kinetics and mechanism of bacterial inactivation by

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The Pennsylvania State University The Graduate School Department of Food Science KINETICS AND MECHANISM OF BACTERIAL INACTIVATION BY ULTRASOUND WAVES AND SONOPROTECTIVE EFFECT OF MILK COMPONENTS A Thesis in Food Science by Neetu Motwani © 2008 Neetu Motwani Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2008

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Page 1: KINETICS AND MECHANISM OF BACTERIAL INACTIVATION BY

The Pennsylvania State University

The Graduate School

Department of Food Science

KINETICS AND MECHANISM OF BACTERIAL INACTIVATION BY ULTRASOUND

WAVES AND SONOPROTECTIVE EFFECT OF MILK COMPONENTS

A Thesis in

Food Science

by

Neetu Motwani

© 2008 Neetu Motwani

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Master of Science

May 2008

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The thesis of Neetu Motwani was reviewed and approved* by the following:

Stephanie Doores Associate Professor of Food Science Thesis Adviser Stephen J Knabel Professor of Food Science John Coupland Associate Professor of Food Science Kelli Hoover Associate Professor of Entomology John D Floros Professor of Food Science Head of the Department of Food Science *Signatures are on file in the Graduate School

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ABSTRACT

The need for alternative food processing technologies that can be used to

pasteurize foods while maintaining their fresh-like qualities is increasing due to

increasing consumer demand for minimally processed foods. Ultrasound technology is

one of the emerging alternate food processing technologies and uses sound waves with

frequencies higher than 20 kHz to inactivate microorganisms. Microbial inactivation by

ultrasound waves (USW) is attributed to cavitation which involves formation, growth and

collapse of bubbles in a liquid medium. Although bacterial inactivation by USW has been

studied extensively in buffers, the potential of USW to inactivate bacteria in real foods

like milk still needs to be investigated. The first objective of this study was to determine

the impact of sonication medium and growth phase on inactivation kinetics of E. coli and

L. monocytogenes. Preliminary studies to select sonication frequency were conducted

by comparing the inactivation of mid-log phase cells of E. coli upon treatment with high

frequency (650, 765 and 850 kHz) and low frequency (24 kHz) USW in phosphate

buffer. Ultrasound waves with high frequency were ineffective in inactivating E. coli,

while low frequency ultrasound waves decreased the number of survivors by ~ 3 logs in

2.5 minutes. Therefore, 24 kHz ultrasound waves were used for this study. Mid-log or

mid-stationary phase cells of E. coli and L. monocytogenes were treated with 24 kHz

ultrasound waves in phosphate buffer, whole milk or skim milk. Log phase cells of E. coli

and L. monocytogenes were more sensitive to ultrasound treatment than stationary

phase cells in all three sonication media. Escherichia coli exhibited non-log linear

inactivation kinetics with tailing while L. monocytogenes exhibited log linear inactivation

kinetics throughout. Ultrasound treatment exhibited minimal injury to bacteria suggesting

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that inactivation of bacteria by USW was following all or nothing phenomenon. The D

values for inactivation of mid stationary-phase cells of E. coli were 2.19, 2.43 and 2.41

min in phosphate buffer, whole milk and skim milk, whereas those for L. monocytogenes

were 7.63, 9.31 and 8.61 min, respectively. Significantly higher (p < 0.05) D values for

inactivation of E. coli and L. monocytogenes in whole and skim milk as compared to

phosphate buffer suggested that milk exerts a sonoprotective effect on these bacteria.

However, the D values for both the organisms were not significantly different between

whole and skim milk indicating that fat in milk does not exert a sonoprotective effect on

these bacteria.

In the second part of the study the sonoprotective effect of milk components,

lactose, casein and β lactoglobulin, on E. coli and L. monocytogenes was investigated.

Lactose, casein, or β lactoglobulin was added to simulated milk ultrafiltrate (SMUF) at a

concentration of 5, 3 and 0.3g/100 ml, respectively. Casein was added to SMUF as a

non-micellar, sodium caseinate or a micellar, phosphocasein, to investigate the

differences in protection conferred due to its physical form. In another experiment, all

three components were added to SMUF to determine the combined protective effect of

these components on bacteria. Presence of casein in micellar and non-micellar forms

did not result in a significant change in the D values compared to SMUF for inactivation

of both organisms. Addition of whey protein β lactoglobulin to SMUF also did not result in

a significant change in D value for E. coli while that for L. monocytogenes increased

significantly (p < 0.05). Presence of lactose in SMUF, however, resulted in significant

increase (p < 0.05) in D values for inactivation of both the organisms. The D values for

E. coli in SMUF and SMUF + lactose were 2.84 and 3.42 min; while those for L.

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monocytogenes were 7.3 and 8.52 min, respectively, suggesting that lactose was

conferring a protective effect on these bacteria. Moreover, D values obtained in SMUF +

lactose were not significantly different from those in skim milk for either organism,

suggesting that amongst the components tested lactose alone was conferring a

significant protective effect to bacteria. There was no significant increase in D values

when E. coli and L. monocytogenes were sonicated in SMUF containing all the three

components suggesting that milk components do not exert any additive or synergistic

protective effect against ultrasound treatment.

Scanning electron microscopy of ultrasound-treated cells of E. coli and L.

monocytogenes was conducted to see the morphological changes in cell structure upon

sonication. The SEM images demonstrated that ultrasound treatment resulted in

physical damage to cell wall and cell membrane of E. coli and L. monocytogenes cells,

leading to their death.

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TABLE OF CONTENTS

LIST OF FIGURES.....................................................................................................ix

LIST OF TABLES.......................................................................................................xi

ACKNOWLEDGEMENTS ..........................................................................................xii

CHAPTER 1 INTRODUCTION .................................................................................1

CHAPTER 2 LITERATURE REVIEW .......................................................................3

2.1 Ultrasound waves (USW) and their physical nature......................................3 2.1.1 Ultrasound waves (USW) and cavitation.............................................3

2.2 Ultrasound waves in food processing ...........................................................5 2.2.1 Food industry applications of ultrasound waves .................................6

2.3 Microbial inactivation by ultrasound waves (USW) .......................................7 2.3.1 Properties of waves.............................................................................7

2.3.1.1 Frequency of Ultrasound waves ...............................................7 2.3.1.2 Amplitude of ultrasound waves .................................................8 2.3.1.3 Energy of ultrasound waves......................................................9

2.3.2 Sonication conditions ..........................................................................10 2.3.2.1 Effect of temperature ................................................................10 2.3.2.2 Effect of pressure......................................................................11

2.3.3 Type of microorganisms......................................................................12 2.3.4 Sonication medium..............................................................................13

2.3.4.1 Composition of foods ................................................................13 2.3.4.2 Water activity (aw) of the sonication medium ............................14 2.3.4.3 pH of the sonication medium ....................................................15 2.3.4.4 Amount of solids in sonication medium.....................................15 2.3.4.5 Volume of the sonication medium.............................................16 2.3.4.6 Presence of chemicals and antimicrobials ................................16

2.4 Mechanism of inactivation.............................................................................17 2.4.1 Mechanical damage to bacteria by USW............................................17 2.4.2 Free radical generation by USW leading to cell death ........................18 2.4.3 High temperatures generated by USW leading to cell death ..............19

2.5 Injury of bacteria by USW .............................................................................19 2.6 Kinetics of bacterial inactivation ....................................................................20 2.7 Improved shelf life and stability of foods .......................................................20 2.8 Improved functional properties of food products ...........................................24 2.9 Enzyme inactivation by ultrasound waves ....................................................25 2.10 Scale up ......................................................................................................26 2.11 Other applications of ultrasound in food industry ........................................27 2.12 Composition of milk.....................................................................................30

2.12.1 Foodborne disease outbreaks associated with raw milk and milk products ................................................................................................31

2.13 Protective effect of food components..........................................................34

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2.14 References..................................................................................................37

CHAPTER 3 ...............................................................................................................44

EFFECT OF SONICATION MEDIUM AND GROWTH PHASE ON INACTIVATION OF E. coli AND L. monocytogenes BY ULTRASOUND WAVES ...............................................................................................................44

3.1 Abstract ................................................................................................................45

3.2 Introduction ...................................................................................................46 3.3.1 Bacterial cultures and culture conditions.............................................49 3.3.2 Preparation of culture for sonication ...................................................50 3.3.3 Ultrasound treatment of sonication sample.........................................50 3.3.4 Enumeration of bacteria and D values ................................................51 3.3.5 Statistical analysis...............................................................................52

3.4 Results and Discussion.................................................................................52 3.4.1 Effect of frequency on inactivation of E. coli .......................................52 3.4.2 Inactivation kinetics of E. coli and L. monocytogenes.........................55 3.4.3 Effect of growth phase and sonication medium on inactivation of E.

coli and L. monocytogenes ...................................................................62 3.6 References...................................................................................................67

CHAPTER 4 ...............................................................................................................71

SONOPROTECTIVE EFFECT OF MILK COMPONENTS ON E. coli AND L. monocytogenes...................................................................................................71

4.1 Abstract .........................................................................................................72 4.2 Introduction ...................................................................................................73 4.3 Materials and Methods..................................................................................75

4.3.1 Bacterial cultures and culture conditions.............................................75 4.3.2 Preparation of sonication media..........................................................75 4.3.3 Preparation of culture for sonication ...................................................76 4.3.5 Enumeration of bacteria and D values ................................................78 4.3.6 Statistical analysis...............................................................................79

4.4 Results and Discussion.................................................................................79 4.4.1 Sonoprotective effect of milk components ..........................................79 4.4.2 Sonoprotective effect of salts of milk...................................................83 4.4.3 Synergistic or additive sonoprotective effect of milk components.......83 4.4.4 Effect of different sugars on protection of E. coli.................................85

4.5 Conclusions...................................................................................................88 4.6 References....................................................................................................90

CHAPTER 5 ...............................................................................................................93

MECHANISM OF INACTIVATION OF E. coli AND L. monocytogenes BY ULTRASOUND WAVES .....................................................................................93

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5.1 Abstract .........................................................................................................94 5.2 Introduction ...................................................................................................94 5.3 Materials and Methods..................................................................................95

5.3.1 Measurement of cellular leakage upon ultrasound treatment .............95 5.3.2 Scanning electron microscopy (SEM) .................................................96

5.4 Results and Discussion.................................................................................96 5.4.1 Cellular leakage upon ultrasound treatment .......................................96 5.4.2 Changes in cell morphology by USW..................................................98

5.5 Conclusions...................................................................................................102 5.6 Acknowledgements .......................................................................................102 5.7 References....................................................................................................103

CHAPTER 6 CONCLUSIONS AND FUTURE RESEARCH......................................104

6.1 Conclusions...................................................................................................104 6.2 Future Research ...........................................................................................105

APPENDIX A COMPOSITION OF BUFFERS ..........................................................107

A.1 Preparation of phosphate buffer ...................................................................107 A.1.1 Stock phosphate solution....................................................................107 A.1.2 Phosphate dilution water (Class O) ....................................................107

A.2 Simulated Milk Ultrafiltrate (SMUF) ..............................................................108

APPENDIX B ADDITIONAL EXPERIMENTAL RESULTS........................................109

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LIST OF FIGURES

Figure 2.1: Cavitation under the influence of ultrasound field ....................................4

Figure 3.1: Effect of ultrasound frequency on inactivation of mid log-phase cells of E. coli (ATCC 25922) in phosphate buffer (pH 7.2) by different frequency ultrasound waves. ...............................................................................................54

Figure 3.2: Temperature profiles with and without temperature control during sonication with 765 kHz and 24 kHz ultrasound waves in phosphate buffer (pH 7.2) ...............................................................................................................56

Figure 3.3: Inactivation kinetics of mid stationary-phase cells of E. coli ATCC 25922(●) and L. monocytogenes ATCC 19115 (▲) in phosphate buffer (pH 7.2) upon sonication with 24 kHz ultrasound waves ...........................................57

Figure 3.4:Survival curves of mid stationary-phase cells of E. coli ATCC 25922 on TSA, a non-selective (-■-) and VRBA, a selective medium (-∆-) upon sonication with 24 kHz ultrasound waves in whole milk......................................59

Figure 3.5:Survival curves of mid stationary-phase cells of L. monocytogenes ATCC 19115 on TSA, a non-selective (-■-) and MOX, a selective medium (-∆-) upon sonication with 24 kHz ultrasound waves in whole milk .......................60

Figure 3.6: Effect of growth phase on inactivation of E. coli ATCC 25922 by 24 kHz ultrasound waves in phosphate buffer (pH 7.2), whole milk and skim milk......................................................................................................................63

Figure 3.7: Effect of growth phase on inactivation of L. monocytogenes ATCC 19115 by 24 kHz ultrasound waves in phosphate buffer (pH 7.2), whole milk and skim milk. .....................................................................................................64

Figure 4.1: Inactivation of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 by 24kHz ultrasound waves in different sonication media .......................80

Figure 4.2: Inactivation of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 in SMUF + NaCN + lactose + β lactoglobulin and Water + NaCN + lactose + β lactoglobulin by 24 kHz ultrasound waves........................................84

Figure 4.3: Comparison of D values for inactivation of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 in skim milk and SMUF + lactose upon sonication with 24 kHz USW ...............................................................................86

Figure 5.1: Change in 260 nm absorbance of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 sample upon treatment with 24 kHz USW ..........97

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Figure 5.2: Scanning Electron microscopy images of E. coli ATCC 25922 upon treatment with 24 kHz USW in SMUF; Control E. coli ATCC 25922(A), Treated E. coli ATCC 25922 (B, C, D) ................................................................99

Figure 5.3: Scanning Electron microscopy images of L. monocytogenes ATCC 19115 upon treatment with 24 kHz USW in SMUF; Control L. monocytogenes ATCC 19115(A), Treated L. monocytogenes ATCC 19115(B, C, D) ....................................................................................................100

Figure B.1: Growth curve of E. coli (ATCC 25922) in Tryptic Soy Broth (TSB) incubated in a shaking incubator set at 37°C and 200 rpm.................................109

Figure B.2: Growth curve of L. monocytogenes (ATCC 19115) in Tryptic Soy Broth (TSB) incubated in a incubator set at 37°C ...............................................110

Figure B.3: Survival curves of mid stationary-phase cells of E. coli ATCC 25922 on TSA (-■-), a non-selective and VRBA (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in phosphate buffer (pH 7.0)..............111

Figure B.4: Survival curves of mid stationary-phase cells of E. coli ATCC 25922 on TSA (-■-), a non-selective and VRBA (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in skim milk........................................112

Figure B.5: Survival curves of mid stationary-phase cells of L. monocytogenes ATCC 19115 on TSA (-■-), a non-selective and MOX (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in phosphate buffer (pH 7.0). ..............................................................................................................113

Figure B.6: Survival curves of mid stationary-phase cells of L. monocytogenes ATCC 19115 on TSA (-■-), a non-selective and MOX (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in skim milk .................114

Figure B.7: Survival curve of mid-log phase cells of E. coli ATCC 25922 by 24 kHz USW in phosphate buffer.............................................................................115

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LIST OF TABLES

Table 2.1: A comparison of fat globule size obtained in conventional and ultrasonic homogenization of milk at different power levels and sonication times....................................................................................................................22

Table 2.2: : Examples of low intensity ultrasound wave applications in food industry................................................................................................................29

Table 2.3: Composition of cow’s milk........................................................................30

Table 2.4: Dairy related incidents and outbreaks associated with E. coli O157:H7 and L. monocytogenes in the United States ......................................................32

Table 3.1: Effect of sonication medium on D values for inactivation of stationary-phase cells of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 by 24 kHz ultrasound waves ....................................................................................65

Table 4.1:Suspending media used for ultrasound treatment of E. coli and L. monocytogenes...................................................................................................77

Table 4.2: Effect of sugar concentration and sugar type on inactivation of E. coli ATCC 25922 upon sonication with 24 kHz USW. ...............................................87

Table A.1: Composition of dry blended mix ...............................................................108

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ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Stephanie Doores, for her guidance and

support throughout my study at The Pennsylvania State University. She always gave me

independence to pursue my research ideas and helped me shape them into my thesis

objectives.

