with standard deviation in the ﬁgures.

Student’st-tests were used for the analysis of statistical signiﬁcance.

2.3. Microscopic visualization of PEF treated potato tissue

Potato tissue (Ø 20 mm, thickness 30

m) was stained with 20

of ruthenium red (SigmaeAldrich, Germany, dry content 50%) and

washed with distilled water after 15 min. Subsequent PEF treat-

ment was applied with a PEF microscope, constructed in the

Department of Food Biotechnology and Food Process Engineering

(TU Berlin). The microscope (Zeiss Optik, Jena, Germany) enabled

the study of direct cell structure changes during PEF treatment.

Main components were a camera (Nikon E 8700, Japan), which was

ﬁxed to the microscope, 3 objectives, with a maximum magniﬁ-

cation of 400 fold, and a glass slide with two copper foil electrodes

(gap 2 mm, length 3 mm, thickness 0.2 mm, area 0.6 mm

). The

treatment chamber was connected to the micro pulse modulator,

consisting of a power supply FUG HCK, 800 M- 20.000, 20 kV,

80 mA (FUG, Rosenheim, Germany) to a capacitor bank of three

capacitors with 6.8 nF each. The pulse parameters were examined

by a high voltage and a current probe, coupled to a TDS220 (Sony

Tektronix, Beaverton, US) oscilloscope. A PC was used to control PEF

treatment intensities. The images obtained with the microscope

from the samples were recorded with the camera and single

pictures of untreated and PEF treated potato tissue were selected.

Camera was activated manually before treatment.

2.4. Determination of cell vitality through impedance measurement

Cell disintegration index (CDI) was analyzed after Angersbach,

Heinz, and Knorr (1999). The method based on the frequency

depending conductivity of intact and permeabilized tissue. The cell

disintegration index CDI analysis was carried out via impedance

measurement equipment (Biotronix GmbH, A. Angersbach, Hen-

nigsdorf, Germany).

CDI was calculated by following equation:

CDI ¼1bK0

hK0

l

KhKl

b¼Kh

0CDI 1 (1)

where K

and K

΄indicate the electrical conductivity of untreated

and treated cell material in a low- frequency ﬁeld (1e5 kHz),

respectively; and K

and K

΄indicate the electrical conductivity of

untreated and treated material in a high- frequency ﬁeld

(3e50 MHz).

The CDI varies between 0 for intact cells and 1 for total disin-

tegration. Cylindrical pieces were cut out of the potato sample and

placed into a plastic test tube. The electrode area of the measuring

cell was 2 cm

. The gap was adjusted to 1.0 cm.

2.5. Determination of sugar content (sucrose,

- glucose,

- fructose)

Sugar analyses were performed with an enzymatic uv test

(Boehringer Mannheim/R-Biopharm, Germany). PEF processed

potato slices were washed after treatment in tap water (500 ml),

cut, 50 g were mixed with 50 ml distilled water and homogenized

with an Ultra- Turax for 3 min 5 ml Carrez I (3.60 g K4 [Fe(CN)

6] 3H2O (potassium hexacyanoferrate/100 ml) and 5 ml Carrez II

solution (7.20 g ZnSO4 7 H2O (zinc sulfate hepta hydrate/100 ml)

were added to potato mash (pH ¼7.0e7.5). In a volumetric ﬂask

0.3 ml n- Octanol were added to the sample and shook till foam was

dissolved. Filtration was performed after addition of distilled water

Table 1

Parameter of different potato PEF treatments.

PEF parameter Section Section Section

2.3 2.4e2.7 2.8

Field strength E [kV/cm] 5.0 1.5; 2.5 1.8

Pulse number n 20 20 40

electrode distance d [cm] 0.2 8 5

Pulse form exponential exponential exponential

Pulse duration

[

] 100 400 400

Pulse frequency [Hz] 2 2 2

A. Janositz et al. / LWT - Food Science and Technology 44 (2011) 1939e1945 1941

to the mark of 250 ml. Sugar content was analyzed spectrophoto-

metrically (Kontron 25/Germany) at 334 nm wavelength.

2.6. Determination of salt uptake

Surface water of sliced PEF and untreated potatoes was dabbed

with a cloth. Each kind of sample was immersed in 1 g/100 mL

solution of Sodium Chloride (NaCl) for 0 (no immersion), 15 and

30 min, respectively. Subsequent, potatoes were mashed in

a blender and conductivity was measured.

2.7. Determination of drying efﬁciency

Potato slices were weighted before PEF treatment. Pre drying of

untreated and PEF treated samples was performed in a drying oven

(Heraeus, Hanau, Germany) for 10 min at 100



C. After subsequent

weighting, slices were sprayed with 200

l (0.177 g) rape oil. Last

drying cyclewas carried out for another 20 min at 200



C and output

weight was determined. The degree of dryness was calculated by

DM½%¼ DM

ðWC þDMÞ100 (2)

Water Content WC ¼initial weight of untreated sampleeoutput

weight after drying [g].

Dry Matter DM ¼output weight after dryingetara [g].

2.8. Analysis of fat content

Blanching and PEF processing were performed for the compar-

ison of different pre-treatments to reduce fat content during frying.

Warm water blanching (potato/water ratio 1:3) was accomplished

for 2 min at 80



C. Blanched potatoes were cooled in tab water for

10 min and dripped of water. After cutting, 100 g potato stripes

were fried in 2 L rapeseed oil for 13 min at 190



C. The frying sieve

was shaken to release the surface oil and cooling of the fries was

performed for 10 min at room temperature. Oil content of potato

stripes was determined by 3 h Soxhlet extraction using petroleum

ether as a solvent (AOAC, 1995).

