SALUBRITÉ DES LÉGUMES EN CONSERVE PAR TRAITEMENT … · les barèmes de stérilisation et les...

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SALUBRITÉ DES LÉGUMES EN CONSERVE

PAR TRAITEMENT THERMIQUE COMBINÉ À

L’ÉLECTRO-ACTIVATION

ANALYSE DE L’EFFICACITÉ DU SYSTÈME ET

DÉTERMINATION DES ÉCONOMIES D’ÉNERGIE

Thèse

Viacheslav Liato

Doctorat en sciences et technologie des aliments

Philosophiae Doctor (Ph. D.)

Québec, Canada

© Viacheslav Liato, 2015

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Résumé

La salubrité et la qualité des aliments en conserve dépendent toujours des paramètres

appliqués lors de la stérilisation (le temps, les températures). L'application de températures

élevées peut détériorer la qualité des produits et augmenter les coûts énergétiques associés

au procédé de stérilisation. De plus, l’attente des consommateurs se dirige, de nos jours, vers

des produits sains et frais, ce qui encourage les chercheurs à découvrir de nouvelles approches

et techniques pour améliorer la conservation des aliments. Les technologies émergentes

combinées à d'autres approches classiques ont démontré des résultats probants (effet barrière

ou «hurdle effect ») quant à la préservation de produits non acidifiés. Récemment, des études

sur l'électro-activation (EA) ont mis en évidence son fort potentiel antibactérien applicable

pour une large gamme de produits alimentaires. En outre, cette technologie a été reconnue

comme étant sécuritaire et peu coûteuse. Le but de ce projet était d'étudier l'EA comme une

barrière efficace afin de préserver les qualités nutritionnelles des aliments tout en réduisant

les barèmes de stérilisation et les coûts de production dans un respect total de la salubrité du

produit. Le premier objectif était d'étudier les propriétés des solutions électro-activées (SEA)

ainsi que la dynamique de leurs modifications en fonction de plusieurs paramètres comme la

densité de courant appliqué au système, le temps d'excitation, le type de sels utilisés, la

concentration en sel et les configurations du réacteur d’électro-activation. De plus, les

paramètres optimaux comme le pH, le potentiel redox et la résistance électrique du système,

qui est un indice d’efficacité énergétique, ont été déterminés pour la production des SEA à

l'aide de la méthode de surface de réponse. Par la suite, les SEA optimisés ont été étudiés sur

la corrosivité dans des boîtes de conserve en métal et les résultats obtenus ont montré que la

SEA n'a aucun effet significatif sur l'oxydation des contenants de maïs en conserve. Le

deuxième objectif a permis de mettre en évidence l'activité sporicide des SEA. En effet,

l’étude a montré que les SEA ont une forte capacité d'inhibition de la croissance des spores

de Clostridium sporogenes et de Geobacillus stearothermophilus. La combinaison de

traitements modérés (T � 100 °C) avec différentes SEA dans la purée de légumes a entraîné

une destruction importante ou totale des spores de C. sporogenes. Une diminution

considérable de la résistance à la chaleur de G. stearothermophilus a été également observée.

Un effet synergique d’une grande efficacité a été obtenu lorsque la SEA a été utilisée en

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combinaison avec un traitement thermique lors de la stérilisation. Sur le plan pratique, cela

résulte en une modification significative des barèmes de stérilisation en utilisant des

températures modérées. Le troisième objectif consistait à étudier l'effet combiné des SEA et

de la stérilisation à des températures modérées sur la qualité des conserves. Des petits pois et

du maïs en grain ont été utilisés comme légumes modèles. L'analyse organoleptique a montré

qu'un temps de stérilisation écourté conserve mieux les attributs sensoriels du produit. En

plus, les températures de stérilisation utilisées avec les SEA étaient adéquates pour la

préservation de la vitamine C qui est utilisée comme indicateur de préservation de la qualité.

Aussi, la différence entre la technologie de stérilisation utilisant l’effet combiné de barrières

composées de SEA et d’un traitement thermique modéré, comparativement à la technologie

de stérilisation classique, laisse entrevoir la possibilité de réaliser d'importantes économies

énergétiques. De plus, il sera possible de préserver la qualité nutritionnelle du produit et tout

cela sans compromettre sa salubrité. Finalement, ce projet a apporté une contribution

significative aux connaissances applicables à l’amélioration de la technologie classique de

mise en conserve des légumes via une meilleure compréhension du comportement des spores

thermophiles sous l'action des SEA combinées à des températures nettement inférieures à

celles utilisées dans le procédé conventionnel de stérilisation des légumes en conserve.

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Abstract

The safety and quality of low-acid canned food always depends on the parameters of

sterilization. However, high temperatures may lead to a deterioration of product quality and

to an increase of energy costs. In addition, today’s the increase of consumer demand for more

«fresh-like» foods is challenging researchers to discover innovative approaches and

techniques to improve methods of food preservation. Emerging technologies in combination

with classical approaches exhibited high effectiveness (hurdle effect) in preservation of low-

acid products. Recently, electro-activation (EA) convincing results by demonstrating high

antibacterial potential on food products at large scale; furthermore, it has been recognized as

a safe and an inexpensive hurdle. Taking sense from the hurdle approach, the aim of this

project was to study EA as an effective and potent hurdle, so that it could result in a decrease

in sterilization temperatures, thereby decreasing energy costs, increasing food quality and

ensuring sterility. The first objective was to study the properties of EA solutions as well as

the dynamics of their changes using variety of parameters (current density, excitation time,

type and concentration of salts, configurations). In addition, by using the response surface

methodology the optimum parameters (pH, redox potential, resistance) for the production of

EA solutions (EAS) were found. Thereafter, the optimized EAS were studied for

corrosiveness in canned containers showing no significant oxidizing effect on container filled

with canned corn which was within the acceptable limits according to the stipulated

regulations. The next objective focused on its sporicidal activity. The study showed that EAS

has strong inhibiting capacity on the growth of Clostridium sporogenes and Geobacillus

stearothermophilus spores. The combination of mild treatments (�100°C) and EAS in

vegetable puree resulted in significant or total destruction of putrefactive spores of

C. sporogenes. A considerable decrease in heat-resistance of G. stearothermophilus was also

observed. A synergistic effect was observed when EAS was used in combination with heat

treatment during sterilisation, which allows changing the temperatures of sterilization. Thus,

the last objective investigated the combined effect of EAS and sterilization at mild

temperatures on the quality of canned peas and corn. The sensorial analysis showed that a

shorter sterilization time increase the preservation of sensorial attributes. Indeed, the lowest

temperatures appeared to be more favourable for vitamin C preservation. Nevertheless the

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difference between classical sterilization and hurdle technologies using EAS displayed

significant energy savings and quality preservation. Overall, this project proposed a new

approach to improve food canning technology; furthermore, it allowed a better understanding

of the thermal behaviour of thermophilic spores under action of EAS as well as expands the

scientific knowledge of EA technology.

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Table of contents

Résumé .............................................................................................................................................. iii

Abstract ............................................................................................................................................... v

Table of contents .............................................................................................................................. vii

List of tables ....................................................................................................................................... xi

List of figures .................................................................................................................................. xiii

List of abbreviations ....................................................................................................................... xvii

Dédicaces ......................................................................................................................................... xix

Acknowledgments ............................................................................................................................ xxi

Preface ........................................................................................................................................... xxiii

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

1. CHAPTER 1: Literature review .................................................................................................. 3

1.1. Canning................................................................................................................................ 3

1.1.1. Fundamentals .............................................................................................................. 3

1.1.2. Process challenges .................................................................................................... 19

1.1.3. Emerging technologies .............................................................................................. 26

1.2. Technology of electro-activation ...................................................................................... 32

1.2.1. Origin ......................................................................................................................... 32

1.2.2. Principles ................................................................................................................... 32

1.2.3. Electrodes process .................................................................................................... 33

1.2.4. Proprieties of EAS ...................................................................................................... 35

1.2.5. Technical aspects of EA reactor ................................................................................ 44

1.2.6. Applications of EAS.................................................................................................... 53

2. CHAPTER 2: Problematic, research hypothesis and objectives ............................................... 63

2.1. Problematic ....................................................................................................................... 63

2.2. Research hypothesis ......................................................................................................... 63

2.3. Main objective................................................................................................................... 63

2.4. Specific objectives ............................................................................................................. 64

3. CHAPTER 3: Ion exchange membrane-assisted electro-activation of aqueous solutions: Effect

of the operating parameters on solutions properties and system electric resistance ......................... 65

3.1. Contextual transitions ....................................................................................................... 66

3.2. Résumé .............................................................................................................................. 67

3.3. Abstract ............................................................................................................................. 68

3.4. Introduction ...................................................................................................................... 69

3.5. Materials and methods ..................................................................................................... 71

3.5.1. Chemicals .................................................................................................................. 71

3.5.2. Ion exchange membranes ......................................................................................... 71

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3.5.3. Electrodes .................................................................................................................. 72

3.5.4. Electro-activation reactor design .............................................................................. 72

3.5.5. Measurements of chemical characteristics ............................................................... 73

3.5.6. Configuration of the electro-activation reactor ........................................................ 74

3.5.7. Protocol of electro-activation ................................................................................... 75

3.5.8. Statistical analysis ...................................................................................................... 75

3.6. Results and discussion ....................................................................................................... 76

3.6.1. Configuration #1: Anolyte ......................................................................................... 76

3.6.2. Configuration #1: The central section ....................................................................... 79


3.6.4. Configuration #2: The central section ....................................................................... 87



3.6.7. Configuration #4: The middle section ....................................................................... 95

3.6.8. Catholyte ................................................................................................................... 96

3.6.9. Electric resistance of system ................................................................................... 100

3.7. Conclusion ....................................................................................................................... 103

3.8. ACKNOWLEDGMENTS ..................................................................................................... 104

4. CHAPTER 4: Application of response surface methodology for the optimization of the

production of electro-activated solutions in a three-cell reactor .................................................... 105

4.1. Contextual transitions ..................................................................................................... 106

4.2. Résumé ............................................................................................................................ 107

4.3. Abstract ........................................................................................................................... 108

4.4. Introduction ..................................................................................................................... 109

4.5. Materials and methods ................................................................................................... 112

4.5.1. Chemicals and materials .......................................................................................... 112

4.5.2. Electro-activation reactor ........................................................................................ 112

4.5.3. Protocol of the electro-activation ........................................................................... 113

4.5.4. Global system resistance ......................................................................................... 114

4.5.5. Experimental design and statistical analysis ........................................................... 114

4.6. Results and discussion ..................................................................................................... 116

4.6.1. Model fitting from RSM ........................................................................................... 116

4.6.2. Effect of salt concentration, current and time ........................................................ 120

4.6.3. Optimization using desirability functions ................................................................ 128

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4.7. Conclusions ..................................................................................................................... 130

4.8. Acknowledgments ........................................................................................................... 131

5. CHAPTER 5: Effect of electro-activated solutions (EAS) on the corrosion of metallic cans used

for food preservation ....................................................................................................................... 133


5.2. Résumé ............................................................................................................................ 135

5.3. Abstract ........................................................................................................................... 136

5.4. Introduction .................................................................................................................... 137


5.5.1. Chemicals ................................................................................................................ 138

5.5.2. EAS generation ........................................................................................................ 138

5.5.3. Relaxation period .................................................................................................... 139

5.5.4. Corrosion dynamics of tinplate can ........................................................................ 139

5.5.5. Statistical analysis ................................................................................................... 140

5.6. Results and discussion..................................................................................................... 140

5.6.1. Effect of EAS relaxation time during storage .......................................................... 140

5.6.2. Effect of EAS on the corrosion of tinplate containers ............................................. 143

5.6.3. Changing of metals concentration of the canned corn produced with EAS ........... 148

5.7. Conclusions ..................................................................................................................... 151


6. CHAPTER 6: Study of the combined effect of electro-activated solutions and heat treatment on

the destruction of spores of Clostridium sporogenes and Geobacillus stearothermophilus in model

solution and vegetable puree ........................................................................................................... 153


6.2. Résumé ............................................................................................................................ 155

6.3. Abstract ........................................................................................................................... 156

6.4. Introduction .................................................................................................................... 157


6.5.1. Chemicals ................................................................................................................ 159

6.5.2. Vegetable puree preparation .................................................................................. 160

6.5.3. Spores production of Clostridium sporogenes ATCC 7955 ...................................... 160

6.5.4. Spores production of Geobacillus stearothermophilus ATCC12980 ....................... 161

6.5.5. Control on the quality of spore suspensions. ......................................................... 161

6.5.6. Preparation of electro-activated solutions ............................................................. 162

6.5.7. Treatment of spore suspensions ............................................................................. 163

6.5.8. Heat resistance of spores in pea and corn purees with EA solutions ..................... 163

6.5.9. Calculations and statistical analysis ........................................................................ 164

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6.6. Results ............................................................................................................................. 165

6.6.1. Effect of EAS on inactivation of spores in moderate temperatures ........................ 165

6.7. Discussion ........................................................................................................................ 178

6.8. Conclusions ...................................................................................................................... 186


7. CHAPTER 7: Production of canned pea and corn by using a hurdle technology composed of

electro-activation and low heat treatment....................................................................................... 187


7.2. Résumé ............................................................................................................................ 189

7.3. Abstract ........................................................................................................................... 190

7.4. Introduction ..................................................................................................................... 191


7.5.1. Materials and chemicals .......................................................................................... 193

7.5.2. Preparation of electro-activated brine solutions .................................................... 193

7.5.3. Thermal processing ................................................................................................. 194

7.5.4. Sterility considerations ............................................................................................ 195

7.5.5. Lethality of heat at a single point ............................................................................ 196

7.5.6. Texture profile analysis (TPA) .................................................................................. 198

7.5.7. Measurement of color ............................................................................................. 199

7.5.8. Vitamin C analysis .................................................................................................... 199

7.5.9. Energy consumption of sterilization ........................................................................ 200

7.5.10. Statistical analysis .................................................................................................... 201

7.6. Results and discussion ..................................................................................................... 201

7.6.1. Texture profile analysis (firmness) .......................................................................... 201

7.6.2. Treatment effect on the color change .................................................................... 204

7.6.3. Effect on the vitamin C ............................................................................................ 209

7.6.4. Analysis of the energy consumption ....................................................................... 211

7.7. Conclusion ....................................................................................................................... 213


General conclusions and perspectives ............................................................................................ 215

References ...................................................................................................................................... 221

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List of tables

Table 1.1: Effect of pH on thermal processing of foods. Represented by (Lange, 1983). ..... 6

Table 1.2: Some typical data of decimal reduction times for bacterial spores relevant to

low-acid canned foods. Adapted from (Ababouch, 1999; Barbosa-Canovas et al., 2004;

Stumbo, 1973). ...................................................................................................................... 11

Table 1.3: Calculation of model process using general method (the reference thermal

parameters are: �121.1°C = 0.21min,Z = 10°C,TD121.110 = 2.52min). Adapted from

(Valentas et al., 1997). .......................................................................................................... 18

Table 1.4: The list of z-value for heat-vulnerable constituents. Adapted from (Holdsworth

and Simpson, 2007). ............................................................................................................. 22

Table 1.5: Main types of fixed charges used in ion-exchange membranes. Adapted from

(Strathmann, 2010b). ............................................................................................................ 53

Table 1.6: Some application of EAS in combination with other treatments ........................ 59

Table 3.1: The power produced by the reactor with different salt concentrations and electric

current in kilowatt per hour. ............................................................................................... 101

Table 4.1: Response functions for corresponding variables of redox potential (E, mV),

minimum resistance (R, Ohm) and pH. .............................................................................. 115

Table 4.2: ANOVA showing the linear and quadratic interactions and the lack of fit of the

response variables for each cell configuration. ................................................................... 118

Table 4.3: Estimated regression coefficients of the fitted second-order polynomial for the

response variables. .............................................................................................................. 119

Table 4.4: Predicted and experimental values of the response variables at optimum

formulation. ......................................................................................................................... 130

Table 5.1: Properties of tested solutions ............................................................................. 140

Table 6.1: The properties of EAS after optimization. ......................................................... 163

Table 6.2: The Log reduction of pure culture of C. sporogenes spores due to the combined

effect of temperature, exposure time and EAS. .................................................................. 168

Table 6.3: The Log reduction of pure culture of G. stearothermophilus spores due to the

combined effect of temperature, exposure time and EAS. ................................................. 170

Table 6.4: Comparison of D values for C. sporogenes spores heated in different

temperatures and different solutions in pea and corn puree. .............................................. 174

Table 6.5: Comparison of D-values for G. stearothermophilus spores heated in different

temperatures and different solutions in pea and corn puree. .............................................. 177

Table 6.6: Destruction kinetics of C. sporogenes and G. stearothermophilus in pea and corn

purees .................................................................................................................................. 185

Table 7.1: Characterization of the fresh and blanched pea and corn grains ....................... 193

Table 7.2: The properties of the brine solutions ................................................................. 194

Table 7.3: The required lethal heat time of products in different brine solutions .............. 196

Table 7.4: Changes in surface color of pea samples ........................................................... 205

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Table 7.5: Changes in surface color of corn samples ......................................................... 208

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List of figures

Figure 1.1: Scanning electron micrograph of C. botulinum flagella. Pictures A and B

correspond to the lengths of the scale equal to 4 µm and 0.4 µm, respectively. Adapted

from (Hanna Skarin and Ljung, 2013). ................................................................................... 5

Figure 1.2: Schematic flowchart for establishing a thermal process. Adapted from (Berry

and Pflug, 2003). ..................................................................................................................... 7

Figure 1.3: Graphical presentation of thermal parameters of �� (A) and Z (B) values.

Represented by (Berry and Pflug, 2003). ............................................................................... 9

Figure 1.4: Model illustration of the heating curves of liquid (black dots) and dense (red

dots) food inside of container. Adapted from (Flaumenbaum et al., 1982). ......................... 15

Figure 1.5: The curves of temperature progression (dotted line) in the geometrical centre of

product (pea in 300x407 cans) and the lethality curve (solid line) during the process (retort

temperature is 121.1°C). Adapted from (Flaumenbaum et al., 1982). ................................. 16

Figure 1.6: Schematic presentation of quality changes during the heat treatment are

displayed by the curves of degree of sterilization (A) and degree of nutrition loss (B).

presented by (Ramesh, 2003). ............................................................................................... 20

Figure 1.7: The schematic presentation of log10time versus temperature of microbial

inactivation (F) and cooking (C) of food product. Adapted from (Holdsworth and Simpson,

2007). .................................................................................................................................... 23

Figure 1.8: Model curve of typical steam consumption. Adapted from (Lopez, 1987) ....... 24

Figure 1.9: Schematic presentation of electrochemical reaction, where Ox and Red are

oxidized and reduced species, respectively. Adapted from (Bazinet, 2004). ....................... 33

Figure 1.10: Schematic presentation of electrochemical cell with diaphragm (ion-exchange

membrane or neutral membrane), where the circled (+) and (-) present anions and cations of

electrolyte, respectively. Adapted from (Zeng and Zhang, 2010). ....................................... 36

Figure 1.11: Possible electrochemical reactions and their compounds which may take place

on the anode (pH~3.5) and cathode (pH~11) with shown standard electrode potentials.

Adapted from (Chaplin, 2014). ............................................................................................. 38

Figure 1.12: The standard hydrogen electrode (Anonymous, 2015). ................................... 41

Figure 1.13: Potential-pH Pourbaix diagram at 25°C for Cl–H2O (1M) system of chlorine

compounds. Adapted from (Radepont et al., 2015). ............................................................. 43

Figure 1.14: Principle of production methods of electrochemical water activation. Presented

by (Gnatko et al., 2011). ....................................................................................................... 46

Figure 1.15: Cylindrical flow electrolyzer with ion-exchange membrane (IEM) where IN

and Out correspond to inlet and outlet of the anolyte (A) and catholyte (C) solutions flows,

respectively. Adapted from (Leonov et al., 1999b). ............................................................. 47

Figure 1.16: Representation of electrochemical reactor. Adapted from (Aït Aissa and Aïder,

2014). .................................................................................................................................... 48

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Figure 1.17: The structure of the cation-exchange membrane (A) and an anion-exchange

membrane (B). Adapted from (Strathmann, 2010a). ........................................................... 50

Figure 1.18: Schematic presentation of the structure of a bipolar membrane. Adapted from

(Strathmann, 2010a). ............................................................................................................ 51

Figure 1.19: Schematic presentation of ion-transport function of cationic membrane with

fixed negative groups excludes negative ions (chloride) but is freely permeable to positively

charged cations (sodium). Adapted from (Baker, 2012). ..................................................... 52

Figure 1.20: The destruction of the cellular protective barriers of C. albicans by EAS

during 0 (A), 1 (B), 5 (C) and 10 (D) min. Adapted from (Zeng et al., 2011). .................... 55

Figure 1.21: Disinfection vs free available chlorine residual.Time scale is for 99.6-100%

kill. Temperature was ranged beween 20-29°C, with pH as indicated. Adapted from (Faust

and Aly, 1998). ..................................................................................................................... 56

Figure 1.22: Scanning electron microscopy (SEM) of E. coli population of cilantro (A) after

treatment by anolyte (B), combination of catholyte and anolyte (C). Adapted from (Hao et

al., 2015) ............................................................................................................................... 58

Figure 3.1: The schematic representation of the electro-activation reactor. AEM: Anion-

exchange membrane. CEM: Cation-exchange membrane. A: Ammeter. V: Voltmeter. DC:

Direct current source. ........................................................................................................... 73

Figure 3.2: Influence of different salt concentrations and current values on pH and ORP of

anolyte in cell configuration #1. ........................................................................................... 78


solution in central cell configuration #1. .............................................................................. 81

Figure 3.4: Mechanisms of OH- and H2 leakages through CEM during the treatments ...... 84


anolyte in cell configuration #2. ........................................................................................... 88


NaHCO3 solutions in middle cell, configuration #2. ........................................................... 89


anolyte in cell configuration #3 ............................................................................................ 91

Figure 3.8: General schematization of the mechanisms of ion transfer through exchange

membranes during the treatments of configuration #4. ........................................................ 93


contactless anolyte in cell configuration #4. ........................................................................ 94

Figure 3.10: Influence of different configuration on pH of catholyte. ................................. 97


catholyte. .............................................................................................................................. 98


catholyte. .............................................................................................................................. 99

Figure 3.13: Influence of different salt concentrations and current values on resistance of

system. ................................................................................................................................ 102

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Figure 4.1: Prototypical electro-activation reactor used for generating solutions. AEM-

anion-exchange membrane. CEM-cation-exchange membrane. A- - anions. C+ - cations.

............................................................................................................................................ 113

Figure 4.2: Response surface plots for the effect of salt concentration vs. current (A), salt

concentration vs. time (B) and current vs. time (C) on the redox potential of anolyte

(configuration #1). .............................................................................................................. 121


concentration vs. time (B) and current vs. time (C) on the pH of anolyte (configuration #1).

............................................................................................................................................ 122


concentration vs. time (B) and current vs. time (C) on the resistance of anolyte

(configuration #1). .............................................................................................................. 123

Figure 4.5: Response surface plots for the effect of concentration of the salt vs. current (A),

concentration of the salt vs. time (B) and current vs. time (C) on the redox potential of

anolyte (configuration#2). .................................................................................................. 125


concentration of the salt vs. time (B) and current vs. time (C) on the pH of anolyte

(configuration #2). .............................................................................................................. 126


concentration of the salt vs. time (B) and current vs. time (C) on the resistance of anolyte

(configuration #2). .............................................................................................................. 127

Figure 4.8: Response optimizer on individual desirability (d_i) of all responses in

correspondence with combined desirability (D), where graph A represent configuration#1,

graph B represent configuration#2. .................................................................................... 129

Figure 5.1: The effect of storage on the change of free residual chlorine (bars) and of redox-

potential (curves). ............................................................................................................... 143

Figure 5.2: Changes in zinc (A), iron (B) and copper (C) concentrations during four weeks

storage with different tested solutions. ............................................................................... 145

Figure 5.3: Changes in zinc (A), iron (B) and copper (C) concentrations during 4 and 52

weeks of storage in canned corn with different tested solutions. ....................................... 150

Figure 6.1: Survivor curves of the spore of C. sporogenes in pea (A, C, E) and corn (B, D,

F) purees with NaCl solution (A, B), at different temperatures ( - 100°C, - 105°C, -

110°C, - 115°C, - 120°C). The reduced temperatures ( - 70°C, - 80°C, - 90°C,

- 100°C, - 105°C) for EA solutions A2 (C, D) and A6 (E, F). The linear curves are

fitted to the first order model. ............................................................................................. 172

Figure 6.2: Survivor curves of the spore of G. stearothermophilus in pea (A, C, E) and corn

(B, D, F) puree, at different temperatures ( - 110°C, - 115°C, - 120°C, - 125°C,

- 130°C) with NaCl solution (A, B) and EA solution A2 (C, D) and A6 (E, F). The linear

curves are fitted to the first order model. ............................................................................ 175

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Figure 6.3: The linear fitted curves of the heat resistance (Log D versus temperature) of C.

sporogenes (A, B) and G. stearothermophilus (C, D) in pea (A, C) and corn (B, D) purees

with different brine solutions. ............................................................................................ 179

Figure 6.4: Electron microscopy of the spores of C. sporogenes and G. stearothermophilus

before (control) and after treatment with EAS and temperature (A6, 60 °C, 15 min). ...... 180

Figure 7.1: The process lethality curves of the heat effecting pea (A, B) and corn (C, D)

cans at different processing temperatures ( - 121°C, - 115°C, - 110°C, - 105°C, -

100°C) as well at different brine solutions : A2 (A, C), A6 (B, D) and NaCl (A, B, C, D;

presented at one temperature - 121°C). ............................................................................ 197

Figure 7.2: The temperature curves of the autoclave used for the difference time profile.

Regimes presented for pea (A) and corn (B) cans at different temperatures for NaCl ( -

121 °C) and EABS ( - 121 °C, - 115 °C, - 110 °C, - 105 °C, - 100 °C). ........ 198

Figure 7.3: Effect of heat treatments on the firmness of canned peas (A) and corn (B) The

grouping information labeled with large letters indicates a significant difference (p � 0.05)

between all EABS; small letters presents the difference (p � 0.05) of one type of EABS

between subgroups. ............................................................................................................ 203

Figure 7.4: The bars groups indicate the effect of heat treatments on the changes of ascorbic

acid of canned peas (A) and corn (B) representing mean ± standard error of mean.The



between subgroups. ............................................................................................................ 210

Figure 7.5: Energy consumption during sterilization of canned pea and corn with EABS

and NaCl at different temperatures. ................................................................................... 213

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List of abbreviations

A: The duration of heating phase toT�, min

AC: alternating current

AEM: Anion-exchange membrane

a∗: CIE color space co-ordinate: degree of greenness/redness

B: Holding phase duration atT�, min

b∗: CIE color space co-ordinate: degree of blueness/yellowness

C: Cooling phase duration, min

CEM: Cation-exchange membrane

C�: Initial number of viable spores

C�: Number of viable spores in the population after treatment time

C�: Specific heat, kJ/kg-1K-1 (3.56 – pea, 4.18 – water, 0.46 – metal containers, cans)

DC: Direct current

D�� : Time, in minutes, to kill 90% of the spore population, min

DO: Dissolved oxygen, mg/L

E: Oxidation-reduction potential, mV

EA: Electro-activation

EABS: Electro-activated brine solutions

EAS: Electro-activated solutions

EAT: Electro-activated technology

F��

" : Equivalent time of all lethal heat in a coldest point of can during the process at T�#$,

with respect to the destruction of organisms characterized by a Z time, in minutes, of heating

at reference temperature with respect to its capacity to destroy the spores, min

I: Electric current, A

IEM: Ion exchange membrane

L: Reciprocal of time at any lethal temperature equivalent to any time period at reference

temperature, min-1

L∗: CIE color space co-ordinate: degree of lightness

': Mass of product, kg (27 cans per 240g)

m(: The mass of ascorbic acid, g

xviii�

m): The mass of grains, g

Q: Thermal energy of heat flux, kJ

Q+: Energy of heating and holding phase, kJ

Q,: Energy for producing the electro-activated solutions, kJ

Q�-: Thermal energy at temperature T., kJ

RC : Total residual chlorine concentrations, mg/L

RSM: Response surface methodology

dt: Time difference, min

T.: Temperature at given moment, °C

T�: Required temperature of retort, °C

T�#$: Reference temperature (normally, the highest lethal temperature), °C

T1: Temperature of time dt, °C

T2: Beginning temperature of heating, °C

T3: Final temperature of heating, °C

T4 : Titer, mg/L

U.: Voltage, V

V2: The volume of 2,6-dichloroindophénol Na used to titer the sample, ml

V3: The volume of total volume of extract solution of the sample (100), ml

V7: The volume of the sample solution after filtration, ml

V8: The volume of 2,6-dichloroindophenol Na used to titer the ascorbic acid standard

solution, ml

V9: The volume of 2,6-dichloroindophenol Na used to titer the acids mixture solution, ml

W.: Power, Watt

Z: Temperature required for the thermal destruction curve to traverse one log cycle, °C.

�

xix�

Dédicaces

Je dédie cette thèse à mes parents

xxi�

Acknowledgments

First of all I would like to express my sincere gratitude to my mentor and teacher,

director Dr. Mohammed Aider. I’m grateful for his aspiring guidance, constructive criticism,

patience and the time spent to give me his valuable advices and encouragements. Exceptional

thanks to my co-supervisor Dr. Steve Labrie, for his inappreciable contribution to my thesis,

whose inextinguishable optimism always revived and inspired me. I have always been

amazed by the knowledge discovered during the meetings. I am especially grateful to my co-

supervisor Dr. Marzouk Benali, for his suggestions contributed to this project and his support.

I would like to thank all people without whom my work would have no chance of

being implemented. I am eternally grateful to Catherine Viel, her exceptional support in

microbiology helped me a lot. In addition big thanks to Marine Béguin for her constant

counselling in laboratory. The best wishes to all Steve’s research team for their inexhaustible

motivation giving the strength. I will never forget the «sweetest» support of Diane Gagnon

as well as her vital advice for my experiments. I would also like to warmly thank to my

interns (Amandine Desby, Pauline Delagarde and Fanny Scheichtele) for their collaboration

and for their young, insatiable enthusiasm.

Here, I want to thank from the bottom of my heart to all of my lovely and close people

who supported and inspired me during these days and nights: Alina Gerzhova, my darling

wife, whose patience has no boundaries, my family in Odessa, Ukraine, who in spite of the

distance has always been in my thoughts and my heart. My endless thanks to all the friends

I met here, in Quebec city, the place that unites people from all over the world: Valerie

Carnovale, Allison Wimont, Ourdia Khabeb, Mayank Pathak, Shyam Suwal, Sergrey

Michaylin, Alexey Kastuchik, Mathieu Persico and many many other, you, guys made my

life happier during my Ph.D. studies.

I do gratefully thank Dr. Alain Doyen for the pre-reading of this thesis.�

A huge thank to all of you...

xxiii�

Preface

This doctoral thesis is comprised of seven chapters where five chapters are presented

in form of scientific articles containing all the results obtained during the realization of the

project. In the first chapter, the literature presents the current state of canning technology,

with followed modern hurdle approaches. The literature review finishes by a description of

the emerging technology of electro-activation and leads to the second chapter of hypothesis

and objectives.

The first article of this thesis is entitled « Ion exchange membrane-assisted electro-

activation of aqueous solutions: Effect of the operating parameters on solutions properties

and system electric resistance» which is published in «Process Safety and Environmental

Protection» in 2015. Authors: Viacheslav Liato, Steve Labrie, Marzouk Benali and

Mohammed Aider.

The second article «Application of response surface methodology for the

optimization of the production of electro-activated solutions in a three-cell reactor » is

published in «Engineering in Agriculture, Environment and Food». Authors: Viacheslav

Liato, Steve Labrie, Marzouk Benali and Mohammed Aider.

The third article « Effect of electro-activated solutions (EAS) on the corrosion of

metallic cans used for food preservation » is pending for submission in «Food Bioscience».

Authors: Viacheslav Liato, Steve Labrie, Marzouk Benali and Mohammed Aider.

The fourth article « Study of the combined effect of electro-activated solutions and

heat treatment on the destruction of spores of Clostridium sporogenes and Geobacillus

stearothermophilus in model solution and vegetable puree» is submitted in «Anaerobe».

Authors: Viacheslav Liato, Catherine Viel, Steve Labrie, Marzouk Benali and Mohammed

Aider.

The fifth article «Production of canned pea and corn by using a hurdle technology

composed of electro-activation and low heat treatment» is in preparation for a submission.

Authors: Viacheslav Liato, Steve Labrie, Marzouk Benali and Mohammed Aider.

1�

Introduction

Canning, as a method for preserving food spoilage, has been known to mankind even

in the earliest stages of its development, when in the struggle for survival it faced with the

need to extend the storage period of the procured or produced foods. In this context, the

simplest methods of food preservation appeared. However, the use of canned food has only

been developed in the beginning of XIX century (Flaumenbaum et al., 1993).

Today commonly understood canned foods are those packed in a sealed container and

subjected to high temperature processing i.e. sterilization, during a specific time-temperature

profile. The principal objective of sterilization is the destruction of foodborne pathogenic and

spoilage bacteria as well as enzymes which promote food product deterioration during

storage. The general idea of food sterilization was born when Nicolas Appert discovered for

the first time, in 1810, the art of canning (Valentas et al., 1997). Nowadays consumers require

not only the production of safe and stable during storage food, but also high quality food

products (Heldman, 2011).

In the production of low-acid canned food, the pathogenic toxins produced by the

anaerobic bacteria Clostridia botulinum are dangerous for human and represents the main

challenge during thermal processing of canned foods. Although the bacterium itself is not

thermostable, its spores are resistant on prolonged boiling. Therefore, the microbiologists by

studying its thermal stability, determined a specific profile between the thermal death time

and the temperature. On the basis of these laws, new opportunities of improving the quality

of products and technology in general arose (Stumbo, 1973).

Today, to provide adequate food safety while minimizing losses in the quality of

products and unnecessary costs during heat treatment, various technological tools and

techniques have been developed. Also, current trends towards products of higher quality

stimulate scientists to seek new ways of improving technology (Sun, 2014). One of the most

effective methods used in canning is hurdle technologies. The heart of the hurdle technology

is the combinative effects of several approaches or techniques in order to reduce the impact

of a single factor. The latest thermal technologies allowed an important reduction in the

duration of heat treatment, whereas the chemical modification of the product helped to

slightly reduce the temperature of heat treatment. Nevertheless, these methods still lead to

2�

undesired losses in food quality and consume a relatively large amount of energy during

sterilization process. A relatively recent technology of EA has proved to be a powerful anti-

bacterial agent and has found wide utilization in the food industry as well as other fields

(Aider et al., 2012). The combined effect of its physicochemical properties allows the

inactivation of a broad spectrum of microorganisms on different food products (Gomez-

Lopez, 2012). The principle of this technology is based on the exposure of diluted salt

solutions to the electric current by changing their properties (pH, redox potential, dissolved

oxygen), which appear to be fatal for microorganisms. The peculiarity of this technology is

its environmental safety and low manufacturing cost (Hung et al., 2008). Given the fact that

the canned food market foresees a growth of consumers in the nearest future, a new approach

to increase the food safety and quality is of utmost importance (IBISWorld, 2014).

This project has been aimed to explore the concept of the production of canned

vegetables using new technological hurdle such as the technology of EA. In addition, the

objective of this Ph.D. thesis was to improve the current knowledge about the phenomenon

of EA and on the basis of scientific capacity to try to explain the interaction mechanism of

electro-activation solutions with the subjects of research which will be presented in this

manuscript.

3�

1. CHAPTER 1: Literature review

1.1. Canning

1.1.1. Fundamentals

1.1.1.1. Emergence

Historically a pioneer in the field of food preservation by heat treatment in the

containers was Nicolas Appert. In 1810, for the first time, he successfully presented a variety

of perishable food products preserved in glass jars by heat-processing (Stumbo, 1965). Later

this method eventually became known as « canning ». Though Appert did not understand

why preserved food prevented the spoilage (Lopez, 1987), his method was initially aimed to

eliminate the use of large quantities of preserving agents (sugar, salt, vinegar, etc.) due to

their negative effect on quality and natural flavour of the food (Berry and Pflug, 2003). Since

1864, Louis Pasteur, working on the action of heat on the destruction of bacteria, discovered

the relationship between canning techniques on a scientific basis (Lopez, 1987).

1.1.1.2. Conception

Today the « canned food means commercially sterile food in hermetically sealed

containers » (Commission, 1993). This definition includes a complex of plural technological

operations. According to the decree of 10 February 1955, canning technology defines the

canned product as a food of vegetable or animal origin where preservation is ensured by the

combination of two main techniques. At first, the product should be packed in the sealed

containers resistant to liquids, gases and microorganisms at temperature below 55°C.

Secondly, the product should be heat treated, or treated by any other method which aim is to

destroy or inhibit completely enzymes, microorganisms and their toxins whose presence or

proliferation could alter the food and make it unsuitable for human consumption (Thomas

and Cheftel, 1963).

1.1.1.3. Principles of heat treatment

Heat treatment in hermetic packaging may involve different processes depending on the

goal. However, it should be noted that heat treatment does not aim to destroy all the

microorganisms in packed product because that will require a very long heating duration

4�

which could affect product quality (Valentas et al., 1997). For microbial control, the heating

process known as pasteurization is used which requires heating the product between 65 to

95°C. This process is designed to kill pathogenic microorganisms and extend product life

under refrigerated storage. In certain cases, a process called sterilization used heat treatment

at temperatures between 100 to 150°C which extends product shelf-life indefinitely at

ambient temperatures (Berry and Pflug, 2003). In order to determine the type of heat

treatment required, it is necessary to know the following factors (Barbosa-Canovas et al.,

2004):

• Type and thermal resistance of microorganisms, spores or enzymes present in the

product;

• pH of product;

• The conditions of heat treatment;

• Thermophysical properties of product and the shape and dimensions of the package;

• The storage conditions of the food product after processing.

Due to the fact that the food is still contaminated by a more or less large variety of

microorganisms, the heat treatment employed should be capable to destroy them and their

spores which may cause food decomposition and food-borne diseases. It should be noted that

the oxygen level in the hermetical containers is kept to a minimal, making it near impossible

for the growth and/or development of mold and/or pathogenic aerobic microorganisms.

However the spores of anaerobic microorganisms could survive during the heat treatment

and be the cause of large economic loss associated with microbial spoilage and illnesses or

even death in humans (Berry and Pflug, 2003).

The biggest challenge of producing sterile low-acid products is the inactivation of

thermally resistant spores of Clostridium botulinum. The micrograph below shows the spore

forming rods of Clostridium botulinum (Fig. 1.1.). The strain of C. botulinum could produce

a most potent biological exotoxin known as botulinum neurotoxin which causes a serious

flaccid paralysis disease if ingested by the consumer (Diao et al., 2014). When the pH of the

product is 4.5, C. botulinum is incapable of producing the toxin and therefore pH level is

taken as a « dividing line » between low acid and medium acidic foods (Valentas et al., 1997).

Currently, a minimal acidity of pH 4.6 for the control of C. botulinum is validated by

5�

stipulated regulations (Austin, 2003). As seen in table 1.1, pH affects the severity of thermal

process (Ababouch, 1999). Thus it becomes important to correctly differentiate between low

acid (pH > 4.6) and medium acid (pH 4.0-4.6) foods.

Figure 1.1: Scanning electron micrograph of C. botulinum flagella. Pictures A and B

correspond to the lengths of the scale equal to 4 µm and 0.4 µm, respectively. Adapted from

(Hanna Skarin and Ljung, 2013).

Another obstacle in the production of low-acid products is the presence of other heat

resistant microorganisms whose endospores can survive to the heat treatment applied to

eliminate the spores of C. botulinum. Thus, bacteria strains such as Geobacillus

stearothermophilus or Clostridium thermosaccolyaticum, are naturally extreme heat resistant

(optimal growing temperature range 50 – 55°C). However if the product is stored at 30°C

they cannot cause spoilage (Valentas et al., 1997). In 1977 the commission of Food and Drug

Administration (FDA) introduced the term “minimum process” for low-acid products. It

defines the scientifically approved specific time and temperature at which the food product

in a hermetically sealed container is heated, before and after packaging, sufficient to

completely destroy microorganisms and pathogens dangerous to humans. Since C. botulinum

is the most dangerous human pathogen in low-acid food, the process requires heating

between 115 to 125 °C which is considered enough to kill them (Lopez, 1987).

�

6�

Table 1.1: Effect of pH on thermal processing of foods. Represented by (Lange, 1983).

�

The product category of medium acidic foods (e.g. tomatoes, tomato juice, fruit jam,

vegetable juice, etc.) requires less severe heat treatment (pasteurization) to achieve

preservation.

1.1.1.4. Establishment of thermal process

A proper and a scientifically validated combination of microbiological and physical aspects

are kept in mind while designing the process of sterilization of canned foods. Figure 1.2

highlights a schematic flowchart of the thermal process employed during

.

7�

Figure 1.2: Schematic flowchart for establishing a thermal process. Adapted from (Berry and Pflug, 2003).

8�

1.1.1.5. Thermobacteriology

Development of a process design starts with the determination of the relationship between

temperature and time required to destroy the potential microbiological contaminants in food.

The branch of food science dealing with this is known as thermobacteriology which helps to

identify the patterns and parameters of thermal destruction and thermal resistance of

microorganisms during the heating process.

1.1.1.5.1. Thermal destruction rate and « survival curve »

Heat treatment causes denaturation of membrane proteins, which destroys enzyme activity

and enzyme-controlled metabolism in microorganisms (Al-Baali and Farid, 2007). Thermal

destruction rate of microorganisms is taken as a first order reaction, indicating that bacterial

death is of a logarithmic order (Bigelow et al., 1920). The rate of destruction of

microorganisms is mentioned as decimal reduction time or « �; ». In the graph �; could be

seen as the time required for a one-log cycle reduction at constant temperature (T). In other

words, it means the time needed to destroy 90% of the microorganisms (to reduce their

numbers by a factor of 10) in a given microbial population. The plot of time and semi-log

coordinates presented below displays the survival curve (Fig. 1.3A). The data of survival

curve is unique for each species of microorganisms, for the medium where it was treated and

for the temperature. Mathematically decimal reduction time may be calculated as:

<= = (?@ − ?B) (DEFG −DEFH)I (Eq. 1.1)

where the values of a and b correspond to the amount of microorganism in the beginning and

in the end of heating at the time J2 and J3, respectively.

Another concept in thermobacteriology is that of «thermal death time» (TDT), which

contradicts the logarithmic death of microorganisms. TDT is the time necessary to kill a given

number of organisms at a specified temperature. By this method, the temperature is kept

constant and the time necessary to kill all cells is determined, while the first one claims that

it is not possible to completely destroy a given microbial population. In practice logarithmic

death of microorganisms is calculated as a residual population with a certain probability of

survival. For example, a heat treated population of 10–8 signifies the probability of one viable

9�

microorganism in a population of 10 8. For example, if TDT is the time required to reduce a

bacterial population from 10 9 to 10– 3, it is the measure of 12D values, more specifically;

TDT = n * �; (Valentas et al., 1997).

Figure 1.3: Graphical presentation of thermal parameters of <= (A) and Z (B) values.

Represented by (Berry and Pflug, 2003).

The TDT concept is important due to the delay in heating (come-up) period during heat

treatment. This effect is usually observed at the beginning and at the end of treatment and it

10�

does not correspond to first order reaction. The factors inducing significant deviations from

logarithmic concept are well studied (Stumbo, 1965), though the most typical curves are:

• activation by heat during germination;

• mixed microflora;

• cluster of cells;

• flocculation during the heat treatment;

• deflocculation during the heat treatment;

• the nature of the culture medium;

• anaerobiosis.

1.1.1.5.2. Thermal resistance

The thermal resistance (Z value) refers to the temperature required for the thermal destruction

curve to traverse one log cycle and it is equal to the reciprocal of the slope of the curve (Fig.