I feel lucky to have such helpful committee members who have always been

willing to help me in my research. I would like to thank Dr. Steve Knabel and Dr. John

Coupland for insightful discussions and invaluable inputs throughout my research. I

would also like to thank Dr. Kelli Hoover for her suggestions.

I feel extremely lucky to have had lab mates like Donna Miller and Denise

Gardner. Their camaraderie and encouragement have perhaps been the highlight of my

graduate school experience. Especially Donna, who was always there to help me with

matters related to and even beyond the laboratory.

I would also like to acknowledge the support of main office staff members

especially Robert Lumely-Sapanski, Juanita Wolfe, Sue Kelleher and Melissa Ann

Strouse. I am thankful to the graduate students in the department for their friendship and

support whenever I needed it. I greatly appreciate help from Ibrahim Gϋlseren who

assisted me with ultrasound equipments.

This thesis would not have been possible without the encouragement and

support of my mother and brother. I am grateful to them for their perseverance and

enthusiasm for my higher studies despite any circumstances. Lastly, I would like to thank

my husband, Tanuj for his love, support and friendship, and patiently listening to me.

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CHAPTER 1 INTRODUCTION

Milk is an indispensable part of the human diet with per capita consumption of

83.9 liters recorded in 2006 in the United States (Goff, 2008). Milk contains nine

essential nutrients required for the growth and maintenance of life functions. However,

raw milk and cheese products may be the source of pathogens such as Escherichia coli

O157:H7 and Listeria monocytogenes which have been associated with foodborne

outbreaks (Headrick et al., 1998, Ramsaran et al., 1998). In fact, raw milk has been

found to be the second most important vehicle for transmission of E. coli O157:H7

(Reitsma and Henning, 1996).

Heat pasteurization is the most common processing technique currently used in

the food industry to kill pathogens and extend the shelf life of milk. The process

however, results in loss of nutritional and sensory properties of milk. With the increase in

demand of minimally processed foods, the importance for alternative processing

technologies that render food safe while retaining nutritional and sensory properties of

foods is increasing. Ultrasound technology is one of the emerging alternate technologies

for food processing. It uses ultrasound waves (USW) with frequency higher than 20 kHz

to inactivate microorganisms. However, it is still in its infancy as a potential

pasteurization technology.

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Until now, most of the studies for inactivation of microorganisms by USW have

been conducted in buffers. However, for its application in the food industry, the potential

of ultrasound waves (USW) to inactivate bacteria in real foods like milk needs to be

investigated.

Milk is more complex than buffer systems and the kinetics of inactivation of

bacteria could be different in milk than buffer. Also, the study of protective effects of milk

components and their interactions might help the food industry to develop and optimize

the commercial technology to inactivate microorganisms by ultrasound. Further, since

the exact composition of bovine milk is known, it is easier to simulate milk using an

artificial milk system in order to study the effect of milk components on sonoprotection.

The objectives of the project were:

1. To investigate the effect of frequency of USW on inactivation of E. coli in

phosphate buffer.

2. To determine the inactivation kinetics of E. coli and L. monocytogenes by USW in

phosphate buffer, whole milk and skim milk.

3. To determine the effect of growth phase of E. coli and L. monocytogenes on

inactivation by USW in phosphate buffer, whole milk and skim milk.

4. To investigate the sonoprotective effect of milk components and their interactions

on E. coli and L. monocytogenes.

5. To examine the structural changes in cell morphology upon ultrasound treatment

using Scanning Electron Microscopy (SEM)

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CHAPTER 2 LITERATURE REVIEW

2.1 Ultrasound waves (USW) and their physical nature

Ultrasound waves (USW) are sound waves with frequencies above 20 kHz,

which is above the threshold frequency for human hearing. Ultrasound waves are

mechanical waves generated by piezoelectric materials such as barium titanate under

the influence of a high frequency electric field. After conversion of electric field to

mechanical oscillations, the sound waves are transmitted to an amplifier (horn) and

finally to the medium (Cartwright, 1998). In the medium, they travel as longitudinal

waves with alternate compression and rarefaction regions characterized by positive and

negative pressure zones, respectively (Suslick, 1988)

2.1.1 Ultrasound waves (USW) and cavitation

Cavitation is the process of formation, growth and collapse of bubbles by USW in

the medium (Figure 2.1). As the wave passes through the medium, continuous pressure

changes in the medium lead to the process known as cavitation. Cavitation bubbles are

formed in the rarefaction region of the sound wave due to negative pressure at this site.

As the wave front passes, the cavitation bubbles oscillate and grow in size in the

compression region, a positive-pressure region. These bubbles grow to a maximum

unstable size over many alternating compression and rarefaction cycles, beyond which

they can not expand. The bubbles finally collapse resulting in radiation of shock waves

from the site of collapse (Suslick, 1990).

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Adapted from

http://www.chemsoc.org/ExemplarChem/entries/2004/bristol_eaimkhong/cavitation.gif

When the collapse occurs near a solid surface such as the cell walls of bacteria,

these shock waves might cause mechanical damage to the cell surface (Frizzell, 1988).

Also, the collapse of the bubble produces short-lived intense local temperatures of

~5000°C and high pressures of ~500 atm. These localized effects can initiate chemical

reactions leading to the formation of free radicals (Suslick, 1990). The shock waves and

free radicals produced during cavitation have been proposed to be the cause of cell

destruction by USW (El’piner, 1964, Suslick 1988).

Figure 2.1: Cavitation under the influence of ultrasound field

Maximum

bubble size

Bubble collapse

in compression

Cycle repeats

New bubble growth

Cavitation bubble

growth in negative

pressure

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2.2 Ultrasound waves in food processing

Prior to the 20th century ultrasound energy was used to detect objects under

water. It was soon discovered that ultrasound is not solely a passive agent like that used

in detection, but could also initiate many chemical reactions. USW were demonstrated to

be used for emulsification (formation of a colloid from water and oil), atomization

(production of a mist consisting of small droplets of liquids in air) and biological damage

of cells (Suslick, 1990). The inactivation potential of ultrasound has been known since

the first study of Harvey and Loomis (1929) on luminescent bacteria. However, it is a

fairly new technology for food processing applications. For a technology to be accepted

as the alternate technology of pasteurization, it should be able to kill pathogens and

lower the bacterial load on the foods as well as to preserve the nutritional and functional

properties of foods (NACMF, 2006).

In thermal processing, the D value is typically used as a measure of reduction of

bacterial cell numbers by heat treatment. D value is defined as the time of heat

treatment required at a certain temperature to reduce the number of viable bacterial

population by 90% or a one-log reduction (Frazier and Westhoff, 1988). The term, D

value, it is not limited to heat treatment however, but can also be used to measure

bacterial inactivation by alternate technologies.

The following terms are generally used for ultrasound treatment under different

conditions:

• Sonication – Ultrasound treatment at sublethal temperatures (<45°C) with the

system open to the atmosphere

• Mano-sonication (MS) – Ultrasound treatment at sublethal temperatures (<45°C)

under pressure (100-600 kPa)

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• Thermosonication (TS) - Ultrasound treatment at lethal temperatures (>45°C) in

an open system

• Mano-thermosonication (MTS) - Ultrasound treatment at lethal temperatures

(>45°C) under pressure (100-600 kPa)

Ultrasound holds the promise of being established as an alternate pasteurization

technology. When compared with conventional heat treatment, ultrasound was found to

inactivate a wider range of bacterial species. The D62°C value of Streptococcus faecium

was 700 times higher than Aeromonas hydrophila (7.1 and 0.0096 min, respectively) in

McIlvaine citrate-phosphate buffer. However, for mano-sonication (MS) at 200 kPa in the

same medium, the D40°C value for S. faecium was greater than 4 times the value for A.

hydrophila (4.0 and 0.9 min, respectively) suggesting that while a single manosonication

treatment at 200 kPa would be adequate to inactivate these bacteria, the amount of heat

required might vary considerably (Pagan et al., 1999b).

2.2.1 Food industry applications of ultrasound waves

USW are used for various applications in the food industry and are generally

classified into two categories –

• Low Energy Ultrasound: Low energy USW are defined as waves with

intensities lower than 1 W/cm2 and frequencies higher than 100 kHz. These

have been used for non-invasive detection and characterization of

physicochemical properties of food material such as crystallization and

viscosity of the food material (Coupland and Saggin, 2003, McClements,

1997).

• High Energy Ultrasound: High energy USW are defined as waves with

intensities higher than 1 W/cm2 (typically 10-1000 W/cm2) and frequencies

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between 18 and 100 kHz. These have been used to break open cells for

extraction of enzymes and proteins, induction of oxidation reduction reactions

and inactivation of enzymes and microorganisms (Knorr et al., 2004,

McClements, 1997).

2.3 Microbial inactivation by ultrasound waves (USW)

High energy USW have also been shown to inactivate a wide range of

microorganisms. The lethality of USW against microorganisms however depends upon

many factors that can be categorized into the following groups -

1. Properties of the waves (frequency, amplitude and energy)

2. Sonication conditions (temperature and pressure)

3. Type of the organism (Gram-positive or Gram-negative, vegetative or spore

forms and log-phase or stationary-phase cells)

4. Sonication medium (buffer, distilled water and food systems)

2.3.1 Properties of waves

2.3.1.1 Frequency of Ultrasound waves

Frequency of ultrasound has been found to have a great impact upon microbial

destruction by sonication. In general, as the frequency of USW increases, bacterial

killing by USW decreases. Hua and Thomson (2000) found that the most effective

frequency for E. coli inactivation was 205 kHz, which was the lowest frequency in the

tested range of 205 to1017 kHz. The rate constants for inactivation of E. coli by 205 kHz

and 1017 kHz USW were 0.078 min-1 and 0.030 min-1, respectively suggesting that high

frequency USW is slower in killing bacteria than low frequency USW. Not only the rate,

but killing potential of low frequency USW has also been found to be greater than high

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frequency USW. Application of USW at frequencies of 20 and 38 kHz showed a

noticeable decrease in number of survivors of Bacillus subtilis after 15 min of treatment

in nutrient broth, whereas USW frequencies of 512 and 850 kHz resulted in an increase

in the number (150%) of cells suggesting that USW at high frequencies result in

declumping of cells rather than killing of bacteria (Joyce et al., 2003). The decreased

inactivation observed at higher frequencies has been attributed to decrease in time

needed for bubble formation leading to less intense cavitation and hence less

inactivation by high frequency USW (El'piner, 1964). The ultrasonic frequencies above

2.5 MHz do not produce cavitation bubbles and hence are not effective for microbial

destruction (Piyasena et al., 2003). The optimal range for microbial inactivation has

generally been found to be between 20 and 100 kHz. Most of the studies with bacterial

inactivation have been done using 20 or 24 kHz USW. Therefore, for the remainder of

the literature review, the frequencies of USW refer to either of these frequencies unless

otherwise stated.

2.3.1.2 Amplitude of ultrasound waves

Amplitude of USW has also been found to have a major effect on inactivation

potential of USW. In general, an increase in amplitude results in an increase in

inactivation of the microorganisms by USW. A 6-fold decrease in DMS values for

inactivation of S. faecium, Listeria monocytogenes, Salmonella Enteritidis and A.

hydrophila was seen when the amplitude of 20 kHz USW was increased from 62 to 150

μm. The magnitude of the inactivation in response to amplitude was the same for both

Gram-positive and Gram-negative organisms (Pagan et al., 1999b). The DMS 200 kPa value

for Yersinia enterocolitica upon ultrasound treatment decreased from 4.0 to 0.37 min

when the amplitude of USW was increased from 21 to 150 μm (Raso et al., 1998a). At

higher amplitudes, the effective size of the zone of liquid undergoing cavitation

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increases, thereby increasing the cavitation and hence, rate of inactivation by USW

(Suslick, 1990). Also, at higher amplitudes the power output increased exponentially

independent of temperature and pressure of sonication; the value being doubled with

every 63 µm increment of amplitude (Raso et al., 1999).

2.3.1.3 Energy of ultrasound waves

Intensity of ultrasound waves is a measure of its energy and also affects the

inactivation of bacteria by ultrasound. The energy output of ultrasound can be given in

terms of intensity (Wcm-2) or Acoustic Energy Density, AED (Wml-1). The advantage of

using AED compared to intensity is that it could be used for scale-up of the technology

(Loning et al., 2002). The inactivation of bacteria has generally been found to increase

with increase in intensity of ultrasound. A 3-fold increase in AED from 0.49 to 1.43 Wml-1

resulted in ~3.5 and ~4.5-fold decrease in D22.3 kHz value of inactivation of Shigella boydii

and L. monocytogenes in 0.85% NaCl solution (Romero et al., 2007). Inactivation of E.

coli in distilled water by 20 kHz USW was also reported to increase as the power

intensity was increased from 4.6 to 74 Wcm-2 (Hua and Thompson, 2000). Stanley et al.

