NONTHERMAL PROCESSING OF MILK PROCESSING OF MILK By LUZ DANIELA BERMÚDEZ A dissertation submitted in partial fulfillment of the requirements …

NONTHERMAL PROCESSING OF MILK

By

LUZ DANIELA BERMÚDEZ

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY (Engineering Science)

WASHINGTON STATE UNIVERSITY Department of Biological Systems Engineering

MAY 2008

ii

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation/thesis of

LUZ DANIELA BERMÚDEZ find it satisfactory and recommend that it be accepted.

___________________________________

Chair

___________________________________

___________________________________

___________________________________

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ACKNOWLEDGEMENTS

I would like to express my gratitude to my advisor, Dr. Gustavo V. Barbosa-Cánovas for

giving me the chance to participate in his projects, which has allowed me to grow

professionally, and for his patience and advice during these four years. My gratitude also

goes to my committee members, Dr. Barry G. Swanson, Dr. Juming Tang and Dr. Ralph

Cavalieri, for their time, enthusiasm and interest in this research project.

I would like to thank Dr. Kees Versteeg and Dr. Raymond Mawson (Food Science

Australia) and Dr. Maria G. Corradini (University of Massachusetts) for their comments

and suggestions during this research. In addition, I greatly appreciate the financial

support from US Army Natick during the Pulsed Electric Fields research.

Indeed, this dissertation could not be possible without the very valuable help of all the

administrative and technical staff from Biological Systems Engineering at WSU, the staff

from the Francheschi Microscopy and Imaging Center, Frank Younce at the Pilot Plant,

Jeannie Bagby and Sharon Himsl.

I would like to thank my former professors and great friends Maru Bárcenas, Fidel

Vergara, José Angel Guerrero-Beltrán, Pedro and Monserrat Wesche for being as my

second family. I also express my gratitude to my friends in Pullman: Thank you for

sharing your life with me for the last four years. I am still learning many things from all

of you, and you have made my life very nice.

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My heartfelt gratitude to my mother, who understands that sometimes dreams are far

away from home, for her unwavering support and encouragement.

Finally, I wish to express my deepest gratitude to my former advisor and professor, Dr.

Jorge Welti-Chanes. Thank you for your friendship, steadfast support and advice,

patience, and enthusiasm. You always have been a great example of a good researcher for

me and you have shown me how to enjoy each project and research task.

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NONTHERMAL PROCESSING OF MILK

Abstract

by Luz Daniela Bermúdez, Ph.D. Washington State University

May 2008

Chair: Gustavo V. Barbosa-Cánovas

Low frequency ultrasound at different intensities (24 kHz, 400 W, 120 μm) plus

heat (63ºC) was used to inactivate Listeria innocua in milk; the most intense treatment

was useful to pasteurization standards in shorter times. Inactivation kinetics did not

follow a first order model; Weibullian and four parameters model better fitted survivor

curves. Composition and physicochemical parameters of milk changed after sonication,

but are still within reported ranges for processed milk; e.g., protein content remained at

3%. Butter fat content was shown to be an important hurdle in cell inactivation with

ultrasound, as lower fat content resulted in faster and higher inactivation. Microscopy

studies performed with Transmission (TEM) and Scanning Electron Microscopy (SEM)

revealed that cell inactivation with ultrasound is caused by disruption of the cell

membrane, pore formation and break-down of cells. With SEM fat globules showed

smaller size (<1μm) after sonication, producing a whiter milk color and a more

homogenized and stable product matrix. Yogurt was processed with thermo-sonicated

milk, showing more compact structure with minor syneresis problems; microwave energy

was successfully used as a catalyst during long-term sample preparation for SEM,

reducing the required time.

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Pulsed electric fields (PEF) was used to inactivate spores of Bacillus cereus in

milk; processing conditions ranged from 0 to 240 pulses (2.5 μs), 20 to 40 kV/cm; room

temperature to 65ºC; recirculation/refrigeration systems and nisin (10 and 50 IU/ml) were

used selectively to reduce spore load in whole and skim milk. Spores showed resistance

to temperature, PEF and the presence of antimicrobials. After 40 kV/cm, 65ºC, 140

pulses (2.5 μs) 3.5 log reduction of spores were achieved. During processing, electrode

fouling was observed to generate arcing problems because of the presence of milk

deposits and bubble formation; the milk layer in the electrode was composed mainly of

protein, significantly reducing this milk component. Studies in strawberry flavored milk

and model systems using PEF were conducted to study the stability of coloring agent

Allura Red. HPLC showed the behavior of this dye, which has better stability when

combined with other additives and it is present in high concentrations in model systems.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS……………………………………………………………....iii

ABSTRACT……………………………………………………………………………….v

LIST OF TABLES………………………………………………………………………xxi

LIST OF FIGURES…………………………………………………………………..xxviii

INTRODUCTION……………………………………………………………………….40

CHAPTER ONE…………………………………………………………………………43

Thermal and nonthermal pasteurization of milk: a review

1. Introduction……………………………………………………………………...43

2. Pasteurization……………………………………………………………………45

2.1 Basic theory………………………………………………………………….45

2.2 Pasteurization process……………………………………………………….47

2.3 Vat pasteurization……………………………………………………………50

2.4 HTST pasteurization…………………………………………………………51

2.5 UHT pasteurization/sterilization……………………………………………..52

3. Nonthermal technologies………………………………………………………...55

3.1 Ultrasound …………………………………………………………………..56

3.1.1 Basic concepts of ultrasound technology…………………………….56

3.1.2 Power ultrasound…………………………………………………….56

3.1.3 Pasteurization of milk with power ultrasound……………………….57

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3.1.4 Enzymes……………………………………………………………...60

3.1.5 Nutritional properties………………………………………………...62

a) Proteins

b) Vitamins and minerals

3.1.6 Physicochemical characteristics……………………………………...64

a) Viscosity

b) Density

c) pH

d) Color

3.1.7 Processing of dairy products with ultrasound technology…………...68

3.1.7.1 Milk………………………………………………………………..68

a) Milk as beverage

b) Lactose free milk

c) Human milk

3.1.7.2 Yogurt……………………………………………………………..71

3.1.7.3 Cheese……………………………………………………………..73

3.1.8 Other uses of power ultrasound in the dairy industry………………..75

3.2 Pulsed electric fields: basic concepts………………………………………...76

3.2.1 Pasteurization of milk with Pulsed Electric Fields…………………..78

3.2.2 Enzymes……………………………………………………………...79

3.2.3 Nutritional properties………………………………………………...80

4. Final remarks…………………………………………………………………….81

References……………………………………………………………………………82

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Tables and figures……………………………………………………………………95

CHAPTER TWO……………………………………………………………………….102

Modeling the inactivation of Listeria innocua in raw whole milk when treated by thermo-

sonication

Abstract…………………………………………………………………………………102

1. Introduction……………………………………………………………………..103

2. Materials and methods………………………………………………………….107

2.1 Milk samples………………………………………………………………..107

2.2 Microbiological analysis……………...………………..…………………...108

2.2.1 Growth and inoculum of Listeria innocua cells….………………108

2.2.2 Enumeration of Listeria and mesophilic bacteria……..………….108

2.3 Thermal and thermo-sonication treatments……………...………………….109

2.3.1 Thermal treatment………………………………………………...109

2.3.2 Thermo-sonication treatments……………………………………109

2.4 Physicochemical characteristics……………………………………………...110

2.4.1 pH and titratable acidity …………………………………………110

2.4.2 Color…….………………………………………………………..110

2.5 Modeling and statistical analysis…………………………………………….111

3. Results and discussion………………………………………………………….112

3.1 Microbial inactivation………………………………………………………..112

3.2 Modeling……………………………………………………………………..114

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3.2.1 First model: Weibull distribution…………………………………115

3.2.2 Four-parameter model……………………………………117

3.3 Physicochemical characteristics……………………..……………………….118

4. Conclusions…………………………………………………………………….120

References………………………………………………………………………………120

Tables and figures………………………………………………………………………127

CHAPTER THREE…………………………………………………………………….137

Composition properties, physicochemical characteristics and shelf-life of whole milk

after thermal and thermo-sonication treatments

Abstract…………………………………………………………………………………137

1. Introduction……………………………………………………………………..138


2.1 Milk samples………………………………………………………………..140

2.2 Thermo-sonication treatments……………………………………...............140

2.3 Proximal analysis…………………………………………………...............140

2.4 Microbiological analysis……………………………………………………141

2.5 Physicochemical analysis…………………………………………………...141

2.5.1 pH and titratable acidity ………………………………….141

2.5.2 Density and freezing point………………………………..142

2.5.3 Color ……………………………………………………..142

2.5.4 Storage life…………………….…………………………..143

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2.7 Statistical analysis....………………………………………………………..143


3.1 Proximal analysis……………………………………………………….144

3.1.1 Protein content……………………………………………144

3.1.2 Fat content………………………………………………...145

3.1.3 Added water………………………………………………145

3.1.4 Solids Non-Fat (SNF)…………………………………….146

3.2 Physicochemical properties……………………………………….........147

3.2.1 pH, acidity, density and freezing point…………………...147

3.2.2 Color……………………………………………………...148

3.3 Storage life……………………………………………………….……..150

3.3.1 Microbiological aspects…………………………………..150

3.3.2 pH and lactic acid content………………………………..152

3.3.3 Color……………………………………………………...153

3.3.3.1 Net change in color

3.3.3.2 Hue angle (h*)

3.3.3.3 Chroma/saturation index (C*)

4. Conclusions…………………………………………………………………156

References………………………………………………………………………………157


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CHAPTER FOUR………………………………………………………………………174

Study of the mechanism of inactivation of Listeria innocua cells in whole milk under

thermo-sonication treatments using Scanning Electron Microscopy and Transmission

Electron Microscopy

Abstract…………………………………………………………………………………174

1. Introduction………………...……………………………………………….176

2. Materials and methods……………………………………………………...180

2.1 Listeria innocua cells……………………………………………….180

2.2 Milk samples………………………………………………………..180

2.3 Thermal and thermo-sonication treatments………………………...180

2.3.1 Ultrasound equipment……………………………….……180

2.3.2 Thermal treatment…………………………………….…..181

2.4 Sample preparation for electron microscopy……………………….181

2.4.1 Method with organic solvents …………………………...182

2.4.2 Freeze-Drying (FD) method……………………………...183

2.5 Scanning electron microscopy (SEM)……………………………...183

2.6 Transmission Electron Microscopy (TEM)………………………...183

3. Results and discussion……………………………………………………….184

3.1 SEM with HMDS dehydration……………………………………...184

3.2 Thermal treatment…………………………………………………..185

3.3 Thermo-sonication treatment……………………………………….186

3.4 SEM freeze drying………………………………………………….188

3.5 TEM………………………………………………………………...190

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3.6 Possible mechanism of inactivation of cells under thermo-sonication

treatments…………………………………………………………………...192

4. Conclusions…………………………………………………………………..193

References………………………………………………………………………………194

Figures………………………………………………………………………………….197

CHAPTER FIVE……………………………………………………………………….210

Microstructure of fat globules in whole milk after thermo-sonication treatment

Abstract…………………………………………………………………………………210

1. Introduction…………..……………………………………………………..212

2. Materials and methods…………..………………………………………….214

2.1 Milk samples………………………………………………………..214

2.2 Thermal and thermo-sonication treatments………………………...215

2.2.1 Ultrasound equipment…………………………………….215

2.2.2 Heat treatment…………………………………………….215

2.3 Scanning electron microscopy……………………………………...216

2.3.1 Sample preparation

2.4 Color………………………………………………………………..217

2.5 Fat content……..……………………………………………………217

2.6 Statistical analysis…………………………………………………..218

3. Results and discussion……………………………………………………...218

3.1 Microstructure of milk: fat globules………………………………..218

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3.2 Net Color Change and Fat Content…………...…………………….224

4. Conclusions…………………………………………………………………..225

References………………………………………………………………………………225


CHAPTER SIX…………………………………………………………………………241

Study of butter fat content in milk on the inactivation of Listeria innocua ATCC 51742

by thermo-sonication

Abstract…………………………………………………………………………………241

1. Introduction……………………………………………………………………..242


2.1 Milk samples………………………………………………………..244

2.2 Listeria innocua analysis…………………………………………...244

2.2.1 Growth of Listeria innocua cells………………………....244

2.2.2 Enumeration of Listeria and mesophilic bacteria………...245

2.3 Thermo-sonication treatments……………………………………...245

2.4 Proximal analysis…………………………………………………..246

2.5 Physicochemical characteristics……………………………………246

2.5.1 pH and titratable acidity …………………………………246

2.5.2 Density and freezing point……………………………….247

2.5.3 Color……………………………………………………..247

2.5.4 Shelf-life studies…………………………………………………247

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2.7 Statistical analysis………………………………………………….248


3.1 Inactivation rate of Listeria innocua……………………………….248

3.2 Proximal analysis…………………………………………………..251

3.2.1 Butter fat content and protein……………………………251

3.2.2 Solids-non-fat……………………………………………252

3.3 Physical-chemistry characteristics………………………………….253

3.3.1 pH and acidity…………………………………………….253

3.3.2 Density …………………………………………………...254

3.3.3 Freezing point…………………………………………….255

3.3.4 Color…………….………………………………………..255

3.4 Shelf-life……………………………………………………………256

3.4.1 Total plate count………………………………………….257

3.4.2 pH…………………………………………………………257

3.4.3 Color……………………………………………………...259

4. Conclusion…..…………………………………………………………………...260

References………………………………………………………………………………260


CHAPTER SEVEN…………………………………………………………………….283

Evaluation of the microstructure of thermo-sonicated yogurt with Scanning Electron

Microscopy using a shorter (new) sample preparation procedure

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Abstract…………………………………………………………………………………283

1. Introduction……………………………………………………………………..285


2.1 Milk and yogurt samples……………………………………………286

2.2 Milk pasteurization…………………………………………………287

Thermo-sonication of milk…………………………………………287

2.3 Yogurt preparation………………………………………………….287

2.4 Microscopy studies…………………………………………………288

2.4.1 Sample preparation…………………………………………...228

2.4.1.1 HMDS dehydration……………………………………..288

2.4.1.2 Freeze-drying…………………………………………...289

2.4.1.3 Microwave dehydration………………………………...290

3. Results and discussion…………………………………………………….........291

3.1 HMDS dehydration…………………………………………………291

3.2 Freeze-drying……………………………………………………….292

3.3 Microwave dehydration…………………………………………….292

4. Conclusions……………………………………………………………………..295

References………………………………………………………………………………295

Figures…………………………………………………………………………………..298

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CHAPTER EIGHT……………………………………………………………………..301

Inactivation of Bacillus cereus spores in milk using Pulsed Electric Fields-Enhanced

Thermal Pasteurization and nisin as a natural antimicrobial

Abstract…………………………………………………………………………………301

1. Introduction……………………………………………………………………..303


2.1 Milk samples………………………………………………………..305

2.2 Microbiological analysis……………………………………………305

2.2.1 Growth and inoculum of Bacillus cereus spores…………305

2.2.2 Enumeration of mesophilic bacteria and Bacillus cereus

spores……………………………………………………………….305

2.3 Nisin ………………………………………………………………..306

2.4 Pulsed electric field treatment………………………………………306

2.4.1 Experiments without recirculation/refrigeration cycles…..307

2.4.2 Experiments without recirculation/refrigeration cycles…..307

2.5 Thermal treatment…………………………………………………..308


4. Conclusions…………………………………………………………………….314

References………………………………………………………………………………314


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CHAPTER NINE……………………………………………………………………….327

Electrodepositing of Milk Materials during Pulsed Electric Fields Processing

Abstract…………………………………………………………………………………327

1. Introduction……………………………………………………………………..329


2.1 Samples……………………………………………………………. 331

2.2 PEF processing……………………………………………………...331

2.3 LactiCheckTM milk analyzer………………………………………..332

2.4 Color………………………………………………………………..332

2.5 pH and titratable acidity ……………………………………………333

2.6 Protein assay………………………………………………………..333

2.7 Statistical analysis…………………………………………………..334


3.1 Physicochemical and composition parameters of milk after PEF

processing…………………………………………………………………..335

3.2 Protein analysis……………………………………………………..337

4. Conclusions……………………………………………………………………..339

References………………………………………………………………………………339


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CHAPTER TEN………………………………………………………………………...349

Determination of Allura Red (Red # 40) by Reverse-Phase High-Performance Liquid

Chromatography (RP-HPLC) and physicochemical changes in strawberry flavored milk

under Pulsed Electric Field Processing

Abstract…………………………………………………………………………………349

1. Introduction……………………………………………………………………..351


2.1 Milk samples and model systems…………………………………..353

2.2 Pulsed electric fields treatment……………………………………..354

2.3 Microbiological analysis……………………………………………355

2.4 pH …………………………………………………………………..355

2.5 Color………………………………………………………………..355

2.6 High Performance Liquid Chromatography (HPLC) analysis……...356

2.6.1 Materials …………………………………………………356

2.6.2 Allura Red Extraction…………………………………….356

2.6.3 Chromatographic conditions……………………………...357

2.7 Storage life………………………………………………………….357


3.1 pH …………………………………………………………………..358

3.2 Color………………………………………………………………..358

3.2.1 Net change of color……………………………………….360

3.2.2 Hue angle (h*)…………………………………………….360

3.2.3 Chroma (C*)………………………………………………361

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3.3 Microbiological counts……………………………………………..361

3.4 Concentration of Allura Red………………………………………..362

4. Conclusions……………………………………………………………………..364

References………………………………………………………………………………364


CONCLUSIONS……………………………………………………………………….379

FUTURE RESEARCH…………………………………………………………………382

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LIST OF TABLES

CHAPTER ONE

1. Heat resistance of selected microorganisms……..........................................................95

2. Standard time-temperature combinations for pasteurization processes……………….96

3. Inactivation of microorganisms by ultrasound in milk and buffer solutions………….97

4. Physicochemical properties of milk…………………………………………………...98

5. Microbial inactivation in milk and dairy products under PEF…………..…………….99

CHAPTER TWO

1. Inactivation of mesophilic bacteria in raw whole milk with thermal and thermo-

sonication treatments…………………………………………………………………...127

2. Parameters of the Weibull equation fitted to thermal inactivation and thermo-

sonication treatments for Listeria innocua in raw whole milk…………………………128

3. Parameters of the Sigmoid or four parameter model (equation 4) fitted to thermal

inactivation and thermo-sonication treatments of different intensity for Listeria innocua

in raw whole milk…………………………....................................................................129

4. Physical-chemical characteristics of milk samples before and after thermal and thermo-


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CHAPTER THREE

1. Proximal analysis of raw milk, heat pasteurized milk and thermo-sonicated pasteurized

milk……………………………………………………………………………………..163

2. Physicochemical characteristics of raw milk, heat pasteurized milk and thermo-

sonicated pasteurized milk……………………………………………………………...164

CHAPTER FIVE

1. Color parameters, net change of color and fat content of raw whole milk, thermally

treated and thermo-sonicated milk……………………………………………………...233

CHAPTER SIX

1. Nutritional attributes reported and measured for four different butter fat content UHT

milk samples before and after thermal and thermo-sonication treatments……………..267

2. Physicochemical characteristics of different butter fat content milk samples (fat free,

1%, 2% and whole) before and after thermo-sonication (TS) treatments (63ºC, 120 μm by

30 min)……………………………….............................................................................268

CHAPTER NINE

1. Physicochemical and composition parameters of raw whole milk and PEF processed

milk……………………………………………………………………………………..344

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2. Color parameters and color functions of raw and PEF processed milk……………...345

CHAPTER TEN

1. List of ingredients and codification of samples used for the experiment....................367

2. Gradient program for HPLC analysis and determination of Allura Red in strawberry

flavored milk samples…………………………………………………………………..368

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LIST OF FIGURES

CHAPTER ONE

1. Color parameters (L, a, b) in raw, thermal treated and sonicated milk at different

intensities. L and a were significantly different from each other; b was not significantly

different…………………………………………………………………………………100

2. Dielectric rupture theory, E = electrical field strength, Ec = critical electrical field

strength………………………………………………………………………………….101

CHAPTER TWO

1. Inactivation of Listeria innocua in raw whole milk under thermal and thermo-


2. Weibull distribution fitted to thermal treatment survivor curve……………………..132

3. Four parameter model fitted to thermo-sonicated survivor data at 30% of amplitude

wave…………………………………………………………………………………….133

4. Weibull distribution fitted to survivor curve of thermo-sonication at 60% of amplitude

wave…………………………………………………………………………………….134

5. Weibull distribution fitted to survivor curve of thermo-sonication at 90% of amplitude

wave……………………………………………………………………………………135

6. Weibull distribution fitted to survivor curve of thermo-sonication at 100% of

amplitude wave…………………………………………………………………………136

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CHAPTER THREE

1. Luminosity (L*) of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-

sonicated Milk at different ultrasound intensities (30%, 60%, 90% and 100%) during

storage…………………………………………………………………………………..165

2. The a* value of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-sonicated

milk at different ultrasound intensities (30%, 60%, 90% and 100%) during

storage…………………………………………………………………………………..166

3. The b* value of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-sonicated


storage…………………………………………………………………………………..167

4. Mesophilic growth behavior of Raw Milk (RM), Heat Pasteurized Milk (TT), and

Thermo-sonicated milk during storage at 4ºC, at different intensities (30%, 60%, 90%

and 100%)………………………………………………………………………………168

5. The pH behavior of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-

sonicated Milk at different amplitude intensities (30%, 60%, 90% and 100%) during

storage…………………………………………………………………………………..169

6. Lactic acid content (%) Of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-

sonicated milk at different amplitude intensities (30%, 60%, 90% and 100%) during

storage………………………………………………………………………………….170

7. Net Change of Color of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-

sonicated milk at different ultrasound intensities (30%, 60%, 90% and 100%) during

storage using raw milk color at time zero as control…………………………………...171

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8. Hue Angle of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-sonicated


storage…………………………………………………………………………………..172

9. Chroma/Saturation index of Raw Milk (RM), Heat Pasteurized Milk (TT), and

Thermo-sonicated milk at different ultrasound intensities (30%, 60%, 90% and 100%)

during storage…………………………………………………………………………...173

CHAPTER FOUR

1. Listeria innocua without any treatment inoculated in raw whole milk and using HMDS

dehydration for SEM. Control sample, 20 kV, magnification 5.90 K, 5.1

μm………………………………………………………………………………………197

2. Streptococcus chains present as natural flora in raw whole milk and using HMDS

dehydration for SEM. 20 kV, magnification 5.90 K, 5.1 μm…………………………..198

3. Microccocus and Bacillus present as natural flora in raw whole milk and using HMDS

dehydration for SEM. 20 kV, magnification 5.90 K, 5.1 μm…………………………..199

4. Listeria innocua cells after thermal treatment (63ºC by 30 min) in raw whole milk and

using HMDS dehydration for SEM. Cell wall shows thinning compared with the control

sample. 20 kV, magnification 5.90 K, 5.1 μm………………………………………….200

5. Listeria innocua cells after 10 min of thermo-sonication (63ºC and 120 μm) in raw

whole milk and using HMDS dehydration for SEM. Cells show puncturing outside the

cell wall. 20 kV, magnification 6.00 K, 5.0 μm………………………………………..201

xxxi


whole milk and using HMDS dehydration for SEM. Cells show some breakage lines in

the middle, suggesting a possible rupture………………………………………………202


whole milk and using HMDS dehydration for SEM. Some cells show puncturing outside

the cell wall in addition to a broken cell in the middle of the image. 20 kV, magnification

6.00 K, 5.0 μm………………………………………………………………………….203


whole milk and using HMDS dehydration for SEM. Some cells show pitting outside the

cell wall in addition to a broken cell in the middle of the image. 20 kV, magnification

6.00 K, 5.0 μm………………………………………………………………………….204

9. Listeria innocua cells after 30 min of thermal treatment (63ºC) in raw whole milk and

using freeze-drying as sample preparation for SEM. Cells show thinning in the cell wall.

20 kV, magnification 6.00 K, 5.0 μm…………………………………………………..205


whole milk and using freeze-drying as sample preparation for SEM. Cell clearly shows

the disruption and breakage of its membrane. 20 kV, magnification 6.00 K, 5.0

μm……………………………………………………………………............................206


whole milk and using freeze-drying as sample preparation for SEM. Broken cells are

shown in the right corner of the image. 20 kV, magnification 6.00 K, 5.0

μm………………………………………………………………………………………207

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Figure 12. Transmission Electron Microscopy for Listeria innocua cells: (a) control

sample; (b) thermo-sonicated (63ºC and 120 μm) after 10 min, showing roughness in the

cell wall; (c) thermo-sonicated (63ºC and 120 μm) after 30 min, showing pore formation;

(d) thermo-sonicated (63ºC and 120 μm) after 30 min, showing pore formation and

disruption of the cell wall; (e) thermo-sonicated (63ºC and 120 μm) after 30 min,

showing lack of cytoplasm; (f) thermo-sonicated (63ºC and 120 μm) after 30 min,

showing unseen effects; (g) thermo-sonicated (63ºC and 120 μm) after 30 min with the

cell membrane broken; (h) thermo-sonicated (63ºC and 120 μm) after 30 min, showing

cytoplasm clumping. Scale 1000 to 2000

nm………………………………………………………………………………………208

CHAPTER FIVE

1. Fat globule in raw whole milk used as a control sample. The globule shows its

integrity; no important changes on the surface or membrane are detected. Magnification

7,000x…………………………………………………………………………………..234

2. Microstructure of thermally treated fat globule after 30 min at 63°C in whole milk.

Magnification 6,000x…………………………………………………………………...235

3. Thermo-sonicated (63ºC, 120 μm for 30 min) fat globules of whole milk showing the

disintegration of the milk fat globule membrane (MFGM) Magnification 7,900x and

6,000x (top); 5,000x and 6,000x (bottom)……………………………………………...236

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4. Graphic representation of the changes in the MFGM in a fat globule after

homogenization (top) and general microstructure of milk after homogenization

(bottom)…………………………………………………………………………………237

5. Thermo-sonicated (63ºC, 120 μm for 30 min) fat globules in whole milk showing the

presence of casein micelles added to the main structure. Magnification 10,000x and

8,000x…………………………………………………………………………………...238

6. Microstructure of fat content in thermo-sonicated (63ºC, 120 μm for 30 min) showing

size reduction in fat globules and the presence of thousands of casein micelles.

Magnification 7,100x…………………………………………………………………...239

7. A comparison of the microstructure of fat globules after three different treatments

using Scanning Electron Microscopy (SEM): a) Thermally treated (63ºC) fat globule

structure, b) thermo-sonicated (63ºC, 120 μm) fat globule structure after 10 min of

treatment and c) thermo-sonicated (63ºC, 120 μm) fat globule after 30 min of treatment

in whole milk. Magnification a) 5,900x, b) 5,900x, c) 6,000x………………………...240

CHAPTER SIX

1. Inactivation of Listeria innocua ATCC 51742 using thermo-sonication treatments

(63ºC, 120 μm by 30 min) in four different butter fat content milks…...........................269

2. L value of different butter fat content milk samples (fat free, 1%, 2% and whole)

before (control) and after thermo-sonication (TS) treatments (63ºC, 120 μm by 30

min)……………………………………………………………………………………..270

xxxiv

3. a value of different butter fat content milk samples (fat free, 1%, 2% and whole) before

(control) and after thermo-sonication (TS) treatments (63ºC, 120 μm by 30

min)……………………………………………………………………………………..271

4. b value of different butter fat content milk samples (fat free, 1%, 2% and whole) before

(control) and after thermo-sonication (TS) treatments (63ºC, 120 μm by 30

min)……………………………………………………………………………………..272

5. Growth of mesophilic bacteria in milk with different butter fat content (fat free, 1%,

2% and whole) in control (C) and thermo-sonicated (TS) samples (63ºC, 120 μm by 30

min) stored under refrigerated conditions (4ºC)………………………………………..273

6. Growth of mesophilic bacteria in milk with different butter fat content (fat free, 1%,

2% and whole) in control (C) and thermo-sonicated (TS) samples (63ºC, 120 μm by 30

min) stored under ambient temperature (21ºC)…………………………………………274

7. pH evolution during storage life of thermo-sonicated (TS) and control (C) milk

samples with different butter fat content (fat free, 1%, 2%, and whole) under refrigerated

conditions (4ºC)………………………………………………………………………...275

8. pH evolution of thermo-sonicated (TS) and control (C) milk samples with different

butter fat content (fat free, 1%, 2%, and whole) during storage life under ambient

temperature (21ºC) conditions………………………………………………………….276

9. L value evolution during the storage life of thermo-sonicated (TS) and control (C) milk


conditions (4ºC)………………………………………………………………………...277

xxxv

10. a value evolution during the storage life of thermo-sonicated (TS) and control (C)

milk samples with different butter fat content (fat free, 1%, 2%, and whole) under

refrigerated conditions (4ºC)……………………………………………………………278

11. b value evolution during the storage life of thermo-sonicated (TS) and control (C)

milk samples with different butter fat content (fat free, 1%, 2%, and whole) under

refrigerated conditions (4ºC)……………………………………………………………279

12. L value for thermo-sonicated (TS) and control (C) milk samples with different butter

fat content (fat free, 1%, 2%, and whole) during the storage life under ambient


13. a value for thermo-sonicated (TS) and control (C) milk samples with different butter



14. b value for thermo-sonicated (TS) and control (C) milk samples with different butter



CHAPTER SEVEN

1. Microstructure of commercial yogurt using HMDS as sample preparation technique

(top); microstructure of commercial yogurt using microwave dehydration as sample

preparation technique (bottom). In all the pictures the protein and fat structure can be

observed………………………………………………………………………………...298

xxxvi

2. Microstructure of thermo-sonicated yogurt using HMDS for sample preparation

technique showing clusters of yogurt in a more compact structure……………….........299

3. Microstructure of thermo-sonicated yogurt using microwave dehydration for sample

preparation, showing these images the internal composition and structure of this dairy

product, lactose crystals, protein structures and fat globules modified in their structure

because of the sonication……………………………………………………………….300

CHAPTER EIGHT

1. Inactivation of Bacillus cereus spores in skim milk using pulsed electric fields of 35

and 40 kV/cm without refrigeration (top); temperature profile for the same experiment

(bottom)………………………………………………………………............................319


and 40 kV/cm plus 40ºC as mild thermal treatment (top); temperature profile for the same

experiment (bottom)…………………………………………………………………….320


and 40 kV/cm plus 50ºC as thermal treatment (top); temperature profile for the same

experiment (bottom)…………………………………………………….........................321

4. Inactivation of Bacillus cereus spores in skim milk thermal treatments at 45ºC, 55ºC,

65ºC and 75ºC (top); temperature profile for the same experiment

(bottom)…………………………………………………………………………………322

xxxvii


and 40 kV/cm plus 55ºC as thermal treatment (top); temperature profile for the same


6. Inactivation of Bacillus cereus spores in skim and whole milk using pulsed electric

fields of 40 kV/cm plus 55ºC as thermal treatment (top); temperature profile for the same


7. Inactivation of Bacillus cereus spores in skim and whole milk using pulsed electric

fields of 30 and 40 kV/cm plus 65ºC as thermal treatment (top); temperature profile for

the same experiment (bottom)………………………………………………………….325

8. Inactivation of Bacillus cereus spores in skim (SM) and whole milk (WM) using

pulsed electric fields of 40 kV/cm plus 65ºC as thermal treatment and Low Concentration

(LC) of nisin (10 IU/ml) and High Concentration (HC) of nisin (50 IU/ml) (top);

temperature profile for the same experiment (bottom)…………………………………326

CHAPTER NINE

1. Standard curve of Bovine Serum Albumin…………………………………………..346

2. a) Fouling of the electrode after processing raw whole milk using PEF at 40 kV/cm,

240 pulses of 2.5 μs and 50ºC; b) Erosion of the stainless steel electrode after processing

using the above mentioned conditions of PEF treatment……………………………….347

3. Total protein content in milk, raw milk, PEF milk and the electrodeposited

material…………………………………………………………………………………348

xxxviii

CHAPTER TEN

1. Chemical structure of Allura Red (Red #40) with chemical name disodium (5Z) -5-[(2-

methoxy – 5 – methyl-4-sulfonatophenyl) hydrazinylidene] – 6 – oxonaphthalene -2-

sulfonate………………………………………………………………………………...369

2. Inlet and outlet temperatures of commercial flavored strawberry milk samples (N, L)

and the model systems (M1, M2) during the Pulsed Electric Fields processing

treatment………………………………………………………………………………..370

3. pH behavior during storage (4ºC) of untreated (C) and PEF treated (P) samples of

commercial strawberry flavored milk (N, L) and model systems (M1, M2)…………...371

4. a* color parameter for samples during storage (4ºC) of untreated (C) and PEF treated

(P) samples of commercial strawberry flavored milk (N, L) and model systems (M1,

M2)……………………………………………………………………………………...372

5. Net change of color (ΔE) for samples during storage (4ºC) for samples of commercial

strawberry flavored milk (N, L) and model systems (M1, M2)………………………...373

6. Hue angle for samples during storage (4ºC) of untreated (C) and PEF treated (P)

samples of commercial strawberry flavored milk (N, L) and model systems (M1,

M2)……………………………………………………………………………………...374

7. Chroma or saturation index for samples during storage (4ºC) of untreated (C) and PEF

treated (P) samples of commercial strawberry flavored milk (N, L) and model systems

(M1, M2)…………………………………………………………..................................375

8. Microbial growth for samples during storage (4ºC) of untreated (C) and PEF treated

(P) samples of commercial strawberry flavored milk (N, L) and model systems (M1,

M2)……………………………………………………………………………………...376

xxxix

9. Allura Red Concentration (mg/mL) for samples during storage (4ºC) of untreated (C)

and PEF treated (P) samples of commercial strawberry flavored milk (N, L) and model

systems (M1, M2)………………………………………………………………………377

40

INTRODUCTION

The search for new alternatives for pasteurizing and processing milk has led food

scientists and technologists to use preservation and processing factors other than heat in

order to ensure microbiological safety and to preserve quality characteristics of the food.

Most of the so-called nonthermal technologies are still undergoing research, with

encouraging results in many aspects. This dissertation deals with two of these emerging

technologies, the use of ultrasound and pulsed electric fields to pasteurize and process

milk.

Chapter One represents a review of state-of-the-art ultrasound and pulsed electric fields

technologies focused in milk pasteurization and the processing of dairy products such as

cheese and yogurt. The first part of this chapter assesses the basic theory of thermal

pasteurization, with the different processing variables of this method showing why the

use of heat has been used for years in the food industry to inactivate microorganisms.

Chapter Two presents inactivation studies of Listeria innocua as a surrogate for a

pathogenic microorganism in raw milk using the combination of mild thermal treatment

with different ultrasound intensities. The use of mathematical models to describe a non-

first-order kinetic of inactivation curves is addressed in this chapter. Composition and

physicochemical characteristics of thermo-sonicated milk with similar processing

conditions used during inactivation experiments are studied in Chapter Three.

41

Chapter Four presents some of the first studies of the possible mechanism of cell

inactivation because of the use of ultrasound. Transmission and Scanning Electron

Microscopy were used as a tool to analyze Listeria cell structure after sonication,

showing the disruption of the cell membrane, breakdown of cells and formation of pores

outside the cell wall.

Chapter Five deals with the structure of milk after thermo-sonication. Scanning Electron

Microscopy was useful to highlight changes, mainly in the fat globules structure, that

take place from the beginning to the end of the treatment with ultrasound. Smaller size,

rugged surface and disruption of the milk lipid globule membrane were found in the

images of sonicated fat globules.

Chapter Six represents the inactivation studies of the same surrogate microorganisms, but

using milk with different butter fat content, showing the obstacle that this component

represents for microbial inactivation, along with some of the physicochemical changes

and the shelf-life of milk after sonication.

With the acquired knowledge from the previous chapters, the processing of yogurt with

thermo-sonicated milk is shown in Chapter Seven; the study was also extended to provide

a comprehensive analysis of the microstructure of this yogurt using Scanning Electron

Microscopy, while at the same time developing an innovative and faster sample

preparation method for microscopy using microwave energy.

42

Chapter Eight shows the different combinations of electric field strengths, number of

pulses, temperature, kind of milk and presence or absence of nisin as an antimicrobial

factor in the inactivation of Bacillus cereus spores. Even with the most severe processing

conditions, spores were not totally inactivated in milk.

During previous spore inactivation studies, fouling of the electrode and arcing problems

were observed through pulsed electric fields processing, so Chapter Nine represents a

detailed study of milk composition and the components of the milk deposited into the

electrode after using the equipment under strong conditions to inactivate spores.

Finally, Chapter Ten shows the influence of pulsed electric fields during the processing

of strawberry flavored milk and the stability of this product and its coloring agent (Allura

Red) during storage. High Performance Liquid Chromatography was used to assess the

dye behavior during the experiments.

43

CHAPTER ONE

THERMAL AND NONTHERMAL PASTEURIZATION OF MILK:

A REVIEW

Daniela Bermúdez-Aguirre and Gustavo V. Barbosa-Cánovas

1. Introduction

Conventional methods of pasteurizing milk involve the use of elevated temperatures

regardless of treatment (batch, HTST or UHT pasteurization), and the quality of the milk

is affected because of the use of high temperatures. Consequences of thermal treatment

include a decrease in nutritional properties such as vitamins or denaturation of proteins,

and sometimes the flavor of milk is undesirably changed. These changes are produced

simultaneously that the goal of the pasteurization processed is achieved, which is to have

a microbiologically safe product free of pathogenic bacteria and to reduce the load of

deteriorative microorganisms and enzymes, resulting in a product with a longer storage

life.

During the processing of dairy products, milk must be pasteurized under specific

conditions to achieve the ideal characteristics of flavor, color, or texture for the final

product such as yogurt, cheese or ice cream, while at the same time maintaining the

microbiological limits established for these products.

Currently, food science and technology is providing new alternatives for

processing food with the aim of have fresh-like characteristics and reduce the decrease of

quality attributes, while at the same time producing a safe and stable product with low

cost and availability for consumers. One of these emerging technologies is ultrasound

44

(the use of ultrasonic waves applied to the food as an alternative to traditional thermal

treatment) to inactivate microorganisms and enzymes while producing fewer changes in

the product. Ultrasound technology is not new in food processing; this technology is

widely used, for example, in quality control when the range of high frequency of

ultrasound is used, this being a non-destructive technique. However, the use of ultrasound

at low frequencies to promote cell disruption and chemical reactions is now under study,

with many favorable results.

Another nonthermal technology that is studied in milk is Pulsed Electric Fields

(PEF) in which contact between the food and the electrical discharges between electrodes

allows reduction of the microbial and enzymatic activity in milk in microseconds with

important energy and money savings. Treatment of some pathogenic bacteria and

enzymes by PEF has been tested in milk with successful results, while studies related to

spore inactivation or in quality aspects such as nutritional characteristics or stability of

other dairy products under this technology are ongoing.

This chapter includes a summary of pasteurization technologies beginning with

the three commercially used time-temperature thermal processing technologies followed

by two sections in which nonthermal technologies for processing milk are discussed. The

nonthermal technologies that will be discussed are the use of ultrasound at low frequency

and also the so-called “power ultrasound.” A brief review of current progress in the

processing of some dairy products such as sonicated cheese and sonicated yogurt will

also be included, remarking on the advantages and potential of this technology for the

dairy industry. The second part will include discussion of state-of-art of the Pulsed

45

Electric Fields technology used in milk, the target pathogens tested in milk, as well as

enzymes and other milk related studies with PEF.

2. Pasteurization

2.1 Basic theory

Pasteurization is a thermal process applied to different foods with the goal of destroying

target non-spore pathogenic microorganisms (vegetative cells) such as Salmonella,

Coxiella burnetti, Escherichia coli, Listeria monocytogenes, or Mycobacterium

tuberculosis. Pasteurization extends the shelf-life of the product by reducing the initial

load of microorganisms (deteriorative) and inactivating key enzymes that could

deteriorate the quality of the food.

After pasteurization, some viable microorganisms remain in the product. These

microorganisms are thermoduric and thermophilic bacteria, and represent the

deteriorative genre of organisms. Thermoduric bacteria resist high temperatures, while

thermophilic bacteria need high temperature for optimal growth. Examples of

thermoduric bacteria are some species of Streptococcus and Lactobacillus; examples of

thermophilic bacteria are species of Bacillus and Clostridium (Jay, 1992). The presence

of these microorganisms is responsible for the spoilage of milk even under refrigeration.

Lactobacillus produce an increase of lactic acid in milk during storage, decreasing the pH

of milk and resulting in spoilage. Other heat resistant microorganisms and enzymes are

related to the proteolysis of milk during storage, generating undesirable changes in flavor,

taste and consistency. In other products such as juices, mayonnaise or mustard, spoilage

is related to other deteriorative microorganisms such as yeasts and molds; pasteurization

46

is an adequate method to inactivate or reduce the counts of these groups of non-

pathogenic bacteria. Generally, pasteurized products are stored under refrigerated

conditions to extend their freshness; shelf-life is limited to a few weeks (generally up to

two weeks), but freshness will depend on several factors, for example the quality of the

raw material, Good Manufacturing Practices after pasteurization, handling, transportation

and storage conditions, among others.

Some commercial and commonly pasteurized foods include milk, liquid eggs,

shell eggs, beer, wine, fruit and vegetables juices, ciders, vinegars, meat products such as

ham, oysters, honey, mustard, mayonnaise, water, and sous-vide and chilled foods. Many

more pasteurized products are consumed that are restricted to specific geographic areas

around the world.

The final microbiological quality of a food will depend on the initial quality of the

raw ingredients; for example, in the case of milk, in some industrialized nations raw milk

has an initial microbial load close to 103 cfu/ml; after pasteurization, which requires at

least a 5 log reduction of the bacterial load to fulfill legal and sanitary regulations, the

microbial quality of pasteurized milk will be excellent, because the counts of mesophilic

bacteria will probably be smaller than can be detected with conventional and standard

microbiological assays, and pathogenic bacteria will not be present in the product.

According to standards in the United States, Grade A raw milk may not exceed 300,000

cfu/ml, and in the European Community the bacterial count should be smaller than

100,000 cfu/ml. In the United States, milk after pasteurization should contain less than

20,000 cfu/ml (Jensen et al., 1995). Nevertheless, in sub-developed countries the initial

count of mesophilic bacteria is sometimes as high as 105 or 106 cfu/ml, and although the

47

inactivation of high loads of microorganisms is provided by pasteurization, small counts

of bacteria can remain in the product, shortening the shelf-life even under refrigerated

conditions. The reason of these elevated counts in some countries is related to the health

of the animal and the use of incorrect sanitary practices during milking, which generates

cross-contamination by adding bacteria from the cow, the ground, the milker and the

milking equipment. This is why the storage life of pasteurized milk is different around

the world. Similar principles apply to other products such as juices; if sanitary practices

are not observed throughout production, pasteurization might be not adequate to

guarantee a long storage life.

2.2 Pasteurization process

Pasteurization, as mentioned previously, is the heating of foods (mainly beverages) under

specific time-temperature conditions to destroy disease microorganisms. The first

pasteurization process carried out by Louis Pasteur used temperatures ranging from 55°C

to 60ºC. He noticed that when heated some products such as wine or vinegar to a certain

temperature and then held the products for a length of time, fermentation was stopped

and no spoilage was observed in the products in the following days. These experiments

were conducted from 1860-1864, and Pasteur realized that food spoilage was produced

by certain microorganisms. This is probably the first time in food microbiology that

spoilage was attributed to specific organisms and not to unknown substances as was

thought previously. Pasteur related human diseases to the production of some substances

generated by fermentation of foods such as wine or beer, but he found that if he boiled

the product, quality characteristics such as flavor often disappeared. Because of that,

48

Pasteur used lower temperatures below the boiling point (100ºC) just to stop the

fermentation of the product. He applied the process to products such as beer (Satin,

1996). Lewis and Heppell (2000) describe how Pasteur developed the first type of

pasteurization using a long time and low temperature batch system when he realized that

tuberculosis was produced by microorganisms present in milk. In 1880, in Germany,

pasteurization was used commercially to extend the shelf-life of milk, and Soxhlet was

the first person to recommend the pasteurization of milk to avoid diseases related to

pathogenic microorganisms associated with milk. Gradually, pasteurization was

transferred to other countries despite being confronted by obstacles such as being

considered a method to hide or mask undesirable conditions of milk related with low

quality. The first pasteurization system in America was used in New York by Charles

North in 1907 and this was also a batch system matching Pasteur’s conditions. By the

1920s, pasteurization had become a common method to extend the shelf-life of milk in

Canada and the United States, although in some countries of Europe it was not yet

accepted. Not until 20 years later, in England and Wales, did pasteurization start to

become a common way to process milk, although at the present time in some parts of the

United Kingdom raw milk is permitted to be sold if it is accordance with Satin (1996). In

Scotland, the establishment of regulations to pasteurize milk began in 1983.