3. Results & discussion

PEF application on plant cell material results in an improved

mass transfer of intracellular substances. In Fig.1 untreated and PEF

treated (E¼5 kV/cm, n¼20) potato tissues with stained cell wall

pectin are shown. The dye ruthenium red binds to deesteriﬁed

carboxyl groups and stains pectin in cell wall and middle lamellae. It

is seen that the tissue compartment is slightly changed. The obser-

vation that the cell wall is effected by PEF treatment brings novel

aspectsintheresearchofnon-thermaltechnology.Still,itisnotclear

whether cell wall components are changed directly due to the PEF

treatment or due to cell membrane disintigration and the release of

cytoplasm. However, it was shown in a recent study (Janositz et al.,

2011) that the content of the cell wall biopolymer lignin reduces

after PEFapplication. Lignin degradation may be occurred due to the

effective break of intermolecular and intramolecular bonds within

or between the cellulose, hemicellulose, and lignin.

3.1. Effect of PEF on diffusion of low molecular substances

3.1.1. Reducing sugars (glucose, fructose), sucrose

It could be demonstrated that pre-treatment of PEF on potato

slices in technical scale led to a reduction of sugar content (Fig. 2). A

signiﬁcant increase in the release of glucose and fructose was

observedafterPEFapplicationof1kgpotatoeswiththeﬁeldstrength

E¼1.5 kV/cm and 20 pulses. The enhanced diffusion characteristics

after PEF induced electroporation resulted in one third reduction of

fructose content and a nearly bisection of glucose rate. The obser-

vations correspond with the study of Jaeger et al. (2010). The authors

demonstrated an enhanced release of reducing sugars in potatoes

after PEF processing in lab scale. It can be considered that PEF pre-

treatment is a capable assistance or alternative to conventional

thermal processing for the removing of reducing sugars, which

represents relevant substrates for the Maillard reaction and acryl-

amide formation. The performed investigations in technical scale

shall help to conduct the novel processing technology in industrial

scale for the PEF assisted production of potato chips.

3.1.2. Sodium chloride

Better diffusion characteristics after PEF processing were not

only noticed by the enhanced sugar release out of the cell but also

Fig. 1. Untreated and PEF treated (E¼5 kV/cm, n¼20, 5 min after treatment) potato tissue stained with ruthenium red.

Fig. 2. Sugar content in potato slices after PEF treatment (E¼1.5 kV/cm, n¼20) in

technical scale in comparison to untreated samples. ,¼PEF treated potato samples,

-¼untreated potato samples. Statistical signiﬁcance (*P<0.05, **P<0.01,

***P<0.001).

A. Janositz et al. / LWT - Food Science and Technology 44 (2011) 1939e19451942

by the increased infusion of sodium chloride in the sample. In Fig. 3

conductivity of untreated and PEF treated potatoes after soaking in

sodium chloride solution is presented. It was observed that

conductivity of PEF treated samples was higher and increased with

residence time, indicating the higher uptake of sodium chloride in

the tissue. Two mass processes occurred, water release out of the

cells as well as salt diffusion into the tissue dependent on the

applied concentration gradient. The observations of enhanced salt

uptake correlated with investigations published by Toepﬂand

Heinz (2007). The authors reported improved diffusion of salt

and nitrite into pork haunches after PEF treatment of E¼3 kV/cm

and 5 kJ/kg. The research work shows that PEF application provides

a potential for the target uptake of ﬂavor and color components not

only in animal but also in plant tissue. In case of potato tissue, PEF

treatment might be applied as a pre-treatment before frying in

potato chips processing.

3.2. Effect of PEF on drying efﬁciency

PEF treatment decreased water content of potato slices after

baking in drying oven. Fig. 4 presents different moisture degrees of

untreated and PEF treated potato slices after hot air drying. In

comparison to untreated samples, PEF processed potatoes (Field

strength E¼1.5 kV/cm, n¼20, variety `Saturna’)show3.89g/100g

higher loss of water content after 10 min baking at 100



C and 8.15 g/

100 g higher water reduction after further 20 min baking at 200



Faster drying of PEF treated potato samples is based on cell

membrane permeabilization due to the electric ﬁeld and subsequent

improved diffusion of intracellular liquid out of the tissue. It was

noticed that stronger treatment conditions (E¼2.5 kV/cm, n¼20)

result in greater water loss during potato baking. The degree of cell

disintegration was calculated by impedance measurement and clar-

iﬁedtherelationbetweenﬁeldstrengthandwaterdecrease(Table 2).

Higher treatment intensities cause higher degree of cell disintegra-

tion.Celldisintegrationindexamountedto0.37 (0.064)fortheﬁeld

strength of E¼1.5 kV/cm and increased to 0.57 (0.041) after PEF

treatment with E¼2.5 kV/cm. However, it became evident that the

effect ofPEF pre-treatmentonpotato slices isless pronounced thanit

was demonstrated in previous studies dealing with PEF induced

improvement of potato samples during drying (Angersbach & Knorr,

1997; Lebovka, Shynkaryk, El-Belghiti, et al., 2007; Lebovka, Shyn-

karyk, & Vorobiev, 2007). This might be referred to the minor thick-

nessandthelargesurfaceof thecrispsaffectingtheprocessofdrying.