(2004) reported that upon sonication with 20 kHz USW at 40°C for 10 min, log

reductions of E. coli increased from 0.5 to 8.0 when ultrasound intensity was increased

from 9.5 Wcm-2 to 49.2 Wcm-2. The inactivation of L. monocytogenes has also been

found to be linearly related to power delivered to the treatment medium (Manas et al.,

2000).

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2.3.2 Sonication conditions

2.3.2.1 Effect of temperature

The temperature of the medium, however, is perhaps a more critical factor

affecting the percentage of surviving cells during sonication. Thermo-sonication has

been found to have more killing effect than heat treatment alone (Cabeza et al., 2004).

Pagan et al. (1999b) found that the ultrasound inactivation rate of A. hydrophila

suspended in McIlvaine citrate-phosphate buffer increased from 1.28 min-1 to 7.38 min-1

as temperature increased from 40 to 62°C. Also, an additive effect of ultrasound and

temperature was observed on inactivation of L. monocytogenes, S. Enteritidis and A.

hydrophila in a temperature range between 40 to 62°C. In the case of Yersinia

enterocolitica, the effect of temperature and USW was found to be additive in a range of

temperature between 50 and 58°C, above which the effect of heat became predominant

(Raso et al., 1998a).

However, in other studies, the inactivation by USW and heat has been attributed

to the synergistic action of both treatments. The rate of inactivation of vegetative cells of

S. faecium upon MTS treatment in McIlvaine citrate-phosphate buffer was found to be

more than the expected rate of addition of ultrasound and heat (62°C) demonstrating

that the inactivation by MTS was the result of a synergistic effect of heat and ultrasound

(Pagan et al., 1999b). The synergistic mode of killing was also reported upon sonication

of S. enterica in distilled water and eggs (Cabeza et al., 2004). It has also been found to

be dependent upon water activity of the sonication medium. The synergistic effect of

inactivation of USW on Salmonella enterica increased when the water activity of

McIlvaine citrate-phosphate buffer was decreased from 0.99 to 0.96 using sucrose

(Alvarez et al., 2003). Not only the vegetative cells, but spore inactivation has also been

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shown to follow synergistic kinetics of heat and ultrasound. Raso et al. (1998b) reported

that inactivation of spores of Bacillus subtilis between 70 and 95°C was due to the

synergistic effect of heat and USW. However, there is a limit of temperature up to which

USW and heat work in a synergistic or additive manner and above this temperature

thermosonication behaves as a heat treatment only. At higher temperatures, generally

starting from 70°C, the violence of bubble collapse decreases due to high vapor

pressure of gas in the bubble which acts as a cushion and lowers the intensity of

collapse (Ashokkumar et al., 2007, Suslick, 1988).

2.3.2.2 Effect of pressure

The D values for inactivation of vegetative cells of S. faecium, L. monocytogenes

S. Enteritidis and A. hydrophila by sonication decreased with the increase in static

pressure during sonication. The DMS values for S. faecium, L. monocytogenes, S.

Enteritidis and A. hydrophila at 0 kPa were 2.46, 1.49, 1.34 and 1.12 min, while those at

300 kPa were 1.4, 0.9, 0.78 and 0.88 min, respectively. However, a further increase in

pressure from 300 to 400 kPa did not result in significant decrease in DMS value (Pagan

et al., 1999b). The DMS value of sonication of Y. enterocolitica also decreased from 1.5 to

0.22 min when pressure during sonication was increased from 0 to 600 kPa (Raso et al.,

1998a). Similar trends have also been observed during manosonication inactivation of

spores. Inactivation of Bacillus subtilis spores increased with increase in static pressure

of sonication treatment, with a maximum 2 log reduction being achieved in 12 min at 500

kPa. However, increase in pressure above 500 kPa did not result in any significant

increase in inactivation of B. subtilis spores by USW (Raso et al., 1998b). At high

pressure the time required for bubble collapse decreases and the intensity of implosion

increases leading to more inactivation at high pressures (Suslick, 1988). However,

above a certain pressure, the ultrasound field is incapable of overcoming the combined

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forces of pressure and cohesive forces of liquid leading to less cavitation and hence

lowered inactivation of bacteria (Raso et al., 1999).

2.3.3 Type of microorganisms

Generally, bacterial spores are the most resistant microorganisms to any

physical or chemical treatment. Among vegetative forms, Gram-positive bacteria are

usually more resistant than Gram-negative bacteria due to their thicker peptidoglycan

cell wall. The resistance of bacteria also depends upon its shape, with cocci being more

resistant than rods forms (Frazier and Westhoff, 1988). However, it is difficult to compare

the resistance of different organisms to ultrasound due to the difference in experimental

conditions such as the type of instrument, strain of the organism and sonication medium

used in different studies. Pagan et al. (1999b) compared the inactivation behavior of S.

faecium, L. monocytogenes, S. Enteritidis and A. hydrophila. They found that the DMS

values for the sonication for Gram-positive organisms S. faecium and L.

monocytogenes, were higher (4.0 and 1.5 min, respectively) than the Gram-negative

organisms S. Enteritidis and A. hydrophila (0.86 and 0.9 min, respectively). Similarly, L.

monocytogenes, a Gram-positive rod (DMS = 7.3 min) was more resistant to sonication

than S. boydii, a Gram-negative rod (DMS = 2.5 min) under the same sonication

conditions (Romero et al., 2007). In another study, S. thermophilus, a Gram-positive

coccus, showed a 2.7 log reduction as compared to a 4.7 log reduction observed with P.

fluorescens, a Gram-negative rod upon sonication with 20 kHz USW (Villamiel and de

Jong, 2000). These differences in resistance can be attributed to the type and shape of

the bacteria. Gram-negative and rod shape bacteria are more sensitive to sonication

than Gram-positive and coccus-shaped bacteria (Manas and Pagan, 2005).

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A comparison of vegetative cells to spores can be made in the studies of Raso et

al. (1998b) and Pagan et al. (1999b) where they used the same sonication machine and

parameters. Calculations from graphs showed that even for the most resistant organism

among those studied (S. faecium) it took ~3 min to reduce the population by 1 log at 200

kPa and 60°C, whereas, for B. subtilis spores the same amount of reduction was

achieved in 12 min at 500 kPa and 70°C. The latter, mano-sonication process is

conducted at a higher temperature and pressure for a longer time compared to S.

faecium, demonstrating that spores are more resistant than vegetative cells. The

increased resistance of bacterial spores has been attributed to the thick cortex and low

internal water content of the core of the spores (Frazier and Westhoff, 1988).

Raso et al. (1998b) reported that 98% of B. subtilis spores survived a heat shock

of 80°C for 10 min, whereas the number of survivors after MTS at 70°C for 10 min was

30% suggesting that spores are more sensitive to MTS treatment than heat alone.

Similar results were reported by Gracia et al. (1989), where the D value for heat

inactivation of B. subtilis spores in distilled water at 70.1°C was 21.23 min while that for

thermosonication at the same temperature was 11.04 min. In addition, USW have also

been shown to decrease the heat resistance of spores. Burgos et al. (1972) found that

D110°C for heat inactivation of B. cereus spores decreased from 11.5 to 1.5 min and D99°C

of B. licheniformis decreased from 5.5 to 3 min following sonication.

2.3.4 Sonication medium

2.3.4.1 Composition of foods

Owing to their heterogeneous nature, foods exert a protective effect on microbes

when they are subjected to inactivation treatments such as heat, chemical and high

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pressure treatments (Black et al., 2007, Moats et al., 1971). This protective nature of

foods makes the task of achieving safe levels of microorganisms in food more difficult.

Food matrices have also been shown to protect organisms against ultrasound. The

protective nature of foods was reported by comparing the inactivation of different species

of Salmonella upon sonication in peptone water and chocolate milk. A 10-min sonication

treatment of S. Eastbourne in peptone water with 160 kHz USW reduced the population

by 2.5 logs whereas only a 0.13 log reduction was achieved in chocolate milk indicating

that chocolate milk provided more protection to the bacteria than peptone water under

the same sonication conditions (Lee et al., 1989). Wrigley and Llorca (1992) compared

inactivation of S. Typhimurium upon sonication in three different media (brain heart

infusion broth; BHIB skim milk and liquid whole egg). Salmonella Typhimurium

suspended in BHIB exhibited 4.0 log reductions upon sonication at 40°C for 30 min,

while those obtained in skim milk and liquid whole egg were 3.0 and 0.75 log reductions,

respectively under the same sonication conditions. The protective effect of the egg was

found to be highest followed by skim milk and BHIB. This can be attributed to the

viscous nature of eggs leading to decreased cavitation in eggs (Raso et al., 1999) or

interaction of food components with bacteria (Manas et al., 2001).

2.3.4.2 Water activity (aw) of the sonication medium

Water activity (aw) of the sonication medium has been found to affect the

inactivation of microorganisms by sonication but to a much less extent than heat

treatment. The D60°C value for heat inactivation of Salmonella Enteritidis increased from

0.10 to 2.70 min (27 times) when the aw was decreased from >0.99 to 0.96. However,

the DMS value for sonication increased from 0.89 to 1.37 min (~1.5 times) with the same

decrease in aw suggesting that while a wide range of heat treatments would be required

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for processing of foods with different water activities, one sonication treatment would be

sufficient to inactivate bacteria in a wide range of food products (Alvarez et al., 2003).

2.3.4.3 pH of the sonication medium

The pH of the medium also influences inactivation of microorganisms by

ultrasound waves. Mack and Roberts (2007) showed that as the pH of malic and citric

acid solution was decreased from 4.0 to 2.5, the log reductions of E. coli upon sonication

were increased by 0.6 and 1 log, respectively. However, the type of organic acid did not

have any influence on inactivation by ultrasound at pH 4.0. Moreover, in a 0.85% NaCl

solution adjusted to pH 3.4 with malic acid, an additive inactivation effect of pH and

sonication was reported for L. monocytogenes (Baumann et al., 2005). Also, a 5 log

reduction of E. coli was achieved in apple cider at 50°C as compared to 2.25 log

reduction in the case of L .monocytogenes again demonstrating that Gram-negative

organisms are more sensitive than Gram-positive organisms to combined pH and

sonication treatment (Ugarte et al., 2006).

2.3.4.4 Amount of solids in sonication medium

Increase in soluble solids has been reported to decrease the inactivation of E.

coli by sonication. Mack and Roberts (2007) found that the time required to achieve a 5

log reduction upon sonication with 24 kHz USW increased from 10 to 12 min when the

amount of soluble solids (D-glucose) in distilled water was increased from 0 to

16g/100ml. This was attributed to decrease in tensile strength of the medium in the

presence of particulate matter leading to decrease in amount of energy required for

bubble formation. Hence there is less energy in the bubble and implosion is less intense

leading to the decrease in efficiency of inactivation by ultrasound waves (Mack and

Roberts, 2007). Valero et al. (2007) reported that presence of pulp in orange juice

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decreased the amount of inactivation achieved upon sonication with 500 kHz USW. The

authors reported that ~1 log reduction was achieved when 0.1% pulp was present in

orange juice. However, when the concentration of pulp was 1% and 10%, no significant

decrease in number of survivors was observed suggesting that pulp had a protective

influence on microbes.

2.3.4.5 Volume of the sonication medium

Volume of the sonication medium also affects the outcomes obtained by the

sonication treatments. An initial increase followed by decrease in number of Bacillus

subtilis has been reported upon ultrasound treatment in larger volumes. The number of

survivors of B. subtilis decreased to 20, 60 and 95% during 15 min sonication in 100,

150 and 250 ml samples, respectively suggesting that more bacteria are killed when the

volume of sample is less (Joyce et al., 2003).

2.3.4.6 Presence of chemicals and antimicrobials

Chemicals and antimicrobial agents have been combined with ultrasound

treatment to kill bacteria more efficiently. Ferrante et al. (2007) reported that 20 kHz

USW alone was able to reduce the number of L. monocytogenes in orange juice by ~1

log in 15 min. However, sonication of L. monocytogenes in orange juice supplemented

with vanillin (a natural antimicrobial substance) at concentrations of 1000 and 1500 ppm,

resulted in 1.8 and 3.5 log reductions, respectively. Further addition of another natural

antimicrobial compound, citral, at concentrations of 50 and 100 ppm resulted in a 6 log

reduction in 10 and ~7.5 min, respectively.

Similarly, the efficiency of the antibiotic, gentamicin, in killing P. aeruginosa and

E. coli increased in the presence of 67 kHz USW. A 6 h culture of P. aeruginosa was not

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susceptible to 67 kHz USW. However, when P. aeruginosa culture was sonicated in 12

µg/ml gentamicin solution, it exhibited 3 log reductions in 2 h. E. coli also showed ~2.5

log reductions under the same conditions in the presence of antibiotic (Pitt et al., 1994).

Ultrasound has also been combined with other chemicals to kill bacteria during

food processing. Rodgers and Ryser (2004) reported that ultrasound (44 to 48 kHz) was

able to reduce the number of E. coli O157:H7 and L. monocytogenes in apple cider by

1.9 and 1 log, respectively. However, when ultrasound treatment was combined with the

sodium hypochlorite and copper ion water during the process of apple cider production,

a 5 log reduction in number of both E. coli O157:H7 and L. monocytogenes was

achieved suggesting that using ultrasound during apple cider processing could reduce

the risk of foodborne illness associated with consuming unpasteurized apple cider.

2.4 Mechanism of inactivation

The mechanism of ultrasound inactivation of microorganisms is still unknown;

though various theories have been put forward. The most widely accepted theory is that

the shock waves generated by cavitation cause mechanical damage to outer structures

of the cell leading to cell destruction (El'piner, 1964). Other alternate mechanisms

attributed to microbial destruction by ultrasound include increase in temperature leading

to formation of free radicals in the sonication medium (Manas and Pagan, 2005).

2.4.1 Mechanical damage to bacteria by USW

The most widely accepted mechanism of destruction by ultrasound has been

attributed to mechanical damage to the cells by USW. The high pressure shock waves

generated by implosion of the cavitation bubbles can cause mechanical damage to

membranes of the cells thus leading to its destruction. The destruction could however,

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be influenced by the differences between the cell wall structures of Gram-negative and

Gram-positive bacteria. The cell wall of Gram-negative bacteria is more complex than

Gram-positive bacteria. It constitutes a thin peptidoglycan layer surrounded by an outer

membrane. Whereas, the cell wall of Gram-positive bacteria is a thick peptidoglycan

layer which is not surrounded by an outer membrane (Frazier and Westhoff, 1988).