It is amazing that pasteurization, as practiced since 1860, which has shown such

positive effects in the control of human diseases caused by pathogenic bacteria mainly

present in milk, is not regulated in the entire world. Some reasons for this for this

resistance are related to loss of quality of the product in flavor and taste, but according to

Pasteur’s principle, pasteurization should be used to inactivate microorganisms related to

49

fermentation and food spoilage, while avoiding boiling the product to maintaining quality

characteristics. However, microorganisms have become more thermally resistant, and the

presence of some of these that commonly should not be present in specific foods, the so-

called emerging pathogens, are forcing the food industry to maintain very high

temperatures, and even to use sterilization conditions in products such as milk and juices.

Recent outbreaks all over the world show how some microorganisms have adapted to

survive even in products that do not offer an ideal growth medium.

Target microorganisms in pasteurization include vegetative cells of pathogenic

bacteria, yeast and molds. The heat resistance and conditions of inactivation for these

genres are shown in Table I. In the food industry there are three main types of

pasteurization processes, which differ mainly in their combinations of time and

temperature. Although the International Dairy Foods Association (IDFA) in 2006 defined

eight pasteurization processes, the three most commercially used and well known

processes—old or conventional pasteurization, High Temperature Short Time (HTST)

and Ultra-High Temperature (UHT) pasteurization/sterilization—will be covered in this

chapter. The five remaining types of pasteurization mentioned by IDFA are cited here.

The common name is Ultra Pasteurization (UP), which refers to thermal treatments

between 89ºC (191ºF) and 100ºC (212ºF) in various intervals of time in seconds and

microseconds. In Table II, the conventional and most common combination time-

temperature parameters of pasteurization are presented.

50

2.3 Vat pasteurization

Vat pasteurization is a very old process that was used successfully in the middle of the

19th century. Vat pasteurization is often called batch pasteurization, low temperature long

time (LTLT), or holding pasteurization because of the conditions during the processing.

Generally, the process is performed in a vat with a jacket in which hot water, steam, or

another heating source is incorporated to heat the food contained inside the vat. The fluid

food is pumped into the vat and heated quickly until the temperature rises; in this

pasteurization process 63ºC is the required temperature, which must be held for at least

30 minutes. Continuous agitation with an industrial stirrer is required during the process

in order to ensure uniform heat transfer to each particle of the food. Time elapsed before

reaching the required temperature is known as the heating come-up time and is not

considered part of the holding time. After 30 minutes the product must be cooled inside

the vat or pumped to a plate or tubular heat exchanger to cool to avoid the growth of

surviving bacteria and to proceed to the packaging process, e.g., to bottling or cartoning.

After pasteurization it is important to observe Good Manufacturing Practices (GMP) to

avoid post-contamination of the product and to extend its shelf-life. Some of the

disadvantages of vat pasteurization are characteristics of the batch process; like many

other operations in food processing, high production is limited to the number of available

vats and space in the industry. However, this pasteurization is widely used by small

farmers and dairy industries to pasteurize milk.

51

2.4 HTST pasteurization

High Temperature Short Time (HTST) pasteurization is the most commonly used method

to pasteurize food in many countries, mainly because this process reduces the time

compared to vat pasteurization, and it uses lower temperatures than UHT sterilization,

avoiding some of that technology’s undesirable effects on the quality of the product.

HTST process is commonly carried out at 72ºC for 15 s; in some countries, such as

Canada, the time of treatment is longer, and usually 16 s is employed as the official

holding time at 72°C. HTST pasteurization is a continuous process that takes place in a

heat exchanger (commonly in a plate heat exchanger). The fluid food is pumped from a

tank to the regeneration system in which the product is pre-heated to around 60°C to

65ºC. After that, the food is transported to the main heating unit in which the required

temperature is reached with heat transfer from hot water flowing through the plates.

Temperature is maintained constant at 72ºC inside each point of the product for the

holding time of at least 15 s. It is common to exceed the residence time to ensure the

safety of the product. After that, food is sent to the refrigeration system to cool the

product, at the first stage from 32°C to 9ºC, then in the last section to 4ºC and then on to

the packaging system. The flow rate must be maintained constant to deliver the same

temperature-time process to all of the food. The advantage of HTST versus vat

pasteurization is the time, which is shortened from 30 min to 15 s, so larger volume can

be produced in the same installation with considerable energy savings.

52

2.5 UHT pasteurization/sterilization

Ultra High Temperature (UHT) pasteurization/sterilization is an alternative pasteurization

process; not only is it focused on the inactivation of vegetative cells of pathogenic

microorganisms, but also the goal of this process is to destroy most of the

microorganisms (mainly heat resistant spores) in acidic foods and milk. A high

temperature (138ºC) is used, which is greater than the sterilization temperature used in

canning (121ºC). But, as will be discussed later, this process can be conducted under

regular sterilization conditions, even in canned foods such as milk. The official time of

treatment recognized in the United States is at least 2 s, although some authors mention

that the time should be at least one second at 138ºC. Other countries require 4 s and

temperatures of 140ºC, depending on the regulations. UHT pasteurization can be

performed in plate or tubular heat exchangers in which the main medium of heat transfer

is steam, or by direct injection of steam. This last option in some countries has many

restrictions, because the saturated steam will be in direct contact with the food, diluting

the product and releasing the vapor with a very fast cooling process. Often these types of

pasteurization and cooling processes are called flash pasteurization and flash cooling

because of the very rapid decrease and increase of temperature. Restrictions are focused

on the total elimination and quality of the steam. This process requires just a few seconds

to sterilize the product (usually milk), and thus avoids spoilage under non-refrigerated

conditions. After pasteurization, an aseptic packaging system is required to extend the

storage life even more. An important critical control point in UHT sterilization is to avoid

post-contamination of the product. Following usual UHT sterilization processing, the

product can have a shelf life of two or three months without refrigeration and without

53

aseptic packaging, but by using UHT, the shelf life is longer. According to data from

Tetra Pack Company (2006), UHT sterilized products can have a storage life of at least

six months without the use of refrigeration or preservatives. For that reason, some of

those products can retain their color, texture, taste and nutritional value.

The process of UHT plus aseptic packaging is carried out in a closed system

under aseptic conditions. The UHT pasteurized product goes directly to the packaging

machine in which the packaging material is previously sterilized with ultraviolet light,

hydrogen peroxide, and heat to remove any residue to achieve a dry and sterile container.

Furthermore, there is no air space in the package and the packing material is maintained

far away from light. Aseptic containers are made mainly of paper combined with layers

of aluminum foil and polyethylene, although some reports (Metha, 1980) show that

aseptic systems are available to fill containers such as cans, cartons and bottles.

Independent of the time-temperature combination for pasteurization, the use of aseptic

packaging is increasing in the food industry. In prepared food, UHT pasteurization is

conducted in conjunction with indirect systems to avoid micro-structural damage when

the steam is forced to pass through the product, which will likely affect the food texture.

Nevertheless, even though the process of UHT pasteurization is used to extend the

shelf life of milk for two or three months, or even longer when an aseptic packaging

system is used in combination, the main inconvenience of the process is an undesirable

change of the flavor of the product, generating a burned or cooked taste and undesirable

appearance. Despite that drawback, UHT milk is highly consumed is some countries in

different presentations according to the butter fat content and even the use of some

artificial flavorings like chocolate, strawberry and vanilla flavors. In some cases, the

54

addition of the flavorings is an attempt to mask the burned flavor and to create better

acceptability by consumers.

It is important to mention the difference between sterilized milk and UHT

sterilized milk. In many reports, websites, books and research articles the term UHT milk

is not specified, and confusion is generated because of the ambivalence of the word.

Currently, consumers may recognize UHT milk as pasteurized milk with long storage life

and, depending on the country, UHT milk may be called sterilized milk, ultra-pasteurized

milk, aseptic milk or shelf-stable milk, among others. But, according to Lewis and

Heppell (2000), there is sterilized milk that was popular some years ago in the United

Kingdom and which is still common in other European countries such as France, Italy,

Belgium and Spain. This “sterilized” milk is bottled in glass containers that are sealed

with cork or foil caps and subjected to a conventional sterilization process to destroy

microorganisms and spores. Bottles are sterilized in a retort at high temperatures from

110ºC to 116ºC and held at that temperature for 20 to 30 min. This is close to the

traditional sterilization temperature of 121.1ºC (250ºF), although heat transfer is much

faster in a liquid than in a solid or a liquid containing particles. This is one reason why

the temperature of sterilization for milk is lower than 121ºC. Obviously, after this very

long process, the quality of the milk drastically decreases; color, taste, and denaturation

of proteins and vitamin content are affected. In UHT pasteurized milk, few

caramelization reactions take place, changing the flavor and color in only a few seconds.

Although the target microorganism in conventional sterilization is the Clostridium

botulinum spores that find their optimum pH in low-acid food (pH > 4.5), this

microorganism is rarely found in milk. Generally, sterilization processes in milk are

55

focused on the inactivation of thermoduric and thermophilic bacteria and their heat

resistance spores, such as Bacillus stearothermophilus and Bacillus cereus.

Mehta (1980) mentions that the term “UHT-pasteurization” was used in North

America since 1965 and officially accepted by the U.S. Public Health Service relating to

the process in which milk is heated at 137.8ºC for at least 2 sec. According to Mehta

(1980), UHT milk is not sterile, but exhibits longer storage life than milk processed by

conventional pasteurization methods.

3. Nonthermal technologies

To reduce undesirable effects associated with thermal processing on quality factors, food

scientists and food engineers are looking for alternatives for processing and preserving

food products. Some of the novel technologies are using preservation factors other than

elevated temperatures to preserve food. Some of these factors are the use of high

hydrostatic pressure, electricity, sound, or light. For milk and dairy products, some of the

nonthermal processes that have been explored are high pressure, ultrasound and pulsed

electric fields.

56

3.1 Ultrasound

3.1.1 Basic concepts of ultrasound technology

Ultrasound is an emerging technology under research in the food engineering field. There

are two broad ranges of application of ultrasound according to the frequency: high

frequency uses from 2 to 10 MHz, and low frequency or power ultrasound uses

frequencies from 20 to around 100 kHz. The main difference between these technologies

is the physical effect generated in the medium. When ultrasound at high frequency is

applied to a food, a non-destructive effect is generated and the different parameters of

this technology, such as the attenuation coefficient, relate important information, e.g., the

structure or internal properties of the product. However, when the ultrasound is supplied

at low frequency, the passage of sound waves through a liquid medium causes the

vibration of molecules, generating physical effects into the food. Ultrasound at low

frequency is the technique that is used for disrupting purposes.

3.1.2 Power ultrasound

Power ultrasound is responsible for physical disruption in some materials, such as cells,

and also for promoting chemical reactions in liquid media. This type of ultrasound is used

in processes in which the breakage of cells or material is required for selected goals, such

as inactivation of microorganisms, extraction of components from cells or tissues, or

when a chemical reaction must be sped up or stopped, for example to accelerate or

inactivate the enzymatic activity in a food.

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Power ultrasound is characterized by the use of low frequencies, continuous mode

of operation, and high power levels such as 10 and 10 000 W/cm2 (Carcel et al., 1998).

The main effect of power ultrasound is called cavitation, the generation of thousands of

bubbles during the passage of sound waves through the medium. These bubbles have

cycles of implosion and explosion that generate micro-currents and micro-climates. Each

time bubbles collapse, increases in temperature and pressure are produced in the medium.

The intensity of the cavitation will depend on the temperature, pressure, amplitude of the

ultrasound wave, medium composition, etc.

This part of the chapter will focus on the applicatoion of ultrasound technology in

the dairy industry and advantages and disadvantages.

3.1.3 Pasteurization of milk with power ultrasound

Pasteurization is used in milk with the main goal of inactivating pathogenic bacteria,

reducing the number of deteriorative microorganisms and reducing enzymatic activity.

This food contains a rich medium for bacterial growth: proteins, fat, carbohydrates,

minerals, vitamins and a high percentage of water make it an excellent substrate for the

growth of bacteria, not only natural flora, but also the pathogenic bacteria that can exist

in the environment and be a prosperous medium for enzymatic activity (Pelczar and Reid,

1972). However, through time, the concept of pasteurization changed with new

technologies and the presence of more resistant microorganisms. The original concept of

pasteurization was based on the time-temperature relation to inactivate the most heat-

resistant pathogen in milk, Mycobacterium tuberculosis. However, more than thirty years

ago, the relationship of time-temperature changed because of the discovery of a new

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bacterium transmitted to humans via milk ingestion, generating Q fever: Coxiella burnetti

(Pelczar and Reid, 1972).

Nowadays, pasteurization of milk is also based on some microorganisms that have

been shown to be more heat resistant and which have been found in several recent

infamous outbreaks in the dairy industry. These outbreaks are related to the

contamination of food after processing and during handling and transportation, but also to

inadequate and insufficient thermal treatment conditions during processing. The new

emerging pathogens are under study with different technologies. Motarjemi and Adams

(2006) briefly define the emerging pathogens, including the microorganisms that have

recently appeared and those that are increasing in incidence. Many foodborne outbreaks

are reported in the food industry (Mohan Nair et al., 2005; Ko and Grant, 2003; Kozak et

al., 1996; Klima and Montville, 1995) related to the presence of pathogenic bacteria due

to underprocessing or post-pasteurization contamination. Listeria monocytogenes is one

of the leading foodborne pathogenic microorganisms generating problems in the food

industry, followed by Salmonella, Escherichia coli 0157:H7, Clostridium botulinum,

Campylobacter and Staphylococcus aureus (Banasiak, 2005). Listeria monocytogenes

was discovered in the early part of the last century, but in the 1980s there was a large

increase in the incidence of this microorganism in food, making it one of the most

important food pathogens found in raw and processed meat, dairy products, vegetables

and seafood (McLauchlin, 2006). However, the facilities of the dairy industry are often a

good source for Listeria contamination; soil, water, and even the cows can transmit the

bacteria to unprocessed and processed products.

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Several studies were conducted with ultrasound to inactivate bacteria such as

Saccharomyces cerevisiae (Tsukamoto et al., 2004a, b; Guerrero et al., 2005),

Escherichia coli (Furuta et al., 2004; Ananta et al., 2005; Ugarte-Romero et al., 2006),

Listeria monocytogenes (Mañas et al., 2000; Ugarte-Romero et al., 2007), Salmonella

(Cabeza et al., 2004), and Shigella (Ugarte-Romero et al., 2007) in different media, but

only a few were carried out on milk (Table III). Microorganisms such as Staphylococcus

aureus, Bacillus subtilis (Carcel et al., 1998), Salmonella typhimurium (Wrigley and

Llorca, 1992), Escherichia coli (Zenker et al., 2003), Listeria monocytogenes (Pagán et

al., 1999; Earnshaw et al., 1995) and total count plate and coliforms (Villamiel et al.,

1999) were tested in milk under sonication.

Results of inactivation studies under sonication are favorable, showing a positive

effect from the use of sound waves to inactivate cells. For example, in studies performed

with Listeria monocytogenes in skim milk, the decimal reduction value was reduced from

2.1 min (D60ºC) from thermal treatment up to 0.3 min when ultrasound was applied in

combination with elevated temperature (D60ºC&US) (Earnshaw et al., 1995); this treatment

is called thermo-sonication. When pressure is used in combination with ultrasound

(mano-sonication), important reductions are achieved in the inactivation of Listeria

monocytogenes in skim milk. Using ambient pressure and temperature, the decimal

reduction value under the processing conditions of ultrasound in this study was 4.3 min;

increasing the pressure to 200 kPa led to a reduction of the D value to 1.5 min; and using

two times the previous pressure (400 kPa) brought the D value to 1.0 min. When the

temperature was increased above 50ºC, the lethality of ultrasound on Listeria cells was

enhanced (Pagán et al., 1999).

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Zenker et al. (2003) studied the inactivation of Escherichia coli K12DH5α in

UHT milk using thermal treatment at 60ºC and combining an equivalent thermal

treatment with ultrasound, reducing the D value from 77 s to 23 s. These results are

examples of the additive effect of the combination between ultrasound and elevated

temperatures, leading to microbial inactivation.

It is important to highlight that ultrasound has the capability to pasteurize milk,

but not to sterilize it. According to the FDA, a pasteurization process is one that achieves

at least 5 log reductions of most pathogenic microorganisms, whereas sterilization is a

process that achieves at least 12 log reduction of the target microorganism, using in both

cases different time-temperature parameters. Ultrasound, at this moment, regardless of

the use of different temperatures, amplitudes or pressures has been shown to be a viable

process only to pasteurize some liquid foods.

3.1.4 Enzymes

Few experiments are reported on enzyme inactivation by ultrasound in milk. Ultrasound

has the ability to accelerate chemical reactions because of an increase in mass transfer

(Wang et al., 1996) or denaturation of proteins, stopping or decreasing the enzymatic

activity. The main chemical reactions activated with power ultrasound can be classified

as generation of free radicals, single electron transfer, and electron transfer catalysis. The

generation of free radicals is the mechanism related to denaturation of proteins because of

the effect on disulphur bonds (Sinisterra, 1992). The main studies conducted in enzyme

inactivation in milk with ultrasound are related to its curdling properties. Previous

research was conducted to study the activity of clotting enzymes of milk under

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sonication. Chymosin, pepsin, and fungal enzymes (proteases) were sonicated in milk and

buffer solutions, with enzymes from fungi microorganisms showing the highest

resistance. As the sonication time was increased, the curdling activity of chymosin

decreased and the buffered media showed a protective effect on the enzymatic

denaturation. The presence of H2O2 was reported in the solutions after sonication because

of the action of ultrasound to generate free radicals (Raharintsoa et al., 1977). When raw

and reconstituted milk were used as media to test clotting enzymes after sonication,

reconstituted milk showed less curdling capacity (Raharintsoa et al., 1978). These were

some of the first studies in enzymatic inactivation with ultrasound at 27 kHz; non-

pressure or temperature values were reported at this time. However, as ultrasound

technology developed, new variables were combined to enhance the effect of cavitation.

Other studies that used ultrasound at 20 kHz and 25ºC for 80 min found an increase in the

yield and activity of chymosin extraction, reducing the processing time considerably and

offering an alternative to the high demand of chymosin for the cheese-making industry

(Kim and Zayas, 1989). Another application of ultrasound is manothermosonication,

which was applied to study inactivation of lipoxygenase, peroxidase, polyphenol oxidase,

lipase and protease. Chymosin and pepsin (important in milk coagulation) were studied

under sonication with an apparent decrease of the activity in model systems, but when

chymosin was mixed with milk, the inactivation was almost null, and the change in

coagulation time was minimal (Villamiel and de Jong, 2000; Raharintsoa et al., 1978). In

general, the proteolytic activity of chymosin, pepsin and fungal enzymes decreases with

sonication as reported by Villamiel et al. (1999).

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Protease and lipase, enzymes related to quality problems during the storage of

milk under refrigerated conditions were also studied after manothermosonication. Both

enzymes are released from Pseudomonas fluorescens, and when ultrasound is applied in

combination with temperature (110 to 140ºC) and pressure (650 kPa), enzyme

inactivation was more favorable than when using only heat (Carcel et al., 1998).

3.1.5 Nutritional properties

a) Proteins

Some changes are reported in protein content in milk after sonication. Indeed,

emerging technologies are under research to improve the quality of the product and to

minimize damage to the product. However, the advantages that these new technologies

offer are several, not just in the final product, but also as a component for further food

processing products in addition to minimal changes in nutritional properties.

For example, in skim milk there is an increase of antioxidant activity when the

product is subjected to sonication, probably because of the action of cavitation in the

disruption of the quaternary and tertiary structure of the proteins (Villamiel and de Jong,

2000).

Some studies performed with raw whole milk under thermo-sonication showed a

significant difference (p<0.05) between the crude protein of thermally treated (63ºC)

samples and thermo-sonicated (63ºC plus ultrasound) samples compared with untreated

milk. A synergistic effect was reported in milk when heat plus ultrasound were used

together in the inactivation of enzymes and the study of the potential denaturation of

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proteins, i.e. alkaline phosphatase, γ-glutamyltranspeptidase and lactoperoxidase, α-

lactalbumin, β-lactoglobulin at temperatures of 61ºC, 70ºC and 75.5ºC. However, under

these processing conditions, casein content did not show any important change (Villamiel

and de Jong, 2000). The use of higher temperatures (greater than 60ºC) in combination

with ultrasound is responsible for the denaturating effect in proteins; in a contrasting

study with skim milk, when it was thermo-sonicated at temperatures below 50ºC, the

soluble protein content was not different from the control (Wrigley and Llorca, 1992).

One of the keys to processing milk under thermo-sonication is to find the ideal

conditions for microbial inactivation, while at the same time generating the minimal

denaturation of protein content; more studies must be performed with this technology,

focusing on protein denaturation.

b) Vitamins and minerals

Scarce information exists related to the vitamin or mineral content of milk after

sonication. Indeed, one of the most important parameters after milk treatment with

ultrasound is protein content because of its caloric and nutritional importance in the

human diet. However, milk also contains important vitamins such as riboflavin and

niacin, and minerals such Ca, P, Na, K or Cl (Walstra et al., 2006).

One of the few studies related to milk after sonication concerned the calcium

content after treatment at 27 kHz. The soluble Ca ultra-filterable value changed from

0.420 g/l in raw milk to 0.460 g/l in processed milk regardless of the time of sonication,

whereas the non-sedimentable Ca changed from 0.482 g/l (untreated milk) up to 0.504 g/l

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after 60 min of sonication. According to the authors, this change of Ca in milk is not

significant (Raharintsoa et al., 1978).

3.1.6 Physicochemical characteristics

Some important physicochemical characteristics in milk are those that will show

whether a new process changes the flow behavior or chemistry of milk in a significant

way. Although many of the studies with ultrasound in milk were conducted to study the

microbial inactivation of target bacteria, there are some available data related to

physicochemical properties such as viscosity, density, pH and color. Table IV presents a

comparison between fresh or raw milk, pasteurized milk with conventional thermal

treatment and sonicated skim and whole milk.

a) Viscosity

Viscosity can be defined as the resistance of a liquid to flow related to the

attraction forces between the molecules of a liquid. The viscosity reported for fresh milk

is 1.9 mPa*s (Table IV). However, as can be observed in the same table, skim milk (low

pasteurized) shows a lower value because of the absence of fat content. As the viscosity

value is higher, the liquid flows slowly, so in this case, the fresh milk will flow slowly

because of the fat content, while the skim milk will flow faster, being mainly a solution

of proteins and minerals. There is a difference between the viscosity of the fresh milk and

sonicated milks, with viscosity decreasing from 1.9 mPa*s to 1.76 and 1.77 mPa*s,

regardless of the butter fat content. One possible explanation for this decrease could be

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the effect of homogenization that the ultrasound generates in the fat content, making the

milk more fluid because of the smaller size of the fat globules. However, the viscosity for

both sonicated milks is in the range for beverage milk.

b) Density

Density is the ratio between the mass of an object per given unit of volume; in

other words, density is the measure of how the molecules of a substance are packaged

together to occupy a certain volume. According to Table IV, the density of whole milks,

both fresh and sonicated, is very similar (1029 and 1025 kg/m3, respectively), whereas

skim milks, low pasteurized and sonicated are also in the same range (1035 and 1033

kg/m3, respectively). In summary, density is a property of milk that does not change in an

important way during sonication, regardless of the butter fat content.

c) pH

The pH of milk is a useful parameter to express the degree of acidity of milk.

Although it is not a routinely measured parameter in milk, this parameter provides

information about the degree of lactic acid that milk contains at specific times. The

reported pH of fresh milk is 6.7, is seen in Table IV, although this value can change

slightly because of many situations related to the milk source, such as the kind of

mammal, season, feeding, etc.

Sonicated milk showed lower pH after treatment, 6.5 and 6.6 for skim and whole

milk, respectively. However, these values do not represent a significant change in the pH

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of milk. There are some possible explanations as to why pH decreases in milk after

sonication. Explanations are proposed for water; considering that milk has a high

percentage of water, these theories may be extrapolated to milk.

One of the former theories concerning chemical changes in a sonicated medium

was related to formation of free radicals from water molecules (Tsukamoto et al., 2004a,

2004b). Water molecules are divided into free radicals such as OH- and H+, and Sochard

et al., (1997) mention the presence of radicals O- between the most abundant components

of the sonolysis of water after treatment. Sonolysis is the breakage of water molecules

into free radicals because of the violent cavitation generated by the sound waves in a

liquid medium. These free radicals are responsible for lowering the pH of the medium,

but are not necessarily the only factor that increases the acidity of the solution. Studies

reported by Supeno (2000) showed how the sonication of aqueous solutions in the range

of ultrasound frequency from 41 to 3217 kHz generated the highest production of nitrite

and hydrogen peroxide as primary products in the liquid at 360 kHz, with the successive

generation of nitrate as a product of the oxidation of nitrite.

Walstra et al. (2006) mention that the decrease of pH in milk could be due to the

hydrolysis of phosphoric esters because of enzymatic action. Ultrasound is responsible

for promoting the activity of some enzymes, and cavitation probably accelerates some

chemical reactions, lowering the pH. However, at this time, there are no available data to

exactly determine the reason for slightly lower pH in milk after sonication.

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d) Color

One of the main characteristics of milk is color; consumers look for the

conventional white color, and some studies show that the whiter the milk, the higher the

consumer preference. After sonication cavitation modifies the color of milk, because it

will also affect the final color of other dairy products, such as cheese or yogurt.

Sonicated milk showed a significant change in luminosity after treatment in

comparison with raw and thermal treated milk. Depending on the extent of cavitation,

milk was whiter as the intensity was increased. In addition to L value, a value changed

significantly in thermo-sonicated milk, having an important contribution to the blue

region, whereas b value did not change significantly between the raw, thermal and

thermo-sonicated milk (Bermúdez-Aguirre et al., 2005). The reason for this is the

reduction of fat globule size (Ertugay et al., 2004). The color of milk is due to the fat

globules in the milk which scatter the light, making it a white fluid; however, when these

globules undergo cavitation, their size is reduced and the agglomerate of the globules

become more homogeneous, modifying the light reflection and resulting in whiter milk.

In Figure 1, the variation in color parameters (L, a, b) between raw milk, thermal treated

milk and sonicated milk at selected intensities is shown. L and a are the parameters that

changed significantly with respect to the raw milk, whereas b did not change in any

important way.

The whiter color and homogenization of sonicated milk is maintained during the

storage life of the milk. Some studies showed that even after 16 d under refrigerated

conditions, the whiter color remained and no problems related with gelation or syneresis

were observed (Bermúdez-Aguirre et al., 2005).

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Indeed, sonication offers many advantages at the time that milk is being

pasteurized; white color is enhanced and homogenization allows milk to have a better

appearance through the storage life. All of these positive effects are generated during the

one-step thermo-sonication process.

3.1.7 Processing of dairy products with ultrasound technology

3.1.7.1 Milk

a) Milk as beverage

At this time, ultrasonicated milk is not commercially available. Because of all the new

technologies, a series of experiments must be conducted to show the efficacy of the

technology and to achieve the sanitary, microbiological and governmental regulations in

each country. However, sonicated milk could be a potential product that offers many

advantages to the consumer as a beverage or to the dairy industry as an ingredient of

other products.

Milk as a beverage after sonication exhibits many advantages compared to heat

treated milk or conventional pasteurized products. Color, homogenization and appearance

are better. Furthermore, for milk processors, homogenization and pasteurization can take

place in one step instead of two (homogenization and further pasteurization), ensuring

better stability throughout storage.

Although only a few experiments were carried out in milk stability during storage,

quality problems such as syneresis, “sweet-curdling” because of the enzymatic action

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(protease and lipase from Bacillus spores), oxidation, etc. could be minimized or avoided

with ultrasound.

b) Lactose free milk

A common health problem is the intolerance of lactose by a large part of the population.

Lactase, the enzyme responsible for metabolizing lactose in milk, is segregated in low

levels in some people, depending mainly on ethnicity. Lactose free milk is successfully

commercialized in many countries for lactose intolerant consumers. This milk without

lactose can be produced by fermentation of lactose-hydrolyzed milk or by the

simultaneous addition of β-galactosidase and lactic acid bacteria (Carcel et al., 1998;

Wang et al., 1996). These bacteria produce β-galactosidase which hydrolyzes the lactose

in fermented milk (Villamiel et al., 1999).

Ultrasound has the capability of raising the reaction activity of cells or to

stimulate a new action into the cells, for example in sterol synthesis with baker’s yeasts

or in lactose-hydrolyzed fermented milk. Using ultrasound in the processing of lactose

free milk, the lactose hydrolysis was around 55%; using traditional methods to produce

lactose free milk (fermentation), hydrolysis was around 36% (Wang et al., 1996). In

general, studies demonstrated that hydrolysis of lactose by ultrasound can be enhanced by

up to 20% compared with conventional methods (Villamiel et al., 1999; Toba et al.,

1990). β-Galactosidase is released from lactobacillus, shortening fermentation time

considerably and hydrolyzing the lactose (Carcel et al., 1998).

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c) Human milk

Sometimes newborns need to be fed by a source of milk other than human milk directly

from their own mothers, because of illness problems or early childbirth. In such special

cases, newborns are fed with stored milk that was previously pasteurized, homogenized

and frozen or lyophilized. However, when the babies are fed with this milk using a

feeding tube (gavage or ostomy tube), almost 47% of the fat content that is added to the

tube is lost from the milk (Dhar et al., 1995; Carcel et al., 1998). But not only is the fat in

milk lost, but also lost are liposoluble vitamins, proteins and minerals, leading to a high

degree of nutritional losses. When sonication is used to homogenize the milk, the final

product is much more stable because of the reduction of fat globules and the longer

maintenance of the emulsion, which considerably reduces nutrient losses in milk

(Martínez et al., 1992).

Some current experiments in human milk homogenization demonstrated that

using sonication at temperatures below 45ºC reduces fat losses during feeding because of

an increase in lipolytic activity, which at the same time protects immunoprotective

substances such as immunoglobulin A (IgA) and immunoglobulin G (IgG) and

lactoferrin. Temperatures higher than 55ºC totally inhibited the lipolytic activity and the

bacteria load, but also decreased the IgA and IgG (Martínez et al., 1992), which is not

convenient for newborns.

Thus, one of the many alternatives that ultrasound offers is the homogenization of

human milk at low temperatures, providing a better consistency and avoiding the loss of

vitamins, minerals, proteins and fat, an important component in the caloric diet for

newborns.

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3.1.7.2 Yogurt

Regular yogurt is a very common and demanded dairy product. Although yogurt may be

processed at home, nowadays there are many commercially available varieties of yogurt.

Addition of special flavors, fruits, cereals or specific beneficial bacteria are examples of

current products. However, one of the biggest issues in yogurt production is the presence

of lactose in the milk that is used to process it; the segment of the population that is

intolerant to lactose needs to find a better option for yogurt consumption. Hydrolyzed

lactose yogurt is focused on consumers with zero tolerance to lactose, providing at the

same time a sweetener effect without calories. As in the case of lactose free milk,

fermented milk to process yogurt can be prepared by ultrasound, releasing the β-

galactosidase from the starter bacteria and hydrolyzing the lactose (Carcel et al., 1998;

Toba et al., 1990). The conventional processing of yogurt requires a long time. In order to

allow fermentation to take place, a starter culture is added to milk, and lactic acid

production starts to lower the pH of the product. After some hours, when lactic acid

reaches the desirable value for yogurt (0.65 – 0.70%), fermentation is stopped by

immediately cooling the product. Fermentation times are also reduced in sonicated yogurt

because of faster acid production and pH drop enhanced by the use of ultrasound.

Cavitation stimulates acid production because of the release of intracellular enzymes

from microorganisms. Lactose exists inside the cells of the starter culture for yogurt, and

the enzyme β-galactosidase increases its activity because the ultrasound hydrolyzes this

sugar and accelerates acid production (Wu et al., 2001).

Some common quality problems of yogurt are related to the separation of the

serum from the main matrix containing the protein and fat; this phenomenon is known as

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syneresis. This problem occurs during the transportation and/or storage of the product

and is a non-desirable characteristic of yogurt. Some studies were performed using

manothermosonication of milk (12 s, 20 kHz, 2 kg pressure and 40ºC) to process yogurt,

and the final characteristics of the product were improved. For example, viscosity was

much better in sonicated yogurt and fermentation time was reduced when milk was

previously homogenized with ultrasound. The final structure of manothermosonicated

yogurt was stronger than the control because of a better interaction of the components of

the network. Casein micelles captured serum and fat globules, showing a significant

difference (p < 0.05) in the water holding capacity between the control and the

manothermosonicated samples, releasing 18.8% and 14.8% of serum, respectively.

Rheological parameters of sonicated yogurt such as penetration and relaxation forces,

texture profile analysis parameters, consistency, flow behavior, yield stress, storage

modulus (G’) and loss modulus (G’’), among others, were improved in the product after

the use of ultrasound in milk and prior to the fermentation process (Vercet et al., 2002).

Similar studies were conducted in milk used to process yogurt, with ultrasound alone

without pressure; results of this study showed good homogenization of the yogurt,

reduction in fermentation time and important and general improvements in water holding

capacity, viscosity and reduction of syneresis (Wu et al., 2001).

The result of having better water holding capacity and minimum syneresis could

be explained by examining the microstructure and physicochemical points of view.

Cavitation is responsible for the disruption of fat globules and decreases the size of fat

globules while at the same time conferring a new surface. All these globules together

represent a bigger surface area than the original surface area, and in this new area there

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will be new attached casein micelles (Wu et al., 2001) because of the action of the

cavitation regrouping the milk components. These micelles are hydrophilic, binding

water molecules in the new structure, leading to a more compact network of proteins, fat

globules and water (serum), presenting better water holding capacity, better structure and

a low degree of syneresis.

Indeed, ultrasound technology offers many advantages in yogurt processing.

Some of the common quality defects such as syneresis could be reduced considerably,

and better rheological properties for the consumer can be achieved after the use of

ultrasound.

3.1.7.3 Cheese

Cheese is one of the most demanded products around the world. Each year, the cheese

industry reports considerable sales increases; in 2003 cheese production in the United

States was more than 8,600 pounds of cheese. Varieties of this dairy product with the

highest demand include Cheddar, colby, monterrey jack, mozzarella, and ricotta (Miller

et al., 2007). The main processing steps of cheese making include clotting or curdling of

the milk, removal of whey, acid production, salting, shaping and ripening (Walstra et al.,

2006). The essential step during cheese processing is the formation of the curd that

occurs by enzymatic and microbial activity. Many environmental conditions such as

temperature, enzyme concentration and microbial load are very important during the

process to generate a firm curd at a specific time. One of the desirable aspects in cheese

making is to reduce the curdling time and increase the yield, because the final yield of

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cheese is around 10%. New technologies are being tested in cheese making in order to

improve these aspects while at the same time improving the overall quality of cheese.

When sonication was applied to milk to study the proteolytic activity of the

enzymes related to curdling, the main observable effect was that ultrasound speeds up the

hardening of the curd, and the final product showed a better firmness because of the

activity on the chymosin, pepsin and other related enzymes (Villamiel et al., 1999).

Chymosin is the main enzyme related to the curdling formation in the cheese making

industry. This enzyme is produced naturally in the stomach of bull calves, and the

conventional extraction of the enzyme with salted and acid solution takes a long time and

the quality and yield are not favorable after the process. Chymosin has a proteolytic

activity in liquid milk, coagulating the protein and conferring a solid-like coagulated gel

(Kim and Zayas, 1991). When ultrasound (20 kHz) was used to enhance the extraction,

the yield extract and enzyme activity was increased considerably, but also extraction

times were much shorter than without sonication. The reason for that could be attributed

to the destruction of cellular structure because of the action of ultrasound, increasing the

activity of the substances contained in the cells and the migration of proteins and

minerals from the cells to the solution (Carcel et al., 1998). The activity of the chymosin

increased with sonication while the nitrogen content of the extract decreased at the same

time (Kim and Kayas, 1991).

Other uses of power ultrasound in the cheese making industry are in the flavor

arena. Proteolysis is the main process that takes place during cheese ripening, and it can

be divided into two main phases: the primary phase in which the residues of chymosin

interact with the casein in the curd, and the secondary phase in which proteins and

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polypeptides are broken into amino acids by enzymatic action released from bacteria

(Engels and Visser, 1996). When Lc. Lactis subsp. cremoris was sonicated, a “cell free

extract” was obtained by cell disruption (Engels and Visser, 1996) and the extraction of

peptides and amino acids was easier; these components were used in the development of

flavor of the cheese (Villamiel et al., 1999).

In addition to all of these benefits of ultrasound technology, the process of “queso

fresco” (a typical product from Hispanic countries) made with sonicated milk showed

better characteristics, such as whiter color, better texture (hardness), higher water holding

capacity, higher yield and, in general, better acceptance by consumers. This is one area

that deserves further research in ultrasound technology.

3.1.8 Other uses of power ultrasound in the dairy industry

Ultrasound offers a great variety of uses in the industry; the specific case of applications

for the dairy industry is wide enough to satisfy different requirements in processing and

preservation. Some additional examples of the use of ultrasound in the dairy industry are

described below.

The topic of edible films is interesting for many researchers in food preservation

worldwide; however, some specific characteristics of the films are required to be used in

food, as well as their mechanical properties. Milk proteins are used for manufacturing

edible films, and their mechanical properties were improved by using sonication in the

process of elaboration of films based on sodium caseinate and whey proteins (Carcel et

al., 1998).

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Other uses of power ultrasound reported in literature are related to cleaning

operations. Power ultrasound was used to disperse cumulus of bacteria in raw milk, and

to clean cheese moulds in the cheese-making industry (Villamiel et al., 1999).

3.2 Pulsed electric fields: Basic concepts

The use of electricity to pasteurize milk started in the early 1900s, and some research

reports the use of electric discharge through carbon electrodes and raising milk

temperature up to 70ºC in order to inactivate bacteria. This first electrical process to

pasteurize milk was named Electropure (Vega-Mercado et al., 1999), and inactivated

bacteria such as Tubercle bacilli, Escherichia coli and other thermal resistant

microorganisms (Barbosa-Cánovas et al., 1999). Today, electricity is applied into

microorganisms not only to destroy them, but also to facilitate electroporation,

electrofusion and food preservation (Barbosa-Cánovas et al., 1999).

Pulsed electric fields (PEF) technology consists of the passage of a liquid food

through a chamber with high voltage electrodes that will transfer an electric current into

the food. The intensity will depend on the electric field strength or the voltage applied

between electrodes (Ruhlman et al., 2001). PEF treatment is the application of short

pulses (from micro to milliseconds) of selected electric field intensity (10 – 80 kV/cm).

When an electric field is applied to the cell, a transmembrane potential across the

membrane is induced, but when this potential exceeds the natural potential of the cell (1

V), the expansion of existing pores or the formation of new pores in the cell membrane

occurs, with an imminent increase of the cell permeability. This phenomenon is called

electroporation, and this process is based on dielectric rupture theory (Figure 2). This

77

theory establishes that when electric fields are applied within the cell, there is an

accumulation of positive and negative charges at the membrane, and when the threshold

value is exceeded pores are formed with a posterior breakage of the cell membrane.

Some studies reported that this process can be reversible in some cases, but other

collateral effects have also been observed, such as shrinkage of the cytoplasmic

membrane, the formation of pearl chains and cell fusion (electrofusion). Physical and

electrical properties of the food, as well as some specific characteristics of the

microorganism, will be important to the success of the process. For example, electrical

conductivity, density, specific heat, pH, ionic strength and viscosity of the product are

some of the basic parameters considered before processing the product (Ruhlman et al.,

2001). Milk is considered in PEF technology as one of the most electrically conductive

liquid foods (Zhang et al., 1995). Processing parameters to be controlled during PEF

treatment are electric field intensity, treatment time, frequency (when it is increased, the

treatment time is decreased), processing temperature (higher temperature enhances PEF

inactivation), specific energy, and pulse wave shape (most lethal is the square wave),

among others (Barbosa-Cánovas et al., 1998; Bendicho et al., 2002a; Vega-Mercado et

al., 1997).

The use of pulsed electric fields to process food has many advantages compared

with traditional pasteurization methods, such as treatment times of microseconds,

important energy and cost savings, as well as minimal changes in food quality, such as

color, flavor or nutritive value of the product (Ruhlman et al., 2001).

78

3.2.1 Pasteurization of milk with Pulsed Electric Fields

Some microorganisms are successfully inactivated in milk using electric field strengths at

refrigerated temperatures, achieving important reductions and retaining the quality of

milk. Some of these microorganisms in milk and other dairy products, shown in Table V,

represent pathogenic, surrogate and deteriorative organisms present in milk. Escherichia

coli, Salmonella dublin, Lactobacillus brevis, Bacillus subtilis, Staphylococcus aureus

and Saccharomyces cerevisiae are some of the tested microorganisms in milk and related

products. Different processing conditions tested and media such as milk, skim milk,

SMUF (simulated milk ultra filtrated) and yogurt were subjected to PEF treatment with

important log reductions. However, as in other technologies, there are some

microorganisms that exhibit persistent resistance to inactivation, in this case with PEF;

some of these resistant microorganisms are Corynebacterium spp. and Xanthomas

malthophilia in inoculated raw skim milk (Odriozola-Serrano et al., 2006).

The factors that can affect the degree of inactivation by PEF related to the

microorganism, which can be called biological factors, are:

a) Genre

b) Species

c) Size

d) Shape

e) Growth stage

Other factors to influencing inactivation are the intrinsic resistance of the

microorganism, growth conditions, cell concentration and recovery conditions, if the cell

79

is partially injured (Barbosa-Cánovas et al., 1998; Pagán and Mañas, 2006; García et al.,

2007; García et al., 2005).

Some advantages that PEF offers to conventional treatments of pasteurization of milk

are:

a) Sensorial and nutritional properties are not or minimally degraded during the

process, showing fresh-like characteristics.

b) It is a safe process because no dangerous chemical reactions have been detected.

c) PEF requires minimal energy and has greater energy efficiency compared to

thermal treatments (PEF requires 90% less than HTST).

d) PEF processing costs less than conventional treatments as well as the low

maintenance (i.e. no steam required) after the initial acquisition of the equipment.

e) The shelf-life of the products can be extended considerably compared to

conventional pasteurization

(Barbosa-Cánovas et al., 1998; Zhang, 2007).

3.2.2 Enzymes

Under conventional thermal treatment, enzymes are slightly inactivated and most of the

native enzymes in milk remain active. This is one cause for milk spoilage even when this

product is subjected to pasteurization. Depending on many factors, enzyme inactivation

can be feasible with the use of pulsed electric fields; some of these factors are the enzyme

itself, the medium of treatment and the processing conditions. However, general

conditions to inactivate enzymes are more severe than those to inactivate

microorganisms, and the process is more effective when temperature is applied at the

80

same time of the PEF treatment (Martínez et al., 2007). There are only a few reports of

enzyme studies under PEF in milk (Odriozola-Serrano et al., 2006; Bendicho et al.,

2002a). Some of the milk enzymes that were tested under selected PEF treatments are

peroxidase, alkaline phosphatase, plasmin, lactoperoxidase, protease and lipase (Van

Loey et al., 2002). However, in all of these studies total inactivation is not reported under

selected processing conditions (Martínez et al., 2007).