Hotairdryingisarapiddehydrationmethodinwhichthedryingrate

seems to be inﬂuenced primarily by baking temperature and thick-

ness of the potato slices, and to a lesser extent by PEF pre-treatment.

Nevertheless, PEF assisted drying of potato slices seems to be

promisingbecauseit allowstheuseofmilderthermalconditions and

thus,avoidsundesirablechangesinpigments,vitaminsandﬂavoring

agents (Aguilera, Chiralt, & Fito, 2003).

3.3. Effect of PEF on fat uptake

Inthepresentinvestigation,theeffectofPEFpre-treatmentonthe

fat uptake of potato strips during frying was examined. As presented

in Fig. 5 PEFpre-treated samples contained 4.69 g/100 g less fat than

untreated fried strips,which equates a fat reduction of 38.66% due to

PEFtreatmentcomparedtountreatedsamples.Thisdistinctdecrease

of fat content could not be found for blanched samples, which

showed with 0.46 g/100 g lower oil content (3.79% reduction) only

minimal fat reduction regarding to the reference samples. The

blanching-inducedlayerofgelatinizedstarch(Moreira,Castell-Perez,

& Barrufet, 1999, p. 202) was shown to be less efﬁcient regarding

limitation of oilabsorption in comparison to PEF pre-treatment. This

Fig. 3. Conductivity of PEF treated (E¼1.5 kV/cm, n¼20) and untreated potato

samples without NaCl immersion and after soaking in 1 g/100 mL NaCl solution for 15

or 30 min ,¼PEF treated potato samples, -¼untreated potato samples. Statistical

signiﬁcance (*P<0.05, **P<0.01, ***P<0.001).

Fig. 4. Water loss of PEF treated (E¼1.5 kV/cm, E¼2.5 kV/cm, n¼20) and untreated

potato slices after baking in drying oven (100 C for 10 min þ200 C for 20 min).

,¼PEF treated potato samples (E¼1.5 kV/cm, n¼20), ¼PEF treated potato

samples (E¼2.5 kV/cm, n¼20), -¼untreated potato samples. Statistical signiﬁcance

(*P<0.05, **P<0.01, ***P<0.001).

Table 2

Cell disintegration index (CDI) of PEF treated potatoes (E¼1.5; 2.5 kV/cm, n¼20).

PEF PEF

E¼1.5 kV/cm E¼2.5 kV/cm

n¼20 n¼20

CDI 0.37 0.57

(0.064) (0.041)

Fig. 5. Comparison of blanching (T¼80 C, t¼2 min) and PEF (E¼1.8 kV/cm, n¼40)

pre-treatment with untreated potato stripes concerning fat uptake during frying.

,¼PEF treated potato samples, ¼blanched potato samples, -¼untreated potato

samples. Statistical signiﬁcance (*P<0.05, **P<0.01, ***P<0.001).

A. Janositz et al. / LWT - Food Science and Technology 44 (2011) 1939e1945 1943

ﬁnding was attributed to the modiﬁed frying characteristics of the

PEF treated potato strips. Frying is mainly a drying process that

involves heat and mass transfer. After initial heating of the food

through the surrounding oil, surface boiling begins including water

vaporizingandtheformationofbubbles.Moistureistransferredfrom

the surface to the oil and later by diffusion of inner cellular liquid to

the surface. The water vapour layer on the potato surface acts as

a barrier against the oil and depends on the vapour pressure differ-

ence between food moisture and oil, which inﬂuence the rate of

drying (Jason,1958). DuetothepermeabilizedcellmembranesofPEF

treated tissue cell liquid diffusion from the core to the surface is

enhanced,whichresultinhighervapourpressuredifferenceandthus

thicker water vapour layer, reducing dehydration and fat uptake. As

revealed visually and haptically the surface of PEF treated potato

strips is smooth and ﬂat, which assist additionally the decreased oil

uptake during frying and post-frying (Thanatuksorn, Pradistsuwana,

Jantawat, & Suzuki, 2005). Due to the even cut, oil absorption during

frying can be reduced in contrast to the more distinct roughness of

nonPEFtreatedtissue.DuringthecoolingperiodPEFtreatedsamples

werelesssusceptibletooilabsorptionoftheadversecrustoilbecause

of the smooth and even outer surface, causing better oil draining

(Bouchon & Pyle, 2006).

4. Conclusions

PEF processing can be applied in potato processing to improve

diffusion characteristics of intracellular liquid and low molecular

substances into and out of the tissue. Technical scale PEF treatment

of potatoes decreases the content of reducing sugars and disac-

charides and thus removes substrates for the Maillard reaction. PEF

assisted infusion of molecules into potato tissue was demonstrated

by enhanced salt uptake after electroporation. Therefore, PEF

treatment can be considered as a novel method to insert color and

ﬂavor carrier into potato tissue. The innovation in the study of PEF

treated potato slices lies in the adaptation of the industrial used

geometry of the samples for potato chips, the special surface/

volume ratio, which inﬂuences the effect of PEF. Improvement of

drying efﬁciency due to PEF was additionally analyzed. It was

detected that water loss of PEF treated potato slices after baking in

a convection oven increased with the process conditions intensity.

Concerning oil uptake during deep fat frying, it could be revealed

that PEF application on potato strips leads to a reduction of fat

content, more effective than hot water blanching. This observation

offers new possibilities to produce low-fat French fries without the

use of special coatings or thermal pre-treatments.

Thus, PEF treatment might provide a potential to be imple-

mented in potato chips or French fries processing in order to

provide a non-thermal method for structural modiﬁcations of food

matrix with simultaneously avoiding heat induced nutritional and

sensorial degradations.