Ananta et al. (2005) reported that death of Lactobacillus rhamnosus and E. coli occurred

without any damage to the cytoplasmic membrane of the cells. The authors used

propidium iodide (PI) to indicate membrane integrity, as a method to elucidate the

mechanism of inactivation. Cells take up PI when the cell membrane is disrupted.

Researchers found that only 7% of cells of L. rhamnosus were labeled with PI, while

92% inactivation was achieved upon sonication suggesting that the major site of action

of ultrasound inactivation could not be the cytoplasmic membrane. However, there was

considerable damage to the outer membrane of Gram-negative bacteria. This change in

physical/physiological nature of organisms has also been shown in spores of Bacillus

thuringiensis where the spores fragmented and fused together leading to a decrease in

number of spores upon sonication with a non-contact transducer with frequencies of 93

and 161 kHz (Hoover et al., 2002).

2.4.2 Free radical generation by USW leading to cell death

When bubbles implode during cavitation very high localized temperatures and

pressures are generated leading to the formation of free radicals (Suslick, 1990). These

free radicals could inactivate bacterial cells. However, it has been demonstrated that the

effect of free radicals towards inactivation of Y. enterocolitica was negligible. Raso et al.

(1998a) used the free radical scavenger, cysteamine, in sonication medium to determine

the difference in inactivation kinetics of Y. enterocolitica by ultrasound. D values for cells

in the presence of free radical scavenger should increase if free radicals are causing the

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destruction in microbial cells. However, it was found that the D value did not change

significantly showing that free radicals are not involved in the inactivation of Y.

enterocolitica by USW. Furuta et al. (2004) compared the formation of hydrogen

peroxide and inactivation of E. coli by USW in water. They reported that even though the

inactivation of E. coli increased with the increase in amplitude starting at 1 µm, there was

no increase in concentration of hydrogen peroxide until the amplitude exceeded 3 µm,

suggesting that shock waves might be a more important phenomenon of cell destruction

than free radical formation.

2.4.3 High temperatures generated by USW leading to cell death

The increase in temperature during sonication has also been thought to be one

of the reasons of ultrasound inactivation. However, it was demonstrated that when the

temperature of sonication was maintained below 30°C, a sublethal temperature for E.

coli, a 7 log reduction was achieved in 12 min (Mack and Roberts, 2007). Similarly,

inactivation of S. faecium, L. monocytogenes, S. Enteritidis, A. hydrophila and Y.

enterocolitica has been reported upon MS treatment at 40°C and 200 kPa with D values

of 4.0, 1.5, 0.86, 0.90 and 0.7 min, respectively (Pagan et al., 1999b, Raso et al.,

1998a). The high localized temperatures (5000°C) generated during ultrasound

treatment could not be the cause of bacterial cell death because the cooling rate for

those points is as high as 109 K/s to even react with any cell molecules (Suslick, 1990).

2.5 Injury of bacteria by USW

Among all alternate food processing technologies, ultrasound is the only

technology that has been shown to have an “all or nothing” principle of inactivation. No

injured cells were found in the case of sonication of L. monocytogenes at sublethal

temperatures (Pagan et al., 1999a). Numbers of Salmonella spp. sonicated with 160 kHz

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USW were similar when recovered on Brilliant Green agar (BGA), a selective medium

and Tryptic Soy Agar (TSA), a non-selective medium indicating that there was no injury

of the bacteria upon sonication (Lee et al., 1989). Also, survival curves of Salmonella

enterica obtained by recovery on Nutrient Agar and Nutrient Agar + 3% NaCl were

similar showing that there is no sublethal injury to the cells during mano-sonication at

175 kPa in phosphate buffer (pH 7) (Alvarez et al., 2003).

2.6 Kinetics of bacterial inactivation

First order inactivation kinetics was reported for L. monocytogenes upon

sonication with 20 kHz USW in 0.85% NaCl solution. However, tailing was observed

after 15 min of sonication for Shigella boydii under the same set of conditions. Also, a

2.7 log reduction was observed in counts of L. monocytogenes compared to 5.5 log

reduction for Shigella boydii (Romero et al., 2007). Baumann et al. (2005) also reported

first order inactivation kinetics of L. monocytogenes at sublethal temperatures in apple

cider (20 to 24°C) but a biphasic curve was seen at lethal temperatures (50 to 60°C).

D’Amico et al. (2006) also reported in their work that ultrasound inactivation follows a

polynomial kinetics when L. monocytogenes and E. coli were sonicated in milk and apple

cider at 57°C. This non linear kinetics was also reported by (Hua and Thompson, 2000)

during the sonication of E. coli in distilled water.

2.7 Improved shelf life and stability of foods

Ultrasound waves have not only been found to inactivate bacteria but also show

potential to enhance the shelf life and improve the sensory properties of foods. Milk has

been reported to be homogenized upon sonication. A reduction in fat globule size of

sonicated milk samples was observed as compared to control milk samples. The size of

the fat globule was reduced by 81.5% upon thermosonication of milk for 102.3 s with 20

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kHz USW at 75.5°C More reduction in size was further observed during longer periods of

sonication. Particle size distribution of samples showed that the thermosonicated sample

had fat globules in the range of 0.57 to 0.95 μm (Villamiel and de Jong, 2000b). The

typical range of fat globule size obtained from conventional homogenization varies from

0.1 to 0.4 µm with an average globule size of 2.38 µm depending upon various factors

like type of valve, pressure and fat content etc. When comparing the fat globule size of

manothermosonicated milk to untreated milk, it was found that 99.9% of the fat globules

of manothermosonicated milk are in the size range of 0 to 1.6 μm, whereas the number

for untreated milk samples is only 31.5% (Vercet et al., 2002b). Ertugay et al. (2004)

also compared homogenization of milk by ultrasound and conventional methods. USW

at four power levels of 90, 180, 360 and 450 W were tested for their ability to

homogenize the milk. Maximum homogenization and lowest fat globule size (0.725 µm)

was achieved by 450 W ultrasound waves in 10 min. This globule size was found to be

smaller than the globular size of 2.625 µm obtained by conventional homogenization.

However, a comparable globule size (2.375 µm) to conventional homogenization was

achieved by using 180 W for 10 min. In addition to globule size, the effectiveness of

ultrasound homogenization was also measured by homogenization efficiency of

ultrasound.

Homogenization efficiency was calculated as –

Where

a = fat content from the upper part of milk in graduated cylinder (1/10)

b = fat content from the bottom of milk in graduated cylinder (9/10)

The lower the value of homogenization efficiency, the more efficient is the

process. The efficiency of homogenization by USW was found to increase with increase

abaHE 100×−

= 2.1

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in power level and sonication time; with the maximum efficiency at 450 W having HE

value of 3%. Similar results were reported by Wu et al. (2001) in a separate study, where

they also found that increasing the level of power of USW results in finer fat globule size

and more homogenization (Table 2.1).

Fat globule size similar to conventional homogenization was achieved either by

sonication of milk for 1 min at 450 W or by using 180 W ultrasound waves for 10 min

(Table 2.1). In both studies, however, sonication with 450 W ultrasound waves for 10

min resulted in fat globule size of <1 µm and the globules were not visible

microscopically. At a diameter below 0.8 μm, creaming effect during storage is known to

decrease leading to an increase in the shelf life of milk. Moreover, the reduction in size

of fat globules in milk imparted improved sensory properties (Villamiel and de Jong,

2000b).

Undesirable enzymes in milk also limit its shelf life. Thus, the inactivation of these

enzymes is necessary to extend the shelf life of milk. Alkaline phosphatase is a native

enzyme of milk and is inactivated during the thermal pasteurization treatment. Absence

Table 2.1: A comparison of fat globule size obtained in conventional and ultrasonic homogenization of milk at different power levels and sonication times

Mean Diameter (µm)*

Treatment Time (min) Ertugay et al.

(2002) Wu et al. (2001)

Non-homogenized 5.5 ~5.0 Conventional homogenization 2.625 2 or lessa Ultrasonic homogenization 180W 10 2.375 225W 10 < 2 450W 1 2 or lessa 450W 10 0.725 (<1)

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of alkaline phosphatase confirms the successful pasteurization process. Alkaline

phosphatase and lactoperoxidase activity were found to decrease ~2.5 and ~6 times,

respectively, upon sonication with 20 kHz USW at 40°C (Ertugay et al., 2003). Villamiel

and de Jong (2000a) also demonstrated that the native enzymes of milk such as alkaline

phosphatase, glutamyltranspeptidase and lactoperoxidase were inactivated more rapidly

by combined heat and ultrasound treatment than heat or sonication treatment alone. No

enzyme activity of alkaline phosphatase was observed after 102 s in continuous flow

ultrasonic treatment of milk suggesting that ultrasound might be used on a commercial

scale for milk processing.

Milk is susceptible to oxidative deterioration which also limits its shelf. Milk fat is

autooxidized in the presence of metals and air leading to oxidized flavor in milk thus

decreasing its consumer acceptability. Sonication has been found to increase the

antioxidant activity in milk. Ultrasound disrupts the casein micelles leading to the

increased concentration of casein and hence increasing the antioxidant activity of skim

milk (Taylor and Richardson, 1980).

Ultrasound also seems promising to extend the shelf life of juices.

Pectinesterases are the enzymes that hydrolyze pectin, a major constituent of juice, and

limit the shelf life of the juices. The activity of pectinmethylesterase (PME) in lemon juice

was effectively decreased by sonication thereby improving cloud stability when stored for

18 d at 4ºC (Knorr et al., 2004). Also, the thermoresistant isozyme of PME was found to

be more sensitive to manothermosonication than heat. Vercet et al. (1999) reported that

the D72°C value for heat inactivation of the thermoresistant fraction of PME in citrate

buffer was 20 min, whereas the D72°C value for MTS treatment at 200 kPa was 0.8 min.

In addition, pectin was also found to confer protection to the enzyme as suggested by

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D72°C values, making it more heat resistant. D72°C value for inactivation of thermoresistant

PME was 125 min in citrate and 1000 min in the same buffer supplemented with 0.01%

pectin, suggesting that heat resistance is not only due to the structure of the enzyme but

also the presence of the solutes in buffer. Similarly, PME from tomato has been found to

be sensitive to MTS treatment. Raviyan et al. (2005) found that the D50°C value for heat

inactivation of tomato PME was 1571 min while that for sonication treatment with 20 kHz

USW at the same temperature was 24 min. As the temperature of sonication was further

increased to 61 and 72°C, the D values were found to be 0.8 and 0.3 min, respectively.

Also, inactivation increased with the increase in intensity of ultrasound, with the lowest D

values obtained at highest sound intensity (0.020 mgL-1min-1) tested. The authors found

a synergistic effect of ultrasound and heat for the inactivation of PME and the

inactivation followed first order kinetics. This is promising for the tomato pulp industry

since the enzyme can be completely inactivated at lower temperatures without the flavor

loss of the product. Kuldiloke et al. (2007) also found a 70% decrease in activity of

pectinesterase upon sonication at 50°C. Thus, ultrasound ensures more shelf stable and

tastier food products.

2.8 Improved functional properties of food products

Ultrasound has also been reported to improve the functional properties of foods.

Vercet et al. (2002b) found that milk treated with MTS resulted in the formation of

superior yogurt as compared to control milk sample. The manothermosonicated yogurts

were found to have a firmer structure than the control yogurt. This was attributed to

changes in fat globule membrane caused by MTS leading to the interaction of fat

globules with casein micelles thus improving their gelling properties of yogurt. Sonication

not only improved the water holding capacity and reduced syneresis (collection of whey

on the surface of yogurt) in the yogurt, but also reduced the fermentation time by 0.5 h.

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Sener et al. (2006) demonstrated that a higher level of lactose hydrolysis (90%) by β-

galactosidase was achieved with sonication as compared to other studies conducted

without sonication (7-70%). The authors also reported that the amount of the enzyme

required was half the amount needed with other methods and 75% of the enzyme

remained active after the treatment. This forms an efficient and cost effective process of

lactose hydrolysis for the production of lactose free milk.

Ugarte et al. (2006) demonstrated that the physical properties of apple cider such

as titratable acidity, pH and °Brix were not affected by ultrasound treatment. Ultrasound

has also been found to be helpful in retaining nutritional and organoleptic properties of

juices. Degradation of ascorbic acid in orange juice during storage at 20ºC has been

found to be lesser after thermosonication than after temperature treatment alone (Knorr

et al., 2004).

2.9 Enzyme inactivation by ultrasound waves

Ultrasound has been shown to be effective against various other enzymes

including heat labile and thermostable enzymes. The enzyme activity of the

thermostable enzyme lysozyme was found to decrease by ~16-fold (from 100% residual

activity to 6% residual activity) in 30 sec upon MTS treatment at 80°C and 200 kPa in

phosphate buffer. However, there was no decrease in enzyme activity upon sonication at

lower temperatures (40 and 50°C). The enzyme inactivation kinetics showed a biphasic

curve with shoulders followed by exponential decrease in activity suggesting that

inactivation of enzymes by MTS is a two step process wherein the protein unfolds and

loses its physical stability by the action of bubbles generated by USW rendering the

enzyme more susceptible to another denaturing agent such as heat. This leads to a

second step of enzyme inactivation where it loses its catalytic activity by the breakage of

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disulfide bonds. Vercet et al. (2002a) also suggested the same mechanisms of

inactivation of lipase and protease by MTS. The authors suggested that shear stresses

and interface unfolding of the enzyme could be the possible mechanism of inactivation of

enzymes produced by Pseudomonas fluorescens. The inactivation of lysozyme by MTS

was found to be synergistic in action (Manas et al., 2006). Similar synergistic inactivation

by MTS was also observed in the case of peroxidase, lipoxygenase and polyphenol

oxidase enzymes (Lopez et al., 1994).