Two of the above mentioned enzymes are inactivated in high percentages.

Plasmin from bovine milk treated in SMUF in a continuous system at 30 kV/cm with 50

pulses of 2 μs and 15ºC was inactivated 90% after treatment. Alkaline phosphatase in raw

milk which was inactivated 96% after 18.8 kV/cm, 20 pulses (2 μs) and 22ºC. Protease

and lipase activity in raw milk was to 60% under selected processing conditions (Van

Loey et al., 2002).

3.2.3 Nutritional properties

As a relatively new technology, studies in nutritional properties of milk after PEF

processing are scarce; most of the reports are focused on microbial inactivation and only

a few examine enzyme inactivation. Although some of these reports include brief studies

on sensorial quality such as taste and flavor of milk, few are related to nutritional

properties such as vitamin or protein content after PEF processing. One of the few reports

was conducted on vitamin content in milk after processing with electric field strengths.

Water-soluble vitamins (riboflavin, thiamin and ascorbic acid) and fat-soluble vitamins

(cholecalciferol and tocopherol) were analyzed after 400 μs at 18.3 to 27.1 kV/cm, and

81

changes were not reported for vitamin contents in milk after processing, although there

were some variations in ascorbic acid (Bendicho et al., 2002b).

4. Final remarks

Ultrasound offers many advantages in dairy processing. Although this is a new

technology that is still being tested at the lab and pilot plant scale, sonication has the

potential to be scaled up to the industry level in order to take advantage of its properties.

Inactivation of bacteria and enzymes is possible with ultrasound, achieving pasteurization

standards and conferring better quality attributes to milk. Beverage milk, yogurt and

cheese were processed successfully under ultrasound activity, and other dairy products

such as cream or ice cream could make use of the beneficial effects of sonication on

texture and rheological characteristics to improve their quality as well. Whereas pulsed

electric fields technology is more advanced with regard to legal aspects, most of the

research conducted with milk and pathogenic bacteria and enzymes demonstrates positive

results that could allow approval from regulatory agencies for use as a pasteurization

method for milk, saving energy, time, and money and providing fresh-like characteristics

in the final product. Research on the development of new and better dairy products with

PEF treated milk provides a huge world of opportunities for the food scientist.

82

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95

Table I. Heat resistance of selected microorganisms (Source: Holdsworth, 1997)

Microorganism Conditions for inactivation Vegetative cells 10 min at 80ºC Yeast 5 min at 60ºC Fungi 30-60 min at 88ºC Thermophilic genre Bacillus stearothermphilus 4 min at 121.1ºC Clostridium thermosaccharolyticum 3-4 min at 121.1ºC Mesophilic genre Clostridium botulinum spores 3 min at 121.1ºC Clostridium botulinum toxins Types A

and B 0.1-1 min at 121.1ºC

Clostridium sporogenes 1.5 min at 121.1 ºC Bacillus subtilis 0.6 min at 121.1ºC

96

Table II. Standard time-temperature combinations for pasteurization processes (Source: Adapted from IDFA, 2006)

Process Temperature Time

Vat pasteurization 63ºC (145F) 30 minutes HTST pasteurization 72ºC (161F) 15 seconds

UHT sterilization 138ºC (280F) 2.0 seconds

97

Table III. Inactivation of microorganisms by ultrasound in milk and buffer solutions

Target microorganism Processing conditions Medium Log Reduction Reference

Listeria monocytogenes 20 KHz, 117 μm, ambient temperature

Not specified D=4.3 min Pagan et al., 1999

Listeria monocytogenes 20 KHz, 117 μm, ambient temperature, 200 kPa or 400

kPa

Not specified D200=1.5 min D400=1.0 min

Pagan et al., 1999

Listeria monocytogenes Heat @ 60ºC Heat @ 60ºC with sonication at

20 kHz

UHT milk D60= 2.1 min D60&S= 0.3 min

Earnshaw et al., 1995

Salmonella spp. 160 KHz, 100 W by 10 min Peptone water 4 log reductions Lee et al., 1989 Salmonella typhimurium 30 min at 50ºC and 40ºC Skim milk 3 and 2.5 log reductions,

respectively Wrigley and Llorca, 1992

Escherichia coli 700 kHz, 32ºC, 10 and 30 min Saline solution 0.83% and 0.2% survival Utsunomiya and Kosaka, 1979

Escherichia coli K12 DH5 α

Heat at 60ºC Ultrasound (110 μm) assisted

with temperature (60ºC)

Phosphate buffer (pH 7)

D60= 84.6 s D60&S= 23.1 s

Zenker et al., 2003

Escherichia coli K12 DH5 α


with temperature (60ºC0

UHT milk (pH 6.7) D60= 77 s D60&S= 23 s

Zenker et al., 2003

Bacillus subtilis 20 kHz and 150 W, 100ºC Distilled water, milk and glycerol

63 and 74% reduction in glycerol 79 and 40% reduction in milk

70 and 99.9% in distilled water (depending of the specie of

Bacillus)

Garcia et al., 1989

Lactobacillus acidophilus


with temperature (60ºC0

Phosphate buffer (pH 7)

D60= 70.5 s D60&S= 43.2 s

Zenker et al., 2003

98

Table IV. Physicochemical properties of milk

Physicochemical properties

Fresh milk Beverage milk (Low

pasteurized)

Skim milk (Low

pasteurized)

Sonicated skim milk

Sonicated whole milk

pH 6.7 6.7 6.7 6.5a 6.6c Viscosity (mPa*s)

1.9 1.8 1.65 1.76b 1.77d

Density (kg/m3) 1029 1030 1035 1033a 1025c Fat globule size

(μm) 3.4 0.5 0.4 - < 0.5 c

Reference Walstra et al., 2006

Walstra et al., 2006

Walstra et al., 2006

a Bermúdez-Aguirre and

Barbosa-Cánovas 2008; bRaharintsoa et

al., 1978

c Bermúdez-Aguirre et al.,

2007; d Raharintsoa et

al., 1978

a Skim milk previously UHT pasteurized after 30 min of sonication at 63ºC b Skim milk after 60 min of sonication, viscosity @ 20ºC c Whole and raw milk after 30 min of sonication at 63ºC d Whole milk after 60 min of sonication, viscosity @ 20ºC - Data not available

99

Table V. Microbial inactivation in milk and dairy products with PEF

Target microorganism

Medium Processing conditions

Log reduction

Reference

Saccharomyces cerevisiae

Yogurt 1.8 V/μm, 55ºC, batch system

3 Dunn and Pearlman, 1987

Escherichia coli Milk 3.3 V/μm, 43ºC, 35 pulses, batch

system


Escherichia coli Skim milk 4.0 V/μm, 15ºC, 3 μs, 64 pulses, batch system

3 Zhang et al., 1994a

Escherichia coli SMUF* 2.5 V/μm, 25ºC, 20 pulses, batch

system

3 Zhang et al., 1994b

Salmonella dublin Milk 3.67 V/μm, 63ºC, 36 μs, 40 pulses,

batch system


Lactobacillus brevis Yogurt 1.8 V/μm, 50ºC, batch system


Bacillus subtilis SMUF 1.6 V/μm, monopolar, 180 μs,

13 pulses, batch system

4.5 Qin et al., 1994

Pseudomonas fragi Milk 9.0 V/μm, 1 μs, batch system

4.5 Gupta and Murray, 1989

Staphylococcus aureus ATCC 6538

SMUF 30ºC, 1.6 V/μm, 200-300 μs, 60

pulses batch system

3-4 Pothakamury et al., 1995

* SMUF: Simulated Milk Ultra Filtrated

100

-20

0

20

40

60

80

100

Raw Milk

Thermal treatment

30% US

60% US

90% US

100% US

Sample

L, a

, b

Lab

Figure 1. Color parameters (L, a, b) in raw, thermal treated and sonicated milk at selected

intensities. L and a were significantly different from each other; b was not significantly

different.

101

Figure 2. Dielectric rupture theory, E = electrical field strength, Ec = critical electrical

field strength (Zhang, 2007).

102

CHAPTER TWO

MODELING THE INACTIVATION OF Listeria innocua IN RAW WHOLE MILK

WHEN TREATED BY THERMO-SONICATION

Daniela Bermúdez-Aguirre, Maria G. Corradini, Raymond Mawson

and Gustavo V. Barbosa-Cánovas

Abstract

Ultrasound was used to pasteurize milk and to study inactivation of Listeria innocua.

Five systems were evaluated in an ultrasonic processor (24 kHz, 120 μm, 400 W). Tested

amplitudes of ultrasonic waves were 0%, 30%, 60%, 90% and 100%, with a constant

temperature of 63ºC and treatment time of 30 min. pH, acidity and color were measured.

After 10 min of treatment, thermal pasteurization achieved 0.69 log-reduction. Using

ultrasound at 60, 90 or 100% in combination with temperature, a 5 log-reduction was

obtained after 10 min. For thermal treatment, a 5.3 log-reduction was reached after 30

min. The heat and the toughest thermo-sonication survival curves were best fitted using a

Weibullian model. However, an alternative-four parameter model was selected for the

mildest thermo-sonication treatment (30%). pH was slightly lower (6.64), acidity was

increased (0.141%) and color of samples were whiter (92.37) as the intensity of thermo-

sonication increased.

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1. Introduction

Throughout the world, milk is a common product that is highly consumed, regardless its

source. Raw milk is still consumed in rural communities in some underdeveloped

countries despite the health risks associated to this practice. Pasteurization of milk is the

most effective technique to destroy pathogenic bacteria, to inactivate some enzymes and

to extend the shelf-life of the product. However, with the higher temperatures used during

inactivation of pathogenic bacteria, in conventional processes using plate heat

exchangers, many quality factors, such as flavor and nutritional content are affected. For

this reason, it is necessary to look for alternative technologies to pasteurize milk avoiding

the thermal treatment adverse effects, while still assuring food safety. According to

United States’ standard, bacterial count in Grade A raw milk may not exceed 300,000

cfu/ml, and in the European Community the bacterial count should be lower than 100,000

cfu/ml. After pasteurization, in the United States the milk should contain less than 20,000

cfu/ml (Jensen, Blanc & Patton, 1995). When milk has a microbial population between

500,000 and 10,000,000 cfu/ml, it is clear that hygienic practices have failed (Paci,

1953). Above 50ºC, inactivation of sensitive enzymes in milk begins. During

conventional high temperature, short time pasteurization (HTST) at 71.8ºC for 15 s, two

of the main enzymes in milk, milk lipase and alkaline phosphatase, are inactivated

(Keenan & Patton, 1995). In addition to nutritional properties, sensorial characteristics of

milk are very important for consumer acceptance; one of these characteristics is color.

Whiteness in milk has been shown to have a positive influence on consumer preference.

White color of milk is due to the presence of fat globules and casein micelles, which

scatter the light (Owens, Brewer & Rankin, 2001; Phillips, McGiff, Barbano & Lawless,

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1995). In order to pasteurize food without the use of high temperature, some alternative

nonthermal technologies have been proposed, such as high hydrostatic pressure, pulsed

electric fields, ultrasound or ultraviolet treatment; some of them appear to be viable. But

to date, only high hydrostatic pressure has been approved by the USDA for use in food

products such as juices and other liquid beverages and pulsed electric fields and

ultraviolet treatment for some specific juices.

Ultrasound is a novel technology in food engineering that is currently being

tested, focusing on microbial and enzymatic inactivation; but ultrasound as a technique to

destroy bacteria is not new and is widely used laboratory method for cell lysis. Examples

closer to food processing include the application of ultrasound to inactivate bacteria in

milk targeting coliform microorganisms Staphylococcus aureus, Bacillus subtilis and

Salmonella typhimurium; enzymes that have been studied include peroxidase, lipase and

protease (Villamiel, van Hamersveld & de Jong, 1999; Carcel, Benedito, SanJuan &

Sánchez, 1998). Other microorganisms tested in different media and studied under

sonication conditions include some species of Salmonella (Cabeza, Ordoñez, Cambero,

De la Hoz & García, 2004; Lee, Kermasha & Baker, 1989; Wrigley & Llorca, 1992);

Escherichia coli (Furuta et al., 2004; Ugarte-Romero, Feng, Martin, Cadwallader &

Robinson, 2006); Saccharomyces cerevisiae (Guerrero, López-Malo & Alzamora, 2001;

Guerrero, Tognon & Alzamora, 2005); Listeria monocytogenes (Pagán, Mañas, Alvarez

& Condón, 1999); among others. Many foodborne outbreaks have been reported in the

food industry (Mohan Nair, Vasudevan & Venkitanarayanan, 2005; Ko & Grant 2003;

Kozak, Balmer, Byrne & Fisher, 1996; Klima & Montville, 1995) related to

underprocessing and consequently survival of pathogenic bacteria or post-pasteurization

105

contamination. Listeria monocytogenes is one of the leading foodborne pathogenic

microorganisms generating problems in the food industry, followed by Salmonella,

Escherichia coli 0157:H7, Clostridium botulinum, Campylobacter and Staphylococcus

aureus (Banasiak, 2005). Listeria monocytogenes can survive under a broad range of

temperatures (1ºC to 45ºC) and pH (4.1 to 9.6) conditions. Affected products include raw

milk, pork, raw poultry, ground beef, and some vegetables (Jay, 1992), cheese, seafood,

and fruits (Mohan Nair, Vasudevan & Venkitanarayanan, 2005). In addition to being a

widespread bacterium in the environment, L. monocytogenes can compete with other

microorganisms, grow at refrigeration temperatures, and survive freezing and high salt

concentrations (Kornacki, 2005). In the United States, 2,500 cases of listeriosis from

food are reported every year; 500 of them end in death, not to mention associated severe

economic losses. One of the main sources of Listeria is recontamination of food in the

processing environment (Kornacki, 2005; Kozak, Balmer, Byrne & Fisher, 1996). Dairy

products are more likely to be contaminated with Listeria because of the conditions in the

production facilities, the environment (animals and farms) and storage temperatures

(Kozak, Balmer, Byrne & Fisher, 1996).

Thermal inactivation of microorganisms is usually assumed to follow first order

kinetics; this assumption is based on the idea that all cells or spores have the same

sensitivity to heat (Peleg & Cole, 1998; McKellar & Lu, 2004; Hassani, Álvarez, Raso,

Condón & Pagán, 2005). If so, the typical relationship between the number of

microorganisms (N) and the treatment time (t) is shown in equation (1), where k

represents the reaction constant:

106

kNdtdN

=− (1)

Several studies (e.g. van Boekel, 2002) have shown that the survival curves of some

microorganisms under different conditions of inactivation have downward or upward

concavities and that the first order kinetics or a linear relationship between N and t is not

followed for a number of reasons. McKellar and Lu (2004) classify the explanations for

nonlinear behavior throughout thermal inactivation into limitations in the experimental

procedure (variability in heating procedures, mixed cultures or populations, clumping,

protective effect of dead cells, method of enumeration, poor statistical design) and normal

features of the inactivation process (heat sensitivity, heat adaptation, heat transfer)

(Chung, Wang & Tang, 2007). Mathematical models to describe the death of

microorganisms in foods have been proposed since the 1920s; log normal distributions

have been used since 1942 in an attempt to explain the nonlinearity in survival curves

(McKellar & Lu, 2004). Several equations have been proposed to characterize the growth

and inactivation behavior of bacteria. For example, for Listeria monocytogenes, logistic

and log-logistic equations have been used to model heat sensitivities (Peleg & Cole,

1998). But seldom any particular model has been proposed to address the nonthermal

inactivation of Listeria monocytogenes (Buchanan, Golden, Whiting, Phillips & Smith,

1994). A model proposed by Peleg and Cole (1998) based on Weibull distribution has

been proven very useful to fit survival curves with upward or downward concavities or

linear trends. This equation has been used to model inactivation data from some emerging

technologies such as high pressure or pulsed electric fields that do not follow a first order

kinetics (Hassani, Álvarez, Raso, Condón & Pagán, 2005; Rodrigo, Ruíz, Barbosa-

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Cánovas, Martínez & Rodrigo, 2003). Emerging technologies such as high pressure,

pulsed electric fields, microwave and ohmic heating now face the challenge of producing

fresh-like products, while at the same time ensuring food microbiology safety (Peleg &

Cole, 1998; Gaze 2005).

In this study, the inactivation of Listeria innocua ATCC 51742 as a surrogate of

Listeria monocytogenes was tested in raw whole milk because of its importance in dairy

products. The objective of this research was to evaluate the use of different ultrasound

intensities in combination with heat treatment in the inactivation of Listeria cells

compared with conventional batch pasteurization in raw whole milk, and to evaluate

some quality characteristics such as pH, acidity and color. At the same time, adequate

mathematical models were sought to describe the inactivation of bacteria under this

emerging technology.

2. Materials and methods

2.1. Milk samples

Raw and whole cow’s milk was obtained from Washington State University’s Creamery

(Pullman, WA). Milk was kept in refrigeration at 4ºC until it was used. Characterization

of raw milk and thermal and thermo-sonicated milk included pH, lactic acid content and

color. The initial microbial load (mesophilic bacteria) and the absence of Listeria cells

were tested for raw milk.

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2.2. Microbiological analysis

2.2.1. Growth and inoculum of Listeria innocua cells

Two ml of thawed Listeria innocua cells (ATCC 51742) (one ml of microorganism

grown in the early stationary phase plus one ml of sterile glycerol) stored at -21ºC were

added to 100 ml Tryptic Soy Broth (TSB, Bacto: Becton, Dickinson and Co., Sparks,

MD) with 0.6% Yeast Extract (YE, Bacto, Becton, Dickinson and Co., Sparks, MD).

Microorganisms were kept in a bath shaker at 37ºC and 218 rpm until they reached the

early stationary phase, approximately after 11 hr. Milk samples were inoculated with

Listeria innocua ATCC 51742 in a ratio of 1:100 (bacteria:milk) (V/V).

2.2.2. Enumeration of Listeria and mesophilic bacteria

Serial dilutions were made in Peptone water (0.1%) with samples taken from thermal,

thermo-sonicated and raw milk to evaluate the microbial load of mesophiles. After that,

samples were pour-plated in Plate Count Agar (Difco, Becton, Dickinson and Co.,

Sparks, MD), dishes were incubated at 35ºC for 48 h, and bacteria were counted. For

Listeria innocua, after the serial dilutions, samples were pour-plated in Tryptic Soy Agar

(TSA Difco, Becton, Dickinson and Co., Sparks, MD) with 0.6% of Yeast Extract (Bacto,

Becton, Dickinson and Co., Sparks, MD). Dishes were incubated at 35ºC for 48 h, and

then bacteria loads were counted.

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2.3. Thermal and thermo-sonication treatments

2.3.1. Thermal treatment

The thermal treatment was carried out in a double-walled vessel of 500 ml with an

internal diameter of 8 cm and a depth of 13.5 cm. Equal volumes of milk were used for

each treatment, and the temperature of the medium was kept at 63 ± 0.5ºC with the use of

a heating bath maintained at 65ºC to allow heat transfer. Temperature was monitored by a

pre-calibrated thermocouple. A magnetic stirrer was used inside of the vessel to assure

the homogeneity of the samples throughout the treatment. Samples were taken every 2

min of treatment for microbiological analysis and transferred to tubes with peptone water.

After 30 min of thermal treatment, samples were transferred to sterile bottles and held at

4ºC until they were used for color measurements and pH and acidity content.

2.3.2. Thermo-sonication treatments

500 ml of raw whole milk were placed in a double-walled vessel (500 ml) that served as a

treatment chamber.

An ultrasonic processor (Hielscher USA, Inc., Ringwood, NJ, model UP400S),

400 W, 24 kHz, 120 microns with a 22 mm diameter probe introduced into the vessel. A

magnetic stirrer was used inside the treatment chamber to assure homogeneity of the

samples. Ultrasonication was carried out at 30%, 60%, 90% and 100% (40μm, 72 μm,

108 μm and 120 μm, respectively) of amplitude of the ultrasound wave and 63 ± 0.5 ºC

of temperature. Temperature was set up and kept constant via a refrigerated bath (VWR

Scientific Model 1166, Niles IL). A thermocouple was used in the treatment chamber to

monitor the temperature (63 ± 0.5ºC) throughout the experiments. As in the case of

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thermal treatment samples were taken every 2 min of thermo-sonication and transferred

to tubes with peptone water to perform microbiological analysis. After 30 min, the

samples were transferred to sterile bottles and held at 4ºC until they were used for color

measurements and physicochemical analysis.

2.4. Physicochemical characteristics

2.4.1. pH and titratable acidity

pH was determined by direct immersion with a potentiometer (Orion Research, Inc.,

Boston, MA). Following that step, titratable acidity was determined using a

potentiometer. Ten ml of raw or processed milk were poured into a beaker with 20 ml of

distilled water with constant agitation. Milk was titrated at room temperature with a 0.1 N

NaOH solution. The end point was reached when the potentiometer showed 8.3. Acidity

was expressed as a percentage of lactic acid (1 ml of 0.1 N NaOH = 0.009 g of lactic

acid). The experiments were preformed in triplicate.

2.4.2. Color

Lightness to darkness (L*) (100 to 0), redness(+) to greenness(-) (a*), and yellowness(+)

to blueness(-) (b*) color parameters were determined using a Minolta CM-2002

spectrophotometer (Minolta Camera Co., Osaka, Japan) in the reflection mode. Twenty

ml of raw and thermo-ultrasonicated milk samples were poured into sterile plastic bags.

A white ceramic plate was used for standardizing the instrument (L* = 93.4, a* = -0.67,

b* = 0.78). Two bags were used for each system, and each measurement was performed

in triplicate.

111

2.5. Modeling and statistical analysis

Non-linear models, chosen on the basis of the general appearance of the survival curves,

were used to characterize the inactivation data of Listeria innocua cells.

Firstly, the experimental data was fitted with the Weibullian model, shown in

equation (2):

ntbN

tNLog *)(

0

−= (2)

where N(t) and N0 are the momentary and initial microbial counts respectively, t is the

treatment time, b is a rate parameter related to the velocity of inactivation of the

microorganism (min-1) and n is a shape factor; a measure of the semilogarithmic survival

curve’s concavity.

A four-parameter mathematical model [equation (3)] was used to describe

sigmoidal survival curves

2121

0

)( nn tktkN

tNLog = (3)

where k1, k2, n1 and n2 are constants.

All models were fitted using Mathematica® (Wolfram Research, Champaign, IL), and

the statistical analysis was conducted with Microsoft Excel®.

112

3. Results and discussion

3.1. Microbial inactivation

In Figure 1 microbial inactivation for thermal and thermo-sonication treatments is

depicted. The graph corresponds to the inactivation of Listeria innocua in which the

effect of conventional batch pasteurization (63ºC and 30 min) is observed; after

completing the total treatment time, 5.3 log reductions are achieved. In this case, as

observed in Figure 1, an almost linear relationship between the number of survivors and

treatment time is generated with just the thermal treatment, only a slight upward

concavity is observed. In this case, the first order kinetics could be considered as a

modeling option in order to obtain the D values for Listeria inactivation. Nevertheless, in

thermo-sonication treatments, different behaviors are observed through inactivation; with

the mildest thermo-sonication treatment (30%), cells maintain a constant period of

inactivation at the first stage that lasts from 10-20 min of treatment. This lapse of time

seems to be a crucial time for inactivation and weakening of the cells; at the end of the

treatment, the inactivation of more than a 5 log reduction is achieved. In the case of

stronger thermo-sonication treatments (intensities of 60, 90 and 100%), a similar

behavior can be observed through inactivation; after 10 minutes of treatment, more than a

5 log reduction is obtained, although in the case of treatment at 60% of amplitude wave at

the end of the process, there is a point outside of the inactivation trend, known as a “tail.”

Tailing is related with the presence of “artifacts” during the process, but also with the

mechanism of inactivation or resistance. The term “artifact” refers to anything that

interferes with the linear survival curve. The expression of heat shock proteins due to the

induction of heat resistance is one of the attributed causes of tailing (McKellar & Lu,

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2004; Chung, Wang & Tang, 2007). The presence of subpopulations that are genetically

more resistant is another cause of tailing (Buchanan, Golden, Whiting, Phillips & Smith,

1994). In this case, the last point of treatment at 60% of ultrasound intensity could be

due to a protective effect from the dead cells, clumping or even the medium, because this

milk contains more than 3.5% butter fat content. If survival cells were clumped and

surrounded by dead cells, or even protected by the fat globules of milk, after 20 min this

clump could be broken and release live cells, generating the tailored shape of the curve.

For the last two thermo-sonication treatments (90 and 100%), it is clear that the

combination of ultrasound, acting together with heat in the inactivation of cells, reduces

the treatment time from 30 min to 10 min, compared with thermal treatment, which could

be an advantage in many aspects of processing. It is important to highlight the fact that

ultrasound is considered as a nonthermal technology because the main lethality effect in

cells is related to preservation factors other than heat. In this case, heat is probably

facilitating inactivation by weakening the cell, but the key reason is the effect of

cavitation, generated by ultrasound, which promotes cell disruption.

In Table 1, the inactivation of mesophilic bacteria is shown. The results observed

for the combined treatments (ultrasound & heat) are similar to those observed for Listeria

cells. Ultrasound in this case applied at 60, 90 or 100% intensity in addition to the

conventional thermal treatment (63ºC); speeds up the inactivation rate. All of these

treatments reduce the natural flora of raw milk by 3 log - reductions in the first 10

minutes of treatment compared to batch pasteurization, in which, after 10 minutes, only

about 1.89 log - reductions are achieved. For the treatment performed at the lowest

ultrasound intensity (30%), an “activation shoulder” is shown to “increase” the

114

population of microorganisms. The reason for the shoulder could be attributed to the

presence of dormant spores in the initial population of milk, followed by the stimulation

of the cells due to sonication, generating a higher number of mesophiles compared with

the initial population. According to the literature, low intensity ultrasound has been used

in the stimulation of living cells and enzymes (Knorr et al., 2004; Chemat & Hoarau,

2004). But even though power ultrasound was used as an inactivation factor in this work,

it is questionable whether 30% ultrasonication was enough to activate the spores, to

promote their growth in the first minutes of treatment and to injure the cells at the end of

the treatment. Earnshaw, Appleyard & Hurst (1995) mention that as the amplitude of the

ultrasound wave is increased, the intensity of cavitation also increases. This “tailing,” as

observed for treatment at 30%, corresponds to the linear type of “activation shoulder”

according to Corradini and Peleg (2003).

3.2. Modeling

As mentioned previously, thermal processing is based on assumptions of linear

relationships between survival fractions and time. In this work, an important focus is the

modeling of the destruction rate of Listeria innocua, because the pathogenic

microorganism is of great interest in food science due to its wide range of growth

conditions, especially in refrigerated products. Currently, there is scarce information in

predictive microbiology related to ultrasound inactivation. Because of the recent interest

in that technology for food preservation the inactivation data for Listeria innocua was

chosen to be fitted with some available models.

115

Using the thermal treatment data for Listeria inactivation, it behaves in a nearly

linear fashion a D value can be calculated (5.43 min), and the fit of the model (data not

shown) is r2 0.9298. Deviations in the linearity of inactivation of Listeria cells is related

to heat shock, that is heat damage and repair mechanisms of the microorganism (Hassani,

Álvarez, Raso, Condón & Pagán, 2005). In order to compare the effectiveness of all

treatments, D values cannot be calculated in the thermo-sonication processes because of

the non-linearity of the plots. Applying first order kinetics to sonication curves would

violate the basic concepts of thermal processing theory. In order to achieve a

mathematical approach to explain and describe the behavior of Listeria cells under

thermo-sonication treatments, various mathematical equations were fit to the data. The

distributions shown for each treatment are the best fit for each one.

3.2.1. First model: Weibull distribution

In Figure 2 the thermal treatment curve is fitted using equation (2) as a model. The values

of the model’s parameters, namely b and n for this model are shown in Table 2. For heat

inactivation of Listeria innocua, the shape factor n is 1.73 and the rate parameter b is

0.016. For the toughest thermo-sonication treatment (100%), the shape factor n was lower

(0.23), and the rate parameter b was higher (2.7). If n < 1, the shape of the survival curve

is concave upwards (tailings), while when n > 1, the shape is concave downward

(shoulders). Both shape factors show a concavity downward in the survival curves, being

more prominent in thermal treatment. Some survival curves of Listeria monocytogenes

under elevated temperature conditions and chemical agents also showed downward

concavity. The fitted model at 62ºC had b equal to 1.722 and n of 0.900 (Peleg &

116

Penchina, 2000). Ugarte-Romero, Feng, Martin, Cadwallader and Robinson (2006)

reported an n value of 1.95 for heat-treated Escherichia coli at 55ºC in apple cider and a

range of shape factors (n) from 0.547 to 0.720 of the same bacteria subjected to thermo-

sonication (20 kHz and 0.46 W/ml) from 40ºC to 60ºC. “Shoulders” in survival curves of

bacteria subjected to different treatments are reported by many authors using the Weibull

model; for example, Escherichia coli under treatment of Pulsed Electric Fields had n

values from to 0.052 to 0.72 (Rodrigo, Barbosa-Cánovas, Martínez & Rodrigo, 2003) and

from 0.483 to 0.579 (Álvarez, Virto, Raso & Condón, 2003); thermal inactivation of

spores had n values from 0.97 to 2.03 (Fernández, Salmerón, Fernández & Martínez,

1999). The rate parameter b shows the velocity of inactivation of the cells, and according

to the data derived from the model, the inactivation is faster in thermo-sonication than in

thermal treatment, confirming the positive effect of sound waves on inactivation rate.

Some references (Ugarte-Romero, Feng, Martin, Cadwallader & Robinson, 2006) show

that by increasing the temperature, ultrasound is more effective and faster than thermal

treatment alone for inactivation, according to the b values reported, increasing the lethal

effect of the treatment (Álvarez, Virto, Raso & Condón, 2003; Rodrigo, Barbosa-

Cánovas, Martínez & Rodrigo, 2003). According to the MSE for that research, the

Weibull model best fitted the survival curve obtained during the thermo-sonication at

90%. Concavity in survival curves is attributed to the properties of the primary

distribution of the lethal effects in addition to the accumulative effect of the heat or lethal

agent in the cells. A downward concavity might be related to weakening of the cells

because of the exposure to the lethal agent and the speeding up of the destruction rate of

them (Peleg & Penchina, 2000).

117

This behavior, as shown in Figure 3, corresponds to the mildest thermo-sonication

inactivation (30%) of Listeria innocua, although this data for sonication does not fit very

well according to the Weibull model. The shape of the survival curve shows two trends:

an upward concavity followed by a downward concavity, which can be described as

sigmoid shaped. For that reason, a second model was proposed in order to describe the

inactivation behavior.

3.2.2. Four-parameter model

In Figure 3 the sigmoid shape of the thermo-sonicated (30%) inactivation of Listeria

innocua is shown. This curve shows a first stage that has an upward concavity at the

beginning of the treatment, followed by a change toward a downward concavity at the

end of the inactivation. The upward concavity at the beginning is a clear indication that

the weakest cells are inactivated in the first minutes of treatment, followed by an almost

stable period in the middle of the treatment in which the toughest cells are showing

resistance. However, those cells are accumulating damage from the heat and ultrasound

effects, ending in a faster inactivation because of the increase of the damage. This type of

survival curve is common, but not typical (Peleg & Penchina, 2000).

In Figures 4, 5 and 6 the survival curves corresponding to higher intensities of thermo-

sonication (60%, 90% and 100%) are shown. Weibull equation can be fitted to these data

with good results as shown in Table 2. However the four parameter model can also be

used. There is a transition between sigmoid shape to upward concavity as the intensity is

increased. Fitting equation (3) to the survivors data at 60%, 90% and 100%, k2 become 0,

so equation (2) and (3) are equivalent because k2tn2 is zero. The fitted parameters for

118

these three survival curves are shown in Tables 2 and 3. As the intensity of the treatment

increases, k1 also is higher; meanwhile, for n1 the value decreases with higher intensities.

k1 is related to the inactivation rate (min); in this case, as the intensity of the ultrasound

increases, the rate decreases, although the difference is just a few seconds, meaning that

they are similar treatments. The expected trend was the decrease of k1 as the intensity was

increased, as reported in other inactivation studies, such as with the increase of

temperature in the inactivation of Clostridium botulinum spores (Peleg & Penchina,

2000) and Bacillus stearothermophilus (Corradini & Peleg, 2003). k2 values show that

with higher intensity, the upward concavity is decreased. Some references have shown

this equation fitted to heat inactivation data of Clostridium botulinum spores without a

defined trend in k2 parameter as the temperature was increased (Peleg & Penchina, 2000),

but also without a defined trend in the survival curves of Bacillus stearothermophilus

because of the scattering of the data and the few parameters evaluated (Corradini &

Peleg, 2003).

3.3. Physicochemical characteristics

In Table 4 the initial and final conditions of pH, acidity and color parameters are reported

for all systems. The pH of milk decreased regardless of the treatment, from the initial

value of 6.80 to 6.74 for heat treated milk and 6.64 (average) for the thermo-sonicated

milk. Respectively, acidity was increased in samples from 0.109% (raw milk) to 0.126%

(thermal) and 0.141% (thermo-sonicated). According to Neville and Jensen (1995), the

physical-chemical properties reported for bovine milk are pH in the range of 6.22 to 6.77,

119

and the titratable acidity as 0.16 ± 0.02. Both pH and acidity of thermo-sonicated milk are

inside the range considered as permissible for milk quality standards.

According to the color data shown in Table 4, thermo-sonicated milk has an

important change in luminosity as ultrasound intensity is increased. L values increased as

the amplitude of the sound wave was higher, that is the intensity of the cavitation inside

the medium was stronger. The reason for that is the homogenization that the sonication is

generating in the fat content of the milk; this increase in the luminosity was appreciated

by sight as a whiter color. The heated milk had a similar luminosity as that in the raw

milk. In apple cider treated with thermo-sonication treatments, the L value slightly

increased after the process in comparison with the original product (Ugarte-Romero,

Feng, Martin, Cadwallader & Robinson, 2006). Also, some studies have shown that as

the fat content in milk is increased, the samples are whiter (Phillips, McGiff, Barbano &

Lawless, 1995); in the case of the present experiment, the reason for higher L values is

the possible breakage of fat globules in milk due to the disruption of membranes

generated by ultrasound, releasing the triacylglycerols content of the globules and making

the fat content more “available.” According to the a values, the milk is closer to the

greenness region of the Hunter’s color scale; specifically, the heated milk and the b

values are very similar among all of the samples, indicating that there is an important

yellow contribution to the color of milk. The most yellow sample is raw milk in all color

measurements, a higher standard deviation was observed in raw milk because of the non

homogenization of the product; even though the sample was agitated before each

analysis, the presence of some fat clumps was observed.

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4. Conclusions

Ultrasound is a viable technology to inactivate pathogenic bacteria in milk in shorter

times without important changes in the pH and acid lactic content, along with better

appearance and homogenization. Modeling of survival curves of thermal and thermo-

sonicated bacteria showed good fit with the Weibull distribution and the four parameter

model, but no extrapolation could be conducted because of the uncertainty of the data

trends shown in many research works. This modeling is just one of the first attempts to

find some mathematical equations that may describe the inactivation rates of bacteria

under an emerging technology such as ultrasound.

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127

Table 1. Inactivation of mesophilic bacteria in raw whole milk with thermal and thermo-

sonication treatments

Treatment time (min)

Thermal treatment

30% 60% 90% 100%

0 0 0 0 0 0 10 -1.89 0.53 -2.98 -3.23 -3.13 20 -4.00 0.87 -3.38 -3.37 -3.45 30 -3.75 -3.86 -4.25 -3.62 -3.58

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Table 2. Parameters of the Weibull equation fitted to thermal inactivation and thermo-sonication treatments for Listeria innocua in raw whole milk

Thermal

treatment 30% 60% 90% 100%

B 0.016 - 2.4 2.9 2.7 N 1.73 - 0.28 0.21 0.23

MSE 0.0614 - 0.152 0.048 0.069

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Table 3. Parameters of the Sigmoid or four parameter model (equation 4) fitted to thermal inactivation and thermo-sonication treatments of different intensity for Listeria innocua

in raw whole milk

Thermal treatment

30% 60% 90% 100%

K1 0.016 1.45x10-9 2.4 2.9 2.7 N1 1.73 6.37 0.28 0.21 0.23 K2 0 0.995 0 0 0 N2 - 0.196 - - -

MSE 0.0614 0.124 0.152 0.048 0.069

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Table 4. Physical-chemical characteristics of milk samples before and after thermal and

thermo-sonication treatments

Sample pH Acidity (%)* L A b Control (raw milk) 6.80 ± 0.04 0.109 ± 0.012 87.82 ± 0.18 -1.70 ± 0.13 5.91 ± 0.25 Thermal treatment 6.74 ± 0.06 0.126 ± 0.008 88.25 ± 0.67 -1.97 ± 0.07 5.61 ± 0.06

30% 6.64 ± 0.02 0.142 ± 0.014 91.92 ± 0.36 -1.67 ± 0.03 5.54 ± 0.05 60% 6.66 ± 0.05 0.141 ± 0.009 91.99 ± 0.34 -1.57 ± 0.04 5.65 ± 0.06 90% 6.65 ± 0.02 0.136 ± 0.012 92.02 ± 0.37 -1.63 ± 0.04 5.61 ± 0.05 100% 6.61 ± 0.01 0.146 ± 0.009 92.37 ± 0.20 -1.55 ± 0.02 5.64 ± 0.09

* Expressed as lactic acid

131

-7

-6

-5

-4

-3

-2

-1

00 5 10 15 20 25 30

TIME (min)

LOG

(N/N

o)

TT 30% 60% 90% 100%

Figure 1. Inactivation of Listeria innocua in raw whole milk under thermal and thermo-

sonication treatments

132

Figure 2. Weibull distribution fitted to thermal treatment survivor curve

0 10 20 30 40

0

-1

-2

-3

-4

-5

-6

THERMAL TREATMENT

LOG

N(t)

/N0

Time (min)

133

Figure 3. Four parameter model fitted to thermo-sonicated survivor data at 30% of

amplitude wave

0 10 20 30 40

0

-1

-2

-3

-4

-5

-6

TIME (min)

30% INTENSITY

LOG

N(t)

/N0

134

Figure 4. Weibull distribution fitted to survivor curve of thermo-sonication at 60% of

amplitude wave

0 10 20 30 40

0

-1

-2

-3

-4

-5

-6

-7

TIME (min)

60% INTENSITY

LOG

N(t)

/N0

135


amplitude wave

0 10 20 30 40

0

-1

-2

-3

-4

-5

-6

-7

TIME (min)

LOG

N(t)

/N0

90% INTENSITY

136


amplitude wave

0 10 20 30 40

0

-1

-2

-3

-4

-5

-6

-7

LOG

N(t)

/N0

TIME (min)

100% INTENSITY

137

CHAPTER 3

COMPOSITION PROPERTIES, PHYSICOCHEMICAL CHARACTERISTICS

AND SHELF-LIFE OF WHOLE MILK AFTER THERMAL AND THERMO-

SONICATION TREATMENTS

Daniela Bermúdez-Aguirre, Raymond Mawson, Kees Versteeg and

Gustavo V. Barbosa-Cánovas

Abstract

Raw whole milk (RM) was pasteurized with thermal (TT) and thermo-sonication (US)

treatments. Batch pasteurization was used for TT and 36 μm, 72 μm, 108 μm and 120 μm

of the ultrasound wave intensity (24 kHz, 400W). In addition, the heat conditions were

applied together for US. Apparent protein content of US milk decreased; butter fat

content was increased by US treatment. Proximal analysis showed the presence of added

water in US milk and a decrease of non-fat solids, whereas pH was decreased for US

milk, lactic acid was increased and density was decreased from RM to US; also, the color

of US milk was whiter. The tested parameters for the TT samples were often intermediate

between RM and US samples. Statistical analysis showed a significant difference (p <

0.05) among all treatments. After 16 d, US samples (4ºC) did not show mesophilic

growth higher than 2 log; pH, acidity and color remained constant.

138

1. Introduction

Nonthermal technologies offer advantages in the final product besides microbiological

safety. The products obtained are fresh-like, and losses in color, flavor and nutrients are

minimal. Technologists and industries are interested in novel nonthermal technologies

such as high pressure, pulsed electric fields, ultrasound, high intensity light, and

irradiation, among others (Knorr et al. 2002). Conventional methods to pasteurize milk

include the use of low temperature and long-time application (63ºC for 30 min), High

Temperature Short Time (HTST) (72ºC for 15 sec), and Ultra High Temperature (UHT)

pasteurization (138ºC for 2-4 sec), which is referred to in some countries as UHT

sterilization. Regardless of treatment, the microbiological safety of the milk is ensured,

but the quality of milk is decreased to some degree due to the combination of the time-

temperature processes acting together. Protein denaturation, vitamin destruction, enzyme

inactivation, Maillard reaction and production of lysinoalanine are some of the common

chemical changes that take place in milk following traditional heat pasteurization

methods (Efigênia et al. 1997). The consumer looks for special sensorial characteristics

of milk such as appearance, aroma, texture and flavor (Phillips et al. 1995) along with, of

course, the important protein source that milk represents. During conventional

pasteurization, heat affects the chemical structures of water-soluble vitamins and

proteins, e.g. denaturization of whey proteins (Mehta 1980).

Most novel nonthermal technologies present a uniform temperature distribution in

the food and, as a consequence, the intensity of the process is reduced and the quality of

the food is increased (Knorr et al. 2002). For some humans, such as newborns, milk

represents not only a source of protein, but also of fat, vitamins, minerals and sugars;

139

often, milk is the only caloric source for infants. Proximal analysis of bovine milk shows

the following composition: 3.4% protein (2.8% casein), 3.7% fat, 4.6% lactose and 0.7%

ash (Jensen 1995). The white color of milk is due to the presence of fat globules and

casein micelles that disperse light in the visible spectrum (Owens et al. 2001). Some of

the appearance characteristics of milk based on butter fat content include transparency,

gloss, and whiteness, which have been evaluated from 0 to 4% of fat content (Phillips et

al. 1995). The energy generated by ultrasound can be absorbed or reflected by colloidal

milk proteins and fat globules (Villamiel and de Jong 2000). Some sensory studies have

shown that there is a correlation between the parameters of color L*, a* and b* with

perceived color and mouthfeel (Phillips et al. 1995). Some of the factors that are

responsible for the alteration of the quality of pasteurized milk during storage include the

quality of the raw milk, the effectiveness of the treatment, contamination after

pasteurization, storage temperature and packaging material (Zygoura et al. 2004).

The objective of this work was to study the proximal composition (protein, butter

fat content, added water, solids non-fat) and physicochemical characteristics (pH, lactic

acid content, freezing point, density, color) of milk samples after different thermal and

thermo-sonication treatments as a viable option for pasteurizing milk. The shelf-life of

these milk samples under refrigerated conditions was evaluated with respect to

microbiological and physicochemical aspects.

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2. Materials and Methods

2.1 Milk samples

Raw cow’s milk was obtained from the Washington State University Creamery. Raw

milk was characterized by the following parameters: pH, acidity, color, proximal analysis

(butter fat, protein, added water, solids non-fat, density and freezing point) and the initial

microbial load. Milk was kept under refrigerated conditions (4ºC) until used.

2.2 Thermo-sonication treatments

An ultrasonic processor (Hielscher USA, Inc., Ringwood, NJ; model UP400S, 400 W, 24

kHz, 120 μm) with a 22 mm diameter probe was used. A 500 ml double-walled vessel

was used as a treatment chamber (8 cm internal diameter and 13.5 cm depth).

Temperature was kept constant via a refrigerated bath (VWR Scientific Model 1166,

Niles, IL). A thermocouple was used in the treatment chamber to monitor the temperature

(63 ± 0.5ºC) throughout the experiments. Five systems were tested, in which the

temperature was constantly maintained (63ºC ± 0.5ºC), while five different amplitudes of

ultrasonic waves (0% - 0 μm; 30% - 36 μm; 60% - 72 μm; 90% - 108 μm; 100% - 120

μm) were used. Amplitude was 0% in conventional batch pasteurization (63ºC by 30

min); that is, no sonication was applied. The treatment time for each system was 30 min.