1.3B); Z can be calculated as:

K = (?@ − ?B) (DEF<B − DEF<@)I (Eq. 1.2)

The value of Z could be also interpreted as the temperature increase required to reduce the

treatment time by a factor of ten while having the same lethal effect.

The Z and �; values can be both different for all microbial species (Table 1.2), and also

within the same microbial species (Brown, 2012). In his work, Stumbo highlighted a number

of factors which could influence the thermal resistance of existing population during

sporulation (e.g. temperature, ionic nature of medium, organic compounds, lipids, age,

growth phase) and heat treatment (e.g. acidity and buffer components, water activity,

composition of medium) (Stumbo, 1973). Currently, the D value is accepted as 0.21 minutes

at 121.1 °C in a phosphate buffer. It is interesting to note that the Z value of 10 °C was

generally used for over 80 years (Berry and Pflug, 2003).

11�

Table 1.2: Some typical data of decimal reduction times for bacterial spores relevant to low-

acid canned foods. Adapted from (Ababouch, 1999; Barbosa-Canovas et al., 2004; Stumbo,

1973).

Microorganism Optimal growth

temperature (° C)

<= value,min

Geobacillus stearothermophilus 55 D232.2�L,4-5;Clostridium thermosaccharolyticum 55 D232.2�L,3-4;D22R�L,5;Desulfotomaculum nigrificans 55 D232.2�L, 2-3 ; D22R�L,

13-55;

Clostridium botulinum types A, B and F 37 �232.2�L, 0.1-0.23;

Clostridium sporogenes PA 3679 37 �232.2�L, 0.1-1.5;

Bacillus coagulans 37 �232.2�L, 0.01-0.07;

Clostridium perfringens 37 �22R�L, 0.2-2;

Clostridium botulinum type E 30-35 �S3.3�L, 0.3-3;

1.1.1.5.3. Methods of measuring of thermal values

A lot of methods have been used to measure the thermal destruction and thermal resistance.

They can be technically divided in cans and capillary methods. For example « Thermal death

time tubes » (Bigelow and Esty, 1920), «Unsealed thermal death time tube » (Schmidt, 1950)

or « capillary tube » methods (Stern and Proctor, 1954). All of these methods have some

general and individual advantages and disadvantages. The common disadvantage of capillary

methods is that it tests only on liquid or homogeneous samples. However methods such as

« thermal death time can » (Ravenel, 1939), « tank » (Williams et al., 1937) or

« thermoresistometer » (Stumbo, 1948) can compensate for this disadvantage. Both methods

are mainly conducted using the same devices capable of rapidly heating the sample to a

required temperature, holding it for a desired time and then immediate cooling it to a sublethal

temperature (Berry and Pflug, 2003).

12�

1.1.1.5.4. Unit of lethality

This parameter of lethality is important in order to compare the sterilization capacity. The

total process lethality (conventionally symbol « F » chosen due to unite of one minute

sterilization at 250°F) understands the sum of all lethal effects while food is heated and

cooled during thermal processing , with respect to its capacity to destroy spores or vegetative

cells of a particular organism (Stumbo, 1965). For example, during sterilization, the container

with food couldn’t achieve instantly the required temperature; however at each given moment

presented each degree of heat in sum have a lethal effect on the microorganisms

(Flaumenbaum et al., 1982).

1.1.1.5.4.1. Sterilization value

Sterilization value or lethality (TR) is measured as the equivalent of time (usually displayed

in minutes) of the reference temperature 121.1°C heat treatment and with the Z value being

10°C. The T; value represents the derivative of the value �; (described below) and can be

calculated as:

UV = U=/BV(=X=V) KI (Eq. 1.3)

In this case TR is equal to T; at the reference temperature �R. It should be noted that eq. 1.3

displays the process when the heating and cooling could be implemented instantaneous

(Valentas et al., 1997). In the real process the product is subjected to a gradual change of

temperature and the integration of the lethal effect should be achieved. Thus it is imperative

for the real thermal process to be mathematically confirmed before validation.

As mentioned above the criteria of safety and economic adequacy of process include two

types of bacterial population for low-acid foods. First type of population (C. botulinum) is

the most significant and requires the design of treatment process in accordance with public

health security, known as commercial sterility. Another type (C. sporogenes and

G. stearothermophilus) follows economic viability and profitability of the canning process


13�

1.1.1.5.4.2. Commercial sterility

Based on the probabilistic theory that spores dies at all temperatures (the higher temperature

the higher the death occurs rapidly) it is necessary to establish a very low probability of spore

survival of C. botulinum (Barbosa-Canovas et al., 2004). In this context, the term of

« minimum process » was introduced. This is to say that one spore of C. botulinum in each

1012 containers of low-acid food may survive, assuming that in the beginning of the

sterilization process at least one spore of C. botulinum was present in all containers, in other

words « minimum process » reduces the C. botulinum population to 10-12. Thus, it leads to

the relationship between order of bacterial death and the change in the concentration of

microorganism in foods at beginning and end of process (eq. 1.4) (Valentas et al., 1997). This

process also known as 12D concept for botulinum cook (Sun, 2014).

U= = <= ∗ (DEFB − DEFBVXB@) = B@<= (Eq. 1.4)

Considering the heat parameters of C. botulinum, the minimum process requirement in terms

of its equivalent minutes at 121.1°C is equal to 2.52 minutes (12 * 0.21 = 2.52) (Stumbo,

1973). Canadian Food Inspection Agency requires 3.0 minutes of the lowest sterilizing value

(TR) resulting in a commercially sterile product (CFIA, 2014).

1.1.1.5.4.3. Economical sterility level

The application of 12D process may still result in spoilage due to some mesophilic

(C. sporogenes) or thermophilic (G. stearothermophilus) spores which are more heat

resistant than C. botulinum. However a 12D process employed for C. botulinum may not be

required for the aforementioned bacteria, as it may lead to over processing and excessive loss

of quality. According to the probabilistic theory the accepted reduction in bacterial

population is 10-5 and 10-3 in 1 ml of a given product for mesophilic and thermophilic spores,

respectively. In practice these prescriptions, considering the appropriate heat parameters of

microorganisms, result in 5 and 19 minutes of lethality, for mesophilic and thermophilic

spores, respectively (Stumbo, 1973).

14�

1.1.1.5.5. Thermal process calculation

There are two principal methods for sterilization value calculation of the process needed to

confirm that process is established as adequate. According to the thermal parameters of

referred microorganisms and the data of heat penetration in the container, the methods could

be classified as general and mathematical. Basically the aim of these methods is to achieve

or estimate the sterilizing capacity of heat process. Though they use the same principles, the

methodologies differ considerably.

1.1.1.5.5.1. Heat penetration

Estimation of heat penetration (HP) is important step in process calculation due to the

necessity to detect the temperature of the slowest heating zone in the food container (Ball,

1928). The processes of heating and cooling of the packed products require accurate

laboratory measurements resulting in mathematically complicate interpretations due to

unstable heat transfer (HT) process (Berry and Pflug, 2003). There are three principal ways

of HP: conduction (dense texture product), convection (product with liquid consistency) or

combined convection- and conduction-heated mechanism (heterogeneous composition

product) (Flaumenbaum et al., 1982). Though velocity of HP mostly depends on the shape

of container and the heat exchange properties of container and food it could be forced by

mechanical agitation of product. The HP data collection is usually carried out by

thermocouples (TC) installed in the centre (product with conduction HT) or in the lowest

part, such as top and bottom tiers (product with convection HT) of container (Al-Baali and

Farid, 2007). The difference of the HP intensity between liquid and dense food could be

schematically presented through a the semi-log plot (Fig. 1.4) where the ordinate scale

represents the temperature difference between the heating agent (�Y) and the container (�Z)


15�

Figure 1.4: Model illustration of the heating curves of liquid (black dots) and dense (red

dots) food inside of container. Adapted from (Flaumenbaum et al., 1982).

�

The obtained time-temperature data could be plotted directly on the lethal-paper to estimate

the lethality of process by the general method. For the mathematical method the heat process

is usually analyzed by the use of the parameter functions: time required for straight-line

portion of the heat penetration heating/cooling curve to traverse one log cycle ([\) and

heating lag factor (]) (Stumbo, 1973). The heating curves for product with conduction or

forced conduction HP normally show a straight single-line (Fig. 1.4.) while the product

which may change their thermo-physical properties could display broken-heating curves

(Berry and Pflug, 2003).

It also should be noted that before estimation of heat process, the HP history of product may

be also assessed, which includes consideration of the following (Valentas et al., 1997):

• process technology (processing temperature, time)

• type of heat transfer agent (vapor, water, etc.)

• heat treatment conditions (initial temperature of product, method of loading)

• the type of product (solid, liquid, thermal properties)

• type of container (shape and dimensions)

16�

1.1.1.5.5.2. General method

The classic general method proposed by Bigelow is a graphical procedure where the lethal

effects of time-temperature data are plotted during the heat process (Bigelow and Esty, 1920).

The data of time-temperature relationships codifying from the thermocouple installed in the

container displays the heating-cooling curves during the process (Fig 1.5). The lethal rates

(eq.1.3) present reciprocal number of minutes required to destroy given percentage of

microorganisms at each assigned temperature (T). Author assumed that the inverse value of

the TDT is the indicator of lethality at corresponding temperature and the sum of these values

gives the anticipated TR. In this case, the sterilization value is considered appropriate when it

is equal to one (Valentas et al., 1997). Later Ball improved this method by plotting the curve

of thermal resistance (eq. 1.5.) while previous method traced the hypothetical curve of

thermal destruction only (Ball, 1928).

^ = BV_=X=`abc/K (Eq. 1.5)

Where equation 1.5 displays the ratio of the �;-value at a particular temperature �, to the

�def-value at the reference standard temperature �defsterilization (conventionally 121.1°C)

(Barbosa-Canovas et al., 2004).

Figure 1.5: The curves of temperature progression (dotted line) in the geometrical centre of

product (pea in 300x407 cans) and the lethality curve (solid line) during the process (retort

temperature is 121.1°C). Adapted from (Flaumenbaum et al., 1982).

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0

20

40

60

80

100

120

0 10 20 30

Leth

al r

ate

, m

in

Te

mp

era

ture

, °C

Time, min

17�

In comparison with first method, this one improved on it by plotting of L-curve and the

calculation of area of the shape by fragmentation into time intervals which are equivalent to

the unit of lethal rate (Fig. 1.5.) and by integration of lethal rate multiplied to the time interval

(eq.1.6.),

UVg = h ^i??V (Eq. 1.6)

the summing the values of lethality (eq. 1.7) (Patashnik, 1953).

UV = j ^i??V (Eq. 1.7)

This method using the actual data of time and temperature demonstrated the most accurate

and simplest way to determine sterilization value (TR) during the process (Holdsworth and

Simpson, 2007). An example of calculating TR by the general methods is presented in Table

1.3

�

18�

Table 1.3: Calculation of model process using general method (the reference thermal

parameters are: �[email protected]�k = V. @Blmn,o = BV�k,p�[email protected] = @. q@lmn). Adapted from

(Valentas et al., 1997).

Time during

process,

min

Temperature of

retort,

°C

Temperature inside

container,

°C

TDT

(eq. 1.3.),

min

TDT -1,

min

Lethal rate

(eq. 1.5.),

min

Lethality

(eq. 1.6.),

min

Cumulative

lethality (eq. 1.7),

min

0 25.01 54.76 >10000 0.000 0.00 0.00 0.00

2 46.01 54.89 >10000 0.000 0.00 0.00 0.00

4 77.04 69.62 >10000 0.000 0.00 0.00 0.00

6 114.40 93.64 1670 0.001 0.00 0.00 0.00

8 121.36 107.54 68 0.015 0.04 0.09 0.09

10 121.36 114.10 15 0.066 0.20 0.40 0.49

12 121.36 117.44 7 0.143 0.43 0.86 1.35

14 121.36 118.68 5 0.191 0.57 1.14 2.50

16 121.36 119.54 4 0.233 0.70 1.40 3.89

18 115.89 115.34 11 0.088 0.27 0.53 4.42

20 57.56 95.64 1056 0.001 0.00 0.01 4.42

22 36.54 77.98 >10000 0.000 0.00 0.00 4.42

24 31.53 65.51 >10000 0.000 0.00 0.00 4.42

26 30.53 57.01 >10000 0.000 0.00 0.00 4.42

28 30.53 51.01 >10000 0.000 0.00 0.00 4.42

30 31.03 46.75 >10000 0.000 0.00 0.00 4.42

32 31.53 43.62 >10000 0.000 0.00 0.00 4.42

34 31.53 41.24 >10000 0.000 0.00 0.00 4.42

1.1.1.5.5.3. Mathematical methods

Nowadays, the mathematical methods have found numerous modifications but they are

mainly based on the formula method proposed by Ball (Berry and Pflug, 2003). In these

methods the equations describe the substitution of an empirical model of the temperature

distribution (e.g. heating, holding and cooling phases utilizing data from HP experiments)

and mathematically have their origin in the use of the simple TR integral (eq. 1.8.) (Barbosa-

Canovas et al., 2004).

UV = h BV_=`abX=c/K?V (Eq. 1.8)

These methods could help to vary process temperature and the heating time and also it makes

possible to design TR when the can size is changed using the HP data. However in practice to

19�

calculate TR values by numerical procedures, supplemental tabulated data or high-speed

computing equipment are required. The process evaluation for products for which the heating

curve shows only break the mathematical procedure should be used with a great deal of

caution (Berry and Pflug, 2003; Stumbo, 1965; Valentas et al., 1997). The process evaluation

for products for which the heating curves are calculated based on mathematical procedure;

they should be used with a great deal of caution.

1.1.1.5.6. Process validation

Confirmation of process design following mathematical validation is performed by

subjection of the canned food to microbes by inoculating them with bacterial spores.

Microbiological validation method is required due to frequent inconsistency between the

measurements of physical parameters (HP and TDT) and actual microbial survivability as

measured through biological methods (Berry and Pflug, 2003).

Although the first aim of validated process is destruction of C. botulinum spores (12D or

botulinum cook), the tests are performed on a nonpathogenic surrogate microorganism

(C. sporogenes PA3679) which also has high thermal resistance of �232.2�Z = 1 − 1.5'rs

(Thomas and Cheftel, 1963). An acceptable process should reduce the population of PA3679

by more than 5-log after the process (Flaumenbaum et al., 1982).

Official methods of process validation also involve aseptic opening of processed cans and

content analysis in appropriate mediums at 30-35°C during 14 days for mesophilic

microorganisms and at 55°C during 7 days for thermophilic microorganisms (SC-HC, 2001).

1.1.2. Process challenges

Optimal thermal sterilization of food primarily comprises of attaining a desired degree of

process sterility while ensuring minimization of quality losses and maximization of energy

savings. Process optimization can be reached as a result of a compromise between the strong

temperatures required for bacterial inactivation and the rate of quality destruction (Al-Baali

and Farid, 2007).

20�

1.1.2.1. Product quality retention problem

The vegetable tissue contains carbohydrates, proteins, lipids, vitamins and minerals which

provide energy value to the canned product. However during the sterilization process, due to

the heat, several types of chemical and physico-chemical reactions occur at the same time

which can be favorable; enzyme destruction, cooking, texture softening, and unfavorable;

loss of organoleptic quality, color, texture and flavor, which are often inevitable (Holdsworth

and Simpson, 2007; Larousse, 1991). Schematically, the relation between degree of heating

and degree of quality losses can be represented as in (Fig. 1.6.)

Figure 1.6: Schematic presentation of quality changes during the heat treatment are

displayed by the curves of degree of sterilization (A) and degree of nutrition loss (B).

presented by (Ramesh, 2003).

During sterilization, there is not any significant deterioration of carbohydrates, proteins and

lipids of vegetable foods, however, the reduction in vitamin content and sensorial

characteristics have been observed (Çinar, 2004; Larousse, 1991; Platonova, 2009; Rickman

et al., 2007; Serpen et al., 2007). Furthermore, no specific changes in mineral contents of

canned food have been reported, however the losses detected in the brine solution were

mainly due to the minerals leaching into the brine (Flaumenbaum et al., 1993). It is important

to note that heat treatment could result in an increased starch digestibility as well as

mastication and digestion of fiber (Larousse, 1991), however starch dilution could also cause

turbidity of product (Weier and Stocking, 1949), furthermore, the carbohydrates (sugars)

�%

Time

��

A

B

21�

getting dissolved in the brine could decrease the energy value of the product (Buera et al.,

1987).

One of the most negative outcomes of high temperature process is over-processing of the

canned food which may result in the color changing due to Maillard reaction involving an

interaction between the dissolved amino acids (amine (-NH2) functional groups) and reduced

sugars (carboxylic acid (-COOH) functional groups) (Buera et al., 1987; Nicoli et al., 1999).

This reaction could also further accelerate the degradation of vitamins in the canned product

which are already heat-labile and its losses could vary between 10 to 100% depending on the

vitamin (Finot, 1990; Ryley and Kajda, 1994). The other negative effect of sterilization is

softness of vegetable tissue due to increase in protopectin hydrolysis (Simpson and Halliday,

1941). It was also found that long heating is related with initiation of hydrolysis of cellulose

and leads to reduction in tissue thickness (Weier and Stocking, 1949). The color change of

the green vegetables observed during thermal processing occurs due to the conversion of

chlorophyll to pheophytin, as a result of the magnesium in the tetrapyrrole ring of the

chlorophyll being replaced by hydrogen (Lau et al., 2000; Shin and Bhowmik, 1995). Other

groups responsible for pigmentation in fruits and vegetables, such as carotenoids, responsible

for the yellow, orange, and red colors could degrade and result in loss of nutrients via heat

degradation or leaching (Scott and Eldridge, 2005). It therefore can be said that most of heat

processes generally influence the organoleptic and nutritional quality of canned food.

Interestingly, the destruction of product quality factors due to heat is also a first-order reaction

and uses similar kinetic parameters which are used to describe the first-order chemical

reactions relating microbial death kinetics and time (Al-Baali and Farid, 2007).

1.1.2.1.1. Kinetics of food quality changes

The destruction rate of most reactions occurring in food are frequently represented as a

“logarithmic order of inactivation or destruction” which could be expressed by same

mathematically� –t concept. However, both �Z and tZ values of chemical constituents of

food are higher than those of microorganisms and enzymes (table 1.3) that result in better

retention of nutritional properties (Al-Baali and Farid, 2007; Holdsworth and Simpson,

2007).

22�

Table 1.4: The list of z-value for heat-vulnerable constituents. Adapted from (Holdsworth

and Simpson, 2007).

Heat-vulnerable constituents Approximate value of Z (°C)

Bacterial spores 7 – 12

Vegetative cells 4 – 8

Enzymes 10 – 50

Vitamins 25 – 30

Proteins 15 – 37

Overall sensory factors 25 – 47

Texture and softening 25 – 47

Color degradation 25 – 47

1.1.2.1.2. Cook value

The concept of « cook » value proposes that the amount of heat received by product during

the process and resulting tZ–values of corresponding biochemical reactions could be

represented by u –value equation (eq. 1.9.) which is similar to the T –value equation (eq. 1.8.)

(Mansfield, 1962).

v = h BV_=`abX=c/Kv?V (Eq. 1.9)

Where tZ corresponds to the analogous thermal destruction rate of the t-value for microbial

inactivation (eq. 1.2.).

Although Ball (Ball and Olson, 1957) and Reichert (Reichert, 1977) determined other

methods of quality assessment, this concept was accepted as standard nomenclature for

analyzing the optimum balance between the requirements for microbiological inactivation

and thermal cooking of various canned products (Abbatemarco and Ramaswamy, 1994; Miri

et al., 2008; Rattan and Ramaswamy, 2014; Silva et al., 1993). Graphical assessment of

processing conditions may be determined by lines representing values of T and u at plot of

log time versus process temperature (Fig.1.7.). These graphs are useful for determining the

23�

suitability of various combinations of temperature and time of processing (Holdsworth and

Simpson, 2007).

Figure 1.7: The schematic presentation of wxyBVtime versus temperature of microbial

inactivation (F) and cooking (C) of food product. Adapted from (Holdsworth and Simpson,

2007).

�

1.1.2.2. Energy costs

Sterilization is a very energy-intensive process therefore estimation of the energy costs

includes periodic heating and cooling of packed product, however it may also encompass

procedures of ionizing radiation, extreme pressure and other methods of preservation by

minimal use of heat (Valentas et al., 1997).

To provide the required lethal rate inside of heated container, the plants use as heating

medium the saturated steam which has the most important property of a very high surface

heat-transfer coefficient that make a negligible resistance while heating by surface of the

24�

food container (Holdsworth and Simpson, 2007). Steam fulfills two main objective: make a

temperature change in the product, that is providing a 'heating up' of product and maintains

the product temperature for the required time (Lopez, 1987). In practice, for adequate process

operations, the advised steam line pressures of 100 to 125 psi are used (Downing, 2013). The

steam generates in the water tube boilers by burning of combustibles (natural gas – 37077

kJ/m3; heavy fuel oil – 41770 kJ/L; coal – 32505 kJ/kg, etc.) (Larousse, 1991). The following

steam consumption values are based on actual flow meter measurements which are used in

analysis of the height of peak demands at the beginning when it is necessary to heat up a

retort chamber (Fig. 1.8.). This information can be relatively simply assessed by summing

up all of the peaks demands which occur at given process time, therefore the heaviest

demands usually occur during retort come-up (Downing, 2013).

Figure 1.8: Model curve of typical steam consumption. Adapted from (Lopez, 1987)

As shown in Fig. 1.8., the principal steam demand of batch retorts occurs during the venting,

when the required temperature is attained, the steam flow falls progressively to a relatively

constant value for the rest of the process.

25�

The steam consumption of vegetable canning operation can be about 0.8 kg per kilogram of

product, meat canning requires a higher amount to about 1 kg per kilogram of product. In

one such report, it was seen that for canning 20,000 tons of vegetables about 7 tons/h of steam

(Holdsworth and Simpson, 2007).

1.1.2.3. Steam consumption

For designing an optimum steam system, the steam consumption rate should be established

by calculating the pipe sizes, the control valves and steam traps can be appropriately sized to

give the best possible results. In practice steam demand could be determined by calculation,

measurement or thermal rating methods. The first one involves the application of heat

transfer equations which may have different unknown variables and should be treated with

caution. Measurement method involves determining by direct measurement and provides

relatively accurate data; however, this method is of little use because its application would

be only at a real plant and it would not be particularly useful at the project designing stage.

The last one, thermal rating or design rating method uses the anticipated heat output provided

by the manufacturers in kilowatt (kW), however the steam consumption is normally required

in kilogram per hour (kg/h) which depends on the recommended steam pressure. Thus the

eventual parameter change may significantly depend on the anticipated heat output and the

manufacturer's rating doesn’t always equate to the connected load (Sarco, 2015).

Thus for steam consumption assessment calculation method can be used to display the

amount of heat required to raise the temperature of a product and could be applied to a range

of heat transfer processes. Next equation (eq. 1.10.) presents the original form that can be

used to determine a total amount of heat energy but does not take into account the rate of heat

transfer (Sarco, 2015).

z = {v|i= (Eq. 1.10)

Where } is the supplied quantity of energy (kJ), ~� is the temperature rise of the canned

product (°C), u� is the specific heat of canned product (J kg-1 °C-1) and ' is the mass of the

canned product (kg).

26�

It should be noted that steam flow rate demands accurate calculation with more information

about heat parameters, nevertheless there are two principal types of heat exchange application

which can establish the rates of heat transfer. They can be divided into flow type application

which involves the product being heated by constantly flowing fluid over the heat transfer

surface and non-flow type where the processing is fixed and the product is heated in a vessel

(Barbosa-Canovas et al., 2004). An interesting optimization study for the estimation of

energy of processes was also presented by (Barreiro et al., 1984; Bhowmik and Hayakawa,

1983; Van Loey et al., 1994) and others.

1.1.3. Emerging technologies

Production of low-acid products provides significant challenges to design and choice of

process system. The choice of equipment such as retorts, cookers or autoclaves, for packaged

foods depends on a number of factors and a careless selection may result in an energy overrun

and undesirable effect on the quality of the product (Barbosa-Canovas et al., 2004).

It was shown that there is a necessity in the research of novel process technologies, which

called as emerging technologies. This area presents wide-ranging research but includes two

main trends such as thermal and non-thermal technologies (Bermúdez-Aguirre and Barbosa-

Cánovas, 2011).

1.1.3.1. Thermal technologies

The current consumer trends are pushing for an increased production of high quality, safe

and enhanced shelf-life products which are also easy to use. Aligning with this point of view,

popular liquid food products (e.g. juices) may be treated through a series of heat exchangers

or holding tubes that deliver a high temperature (HTST) or ultra-high temperature (UHT) and

short time that result in better product quality retention (Teixeira, 2011). High-viscosity

products (e.g. beans in pea soup, cream-style corn) packed in large size cylindrical metal

containers (cans) may require agitation, for even heating which is achieved through an

agitation mechanism (Barbosa-Canovas et al., 2004). Introduction of an agitation mechanism

ensures and equitable rate of heat penetration, avoiding problems of overcooking in outside

27�

layer of canned product, while the inside remains undercooked and potentially hazardous

from a microbiological point of view. It is necessary to control the headspace, solid–liquid

ratio and consistency during agitating process (Holdsworth and Simpson, 2007). In the last

few decades, various agitation and rotation mechanisms have been introduced and

investigated, among them the most widespread include; end-over-end agitation (Tucker and

Richardson, 2004), axial rotation (Roberts and Sognefest, 1947), spin cooking

(Anantheswaran and Rao, 1985), ShakaTM retort (Walden and Richardson, 2008), Steritort

(Fernandez et al., 1988) and continuous cookers (Ball and Olson, 1957).

One of the governing factors ensuring product quality, stability and safety is product

packaging. Any food container should be made from nonhazardous and safe packaging

material and should have the capacity to stay intact during the sterilization process and

thereafter during storage (Bukin et al., 2008). Tinplate is the most widely used material for

canned food packaging. Cans are manufactured with a thin layer that ensures higher heat

penetration rate and high stability to vapor pressure during heat treatment. The flip-side of

the thin tin plate being used for caning is that an imbalance of inner and outer pressure caused

by the pressurized steam or sudden change of temperature during the cooling phase, may

compromise the integrity of the container. This particular problem is avoided by counter-

balancing; especially for rigid containers and larger cans (Holdsworth and Simpson, 2007).

One other major and potential disadvantage of tinplate can is the probability of poisoning by

heavy metals (tin, cooper, iron) (Blunden and Wallace, 2003; FAO\WHO, 2002; Platonova,

2009), for avoiding this the internal side is lacquered (Foças, 2003).

For the less robust plastic containers which are unable to withstand the pressure difference,

the additional pressure is achieved by the use of steam–air mixtures or in some other cases

steam is altogether avoided and pressurized hot water is used for processing (Holdsworth and

Simpson, 2007). A very suitable and advantageous alternative to tin plate cans or glass

containers are flexible containers, more commonly referred to as retort pouches. They

provide superior barrier properties for a long shelf life, warranted seal-integrity, toughness,

and puncture resistance and most importantly can withstand the rigors of sterilization. The

28�

technology of flexible containers is perhaps the most important innovation in the food

packaging industry after the metal can (Al-Baali and Farid, 2007).

1.1.3.2. Non-thermal technologies

There is an increase of consumer demand for ready-to-eat minimally processed fresh food

products with extended shelf life. Nowadays, consumers expect products with extra nutrients,

enhanced flavor and texture. This has resulted in the advent of a number of non-thermal

processing technologies (Aguilera et al., 2010).

The term «non-thermal» is used because these processes use pressure, electricity, light and

sound as means of processing the product, affecting the microorganisms and other

components of the food (Bermúdez-Aguirre and Barbosa-Cánovas, 2011). One of the most

widely used non-thermal technologies is high hydrostatic pressure (HHP) which treats the

products at pressure levels up to 700 MPa. HHP was successfully used to inactivate

vegetative forms of microorganisms (Smelt et al., 2001), spores (in combination with

additional treatment) (Heinz and Knorr, 2001). This technology showed a high level of

quality retention including other advantages, when used for foods, such as juices and selected

meat products (Ramaswamy and Shao, 2010; Shao, 2008).

Electrical technology is treating food by using electrodes and passing pulses of high voltage.

This process, called pulsed electric fields, demonstrated the impact on vegetative micro-

organisms to control permeabilization (reversible or irreversible) of biological membranes

(Anon, 2001; Heinz and Knorr, 2001; Wouters et al., 2001). Another technology which uses

ultrasound waves was investigated but found limited application regarding microorganism

inactivation, but has found wide spread application in medicine and biotechnology

(Bermúdez-Aguirre and Barbosa-Cánovas, 2011; Merino et al., 2003). The ultraviolet (UV)

light treatment was found to be effective and has found applications for food manufacturers

due to its ability to significantly inactivate micro-organisms (Peter Zeuthen, 2003). The cold

plasma food treatment is a low temperature process which was found effective and is known

to cause a reduction in microbial population by generating free radicals (Bermúdez-Aguirre

and Barbosa-Cánovas, 2011). Another wide spread and not so new technology involving

29�

application of chemical and biochemical antimicrobial preservatives has been used in food

preservation, especially to preserve, flavor, color and to provide required texture,

furthermore, it is also effective in preventing chemical or biological deterioration of foods

(Sun, 2014). A few of the chemical and biochemical food preservatives include organic acids

(such as acetic, propionic, lactic, sorbic, benzoic, citric, malic, fumaric, tartaric, p-

hydroxybenzoic, long chain fatty phenolic acids and glucono-delta-lactone etc), plant-

derived antimicrobials (alliin, sinigrin, phytoalexins, etc), antimicrobial enzymes (lysozyme,

glucose oxidase, lactoperoxidase, etc), chitin/chitosan, nisin, lactoferrin, ozone, reuterin and

electrolyzed water, also called electro-activated water (Aguilera et al., 2010; Robinson et al.,

2012; Søltoft-Jensen and Hansen, 2005).

A recent and state of the art research pertaining to food preservation against chemical and

microbiological spoilage focused on combining traditional inactivation technologies, here

known as « hurdles » (heat, pH, redox-potential, preservatives etc.), with emerging non-

thermal technologies (Juneja, 2003; Leistner, 2004).

1.1.3.3. Hurdle effect

Emerging preservation methods (e.g. high hydrostatic pressure, high-intensity pulsed electric

fields, etc) showed the most effective results when they are combined with additional hurdles

(Leistner, 1992). An intelligent combination of preservation factors has often been proposed

as the way forward, minimizing adverse effects of individual treatments on the organoleptic

properties while maximizing their (combined) antimicrobial effect on the microorganisms

and their spores (Juneja, 2003; Leistner, 2004). The effect of the combined intervention is

either additive, or synergistic action which could act via different mechanisms on the

microorganisms by disturbing their homoeostasis in several aspects (pH, organic acid

preservatives, reduced water activity, and low/high temperatures) (Gould and Leistner,

2002). The most important aspect of hurdle technology is that the interaction of various

technologies used simultaneously leads to a combined effect which is of a greater magnitude

than the sum of the constraints applied individually (Juneja, 2003).

30�

A number of emerging hurdles combination (also called «hurdle technology») have found

their application in low-acid food production due to high effectiveness against undesirable

microorganisms and quality retention (Peter Zeuthen, 2003). However these types of

products have strict regulations pertaining to safety and spoilage-free shelf life, as per the

data of potential growth of a particular microorganism in a particular type of food.

The disadvantage of these preservation methods is that it can be used only for type of food

with specific composition and under the particular conditions of storage that were strictly

selected (Gould and Leistner, 2002). In addition, the risks in terms of public safety and

foodborne infections still appear (Silva and Gibbs, 2010; Sun, 2014). There is always a

concern about the growth of anaerobic pathogens such as C. botulinum due to the violations

of established process design (Ngadi et al., 2012; Silva and Gibbs, 2010), thus the

sterilization in canning provides the best security level of low-acid foods.

1.1.3.4. Hurdles in low-acid canning

Although nowadays botulism is less prevalent due to the establishment of the strict 12D

process in canned and emerging low-acid food products, still occasional incidences of

botulism are reported annually (Codina, 2006; Peter Zeuthen, 2003; Rocourt et al., 2003).

Process design for canned vegetables has been considerably improved by emerging thermal

technologies (agitation, packing, etc.), however the sterilization regime is still in practice at

high temperatures (Flaumenbaum et al., 1993). Some emerging hurdles, e.g. preservatives,

have resulted in a significant increase in quality retention due to a successful combination of

the hurdle being used with knowledge of product properties. The most common challenge

concerns the retention of green color in canned green vegetables. This problematic was

resolved by scientists by an accurate estimation of the chemical properties involving the

sterilization process and combining it with the product phytomorphology (Blair and Ayres,

1943a; Koca et al., 2007; Malecki, 1978). The studies of tissue histology helped to prevent

the cellular adhesion and softening of canned vegetables by the use of additives (Weier and

Stocking, 1949). Indeed, the application of ascorbic acid in canned food helps to prevent

flavor and color changes during the storage (Bauernfeind, 1953). In addition some chemical

preservatives as hurdles used in food processing can effectively inhibit the growth of

31�

C. botulinum spores, although even this has achieved only a slight reduction in the process

length (Herzberg, 1970). Historically, sodium chloride and sucrose are used at inhibitory

concentrations ranged from 4.8% and 30%, respectively (Flaumenbaum et al., 1993; Lund et

al., 2000). Some experimental polypeptide antibiotics such as neomycin, celiomycin, streptin

and circulin have no effect on inhibition of the outgrowth of C. botulinum. Although subtilin

provided promising inhibition at 4 µg/ml, eventually the strain mutated and acquired

resistance to the antibiotic (Campbell Jr and Winiarski, 1959). The bacteriocin nisin has been

found as effective as subtilin, but it was not effective in the non-acid foods (pH close to 7) as

molecules of nisin becomes more vulnerable to the effect of heat with increase in pH values

(from 3 to 7) (Delves-Broughton, 1990; Denny et al., 1961; Thorpe, 1960). The bacteriostat

feed tylosin (or tylosin lactate) was used as an additive to minimize economic spoilage of

canned foods (Sheneman, 1965). The chemicals as sodium benzoate, nitrite and nitrate were

found effective in combination with sodium chloride and appropriate pH (5.9 – 7.6) (Pivnick

et al., 1967; Tarr, 1953). The study of chlorine and chlorine compounds against C. botulinum

performed by the National Caner’s Association of the United States showed that it had a

significantly sporocidical effect (Ito et al., 1966). Successful tests were also made with EDTA

(ethylene-diamine-tetra-acetic acid), which showed effect on chelating essential cations

responsible to the spore growth (Nygaard et al., 2013). Numerous tests of chemicals in

combination with solutes responsible for changing the water activity (��) showed a

beneficial effect against C. botulinum (e.g. 5 % NaCl ��- 0.975, 6 % KCl ��- 0.974, 5.5 %

NaCOOH ��- 0.971, 22.5 % glucose ��- 0.970) (Lechowich, 1970). Finally, a large scale

of edible plant extracts such as allylisothiocyanate and auxins have also shown promise for

the reduction of spore thermal resistance of food spoilage microorganisms (Stumbo, 1965).

As previously mentioned, hurdles represent a great opportunity for low-acid foods

preservation. The principal mechanism involves using individual hurdles for controlling a

specific physico-chemical property (relative humidity, acidification, water activity),

furthermore the added substance could also change the redox potential of the medium.

However, the modern trends are directed to the development of hurdles for decreasing the

application of high temperature treatments such as sterilization and to inhibit the action of

undesirable microorganisms. Electro-activation (EA) technology has been recently described

32�

as interesting hurdle with high antimicrobial effects. The mechanism of antimicrobial action

of these solutions is still under investigation, however it can be safely said that the effect is

due to the combined action of pH, redox potential and mixed radicals acting as strong but

safe disinfectants (Hung et al., 2008).

1.2. Technology of electro-activation

1.2.1. Origin

The backgrounds of electro-activation (EA) as disinfectant produced by electrolysis was

discovered for over 100 years (Shaposhnik, 1999). The founders of modern EA technology

were russian scientists studied the ways to improve the drilling mud (Prilutsky and Bakhir,

1997; Waters, 2014). Since 90s, the EA as a sub-branch of science found wide application in

medicine, agriculture, industry, water disinfection, disinfection and physics amongst others

(Aider et al., 2012; Anonymous, 1997). In the past few years owing to various technical

improvements and modernization of EA technology, electro-activated water has come to be

known as superoxidized water (Tanaka et al., 1996), strongly acidic electrolyzed water

(Nakamura and Iwasawa, 1997), electrolyzed strong acid aqueous solution (Sekiya et al.,

1997), Sterilox (Shetty et al., 1999), electrolyzed oxidizing water (Venkitanarayanan et al.,

1999), ECASOL (Rogers et al., 2006) and by other names (Hung et al., 2008).

1.2.2. Principles

The principle of EA technology involves subjecting of the aqueous solution to an external

electric field through the immersed oppositly charged electrodes such as anode (positive

charge) and cathode (negative charge) (Leonov et al., 1999b). The electrochemical processes

at the electrodes surfaces generate the electro-activated particles when the direct current is

applied. These particles correspond to an activated («metastable») state of solutions

characterized by an anomalous physico-chemical activity decreasing with time

(«relaxation») (Prilutsky and Bakhir, 1997). Electro-activated solutions (EAS) display

chemical reactivity and resulting in the modification of molecular ionic structures (Thorn et

al., 2012). There are various other processes which occur at the electrode surface under action

of direct current, however the basic phenomenon of EA process may be attributed to

electrolysis reaction (Shaposhnik and Kesore, 1997).

33�

1.2.3. Electrodes process

The electrodes can act as reducers (cathode) or oxidisers (anode) of electrons transferred to

or from electrolyte species in the bulk solution (Bazinet, 2004). The reactions occuring in an

electrochemical cell may involve a series of intermediate stages; firstly, mass transfer from

the bulk solution to the electrodes followed by an electronic transfer at the electrode surface

and subsequently mass transfer form electrode surface to the bulk solution (Fig. 1.9)

(Prokhorova, 1995).

Figure 1.9: Schematic presentation of electrochemical reaction, where Ox and Red are

oxidized and reduced species, respectively. Adapted from (Bazinet, 2004).

34�

In addition, electrode processes are strongly affected by the composition of the electrolyte,

solvent, electrode material, and EA mode (voltage, current density, temperature, etc.) (Hsu,

2003; Prokhorova, 1995).

1.2.3.1. Anode

As mentioned before, the oxidation reactions of anions occur at the anode. If the solution

contains a mixture of anions (e.g. Cl–, Br–, S2–, etc.) based on their electronegativity, the order

in which they would get oxidized at the anode would be: I–, Br–, Cl–, S2– (Prokhorova, 1995).

Oxidation reactions at the anode may occur as follows :

2�l- (aq) – 2� � �l2 (g) (Eq. 1.11)

�

Anions could be also oxygen-containing ions (oxoanions), therefore they are not oxidized

at the anode but the oxygen is liberated from the water molecules as follows:

2H2O (l) – 4� � 4H+ (aq) + O2 (g) (Eq. 1.12)

4OH- (aq) – 4� � 2H2O (l) + O2 (g) (Eq. 1.13)

�

where the symbols (l), (g), (aq) or (s) are showing the states material as liquid, gas, aqueous,

or solid, respectively.

1.2.3.2. Cathode

Reduction reactions occur at the cathode, however for certain metals, it depends on their

reactivity (Greenwood and Earnshaw, 1984). All metals can be divided into three according

to the reactivity series; highly active metals (from Li to Al), moderate activate metals (from

Al to H) and less active metals (after H). The cations of the active metals (all metals before

H) are not reduced on the cathode; instead the hydrogen is liberated from the water according

to the following reactions:

2�2O (l) + 2Na+ (aq) + 2� � 2NaOH (aq) + H2 (g) (Eq. 1.14)

2O�- (aq) + 2� � 2O�- (aq) + �2 (g) (Eq. 1.15)

�

35�

Near and at electrode surface, the local electric field intensity can reach thousands of volts

per centimeter, a prime causative factor for the different electrochemical reactions involving

electrolytes. These reactions involve a series of electron and proton transfers to and from the

electrode surfaces which results in changes of the physico-chemical properties of the solution

(Aider et al., 2012; Gnatko et al., 2011).

1.2.4. Proprieties of EAS

The physico-chemical properties of EAS are subject to possible modifications during

treatement in the electrochemical cell. When direct current is applied, principal modifications

regarding pH, redox potential, and liberation of chemical compounds occur at the interface

of the electrode/electrolyte (Fig. 1.8.) (Prilutsky and Bakhir, 1997).

1.2.4.1. Changes of pH in EAS

As mentioned earlier, the principle of EA technology involves the electrolysis reactions

occurring at electrode interfaces. The electrolysis of water can change the pH change of a

solution. Indeed, oxidation of water at the anode (eq. 1.12) results in liberation of one oxygen

(O2) molecule and four protons (H+) resulting in an appreciable decrease in pH to a value

below 2.5. Simultaneously, the reactions occurring at the cathode interface (eq. 1.15.) lead to

a saturation of hydrogen molecules (H2) and liberation of two hydroxyl ions (O�-) which

increases the pH to a value of about 11 (Hung et al., 2008). In order to obtain desired pH and

to avoid the mixing of both solutions, the electrochemical cell is usually equipped with a

selective ion-exchange membrane (Fig. 1.10.).

36�

Figure 1.10: Schematic presentation of electrochemical cell with diaphragm (ion-exchange

membrane or neutral membrane), where the circled (+) and (-) present anions and cations of

electrolyte, respectively. Adapted from (Zeng and Zhang, 2010).

The electrodes also react with electrolyte ions which could result to a series of complex

reactions generating different chemical compounds, including highly-active oxidizers and

reducers at the anode and cathode respectively (Gnatko et al., 2011).

1.2.4.2. Chemical compounds release during EA treatment

Diluted or moderately concentrated salt solutions (from 0.1 to 120 g/L) are normally used

during EA treatment in order to limit power losses (Prilutsky and Bakhir, 1997). Different

salts may have different redox reactions at electrodes and can produce different solutes which

may consequently react with each other to produce other new transient solutes and/or gases.

The example in Figure 1.11 presents reactions occurring in an aqueous NaCl solution

subjected to EA. A multitude of reactions may take place simultaneously on the electrodes

37�

during the passage of electric current. However it is not the objective of this work to focus

on them and only the principal ones are described. It should be noted that when the applied

voltage in the cell is greater than level required by the reactions, the nature of ensuing

reactions can be even more complex (Chaplin, 2014).

Highly active substances arising from electrolytic water splitting during EA are responsible

for the catalytic properties of EAS. However these substances �2-, �O2, �O2

-, �O�2, �2O2

-,

O�� and their associations cannot exist in any other state of aggregate beyond water

(Belovolova et al., 2006). Electrolytes commonly present in ordinary water also form side

products (ClO�; ClO2�; S2O

2-; SO2-, etc.) (Ignatievich, 2012; Miroshnikov, 2013; Prilutsky

and Bakhir, 1997).

When an electrolyte is dissolved in water, its dissolution is accompanied by a heterolytic

bond rupture, e.g. ionization. The level of ionization depends on the reducing and oxidizing

properties of the solute and water. For example; when HCl is dissolved in water, HCl is the

electron acceptor and H2O is the electron donor, as a result along with HCl getting ionized

��3+ is also formed. The free chlorine anion engages water molecules in a complex

formation via hydrogen bonding: Cl- + 4H2O�[Cl(H2O)4]4- (Leonov et al., 1999b).

The electroactivated solutions (EAS) could temporarily exist in a metastable state possessing

specific physicochemical properties and distinct biological activities, which are decreased

during the relaxation period lasting for days or even weeks (Gnatko et al., 2011). These

reactive transient substances, due to their metastable state, are prone to spontaneous

degradation during the relaxation period; in fact the redox values of EA brine solutions prove

this and have been observed to behave accordingly (Prilutsky and Bakhir, 1997).

38�

Figure 1.11: Possible electrochemical reactions and their compounds which may take place

on the anode (pH~3.5) and cathode (pH~11) with shown standard electrode potentials.

Adapted from (Chaplin, 2014).