The effect of sonication medium and properties of USW on enzyme inactivation

were also studied. Inactivation of lipase and protease by MTS was found to be

independent of the pH of the buffer. As the pH was increased from 5.75 to 8.0, D110°C

values for heat inactivation decreased from 3 min to 1 min. However, for MTS at 110°C,

the same increase in pH did not result in any significant change in D value for

inactivation of enzymes suggesting that lipase and protease can be inactivated by MTS

over a wide range of food products. The inactivation of the enzymes by USW was also

found to be dependent upon the amplitude of USW used. D values for inactivation of

lipase and protease were found to decrease with increase in the amplitude of USW. The

D76°C value decreased from 1.15 to 0.16 min for lipase and 1.15 to 0.77 min for protease

(calculated from rate constant plots) when the amplitude of USW was increased from 60

to 140 µm for MTS at 350 kPa (Vercet et al., 2002a). Similarly, the activity of peroxidase,

lipoxygenase and polyphenol oxidase was found to decrease (~100 times) as the

amplitude of USW was increased (Lopez et al., 1994).

2.10 Scale up

Ultrasound is one of the emerging technologies that can be used for processing

of food products while retaining desirable sensory and functional properties of foods.

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Ultrasound alone or combined with heat (thermosonication) or pressure

(manosonication) can be applied to heat-sensitive foods to achieve the required degree

of microbial inactivation at lower temperatures.

The practical aspect of ultrasound as an alternate technology is found in the fact

that it can be scaled up for industrial purposes. D’Amico et al. (2006) reported 5 log

reductions in the aerobic plate count in raw milk. Also, a continuous flow ultrasound

system was shown to be effective against P. fluorescens and S. thermophilus in tryptic

soy broth and milk when the samples were sonicated with 20 kHz USW at varying flow

rates. It was found that at low flow rates (20 and 11 ml/min), the reduction in number of

bacteria was high as compared to high flow rates (50 and 33 ml/min). Continuous flow

sonication at flow rate of 11 ml/min reduced the number of P. fluorescens and S.

thermophilus by 4.2 and 2.7 logs, respectively. The sonicated milk showed no

phosphatase activity after 6 days of storage at 5°C suggesting that ultrasound is a

promising technology for the processing of milk and other food products (Villamiel and

de Jong, 2000a).

Besides milk, sonication has been found to be effective for the processing of

juices. A mild thermosonication (TS) treatment of L. monocytogenes in apple cider for 20

min at 60ºC resulted in a 2.4 log-reduction in number of bacteria (Baumann et al., 2005).

2.11 Other applications of ultrasound in food industry

Low intensity USW (< 1 W/cm2) has been used for a long time for process control

and characterization of physicochemical properties of food products. The major

advantage of using ultrasound over other techniques is that it is non-destructive, rapid,

can be used with opaque systems and upgraded for on-line measurements. The

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measurable properties of USW are related to the physicochemical property of the food

product either empirically or theoretically and these are used to characterize the food

products. The most commonly used USW parameters for food characterization are

velocity, attenuation coefficient and acoustic impedance

Ultrasound velocity (c) refers to the velocity with which the wave travels in the

medium and is measured by wavelength or time required by the wave to travel a certain

distance.

Attenuation coefficient (α) measures the decrease in amplitude of the wave as

it travels through a medium. It is measured either by adsorption or scattering of the

waves.

Acoustic impedance (Z) is the ratio of amplitude of reflected wave and incident

wave and is helpful in determining the interface difference between two materials.

Time (t) It is the time required for the pulse to travel through the sample and be

reflected back to the transducer

All of these properties of waves depend upon the composition and structure of

the food material and therefore are useful to measure the properties of food material

(McClements, 1997). Table 2.2 summarizes various applications of USW used in the

food industry.

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Adapted from McClements, 1995 with additional information from Coupland and Saggin, 2003

Table 2.2: : Examples of low intensity ultrasound wave applications in food industry

Application Food material Property measured Ultrasound Frequency (MHz)

Composition Composition of milk c, α

Sugar concentration of juices and beverages c 2.5

Alcohol content of beverages c Biopolymer concentration in gels c, α 0.6 - 9

Fat, protein and moisture content of sausages c, α 1

Presence of bubbles in aerated foods c, α, Z Crystallization Solid fat content of cocoa butter c Particle sizing Emulsion stability c, α Properties of foods Texture of cheese c, α 1 Texture of biscuits c, α Viscosity of foods c, α Process measurements Flow rate and level measurement t* Temperature measurement c

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In addition, high intensity (10 to 1000 W/cm2) ultrasound waves have also been

used widely for degassing of liquids, disruption of cells in laboratories, formation of

emulsions, enhancement of crystallization and promotion of oxidation reduction

reactions (Suslick, 1990). However, the use of USW to inactivate microorganisms for

food processing is comparatively recent and there are many unexplored research areas

such as the potential of USW to inactivate bacteria in real foods like milk needs to be

investigated before it could be established as an alternate technology for food

processing.

2.12 Composition of milk

Milk is a complex, nutritious food product that contains its components in

solution, suspension or emulsion form (Table 2.3)

Water is the main constituent of milk comprising ~90% of the body of milk. Milk is

an oil-in-water emulsion, the milk fat present in the form of small globules (3 to 4 µm)

dispersed in water. Milk fat is in the form of triglycerides formed by bonding of fatty acids

with glycerol. The unique character of milk fat is that it is mostly composed of short chain

fatty acids (butyric and caproic acid).

Table 2.3: Composition of cow’s milk

Milk Constituent Amount present (g/100 g )

Water 87.5 Fat 3.9 Proteins 3.4 Lactose 4.8 Minerals 0.8

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The proteins of milk can be divided into two groups – caseins and whey proteins,

with caseins constituting ~80% of total proteins. Caseins form micelles in milk and are

present as a colloidal solution in milk. The micelles are stabilized by calcium phosphates

and hydrophobic interactions between sub-micelles. Whey proteins, on the other hand

are present in solution in milk. These are the proteins that remain after precipitation of

casein proteins from skim milk. Whey proteins have high nutritive value, with β

lactoglobulin constituting a major portion. In addition some membrane proteins are also

found in milk around fat globules and stabilize the emulsion. Lactose, another major

component of milk, is the only sugar found in milk. The rest of the milk component

percentage is composed of minerals, salts and vitamins (Bylund 1995).

2.12.1 Foodborne disease outbreaks associated with raw milk and milk products

Raw milk and products made from it have been found to be a source of

pathogens including Escherichia coli O157:H7 and Listeria monocytogenes. Raw milk

and soft cheeses have been associated with the foodborne disease outbreaks due to E.

coli O157:H7 and L. monocytogenes (Table 2.4) (Headrick et al., 1998, Ramsaran et al.,

1998).

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Adapted from Farber et. al. (1991) with additional information from cdc.gov

L. monocytogenes is a Gram-positive, facultative anaerobic, non-sporeforming

bacteria that can grow over a wide range of temperature (0 to 45°C) and pH (4.4 to 9.6).

It can also grow in saline environments of 10% NaCl and low water activity environments

as low as 0.97. Because of its resilient nature and ubiquitous presence, it can enter into

food processing plants by various routes and survive there for long periods of time, and

can be a cause of post-process contamination of food products (Swaminathan, 2001).

Listeria monocytogenes has been detected in various food products such as meats,

eggs, poultry, raw milk, cheese and vegetables. In the dairy industry, it has been of great

concern in association with soft cheeses since it can grow and survive during the

Table 2.4: Dairy related incidents and outbreaks associated with E. coli O157:H7 and L. monocytogenes in the United States

Organism Year Location Number of cases Dairy food vehicle

E. coli O157:H7 2006 California and Idaho Raw milk 2005 Washington 18 Raw milk 2004 Washington 3 Queso fresco, unpasteurized 1998 California 28 Milk 1998 Wisconsin 63 Cheese curds 1992 Oregon 9 Raw milk Listeria monocytogenes 2005 Texas 12 Queso fresco, unpasteurized 2003 Texas 12 Queso fresco, unpasteurized 2000 North Carolina 12 Queso fresco 1994 Multiple locations 56 Pasteurized chocolate milk 1987 Los Angeles 11 Butter 1986 Philadelphia, PA 36 Ice cream 1985 California 142 Jalisco cheese

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manufacture of some cheeses. It is usually concentrated in curd, with only a small

number present in whey. L. monocytogenes has also been found in butter, butter-milk

and yogurt. However, L. monocytogenes cannot survive pasteurization and is inactivated

upon proper pasteurization treatment (Farber and Peterkin, 1991).

Listeria monocytogenes is a pathogen that causes listeriosis in humans, primarily

in individuals with compromised T-cell mediated immunity. The disease affects pregnant

women, new born babies (neonatal), elderly people, and people with a weakened

immune system. The mortality rate due to listeriosis ranges from 10-50% with neonatal

mortality rate being the highest. The infectious dose of L. monocytogenes varies widely

depending upon the strain of organism and susceptibility of the host. The symptoms of

listeriosis include muscle aches, fever, and sometimes gastrointestinal symptoms such

as nausea or diarrhea. When infection spreads to the nervous system, symptoms such

as headache, stiff neck, loss of balance, or convulsions can occur. In pregnant women

though, it may lead to miscarriage or stillbirth, premature delivery, or infection of the

newborn (Farber and Peterkin, 1991, Swaminathan, 2001).

Escherichia coli O157:H7 is a Gram-negative, non-sporeforming bacterium that

can grow over a wide temperature range (7 to 50°C) and even in acidic environments

(pH 4.0 to 4.5). Cattle act as reservoirs of E. coli O157:H7. However, it does not cause

disease in these animals. Various foods such as ground beef, lettuce, raw milk, cheese,

yogurt and unpasteurized apple cider have been associated with outbreaks caused by of

E. coli O157:H7. The organism enters the food system via cross contamination with

meats or feces contaminated with E. coli O157:H7. Escherichia coli O157:H7 can also

be transmitted by person to person contact and has been associated with outbreaks in

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day care centers (Meng et al., 2001). Because of its ability to survive low pH and

refrigeration temperatures, as well as tolerate fermentation products, it can survive in

cheese. Raw milk has also been found to be the second most important vehicle for

foodborne infections of E. coli O157:H7 (Reitsma and Henning, 1996).

All age groups are sensitive to infection with E. coli O157:H7 especially young

children under age 5 and the elderly people. It can lead to complications such as

hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP)

which involve failure of kidneys and neurological fluctuations, respectively (Meng et al.,

2001). The infectious dose of E. coli O157:H7 is very low (<100 cells) with even as few

as 10 cells causing disease in highly susceptible populations. However, the fatality rate

from E. coli O157:H7 is only 1% and the infection might be asymptomatic or result in

mild symptoms such as non-bloody diarrhea and abdominal cramps.

2.13 Protective effect of food components

Historically, inactivation of bacteria by physical treatments has been found to be

more pronounced in buffers than in food systems, suggesting that properties of foods

somehow protect bacteria against these treatments. Various constituents of food

systems have been shown to protect bacteria against physical and chemical stresses.

However, it is also known that all components of a food system might not protect

bacteria in the same way.

Disaccharides such as trehalose and maltose were found to stabilize bacterial

membranes more than other sugars such as raffinose and inositol. The stabilization of

membranes was attributed to interaction of sugar with the phospholipid layer of the

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membrane wherein the polar head group (phosphate group) of phospholipids attaches to

the free –OH group of trehalose as suggested by infrared spectrum of the membrane

(Crowe et al., 1987). Sugars have also been reported to stabilize the proteins of bacteria

by preferential exclusion of sugar molecules from protein surface. Addition of sugar to

protein solution is a thermodynamically unfavorable phenomenon. To avoid this, the

protein structure stabilizes in the presence of sugars because the denaturation of the

protein molecule will lead to a larger contact surface between the protein and the solvent

(Timasheff et al., 1975). Kilimann et al. (2006) also found that sucrose protects

Lactococcus lactis against heat inactivation by stabilizing the secondary protein structure

of bacteria. Moreover, microorganisms produce compatible solutes like betaines or

sugars to maintain osmotic balance of cells in the presence of high levels of sugars in

the environment. In addition, these solutes also provide protection to bacteria against

physicochemical treatments such as heat, freezing, drying and high pressure (Welsh,

2000). Studies show that in the presence of sucrose and lactose in the environment, the

concentration of sucrose and lactose present in cell cytoplasm increased suggesting that

sugars were transported into the cells (Glaasker et al., 1998, Molina-Hoppner et al.,

2004).

Fat, a major constituent of animal products, has also been shown to influence the

inactivation of bacteria by physical processes such as heat. D values for inactivation of

E. coli O157:H7 by heat at 50, 55 and 60°C increased with increase in fat content of

meat products. The D value increased about ~1.1 to ~1.6 times when the fat content of

chicken increased from 3 to 11% at all the three temperatures (Ahmed et al., 1995).

Kaur et al. (1998) also found that the resistance of E. coli O157:H7 to heat increased in

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the presence of lipids. The protective effect of lipids has been attributed to the reduced

water activity of lipid systems and dehydration of cells immersed in lipid phase.

Proteins and peptides have also been known to increase the heat resistance of

bacteria. However, the mechanism of protection is not clear yet. This could possibly be

due to the formation of aggregates of bacteria due to the interactions of peptides and

bacteria (Moats et al., 1971). Divalent cations were found to increase the resistance of

Salmonella Senftenberg to heat by stabilizing ribosomes or cell envelops (Manas et al.,

2001). Caseins were also found to increase resistance of Listeria innocua against high

pressure treatment. This was attributed to the minerals associated with micelle of casein.

The pH of the system decreases during high pressure treatment and the presence of

minerals increases the buffering capacity of the system, thereby increasing the

resistance of bacteria to high pressure treatment (Black et al., 2007).

Review of literature indicates that most of the microbial inactivation studies with

USW have been done in buffers. The effect of different sonication media and the

protective effect of food components have not been investigated in detail. Further, the

effect of cell growth phase and the mechanism of inactivation have not been studied.

Hence, the objectives of this thesis have been designed in an attempt to fill these gaps

in literature.

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Escherichia coli O157:H7 during the manufacture and curing of cheddar cheese. J Food

Prot 59(5):460-464.

Rodgers, S. L. and E. T. Ryser. 2004. Reduction of microbial pathogens during

apple cider production using sodium hypochlorite, copper ion, and sonication. J Food

Prot 67(4):767-771.

Romero, E., H. Feng, and S. E. Martin. 2007. Inactivation of Shigella boydii 18

IDPH and Listeria monocytogenes Scott A with power ultrasound at different acoustic

energy densities and temperatures. J Food Sci 72(4):M103-M107.