2.3 Proximal analysis

Analysis of butter fat and protein content, added water and solids non-fat were carried out

using a LactiCheck™ LC-01 Milk Analyzer (Page & Pedersen, International Ltd.,

Hopkinton, MA). The performance of this equipment was based on high frequency

141

ultrasound. The milk analyzer was validated to determine butter fat content (Gerber) and

protein (Kjeldahl) in milk, according to the Association of Analytical Chemists (AOAC

1986) methodology. The equipment was previously calibrated with raw cow’s milk, and

samples were adjusted to room temperature (20ºC). Each sample was analyzed in

triplicate. No previous preparation was required for the thermo-ultrasonicated samples.

2.4 Microbiological analysis

The initial load of microorganisms was evaluated for raw milk. Plate Count Agar (Difco

Becton, Dickinson and Co., Sparks, MD) was used for mesophiles aerobes. Serial

dilutions were made with peptone water before pouring samples into dishes, which were

then incubated at 35°C for 48 h.

For the thermal and thermo-sonication treatments, the microbial counts were

evaluated after 5 min of treatment, and the final count after 30 min of treatment was used

as initial load of microorganisms for the storage life. Serial dilutions were made with

peptone water, by pouring samples into Plate Count Agar (Difco ®). Dishes were

incubated at 35ºC for 48 hr.

2.5 Physicochemical analysis

2.5.1 pH and titratable acidity

The pH was determined by direct immersion of the electrode with a potentiometer (Orion

Research Inc., Boston, MA), and titratable acidity was also determined using a

potentiometer. Ten ml of raw or processed milk was poured into a beaker with 20 ml of

distilled water, undergoing constant agitation. Milk was titrated at room temperature with

142

0.1 N NaOH solution. The end point was reached when the potentiometer showed 8.3.

Acidity was expressed as a percentage of lactic acid (1 ml of 0.1 N NaOH = 0.009 g of

lactic acid). Each sample was again measured in triplicate.

2.5.2 Density and freezing point

Density and freezing point were evaluated in each milk sample after the different

treatments and compared with the raw milk used as a control. A LactiCheck™ LC-01

Milk Analyzer (Page & Pedersen, International Ltd., Hopkinton, MA) was used for this

purpose. It was calibrated with raw milk and samples were analyzed at 20ºC. Each

sample was analyzed in triplicate.

2.5.3 Color




ml of raw and thermo-ultrasonicated milk samples were poured into sterile plastic bags.


b* = 0.78).

The net color difference was evaluated with the following equation, using the

parameters L*, a* and b* and comparing the different treatments with the raw milk from

the beginning to end of storage life.

( ) ( ) ( )2*2*2** baLE Δ+Δ+Δ=Δ

143

Hue angle (h*) was determined using the following relationship:

⎟⎟⎠

⎞⎜⎜⎝

⎛= −

*

*1* tan

abh

and the chroma or saturation index (C*) was evaluated using this equation:

( ) 21

*** 22

baC +=

2.6 Storage life

Thermo-ultrasonicated milk samples were stored at 4ºC for 16 d. Samples were placed in

sterile plastic bags, using and discarding one bag daily for each treatment. Every other

day, the quality characteristics (pH, acidity and color) were evaluated, and the microbial

loads were quantified. The storage period was 16 days.

2.7 Statistical analysis

All treatments were performed at least in duplicate, and the physicochemical and

composition characteristics were evaluated in triplicate for each sample. Microbial

analyses were performed in duplicate for each sample.

Statistical analysis of the data was performed using a Microsoft Excel program. Analysis

of variance (ANOVA) was calculated with the SAS program (SAS Institute, Cary, NC

1999), and a confidence level (α) of 0.05 was used to evaluate significant differences.

144



Proximal analysis was performed in thermo-sonicated milk to evaluate the possible

changes (i.e. protein denaturation) in milk composition after treatments.

3.1.1 Protein content

Thermo-ultrasonicated milk samples showed an apparent decrease in protein content

(Table 1). Raw milk had 3.28% protein and thermal pasteurized milk had 3.55% protein,

while samples of milk pasteurized with ultrasound showed a final value of 3.02% protein

content. Similar values have been reported for raw milk (3.27%) and thermal pasteurized

milk at temperatures over 72ºC (3.35%), as shown in previous experiments (Rynne et al.

2004). In accordance with the statistical analysis, there was a significant difference (p<

0.05) in protein content among all samples from different treatments. The average

decrease of protein content after thermo-sonication in milk samples was around 0.26%.

The decrease can be attributed to the use of elevated temperature plus ultrasound;

however, studies of protein content in milk after sonication are scarce. In one study,

denaturation of whey protein occurred in ultrasonicated milk and the effect increased

when heat and ultrasound were used together. Sonication is responsible for the

modification of the tertiary and/or quaternary structures of casein, but does not

completely alter the micelle (Villamiel and de Jong 2000). In this study, protein content

after sonication was around 3.02%, which is inside the range of protein content for cow's

milk intended for human consumption. However, further studies of protein content are

145

recommended to specifically analyze the type of protein denaturized by ultrasound and

possible modifications to its structure.

3.1.2 Fat content

Butter fat analysis showed an increase of butter fat in the thermo-ultrasonicated milk

samples (Table 1). The raw milk had a high butter fat content of 4.04%; meanwhile,

thermal treated and thermo-ultrasonicated milk samples showed a higher butter fat

content; the former contained 4.22% butter fat, while the average content in the latter was

around 4.25%. Significant difference (p < 0.05) was found between the butter fat contents

in all samples, meaning that the intensity of cavitation has a proportional effect in the

breakage of globules and release of triacylglycerols. Cavitation is the main mechanism

for homogenization of fat globules in milk treated with ultrasound. Breakage of large fat

globules is produced during cavitation, making the butter fat more available for analytical

quantification. In cow's milk, over 95% of the butter fat content corresponds to fat

globules, and 95% of the globule lipids are triacylglycerols (Keenan and Patton 1995).

When heat is used in combination with ultrasound, smaller fat globule sizes are achieved

(Villamiel and de Jong 2000). Through this size reduction, the fat globules release their

contents to the medium, resulting in the triacylglycerols generating a higher butter fat

content for the sonicated samples, as shown in Table 1.

3.1.3 Added water

The added water values in milk samples are shown in Table 1; this parameter has long

been related with milk quality; the addition of water in milk is considered as adulteration

146

of the product, and it is intrinsically related to the freezing point. The highest recorded

value for the sonicated milk samples was 4.92%, which represents a significant difference

(p < 0.05) compared with the raw and heat treated milk samples. This parameter suggests

the denaturation of proteins, because release of water must occur during thermo-

sonication due to the rearrangement of macromolecules, such as proteins. Also, the

production of some chemical reactions may generate water molecules in the medium.

With higher intensities of cavitation, higher apparent added water content is generated in

the medium. From the point of view of quality, the apparent addition of water is not a

desirable characteristic. However, an in-depth chemical analysis of milk after sonication

is recommended in order to study the possible breakage and chemical activity in milk

after treatment.

3.1.4 Solids Non-Fat (SNF)

The solids non-fat (SNF) present in milk include protein (casein and whey protein),

carbohydrates (lactose), minerals (calcium and phosphorus), and some soluble vitamins,

such as riboflavin (IDFA 2006). The content of SNF in milk samples is shown in Table 1.

Raw milk has an SNF content of 9.15%, while heat pasteurized milk shows a higher

value (9.50%), probably because the pasteurization process results in a slight

concentration due to water evaporation. In thermo-sonicated samples, inverse behavior

was demonstrated. There was an apparent reduction of the solids non-fat content to

8.03% for the highest ultrasound intensity. This result could be due to a new and

complete regrouping of some components; if the ultrasound is generating a

homogenization effect in milk, disrupting fat globules and creating new microstructures,

147

the solids non-fat could be adhering to the new fat matrices, reducing the apparent fat

content.

3.2 Physicochemical properties

3.2.1 pH, acidity, density and freezing point

In Table 2, physicochemical properties are shown for all milk samples. The first

parameter, pH, was evaluated in raw milk and in the thermal and thermo-ultrasonicated

samples after 30 min of treatment. Clearly, after treatment with ultrasound, the pH of the

samples was lower than for either raw milk or heated milk; nevertheless, the average

value (6.64) is in the range acceptable for commercial milk. Decrease of pH could be

because of the enzymatic action promoted by the cavitation producing the hydrolysis of

esters (Walstra et al. 2006). Titratable acidity (expressed as lactic acid) increased from

0.109% to 0.141% (average) after sonication, but this value is also within the reported

range. This increase in the acidity could be attributed to two facts. The first fact relates to

lipolysis, because of the enzymatic action on triglycerides (Walstra et al., 2006) which is

enhanced by ultrasound and the release of fat free acids to the medium. The second result

could be due to the formation of nitrite, hydrogen peroxide and nitrate in milk after

sonication; some studies have shown the formation of these products in aqueous media at

different frequencies and ultrasound intensities (Supeno 2000). The physicochemical

properties reported for bovine milk are: pH in the range of 6.22 to 6.77; specific gravity

between 1.021 and 1.037, and titratable acidity of 0.16 ± 0.02 (Neville and Jensen 1995).

Agnihotri and Pal (1996) reported a pH from 6.40 to 6.68 for goat's milk. Density

148

decreased in raw milk from 1.0303 g/cm3 to 1.0257 g/cm3 (100%), probably because of

the homogenization achieved with ultrasound treatment, making a more fluid product.

The freezing point of the milk samples is shown in Table 2. Raw milk showed a

value of -0.5160ºC; the equipment was calibrated in accordance with that value. Some of

the reported values in the literature are in the range -0.520ºC to -0.531ºC, depending on

the country (Slaghuis 2001). The freezing point values after sonication are shown in

Table 2; the average value was -0.5317ºC. In this case, there was no tendency related to

the intensity of the treatments.

The addition of water in milk was discussed previously, and the depression of the

freezing point data after thermo-sonication matched very well, confirming that the

freezing point temperature is a very useful measurement in quality assessment as it shows

if water has been added to the milk.

The statistical analysis performed for each physicochemical property (pH, lactic

acid content, density and freezing point) evaluated in this section showed a significant

difference (p < 0.05) between each treatment, meaning that thermo-sonicated milk has

completely different and new properties compared to the raw and heat treated fluids.

3.2.2 Color

Color parameters after thermal treatment and the different sonication treatments are

shown in Figures 1, 2 and 3, corresponding to time zero during storage life in each plot.

Raw milk and heated milk showed lower luminosity (87.77 and 88.59, respectively)

compared to the sonicated samples, which showed an increase in this value (from 91.69

to 92.37). All samples processed with ultrasound showed higher L* values; thus, with the

149

increase of cavitation (corresponding to higher ultrasonic wave amplitudes and stronger

treatments) the luminosity increases; the resulting color improvement is due to the

enhanced homogenization of milk. Similar results were observed by Ertugay et al.

(2004); excellent homogenization occurred in milk due to the reduction of fat globule

size with the highest ultrasound power used and a long treatment time (10 min).

Ultrasonic treatment plus temperature of 60ºC reduced the fat globule size to 1 μm. Large

changes in particle size are enough to modify light reflection (Owens et al. 2001). The

size of fat globules, distribution of milk proteins, and the Maillard reaction are

responsible for the color of milk. Whitening in milk can occur because of the

denaturation and coagulation of soluble protein in milk, increasing the opaque particles

(Mehta 1980); all of these reasons may account for changes in the color of milk before

and after sonication.

Color parameter a* changed from -1.81 (in raw milk) to -1.92 (in thermally

treated milk) and to -1.59 (in thermo-sonicated milk), meaning that the unprocessed

product has a higher contribution in the green region than the treated milk. The b* value

readings were very close to each other: raw milk (5.68), thermal treatment (5.60) and

thermo-sonicated (5.58 to 5.64), which is related to an approximately equal contribution

from the yellowness region. This similarity was confirmed by statistical analysis; the b*

value was the only color parameter that did not show a significant difference (p < 0.05)

between each milk treatment.

150

3.3 Storage life

3.3.1 Microbiological aspects

In Figure 4 the mesophilic bacteria growth behavior in the different samples is shown.

The growth of mesophiles is very fast in raw milk, starting with approximately 103

cfu/ml. After 5 d there was a 5.4-log growth, and after 9 d the growth had reached 6.7 log

cycles. Similar results are reported by Zygoura et al. (2004), who found an initial load of

mesophilic bacteria of 4.65 log cfu/ml in raw milk. After 7 d of storage at 4ºC the counts

were between 6.56 and 7.16 log cfu/ml, very close to the allowable limit (106 – 107

cfu/ml). The initial count of mesophilic bacteria in this experiment is very important

because it determines the final quality of the product and the storage life length.

According to U.S. standards, Grade A raw milk may not exceed 300 000 cfu/ml, and in

the European community the bacterial count should be lower than 100 000 cfu/ml. After

pasteurization, in the United States, milk should contain less than 20 000 cfu/ml (Jensen

et al. 1995). The raw milk used in this experiment achieves the standards reflecting good

sanitary practices; after pasteurization with heat and ultrasound, microbial loads were

reduced to 102 cfu/ml (data not shown), which is also under the established limit.

Thermal treated milk showed a slight increase of bacteria through storage life; as

can be seen in Figure 4, the refrigerated conditions retarded the growth of

microorganisms compared with raw milk. After 16 d the microbiological count for the

heat treated product was 5.8 log cycles. The behavior of the bacteria in thermo-sonicated

milk showed an interesting increase/decrease of cells under storage conditions. The

lowest amplitudes (30% and 60%) showed a decrease in the bacteria population during

151

storage. This decrease could be attributed to many factors, such as the progressive dying

off of injured cells that were not totally inactivated during the sonication.

In some inactivation studies the effect of the amplitude of the ultrasound wave has

been shown to be a crucial parameter related to the degree of inactivation and, hence,

with the degree of injury to the cells. Lower amplitudes do not generate enough

inactivation, and in other cases these amplitudes can even promote cell growth (Knorr et

al. 2004; Chemat and Hoarau 2004). Higher amplitudes do generate total inactivation and

the possibility of cell recovery after treatment is scarce. In addition, the action of

ultrasound in an aqueous medium generates the sonolysis of water, which is the breakage

of water molecules, H+ and OH-, and other free radicals, and their recombination. These

radicals and new compounds could show a bactericidal effect in cells (Tsukamoto et al.

2004a, 2004b; Furuta et al. 2004). The mechanism of inactivation in cells due to

ultrasound cavitation seems to be the formation of pores outside the cell membrane,

disruption of cell structures, and breakage of cells (Bermúdez-Aguirre and Barbosa-

Cánovas 2006; Ugarte-Romero et al. 2006). Thus, the reason for possible “inactivation”

of cells during storage life for 30% and 60% of treatments is a combination of cells

injured and free radical formation.

Treatment time (30 min) did not inactivate more mesophiles, but was long enough

to injure cells with the characteristics described above. The presence of radicals and new

compounds could be acting as bactericidal agents freely entering the inside cell structures

and making these cells unavailable for microbial counts during storage. The disruption of

the tertiary and quaternary structures of the casein generated by ultrasound seems to be

the cause of the increase in the antioxidant activity of milk (Villamiel and de Jong 2000).

152

This antioxidant activity produced in milk via sound waves could also act as a natural

antimicrobial.

With 90% treatment intensity there was a decrease in the growth of mesophiles

during storage life until day 16. The initial count was 2 log cycles, finishing with counts

of less than 1 log cycle. The highest intensity (100%) showed a maximum growth of 1

log cycle at the beginning of storage, followed by a decrease of the microorganism’s

viability during the remaining storage life. The decrease of the cell growth could be due

to the adverse conditions of the cell environment, in addition to cell injury through

cavitation, making it impossible for cell recovery in the medium after treatment.

In all samples of sonicated milk, no more than 2 log cycles of growth was

detected up to 16 d at 4ºC, making ultrasound a viable alternative to pasteurized milk,

with longer storage life than for heat treated milk. Ultrasonic technology deserves further

research; for example, the study of psychrotrophic bacteria and enzyme behavior

throughout storage life could provide valuable information about the stability of milk. In

addition, encouragingly, e.g. after 16 d of storage, milk samples did not show visible

spoilage signs from enzymatic or microbiological origin.

3.3.2 pH and lactic acid content

Although pH and acid lactic content are not routinely evaluated as quality indicators of

milk's storage life, it is important to monitor them as they are an indirect measure of the

microbiological growth of lactobacillus, which is responsible for acid production in milk.

Lactic acid is measured by titratable acidity and it is often used to determine bacterial

growth in fermentation or contamination processes (Neville and Jensen 1995). The pH

153

behavior of different milk samples is shown in Figure 5. The raw milk value drastically

decreased after 5 d at 4ºC; meanwhile, the heat treated and thermo-sonicated samples

stayed almost constant after 16 d. Similar behavior was observed for all samples with

regard to lactic acid content, as shown in Figure 6. The pH and acidity values for both

thermal and thermo-sonicated milk samples are within the range reported for cow's milk

by Neville and Jensen (1995): 6.22 – 6.77 and 0.16 ± 0.02, even after 16 d of storage.

3.3.3 Color

Thermo-sonicated milk maintained a very homogeneous color throughout its storage life.

The luminosity of samples observed after sonication was a very white fluid. After storage

at 4ºC for more than 16 d, the color remained in the samples. Even in the sample treated

at 100%, the L* value increased, as seen in Figure 1. Raw and heat treated samples

always showed lower luminosity, and L* values decreased with the passage of time

because of the different physicochemical reactions and microbiological growth that took

place in the milk during storage. One chemical reaction that occurs during processing and

storage is the Maillard reaction because of the presence of lactose and caseins and whey

proteins. When milk is heated, the aldehyde group of lactose is bound with the ε-amino

group of lysyl residues from proteins generating brown pigmented products such as

pyrazines and melanoidins (Gaucher et al. 2008), and continues to develop during

storage. This color can be observed in the heat treated samples in Figure 3, with an

increase of the b* values. Also, when solutions of D-glucose and lactose are treated with

ultrasound, one of the main products is malonaldehyde (Heusinger 1986; O’Brien 1997)

in addition to the known free radicals OH- and H+ from the lysis of water molecules. In

154

neutral solutions, the amount of malonaldehyde is not very high (2-5 x 10-7 g/ml)

compared with alkaline solutions (Heusinger 1986), indicating that in sonicated milk the

amount of malonaldehyde is probably not very high because of the pH of milk (6.6).

However, the effect of pH on the Maillard reaction in milk has been related with the

amount of H+ ions that depress and OH- ions that enhance discoloration (Patton 1995). As

previously mentioned, during the sonication of solutions, the generation of other free

radicals, besides H+ and OH-, is produced (Supeno 2000), which could have a

discoloration effect that would prevent the browning pigments generated. This is because

of the heat treatment applied together with ultrasound, which diminishes the brown color

in milk and retains a lower b* value in the samples.

Whiteness in milk is a very important characteristic for the consumer, which can

often increase sales (Owens et al. 2001). One advantage of thermo-sonicated milk for the

consumer is a whiter color after treatment and during storage. The color parameter a*,

which is related with the redness (+) or greenness (-) of the sample, remained constant in

the thermo-sonicated milk after 16 d of storage, as shown in Figure 2. However, in the

case of thermal treatment and raw milk, the values were more negative, indicating a green

contribution to the milk color. Samples that were sonicated showed a constant value in

the storage time and the value was in the yellowness (+) area; but again the samples with

thermal pasteurization and raw milk showed an increase of the b* value as storage time

passed. Finally, the combination of green and yellow colors indicated the spoilage aspect

of milk because of the different chemical reactions taking place, such as the separation of

whey and the growth of bacteria.

155

Gelation and bitterness occur during the storage of milk because of proteolysis;

this phenomenon is related to the resistance of some heat-stable enzymes and

microorganisms that survive after pasteurization (Valero et al. 2001). Occurrence of

gelation signifies the end of storage life, and this process is the result of the formation of

hydrophobic bonding between casein and lactose (Mehta 1980). Gelation was observed in

the raw milk sample and was initially observed in the heat treated milk. No visible

changes, such as separation of phases in the thermo-sonicated milk, were observable after

16 d of storage at 4ºC; regardless of the intensity of the treatment, the samples displayed

very good appearance.

When milk is homogenized after processing at temperatures lower than normal,

sediment formation is reduced (Mehta 1980). In this experiment at temperature of 63ºC,

which is conventionally used in batch pasteurization, no sedimentation was observed in

thermo-sonicated samples.

3.3.3.1 Net change in color

A very useful parameter to evaluate the differences in color is the net change in color; in

this research, this mathematical function was used to evaluate the difference in the color

of milk samples after sonication of raw milk. The values were also calculated throughout

storage life showing an important difference between raw milk and the other samples. In

Figure 7, the net change in color for all tested samples is shown. Even the raw milk,

compared with its initial value, showed a change in color during storage. However, when

comparing the raw product with the sonicated product, the difference was more

noticeable after treatment. Results were also statistically compared, showing that there is

156

a significant difference (p < 0.05) among the net change in color for the different

treatments.

3.3.3.2 Hue angle (h*)

The hue angle for all samples is shown in Figure 8; according to the Hunter color scale,

h* is a quality indicator; it allows classification of the product as: reddish (0º), yellowish

(90º), greenish (180º) and bluish (270º). Raw and thermally treated milk samples showed

at time zero a yellowish color, with some degree of reddish, but presenting variations

during storage. However, sonicated milk samples kept almost a constant hue angle

between 73º and 75º from the beginning to end of storage time, regardless of intensity of

treatment.

3.3.3.3 Chroma/saturation index (C*)

Regarding the chroma C* of milk samples, this value that indicates the degree of

saturation, purity or intensity of color, which remained almost constant after all

treatments (Figure 9). However, during storage life, C* was slightly increased for the raw

and heated milk samples because of the enzymatic reactions and microbiological growth

that were taking place in the samples, thus intensifying the color. Meanwhile, thermo-

sonicated samples maintained a constant saturation index.

4. Conclusions

Ultrasound technology offers the chance to produce a safe and stable product from the

microbiological point of view for more than 16 d at 4ºC. Despite the apparent slight

157

decrease in protein content, other characteristics are improved, such as a better

availability of butter fat content, and better color, appearance and homogenization. Minor

changes in other physical-chemical characteristics of thermo-sonicated milk allow the

product to be available, not just as a beverage, but also as a potential ingredient for the

dairy industry by improving and developing new products.

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163

Table 1. Proximal Analysis of Raw Milk, Heat Pasteurized Milk and Thermo-Sonicated Pasteurized Milk

Milk sample Protein

content (%) Butter fat

content (%) Added water to milk (%)

Solids Non-Fat (%)

Raw 3.28 ± 0.04 4.04 ± 0.05 0.00 ± 0.00 9.15 ± 0.04 Thermal treatment

3.55 ± 0.01 4.22 ± 0.02 0.00 ± 0.00 9.50 ± 0.03

30% 3.02 ± 0.03 4.21 ± 0.04 4.35 ± 0.92 8.08 ± 0.09 60% 3.04 ± 0.03 4.28 ± 0.05 3.88 ± 0.91 8.13 ± 0.08 90% 3.03 ± 0.04 4.29 ± 0.05 4.28 ± 1.12 8.36 ± 0.43

100% 3.00 ± 0.01 4.24 ± 0.02 4.92 ± 0.39 8.03 ± 0.04 a All analyses were performed on the same day of processing.

b All characteristics were significantly different for each treatment (p < 0.05).

164

Table 2. Physicochemical Characteristics of Raw Milk, Heat Pasteurized Milk and Thermo-Sonicated Pasteurized Milk

Milk sample pH Acidity* Density (g/cm3) Freezing point (ºC)

x 10-2

Raw 6.80 ± 0.04 0.109 ± 0.012 1.0303 ± 0.0014 - 51.60 ± 0.031 Thermal treatment 6.74 ± 0.06 0.126 ± 0.008 1.0317 ± 0.0010 - 61.73 ± 0.015

30% 6.64 ± 0.02 0.142 ± 0.014 1.0260 ± 0.0032 - 53.20 ± 0.053 60% 6.66 ± 0.05 0.141 ± 0.009 1.0261 ± 0.0029 - 53.47 ± 0.049 90% 6.65 ± 0.02 0.136 ± 0.012 1.0259 ± 0.0038 - 53.20 ± 0.061

100% 6.61 ± 0.01 0.146 ± 0.009 1.0257 ± 0.0015 - 52.83 ± 0.021 * Expressed as lactic acid.

a All analyses were performed on the same day of processing.

b All characteristics were significantly different for each treatment (p < 0.05).

165

84.00

85.00

86.00

87.00

88.00

89.00

90.00

91.00

92.00

93.00

94.00

0 2 4 6 8 10 12 14 16 18

Time (days)

L*

RMTT30%60%90%100%

Figure 1. Luminosity (L*) of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-

sonicated Milk at different ultrasound intensities (30%, 60%, 90% And 100%) during

storage.

166

-2.20

-2.00

-1.80

-1.60

-1.40

-1.20

-1.000 2 4 6 8 10 12 14 16 18

Time (days)

a*

RMTT30%60%90%100%

Figure 2. The a* value of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-


storage.

167

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 2 4 6 8 10 12 14 16 18

Time (days)

b*

RMTT30%60%90%100%

Figure3. The b* value of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-


storage.

168

-2-1012345678

0 5 10 15

Time (days)

LOG

(N/N

o)

RM TT 30% 60% 90% 100%

Figure 4. Mesophilic growth behavior of Raw Milk (RM), Heat Pasteurized Milk (TT),

and Thermo-sonicated milk during storage at 4ºC, at different intensities (30%, 60%,

90% and 100%).

169

6.006.106.206.306.406.506.606.706.806.907.00

0 5 10 15 20

Time (days)

pH

RM TT 30% 60% 90% 100%

Figure 5. The pH behavior of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-

sonicated Milk at different amplitude intensities (30%, 60%, 90% and 100%) during

storage.

170

0.000.050.100.150.200.250.300.350.40

0 5 10 15 20

Time (days)

Lact

ic a

cid

cont

ent (

%)

RM TT 30% 60% 90% 100%

Figure 6. Lactic acid content (%) Of Raw Milk (RM), Heat Pasteurized Milk (TT), and

Thermo-sonicated milk at different amplitude intensities (30%, 60%, 90% and 100%)

during storage.

171

0

1

2

3

4

5

6

0 5 10 15 20

Time (days)

Net

cha

nge

of c

olor

(Del

ta E

)RMTT30%60%90%100%

Figure 7. Net Change of Color of Raw Milk (RM), Heat Pasteurized Milk (TT), and


during storage using raw milk color at time zero as control.

172

69

70

71

72

73

74

75

76

77

78

0 5 10 15 20

Time (days)

Hue

ang

le (h

*) RMTT30%60%90%100%

Figure 8. Hue Angle of Raw Milk (RM), Heat Pasteurized Milk (TT), and Thermo-


storage.

173

0

1

2

3

4

5

6

7

8

9

0 5 10 15 20

Time (days)

Chr

oma/

satu

ratio

n in

dex

(C*)

RMTT30%60%90%100%

Figure 9. Chroma/Saturation index of Raw Milk (RM), Heat Pasteurized Milk (TT), and


during storage.

174

CHAPTER FOUR

STUDY OF THE MECHANISM OF INACTIVATION OF Listeria innocua CELLS

IN WHOLE MILK UNDER THERMO-SONICATION TREATMENTS USING

SCANNING ELECTRON MICROSCOPY AND TRANSMISSION ELECTRON

MICROSCOPY

Daniela Bermúdez-Aguirre, Raymond Mawson and


Abstract

Listeria innocua ATCC 51742 cells were studied under thermal (63ºC) and thermo-

sonciation treatments (63ºC, 400 W, 24 kHz, 120 μm) in raw whole milk. Heat-treated

cells were collected after 30 min, and thermo-sonicated cells were analyzed at 10 and 30

min of treatment. Scanning Electron Microscopy (SEM) and Transmission Electron

Microscopy (TEM) were used to evaluate the physical effects of the cavitation produced

in the cells and to establish a possible mechanism of inactivation. Two different sample

preparation techniques, hexamethyldisilazane (HMDS) dehydration, and freeze-drying

(FD), were evaluated for SEM in an attempt to preserve the cell structure as well as

possible in order to evaluate the effects of ultrasonic treatment. Heat-treated cells showed

a thinning in the cell wall after thermal treatment. Thermo-sonicated cells showed the

common characteristic of the formation of pores outside the cell wall, regardless of the

time of treatment. After 10 min of treatment some cells showed a breakage line,

suggesting a possible breakdown of the membrane with longer treatment times

175

confirming that after 30 min of exposure to ultrasonic treatment, several cells were

broken into pieces. Differences between HMDS and FD techniques in images were not

noticeable. In both cases the microstructure of cells was preserved, and FD was a faster

technique. TEM images matched with SEM images, showing valuable information such

as lack or clumping of cytoplasm content, as well as perforation, disruption, and

roughness of the cell membrane after thermo-sonication.

176

1. Introduction

Studies about the use of sound waves to kill bacteria have been reported since 1929.

Harvey and Loomis (1929) exposed cells to high power, high frequency sound waves,

combined with elevated temperatures in some experiments. Heat was responsible for

injuring cells, but results showed that luminous bacteria were killed after 30 min of

thermal treatment plus sound waves. In the same experiment needle crystals of

benzopurpurin were radiated with sound waves; when observed under a microscope, the

crystals had been broken into shorter lengths. These were the first studies to show how

bacteria are disrupted by sound waves. But because of the expensive material used at that

time, no commercial use was developed.

The mechanism of interaction between cells and ultrasound was under research in

the 1960s when free radical formation was found; later, in 1975, the release of the

cytoplasmic membrane from the cell wall generated by ultrasound was observed as a

thinning of the cell wall (Earnshaw et al., 1995). Low frequency ultrasound or high

power ultrasound generates physical, chemical and mechanical effects in the process or

product. Sonochemistry is used to study some of these effects such as increasing the rate

of chemical reactions, inactivation of microorganisms and enzymes, breakage of cells,

improving heat transfer and extraction processes, among others (Carcel et al., 1998).

When sound waves pass through a liquid, changes in pressure and temperature take place,

and thousands of bubbles are produced, causing a physical phenomenon called cavitation

(Hughes and Nyborg, 1962). Cavitation is generated by alternate rarefaction and

compression cycles in the medium, producing bubbles or cavities (Earnshaw et al., 1995),

and this is assumed to cause a lethal effect of ultrasound in cells (Mañas and Pagán,

177

2005). Implosion occurs when the amplitude is great enough to generate instability and

collapse of bubbles. The bubbles are in extreme movement and collapse violently,

generating an increase of temperature on the order of 104 K and pressures of 101.325 x

106 kPa (106 atm). Some of the effects of cavitation phenomenon are heating, electrical

discharges, sonoluminescence, chemoluminescence, formation of free radicals (Hughes

and Nyborg, 1962), acceleration of chemical reactions, improvement of extraction

processes in animal and vegetable tissues, yeasts and bacteria, and breakage of

microorganisms and viruses (Carcel et al., 1998). The type of effect of ultrasound on cells

is related to the type of cavitation. This physical phenomenon has been classified as

either stable or transient. Stable cavitation is related to the production of small bubbles

dissolved in a liquid that are produced during thousands of cycles. These bubbles have

strong forces and create microcurrents inside the medium that are often called

microstreaming; this phenomenon rubs the membrane surface, causing shear at the cell

membrane and breaking it down. Implosion is not generated in this cavitation. In contrast

with stable cavitation, in transient cavitation the size of bubbles changes quickly in just a

few oscillatory cycles, generating an important increase in temperature and pressure

because of the violent collapse of bubbles; this physical phenomenon attacks the cell

membrane, and it is enough strong to disrupt the cell structures in the cell wall, or even to

remove particles from the surface (Earnshaw et al., 1995). Much of the physical

phenomena associated with cavitation start to be produced when pressures are in the

range of 1013.25 – 101 325 Pa (Hughes and Nyborg, 1962), and much of the lethal effect

in cells is attributed to these high pressures (Earnshaw et al., 1995). Because of the

formation of free radicals in the medium caused by ultrasound, the products of the

178

sonolysis of water, such as OH- and H+ and hydrogen peroxide, could contribute to a

bactericidal effect (Earnshaw et al., 1995). However compared with the mechanical

forces produced in the medium, the free radical effect is almost insignificant (Mañas and

Pagán, 2005).

In the past, one technique for estimating cell breakage generated by physical

alteration methods, such as the use of vibrating needles, was the use of a protein

determination method. In the specific case of erythrocytes, after a treatment involving a

micro-storming phenomenon, measurement of the released hemoglobin in the medium

that was related directly to the breakage of cells was useful. Some micrographs showed

some empty cells of Escherichia coli after treatment, but the damage induced with the

previously cited treatment was insignificant compared to the damage created with

ultrasound (Hughes and Nyborg, 1962). Recently, the use of flow cytometry has been

used to study the effect of ultrasonic treatments on cellular injuries (Ananta et al., 2005).

Thermal treatment has been used in the food industry to inactivate bacteria for

more than 200 years. Some target sites in the cell that are considered as heat resistant

points because of their intrinsic stability are RNA, ribosomes, nucleic acids, enzymes and

proteins (Earnshaw et al., 1995). Damage in membranes, along with loss of nutrients and

ions also contribute to cell death (Mañas and Pagán, 2005), although the main reason for

cell death is still not clear (Earnshaw et al., 1995). Mechanisms of cell inactivation by

ultrasound are not totally clear, but may be attributed to the cavitation of the medium,

heating, shear forces, turbulence or structural effects, among others (Carcel et al., 1998;

Villamiel and de Jong, 2000). As a new technology, most research must be conducted in

an attempt to understand the physiological basis of ultrasound in the process (Earnshaw

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et al., 1995) and the effects of environmental factors, stress adaptation mechanisms or

sublethal injuries in cells (Mañas and Pagán, 2005).

Listeria monocytogenes is a gram-positive bacteria, non-spore forming, with a rod

shape. It can survive under a broad range of temperature (1ºC to 45ºC) and pH (4.1 to

9.6) conditions some products that are affected are raw milk, pork, raw poultry, ground

beef and some vegetables (Jay, 1992). The size of Listeria cells ranges from 0.8 to 2.5

μm in length and 0.5 μm diameter (Pelczar and Reid, 1972; Rocourt, 1999). Listeria

monocytogenes is considered an emerging pathogen because of the high incidence in

outbreaks reported recently in the United States. Listeria monocytogenes is currently

highlighted as the main foodborne pathogenic microorganism that is generating problems

in the food industry (Banasiak, 2005). Reports show that these foodborne diseases

involve millions of people, with a significant number of deaths every year; in addition,

billions of dollars are spent on treatments (Kozak et al., 1996; Ko and Grant 2003;

Mohan Nair et al., 2004 and 2005). Many of the current outbreaks involve the presence of

Listeria cells in dairy products. According to some reports, species of this microorganism

can be present in 3-4% of raw milk samples, and post-pasteurization contamination could

occur easily in dairy industry facilities because of the environment. Other examples of

dairy products that have already generated listeriosis are pasteurized milk, Mexican-style

cheese, chocolate milk, frozen desserts, ice cream (Kozak et al., 1996), sour milk, cream,

and soft-cheeses such as cottage cheese (Ryser, 1999), showing its ability to survive

under many conditions.

The objective of this work was to study the possible mechanism of inactivation of

Listeria innocua cells under thermo-sonication treatments, using raw milk as a medium

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of treatment, and to evaluate the physical effects in cells generated by cavitation with

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM).


2.1. Listeria innocua cells

Two ml of thawed Listeria innocua cells (ATCC 51742) (one ml of microorganism

grown in the early stationary phase plus one ml of sterile glycerol (20 ml glycerol/100 ml

water) stored at -21ºC were added to 100 ml Tryptic Soy Broth (TSB) with 0.6% Yeast

Extract (TSBYE). The microorganism was kept in a bath shaker at 37ºC and 218 rpm

until it reached the early stationary phase, approximately after 11 h.

2.2. Milk samples

Raw and whole cow’s milk was obtained from Washington State University’s Creamery.

Milk was kept in refrigeration at 4ºC until it was used. The inoculation of Listeria

innocua cells was made directly in a ratio of 1:100 (inoculum: milk; V/V). Fifteen ml of

inoculated raw milk was transferred to a disposable 15 ml sterile plastic conical test tube

to be used as a control sample through microscopy sample preparation.

2.3. Thermal and thermo-sonication treatments

2.3.1. Ultrasound equipment

500 ml of raw milk was used in a sterile double-walled vessel (500 ml) as a treatment

chamber with an internal diameter of 8 cm and depth of 13.5 cm. An ultrasonic processor

Hielscher USA, Inc. (Ringwood, NJ) model UP400S (400 W, 24 kHz, 120 μm) with a 22

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mm diameter probe was used. A magnetic stirrer was used inside the treatment chamber

to assure the homogeneity of the samples. Thermo-sonication was carried out at 100%

(120 μm) of amplitude of the ultrasound wave and a constant temperature of 63 ± 0.5ºC.

Temperature was set up and kept constant via a refrigerated bath (VWR Scientific Model

1166, Niles, IL). A thermocouple was used in the treatment chamber to monitor the

temperature (63 ±0.5ºC) throughout the experiments. Samples were taken after 10 and 30

min of thermo-sonication and transferred to disposable 15 ml sterile plastic conical test

tubes.

2.3.2. Thermal treatment

Thermal treatment was carried out in the same double-walled vessel of 500 ml. The same

volume of milk was used, and temperature of the medium was kept at 63 ± 0.5ºC with the

use of a heating bath (VWR Scientific Model 1166, Niles, IL) maintained at 65ºC. A

magnetic stirrer was used to ensure that all cells received the same thermal treatment.

Temperature was monitored by a thermocouple. After 30 min of treatment samples were

transferred to disposable 15 ml sterile plastic conical test tubes.

2.4. Sample preparation for electron microscopy

Two different sample preparation techniques were used to evaluate for the less invasive

method throughout the process until the final observation of samples at SEM. The first

technique corresponds to conventional sample preparation for biological specimens that

uses several organic solvents, with the HMDS dehydration as the final step. The second

technique was freeze drying (FD), in an attempt to evaulate the feasibility of this

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technique for preserving structures as well as the shorter time required, but also as an

alternative sample preparation for comparison with the conventional method.

2.4.1. Method with organic solvents

Disposable 15 ml sterile plastic conical test tubes containing samples of raw milk,

thermo-sonicated and thermally treated milk were centrifuged at 1500 rpm for 5 min at

10ºC. The supernatant was discarded and the cells were resuspended in 15 ml of sterile

TSBYE (Tryptic Soy Broth Yeast Extract). After that, centrifugation was performed

again under the same conditions described above. The supernatant was discarded and the

cells were transferred with a sterile Pasteur pipette to disposable 1.5 ml sterile plastic

microcentrifuge tubes. 0.5 ml of a solution of glutaraldehyde (2%) paraformaldehyde

(2%) in 0.1 M phosphate buffer (pH 7.2) was added to each microtube and allowed to

undergo the fixation process for 24 h at 4ºC. Next, the fixation solution was washed with

phosphate buffer (0.1M) for 10 min, followed by two consecutive 10 min washes with

cacodylate buffer (0.1M). Post-fixation procedure consisted of adding 2% osmium

tetroxide in cacodylate buffer (0.1M) at 4ºC for 24 h. Cells were washed three times with

cacodylate buffer (0.1M) for 10 min each time.

Dehydration of cells was achieved with serial dilution solutions of ethanol (30%,

50%, 60%, 70%, 95% and 100%). Each solution was maintained in contact with cells for

10 min and the last solution (100% ethanol) was used three consecutive times. Samples

after the last dehydration solution and the last time were then prepared for each type of

microscope (SEM or TEM).

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2.4.2. Freeze-Drying (FD) method

An alternative method using freeze-drying was employed to compare the results of the

traditional lengthy sample preparation for scanning electron microscopy, and also to see

the effect of freeze-drying as a dehydration technique. The procedure was the same until

after the centrifugation steps. Cells in the microcentrifuge tubes were frozen at -37ºC

overnight and then freeze dried in a freeze dryer (Freeze Mobile 24 Virtis, Gardiner, NY)

for 24 h.

2.5. Scanning electron microscopy (SEM)

After dehydration of samples with ethanol, the second dehydration procedure with

hexamethyldisilazane (HMDS) was carried out with the cells. Consecutive 15 min

periods of contact of cells with ethanol/acetone/HMDS solutions in different ratios (1:0:0,

1:1:0, 0:1:0, 0:1:1, 0:0:1, 0:0:1) were used. Air drying was used as a final step, leaving

the microcentrifuge tubes with open lids inside a hood for at least one night.

Samples were mounted on aluminum stubs, and gold plating (Sputter coater, Technics

Hummer V, San José, CA) was used as a final step for freeze-dried and organic

dehydrated samples prior to viewing the samples in a Hitachi S-570 (Japan, Tokyo)

Scanning Electron Microscope (SEM) operating at 20 kV.

2.6. Transmission Electron Microscopy (TEM)

Dehydrated cells were infused in 1:1 Acetone:SPURRS resin and kept overnight. The

concentration of resin was increased every 24 h for 2 d. Next, the polymerization of resin

was accomplished in an oven (Fisher Scientific, Tustin CA) at 70ºC for 24 h in order to

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form specimen blocks. Sectioning procedure started with hand trimming of the blocks

with a razor blade, after which the samples were sectioned (90 nm) with a glass knife in a

Reichert Ultracut R (Leica, West Germany). Sectioned samples were placed on 200 mesh

copper grids (previously prepared for TEM) and stained according Sato’s Lead Stain

Procedure. 4% Uranyl Acetate and Sato’s Lead Stain were used as the main chemical

reactives in the procedure. Samples were viewed in a JEOL 1200 EX II (Japan, Tokyo)

Transmission Electron Microscope (TEM) operating at 100 kV.


3.1. SEM with HMDS dehydration

Scanning Electron Microscopy was used to observe the changes in the surface of cells

subjected to thermo-sonication compared to thermally treated cells. The conventional

HMDS dehydration of cells was followed as the preparation sample technique because of

the feasibility of this technique for preserving biological specimens for SEM.

The control sample can be observed in Figure 1. The image corresponds to

Listeria innocua cells in raw milk without any treatment. As can be seen in the picture,

the cell surface shows total integrity and a smooth surface, and no changes in the cell

membrane or exterior can be observed. Listeria cells show the traditional rod shape and

the typical dimensions for these cells, 0.8 to 2.5 μm in length and 0.5 μm in diameter

(Pelczar and Reid, 1972; Rocourt, 1999).

As a complement to this study, pictures of the natural flora in raw milk are shown

in Figures 2 and 3. These bacteria were also under thermo-sonication because of their

presence in the milk before starting the treatments. Some of the common microorganisms

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found in raw milk are Enterococcus, Lactococcus, Streptoccocus, Leuconostoc,

Lactobacillus, Microbacterium, Propionibacterium, Micrococcus, coliforms, Proteus,

Pseudomonas, Bacillus (Jay, 1992). In Figure 2, chains of Streptoccocus can be observed.

Meanwhile, in Figure 3, Micrococcus and Bacillus are observed as a mix of several

microorganisms in raw milk. These pictures are presented in this work because through

the inactivation of bacteria with ultrasound, some of these cells show characteristics of

cell injury by cavitation in addition to the Listeria cells.