39�

Among the properties which are prone to change, a change in the oxido-reduction potential

is the most important parameter in context of EAS (Aider et al., 2012).

1.2.4.3. Redox of EAS

The electrochemical reactions occurring during electrolysis also result in a reduction-

oxidation process (Redox) occuring on the electrodes while the electric energy is transformed

in chemical energy (Weaver, 1988). Electrochemical redox reactions are also known as «half-

reaction» and could be described as follows:

Ox + � � Red (Eq. 1.16)

�

Where the Ox and Red represents the oxidize and reduce forms of the redox couple in bulk

solution. In addition the general half-reaction for reversible half-cell electrode potential (�d)

can be given by the Nernst equation (eq. 1.17.) (Rubinstein, 1995).

�` = �° +�=

�UD�

��

��ai (Eq. 1.17)

Where:

�°is the standard potential at 25 °C and unit activity;

� is the universal gas constant (8.314 J/mol.K);

� is the absolute temperature (Kelvin);

s is the overall number of electrons participating in the reaction

T is the Faraday's constant (96,486 C mol− 1);

��is the activity of the oxidative species;

�Ye� is the activity of the reduce species;

In addition, it should be noted that in an open circuit, the �d equals �° when the activities of

all species (Ox and Red) are equal, however such standard state conditions are often difficult

to achieve in practice. Normally it is physically impossible to measure the potential of an

electrode; therefore it is estimated by measuring difference between the potentials of two

electrodes (Anonymous, 2015).

40�

1.2.4.3.1. Standard electrode potentials

The electrochemical cell is composed of two electrochemical half-cells, thus the sum of two

individual half-cell potentials is equal to the overall cell voltage. For the estimation and

calculations of these values, the reduction potentials and the hydrogen scale are used. The

standard hydrogen electrode (SHE) is arbitrarily defined as zero under standard conditions

(25°C) and could be used for this purpose and is assigned as a standard potential of aqueous

solution (Rubinstein, 1995). This electrode consists of a strip of platinum wire that is in

equilibrium with the aqueous solution containing 1 M of H+/H2, at a pressure of 1 atm.

This electrode consists of a strip of platinum wire that is connected to a Pt surface with an

aqueous solution containing 1 M of H+/H2 at a pressure of 1 atm (Fig. 1.12.). The protons are

reduced or hydrogen molecules are oxidized at the platinum surface (eq. 1.18.). Thus the

SHE is arbitrarily assigned as value �°(H+/H2) equal to 0.00 V for the standard potential.

Another important oxidative/reduced potential couple of electrolysis is �°(�3/�3�) = +

1.23 V (Anonymous, 2015).

@��(��) + @a� ↔ �@(F) (Eq. 1.18)

A schema of a cell with the aqueous SHE as reference and an Ox/Red couple in solvent «S»

is as follows:

Pt | ��(aq) (�� =1); �@(g) (��@=1 atm) | Ox(S) (��); Red (��ai) | Pt (Eq. 1.19)

This equation (eq. 1.12.) demonstrates that SHE acts as an anode while the Ox/Red couple is

the cathode where vertical lines are phase boundaries (Ho et al., 2012).

41�

Figure 1.12: The standard hydrogen electrode (Anonymous, 2015).

Another common reference electrode is the saturated calomel electrode (SCE). A schematic

equation of a cell with the aqueous SCE is as follows:

Pt (s) ∣ Hg2Cl2 (s) ∣ KCl (aq, sat) (Eq. 1.20)

At standard conditions, the potential of the SCE is 0.2415 V versus the SHE, which means

that calomel potential reaction (eq. 1.20.) 0.2415 V must be subtracted from the potential

versus an SCE to obtain the standard electrode potential (Anonymous, 2015).

�F@vD@(�) + @a� → @�F + @vDX(��) (Eq. 1.21)

In practice the reduction potential is a direct measure of the thermodynamic feasibility of an

oxidation–reduction half-reaction. It should also be noted that high reactivity of many species

42�

(e.g., free radicals, oxygen species) participating in electrochemical reactions can lead to

tissue injury and even cell death (Ho et al., 2012).

The peculiarity of EA is its ability to change the chemical composition of the solution with

the formation of various metastable complex compounds such as oxidizing agents, free

radicals, etc. Thus on the anode, the generated anolyte solution is characterized by a high

oxidation potential which is higher than +1200 mV. It is also characterized by the acidic

environment where pH can reach values around 1.5 or less. On the cathode the catholyte is

generated having properties radically opposite to those of the anolyte. Potential and pH of

the catholyte can reach -900 mV and 12, respectively (Marais and Brozel, 1999).

In complex multi-component chemical and biological systems, the redox couples are in

unstable complex combinations and pH values depending on the balance of oxidized and

reduced forms may affect the redox potential (Prilutsky and Bakhir, 1997).

1.2.4.3.2. Potential – pH relation

The redox potential values of EAS are strongly dependent on the pH of the solution and this

relation is established as a redox potential (E) versus pH diagram of Pourbaix (Fig. 1.13).

The diagram clearly shows the existence of thermodynamically stable forms of elements

(ions, molecules, atomic crystals, and metals), in solutions at different pH values and redox

potentials of the medium. Pourbaix diagrams are based on the Nernst equation and standard

redox potentials (Pourbaix, 1974a).

Nature of chemical potential has a great influence on the kind of reaction it favours. Higher

potential always promotes the oxidising behaviour of any system, which can be easily

interpreted from graph (eq. 12, 13). While, lesser potential changes the system nature to

reducing (eq. 13, 14) (Mazza, 2011). It is notable that such reactions depends are possible

only under specific conditions, such as pH, redox potential. Various existing forms normally

do not depend on the concentration of dissolved ions (Morozov et al., 2003).

43�

Figure 1.13: Potential-pH Pourbaix diagram at 25°C for Cl–H2O (1M) system of chlorine

compounds. Adapted from (Radepont et al., 2015).

This diagram shows only the thermodynamically stable forms but does not take into

consideration quasi-stable (metastable) forms. Therefore, it cannot predict the formation of

metastable forms such as the formation of the hypochlorite ion OCl- (Morozov et al., 2003).

Since EA is a relatively new approach in science, the thermodynamics of this process is still

not fully understood (Shirahata et al., 2012).

1.2.4.3.3. Phenomenon of EAS

After the excitation by an external electric field, EAS are transformed into a metastable state

and their reactivity increases significantly in comparison with conventional water. Pastukhov

and Morozov (2000) studied the Raman spectra of infrared radiation of EAS, which reflects

the state of hydrogen bonds in its clusters and their structures (Pastukhov and Morozov,

2000). In this study, the authors compared the spectra of EAS with chemically similar

acidified or alkalized water samples. To investigate the Raman spectra, the probes were taken

from the near-electrode (anode -anolyte and cathode -catholyte) layers (Pastukhov and

Morozov, 2000).

44�

The results showed that the Raman spectra of the EA water were significantly different from

those of chemically acidified water. The addition of sulphuric acid to the water led to the

formation of vibrational bands in the spectrum. Also it was noted that the intensity of

radiation scattering is time dependent. Thus, infrared radiation scattering of the catholyte was

significantly reduced after 24 hours, which was not observed in the anolyte (Pastukhov and

Morozov, 2000).

Infrared absorption spectra of concentrated acids and alkalis showed the same absorption

bands as the Raman spectra of the anolyte and catholyte. This led to the assumption that EAS

had properties similar to concentrated acids and alkalis, even if their concentration was very

low (Plutakhin et al., 2013a).

In addition, electrochemical dissociation of water molecules in the near-electrode layer leads

to the formation of unstable complexes. These complexes are considered as intermediates,

and their vibrational modes can contribute to the scattering in the relevant regions of the

spectrum. Addition of a small amount of sodium sulphate does not affect the Raman spectra

of EA solutions. This observation confirms the metastable state of the near-electrode solution

(Aider et al., 2012; Belovolova et al., 2006; Plutakhin et al., 2013a).

Three principal factors explained the anomalous nature of EAS (Gnatko et al., 2011). Firstly,

the formation of metastable intermediate products of electrolysis, such as hydrogen peroxide

and free radicals, are able to exist in the medium for a long time with untibactericidal

proprietes. Secondly, EA treatement might modify the structural and energetic properties of

electrolytes. Finally the formation and prolonged existence of gaseous microbubbles also

supports the properties exhibited by EAS (Gnatko et al., 2011; Kikuchi et al., 2009; Nisola

et al., 2011; Petrushanko and Lobyshev, 2000; Prilutsky and Bakhir, 1997; Shirahata et al.,

2012; Thorn et al., 2012).

1.2.5. Technical aspects of EA reactor

Electro-activation is performed using aqueous solutions in the electrolysis devices, so-called

reactor, electro-activator or electrolyser. The reactor consists of a pair of electrodes immersed

45�

in electrolyte and connected to an external electric field (DC or AC). The continuity of the

flow of electric current is ensured by positively and negatively charged ions and molecules

of the electrolyte. In the chamber of a reactor, different types of ions continuously migrate

toward the opposite charge. In order to ensure the production of electro-activated aqueous

solutions in a metastable state, it is necessary to keep different kinds of ions in a separation

section by means of non-selective or ion-exchange membrane (Plutakhin et al., 2013a).

1.2.5.1. Electrochemical cell

In general, the devices used to produce EAS differ from each other based on the methods

they utilize for treating aqueous solutions (Fig. 1.14).

46�

Figure 1.14: Principle of production methods of electrochemical water activation. Presented

by (Gnatko et al., 2011).

EA of aqueous solutions is carried out in the electrochemical reactors, i.e. electrolysers, and

their designs vary widely. Over the past 20 years, economic and environmental requirements

in the industry caused a significant increase in scientific research and publications in this

field (Bakhir V.M. et al., 1986.; Bakhir et al., 1995; Hung et al., 2008; Patermarakis and

Fountoukidis, 1990; Plutakhin et al., 2013a).

47�

There is a lack of studies on the relationship between the structural characteristics and process

parameters of the electrolytic process, on the one hand, and functional properties of EA

solutions, on the other hand. Typically, the desirable process parameters are adjusted

empirically. It is important to choose appropriate process parameters such as temperature,

voltage, concentration and flow of the electrolyte which is fed into the reactor, the duration

of the process, the final values of pH, redox, etc (Hsu, 2003). However, it is quite difficult to

standardize the process of electrolysis (Pastukhov and Morozov, 2000).

There are periodic and continuous systems (Kharchenko, 2008; Plutahin G.A., 2005 ) but

machinery operating in the continuous flow mode is the most commonly used (Fig. 1.15).

Figure 1.15: Cylindrical flow electrolyzer with ion-exchange membrane (IEM) where IN

and Out correspond to inlet and outlet of the anolyte (A) and catholyte (C) solutions flows,

respectively. Adapted from (Leonov et al., 1999b).

EA devices operating in a closed mode (batch-type) (Fig. 1.16) are mainly used in

laboratories for the preparation of large amounts of water in experimental studies (Aider et

al., 2012).

48�

Figure 1.16: Representation of electrochemical reactor. Adapted from (Aït Aissa and Aïder,

2014).

EA systems produce EAS with specific physicochemical and biological properties, based on

the objective, they can either preferably be used to produce solutions with oxidizing

properties (anolyte) which are obtained in the anode compartment of the electro-activator.

However, the versatility of the technology allows obtaining the solution with reducing

properties (catholyte) at the same time. In such cases, where the anolyte and catholyte are

produced concurrently, the EA unit consists of two sections (anode and cathode section),

divided by an ion specific membrane. However, there are electro-activation processes which

do not require the separation by the membrane (Kharchenko, 2008). In this case, the electro-

activator consists of two parts, one part with separated anolyte and catholyte, and another

without separation. On the basis of this feature, the system can have reversible electrodes

(with reversible polarity) and irreversible electrodes (Strekalov et al., 2015). In addition, EA

systems with electrochemically inert electrodes (anode and cathode) have been developed

(Yakimenko, 1977).

1.2.5.2. Electrodes

Electrodes are one of the structural elements of the cell. Due to the fact that the highest

voltage is observed at the electrode surface, the electrode material should correspond to the

following parameters (Yakimenko, 1977):

• The material must have good electrical conductivity and selectivity;

49�

• Electrodes must be chemically inert and insoluble in the products of electrolysis to

be suitable for use in the food industry;

• Electrode must have sufficient mechanical strength;

• Arrangement of electrodes in the cell and their shape should provide a uniform

distribution of electric current density;

• It should have a low cost.

�

Chemical resistance of the anode is important for the production of solutions of high quality,

so anticorrosive materials produced from more active metals (platinum, platinum with

addition of iridium and rhodium oxides, titanium coated with Fe3CO4, active RuO2 or porous

layers of a metal oxide catalyst) should be used. Same materials can be used for the cathode

(Aider et al., 2012).

Random-oriented graphite-epoxy matrix composite materials are also used for the

manufacture of the anode and cathode. They represent graphite randomly oriented in an

epoxy matrix. However, chemically inert graphite has some disadvantages such as its

porosity which makes it crumble and accumulate hydrogen and oxygen in the pores, thus

reducing the active surface of electrodes (Plutakhin et al., 2013b). Titanium coated electrodes

are commonly used (Thorn et al., 2012).

1.2.5.3. The ion-exchange membranes

As mentioned before, electrochemical activation of water solutions was performed in non-

flow and flow reactors with and without a porous non-selective membrane (Kharchenko,

2008; Selyunin Elena, 2002). Membranes are used to produce EA solutions with desired pH,

such as acid (anolyte), alkaline (catholyte) or neutral solutions (Plutakhin et al., 2013b).

Ion-exchange membrane (IEM) is a thin (0.17-0.65 mm thickness) sheet that provides a

selective transfer of cations (cation exchange membrane) or anions (anion exchange

membrane) (Shaposhnik, 1999).

50�

There are homogeneous IEM which are entirely made of the ion exchange material and

heterogeneous membranes which apart from the ion exchange material contain the inert filler

to improve the mechanical strength (Shaposhnik, 1999).

Homogeneous membranes are prepared by copolycondensation or copolymerization of the

monomers which provides a uniform distribution of a polymeric material. Heterogeneous

membranes include macroparticles (1-50 micron size) of different polymeric materials. For

example, heterogeneous cation exchange membrane MK-40 is a composite of a cation

exchange resin KU-2, which is a three-dimensional sulfonated polystyrene, and polyethylene

(Fig. 1.17A) (Nikonenko et al., 2010). The heterogeneous anion-exchange membrane MA-

41 is manufactured from AV-17 anion exchanger derived from three-dimensional

polystyrene by introducing the benzyltrimethylammonium ionic group into the polymer

matrix during the synthesis (Fig. 1.17B) (Shaposhnik, 1999).

Figure 1.17: The structure of the cation-exchange membrane (A) and an anion-exchange

membrane (B). Adapted from (Strathmann, 2010a).

It is important to mention that bipolar membranes have specific properties. They consist of

two layers of different membrane materials (often cation and anion exchange) where the

transport of electrical charges is accomplished by protons and hydroxyl ions of the

dissociated water from the interphase layer (Fig 1.18.) (Nikonenko et al., 2010).

51�

Figure 1.18: Schematic presentation of the structure of a bipolar membrane. Adapted from

(Strathmann, 2010a).

�

It is known that in the polymer matrix, atoms are connected by covalent bond whereas ionic

groups are linked by ionic bond. When the ion-exchange membrane swells, water molecules

penetrate between the fixed ions, attached to a matrix and counterions. This leads to a

weakening of the electrostatic interactions between ions and their capability to transport the

charge when electric current is on. As a result ion-exchange membranes turn into unipolar

electrical conductors of the second order, as the cation exchange membrane selectively

transmit cations and anion-exchange membranes selectively transmit anions (Fig. 1.19)

(Shaposhnik, 1999).

52�

Figure 1.19: Schematic presentation of ion-transport function of cationic membrane with

fixed negative groups excludes negative ions (chloride) but is freely permeable to positively

charged cations (sodium). Adapted from (Baker, 2012).

High-molecular weight exchange membranes consist of flexible polymer chains whose

repeating units with aliphatic cyclic, aromatic hydrocarbon moieties or perfluorinated units

contain functional groups (Table 1.5). These functional ionic groups are largely responsible

for the conductivity of the membrane, as well as its selectivity and transport processes

(Nikonenko et al., 2010).

�

The basic requirements of membranes stacked into EA reactors are chemical stability,

hydrophilicity, high porosity, high electrical conductivity and low electrical resistance (Aider

et al., 2012; Middleton et al., 2000; Plutakhin et al., 2013b). The diversity of requirements

and practical applications of membranes determines and ultimately promotes development

of a wide range of membrane materials for different processes (Nikonenko et al., 2010;

Strathmann, 2010a).

53�

Table 1.5: Main types of fixed charges used in ion-exchange membranes. Adapted from

(Strathmann, 2010b). Name and fixed ionic groups

CEM AEM

Sulfonic –SO3- Alkyl-ammonium –NR3

+, – NHR2+,–NH2R

+

Phosphoric – PO32- Alkyl-sulfonium –SR2+

Carboxylic –COO- Alkyl-phosphonium –PR 3+

Arsenic –AsO32- Vinyl-pyridinium –C5H4NH+

1.2.6. Applications of EAS

The range of application of EA includes many diverse technological areas. In the food

industry apart from being used for its disinfectant properties, EA could be applied in order to

improve existing technological and biotechnological processes. A number of research

proposals have been made utilising EA; to improve the consistency of bread and its

nutritional value (Bogatova O.V., 2003; Nabok, 2009; Ponomarev et al., 2011; Ponomareva

and Voropaeva, 2008; Sanin et al., 2005). The activation of whey (Petrovna et al., 2014;

Sprinchan et al., 2011), activation of enzymes (Khrapenkov et al., 2005), protein production

(Koschevoi et al., 1997), lactose electroisomerization (Aider and Gimenez-Vidal, 2012; Aït

Aissa and Aïder, 2013), drinking water disinfection (Bergmann, 2010), green fodder

preservation (Osadchenko Ivan et al., 2009), and starch retrogradation (Xijun et al., 2012)

were studied in relation to EAS. In agricultural field, it was reported to accelerate the

germination of wheat seeds (Osadcenco et al., 2014), and barley (Gennady et al., 2014),

improve grain and vegetable crops yield (Semenenko S.Y., 2013), hop extraction

(Khrapenkov et al., 2004) and in other fields (Aider et al., 2012; Anonymous, 1997; Plutakhin

et al., 2013b).

In the field of medicine EA has has been used for its its antiviral effects (Selkon et al., 1999;

Tagawa et al., 2000), for treatment of various animal diseases (Lee et al., 2004; Tanner and

Tanner, 2000) and also for human beings (Inoue et al., 1997; Leonov et al., 1999b; Sekiya et

al., 1997). It should be noted that EA has successfully been used as a disinfectant of medical

(Marais and Brozel, 1999; Tanaka et al., 1999b, c; Vorobjeva et al., 2004) and technological

equipment and surfaces (Ayebah and Hung, 2005; Park et al., 2002; Walker et al., 2005)

54�

unlike other disinfectants (Bakhir et al., 2003; Granum and Magnussen, 1987; Kiura et al.,

2002; Sakashita et al., 2002; Tanaka et al., 1996). Successful application of EAS as disinfect

and their antimicrobial properties give a stimulus to new discoveries.

1.2.6.1. Mechanism of EAS on the microorganism inactivation

A large number of studies proved that EAS is a powerful disinfectant and may inactivate

various bacteria, fungi, spores and viruses (Hung et al., 2008; Tanaka et al., 1996; Thorn et

al., 2012). However microorganism inactivation mechanism of EAS has not been fully

understood or agreed upon (Aider et al., 2012; Al-Haq and Gómez-López, 2012). Thus, there

are different hypotheses about the mode of action of its disinfecting property. The principal

factors acting against microorganism are pH, redox potential, chlorine and different chemical

metastable species, such as superoxide anion, hydroxyl radical and other (Al-Haq and

Gómez-López, 2012; Prilutsky and Bakhir, 1997). Although there is a number of suggestions

that all of these factors contribute towards disinfection, the metastable species and redox

potential are mostly contributed in the antimicrobial capacity of EAS (Al-Haq et al., 2002).

It well known that favourable redox potential of aerobic microorganism may vary from +200

to +800 mV while for anaerobic microorganisms an optimum range is about of −200 to −400

mV (Best et al., 1985). Thus, redox, as an indicator of its ability to oxidize or reduce, may

disturb the state of microorganisms by extreme value of EAS (Kim et al., 2000b; Liao et al.,

2007). In addition, bacteria generally grow in a pH range of 4–9 therefore the pH also has its

own role in restricting the microbial growth (Al-Haq and Gómez-López, 2012) . For example,

low pH may sensitize the outer membrane of bacterial cells to the entry of HOCl into bacterial

cells and leading to the death (Hung et al., 2008). Figure below (Fig. 1.20) presents the action

of EAS against Candida albicans. Transmission-electron microscopy showed inactivation of

fungi where white and black arrows indicate the destruction of wall/membrane layers and

cytoplasm leakages of fungi, respectively (Zeng et al., 2011).

55�

Figure 1.20: The destruction of the cellular protective barriers of C. albicans by EAS during

0 (A), 1 (B), 5 (C) and 10 (D) min. Adapted from (Zeng et al., 2011).

It is important to remember that all acting factors of EAS are interrelated thereby it is

displayed on the disinfecting capacity (Granum and Magnussen, 1987). As observed in

Fig. 1.13, the low pH of EAS is led to non-equilibrium HOCl, which is a very weak but

effective sanitizer, furthermore it was reported that hypochlorous acid (HOCl) may penetrate

into microbial cell membranes and subsequently exerts its antimicrobial action through the

oxidation of key metabolic systems (Al-Haq et al., 2002; Suzuki et al., 2001). Antimicrobial

action of chlorine has been well studied thereby can give some explanation of inactivation

56�

mechanism, like disruption of protein synthesis, reactions with nucleic acids, purines, and

pyrimidines, etc (Hung et al., 2008). As an example, the figure below (Fig. 1.21.)

demonstrates the antimicrobial capacity of chlorine via pH and concentration on the different

microorganisms. However, it should be noted that EAS could be more effective than chlorine

to inactivate microorganisms per available chlorine concentration, this is to say that with the

same amount of chlorine EAS would perform better (Al-Haq and Gómez-López, 2012).

Figure 1.21: Disinfection vs free available chlorine residual.Time scale is for 99.6-100%

kill. Temperature was ranged beween 20-29°C, with pH as indicated. Adapted from (Faust

and Aly, 1998).

57�

Higher multicellular organisms, including humans, synthesize hypochlorous acid, chlorine-

oxygen and highly metastable hydroperoxide compounds in the cell structures to control

microorganisms and foreign substances (Archakov and Karuzina, 1988; Bakhir et al., 2003).

Bakhir et al. (2003) stated that EAS contain similar mixture of oxidants mentioned above. In

addition, only metastable active substances may prevent the development of microbial

resistance to liquid antimicrobial agents due to spontaneous decay of the former with multiple

and unpredictable (for microorganisms) reactions violating their metabolic processes. Thus

the combination of active ingredients ensures the absence of microbial adaptation to the

biocidal effect of EAS (Bakhir et al., 2003).

1.2.6.2. Application of EAS in foods

In recent years, EAS has gained particular interest as a disinfectant of food due to the

potential to be more effective and inexpensive than traditional antimicrobial agents

demonstrating less adverse impact on the environment as well as no harm to the human (Hung

et al., 2008). In addition, investigations were made on EAS application on various products

and with different methods of treatments. Investigations made on applicability of EAS

demonstrated different effectiveness due to different physiological types of product and their

surfaces (smooth, rough, etc) (Al-Haq and Gómez-López, 2012). As an example, the studies

of microbial inhibition from the cilantro leaves showed significant reduction of E. coli O78

after treatment with EAS. However, the comparison of the inoculated control (Fig. 1.22A)

and anolyte (pH = 2.48 ± 0.07, E = 1127 ± 10 mV, ACC = 68.35 ± 0.53 mg/l), on the figure

Fig. 1.22B, showed that the contamination bacteria still presents on the leaves which

suggesting that the flora was adhered on the surface (Hao et al., 2015). Next step of EAS

improvement found the application in combination with other treatments (or hurdles)

allowing the study of innovative applications (Table 1.6). An example with cilantro clearly

shows that the washing leaves in combination with catholyte (pH = 11.65 ± 0.07, E = - 824

± 5 mV) and anolyte (Fig. 1.22C) gave the best result leading to no detection of any

contamination after treatment (Hao et al., 2015).

58�

Figure 1.22: Scanning electron microscopy (SEM) of E. coli population of cilantro (A) after

treatment by anolyte (B), combination of catholyte and anolyte (C). Adapted from (Hao et

al., 2015)

59�

Table 1.6: Some application of EAS in combination with other treatments

Product

EAS paramters Conditions of treatement

Microbiological indicator Reference pH

E,

mV

ACC,

mg/l Temperature, °C

Experience

Time Additionnal hurdle

Apple 7.76 786 191 20 1-5 (min) Sodium hypochlorite Alicyclobacillus spores (Torlak, 2014)

Alfalfa – – – 50 – Dry-heat treatment E. coli O157:H7, L. monocytogenes (Bari et al., 2009)

Radish – – – 50 – Dry-heat treatment E. coli O157:H7, L. monocytogenes (Bari et al., 2009)

Broccoli – – – 50 – Dry-heat treatment E. coli O157:H7, L. monocytogenes (Bari et al., 2009)

Mung bean seeds – – – 50 – Dry-heat treatment E. coli O157:H7, L. monocytogenes (Bari et al., 2009)

Lettuce 2.6 – 40 4, 20, 50 1 - 5 (min) NaOCl (200 ppm), temperature E. coli O157:H7, L. monocytogenes

(Caballero et al.,

2004)

Lettuce 11.4 – – 4, 20, 50 1 - 5 (min) NaOCl (200 ppm), temperature E. coli O157:H7, L. monocytogenes

(Caballero et al.,

2004)

Lettuce 2.83 29 15 5, 10, 15, 20 (min) 6% acetic acid Listeria monocytogenes

(Casadiego Laíd et

al., 2005)

Cherry tomatoes 6.49 853 34.33 – – Ultrasonication 40khz Total bacterial, mold and yeast (Ding et al., 2015)

Strawberries 6.49 853 34.33 – – Ultrasonication 40khz Total bacterial, mold and yeast (Ding et al., 2015)

Chinese cabbage 5.5 600 22 23 3 (min) Ultrasonication E. coli O157:H7, L. monocytogenes

(Forghani and Oh,

2013)

Lettuce 5.5 600 22 23 3 (min) Ultrasonication E. coli O157:H7, L. monocytogenes

(Forghani and Oh,

2013)

Sesame leaf 5.5 600 22 23 3 (min) Ultrasonication E. coli O157:H7, L. monocytogenes

(Forghani and Oh,

2013)

Spinach 5.5 600 22 23 3 (min) Ultrasonication E. coli O157:H7, L. monocytogenes

(Forghani and Oh,

2013)

Chinese cabbage 5.5 600 22 23 3 (min) Ultrasonication E. coli O157:H7, L. monocytogenes

(Forghani and Oh,

2013)

60�

Lettuce – – – – – Ultrasonication 40khz L. monocytogenes

(Forghani and Oh,

2013)

Sesame leaf – – – – – Ultrasonication 40khz L. monocytogenes

(Forghani and Oh,

2013)

Spinach – – – – – Ultrasonication 40khz L. monocytogenes

(Forghani and Oh,

2013)

Carrots 6.8 – 20 23 4 (d) Mechanical washing Aerobic bacteria counts (Izumi, 1999)

Spinach 6.8 – 21 23 4 (d) Mechanical washing Aerobic bacteria counts (Izumi, 1999)

Cucumber 6.8 – 22 23 4 (d) Mechanical washing Aerobic bacteria counts (Izumi, 1999)

Pepper 6.8 – 23 23 4 (d) Mechanical washing Aerobic bacteria counts (Izumi, 1999)

Potato 6.8 – 24 23 4 (d) Mechanical washing Aerobic bacteria counts (Izumi, 1999)

Radish 6.8 – 25 23 4 (d) Mechanical washing Aerobic bacteria counts (Izumi, 1999)

Date palm 6.99 870 100 20 30 (d) Ozone,uv-c Coliforms, mold, yeast, total mesophilic (Jemni et al., 2014)

Date palm 11.2 -880 1.83 20 30 (d) Ozone,uv-c Coliforms, mold, yeast, total mesophilic (Jemni et al., 2014)

Cilantro 11.6 -824 – – – EAS mixing Total aerobic, coliforming, moulds, yeasts (Hao et al., 2015)

Cilantro 5.85 809 19.85 – – EAS mixing Total aerobic, coliforming, moulds, yeasts (Hao et al., 2015)

Cilantro 2.48 1127 68.35 – – EAS mixing Total aerobic, coliforming, moulds, yeasts (Hao et al., 2015)

Pineapple – – 100 – 0, 10, 30, 60 (min) 108, 400, 700, 1000 khz ultrasonic Aerobic microorganism, Fusarium sp.

(Khayankarn et al.,

2013)


(Khayankarn et al.,

2013)


(Khayankarn et al.,

2013)

Carrot 5.5 – 23 18, 45 – Mechanical washing, temperature Total bacterial, mold, yeast (Koide et al., 2011)

Brown rice 3 1024 25 25 4 (d) EAS mixing, ultrasonication (40 khz 30 min) Total aerobic count (Liu et al., 2013)

Brown rice 7 860 25 25 4 (d) EAS mixing, ultrasonication (40 khz 30 min) Total aerobic count (Liu et al., 2013)

Brown rice 11.9 -845 – 25 4 (d) EAS mixing, ultrasonication (40 khz 30 min) Total aerobic count (Liu et al., 2013)

61�

Broccoli 7 900 100 10 14

Peroxyacetic acid, uv light, uperatmospheric),

MAP, NaClO E. coli O157:H7, S. enteritidis

(Martínez-

Hernández et al.,

2015)

Lettuce 2.5 1100 45 4, 22 1, 3 (min) Hydrochloric acid (45 ppm) with different pH E. coli O157:H7, L. monocytogenes (Park et al., 2001)

Cereal grains 2.4 1150 – 25, 30, 40, 50, 60 3, 6 (h) 1% citric acid, temperatures B. cereus (cells and spores) (Park et al., 2009)

Cereal grains 11 -795 – 25, 30, 40, 50, 60 3, 6 (h) 1% citric acid, temperatures B. cereus (cells and spores) (Park et al., 2009)

Lettuce 6.2 520 5

4, 15, 23, 35, 50 1, 3, 5, 7, 10 (min) Mechanical washing, temperatures

L. monocytogenes, S. typhimurium, E. coli

O157:H9

(Rahman et al.,

2010b)

Lettuce 2.54 1120 50

4, 15, 23, 35, 50 1, 3, 5, 7, 10 (min) Mechanical washing, temperatures

L. monocytogenes, S. typhimurium, E. coli

O157:H9

(Rahman et al.,

2010b)

Cabbage 2.4 1100 60 1, 20, 40, 50 3, 5, 10 (min) 1% citric acid, temperatures E. coli O157:H7, L. monocytogenes

(Rahman et al.,

2010b)

Cabbage 11.2 -850 – 1, 20, 40, 50 3, 5, 10 (min) 1% citric acid, temperatures E. coli O157:H7, L. monocytogenes

(Rahman et al.,

2010b)

Lettuce 2.6 1100 60

1, 20, 40, 50 1, 3, 5 (min) Mechanical washing, temperatures E. coli O157:H7, L. monocytogenes

(Rahman et al.,

2011)

Carrot 11.2 -850 –

1, 20, 40, 50 1, 3, 5 (min) Mechanical washing, temperatures E. coli O157:H7, L. monocytogenes

(Rahman et al.,

2011)

Growth medium – – 80 10, 12.5, 15 16 (d) MAP, chloride dioxide, lactic acid E. coli O157:H7 (Smigic et al., 2009)

Alfa seeds 2.54 1073 – – – MAP Salmonella enterica

(Stan and Daeschel,

2003)

Apple 7.76 786 191 20 1, 5 (min) Sodium hypochlorite Alicyclobacillus spores (Torlak, 2014)

E - Redox potential

ACC - available chlorine concentration

MAP - modified atmosphere packaging

62�

Nevertheless problems of canning technology such as microbial spoilage, nutrient

degradation and damage to sensorial attributes after processing still exists and the main

challenge of food scientists is not only to search for solutions, but also to explore new avenues

in order to create innovative and high quality products at an affordable price and with

extended shelf- life (Bermúdez-Aguirre and Barbosa-Cánovas, 2011).

63�

2. CHAPTER 2: Problematic, research hypothesis and objectives

2.1. Problematic

The highlighted information about the food preservation and especially vegetable canning

evidently demonstrates that the heat sterilization methodology as currently used has much

room for improvement, as it has detrimental effects on nutritional value and organoleptic

properties of canned food. Therefore, in order to optimise emerging food preservation

technology, substantial research is required to confirm that new process reduces the negative

effects of conventional sterilization. Hurdle technology is a promising approach to decrease

food spoilage by using various barriers together providing the microbial safety and stability

as well as the product quality and the economic viability. In this context, the application of

electro-activation, as an additional hurdle in the treatment of canned vegetables, combined

with moderate heat treatments could be an effective solution for the production of canned

vegetables with increased and sustained nutritional and organoleptic quality, simultaneously

reducing the temperature of the sterilization regime.

2.2. Research hypothesis

The combination of electro-activated solutions as hurdle with conventional canned

sterilization would preserve the maximum organoleptic and nutritional quality as well as

reduce the energy costs by using moderate heat treatment.

2.3. Main objective

The main goal of this project is to study and understand the possibility of reducing the

maximum temperature and duration of heat treatment currently used for sterilization of

canned peas and corn. Furthermore, the developed method must ensure microbiological

safety of the canned food and preserve the nutrional and the organoleptic properties while

minimizing energy costs.

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2.4. Specific objectives

In order to achieve the main objective and verify the research hypothesis, this study has been

performed by specific objectives:

1. To study different production methods of electro-activated solutions in a three cells-

compartment of electro-activation reactor in order to assess and modulate their

proprieties.

2. To evaluate the interaction of electro-activated solutions with tinplate cans regarding

to its corrosive activity and the assessment of the properties during the time of

relaxation.

3. To investigate the behavior of undesirable food spoilage spores of thermo-resistant

Clostridium sporogenes and Geobacillus stearothermophilus using the hurdles

approaches of electro-activation and with moderate heat treatments and sterilization.

4. To assess the quality changes of canned vegetables (pea and corn) during the

sterilization at moderate temperatures with electro-activated solutions as well to

evaluate the energy costs after integration of hurdle approach in conventional canning

sterilization.

65

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3. CHAPTER 3: Ion exchange membrane-assisted electro-activation of

aqueous solutions: Effect of the operating parameters on solutions

properties and system electric resistance

Viacheslav Liato1,2, Steve Labrie1,2, Marzouk Benali3, Mohammed Aïder1,4

1) Institute of Nutrition and Functional Foods (INAF), Université Laval, Quebec, QC,

G1V 0A6, Canada.

2) Department of Food Sciences, Université Laval, Quebec, QC, G1V 0A6, Canada.

3) Natural Resources Canada/CanmetENERGY, 1615 Lionel-Boulet Blvd., P.O. Box

4800, Varennes, QC, Canada, J3X 1S6.

4) Department of Soil Sciences and Agri-Food Engineering, Université Laval, Quebec,

QC, G1V 0A6, Canada.

This article is published in Process Safety and Environmental Protection.

The authors are: Viacheslav Liato (The Ph. D. candidate: planning and performing of the

experiments, results analyzing and manuscript writing), Steve Labrie (Co-supervisor of

thesis: scientific supervision of the student, correction and revision of the manuscript),

Marzouk Benali (Scientific collaborator of the project: correction and revision of the

manuscript) and Mohammed Aïder (Supervisor of thesis: scientific supervision of the

student, correction and revision of the manuscript).

66�

3.1. Contextual transitions

The literature review showed the main problems and challenges of canning technology, as

well as modern ways of improving the situation. EA of solutions (EAS), as an effective

antibacterial method was proposed for improving the canning technology. Although there is

an ample scope of scientific works about production and optimization of EA, the literature

review failed to provide with the information about the physicochemical changes of the

solutions during EA treatment (EAT). In addition there is no information about EAT in three

compartment electro-chemical cell. Thus, the goal of this work was to study the influence of

various factors (time, current intensity, cell configuration, type of salt and it concentration)

on the physicochemical properties of produced EAS.

67

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3.2. Résumé

Les propriétés des solutions électro-activées (SEA) ainsi que les changements de leurs

propriétés dynamiques sont décrites dans le présent travail en utilisant des solutions aqueuses

de NaCl et NaHCO3. Les concentrations de sels suivantes : 0,5, 0,25 0,125 et 0,05 M ont été

évaluées sous l’apport externe de différentes valeurs de densité électrique : 25, 37,5 et 50

A/m2. Le réacteur d’électro-activation est composé de trois compartiments assemblés et

séparés par des membranes échangeuses d’anions et de cations (MEA) et (MEC),

respectivement. Quatre différentes configurations en termes de positionnement des

membranes échangeuses d’ions ont été choisies pour cette étude. Les résultats obtenus ont

montré l’effet des paramètres étudiés sur les principales propriétés des SEA; à savoir le pH

et le potentiel d’oxydoréduction (POR). Des solutions électro-activées ont été générées dans

le compartiment anodique avec des valeurs de pH de 3, 3,5 et 4 ainsi qu’un POR de + 1100

± 15 mV en utilisant des solutions de NaCl comme électrolytes. De plus, dans cette étude,

plusieurs solutions électro-activées caractérisées par des valeurs élevées de POR (+921 ± 12

mV) et un pH neutre (6,48 ± 0.05) ont été générées dans le compartiment anodique lorsque

des solutions de carbonate de sodium sont utilisées. Dans le même ordre d’idée, deux

solutions, l’une acide (pH de 2,14 ± 0,14) et l’autre alcaline (pH avoisinant 10,46 ± 0,03)

caractérisées par un POR de + 689 ± 10 et 110 ± 21 mV, respectivement, ont été obtenues

dans le compartiment central de la cellule et sont considérées comme des solutions électro-

activées sans contact direct avec le processus d’électro-activation.

68�

3.3. Abstract

The properties of electro-activated (EA) aqueous solutions as well as the dynamics of their

changes were considered in the current study using aqueous solutions of NaCl and NaHCO3.

The concentrations of the salt solutions were 0.5, 0.25 0.125 and 0.05 . The tests were

performed at the DC current densities of 25, 37.5, and 50 A/m2. The electro-activation reactor

consisted of three individual cells assembled together and separated by anion-exchange

(AEM) and cation-exchange (CEM) membranes. During the experiments, four

configurations of the membrane placements and solutions concentrations were studied. The

obtained results showed the dynamics of the electro-activation process that allows obtaining

electro-activated solutions with targeted properties such as pH and oxydo-reduction potential

(ORP). It was possible to obtain electro-activated solutions at the anodic side (acid anolyte)

with pH of 3.0, 3.5, and 4.0 and ORP of +1100 ± 15 mV when NaCl solution was used as

electrolyte. Furthermore, several types of electro-activated solutions with high redox

potential (ORP = +921 ± 12 mV) and neutral pH (6.48 ± 0.05) were obtained on the anode

side when sodium carbonate was used. At the same time, two types of solutions, one with

acid pH (2.14 ± 0.14) and the other one with alkaline pH (10.46 ± 0.03) with ORP = +689 ±

10 and 110 ± 21 mV, respectively, were obtained in the central compartment which

considered as electro-activated solutions obtained by means of noncontact electro-activation.

69

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3.4. Introduction

Electro-activated aqueous solutions (EAS) can be generated by the electrolysis of a dilute

salt solution in an electro-activation reactor by direct or indirect contact with electrode

surface. Such contact with the anode surface results in strong acidic electro-activated solution

which is known as anolyte. This solution is generally characterised by a high oxidoreduction

potential (ORP) which can exceed values of +1000 mV (Al-Haq et al., 2002). At the same

time, a contact between dilute salt solution with the cathode surface of the electro-activation

reactor yields alkaline electro-activated solution. This solution is characterised by alkaline

pH and negative ORP which can reach such values as -900 mV. This electro-activated

solution is generally known as catholyte. Other electro-activated solutions can also be

obtained without direct contact with the electrodes. The use of ion exchange membranes in

the stack of an electro-activation reactor gives the possibility to generate acidic (anolyte) and

alkaline (catholyte) directly in the anodic and cathodic sides, respectively, as well as electro-

activated solutions in the compartments separated from the anode and cathode by means of

anion and/or cation exchange membranes (Aider et al., 2012).

Electro-activated solutions have been introduced to the food industry as a strong and

promising disinfecting agent. Several research works have reported that EAS are effective to

reduce or eliminate foodborne pathogens in different food materials. Acidic electro-activated

solution can be used against different pathogens such as Clostridiums. The main advantage

of such electro-activated solution obtained on the anodic side of an electro-activation reactor

is its highly acidic pH and extremely high ORP (Prilutsky and Bakhir, 1997). Moreover, the

anolyte is also well saturated with oxygen; a factor which is extremely no favourable for

Clostridium growth (Rahman et al., 2010a). Thus, this solution can be a valuable component

in the canned foods. Acidic electro-activated solution can be obtained as both strong and

slightly acidic. Slightly acidic electro-activated water has been permitted as a food additive

in Japan since 2002 because it has been proven as a biologically safe and effective

bactericide. However, although several studies have been carried out on the application of

electro-activated (electrolysed) water and aqueous solutions, there is a serious lack of specific

scientific data on the methods of EAS production and their properties under different

conditions (Zeng et al., 2011).

70�

Electro-activation of dilute salt solutions can be attributed to the category of emerging

technologies and can be considered as a reagentless technology. This is because such an

innovative approach makes it possible to obtain acidic and alkaline media without acid or

base addition. These chemicals are auto-generated by the electro-activation reactor itself.

Moreover, the use of appropriate ion exchange membranes allows the modulation of the

electro-chemical and physico-chemical properties of these solutions. Electro-activation as a

method of treatment is widely spread in various scientific and industrial fields (Aider et al.,

2012). The particular attention to such a reagentless technology has been paid in view of

numerous studies reviewed by (Gnatko et al., 2011). One of the main features of an electro-

activated water or aqueous solution is its metastable state which makes it highly reactive and

useful in physicochemical and biological reactions. The modified redox potential and critical

pH of the electro-activated solutions are responsible for its metastable state (Aider et al.,

2012). The redox potential (ORP) and pH are the main characteristics of these solutions. The

intensity of the solution ORP is directly dependent of the concentration of oxidative and

reductive forms of different ionic species. Changes in ORP might provide the information

about the speed and the direction of chemical reactions during the contact between EAS and

work samples (Hsu and Kao, 2004).

Technically, electro-activated solutions are aqueous solutions with modified

physicochemical properties and they are obtained under the influence of an external electric

field which is applied to this solution in an electro-activated reactor with given geometry and

configuration. The process takes place in the reactor of which one side is anodic while the

other one is cathodic. Due to applied electric field, the oxido-reduction processes on the

anode and cathode sides take place with intensities which depend on the electric current

density, membrane types used to separate the compartments of the reactor and other

parameters such as salt concentration, temperature and intensity of agitation. These results in

changes of pH of the solution, oxidizing reactions take place at the anode side with a decrease

of the solution pH in this section. Moreover, during an anodic electrochemical treatment, a

slight decrease in the solution surface tension is observed while its electro- conductivity

grows along with the amount of solubilized oxygen and chlorine. The amount of hydrogen

and nitrogen decreases and the water structure changes. Electro-reduction reactions on the

71

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cathode side cause an increase of the solution pH and a decrease of the ORP (Brewer and

Begum, 2003). The processes can be controlled by using appropriate ion exchange

membranes and cell configurations of the reactor, the factor which permits obtaining desired

pH and ORP of the solution (Aider and Gimenez-Vidal, 2012). By varying the DC electric

current applied to the electrolyser, it is possible to modulate other characteristics of the EA

aqueous solution on the cathode side such as the modification of the solution surface tension

and the amount of solubilised oxygen. The concentration of the hydrogen and free hydroxyl

groups is increased on the cathodic side. At the same time, changes of the hydration shells of

the ions can also be noticed. The formation of poorly soluble calcium carbonate and

magnesium as well as almost insoluble hydroxides of iron and other metals occur under

alkaline conditions. This factor must be seriously considered when catholyte from aqueous

solutions of bivalent electrolytes is to be obtained (Aider et al., 2012).