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Sener, N., D. K. Apar, and B. Ozbek. 2006. A modeling study on milk lactose

hydrolysis and β-galactosidase stability under sonication. Process Biochem 41:1493-

1500.

Stanley, K. D., D. A. Golden, R. C. Williams, and J. Weiss. 2004. Inactivation of

Escherichia coli O157:H7 by high-intensity ultrasonication in the presence of salts.

Foodborne Pathog Dis 1(4):267-280.

Suslick, K. S. 1988. Homogenous sonochemistry. Pages 123-159 in

Ultrasound:Its chemical, physical and biological effects. K. S. Suslick, ed. VCH

Publishers, New York.

Suslick, K. S. 1990. Sonochemistry. Science 247(4949):1439-1445.

Swaminathan, B. 2001. Pages 383-410 in Fundamentals of Food Microbiology

2nd ed. M. P. Doyle, L. R. Beuchat, and T. J. Montville, ed. ASM Press, Washington D

C.

Taylor, M. J. and T. Richardson. 1980. Antioxidant activity of skim milk: effect of

sonication. J Dairy Sci 63(11):1938-1942.

Timasheff, S. N., J. C. Lee, E. P. Pitts, and N. Tweedy. 1975. The interaction of

tubulin and other protein structure-stabilizing solvents J Colloid Interface Sci 55(3):658-

663.

Ugarte, R. E., H. Feng, S. E. Martin, K. R. Cadwallader, and S. J. Robinson.

2006. Inactivation of Escherichia coli with power ultrasound in apple cider. J Food Sci

71(2):E102-E108.

Valero, M., N. Recrosio., D. Saura., N. Munoz., N. Marti and V. Lizama 2007

Effect of ultrasonic treatments in orange juice processing. J. Food Eng 80:509-516.

Vercet, A., J. Burgos, and P. Lopez-Buesa. 2002a. Manothermosonication of

heat-resistant lipase and protease from Pseudomonas fluorescens: effect of pH and

sonication parameters. J Dairy Res 69(2):243-254.

Vercet, A., R. Oria, P. Marquina, S. Crelier, and P. Lopez-Buesa. 2002b.

Rheological properties of yoghurt made with milk submitted to manothermosonication. J

Agric Food Chem 50(21):6165-6171.

Vercet, A., P. Lopez, and J. Burgos. 1999. Inactivation of heat-resistant

pectinmethylesterase from orange by manothermosonication. J Agric Food Chem

47:432-437.

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Villamiel, M. and P. de Jong. 2000a. Inactivation of Pseudomonas fluorescens

and Streptococcus thermophilus in Trypticase Soy Broth and total bacteria in milk by

continuous-flow ultrasonic treatment and conventional heating. J Food Eng. 45:171-179.

Villamiel, M. and P. de Jong. 2000b. Influence of high-intensity ultrasound and

heat treatment in continuous flow on fat, proteins, and native enzymes of milk. J Agric

Food Chem 48(7):3068.

Welsh, D. T. 2000. Ecological significance of compatible solute accumulation by

micro-organisms: from single cells to global climate. FEMS Microbiol Rev 24(3):263-290.

Wrigley, D. M. and N. G. Llorca. 1992. Decrease of Salmonella typhimurium in

skim milk and eggs by heat and ultrasonic wave treatment. J Food Prot 55(9):678-680.

Wu, H., G.J. Hulbert, and J. R. Mount. 2001. Effects of ultrasound on milk

homogenization and fermentation with yogurt starter. Innov Food Sci Emerg Technol

1:211-218.

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CHAPTER 3

EFFECT OF SONICATION MEDIUM AND GROWTH PHASE ON

INACTIVATION OF E. coli AND L. monocytogenes BY ULTRASOUND WAVES

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3.1 Abstract

Inactivation kinetics of log and stationary phase cells of Escherichia coli ATCC

25922 and Listeria monocytogenes ATCC 19115 were studied in phosphate buffer,

whole and skim milks upon treatment with 24 kHz ultrasound waves (USW) at sublethal

temperatures. The effect of high frequency ultrasound waves (650, 765 and 850 kHz) on

inactivation of log phase E. coli cells was also investigated. D values for inactivation of

E. coli (2.43, 2.41 and 2.19 min) and L. monocytogenes (9.3, 8.61 and 7.63 min) were

significantly higher (p < 0.05) in whole and skim milks than phosphate buffer,

respectively suggesting that milk exerts a sonoprotective effect on these bacteria. There

was no significant difference in D values between in whole and skim milk for both

organisms suggesting that presence of fat in milk does not influence inactivation of E.

coli and L. monocytogenes by USW. Escherichia coli exhibited non-log linear inactivation

kinetics with tailing in all three sonication media while L. monocytogenes exhibited log

linear inactivation kinetics throughout. Log phase cells of E. coli and L. monocytogenes

were more sensitive to ultrasound treatment than stationary phase cells in all three

media. Ultrasound treatment exhibited minimal injury to bacteria suggesting that

inactivation of bacteria by USW follows all or nothing phenomenon. High frequency

ultrasound waves (650, 765 and 850 kHz) did not significantly reduce the number of E.

coli upon sonication for up to 3 min, whereas the number decreased by ~ 3 logs in 2.5

min with low frequency (24 kHz) USW. This study shows that ultrasound waves can

inactivate these organisms in milk.

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3.2 Introduction

Milk is an indispensable part of the human diet with per capita consumption of

83.9 liters recorded in 2006 in the United States (Goff, 2008). Milk contains nine

essential nutrients required for the growth and maintenance of life functions. However,

raw milk and cheese products may be the source of pathogens such as Escherichia coli

O157:H7 and Listeria monocytogenes, which have been associated with outbreaks of

foodborne illness (Headrick et al., 1998, Ramsaran et al., 1998). In fact, raw milk has

been found to be the second most important vehicle for transmission of E. coli O157:H7

(Reitsma and Henning, 1996). Mexican-style cheeses made from unpasteurized milk

have been associated with outbreaks of L. monocytogenes in California in 1985-86 and

in North Carolina in 2001 (CDC, 2001, Farber and Peterkin, 1991).

Escherichia coli O157:H7 is a Gram-negative, non-sporeforming bacterium that

can grow over a wide temperature range (7 to 50°C) and in acidic environments (pH 4.0

to 4.5). It is a member of the enterohemorrhagic E. coli (EHEC) group and causes

hemolytic uremic syndrome (HUS) in humans. The infectious dose of E. coli O157:H7 is

very low (<100 cells); even as few as 10 cells can cause disease in highly susceptible

populations (Meng et al., 2001). Various foods such as meats, ground beef, lettuce, milk,

cheese, yogurt and unpasteurized apple cider have been associated with transmission

of E. coli O157:H7 (Meng et al., 2001). It can also survive cheddar cheese manufacture

and the ripening process (Reitsma and Henning, 1996). The organism enters the food

system via cross contamination with feces contaminated with E. coli O157:H7 or raw

foods, equipment and hands contaminated with those feces.

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Listeria monocytogenes is a Gram-positive, facultative anaerobic, non-

sporeforming bacterium that can grow over a wide range of temperature (0 to 45°C) and

pH (4.4 to 9.6). It can also grow in saline environments up to 10% NaCl and

environments of low water activity down to 0.97 (Swaminathan, 2001). Listeria

monocytogenes causes listeriosis in humans which mainly affects pregnant women, new

born babies (neonatal), elderly people, and people with weakened immune systems.

Listeria monocytogenes has been detected in various food products such as meats,

eggs, poultry, raw milk, cheese and vegetables (Farber and Peterkin, 1991). Because of

its resilient nature and ubiquitous presence, it can enter into food processing plants by

various ways, survive there for long periods of time and cause post-processing

contamination of food products (Swaminathan, 2001).

Heat pasteurization is the most common processing technique currently used in

the food industry to kill pathogens and extend shelf life of milk. However, this process

results in loss of nutritional and sensory properties of milk. With increased demand for

‘fresh-like’ foods, the importance of alternative food processing technologies is

increasing. In recent years, several alternative technologies such as high pressure

processing, pulsed-electric field and irradiation have been explored for food processing

(Piyasena et al., 2003). Ultrasound technology is also one of the emerging alternative

food processing technologies and uses sound waves with frequencies higher than 20

kHz (Mason, 1998). Ultrasound waves (USW) have been used in the food industry since

the late 1920s. Low energy ultrasound waves with intensities less than 1 W/cm2 and

frequencies higher than 100 kHz have been used for process control and

characterization of physicochemical properties such as viscosity, sugar content and

texture of foods (McClements, 1997). High energy ultrasound waves with intensities

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higher than 1 W/cm2 have been used to break open microbial cells for extraction of

enzymes and proteins, induce oxidation-reduction reactions and inactivate enzymes

(Mason, 1998). Ultrasound has been reported to preserve the flavor and color of juices

(Valero et al., 2007), improve homogeneity (Ertugay et al., 2004, Villamiel and de Jong,

2000b, Wu et al., 2001) and enhance functional properties of foods (Sener et al., 2006,

Vercet et al., 2002).

Microbial inactivation by ultrasound waves is attributed to the process known as

cavitation. Cavitation is the process of formation, growth and collapse of bubbles in a

liquid medium. As the ultrasound waves pass through a liquid medium, continuous

pressure changes lead to cavitation. Cavitation bubbles form in the rarefaction region of

the sound wave due to negative pressure and grow in size in the compression region, a

positive pressure region. The bubbles grow to a maximum unstable size over many

alternating compression and rarefaction cycles, beyond which they are unable to sustain

themselves and finally collapse. This collapse results in radiation of shock waves from

the site of collapse. The collapse also generates high local temperatures leading to the

formation of free radicals in the medium (Frizzell, 1988, Suslick, 1990). The free radicals

and shock waves produced by ultrasound waves are thought to be involved in the

inactivation of microorganisms (Piyasena et al., 2003). However, the exact reason for

the lethality of ultrasound is not yet completely understood.

Further, when combined with temperature and pressure USW were more

effective in killing vegetative cells of Streptococcus faecium, L. monocytogenes,

Salmonella Enteritidis, Aeromonas hydrophila, Yersinia enterocolitica, Shigella boydii

and spores of Bacillus subtilis (Pagan et al., 1999b, Raso et al., 1998a, Raso et al.,

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1998b, Romero et al., 2007). However, ultrasound as a stand-alone food processing

technology has not been fully explored. Moreover, most of the studies for inactivation of

microorganisms by USW have been conducted either in distilled water or buffers. Foods

like milk are more complex than buffers and could influence the results of bacterial

inactivation by USW. Furthermore, there is scant literature data available regarding the

aspect of injury of bacteria during sonication, effect of growth phase of bacteria and

frequency of USW on efficacy of ultrasound waves to kill bacteria.

The objectives of this study were to compare the inactivation kinetics of E. coli

and L. monocytogenes in phosphate buffer and milk by ultrasound treatment and

determine the effect of ultrasound frequency and growth phase of bacteria on

inactivation. Also, the injury of these bacteria upon sonication was investigated.

3.3 Materials and Methods

3.3.1 Bacterial cultures and culture conditions

Escherichia coli ATCC 25922, a clinical isolate, was obtained from American

Type Culture Collection (Manassas, VA) and L. monocytogenes ATCC 19115, a human

isolate, was obtained from Dr. Stephen J. Knabel, Department of Food Science, Penn

State University. The cultures were activated by transferring a loopful of frozen culture to

10 ml Tryptic Soy Broth (TSB; Becton Dickinson and Company, Sparks, MD; BD) and

incubating the tube for 24 h at 37±2°C. The activated cultures were then streaked onto

Tryptic Soy Agar (TSA; BD) plates and incubated for 24 h at 37±2°C. Isolated colonies

from plates were transferred to TSA slants and incubated for 18 h at 37±2°C, then stored

at 4°C until needed. The cultures were periodically transferred to fresh TSA slants.

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3.3.2 Preparation of culture for sonication

A loopful of E. coli or L. monocytogenes culture was transferred from the TSA

slant to 10 ml of TSB and incubated for 18 h at 37±2°C. The resulting culture was diluted

to ~107 CFU/ml in sterile phosphate buffer (pH 7.2±0.2, Appendix A.1) and 1 ml of the

diluted culture was added to 250 ml and 100 ml TSB for E. coli and L. monocytogenes,

respectively. For E. coli, the flasks were incubated for 24 h at 37±2°C in a shaking

incubator (Model 3597, Lab Line Instrument Inc., Melrose Park, IL) set at 200 rpm, while

for L. monocytogenes, the flasks were incubated under static conditions for 20 h at

37±2°C to achieve mid-stationary phase cells of bacteria. For mid-log phase cells, the

flasks of E. coli and L. monocytogenes were incubated for 5 and 8 h, respectively. The

times to achieve mid-log and mid-stationary phase cells were determined by growth

curves of bacteria performed previously by plating and absorbance measurements at

600 nm (Appendix B, Figures B.1 and B.2). Growth phase was determined by

absorbance at 600 nm for subsequent experiments. The mid-log or mid-stationary phase

cells were added to 100 ml sonication medium to achieve a final concentration of ~105

and ~107 CFU/ml, respectively. The sonication media were phosphate buffer (Appendix

A.1), UHT whole milk and UHT skim milk (Parmalat, Wallington, NJ). The sonication

medium inoculated with bacterial cells constituted a sonication sample.

3.3.3 Ultrasound treatment of sonication sample

Sonication samples were treated with 24 kHz pulse (80% pulse/sec) USW

(Model UP400S, Hielscher, Ringwood, NJ) with an amplitude of 100 µm and energy

density of 85 W/cm2, respectively. The probe was immersed in 70% alcohol for 15 min

and left to air dry prior to sonication of the sample in order to avoid any contamination of

the sample during sonication. The probe was rinsed with 2 ml of sterile phosphate buffer

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and plated onto TSA plates to confirm that the probe was not contaminated with

bacteria.

An aliquot of the sonication sample was withdrawn and placed into a 2 ml sterile

vial before starting the sonication; this was the 0 time sample. The rest of the sonication

sample was then transferred to a 250 ml reaction beaker (Cole Parmer, Vernon Hills, IL)

connected to a water bath (Model 9610, Fisher Scientific, Pittsburgh, PA) maintained at

5°C. The probe was inserted into the center of the beaker, immersed into the sonication

sample and the ultrasound was switched on. Aliquots (2 ml) were withdrawn and

transferred into 2 ml sterile vials at set time intervals during sonication. Temperatures of

the sonication sample were recorded with a digital thermocouple at various time intervals

throughout the sonication treatment. The temperature during sonication never exceeded

35°C which is a sublethal temperature for E. coli and L. monocytogenes (Mack and

Roberts, 2007, Romero et al., 2007).