3.2. Thermal treatment

Although the main objective of this research was to clarify the mechanism of inactivation

of Listeria cells after sonication, some images of cells subjected to thermal process

conditions were used to compare with those that were sonicated and to establish whether

the lethal effects can be attributed to sound waves or only to the thermal process. As

many references mention, the effects of cell damage with temperature are not clear, but it

is known that elevated temperatures has a weakening effect on cells until they die. In

Figure 4, thermally treated cells are shown. The image corresponds to batch pasteurized

cells after 30 min of treatment. As can be seen, cell integrity is maintained in all cases;

the only observable difference in these images compared to the control sample (Figure 1)

is a thinning in the cell wall, making the cells look “transparent.” It is not clear which

event taking place under thermal treatment contributes most to lethality. Some studies in

thermally treated cells with phase contrast microscopy and environmental scanning

electron microscopy have shown cellular integrity (Mañas and Pagán, 2005) and loss of

the granular surface of some Escherichia coli cells (Ugarte-Romero et al., 2006),

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respectively. According to microbiological counts, at 63ºC 5 – log reduction of cells is

achieved after 30 min of thermal treatment (data not shown), so probably the cells shown

in Figure 4 correspond to dead cells despite the apparent integrity. Some heat effects in

cells are related to RNA modification, depletion of Mg+ (which usually works as a

stabilizer in cells), protein coagulation, reduced ability to produce osmolytes (amino

acids and related compounds that increase the thermal stability of proteins), and damage

of the cytoplasmic and plasma membranes (Earnshaw et al., 1995). Probably, the

apparent “transparent” effect is caused by the protein coagulation that takes place in the

proteins of cell membrane.

3.3. Thermo-sonication treatment

Thermo-sonicated cells showed common characteristics after 10 and 30 min of treatment

when the images were analyzed with SEM. After 10 min of treatment, the presence of

small pores along the outside of the cells could be observed at the surface; this physical

effect was observed not only in Listeria innocua cells, but also in some cocci present in

the raw milk, as can be seen in Figures 5 and 6. The formation of pores could have

occurred through transient cavitation resulting from the increase of temperature and

pressure in the medium generated by the violent collapse of unstable bubbles, imploding

close to the cells. As Earnshaw et al. (1995) mentioned, these physical phenomena attack

the cell by removing particles from its surface. In addition to these pores, lines were

observed in the surface of some cells, as shown in Figure 6, suggesting a possible rupture

of the cell if the heat and sound conditions in the medium continued to bombard them. As

the sound waves transverse the medium of treatment, the temperature increases because

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of the absorption of acoustic energy (Carcel et al., 1998). Pore formation and the

presence of breakage lines could be responsible for the release of cytoplasmatic material

from the cell to the medium, even allowing the entrance of some material present in the

medium to the inside of the cell, thus generating a lethal effect. According to the images

shown in this work of heat-treated and thermo-sonicated cells, an assumption could be

made relating weakening of the cells with heat treatment, facilitating cell disruption, with

the sound waves generating the bacteria death.

Thermo-sonication has been more effective in bacteria inactivation than

sonication at ambient temperature because high temperature speeds up the bubble

formation in the medium due to the increase of vapor pressure and the decrease of tensile

strength. If treatments are conducted at atmospheric temperature, the lethal effect from

the heat generated in the medium is not very significant, since, according to Earnshaw et

al. (1995), this increase of temperature occurs for a short time and only in the part of the

liquid close to the sound waves, and not all of the cells are affected.

In Figures 7 and 8, thermo-sonicated cells after 30 min of treatment are observed.

The common pore formation after 10 min of treatment can be seen in the images;

however, while the breakage line is no longer observed, broken cells in two or more

pieces are present, confirming the suggestion that cells would be broken with longer

treatment times. In Figure 7, a broken cell can be clearly seen in the middle of the image,

and this breakage of cells probably takes place because of stable cavitation, in which the

micro-currents described by Earnshaw et al. (1995) rub against the membrane surface,

generating breakdown of the cell membrane. Other cells shown in Figure 7 have pores,

confirming that through sonication, transient and stable cavitation take place at the same

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time. Rubbing the surface, bubbles bombard the structures, and the removal of particles

from the surface take place at the same time as ultrasound effects. In a study conducted

with Escherichia coli cells, after thermo-sonication (60ºC, 20 kHz, 0.46 W/ml) for ≤ 3

min, Environmental Scanning Electron Microscopy (ESEM) images showed the presence

of holes in the surface, shrinkage, cell deformation, cell collapse and wrinkled areas

(Ugarte-Romero et al., 2006). The advantage of ESEM is that sample preparation is

minimal, but one disadvantage of ESEM is the possibility of shrinkage of some biological

tissues. According to Carcel et al. (1998), ultrasonic cavitation has a lethal effect in

tissues, not only because it makes heat transfer easier, but also because of the

compression and decompression cycles that can erode the cells. If ultrasound improves

heat transfer and at the same time increases the temperature, the lethal effect is enhanced.

3.4. SEM freeze drying

Freeze drying was used as a method of sample preparation to observe the cells in SEM in

an attempt to take advantage of this method to preserve the cell structure. In addition, in

this work freeze drying was compared to the conventional sample preparation to see if

there was a difference between the images. Figure 9 shows heat-treated cells prepared by

freeze drying; the picture shows the same “transparent” effect and integrity in cells after

30 min of thermal treatment. After evaluating numerous images prepared with freeze

drying, non-shrinkage of cells was observed in every cell, making FD a viable technique

for sample preparation, and reducing the time from four days (HMDS drying) to one day.

In Figures 10 and 11, thermo-sonicated cells after 30 min of treatment are shown.

In Figure 10, the effect of the sound waves is clearly observed in the disruption of the cell

189

membrane; the continuity of the cell wall is broken and the free interchange of

cytoplasmatic content and medium material is generated. The loss of some organelles and

lack of cytoplasmatic material from the outside of the cell most likely occurred because

of the separation of the membrane from the cell. In this cell, the breakage seems to have

occurred with longer times than as shown in other pictures, because of the apparent

fragility of the attachment of the disrupted membrane to the rest of the cell. In Figure 11

some broken cells can be observed after 30 min of treatment in which the release of the

cytoplasmatic content occurred and the total structure of the cells was modified. As

Hughes and Nyborg (1962) affirm, one theory about the effect of ultrasound on cells is

the fragmentation of microorganisms. Micro-storming or whirlpools inside the medium

of treatment are associated with the speeding up of some reactions, modifying the

exterior side of cells or breaking them. As shown in both Figures 10 and 11, transient and

stable cavitation again were responsible for the lethal effects. The frequency of the

ultrasound is a parameter that defines the bubble size; at low frequency, bigger bubble

sizes and higher collapse transfer more energy (Earnshaw et al., 1995). This is probably

another reason for different inactivation rates with ultrasound technology in microbial

inactivation, in addition to the kind of bacteria, medium composition, and other variables

such as pressure and use of antimicrobials. In this research, the frequency used was 24

kHz; in some related studies, the frequency used was 20 kHz, but under different

conditions of temperature, medium of treatment and intensity of the cavitation (Guerrero

et al., 2005; Ugarte-Romero et al., 2006).

Although in this study the main focus was the inactivation of Listeria cells, and

many of the shown images correspond to this rod shaped microorganism, much research

190

must be done on the study of cell inactivation with ultrasound. In other novel

technologies such as Pulsed Electric Fields, the size, shape and kind of bacteria is a target

factor for their inactivation. According to Earnshaw et al. (1995), the size of the bacteria

is an important parameter in ultrasound inactivation because the larger surface exposed to

cavitation, leads to more attacked sites. Cocci are more resilient than rod shaped

microorganisms to ultrasound effects, and their smaller size gives them higher resistance

to ultrasound, high pressure and pulsed electric fields (Mañas and Pagán, 2005).

Comparing the images observed under SEM with two different sample

preparations such as HMDS dehydration and freeze drying, in this study both techniques

worked very well to reach the objective of seeing what happens in cells during thermo-

sonication and trying to establish the mechanism of inactivation. Since HMDS

dehydration has been used for a long time as the standard preparation sample, comparing

these images with those obtained using freeze drying showed that both techniques

delivered similar images of inactivation effects, although the time of sample preparation

was reduced considerably with the latter.

3.5. TEM

Some of the images obtained by Transmission Electron Microscopy matched those

observed with Scanning Electron Microscopy. Figure 12 shows thermo-sonicated cells

after 10 and 30 min of treatment, demonstrating similar inactivation effects as those

shown previously in the SEM images. The control cell is shown in Figure 12a; it is

important to note that TEM uses a sectioning procedure in which different layers of the

cell are observed, in contrast with SEM, which analyzes the whole cell surface. In Figure

191

12b, the membrane of the cell shows roughening and thinning compared to the control

cell (Figure 12a), as well as a partial loosening of the cytoplasmic membrane from the

cell wall, which is in agreement with the event described in ultrasonicated cells in 1975

by Earnshaw et al. (1995). Other lethal effects observed by TEM in cells are shown in

Figures 12c, 12d and 12e, including the formation of small holes, the disruption and

breakage of cell membranes, the lack of cytoplasm and the development of cytoplasm

clumps. The formation of pores and breakage of membranes were observed previously

with SEM images, but the advantage of TEM in the analysis of cells was that some

effects in the cytoplasm content can be observed, such as the lack of cytoplasm and the

clumping of cytoplasm due to both types of cavitation (stable cavitation that breaks down

the cell membranes, and transient cavitation that removes particles that could be

cytoplasmatic content, such as some organelles). Cell breakage is related to the shearing

action generated by the whirlpools generated from bubbles (Hughes and Nyborg, 1962).

As Transmission Electron Microscopy can deliver very valuable information about the

cell structure because of the sectioning technique used to prepare the samples, some cells

showed unseen effects, as shown in Figure 12f. This cell was thermo-sonicated for 10

min, but the damage was not visible in the analyzed section of the whole cell. It is likely

that this cell was injured on the very upper surface and so the TEM image did not show

the effects, although the physical effects of cavitation are evident in much of the image.

In this case, SEM would probably be most useful for observing the injured surface. In

Figures 12g and 12h, other effects are shown, such as the breakage of the cell membrane

in the upper left corner in Figure 12g and the presence of cytoplasm clumps in Figure

12h. In some studies conducted with yeasts (Saccharomyces cerevisiae) under thermo-

192

sonication (45ºC, 95.2 μm, 20 kHz), after 20 min TEM showed images of puncturing in

the cell wall, the disruption of organelles and a discontinuity of the plasmalemma. The

use of chitosan enhanced the thermo-sonication treatments, improving the inactivation

rate and the lethal effects observed in cells (Guerrero et al., 2005). Because of cavitation,

free radicals are formed in the medium. These free radicals can act in biological materials

such as DNA (Villamiel et al., 1999), breaking the hydrogen bonds between bases or

even breaking DNA molecules by liquid shear, thus reducing the molecular size (Hughes

and Nyborg, 1962). Future studies in cavitation could be done with hybridization in situ

that will deliver important information.

3.6. Possible mechanism of inactivation of cells under thermo-sonication treatments

On the basis of the previous images and related references (Guerrero et al., 2005; Ugarte-

Romero et al., 2006) that describe the possible effects of cavitation of cells, a summary of

some of the main physical effects of thermo-sonication as lethal agents in Listeria cells

could include the weakening of the cell membrane due to heating action, followed by the

formation of pores along the outside of the membrane, the disruption of the cell

membrane, the lack or clumping of cytoplasmic material generated by transient

cavitation, and finally the breakdown of the cell membrane due to erosion caused by

stable cavitation. This could be described as the mechanism of inactivation for Listeria

innocua cells during and after thermo-sonication at 63ºC and 120 μm; although the same

conclusion could be generalized for most microorganisms, it is important to note that

every microorganism has a different response to the same inactivation method. For

example, the type of bacteria is another factor of interest in ultrasound inactivation; in

193

accordance with some studies, gram-positive microorganisms are more resistant to

ultrasound than gram-negative microorganisms, probably because the cell wall between

them is different. Gram-positive cell walls are thicker than gram-negative cell walls,

which contain a tight layer of peptidoglycans (Earnshaw et al., 1995), making a rigid

envelope of the cell (Mañas and Pagán, 2005). Listeria species are gram-positive bacteria

and, because of that, they are more resistant and their cell wall is stronger against

sonication. Thus, the inactivation of gram-negative bacteria may occur faster, and the

cells might present more visible effects in the first minutes of treatment. Thermo-

sonication seems to be a viable method to inactivate cells, although extremely high

temperatures should not be used, since cavitation looses intensity as temperature

increases; when the temperature is close to the boiling point, the so-called micro-

storming inside the medium decreases, because the vapor pressure acts as a cushion

(Villamiel et al., 1999).

4. Conclusions

Thermo-sonication is a viable technology to inactivate bacteria; the heating effect in cells

seems to weaken the microorganisms, making it easier for the cavitation to disrupt and

break down the cells. The main effects of cavitation in cells are the formation of small

pores, the disruption of cell membranes, and lack or clumping of cytoplasmic material

with the final breakdown of cell membranes inactivating the microorganism. Stable and

transient cavitation acting together on cells causes the erosion of the cell surface and the

disruption of cell structures.

194

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Figure 1. Listeria innocua without any treatment inoculated in raw whole milk and using

HMDS dehydration for SEM. Control sample, 20 kV, magnification 5.90 K, 5.1 μm.

198

Figure 2. Streptococcus chains present as natural flora in raw whole milk and using

HMDS dehydration for SEM. 20 kV, magnification 5.90 K, 5.1 μm.

199

Figure 3. Microccocus and Bacillus present as natural flora in raw whole milk and using

HMDS dehydration for SEM. 20 kV, magnification 5.90 K, 5.1 μm.

200

Figure 4. Listeria innocua cells after thermal treatment (63ºC by 30 min) in raw whole

milk and using HMDS dehydration for SEM. Cell wall shows thinning compared with the

control sample. 20 kV, magnification 5.90 K, 5.1 μm.

201

Figure 5. Listeria innocua cells after 10 min of thermo-sonication (63ºC and 120 μm) in

raw whole milk and using HMDS dehydration for SEM. Cells show puncturing outside

the cell wall. 20 kV, magnification 6.00 K, 5.0 μm.

202


raw whole milk and using HMDS dehydration for SEM. Cells show some breakage lines

in the middle, suggesting a possible rupture. 20 kV, magnification 6.00 K, 5.0 μm.

203


raw whole milk and using HMDS dehydration for SEM. Some cells show puncturing

outside the cell wall in addition to a broken cell in the middle of the image. 20 kV,

magnification 6.00 K, 5.0 μm.

204


raw whole milk and using HMDS dehydration for SEM. Some cells show pitting outside

the cell wall in addition to a broken cell in the middle of the image. 20 kV, magnification

6.00 K, 5.0 μm.

205

Figure 9. Listeria innocua cells after 30 min of thermal treatment (63ºC) in raw whole

milk and using freeze-drying as sample preparation for SEM. Cells show thinning in the

cell wall. 20 kV, magnification 6.00 K, 5.0 μm.

206


raw whole milk and using freeze-drying as sample preparation for SEM. Cell clearly

shows the disruption and breakage of its membrane. 20 kV, magnification 6.00 K, 5.0

μm.

207


raw whole milk and using freeze-drying as sample preparation for SEM. Broken cells are

shown in the right corner of the image. 20 kV, magnification 6.00 K, 5.0 μm.

208

Figure 12. Transmission Electron Microscopy for Listeria innocua cells: (a) control

sample; (b) thermo-sonicated (63ºC and 120 μm) after 10 min, showing roughness in the

209

cell wall; (c) thermo-sonicated (63ºC and 120 μm) after 30 min, showing pore formation;

(d) thermo-sonicated (63ºC and 120 μm) after 30 min, showing pore formation and

disruption of the cell wall; (e) thermo-sonicated (63ºC and 120 μm) after 30 min,

showing lack of cytoplasm; (f) thermo-sonicated (63ºC and 120 μm) after 30 min,

showing unseen effects; (g) thermo-sonicated (63ºC and 120 μm) after 30 min with the

cell membrane broken; (h) thermo-sonicated (63ºC and 120 μm) after 30 min, showing

cytoplasm clumping. Scale 1000 to 2000 nm.

210

CHAPTER FIVE

MICROSTRUCTURE OF FAT GLOBULES IN WHOLE MILK AFTER

THERMO-SONICATION TREATMENT

Daniela Bermúdez-Aguirre, Raymond Mawson and


ABSTRACT

The structure of fat globules in whole milk was studied after heat and thermo-

sonication treatments to observe what happens during these processes at the microscopic

level (using Scanning Electron Microscopy). Raw whole milk was thermo-sonicated in an

ultrasonic processor—Hielscher® UP400S (400 W, 24 kHz, 120 μm amplitude), using a

22 mm probe at 63ºC for 30 min. Heat treatment involved heating the milk at 63ºC for 30

min. Color and fat content were measured to correlate the images with analytical

measurements. Results showed that the surface of the fat globule was completely

roughened after thermo-sonication. Ultrasound waves were responsible for disintegrating

the milk fat globule membrane (MFGM), by releasing the triacylglycerols. Furthermore,

the overall structure of milk after sonication showed smaller fat globules (smaller than 1

μm) and a granular surface. This was due to the interaction between the disrupted MFGM

and some casein micelles. Minor changes in the aspect of the globules between thermal

and raw milks were detected. Color measurements showed higher L values for sonicated

samples. Sonicated milk was whiter (92.37 ± 0.20) and generally showed a better degree

of luminosity and homogenization compared to thermal treated milk (88.25 ± 0.67) and

211

raw milk (87.82 ± 0.18). Fat content was higher after sonication (4.24%) than in

untreated raw milk (4.04%) because of the release of triacylglycerols from the core of the

globules. The advantages of thermo-sonicated milk is that it can be pasteurized and

homogenized in just one step, it can be produced with important cost savings, and it has

better characteristics, making thermo-sonication a potential processing method for milk

and most other dairy products.

212

1. Introduction

Milk fat or the milk lipid globule membrane (MFGM) is related to the membrane

and the material associated with it that surrounds the milk fat globules. The core of these

micro-droplets contains triacylglycerols. The surface coating material contains

cholesterol and phospholipids such as sphingomyelin and phosphoglycerides of choline,

ethanolamine, inositol and serine (Keenan and Patton, 1995), along with lipoproteins,

glycoproteins and enzymes (Michalski and others, 2002a) such as xanthine oxidase

(Wiking and others, 2003); butyrophillin, γ-glutamyl transpeptidase, among others

(Wiking and others, 2006). The MFGM and lipid droplets represent 80% of the

cholesterol supplied by milk (Mather and Keenan, 1998). The triacylglycerol content of

the fat globule’s core is mainly composed of lauric, myristic, oleic, stearic and linoleic

acids (Michalski and others, 2005). In cow’s milk, the size of a fat globule is between 0.2

to 10 μm in diameter. Almost 90% of the fat content in milk corresponds to fat globules

that are between 1 and 8 μm (Keenan and Patton, 1995), with an average diameter of 4

μm (Michalski and others, 2005; Briard and others, 2003). However small and large fat

globules differ a little in composition (Michalski and others, 2004); smaller globules (1.5

μm) have fewer short chain fatty acids, less stearic acid, and more oleic acid (Briard and

others, 2003). In fat globules smaller than 0.8 μm, the creaming effect of milk during

storage is very slow (Villamiel and de Jong, 2000). Experimental results show that during

homogenization of milk the mechanical alteration of fat globules is generated by pressure

differences due to shear, turbulence and cavitation; this last phenomenon is sometimes

present in the valves (Walstra, 1969; Thiebaud and others, 2003). Some operations in

milk processing include pumping, agitation, pooling, cooling, clarification, pasteurization

213

and homogenization (Jensen and others, 1995). Stabilization of fat, creamy taste, and

resistance of milk to oxidized flavor are some of the advantages of the homogenization

process, which consists of pumping the milk at high pressures through small eddies at

pasteurization temperatures (Keenan and Patton, 1995). This operation requires a high

volume of milk for processing (Carcel and others, 1998) and uses pressures from 20 to 50

MPa to reduce the size of fat globules from 0.3 to 0.8 μm (Thiebaud and others, 2003),

thus changing the structure of the globules (Corredig and Dalgleish, 1996) and increasing

the surface area of fat more than 10-fold (Sharma and Dalgleish, 1993). There are three

constituent parts in homogenized milk, regular milk fat globules, tiny and native fat

globules (100 nm), and small and new lipid-protein complexes (<500 nm) (Michalski and

Januel, 2006; Michalski and others, 2002b). The size of fat globules and the composition

of the membrane are of tremendous importance in the technical and sensory properties of

dairy products (Fauquant and others, 2005; Michalski and others, 2004; Michalski and

others, 2002a). Novel nonthermal technologies, such as high pressure, pulsed electric

fields or ultrasound, are of great interest in the research and development of food

processing, not only because of the advantages they present compared with thermal

processing, but also because of the potential generation of new ingredients and products

with specific features due to action in the food matrix (Knorr and others, 2002).

Ultrasound is responsible for the breakage of fat globules in milk, which releases

liposoluble components such as phenilureas; sometimes sonication is used with the goal

of extraction in the dairy industry (Carcel and others, 1998). The main mechanism of

action in ultrasound technology is called cavitation, a phenomenon that can be either

stable or transient. Stable cavitation is associated with the small bubbles dissolved in a

214

liquid, while transient cavitation occurs when the bubble size changes quickly and

collapses, and as a result produces very high pressure (100 MPa) and high temperature

(5000 K) (Earnshaw and others, 1995). One of the current applications of ultrasound is

human milk homogenization in hospitals; some nutrients such as liposoluble vitamins,

proteins, and minerals are usually bound to fat globules; if milk is not homogenized these

nutrients are lost when the fluid circulates through the feeding system designed for

newborn infants (Carcel and others, 1998). The nanometer resolution of scanning

electron microscopy (SEM) enables detailed analysis of casein micelles, fat globule

membranes, bacteriophages, very small particles, and the changes produced during

processing (Kaláb, 1981, 1993).

The objective of this research was to study the changes in microstructure of the fat

globules after heat and themo-sonication treatments, by using SEM to understand these

changes at the microscopic level. Also, change in color was studied and quantification of

fat content performed in order to correlate these parameters with microscopic images.


2.1 Milk samples

Raw and whole cow’s milk was obtained from the Washington State University

Creamery. Milking process was performed previously using automatic milkers for

different cows. Milk was kept in refrigeration at 4ºC until used. The raw milk (15 ml)

was then transferred to a disposable 15 ml PET conical and sterile test tube (Corning®)

and used as a control sample during microscopy sample preparation.

215

2.2 Thermal and thermo-sonication treatments

2.2.1 Ultrasound equipment

An ultrasonic processor—Hielscher® USA, Inc. (Ringwood, NJ), model UP400S

(400 W, 24 kHz, 120 μm amplitude), was used with a 22 mm diameter probe. Raw milk

(500 ml) was placed in the treatment chamber, a double-walled vessel (500 ml), with an

internal diameter of 8 cm and a depth of 13.5 cm. A magnetic stirrer was used inside the

treatment chamber to assure the homogeneity of the treatment. Ultrasonication was

carried out at 100% (120 μm amplitude) and 63 ± 0.5 ºC temperature. Temperature was

kept constant with a refrigerated bath (VWR Scientific Model 1166, Niles, IL). A

thermocouple was used in the treatment chamber to monitor the temperature (63 ±0.5ºC)

throughout the experiments. Treatment times were 10 and 30 min. Samples were

collected and transferred to disposable 15 ml PET conical and sterile test tubes (Corning

®) and kept at 4°C.

2.2.2 Heat treatment

Heat treatment was carried out in the same 500 ml double-walled vessel. The

same volume of milk was used, and temperature of the medium was kept at 63 ± 0.5ºC by

using a heating bath maintained at 65ºC. Temperature was monitored with a

thermocouple. After 30 min of treatment samples were transferred to disposable 15 ml

PET conical, sterile test tubes (Corning®).

216

2.3 Scanning electron microscopy


Disposable 15 ml PET conical, sterile test tubes (Corning®) containing raw milk

samples (thermo-sonicated and heated) were centrifuged at 1500 rpm for 5 min at 10ºC.

The supernatant fat layer of the sample was transferred to disposable 1.5 ml sterile plastic

microcentrifuge tubes (Fisherbrand®). A 0.5 ml solution of glutaraldehyde (2%)

paraformaldehyde (2%) in 0.1 M phosphate buffer (pH 7.2) was added to each microtube;

the fixation process was allowed to proceed for 24 hours at 4ºC. After fixation, the fat

was washed for 10 min with phosphate buffer (0.1 M), followed by two consecutive 10

min washes with cacodylate buffer (0.1 M). The post-fixing procedure consisted of

adding 2% osmium tetroxide in cacodylate buffer (0.1 M) at 4ºC for 24 hours. Samples

were then washed three times with cacodylate buffer (0.1M) for 10 min each time.

Dehydration of samples was achieved with serial dilution solutions of ethanol (30,

50, 60, 70, 95 and 100%). Each solution was maintained in contact with the sample for 10

min; the last solution (100% ethanol) was repeated three times.

After dehydrating the samples with ethanol, a second dehydration procedure with

hexamethyldisilazane (HMDS) was carried out on the samples. Consecutive contact (15

min each time) was used between the samples and ethanol/acetone/HMDS solutions at

different ratios (1:0:0, 1:1:0, 0:1:0, 0:1:1, 0:0:1, 0:0:1). Air drying was used as a final

step, leaving the microcentrifuge tubes with an open lid for at least 24 hours. Samples

were mounted on aluminum stubs, and gold plating was used as a final step to view the

samples with a Hitachi S-570 (Japan, Tokyo) scanning electron microscope (SEM)

operating at 20 kV.

217

2.4 Color

Lightness to darkness (L*) (100 to 0), redness(+)-greenness(-) (a*), and

yellowness(+)-blueness(-) (b*) color parameters were determined using a Minolta CM-

2002 spectrophotometer (Minolta Camera Co., Osaka, Japan) in reflection mode.

Samples (20 ml) of raw and thermo-ultrasonicated milk were poured into sterile plastic

bags. A white ceramic plate was used for standardizing the instrument (L* = 93.4, a* = -

0.67, b* = 0.78). The net color difference (ΔE*) was calculated as follows:

222 *)(*)(*)(* baLE Δ+Δ+Δ=Δ

2.5 Fat content

Fat content was determined using a LactiCheck™ Milk Analyzer (Page &

Pedersen, International Ltd., Hopkinton, MA). The performance of this equipment was

based on high frequency ultrasound. The milk analyzer was validated to determine butter

fat content (Gerber) according to the Association of Analytical Chemists (AOAC 1986)

methodology. The equipment was previously calibrated with raw cow’s milk, and


triplicate. No previous preparation was required for heat and thermo-ultrasonicated

samples.

218


All treatments were performed at least in duplicate, and the physicochemical and

composition characteristics were evaluated in triplicate for each sample. Statistical

analysis of the data was performed using a Microsoft Excel program. Analysis of

variance (ANOVA) was calculated with the SAS program (SAS Institute, Cary, NC,

1999) and a confidence level α of 0.05 was used to evaluate significant differences.


3.1 Microstructure of milk: fat globules

Fat globules are delimited with a membrane, derived from the apical plasma

membrane (Corredig and Dalgleish, 1998). In Figure 1, the structural appearance of a fat

globule in raw and whole cow’s milk is shown. This structure represents the control

sample and shows the native form of the globule structure. Despite the fact the milk fat

globule membrane (MFGM) shows no important changes or disruption, little damage can

be present in the globule because of the milk handling. Changes into composition or

structure of the MFGM occur as a result of milk handling and treatment during and after

milking; these changes could be because of the induced motion of fat globules, changes

in temperature, microbial growth or biochemical reactions (Evers, 2004). The scale of the

image in Figure 1 is 4.3 μm; the fat globule has a diameter slightly bigger than the

average size generally recorded in the literature, although it is inside the range reported

by Keenan and Patton (1995) for most of the fat globules present in milk. According to

Wiking and others (2004), large fat globules are more predisposed to coalescence and

lipolysis during some milking operations, such as pumping, so the size of the globules is

219

of high importance in the stability of milk. In cheese–making, smaller fat globules are

preferred because this physical characteristic can reduce the rennet coagulation time as

well as the curd firming rate (Tosh and Dalgleish, 1998).

Figure 2 shows the microstructure of another fat globule after heat treatment at

63ºC for 30 min. The general appearance of the fat globule shows a surface without

changes compared to Figure 1. According to Corredig and Dalgleish (1998), there are no

important changes in the size of the fat globules taking place during heating. However, a

more resistant membrane is developed outside the fat globules, which is probably due to

the interaction with proteins (β-lactoglobulin and MFGM proteins) generating a

polymerized new surface that is more resistant to coalescence; these complexes are

formed by disulphide bridges. The appearance of heated fat globules is characterized by

fairly thick deposits. These deposits could be protein in the membrane (Corredig and

Dalgleish, 1996), which coincides with the appearance of the globule shown in Figure 2.

In Figure 3, some examples of fat globules after thermo-sonication are shown;

treatment time was 30 min. A visible orifice on the surface of each globule is shown,

allowing observation of the disruption and cracking of the fat globule membrane, which

is composed of cholesterol, phospholipids, proteins and enzymes. Despite the damage

shown in these fat globules, they can be considered as not totally disrupted by cavitation.

Heertje and others (1987) showed the structure of fat globules in butter after a de-oiling

process as being hollow spheres, which is similar to the globule shown in Figure 3

(bottom right). However, MFGM protects the globule's fat content from lipolysis and

oxidation; when the membrane is damaged and disrupted due to physical stress,

coalescence of the milk fat globules occurs and presence of casein is observed in the new

220

interface (Wiking and others, 2003). When a fat globule has lost part of its membrane, a

granular material can be seen covering the surface.

During conventional homogenization there is turbulence in the medium, and

cavitational forces are generated by the high pressures. These forces disrupt the fat

globules, disintegrating them and reducing their size to less than 1 μm (Keenan and

Patton, 1995). In Figure 4 a graphic representation of the changes occurring in the fat

globule and milk after homogenization is presented. First, it can be seen that after the

disruption of the fat globule membrane, a number of casein micelles are added to the new

surface as well as some fragments of this protein. Some whey proteins can be present as

well in forming the new structure of the homogenized fat globules. In the same figure

(see bottom) a possible depiction of how the new microstructure of milk would look can

be seen, showing: the lipoprotein complexes (< 500 nm) with membranes composed

mainly of caseins; the tiny native fat globules (100 nm), which are not affected during

homogenization because of their size; the homogenized fat globules (disrupted and

covered by casein micelles); and some fragments of MFGM (Michalski and others, 2002;

Michalski and Januel, 2006).

In Figure 5, two examples of fat globules after thermo-sonication (basically a

homogenization process) can be seen. Both globules were thermo-sonicated for 30 min;

the effect of high pressurization generated through cavitation totally disintegrated the

MFGM, resulting in a granular interface. This effect is a result of the increase in pressure

caused by the violent collapse of bubbles (Villamiel and de Jong, 2000). In Figure 5 (left

top) some free casein micelles are also shown; according to Dalgleish (1998) these are

colloidal particles and are considered to be solid spheres with a protein coating. SEM

221

images of untreated, heated, and homogenized milk with ultra-high pressure (Sandra and

Dalgleish, 2005; Dalgleish and others, 2004; Martin and others, 2006) show similarity

with the casein micelles observed in Figure 5. In the same figure (right side), the fat

globule is can be seen after thermo-sonication, showing a more granular surface

compared to the previous one. This could be due to the adhesion of casein micelles

present in the environment after sonication; but also a stronger effect of cavitation could

be the reduction in size of the fat globule, giving off smaller structures composed mainly

of triacylglycerols micro-droplets. When a fat globule is disrupted, the new area is

covered with the hydrophobic part of casein particles, generating a new membrane for the

fat globule with a different composition (Meyer and others, 2006) and binding mainly

casein micelles with fat globules (Tosh and Dalgleish, 1998). Part of the original MFGM

remains on the globule but it is insufficient to cover the new surface. For that reason

casein semi-intact micelles and micellar fragments completely wrap the new surface and

avoid the coalescence of fat globules (Sharma and Dalgleish, 1993), although the

presence of some whey proteins is also possible in minor proportion (Michalski and

others 2002b).

Figure 6 the effect on size reduction of fat globules can be observed. Two main

fat globules can be seen at the top of the image. Their diameters are approximately 2 μm

each, showing a reduction in dimension from the native fat globule (4.3 μm). Changes in

MLGM are generated by disruption of the fat globules (Ye and others, 2004). Further, in

Figure 6 there are thousands of casein micelles with sizes below 0.5 μm.

Ultrasound homogenization showed that the fat globule's size can be reduced

below 1 μm when the treatment is carried out at 60ºC. At the same time, the globules

222

have more binding sites on the membrane, favoring the amalgamation of casein and

serum proteins, and thus producing an ideal ingredient for cheese-making (Villamiel and

others, 1999) because of the enhancement of the large surface area (fat globules-casein

network) (Michalski and others, 2002b). Heating the milk at the same time as sonication

reduces viscosity, enhancing the action of ultrasound in the size reduction of globules.

With temperatures between 55 and 75.5ºC plus continuous flow sonication, a reduction of

up to 81.5% in fat globule size was achieved by Villamiel and de Jong (2000).

The localized transient pressure that could be achieved with ultrasound (up to 100

MPa) is substantially higher than the conventional lower duration pressures used in

homogenization (20-50 MPa). Thiebaud and others (2003) found that pressures above

200 MPa demonstrated certain benefits: in the stability of milk, size of fat droplets,

microbial and enzymatic inactivation, and modification of rheological properties in some

emulsions. However, pressures up to or above 300 MPa were responsible for protein

denaturation and poor emulsification characteristics. Emulsification at 150 MPa

generated oil-in-water emulsions with a droplet size below 1 μm (Thiebaud and others,

2003). The beneficial effects observed in milk after sonication could be a potential tool

in future research, development, and improvement of dairy products; thermo-sonicated

milk could also be a viable option for milk beverages. With sonication, the volume of fat

globules is reduced, and a larger contact surface for lipolytics enzymes is generated,

making the digestion process easier (Paci, 1953) for consumers, as pointed out by

Michalski and Januel (2006) in comparing homogenized milk with untreated milk.

Finally, in Figure 7, a comparison between heat-treated and thermo-sonicated fat

globules is shown. Figure 7a shows the appearance of MFGM, followed by initial

223

disruption of the surface of the fat globule after 10 min of thermo-sonication (Figure 7b);

the final microstructure of the fat globule, subjected to 30 min of ultrasound technology,

shows a rough surface, and loss of MFGM with subsequent adhesion of casein micelles

(Figure 7c). In this comparison, the effects of ultrasound clearly allow the possibility of

research and development of new products based on this technology. Power ultrasound

has the ability to disrupt cells and membranes, and to promote chemical reactions.

Depending on the intensity of treatment, the effects may or may not be more noticeable.

Cavitation is the main mechanism behind globule disruption in an ultrasound

homogenizator (Walstra, 1969). In Figure 7b, a fat globule of milk after 10 min of

thermo-sonication is shown. The MFGM shows a high degree of disruption; the smooth

surfaces shown in the native and heated globules have almost disappeared, generating a

new structure with a rough surface, and releasing the internal tryacylglycerol content of

the fat globules. Cavitation is not only responsible for disruption of fat globules with

subsequent adhesion of casein micelles; other small particles can be observed after

ultrasonication because of the mechanical stress and strong forces involved (Michalski

and others, 2002).

In addition to the good results achieved with power ultrasound in microbial

inactivation (still under research) (Pagán and others, 1999; Ugarte-Romero and others,

2006; Bermúdez-Aguirre and others, 2005) and which could make thermo-sonication a

viable option for pasteurization-homogenization of milk in the near future, there are

positive effects due to cavitation in the microstructure of milk.

224

3.2 Net Color Change and Fat Content

Because of the new and improved physical characteristics (e.g., appearance, color,

homogenization, and stability) of thermo-sonicated milk, the color and fat content were

quantified to analyze how these parameters change in the product with heat and thermo-

sonication, and how they can be correlated with changes in the microstructure of milk.

In Table 1 the color parameters L, a and b are shown for the three different

samples: raw milk, heated milk and thermo-sonicated milk. Raw and heated milk have

similar values for Hunter’s parameters, as can also be seen in the net change of color ΔE

(0.87), confirming that heat treatments do not change milk characteristics to a high

degree. The color parameters for thermo-sonicated milk show an important variation with

respect to the raw milk values, especially for the L value. The whiter color of milk

observed at first sight in the samples after thermo-sonication is confirmed with a higher L

value, meaning a greater luminosity of the sample. This value could be related to the

better homogenization observed in the samples after sonication. Smaller fat globules are

produced by cavitation, and apparently create a more uniform sample. The net change in

color (ΔE) for the thermo-sonicated sample compared to raw milk also showed a high

value (4.56). According to these studies on whole milk, the homogenization by cavitation

could be eliminating or destroying smaller size clumps of butter fat that are sometimes

present in raw milk.

The release of triacylglycerols from the core of the fat globules is confirmed by

proximal analysis, as shown in Table 1. Fat content was 4.04% for raw whole milk; after

30 min of thermo-sonication the final result for lipids content was increased to 4.24%.

This value demonstrates that the triacylglycerols content encapsulated inside the MFGM

225

of the fat globules is released to the environment, making their quantification easier after

cavitation. Other lipids, such as cholesterol and phospholipids, which usually wrap the

core of the fat globules, are also quantified after sonication because of the release of this

membrane and later emulsification in the medium. Ultrasound generates the transfer of

particles inside the medium. Villamiel and others (1999) mention that the mechanical

effects generated by cavitation enhance mass transfer. For this reason thermo-sonicated

milk has better homogenization and stability.

4. Conclusions

Ultrasound was responsible for the disruption of MFGM in the fat globules,

generating a roughened surface and smaller size of droplets, as well as the adhesion of

casein micelles to the main globule structure. Better homogenization, stability, and

appearance than in heat treated milk were observed in thermo-sonicated milk. The color

was improved and the fat content was more available because of the cavitation effect.

These new properties of milk following sonication treatment can be used in developing

and improving the sensorial and quality characteristics of commercial and new dairy

products.

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characteristics of the synthetic membrane on the fat globules of microfluidized milk.

Journal of Dairy Science. 81:1840-1847.

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Ugarte-Romero E, Feng H, Martin SE, Cadwallader KR, Robinson SJ. 2006.

Inactivation of Escherichia coli with power ultrasound in apple cider. Journal of Food

Science. 71(2): E102-E108.

Villamiel M, van Hamersveld EH, de Jong P. 1999. Review: Effect of ultrasound

processing on the quality of dairy products. Milchwissenschaft 54(2):69-73.

Villamiel M, de Jong P. 2000. Influence of High-Intensity Ultrasound and Heat

Treatments in Continuous Flow on Fat, Proteins, and Native Enzymes of Milk. Journal of

Agriculture and Food Chemistry. 48:472-478.

Walstra P. 1969. Preliminary note on the mechanism of homogenization.

Netherlands Milk and Dairy Journal. 23:290-292.

Wiking L, Björck L, Nielsen JH. 2003. Influence of feed composition on stability

of fat globules during pumping of raw milk. International Dairy Journal. 13:797-803.

Wiking L, Stagsted J, Björck L, Nielsen JH. 2004. Milk fat globule size is

affected by fat production in dairy cows. International Dairy Journal. 14: 909-913.

Wiking L, Nielsen JH, Båvius AK, Edvardsson A, Svennersten-Sjaunja K. 2006.

Impact of milking frequencies on the level of free fatty acids in milk, fat globule size, and

fatty acid composition. Journal of Dairy Science. 89:1004-1009.

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Ye A, Singh H, Taylor MW, Anema SG. 2004. Interactions of fat globule surface

proteins during concentration of whole milk in a pilot-scale multiple-effect evaporator.

Journal of Dairy Research. 71:471-479.

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Table 1. Color parameters, net change in color and fat content of raw whole milk,

thermally treated milk, and thermo-sonicated milk

Sample L a b ΔE Fat contenta

(%)

Raw milk 87.82* ± 0.18 -1.70* ± 0.13 5.91 ± 0.25 4.04* ± 0.05

Thermal

treatment

88.25* ± 0.67 -1.97* ± 0.07 5.61 ± 0.06 0.87 ± 0.38 4.22* ± 0.02

Thermo-

sonication

92.37* ± 0.20 -1.55* ± 0.02 5.64 ± 0.09 4.56 ± 0.03 4.24* ± 0.02

* Significantly different results (p < 0.05)

a Accuracy ± 0.1%

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Figure 1. Fat globule in raw whole milk used as a control sample. The globule shows its

integrity; no important changes on the surface or membrane are detected.

Magnification 7 000x.

235

Figure 2. Microstructure of thermally treated fat globule after 30 min at 63°C in whole

milk. Magnification 6 000x.

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Figure 3. Thermo-sonicated (63ºC, 120 μm for 30 min) fat globules of whole milk

showing the disintegration of the milk fat globule membrane (MFGM). Magnification 7

900x and 6 000x (top); 5 000x and 6 000x (bottom).

237

Figure 4. Graphic representation of the changes in the MFGM in a fat globule after

homogenization (top) and general microstructure of milk after homogenization (bottom)

(Adapted from Michalski and Januel, 2006).

238

Figure 5. Thermo-sonicated (63ºC, 120 μm for 30 min) fat globules in whole milk

showing the presence of casein micelles added to the main structure. Magnification 10

000x and 8 000x.

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Figure 6. Microstructure of fat content in thermo-sonicated milk (63°C, 120 μm for 30

min), showing size reduction in fat globules and the presence of thousands of casein

micelles. Magnification 7 100x.

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Figure 7. A comparison of the microstructure of fat globules after three different treatments using scanning electron microscopy

(SEM): a) Thermally treated (63ºC) fat globule structure, b) thermo-sonicated (63ºC, 120 μm) fat globule structure after 10 min of

treatment, and c) thermo-sonicated (63ºC, 120 μm) fat globule after 30 min of treatment in whole milk. Magnification a) 5 900x, b) 5

900x, c) 6 000x.

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CHAPTER SIX

STUDY OF BUTTER FAT CONTENT IN MILK ON THE INACTIVATION OF

Listeria innocua ATCC 51742 BY THERMO-SONICATION

Daniela Bermúdez-Aguirre and Gustavo V. Barbosa-Cánovas

Abstract

Ultrasound combined with heat treatment has yielded favorable results in the inactivation

of microorganisms; however, the composition of food influences the rate of microbial

inactivation. The objective of this research was to study the effect of butter fat content in

milk on the inactivation of Listeria innocua and compositional parameters after thermo-

sonication. Four butter fat contents in milk were evaluated at 63ºC for 30 min of

sonication (Hielscher® UP400S, 400 W, 24 kHz, 120 μm amplitude). Results showed

that inactivation of Listeria cells occurs first in fat free milk, and that the rate of

inactivation decreases with increasing fat content. No degradation of protein content or

color variation was observed after the treatments. The pH dropped to 6.22, and lactic acid

content showed an increase of 0.015% after the treatment; solids-non-fat, density and

freezing point decreased. During storage life, growth of mesophiles was retarded with

sonication.

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1. Introduction

Milk is a common worldwide product with high commercial demand, regardless of the

source, adding important protein content to the human diet. The sensory and

microbiological characteristics of milk are also very important to the consumer.

Pasteurization is the common thermal process used to inactivate pathogenic bacteria and

some enzymes in milk, however undesirable effects on nutritional and sensory properties

may be found in pasteurized milk because of the high temperature used. Further, high salt

concentrations in the medium have been shown to increase the heat resistance of

microorganisms (Earnshaw, Appleyard & Hurst, 1995), while the butter fat content of

milk can also be a barrier to the inactivation of bacteria (Zapico, de Paz, Medina &

Nuñez, 1999). Even the mode of thermal treatment batch or continuous, has a different

mechanism of inactivation in cells. Although the supplied heat is the same, cells do not

receive the same physical effects in batch mode, as compared to continuous processes

such as pressure and shear forces (Fairchild, Swartzel & Foegeding, 1994).

Listeria monocytogenes has been reported as one of the most common pathogenic

microorganisms related to foodborne outbreaks in the dairy industry (Zapico et al., 1999;

Powell, Tamplin, Marks & Campos, 2006; Piyasena, Liou & McKellar, 1998). This

microorganism is a short rod that can grow under aerobic and microaerophilic conditions.

The optimum temperature for growth is 35-37ºC, but it can also grow under refrigerated

conditions, over a wide range of pH (4-9), and with salt concentration up to 10% (Yousef

& Carlstrom, 2003; Zapico et al., 1999). Although the dairy industry is most concerned

about the presence of Listeria monocytogenes in products such as cheese, other sources of

this microorganism are frankfurters and sliced meats (Yousef & Carlstrom, 2003).