Thus, the aim of the present work was to study the possibility of obtaining electro-activated

aqueous solutions (anolyte and catholyte) in an electro-activation reactor built with anion and

cation exchange membranes in order to modulate the solutions pH, ORP and soluble oxygen.

The dynamics of the changes in the electro-activated solutions properties as a function of the

storage time were also studied. The study on the effect of storage time is justified by the lack

of data on the characteristics of these solutions as affected by the relaxation time. Also, the

present work was aimed to understand the processes conditions of obtaining EA solutions

under the best economic conditions.

3.5. Materials and methods

3.5.1. Chemicals

The aqueous solutions were prepared by dissolution of the NaCl and NaHCO3 crystals in 1

L of distilled water to obtain final molar concentrations from 0.05-0.5 mole/L. All chemicals

were of analytical/HPLC grade.

3.5.2. Ion exchange membranes

New and non-prepared for direct use anion (AM-40) and cation (CM-40) exchange

membranes were purchased from the Publicly Traded Company Schekina-Azot (Shchekina,

72�

Russian Federation). Before use, the anion (AM-40) and cation (CM-40) membranes were

gently wiped with 96%-ethanol to induce their activation. Then the membranes were soaked

in different NaCl solutions in 3 steps as follows: First, the membranes were soaked in a

320 g L−1 NaCl solution (saturated solution) for 24 h. After that, they were washed with

distilled water and soaked in a 160 g L−1 NaCl solution for another 24 h. Then, they were

again washed with distilled water and soaked in an 80 g L−1 NaCl solution for 24 h. Finally,

the membranes were stored in a diluted NaCl solution (40 g L−1) until use.

3.5.3. Electrodes

To carry out the experiments for electro-activation of aqueous solutions and to avoid

corrosion of the metallic electrodes, Ruthenium-Iridium coated titanium (RuO2–IrO2–TiO2)

anode and cathode were used (Qixin Titanium Co. Ltd, Baoji, Shaanxi, China). Indeed,

RuO2–IrO2–TiO2 anode is an ideal anti-fouling electrode and dimensionally stable material

against corrosion in the electrolysis processes of brine solutions.

3.5.4. Electro-activation reactor design

The reactor for the electro-activation of aqueous salt solutions is shown in Fig. 3.1 A, B. It

is composed of three Plexiglas columns with the dimensions of L50xW50xH120 mm.

Plexiglas was used to avoid any interference between the electric current and the reactor. The

electro-activation reactor is composed of anodic, central and cathodic compartments. The

central compartment is separated from the anodic and cathodic compartments by means of

anion and cation exchange membrane, respectively. This configuration was chosen to keep

the H+ ions in the anodic side while the OH- ions were kept in the cathodic compartment. The

three compartments have a perforation of 24 mm in diameter and 10 mm in depth. The central

compartment has two perforations where the anion and cation exchange membranes were

applied from anodic and cathodic sides, respectively. The RuO2–IrO2–TiO2 electrodes (anode

and cathode) with an active surface area of 40 cm2 were introduced into the reactor so as to

give a working active area of 40 cm2 (Qixin Titanium Co. Ltd, Baoji, Shaanxi, China). They

were installed at each side of the extreme chambers of the reactor. The anodic compartment

was connected to the positive side of a direct electric current power source (Lambda, GR.260,

Electronics Corp. Melville, NY, USA), whereas the cathode one is connected to the negative

73

�

side. The values of the applied voltage and the intensity of the electric current were measured

using a digital Volt-Ohm-Milliammeter (Keithley, Integra Series, Cleveland, Ohio, USA)

connected to the power source. All three compartments were stirred mechanically.

Figure 3.1: The schematic representation of the electro-activation reactor. AEM: Anion-

exchange membrane. CEM: Cation-exchange membrane. A: Ammeter. V: Voltmeter. DC:

Direct current source.

�

3.5.5. Measurements of chemical characteristics

The pH and dissolved oxygen (DO) of treated solutions were measured immediately after

treatment with a pH-DO meter (Model SR 601 C SympHony, VWR Scientific Products,

74�

USA). The oxidoreduction potential (ORP) was measured with an ORP-meter (Eco Sense

ORP15A). ZoBell’s standard solution (Hach Company, Loveland, CO, USA) was used for

the calibration of the ORP-meter.

3.5.6. Configuration of the electro-activation reactor

The electro-activation of the studied aqueous solutions was carried out as a function of the

configuration (design, Fig. 3.1: B) of the electro-activation reactor as follows:

Configuration #1

In this configuration, the electro-activation reactor consisted of 3 cells in which the central

compartment was separated from the anodic side by an anion exchange membrane (AEM)

and from the cathodic side by a cation exchange membrane (CEM). The used NaCl

concentrations were 0.5, 0.25, 0.125, 0.05 M in all compartments during each test.

Subsequently, NaHCO3 salt solution was treated in the same configuration.

Configuration #2

The membrane disposition was the same as in the configuration #1 but in the anodic and

central cells, the solutions of NaHCO3 of the similar molar concentrations were used. In the

cathode compartment, the NaCl solution with the concentration of 0.1 M was used for all the

tests.

Configuration # 3

The same disposition of the membranes and cells as in previous tests but 0.1 M NaCl solution

was put into the central and cathodic cells. At the same time, in the anode cell the solutions

of NaHCO3 with molar concentrations of 0.5, 0.25, 0.125, 0.05 M were used.

Configuration # 4

In this case, the AEM was placed in the side of the cathodic section while the CEM was

placed in the anodic side of the electro-activation reactor. All the compartments contained

NaHCO3 solutions with the molar concentrations of 0.5, 0.25, 0.125, 0.05 M.

75

�

3.5.7. Protocol of electro-activation

The electro-activation reactor was designed as an electrochemical system for synthesizing of

electro-activated aqueous solutions; namely the anolyte and catholyte. During the treatment,

it was necessary that each micro-volume of the treated aqueous solution will be sufficiently

exposed to the surface of the electrode. As a result of such a contact, non-equilibrium changes

of the water structure and consequent enrichment of such solution with the products of electro

chemical reactions take place. To determine the factors which influence the intensity of the

output parameters such as pH, ORP and dissolved oxygen of the EA solutions, measurements

were carried out as a function of time, current density, type and molarity of the used

electrolyte. Specifically, the influence of NaCl and NaHCO3 aqueous solutions with different

concentrations and combinations was determined. The changes of the output voltage were

also measured in order to estimate the power consumption of the electro-activation reactor.

At time zero, a volume of 300 mL of the studied aqueous solution was put into the reactor.

Taking into consideration that the galvanic mode was used as a source of electricity the

following current values were used 100, 150 and 200 m corresponding to 2.5, 3.75 and 5

mA/cm2 of the electrode. At each time interval of 5 min, 10 mL of a solution were collected

from each of the three compartments for measurements. After each measurement, they were

returned back to the reactor so as to keep the volume unchanged. All the measurements were

carried out at 23 ± 1 °C for total experiment duration of 60 min.

3.5.8. Statistical analysis

A full factorial experimental design was used; each experiment was carried out in triplicate

and mean arithmetic values were used for data analysis. The independent variables were:

electric current density, electro-activation reactor, electrolyte type, electrolyte molar

concentration, running time. The dependent variables were: solution pH in each compartment

of the reactor, ORP of each solution, and total dissolved oxygen in the anodic and central

compartments. One-way analysis of variance (ANOVA), by using SAS software and Systat

v.11, was applied to investigate the differences between the mean values at 5% significant

level.

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3.6. Results and discussion

3.6.1. Configuration #1: Anolyte

The following configuration #1 is a three-compartment cell. In this case the central unit

serves as a barrier that prevents the penetration of the reaction products from anodic and

cathodic units. Such cell configuration has been successfully applied in publically available

literature (Aider and Gimenez-Vidal, 2012; Yahagi et al., 2000) and as a result, it is possible

to obtain a highly acidic anolyte and to prevent the possibility of transferring the reaction

products from the cathodic compartment. According to the reported data during electro-

activation, the solution passes into a metastable state and significant changes in its pH and

ORP values occur. As it is shown in Fig. 3.2 under different current values and different salt

concentrations, the solution changes its properties with similar tendency. The speed of EA is

relatively high so within one minute the pH of the solution decreases significantly.

Particularly, the 0.05 M NaCl solution reduces its pH from 6.00 ± 0.04 to highly acid pH of

2.74 ± 0.05 after 10 min electro-activation under 0.2 A electric current. Within an hour of

electro-activation, these pH values reached 2.20 ± 0.07 under 0.1 , 2.23 ± 0.16 under 0.15,

and 1.96 ± 0.02 under 0.2 . Redox potential drop may also vary in a wide range from 300

± 15mV to the maximum of 1100 ± 50 mV. Such a drop is related to the reactions of

acidification on the anode interface due to water electrolysis and formation of H+ ions

responsible for the medium acidification (Jung et al., 2010). During the electro-activation

treatment, several electro-chemical reactions between NaCl and H2O take place at the

anode/solution interface. Among them, the most abundant and significant ones are the

oxidoreduction reactions; namely the reactions between oxygen and chlorine (Bergmann,

2010). The sharp decrease in pH of the medium in the anodic cell is in-turn caused by the

reaction of water electrolysis which also has strong redox potential; as shown in (Eq. 3.1)

(Aider et al., 2012):

2H2O (l) � O2 (g) + 4H+(aq) + 4e− E° = +810 mV (Eq. 3.1)

Nevertheless, the reactions with chlorine on the anode occur first, followed by the reactions

with oxygen. This was explained by the anion properties due to the location of Cl− on the

right in comparison with OH− in the range of electronegativity (Pourbaix, 1974b). Thus,

chlorine and its derivatives are the main products of the reactions in the anodic compartment,

77

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which is generally responsible for the high positive ORP values of the anodic solution,

commonly named anolyte (Eq.3. 2) (Baker, 2012; Bergmann, 2010):

2Cl−(aq) �Cl2(g) + 2e−; E° = +1360 mV (Eq. 3.2)

Taking into account the fact that the anodic compartment is separated from the central

compartment by the AEM, the amount of chlorine decreases slowly. Owing to ion-exchange

processes and under applied electric current, the chlorine ions migrate from the central

compartment (central cell) to the anodic one and thus increasing its concentration in available

chlorine (Baker, 2012). The Redox potential of the anodic cell reaches its maximum value of

+1100 mV under all the concentrations and current values tested. Equations 1 and 2 show the

high redox potential which was experimentally reached (Fig. 3.2). Therefore, the most

abundant reactions on the anolyte are the reactions of oxidation with subsequent gas release

in the form of free O2 and Cl2 (Tilley, 2004). In addition, highly active oxidants such as

Cl2O, ClO2, ClO–, HClO, Cl*, O3, HO2, and OH* take place on the anode/solution interface

(Gnatko et al., 2011; Sprinchan et al., 2011). The presence of these substances allowed using

this solution as a unique disinfectant in different applications such as surface and vegetables

cleaning and disinfection. Moreover, due to its high content in metastable oxidants, the

anolyte perfectly works against microorganisms. It was supposed that the high ORP of the

anolyte could influence the ATP production of bacterial cells as well as metabolic fluxes.

Low pH values make the outer membrane of bacterial cell permeable to the HOCl so the

anolyte can be used in the food industry as a disinfectant solution (Hung et al., 2008).

78�

Figure 3.2: Influence of different salt concentrations and current values on pH and ORP of anolyte in

cell configuration #1.

79

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3.6.2. Configuration #1: The central section

The innovative character of such a configuration lies in the central section equipped with

AEM and CEM which protect the solution against the penetration of reaction products from

the adjacent anodic and cathodic compartments. The AEM is placed near the anode section

so as to prevent the transition of cations from the anode section while the CEM protects this

section from anions generated in the cathodic compartment, respectively. Consequently, the

solution in the middle section is not in contact with the electrodes. Thus, it does not have to

deal with reaction products such as chlorine salts, oxidants and radicals (Bergmann, 2010;

Gnatko et al., 2011).

During the EA of NaCl solution with higher concentration, as it is shown in Fig. 3.3, the

solution in the central section tends to exhibit alkaline properties. On the other hand, lower

concentrations resulted in acidification of the medium. The central cell is separated by an

AEM from the anode section and a CEM from the cathode section. During the ion transfer

under the applied current, other processes take place such as membrane transport and mass

transfer (Bazinet, 2004; Nikonenko et al., 2010). As it might be seen for the solutions with

concentrations of 0.500, 0.250, and 0.125 , the pH is maintained at a slightly acid value

during the first 20 min of electro-activation. However, after that time it becomes alkaline. In

the beginning of the treatment, there are sufficient ions in the central section for current flow

but their quantity decreased significantly after some time which leads to changes in the

solution properties. Thus, 0.5 M solution after 60 min of electro-activation treatment reached

pH 7.75± 0.26 under 0.1 A, 9.43 ± 0.27 under 0.15 and 9.96 ± 0.15 under 0.2 ,

respectively. The solution of 0.125 and current 0.1 A remained neutral. However, it

becomes obvious that with an increasing of the applied electric current intensity, the pH tends

to drop down. At the same time, the solutions with lower concentrations (under 0.1 A)

showed a decrease of their pH to become acid. Specifically, the 0.05 M solution similarly to

anolyte compartment reached pH 3.34 ± 0.05 under 0.1 , 3.44 ± 0.16 under 0.15 , and

2.82 ± 0.13 under 0.2 , respectively. After 60 min of electro-activation, the same solution

becomes highly acidic with a pH value of 2.65 ± 0.07 under 0.1 , 2.62 ± 0.14 under 0.15 ,

and 2.14 ± 0.14 under 0.2 , respectively. The increase in hydroxyl ions concentration might

occur due to several reasons. The first reason is water splitting on the membranes surfaces,

as confirmed by several studies (Nikonenko et al., 2010; Strathmann, 2010b; Tanaka, 2010):

80�

2H2O (l) � H3�- (aq) + OH- (aq) (Eq. 3.3)

As it is generally recognised, under the influence of an external electric field the anions and

cations are heading for an anode and cathode. During the ion transfer, a small amount of ions

situated close to the membrane crosses it immediately resulting in the so-called ion

deficiency. In other words, the lack of current carriers occurs in the boundary layer due to

ion transfer. Therefore, to compensate such a deficiency the water splitting takes place (Fig.

3.4 A). During the depletion of the solution on the boundary layers of the middle section,

hydroxyl ions of the split water rush to reach the anode, simultaneously saturating the

solution and raising the pH. However, the same situation on the boundary layer of the AEM

might lead to the formation of hydronium ion, as seen in Fig. 3.4 A. In this case, the cations

will seek the cathode interacting simultaneously with hydroxyl ions.

Another situation is observed in the cathode compartment. It is known that during the electro-

activation treatment of NaCl solution the following reactions occur (Tilley, 2004):

2Na (aq) + e− � Na (s) E° = - 2170 m V (Eq. 3.4)

2H2O (l) + 2 e− � H2 (g) + 2OH- (aq) +4e− E° = - 830 mV (Eq. 3.5)

Regarding equation 3.5, the cathode cell contains high concentration of hydroxyl ions, which

grows with an increase in the applied current (Rosenberg and Tirrell, 1957). Ideally, the

transfer of ions through the membrane of similar charge is not possible (Tanaka, 2010),

though the high concentrations, alkaline medium and the effect of the current lead to inter-

membrane diffusion and penetrating of the hydroxyl anions to the central section (Fig. 3.4

B) (Cifuentes-Araya et al., 2012). As shown in Fig. 3.3, at solution concentrations of 0.5 and

0.25 M, the pH of the solution increases while the ORP decreases. At the same time, in the

middle section there are almost no reduction reactions, only exchange reactions occur. It

would have been clear why the ORP in the central section drops if the reaction shown in (Eq.

3.5) was observed, however this was not the case. According to the reaction illustrated in

(Eq. 3.5), a considerable amount of reduced hydrogen is liberated on the cathode with

potential of – 830 mV. During the electro-activation treatment, in all the tests in which

configuration #1 was used under various current and concentration values, the catholyte

reached pH 12 ± 0.2 and ORP of -900 ± 30mV(data not shown).

81

�

Figure 3.3: Influence of different salt concentrations and current values on pH and ORP of solution

in central cell configuration #1.

82�

Reduced oxygen is an electro neutral particle which finds itself in a free form and tends to be

released from the system. As the experiment showed, the hydrogen when passed through the

solution provoked the changes in ORP which reached the values from - 500 to -700 mV. At

the same time, the changes in pH values were not observed. Therefore, an increase in

concentration of the hydrogen in the cathode section enables it to diffuse through the CEM,

decreasing the ORP of the solution (Fig. 3.4 C). The concentration of hydrogen cations

increases in 0.125 and 0.050 M solutions of sodium chloride (Fig. 3.3). This growth is

provided by the water splitting on the membranes, as shown in equation 3 and Fig. 3.4 A. In

this case, as it was previously shown, the water splitting is caused by the lack of the ions

which are needed to transfer the electric current. However, in this case due to lower

concentrations of the solution, the hydrogen cations predominate in the solution. This might

be explained by the phenomenon that on the interface of the AEM the water dissociation

process runs faster than on the CEM (Tanaka, 2007, 2010). As it is shown in Fig. 3.3, the

lack of electrons in the case of 0.125 M concentration occurs within the first minutes under

high current values. The pH of the solution was equal to 5.82 ± 0.18 under 0.1 , 3.57 ± 0.27

under 0.15, and 3.94 ± 0.04 under 0.2 .

ORP readings show the dynamics of the oxidoreduction reactions. In the central section, and

as it might be observed under 0.1 A, the solution oxidoreduction potential is nearly stable

and was close to initial ORP values. This is related to the penetration of the solubilized

hydrogen from the cathode compartment. Thus, during one hour of electro-activation, the

ORP of the solutions with concentrations of 0.500, 0.250, and 0.125 were 177 ± 40 mV,

210 ± 21 mV, and 246 ± 27 mV, respectively. The ORP of the 0.05 M solution was 488 ± 27

mV. With an increase in applied electric current, the ORP readings of the solutions with

concentrations of 0.5 and 0.25 M were 113 ± 15 and 214 ± 34mV and with 0.25 and 0.05

they were of 471 ± 17 mV and 734 ± 26 mV, respectively. In the case when an electric current

intensity of 0.2 A was applied, the aqueous solutions with salt concentrations of 0.5, 0.25,

0.125 and 0.05 showed ORP readings of 88 ± 36 mV, 110 ± 37 mV, 530 ± 39 mV, and

689 ± 11 mV, respectively.

All the aforementioned data support the theory that the present configuration # 1 of the

electro-activation reactor allows to obtain the solutions with both acid and alkaline properties.

Such solutions are called non-contact electro-activated solutions because in spite of similar

83

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properties (pH & ORP), they were obtained without a direct contact with electrodes.

Although there are various methods of production of EA solutions, all of them have a

substantial disadvantage, notably the changings of the chemical composition due to the direct

contact with electrode surfaces. There were several attempts to create an activator which

would allow a creation of EA solutions without a direct contact with electrode surface. The

cells or a single cell containing an anode and a cathode immersed into EA solution would be

separated by a semi-permeable membrane or a thin layer of diaphragm (Chironosov V.G,

2002). The suggested technical decision allows not only solving this problem but also helps

to control the process of activation. Furthermore, it can widen the operational functionality

of the device and improve the efficiency of the treatment. Specifically, it will make possible

the acquisition of anolyte and catholyte by solution electro-activation as the

electrode/solution interface as well as contactless anolyte and catholyte as those obtained in

the central compartment of the electro-activation reactor, depending on the technical or

application needs. This device also helps to get solutions with predetermined properties such

as pH and ORP.

84�

Figure 3.4: Mechanisms of OH- and H2 leakages through CEM during the treatments

�

85

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In order to study the EA process of solutions that do not release toxic substances such as

chlorine, the solution of NaHCO3 in the anode compartment has been chosen. It is also known

as soda in the food industry and does not produce toxic substances. Another advantage of

such a solution is its fungicide property. Taking into consideration the fact that during EA,

the substances pass into metastable state (Prilutsky and Bakhir, 1997), the main aim of this

configuration was to investigate the changes in the sodium bicarbonate solution during the

electro-activation treatment. Provided that the process of EA using sodium bicarbonate

solution was not described in the literature, the NaHCO3 treatment was held in the same

conditions with the addition of 3 other configurations.

In the first case, the solution was poured into the anode and the central compartments at

concentrations of 0.5, 0.25, 0.125 and 0.05 . In the second case, to avoid the accumulation

of ��3- anions in the central sections, the 0.2 M NaCl solution was used in this section. To

ensure the electro-conductivity, in both cases 0.1M NaCl solution was used in the cathode

compartment. The Fig. 3.5 shows that the same tendency of the oxido-reaction processes

occur as in the anode compartment containing NaCl solution. As it was shown in Fig. 3.5 the

lowest pH after 60 min electro-activation was attained by using the 0.05 M solutions with

mean values of 7.01 ± 0.05, 6.81 ± 0.03 and 6.76 ± 0.01 under the applied electric current

intensities of 0.1, 0.15 and 0.2 , respectively. The ORP values in the same configuration

increased up to 424 ± 15, 655 ± 14 and 724 ± 16 mV under 0.1, 0.15 and 0.2 , respectively.

Thus, the 0.5 M solution of sodium bicarbonate changed its pH by 0.26, 0.4 and 0.56 units

under the current of 0.1, 0.15 and 0.2 , respectively. Thus, it might be concluded as a general

tendency that the ORP values of the NaHCO3 solutions underwent minor changes during the

electro-activation treatment.

When NaHCO3 solutions were used as substrate of electro-activation, the reaction which

takes place on the anode interface is the exchange reaction with the formation of carbonic

acid. Carbonic acid is not stable by its nature, its decay rate is rather high with a mean value

of approximately 23 s−1 (Welch et al., 1969). When CO2 is dissolved in water it tends to reach

the equilibrium according to the following equations:

CO2 (g) + H2� (aq) � HCO3− (aq) + H+ (aq) (Eq. 3.6)

HCO3− (aq) � CO3

2- (aq) + H+ (aq) (Eq. 3.7)

86�

The dissociation constants for the reactions (Eqs. 3.6 and 3.7) are pKa1 = 6.35 and pKa2 =

10.33, respectively. In the NaHCO3 solution, the major part of the dissolved carbon dioxide

is in the form of CO2 and only a small part is found as H2CO3. At the same time, there is a

balance between the carbon dioxide in the solution and in the surrounding atmosphere, and

the CO2/HCO3-/CO3

2- equilibra are the major buffering compounds affecting the solution pH.

Therefore, and as shown in the equations 6 and 7, in the range of pH lower than 6.35, CO2

form predominates, at pH values 6.35-10.33 it is the HCO3- form which is the dominant one

and finally, at the pH values higher than 10.33, the CO32- form is the most abundant one.

Within the pH values of 7 and 9, it is the bicarbonate ion that is responsible for buffering

capacity of the aqueous solution. With the addition of bicarbonate to the water, the pH value

of the solution is equal to 8.34. The value of 8.34 is equal to the (pKa1 + pKa2)/2.

In this study and under the applied electric current, the first reaction occurring on the anode

interface is water dissociation (Eq. 3.1) due to the slow discharge of the anions of the oxygen-

containing acids. Therefore, the water electrolysis with the liberation of the oxygen is

observed (Pourbaix, 1974b). As the sodium bicarbonate possesses high buffering capacity,

the solution with the adequate concentration (0.5 M) almost does not change its properties.

This is mainly due to the fact that there are enough bicarbonate groups in the solution which

absorb the released H+ cations. This was confirmed by the observation that at the moment

when there is a lack of bicarbonate anions, the pH of the solution starts to decrease. Fig. 3.5

indicates the relation between the decrease in pH and the concentration of the NaHCO3

solution. As it might be observed, the ORP values change insignificantly. The ORP values

of the 0.5 M sodium bicarbonate solutions vary from 226 ± 18 mV to 287 ± 12, 314 ± 13,

and 322 ± 12 mV, respectively. Regarding Eq. (3.1), the oxygen is released on the anode

interface with its ORP equal to +810 mV. The solutions of higher concentrations are able to

keep unchanged the redox potential of the medium. The solutions of lower concentrations

tend to change their potential from 226 ± 18mV to 424 ± 15, 655 ± 14, and 724 ± 14 mV

under the applied electric current of 0.10, 0.15 and 0.20 , respectively. This means that the

buffering capacity of a solution with such concentration is insufficient to maintain stability

of the system. Moreover, with the increase in current up to 0.2A and as a consequence of

faster formation of O2 on the anode interface, the ORP changed significantly. Thus, the

concentrations of 0.500, 0.250, 0.125, and 0.050 M showed an increase of their ORP from

87

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260 ± 42 mV up to 322 ± 18, 451 ± 15, 573 ± 13, and 724 ± 16 mV, respectively. In addition,

Fig. 3.5 shows the relation between the ORP of the solution, changes in concentrations, and

electric current intensity. To sum up, with an increase in the applied electric current, the

dynamics of the oxidation-reduction reactions increases with a tendency to change the

properties of the medium.

3.6.4. Configuration #2: The central section

The central section with NaHCO3 solutions of molar concentrations of 0.5, 0.25 and 0.125

did not undergo significant changes (Fig. 3.6). Within an hour of electro-activation, the

solution with 0.5 M concentration changed its pH from 8.26 ± 0.13 to 8.35 ± 0.04 under 0.1

, 8.5 ± 0.006 under 0.15 , and 8.14 ± 0.08 under 0.2 . Considering the fact that the

solution of NaHCO3 possesses a good buffering capacity, it is possible to conclude that

changes which took place during the electro-activation treatment on the membranes were

immediately substituted by the ions of high buffering capacity of dissociated NaHCO3. It is

generally recognized that the buffering capacity is a measure of a buffer activity and depends

on the concentration and the components ratio. Therefore, the 0.05 M NaHCO3 solution after

one hour of electro-activation treatment although not significantly experienced significant

variations among other concentrations. Its pH only changed from 8.48 ± 0.03 to 8.13 ± 0.05

under 0.1 , 8.15 ± 0.01 under 0.15 , and from 8.53 ± 0.1 to 7.95 ± 0.06 under 0.2 . These

results indicate that the effect of the applied electric field intensity was significant but not

enough to cause drastic changes in the solution properties. The ORP values were not

influenced as there are no oxidoreduction reactions in the system. Only the exchange

reactions took place as illustrated by the Eq. 3.6 and 3.7, respectively. Thereby, the data from

this experiment are not sufficient to conclude which processes have the major influence on

the solution properties after one hour of electro-activation. Considering the fact that in the

cathode compartment there was dilute NaCl solution (0.1 M), the excess and the penetration

of hydroxyls cannot have the impact on the central cell. Also no oxygen is transferred from

the anode section because during the whole electro-activation the steady values of diluted

oxygen (O.D.) 5.02 ± 1.1 mg/L were maintained in the central cell.

88�

Figure 3.5: Influence of different salt concentrations and current values on pH and ORP of anolyte

in cell configuration #2.

89

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NaHCO3 solutions in middle cell, configuration #2.

�

90�


In the case of the configuration #3, the tendency observed was quite similar to the previous

configuration (Fig. 3.7). The pH values of the 0.05 M solution after one hour of the electro-

activation treatment under 0.1, 0.15 and 0.2 indicated the following changes: from 8.57 ±

0.06 to 6.83 ± 0.02, from 8.6 ± 0.03 to 6.64 ± 0.04, and from 8.68 ± .06 to 6.48 ± 0.05,

respectively. The major difference between the configuration #3 and #2 was the high ORP

of the anolyte solution and the dynamics in the ORP changes within the first 5-10 min of

electro-activation. Under the applied electric current of 0.1 A, the solutions of 0.500, 0.250,

0.125, and 0.050 after one hour treatment showed a change of their ORP which reached

the values of to 674 ± 21, 717 ± 11, 804 ± 13 and 804 ± 18 mV, respectively. This means an

increase of 2.72, 2.95, 3.15, and 3.39 times comparing to the initial ORP values of sodium

bicarbonate solution. As it was previously described, the central section of the configuration

# 2 was filled with sodium bicarbonate solution and its anions migrated/diffused to the anodic

compartment in order to maintain its buffering capacity. The configuration # 3 contained the

0.1 M NaCl solution in the central cell, and its anions pass quickly to the anodic cell

saturating it with chlorine molecules. The same situation was observed in the configuration

# 1. As it was mentioned previously, the first reaction to occur on the anode interface is

chlorine reduction with its ORP of +1360 mV. Therefore, the high acidification rate of the

reactions # 1 and # 2 provides high ORP of the anolyte in the configuration # 3. Its ORP was

higher than the ORP of the oxygen by the value of 550 mV. The buffering capacity of the

NaHCO3 solution which equilibrates the variations in pH also had a great impact on the ORP

changings of the medium (Pourbaix, 1974b). Consequently, there were two oxidoreduction

reactions between oxygen and chlorine which caused such a high ORP value in the middle

section.

In literature, the attempts have been reported on creating an anolyte that would have a pH of

7.4 and high ORP values. The production of the anolyte in the anode cell by adding NaOH

solution just until the neutral pH is one of such methods (Yahagi et al., 2000). Another

method used the initial treatment in the cathode compartment with subsequent treatment of

the obtained catholyte by solubilized hydrogen in the anode cell. The authors named the

obtained solution as “neutral anolyte” for its high ORP and the amount of available chlorine

typical for anolyte but neutral pH. The proposed method also allows obtaining the anolyte

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with high ORP values and low pH so it might be called “neutral anolyte”. However, further

investigations of its properties and the behavior while being in contact with various agents is

needed.

Figure 3.7: Influence of different salt concentrations and current values on pH and ORP of anolyte

in cell configuration #3

92�


During the electro-activation treatment of NaHCO3 solutions in the reactor according to the

configuration # 4, the swapped membranes allowed the anions and cations to migrate to the

central section. In addition, this configuration of the electro-activation reactor allowed

isolating the anode cell from the possible penetration of the bicarbonate anions. As illustrated

in Fig. 3.9, the relation between pH variation and concentration as well as the applied electric

current remains the same as in previous configurations of the reactor. For 0.05 M

concentration, the pH values changed from 8.31 ± 0.02 to 6.82 ± 0.06 under 0.1 , 8.46 ±

0.06 to 6.56 ± 0.02 under 0.15 , and 8.47 ± 0.06 to 6.35 ± 0.06 under 0.2 . In the anode

cell the water dissociation processes with the release of oxygen and hydrogen cations as well

mass transfer of the cations take place (Fig. 3.8). Hydrogen ions, similarly as in the previous

case, interacted with bicarbonate ions to form carbon dioxide and water. In the cathode cell,

water dissociation caused the formation of hydroxyl ions and molecular hydrogen. The

bicarbonate anions under the influence of the applied electric current migrated to the central

compartment. In the case of this configuration, the ORP of the anolyte indicated the oxidative

reactions such as water splitting and oxygen formation. However, as shown in Fig. 3.9, the

ORP decreased during the electro-activation treatment even though the dissolved oxygen

(DO) values indicated a significant amount of decomposed water. Therefore, the ORP values

of 0.5 M solution within an hour of electro-activation changed from 219 ± 11 to 160 ± 12

under 0.1 , from 214 ± 18 to 127 ± 12 under 0.15, and from 199 ± 14 to 140 ± 18 mV.

93

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Figure 3.8: General schematization of the mechanisms of ion transfer through exchange

membranes during the treatments of configuration #4.

94�

�

�


contactless anolyte in cell configuration #4.

95

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3.6.7. Configuration #4: The middle section

Fig. 3.8 shows the theoretical phenomena that can take place in the central cell of the electro-

activation reactor. It can be seen that the ions migrate towards this compartment from both

the cathode and anode compartments. Cations of sodium and hydrogen penetrated through

the CEM from the anode cell while the anions of bicarbonate and hydroxyls diffused through

the AEM from the cathode cell. During the treatment of NaHCO3 solution, the pH of the

solution in the central compartment varied slightly (Fig. 3.9). Within one hour of electro-

activation, the pH values of the 0.5 M solution were almost unchanged and had the following

mean values: 8.12 ± 0.05 to 8.18 ± 0.04 under 0.1 , 8.12 ± 0.09 to 8.18 ± 0.02 under 0.15

, and 8.23 ± 0.1 to 8.26 ± 0.07 under 0.2 . This fact showed the intensity of the buffering

capacity of the solution with such concentration as well as the alteration in the buffering

capacity with a decrease in concentration and increase in the applied electric current. Thus,

the 0.5 M solution changed its pH from 8.31 ± 0.05 to 8.63 ± 0.05 under 0.1 , from 8.56 ±

0.06 to 8.92 ± 0.04 under 0.15 , and from 8.47 ± 0.1 to 8.93 ± 0.06 under 0.2 . However,

in comparison with the tendency of the electro-activation treatment in the configuration # 2,

the pH of the bicarbonate solution had an opposite trend. If an aqueous solution with lower

concentrations (0.05 M) is considered at applied electric current of 0.2 A, the pH value of the

configuration # 2 will decrease to some extent (from 8.53 ± 0.1 down to 7.95 ± 0.06) while

in the configuration # 4, it will increase from 8.47 ± 0.1 up to 8.93 ± 0.06. Although there

were minor changes, it is reasonable to assume that during the electro-activation treatment,

the hydroxyl ions migrated to the central compartment when the electro-activation reactor

was used in the configuration #4. Together with hydroxyl ions, there was a migration of

molecular hydrogen which had significant impact on the solution ORP values. Under the

applied electric current of 0.1 A and electrolyte concentrations of 0.5, 0.25, 0.125 and 0.05

M, the ORP changed from 213 ± 15 to 64 ± 14, -9 ± 16, -56 ± 21and -31 ± 22 mV,

respectively. With an increase in the applied electric current, the dynamics of hydrogen

formation on the cathode interface also changed as well as its migration to the middle cell.

Thus, the 0.05 M solution changed its ORP from 210 ± 15 down to -31 ± 12, from 191 ± 19

down to -130 ± 12, and from 195 ± 12 down to -136 ± 19mV under 0.10, 0.15, and 0.20 ,

respectively. If comparing two middle cells in the configuration # 1 and configuration # 4, in

spite of different solutions used, it is possible to observe the negative correlation with the pH

96�

variation. At high electrolyte concentrations (0.50 and 0.25 M) in the configuration # 1 there

was a tendency to decrease the ORP. On the contrary, there was an increase in the ORP values

at lower concentrations (0.125 and 0.050 ). Such results demonstrated that at lower

electrolyte concentrations, the buffering capacity of NaHCO3 solution does not maintain the

pH level which leads to saturation of the solution by hydroxyl ions. NaCl solution while

having another membrane disposition showed that pH values changed due to the penetration

of hydroxyl groups through the CEM at higher concentrations. ORP values observed in case

of the configuration # 4 mean that either the solution is saturated by the hydrogen or the

oxidoreduction reactions occurred. This experiment is another proof of the concept that

during the electro-activation treatment, the middle compartment of the used electro-activated

reactor is essential for prevention of ion and molecule migration formed on both electrodes.

3.6.8. Catholyte

The catholyte is the electro-activated solution which was obtained at the cathode/solution

interface in the cathodic compartment of the electro-activation reactor. During the EA the

obtained catholyte had high alkaline pH and highly negative ORP values. As it was

previously mentioned (Eqs. 3.4 and 3.5), the catholyte is saturated with reducers leading to

high absorption–chemical activity. In addition, dissolution of the electrolyte in water leads

to its ionization which depends on the donor and acceptor properties of electrons in the

solution. Ions in the solution exist in the form of hydrates (Sprinchan et al., 2011). Under

those circumstances, the solutions obtained on the cathode are supposed to have high pH and

negative ORP values (Tilley, 2004). Fig. 3.10 characterizes the EA process of the catholyte

of two configurations such as configuration # 1 with NaCl solutions and configuration # 4 in

which the cathodic section was filled with NaHCO3. It should be mentioned that the

concentrations of NaCl used in the cathodic cell were 0.500, 0.250, 0.125, and 0.050 .

Configurations # 2 and # 3 are not showed in Fig. 3.10 as during the treatment only 0.1 M

NaCl solution was used. Under those circumstances, in the case of catholyte with NaCl

solution the increase in pH might be observed from the first minutes of electro-activation and

it keeps growing during the whole treatment. Therefore, it is possible to state that the

dynamics and the tendency in pH variations were equal for all concentrations and current

intensities used in this study. The standard deviations between the initial and the final pH

97

�

values after one hour of treatment in configuration # 1 ranged from 5.71 ± 0.28 to 12.01 ±

0.22, which is less than 5%. In other words, the concentration and electric current intensity

have only a little impact on the increase in concentration of hydroxyl ions on the cathode

using this type of salt. In configuration # 4 where the cathode cell was filled with the NaHCO3

solutions of the concentrations of 0.500, 0.250, 0.125, and 0.05 , the standard deviation

was higher than 5%.

Figure 3.10: Influence of different configuration on pH of catholyte.

As there is a significant deviation in pH values in configuration # 4 (Fig. 3.10), the changes

during the electro-activation treatments should be scrutinized more carefully. Although

having the same tendency, the pH in configurations # 4 and # 1drastically differ by its values

and its evolution dynamics (Fig. 3.11). Regarding the situation on the anode side of the

electro-activation reactor, the NaHCO3 solution on the cathode also compensates the pH

variations owing to its buffering capacity. The NaHCO3 solution at a concentration of 0.5 M

over an hour and under 0.1 A electric current changed its pH from 8.12 ± 0.03 to 8.50 ± 0.02

while the 0.05 M solution under the same electric current resulted in pH changes from 8.31

± 0.05 to 9.79 ± 0.07.

98�

Figure 3.11: Influence of different salt concentrations and current values on pH and ORP

of catholyte.

The concentration and the current intensity influenced the buffering capacity of the

bicarbonate solution. The pH of a 0.5 M solution changed its pH to 8.5 ± 0.02 under 0.1 ,

8.63 ± 0.05 under 0.15 , and 8.78 ± 0.02 under 0.2 . Bigger changes were observed during

the treatment of the 0.05 M solution where pH attained the values of 9.79 ± 0.07 under 0.1

, 10.13 ± 0.16 under 0.15 , and 10.3 ± 0.02 under 0.2 . It is also important to mention

that at pH = 10.33, the CO3-2 becomes the predominant ionic species (Tanaka, 2010). Thus,

99

�

it is possible to obtain the catholyte with high pH values using bicarbonate solution of even

lower molar concentrations. The changes in the evolution dynamics of the ORP values of the

catholyte during the EA process as opposed to pH values considerably depended on the

applied electric current intensity, solute concentration and the type of salt used (Fig. 3.12).

The ORP values become more stable after one hour of electro-activation when it reached its

maximum reductive value of -905.22 ± 40 mV. However, during the electro-activation

process, significant deviations were observed at 5th and 10th minutes when the solution only

started to stabilize itself due to oxidoreduction processes. At the 5th minute of electro-

activation, the ORP values were -378.43 ± 27.5 mV, which was related to the fact that at

different concentrations and electric current intensities these values vary considerably (data

not shown). According to the observed changes in the dynamics of bicarbonate EA, it was

possible to conclude that the major part of redox potential of reactions is compensated by the

salt. As a result, greater changes in ORP values occurred over an hour of electro-activation

(468.7 ± 36.6 mV).

Figure 3.12: Influence of different salt concentrations and current values on pH and ORP

of catholyte.

�

100�

3.6.9. Electric resistance of system

The results of this study were used for the evaluation of a resistance during the electro-

activation process of different salt solutions. This factor is essential for the system evaluation

in terms of mass and electron transfer. Table 1 allows characterizing the consumed energy

by the EA reactor as a function of various concentrations and types of salt under various

applied electric current intensities. Accordingly, with higher current intensity the

consumption of the energy was increased. In fact, according to the Ohm law, an increase in

the system resistance at the static mode is caused by an increase in voltage. As it might be

noticed from Fig. 13 the solutions with the lowest molar concentration (0.05 M) have the

highest electric resistance. This behavior shows that probabely there is a lack of current

carriers (ions) at this concentration. Therefore, under the fixed current intensity and high

voltage, the limiting current density occurs and this phenomenon could cause the water

splitting in order to compensate such a lack of ions that are needed to carry the electric

current.

101 �

Table 3.1: The power produced by the reactor with different salt concentrations and electric

current in kilowatt per hour.

0.1 A 0.15 � 0.2 �

C,, M 0.5 0.25 0.125 0.05 0.5 0.25 0.125 0.05 0.5 0.25 0.125 0.05

Config. # 1 3.24 3.6 6.84 15.84 5.4 10.26 15.66 32.4 8.64 17.3 24.48 57.6

Config. # 2 4.32 6.12 10.08 32.4 10.8 13.5 25.92 62.6 18 24.5 51.84 86.4

Config. # 3 3.6 8.64 10.8 12.24 16.2 18.36 21.6 25.9 27.36 31.7 36 44.64

Config. # 4 3.6 5.4 11.16 20.16 8.64 11.34 19.98 42.1 13.68 23 36 72

*C: Molar concentration of the electro-activated salt.

*Config.: Configuration of the electro-activation reactor.

102�

Figure 3.13: Influence of different salt concentrations and current values on resistance of

system.

Not only insufficient salt concentrations and high current intensity values lead to an increase

in electric resistance of the system but the type of salt, intra-membranous and inter-electrode

space also have their impact. As the intra-membranous and inter-electrode spaces were

constant, the conditions at which the cells configurations, current intensity, type and salt

concentration were changed should be analyzed. Hence, Fig. 3.13 shows that the

configuration # 2 had the highest electric resistance at 0.05 M salt concentration. This

configuration also exhibited high resistance values at different concentrations and under

various applied electric currents. Considering the fact that this configuration consists of the

cathode compartment filled with 0.1 M NaCl solution, the central and the anode

compartments were filled with NaHCO3 solution, their electric resistances at 25°� should be

regarded (10.74 and 7.59 Ohm, respectively). Because the resistance of the bicarbonate

solution is lower comparing to the NaCl solution, this means that under the applied electric

current the system resistance should be lower likewise, and the first reaction to take place on

the electrodes is the water splitting. Hence, there is a formation of �+ ions on the

0

100

200

300

400

500

600

700

800

900

1000

0,5 0,25 0,125 0,05 0,5 0,25 0,125 0,05 0,5 0,25 0,125 0,05

Re

sist

an

ce o

f sy

ste

m,

Oh

m

Salt concentration, Mole/l

Config.1 Config.2 Config.3 Config.4

��

103 �

anode/solution interface as well as ��- on the cathode/solution interface. Considering what

was mentioned before, the water splitting will take place as the anions of oxygen containing

acids discharge with difficulty. Consequently, the electric resistance of the configuration # 4

will also be not very high due to the low quantity of current carriers and the big amount of

split water. Another tendency of decrease in the electro-activation reactor electric resistance

with an increase in the applied electric current might be observed. Consequently, the

resistance of the configuration # 4 will also be not very high due to the low quantity of current

carriers and the big amount of split water.

Another phenomenon is a decrease in the reactor electric resistance with an increase in the

applied electric current. Thus, the configuration # 4 with a concentration of 0.05 M showed

a decrease of its resistance from 560 at 0.1 A to 500 Ohm under 0.2 A. Such phenomenon is

related to an increase in double diffusion layer which leads to an increasing rate of water

splitting and the formation of additional current carriers (Welch et al., 1969). With an

increase in the applied electric current, the limiting current density increases likewise which

enables water splitting but which in-turn leads to an increase in resistance and, consequently,

to higher energy consumption (Bard A.J. et al., 1987).

3.7. Conclusion

The present study showed that electro-activation of both NaCl and NaHCO� solutions under

different conditions permitted to obtain solutions with different targeted acidic and alkaline

pH values with positive and negative ORP values. Moreover, a neutral anolyte with high

ORP values (700 ± 50 mV) and pH 6.5 ± 0.3 was obtained while using the sodium

bicarbonate solution. The power consumption of the electro-activation reactor expressed

through the system electric resistance was dependent of the cell configurations, types of

solutions, their concentrations and current densities. It was shown that at extremely low salt

concentration, the system electric resistance is high and must be optimized.