For high frequency ultrasound treatment, continuous USW (Ultran, Boalsburg,

PA) at frequencies of 650, 765 and 850 kHz with 200 mV amplitude were used to

sonicate mid-log phase cells of E. coli in phosphate buffer. Sonication with high

frequency USW was carried out in 250 ml beakers and temperature was not controlled

during sonication. The remainder of the procedure was the same as that for low

frequency ultrasound treatment.

3.3.4 Enumeration of bacteria and D values

The previously collected aliquots were serially diluted in sterile phosphate buffer

(pH 7.2) and surface plated onto duplicate TSA plates. The dilutions were also surface

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plated onto duplicate plates of Violet Red Bile Agar (VRBA; BD) and Modified Oxford

Agar (MOX; Oxoid, Remel, KS) to estimate injury in E. coli and L. monocytogenes,

respectively. The plates were incubated at 37±2°C and counted after 24 h for E. coli and

48 h for L. monocytogenes. The survival curves were drawn by plotting Log10 CFU/ml

against time. A trendline was drawn on the straight line portion of the survival curve and

D values were calculated by taking the negative inverse of the slope of the trendline.

Percent injury of the cells was calculated as Eq. 3.1

Where,

A = CFU/ml on TSA after sonication

B = CFU/ml on VRBA or MOX after sonication

3.3.5 Statistical analysis

All experiments were performed in triplicate unless otherwise indicated and the D

values from all three replicates were used for data analysis. Data were analyzed using

one way ANOVA followed by Tukey’s pairwise comparison test at p = 0.05 using SPSS

version 11 (SPSS Inc. Chicago, IL).

3.4 Results and Discussion

3.4.1 Effect of frequency on inactivation of E. coli

Log-phase cells of E. coli were exposed to four different USW frequencies. No

decrease in number of E. coli was observed upon sonication with 650, 765 and 850 kHz

(high frequency) USW for up to 3 min. However, the number of E. coli decreased by ~ 3

100% ×−

=A

BAinjury 3.1

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logs in 2.5 min with 24 kHz (low frequency) USW (Figure 3.1). Joyce et al. (2003) also

reported that high frequency USW (512 and 850 kHz) did not result in any decrease in

number of Bacillus subtilis cells whereas low frequency USW (20 and 38 kHz) resulted in

a noticeable decrease in number of bacteria. The differential bactericidal effect of high

and low frequency USW could either be due to the different frequencies employed or

differences in power ratings of the instruments used. To unambiguously infer the effect

of USW frequency only, an instrument that can be operated over a wide range of

frequencies is needed. Hua and Thompson (2000) used one ultrasound (Allied-signal

ELAC nautic reactor) instrument to generate USW with 205, 358, 618 and 1071 kHz

frequencies. They reported that with the increase in frequency of ultrasound waves the

rate of inactivation of E. coli decreased, suggesting that more killing is observed at lower

frequencies than higher frequencies.

The lowered inactivation of E. coli by high frequency USW could be due to

reduced and less intense cavitation at high frequencies. Cavitation bubbles need a finite

time to form in the rarefaction region of USW. At high frequencies, the duration of the

rarefaction phase is reduced leading to a decrease in the number of cavitation bubbles

formed. Moreover, at high frequencies, the bubble collapse is not intense and hence the

hydraulic shock produced by collapsing bubbles is less as compared to bubbles

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0

1

2

3

4

5

6

0 0.5 1 1.5 2 2.5 3 3.5Time (min)

Log 1

0CFU

/ml 850 kHz

765 kHz

650 kHz

24 kHz

The plots for inactivation with high frequency USW (850, 765 and 650 kHz) show the values of one experiment only.

Figure 3.1: Effect of ultrasound frequency on inactivation of mid log-phase cells of E. coli (ATCC 25922) in phosphate buffer (pH 7.2) by different frequency ultrasound waves.

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produced in a low frequency ultrasound field, leading to lowered bactericidal effect of

USW at high frequencies (El'piner, 1964). Cavitation is associated with a hissing sound

produced by collapsing bubbles and an increase in temperature of the sonication

medium. The temperature profiles show that the temperature rose to 80°C with 24 kHz

USW in 5 min when the temperature was not controlled whereas the temperature did not

rise beyond 35°C for 765 kHz USW (Figure 3.2). Moreover, during this study no hissing

sound was audible during sonication with high frequency USW while it was apparent

with 24 kHz USW, suggesting that bubble collapse was not intense at high frequencies

(El'piner, 1964). As a result, low frequency (24 kHz) USW was used for the remaining

studies.

3.4.2 Inactivation kinetics of E. coli and L. monocytogenes

Inactivation kinetics of E. coli and L. monocytogenes were investigated in

phosphate buffer, whole milk and skim milk. Escherichia coli exhibited non-log linear

inactivation kinetics in all three media with inactivation being relatively rapid during the

first 12 min of sonication followed by tailing in the case of mid-stationary phase cells

(Figure 3.3). However, L. monocytogenes followed log linear inactivation kinetics

throughout (Figure 3.3). These results are in accordance with Hua and Thompson

(2000) who also observed pseudo-first order inactivation kinetics of E. coli upon

sonication in distilled water using 20 kHz USW and power intensity of 74 W/cm2 which is

closer to the sonication frequency and power of our instrument. Non-linear inactivation

kinetics of E. coli has also been reported in apple cider and sterile water (D'Amico et al.,

2006, Furuta et al., 2004). Furthermore, Shigella boydii also exhibited tailing upon

sonication up to 30 min with the tail starting to appear at 15 min, a time closer to when

tailing with E. coli was seen in this study.

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0

10

20

30

40

50

60

70

80

90

0 2 4 6 8 10 12Time (min)

Tem

p. (°

C)

24 kHz without temperature control

24 kHz with temperature control

765 kHz without temperature control

Figure 3.2: Temperature profiles with and without temperature control during sonication with 765 kHz and 24 kHz ultrasound waves in phosphate buffer (pH 7.2)

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0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30 35 40 45 50 55Time (min)

Log 1

0CFU

/ml

Figure 3.3: Inactivation kinetics of mid stationary-phase cells of E. coli ATCC 25922(●) and L. monocytogenes ATCC 19115 (▲) in phosphate buffer (pH 7.2) upon sonication with 24 kHz ultrasound waves

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The first order inactivation kinetics of L. monocytogenes observed in this study is

in accordance with results of D’Amico et al., (2006) who studied inactivation kinetics of L.

monocytogenes in UHT whole milk by USW. Studies done in 0.85% NaCl solution

(Romero et al., 2007) and apple cider (Baumann et al., 2005) also reported first-order

inactivation kinetics of L. monocytogenes by USW.

The tailing of E. coli during sonication has been attributed either to decrease in

the level of dissolved gas in the medium or the presence of more resistant cells in the

medium (Baumann et al., 2005). The decrease in level of dissolved gases leading to

tailing can be ruled out in this study since L. monocytogenes did not show any tailing

under the same sonication conditions. Also, Villamiel et al. (2000) showed that high

intensity ultrasound does not decrease the level of dissolved gases in the medium when

sonicated up to 20 min. In this study, E. coli started showing tailing after 12 min,

suggesting that decrease in level of dissolved gases might not be the reason for tailing.

It is possible that some cells in the population are more resistant than others leading to

the tailing. Tailing could also be due to clumping of bacterial cells at acoustic pressure

nodal planes where cavitation is absent due to standing wave formation (Tsukamoto et

al., 2004). Ultrasound treated cells in this study showed clumps of cell debris under

phase contrast microscope.

Survival curves of E. coli and L. monocytogenes sonicated in whole milk showed

no significant difference in survivors on non-selective (TSA) and selective (VRBA or

MOX) media suggesting that minimal injury occurred to these bacteria upon sonication

with 24 kHz USW (Figures 3.4 and 3.5). Similar results were obtained for both

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0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16 18Time (min)

Log 1

0CFU

/ml

TSA

VRBA

Figure 3.4:Survival curves of mid stationary-phase cells of E. coli ATCC 25922 on TSA, a non-selective (-■-) and VRBA, a selective medium (-∆-) upon sonication with 24 kHz ultrasound waves in whole milk

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0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30 35 40 45Time (min)

Log 1

0CFU

/ml

TSA

MOX

Figure 3.5:Survival curves of mid stationary-phase cells of L. monocytogenes ATCC 19115 on TSA, a non-selective (-■-) and MOX, a selective medium (-∆-) upon sonication with 24 kHz ultrasound waves in whole milk

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organisms when skim milk and phosphate buffer were the sonication media (Appendix

B; Figures B.3 to B.6). The injury observed in E. coli and L. monocytogenes was never

more than 35 and 61%, respectively. Pagan et al. (1999a) also observed almost

overlapping survival curves obtained by plating of ultrasound-treated L. monocytogenes

on TSA with Yeast Extract (TSAYE), a non-selective and TSAYE + 3% NaCl, a selective

medium. Similar survival curves were also obtained for sonicated Salmonella enterica

upon plating on nutrient agar and nutrient agar + 3% NaCl (Alvarez et al., 2003). Lee et

al. (1989) also found no significant difference in counts of Salmonella spp. on TSA and

Brilliant Green agar (BGA) suggesting that there was no injury of the bacteria upon

sonication. Our results are consistent with the literature suggesting that there is an “all or

none” principle of inactivation by ultrasound. It is also possible that the level of USW

used in this study was high enough to cause no sublethal injury to bacteria.

It is noteworthy that while a 4-log reduction of E. coli was achieved in 12 min, it

took 40 min to achieve the same log reduction for L. monocytogenes in whole milk. Four

log reductions were also achieved in skim milk for both organisms (Appendix B; Figures

B.4 and B.7). D’Amico et al., (2006) reported a 4 log reduction in aerobic plate count of

bacteria in raw milk by continuous flow ultrasound treatment of raw milk at 20°C in 17

min. These results suggest that ultrasound could potentially be used as an alternate

pasteurization technology to reduce number of microorganisms in milk.

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3.4.3 Effect of growth phase and sonication medium on inactivation of E. coli and

L. monocytogenes

The D values for inactivation of E. coli and L. monocytogenes were significantly

higher (p < 0.05) for stationary-phase cells than log-phase cells in all three sonication

media (Figure 3.6 and Figure 3.7). The results are in agreement with the generally

accepted fact that log-phase cells are more sensitive to any physical/chemical treatment

than stationary-phase cells (Frazier and Westhoff, 1988). However, the results are

contradictory to the report of Wrigley and Llorca (1992), who reported that the age of the

Salmonella Typhimurium culture did not affect the susceptibility to ultrasound treatment.

The effect of sonication medium on inactivation of E. coli and L. monocytogenes

was investigated by sonication of these bacteria in phosphate buffer, skim milk and

whole milk. D values for inactivation of E. coli and L. monocytogenes were significantly

higher (p < 0.05) in whole and skim milk as compared to phosphate buffer (Table 3.1).

There was no significant difference in D values, however, for both the organisms in

whole and skim milk suggesting that presence of fat in milk does not influence the

inactivation kinetics of these bacteria by 24 kHz USW. This could be due to very few

bacterial cells being immersed in the lipid phase. Fat has been known to increase the

resistance of bacteria either by decreasing the water activity of the system or by

dehydration of cells immersed in the lipid phase of the system (Kaur et al., 1998). Manas

et al. (2001) found similar heat inactivation kinetics of Salmonella Senftenberg in whole

and skim milk. They argued that the fat content in whole milk was not enough to confer a

protective effect on bacteria. This could be true for this study as well.

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a

a a

b b

2.43 2.41

1.381.30

0.72

2.19

0

0.5

1

1.5

2

2.5

3

Phosphate Buffer Whole Milk Skim milk

D v

alue

(min

)

Log Phase Stationary Phase

b

D values within a sonication medium with same superscript are not significantly different at p = 0.05

Figure 3.6: Effect of growth phase on inactivation of E. coli ATCC 25922 by 24 kHz ultrasound waves in phosphate buffer (pH 7.2), whole milk and skim milk

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2.953.51

3.89

7.63

9.318.61

0

2

4

6

8

10

12

Phosphate Buffer Whole Milk Skim milk

D V

alue

(min

)

Log Phase Stationary Phase

a

a a

b

b b

D values within a sonication medium with same superscript are not significantly different at p = 0.05

Figure 3.7: Effect of growth phase on inactivation of L. monocytogenes ATCC 19115 by 24 kHz ultrasound waves in phosphate buffer (pH 7.2), whole milk and skim milk.

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D values for inactivation of E. coli were significantly lower than L. monocytogenes

in all three media suggesting that E. coli is more sensitive to ultrasound treatment than

L. monocytogenes (Table 3.1). This could be due to the difference in the cell wall

structure of E. coli, a Gram-negative bacterium and L. monocytogenes, a Gram-positive

bacterium. The cell wall of Gram-positive bacteria contains more peptidoglycan and is

thicker than the wall in Gram-negative bacteria, thus making them more resistant to

physical and chemical treatments (Frazier and Westhoff, 1988). Also, several other

studies have reported that Gram-negative and rod-shaped bacteria are more sensitive to

ultrasound treatment than Gram-positive and coccus-shaped bacteria (Pagan et al.,

1999b, Romero et al., 2007, Villamiel and Jong, 2000a).

D values ± standard deviation having the same upper case superscript within the organism grouping are not significantly different at p = 0.05. D values ± standard deviation having the same lower case superscript within the sonication medium grouping are not significantly different at p = 0.05.

Table 3.1: Effect of sonication medium on D values for inactivation of stationary-phase cells of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 by 24 kHz ultrasound waves

Sonication Medium D Value (min) E. coli L. monocytogenes Whole Milk 2.43±0.09Ab 9.31±0.26Aa Skim Milk 2.41±0.08Ab 8.61±0.63Aa Phosphate Buffer 2.19±0.10Bb 7.63±0.22Ba

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3.5 Conclusions

The effect of sonication medium, ultrasound frequency and bacterial growth

phase upon inactivation of E. coli and L. monocytogenes by ultrasound waves were

evaluated in this study. The rate of inactivation of E. coli and L. monocytogenes was

significantly slower in whole and skim milk as compared to phosphate buffer suggesting

that milk exerts a sonoprotective effect on bacteria. Mid stationary-phase cells of E. coli

exhibited non log-linear inactivation kinetics with tailing, whereas L. monocytogenes

showed linear inactivation kinetics throughout. Maximum inactivation of E. coli was

observed with an instrument generating 24 kHz ultrasound waves while the instrument

generating high frequency ultrasound waves (650, 765 and 850 kHz) was ineffective in

killing E. coli. Log-phase cells of both bacteria were more sensitive to ultrasound

treatment than stationary phase cells. This study shows that ultrasound technology can

potentially be used for milk pasteurization. However, further studies regarding effect of

ultrasound waves on nutritional and sensory profiles of the milk and its scale up to an

industrial level are required for its effective implementation in the food industry.