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Listeria innocua, used as a surrogate of Listeria monocytogenes for microbiological

studies, has shown similar behavior against heat and radiation in different media (Zapico

et al., 1999), and a very high resistance to thermal treatment in skim milk (Fairchild et al.,

1994) and to electric pulses (Picart, Dumay & Cheftel, 2002).

The use of sound waves in liquid media to disrupt cells, either alone (sonication),

or in combination with heat (thermo-sonication), is an alternative approach to the

inactivation of such bacteria. Some studies have been conducted on milk under sonication

in order to analyze its functional properties after processing (Ertugay, Şengül & Şengül,

2004; Vercet, Oria, Marquina, Crelier & Lopez-Buesa, 2002; Villamiel & De Jong, 2000;

Wu, Hulbert & Mount, 2001) and to test the inactivation of bacteria such as Salmonella,

Staphylococcus aureus, Bacillus subtilis and coliforms (Villamiel, Van Hamersveld & De

Jong, 1999; Carcel, Benedito, SanJuan & Sánchez, 1998). Several studies have also

reported the use of ultrasound for microbial inactivation in other media. Some of the

tested microorganisms are Saccharomyces cerevisiae (Guerrero, Tognon & Alzamora,

2005; Tsukamoto, Constantinoiu, Furuta, Nishimura & Maeda, 2004a; Tsukamoto, Yim,

Stavarache, Furuta, Hashiba & Maeda, 2004b; Guerrero, López-Malo & Alzamora,

2001); Escherichia coli (Ugarte-Romero, Feng, Martin, Cadwallader & Robinson, 2006;

Ananta, Voigt, Zenker, Heinz & Knorr, 2005; Furuta, Yamaguchi, Tsukamoto, Yim,

Stavarache, Hasiba & Maeda, 2004); Listeria monocytogenes (Mañas, Pagán & Raso,

2000; Pagán, Mañas, Alvarez & Condón, 1999); Salmonella (Cabeza, Ordóñez, Cambero,

De la Hoz & García, 2004); Lactobacillus rhamnosus (Ananta et al., 2005); Yersinia

enterocolitica (Raso, Pagán, Condón & Sala, 1998), among others.

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The objective of this work was to study the inactivation rate of Listeria innocua in

milk with different butter fat content (fat free, 1%, 2% and whole milk) while exposed to

pre-selected thermo-sonication treatment. Nutrient retention (butter fat and protein

content) and physical-chemical properties (pH, acidity, color, density, solids-non-fat and

freezing point) were evaluated after completion of the treatment.


2.1 Milk samples

Four different commercial milks with different butter fat content (fat free, 1%, 2% and

whole) were purchased from Farmland Dairies, LLC. (Wallington, NJ). Samples were

previously processed with Ultra-High-Temperature (UHT) and packaged in Tetra Brik®

containers. Samples were maintained at room temperature until use. Characterization of

all samples consisted of sterility testing (no presence of bacteria), pH, acidity, color,

butter fat and protein content, density, solids-non-fat, and freezing point.

2.2 Listeria innocua analysis

2.2.1 Growth of Listeria innocua cells

Listeria innocua cells (ATCC 51742) from early stationary phase growth were stored at -

21ºC as a 50% volume suspension in sterile glycerol solution (20 ml glycerol/100 ml

water). Two ml of this suspension were unfrozen and added to 100 ml Tryptic Soy Broth

(Bacto: Becton, Dickinson and Co., Sparks, MD) with 0.6% Yeast Extract (YE) (Bacto,

Becton, Dickinson and Co., Sparks, MD). The microorganism was kept on a bath shaker

at 37ºC and 218 rpm until it reached the early stationary phase, approximately after 11 h.

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Inoculation of Listeria cells was made in each kind of milk in a ratio of 1:100 (cells:milk)

(V/V).

2.2.2 Enumeration of Listeria and mesophilic bacteria

The sterility of each kind of milk was evaluated with plate count agar (Difco: Becton,

Dickinson and Co., Sparks, MD). Samples were taken directly from each container. After

serial dilution in aqueous peptone solution (0.1%), the samples were plated. Dishes were

incubated at 35ºC for 48 h and cells were counted.

Samples inoculated with Listeria innocua were plated in Tryptic Soy Agar (Difco:

Becton, Dickinson and Co., Sparks, MD) with 0.6% of Yeast Extract (Bacto, Becton,

Dickinson and Co., Sparks, MD). Dishes were stored at 35ºC for 48 h, after which

bacteria loads were counted.

2.3 Thermo-sonication treatments

An ultrasonic processor (Hielscher USA Inc., Ringwood, NJ) model UP400S (400 W, 24

kHz, 120 μm amplitude) with a 22 mm diameter probe was used. A double-walled vessel

of 500 ml (8 cm internal diameter, 13.5 cm depth) was used as a treatment chamber.

Temperature was set up and kept constant via a refrigerated bath (VWR Scientific Model

1166, Niles IL). A thermo-couple was used in the treatment chamber to monitor the

temperature (63 ± 0.5ºC) throughout the experiments. The acoustic intensity was kept

constant at 100% (120 μm) in all treatments. A magnetic stirrer was used inside the

vessel to assure the homogeneity of the samples throughout the sampling. The treatment

time was 30 min for all systems, and samples were taken after 0, 5, 10, 15, 20, 25 and 30

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min. All treatments were performed in duplicate, and the analysis for each treatment was

carried out in triplicate.

Treatments were carried out in batch mode to simulate the conventional batch

pasteurization (63ºC by 30 min) and to compare with previous studies (Bermúdez-

Aguirre & Barbosa-Cánovas, 2005 and 2006).


Butter fat, solids-non-fat and protein content were evaluated using a LactiCheck™ milk

analyzer (Page & Pedersen, International Ltd., Hopkinton, MA). The performance of this

equipment is based on high frequency ultrasound. The milk analyzer was validated with

official methods to determine butter fat content (Gerber) and protein (Kjeldahl) in milk,

according to Association of Analytical Chemists (A.O.A.C.) methodology. Solids-non-fat

measurements were simultaneously performed with the same equipment. The

LactiCheckTM analyzer was previously calibrated with each kind of milk (according to

butter fat content) and samples were adjusted to room temperature (20ºC). Each sample

was analyzed in triplicate. No prior preparation was required for the different butter fat

content samples and thermo-ultrasonicated samples.

2.5 Physicochemical characteristics

2.5.1 pH and titratable acidity

pH was determined by direct immersion with a potentiometer (Orion Research Inc.,

Boston, MA). Acidity was determined by titration at room temperature with 0.1 N NaOH

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solution to pH 8.3 and expressed as a percentage of lactic acid (1 ml of 0.1 N NaOH =

0.009 g of lactic acid). Each sample was measured in triplicate.

2.5.2 Density and freezing point

Density and freezing point were evaluated in each milk sample after treatment. A

LactiCheck™ milk analyzer (Page & Pedersen, International Ltd., Hopkinton, MA) was

used for this purpose; it was calibrated with each kind of milk before treatments, and

samples were analyzed at 20ºC, each one in triplicate.

2.5.3 Color




ml of UHT and thermo-ultrasonicated milk samples were poured into sterile plastic bags.


b* = 0.78).

2.6 Shelf-life studies

In order to study the effect of thermo-sonication as a method for extending the shelf-life

of UHT milk, four batches of milk (fat free, 1%, 2% and whole milk) were treated with

the same sonication conditions described above (63ºC, 120 μm for 30 min), but without

the addition of Listeria cells. Milk samples of 25 ml each (with and without treatment)

were poured into sterile plastic bags. Multiple samples were prepared in order to use a

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different bag of each sample on each testing day, to avoid reusing the same sample for

different purposes. Samples were stored at room temperature (21ºC) and refrigeration

conditions (4ºC) for 9 days to evaluate their physicochemical and microbiological

characteristics, such as pH, color and total plate count. Each batch of shelf-life studies

was conducted in duplicate.


Statistical analysis of the data was performed using a Microsoft Excel program. The

analysis of variance (ANOVA) was calculated with the SAS program (SAS Institute,

Cary, NC, 1999). A confidence level (α) of 0.05 was used to evaluate significant

differences.


3.1 Inactivation rate of Listeria innocua

A sterility test of samples showed that the initial count of bacteria in milk was lower than

10 cfu/ml for all milks. Afterwards, the inoculation of different milks was performed with

initial cell counts of 107 cfu/ml of Listeria innocua ATCC 51742. The inactivation curves

for the four samples tested are shown in Figure 1. The effect of butter fat content seems

to be a hurdle that lowers the rate of inactivation. After 30 min of treatment, whole milk

(3.47% butter fat content) showed a low degree of cell inactivation; a 2.5 log reduction

was achieved. As the butter fat content decreased, the inactivation was faster, as shown in

the 1% and 2% butter fat content milks, where 4.5 and 3.2 log reductions were achieved.

In these experiments, the highest inactivation (4.9 log reduction) was achieved in fat free

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milk after 30 min of treatment. It is clear that the presence of fat globules in milk creates

a protective effect for the cells. Some factors that affect the heat resistance of

microorganisms are high protein and fat content or high total solids (Gaze, 2005). In the

case of whole milk, the butter fat content is mainly distributed as fat globules of average

size (4 μm). Bacteria can adhere to these fat globules, but ultrasound has the ability to

disrupt the Milk Lipid Globule Membrane (MLGM), generating globules of smaller size.

Some of these smaller globules appear as hollow spheres with a completely new

roughness surface (Bermúdez-Aguirre & Barbosa-Cánovas, 2006). These

microorganisms may adhere to the surface of the newest globules, but they can also be

hidden within the rough surface and inside the globules that were disturbed with

ultrasound providing a protective fat layer against the heat and cavitation generated with

the sonication. Fat free milk, having few or no fat globules, does not offer a protective

effect to the Listeria cells.

Other workers have found that the inactivation of bacteria is reduced in food with

significant fat content because of changes in ultrasound penetration and energy

distribution (Earnshaw et al., 1995). Cavitation is the mechanism of action of ultrasound;

when a sound wave passes through a liquid medium thousands of bubbles are produced,

which increase and decrease in size very quickly. Intensity of cavitation depends on the

amplitude wave, pressure, temperature, and medium of treatment, among others. The

explosion and implosion of the bubbles are responsible for the erosion in food and

formation of free radicals (Povey & Mason, 1998). The intensity of cavitation is related

to the boiling point of the product; in milk, some of the soluble solids can affect the

intensity of the cavitation (Carcel et al., 1998). This effect of inactivation according to the

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medium of treatment has been reported in previous studies. For example, inactivation of

Salmonella was found to be higher in aqueous peptone (2.5 log) than in milk chocolate

(0.1 log) after 10 min of treatment, suggesting the limitation of cavitation in milk samples

(Villamiel et al., 1999). A strain of Staphylococcus aureus was studied under thermo-

sonication treatments (50.3 – 56.7ºC, 20 kHz, 3.75 W/ml) in a phosphate buffer and UHT

whole milk, and again showed the protective effect of the whole milk in the inactivation

of the microorganism (Villamiel et al., 1999; Carcel et al., 1998). Some studies conducted

with Listeria monocytogenes have shown that the butter fat content of milk is an obstacle

for microbial inactivation, using nisin as an antimicrobial. When skim milk (0% fat) was

used in combination with 50 IU/ml of nisin, 7 log reductions were achieved; in milk with

4% fat content, 3 log reductions were obtained; but just 1 log was achieved in milk

containing 12.9% fat. The suggested explanation is that fat globules might absorb the

nisin molecules, reversing the antimicrobial effect of this bacteriocin (Zapico et al.,

1999).

Research conducted with Listeria innocua under pulsed electric fields treatment in

milk has also shown how the butter fat content of this fluid can enhance or reduce the

inactivation rate, although in this case other operating parameters, such as pulse

frequency, also influenced the results. At 1.1 MHz, 0.67 log reductions were achieved in

whole milk, whereas 0.95 log reduction was observed in skim milk under the same

processing conditions (Picart et al., 2002). The butter fat content of milk also influences

other properties of milk, such as electrical conductance. When the fat content in milk

increases, the conductance decreases because the fat globules are nonconductive

materials (Mabrook & Petty, 2003).

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However, not only does butter fat content protect the cells, the use of UHT milk,

which is considered as sterile, also has an effect on the rate of inactivation. Even though

milk was tested for sterility (count was lower than 10 cfu/ml), it is clear that the absence

of the natural flora in raw milk has an effect. When raw milk was tested under the same

conditions, as those presented in this research, but with an initial count of mesophiles of

103 cfu/ml and inoculation with a similar count of Listeria innocua cells (108 cfu/ml),

inactivation was higher after 30 min of treatment, up to 5.5 log reductions (Bermúdez-

Aguirre & Barbosa-Cánovas, 2005), suggesting a possible effect of competitive flora. In

addition, the characteristics of UHT milk after processing at very high temperature

(138ºC for 2 sec; IDFA, 2006) including the aseptic conditions of the packaging process

and material used provide a different environment for Listeria cells compared to the rich

microbiological and enzymatic medium of fresh (raw) milk.


3.2.1 Butter fat content and protein

Nutrient retention for all samples is shown in Table I. Regarding butter fat content, the

reported value listed on the package was slightly different from that measured for this

experiment. After thermo-sonication, butter fat content remained equal, with insignificant

variations, as seen in Table I. It is quite apparent that the effect of previous

homogenization of UHT milk does not generate significant changes in the fat content of

milk with thermo-sonication. When raw milk is thermo-sonicated significant change in

the butter fat content has been reported; which could be due to disruption of the fat

252

globules and consequent release of triacylglycerols to the medium (Bermúdez-Aguirre &

Barbosa-Cánovas, 2006).

As to protein content, the difference between control samples and the thermo-

sonicated samples was found to be minimal, which could be attributed to the normal

variation and accuracy of the equipment used to measure protein. The analyzer used to

detect protein content is based on ultrasound spectroscopy, with an accuracy of 0.2% for

protein, so the differences shown in Table I could be related more to the accuracy of the

analyzer; otherwise, the difference in protein content due to ultrasound was minimal (i.e.

minimal denaturization of protein). Although total protein is being evaluated here, these

results agree with some studies on skim milk, which showed that after thermo-sonication

treatments (temperatures up to 50ºC), and the soluble protein content of the sonicated

samples was not significantly different from the control samples. When the temperature

was increased to 75.5ºC and the samples were sonicated for 102.3 s, the whey proteins

were denaturated in whole and skim milk, with a higher effect in whole milk. One theory

about protein denaturation states that the energy supplied from ultrasound can modify the

quaternary and/or tertiary structure of the casein, although it does not totally disturb the

micelle casein (Villamiel & De Jong, 2000).

3.2.2 Solids-non-fat

The solids-non-fat did not change significantly after thermo-sonication (Table II). There

was a trend in milk with fat (1%, 2% and whole) to show a slightly lower value after

thermo-sonication compared with the control sample, but again these values are in the

range of the accuracy of the milk analyzer for SNF (± 0.2%). The total solids in cow’s

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milk are around 12.6%, of which approximately 9% are solids-non-fat and the rest is fat.

The solids-non-fats are usually called serum solids (Potter, 1986) and include casein,

lactalbumin, lactose, calcium, phosphorus, and riboflavin, among others (IDFA, 2006).

3.3 Physical-chemistry characteristics

3.3.1 pH and acidity

For all control milk samples, the pH values are inside the range reported for bovine milk

(pH 6.22 to 6.77), and as expected, the presence of fat globules decreases the pH of the

milk (Table II). The reported acidity for milk is 0.16% (± 0.02) (Neville & Jensen, 1995).

The pH of all samples decreased after thermo-sonication, with a consequent increase in

the acidity of the milks. This increase in acidity could have occurred because of

lipolysis, that is, the breakdown of triglycerides to free fatty acids due to enzyme activity

(Walstra, Wouters & Geurts, 2006). Ultrasound can speed enzymatic reactions in the

milk in addition to the breakage of fat globules that contain triglycerides, making these

acids more available to the enzyme. The butter fat content of the milk affects the acidity,

and according to the results of this study, the whole milk sample showed the greatest

change in acidity content. Another possible theory as to why the acidity increased in milk

could be related to the nitrite and nitrate formation on air-saturated water after sonication.

Studies reported by Supeno (2000) showed how the sonication of aqueous solutions in

the range of ultrasound frequency from 41 to 3217 kHz generated the maximum

production of nitrite and hydrogen peroxide as primary products in the medium at 360

kHz, with the subsequent formation of nitrate as a product of the oxidation of nitrite.

However, this is only a theory in the case of milk, because the reported studies were

254

performed in water; also, the frequency used for the current experiment (24 kHz) is lower

than the reported range by Supeno (2000). It is likely that the decrease in acidity in milk

could be a combination between the formation of fat free acids and the formation of

nitrite/nitrate. Further studies on thermo-sonicated milk must be performed in order to

establish the new chemical products that may develop after sonication. According to

Walstra et al. (2006), the decrease in pH could be due to the hydrolysis of phosphoric

esters because of enzymatic action. It is also possible that this change can be attributed to

ultrasound action; however changes in pH and acidity could be reflected in taste when

these changes are higher than the established limits for milk.

3.3.2 Density

After thermo-sonication, milk samples with higher butter fat content showed a slight

decrease in density. In previous experiments, the real effect of ultrasound on milk

homogenization and change in milk properties such as density was clearer when raw

whole milk was used. The milk used for the current experiments was previously

homogenized during UHT processing, so the minor observable changes in density could

be attributed to this previous process. According to the data shown in Table II, the trend

after thermo-sonication was that the density of the samples increased as the content of

solids-non-fat was higher, agreeing with the report by Walstra et al. (2006). Meanwhile,

the density of the samples was higher when the milk had less butter fat content. The

density of fresh whole milk is 1029 kg/m3 while the density of whole milk at 10ºC is

1031 kg/m3.

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3.3.3 Freezing point

In previous studies, production of a small amount of water was generated after sonication

treatment of milk, probably because of the breakage of some macromolecules and

chemical reactions under the action of the cavitation releasing water molecules to the

medium. One of the usual quality assurance parameters in milk to examine if water has

been added to milk is to test the freezing point; the depression of this temperature

indicates adulteration of milk with water. The reported freezing point for bovine milk is

within the range of -0.512 to -0.550 ºC (Jensen & Newberg, 1995). The values of the

current experiment ranged from -0.584ºC to -0.504ºC for the control sample (from fat

free to whole milk), and from -0.599ºC to -0.493ºC for the thermo-sonicated samples

(from fat free to whole milk). As in the case of other physicochemical properties, fat free

milk did not show common behaviors in comparison to the other milks. However, for the

rest of the samples (1%, 2% and whole milk) the freezing point was slightly increased

after thermo-sonication.

3.3.4 Color

The color of the milks before and after thermo-sonication is shown in Figures 2, 3 and 4.

As shown in Figure 2, insignificant changes (p < 0.05) were observed in the L value. The

a value was more negative for all samples after sonication, except for the fat free milk

(Figure 3). In this case, a greenish color was generated. In Figure 4 the change in b value

is shown for all samples. Regardless of butter fat content the b value decreased with

sonication, meaning that the yellow contribution declined in the milk and the parameter

moved into the bluish region. As reported by Walstra et al. (2006) the bluish color of

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skim milk is due to small casein micelles, while the yellowish color of milk fat, milk

serum and whey is due to the presence of β-carotene. As described in more detail later,

the whiter color remained during the days that these samples were kept for shelf life

studies.

3.4 Shelf-life

The storage life of UHT milk packaged in aseptic conditions can be up to 6 months or

more because of the extreme conditions of temperature used in its processing and

packaging. Although in some countries UHT milk is referred to as sterile milk, UHT

processing does not warrant the sterility of the product. While UHT reduces the number

of bacteria to a minimum, some thermo-resistance spores and enzymes can remain in the

milk, generating spoilage of the product in a matter of months. Once the aseptic package

of UHT milk has been opened, the storage life of milk is not longer than 10 days because

of the presence of bacteria and new environmental conditions.

In this research, thermo-sonication was used in UHT milk to see if this technology

could extend the shelf life of milk after the package is opened. No bacteria were added to

the milk samples in order to test the growth of microorganisms that survived the UHT

process and the growth of common flora in the spoilage of milk packaged under non

extreme aseptic conditions. Samples were poured into sterile plastic bags. Two different

storage conditions were evaluated, first under refrigeration temperature (4ºC), and

secondly corresponding to the ambient temperature of the lab (21ºC).

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3.4.1 Total plate count

In Figure 5, the growth of mesophilic bacteria is shown for the four kinds of milk

(according to butter fat content) in the control and thermo-sonicated samples. First, the

presence of butter fat in milk accelerated the growth of bacteria; milk with fat is a good

microbiological medium, and as can be observed in the plot, whole milk presented more

than 3 log of growth on the second day, rising up to 5.23 log on day 9. Meanwhile, fat

free milk reached 3 log of growth after 9 days in the control sample. When the milk was

treated under thermo-sonication, the storage life was extended considerably. After 9 days

of storage under refrigerated conditions, fat free milk showed a growth of 1.6 log, while

whole milk had 3.6 log of total plate count. The growth in the 1% and 2% milks was

intermediate between fat free and whole milk, showing again how the presence of fat

increases or decreases the growth of bacteria proportionally.

In samples stored at ambient temperature, the use of thermo-sonication also

retarded the growth of bacteria, as can be observed in Figure 6. After two days, no

growth was detected in the thermo-sonicated samples regardless of butter fat content, but

on day six, growth was evident in all samples, reaching the bacterial limit for Grade “A”

pasteurized milk (HHS/PHS/FDA, 2001) on day 9 for milks with fat.

3.4.2 pH

The pH of the control and thermo-sonicated samples was evaluated during storage. In

Figure 7 the pH behavior for all samples stored under refrigerated conditions is shown. In

this case, there is no important difference between the pH of the control samples and the

258

thermo-sonicated samples. The lowest pH values were again found for the whole milk

because of the presence of fat globules.

For samples stored under ambient temperature, the pH behavior is shown in

Figure 8. Control samples exhibited a small drop in pH because of the growth of bacteria

that produced lactic acid in the milk. However, the drop of pH in the thermo-sonicated

samples was more evident; fat free milk decreased its pH to as low as 3.77 by day 9, and

the rest of the milks showed pH around 5.0. This drop in pH could be attributed in part to

the growth of bacteria, but in the case of fat free milk the growth was retarded because of

the absence of fat. This suggests a possible enzymatic or chemical activity in milk that is

accelerated with ultrasound, but which can be delayed with the use of refrigeration.

During sonication in an aqueous medium, cavitation breaks the water molecules into free

radicals such as H+ and OH- that hit the cell membrane having a chemical effect on

microorganisms. However, the possible recombination of these free radicals can generate

oxidants that will also have an antimicrobial effect, which could be lethal for cells after

they have been injured by ultrasound (Tsukamoto et al., 2004a; 2004b). Supeno (2000)

confirms the theory of the formation of free radicals such as H+, NO3- and NOx

- in

aqueous medium after sonication. This researcher confirms the presence of nitrite and

hydrogen peroxide such as primary products in water and the production of nitrate

because of the oxidation process. Free radicals can be recombined in toxic products for

cells; even hydrogen peroxide can act as an antimicrobial in the bacteria, and based on

the present research, it is possible that these radicals could have little effect on bacteria

inactivation. In the case of fat free storage at 21ºC, some of the chemical reactions that

are taking place could be due to the sonolysis of water.

259

3.4.3 Color

The color of samples stored under refrigerated conditions is shown in Figures 9, 10 and

11. No important color changes (p < 0.05) were observed throughout the storage life.

Again, samples with lower fat content showed lower luminosity and an important

contribution to the bluish region. This finding is in agreement with that reported by

Owens, Brewer & Rankin (2001) for fat free milks. The whiteness of milk is a result of

the suspended particles that scatter light in the visible spectrum. These particles are fat

globules and casein micelles, but when fat globules are scarce in the medium (e.g. in fat

free milk) the light is scattered in a different way and it generates a blue-gray coloration.

A study to evaluate the color of milk with different butter fat content showed the

following values for the three parameters L 81.11, a -3.74 and b 2.99 for milk with 2%

butter fat content (Phillips, McGiff, Barbano & Lawless, 1995).

Samples stored at 21ºC showed changes in the color parameters L, a and b, as

shown in Figures 12, 13 and 14, respectively. A decrease of luminosity was observed in

all samples. The a values moved to the green region for the thermo-sonicated samples,

especifically for fat free milk. Actually, the change in the color of the thermo-sonicated

sample without fat was observed at first sight. The b value also registered an increase in

its value during storage life, meaning that according to the Hunter color scale, the

samples had a yellow color. The changes in b value could be because of microbial growth

and enzymatic activity in the samples. Owens et al., (2001) showed that in milk

inoculated with bacteria such as Lactococcus lactis ssp. or Propionibacterium

freudenreichii ssp. Shermanii, the b value was increased.

260

4. Conclusion

The butter fat content of milk was a hurdle in the inactivation of Listeria cells in milk

under thermo-sonication treatments, lowering the inactivation rate. Minor changes were

reported in physicochemical and nutritional properties after sonication. The storage life of

UHT milk was extended by the use of thermo-sonication and refrigerated conditions,

with the storage life increasing with lower butter fat content.

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267

Table I. Nutritional attributes reported and measured for four different butter fat content UHT milk samples before and after thermal and thermo-sonication treatments

Milk Control

(reported*) Butter fat content

Control (measured)

Butter fat contenta (%)

After TS Butter fat

contenta (%)

Control (reported*)

Protein content

Control (measured)

Protein contentb (%)

After TS Protein contentb

(%)

Fat free 0g/240 ml (0.00 %)

0.00 (± 0.00)

0.00 (± 0.00)

8 g/240ml (3.33%)

3.10 (± 0.00)

3.17 (± 0.00)

1% 2.5g/240 ml (1.04%)

0.76 (± 0.01)

0.76 (± 0.01)

8 g/240 ml (3.33%)

2.95 (± 0.00)

2.92 (± 0.01)

2% 5 g/240 ml (2.08%)

1.94 (± 0.01)

1.84 (± 0.01)

8g/ 240 ml (3.33%)

2.87 (± 0.01)

2.84 (± 0.01)

Whole 8 g/240 ml (3.33%)

3.47 (± 0.01)

3.40 (± 0.00)

8g/240 ml (3.33%)

2.72 (± 0.00)

2.70 (± 0.00)

* Taken from nutritional information reported on individual containers TS = Thermo-sonication treatment (63ºC, 120 μm, 30 min) a Accuracy ± 0.1% b Accuracy ± 0.2%

268

Table II. Physicochemical characteristics of different butter fat content milk samples (fat free, 1%, 2% and whole) before and

after thermo-sonication (TS) treatments (63ºC, 120 μm by 30 min)

Milk Control Solids-

Non-Fata

After TS Solids-

Non-Fata

Control pH

After TS pH

Control Acidity (%)*

After TS Acidity (%)*

Control Density (g/cm3)b

After TS Density (g/cm3)b

Control Freezing

Point (ºC)

After Freezing

Point (ºC) Fat free 8.79 ±

0.00 8.98 ± 0.01

6.56 ± 0.02

6.46 ± 0.01

0.132 ± 0.005

0.148 ± 0.003

1.0326 ± 0.0000

1.0334 ± 0.0000

- 0.584 ± 0.000

- 0.599 ± 0.001

1% 8.36 ± 0.01

8.27 ± 0.02

6.57 ± 0.02

6.30 ± 0.03

0.132 ± 0.001

0.140 ± 0.002

1.0302 ± 0.0000

1.0294 ± 0.0035

- 0.551 ± 0.000

- 0.544 ± 0.017

2% 8.11 ± 0.02

8.04 ± 0.02

6.46 ± 0.02

6.30 ± 0.02

0.140 ± 0.003

0.126 ± 0.001

1.0282 ± 0.0006

1.0279 ± 0.0006

- 0.536 ± 0.059

- 0.528 ± 0.012

Whole 7.65 ± 0.01

7.5 ± 0.00

6.43 ± 0.02

6.23 ± 0.01

0.132 ± 0.001

0.154 ± 0.001

1.0250 ± 0.0000

1.0243 ± 0.0001

- 0.504 ± 0.006

- 0.493 ± 0.001

* Expressed as lactic acid content a Accuracy ± 0.2% b Accuracy ± 0.0005

269

-6

-5

-4

-3

-2

-1

00 5 10 15 20 25 30

Time (min)

log

(N/N

o)

Whole 1% 2% Fat free

Figure 1. Inactivation of Listeria innocua ATCC 51742 using thermo-sonication

treatments (63ºC, 120 μm by 30 min) in four different butter fat content milks

270

82

83

84

85

86

87

88

89

90

91

92

93

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

L

Control TS

Fat free Whole2%1%

Figure 2. L value of different butter fat content milk samples (fat free, 1%, 2% and

whole) before (control) and after thermo-sonication (TS) treatments (63ºC, 120 μm by 30

min)

271

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

00 0

.

5

1 1

.

5

2 2

.

5

3 3

.

5

4 4

.

5

a

Control TS

Fat free Whole2%1%

Figure 3. a value of different butter fat content milk samples (fat free, 1%, 2% and


min)

272

0

1

2

3

4

5

6

7

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

b

Control TS

Fat free Whole2%1%

Figure 4. b value of different butter fat content milk samples (fat free, 1%, 2% and


min)

273

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8 9 10

Time (days)

Log

(N/N

o)

Fat free C 1% C 2% C Whole C Fat free TS 1% TS 2% TS Whole TS

Figure 5. Growth of mesophilic bacteria in milk with different butter fat content (fat free,

1%, 2% and whole) in control (C) and thermo-sonicated (TS) samples (63ºC, 120 μm by

30 min) stored under refrigerated conditions (4ºC)

274

-1

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6 7 8 9 10

Time (days)

Log

(N/N

o)

Fat free C 1% C 2% C Whole C Fat free TS 1% TS 2% TS Whole TS

Figure 6. Growth of mesophilic bacteria in milk with different butter fat content (fat free,

1%, 2% and whole) in control (C) and thermo-sonicated (TS) samples (63ºC, 120 μm by

30 min) stored under ambient temperature (21ºC)

275

5.5

5.7

5.9

6.1

6.3

6.5

6.7

6.9

0 2 4 6 8 10 12 14

Time (days)

pH

Fat free TS 1% TS 2% TS Whole TS Fat free C 1% C 2% C Whole C

Figure 7. pH evolution during storage life of thermo-sonicated (TS) and control (C) milk


conditions (4ºC)

276

3

3.5

4

4.5

5

5.5

6

6.5

7

0 2 4 6 8 10 12 14

Time (days)

pH


Figure 8. pH evolution of thermo-sonicated (TS) and control (C) milk samples with

different butter fat content (fat free, 1%, 2%, and whole) during storage life under

ambient temperature (21ºC) conditions

277

80

82

84

86

88

90

92

94

96

98

0 2 4 6 8 10 12 14

Time (days)

L

Fat free TS 1% TS 2% TS Whole TS Fat free C 1% C 2% C Whole

Figure 9. L value evolution during the storage life of thermo-sonicated (TS) and control

(C) milk samples with different butter fat content (fat free, 1%, 2%, and whole) under

refrigerated conditions (4ºC)

278

-4.5

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

00 2 4 6 8 10 12 14

Time (days)

a


Figure 10. a value evolution during the storage life of thermo-sonicated (TS) and control



279

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14

Time (days)

b


Figure 11. b value evolution during the storage life of thermo-sonicated (TS) and control



280

76

78

80

82

84

86

88

90

92

94

96

0 2 4 6 8 10 12 14

Time (days)

L


Figure 12 . L value for thermo-sonicated (TS) and control (C) milk samples with different

butter fat content (fat free, 1%, 2%, and whole) during the storage life under ambient

temperature (21ºC) conditions

281

-5

-4

-3

-2

-1

0

1

2

3

0 2 4 6 8 10 12 14

Time (days)

a


Figure 13. a value for thermo-sonicated (TS) and control (C) milk samples with different



282

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14

Time (days)

b


Figure 14. b value for thermo-sonicated (TS) and control (C) milk samples with different



283

CHAPTER SEVEN

EVALUATION OF THE MICROSTRUCTURE OF THERMO-SONICATED

YOGURT WITH SCANNING ELECTRON MICROSCOPY USING A SHORTER

(NEW) SAMPLE PREPARATION PROCEDURE

Daniela Bermúdez-Aguirre, Valerie Lynch-Holm and


Abstract

Ultrasound has been used in food research mainly in microbial inactivation, but at the

same time it has shown positive effects in the processing of some foods. In electron

microscopy, considerable sample preparation time is required to make meaningful

observation of the product under study. In the case of dairy products, some artifacts might

occur, making sample preparation even longer and more complicated. The objective of

this work was to study the microstructure of thermo-sonicated yogurt with three different

sample preparation techniques used in electron microscopy. The thermo-sonication

treatment was applied with an ultrasonic processor (Hielscher® UP400S, 400W, 24 kHz,

120 μm) in raw whole milk at 63ºC for 30 minutes. Yogurt preparation followed the

thermo-sonication of milk with a starter culture of lactobacillus. The first sample

preparation consisted of the conventional sample dehydration with organic solvents

(ethanol, acetone and HMDS). Technique number two consisted of freeze-drying the

samples, freezing with liquid nitrogen and using a freeze-drier for 24 h. The third

technique consisted of dehydration of the samples with the same organic solvents as in

284

the first technique, but assisted with the use of microwave (MW) energy. Samples were

observed with a Scanning Electron Microscope Hitachi® S-570 at 20 kV. Results showed

that freeze-drying is not a technique suitable for yogurt because of the low resolution of

the images. However, high resolution and defined images were obtained with the MW

technique showing very good homogenization of the components of the thermo-sonicated

yogurt (casein and fat globules) while avoiding the syneresis of the product compared

with the protein network with interstitial spaces filled with whey of the conventional

yogurt. MW technique shortened the time in sample preparation to 3 h compared with the

conventional dehydration technique that takes 5 d, and at the same time provided images

with high resolution.

285

1. Introduction

One of the keys in the use and performance of electron microscopy to analyze samples is

the sample preparation procedure. Each step in the sample preparation protocol must be

followed as closely as possible in order to obtain images with high resolution, images that

represents the real microstructure of the food without damages generated through the

preparation. Some of the techniques that are available in food science to study the

structure of food products are electron and optical microscopy, elemental analysis such as

X-ray microanalysis and electron loss spectrometry, magnetic resonance imaging,

acoustic microscopy and ultrasoft X-ray microscopy (Kaláb et al., 1995). Some of the

methods of sample preparation for food products that have been used for electron

microscopy include air drying, freeze drying and fixation and chemical dehydration

(Chabot, 1979). Conventional techniques in sample preparation for food microstructure

have shown some disadvantages such as obscuring ultra-fine details because of the thick

metal coating, addition of metal particles in the specimen modifying the original

structure, or the presence of edge-effect brightness (McManus et al., 1993). Sample

preparation for Scanning Electron Microscopy (SEM) requires removal of the water from

the sample without minor change in the structure, but also the sample must be conducive

to be observed under the microscope (Chabot, 1979; Kaláb, 1981). Staining techniques

are currently used in the study of food microstructure because these techniques show the

chemical composition of the structures such as proteins, fats, ribonucleic acids and some

salts (Kaláb, 1993). In general, sample preparation procedures for SEM take considerably

longer times to obtain the required characteristics of the specimen before analyzing it

under the microscope, as is shown in some references (McManus et al., 1993).

286

Examination of dairy products with SEM has been a difficult area because of several

artifacts that occur through the sample preparation procedure (McManus et al., 1993).

The main constituents of milk that can be visible are the casein micelles, fat globules and

submicellar casein. In raw milk the size of the casein micelles is between 100-300 nm in

diameter and that of the submicelles from 10-20 nm in diameter (Kaláb, 1993); the size of

fat globules is between 0.2 to 10 μm in diameter (Keenan and Patton, 1995). Yogurt is a

fermented dairy product made from milk that is usually heated at 85ºC for 30 min then

cooled to 40ºC and the addition of the starter cultures that usually are lactobacillus.

Studies of yogurt with electron microscopy have shown the protein network as a chain of

casein micelles containing in the interstitial spaces whey protein (liquid), this matrix

being determinant in the phase separation or syneresis of the product (Kaláb, 1993). This

physical change in the product is an undesirable phenomenon in the product that can take

place through transportation and storage.

The objective of this research was to evaluate three different sample preparation

protocols for Scanning Electron Microscopy of thermo-sonicated yogurt and to decide

which protocol is optimal for the purpose of analyzing the microstructure of this dairy

product on the basis of time effectiveness-image resolution.


2.1 Milk and yogurt samples

Two different systems were evaluated. System one corresponds to the control and it used

commercial plain natural yogurt bought in the local supermarket. This yogurt had 3.25%

milk fat and belongs to grade A. For the second sample, raw milk was used. The raw milk

287

was bought in the local creamery of Washington State University; this milk had an

average fat content of 4.25%.

2.2 Milk pasteurization

Thermo-sonication of milk

Thermo-sonication pasteurization was performed with an ultrasonic processor Hielscher

USA Inc. (Ringwood, NJ) model UP400S (400 W, 24 kHz, 120 microns) with a 22 mm

diameter probe. A double-walled vessel (500 ml) was used as a treatment chamber with

an internal diameter of 8 cm and depth of 13.5 cm. A magnetic stirrer was used inside the

treatment chamber to assure homogeneity of the treatment. The amplitude of the

ultrasonic wave was constant for the thermo-sonication treatment, 100% (120 μm), and

the temperature was maintained at 85ºC (±0.5). Temperature was kept constant via a

refrigerated bath (VWR Scientific Model 1166, Niles IL). A thermo-couple was used in

the treatment chamber to monitor the temperature throughout the experiments. Treatment

time was 30 min and milk samples were kept at 4ºC until they were processed.

2.3 Yogurt preparation

In order to process yogurt, milk samples were heated at 45ºC and milk powder (2.8%)

was added and stirred until total solubilization was achieved. Plain commercial yogurt

(2.4%) containing Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus

acidophilus, Lactobacillus bifidus and Lactobacillus casei was used as starter culture.

Samples were incubated at 45ºC and the acidity (lactic acid) and pH were monitored from

the beginning of the process until it reached 0.65 to 0.70% and the appearance of the

288

yogurt was homogeneous. Afterwards, samples were stored at 4ºC for 24 h to proceed

with the following analysis. For the commercial yogurt, sample 1, acidity and pH were

measured as a control parameter.

2.4 Microscopy studies


Three different sample preparations were used as steps to look at the yogurt

microstructure in the microscope. The main difference between the procedures was the

time to prepare the sample. The HMDS (Hexamethyldisilazane) dehydration was used as

a control because that is the conventional procedure to prepare samples for microscopy

studies. The freeze-drying procedure was used in order to compare a faster method that

does not require the use of a fixative, and in which post-fixative and sample dehydration

takes place mainly by sublimation. Finally, the third method was similar to HMDS

dehydration, but took advantage of the microwave energy to dehydrate the samples faster.

2.4.1.1 HMDS dehydration

100 μg of yogurt was transferred to disposable 1.5 ml sterile plastic microcentrifuge

tubes. 0.5 ml of a solution of glutaraldehyde (2%) paraformaldehyde (2%) in 0.1 M

phosphate buffer (pH 7.2) was added to each microtube and allowed to undergo the

fixation process for 24 h at 4ºC. After that the fixation solution was washed for 10 min

with phosphate buffer (0.1M) and then two consecutive 10 min washes with cacodylate

buffer (0.1M). Post-fixed procedure consisted of adding 2% osmium tetroxide in

289

cacodylate buffer (0.1M) at 4ºC for 24 h. Samples were washed three times with

cacodylate buffer (0.1M) for 10 min each time.

Dehydration of samples was achieved with serial dilution solutions of ethanol

(30%, 50%, 60%, 70%, 95% and 100%). Each solution was maintained in contact with

the sample for 10 min and the last solution (100% ethanol) was used three consecutive

times.

After dehydration of the samples with ethanol, the second dehydration procedure

with Hexamethyldisilazane (HMDS) was carried out with the samples. Consecutive 15

min contact of samples with ethanol/acetone/HMDS solutions in different ratios (1:0:0,

1:1:0, 0:1:0, 0:1:1, 0:0:1, 0:0:1) were used. Air drying was used as a final step, leaving

the microcentrifuge tubes with the open lid inside of a hood for at least one night.

Samples were mounted in aluminum stubs and Gold plating (Sputter coater, Technics

Hummer V, Anatech, San José, CA) was used as a final step to view the samples in a

Hitachi S-570 (Japan, Tokyo) Scanning Electron Microscope (SEM) operating at 20 kV.

2.4.1.2 Freeze-drying

Yogurt samples were placed in disposable 1.5 ml sterile plastic microcentrifuge tubes.

Two batches of samples were used; the first batch without any fixative was placed

directly to freeze. In the second batch of yogurt samples a solution of glutaraldehyde

(2%) paraformaldehyde (2%) in 0.1 M phosphate buffer (pH 7.2) was used as a fixative

for one hour. After that samples were rinsed twice with filtered and deionized water for 5

min each time. Water was taken out and samples were placed to freeze. The top of the

tubes was covered with small paper balls. Liquid nitrogen was used as a freezing

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medium; microtubes were placed inside the medium for a few seconds in order to freeze

the samples. Samples were put together inside the jar of a freeze dryer (Virtis Model

#6201-3130, Gardiner, NY) at 13.33 Pa (100 millitorr) and -50ºC and held overnight.

Samples were taken out of the freeze dryer and placed in a vacuum dissicator. Samples

were mounted in aluminum stubs and Gold plating (Sputter coater, Technics Hummer V,

Anatech, San José, CA) was used as a final step to view the samples in a Hitachi S-570

(Japan, Tokyo) scanning electron microscope (SEM) operating at 20 kV.

2.4.1.3 Microwave dehydration

Microwave dehydration was used as a third sample preparation method. All the solutions

to be used in this procedure were placed on ice before starting in order to lower their

temperature. A lab Microwave Processor Pelco 3450 (Redding, CA) was used and the

temperature restriction was set at 30ºC. The standard fixative solution was glutaraldehyde

(2%) paraformaldehyde (2%) in 0.1 M phosphate buffer (pH 7.2), 300 μl of the solution

was added to the microtubes (1.5 ml sterile plastic microcentrifuge tubes) and 100 μl of

yogurt were placed in each disposable microtube. After that another 300 μl of the fixative

solution was added on top of the sample to fix it. In total, 600 μl of fixative solution was

used for each sample. Samples were placed inside of the microwave and fixed for 2 min

and 30 seconds at 100% of power. Afterwards, samples were taken out, the fixative was

removed and the samples were rinsed twice with 0.1 M Phosphate buffer, each time

during 5 min. Dehydration was carried out with serial dilution solutions of ethanol (30%,

50% and 60%) that were added to the tubes, and each solution in contact with the yogurt

sample was microwaved once for 40 s. Higher serial dilution solutions of ethanol (70%,

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80%, 90% and 100%) were used and each one was microwaved for 40 s twice. Each time

before the microwave dehydration the solution was replaced with a new one. Acetone

was used in combination with ethanol, the first time in a ratio of 1:1 and microwaved for

40 s; afterwards, acetone was used and microwaved for 40 s twice. HMDS and acetone

were used in a ratio of 1:1 for 15 min without microwave dehydration and this solution

was replaced with fresh HMDS and kept in the samples overnight to allow air drying of

the samples. Samples were mounted in aluminum stubs and Gold plating (Sputter coater,

Technics Hummer V, Anatech, San José, CA) was used as a final step to view the

samples in a Hitachi® S-570 (Japan, Tokyo) scanning electron microscope (SEM)

operating at 20 kV.