This study also showed that the obtained anolyte and catholyte were saturated by oxygen and

hydrogen, respectively. When NaCl was used in the anodic cell, free chlorine was also

generated, suggesting a potential antibacterial used of this solution. Other studies are under

consideration to understand the mechanisms of chlorine and other chlorine-based radicals

formation. Moreover, a study of the possible formation of electro-activated solutions with

104�

different pH, ORP values, as well as oxygen, hydrogen and chlorine content in a continuous

flow mode are considered.

Further studies are required to understand the stability of the electro-activated solution, the

mechanisms of formation of the reactive species at the electrode interfaces as well as the

relaxation time during which the solution maintain the reactivity in a metastable state. Studies

on the conception of devices for the generation of electro-activated solutions at large scale

are also required. The life cycle of the process for electro-activation of different aqueous

solutions for targeted applications must be also studied.

3.8. ACKNOWLEDGMENTS

This work was financially supported by the innovation in food support program that was

funded by contracts through the "Growing Forward" Program that occurred between the

Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (Ministry of

Agriculture, Fisheries and Food of Quebec) and "Agriculture and Agri-Food Canada".

105�

�

4. CHAPTER 4: Application of response surface methodology for the

optimization of the production of electro-activated solutions in a

three-cell reactor



G1V 0A6, Canada.

2) Department of Food Sciences and Nutrition, Université Laval, Quebec, QC,

G1V 0A6, Canada.





This article is published in Engineering in Agriculture, Environment and Food.







106�


The previous chapter showed the impact of different factors on the changes in

physicochemical properties of EAS during the treatment. It was found that sodium chlorine

(NaCl) solutions just within a few minutes, generate solutions with a lower pH than do

solutions of sodium bicarbonate (NaHCO3). Although EA of NaHCO3 solutions generate

slightly acidic solutions (pH ~6,5) their redox potential changes considerably. Obtained data

showed the many factors which may depends on the EA production, thereby analysis of those

variables was the goal of the following chapter. Thus this chapter using response surface

methodology aims to optimize the production of EAS in order to obtain the solutions with

required proprieties (pH, redox etc).

107�

�

4.2. Résumé

Cette étude vise à étudier la possibilité de produire des solutions électro-activées (SEA) de

façon optimale en utilisant l’approche statistique «response surface methodology » (RSM).

La méthode RSM a donné des modèles pour prédire les propriétés de la solution sous forme

de fonctions des paramètres du procédé. Les analyses statistiques des données ont montré

que les variables dépendantes sont corrélées à ces paramètres avec des coefficients de

détermination très élevés (R2 = 0,9393 à 0,9974); indiquant une grande corrélation entre les

valeurs observées et prédites. L'effet des paramètres de production sur les propriétés des

solutions, tels que la concentration en sel, le courant électrique et le temps d'excitation a été

étudié. La concentration en sel a été le facteur le plus significatif sur les valeurs du potentiel

d'oxydo-réduction de la SEA, son pH et la résistance électrique globale du réacteur d’électro-

activation. L’effet du temps d’excitation et du courant électrique ont été également

significatifs, mais dans une moindre mesure en comparaison avec l’effet du sel. La fonction

de Derringer a permis d’optimiser la production des solutions électro-activées. Les meilleurs

résultats ont été trouvés avec une valeur du courant électrique nominal de 200 mA, dans une

solution d’électrolyte à 0,05 M pour les deux types de configurations du réacteur avec un

temps d’excitation de 38 et 60 min; dépendamment du type de configuration. Les paramètres

prédits ont été confirmés par des tests additionnels qui ont confirmé que l'équation du modèle

développé peut être utilisée pour la prédiction des propriétés des solutions électro-activées.

108�

4.3. Abstract

This study examines the possibility of producing optimally electro-activated (EA) solutions

using response surface methodology (RSM). RSM yielded models to predict solution

properties as functions of the process parameters studied with very high coefficients of

determination (R2 = 0.9393 to 0.9974), indicating the models provided high correlation

between observed and predicted values. The effect of production parameters, such as salt

concentration, current and exposure time on the properties of the solutions, was investigated.

For oxidation-reduction potential, pH and resistance, salt concentration was the most

significant factor followed by exposure time and current. Using a Derringer’s function, the

best performance for the production of electro-activated solution was obtained at a current

intensity of 200 mA in the presence of 0.05 M electrolyte after 38 and 60 min of treatment

time, depending on which reactor configuration was used. The current investigation shows

an agreement with the addition test of predicted data and confirms that the developed model

equation can be used for prediction.

109�

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4.4. Introduction

Electrochemical activation of aqueous solutions is a type of electro-membrane technology

that enables technological processes that are chemical-free and only generate

environmentally friendly solutions (Aider et al., 2012; Gnatko et al., 2011; Hung et al., 2008).

The phenomenon of electrochemical activation is based on a phenomenon in which dilute

solutions of mineral salts (including tap water) are transformed into a metastable state, which

is characterized by abnormal physical and chemical activity, by means of electrode (unipolar)

treatment in a reactor (Aider et al., 2012; Leonov et al., 1999a). The word "metastable" means

that the solution is in a very high reactive state that depends on the relaxation time. This state

makes the electro-activated solution much more reactive than its equivalent obtained by a

simple acid addition to the solution. Thus, at similar pH, solutions acidified by electro-

activation are at least an order of magnitude much more reactive than any similar aqueous

solution which is simply acidified by an acid, such as HCl. Electrochemically activated

solutions are implemented/produced by treating a solution in the electro-activation cell of the

reactor, which is a variation of a diaphragm electrolyzer (Aider et al., 2012; Bogatova O.V.,

2003). During the anodic electrochemical treatment, the acidity of the solution increases to

reach pH values of 2 or lower, and the solution redox potential (E) increases up to +1200

mV. Meanwhile, in the anodic compartment, the surface tension decreases and the

conductivity and amount of dissolved oxygen increase, and in the cathodic compartment, the

concentration of hydrogen and nitrogen decreases. The concentration of the dissolved oxygen

increases in the anodic side of the electro-activated reactor because of oxygen generation by

intensified water dissociation at the solution/anode interface. As a result of cathodic

treatment, the solution becomes alkaline and the pH reaches 10 or higher, whereas the redox

potential is sharply reduced to a value of -900 mV or lower. In addition, there is a decrease

in the surface tension of the solution, and a reduction of the concentration of dissolved

oxygen and nitrogen. In the cathodic solution, the insoluble compounds of calcium carbonate

and magnesium oxide are formed, and additional metal ions are almost completely converted

to insoluble hydroxyls (Bahir et al., 2002; Latysheva Y.N. et al., 2009).

Research on the properties of electro-activated solutions has vast application in numerous

domains. It was found that EA solutions possess a high fungicidal effect. They are used for

110�

the inactivation of bacteria, viruses, bacteriophages and numerous other types of

microorganisms (Aider et al., 2012). EA solutions have been investigated and used as

disinfectants for food preservation and safety for non-thermal processes. Additionally, they

have been regarded as sanitizers with numerous advantages such as a strong disinfecting

character, ease of implementation, relatively low cost and environmentally friendly

procedure (Bakhir et al., 2003). Apart from the sanitizing character, EA solutions have also

been investigated for their effect on antioxidant enzyme activation (Podkolzin et al., 2001),

poultry spraying, chilling of meat and preservation of green fodder baking wheat bread

(Ponomareva and Voropaeva, 2008; Sanin et al., 2005). Furthermore, EA solutions have also

found wide application in medicine and other domains (Bakhir et al., 2003; Leonov et al.,

1999a).

Today, there are many types of electrolyzers and reactors for aqueous solution activation.

The use of mono- or multi-chamber reactors, which are separated by a diaphragm

(membrane), underlies the existing technology of electro-activation (Gnatko et al., 2011).

The main objective of using such reactors is the production of solutions (anolyte) with acidic

pH and high redox potential on one side and alkaline pH with a negative potential on the

opposite side (catholyte). However, there are also other methods of obtaining EA solutions.

For example, there are reactors that allow for the production of solutions with a neutral pH

but high redox potential (Latysheva Y.N. et al., 2009). In this case, the main disinfecting

element is remaining metastable chlorine, which is dependent on the pH value, leading to a

shift in the equilibrium reaction of chlorine directed towards increasing concentrations of

hypochlorite anions (Zaviska et al., 2012).

Electrical activation is conducted by passing a direct current through an aqueous solution

(electrolyte) in which electro-activation of molecules, atoms and ions, as well as the

redistribution of ions in an electric field, takes place (Bahir et al., 2002; Leonov et al., 1999b).

However, to obtain a solution with the given metastable characteristics, the parameters

influencing the process should be considered. These parameters include processing time,

current density, electrolyte type and concentration, membrane type, distance between the

membranes and electrodes, type of the electrode and the working electrode area where the

111�

�

unipolar treatment of solutions takes place. It should also be noted that the flow velocity in

continuous flow reactors is also as important as the above parameters. However, while this

option depends on the aforementioned factors, it also depends on the time of contact of the

electrolyte with the electrode. By tuning these parameters, solutions with the desired

properties may be prepared. Given that a measure of the redox balance is one of the most

vital indicators for the electro-activated solutions (Bagramyan et al., 2000; Kim et al., 2000b),

the process optimization of this parameter is considered to be of utmost importance.

Consequently, to obtain adequately electro-activated solutions, it is necessary to

maximize/optimize the redox potential value of the solution. The amount of active hydrogen

in an environment is another crucial parameter that has a significant effect on the inhibition

of pathogens. These parameters are most commonly controlled in commercial electrolyzers

(Hsu, 2005; Hsu, 2003). One more important parameter in terms of economic efficiency for

the optimization of the electric-activation technology in a three-cell reactor is the electrical

resistance of the system, which should also be analyzed in the process. Response surface

methodology (RSM), a type of Design of Experiments (DoE) technique, is an efficient

experimentation technique and is well known for effectively assessing the effect of

parameters on treatment results (Li et al., 2010). The optimization of processing parameters

is essential in product development (King and Zall, 1992). It is considered to be more

efficient than most other classical approaches in seeking process optima and is useful for

investigating complex, multivariable research problems. This technique has been

successfully applied in a wide range of applications, such as the optimization of analytical

procedures (Bezerra et al., 2008) and chemical and biological processes in food processing

(Gan et al., 2007; Switzar et al., 2011), including ozonation of drinking water (Li et al., 2010)

and active chlorine formation (Ezeike and Hung, 2004; Hsu, 2005; Hsu, 2003; Hung et al.,

2008; Zaviska et al., 2012). In the study of optimization methods in regards to flow rate,

amperages and/or voltages, water temperature and salt concentration for water electrolysis

are also important.

However, to our knowledge, the production of EA solutions in three-cell reactors with

different control properties has not been yet reported. Thus, in this paper we consider an

approach to optimize the activation of the solution in a three-cell reactor through RSM. The

112�

goal was to investigate the processing parameters that determine the properties of EA

solutions during activation. As soon as the factors affecting the properties of EA solutions

during electro-activation were understood, the efficiency of EA solutions could be optimized

for every single case of a given application.


4.5.1. Chemicals and materials

Solutions were prepared by the dissolution of crystalline sodium chloride (NaCl) and sodium

bicarbonate (NaHCO3) (Laboratory MAT, Montreal, Qc, Canada) in 1 L of distilled water to

give final concentrations from 0.5 to 0.05 M. In this study, NaCl was compared with NaHCO3

for two main reasons. The first one is related to the high buffering capacity of the sodium

carbonate in comparison with NaCl. This characteristic can affect the evolution of the electro-

activated solution pH and redox potential. Indeed, it was found that NaCl activates more

quickly than NaHCO3. The second reason is related to the presence of chloride ion in NaCl.

After electro-activation in the anodic side of the reactor, NaCl can yield Cl2, which is not

suitable for food applications at high concentrations, but it can play a very important role as

a disinfectant. Therefore, the salt concentration was optimized to establish the behaviour of

this particularity.

4.5.2. Electro-activation reactor

In this work, a three-cell reactor was used to study the factors that affect the properties of the

EA solutions, such as pH, dissolved oxygen and redox potential. The electro-activation

reactor is made of Plexiglas and is composed of three sections: the anodic, central and

cathodic compartments (Fig. 4.1). The volume of each compartment is 300 mL. The

ruthenium–iridium coated titanium electrodes (100 x 40 mm) were placed in the anodic and

cathodic compartments with a distance of 150 mm between the electrodes. The anodic and

cathodic compartments were separated from the central compartment by anion (AM-40) and

cation (CM-40) exchange membranes, respectively. The membranes were purchased from

Schekinaazot (Shchekina, Russia). The original anion exchange membrane (MA-40) is

formed using EDE-10P resin, which is a product of the condensation of normal diamine with

epichlorohydrin, polyethylene and nylon. The density of the fixed charge on this membrane

113�

�

is 2.5-3 mol/l. The original cation exchange membrane (MC-40), with a density of fixed

active charges of 1.5-2 mol/l, consists of KU-2 cation exchange resins (polystyrene (PS)

matrix cross linked with divinylbenzene (DVB) and functional groups), polyethylene and

nylon. The electrodes were connected to a direct current power supply (Lambda, GR.260,

Electronics Corp. Melville, NY, USA).

Figure 4.1: Prototypical electro-activation reactor used for generating solutions. AEM-

anion-exchange membrane. CEM-cation-exchange membrane. A- - anions. C+ - cations.

�

4.5.3. Protocol of the electro-activation

The treatments were carried out at 23 ± 1 °C for a total duration of 60 min. Samples from

each compartment were collected every 5 min and were analyzed immediately after

collection for pH, dissolved O2 and redox potential using a pH-meter (Model SR 601C

SympHony, VWR Scientific Products, USA) and an ORP-meter (Eco Sense ORP15A, VWR

Scientific Products, USA). ZoBell’s standard solution (Hach Company, Loveland, CO, USA)

was used for the calibration of the latter. For each applied current density value, the

corresponding values of the electric field (voltage) were directly recorded on the power

supply in use. The intensity of the current was controlled by using a circuit-test (DMR-1000,

ON, Canada), which was connected to the power source. Two different reactor configurations

were used. In configuration #1, each compartment had the same type of salt (NaCl) with

concentrations ranging from 0.05 to 0.5 M. For configuration #2, the cathodic and central

compartments were filled with the same NaCl solution (0.1 M), whereas the anodic

114�

compartment was filled with a NaHCO3 solution at various concentrations ranging from 0.05

to 0.5 M.

4.5.4. Global system resistance

The global system resistance R (�) of the electro-activation system was calculated from the

values of the current intensity I (A) and the applied voltage U (V) using Ohm's law as follows

� =�

� (Eq. 4.1)

�

4.5.5. Experimental design and statistical analysis

Data from the experiments with different levels of one or more factors were investigated to

compare the effects of the studied factors and their interactions. In this study, a response

surface methodology (RSM) was applied to obtain the optimal response settings to produce

electro-activated solutions (Li et al., 2010). To determine the optimal design given a specified

set of design parameters, the D-optimality technique was used. This technique, which

consists of minimizing the variance of the full factorial design in the regression coefficients

of the fitted design model, thereby providing the most precise estimate of the effects that also

can be used to determine an optimal design for a full-factorial experimental design (Ghanekar

et al., 2003). The electro-activation process used to generate the target solutions involves the

interaction of several process parameters. Three influencing operating parameters, salt

concentration, current and exposure time, were chosen as the independent variables and

designated as X1 (0.050, 0.125, 0.250, 0.500 M), X2 (100, 150, 200 mA) and X3 (0 to 60 with

5 min interval), respectively. Each of these parameters was evaluated in each of the two

different reactor configurations.

Based on this function, the response optimizer (RO) identifies the combination of input

variable settings that jointly optimizes a single response or a set of responses (Y). The RO

method was used to determine the effects of major operating variables on the formation of

EA solutions and to find the combination of variables resulting in minimum resistance (R),

changing concentration of active hydrogen (pH) and increasing of redox potential (E). The

following model (Eq. 4.2) was employed to explore a quadratic relationship between the

response variables. This includes two empirical second-order quadratic terms that are

commonly used in RSM experiments, as shown in the equation:

115�

�

�>�C = �V + j �� B + j ��

@�� B + j j ��¡��¡

��¢B (Eq. 4.2)

£>rC represents the predicted response and exploratory independent variables of ¤¥ and ¤¦.

The values§R, §¥, §¥¥ and §¥¦ are regression coefficients in the intercept, linear, quadratic and

interaction terms, respectively. The value k is the number of design variables (Aljundi, 2011;

Angelopoulos et al., 2013; Bezerra et al., 2008; Quanhong and Caili, 2005). The response

functions for values of redox potential (E), pH and resistance (R) of EA solution production

are presented in Table 4.1.

Table 4.1: Response functions for corresponding variables of redox potential (E, mV),

minimum resistance (R, Ohm) and pH.

Function Cell configuration Type of solution Dependent variable

£2 1 Anolyte E

£3 1 Anolyte pH

£7 1 Anolyte R

£8 2 Anolyte E

£9 2 Anolyte pH

£ 2 Anolyte R

This optimization process involves a determination of the factors and levels that affect the

response variable. The optimization must satisfy the requirements for all of the responses in

the set, which is measured using Derringer’s desirability function (D), also called the

composite desirability or utility transfer function. For each response, they are combined to

provide a measure of the overall desirability of the multi-response system (G. Derringer,

1980) and (Bezerra et al., 2008). Predicted values obtained from each response surface are

transformed to a dimensionless scale ~¥. The scale of the desirability function ranges between

~¥=0 (for an unacceptable response value) and ~¥=1 (for a completely desirable one) (Ferreira

et al., 2007). The individual desirabilities (~¥) are calculated as follows:

i� = aX|��ª|«

(Eq. 4.3)

Where e is the natural logarithm constant, c is a positive number subjectively chosen for

curve scaling, and £¥g is the linear transformation of the univariate response £¥ whose

116�

properties link the desirability values with product specifications. The measure of D is the

weighted geometric mean of the individual desirabilities for the responses. Each individual

desirability is weighted according to the importance that is assigned to each response.

< = > iB�B ∗ i@

�@ ∗ … ∗ i��CB/>�B��@�⋯��C (Eq. 4.4)

where ~¥ is the individual desirability for response r, and ®¥ is the importance for the response

r >r = 1,2 … sC (Ferreira et al., 2007).

In the present study, each treatment was performed in triplicate. Minitab software (V16.0,

Minitab Inc., USA) was used for all statistical analysis of the obtained data, such as

evaluation of the process efficiency or comparison between the mean values. ANOVA was

at a confidence interval 95%.


4.6.1. Model fitting from RSM

The regression coefficients obtained by fitting the experimental data to the second-order

response surface models and the equations for each of the response variables (Eq. 4.2) were

analyzed by applying t-test and p-values for each coefficient of each parameter (Tables 4.2,

4.3). Table 4.2 shows an empirical relationship between the response function and the

independent variables of the treatments in the three-cell reactor obtained by the application

of RSM. The independent and dependent variables were fitted to the second-order model

equation and examined for the goodness of fit. The analyses of variance were performed to

determine the lack of fit and the significance of the linear, quadratic and interaction effects

of the independent variables on the dependent variables (Table 4.2) and the significance of

the estimated regression coefficients for the response variables (Table 4.3). The lack of fit

test is a measure of the failure of a model to represent data in the experimental domain at the

points which were not included in the regression (Gan et al., 2007; Li et al., 2010).

Furthermore, statistical significance of the quadratic model was evaluated by the ANOVA-

test. The results in Table 4.2 show that the regression was statistically significant at F value

and values of probability F (p � 0.001).

The coefficient of determination (R2) is the proportion of variation in the response attributed

to the model rather than to random error. It was suggested that for a good fit model, R2 should

be as close to the 100% as possible but not less than 80%, meaning that the stronger the

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model, the better it predicts the response (Amini et al., 2008). The results showed that the

models for all of the response variables were highly adequate because they have satisfactory

levels of R2 of more than 80%. The values of adjusted determination coefficients (R2 adj.)

were also high, showing a high significance of the model. Response function predictions

were also consistent with the experimental data (R2 > 99). The lowest predicted value (R2

pred.) was 93.93, showing that the model does not explain only 6.07% of the total variations.

The predicted R2 of 93.93 is also in good agreement with the adjusted R2 of 94.36. The R2

values of all of the responses exceeded 80% demonstrating a high proportion of variability,

meaning the developed response surface models appear to be adequate as explained by the

data (Table 4.2).

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Table 4.2: ANOVA showing the linear and quadratic interactions and the lack of fit of the response variables for each cell configuration.

Source of

variation

DF Configuration 1 Configuration 2

£2 (E) £3 (pH) £7 (R) £8 (E) £9 (pH) £ (R)

Seq. SS F Seq. SS F Seq. SS F Seq. SS F Seq. SS F Seq. SS F

Regression 9 21147413 5529,95*** 420,303 42245,13*** 3672138 707,68*** 10656373 439,35*** 57,1269 255,6*** 358107 347,52***

Linear 3 17454621 3517*** 361,122 1208,57*** 3123124 535*** 9523784 168,74*** 39,6632 76,65*** 251415 290,2***

Square 3 3634978 2851,92*** 58,149 932,8*** 491985 282,85*** 689818 85,27*** 2,3937 32,33*** 85515 249,72***

Interaction 3 57815 45,35*** 1,032 16,53*** 57028 32,97*** 442771 54,77*** 15,07 202,28*** 21177 61,65***

Residual Error 146 56938 2,787 77258 361127 3,3277 15342

Lack-of-Fit 17 56938 11,02* 2,787 18,05* 77258 77,27* 361127 18,47* 3,3277 32,23* 15342 16,26*

Pure Error 129 0 0 0 0 0 0

Total 155 21204351 423,091 3749396 11017500 60,4546 373449

R2 (%) 99,74 99,37 97,94 96,69 94,69 96,06

R2 (adj.) (%) 99,72 99,33 97,81 96,48 94,36 95,82

R2 (pred.) (%) 99,70 99,28 97,65 96,22 93,93 95,50

*Significant at p�0.05; **significant at p�0.01; ***significant at p�0.001; DF = degree of freedom; F = ratio of variance estimates; Seq.

SS = Sequential sum of squares.

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Table 4.3: Estimated regression coefficients of the fitted second-order polynomial for the response variables.

Model terms Coefficients Sum of squares

Configuration #1 Configuration #2

E pH R E pH R

Constant §R 326,396*** 5,06599*** 823,63*** 286,390** 9,05458*** 348,82***

Concentration, M §2 133,422* 2,17495*** -2609,49*** -496,629*** 0,76081ns -863,86***

Current, mA §3 0,112ns 0,00557 ns -2,97*** -0,277ns -0,00824* -0,29ns

Time, min §7 38,070*** -0,15604*** -1,29* 20,453*** -0,04142*** 0,16ns

Concentration,

M*Concentration, M

§22

-261,135** -1,99875*** 2823,83***

476,599* -3,87786*** 1127,48***

Current, mA*Current, mA §33 0,001ns -0,00002 ns 0,01*** -0,001ns 0,00003* 0*

Time, min*Time, min §77 -0,433*** 0,00176*** 0,01ns -0,183*** 0,00028*** 0,01***

Concentration, M*Current, mA §23 -1,106*** 0,00137ns 1,66*** 1,647*** 0,00388** 0,21*

Concentration, M*Time, min §27 3,255*** -0,01387*** 2,36*** -9,276*** 0,05768*** 0,29ns

Current, mA*Time, min §37 0,001ns -0,00003** 0ns 0,016*** -0,00006*** -0,01***

*Significant at p �0.05; **significant at p �0.01; ***significant at p�0.001; ns - not significant.

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4.6.2. Effect of salt concentration, current and time

The effect of different concentrations of electrolytes, current and the exposure time on the

variables (redox potential, pH and resistance) of the three-cell reactor was made by

determining the coefficient of the second-order polynomials (Table 4.3). The response

surfaces plots were drawn to illustrate the main and interactive effects of the independent

variables on the dependent ones. Taking into account that there is a full factorial design with

numerous response levels, the graphs are represented by averaging hold values, such as

concentration (0.125 M), current (150 mA) and time (30 min). These graphs were drawn by

imposing a constant value for these response variables, as shown in Figs. 4.2–4.7.

During electrochemical treatment of solutions in the three-cell reactor (configuration #1), one

of the most important factors affecting the properties of the anode section was dosage of

NaCl and exposure time (Figs. 4.2, 4.3). As seen in Table 4.3, the analyses show that the

parameters of dosage of NaCl (p � 0.05) and exposure time (p � 0.001) have the most

significant effect, whereas the effect of the current is not significant. These conditions are in

agreement with other reported investigations (Ezeike and Hung, 2004), where the changing

of properties was slowed down with an increase in flow rate, reducing the time of contact

between the electrolyte and electrode. The major electrochemical reactions involved in the

electrochemical treatment are:

2H2O (l) � O2 (g) + 4H+ + 4e− (Eq. 4.5)

2Cl−�Cl2 (g) + 2e− (Eq. 4.6)

HClO � ClO− + H+ (Eq. 4.7)

Cl2 + H2O � HClO + H+ + Cl− (Eq. 4.8)

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concentration vs. time (B) and current vs. time (C) on the redox potential of anolyte

(configuration #1).

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concentration vs. time (B) and current vs. time (C) on the pH of anolyte (configuration #1).

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concentration vs. time (B) and current vs. time (C) on the resistance of anolyte (configuration

#1).

124�

As seen in Fig. 4.3 A, pH variation reaches its lowest value when the current increases and

the concentration of the electrolyte decreases. The reduction of electrolyte concentration

decreases the number of current carriers which results in voltage and resistance increases,

(Fig. 4.4 �). In this case, the lack of carriers is compensated by water electrolysis (Eq. 4.5),

which accelerates the oxidation of the solution. The same conditions also lead to a change in

the redox potential of the medium (Fig. 4.2 �). The water electrolysis reaction results in high

oxidative charge of solution (+810 mV) (Pourbaix, 1974b). In addition, the redox potential

value becomes higher due to reactions with chlorine (Eq. 4.6-4.8), where only one reaction

(Eq. 4.6) of chlorine gas formation gives charge up to +1360 mV. It is well known that the

oxidation of chloride ions takes place at a potential scale very close to that of the electrolysis

of water (Eq. 4.5), so there is a competition between oxygen formation and chloride reactions.

It should be noted that during the activation process, the formation of highly active oxidizers

in the anode chamber (ClO2, O3, H2O2) is also important, providing strong disinfectant

properties, which are not, however, stable over long-term storage (Bahir et al., 2002; Gnatko

et al., 2011). Electrons located in the diffuse electrical layer of the electrode (anodic and

cathodic compartment) are transformed into a so-called excited metastable state, after which

there is a period of relaxation of the solution. It was reported that the process of electro-

activation occurs in the fields of high tension in the areas of the unipolar electrode layer

(electrodes) (Prilutsky and Bakhir, 1997). From the obtained data, one can reach the

conclusion that the most significant factors in the three-cell reactor are dosage of NaCl and

exposure time. These factors were also confirmed by others (Hsu, 2005; Hsu, 2003) to

influence the formation of by-products, as indicated by the total chlorine concentration and

the change in electrical conductivity. It should also be noted that although the applied current

is not significant in the activation process, its accelerates the rate of reactions taking place

over the entire period of time of the activation process (Figs. 4.2, 4.3) (Randdle, 1947).

The resistance in the closed circuit configuration #1 depends on all of the factors (Table 4.4).

However, unlike other indicators, current had a significant effect (p�0.001), whereas

exposure time is less significant (p �0.05). Given that the process was under galvanostatic

conditions, the effect of the concentration complies with the current number of available

carriers where lower concentration leads to a greater voltage in the network and greater

resistance, according to Ohm's Law (Fig. 4.4). As seen in Figs. 4.4 �, �, a change in

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concentration significantly affects the resistance. Current intensity also has the same effect

on the resistance. As noted before, an increase in current correspondingly increases the

reaction rate and the electro-migration in the system. This is supported by the results in Fig.

4.4 �, which shows that higher current corresponds with lower resistance.


concentration of the salt vs. time (B) and current vs. time (C) on the redox potential of anolyte

(configuration#2).

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concentration of the salt vs. time (B) and current vs. time (C) on the pH of anolyte

(configuration #2).

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�


concentration of the salt vs. time (B) and current vs. time (C) on the resistance of anolyte

(configuration #2).

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In previous work (Liato et al., 2014), reactor configuration #2 was investigated. This work

showed that this reactor configuration enables the production of solutions with a high redox

potential and neutral pH (Figs. 4.5-4.6), as the goal was to develop a neutral anolyte, similar

to other study (Bahir et al., 2002). The main parameter influencing the change in redox

potential and pH of the medium is the exposure time (p � 0.001). The following equations

show the main reactions of NaHCO3 that occur in the anodic section:

CO2 (g) + H2� (aq) � HCO3− + H+ (aq) pKa1 = 6.35 (Eq. 4.9)

HCO3− � CO3

2- (aq) + H+ (aq) pKa2 = 10.33 (Eq. 4.10)

�

These equations show that during the electrolysis of water at the anode, the resulting

hydrogen cations react with bicarbonate ions (HCO3−) to form carbonic acid. Because

carbonic acid is unstable, it rapidly decomposes to form water and carbon dioxide, thereby

not having a significant effect on the change in pH (Lakhanisky, 2002). The change of

current, as mentioned before, can accelerate the flow of anodic electrolysis (Figs. 4.6 �, �),

but not significantly (p > 0.05). This can also induce the buffer capacity of NaHCO3.

The redox potential of the medium except the time also depends on the concentration of salt

(p � 0.001). As seen in Fig. 4.5 �, lower salt concentration enhances the capacity. Such

dynamics are related to two phenomena: first, the migration of Cl- ions from the middle

section of the anode through the anion-exchange membrane (Eq. 4.6 bringing a potential of

the solution to a value of +1360 mV) and secondly, the water electrolysis reactions (Eq. 4.5)

which liberate oxygen and increase the redox potential to +810 mV due to low salt

concentration (Pourbaix, 1974b). As seen in Table 4.4, the resistance change in reactor

configuration #2 is affected only by the concentration of salt (p�0.001) because of the lack

of current carriers in the electrolyte. Considering that in configuration #2, the NaCl

concentration was constantly high (0.1 ) in the middle and cathode sections, the amount of

current carriers was sufficient to maintain a low resistance, compared with configuration #1.

4.6.3. Optimization using desirability functions

For the numerical optimization of the process parameters, the Minitab software was chosen

to find the specific point prediction option that maximizes the desirability function. The

obtained goals were selected by adjusting the weight importance that affects the

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characteristics of a goal. The main objective of the applied optimization was to maximize the

redox potential and to minimize the pH of solutions as well as to minimize the resistance of

the system. Using desirability functions, the ‘goals’ for dependent variables were assigned

with a corresponding ‘weight’ of 1.0 for ‘minimize’ and of 0.1 for ‘maximize’. The same

criteria are used for the corresponding ‘importance’. Fig. 4.8 represents the response

optimizer searcher, which combines the input variables that accomplish the individual

desirability (d.) functions as well as the calculated geometric mean for overall desirability

(D) for each of the responses.

Figure 4.8: Response optimizer on individual desirability (d_i) of all responses in

correspondence with combined desirability (D), where graph A represent configuration#1,

graph B represent configuration#2.

By using this desirability function with the preselected goals for each of the factors, the

software gave specific values for each of the responses. As observed, the lowest concentration

and highest values of current were predicted as the most desirable; however, time had a

different level for this function. The composite desirability, as the weighted geometric mean

of the individual desirabilities, showed a high significance of desirability of each response

(D = 1.0000), which indicates that the settings appear to achieve results for all responses as

a whole. To verify the suitability of the model for predicting the optimum response values,

the recommended optimum conditions were tested. The additional experimental values were

found to be in accordance with the predicted ones. Consequently, the optimized conditions

A B

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showed the adequacy of this model and proved that it can be used for the production of

anolytes with given parameters in a three-cell reactor (Table 4.4).

Table 4.4: Predicted and experimental values of the response variables at optimum

formulation.

Global solutions Predicted values Experimental values a

Config. C,

M

I,

mA

T,

min

E,

mV pH

R,

Ohm

E,

mV pH

R,

Ohm

#1 0,05 200 38,18 1207,49 1,73 323,63 1200

± 5

1,89

± 0,12

320

± 2

#2 0,05 200 60 987,357 6,43 261,56 985

± 7

6,3

± 0,07

260

± 2

�

a Mean value of five determinations

*Config.: Configuration of the electro-activation reactor.

4.7. Conclusions

The production of electro-active solutions from a three-cell reactor was optimized using

Minitab version 16 software. The three independent variables studied for process

optimization were salt concentration (¤2), current (¤3) and exposure time (¤7). The F-test and

p value indicated that all variables have a significant effect on the responses. However, salt

concentration is the dominant factor, having the highest significance on the process of

electro-activation, which was also demonstrated by other researchers. This factor is also

followed by the quadratic effect of concentration (¤2*¤2) and different interaction effects of

time and current on the predicted values. RSM appears to be a useful tool in optimization of

the solutions. The results of this study suggest that for this system, the provided optimized

conditions were the same for current (200 mA) and for the salt concentration (0.05 M) for

both types of reactor configurations, although the exposure time was different at 38 and 60

min for the first and second configurations, respectively. The time of life (relaxation time) as

well as other factors should be investigated in further studies to have an understanding of the

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effectiveness of electro-active solutions. Regarding the salt concentration, our investigations

showed that with the lowest salt concentration in the anodic side, the highest the reactivity

of the solution is achieved. However, at very low salt concentration, the overall reactor

electric resistance increases drastically and makes the operating costs very high. For this

reason, it is suggested that the salt concentration should be optimized.

4.8. Acknowledgments





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5. CHAPTER 5: Effect of electro-activated solutions (EAS) on the

corrosion of metallic cans used for food preservation



G1V 0A6, Canada.






This article is submitted for publication in Food Packaging and Shelf Life.







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The chapters 3 and 4 were devoted to the first objective, which was to study different

production methods of electro-activated solutions in a three cells-compartment of electro-

activation reactor in order to produce the EAS with required proprieties. The goal of this

chapter is to study the obtained EAS from the point of view of their relaxation modification

during the storage in glass bottles as well as their interaction with tin cans in terms of their

corrosion intensity.

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� �

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5.2. Résumé

Lors de stockage (140 jours), les solutions électro-activées (SEA), produites dans un réacteur

à trois compartiments, ont présenté des propriétés stables (pH, potentiel redox). Pour vérifier

si ces SEA sont plus corrosives que la saumure classique utilisée dans la conservation des

aliments, des boîtes en fer blanc émaillées ont été utilisées. Ces boîtes sont souvent utilisées

dans la conservation des légumes en conserve. L'analyse par ICP des ions métalliques

présents dans les boîtes a été utilisée afin d’évaluer le taux de corrosion dans les boîtes. Les

résultats obtenus ont montré les relations différentes entre les SEA et les boîtes en fer blanc

que ce soit avec ou sans produit. Les SEA acides réagissent fortement avec les boîtes,

principalement à cause de leur pH et leur potentiel d’oxydoréduction (E) qui varie de 2 à 3

et de + 900 à + 1200 mV, respectivement. Dans ces conditions, les concentrations de zinc,

de fer et de cuivre dans la solution étaient de 0,028, 28,814 et 0,0220 ppm, respectivement.

Lorsque des SEA neutres ou acides ont été utilisées, aucune différence significative n'a été

observée en comparant ces derniers avec l’effet corrosif obtenu avec une saumure classique

de NaCl. Aucune migration d’étain n’a pas été observée une fois que des SEA acides ou

neutres ont été utilisées. Les SEA alcalines ayant un pH > 10 et une valeur du potentiel

d’oxydoréduction négative (� -966mV) n’étaient pas réactives non plus en termes de

migration des ions Zn, Fe et Cu. Cependant, il est à noter que les SEA alcalines ont causés

la migration de l’étain dans les boîtes. Néanmoins, il est important de mentionner également

que, la corrosion observée reste dans la limite du niveau de concentration autorisé une fois

que les boîtes sont remplies avec le produit.

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5.3. Abstract

Electro-activated solutions (EAS) produced in a three compartmental reactor demonstrated

stable proprieties during storage (140 days). Tinplate metallic cans that are generally used in

the canning of vegetable foods used to verify if the EAS is more corrosive than conventional

brine used in the food canning industry. The ICP analysis of metal ions in the EAS that are

normally present in the cans was used as a method of monitoring the corrosive effect and

corrosion rate of the cans. The obtained results showed different relativities between the EAS

and the tinplate cans with and without product. The acidic EAS with pH 2-3 and redox

potential (E) +900-1200 mV reacted highly with the cans. In this case, the concentrations of

zinc, iron and copper in the solution was 0.028, 28.814 and 0.0220ppm, respectively. When

neutral or acidic EAS was used, no significant difference was observed in comparison with

the corrosive effect of standard brine composed of NaCl. No tin migration was observed

when acidic or neutral EAS was used. The alkaline EAS with pH > 10 and negative E (� -

966mV) was not reactive in terms of Zn, Fe and Cu migration. However, with the alkaline

EAS, tin migration from the can to the solution was observed. Nevertheless, it is important

to mention that even if some corrosion was observed, it was in the limit of the permitted level

of concentration when the cans were filled with a product.

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5.4. Introduction

Technology of electro-activation is based on passing a direct electric current in the reactor

cell through an aqueous solution (electrolyte) in which the redistribution of ions in an electric

field simultaneously occurs along with the electro-activation of molecules, atoms and ions

(Leonov et al., 1999b). On one hand, during the anode electrochemical treatment the acidity

of solutions (anolyte) increases to pH ≈ 2, the oxidation reduction potential (E) increases up

to +1200 mV; on the other hand, as a result of cathode treatment the solution (catholyte)

becomes alkaline, its pH reaches � 11 while the E is sharply reduced to -900 mV and below

(Bakhir et al., 2002).

Research on the properties of electro-activated solutions (EAS) has a wide array of

applications in numerous domains. EAS have been employed as a sanitizer with numerous

advantages such as strong disinfecting property, easy operability, relatively inexpensive, and

being environmentally friendly (Aider et al., 2012; Bahir et al., 2002; Bakhir et al., 2002;

Gnatko et al., 2011; Huang et al., 2008). By using this type of a sanitizer, the food industry

may reduce microbial numbers to safe levels; however, it may have side effects such as

corrosion of food contact surfaces because of its reactivity. Correct and thorough evaluation

of the reactivity of sanitizers with respect to the equipment to which they will be applied is

therefore extremely important (Ayebah and Hung, 2005).

Work done on EAS reactivity by other authors has shown that the metals (stainless steel,

carbon steel, aluminum, copper) and dental alloys (Au–Ag–Pd and silver) exhibit a higher

rate of corrosion (as measured by mass loss and surface roughness changes) in EAS samples

that have a low pH (<3) (Ayebah and Hung, 2005; Waters et al., 2014); furthermore, pH was

found to play a large role in deciding the corrosion rate (Ayebah and Hung, 2005; Tanaka et

al., 1999a; Waters et al., 2014; Xia et al., 2012). Many studies have also determined that

corrosion rates in chlorine-based sanitizers depends on the chlorine concentration especially

for stainless steel, and that increased chloride concentrations lead to higher rates of corrosion

(Waters et al., 2014). Although, solution components such as chloride ion are an important

factor that determine corrosion rates in chloride-based sanitizers, its role in EAS is still not

clear (Waters et al., 2014). It should be noted there is a lack of information about corrosion

and migration rates of the metal ions from the food containers (cans) in contact with EAS. It

138�

has been reported that internal corrosion of cans may increase the concentration of metal

contents like Zn, Cu, Fe, Sn, and Al. There are significant problems associated with the use

of tinplate cans in corrosive food products, such as corrosion failure, loss of seal integrity, or

discoloration problems that result in their rejection by the consumer (Tuzen and Soylak,

2007). Also, if this increase in heavy metals in the canned food exceeds the prescribed limits,

they may become toxic to human health (Arvanitoyannis, 1990). Thus, in this work we

investigated the relaxation time and the corrosivity of EAS with respect to the type of metallic

cans currently used for canning by the food industry.


5.5.1. Chemicals

Solutions were prepared by the dissolving crystalline sodium chloride (NaCl) and sodium

bicarbonate (NaHCO3) (Laboratory MAT, Montreal, Qc, Canada) in 1 L of distilled water to

give a final concentration ranging between 0.5 to 0.05 M. Sodium hydroxide (NaOH) was

purchased from VWR International LLC, West Chester, PA, USA. Concentrated

hydrochloric acid (HCl) was purchased from Fisher Scientific (Canada).

5.5.2. EAS generation

In this study a three-cell electro-activation reactor was used. The ruthenium–iridium coated

titanium electrodes were connected to a direct electric current (200 mA) power source so that

the anode was connected to the positive side and the cathode was connected to the negative

side of the DC-electric current generator (Lambda, GR.260, Electronics Corp. Melville, L.I.,

NY, USA). The anode and cathode compartments were separated from the central

compartment by an anion (AM-40) and cation (CM-40) exchange membranes (purchased

from Schekinaazot ltd., Russia). The electrodes (100x40 mm) were placed in the anode and

cathode compartments, with the distance between the electrodes being 150 mm.

The EAS was made from two configurations. In the first configuration, all the three

compartments were filled with NaCl (0.05 M), in the second configuration the cathode and

the central compartment were filled with the NaCl (0.1 M) solution, whereas the anode

compartment was filled with NaHCO3 (0.05 M). The resulting EAS from both configurations

were optimized as described in previous work to give the pH value of each solution between

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2 to 6. As result following solutions were chosen: A2, A3, A4 (configuration #1) and A6

(configuration #2), their excitation times were 30, 14, 5 and 60 minutes, respectively. The

solutions were named by the first letter of section from which they were taken (anode

section), followed by the pH of obtained solution (Table 5.1).

During the treatment EAS was analyzed for change in pH and redox-potential (E) using a

pH-meter (Model SR 601C SympHony, VWR Scientific Products, USA) and ORP-meter

(Eco Sense ORP15A). ZoBell’s standard solution was used for the calibrating the ORP-

meter. The values of the applied voltage were measured with a Lambda generator (GR.260,

Electronics Corp. Melville, L.I., New York, USA) and the intensity of the electric current

was measured using a circuit-test (DMR-1000) which was connected to the power source.

5.5.3. Relaxation period

Duplicate sets of EAS were generated and collected in 250 ml narrow mouth glass reagent

bottles (Corning Pyrex). The bottles were subsequently closed with glass stoppers and stored

in dark at ambient temperature (22 ± 3°C). During the storage the change in redox-potential

(E), pH, dissolved oxygen (DO) and total residual chlorine concentrations (RC) of the

solutions were measured periodically. The dissolved oxygen was measured using a DO meter

(Model SB 90M5 SympHony, VWR Scientific Products, USA). Measurement of the redox-

potential and pH was done as detailed above. Total residual chlorine (free and combined

chlorine) was measured with a chlorine colorimeter kit (Orion AQ3070, Thermo scientific

AQUA-fast, Singapore).

5.5.4. Corrosion dynamics of tinplate can

For the corrosion analysis of food surface container, the enamelled white can 300x407

(Dominion & Grimm, Quebec, Canada) was used. The tested solutions were prepared on the

day of experiment, heated to 85 ± 5 °C and seamed (Dixie UVG6MD, USA). The seamed

cans were sterilized at 121 °� for 1 hour in a horizontal autoclave (Gebr.Stork & Co. In,

Holland) without agitation, chilled, dried and stored at ambient temperature (22 ± 3 °C)

(Platonova, 2009). For analysing the dissolution of the metal ions in the stored product,

inductively coupled plasma (ICP, Optima 4300 DV, Perkin-Elmer, Norwalk, CT) was used.

140�

The concentration of zinc (Zn), iron (Fe), copper (Cu), tin (Sn) and aluminum (Al) was

measured at wavelengths 213.857, 239.562, 324.752, 283.998 and 237.313 nm, respectively.