Acknowledgments

This work has been supported by an EU- integrated Project

NovelQ “Novel Processing Methods for the Production and

Distribution of High-Quality and Safe Food”, FP6-CT-2006-015710,

Priority 5 ‘Food Quality and Safety’.

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Impact of PEF treatment on quality parameters of white asparagus

(Asparagus ofﬁcinalis L.)

A. Janositz ⁎, J. Semrau, D. Knorr

Technical University of Berlin, Institute of Food Technology and Food Chemistry, Germany

Technical University of Berlin, Department of Food Biotechnology and Food Process Engineering, Germany

abstractarticle info

Article history:

Received 18 November 2010

Accepted 10 February 2011

Available online xxxx

Keywords:

Pulsed Electric Fields (PEF)

Asparagus

Storage

Lignin

Dry solids

Pulsed Electric Field (PEF) application on white asparagus (Asparagus ofﬁcinalis L.) was exercised to examine

inﬂuence of electroporation on spear characteristics as composition and texture. PEF treated spears showed

altered storage behaviour, which was noticed by increased mass transfer as higher water loss as well as the

decrease of the Maillard reaction substrate glucose. Cell disintegration measurement revealed signiﬁcant

inﬂuence of electric ﬁeld orientation on electroporation. Since the anisotropy of asparagus tissue, PEF

processing in longitudinal direction of the spear axis resulted in 9.06% higher cell membrane permeabilization

than treatment in transverse direction. Furthermore, total solids inclusive lignin content were measured to

obtain textural improvements of asparagus spears. It could be shown that dry weight as well as the amount of

lignin was reduced after PEF implementation. Lignin degradation (−2.4%) might be attributed to the PEF

induced interference of electrostatic dipole–dipole interactions between lignin and cellulose and subsequent

deligniﬁcation.

Industrial relevance: Since three decades the technology of Pulsed Electric Fields (PEF) received considerable

relevance in food —and bioengineering as well as in medicine. Besides the use of PEF to inactivate

microorganisms main focus is put on the disintegration of biological cell material to enhance mass transfer in

drying or extraction processes. Although the effects of PEF on various plant cell materials are well studied only

scarce knowledge exists concerning the impact of PEF on white asparagus. In the study undertaken the PEF-

induced changes in asparagus texture and composition were examined. The investigations shall help to

reduce unfavored intra- and extracellular components to gain food safety as well as softer spear texture.

1. Introduction

From the sixteenth century until now, white and green asparagus

has been gaining global popularity. Over the past decade, global

asparagus production has risen substantially, to around one million

tonnes. The steady consumer demand for asparagus can be seen as a

result of the distinctive ﬂavour devoid of fat or sodium and of the

availability of antioxidants, folic acids, and dietary ﬁbers, which

constitute the high nutritional value (Steinmetz & Potter, 1996). Near

antioxidant activity, texture is an important quality factor of the white

asparagus. During postharvest storage, asparagus texture deteriorates

rapidly and endures hardening processes, causing quality degrada-

tion. Fibrous texture strongly reﬂects the amount of product loss

during peeling, which increases with the extent of hardening and can

affect half the product (Clore, Carter, & Drake, 1976). The degree of

hardening is related to biochemical modiﬁcations of the cell wall

composition and can be affected by ligniﬁcation (Salunkhe & Desai,

1984). Lignin, a phenolic biopolymer, is mainly located in cell wall and

crosslinked with different plant polysaccharides, causing mechanical

strength to cell wall and tissue (Hepler, Fosket, & Newcomb, 1970;

Krogmann, 1973). To reduce post harvest losses, it is required to

process fresh asparagus quickly or preserve it by canning, pickling,

freezing and drying. During treatment with heat, it has to be regarded,

that acrylamide formation can occur as a result of Maillard reaction

between reduced sugars and amino acids (Stadler et al., 2002).

Acrylamide, a toxin that has been found in various heat-processed

foods, is considered to be a health risk to humans caused by its

mutagenic and carcinogenic potential (Dearﬁeld, Douglas, Ehling,

Moore, Sega & Brunsick, 1995; International Agency for Research on

Cancer [IARC], 1994). Due to the high amount of asparagine (11,000–

94,000 mg/kg) in asparagus and the availability of glucose and

fructose, asparagus processing as roasting and frying, which include

high temperatures (≥200 °C) and result in low moisture products has

Innovative Food Science and Emerging Technologies xxx (2011) xxx–xxx

⁎Corresponding author at: TU Berlin, Department of Food Biotechnology and Food

Process Engineering, Koenigin-Luise-Str. 22, D-14195 Berlin, Germany. Tel.: +49 30

314 71411.

E-mail address: [email protected] (A. Janositz).

INNFOO-00762; No of Pages 6

doi:10.1016/j.ifset.2011.02.003

Contents lists available at ScienceDirect

Innovative Food Science and Emerging Technologies

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

Please cite this article as: Janositz, A., et al., Impact of PEF treatment on quality parameters of white asparagus (Asparagus ofﬁcinalis L.),

Innovative Food Science and Emerging Technologies (2011), doi:10.1016/j.ifset.2011.02.003

become a critical view concerning human health aspects (Hurst,

Boulton, & Lill, 1998). An elevated level of acrylamide (143 μg/kg) was

found in roasted asparagus (Friedman, 2003) and it intensiﬁed the

relevance of developing gentle food technologies, to assist or replace

the currently common thermal processing.