3.1 HMDS dehydration

In Figure 1 the comparison between the HMDS and microwave drying methods for

sample preparation is shown. At the top of the Figure the samples are representative of

commercial yogurt following the preparation of HMDS dehydration that usually takes 5

d. In the same Figure at the bottom both images correspond to natural yogurt but follow

the sample preparation for electron microscopy using microwave dehydration. As can be

observed, the images are very similar and microwave dehydration shows a better

resolution of the fat and protein structure of yogurt. This sample preparation takes only 3

h to prepare the sample for observation in the SEM. During sample preparation with

HMDS, chemical fixation takes place in the sample; glutaraldehyde is used first to

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crosslink proteins and the osmium tetroxide solution is useful to stabilize unsaturated

lipids (Kaláb et al., 1995).

3.2 Freeze-drying

It is worth mentioning that the use of freeze-drying sample preparation was not able to

process yogurt samples; despite the fact that this procedure takes only one day to prepare

the sample, images did not have good resolution and for that reason no images of this

methodology are shown in the present work. According to Chabot (1979), freeze-drying

is a sample preparation that presents many artifacts and drawbacks during processing of

the food samples, such as a very quick freezing in an attempt to avoid damage in the

tissue; sometimes cryoprotectors are required to preserve the sample, which is not

suitable for SEM. The images that were obtained with this technique in this research

could have low resolution because of the formation of large crystals that are not the

structure of the yogurt that were intended to be observed. As Chabot (1979) mentions, in

dairy products specifically liquid freeze drying can generate large ice crystals showing an

ice crystal structure in a protein solution. Also, liquid nitrogen is not useful for rapid

freezing when it is used near its boiling point because of the generation of an insulating

layer around the sample avoiding a quick freezing. Melting nitrogen slush is a better and

more recommendable cryo-fixative (Kaláb, 1981).

3.3 Microwave dehydration

In Figure 2 samples are representative of the thermo-sonicated yogurt using the

conventional HMDS dehydration for sample preparation. It is quite interesting to observe

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the new microstructure that yogurt samples have after sonication. The images shown in

Figure 1 have a more relaxed structure without an important differentiation between fat

or protein structure regardless of the sample preparation protocol. But in using thermo-

sonication as a pasteurization method of milk to process yogurt, the internal structure of

yogurt changes in an important way, as can be observed in Figure 2. A more compact

matrix of protein and fat globules is shown with a more homogeneous particle size.

Ultrasound generates a homogenized effect in the fat globules of milk; the main objective

of homogenizing milk is to divide the fat globules and clumps in milk as small as

possible in order to prevent them from rising to the top of the milk as a layer (Wu et al.,

2001). According to Dave and Shah (1998), some of the parameters that can affect the

size of the granules in yogurt are the kind of heat treatment, type of stabilizer, starter

culture and incubation time and temperature. Commercial yogurt has a porous protein

matrix formed of interconnected chains and clusters; the void spaces reflected in

microscopy images signify air compartments (Kaláb et al., 1995).

Finally, Figure 3 shows two important aspects of this research. The first aspect is

the structure in the detail of the thermo-sonicated yogurt. In these images the presence of

reduced size fat globules, proteins structure, lactose crystals and other milk components

can be observed. Structures were reduced in size because the action of ultrasound in the

milk components lead to a more homogeneous network, with other components such as

whey integrated as part of the new structure and avoiding the common problems in

yogurt such as syneresis. Whey separation or syneresis is one the quality aspects in

yogurt that has a main role in consumer preference and quality, as some authors mention

(Lee and Lucey, 2004; Wu et al., 2001; Dave and Shah, 1998). Also because of the

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reduction of fat globules, as observed in the same Figure, the color of yogurt was whiter

after the process, offering another important quality characteristic. Ultrasound by itself

showed a very good homogenization in yogurt and it was better as the treatment time was

longer, generating very small fat globules, higher water holding capacity, higher

viscosity, and changes in syneresis in accordance with the power used in the treatment

(Wu et al., 2001). Some studies conducted with manothermosonication in yogurt showed

that after the treatment at 40ºC, 117 μm of amplitude (20 kHz) and 2 kg/cm2 the samples

had better rheological properties than the control sample; stronger structures with

superior texture and better consistency and viscosity in yogurt were reported after

treatment with ultrasound, pressure and heat (Vercet et al., 2002).

The images presented in Figure 3 were achieved using the short and quick

microwave dehydration process that requires only 3 h to complete. Images are clear and

have high resolution with minor degradation because of the use of microwave energy.

Microwaves accelerate the dehydration process because the energy produced by these

waves are vibrating and accelerating water molecules inside the sample and generating

heat as consequence of the friction between them, drying the sample at the same time. As

Hassan et al. (1995) mention, SEM is a very useful instrument to study dairy gel

microstructure because of the excellent resolution and feasibility to characterize internal

and external surfaces. Combining the positive aspects of SEM with the advantages of

using microwave for dehydration of samples, this sample preparation protocol could be

very useful to successfully evaluate the microstructure of other food items that usually

takes a long time for processing.

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4. Conclusions

HMDS dehydration showed good resolution of yogurt images; however, better resolution

was achieved using microwave dehydration and the time consumed was shorter. Freeze-

drying was not a suitable procedure for sample preparation in yogurt samples. Thermo-

sonication of milk was useful as a pasteurization process to improve yogurt quality,

showing better homogenization and color, in addition to there being no syneresis

problems during storage of the product.

References

Chabot, J.F. 1979. Preparation of food science samples for SEM. Scanning

Electron Microscopy III. 279-298.

Dave, R.I. and Shah, N.P. 1998. The influence of ingredient supplementation on

the textural characteristics of yogurt. Australian Journal of Dairy Technology. 53(3):180-

184.

Hassan, A.N., Frank, J.F., Farmer, M.A., Schmidt, K.A., and Shalabi, S.I. 1995.

Formation of yogurt microstructure and three-dimensional visualization as determined by

confocola scanning laser microscopy. Journal of Dairy Science. 78:2629-2636.

Kaláb, M. 1981. Electron Microscopy of milk products: A review of techniques.

Scanning Electron Microscopy III. 453-472.

296

Kaláb, M. 1993. Practical Aspects of Electron Microscopy in Dairy Research.

Food structure. 12: 95-114.

Kaláb, M., Allan-Wojtas, P., and Miller, S.S. 1995. Microscopy and other

imaging techniques in food structure analysis. Trends in Food Science and Technology.

6:177-186.

Keenan, T.W., and Patton, S. 1995. The structure of Milk: Implications for

Sampling and Storage. In “Handbook of Milk Composition”. R.G. Jensen, Ed. Academic

Press. San Diego, California, pp: 5-50

Lee, W.J., and Lucey, J.A. 2004. Structure and Physical Properties of Yogurt

Gels: Effect of inoculation rate and incubation temperature. Journal of Dairy Science.

87:3153-3164.

McManus, W.R., McMahon, D.J., and Oberg, C.J. 1993. High-Resolution

Scanning Electron Microscopy of Milk Products: A New Sample Preparation Procedure.

Food Structure. 12: 475-482.

Vercet, A., Oria, R., Marquina, P., Crelier, S., and Lopez-Buesa, P. 2002.

Rheological properties of yogurt made with milk submitted to manothermosonication.

Journal of Agricultural and Food Chemistry.50:6165-6171.

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Wu, H., Hulbert, G.J., and Mount, J.R. 2001. Effects of ultrasound on milk

homogenization and fermentation with yogurt starter. Innovative Food Science and


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Figure 1. Microstructure of commercial yogurt using HMDS as sample preparation

technique (top); microstructure of commercial yogurt using microwave dehydration as

sample preparation technique (bottom). In all the pictures the protein and fat structure can

be observed.

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Figure 2. Microstructure of thermo-sonicated yogurt using HMDS for sample preparation technique showing clusters of yogurt in a

more compact structure.

300

Figure 3. Microstructure of thermo-sonicated yogurt using microwave dehydration for

sample preparation, showing these images the internal composition and structure of this

dairy product, lactose crystals, protein structures and fat globules modified in their

structure because of the sonication

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CHAPTER EIGHT

INACTIVATION OF Bacillus cereus SPORES IN MILK USING PULSED

ELECTRIC FIELDS-ENHANCED THERMAL PASTEURIZATION AND NISIN

AS A NATURAL ANTIMICROBIAL

Daniela Bermúdez-Aguirre, C. Patrick Dunne and


Abstract

Pulsed electric fields (PEF) is an emerging technology that is able to pasteurize milk;

however, some thermal resistant spores can survive and generate problems during

storage. The objective of this work was to study the inactivation of Bacillus cereus spores

in skim and whole milk under selected PEF treatments with and without refrigeration

cycles. Peak electric fields were 30, 35 and 40 kV/cm, thermal treatments used in

conjunction with PEF were from 40ºC to 65ºC, and the number of pulses was up to 20

pulses without refrigeration and up to 240 pulses with cooling conditions. The use of

nisin in low (10 IU/ml) and high (50 IU/ml) concentrations was tested in combination

with PEF and thermal treatments. Results showed a high resistance of spores to PEF

alone; mild thermal treatments (40ºC) had an antagonist effect in inactivation and 50ºC

enhanced spore death. Skim milk was a better medium than whole milk to inactivate

spores, and stronger thermal treatments (65ºC) achieved a slight increase in spore

reduction. The use of a high concentration of nisin was effective in combination with 40

kV/cm, 144 pulses and 65ºC with almost 3.5 reductions. Indeed, spores showed their

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resistance to preservation factors alone or in combination regardless of the media of

treatment or the severity of the processing conditions.

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1. Introduction

Bacillus cereus is a spore-forming, Gram-positive bacteria; its optimum growth

temperature is between 28 – 35ºC, the minimum growth temperature is 4 - 5ºC and the

maximum is 48ºC. The pH that allows its growth is 4.9 to 9.3. Its spores are heat resistant

and the strains of this bacillus from milk are responsible for the production of

diarrheagenic enterotoxin (Batt, 2000; Jay, Loessner and Golden, 2005). Bacillus cereus

is present in some foods such as dairy products like ice cream, milk powders, fermented

milks and pasteurized milks (Røssland et al., 2005), meat species (Penna and Moraes,

2002); cereals (Batt, 2000), and sauces and desserts (Van Opstal et al., 2004). Some

foods that are subjected to drying or heating can contain spores of this microorganism.

This microorganism can grow in low-acid foods after pasteurization during refrigerated

storage because of its characteristics (Van Opstal et al., 2004). Often, the presence of

Bacillus cereus in milk is attributed to raw milk and cross contamination because of a

lack of good cleaning practices in the processing equipment. In addition, this

microorganism can survive thermal processing and promote the so-called “broken cream”

that is related to proteolytic activity in milk (Batt, 2000). This quality defect is also called

sweet curdling (Batt, 2000; Hanson et al., 2005) because of the formation of a non-acid or

sweet curd that has a bitter off-flavor due to the precipitation of the casein (Hanson et al.,

2005). The presence of different species of bacillus is responsible for the reduction of the

shelf-life of milk (Hanson et al., 2005).

Pulsed electric fields (PEF) technology has shown positive results in the

inactivation of pathogenic and deteriorative microorganisms in liquid foods, minimally

reducing the fresh-like characteristics of the product and showing a better quality

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compared with the thermal treated product (Amiali et al., 2007; Węsierska and Trziszka,

2007), in addition to low energy requirements (Vega-Mercado et al., 1997). A synergistic

effect between PEF and thermal treatment has been reported in microbial inactivation

(Amiali et al., 2007). The mechanism of inactivation of cells using PEF is known as

electroporation, which generates small pores into the cell membranes and organelles

using high intensities, creating an irreversible process and disintegrating some cell

structures (García et al., 2007).

Nisin is a lantibiotic discovered in 1920, a 34 amino acid peptide produced by

Lactococcus lactis subspecies lactis by fermentation of a modified milk medium. Its

antimicrobial activity against Gram-positive bacteria has been shown in several products

(Guinane et al., 2005; Barbosa-Cánovas et al., 1998; Thomas and Delves-Broughton,

2005). The advantages of nisin as a food preservative are that it is not toxic, is produced

naturally, is heat stable, has excellent storage stability, is destroyed by digestive enzymes,

does not contribute to off-flavors, and it has a narrow spectrum of antimicrobial activity

(Jay et al., 1995). It is added to milk, cheese, dairy products, canned food such as

vegetables, mayonnaise and baby foods (Montville et al., 2001), liquid eggs, pasteurized

soups, crumpets, fruit juices, sauces, meat products such as bologna and frankfurter

sausages, beer, wine, fermented beverages, smoked salmon, shellfish, soymilk, fermented

cabbage, sauerkraut, kimchi, cooked potatoes products, pizza. This bacteriocin does not

change the taste of the product when is added (Thomas and Delves-Broughton, 2005).

The objective of this research was to study selected pulsed electric fields

treatments with and without other preservation factors such as thermal treatments and

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different nisin concentrations in the inactivation of Bacillus cereus spores in skim and

whole milk.


2.1 Milk samples

Pasteurized whole and skim milk were obtained from the Washington State University

Creamery. Milk was characterized in initial microbial loads (mesophilic bacteria and

Bacillus cereus spores) and was kept under refrigerated conditions (4ºC) until used.

2.2. Microbiological analysis

2.2.1. Growth and inoculum of Bacillus cereus spores

Bacillus cereus spores (ATCC 7004) were rehydrated with 5 ml of sterile Nutrient Broth

(Bacto: Becton, Dickinson and Co., Sparks, MD). After 30 min the cell suspension was

inoculated into 100 ml of Nutrient Broth and incubated at 30ºC with continuous agitation

at 225 rpm in an orbital shaker. The spectral absorbance was read each hour at 540 nm

until reach the stationary phase approximately after 9 h. One ml of culture from the early

stationary phase plus one ml of sterile glycerol were stored at -21ºC for not more than 2

months. Milk samples were inoculated with Bacillus cereus spores ATCC 7004 from the

stock suspensions in a ratio to reach 108 and 104 spores/ml depending on the experiment.

2.2.2. Enumeration of mesophilic bacteria and Bacillus cereus spores

Serial dilutions were made in Peptone water (0.1%) with samples taken from unprocessed

milk to evaluate the initial microbial load of mesophiles and quality of milk. After that,

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samples were pour-plated in Plate Count Agar (Difco, Becton, Dickinson and Co.,

Sparks, MD), dishes were incubated at 35ºC for 48 h, and bacteria were counted. For

Bacillus cereus spores, samples were heated at 80ºC for 30 min to germinate the spores

and kill any vegetative cells. After that, samples were cooled down at 50ºC for 5 min and

pour-plated in Nutrient agar (Difco, Becton, Dickinson and Co., Sparks, MD) Dishes

were incubated at 35ºC for 48 h, and then spores loads were counted.

2.3 Nisin

Nisaplin® was obtained from Danisco USA, Inc. (New Century, KS) with a composition

of nisin of 1000 IU per mg. A standard solution of 1000 IU/ml was prepared by

suspending nisin in sterilized distilled water. Two concentrations were used in milk; the

low concentration sample had 10 IU/ml of nisin and the high concentration sample had

50 IU/ml of nisin, using the allowed limits of this bacteriocin in dairy products (Thomas

and Delves-Broughton, 2005). Nisin solution was added to milk samples immediately

before pouring the samples into the PEF chamber.

2.4 Pulsed electric field treatment

A pilot plant PEF system manufactured by Physics International (San Leandro, CA) and a

cylindrical concentric-electrodes treatment chamber (Qin et al., 1995) was used to apply

the desired treatments to the milk. The continuous treatment chamber had a volume of

25cm3 and a gap between the stainless steel electrodes of 0.6 cm. Frequency was kept

constant at 10 Hz. Flow rate of milk was set up and controlled with a rotary pump

(Masterflex 7654-00, Cole Parmer Instruments Co., Chicago, IL).

307

2.4.1 Experiments without recirculation/refrigeration cycles

Peak electric fields of 30, 35 and 40 kV/cm were used in combination up to 20 pulses of

around 2.5 μs pulse-width. Inlet and outlet temperatures were monitored with digital in-

line thermocouples (Cole-Palmer, Vernon Hills, IL) recording the maximum temperature.

Energy consumption, electric field intensity, pulse width and pulse shape were directly

measured by a digital oscilloscope (Hewlett-Packard 54530A, Colorado Springs, CO)

connected to the treatment chamber through high voltage probes. Three different sets of

experiments were run: the first was without any thermal treatment, using only the heat

generated by the PEF treatment; the second experiment used, in addition to the PEF, a

mild thermal treatment at 40ºC; and the last experiment used a more intense heat

treatment at 50ºC together with the PEF process. These experiments were conducted in a

batch system.

2.4.2 Experiments without recirculation/refrigeration cycles

Electric fields of 30 and 40 kV/cm were used in combination with up to 240 pulses of


line thermocouples (Cole-Palmer, Vernon Hills, IL) and recirculation and refrigeration

systems were used between each set of pulses to cool down the milk. Two groups of

experiments were conducted: the first group consisted of maintaining the temperature at

55ºC and applying PEF at the same time; the second group of experiments kept the

temperature at 65ºC while also using PEF treatment. Energy consumption, electric field

intensity, pulse width and pulse shape were directly measured by a digital oscilloscope

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(Hewlett-Packard 54530A, Colorado Springs, CO) connected to the treatment chamber

through high voltage probes.

For experiments using nisin in milk as an antimicrobial, electric peaks of 40

kV/cm, 144 pulses (2.5 μs) in combination with 65ºC were used as processing conditions

to evaluate high and low concentration doses of nisin. Each experiment was performed at

least in duplicate.

2.5 Thermal treatment

For thermal treatments the same PEF continuous circuit system was used but without the

application of electric fields; samples were taken each 5 min of treatment during 60 min

and skim and whole milk were tested for Bacillus spores inactivation.


This research was divided into two parts in order to study and explain the behavior of

Bacillus cereus spores under pulsed electric fields treatments in conjunction with other

preservation factors. The first part corresponds to the use of high electric fields using a

short number of pulses without any refrigeration system or recirculation of the product. In

these experiments the outlet temperature was not controlled; it was only recorded in order

to see the delta of temperature generated because of the intensity of the pulses. Skim and

whole milk were used as media of treatment, and a high concentration of spores (108

spores/ml) was inoculated into the milk before processing it with PEF. In Figure 1, the

inactivation of spores is shown in skim milk as along with the temperature profile at the

inlet and outlet of the PEF chamber. A selected number of pulses was applied with 35

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and 40 kV/cm as electric peaks, showing similar inactivation patterns because of the

similitude in the intensity. However, after 20 pulses (2.5 μs) only 1.5 log reduction of

spores was achieved. As can be observed in the same Figure, the increase of temperature

according to the number of pulses also depends on the applied voltage, showing the

maximum delta of temperature of the treatment at 40 kV/cm and 20 pulses (Δ 52.8ºC).

Low or room temperature did not have an important effect in the inactivation of cells

regardless of the use of pulsed electric fields; Listeria innocua cells in McIllvaine buffer

did not show important inactivation at low temperatures, and the use of mild thermal

treatments with temperatures above 40ºC has been shown to increase the effectiveness of

PEF as an inactivation treatment (Sepúlveda et al., 2005).

In Figure 2, the combination of a mild thermal treatment (40ºC) plus the use of up

to 15 pulses at 35 and 40 kV/cm is shown in the inactivation of spores. According to

some references, the use of heat plus PEF could result in a synergic effect in the

inactivation of some bacteria (Sepúlveda et al., 2005). However, in accordance with the

characteristics of the spores, the use of this mild thermal treatment did not show a

synergic effect. Instead, there was an antagonist effect, even promoting the activation of

spores. As can be observed in the same Figure, the delta of temperature was increased

when samples were heated at the entrance at 40ºC, with a final value of 70.4ºC

(Δ 30.6ºC) at 40 kV/cm and 15 pulses. Similar results were obtained when spores of

Bacillus cereus in skim milk were pressurized up to 200 MPa and temperatures from 30

to 40ºC that caused almost 2 log of spore germination (Van Opstal et al., 2004)

Higher temperature (50ºC) was tested in combination with the PEF treatment in

skim and whole milk, as shown in Figure 3. In this case, the use of 50ºC plus 40 kV/cm,

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10 pulses was able to inactivate almost 3 log reduction of spores in skim milk. However,

some technical problems were observed at the time regardless of the kind of milk. When

the temperature was increased to higher than 80ºC, some arcing problems occurred. The

production of small bubbles inside the milk during processing resulted in explosions in

the chamber because of the null electrical conductivity of the air. Electrical breakdown of

the system is one of the limiting factors in PEF technology and it is produced because of

the presence of bubbles that act as impurities in the liquid (Góngora-Nieto et al., 2003).

In Figure 3, the antagonist effect of mild thermal treatment (40ºC) and the synergistic

effect of more intense thermal treatment (50ºC) can be observed. Similar results were

observed in whole milk.

In order to explore the feasibility of using thermal treatment for enhancing the

PEF treatment, some temperatures were used to again test the Bacillus cereus spores, as

shown in Figure 4. Extreme temperature and time conditions were used to study the

possibility of using some of them in combination with PEF. The lowest tested

temperature (45ºC) again showed the germination of spores through the treatment.

Indeed, 55ºC showed a slight inactivation in spores, but only after 30 min, showing the

best results at 65ºC and 75ºC with more than 4 log reductions after 10 min of treatment.

However, the use of high temperatures (72ºC – 76ºC) for some seconds to pasteurize milk

using High Temperature Short Time can activate the spores of Bacillus present in milk

and even recovery of some of the injured spores during the treatment (Hanson et al.,

2005). Even Bacillus cereus spores have decimal reduction times at 100ºC of 2.2 to 5.4

min (Van Opstal et al., 2004), showing their resistance to heat treatment.

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On the basis of those findings, new PEF experiments were designed, but with

some restrictions. The new series of experiments were conducted with minor spores loads

(104 spores/ml) to try to simulate a more real case scenario; as PEF treatment lasts only

microseconds, 55ºC was chosen to test its ability to inactivate spores together with

electricity while not elevating the temperature too much and trying to keep the treatment

as nonthermal. Finally, 65ºC was chosen as the limit temperature for PEF-thermal

enhanced treatments to ensure the safety and functionability of the PEF chamber and to

avoid any arcing problems because of the increase of temperature. Another new variable

was to increase the number of pulses up to 240 pulses (2.5 μs) using refrigeration cycles

between each set of pulses and recirculation loops of milk.

In Figure 5, the effect of the electric field intensity is shown for skim milk; 30 and

40 kV/cm were tested with 240 pulses, indicating an important difference according to

the strength of the field. 30 kV/cm did not inactivate more than 0.5 log even after the

large number of pulses; meanwhile 40 kV/cm was enough to inactivate almost 2 log of

spores with a few pulses and retained this behavior until the end of the treatment.

Comparing Figure 5 with Figure 3, the same conditions were tested, but the difference in

inactivation could be because of the different initial spore load being lower that shown in

Figure 5. Some studies using PEF processing for inactivation of yeasts (Saccharomyces

cerevisiae) in liquid and solid model systems tested the initial concentration of cells (103

to 108 cfu/ml) and found that inactivation is increased as the initial load of

microorganisms is decreased (Donsì et al., 2007). In Figure 5 the temperature profile is

shown for the corresponding experiment, not going higher than 55ºC.

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In Figure 6, the effect of the type of milk is shown for the inactivation of spores.

Skim milk and whole milk were tested as the media of treatment; the highest voltage (40

kV/cm) was used for this comparison and the same number of pulses. As can be observed

in the same Figure, outlet temperature was kept at 55ºC as well. Inactivation occurred

first in skim milk and then in whole milk, probably because of the protective effect of the

fat in milk; however, after 90 pulses inactivation behavior was very similar in both kinds

of milks, raising 2 log reductions. Although some authors mention the protective effect of

fat for bacteria, others affirm that a high content of fat enhances the microbial

inactivation if different frequencies of PEF are used, although at this moment no theory

has been totally validated (Picart et al., 2002).

In Figure 7, the comparison between two kinds of milk and two different

processing conditions (30 and 40 kV/cm and 240 pulses) is shown using a stronger

thermal treatment at 65ºC. 30 kV/cm was not enough to inactivate spores; even in skim

milk sporulation was observed with a small number of pulses. After the treatment was

completed, only 1 log reduction was achieved with this electric strength. 40 kV/cm plus

65ºC was more effective to inactivate spores, as can be observed in the same Figure; after

240 pulses almost 2.5 log reductions were achieved in skim milk, also showing the effect

of the intensity of the electric field. Other reports have also shown this effect in the

inactivation of different microorganisms; as the intensity of the electric field strength

increases, the inactivation is enhanced (Picart et al., 2002; Sepúlveda et al., 2005)

As can be observed, resistance of spores to PEF treatment even with the use of

thermal treatments and a high number of pulses is very difficult to inactivate them.

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Spores are very resistant regardless the adverse conditions of the process; only in skim

milk do spores seem to be slightly unprotected and favor inactivation.

Finally, the use of the most severe conditions tested in this work was used in

conjunction with nisin added as a natural antimicrobial. 40 kV/cm and 65ºC were tested

in skim and whole milk using two nisin concentrations. The lowest concentration was 10

IU of nisin/ml showing important inactivation in skim milk; almost 3 log reductions were

achieved with a few pulses. Despite the use of the same concentration in whole milk and

application of the same processing conditions, inactivation was limited to only 1.5 log,

probably because of the effect of fat. Some authors mention that the effect of fat in milk

affects the activity of nisin as an antimicrobial; it decreased 33% in skim milk and 50% in

1.29% fat milk (Sobrino-López and Martín-Belloso, 2006) and the effect of heat

treatment in Bacillus cereus was enhanced when nisin was used in milk (Vessoni Penna

and Moraes, 2002). But when high concentrations of nisin (50 IU/ml) were tested under

40 kV/cm, 65ºC and 144 pulses, inactivation was very similar regardless the kind of milk

at the beginning of the treatment, but being favorable at the end for skim milk with

almost 3.5 log reduction of spores. Synergic effects have been reported in other

microorganisms using selected combinations of PEF treatments plus nisin, e.g. Listeria

monocytogenes in liquid whole egg (Calderón-Miranda et al., 1999a) and skim milk

(Calderón-Miranda et al., 1999b); Staphylococcus aureus in skim milk (Sobrino-López

and Martín-Belloso, 2006); and Micrococcus luteus in phosphate buffer (Dutreux et al.,

2000).

314

4. Conclusions

Pulsed electric fields resulted in a favorable inactivation technique for spores of Bacillus

cereus when it was used in combination with temperature (65ºC) and 40 kV/cm as field

strength and concentration of nisin (50 IU/ml) regardless the kind of milk. Despite all the

tested combinations to inactivate spores in this research, the hurdle technology using

three preservation factors such as electricity, temperature and antimicrobials showed a

synergic effect and were useful to achieve a good inactivation level of spores.

References

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effect of temperature and pulsed electric field on inactivation of Escherichia coli

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689-694.

Barbosa-Cánovas, G.V., Pothakamury, U.R., Palou, E., and Swanson, B.G. 1998.

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Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin.


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Donsì, G., Ferrari, G., and Pataro, G. 2007. Inactivation kinetics of

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Dutreux, N., Notermans, S., Góngora-Nieto, M.M., Barbosa-Cánovas, G.V., and

Swanson, B.G. 2000. Effects of combined exposure of Micrococcus luteus to nisn and

pulsed electric fields. International Journal of Food Microbiology. 60:147-152.

García, D., Gómez, N., Mañas, P., Raso, J., and Pagán, R. 2007. Pulsed electric

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Góngora-Nieto, M.M., Pedrow, P.D., Swanson, B.G., and Barbosa-Cánovas, G.V.

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pulsed electric fields. Lebensmittel-Wissenschaft und-Technologie. 38:167-172.

Sobrino-López, A., and Martín-Belloso, O. 2006. Enhancing inactivation of

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1325.

319

-2.0

-1.5

-1.0

-0.5

0.0

0.5

0 5 10 15 20 25

Number of pulses

Log

(N/N

o)

40 kV/cm 35 kV/cm

Electric field

(kV/cm)

Number of pulses Inlet temperature

(ºC)

Outlet temperature

(ºC)

0 13.0 23.0

5 5.6 23.0

10 6.0 34.6

15 6.6 50.1

40

20 9.0 61.8

0 13.0 23.0

5 5.7 21.3

10 6.2 34.6

35

15 7.1 48.9

20 8.2 59.1

Figure 1. Inactivation of Bacillus cereus spores in skim milk using pulsed electric fields

of 35 and 40 kV/cm without refrigeration (top); temperature profile for the same

experiment (bottom)

320

-2

-1.5

-1

-0.5

0

0.5

0 5 10 15

Number of pulses

Log

(N/N

o)

35 kV/cm + 40C 40 kV/cm + 40C

Pulsed electric field

(kV/cm)

Number of pulses Inlet temperature (ºC) Outlet temperature

(ºC)

5 37.4 54.0

10 37.1 65.2

15 37.0 70.0

5 40 57.5

10 40 68.4

35 kV

40 kV

15 39.8 70.4


of 35 and 40 kV/cm plus 40ºC as mild thermal treatment (top); temperature profile for the

same experiment (bottom)

321

-3.5

-2.5

-1.5

-0.5

0.5

0 5 10 15 20

Number of pulses

Log

N/N

o

35 kV/cm + 40C 35 kV/cm 35 kV/cm + 50C

Electric field

(kV/cm)

Number of pulses Inlet temperature

(ºC)

Outlet temperature

(ºC)

35 0 13.0 23.0

5 5.7 21.3

10 6.2 34.6

15 7.1 48.9

20 8.2 59.1

35 5 37.4 54.0

10 37.1 65.2

15 37.0 70.0

35 5 49.8 68.8

7 49.8 72.8

10 49.3 78.0


of 35 and 40 kV/cm plus 50ºC as thermal treatment (top); temperature profile for the


322

-8.0

-6.0

-4.0

-2.0

0.0

2.0

4.0

0 10 20 30 40 50 60

Time (min)

log

(N/N

o)

45C 55C 65C 75C

35

40

45

50

55

60

65

70

75

80

85

0 10 20 30 40 50 60

Time (min)

Tem

pera

ture

(C)

T inlet 45C T outlet 45C T inlet 55C T outlet 55C T inlet 65CT outlet 65C T inlet 75C T outlet 75C

Figure 4. Inactivation of Bacillus cereus spores in skim milk thermal treatments at 45ºC,

55ºC, 65ºC and 75ºC (top); temperature profile for the same experiment (bottom)

323

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

-10 40 90 140 190 240

Number of pulses

Log

(N/N

o)

40 kV/cm SM 30 kV/cm SM

0

10

20

30

40

50

60

0 50 100 150 200

Number of pulses

Tem

pera

ture

(C)

T inlet 40 kV/cm T outlet 40 kV/cm T inlet 30 kV/cmT outlet 30 kV/cm LIMIT


of 30 and 40 kV/cm plus 55ºC as thermal treatment (top); temperature profile for the


324

-2.5

-2

-1.5

-1

-0.5

0-10 40 90 140 190 240

Number of pulses

Log

(N/N

o)

40 kV/cm SM 40 kV/cm WM

0

10

20

30

40

50

60

0 50 100 150 200 250

Number of pulses

Tem

pera

ture

(C)

Whole milk Inlet Whole milk Outlet Skim milk InletSkim milk Outlet LIMIT

Figure 6. Inactivation of Bacillus cereus spores in skim and whole milk using pulsed

electric fields of 40 kV/cm plus 55ºC as thermal treatment (top); temperature profile for

the same experiment (bottom)

325

-3.5-3

-2.5-2

-1.5-1

-0.50

0.51

1.5

0 50 100 150 200 250 300

Number of pulses

Log

N/N

o

30 kV/cm Skim 30 kV/cm Whole 40 kV/cm Whole 40 kV/cm Skim

0

10

20

30

40

50

60

70

80

0 50 100 150 200 250

Number of pulses

Tem

pera

ture

30 kV/cm - Skim Inlet 30 kV/cm - Skim Outlet 30 kV/cm - Whole Inlet30 kV/cm - Whole Outlet 40 kV/cm - Whole Inlet 40 kV/cm - Whole Outlet40 kV/cm - Skim Inlet 40 kV/cm - Skim Outlet LIMIT

Figure 7. Inactivation of Bacillus cereus spores in skim and whole milk using pulsed

electric fields of 30 and 40 kV/cm plus 65ºC as thermal treatment (top); temperature

profile for the same experiment (bottom)

326

-5

-4

-3

-2

-1

00 50 100 150

Number of pulses

Log

(N/N

o)

SM LC SM HC WM LC WM HC

0

10

20

30

40

50

60

70

80

12 32 52 72 92 112 132

Number of pulses

Tem

pera

ture

(C)

SM LC Inlet SM LC Outlet SM HC InletSM HC Outlet WM LC Inlet WM LC OutletWM HC Inlet WM HC Outlet LIMIT

Figure 8. Inactivation of Bacillus cereus spores in skim (SM) and whole milk (WM)

using pulsed electric fields of 40 kV/cm plus 65ºC as thermal treatment and Low

Concentration (LC) of nisin (10 IU/ml) and High Concentration (HC) of nisin (50 IU/ml)

(top); temperature profile for the same experiment (bottom)

327

CHAPTER NINE

ELECTRODEPOSITING OF MILK MATERIALS DURING PULSED

ELECTRIC FIELDS PROCESSING

Daniela Bermúdez-Aguirre, Jaime A. Yáñez, C. Patrick Dunne,

Neal M. Davies, Gustavo V. Barbosa-Cánovas

Abstract

Pulsed electric fields (PEF) treatment of milk has been shown to have positive effects on

the inactivation of pathogenic bacteria and it is considered a viable option for

pasteurization of milk. However, severe conditions are required during processing to

inactivate spores in milk. These processing conditions not only achieve the inactivation

of spores, but can also generate electrodeposition of milk materials as well as erosion of

the PEF chamber electrodes. The objective of this study was to analyze the composition

of milk and the milk layer formed in the electrodes of the chamber during PEF

processing. Tested conditions were those previously required to inactivate spores (40

kV/cm, 50ºC, 240 pulses of 2.5 μs) in raw whole milk. Milk was characterized before and

after processing according to its physicochemical properties, and a proximal analysis was

carried out with a LactiCheckTM analyzer. The Bradford Coomassie blue dye-binding

method was also used to test total protein content. Results showed that after PEF

processing 0.20% of the milk was deposited as a layer inside the electrode and that the

final composition of milk changed significantly. Fat content ranged from 3 (control) to

2.63%; protein content was reduced from 3.26 to 2.85%; density changed from 1029.8 to

328

1025.7 kg/m3; nonfat solids changed from 8.75 to 7.65%; pH ranged from 6.3 to 6.44.

The Bradford Coomassie blue analysis confirmed a decrease in total protein of 1.02%.

These changes in milk are the result of extreme processing conditions that are necessary

to inactivate spores. Therefore, PEF treatment can inactivate pathogens in milk under

milder processing conditions, with minor effects on milk's composition as well as

important energy and cost savings compared to conventional pasteurization, and

avoidance of the electrodeposition of milk materials because of shorter processing times.

Industrial relevance

Pulsed Electric Field processing is currently under use in the industry to pasteurize juice;

this technique also offers important savings in energy, time and money to pasteurize

milk. Nevertheless, some spores can reduce the storage life of milk, and extreme

conditions are required to inactivate them. During the inactivation process of spores, the

severe conditions of the required PEF processing show fouling and erosion of the

electrode. This research deals with the composition of the layer (fouling) of the electrode

and the changes in milk after processing.

329

1. Introduction

Pulsed Electric Fields technology has been used to inactivate target microorganisms such

as Escherichia coli, Listeria innocua, Micrococcus luteus and Staphylococcus aureus in

different media such as skim milk, liquid egg, fruit juices and buffer solutions. In most

cases inactivation with PEF in combination with mild thermal treatments has been

successful in inactivating the microorganisms, as many of the available references show:

Sobrino-López and Martín-Belloso, 2006; Dutreux et al., 2000; Calderón-Miranda et al.,

1999a, b; Sepúlveda et al., 2005; Martín-Belloso et al., 1997; Heinz et al., 2003; Qin et

al., 1998, to name a few. When food is exposed to high-voltage-short pulses through

electrodes into a chamber, pasteurization standards are achieved. The basic components

of the electrical circuit of a PEF system are power supplies, switches, capacitors,

inductors, resistors and treatment chambers containing the electrodes (Góngora-Nieto et

al., 2002). Different inactivation conditions have been tested, including changing the

number of pulses, intensity of the electric field, temperature, treatment medium, using

natural antimicrobials, etc., although the effectiveness of the treatment depends mainly on

the field strength and the number of pulses (Odriozola-Serrano et al., 2006). Electric

fields can be in the range from 2 to 87 kV/cm, showing effectiveness in bacterial

inactivation at 20-50 kV/cm (Góngora-Nieto et al., 2002). However, some studies in

spore and enzyme inactivation report more severe processing conditions than those

mentioned for bacteria. The resistance of spores could be because of the smaller size,

rounder shape and their intrinsic protection against different inactivation factors

(Góngora-Nieto et al., 2002).

330

Chamber electrodes are often made of stainless steel in order to satisfy the

sanitary and chemicals requirements for material that is to be in contact with food

(Góngora-Nieto et al., 2002). Stainless steel electrodes are made of iron (62-69%),

chromium (16-18%), nickel (10-14%) and manganese (2%) (Roodenburg et al., 2005a).

The electric currents can generate electrochemical reactions in the chamber that can

affect food quality, and generate electrode fouling and electrode corrosion. Previous

studies have been conducted with the toxic products formed in food and electrode

corrosion (Morren et al., 2003). Some products, such as milk and eggs, have shown

corrosion or deposit of solids onto the high voltage electrode, probably because of

electrolysis of the product (Góngora-Nieto et al., 2002). The main mechanism of mass

transfer during the use of high voltages is migration (Morren et al., 2003).

Determination of protein in food is commonly tested using the official Kjeldahl

method, but one of the disadvantages of this method is the long time required to process a

sample. However, spectrophotometric methods are easy to perform and are based on

chemical reactions. The Bradford method is useful to determine total proteins because of

the interaction between the dye (Coomassie Blue) and proteins that shows higher

sensitivity for proteins, quantification of protein nitrogen, and easy and fast carry out

(Kamizake et al., 2003).

The aim of this work is to determine the physicochemical and compositional

changes in milk after pulsed electric fields processing, as well as to analyze the

composition of the electrodeposited layer into the chamber electrode when severe

processing conditions are used to inactivate spores in milk.

331


2.1 Samples

Raw cow’s milk was obtained from the Washington State University Creamery. Raw

milk was characterized by the following parameters: pH, acidity, color and proximal

analysis (butter fat, protein, solids non-fat and density). Milk was kept under refrigerated

conditions (4ºC) until used.

2.2 PEF processing

A pilot plant PEF system manufactured by Physics International (San Leandro, CA) and a

cylindrical concentric-electrodes treatment chamber (Qin et al., 1995) was used to apply

the desired treatments to the milk. The continuous treatment chamber had a volume of

25cm3 and a gap between the stainless steel electrodes of 0.6 cm. Frequency was kept

constant at 10 Hz and the flow was 490 ml/min. Flow rate of milk was set up and

controlled with a rotary pump (Masterflex 7654-00, Cole Parmer Instruments Co.,

Chicago, IL). Peak electric field of 40 kV/cm was used in combination with 240 pulses of


line thermocouples (Cole-Palmer, Vernon Hills, IL) maintaining 50ºC as maximum

processing temperature. Energy consumption, electric field intensity, pulse width and

pulse shape were directly measured by a digital oscilloscope (Hewlett-Packard 54530A,

Colorado Springs, CO) connected to the treatment chamber through high voltage probes.

332

2.3 LactiCheckTM milk analyzer

Analysis of butter fat and protein content, solids non-fat and density were carried out

using a LactiCheck™ LC-01 Milk Analyzer (Page & Pedersen, International Ltd.,

Hopkinton, MA). The performance of this equipment is based on high frequency

ultrasound. The milk analyzer was validated to determine butter fat content (Gerber) and

protein (Kjeldahl) in milk, according to Association of Analytical Chemists (A.O.A.C.,

1986) methodology. The equipment was previously calibrated with raw cow’s milk, and


triplicate. No prior preparation was required for the PEF processed samples.

2.4 Color




ml of raw and PEF processed milk samples were poured into sterile plastic bags. A white

ceramic plate was used for standardizing the instrument (L* = 93.4, a* = -0.67, b* =

0.78).


parameters L*, a* and b* and comparing the different treatments with the raw milk from

the beginning to end of storage life:

( ) ( ) ( )2*2*2** baLE Δ+Δ+Δ=Δ

333


⎟⎟⎠

⎞⎜⎜⎝

⎛= −

*

*1* tan

abh


( ) 21

*** 22

baC +=

2.5 pH and titratable acidity

pH was determined by direct immersion of the electrode with a potentiometer (Orion

Research Inc., Boston, MA), and titratable acidity was also determined using a

potentiometer. Ten ml of raw or processed milk was poured into a beaker with 20 ml of

distilled water with constant agitation. Milk was titrated at room temperature with 0.1 N

NaOH solution. The end point was reached when the potentiometer showed 8.3. Acidity

was expressed as a percentage of lactic acid (1 ml of 0.1 N NaOH = 0.009 g of lactic

acid). Each sample was again measured in triplicate.

2.6 Protein Assay

Aliquots of whole milk, milk after PEF treatment and milk residue were collected. The

milk residue was reconstituted in HPLC water and ethyl acetate to account for the

original content of water and fat content (3.2% fat content in whole milk). The Bradford

Coomassie blue dye-binding method (Bradford, 1976), available commercially from Bio-

Rad (Richmond, CA) was used for total protein determination.

334

The Bio-Rad Protein Assay is a dye-binding assay in which a differential color

change of a dye occurs in response to various concentrations of protein. The absorbance

maximum for an acidic solution of Coomassie® Brilliant Blue G-250 dye shifts from 465

nm to 595 nm when binding to protein occurs. The Coomassie blue dye binds to

primarily basic and aromatic amino acid residues, especially arginine.

A bovine serum albumin protein standard (5 g/dL albumin) (Bio-Rad, Richmond,

CA) was used in the protein assay. Standards were diluted with HPLC water to protein

concentrations ranging from 0.05 to 0.4 mg/mL to initially determine the linear working

range of the assay (Figure 1).

To perform the test, 10 μl of each standard and sample solution were aliquoted

into separate microtiter plate wells (samples were run in triplicate). Then, 200 μl of

diluted dye reagent was added to each well. The plate was shaken for 30 s in a plate

orbital shaker. The samples were incubated to at least 5 min and absorbance was read at

595 nm.


The treatment was performed at least in duplicate, and the physicochemical and

composition characteristics were evaluated in triplicate for each sample. Statistical

analysis of the data was performed using a Microsoft Excel program. Analysis of

variance (ANOVA) was calculated with the SAS program (SAS Institute, Cary, NC

1999), and a confidence level α of 0.05 was used to evaluate significant differences.

335


3.1 Physicochemical and composition parameters of milk after PEF processing

Table 1 presents the physicochemical and composition parameters of milk before and

after processing. Regarding pH, raw whole milk showed 6.30 as initial pH; however,

after processing there was an increase in the alkalinity of the medium (6.44) that was

observed also in the lactic acid content of the milk with a decrease of 0.024%. These

results indicate that during PEF processing some alkali products are probably generated

in milk because of the interaction between the food and the electricity. The main changes

to food quality induced by the interaction with electric current discharge into the

electrode are in the chemical structure of liquids, which are mainly generated close to the

electrode surface. Production of some products such as free oxygen, hydrogen, hydroxyl,

hydroperoxyl radicals (Reyns et al., 2004), hydrogen peroxide (H2O2) and small particles

of electrode material are found in the food, although the main products are produced

because of the electrolysis of water molecules (Morren et al., 2003) and other food

components in the interface electrode-food (Saulis et al., 2007). pH and lactic acid

content of milk were significantly different (p < 0.05) before and after the PEF

processing.