Each analysis was carried out in triplicate. Minitab software (V16.0, Minitab Inc., USA) was

used for all statistical analysis of the obtained data, including evaluating the process

efficiency or comparing the mean values. The Tukey's method (one-way ANOVA) with a

confidence interval 95 % was use to adjust the confidence level for each individual interval.


5.6.1. Effect of EAS relaxation time during storage

Table 5.1 demonstrates the proprieties of EAS, acidified (NaCl-2) and non-acidified (NaCl)

control solutions. The physiochemical properties of EAS depends on the characteristics of

the electrochemical cell and its operating parameters (Liato et al., 2015c). Thus, as seen from

table 5.1, configuration one generates solutions (A2, A3 and A4) with low pH and high E

while the second configuration generates solution (A6) with neutral pH and high E. The EAS

differed in their content of RC, D.O., E and pH.

Table 5.1: Properties of tested solutions

Solutions pH E, mV RC, mg/L DO, mg/L

A2 1.98 ± 0.12 >1200 205 ± 10 19 ± 3.5

A3 3.05 ± 0.10 1115 ± 11 34 ± 6 14 ± 6.1

A4 3.98 ± 0.11 915 ± 8 4 ± 2 11 ± 3.3

A6 6.45 ± 0.07 935 ± 7 350 ± 17 25 ± 2.2

NaCl 6.50 ± 0.04 355 ± 11 - 4.6 ± 1.8

NaCl-2 2.00 ± 0.03 395 ± 10 - 4.5 ± 2.9

Several studies involving analysis of physiochemical and antimicrobial properties of EAS

during long-term storage demonstrated that the best method of preserving the solutions is

keeping it sealed in a chemically inert container, like a glass flask (Bordun and Ptashnyk,

2012). During the analysis the solutions A3 and A4 were found to be the most unstable; their

141�

�

redox potential decreased and equaled that of the control solution (NaCl-2) at (380 ± 11 mV)

at the end of the 30 day storage period (Fig. 5.1). Despite the fact that concentration of DO

of EAS was observed to have decreased to 4.6 ± 1.8 mg/L equal to that of the DO of the

control solution (NaCl) after 5 hours a significant change in the redox potential of EAS was

not seen (data not shown). The concentration of residual chlorine decreased below the

detection level (0.02 mg/L) of A4 at 30th day and for A3 at 70th day, respectively. Our results

were in accordance with Hsu and Kao (Hsu and Kao, 2004) which suggests that EAS exposed

to the atmosphere contained less chlorine and oxygen than when kept in a closed systems for

a long time. The authors point out that in their experiment, after 21 days of measurement

(periodic opening only for measurements), they observed significant decrease in residual

chlorine and dissolved oxygen in the EAS, probably due to evaporation. However, it should

be noted that storage experiments with opened bottles, showing decrease in residual chlorine

and dissolved oxygen, demonstrated the evaporation following to first-order kinetics,

whereas it did not correspond to closed conditions (Hsu and Kao, 2004).

The solutions A2 and A6 showed the longest time of relaxation, their redox potential was

stable for more than 140 days (Fig. 5.1). It should be noted that the treatment (generation)

time of A2 and A6 solutions was 30 and 60 minutes; markedly longer than others. Based on

recent investigations it may be assumed that the most activated form of EAS is obtained near

the electrode interface, where a variety of reactions occur, therefore it may be safely assumed

that a longer excitation time may result in generation of more metastable compounds (i.e.

radicals, chlorine compounds, dissolved oxygen etc) in the EAS (Prilutsky and Bakhir, 1997).

This assumption could also explain the longer relaxation period of the redox potential of both

EAS; the E values of A2 and A6 decreased to ∼377 mV after 190 and 270 days (data not

shown). The concentration of residual chlorine (RC) of solutions A2 and A6 decreased

gradually; by the 100th day the RC concentration of A2 decreased below the detectable limit

(Fig. 5.1), solution A6 showed the same result on 140th day, however the E value of both

solutions remained stable throughout the storage duration. A decrease in RC during the long-

term storage test of EAS was observed by Robinson et al. (Robinson et al., 2012), they found

that EAS stored for more than 200 days had a free chlorine concentration of �0.01 mg/L.

They also found that the bactericidal activity did not change after the storage duration even

in the absence of measurable chlorine. However, in the work of Len, S.-V., et al. (Len et al.,

142�

2001), it is seen that during the storage of EAS a reduced bactericidal activity was observed

possibly due to the evaporation of the dissolved chlorine gas and ensuing decomposition of

hypochlorous acid (HOCl). The main disadvantage of EAS is the decrease in its bactericidal

activity after the relaxation period, it reverting to an ordinary salt solution wherein the pH

remains generally unchanged. Thus, it should be noted that pH could also be one of the

important factors influencing the relaxation time of EAS. In our studies the pH of EAS did

not significantly change after relaxation which was in accordance with the work done by Cui

et al. (Cui et al., 2009). They observed that pH of neutral and acidic EAS remained stable till

the end of investigation. Furthermore, authors found that loss of available chlorine was

significantly higher in acidic EAS than in neutral EAS.

Bakhir et al. (2003) suggest that the main disinfecting element in EAS might be the residual

metastable chlorine, depending on the pH value of the EAS the equilibrium reaction of

chlorine may shift and result in an increased concentration of hypochlorite anions (OCl-)

(Bakhir et al., 2003). The generation of chlorine depends upon a specific combination of E

and pH where a minimum pH value of (<3) is required for chlorine gas (Cl2) production while

a pH �3 results in production of only HOCl and OCl- (Pourbaix, 1974b; Zaviska et al., 2012).

Therefore a shift in this equilibrium may result in production of lesser Cl2, favoring formation

of HOCl, which is not volatile and could probably slow the decrease of redox-potential as

well as result in a longer relaxation time. A similar result was shown by Len et al. (Len et al.,

2001), in their study involving EAS and chlorinated water with different pH values ranging

between (6.0 and 9.0.), they reported that the solutions with lower pH displayed significantly

higher loss of chlorine than solutions with alkaline pH. However, in a closed environment,

as in this study, the primary mechanism of chlorine loss may be self-decomposition of

chlorine species in the solution (in the absence of evaporation).

143�

�

Figure 5.1: The effect of storage on the change of free residual chlorine (bars) and of redox-

potential (curves).

�

5.6.2. Effect of EAS on the corrosion of tinplate containers

The data obtained by inductively coupled plasma was used to determine the concentration of

the metal ions which potentially migrated from the container walls to the solution (Fig. 5.2).

The concentration of zinc was more important in solutions 2 and 3 (0.028 and 0.025 ppm)

followed by A4 (0.014 ppm) and the acidified control solution (NaCl-2) at 0.010 ppm (Fig.

5.2A). The solutions 6 and neutral control solution (NaCl) were found to be least corrosive

with the dissolved Zn concentration being at 0.006 and 0.005 ppm, respectively. After 4

weeks of storage, the concentration of iron in solution 2 (28.814 ppm) was observed to be

the highest and significantly different to that of the controls; NaCl-2 (14.5 ppm) and NaCl

(0.0061 ppm) (Fig. 5.2B). Solutions 3 and 4 with 6.992 and 3.883 ppm, did not have a

significantly different concentration of iron to each other but were significantly different to

the values of both the control solutions (�>0.005). Furthermore the neutral EAS (A6) at

0.0062 ppm had the smallest concentration closest to the detection limit of 0.0018 ppm.

Fig. 5.2C shows that in one month the concentration of copper was low and close to the

detection limit (0.0054 ppm) for all solutions except A2, which displayed a significant

corrosion rate ability to corrode the container. The concentration of copper in A2 (0.0220

ppm) was twice as much than in the other samples. Even though the solutions 4, 3, 6

144�

and control samples were less active, the presence of Zn indicated that they had caused

corrosion (0.0112, 0.0098, 0.0091, 0.0089 and 0.0080 ppm, respectively). Corrosion may be

defined as a process dependent on the interaction of a material and its immediate environment

which results in the degradation of the material surface (Waters et al., 2014). The main

characteristic of corrosion is the reaction rate which depends on the environmental impact

(such as contact with water, acids, bases, salts, some chemicals, gaseous compounds like acid

vapors, sulfur-containing gases or ammonia gas) and time. Tinplate of cans is a light gauge,

cold reduced, low-carbon steel sheet or strip, coated on both sides with pure tin. Many studies

have shown that corrosion process of tinplate is complicated due to its scarified structure and

heterogeneity. The corrosion of tinplate in contact with NaCl solutions increases significantly

with an increase in salt concentration (Xia et al., 2012). In our study, all solutions were 0.05

M NaCl concentration, except A6 solution which contained 0.05M NaHCO3. The control

solutions were prepared from NaCl (0.05 M) solution, than adjusted to necessary pH by

0.025M HCl or NaOH. The pH of the media may be significant, due to the complex behavior

of tinplate. If the EAS are able to retain their original properties (pH and redox potential) for

a long time (Fig. 5.1), it is possible that they could stay stable with chemical properties and

levels as at the time of preparation. However at the moment of opening the EAS lost its

activated form and reverted to the normal state with a pH equal to the initial pH before the

commencement of storage (data not shown).

145�

�

�

�

�

�

Figure 5.2: Changes in zinc (A), iron (B) and copper (C) concentrations during four weeks

storage with different tested solutions.

BC

DE

AB A

CD

E

B

C

AB

A

BC

C

BC

D

B

A

BC

D

C

D

A

A

B

CD

0

0,006

0,012

0,018

0,024

0,03

0,036

NaCl2 NaCl A2 A3 A4 A6

Zn

, p

pm

Tested solutions

A 1 week

2 week

3 week

4 week

Detection

limit

B

D

A

B C

D

C

D

A

B

C

D

B

D

A

C

C D

B

D

A

C

C

D

0

5

10

15

20

25

30


Fe

, p

pm

Tested solutions

B 1 week

2 week

3 week

4 week

B

B

A

AB

AB

B

A

B

A

A A

B

AB

B

A

B

B

B

B

B

A

AAB

B

0

0,005

0,01

0,015

0,02

0,025


Cu

, p

pm

Tested solutions

C 1 week

2 week

3 week

4 week

146�

Low pH values, particularly below 5, seemed to be more capable of diluting metals (cooper,

iron, etc), the advantage of tinplate lies in protection of the steel of cans. Some metals like

aluminum, tin and zinc increase in corrosion rate when pH is above 9 (Xia et al., 2012). In

our studies, aluminum and tin was not detected in the solutions or their concentration was

below the limit detection (0.0230 and 0.096ppm, respectively). However alkaline solution

(catholyte, pH=12 ± 0.02, E = -966 ± 13 mV) showed high tin and aluminum dilution (6 and

0.05ppm, respectively) after 4 weeks of experiment (data not shown). Change in metal level

in the solutions is shown in Fig. 5.2, it can be seen that a decrease in the metal concentration

is observed with a decrease in the pH of the respective EAS. The Tukey`s test was used to

show the difference between each tested solution during the same time period. The control

solutions also followed the same trend, with the pH of the solution being inversely co-related

to the metal concentration in the solution. However, studies done on the corrosion of food

cans have demonstrated that even though pH has a significant impact, it is not the only factor,

for example the test with different acids like citric, malic or tartaric showed that the acids

alone were less corrosive than the fruits (Hartwell, 1951). In works of Platonova et al.

(Platonova, 2009), a dissolution of iron and zinc was observed in tomato, grape and apple

juices at pH values ranged between 2.8 and 3.3 and stored in tinplate cans after ten days. The

dissolution of iron was almost the same in all the samples (5.5 ± 0.2 ppm), while the

dissolution of tin was found as following: grape > apple > tomato (47.5, 22.0, 15.0 ppm,

respectively). Despite the fact that the influence of pH is significant, the impact of EA

solutions is more important in terms of corrosion rate with the course of time. In order to

avoid the tin and iron contamination, lacquers or different kinds of corrosion inhibitors can

be used in order to protect the surface of the container (Nin�evi� Grassino et al., 2009).

Although, corrosion can form under the layer of lacquer due to a variety of reasons such as

improper uniform coating, damaged layer during manufacturing and packaging or the

influence of other physical or chemical factors (Platonova, 2009). It is known that EAS

inherently includes reactive components such as active chlorine and dissolved oxygen which

may cause high corrosion. Tanaka et al. (1999) found that EAS (pH2.3-2.7, E=1000 mV, and

available chlorine 10-50 ppm) has a higher rate of dissolving stainless steel of hemodialysis

equipment than 0.1% sodium hypochlorite solution (available chlorine is 1000 ppm) (Tanaka

et al., 1999a). For EAS, the same authors found that the concentration of ferric ions on the

147�

�

36th day of soaking was 0.6 mg/L while sodium hypochlorite solution was less than 0.05

mg/L. In the work of Ayebah et al. (2005) stainless steel showed outstanding corrosion

resistance in EAS (pH = 2.42, E = 1077 mV, chlorine concentration 48.66 mg/L), deionized

water (pH=6.37, E=584 mV, chlorine concentration 0 mg/L), chlorinated water (pH=8.72,

E=656 mV, chlorine concentration 49.16 mg/L), modified EAS (pH=6.12, E=774 mV,

chlorine concentration 50.39mg/L) (Ayebah and Hung, 2005). The highest mass loss of

metals such as carbon steel, aluminum and copper coupons (immersed in tested solutions for

22 hours) was observed in EAS, suggesting that chloride ions could be one of the primary

causes of corrosion. Authors found highly significant correlation between the decreasing

concentration of chlorine and average weight loss. In our study, after the storage duration in

cans, the EAS exhibited changes in chemical properties after the first week of experiment.

After opening the container, no available free chlorine was found, the E value decreased and

was observed to be 380±13 mV (data not shown). However, the pH remained stable and

consequently may be assumed to be a crucial factor causing corrosion of the tinplate in this

study. The work of Waters et al. (2014) also confirmed that pH of chlorine-based sanitizers

is a significant factor in determining the corrosion rate of different types of metals used in a

food processing environment (Waters et al., 2014). However, the high concentrations of

metals in this study indicate that EAS solutions have a significant corrosive effect on certain

types of tinplate. The corrosion of metallic materials in contact with aggressive media

involves a whole range of factors, which may act singly or jointly (Ayebah and Hung, 2005).

The simultaneous reaction of chemical components of EAS at temperatures such as during

sterilization and filling (90 °C) could cause significant damage to the coating layer (lacquer,

tin). After visual observation the majority of cans were found to have initiation points of

corrosion on the side seam and locale sites. These observations suggest that this type of

corrosion is the typical pitting corrosion induced by the breaking of the passivation film or

coatings. The presence of reactive elements (free chlorine, chlorine compounds or dissolved

oxygen) could accelerate the kinetics of reaction which can explain the significant difference

in the corrosivity of acid EAS, neutral EAS and control solutions. In this study the analysis

of corrosiveness of EAS on the tinplate surface was carried out based on the indirect method

of observing the migration of metals from the container to the solution, additional studies are

required to understand the reactivity of chlorine-based solutions such as EAS in contact with

148�

the can surface in combination with different storage temperatures in as well as with different

foods.

5.6.3. Changing of metals concentration of the canned corn produced with EAS

The metal content found in canned corn is presented in the figure 5.3. The Tukey’s test was

used to show the difference between the analyzed samples at one period of time. The corn

samples were purchased from local market, then peeled, blanched and sterilized as described

above. Analyses of metal concentration in the canned corn were carried out at week 4 and

week 52. After opening the container, no visual defects on surface was found but the increase

of concentration of metal content with time indicates that the corrosion took place. The

presence of aluminum and tin was not detected or their concentration was below the limit

detection. At the end of experiment, the highest level of metal was found in A2, where the

concentration of zinc, iron and copper were at 1.73±0.0009, 4.87±0.12 and 0.70±0.04 ppm,

respectively. It should be noted that the canned corn in EAS had a significantly low

concentrations of the different metals, well within the safe consumption limit for tin (�14

mg/kg, Codex, 1998), for zinc between 9-14 mg/day (NRC 2000), for iron 8.7-14.8 mg/day

(Food Standard Agency 2010), copper 2.1-3 mg/day (WHO), and aluminum at �50 mg/day

(WHO).

After one month of storage of the product in the EAS, a change in the properties of EAS was

observed because after contact with any organic matter EAS becomes ordinary water (Huang

et al., 2008). It was observed that A2 solution had significant difference in iron and copper

content after one month and of zinc after one year. The synergy between different factors

(chlorine ions, acidity and high redox-potential) could cause corrosion, especially when the

hot solutions come in contact with the can surface. Besides, the metal concentration detected

partly could be due to their transfer from the product. The metal level in canned vegetables

may vary in a large spectrum, for example the zinc, iron and copper levels have been reported

in the range of 1.0–8.9, 9.3–76.0l and 0.07–7.30 ppm, respectively, significantly higher than

found in our study (Tuzen and Soylak, 2007). The influence of added materials (sugar,

syrups, salt, etc.) or those present in vegetable substrate (amino or organic acids, phenols,

etc.) may also significantly impact the corrosion rate, but these organic and inorganic

substrates may also retard it (Hartwell, 1951). Furthermore, the usual pH of the media is

149�

�

neutral, corrosion rate and related properties in such a condition are still uncertain (Xia et al.,

2012). Corrosion in canned food may also be supported by the trace amounts of oxygen

present due to unsatisfactory commercial practice or leakage, which has been recognized as

a factor responsible for accelerating corrosion (Hartwell, 1951).

150�

Figure 5.3: Changes in zinc (A), iron (B) and copper (C) concentrations during 4 and 52

weeks of storage in canned corn with different tested solutions.

151�

�

5.7. Conclusions

The relaxation time was longer for neutral EAS compared to that of acid EAS. The decrease

in redox-potential as a function of pH showed that neutral medium inhibits the decay of free

chlorine in a relatively acidic medium it is accelerated. A better understanding between the

relaxation time and the change in values of redox-potential is important to clarify the

reactivity of EAS during storage and more importantly the nature and attributes of electro-

activation of solutions. Contrary to assumption it was found that temperature close to the

boiling point did not weaken the EAS but accelerated its reactivity. Storage in glass bottle

provided a good protective and inert environment for the EAS.

This study also showed that corrosion caused by free chlorine is not significant in EAS with

pH near to neutral while in acidic EAS the rate of corrosion was higher which may be

attributed to the combined effect of pH and chemical compounds (free or/and combined

chlorine, radicals etc) present in EAS. Although EAS was found to be significantly more

corrosive than the control solutions (in the canned corn and tinplate cans) the dissolution of

metals in canned corn with EAS (neutral and acid) was equal to or lower than in canned

vegetable food, as it was found in the control solutions. Therefore EAS may be used as a

non-corrosive replacement to the solutions used which in itself are primary agents of

corrosion in canned food. Thus to sum up, EAS is a viable alternative with its corrosiveness

equal to or less than the canning solutions currently employed in the food canning industry.






153�

�

6. CHAPTER 6: Study of the combined effect of electro-activated

solutions and heat treatment on the destruction of spores of

Clostridium sporogenes and Geobacillus stearothermophilus in model

solution and vegetable puree

Viacheslav Liato1,2, Steve Labrie1,2, Catherine Viel1,2, Marzouk Benali3, Mohammed

Aïder1,4


G1V 0A6, Canada.






This article is submitted for publication in International Journal of Food Microbiology.




Catherine Viel (Scientific collaborator of the project: correction and revision of the

manuscript), Marzouk Benali (Technical collaborator of the project: correction and

performing of the experiments, revision of the manuscript) and Mohammed Aïder

(Supervisor of thesis: scientific supervision of the student, correction and revision of the

manuscript).

154�


This chapter extends the information about antimicrobial capacity of EAS. The effect of EAS

optimized in previous chapter (2) was studied on the behaviour of the spores of C. sporogenes

and G. stearothermophilus. These strains are widely spread in canning, causing the food

spoilage problems, and are commonly used like surrogate strains for pathogen C.

botulinum. This chapter demonstrates the study of inhibition effect of EAS combined with

the effect of temperatures on the spores in the product and without it. The hypothesis of this

work assumes that the EAS in combination with mild temperature will have synergetic effect

on inhibition of heat resistant spores, which may allow to reduce the temperatures of

sterilization of canned vegetables.

�

� �

155�

�

6.2. Résumé

L’effet combiné du traitement thermique et des solutions électro-activées (SEA) sous

différentes combinaisons de chauffage et de temps d'exposition ont été testés sur la résistance

thermique des spores de Clostridium sporogenes et de Geobacillus stearothermophilus. Les

SEA neutres et acides ont démontré la plus importante activité inhibitrice qui indique que ces

solutions peuvent être considérées comme de forts désinfectants sporicides. Les SEA ont

causé une réduction des spores C. sporogenes supérieure à 6 uniquement après une minute

d'exposition à la température 60 °C. Les spores de G. stearothermophilus ont été réduites de

4,5 log pendant un traitement d’une minute à la température 60 °C, tandis que le traitement

de 5 minutes a entraîné une réduction des spores supérieure à 7 log. Des purées de petits pois

et de maïs en grain inoculées ont été utilisées comme matrices alimentaires modèles pour la

détermination de la résistance thermique des spores au cours de traitements de chaleur dans

des capillaires en verre. Les cinétiques d'inactivation des spores ont été testées dans un bain

d'huile. L’effet du traitement combiné de SEA et de la température a démontré une

diminution significative de la résistance de C. sporogenes. La valeur �2RR�L de C. sporogenes

a démontré que le traitement de la purée de petits pois inoculée avec une solution de NaCl

était de 66,86 min tandis que cette valeur était réduite à 3,97 et à 2,19 min en utilisant un

traitement avec des SEA acides et neutres, respectivement. Les spores de

G. stearothermophilus ont confirmé leur forte résistance à la chaleur telle que déjà démontrée

dans d’autres études. La valeur �27R�L des spores de G. stearothermophilus dans la purée de

petits pois inoculée a montré une diminution de 1,45 min dans une solution de NaCl et jusqu'à

1,30 et 0,93 min pour les EAS acides et neutres EAS, respectivement. Les différences entre

les spores de ces espèces sont attribuables à leurs sensibilités à l'égard du pH, du potentiel

redox (E) et de l'oxygène.

156�

6.3. Abstract

The combined effect of heat treatment and electro-activation solution (EAS) on the thermal

resistance of Clostridium sporogenes and Geobacillus stearothermophilus spores was

assessed under various heating and exposure time combinations. The acid and neutral EAS

showed the highest inhibitory activity, indicating that these solutions may be considered as

strong sporicidal disinfectants. These EAS were able to cause a reduction of � 6 log of spores

of C. sporogenes at 60 °C in only one minute of exposition. For G. stearothermophilus

spores, a reduction of 4.5 log was observed at 60 °C in one minute, while in 5 min, a reduction

� 7log CFU/ml was observed. Inoculated puree of pea and corn were used as a food matrix

for the determination of the heat resistance of these spores during the treatments in glass

capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined

treatment by EAS and temperature demonstrated a significant decrease in the heat resistance

of C. sporogenes. The D100°C in pea puree with NaCl solution was 66.86 min while with acid

and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G.

stearothermophilus displayed higher heat resistance as confirmed by other similar studies.

Its D130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and

0.93 min for acid and neutral EAS, respectively. The differences between the spores of these

species are attributable to their different sensitivities with respect to pH, Redox potential (E)

and oxygen.

157�

�

6.4. Introduction

One of the most serious foodborne diseases associated with the canning industry is botulism,

resulting from ingestion of preformed Clostridium botulinum neurotoxin (Diao et al., 2014).

C. botulinum is a mesophilic obligate anaerobe and spore-forming bacteria. The bacteria

growing between 10–50 °C with an optimal around 35-40 °C (Taylor et al., 2013). C.

botulinum is heat-resistant and are known to survive in low-acid, ambient-stable canned

products. Apart from being responsible for foodborne botulism, it is also a leading cause of

home and commercially canned food spoilage (Diao et al., 2014; Stumbo, 1973). With its

ability to sustain chemical disinfectant and heat-sterilization processes combined with its

disease causing and food spoilage attributes, C. botulinum poses serious challenges to the

food industry (Setlow, 1994, 2006). The heat-resistant endospores generated in the anaerobic

food environment have been documented to survive in heat-processed foods, and thereafter

germinate and grow in sealed packs where anaerobic conditions develop. In order to

eliminate these spores from foods, a severe heat treatment is required owing to their high

resistance to physical and chemical stresses (Naim et al., 2008).

The two main goals of food processing include quality preservation and safety during shelf-

life storage. Based on the kinetic parameters (D- and z-values) the canning of low-acid foods

conventionally uses the 12-D cook, also known as “botulinum cook” (Anderson et al., 2011).

This widely used process although ensures food safety but not without compromising on the

nutritional value and sensory properties (color, aroma and texture changes) of the end product

(Naim et al., 2008). Furthermore, consumer trends require high-quality, minimally processed

foods, which is not subjected to the traditional thermal processing methods causing loss of

desirable properties related to texture, flavor, color and nutritional value (Stewart et al.,

2000). In this context and in order to satisfy current consumer demand for convenient and

more “natural” foods, without compromising on nutrition or food safety, Leistner et al.

(2000) proposed an approach of application of different preservation treatments at lower

individual intensities. This approach is called `hurdle technology`` (Leistner, 1992; Leistner,

2000). The most important hurdles include temperature (high or low), low water activity (aw)

acidity (pH), redox potential (E), preservatives (e.g., nitrite, sorbate, sulfite), competitive

158�

microorganisms (e.g., lactic acid bacteria) (Leistner, 2000). In this way, it is possible to use

a combination of nonthermal and compositional parameters to ensure the safety of food

products. A similar and feasible approach for the canning industry could be the combined

application of thermal treatment and electro-activation technology, recently recommended as

a strong antimicrobial method finding wide applications in food and biotechnology industry

due to its low cost and ease of application (Aider et al., 2012).

The technology of electro-activation is based on a phenomenon called modulated (controlled)

electrolysis which requires the aqueous solution to be subjected to the effect of an external

electric field in an electrochemical reactor (Aider et al., 2012). The reactor is composed of

three compartments with two ion-exchange membranes placed to separate the solutions in

the anode, central and cathode compartments. This results in the oxidation and reduction

reactions to occur simulataneously at the electrode/solution interface (Liato et al., 2015c).

The acidic solution (anolyte) which is generated at the anode/solution interface has an acid

pH which can vary between 1.5-4.5 and an oxidation reduction potential (E) of � +1150 mV.

A basic solution (catholyte) is generated at the cathode/solution interface and has an alkaline

pH with a Redox potential of < - 950 mV, as shown in (Eqs. 6.1-6.5). The reactions are

presented further:

Anode side:

2H2O�4H++O2�+4e- (Eq. 6.1)

2NaCl�Cl2�+2e-+2Na+ (Eq. 6.2)

Cl2+H2O�HCl+HOCl (Eq. 6.3)

�

Cathode side:

2H2O+2e-�2OH-+H2� (Eq. 6.4)

2NaCl+2OH-�2NaOH+Cl- (Eq. 6.5)

�

The metastable state of the electro-activated aqueous solutions (EAS) was previously

confirmed by Raman scattering spectroscopy analysis. The authors observed that after

excitation by an external electric field, the reactivity of EAS was significantly increased

suggesting that this phenomenon uses the vibrational spectrum of water (Aider et al., 2012).

159�

�

The fact that electro-activation has been found to have no discernable side effects on the

human body and the immediate environment, this procedure found important applications in

food industry such as baking wheat bread (Nabok, 2009), activation of enzymes (Khrapenkov

et al., 2005), activation of antioxidant enzymes (Podkolzin et al., 2001). Furthermore, the

most vital application of EAS involves inactivation of microorganisms at a large scale

(Huang et al., 2008), including viruses and biofilms (Aider et al., 2012). It is known that C.

botulinum is very sensitive to pH below 4.5, an oxidative redox potential and the presence of

oxygen in the medium (Linton et al., 2014). The electro-activated solution at the anode

/solution interface meet these criteria, as they have a pH which can vary from 1.5 to 4.5, a

redox potential which can easily reach +1200 mV and a good saturation by oxygen.

Therefore, they can potentially act as effective barrier against C. botulinum.

According to the aforementioned information on the properties of electro-activated solutions

which are characterized by low pH, high oxidative Redox potential and oxygen saturation,

these solution may have potential inhibitory effect on the spores of C. sporogenes used as a

non-pathogenic surrogate of C. botulinum. To exclude the exclusive effect of oxygen,

Geobacillus Stearothermophilus which is oxygen non-sensitive organisms will be used to

demonstrate the sporicidal effect of EAS against C. sporogenes. Thus, the aim of this work

was to investigate the effect of EAS on food spoiling spore-forming bacteria (Clostridium

sporogenes and Geobacillus Stearothermophilus); alone or combined with different heat

treatment intensities. Furthermore, the combined effect of EAS and heat treatments was

assessed to find the inactivation kinetics of the spores.


6.5.1. Chemicals

Solutions were prepared by dissolving crystalline sodium chloride (NaCl) and sodium

bicarbonate (NaHCO3) (Laboratory MA, Montreal, Qc, Canada) in 1 L of distilled water to

achieve a final concentration 0.05 M. Concentrated hydrochloric acid (HCl) was purchased

from Fisher Scientific (Mississauga, ON, Canada) and used for dilute HCl solutions

preparation. Yeast extract, Brain Heart Infusion and Bacto Agar were from BD (Becton,

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Dickinson & Co., Sparks, MD, USA). L-cysteine was purchased from Amresco (Solon, OH,

USA).

6.5.2. Vegetable puree preparation

Frozen pea (Pisum sativum) and corn (Zea mays) were purchased from a local market and

stored at -18 °C until use. After defrosting, the both products were treated by wet steam to

generate soft granules. Thereafter, pea and corn were blended (New Hartford, USA) to give

a uniform homogeneous puree. The obtained purees were placed in a hermetically sealed

container and sterilized at 121 °C during 45 min including the pre-heating step. Such heat

treatment was used because of the low heat conductivity of the purees. This treatment was

then followed by gradual cooling to room temperature and subsequent storage at 4 °C until

they were used.

6.5.3. Spores production of Clostridium sporogenes ATCC 7955

C. sporogenes (ATCC 7955) freeze-stock spores were prepared according to the method

developed by Sorge and Uehara (Sorg and Sonenshein, 2010; Uehara et al., 1965). Firstly,

the stock culture was inoculated into 10 ml Brain heart infusion-�supplemented (BHIS), Yeast

extract and 0.1 % L-cysteine. After 24 h of incubation at 37 °C, the cells pellet was gently

streaked on BHIS medium (BHIS and Bacto agar) and incubated under anaerobic conditions

in the jar (Oxoid) with added Anaerogen sachet (AN, Thermo-Scientific). Thereafter, the

Petri plates were removed from the incubator to scrape up the surface composed of vegetative

cells, spores and debris. A volume of 5 ml of sterile ice-cold water was poured on every plate

to protect the flora against oxygen and to easily remove the culture. After overnight

incubation at 4 °C, the spores were washed three times in ice-cold sterile water and

centrifuged at 5000xg at 4 °C to separate the mature spores from the cells. The resulting

suspension was separated in 50 % (w/v) sterile sucrose-water solution. It was then

centrifuged for 20 min at 3200xg at 4 °C. The spores were precipitated and harvested from

the pellet. The spores were then re-suspended in sterile water and washed three times in sterile

ice-cold water, as described previously, followed by heating in boiling water for five minutes.

The resulting 1 ml water-spore solution was added with equal volume of BHIS-30 % glycerol

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solution. This spore stock solution was stored at 4 °C. For the subsequent experiments, the

stock solution was diluted in order to obtain 8 log CFU/ml spore solution.

6.5.4. Spores production of Geobacillus stearothermophilus ATCC12980

Freeze-dried culture of G. stearothermophilus (ATCC 12980) was purchased from American

Type Culture Collection and stored at -80 °C until use. The method of the spore preparation

was adapted from the method of reported in. The culture was hydrated in a 5 ml pre-

sporulation medium Nutrient broth (Difco) containing (37.5 % beef extract, and 62.5 %

peptone) at 55 °C for 12-14 h under aerobic conditions on a rotary shaker. After incubation,

the media was centrifuged at 5000xg during 20 min and the resulting 2 ml of pellet was

spread onto the Nutrient Agar (EMD) sporulation medium containing 15 % meat extract, 25

% peptone meat and 60 % agar-agar, and incubated for 10 h at 55 °C. After incubation, the

20 ml of Nutrient broth was poured on the plate surface, and the surface cultures were gently

scraped. The culture suspension was added to 80 ml nutrition broth (Nutrient broth, Difco)

in a 2-liter Erlenmeyer flask. Thereafter, the media was incubated on a rotary shaker for 14

h at 55 °C, and this culture served to inoculate 40 Nutrient Agar Petri plates. The inoculated

plates were incubated for 48 h at 55 °C and subsequently the cultures were scraped from the

surface, suspended in sterile ice cold water and centrifuged at 5000xg, 4 °C for 15 minutes.

The supernatant was discarded and the pellet containing the spores was washed three times

with distilled ice-cold water while maintaining the volume at 1 ml. The final concentration

of spores was 9 log CFU/ml. The spores were stored in sterile water at 4 °C until further use.

6.5.5. Control on the quality of spore suspensions.

The quality of the spores in the corresponding suspensions was systematically controlled to

ensure the validity of the experiments. The quality of C. sporogenes was assured according

to the methods used for spore preparation in which the spores were found to be free of

vegetative cells and debris (Microscope Retiga-2000R, Qlmaging, Canada). In the case of

the G. Stearothermophilus, the quality control was carried out according to the method based

on the lysozyme treatment which is necessary to kill vegetative cells.

162�

The microscope examination showed there were few vegetative cells left. The heat activation

and count of C. sporogenes and G. stearothermophilus (80 °C during 10 minutes) were

provided to verify the initial spore concentration.

6.5.6. Preparation of electro-activated solutions

The electro-activated solution (EAS) was made from 0.05 M NaCl (A2, A3, A4) and 0.05 M

NaHCO3 (A6) solutions to give four types of solutions with different properties,

characterized by the Redox potential (E) and pH (Liato et al., 2015c). The electro-activation

reactor cell was composed of a Plexiglas made three-cell compartments; namely the anodic,

central and cathodic compartments. The central compartment was separated by an anion

(AM-40) and cation (CM-40) exchange membranes (Schekinaazot, Shchekina, Russia) from

the anode and cathode, respectively. The ruthenium–iridium coated titanium electrodes were

immersed in the anode and cathode compartments and connected to the positive and the

negative side of the DC-electric current generator (Lambda, GR.260, Electronics Corp.

Melville, NY, USA), respectively. The solutions required different generation times at a

current density of 50 A/m2 (Table 6.1). The EAS were made immediately before use every

time. The pH and dissolved oxygen (DO) of the EAS was measured using a pH-meter (Model

SR 601C SympHony, VWR Scientific Products, USA) and DO meter (Model SB 90M5

SympHony, VWR Scientific Products, USA). The Redox potential (E) was analyzed using

ORP-meter (Eco Sense ORP15A). The Redox potential (ORP) meter was calibrated using

the ZoBell’s standard solution. Total residual chlorine (RC) was measured with chlorine

colorimeter kit (Orion AQ3070, Thermo scientific AQUA-fast, Singapore). A 0.05 M NaCl

solution (named NaCl) which was not electro-activated was used as a negative control, while

0.05 M NaCl acidified with 0.05 HCl (named NaCl2) was used as a positive acidic control

(Table 6.1).

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Table 6.1: The properties of EAS after optimization.

Solutions pH E, mV RC, mg/L DO, mg/L

A2 1.98 ± 0.12 >1200 205 ± 10 19 ± 3.5

A3 3.05 ± 0.10 1115 ± 11 34 ± 6 14 ± 6.1

A4 3.98 ± 0.11 915 ± 8 4 ± 2 11 ± 3.3

A6 6.45 ± 0.07 935 ± 7 350 ± 17 25 ± 2.2

NaCl 6.5 ± 0.05 255 ± 14 - 5.4 ± 1.2

NaCl2 2.0 ± 0.06 306 ± 9 - 5.5± 1.3

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6.5.7. Treatment of spore suspensions

To investigate the effect of the EAS on the inactivation of the studied spores, the electro-

activated solutions were placed in sterile screw-cap tubes and heated up to 30, 60 and 90 °C.

The solutions were agitated at 400 rpm using a rotary platform heater (Eppendorf AG,

Germany). One micro-liter of spore suspension was individually added to the tubes to achieve

the desired concentration of C. sporogenes and G. stearothermophilus of 6 and 7 log CFU/ml,

respectively in a total volume of 10 ml. Exposure times of 1, 5 and 15 minutes were used.

After each exposure time, the viable count of spore culture was determined by directly plating

(0.1 ml) or after a serial dilution (1:10) in sterile peptone water on appropriate medium in

triplicate. Spores of C. sporogenes were enumerated in BHIS medium after incubation at 37

°C under anaerobic conditions after 48 h. Spores of G. stearothermophilus were enumerated

in sporulation medium (Nutrition agar) after incubation at 55 °C during 48 h. The experiment

was conducted at least three times in each condition.

6.5.8. Heat resistance of spores in pea and corn purees with EA solutions

The heat resistance of the studied spores was analyzed by using glass capillary melting point

tubes (Drummond scientific, USA). Prepared vegetable purees were inoculated with spore

suspension to give a final concentration 7 and 8 Log CFU/ml for C. sporogenes and G.

stearothermophilus, respectively. The prepared EAS along with the positive and negative

controls were heated to 60 °C and mixed with inoculated puree at 1:1 (V/V) dilution and

vortexed to obtain a homogenous distribution of the spores throughout the suspension.

164�

Immediately, a volume of 100 µl of the resulting suspension was placed into the capillary

tubes, with a microdispenser (Drummond, Fisher) and heat sealed with a flame. The tubes

were placed in a thermal bath (Polystat, Cole-Parmer, Canada) with integral pump agitation

to maintain the required temperature and heating (Synthetic oil, Boss products,

Elizabethtown, KY). The tubes were then removed at different time intervals in triplicate.

After removal, the tubes were cooled by immersing in ice-cold water and surface sterilized

by washing in a soap solution and rinsing in 70 % ethanol. The spheres were crushed with a

sterile rasp and the viable spore enumerated using appropriate medium as described above.

The C. sporogenes spores in the 0.05 M NaCl solution (negative control) were heated at 100,

105, 110, 115, and 120 °C, and the spores in the EAS were heated at 70, 80, 90, 100 and 105

°C. For G. stearothermophilus both the EAS and control solutions were heated at 105, 110,

115, 120, 125 and 130 °C. This experiment was repeated three times. For each temperature,

the logarithm of survivors was plotted against respective heating time durations. The time

needed to achieve a 10 fold reduction in the cell count was calculated as the inverse negative

slope of the regression line. The temperature necessary to reduce the D value by 10-fold (Z

value) was determined from the regression as the inverse negative slope when plotting the

logarithm of D-values against the appropriate treatment temperature.

6.5.9. Calculations and statistical analysis

All experiments were carried out at least in triplicate and the mean values ± std were

considered. The obtained data were analyzed and plotted using a general linear model

procedure by using the Systat-10 Software (Systat Software, Inc. San Jose, CA, USA). After

dilution of initial spore stock, the final concentration was around 6 log CFU/ml in all tested

samples (for C. sporogenes), thus the detection limit by a direct plating procedure would be

around 6 log. The mentioned limit provides a �6 log reduction of the spores for C. sporogenes

and �7 log reduction of G. stearothermophilus. Therefore, statistically significant difference

was found between different solutions for a given treatment for a particular time-temperature

profile and accordingly no difference between one (e.g. A6) time-solution at different

temperature was observed. To assess the effect and interaction of EAS, temperature and

exposure time on spores surviving, an ANOVA and a regression analysis were performed.

Data were represented in a semi-logarithmic graph, with the logarithm of the surviving

165�

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organism as the ordinate and the heating time as the abscissa (Bigelow model). According to

this model, thermal destruction of microorganisms is calculated according to first-order

kinetic model at a given constant temperature:

wxy ± ²²V³ = �? (Eq. 6.6)

Where ´ is the number of survival spores after heat treatment, R is the initial number of

microorganisms and J is a temperature of heat treatment. µ is the first-order reaction rate

constant which can also be represented as:

� = </@. ¶V¶ (Eq. 6.7)

Where D is the time required to destroy 90% of the microorganisms or, in other words, is the

decimal reduction time. The D value has been obtained by plotting the average difference of

three experiments (triplicates each time) between log concentration of microorganisms

against the appropriate heat treatment time (time taken to traverse one logarithmic cycle) or

from the linear regression slope of log reduction and time:

< = (?@ − ?B) (ÊF²B −ÊF²@)I = −w/·wx¸¹ (Eq. 6.8)

The curve of destruction of (so-called survivor curves) indicating the logarithmic dependence

of spore death on treatment time duration. Log D value is plotted against temperature

resulting in a graph known as heat resistance curve, through which thermal sensitivity

parameter (Z value) can be calculated:

K = (?@ − ?B) (ÊF<B − ÊF<@)I = −w/·wx¸¹ (Eq. 6.9)

Data were analysed and plotted using general linear model procedures (Systat Software Inc.,

2010). To assess the effect of EAS, temperature and exposure time on spore survival and

their interactions, ANOVA and regression analysis were performed.

6.6. Results

6.6.1. Effect of EAS on inactivation of spores in moderate temperatures

To determine the temperature required for one log reduction in the D-value time which

represents the resistance of the targeted microorganism to differing temperatures (Z-value),

the log D-values were plotted against heating temperatures to obtain straight lines. In this

study, the Z-values obtained by treating spores in various purees at different temperatures

were used to discuss the feasibility of the proposed approach. During the heating time of the

EAS and control solutions, no significant pH and redox potential change were observed for

166�

each solution (data not shown). Owing to the unstable nature of the dissolved oxygen, it was

perceived that the heating of the EAS would have an effect on its concentration. Indeed, when

the temperature of the solution reached 60 °C, the dissolved oxygen concentration decreased

from 25 ± 2.2 (A6), 19 ± 3.5 (A2), 14 ± 6.1 (A3) and 11 ± 3.3 (A4) mg/L down to 5.5 ± 2.1

mg/L (Table 6.1). On the contrary, no change in the concentration of free chlorine was

observed when the EAS was heated up to 90 °C. The pH and the redox potential of all testes

solutions remained stable.

6.6.1.1. Effect of EAS, time and temperature on Clostridium sporogenes spores

The present study showed that C. sporogenes spores are sensitive to the electro-activated

solutions (EAS). Indeed, a treatment with EAS showed significant inhibition effect at all

temperatures and exposure times (Table 6.2). For one minute treatment at 30 °C, the

solutions identified as acidic (A2) and neutral (A6) anolytes displayed the highest inhibitory

activity indicating that these solutions may be used as sporicidal agents. However, the control

solutions; acidified NaCl (pH2) by HCl, neutral NaCl and EAS A3 and A4 did not show any

significant differences in their sporicidal activities. With increasing the exposure time at 30

°C up to 5 and 15 minutes, the control solutions showed a slight reduction in spore count

with time from 0.33 ± 0.02 to 0.59 ± 0.05 and 0.96 ± 0.01 log CFU/ml for the NaCl2 and

NaCl control solutions, respectively. This may have been caused by the constant stirring in

the shaker which could result in the accumulation of oxygen thereby injuring the spores

which are obligate anaerobes. The EAS named A3 and A4 for the 15 minutes exposure time

displayed significantly high spore reduction values compared to the control solutions with

4.90 ± 0.74 and 1.28 ± 0.13 log reduction, respectively. When the heating temperature was

increased up to 60 °C, the spore reduction value for the control solutions did not show

significance difference, however EAS A3 displayed marked improvement at 60 °C when the

exposure time was increased from 1 up to 15 minutes; where at 15 minute there was no

growth on the plate indicating more than 6 log reduction in the spore count. The temperature

was seen to affect the sporicidal potential of EAS, with the surviving spore count higher for

A4 at 60 °C indicating that this solution was less effective. At 90 °C, for all the exposure

times (1, 5 and 15 minutes) EAS A2 and A6 showed similar results as for 30 °C and 60 °C

with more than 6 log reduction in viable spore count. For NaCl control solution, the sporicidal

activity at 90 °C was slightly higher but is not statistically different when compared to that

167�

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observed at 30 °C and 60 °C. Furthermore, for NaCl2 the log reduction values were

significantly higher at 90 °C compared to 30 °C and 60 °C. Also, the log reduction in the

spore count was significantly higher for increasing exposure times of 1, 5 and 15 minutes

(3.88 ± 0.16, � 6 and � 6 log, respectively). In contrast with the destruction at 60 °C, A3 was

less significant at 90 °C (Table 6.2). The A4 solution showed the highest sporicidal activity

at 90 °C (4.87 ± 0.77 log) compared to the observed effect at 60 °C and 30 °C (0.47 ± 0.17

and 1.28 ± 0.13) for 15 minute exposure, respectively.