The application of Pulsed Electric Fields (PEF) on biological cell

material results in permeabilization of the cell membrane. The external

electrical ﬁeld affects thecellin form of short repeatedpulses (μs–ms) of

a high voltage (kV), which induce either temporary or permanent pores

in cell membrane. Temporary pores can be formed when the electric

ﬁeld is removed and the induced pores in cell membrane are small

related to the total membrane area. Thus, the resealing of membrane

pores remains the cell vitality. Irreversible pores are formed at higher

treatment intensities, which induce stable pores in cell membrane and

cause lethal cell damage (Zimmermann, Pilwat, & Riemann, 1974).

Reversible permeabilization is a useful tool to induce stress reactions on

plant systems and stimulate the generation of secondary cell metabo-

lites (Guderjan, Toepﬂ, Angersbach, & Knorr, 2005; Ye, Huang, Chen, &

Zhong, 2004). Irreversible PEF treatment can be applied to inactivate

microorganisms (Barbosa-Cánovas, Góngora-Nieto, Pothakamury, &

Swanson,1999;Heinz,Toepﬂ,&Knorr, 2003;Jaeger,Schulz,Karapetkov,

& Knorr, 2009) or tofacilitate mass transferand improve the diffusion of

intra and extra cellular liquids. Increased extractions efﬁciency of juice

and cell compounds from food plants could be shown by several

researchers (Eshtiaghi & Knorr, 2000; Schilling et al., 2008; Yin, Han, &

Han,2006).Theimprovementofdryingrateswithresulting reductionof

drying time and temperature was reported after application of PEF on

vegetables (Ade-Omowaye, Rastogi, Angersbach, & Knorr, 2001;

Lebovka, Shynkaryk, & Vorobiev, 2007; Taiwo, Angersbach, & Knorr,

2002). Based on the enhanced mass transfer after PEF processing, it

could be demonstrated that PEF cause not only higher release of intra

molecular content but also improve the uptake of low molecular

substances into the tissue. An increased infusion into PEF treated potato

slices was shown for glucoseoxidase to assist the removal of reducing

sugars and for sodium chloride to examine the potential of PEF to insert

ﬂavour carrier into the food matrix (Jaeger, Janositz, & Knorr, 2010;

Janositz et al., submitted for publication).

The objective of this study was to investigate the effects of PEF on

asparagus to ﬁnd a pre-treatment method to prior thermal treatments

or an alternative to heat processing for the production of better quality

asparagus. Lignin content of PEF treated asparagus was analyzed to

clarify impact of PEF on the biopolymer lignin, gaining improved

macroscopic characteristics of the spears. The inﬂuence of PEF on the

asparagus matrix concerning the release of reduced sugars was

investigated to identify the potential of PEF on asparagus for the

removal of Maillard reaction substrates. In addition, post permeabi-

lization changes like water loss and colour changes of PEF treated

spears were studied, identifying the effect of PEF on storage behaviour.

2. Material and methods

2.1. Sampling

White Asparagus (Asparagus ofﬁcinalis) was obtained from Beelitz/

Germany and experiments were performed within 4 to 24 h after

harvest. Samples were stored in a refrigerator at 4 °C. Before treatment

10 g of the spear base section (circular area=circa 7 cm²) was cut and

divided vertically. PEF treatment was performed with one-half of the

spear base; the other half was used as a reference. After treatment,

samples were either directly analyzed, stored at 4 °C in the dark or freeze

dried (Freeze Dryer Modulys; Erwardy High Vacuum International;

West Sussex/England) to perform further studies. Dry solids and water

evaporation were analyzed on days 0, 4 and 6. Sugar content was

analyzed on days 0 and 4. The direct effect of PEF on lignin content was

analyzed immediately after PEF treatment.

2.2. Experimental set-up and electric ﬁeld pulses protocol

PEF treatment was applied with exponential pulses. The cuboid

batch treatment chamber included two parallel-plate electrodes with

a size of 20×7 cm and an electrode gap of 3 cm (420 ml in volume).

The treatment chamber was connected to a capacitor bank of four DP

30560 (GA, San Diego, USA), 15 kV, and 2 μF in series has been used to

achieve a total capacity of 0.5 μF and was charged using an ALE802

(Lambda-Emi, Neptune, USA), 40 kV power supply. The applied PEF

treatment intensities for asparagus were: output voltage: 15,000 V,

electrode gap: d=3 cm; electric ﬁeld strength: E=5 kV/cm; pulse

number: n=20; pulse duration: τ=400 μs and frequency: ƒ=2 Hz.

The speciﬁc energy per pulse was 337.5 J. The energy input per

treatment was 16.0714 kJ/kg.

2.3. Determination of sugar content (D-glucose, D-fructose)

PEF treated asparagus samples were washed in tap water (500 ml)

and freeze dried subsequently or stored before freeze drying. The freeze

dried samples were homogenized in an Ultra-Turrax (T 25 digital Ultra-

turrax, IKA laboratory technology, Germany) at 15,000 rpm for 3 min

at room temperature. Distilled water was added to the mixture in a

ratio 1:1. 5 ml Carrez I solution (3.60 g K4[Fe(CN)6]×3H2O (potassium

hexacyanoferrate/100 ml) and 5 ml Carrez II solution (7.20 g ZnSO4×7

H2O (zinc sulfate hepta hydrate/100 ml) were added to asparagus mash

(pH=7.0–7.5). In a volumetric ﬂask 0.3 ml n-Octanol was added to the

sample and shook until foam was dissolved. Filtration was performed

after addition of distilled water to themark of 250 ml. Sugarcontent was

analysed spectrophotometrically (Kontron 25/Germany) at 334 nm

wavelength.