Other physicochemical properties that were studied in milk after processing were

the density and solids non-fat content. Density remained almost unchanged, as can be

observed in Table 1; whereas the solids non-fat content decreased from the raw milk to

1.1% in the PEF milk. Significant differences (p<0.05) were found between raw milk and

PEF treated milks in both physicochemical properties. This decrease in solids non-fat can

be attributed to the electrodeposition of milk materials into the electrode that was forming

336

a rubber layer. The solids non-fat are usually called serum solids (Potter, 1986) and

include casein, lactalbumin, lactose, calcium, phosphorus and riboflavin, among others

(IDFA, 2006).

Composition parameters are presented in Table 1. Important changes were

reported in fat and protein content of milk after PEF processing. Fat was decreased from

3% to 2.63% and protein from 3.26% to 2.85%. These changes can be attributed to the

electrode fouling during the processing of milk that showed a layer, as observed in Figure

2. In accordance with the composition analysis, the fouling of the electrode that is shown

in Figure 2a would contain protein, fat and solids non-fat. However, other compounds

can be produced during the treatment because of the electrolysis of water, and more when

milk is used because of the high moisture content of this product.

In Figure 2b the erosion of the electrode is shown by the discoloration on its

surface; also, at the end of the treatment some dark solids were observed as precipitated

in milk that are probably metal components of the electrode. Some studies have shown

that the presence of metal particles from electrode material (stainless steel) is increased as

the voltage increases, and as the frequency decreases. At 150 V peak-peak and 10 Hz, the

concentrations of metal in demineralized water with NaCl (conductivity of 385 mS/m)

was Cr 200 μg/l, Fe 940 μg/l, Mn 50 μg/l and Ni 270 μg/l after 2 h of treatment.

Electrode corrosion can be avoided by using short pulses (Morren et al., 2003), and also

by polishing the electrodes because the surface composition (Cr2O3) is more chemical

resistant than Fe2O3, releasing less dissolved iron (Roodenburg et al., 2005a).

Experiments performed in orange juice and electric field of 30 kV/cm and 6.7 pulses at

room temperature showed that the presence of metal particles is below the European

337

limits established for this product, even the high pH could enhance the electrode

corrosion (Roodenburg et al., 2005b). Initial fat and protein content were also

significantly different (p < 0.05) from those reported after PEF processing, as shown in

Table 1.

Regarding color changes in milk, there was a slight decrease in the luminosity

(L*) after processing (Table 2), exhibiting a net color change of 1.12 between the

unprocessed and PEF milks. The quality color indicator, h* shows the point in the color

space according to angle: reddish 0º, yellowish 90º, greenish 180º and bluish 270º; for

raw milk the color was closer to the yellowish region (75.01º), while the PEF milk moved

its hue to the reddish region (71.23º); this change could be attributed to the loss of

riboflavin after processing that produces a yellow color in some products. Finally, the

chroma or saturation index that indicates the degree of saturation, purity or intensity of

color showed a decrease of this value for the PEF processed milk, moving this value to

the neutral gray point (0), probably because of the presence of some metal particles in the

product.

3.2 Protein analysis

In order to test the composition of the layer formed in the electrode after processing, a

second protein analysis was carried out. The total weight of this layer was 2.08 g after

processing one liter of raw whole milk. The Bradford Coomassie method was used to

determine the total protein content. The average protein content in raw milk was 2.35%,

whereas in PEF milk the average protein content was 2.25% (Figure 3). Protein found in

the layer of the fouling electrode was 0.023%, meaning that the protein content in the

338

PEF liquid milk plus the layer was 2.27%. These values are quite different from those

reported in Table 1, in which raw milk had 3.26% and PEF milk had 2.85% of protein

content. The reasons for these differences (significantly p < 0.05) are probably because of

the method of protein determination. The Bradford Coomassie method is useful to

determine total proteins; however, the dye does not react with small peptides or amino

acids (Kamizake et al., 2003). So, if PEF is denaturizing some proteins in peptides and

amino acids, as the value obtained with LactiCheckTM analyzer show in Table 1, these

residues are not going to be quantified with the Bradford Coomassie method. However,

the total protein content in the milk residue (layer) is 1.02% of the total protein content in

milk. Nevertheless, other components such as fat and solids non-fat could be found in the

layer because of the variations in the compositional analysis of milk after processing. The

formation of the deposits layer in the electrode, so-called fouling, could be because of the

electrophoretic concentration of charged molecules in one the outer layers of food that

has direct contact with the electrode (Morren et al., 2003; Kershner et al., 2004). Based

on this fact, a large proportion of the layer could be formed of solids non-fat because

most of them are charged molecules that can interact with the electrode.

The deposits into the electrode can affect the process because the layer can reduce

the intensity of the electric field into the chamber and transport some particles from the

electrode material to the food (Góngora-Nieto et al., 2002). But the electrode fouling can

also produce arcing problems or electrical breakdown in the chamber during the

processing of food (Morren et al., 2003) with the generation of sparks and undesirable

effects in the food and chamber components (Saulis et al., 2007). Some of the

recommended materials for chamber electrodes are gold, platinum and metal oxides

339

(iridium and ruthenium), as well as the use of some high conductive polymer coatings

(Góngora-Nieto et al., 2002). Titanium and glassy carbon are other material options for

building electrodes that are more resistant to corrosion (Roodenburg et al., 2005b). Some

processing variables that would help to reduce the electrodeposition of materials into the

electrode are the use of bipolar pulses, instant reverse pulses, low energy pulses, increase

of flow rate, reduction of build-up area (Góngora-Nieto et al., 2002), and removal of the

charge after each pulse (Morren et al., 2003).

4. Conclusions

Indeed, significant changes in all tested milk parameters were observed after PEF

processing; however, the tested conditions were extreme conditions to inactivate spores

and do not represent the regular conditions for pasteurizing milk using pulsed electric

fields technology. Regular conditions to process milk require a shorter number of pulses

(less than 20) without temperature increase, maintaining the effectiveness of PEF

processing such as alternatives to save energy, time and money.

References


Chemists. USA.

Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye binding. Analytical

Biochemistry. 72, 248-254.

340

Dutreux N., Notermans S., Góngora-Nieto M.M., Barbosa-Cánovas G.V. and

Swanson B.G. 2000. Effects of combined exposure of Micrococcus luteus to nisin and

pulsed electric fields. International Journal of Food Microbiology. 60:147-152.

Calderón-Miranda, M.L., Barbosa-Cánovas, G.V., and Swanson, B.G. 1999a.

Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin.

International Journal of Food Microbiology. 51: 7-17.

Calderón-Miranda, M.L., Barbosa-Cánovas, G.V., and Swanson, B.G. 1999b.

Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin.

International Journal of Food Microbiology. 51: 19-30.

Góngora-Nieto, M.M., Sepúlveda, D.R., Pedrow, P., Barbosa-Cánovas G.V., and

Swanson, B.G. 2002. Food processing by pulsed electric fields: Treatment delivery,

inactivation level, and regulatory aspects. Lebensmittel-Wissenschaft und-Technologie,

35: 375-388.

Heinz, V., Toepfl, S., and Knorr, D. 2003. Impact of temperature on lethality and

energy efficiency of apple juice pasteurization by pulsed electric fields treatment.

Innovative Food Science and Emerging Technologies. 4: 167-175.

IDFA. (2006). International Dairy Foods Association.


341

Kamizake, N.K.K., Gonçalves, M.M., Zaia, C.T.B.V. and Zaia, D.A.M. 2003.

Determination of total proteins in cow milk powder samples: A comparative study

between Kjeldahl method and spectrophotometric methods. Journal of Food Composition

and Analysis. 16: 507-516.

Kershner, R.J., Bullard, J.W., and Cima, M.J. 2004. The role of electrochemical

reactions during electrophoretic particle deposition. Journal of Colloid and Interface

Science. 278:146-154.

Martín-Belloso, O., Vega-Mercado, H., Qin, B.L., Chang, F.J., Barbosa-Cánovas,

G.V., and Swanson, B.G. 1997. Inactivation of Escherichia coli suspended in liquid egg

using pulsed electric fields. Journal of Food Processing and Preservation. 21: 193-208.

Morren, J., Roodenburg, B., and de Haan, S.W.H. 2003. Electrochemical

reactions and electrode corrosion in pulsed electric field (PEF) treatment chambers.

Innovative Food Science and Emerging Technologies. 4: 285-295.

Odriozola-Serrano, I., Bendicho-Porta, S., and Martín-Belloso, O. 2006.

Comparative study on shelf-life of whole milk processed by high-intensity pulsed electric

field or heat treatment. Journal of Dairy Science. 89: 905-911.

Potter, N.N. 1986. Food Science. 4th Ed. (pp. 348-350). New York: Van Nostrand

Reinhold.

342

Qin, B.L., Barbosa-Cánovas, G.V., Swanson, B.G., Pedrow P.D., and Olsen, R.G.

1998. Inactivating microorganisms using a pulsed electric field continuous treatment

system. IEEE Transactions on Industry Applications 34(1):43-50.




Reyns, K.M.F.A., Diels, A.M.J., and Michiels, C.W. 2004. Generation of

bactericidal and mutagenic components by pulsed electric field treatment. International

Journal of Food Microbiology. 93:165-173.

Roodenburg, B., Morren, J., Berg, H.E., and de Haan, S.W.H. 2005a. Metal

release in a stainless steel pulsed electric field (PEF) system. Part I. Effect of different

pulse shapes; theory and experimental method. Innovative Food Science and Emerging

Technologies. 6: 327-336.

Roodenburg, B., Morren, J., Berg, H.E., and de Haan, S.W.H. 2005b. Metal

release in a stainless steel pulsed electric field (PEF) system. Part II. The treatment of

orange juice; related to legislation and treatment chamber lifetime. Innovative Food

Science and Emerging Technologies. 6: 337-345.

343

Saulis, G., Rodaitė-Riševičienė, R., and Snitka, V. 2007. Increase of the

roughness of the stainless-steel anode surface due to the exposure to high-voltage electric

pulses as reveled by atomic force microscopy. Biochemistry. 70:519-523.

Sepúlveda, D.R., Góngora-Nieto, M.M., Guerrero J.A., and Barbosa-Cánovas,

G.V. 2005. Production of extended-shelf life milk by processing pasteurized milk with

pulsed electric fields. Journal of Food Engineering. 67: 81-86.

Sobrino-López, A., and Martín-Belloso, O. 2006. Enhancing inactivation of

Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields

and nisin. Journal of Food Protection. 69(2): 345-353.

344

Table 1. Physicochemical and composition parameters of raw whole milk and PEF processed milk

Sample pH Acidity*

(%) Density

(g/cm3)** Solids non-

fat** Fat

content** (%)

Protein content**

(%) Raw milk 6.30a

(± 0.01) 0.168a

(± 0.010) 1.0298a

(± 0.0002) 8.75a

(± 0.04) 3.00a

(± 0.01) 3.26a

(± 0.02) PEF

processed milk

6.44b

(± 0.01) 0.144b

(± 0.000) 1.0257b

(± 0.0001) 7.65b

(± 0.03) 2.63b

(± 0.02) 2.85b

(± 0.01)

* Expressed as lactic acid ** Quantified with Lacticheck Milk Analyzer a was significantly different from b (p < 0.05)

345

Table 2. Color parameters and color functions of raw and PEF processed milk

Sample L* a* b* ΔE h* C*

Raw milk 91.12a

(± 0.07) -1.46a

(± 0.02) 5.45a

(± 0.03) 75.01a

(± 0.12) 5.65a

(± 0.03) PEF

processed milk

90.35b (± 0.12)

-1.58b

(± 0.02) 4.65b

(± 0.03) 1.12

(± 0.12) 71.23b

(± 0.29 ) 4.91b

(± 0.02)

a was significantly different from b (p < 0.05)

346

y = 0.9186x + 0.2153R2 = 0.9939

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Concentration (mg/ml)

Abso

rban

ce @

595

nm

Figure 1. Standard curve of Bovine Serum Albumin.

347

(a) (b)

Figure 2. a) Fouling of the electrode after processing raw whole milk using PEF at 40 kV/cm, 240 pulses of 2.5 μs and 50ºC;

b) Erosion of the stainless steel electrode after processing using the above mentioned conditions of PEF treatment.

348

0

0.5

1

1.5

2

2.5

3

1

Prot

ein

Con

tent

(%)

Milk before treatment Milk after treatment Accumulate from milk TOTAL

Figure 3. Total protein content in milk, raw milk, PEF milk and the electrodeposited

material.

349

CHAPTER TEN

DETERMINATION OF ALLURA RED (RED # 40) BY REVERSE-PHASE HIGH-

PERFORMANCE LIQUID CHROMATOGRAPHY (RP-HPLC) AND

PHYSICOCHEMICAL CHANGES IN STRAWBERRY FLAVORED MILK

UNDER PULSED ELECTRIC FIELD PROCESSING

Daniela Bermúdez-Aguirre, Jaime A. Yáñez, C. Patrick Dunne,

Neal M. Davies and Gustavo V. Barbosa-Cánovas

Abstract

Few studies have been conducted on flavored milk using pulsed electric fields (PEF).

Despite the positive results this emerging technology has shown in milk pasteurization,

its use in processing other dairy products has not been reported. One of the main concerns

in using PEF is the stability of the product during storage, mainly those with additives.

The objective of this study was to analyze the degradation of Allura Red, the coloring

agent in strawberry milk, under PEF and during storage. Four systems were tested: two

commercial strawberry flavored milks and two model systems prepared with milk,

sucrose, and selected concentrations of Allura Red. Processing conditions were 40

kV/cm, 48 pulses (2.5 μs), and 55ºC. Allura Red extraction was performed using

acetonitrile. The samples were analyzed via RP-HPLC utilizing a Phenomenex Luna C18

analytical column with a mobile phase of sodium acetate and acetonitrile. Separation of

Allura Red was carried out at 25ºC and UV detection at 520 nm. After PEF processing of

350

the milk, only minor changes were observed in color, concentration of Allura Red, and

pH. Commercial samples (with or without PEF) maintained a pH above 6, but model

systems dropped below this pH value after 10 days of storage (4ºC). Color changed in the

PEF samples during storage, showing an important decrease in a*. Hue angle and chroma

also changed during storage. The HPLC analysis reported a bi-phasic effect in Allura Red

concentrations versus time. It was observed that Allura Red concentrations increased

until reaching a maximum concentration during the middle of storage life followed by a

significant decrease and degradation in the samples. These changes can be attributed to

microbial growth, reduction of pH, or changes in protein content. However, PEF

processing was observed to not have a significant effect in Allura Red concentrations

compared to the control samples.

351

1. Introduction

There are different commercially available flavored milks such as chocolate, vanilla,

banana, orange and strawberry. Chocolate milk is the most popular flavored milk in

children (90% of them like its taste), providing high content of protein, vitamins, calcium

and other nutrients. After this, strawberry flavored milk has a great acceptability with the

consumer (Miller et al., 2007). Milk is not only important in the growth of children, in

other life circumstances this beverage is one of the main sources of protein. For example

in the combat rations for soldiers, milk is an important component of the different menus.

Combat rations must accomplish provision of a quality life to the soldiers but at the same

time cover all their nutritional needs. At the R&D department of the Natick Soldier

Center, every day food researchers are looking for better products in order to change and

improve the menus each year. Some of the current milk-based beverages for military

rations are dairy shakes (chocolate, vanilla, strawberry and soon a banana shake will be

included in the 2008 menu) (NSRDEC, 2007). However, the shelf-life sometimes is

limited because of the processing conditions and shipping and storage environment. For

soldiers’ feeding purposes, the product must have a storage life as long as possible under

the most adverse conditions, but at the same time having acceptable organoleptic

characteristics such as flavor, taste, color and appearance. Conventional food processing

operations (i.e. drying, pasteurization, blanching, canning, etc.) have the inconvenience

of decreasing the overall food quality, destroying some vitamins and pigments,

denaturalization of some proteins or volatizing odors, all of which can affect the final

quality of the product. Nevertheless, these unit operations must be carried on in food in

order to inactivate pathogenic microorganisms, reduce the load of deteriorative

352

microorganisms and the enzymatic activity and extending the shelf-life of the product.

Emerging technologies such as high hydrostatic pressure, pulsed electric fields,

ultrasound or ultraviolet are being tested in foods in order to ensure a microbiologically

safe product but with fresh-like characteristics. Pulsed electric fields have shown positive

effects in the extension of the shelf-life of milk, retarding the growth of mesophilic

bacteria up to 80 days and with minor effects in quality (Sepúlveda et al., 2005).

Color in food is an important quality parameter that may determine acceptability

or rejection by the consumer. Color is important in flavor and taste recognition and

identification as well as in food preference (Gifford and Clydesdale, 1986). Children

often prefer to drink beverages and eat products with attractive colors rather than

colorless ones. Color in foods has psychological effects on how the product is perceived

by the consumer. For example, the degree of sweetness was perceived as favored in

cherry-flavored drinks because of the presence of red-colored pigments (Gifford and

Clydesdale, 1986). Allura Red AC or FD&C Red No. 40 is a red powder, soluble in

water, which is used as an additive to color beverages, candy, cereals, confections,

gelatins, puddings, ice cream, condiments, dairy products and others. Allura Red is an

organic molecule that can be classified as monoazo structure (Figure 1) and it has been

listed for use in foods since 1971 (Thorngate, 2002). Regulations of this color are in

accordance with Good Manufacturing Practice and it must be a certified additive to

become used in food products. Experimental reproductive effects have been shown with

an overdose of this additive. The TDL0 (Toxic Dose Low) is the lowest dose over a given

period of time to produce any toxic effect in humans or produce carcinogenic,

neoplastigenic or teratogenic effects in animals or humans; for Red No. 40 the TDL0 is

353

38,500 mg/kg tested in rats via oral ingestion (Lewis, 1989); hyperactivity has been

reported in children as a result of the ingestion of this colorant (Anonymous, 1984);

nevertheless, the reported average daily intake in human beings is 100 mg/kg (Thorngate,

2002). Some of the analytical techniques that have been used for determination of

synthetic colorants in food are thin-layer chromatography, derivative spectrometry,

adsorptive voltammetry (Alghamdi, 2005), capillary electrophoresis, reverse-phase liquid

chromatography (RPLC) and ion-pair RPLC (Chen et al., 1998).

The objective of this research was to study the use of pulsed electric fields in

strawberry flavored milk using two commercial brands and two model systems,

evaluating the physicochemical changes and possible degradation of Allura Red during

storage.


2.1 Milk samples and model systems

Two brands of commercial strawberry flavored milk were purchased at the local

supermarket. Initial characterization of milks consisted of microbial evaluation

(mesophilic load), color and pH. Milk samples were kept at 4ºC until they were used.

Two model systems for strawberry milk were prepared with 2% pasteurized milk

purchased at the local supermarket, sucrose and Allura Red AC (Sigma-Aldrich®

458848) as a coloring agent (80% dye content). Model system 1 (M1C) consisted of 2%

pasteurized milk, sucrose (5.5%) and Allura Red AC (0.01%) using the minimum of

coloring agent recommended for flavored milks according to the patent

(WO/2004/100671). Model system 2 (M2C) consisted of 2% pasteurized milk, sucrose

354

(5.5%) and Allura Red AC (0.2%) in the maximum allowable concentration for flavored

milk according with the same patent mentioned above. Ingredients of each sample are

listed in Table 1. Again, samples were characterized in microbial load, color and pH and

were kept at 4ºC until they were used.

2.2 Pulsed electric fields treatment

A pilot plant pulsed electric fields (PEF) system manufactured by Physics International

(San Leandro, CA) and a cylindrical concentric-electrodes treatment chamber (Qin et al.,

1995) was used to apply the desired treatments to the milk. The continuous treatment

chamber had a volume of 25cm3 and a gap between the stainless steel electrodes of 0.6

cm.

Frequency was kept constant at 10 Hz and the flow was 490 ml/min. Flow rate of

flavored milk was set up and controlled with a rotary pump (Masterflex 7654-00, Cole

Parmer Instruments Co., Chicago, IL). Treatments with peak electric fields of 40 kV/cm

were used in combination with 48 pulses of around 2.5 μs pulse-width. A refrigerated

bath and recirculation system was used to keep the milk temperature at or below 55ºC.

Inlet and outlet temperatures were monitored with digital in-line thermocouples (Cole-

Palmer, Vernon Hills, IL) and registered (Figure 2). Energy consumption, electric field

intensity, pulse width and pulse shape were directly measured by a digital oscilloscope

(Hewlett-Packard 54530A, Colorado Springs, CO), connected to the treatment chamber

through high voltage probes.

355

2.3 Microbiological analysis

The initial load of microorganisms was evaluated for each milk (flavored and

unflavored). Plate Count Agar (Difco Becton, Dickinson and Co., Sparks, MD) was used

for mesophiles aerobes. Serial dilutions were made with peptone water before pouring

samples into dishes. These were incubated at 35°C for 48 h. For the PEF treatments, the

microbial counts were evaluated after 48 pulses. Samples were analyzed in duplicate.

2.4 pH

pH was determined by direct immersion with a potentiometer (Orion Research Inc.,

Boston, MA); each sample was measured in triplicate.

2.5 Color




ml of each milk sample was poured into sterile plastic bags. A white ceramic plate was

used for standardizing the instrument (L* = 93.4, a* = -0.67, b* = 0.78). Each sample

was read in triplicate.


parameters L*, a* and b* and comparing the different treatments with the untreated milk

of each system from the beginning to the end of storage life.

( ) ( ) ( )2*2*2** baLE Δ+Δ+Δ=Δ

356


⎟⎟⎠

⎞⎜⎜⎝

⎛= −

*

*1* tan

abh


( ) 21

*** 22

baC +=

2.6 High Performance Liquid Chromatography (HPLC) analysis

2.6.1 Materials

Allura Red and sodium acetate were purchased from Sigma (St. Louis, MO, USA).

HPLC grade acetonitrile and water were purchased from J. T. Baker (Phillipsburg, NJ,

USA). Commercial samples (N, L) and model systems (M1, M2) were used before and

after PEF treatments to quantify the concentration of Allura Red.

2.6.2 Allura Red Extraction

Stock solutions of Allura Red and kaempferol (internal standard, IS) were prepared in

HPLC methanol at c.a. 100 μg/ml and stored at -20ºC protected from the light. A 5 ml

aliquot of each sample was collected and mixed with ice-cold HPLC acetonitrile,

vortexed for 30 s, and centrifuged at 3,500 rpm for 8 min (Lonnerdal et al. 1987). The

supernatant was transferred to new tubes and any precipitate was discarded. Then 100 μl

of kaempferol was added to each sample, vortexed, and dried to completion under a

357

constant stream of compressed nitrogen gas. Each sample was reconstituted in 500 μl of

HPLC acetonitrile, vortex, centrifuged, and 100 μl was injected into the HPLC.

2.6.3 Chromatographic conditions

For the quantification of Allura Red, the HPLC system utilized was a Shimadzu HPLC

(Kyoto, Japan) consisting of two LC-10ATVP pumps, a SIL-10AF auto injector, a SPD-

M10A VP spectrophotometric diodearray detector, and a SCL-10A VP system controller.

Data collection and integration were accomplished using Shimadzu EZ Start 7.1.1 SP1

software (Kyoto, Japan). The method was adapted from a report by Kirschbaum et al.

(2003); the analytical column used was a Phenomenex Luna (2) C18 column (250 x 4.60

mm, i.d. 5 µm particle size, Torrance CA, USA). The mobile phase consisted of 100 mM

sodium acetate (NaOAc) (mobile phase A) and 100% acetonitrile (MeCN) (mobile phase

B). The gradient program is presented in Table 2. Separation was carried out at ambient

temperature (25 ± 1°C), and UV detection was carried at 520 nm for Allura Red and 370

nm for kaempferol (internal standard). Standard curves were prepared in whole milk with

known concentrations of Allura Red.

2.7 Storage life

PEF treated milk samples were stored at 4ºC for 40 d. Samples were collocated in sterile

plastic bags, using and discarding one bag for each day for each treatment. Every third

day, the quality characteristics (pH and color) were evaluated, and the microbial loads

were poured into Plate Count Agar (Difco®) for mesophiles aerobes. Samples were taken

from a different bag each day and diluted with peptone water. Samples were incubated at

358

35°C for 48 h. Every fifth day the coloring agent concentration was quantified using

HPLC.


3.1 pH

Regarding the pH of samples, in Figure 3 the behavior of this physicochemical parameter

is shown. First, for the commercial and untreated samples NC and LC, the pH remained

almost constant during one month of storage without important changes. However, the

PEF treated samples (NP and LP) showed a decrease of the pH after the first week of

storage going from 6.7 to 6.3 and remaining at this value for most of the storage time,

with a final drop of the value after day 30. However, in all cases, the pH is inside the

range for commercial milk.

Both model systems showed an interesting pH behavior during storage; pH of

samples that were not processed with PEF (M1C and M2C) dropped quickly after 7 d of

storage and the behavior was very similar between them. Samples that were treated with

PEF (M1P and M2P) showed slower decrease of pH than the untreated, but finished at

the end of the storage period with pH below 5. The reason for this drop could be the

growth of lactobacillus and production of lactic acid into the milk, in addition to other

chemical reactions that could be taking place.

3.2 Color

Immediately after processing samples did not show any important change in color; at

least they showed similar appearance than before processing. Analytical measurements

359

showed the same Hunter color parameters L*, a* and b* between control samples and their

respective PEF treated samples after processing. However, during storage at 4ºC some

samples showed important variations. The three Hunter color parameters were quantified,

however the a* is of interest because of the meaning of this one. A more positive value

means that the product is closer to the redness region and a negative number means being

closer to the greenness region. Commercial samples as shown in Figure 4 remained with

a constant color in accordance with the a* value, but these same samples treated with PEF

lost their coloration after 20 d of storage, with a remarked decrease of a* dropping below

zero. One of the physicochemical mechanisms from which color is produced involves the

electron transitions between molecular orbitals. Changes in bond conjugation in an

organic molecule such as delocalization of electrons or presence of electron donor and

acceptor groups affect the absorption of light accomplishing change of colors (Thorngate,

2002). Here it is important to mention that the use of pulsed electricity has an observable

effect on the stability of the coloring agent Red No. 40 in flavored milk. Both commercial

samples, regardless the rest of the ingredients, showed this decrease that kept constant

until the end of storage (day 37).

Model systems also showed an interesting behavior of a* parameter; the model

with the lowest allowed concentration of Red No. 40 (M1) was discolored after 10 days

of storage regardless of whether it was pulsed or not. PEF only retarded the loss of color

a couple of days with a later discoloration. Model system M2, with the highest coloring

agent concentration, kept its color constant during storage regardless of whether the

sample was pulsed or not. This suggests that as the coloring agent is more concentrated,

more stability is shown after PEF processing and during storage.

360

3.2.1 Net change of color

In order to have a better understanding of the changes in color in milk after processing

and during storage, some color functions were evaluated based on Hunter parameters.

First, net change of color was calculated for each sample, using as a reference the sample

without treatment (C) of the same brand or model system. In Figure 5 this behavior is

shown for the four samples. For both commercial samples (N, L) the net change of color

was very high after day 20; meanwhile, model system M1 showed an undefined behavior

during storage, with a peak after 10 days of storage that represented the maximum

difference between treated and untreated sample. Model system M2 did not show any

important change during storage in ΔE probably because of the high concentration of Red

No. 40 present in the sample.

3.2.2 Hue angle (h*)

The hue angle is shown in Figure 6. For commercial untreated samples (NC and LC), h*

remained constant during storage, close to 0ºC, which is close to the reddish h* value. But

samples processed with PEF changed the angle to negative values close to the yellowish

region. This change was observed at first sight like a loss of color in the milk. Model

systems showed similar behavior as in ΔE; the less concentrated sample changed the hue

angle after 10 d of storage with a hysteresis region from day 15 to day 25. Samples with

highest coloring agent did not change h* during storage and remained at a constant value

of around 27º.

361

3.2.3 Chroma (C*)

The saturation index or chroma (C*) was calculated for the samples as shown in Figure 7.

This value, which indicates the degree of saturation, purity or intensity of color, showed a

constant value for commercial samples during storage. Sample N showed a higher value

during the entire time than did sample L, indicating more purity in the color of the milk.

Flavored milk N is a better and more expensive brand of this kind of beverage than brand

L; in fact, that could be a reason to use coloring with high purity and other stabilizing

agents, such as can be observed in Table 1. However, during storage samples treated with

PEF lost the intensity of the Red 40, diminishing the C* value as observed. Model

systems showed similar behavior as in the previous functions, i.e., less concentrated

system, less saturation and faster degradation, with or without PEF. As the concentration

was higher the stability of the color remained despite the treatment, as observed in Figure

7.

3.3 Microbiological counts

Microbial counts of commercial milks before processing were too low (< 10 cfu/ml) to be

detected with conventional microbiological methods. Model systems showed less than 10

cfu/ml before processing with PEF. After processing and during storage the growth of

mesophilic bacteria is shown in Figure 8. In the first case, the commercial samples NC

and LC did not show microbial growth throughout storage, at least at a level able to be

detected with conventional methods. The reason for that is because these milk beverages

were processed with Ultra High Temperature (UHT) pasteurization, eliminating all the

mesophilic bacteria in the product or reducing the bacteria level to very low counts that

362

can not be detected. Commercial samples processed with PEF showed an increase in the

bacterial population throughout storage. One reason for this increase could be a possible

cross contamination, but the microbial quality of the PEF system and all the related

material was tested without evidence of microorganisms; thus, the possibility of

activation of spores of some thermally resistant microorganisms could be the reason for

this growth. After 12 d of storage, milk showed almost 5 log of microbial growth. Model

systems showed a similar behavior in microbial growth during storage, raising 5 log after

14 d of storage. In this case, there is no difference between commercial and model

systems because the milk used for the model systems was not UHT pasteurized, having

the presence of some bacteria that could accelerate the growth of more microorganisms.

3.4 Concentration of Allura Red

In Figure 9 the concentration of Allura Red for all the systems is shown during storage.

Samples were analyzed during 41 d but results are shown only until day 31 for

commercial samples and day 26 for model systems since after these respective days of

storage the HPLC values were not possible to integrate because big interfering peaks

interfered. These peaks were not present originally. The presence of other dyes such as

Sunset Yellow or Tartrazine may cause interference with Allura Red because of similar

azo structure (Alghamdi, 2005). The first part of the Figure shows the behavior for the

commercial brand N; both samples untreated (NC) and PEF treated (NP) showed a

constant concentration during storage, and the PEF sample even showed a slight increase

in the concentration detected. For the commercial sample L, the concentration of the

coloring agent showed a maximum peak of 2.5 g/L after 20 d of storage and an opposite

363

behavior of the untreated sample (LC) and the PEF sample (LP) after day 26, with an

important decrease for the LC sample at the end of the storage period.

The model systems showed different behaviors. Model system M1 showed an

almost constant concentration of coloring agent during storage with a final drop of

coloring agent. Meanwhile, model system M2 presented the same concentration for the

untreated (M2C) and the treated (M2P) samples, although concentration did not follow a

constant trend. Reports on stability data for FD&C Red No. 40 shows that this dye has a

very good stability regardless of the pH (from 3 to 8), and is stable to light, heat (to

105ºC), acid (10% acetic acid) and presence of SO2; this colorant shows fair stability to

the presence of base (Thorngate, 2002).

So, the changes observed in the coloring agent in these samples are not because of

changes in pH or increase of temperature (in all the experiments temperature was kept

below 55ºC); one theory that could describe these changes is the possibility of the

binding between the colorant agent with some proteins and other charged molecules of

milk because of the presence of strong electric fields attracting particles and forming new

matrices, thus changing the electron configuration of the molecules and scattering or

absorbing the light in different ways and producing different colors to the human eye.

However, further research must be conducted in flavored milks in order to have a better

explanation of what is happening with the electrons of the molecules and the new

complexes and chemical reactions that take place during and after PEF processing.

364

4. Conclusions

Pulsed electric fields is a nonthermal technology that offers important energy and cost

savings during milk pasteurization; however, because of the interaction of electricity with

charged molecules in milk with coloring agents and other additives, new matrices were

formed within the product resulting in a lack of color after some days of storage in some

of the PEF processed milk samples. The presence of other ingredients and dyes helped to

retain the color after processing for commercial samples. The effect of the concentration

of Allura Red showed better stability in color at higher concentration regardless of the

treatment.

References

Alghamdi, A.H. 2005. Determination of Allura Red in Some Food Samples by

Adsorptive Stripping Voltammetry. Journal of AOAC International. 88(5):1387-1393

Anonymous. 1984. Allura Red-Developmental and psychotoxic effects? Food and

Chemical Toxicology. 22(11): 913.

Chen, Q., Mou, S., Hou, X., Riviello, J.M., and Ni Z. 1998. Determination of

eight synthetic food colorants in drinks by high-performance ion chromatography.

Journal of Chromatography A. 827:73-81.

365

Gifford, S.R., and Clydesdale, F.M. 1986. The psychophysical relationship

between color and sodium chloride concentrations in model systems. Journal of Food

Protection. 49 (12): 977-982.

Kirschbaum, J., Krause, C., Pfalzgraf, S., and Brückner, H. 2003. Development

and evaluation of an HPLC-DAD method for determination of synthetic food colorants.

Chromatographia. 57(1): S115-S119.

Lewis, R.J. 1989. Food additives. Van Nostrand Reinhold Company. New York.

pp:214.

Lonnerdal, B., Woodhouse, L. R., and Glazier, C. 1987. Compartmentalization

and quantitation of protein in human milk. Journal of Nutrition: 117, (8), 1385-95.

Miller, G.D., Jarvis, J.K., and McBean, L.D. 2007. Handbook of Foods and

Nutrition. 3rd Ed. National Dairy Council. CRC Press. Boca Ratón, Fl. pp: 28-32, 358-

359.

NSRDEC. 2007. US Army Natick Soldier Center. Operational Rations of the

Department of the Defense. 7th Ed. Department of Defense, Natick, MA, USA.

366




Sepúlveda, D.R., Góngora-Nieto, M.M., Guerrero J.A., and Barbosa-Cánovas,

G.V. 2005. Production of extended-shelf life milk by processing pasteurized milk with

pulsed electric fields. Journal of Food Engineering. 67: 81-86.

Thorngate III, J.H. 2002. Synthetic Food Colorants. In “Food Additives”, 2nd Ed.

A. L. Branen, P.M. Davidson, S. Salminen and J.H. Thorngate III, Eds. Marcel Dekker,

Inc. New York. pp: 477-500.

WO/2004/100671. Flavored milk manufacturing processes and compositions.

World Intellectual Property Organization.

367

Table 1. List of ingredients and codification of samples used for the experiment

Commercial

sample N Commercial

sample L Model system

M1 Model system

M2 Composition Reduced fat milk

with vitamin A Palmitate and vitamin D3 added, high

fructose corn syrup, sugar, nonfat milk,

calcium carbonate,

potassium citrate, artificial flavors,

carrageenan, citric acid, salt, Red 40*, Blue 1

Milk, high fructose corn syrup, nonfat

milk, natural and artificial flavors, carragenan, Red 40*, Vitamin D3

2% Milk, Allura Red*

(0.01%), sucrose (5.5%)

2% Milk, Allura Red*

(0.2%), sucrose (5.5%)

Code for control sample

NC LC M1C M2C

Code for sample

treated with PEF

NP LP M1P M2P

*Red 40 and Allura Red are synonyms

368

Table 2. Gradient program for HPLC analysis and determination of Allura Red in

strawberry flavored milk samples

Time (min) Mobile phase A

(%)

Mobile phase A

(%)

Flow rate (ml/min)

0 100 0 0.5

5.5 100 0 0.5

18 89 11 0.5

18.5 89 11 0.9

25 89 11 0.9

30 80 20 0.9

44 70 30 0.9

53 0 100 0.9

53.5 0 100 1.3

62 0 100 1.3

63 100 0 1.3

70 100 0 1.3

369

Figure 1. Chemical structure of Allura Red (Red #40) with chemical name disodium (5Z)

-5-[(2-methoxy – 5 – methyl-4-sulfonatophenyl) hydrazinylidene] – 6 – oxonaphthalene -

2-sulfonate

370

0

10

20

30

40

50

60

10 15 20 25 30 35 40 45 50

Number of pulses

Tem

pera

ture

(C)

N inlet N outlet L inlet L outlet LIMIT

0

10

20

30

40

50

60

10 15 20 25 30 35 40 45 50

Number of pulses

Tem

pera

ture

(C)

M1 inlet M1 outlet M2 inlet M2 outlet LIMIT

Figure 2. Inlet and outlet temperatures of commercial flavored strawberry milk samples

(N, L) and the model systems (M1, M2) during the Pulsed Electric Fields processing

treatment

371

5.9

6

6.1

6.2

6.3

6.4

6.5

6.6

6.7

6.8

0 5 10 15 20 25 30 35

Time (days)

pH

NC NP LC LP

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

0 5 10 15 20 25 30 35

Time (days)

pH

M1C M1P M2C M2P

Figure 3. pH behavior during storage (4ºC) of untreated (C) and PEF treated (P) samples

of commercial strawberry flavored milk (N, L) and model systems (M1, M2)

372

-10

-5

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Time (days)

a*

NC NP LC LP

-10

0

10

20

30

40

50

60

0 5 10 15 20 25 30 35 40

Time (days)

a*

M1C M1P M2C M2P

Figure 4. a* color parameter for samples during storage (4ºC) of untreated (C) and PEF


(M1, M2)

373

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30 35 40

Time (days)

Del

ta E

N L M1 M2

Figure 5. Net change of color (ΔE) for samples during storage (4ºC) for samples of

commercial strawberry flavored milk (N, L) and model systems (M1, M2)

374

-100

-80

-60

-40

-20

0

20

40

0 5 10 15 20 25 30 35 40

Time (days)

h*

NC NP LC LP

-150

-100

-50

0

50

100

150

0 5 10 15 20 25 30 35 40

Time (days)

h*

M1C M1P M2C M2P

Figure 6. Hue angle for samples during storage (4ºC) of untreated (C) and PEF treated (P)

samples of commercial strawberry flavored milk (N, L) and model systems (M1, M2)

375

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Time (days)

C*

NC NP LC LP

0

10

20

30

40

50

60

0 5 10 15 20 25 30 35 40

Time (days)

C*

M1C M1P M2C M2P

Figure 7. Chroma or saturation index for samples during storage (4ºC) of untreated (C)

and PEF treated (P) samples of commercial strawberry flavored milk (N, L) and model

systems (M1, M2)

376

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Time (days)

log

(N/N

o)

NC NP LC LP

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14 16

Time (days)

log

(N/N

o)

M1C M1P M2C M2P

Figure 8. Microbial growth for samples during storage (4ºC) of untreated (C) and PEF


(M1, M2)

377

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35 40

Time (days)

Allu

ra R

ed C

once

ntra

tion

(mg/

mL)

NC NP

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35 40

Time (days)

Allu

ra R

ed C

once

ntra

tion

(mg/

mL)

LC LP

378

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35 40

Time (days)

Allu

ra R

ed C

once

ntra

tion

(mg/

mL)

M1C M1P

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40

Time (days)

Allu

ra R

ed C

once

ntra

tion

(mg/

mL)

M2C M2P

Figure 9. Allura Red Concentration (mg/mL) for samples during storage (4ºC) of untreated (C) and PEF treated (P) samples of

commercial strawberry flavored milk (N, L) and model systems (M1, M2)

379

CONCLUSIONS

This research project tested ultrasound and pulsed electric fields to process milk. Some

general conclusions can be summarized as follows:

Ultrasound technology in combination with thermal treatment was able to inactivate

pathogens and deteriorative microorganisms in milk, considerably reducing the treatment

time and achieving pasteurization standards. Because of the non-linear behavior of the

inactivation curves with ultrasound, Weibull’s and four parameter models were useful to

describe the inactivation kinetics of Listeria innocua in milk by ultrasound.

Despite the changes that ultrasound generated in the composition and physicochemical

parameters of milk after processing, one of the most important nutritional components

protein, remained in the regular range of this milk constituent. Beneficial effects were

observed in color (whiter), stability and homogenization degree in milk after sonication.

After the inactivation and microscopy studies of Listeria cells before and after sonication,

it is possible to suggest as a likely mechanism of cell inactivation the rupture of the cell

membrane, formation of pores, breakdown of cells and the deposition of cell material into

the surface because of the rubbing effect produced by the cavitation of ultrasound. The

presence of free radicals in the milk after the sonication could also have a bactericidal

effect.

380

Caviation generated by sonication homogenizes fat globules of milk, reducing the size to

smaller than one micrometer and generating a more compact and stable structure of milk.

Also, this new structure has a whiter color because of the size reduction of the globules

and the availability of fat, which is creates a new surface area of the oil droplets.

Fat content of milk was shown to be an important hurdle in the inactivation of Listeria

innocua, representing a limiting factor for microbial inactivation using sound waves plus

heat. However, probably because of the presence of new chemical components in milk

caused by the lysis of macromolecules, longer storage life was conferred to the milk.

New and improved dairy products can be developed from thermo-sonicated milk by

taking advantage of the better quality characteristics of the processed milk, having higher

yields, better color, structure and stability. The use of microwave energy was successfully

used to prepare samples for electron microscopy, considerably reducing the preparation

time and improving the resolution of the microscopy images.

Bacillus cereus spores again showed their resistance, not only to thermal treatments, but

also to pulsed electric fields treatment, regardless of the processing conditions. Even with

the use of natural antimicrobials, the spores were resistant, but remained in low counts

after processing milk. Again, the effect of butter fat content in milk was shown with

another microorganism and another technology.

381

The electrodeposition of milk materials during intense treatment with pulsed electric

fields processing is a reality that must be minimized or eliminated. Presence of metals

after processing and decrease mainly of protein content in milk showed the disadvantage

of the processing in order to eliminate spores.

Pulsed electric fields can generate a lack of color in strawberry flavored milks because of

the interaction of the coloring agent (Allura Red) with other components, but this effect is

reduced when other additives are present or the coloring agent is added in higher

concentration but still within the range for food colorants allowed by FDA.

382

FUTURE RESEARCH

After evaluating ultrasound and pulsed electric fields as surrogate technologies to

pasteurize and/or process milk, it is recommended that it would be helpful to widely

document both technologies and to look forward to the possible approval for regulatory

and legal institutions. Some of the future work should be focused on the following:

a) From the engineering point of view, the search for better, more resistant and

sanitary materials for sonotrodes (ultrasound) and electrodes (pulsed electric

fields) would be a priority. Food technologists should work together with

scientists from material science in order to create materials that resist processing

conditions in both technologies without erosion problems and the release of metal

particles into the food.

b) Special sensors should be installed and validated in pulsed electric fields

equipment in order to ensure the absence of bubbles before and during processing

as well as the removal of fouling from the electrode in order to avoid arcing

problems.

c) A detailed analysis of energy consumption in ultrasound processing and pulsed

electric fields to inactivate spores should be carried out in order to show the

economical and energy-saving advantages of these technologies.

d) Better temperature control should be adapted and installed into the ultrasound

equipment in order to avoid overcharge of the system and to facilitate testing of

other processing conditions.

383

e) From the microbiological point of view, more pathogenic bacteria should be

tested under thermo-sonication and other processing conditions such as higher

temperatures and shorter times.

f) A complete chemical study is required in milk after thermo-sonication and pulsed

electric field treatments in order to study the new substances that are generated by

the cavitation of the ultrasound and the contact of electricity with food materials.

Quantification of free radicals, metal particles and other substances are of interest.

g) Studies with pulsed electric fields in other flavored milks, such as chocolate, are

recommended in order to test the stability of the ingredients of these products and

to evaluate the feasibility of this technology to process one of the most consumed

flavored milks.

h) After testing the new chemical composition as well as ensuring the microbial

quality of milk processed with both technologies, sensorial analysis of the product

is highly recommended. Positive effects have been observed in the appearance of

treated milk with ultrasound and PEF, so the next step should be to analyze the

flavor, taste and general acceptance of milk and other dairy products.

i) A huge research world of opportunities for the processing of dairy products with

these new characteristics of milk is available; processing of cheese, yogurt and

other related products with both technologies will lead to improved products for

the consumer.

Documents

NONTHERMAL PROCESSING OF MILK PROCESSING OF MILK By LUZ DANIELA BERMÚDEZ A dissertation submitted in partial fulfillment of the requirements …