168�

Table 6.2: The Log reduction of pure culture of C. sporogenes spores due to the combined effect of temperature, exposure time and

EAS.

ND – no detectable survivors by a direct plating procedure, minimum level of detection was 6 log CFU/ml of solution. Values with

different letter in each column are significantly different (P�0.05), where big letter (A) – significant inhibition mean difference between

the solutions at one temperature by one exposure time, and small letter (a) – significant inhibition mean difference between the one type

of the solutions at one exposure time and different temperatures.

30°C 60°C 90°C

1 min 5 min 15 min 1 min 5 min 15 min 1 min 5 min 15 min

NaCl 0.33±0.12 Ba 0.42±0.14 Ca 0.59±0.05 Ba 0.31±0.12 Ca 0.20±0.17 Ba 0.20±0.21 Ba 0.69±0.12 Ba 0.64±0.92 Ba 0.68±0.18 Ca

NaCl2 0.33±0.19 Bb 0.75±0.53 Cb 0.96±0.01 Bb 0.22±0.02 Cb 0.22±0.09 Bb 0.71±0.52 Bb 3.88±0.16 ABa ND Aa ND Aa

A2 ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa

A3 2.38±1.08 Ba 3.20±0.01 Ba 4.90±0.74 Aa 3.43±0.71 Ba 4.85±0.74 Aa ND Aa 1.59±0.47 Ba 2.52±0.14 Ba 4.70±0.57 Ba

A4 0.61±0.12 Ba 0.62±0.17 Ca 1.28±0.13 Bb 0.31±0.30 Ca 0.35±0.19 Ba 0.47±0.17 Bb 0.44±0.08 Ba 1.29±0.58 Ba 4.87±0.77 Ba

A6 ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa ND Aa

�

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6.6.1.2. Effect of EAS, time and temperature on Geobacillus stearothermophilus spores

The spores of the genus Bacillus are well known to be resistant to extreme stress, most

notably high thermotolerance which also provides the bacterium with resistance to different

kind of chemicals (Brul et al., 2011; Setlow, 2006). At 30 °C, the EAS and control solution

NaCl2 showed a nominal decrease in spore count (Table 6.3). No significant difference was

found between the solutions. However, at the same temperature for an exposure time of 15

minutes, the A2 and A6 solutions reduced the spore count by 3.07 ± 0.38 and 3.57 ± 0.37

log, respectively, conforming with the trend that the A2 and A6 solutions have good

sporicidal activity across the time and temperature profiles tested. Considering the fact that

this bacterium favors a growth at 60 °C, the EAS should have had no effect on the spores.

On the contrary, A2 and A6, after an exposure time of 5 and 15 minutes at 60 °C, reduced

the spore count by �7 log. Furthermore, at 30 and 60 °C, the A3 and A4 solutions had no

significant difference across the exposure time duration on the spore reduction count. It is

noteworthy that in contrast to control solutions, the A3 performed slightly better at 60 °C for

5 and 15 minutes indicating that there might be a slight synergy between the heat treatment

and the EAS. When the temperature was increased up to 90 °C, it seemed to increase the

sporicidal activity of the control and A6. The A2 was observed to be less effective at this

temperature for all the exposure times when compared to 60 °C. The NaCl2 gave better

results at 90 °C with 15 minute exposure time compared to A3 and A4. The A6 was as

effective at 90 °C as it was at 60 °C and reduced the spore count by � 7 log. Furthermore, at

90 °C NaCl2 for 15 minutes gave results similar to that of A2, (3.31 ± 0.28 and 2.95 ± 0.85

log CFU/ml, respectively). Likewise as it was above, the A4 solution at 90 °C had slightly

higher inhibition effect than it was at 60 °C with 0.95 ± 0.12 and 0.53 ± 0.32 log CFU/ml

during 15 minutes, respectively.

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Table 6.3: The Log reduction of pure culture of G. stearothermophilus spores due to the combined effect of temperature, exposure time

and EAS.

30°C 60°C 90°C

1 min 5 min 15 min 1 min 5 min 15 min 1 min 5 min 15 min

NaCl 0.00±0.12 Ab 0.13±0.16 Aa 0.03±0.01 Bb 0.26±0.23 Bb 0.27±0.31 Ba 0.39±.38 Cab 0.07±0.12 Ca 0.73±0.37 Da 0.99±0.34 Ca

NaCl2 0.20±0.19 Aa 0.21±0.23 Bb 0.17±0.16 Bb 0.32±0.43 Ba 0.19±0.20 Cb 0.05±0.04 Cb 0.37±0.08 Ca 1.35±0.41 Ca 3.31±0.28 Ba

A2 0.82±0.28 Ac 1.84±0.47 Ab 3.07±0.38 Ab 4.44±0.46 Aa ND Aa ND Aa 2.12±0.45 Bb 2.51±0.66 Bb 2.95±0.85 Bb

A3 0.40±0.30 Aa 0.25±0.14 Ba 0.53±0.25 Ba 0.15±0.19 Ba 1.19±0.28 Ba 1.33±0.38 Ba 0.12±0.28 Ca 0.63±0.25 Ca 1.01±0.29 Ca

A4 0.50±0.26 Aa 0.29±0.26 Aa 0.46±0.25 Ba 0.34±0.42 Ba 0.33±0.27 Ca 0.53±0.32 Ca 0.57±0.01 Ca 0.88±0.33 Ca 0.95±0.12 Ca

A6 0.44±0.05 Ac 1.56±0.08 Ab 3.57±0.37 Ab 4.22±0.45 Ab ND Aa ND Aa 5.57±1.04 Aa ND Aa ND Aa

ND - no detectable survivors by a direct plating procedure, minimum level of detection was 7 log CFU/ml of solution. Values with

different letter in each column are significantly different (P�0.05), where big letter (A) – significant inhibition mean difference between

the solutions at one temperature by one exposure time, and small letter (a) – significant inhibition mean difference between the one type

of the solutions at one exposure time and different temperatures.

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6.6.1.3. Thermal inactivation of the spores in purees

In the previous section, it has been shown that the synergy between the electro-activated

solutions (EAS) and temperature was effective against the growth of the spores of

Clostridium sporogenes. To validate this effect in a model food, the temperature inside the

capillaries (tubes) during the heat treatment was recorded by K-type thermocouples

connected to a data-logger electronic thermometer (Traceable, VWR). This permitted to

avoid the time difference of the temperature gradient (Ocio et al., 1994). Analysis of the

initial concentration of spores showed no decrease before and after dilution of inoculated

puree with tested solutions, indicating that EAS did not have any effect on the spore count

without the heat treatment. The level of pH, redox-potential and concentration of residual

chlorine and oxygen did not change the proprieties of the vegetable purees. Although the mix

of A2 with corn puree slightly decreased the pH of corn puree from 6.85 ± 0.2 to 6.5 ± 0.11.

6.6.1.4. Thermal destruction of Clostridium sporogenes in vegetable purees

The C. sporogenes strain ATCC 7955 is commonly used as a non-pathogen surrogate of the

proteolytic C. botulinum. The heat resistance of these spores was primarily analyzed with

vegetable purees mixed with NaCl solution to determine the heat survivability (Figs. 6.1 A,

B). All survivor curves were obtained by plotting the logarithm of the inhibited number (log

N/N0) of the C. sporogenes against heating time ranging from 1 min to 20 min at each

temperature treatment ranging from 70 to 120 °C (Fig. 6.1). Regression lines were

established for each time temperature combination and individual D values were deduced as

the inverse negative of the slope of the fitted curve, the coefficients of determination for

individual survivor curves was also analyzed (Table 6.4). It was noted that the survivor

curves could be generally described by the first-order linear regression model with the

coefficients of determination (R2) above 0.8 (Hassan and Ramaswamy, 2011).

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Figure 6.1: Survivor curves of the spore of C. sporogenes in pea (A, C, E) and corn (B, D,

F) purees with NaCl solution (A, B), at different temperatures ( - 100°C, - 105°C, -

110°C, - 115°C, - 120°C). The reduced temperatures ( - 70°C, - 80°C, - 90°C,

- 100°C, - 105°C) for EA solutions A2 (C, D) and A6 (E, F). The linear curves are fitted

to the first order model.

A

Time, min

0 5 10 15 20 25

Log N

/N0

-10

-8

-6

-4

-2

0 B

Time, min

0 5 10 15 20 25

Log N

/N0

-10

-8

-6

-4

-2

0

C

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0D

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0

E

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0F

Time, min

0 10 20 30 40

LogN/N

0

-10

-8

-6

-4

-2

0

�

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In this study, the plotted curves of C. sporogenes in pea puree mixed with NaCl solution gave

a D120°C = 1.09 min which is comparable to the values reported in the literature suggesting a

D121°C value ranging from 0.43 to 3.1 minutes in pea products (Brown et al., 2012). This

variability of the D value may be explained by the different composition and chemical

properties of the various pea products, including different pH and water activity (aw) which

could protect the spores at high temperatures. The heat resistance observed for C. sporogenes

in corn puree was similar (D120°C = 1.45 min) to the results for various corn products as in the

literature, having D121°C value from 0.41 to 2.50 minutes (Brown et al., 2012). It was found

that temperature in some way influenced the effect of EAS mixed with the puree (A2 and

A6) on the heat resistance of the spores, more specifically, the extract was more effective in

reducing the spore count at high temperatures (Figs. 6.1 C, D, E, F). Hence, during the

heating at the temperature higher than 110 °C, no viable spores were detected. The data

obtained for other EAS (A3, A4) mixed with the puree was similar to control (NaCl) solution

(data not shown). The thermal death time for C. sporogenes at 100 °C was 66.86 and 61.66

min in pea and corn puree mixed with NaCl solution, respectively. At the same time, for these

purees mixed with A2 solution D100°C value were 3.97 and 1.81 min; for the A6 solution the

D100°C value were 2.19 and 4.14min, respectively. For A6 solution and for both types of

purees, the corresponding D value was 63.01 min at 70 °C. However, for the A2 solution at

the same temperature, the D value was 50.11 and 42.59 min for pea and corn purees,

respectively (Table 6.4). Our data suggest that there might be synergism between heat, low

pH and the chemical composition of the EAS. The solutions demonstrated different results

with both the vegetable purees, suggesting that the nature of puree could also be an important

parameter to be considered, for e.g. protein content, type of mono and oligosaccharides, and,

presence of vitamins and phenols etc. The acid EAS (A2) at 105 °C in corn puree killed all

the spores (D value not calculated) while the neutral EAS (A6) at the same temperature had

a D value higher in corn puree than in pea puree. It is noteworthy that with increasing

temperature the difference in the activity of EAS mixed with the puree and the control

solution mixed with the puree significantly increased, with the EAS being more effective,

suggesting that temperature as a parameter plays a vital role in this setup.

�

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Table 6.4: Comparison of D values for C. sporogenes spores heated in different temperatures

and different solutions in pea and corn puree.

NaCl A2 A6

Temperature, °C D, min R2 Temperature, °C D, min R2 D, min R2

Pea Pea

100 66.86 0.38 70 50.11 0.75 63.01 0.85

105 27.25 0.96 80 26.14 0.90 36.16 0.68

110 9.25 0.98 90 8.59 0.69 7.14 0.85

115 4.05 0.97 100 3.97 0.96 2.19 0.95

120 1.09 0.95 105 1.81 0.95 1.45 0.95

Corn Corn

100 61.66 0.58 70 42.59 0.87 63.01 0.85

105 18.99 0.99 80 21.91 0.95 36.45 0.73

110 9.11 0.96 90 9.27 0.24 13.47 0.93

115 2.56 0.97 100 1.81 0.55 4.14 0.74

120 1.45 0.97 105 - - 2.53 0.81

6.6.1.5. Thermal destruction of Geobacillus stearothermophilus in vegetable

purees

The survivor curves (logarithm of the survival spores [N/N0] as a function of the heating time

[min] for G. stearothermophilus in pea and corn purees under a thermal treatment ranging

from 110 °C to 130 °C and holding time ranging from 1 min to 35 minutes are shown in Fig.

6.2.

�

�

175�

�

�

�

�

�

�

Figure 6.2: Survivor curves of the spore of G. stearothermophilus in pea (A, C, E) and corn

(B, D, F) puree, at different temperatures ( - 110°C, - 115°C, - 120°C, - 125°C,

- 130°C) with NaCl solution (A, B) and EA solution A2 (C, D) and A6 (E, F). The linear

curves are fitted to the first order model.

�

A

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0 B

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0

C

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0 D

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0

E

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0

F

Time, min

0 10 20 30 40

Log N

/N0

-10

-8

-6

-4

-2

0

176�

The heat resistance curves for G. stearothermophilus in pea and corn purees with NaCl

solution are shown in Figs. 6.2 A,B. For corn puree, the D values for G. stearothermophilus

ranged between 1.45 min and 130.02 min. For pea puree, the D values ranged between 1.45

min and 273.81 min (Table 6.5). No similar studies specific to the thermal destruction kinetics

of G. stearothermophilus spores in pea and corn purees were found in the literature but, there

are some published values for their destruction kinetics in other food bases. G.

stearothermophilus is a common indicator strain used for steam sterilization, with the

recommended minimum D121°C value of 1.5 min, according to FDA (Guizelini et al., 2012).

Brown et al. (1984), by using capillary melting, reported that D121°C values of G.

stearothermophilus in potato, meat and pea alginates were 3.5, 2.8 and 2.5 min, respectively

(Brown et al., 1984). In their study, Hassan et al. (2011), detailed the D110°C, D115°C, D120°C

and D125°C values as 42.6, 12.4, 6.0, and, 1.9 min, respectively, in carrot alginate puree and

40.8, 11.0, 5.7 and 1.9 min, respectively, in meat alginate puree (Hassan and Ramaswamy,

2011). In deionized water and egg patties, the D120°C of G. stearothermophilus was found to

be 6.95 min and 8.50 min, respectively (Rajan et al., 2006). The thermal death time in milk

(pH = 6.5) was reported, where the D110°C, D115°C and D120°C values were 49.4, 16.1 and,

6.3 min, respectively (Shao, 2008). Iciek et al. (2008) reported that D115°C, D121°C and D125°C

values of G. stearothermophilus in red beet juice were 90, 18 and 7 min, while in a tryptone

medium were 108, 25 and, 8 min, respectively (Iciek et al., 2008). Our results are in

accordance with all these reports which suggest that the spores of G. stearothermophilus

show high heat resistance, as obvious in our report for pea and corn purees, albeit, for the

same heating temperature and exposure time the addition of EAS results in lowering of the

heat resistance. Table 5 shows the D values for purees mixed with A2 solution; the D110°C,

D115°C, D120°C, D125°C and D130°C values were 159.72, 23.86, 10.09, 5.53 and 1.30 min for pea

puree, and 102.68, 28.50, 11.97, 6.42 and 0.82 min for corn puree, respectively. For A6

solution the D110°C, D115°C, D120°C, D125°C and D130°C values for pea puree were 184, 32.95,

11.13, 5.69 and 0.93 min. From the results it may be understood that the heat resistance values

being significantly higher in corn puree could due to the chemical composition of substrate.

The D value for corn puree for D110°C, D115°C, D120°C, D125°C and D130°C were 376.49, 88.98,

11.05, 4.96 and 1.21 min, respectively.

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177�

Table 6.5: Comparison of D-values for G. stearothermophilus spores heated in different

temperatures and different solutions in pea and corn puree.

NaCl

A2

A6

Temperature, °C D, min R2 D, min R2 D, min R2

Pea

110 273.81 0.95 159.72 0.91 184.00 0.91

115 24.55 0.95 23.86 0.92 32.95 0.98

120 11.16 0.93 10.09 0.91 11.13 1.00

125 5.64 0.85 5.53 0.91 5.69 0.96

130 1.45 0.89 1.30 0.99 0.93 0.95

Corn

110 130.02 0.85 102.68 0.72 376.49 0.97

115 60.07 0.69 28.50 0.96 88.98 0.89

120 17.02 0.96 11.97 0.97 11.05 0.99

125 6.53 0.93 6.42 0.93 4.96 0.99

130 1.45 0.91 0.82 0.76 1.21 0.95

�

178�

6.7. Discussion

This study shows that the anolyte in combination with a moderate heat treatment has a potent

sporicidal activity (Figs. 6.3A-D and Fig. 6.4). The EAS heated to 60 °C for 1 minute resulted

in a � 6 log reduction of Clostridium sporogenes and � 4 log reduction of Geobacillus

stearothermophilus (Tables 6.2, 6.3). Tanaka et al. (1996) studied the disinfecting potential

of acid anolyte on a large number of bacteria, showing high efficacy of EAS. Using ``super

oxidized water`` (EAS-redox potential of +1000 to +1100 mV and a pH of 2.3–2.7) against

Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Escherichia coli,

Pseudomonas aeruginosa and Burkholderia cepacia, they concluded that the activity of super

oxidized water was equivalent to 80% ethanol and superior to that of 0.1% chlorhexidine or

0.02% povidone iodine (Tanaka et al., 1996). Kim et al. (2000) compared the efficiency of

electrolyzed oxidizing (EAS-pH about 2.5, redox potential about +1150 mV, residual

chlorine 10 mg/L) and chemically modified water (pH about 2.9, redox potential 1160 mV,

residual chlorine 13 mg/L) against foodborne pathogens (Escherichia coli O157:H7, Listeria

monocytogenes and Bacillus cereus). They concluded that chemically modified solutions

were generally as effective as EAS in killing the bacteria. However, the EAS with reduced

redox potential (pH 2.6, redox potential 322 mV, residual chlorine 10 mg/L) was less

effective in inhibiting the bacteria compared to normal EAS or chemically modified water.

Furthermore their study suggests that spore-forming bacteria, and especially their spores, are

more resistant than general foodborne pathogens to EAS or chemicals and that higher

chlorine concentration and longer treatment time are more effective in reducing the number

of bacterial populations (Kim et al., 2000a, b). �

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179�

Figure 6.3: The linear fitted curves of the heat resistance (Log D versus temperature) of C. sporogenes (A, B) and G. stearothermophilus

(C, D) in pea (A, C) and corn (B, D) purees with different brine solutions.

�

A

Temperature, °C

60 80 100 120

LogD

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0NaCl

A2

A6

Curve fit NaCl

Curve fit A2

Curve fit A6

B

Temperature, °C

60 80 100 120

LogD

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

NaCl

A2

A6

Curve fit NaCl

Curve fit A2

Curve fit A6

C

Temperature, °C

105 110 115 120 125 130 135

LogD

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

NaCl

A2

A6

Curve fit NaCl

Curve fit A2

Curve fit A6

D

Temperature, °C

105 110 115 120 125 130 135

LogD

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

NaCl

A2

A6

Curve fit NaCl

Curve fit A2

Curve fit A6

180�

Figure 6.4: Electron microscopy of the spores of C. sporogenes and G. stearothermophilus before (control) and after treatment with

EAS and temperature (A6, 60 °C, 15 min).

C. sporogenes before

treatment (Control)

G. stearothermophilus

before treatment

(Control)

C. sporogenes before

treatment after treatment

(A6 + 60°C)


after treatment

(A6 + 60°C)

�

�

181�

Destruction of Clostridium difficile was observed by Shetty et al. (1999) and they

demonstrated that EAS ``Sterilox`` resulted in > 6 log reduction of spores at 10 min with C.

difficile challenging EAS Sterilox (ratio of spore suspension to Sterilox is 1:10) (Shetty et

al., 1999). On other hand by sampling more frequently, Robinson et al. (2010) demonstrated

that the anolyte (pH < 2.46 and Redox potential > +1170mV) achieved a log reduction of >

2.5 within the first 10 seconds of treatment when diluted to 5% (D-value is 3.9 second) while

the dilution rate is 10% the log reduction of > 2.5 less than 10 seconds (D-value is not more

than 3.68 seconds) (Robinson et al., 2010). In our work we found a reduction of �6 logs

CFU/ml of C. sporogenes with 1 min heating at 30 °C with A2 and A6 solutions (dilute ratio

1:100), while the solution A3 and A4, differing by their properties demonstrated C.

sporogenes spore destruction > 4.5 log in 15 minutes at 90 °C.

The physicochemical nature of ECAS is not clearly understood, but based on recent

investigations, the most activated form of EAS is obtained near-electrode interface, where

the aqueous systems are in a metastable state, making them highly reactive and useful in

physicochemical and biological reactions (Aider et al., 2012). The physiochemical properties

of EAS depend upon the parameters of the treatment in electrochemical cell, where solutions

that occur at the anode interface have proven bactericidal activity resulting from the

formation of HOCl, which is considered to be the primary antimicrobial agent (Robinson et

al., 2012). Liao et al. (2007) investigated the impact of redox potential on the inhibiting effect

of EAS on E. coli, concluding that the mode of action involved damaging inner and outer

bacterial membranes resulting in necrosis thereby killing E. coli O157: H7 (Liao et al., 2007).

A similar result was found by Ronning and Frank (1989) since they observed by microscopy

the growth phases of C. sporogenes injured by sorbate, hydrochloric acid and nitrite in the

medium (Ronning and Frank, 1989). Inhibitory action of sorbate and hydrochloric acid was

found similar; the cells were with distorted shapes characterized by numerous bends and

bulges. Furthermore, cell wall appeared to be thickened and the outer wall was absent in

many areas (Ronning and Frank, 1989). Marais et al. (2006) concluded that different reactive

chemical species (e.g. hydrogen peroxide and hydroxyl radical) could contribute to the

antimicrobial properties of EAS, although their presence cannot be conclusively detected as

in the case of free chlorine (Marais and Brozel, 1999; Rogers et al., 2006). Koseki et al.

(2001) proposed that redox potential may not be the only factor of antimicrobial activity,

182�

other mechanisms proposed by the author include the presence of hypochlorous acid (HOCl),

which produces hydroxyl radical (•OH) which can act on microorganisms. This assumption

was based on the fact that EAS with low redox potential showed higher disinfectant effect

than did the ozonized water with high redox potential. . Furthermore, they concluded that the

higher •OH produced by higher HOCl concentration in EAS water was responsible for its

better disinfectant efficacy than the ozone solution (Huang et al., 2008; Koseki et al., 2001).

Venczel et al. (1997) investigated inactivation of C. perfringens spores using an alternative

EAS disinfection system consisting of an electrochemically produced mixed-oxidant solution

(MIOX). The authors compared the sporicidal potential of mixed-oxidant solution to that of

free chlorine on the basis of equal weight per volume concentration of total oxidants,

achieving a > 99.9% inactivation of spores (Venczel et al., 1997).

As mentioned above, the spores are generally more resistant to disinfecting treatments than

growing cells of the same species and a variety of factors have been identified which

contribute to the elevated resistance of spores; including a thick proteinaceous spore, reduced

permeability of the spore core, reduced water content in the spore core (Loshon et al., 2001).

In our study we showed that the spores of Geobacillus stearothermophilus as other species

of Bacillus genus had high resistance to disinfection treatments such as EAS. A similar result

was obtained by Granum et al. (1987) whose observed that the spores of Bacillus subtilis

were more susceptible when hypochlorite was used at a alkaline pH, the effect of

hypochlorite increased by a factor of about 2.5 from pH 5 to 8 and by a factor 6 from pH 8

to 9 (Granum and Magnussen, 1987). An alternative disinfection system of EAS (ECASOL)

was juxtaposed against Bacillus anthracis spore, demonstrating that 30-min treatment

decreased the viability to � 7 log reductions (Rogers et al., 2006). Rogers et al. (2006)

discussed that a change in pH did not affect the efficacy of EAS, furthermore its sporicidal

potential was also not significantly affected when the concentration of free available chlorine

was between 300 to 460 ppm suggesting that the high efficacy of EAS could be explained by

the presence of other reactive chemical species (Rogers et al., 2006). Robinson et al. (2010),

observed the spore inactivation of Bacillus atrophaeus by EAS. The strain was selected due

to its high resistance to inactivation by disinfectants, nevertheless a rapid reduction of > 6.5

logs at 90 s was observed when treated with EAS. Another studied of Bacillus subtilis with

EAS ``Sterilox`` (pH 6.3, available free chlorine level about 240 mg/L), suggests that the

�

183�

mechanism of action involved oxidation of proteins or fatty acids present in the inner-

membrane of the dormant spores resulting in membrane permeabilization, however the

effects of damage are only seen after spore germination (Loshon et al., 2001; Robinson et al.,

2010).

A combined action of EAS heated at different temperatures and exposure times was observed

to be the most suitable application condition. In their work Walker et al. (2005) studied the

effectiveness of the EAS treatment as a cleaning method by ATP bioluminescence and

microbiological analysis. In the study, the milking system was washed for 10 min with 60 °C

alkaline EAS followed by a 10 min wash with 60 °C acid EAS and finally water; this

procedure successfully removed all detectable bacteria as well as the ATP from the non-

porous milk contact surfaces. However, their results showed that there were no significant

difference between the 10 min and 7.5 min EAS treatment and the conventional treatment by

chlorinated alkaline detergent at 80 °C (pH 11.0, chlorine content 50 ppm), indicating that

EAS has the potential to be used as a cleaning and sanitizing agent for milking systems

(Walker et al., 2005).

As already mentioned, when EAS comes into contact with organic matter, it becomes

ordinary water again, thus having less adverse impact on the environment and the users

health, giving it the distinct advantage of being safe to handle and use (Huang et al., 2008).

Therefore, EAS has been used to inactivate pathogens on fresh products. In one such study

Rahman et al. (2011) studied the synergistic effect of EAS with mild heat treatment against

background and pathogenic microorganisms. They observed the behavior of E.coli O157:H7

and L. monocytogenes on the carrots after various exposure times (1, 3, and 5 min) and

treatment at different dipping temperatures (1, 20, 40, and 50 °C). They concluded that the

optimal combination was 3 min at 50 °C. Furthermore, the combinations with EAS better

maintained the sensory and microbial quality of the fresh-cut carrots and enhanced the overall

shelf-life of the produce (Rahman et al., 2011). A similar study on carrots by Koide et al.

(2011) concluded the same results (Koide et al., 2011). Xie et al. (2012) observed that the

efficacy of EAS could be greatly enhanced when coupled to a heat treatment and was

effective in inactivating Vibrio parahaemolyticus present on the shrimp. According to the

authors, the effective order of heating temperatures on bactericidal activities of EAS was 50

°C>20 °C> 4 °C (Xie et al., 2012).

184�

There are many processes used for making foods safe, for example acidification and thermal

treatment are widely used in preserving canned foods (Leistner, 1992; Lucas et al., 2006).

Similar to that this process could also be used, based on relatively few parameters or hurdles

(i.e. high temperature, low temperature, water activity aw, pH, redox potential (E) and

preservatives) whereby some parameters are of major importance, in others process they are

only secondary hurdles (Leistner, 1992). The results obtained here due to the combined effect

of the EAS heated at different temperatures on the inactivation of pure spore culture and the

spores in vegetable purees as well.

As expected, the Z values of C. sporogenes in pea and corn purees were similar to values

from literature, being at 11.36 and 13.33 °C, respectively (Brown et al., 2012). However,

obtained Z values for the tested purees in this work appeared to be higher than that of the

control (NaCl). One could say that the heat resistance of spores increased due to the mixing

with EAS. However the temperature and time were also observed to decrease (Table 6.4)

possibly due to the fact that the injured spores could be more susceptible to the heat treatment

combined with EAS suggesting a synergistic effect. This agrees with the observations made

by other authors working with C. sporogenes spores. For example, Naim et al. (2008),

observed the combined effect of nisin and pH on mild heat treatment. Their Z values were

found to be 32.2 and 20.6 °C in Sorensen’s phosphate buffer and carrot-alginate particles,

respectively, at the heating temperature less than 100 °C (Naim et al., 2008). Santos et al.

(1993) found that Z values of C. sporogenes in asparagus puree combined with citric acid

could reach > 22 °C (Santos et al., 1993). Later they also found that a combination of

asparagus glucono-�-lactone and citric acid gives Z values of C. sporogenes that can reach

up to 25 °C (Santos and Zarzo, 1995). The main explanations in the scientific literature for

this kinetic behavior ensue the different influence could impact diverse factors of the heating

medium and exert on spore heat resistance otherwise such factors as strain choice, heating

method (i.e. dry or moist heat) may affect the results and have to be considered when

establishing the heating process (Naim et al., 2008; Santos et al., 1993).

�

�

185�

Table 6.6: Destruction kinetics of C. sporogenes and G. stearothermophilus in pea and corn

purees �

Pea Corn

Z,°C

R2

Standard

error

Z,°C

R2

Standard

error

C. sporogenes

NaCl 11.36 0.99 0.057 12.13 0.98 0.090

A2 29.59 0.98 0.076 22.27 0.95 0.153

A6 18.53 0.98 0.116 24.21 0.98 0.086


NaCl 9.63 0.94 0.236 10.27 0.99 0.919

A2 10.38 0.94 0.236 10.33 0.99 0.919

A6 9.33 0.97 0.147 8.03 0.98 0.145

In general, the spores of G. stearothermophilus are very heat-resistant and usually survive

during the sterilization procedure and could cause a “flat-sour spoilage” of low-acid canned

foods if the products are stored at elevated temperatures (Head et al., 2008). When the spore

inactivation kinetics is not known, the method and conditions of thermal treatment during

sterilization are chosen on the basis of experimental data (Iciek et al., 2008). As seen in Table

6.6, the Z values were found to be 9.63 and 10.27 °C for pea and corn puree, respectively

whereas a range of 8.3 to 11.8 °C has been reported for various strains of G.

stearothermophilus (Guizelini et al., 2012; Shao, 2008). Our studies showed that the addition

of stressful conditions as EAS, produced spore injury, thereby making them more susceptible

to subsequent heat shock, as reflected in the results where the treatment with neutral EAS

(A6) decreased the heat resistance to 9.33 and 8.03 °C for pea and corn puree, respectively.

It is important to point out here that the D values associated with the neutral EAS (A6) and

acidic EAS (A2) followed the known trend that a medium with lower pH has a lower D value

as it requires lesser heating time, as seen here for both species tested, however the

corresponding Z values do not follow the same trend, with the acid EAS (A2) showing

increased heat resistance in both puree to 10.38 and 10.33 °C for pea and corn puree,

186�

respectively. We observed that a decreasing time of thermal destruction did not correlate with

the decrease in Z values (Table 6.5). This anomaly could be explained based on the fact that

although some bacterial species are inherently more heat resistant than others, spore heat

resistance is also strongly correlated to the mineral and moisture content, age of the species

or to the composition of the sporulation media (Guizelini et al., 2012). This study warrants

further investigation in this phenomenon so as to find a suitable explanation to it.

6.8. Conclusions

In this study, we showed the evident effectiveness of electro-activated solutions as strong

inhibitors of the growth of spores of Clostridium sporogenes and Geobacillus

stearothermophilus which are considered as highly heat and chemical resistant

microorganisms. The acidic electro-activated solution with low pH, high redox potential and

oxygen content permitted complete inhibition of the growth of spores of Clostridium

sporogenes at temperatures below 121 °C. Furthermore, neutral electro-activated solution

also inhibited the growth of these spores, suggesting their high sensitivity to oxidative

medium with high redox potential. These solutions also inhibited the growth of spores of

Geobacillus stearothermophilus which is not sensitive to oxygen. This result also supports

the sporicidal activity of electro-activated solutions.

Even if these bacteria are considered as non-pathogenic, they are used as a good indication

of a potential effectiveness of novel treatments against Clostridium botulinum. Thus, this

study permitted to highlight a novel and promising hurdle technology to produce low

processed canned vegetables with total ensuring of food safety.






�

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7. CHAPTER 7: Production of canned pea and corn by using a hurdle

technology composed of electro-activation and low heat treatment



G1V 0A6, Canada.






This article is submitted for publication in Journal of Food Engineering.







188�


In the previous chapter (6) it was found that EAS in combination with mild temperatures

showed synergetic effect on the heat resistance spores which are usually responsible for can

spoilage. In this chapter the mathematically validated temperatures of sterilization were

examined on the canned pea and corn. The aim of this chapter was to study the effect of EAS

with different temperatures of sterilization on the product quality as well as the energetic

costs of conventional technology compared to technology combined with EAS.

�

� �

�

189�

7.2. Résumé

La salubrité et la qualité des produits de conserve sont étroitement liées aux paramètres de

stérilisation appliqués. Les hautes températures sont très utilisées dans le secteur

agroalimentaire. Cependant, ces dernières peuvent, non seulement entraîner des altérations

majeures sur la qualité du produit final du point de vue nutritionnel et organoleptique, mais

aussi engendrer d’importantes dépenses énergétiques. Plusieurs barrières technologiques

sont aujourd’hui proposées pour réduire l’impact des traitements de stérilisation

conventionnels menés à haute température. Récemment, l’électro-activation a démontré son

efficacité comme une nouvelle alternative prometteuse afin d’assurer la salubrité des produits

de conserve. L’objectif de ce travail est d’évaluer les qualités nutritionnelles et

organoleptiques des petits pois et du maïs en conserve stérilisés à températures modérées

avec l’utilisation de solutions électro-activées (SEA). Les résultats obtenus montrent que la

teneur en vitamine C dans le produit final subit peu de détérioration à basse température de

stérilisation. Le traitement de stérilisation à haute température et à courte durée a eu un impact

significatif sur les changements de couleur et de texture des produits traités. Le meilleur profil

de texture a été obtenu avec les SEAs acides A2 pour les petits pois et le maïs avec des valeurs

de 5,19 ± 0,27 et 10,21 ± 1,09 N, respectivement. Les SEAs neutres (A6) résultent en une

texture solide pour les petits pois et le maïs avec des valeurs de 4,20 ± 0,06 et 7,63 ± 1,14 N,

respectivement. Cependant, l’éclat de la couleur verte des petits pois est plus important lors

de l’utilisation des A6 (a* = -8,4 ± 0.3) comparée aux A2 (a* = -3,7 ± 0.6). La couleur

jaunâtre du maïs est aussi plus attrayante avec les A6 (b* = 36,32 ± 1.24) comparées aux A2

(b* = 28,44 ± 2,39). Il est intéressant de noter que dans cette étude, une baisse énergétique

de 33 % est obtenue avec l’utilisation des solutions SEAs comparativement aux procédés de

stérilisation conventionnels qui nécessitent des apports énergétiques très élevés, de l’ordre

de 2118,72 ± 42,37 et 2124,73 ± 42,49 kJ par boîte de conserve de 385 ml de petits pois ou/et

de maïs, respectivement.

190�

7.3. Abstract

The safety and quality of canned products mostly depend on the parameters of sterilization.

High temperatures used in the industry cause serious deterioration of the food quality and

result in high energy costs. Various hurdle technologies can reduce the severity of the heat

treatments. Recently, electro-activation (EA) technology was successfully used for the

reduction of the sterilization temperatures. The objective of this work was to evaluate the

quality characteristics (vitamin C, colour and texture) of sterilized canned pea and corn in

electro-activated brine solutions (EABS) at moderate temperatures. It was found that the least

change in vitamin C was associated with the lowest heat treatment, while the short treatment

time resulted in more significant changes in texture and color characteristics for both

vegetables. The best texture profile was obtained with the acid EABS (A2) for pea and corn

with 5.19 ± 0.27 and 10.21 ± 1.09 N, respectively, while neutral EABS (A6) resulted in a

less solid texture for pea and corn demonstrating 4.20 ± 0.06 and 7.63 ± 1.14 N, respectively.

However, the green color brightness of canned pea was higher for neutral EABS (a* = -8.4

± 0.3) than for acid one (a* = -3.7 ± 0.6), also, the yellowness of corn was also significantly

better with neutral EABS (b* = 36.32 ± 1.24) than with acid ones (b* = 28.44 ± 2.39). More

importantly, considerable decrease in energy consumption (at least 33%) was found using

EABS in comparison with the conventional sterilization (121 °C) which utilised 2118.72 ±

42.37 and 2124.73 ± 42.49 kJ per a can of 385 mL for the production of canned pea and corn,

respectively.

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191�

7.4. Introduction

Thermal processing, i.e. heating, is one of the most important and the most widely used

method of preserving and extending the useful shelf-life of foods (Simpson and Abakarov,

2009). Sterilization is a high-temperature thermal treatment performed in a closed container

for a specified amount of time in order to destroy microorganisms of public health and

spoilage concerns (Awuah et al., 2007; Miri et al., 2008). Regarding public health

significance for low-acid foods (pH �4.5), Clostridium botulinum is the organism of concern.

Thus, based on the kinetic parameters (D- and z-values), the thermal processing of low-acid

foods conventionally uses an amount of heat treatment (F-value) equal to 2.58 min, also

known as “12-D” of botulinum cook (Heinz and Hautzinger, 2007). However, most of low-

acid foods are processed beyond the minimum botulinum cooks (F-values ≥4) because of the

mesophilic spore forming bacteria Clostridium sporogenes which is more resistant than C.

botulinum and could cause food spoilage and pose economic concerns (Heinz and

Hautzinger, 2007; Stumbo, 1973). Thus, more severe thermal process causes more

irreversible changes of quality characteristics. Many studies have demonstrated that texture

and color, contributing to the esthetic quality of various foods, deteriorated with an increase

in the severity of the used heat treatment. The most concerning are the softening and

darkening of the canned foods which have been previously observed in several studies (Blair

and Ayres, 1943b; Bourne, 1982; Bourne and Comstock, 1986; Garrote et al., 2008;

Hayakawa and Timbers, 1977; Lathrop and Leung, 1980a; Scott and Eldridge, 2005; Van

Loey et al., 1995). Therefore the parameters of heat processing provide a good indication

about the severity of the treatment and the extent of the damage it may cause (Rattan and

Ramaswamy, 2014).

It is well known that excessive heat treatment should be avoided because it has a detrimental

effect on food quality (Bigelow and Esty, 1920; Simpson and Abakarov, 2009). While,

considering the ever increasing demand for canned foods, consumer requirements for high-

quality and minimally processed foods, industries are always in search of advanced thermal

processing procedures. However, improvement of processing conditions becomes even more

important because of the fact that acceptance or rejection of most foods relies on their texture

and external appearance (Rattan and Ramaswamy, 2014). Based on the conception of hurdle

192�

technology approach, an intelligent combination of different preservation treatments could

be employed to curb microbiological growth and still reduce the temperature of sterilization

(Leistner, 2000). Furthermore, improvement of food processing could bring other benefits

namely; high productivity, reduced consumption of heating medium (steam, water) and lower

labor requirements (Dwivedi and Ramaswamy, 2010).

Recent studies on the behavior of the C. sporogenes spores in contact with electro-activated

solutions (EAS) showed high effectiveness of EAS regarding inhibition of the growth of

spores at moderate temperatures with and without contact with food (Liato et al., 2015d).

The technology of electro-activation has also found important applications in food industry

as an effective disinfectant agent, aiding protein and fiber extraction, in activating enzymes

amongst others (Aider et al., 2012; Hung et al., 2008). Furthermore, the application of this

technology does not require high investment and production cost of EAS is quite low (Hung

et al., 2008; Liato et al., 2015c). New method requires process optimization so as to find the

optimal balance between heating temperature and the process time in order to maximize the

final nutrient retention and assure sufficient microbiological lethality. Furthermore,

maximization of energy efficiency and minimization of total process time should also be

taken into account (Sendín et al., 2010).

Thus, the purpose of this work was to investigate the effect of electro-activated solutions

(EAS) combined to low heat treatment on the quality of canned food (pea and corn) as well

as to compare the energy costs of the modified sterilization treatment with the conventional

one.

�

193�


7.5.1. Materials and chemicals

Fresh pea (Pisum sativum) and corn (Zea mays) were purchased from a local market and

treated the same day. Some characteristics of the used grains are shown in Table 7.1. All

other chemicals used were of analytical or food grade.

Table 7.1: Characterization of the fresh and blanched pea and corn grains

Parameter Pea Corn

Raw Blanched Sterilized 1 Raw Blanched Sterilized 2

Firmness (N) 14.18 ± 0.81 8.58 ± 3.58 1.63 ± 0.05 11.46 ± 0.03 10.37 ± 0.30 4.68 ± 2.45

Lightness L* 41.66 ± 0.80 37.09 ± 0.71 42.73 ± 2.24 52.59 ± 0.97 47.02 ± 2.81 41.69 ± 2.31

Greenness -a* 14.23 ± 0.05 17.16 ± 0.26 3.63 ± 0.62 2.69 ± 0.07 3 1.14 ± 0.68 3 0.25 ± 0.12 3

Yellowness +b* 25.4 ± 0.40 21.23 ± 0.34 22.88 ± 0.91 22.90 ± 2.59 30.39 ± 1.90 25.17 ± 1.86

Ascorbic acid (mg/g) 15.97 ± 0.84 9.93 ± 0.30 0.18 ± 0.03 3.34 ± 0.61 1.62 ±0.07 0.08 ± 0.03

1-sterilized according to lethality process ( F�º»¼�½22.7¨ = 8.27) 2-sterilized according to lethality process ( F�º»¼�½23.27 = 11.00)

3-color parameter presented as redness indicator (+a*)

7.5.2. Preparation of electro-activated brine solutions

The electro-activated brine solutions (EABS) were made from 0.05 M NaCl and 0.05 M

NaHCO3 (Laboratoire MAT, Montreal, Canada) according to the method developed by Liato

et al. (2015a). The electro-activation treatments were carried out in a three compartmental

electro-activation reactor and the yielded solutions were characterised by different proprieties

such as the Redox potential (E), pH and oxygen content. Non-electro-activated 0.05 M NaCl

solutions were used as control (Table 7.2) (Liato et al., 2015a). The reactor was composed

of three cells made of Plexiglas. A ruthenium–iridium coated titanium electrodes (100 x 40

mm) were used and connected to a direct electric current power source (Lambda, GR.260,

Electronics Corp. Melville, NY, USA). The electrodes were placed in the anodic and cathodic

compartments, where an anion (AM-40) and cation (CM-40) exchange membranes

(Schekinaazot, Shchekina, Russia) were used to separate the anode and cathode from the

central compartment, respectively. The EABS were prepared by using two different reactor

194�

configurations. In the first configuration, all compartments were filed with NaCl so that after

the electro-activation treatment, the resulting solution had strong acidic pH and high

oxidizing Redox potential (A2) (Table 7.2) (Liato et al., 2015c). For the second reactor

configuration, the cathodic and central compartments were filled with 0.1 M NaCl solution,

whereas NaHCO3 was used in the anodic compartment in order to obtain a neutral EABS

solution (A6). The electro-activated solutions were obtained by applying a direct electric

current with a current density of 50 A/m2 for 30 min for A2 solution and 60 min for A6

solution (Liato et al., 2015a). The EABS were made and analyzed immediately before use

every time. The pH and dissolved oxygen (DO) of the EABS were measured by using a pH-

meter (Model SR 601C SympHony, VWR Scientific Products, USA) and a DO meter (Model

SB 90M5 SympHony, VWR Scientific Products, USA), the Redox-potential (E) was

analyzed by an ORP-meter (Eco Sense ORP15A). The calibration of ORP-meter was done

with ZoBell’s standard solution. The total residual chlorine (RC) was measured with chlorine

colorimeter kit (Orion AQ3070, Thermo scientific AQUA-fast, Singapore). The EABS were

subsequently heated to 60 ± 5 °C with the help of a magnetic stir plate (VWR, Montreal,

Canada) and added to the can filled with the appropriate grains (pea or corn).