2.4. Determination of cell vitality through impedance measurement

Celldisintegration index (CDI) was analyzed after Angersbach, Heinz,

and Knorr (1999). The method based on the frequency depending

conductivity of intact and permeabilized tissue. The cell disintegration

index CDI analysis was carried out via impedance measurement

equipment (Biotronix GmbH, A. Angersbach, Hennigsdorf, Germany).

CDI was calculated by following equation:

CDI =1−bK′

h−K′



Kh−Kl

b=Kh

K′h0≤CDI≤1ð1Þ

where K

and K′

indicate the electrical conductivity of untreated and

treated cell materials in a low-frequency ﬁeld (1–5 kHz), respectively;

and K

and K′

indicate the electrical conductivity of untreated and

treated materials in a high-frequency ﬁeld (3–50 MHz).

The CDI varies between 0 for intact cells and 1 for total

disintegration. Cylindrical pieces were cut out of the asparagus

spear and placed into a plastic test tube. The electrode area of the

measuring cell was 2 cm². The gap was adjusted to 1.0 cm.

2.5. Colour determination

Colour of PEF treated and untreated asparagus samples was measured

photometrically using a colorimeter (CR-200 Minolta, Japan). L*a*b*-

values are a numerical classiﬁcation of the colours of the different

concentrates. L* represents the lightness of the sample and ranges from

black=0 to white=100, a* indicates sample position between red (+)

and green (−) and b* its position between yellow (+) and blue (−).

2.6. Determination of water content and dry weight

PEF treated and untreated asparagus samples (1–2 g) were dried

to constant weight in a drying oven at 105 °C for 5 h. After cooling in a

2A. Janositz et al. / Innovative Food Science and Emerging Technologies xxx (2011) xxx–xxx

Please cite this article as: Janositz, A., et al., Impact of PEF treatment on quality parameters of white asparagus (Asparagus ofﬁcinalis L.),

Innovative Food Science and Emerging Technologies (2011), doi:10.1016/j.ifset.2011.02.003

dehydrator output weight was determined and dry weight/water

content was calculated by following equations:

DW %½=DW

WC +DWðÞ

× 100 ð2Þ

Water Content WC = initial weight–output weight g½

Dry Weight DW = output weight–tara g½:

2.7. Analysis of lignin

2.7.1. Qualitative

Phloroglucin, a benzentriol (1,3,5-Trihydroxybenzol, Merck,

Darmstadt/Germany), was solubilized in a mixture of ethanol/water

(1:1) (w=5%). For the qualitative detection of lignin, phloroglucin

solution was applied to the asparagus sample and one drop of

hydrochloric acid was added to turn the contained lignin red.

Pictures were recorded by using a light microscope (Nikon Eclipse

E400) equipped with a digital camera (JVC, TK-10070E).

2.7.2. Quantitative

Lignin content determination is based on Association of Ofﬁcial

Analytical Chemists [AOAC] methods (1984) according to the proce-

dures of Goering and Van Soest (1970). The analysis includes the

detection of ADF (Acid Detergent Fiber) and ADL (Acid Detergent

Lignin). The freeze dried samples were homogenized in an Ultra-Turrax

(T 25 digital ultra-turrax, IKA laboratory technology, Germany) at

24,000 rpm for 5 min at room temperature. Detection of ADF content:

100 ml of acid detergent dissolution (20 g of N-trimethyl-ammonium

bromide) was soluted in sulphuric acid (c: 1/2 H

=1 mol/l) and

added with 0.5 ml Octanol. 1 g of grounded sample was weighted out

and mixed with the solutionand boiled for 60 min.After boiling, content

of the glass beaker was vacuum-ﬁltrated through a ﬁlter crucible and

washed afterwards with 250 ml hot water and acetone. Filter crucible

was dried over night in a drying oven at 100 °C and weighted out after

cooling in a dehydrator. The content of ADF was determined by the

formula:

ADF =m2−m1ðÞ100

Eð3Þ

where m1 indicates the mass of the ﬁlter crucible [g], m2 indicates the

mass of the ﬁlter crucible and ADF [g] and Enotiﬁes the initial weight

[g].

The ﬁlter residue can be used for the detection of raw lignin=ADL

(Acid Detergent Lignin).

2.7.2.1. Determination of ADF content. Filter crucible with residue of

ADF analysis was weighted out and placed in a glass beaker. Crucible

content was covered with 72% sulphuric acid, which was cooled to

15 °C. Over a period of three hours, sulphuric acid was reﬁlled and

mixture was stirred hourly at a temperature of 20–23 °C. Suction, hot

water washing, drying and weighting were performed subsequently.

After incineration of organic substances the specimen was weighted

again. The annealing loss equates the amount of raw lignin. The

experiments were performed in duplicates and replicated ﬁve times

for statistical purposes.

Fig. 1. Water evaporation of PEF treated (E=5 kV/cm, n=20) and untreated asparagus

after 4 and 6 days of storage. Day 0 showed no water evaporation. Statistical signiﬁcance

(*Pb0.05, **Pb0.01, and ***Pb0.001).

Fig. 2. Dry weight content of PEF treated (E=5 kV/cm, n=20) and untreated asparagus

after 0, 4 and 6 days of storage. Statistical signiﬁcance (*Pb0.05, **Pb0.01, and

***Pb0.001).

Fig. 3. Glucose (a) and fructose (b) content of PEF treated (E=5 kV/cm, n=20) and

untreated asparagus after 0 and 4 days of storage. Statistical signiﬁcance (*Pb0.05,

**Pb0.01, and ***Pb0.001).

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Please cite this article as: Janositz, A., et al., Impact of PEF treatment on quality parameters of white asparagus (Asparagus ofﬁcinalis L.),

Innovative Food Science and Emerging Technologies (2011), doi:10.1016/j.ifset.2011.02.003

3. Results and discussion

3.1. Effect of PEF on mass transfer

3.1.1. Cell liquid and dry solids

It is knownthat PEF exposure on plant material leads to anenhanced

mass transfer rate via permeabilization of the cell membrane (Ade-

Omowaye et al., 2001; Lebovka et al., 2007). A trend towards the

facilitated release of inner cell liquid was noticed by the higher water

loss of PEF treated asparagus during the storage period of six days. Fig. 1

presents the water evaporation of PEF treated (E=5 kV/cm, n=20)

and untreated asparagus during short-time storage on days 4 and 6. On

day 4, the release of cell liquid from PEF treated tissues amounted twice

as high as for the untreated sample. Additionally, the water evaporation

increased with storage time and both samples showed similar rise

within the two days. Although higher release of cell liquid from

electroporated asparagus tissue indicates advantages during drying

processes in food industry as shown in shorter drying times and lower

drying temperatures (Tedjo, 2003), increased water loss after harvest

constitutes a problem due to product shrivelling and the loss of glossy

appearance. To reduce moisture loss, constant cooling of the spears

should be provided as well as holding the produce in plastic liners.

Otherwise, water loss is not always a detriment. Mechanical damage of

the product inhandlingand processingcan bereduced due to the loss of

turgidity (Suslow, 2000).

The measurement of dry weight indicated an inﬂuence of PEF on

asparagus solids. As shown in Fig. 2, dry matter of untreated asparagus

remained constant over the period of storage, whereas dry solid

content of PEF processed asparagus decreased within the six days by

1.23%. Explanations for the increased reduction of solids after PEF

processing might be found in the enhanced release of intracellular

substances as sugars (see Section 3.1.2), enzymes and antioxidants

(Bazhal, Lebovka, & Vorobiev, 2001; Eshtiaghi & Knorr, 2000; Van

Loey, Verachtert, & Hendrickx, 2002). Although experiments were

performed without additional mechanical compression of the tissue,

PEF-induced cell membrane permeabilization could cause sufﬁcient

cell destruction to transfer particularly low molecular substances out

of the cell. Furthermore, the release of endogenous enzymes as

hydrolases after PEF treatment as well as microbial infection of the

spears could result in increased degradation of dry weight during the

period of storage.

3.1.2. Reducing sugars (D-glucose, D-fructose)

As represented in Fig. 3a no alteration of glucose level was found for

untreated and PEF treated asparagus directly after treatment. However,

on the fourth day of storage, both samples showed signiﬁcant reduction

of glucose content. PEF treated samples amounted 3% less glucose than

the reference.

The lowering in glucose content during storage could be attributed

to respiratory processes, microbial load and/or the enhanced release

of endogenous enzymes due to PEF-induced cell disintegration

causing degradation of glucose (Bisson, Jones, & Robbins, 1926). As

demonstrated in Fig. 3b, only minimal changes in fructose content due

to PEF treatment were observed, but no further decrease of fructose in

untreated as well as in PEF treated asparagus was noticed.

3.2. Effect of PEF direction on cell disintegration index (CDI)

The enhanced diffusion can be regulated by the degree of cell

disintegration. Higher CDI results in higher release of cell content,

causing better diffusion of low molecular substances (Jaeger et al., 2010;

Janositz et al., submitted for publication). Fig. 4 presents the CDI of PEF

processed (E=5 kV/cm, n=20) asparagus, treated and measured in

longitudinal and diagonal direction. CDI increases by 9.06% when

orientating the electrodes longitudinally relative to themajor axis of the

tissue. This observation demonstrates that the direction of the electric

ﬁeld has a signiﬁcant inﬂuence on the effectiveness of PEF treatment.

Asparagus tissue can be seen as distinctly anisotropic, since the cells

have a diameter of approximately 20 μm, but a length of up to 100 μm

(Gassner, Hohmann, & Deutschmann, 1989). Electrical conduction

along the length of cell, ﬁlled with rich ionic intracellular liquid, is

thus easier than conduction between the cells in the less conductive

extracellular matrix and the non-conductive cell membrane.

3.3. Colour determination

Colour coordinates of PEF processed and unprocessed asparagus

were determined to investigate impact of PEF on sensory qualities

directly after treatment and during storage of 0, 4 and 6 days. By

comparing the samples, markedly colour differences were noticed. In

agreement with visual observation, instrumental light measurement

showed higher browning of PEF treated asparagus samples compared

to untreated samples, which was indicated by lower L* values

(Table 1). Analysis of a* and b* measurements showed yellow tint

of PEF treated or green tint of untreated asparagus, respectively. At all

samples intensiﬁed darkening, indicated by higher b* values, was

observed during storage period. The more pronounced darkening of

the PEF treated samples might be referred to the higher release of

polyphenol oxidase (PPO) after cell membrane permeabilization. An

addition of ascorbic acid could help to prevent the oxidation of

phenolic compounds and thus the browning of the samples (Mayer &

Harel, 1979; Vamos-Vigyazo, 1981).