Table 7.2: The properties of the brine solutions

Brine solutions pH E, mV RC, mg/L DO, mg/L

NaCl 6.51 ± 0.05 255 ± 14 - 5.4 ± 1.2

A2 1.98 ± 0.12 >1200 205 ± 10 19 ± 3.5

A6 6.45 ± 0.07 935 ± 7 350 ± 17 25 ± 2.2

7.5.3. Thermal processing

The grains were blanched in a steam blancher (Lee-metal Inc., USA) equipped with stainless

steel basket (cylindrical, 450 mm diameter and 550 mm height). A sample of 1000 g of grain

was immersed for 5 min in a hot water (90 ± 0.5 °C) of the 10 L blancher bath. The blanched

product was drained and then placed into 300 x 407 enameled white cans (Dominion &

Grimm, Montreal, Canada) which were then filled with heated (60 ± 5 °C) brine solution

(Table 7.2), seamed (Dixie UVG6MD, USA) and sterilized according to the results obtained

in model conditions of sterilizing solutions containing spores of Clostridium sporogenes

�

195�

(Liato et al., 2015b). The seamed cans with grains were sterilized in a horizontal non-rotary

retort (Gebr.Stork & Co. In., Amsterdam, Holland), consisting of a cylindrical storage vessel

(0.5 m3), re-circulating and cooling water pumps, a digital thermo-regulator, temperature

recording and control elements. Each batch of cans was treated according to the required

lethality process described later. The heat treatment of canned foods was carried out with

saturated live steam (highest working pressure of 35 psi) which was quickly transferred to

the process vessel so that a come-up time was achieved within 3 min for all processing

temperatures. At the end of sterilization process, the product was cooled by rapidly replacing

the sterilizing steam with cold water. At the end of the heat treatment, the samples were

chilled, dried up and stored at ambient temperature (22 ± 3°C) until use.

Considering the variable heat penetration time for heterogeneous products at different

processing temperatures, the canned products were first heated in order to know the

corresponding thermal history. To determine the time required for heat penetration at the

coldest point of the canned grains (center of the can), electronic thermocouples (iButton/iBee

22E, Alpha Match inc., Montreal, Canada) were used. The iButtons were placed in a small

metal grid box at the center of the samples so that thermocouple did not move from the center

and remained tightly surrounded by the grains and liquid (electro-activated brine solution).

The test was performed with 4 iBottons placed in the cans in different corners of the vessel.

This test was repeated 3 times at each temperature. Another thermocouple was placed in the

retort to measure its temperature. The temperature responses of the thermocouples were

synchronized to periodically collect data at intervals of 1 min at all treatment temperatures

in real time.

7.5.4. Sterility considerations

Thermal order of bacteria death is normally represented by logarithmic thermal destruction

(TD) curve obtained by plotting the time required to destroy the microorganisms in a given

population against heating temperature (Stumbo, 1973). In view of the order of bacterial

death, the survive population is considered per some unit volume, so that a general equation

of the survivor curve was used as follows:

U=`abK = <=`ab ∗ _wxykÀ − wxyk�c (Eq. 7.1)

196�

The criterion of process adequacy for the low-acid product is the extent to which the bacterial

population should be reduced. Thus, for the mesophilic spore forming bacteria which are

more resistant than C. sporogenes, sterility can generally be accomplished by reducing the

population by 10-4 (CÁ= 0.0001). The average contamination load was determined to obtain

CÂ value as one spore per one ml of product (can 300 x 407 � 385 mL). Estimated thermal

destruction time (�;ÃÄÅ) of spores for different brine solutions and products allowed to

determine required lethality process, F value (Table7.3) (Liato et al., 2015d).

Table 7.3: The required lethal heat time of products in different brine solutions

Name and properties of brine solutions Product

Pea Corn

NaCl T;º»¼�Æ22.7¨ = 8.27 T;º»¼�Æ23.27 = 11.00

A2 T;º¼Ç�Æ3È.9È = 9.05 T;º¼¼�Æ33.3Ê = 9.05

A6 T;º¼Ç�Æ2S.97 = 7.25 T;º¼Ç�Æ38.32 = 12.65

7.5.5. Lethality of heat at a single point

In this study, the improved general method of process evaluation was used to evaluate the

time-temperature data plotted directly on the lethal-rate graph giving a process lethality curve

(Fig. 7.1). Using electrical thermocouples (iButton), the lethal-rate value was assigned to

each temperature which was numerically equal to the respective number of minutes required

to destroy a given population of spores at corresponding temperatures. The lethality value of

each process was calculated according to the following equation:

Ì = h BV(pÍXpÎ¹Ï) ÐI i?=Ñ=� (Eq. 7.2)

The sum of destruction rates was analyzed at all points on the TD curve to find the appropriate

regime of required sterility level. The graph (Fig. 7.1) was empirically optimized after

preliminary testing so that it adequately corresponded to the F-value, as recommended by

relation T;ÃÄÅÒ ≦ j L~J1R (Flaumenbaum et al., 1982; Stumbo, 1973).

�

197�

A

T, min

0 10 20 30 40

L, m

in

0

1

2

3

4

5

6

B

T, min

0 10 20 30 40

L, m

in

0

1

2

3

4

5

6

C

T, min

0 10 20 30 40

L, m

in

0

1

2

3

4

5

6

D

T, min

0 10 20 30 40

L, m

in

0

1

2

3

4

5

6

Figure 7.1: The process lethality curves of the heat effecting pea (A, B) and corn (C, D)

cans at different processing temperatures ( - 121°C, - 115°C, - 110°C, - 105°C, -

100°C) as well at different brine solutions : A2 (A, C), A6 (B, D) and NaCl (A, B, C, D;

presented at one temperature - 121°C).

According to the observed time of the heat progression in the container (can 300 x 407) with

grains and lethal values time at each processing temperature, the so-called regime equation

of sterilization was found (Flaumenbaum et al., 1982).

±GXHXv=`³ (Eq. 7.3)

In order to have similar conditions of experiment, the regime for the tested EABS was

equated and insignificant processing time differences were ignored. Thus, the highest regime

time between two types of EABS was selected and has been graphically presented in Fig.

7.2. For example, different regimes of control brine solution (NaCl) are presented in both

198�

graphs meanwhile for both EABS (A2 and A6) the regime is displayed as one (the longest

one) for each product.

A

Time, min

0 10 20 30 40

Tem

pera

ture

, C

20

40

60

80

100

120

140

B

Time, min

0 10 20 30 40

Tem

pera

ture

, C

20

40

60

80

100

120

140

Figure 7.2: The temperature curves of the autoclave used for the difference time profile.

Regimes presented for pea (A) and corn (B) cans at different temperatures for NaCl ( - 121

°C) and EABS ( - 121 °C, - 115 °C, - 110 °C, - 105 °C, - 100 °C).

�

7.5.6. Texture profile analysis (TPA)

Firmness of fresh, blanched and sterilized grains (green pea and yellow corn) was measured

using a texture profile analyzer TA-XT2 (PLUS-UPGRADE, Texture Technologies Corp.,

NY, USA), equipped with a 75 mm aluminum compression plate (P/75) installed on a 50 N

load cell. Around 10 ± 1 grams of grains were spread on the plexi-glass bar (height 30 mm)

with a round neckline (80 mm diameter) placed parallel to the fixed metallic stand. The test

of a single compression measurement was 50% of the original height and ran with a speed of

2 mm per second on each sample. The maximum peak force (firmness, N) was recorded for

�

199�

at least 10 measurements per sample for all the conditions which were tested (Chenoll et al.,

2009).

7.5.7. Measurement of color

Color measurements were made by using a Minolta CR-300 Chroma Meter (Minolta data

processor DP-300, Osaka, Japan), calibrated with a standard white plate. The determining

color space coordinates were expressed in terms of CIE* values for lightness (L*),

greenness/redness (a*), and blueness/yellowness (b*). To carry out this analysis, a sample

composed of 15 grains was placed in the measurement support covered by glass, and

evaluated in triplicate for each tested condition. For green pea, the lightness (L*) and

greenness (a*) parameters were considered as the most important ones, while the (L*) and

yellowness (b*) values were considered for the yellow corn grains.

7.5.8. Vitamin C analysis

The concentration of vitamin C (reduced ascorbic acid, Amresco, Solon, OH, USA) of the

samples was analyzed according to AOAC method 985.33 (Horwitz and Chemists, 2000).

After each run, a can was opened; the brine solution was separated from the grains and

weighed. A sample of 30 g of grain were immersed in 30 ml of 2% glacial hydrochloric acid

(HCl, Fisher scientific, Missisauga, Canada) and crushed with mortar in a porcelain cup.

Subsequently, the crushed sample was placed in a 100 ml graduated cylinder and HCl volume

made up to 50 ml was added and left for extraction for 10 min. After that, 30 ml of 70%

acetate buffer (pH =4.5 ± 0.05) (CH3COONa, Caledon, Montreal, Canada) and 10 ml of

0.005% EDTA (Amresco, Solon, OH, USA) were added and brought to the mark of 100 ml

by HCl. Obtained solution was mixed and then filtered through qualitative paper (18.5 cm,

Whatman No. 541, GE), divided in 10 ml volumes and poured in conical flasks with magnetic

stirrer and estimated by titration of 0.00025% colored oxidation-reduction indicator (2,6-

dichloroindophenol Na, Avantor, Center Valley, PA, USA). The concentration of ascorbic

acid in the samples was obtained by using the following equation:

G�«E`Ñ�«�«�i = ÔB∗=4 ∗Ô@Ô¶∗{F∗ BVV (Eq. 7.4)

200�

To determine the titer, the control samples of 0.1 and 1% of ascorbic acid standard solution

were prepared immediately before use by dissolving in a mixture of hydrochloric acid and

acetate buffer (1:1). The titer was obtained by the following equation:

p4 = lÕÖ×XÖq

(Eq. 7.5)

�

7.5.9. Energy consumption of sterilization

The energy consumption of a heat sterilization process in a stationary retort can be estimated

from a thermal energy balance where the incoming energy is equal to outgoing energy

(Barreiro et al., 1984). The different energy terms could involve a variety of options

according to the thermal energy balance. It is well-known that the total energy consumption

for sterilization consists of three phases: heating, holding and cooling (Barreiro et al., 1984;

Valentas et al., 1997). In this study, two principal energy-intensive phases (heating and

holding) were chosen to analyze the used sterilization regimes. The total energy consumption

was calculated as the sum of the steam energy (}Ø) used to heat the product in the metallic

can and the energy of the production of EA solutions (}Y), excluding the control NaCl

solution:

z=E?�D = z� + z� (Eq. 7.6)

To calculate the energy consumption of heating (come-up) and holding phase of product and

containers, the integration analysis of the heat processing was applied by using the following

equation:

z=� = {h v|=Ñ=� i? (Eq. 7.7)

Analysis of energy consumption was made according to heat progression in the canned

product at each regime of each individual brine solution, based on the data obtained from the

thermal process evolution. Therefore, the sum of the energy of heat at any given time (}Ø =j};Ù) presents the total energy consumption required to provide the lethality of a product at

a single point (Fig. 1.). The loss of heating in the retort and environmental losses were not

included.

The energy consumption of producing the EA solutions was calculated as the sum of electric

power used for each brine solution:

z� = jÚ� (Eq. 7.8)

�

201�

The electric power can be found by using the Ohm's Law. In this study, the three-cell reactor

produced the solutions at the constant current density of 50A/m2 and alternating voltage

(Liato et al., 2015a). Hence, the electric power was calculated as follows:

Ú� =h ��?@?B ∗ �i? (Eq. 7.9)

�


The results of heat penetration analysis were measured in triplicate, vitamin C and color

measurements were performed three times, and texture analysis was done 10 times. The

results were expressed as mean values ± standard deviation. To assess the effect of interaction

of the EABS and heat treatments on the food quality, pairwise multiple comparison procedure

of two-way ANOVA was used. The Fisher’s LSD test was used to determine differences at

� = 0.05. All statistical analyses were performed using Sigma Plot Statistics (Systat Software

Inc., 2010, USA).


7.6.1. Texture profile analysis (firmness)

Texture is conventionally determined as the way in which the structural components of a

food are arranged in a micro- and macrostructure and the external manifestations of this

structure. It is also considered as one of the most important quality attribute (DeMan et al.,

1976; Jaiswal et al., 2012).

In this study, the firmness of conventionally sterilized pea and corn was found to be 1.63 ±

0.05 and 7.68 ± 2.45 N, respectively (Table 7.1). The results of firmness changes of the pea

and corn with different EABS at different processing temperatures are presented in Fig. 7.3.

Conventional sterilization consisted of heating at 121 °C for 27 ± 2 min of the total processing

time (Fig. 7.2) to achieve the necessary lethality level as per the data of kinetic destructions

of reference microorganisms (Table 7.3) obtained in a previous study (Fig. 7.1) (Liato et al.,

2015d). At the same processing temperature of 121 °C, EABS was heated for a shorter time

duration (Fig. 7.2) but it still preserved the grains better with an improved texture and

increased firmness, as observed for pea with a firmness value of 4.93 ± 0.50 N when electro-

activated solution A2 was used and 2.20 ± 0.06 N when the A6 solution was used as the brine

solution. For the corn grains, the firmness at 121 °C processing temperature was found to be

202�

8.11 ± 0.70 and 5.77 ± 1.57 N, when A2 and A6 were used as brine solutions, respectively.

These observation are in good agreement with the reported information in the literature since

it is well known that textural changes that occurring during heat treatment follows first-order

degradation kinetics (Van Loey et al., 1995). Thus, the textural changes almost decrease with

increasing temperature (Bourne and Comstock, 1986). In the present study, the maximum

shearing force of pea was found better at 115 °C in contact with A2 (5.19 ± 0.27 N) and A6

(2.74 ± 0.16 N) solutions. Also, at the lowest processing temperature (100 °C) the firmness

was less with mean values of 2.02 ±0.09 and 1.74 ± 0.08 N, respectively; indicating that a

shorter processing time results in increased firmness and improved texture (Fig. 7.3a). Data

for pea firmness showed no significant difference (p = 0.011) between the sterilizing

temperatures of 121 and 115°C. Nevertheless, it is important to note that the results obtained

with these temperatures were significantly different (p < 0.001) in comparison with those

obtained at 110, 105 and 100 °C. The firmness of corn grains was most significant at 110 and

115 °C with A2 with mean values of 10.21 ± 1.09 and 9.28 ± 1.17 N, respectively. The same

temperatures showed highest shearing force for A2 and for A6 resulting in 7.63 ± 1.14 and

6.97 ± 1.96 N, respectively (Fig. 7.3b).

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203�

Figure 7.3: Effect of heat treatments on the firmness of canned peas (A) and corn (B) The



between subgroups.

Normally, the firmness-temperature plots are rectilinear and the firmness-temperature

coefficient, defined as the percent change in firmness per temperature, decreases with

increasing temperature. However, there are several exceptions that can occur as it has been

previously reported in the literature (Bourne, 1982; Bourne and Comstock, 1986). In this

Aa

Db

Aa

Ca

Bb

Fd

DEcDEFcd DEc

EFcd

0

2

4

6

8

10

12

A2 A6 A2 A6 A2 A6 A2 A6 A2 A6

121°C 115°C 110°C 105°C 100°C

Fir

mn

ess

, N

B.S. / Temperature

A

BCa

DEb

ABa

CDa

Aa

BCa

DEb Eb

Eb

Eb

0

2

4

6

8

10

12

A2 A6 A2 A6 A2 A6 A2 A6 A2 A6

121°C 115°C 110°C 105°C 100°C

Fir

mn

ess

, N

B.S. / Temperature

B

204�

study, the comparison of the sterilization regimes accompanied by the influence of EABS

which in combination with temperature showed different effects on the product firmness.

Indeed, it is known that thermal process causes disruption of cell membranes and results in

diffusion of water and low-weight molecules outside of the vegetable, leading to loss in

turgor pressure (Greve et al., 1994). Thus, a rubbery texture of the product can be developed

(Gonçalves et al., 2007). The most significant softening occurs due to increase in the

solubilization of pectic substances, loss of turgor pressure, and some degree of cell separation

during the heat treatment (Chang et al., 1995; Galindo et al., 2005; Gonçalves et al., 2007;

Waldron et al., 2003). However, the analysis of product texture which was sterilized in

contact with the EABS without heat treatment showed no significant changes, indicating no

effect of the EABS on product texture (Izumi, 1999; Koide et al., 2011; Wang et al., 2004).

Moreover, mild heat treatment at 50 °C with EABS demonstrated better firmness

preservation than untreated product after storage (Rahman et al., 2011). However, in these

studies the additional impact of temperature and EABS showed significant difference

between the used sterilization regimes. The impact of A2 on the firmness of pea grains

showed significant difference (p < 0.001) at 121, 115 and 110 °C in comparison to the A6

solution. For temperatures less than 110 °C, no significant differences were observed

between the solutions. For the corn grains, less significant statistical difference was found

between the EABS at 121, 115 and 110 °C with p values equal to 0.007, 0.008 and 0.003,

respectively. No statistical difference between the effect of EABS at 105 and 100 °C was

found (p =0.779 and 0.821).

7.6.2. Treatment effect on the color change

Color of product is a primary consumer perceived characteristic (MacDougall, 2002). For the

determination of color changes in canned green peas, the parameters L* and –a* are the most

commonly used since they represent the lightness and greenness, respectively. At the same

time, the L* and +b* are the most commonly used to define color changes of canned corn

since b* is the indicator of the product yellowness (Garrote et al., 2008; Smout et al., 2003).

Color plays an important role for food quality and effort is made to ensure that the color of a

processed product is as similar as possible to its raw form (MacDougall, 2002). However,

heat sterilization results in color change. Indeed, in works of Hayakawa et al. (1997)

�

205�

(Hayakawa and Timbers, 1977), it was found that color of green vegetables varies from the

natural green to an olive brown color, which could be explained by conversion of the

chlorophyll found in the green plants to pheophytin through the substitution of the

magnesium ion of the chlorophyll by the hydrogen (Bahçeci et al., 2005). In the present study,

the color changes of canned peas expressed by L*, a*, b* values are shown in Table 7.4.

Table 7.4: Changes in surface color of pea samples

Temperature,

°C

Brine

solutions

Pea

L +a +b

121 A2 40.00±0.06 Bb 3.67±0.65 Da 20.49±2.74 Cbc

A6 37.49±0.69 Da 8.38±0.31 Bb 20.12±0.16 Cb

115 A2 39.02±2.02 BCb 3.41±0.34 DEab 20.10±4.00 Cc

A6 37.85±1.01 CDa 9.49±0.31 Aa 20.36±0.44 Cb

110 A2 40.25±0.70 Bb 3.16±0.22 Eb 21.54±1.70 BCabc

A6 37.07±0.56 Da 5.89±0.54 Cc 20.38±1.48 Cb

105 A2 42.96±1.65 Aa 1.86±0.13 Fc 23.66±3.16 ABab

A6 34.80±1.81 Eb 2.57±0.14 Gd 17.72±1.35 Dc

100 A2 39.84±0.81 Bb 1.63±0.42 Gd 24.64±0.69 Aa

A6 33.03±0.64 Fc 1.97±0.09 Gd 23.31±0.43 ABa

Mean values ± standard deviation. The value of large letters indicates a significant difference

(p � 0.05) between all EABS at column; small letters presents the difference (p � 0.05) of

one type of EABS between temperature subgroups of column.

The measured color data of canned peas showed that the lightness and yellowness was most

significantly distinguished for the electro-activated brine solution A2 at 105 °C and A2 at

100 °C (Table 7.4). The effect of EABS on the lightness (L*) showed significant difference

(p < 0.001) for A2 against A6 at all temperatures except at 115 °C (p = 0.092). The parameter

of yellowness showed significant difference (p < 0.001) only at 110 °C. The other differences

were non-significant, indicating weaker effect of EABS on the yellowness characteristic of

green pea. As mentioned above, the bright green color, which is the most important indicator

of green products, was obtained with the electro-activated brine solution A6 at 115 °C (Table

206�

7.4). The longer time of treatment demonstrated the deterioration of the greenness parameter

which was in accordance with other studies suggesting that better color quality of green peas

may be obtained working for shorter time at higher temperatures (STHT) during sterilization

(Garrote et al., 2006, 2008; Ruiz-Ojeda and Peñas, 2013). In terms of temperature influence,

it is well known that the color-loss effect by overheating is mainly due to thermal degradation

of chlorophyll which is converted to pheophytin (Bahçeci et al., 2005; Ruiz-Ojeda and Peñas,

2013). Therefore, in the studies of thermal degradation kinetics of chlorophyll as a function

of pH value, it has been also reported that chlorophyll degradation corresponds to the first

order kinetic and the temperature dependence of the rate constant may well be described by

the Arrhenius equation (Canjura et al., 1991; Derossi et al., 2011; Hayakawa and Timbers,

1977; Schwartz and Lorenzo, 1991). This information clearly indicates the direct effect of

the temperature on the green color degradation. Thus, based on this established pattern, one

can conclude that EABS also impacts on one of the important color parameter which is the

(-a*) value. It was found that shorter processing times (and higher temperatures) showed

more significant interaction with surface which was inherent to the neutral solution (A6)

meanwhile acid solution impaired the greenish index (Table 7.4). Also, it should be noted

that by using temperatures of 121, 115, 110 and 105 °C, significant differences were observed

between the effect of the two EABS (A2 vs A6), while only at 100 °C the difference between

the effect of A6 and A2 was not significant (p = 0.119).

Conventional attempt of controlling chlorophyll degradation involves selection of cultivars

with reduced or enhanced chlorophyllase activity or chlorophyll concentrations, the addition

of antioxidants, and addition of alkalines or even alkalizing agents in brine solutions (Blair

and Ayres, 1943b; Derossi et al., 2011; Gilpin et al., 1959; Heaton and Marangoni, 1996).

However addition of alkalizing agents to the brine solutions cannot neutralize the organic

acids naturally present in vegetable tissue and may even have a negative impact on the

industrial scale due to the increase in pH which could provide a suitable environment for C.

botulinum spores to germinate (Derossi et al., 2011). Nevertheless, the best method to

minimize chlorophyll degradation has always been the application of high-temperature short-

time treatments and the use of a heat-stable chlorophyllide (Derossi et al., 2011; LaBorde

and von Elbe, 1994; Schwartz and Lorenzo, 1991). In context of the present study, the

�

207�

obtained data suggests that the application of EABS could be used to improve the

manufacturing process, preserving the green color more, considering the fact that better green

color of pea was found with EABS sterilization than with conventional treatment (Tables 7.1

and 7.4).

It is well known that the corn is rich in carotenoids giving it the yellow/reddish color.

However, the crucial problem during processing and storage is the stability of carotenoids

which gives attractiveness and acceptability to the product. Furthermore, degradation of

carotenoid affects not only color, but also its nutritive value and flavor since phytonutrients

such as lycopene, xanthophylls, lutein and zeaxanthin achieved a great deal of attention due

to their relation to eye health (Clinton, 1998; Gonçalves et al., 2007; Siems et al., 1999). In

this study, the color parameter data of the surface of the corn demonstrated that the best hue

retention was achieved with a shorter processing time, as shown in Table 7.5. Parameter such

as lightness (L*) was significantly different at 121 °C for A2 while parameter of redness

(+a*) was significantly different for A2 at 121 and 110 °C. The main parameter of quality

color characteristic of corn which is the yellowness (+b*), also demonstrated significant

difference when the product was sterilized at 121 and 115 °C with the electro-activated brine

solution A6. Less pronounced color of corn was reported at 121, 115 and 110 °C for the

solution A2, indicating different impacts of the EABS. Longer heating at less temperature

(105 and 100 °C) did not show any significant statistical difference between the samples.

208�

Table 7.5: Changes in surface color of corn samples

Temperature,

°C

Brine

solutions

Corn

L +a +b

121 A2 44.84±0.37 EFGbc 2.16±0.22 Aa 28.44±2.39 Ba

A6 50.49±0.61 Aa 0.16±0.03 Dc 36.32±1.24 Aa

115 A2 43.75±0.27 FGc 1.63±0.04 Bb 27.84±1.38 Ba

A6 48.10±1.69 Bb 1.60±0.84 Ba 34.10±0.24 Aa

110 A2 45.06±1.15 BCa 2.36±0.36 Aa 24.45±2.92 Ba

A6 47.29±3.68 DEFcd 1.48±0.17 Ba 28.36±2.93 Cb

105 A2 46.10±1.02 BCDEab 0.90±0.09 Cc 25.13±1.68 Cb

A6 42.77±0.57 Gd 0.77±0.10 Cb 25.16±1.82 Cb

100 A2 47.29±0.04 BCDa 0.88±0.03 Cc 25.11±1.24 Cb

A6 45.06±1.38 CDEFc 0.78±0.36 Cb 24.86±2.93 Cb

Mean values ± standard deviation. The value of large letters indicates a significant difference

(p � 0.05) between all EABS at column; small letters presents the difference (p � 0.05) of

one type of EABS between of temperature subgroups of column.

Carotenoids are known to undergo degradation due to isomerization, oxidation and

fragmentation promoted by heat, light and acids (Çinar, 2004). However, in the study of Scott

and Eldridge (2005), it was shown that canning caused no significant changes in carotenoid

content of corn. When corn is removed from the cob, the pericarp is ruptured resulting in

nutrient loss of carotenoids, sugars and other soluble components into the brine solution

(Scott and Eldridge, 2005). This loss and further carotenoid degradation caused by light,

oxygen, and pH could be minimized by appropriate canning temperatures (Nguyen et al.,

2001; Updike and Schwartz, 2003). In the present study, the impact of EABS on color was

found only for higher processing temperatures, greater than 110 °C, with the duration of

treatment playing the most important and defining role, as mentioned above. Nevertheless, it

was found that the conventional treatment with simple NaCl brine solution causes statistically

more significant color alteration in comparison with EABS (data not shown). It is also worth

noting that other studies reported that EABS had no effect on the surface color of tissue,

�

209�

general appearance, customer acceptance or other quality characteristics of the whole and

fresh-cut vegetables, such as lettuce, carrot, endive and corn salad (Bari et al., 2003; Izumi,

1999; tain et al., 2008).

7.6.3. Effect on the vitamin C

Vitamin C is primarily presented as biologically active L-isomer of the ascorbic acid (Bremus

et al., 2006). In this study, the concentration of vitamin C in fresh pea and corn was found to

be 15.97 ± 0.84 and 3.34 ± 0.61 mg/g, respectively. It is well known that vitamin C is unstable

in foods, therefore, processing and cooking conditions cause significant losses depending on

the specific parameters used, e.g., temperature, presence of oxygen, light, moisture content,

pH, etc. (Lešková et al., 2006). In the present study, it has been shown that the blanching and

subsequent sterilization of pea and corn led to significant reductions in ascorbic acid content

(Table 7.1). Moreover, it is also well known that naturally reduced hydrated form of ascorbic

acid is resistant to heating, but oxidized dehydrated form is unstable because of irreversible

oxidation to the inactive diketogulonic acid (Flaumenbaum et al., 1993). However, it was

found that the most important factor depending on reduced hydrated form of ascorbic acid

during sterilization is not high temperature but residual oxygen in the can. In studies of

Flaumenbaum et al. (1982, 1993), it was reported that the reaction rate constant of thermal

degradation of ascorbic acid is 10-20 times lesser in the absence of the air than in its presence

(Flaumenbaum et al., 1982; Flaumenbaum et al., 1993). A similar study was performed with

a simulated retort operation of vacuum sealed canned peas, at temperatures between 110 and

132 °C, with the authors concluding that both aerobic and anaerobic degradation of ascorbic

acid may occur during heating (Lathrop and Leung, 1980a). In our study, the highest

concentration of ascorbic acid was found at the lowest temperature of treatment (100 °C) for

pea and corn (Fig. 7.4, A, B). Moreover, analysis of the obtained data showed that the

presence of EABS did not have any significant impact. However, the product canned in the

electro-activated brine solution A2 displayed higher ascorbic acid concentration of 0.78 ±

0.05 and 0.33 ± 0.04 mg/g for pea and corn at 100 °C, respectively. The concentration of

ascorbic acid in contact with the electro-activated brine solution A6 was less compared to the

values obtained with the solution A2. This could be associated with the alkali property of the

solution which could highly affect its stability in comparison to an acidic environment.

210�

Moreover, it is very important to consider that even if the thermal degradation of vitamin C

is a significant issue during sterilization, a large proportion of vitamin C is lost due to

leaching into the surrounding brine (Lathrop and Leung, 1980b). Finally, it is possible to

consider that the use of electro-activated brine solution during pea and corn canning can

enhance the retention of vitamin C since the temperature used during sterilization can be

significantly reduced in comparison with conventional sterilization regime.

Figure 7.4: The bars groups indicate the effect of heat treatments on the changes of ascorbic

acid of canned peas (A) and corn (B) representing mean ± standard error of mean.The


BCa BCb

BCa

Cb

ABa

BCbABa

BCab

Aa

ABa

0

0,2

0,4

0,6

0,8

1

A2 A6 A2 A6 A2 A6 A2 A6 A2 A6

121°C 115°C 110°C 105°C 100°C

mg

/g

B.S. / Temperature

A

ABCa

Cbc

ABCa

BCc

ABCa

BCbc

ABa

ABCabcAa

ABCa

0

0,2

0,4

0,6

0,8

1

A2 A6 A2 A6 A2 A6 A2 A6 A2 A6

121°C 115°C 110°C 105°C 100°C

mg

/g

B.S. / Temperature

B

�

211�


between subgroups.

�

�

7.6.4. Analysis of the energy consumption

In this study, energy consumption induced by heating of canned vegetables (pea and corn)

was assessed by using energy balance of 2nd grade operations (Valentas et al., 1997). This

balance considers sterilization as a closed system which is subjected to intensive conditions

(heating). This system has a cumulative internal energy, which presents the heat (}) and heat

transfer operation (Û) when crossing the border of a system, as it is displayed in equations

8 and 9 (Barreiro et al., 1984; Valentas et al., 1997). The heat transfer is the result of the

temperature difference between the system and its environment and may be expressed in the

form of heat conduction, convection and/or radiation (Valentas et al., 1997). This means that

a corresponding temperature decrease results in reduced energy costs of the system and vice

versa (Fig. 7.5). As it was mentioned before, this type of sterilization is conditionally divided

into three main phases where the heating phase or “come-up” time is the most energy

intensive stage of sterilization. This is because when the steam fills the retort volume at

processing conditions, numerous losses take place such as the displacement of air by

condensed steam, losses due to convection, radiation, and surface energy losses (Usov et al.,

1987; Valentas et al., 1997). When the desired temperature is reached, the holding phase is

required to warm up the product in the slowest-heating zone which is the "critical point"

(Berry and Pflug, 2003; Unknown, 2007). In this phase, the steam is fed less intensely as the

required temperature in the retort has already been reached so the energy is used for

maintaining it at the same level in order to heat the product and may include losses to the

environment. The holding phase is crucial because it is important to achieve the desired

lethality level at the moment depending on the product properties, the initial contamination

of the product, and the targeted microorganism (Stumbo, 1973). In most cases, the high

temperature is used to reduce the duration of the holding phase which could cut the overall

time of sterilization and preserve more nutrients (Flaumenbaum et al., 1982).

In a previous work, it was found that by utilizing EABS instead of the conventional brine

solution, we could either lower the temperature of sterilization or shorten the time of

processing (Liato et al., 2015d). In order to achieve the required lethality (Table 7.3) at 121

212�

°C the sterilization time of pea and corn with NaCl solution has to be about 10 and 12 min,

respectively (Fig. 7.1, 7.2). The energy required for the sterilization of pea and corn at 121 °

C under such conditions is 2118.72 ± 42.37 and 2124.73 ± 42.49 kJ per a can of 385 ml,

respectively (Fig. 7.5.). At the same temperature in A2 solution the pea and corn is

maintained about 3 and 6 min, respectively; whereas in A6 solution they are processed during

3 and 4 min, respectively (Fig. 7.1, 7.2). Thus, less energy is required for sterilization with

EABS in accordance with the data presented in Fig. 7.5. An exception is the regime with

corn in A6 brine. In this case, the energy required for sterilization added with the energy

required to generate the electro-activated solution results in overall increased energy

consumption. Therefore, in order to obtain the solutions A2 and A6, one must expend energy

equal to 46.8 ± 0.15 and 93.6 ± 0.19 kJ, respectively. However, as it was mentioned before,

the holding time with A6 is lesser than with NaCl by more than 33%, which could result in

lowered energy consumption in factory conditions. It may be argued that the energy required

for sterilizing pea and corn grains in NaCl brine solution at 100 °C would cost less compared

to EABS, but in order to achieve the required lethality a considerably longer time will be

needed compared to EABS at the same temperature. This hypothesis was checked on pea

puree packed in 307 x 409 mm (DxH) cans between temperatures of 105 and 132 °C. The

results showed that the heating at 105 °C brings the most significant energy losses. This

observation was in good agreement with already reported information in the literature

(Barreiro et al., 1984). The heat treatment with A2 for pea and corn required around 15 and

19 min at 100 °C, respectively (Fig. 7.1 A, C). Treatments with A6 required holding times

of approximately 18 and 24 min, respectively (Fig. 7.1 B, D). Thus, the expended energy is

1453.27 ± 43.59, 1500.07 ± 45.02 kJ for A2 and 1455.67 ± 43.67, 1546.86 ± 46.40 kJ for A6

solutions to sterilize pea and corn grains in a can of 385 ml, respectively (Fig. 7.5).

�

213�

Figure 7.5: Energy consumption during sterilization of canned pea and corn with EABS and

NaCl at different temperatures.

�

7.7. Conclusion

This study showed that the quality of canned products strictly depends on the sterilization

temperatures, which in turn reflects on the energy costs. The EA technology as a hurdle

approach in contrast to conventional methods of sterilization could significantly improve the

canned product quality as well as decrease the energy costs. This work analysed quality

characteristics (vitamin C, colour and texture) by sterilizing the canned pea and corn in EABS

under moderate temperatures. Vitamin C was better preserved at the lowest heat treatment

conducted at 100 °C with the values of 0.78 ± 0.05 and 0.33 ± 0.04 mg/g for canned pea and

corn, respectively. In comparison to conventional sterilization (121 °C), it is 24% and 23 %

better for canned pea and corn, respectively. The best texture profile expressed as firmness

for pea and corn, a value of 5.19 ± 0.27 and 10.21 ± 1.09 N, respectively, were obtained with

electro-activated brine solution A2 for a short treatment time. With a solution A6, a less solid

texture for pea and corn was obtained with mean values of 4.20 ± 0.06 and 7.63 ± 1.14 N,

respectively. The short heating treatment time also resulted in better green color and

brightness of the canned pea with the A6 solution being significantly higher (a* = -9.5 ± 0.3)

than the A2 (a* = -3.7 ± 0.6). The yellowness of the corn was also significantly better with

A6 solution (b* = 36.32 ± 1.24) than with A2 (b* = 28.44 ± 2.39). Though the energy

1400

1600

1800

2000

2200

2400

NaCl A2 A6 A2 A6 A2 A6 A2 A6 A2 A6

121°C 115°C 110°C 105°C 100°C

Q,

kJ/

can

B.S. / Temperature

Pea

Corn

214�

consummation of neutral A6 was more than the acid A2 resulting in 46.8 ± 0.15 and 93.6 ±

0.19 kJ, respectively, the application of EABS could probably replace the sterilization

method.






�

215�

General conclusions and perspectives

The aim of this study was to extend the knowledge available today about electrochemical

technology and electro-activation process. The field of investigation was largely centered on

the canning of low-acid products. The objectives were chosen to reflect and possibly solve

the questions frequently encountered in canning, more specifically to counter the chinks in

the sterilization processes prevailing today which sometimes cause problems during food

production. Research for bettering the food sterilization process has claimed that utilizing

EA solution as a barrier/hurdle in a hurdle approach for sterilizing canned vegetables could

reduce sterilization temperatures resulting in increased organoleptic and nutritional quality

and reduced energy costs.

Before the potential of EA as an efficient hurdle was investigated, the first objective involved

studying the EA production process, physico-chemical properties of EA solutions was

thoroughly investigated. This was done for identifying an efficient and optimized production

process for EA. The first sub-objective, as highlighted in chapter 3, was to understand the

process and governing aspects of EA technology. Analysis were performed by varying salt

concentrations, current densities, type of salts used, furthermore, these tests were done in

combination with modifications to the reactor configuration during the excitation period.

Solution proprieties, such as pH, redox potential, oxygen concentration and the general

resistance of the electrochemical system were assessed. Any and all changes in the properties

of the EA solutions were related with the process parameters and physico-chemical

properties, subsequently they were evaluated by statistic optimization. The principal

requirement of an EA solution (EAS) is a high redox potential which can be related to the

metastable state of the chemical species generated on the electrode surface. The acidic

solution or the anolyte was found to be activated on and near the anode. Interestingly, even

though the choice of salt concentration and current intensity as process parameters did not

significantly affect the EAS, their choice did reflect in a change in the overall property of EA

solution. The type of electrolyte chosen exhibited a significant dependence on the pH of EAS,

it was concluded that NaCl was the preferred choice for achieving the minimum possible pH

of an acidic anolyte while NaHCO3 was selected for the production of a neutral anolyte due

216�

to its buffering capacity. For the production of catholyte, it was concluded that a NaCl based

electrolyte showed significantly better propriety changes than a NaHCO3 based electrolyte.

The required process conditions were subsequently calculated using «response surface

methodology» (RSM). Changing salt concentration and current density influenced the

physico-chemical properties of the solution, which can be attributed to the presence of the

ion-selective membrane. Analysis of the reactor configuration showed that the anion-

exchange membranes at the anodic side showed significant changes in redox potential when

a NaHCO3 based electrolyte was present in the anodic chamber and NaCl based electrolyte

in the middle section. This can be explained by the chlorine ion migration from the middle

section to the anodic chamber across the ion selective membrane. The second sub-objective

as highlighted in chapter 4 was optimizing the process of EAS production. The main goals

of optimization were to find the appropriate conditions and time to obtain the EAS solutions

with required proprieties. The task of optimization was to maximize redox potential and

minimize the global electrochemical resistance; although these tasks countered each other, it

was observed that the solution of this task was found by using the least possible salt

concentration without compromising on any of the underlined objectives. Overall it was

concluded that for producing EA solutions, all process parameters are significant, with the

type of salt and concentration being the most important and governing factors. Our finding

that for having the most active solution, the concentration of electrolyte should be as low as

possible, aligned with relevant research of other authors.

The second objective as stated in chapter 5 was to evaluate the EA solution in its post

excitation phase. The activity of EAS was investigated relative to its corrosive nature and life

time (relaxation) during the storage. It was observed that only the EA and control solutions

with pH <4 were corrosive to the tinplate can (300x407). Although the tested cans are

designed for the low-acid products, i.e. for foods with pH =~ 7, we observed that even with

the addition of the control solution (pH 6.5), there was corrosion in the cans, which was not

expected and is not in acceptable limits according to the stipulated regulations. Furthermore,

the ion metal concentration in the cans filled with product and EAS/control brine was within

limit as per the regulations. Finally, in context of the second objective it was concluded that

low pH EAS solutions similar to acidified chlorinated solutions may be corrosive to metal

�

217�

surfaces during prolonged contact times, however it is important to state that all the EAS

which were tested were not corrosive when they were subjected to standard canning

conditions, also, the relaxation time was observed not to be directly dependent on the

available chlorine concentration, which is contrary to the findings of some authors.

For our third objective (chapter 6), the sporicidal activity of EAS was tested against two

bacterial strains; the anaerobe Clostridium sporogenes PA 3679 (surrogate strain for

C. botulinum) and the thermo-resistant facultative aerobic Geobacillus stearothermophilus.

Obtained results showed that EAS in combination with mild heat treatments increased the

sporicidal capacity of EAS. Furthermore, EAS and a mild sterilization temperature when

used together exhibited synergy and significantly decreased the heat resistance of

C. sporogenes, which was not the case when they were used separately for sterilization. We

were able to determine that EAS could be used as an effective hurdle in a sterilization process

along with other barriers such as pH, salt concentration and heat and the fact that using EAS

in place of currently used brine solution resulted in lowering the sterilization temperature and

also that it exhibited synergy supports this claim. This was further supported by the lowered

thermal resistance of Geobacillus stearothermophilus when tested in EAS. Lastly, here we

report a new thermo-resistance value of Geobacillus stearothermophilus.

Fourth objective (chapter 7) aimed to evaluate the effect of EAS and mild sterilization

temperatures on the quality changes of canned pea and corn. Based on the heat-resistance

coefficients of the food spoilage bacteria C. sporogenes taken from the previous study done

with control and EAS solutions, different time/temperature combinations were assessed for

their effect on the quality based on the lethal value (TR=5D). All the time/temperature profiles

assessed showed increased quality retention with EAS than with the control solutions for

sterilization. The analysis pertaining to the impact of the EAS used at different temperatures

was found to be related to the morphology of vegetables. Short treatment duration in

combination with neutral EAS preserved the color significantly better while acidic EAS in

combination with short treatment duration preserved the texture significantly better of both

the vegetables. The lowest temperature treatment with EAS which we tested resulted in the

least amount of vitamin C being degraded for both the vegetables. Most importantly, our tests

218�

were successful in decreasing the currently used sterilisation temperature of 121 to 100°C for

low acid foods accompanied with relevant modifications in the time duration of the process.

The data obtained through mathematical validation of the process design and the results

observed for quality and nutrient retention suggest that the hypothesis is confirmed. We were

also successful in lowering the average energy costs of sterilization by 30%. Taking the

success of this work further and based on the validated hypothesis it can be safely said, if

supported with studies done about time/temperature profiles and EAS in combination

providing lethality rate, EAS could effectively not only reduce the sterilisation temperature,

it could also be utilised as an efficient and potent barrier in the hurdle approach of food

sterilisation and subsequent preservation.

This study makes major contributions to the knowledge of EA technology. It was the first

time that a three-cell reactor with multiple factors was analyzed. It was also the first attempt

at using response surface methodology to optimize the production of EAS with desired

proprieties. The investigation concerning corrosiveness showed that tin-cans designed to be

resistant to a non-corrosive medium, showed adequate interaction with EAS. The thermal

resistance of C. sporogenes and G. stearothermophilus spores in EAS for different

time/temperature profiles was investigated for the first time; in addition this study provided

new process parameter values of heat-resistant spores, highly relevant with respect to food

spoilage in tropical countries. This study also provides relevant thermal resistance data for

C. sporogenes, a surrogate strain of pathogen Clostridium botulinum, in EAS. Most

importantly, the study highlighted the synergism between EAS and sterilization temperature.

Although this work highlights several important findings, it also poses several questions

which open new avenues and recommendations for future research.

In practice of EAT it is assumed to use the solutions of sodium chloride due to their easy

availability and low price. In this study was at first time showed the proprieties changes of

sodium bicarbonate solutions during EA. Although the modification of cell configuration

allowed to obtain required proprieties, this sodium bicarbonate solution demonstrated strong

buffer properties. It will be also interesting to try other types of salts for EA, such as sodium

acetate, sodium sulfate or sodium benzoate. In addition, considering the trend of decreasing

�

219�

the sodium consumption due to its association with the development of cardiovascular

disease, reduction or total substitution of sodium could be of interest.

Application of EAS in food industry established a reputation as non-corrosive and highly

antibacterial agent for different technical surfaces. In this study for the first time EAS were

tested on the tin-plate cans where it demonstrated promising results for canning application.

In addition, the decreased heat treatment could be an interesting way to replace the metallic

cans for the more convenient and more practical plastic containers or aseptic technology

could be studied. The mathematical validation of thermal process showed that there was a

significant decrease in the sterilization temperature, however, for this to be validated

biological studies are required to be done to assess the microbiological state and stability of

the food where it is being applied. Additional studies with pathogen C. botulinum would be

essential for validating the process; it would be interesting to test this in order to insure the

microbiological safety of the product. Although the application of EAS is legalized in Japan,

the quality and safety of the product needs to be tested to provide information about

interactions between the metastable chemical species of EAS and organic matter.

It will be interesting to try this technology on other low-acid foods such as carrot, beans,

mixed vegetables, beetroots, some fish and meat. For such an extended application, further

tests of microbiological stability, nutritional quality and heat parameters would have to be

carried out before a standard process can be established relevant to the canning industry.

�

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221�

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