Fig. 4. Cell disintegration index of PEF treated asparagus (E=5 kV/cm, n=20) with

electrode orientation in longitudinal or diagonal path direction. Statistical signiﬁcance

(*Pb0.05, **Pb0.01, and ***Pb0.001).

Table 1

A*, b* and light values of PEF treated (E=5 kV/cm, n=20) and untreated asparagus after 0, 4 and 6 days of storage.

L* value A* value B* value

Day 0 Day 4 Day 6 Day 0 Day 4 Day 6 Day 0 Day 4 Day 6

Untreated 82.96

(±0.43)

83.08

(±0.97)

82.93

(±2.04)

−1.30

(±0.14)

−1.47

(±0.09)

−1.51

(±0.26)

5.22

(±0.56)

6.33

(±0.48)

6.97

(±0.96)

PEF treated 78.98

(±1.96)

78.73

(±1.60)

82.09

(±2.33)

−1.51

(±0.19)

−1.22

(±0.24)

−1.68

(±0.29)

4.81

(±0.68)

4.73

(±0.71)

6.08

(±1.19)

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Please cite this article as: Janositz, A., et al., Impact of PEF treatment on quality parameters of white asparagus (Asparagus ofﬁcinalis L.),

Innovative Food Science and Emerging Technologies (2011), doi:10.1016/j.ifset.2011.02.003

3.4. Effect of PEF on lignin content

3.4.1. Qualitative

In Fig. 5 asparagus tissue with red stained lignin is shown. The

chemical reaction with phloroglucin and sulphuric acid was per-

formed to visualize the distribution of lignin in the spear. It is seen

that lignin is particularly abundant in the pod (a) and located in

longitudinal direction of the spear. This can be clariﬁed by viewing

cross-sectional imaging (b). Lignin deposition was noticed as compact

and bundled grown in asparagus tissue.

3.4.2. Quantitative

The application of PEF has an inﬂuence on lignin content in as-

paragus. As represented in Fig. 6, the amount of raw lignin decreased

from 12.6% (±0.08) in untreated asparagus samples to 10.2% (±0.34)

in the PEF treated asparagus base section. The behaviour of mac-

romolecules exposed to an intense electric ﬁeld is not well understood

(Neumann, 1986). Lignin, a complex phenolic polymer, is seen as

highly resistant to biodegradation (Crawford, 1981). Its chemical

structure is branched and the macromolecule is bonded with various

lignin cross-links and also linkaged between lignin and polysacchar-

ides as cellulose and hemicellulose (Eriksson, Goring, & Lindgren,

1980). Application of PEF may be able to enhance separation of

cellulosic material from lignin. High voltage pulses may be effective to

break intermolecular and intramolecular bonds within or between the

cellulose, hemicellulose, and lignin (Navapanich & Giorgi, 2008).

Explanation for the breakage could be that cellulose microﬁbrills

contain large number of hydroxyl groups on the surface causing

interactive force attraction with the hydroxyl and methoxyl groups of

coumaryl, coniferyl, and sinapyl alcohols from lignin (Houtman &

Atalla, 1995). These ﬁndings indicate that the dominant force

connecting lignin and cellulose is caused by electrostatic dipole–

dipole interactions. Subsequent deligniﬁcation can occur when the

covalent bonds are cleaved resulting in solubilization of polymer

fragments (Goring, 1971).

4. Conclusions

The ﬁndings suggest that PEF application on asparagus presents a

promising tool to improve macroscopic characteristics of the spears.

Enhanced mass transfer of PEF treated tissue was noticed by higher

moisture loss of PEF treated spears during post-harvest storage.

Concerning the removal of glucose, increased mass transfer could not

be conﬁrmed. Nevertheless, a higher decrease of glucose after PEF

treatment was observed after storage. A pronounced decrease of the

Maillard reaction substrates glucose and fructose could beshown for PEF

treated potato slices in laboratory as well as in technical scale (Jaeger

etal.,2010;Janositzetal.,submittedforpublication). These observations

mark the different inﬂuences of PEF on different food matrices.

Colour determinations showed darker spears with higher a* and b*

values. Raised water reduction aswell asintensiﬁed browning should be

minimized by fast process control and the addition of citric or ascorbic

acid. The measurement of the cell disintegration index indicated the

anisotropy of asparagus tissue. PEF processing in longitudinal direction

of the major axis caused higher cell rupture than PEF application in

transverse direction. Furthermore, inﬂuence of PEF on dry solids

including lignin was studied. A trend towards the decrease of dry

weight content of PEF produced asparagus within the period of storage

was noticed. Reduction on lignin contentwas found after PEF treatment.

This effect might be attributed to the PEF induced leakage of lignin–

cellulose bonds, enabling the start of deligniﬁcation to gain softer

texture of the spear. The research ﬁndings shall help to include PEF

technology in asparagus processing as a method prior to heat treatment

especially for the production of roasted and fried asparagus. Future

investigations on asparagus tissue shall deal with the inﬂuence of

longitudinal and transverse direction of the electric ﬁeld concerning the

differentabilitytoenhance masstransferprocessesaswellastoalterdry

solid and lignin content.

Acknowledgments

This work has been supported by an EU-founded Integrated

Project NovelQ “Novel Processing Methods for the Production and

Distribution of High-Quality and Safe Food”, FP6-CT-2006-015710,

Priority 5 ‘Food Quality and Safety’.

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Innovative Food Science and Emerging Technologies (2011), doi:10.1016/j.ifset.2011.02.003