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3.6 References

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Salmonella enterica serovar Enteritidis by ultrasonic waves under pressure at different

water activities. Appl Environ Microbiol 69(1):668-672.

Baumann, A. R., S. E. Martin, and H. Feng. 2005. Power ultrasound treatment of

Listeria monocytogenes in apple cider. J Food Prot 68(11):2333-2340.

CDC. 2001. Outbreaks of listeriosis associated with homemade Mexican-style

cheese-North Carolina, October 2000-January 2001. J Am Med. Assoc 286(6):1-5.

D'Amico, D. J., T. M. Silk, J. Wu, and M. Guo. 2006. Inactivation of

microorganisms in milk and apple cider treated with ultrasound. J Food Prot 69(3):556-

563.

El'piner, I. E. 1964. Ultrasound Physical, Chemical and Biological Effects.

Consultants Bureau Enterprises Inc, New York.

Ertugay, M. F., M. Sengul, and M. Sengul 2004. Effect of ultrasound treatment

on milk homogenisation and particle size distribution of fat. Turk J Vet Animal Sci

28:303-308.

Farber, J. M. and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne

pathogen. Microbiol Rev 55(3):476-511.

Frazier, W. C. and D. C. Westhoff. 1988. Food Microbiology. 4th ed. McGraw-

Hill, Inc. USA.

Frizzell, L. A. 1988. Biological effects of acoustic cavitation. Pages 287-301 in

Ultrasound: Its chemical, physical and biological effects. K. S. Suslick, ed. VCH

Publishers, New York.

Furuta, M., M. Yamaguchi, T. Tsukamoto, B. Yim, C. E. Stavarache, K. Hasiba.,

and Y. Maeda. 2004. Inactivation of Escherichia coli by ultrasonic irradiation. Ultrason

Sonochem 11(2):57-60.

Goff, D. http://www.foodsci.uoguelph.ca/dairyedu/intro.html Accessed Jan. 16,

2008.

Headrick, M. L., S. Korangy, N. H. Bean, F. J. Angulo, S. F. Altekruse, M. E.

Potter, and K. C. Klontz. 1998. The epidemiology of raw milk-associated foodborne

disease outbreaks reported in the United States, 1973 through 1992. Am J Public Health

88(8):1219-1221.

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Hua, I. and J. E. Thompson. 2000. Inactivation of Escherichia coli by sonication

at discrete ultrasonic frequencies. Wat Res 34(15):3888-3893.

Joyce, E., S. S. Phull, J. P. Lorimer, and T. J. Mason. 2003. The development

and evaluation of ultrasound for the treatment of bacterial suspensions. A study of

frequency, power and sonication time on cultured Bacillus species. Ultrason Sonochem

10(6):315-318.

Kaur, J., D. A. Ledward, R. W. Park, and R. L. Robson. 1998. Factors affecting

the heat resistance of Escherichia coli O157:H7. Lett Appl Microbiol 26(4):325-330.

Lee, B. H., S. Kermasha, and B. E. Baker. 1989. Thermal, ultrasonic and

ultraviolet inactivation of Salmonella in thin films of aqueous media and chocolate. Food

Microbiol 6:143-152.

Mack, S. Z. and J. S. Roberts. 2007. Ultrasound pasteurization: The effects of

temperature, soluble solids, organic acids and pH on the inactivation of Escherichia coli

ATCC 25922. Ultrason Sonochem 14(323-329):323.

Manas, P., R. Pagan, F. J. Sala, and S. Condon. 2001. Low molecular weight

milk whey components protect Salmonella senftenberg 775W against heat by a

mechanism involving divalent cations. J Appl Microbiol 91(5):871-877.

Mason, T. J. 1998. Power ultrasound in food processing-the way forward. Pages

105-125 in Ultrasound in Food Processing. M. J. W. Povey and T. J. Mason, ed. Blackie

Academic & Professional London.

McClements, D. J. 1997. Ultrasonic characterization of foods and drinks:

Principles, Methods and Application. Cri Rev Food Sci Nutr 37(1):1-46.

Meng, J., M. P. Doyle, T. Zhao, and S. Zhao. 2001. Pages 193-214 in

Fundamentals of Food Microbiology, 2nd ed. M. P. Doyle, L. R. Beuchat, and T. J.

Montville, ed. ASM Press, Washington D C.

Pagan, R., P. Manas, I. Alvarez, and S. Condon. 1999a. Resistance of Listeria

monocytogenes to ultrasonic waves under pressure at sublethal (manosonication) and

lethal (manothermosonication) temperatures. Food Microbiol 16:139-148.

Pagan, R., P. Manas, J. Raso, and S. Condon. 1999b. Bacterial resistance to

ultrasonic waves under pressure at nonlethal (manosonication) and lethal

(manothermosonication) temperatures. Appl Environ Microbiol 65(1):297-300.

Piyasena, P., E. Mohareb, and R. C. McKellar. 2003. Inactivation of microbes

using ultrasound: A review. Int J Food Microbiol 87(3):207-216.

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Ramsaran, H., J. Chen, B. Brunke, A. Hill, and M. W. Griffiths. 1998. Survival of

bioluminescent Listeria monocytogenes and Escherichia coli O157:H7 in soft cheeses. J

Dairy Sci 81(7):1810-1817.

Raso, J., R. Pagan, S. Condon, and F. J. Sala. 1998a. Influence of temperature

and pressure on the lethality of ultrasound. Appl Environ Microbiol 64(2):465-471.

Raso, J., A. Palop, R. Pagan, and S. Condon. 1998b. Inactivation of Bacillus

subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. J

Appl Microbiol 85(5):849-854.

Reitsma, C. J. and D. R. Henning. 1996. Survival of Enterohemorrhagic

Escherichia coli O157:H7 during the manufacture and curing of cheddar cheese. J Food

Prot 59(5):460-464.

Romero, E., H. Feng, and S. E. Martin. 2007. Inactivation of Shigella boydii 18

IDPH and Listeria monocytogenes Scott A with power ultrasound at different acoustic

energy densities and temperatures. J. Food Sci 72(4):M103-M107.

Sener, N., D. K. Apar, and B. Ozbek. 2006. A modeling study on milk lactose

hydrolysis and β-galactosidase stability under sonication. Proc Biochem 41:1493-1500.

Suslick, K. S. 1990. Sonochemistry. Science 247(4949):1439-1445.

Swaminathan, B. 2001. Pages 383-410 in Fundamentals of Food Microbiology

2nd ed. M. P. Doyle, L. R. Beuchat, and T. J. Montville, ed. ASM Press, Washington D

C.

Tsukamoto, I., B. Yim, C. E. Stavarache, M. Furuta, K. Hashiba, and Y. Maeda.

2004. Inactivation of Saccharomyces cerevisiae by ultrasonic irradiation. Ultrason

Sonochem 11(2):61-65.

Valero, M., N. Recrosio, D. Saura, N. Munoz, N. Marti, and V. Lizama. 2007.

Effects of ultrasonic treatments in orange juice processing. J. Food Eng 80(509-

516):509.

Vercet, A., R. Oria, P. Marquina, S. Crelier, and P. Lopez-Buesa. 2002.

Rheological properties of yoghurt made with milk submitted to manothermosonication. J

Agric Food Chem 50(21):6165-6171.

Villamiel, M. and P. de Jong. 2000a. Inactivation of Pseudomonas fluorescens

and Streptococcus thermophilus in Trypticase Soy Broth and total bacteria in milk by

continuous-flow ultrasonic treatment and conventional heating. J. Food Eng. 45:171-179.

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Villamiel, M. and P. de Jong. 2000b. Influence of high-intensity ultrasound and

heat treatment in continuous flow on fat, proteins, and native enzymes of milk. J Agric

Food Chem 48(7):3068.

Villamiel, M., R. Verdurmen, and P. de Jong. 2000. Degassing of milk by high

intensity ultrasound. Milchwissenschaft. 55(3):123-125.

Wrigley, D. M. and N. G. Llorca. 1992. Decrease of Salmonella typhimurium in

skim milk and eggs by heat and ultrasonic wave treatment. J. Food Prot. 55(9):678-680.

Wu, H., G. J. Hulbert, and J. R. Mount. 2001. Effects of ultrasound on milk

homogenization and fermentation with yogurt starter. Innov Food Sci Emerg Technol

1:211-218.

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CHAPTER 4

SONOPROTECTIVE EFFECT OF MILK COMPONENTS ON E. coli

AND L. monocytogenes

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4.1 Abstract

Sonoprotective effects of milk components on Escherichia coli ATCC 25922 and

Listeria monocytogenes ATCC 19115 upon treatment with 24 kHz ultrasound waves

(USW) at sublethal temperatures were investigated. Casein, lactose or β lactoglobulin

was added to simulated milk ultrafiltrate (SMUF) to determine the individual as well as

combined sonoprotective effects of milk components on these bacteria. Addition of

caseins to SMUF in both micellar (phosphocasein) and non-micellar (sodium caseinate)

forms did not significantly change the D values for inactivation of either organism. The D

values for SMUF, SMUF + phosphocasein and SMUF + sodium caseinate were 2.84,

3.15 and 3.05 min for E. coli, while those for L. monocytogenes were 7.3, 8.07 and 7.9

min, respectively. Addition of whey protein β lactoglobulin to SMUF did not result in any

significant change in D value for E. coli, while that for L. monocytogenes increased

significantly (p < 0.05). Addition of lactose to SMUF significantly increased (p < 0.05) D

values for inactivation of E. coli and L. monocytogenes. The D values for E. coli in SMUF

and SMUF + lactose were 2.84 and 3.42 min; while those for L. monocytogenes were

7.3 and 8.52 min, respectively. Moreover, D values obtained in SMUF + lactose were

not significantly different from that in skim milk for either organism, suggesting that

amongst the components tested lactose was conferring protection to bacteria. There

was no significant increase in D values when E. coli and L. monocytogenes were

sonicated in SMUF with the three components, suggesting that milk components do not

exert any additive or synergistic protective effect against ultrasound treatment. Scanning

Electron Microscopy images of ultrasound treated samples showed that ultrasound

treatment resulted in mechanical damage to the cells leading to their death.

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4.2 Introduction

With the increase in the demand of ‘fresh-like’ foods, the importance of

alternative food processing technologies to pasteurization is increasing. In recent years,

several alternative technologies such as high pressure processing, pulsed-electric field

and irradiation have been explored (Manas and Pagan, 2005, Wan et al., 2005).

Ultrasound technology is also one of the emerging alternate food processing

technologies that uses sound waves with frequencies higher than 20 kHz (Piyasena et

al., 2003).

Until now, most of the research on ultrasound inactivation of microorganisms has

been done in buffer systems (Hua and Thompson, 2000, Manas et al., 2000, Pagan et

al., 1999). However, when inactivation in buffers is compared to food systems, the extent

of inactivation differs widely (Lee et al., 1989, Wrigley and Llorca, 1992). Several factors

such as water activity (Alvarez et al., 2003), pH (Baumann et al., 2005, Mack and

Roberts, 2007), viscosity (Wrigley and Llorca, 1992) and composition of foods (Baumann

et al., 2005, Valero et al., 2007) influence inactivation of microorganisms in foods.

However, no study on sonoprotective effect of food components on bacteria has been

conducted until now, whereas several sonication studies have been performed in

conjunction with heat and high pressure treatment (Black et al., 2007, Manas et al.,

2001). Presence of salts and sugars in the treatment medium increases the resistance of

bacteria to heat and high pressure treatments, mostly attributed to the lowering of water

activity of the system (Alvarez et al., 2003, Fujii et al., 1996, Kilimann et al., 2006,

Molina-Hoppner et al., 2004, Sumner et al., 1991). Most foods have a low concentration

of these solutes, such that they do not affect the water activity of the system. Thus the

decrease in water activity of foods would not explain the increased resistance of

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microorganism to physical treatments (Moats et al., 1971, Molina-Gutierrez et al., 2002).

Salts and sugars also protect bacteria by stabilizing the proteins and membranes thus

conferring increased resistance to physical treatments (Leslie et al., 1995, Timasheff et

al., 1975). The presence of lipids in a food system also increases the resistance of

bacteria to heat treatment (Ahmed et al., 1995, Kaur et al., 1998). The protective effect

of lipids is due to the reduced water activity of lipid systems and dehydration of cells

immersed in the lipid phase (Ahmed et al., 1995, Jay et al., 2005). Proteins and peptides

also increase the heat resistance of bacteria. However, the mechanism of protection is

not yet clear. This could possibly result from the formation of aggregates of bacteria due

to the interactions of peptides and bacteria (Moats et al., 1971). Caseins, the proteins

present in milk, protect more bacteria in micellar form than in non-micellar form due to

the presence of minerals in micellar casein (Black et al., 2007). Also, divalent cations

present in milk increase heat resistance of bacteria by stabilizing their ribosomes or cell

envelopes (Manas et al., 2001).

Foods are complex systems, wherein food components as well as their

interactions may affect the sonoprotection of bacteria (Mack and Roberts, 2007, Valero

et al., 2007). To understand the protective effect of individual food components on

microbial inactivation, a model food system that can be mimicked in buffer is desired.

Milk would be an ideal system to investigate the sonoprotective effect of individual food

components, since the exact composition of milk is well known and could be easily

mimicked (Jenness and Koops, 1962). Moreover, many studies show that milk exerts a

sonoprotective effect on bacteria as compared to buffers (Lee et al., 1989, Wrigley and

Llorca, 1992, Zenker et al., 2003). Since each food component may protect different

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organelles or areas of bacterial cells, study of the sonoprotective effect of milk

components on bacteria subjected to USW will help in understanding the target of USW.

Therefore, the objective of this study was to investigate the effect of individual

components of milk such as lactose, casein, whey proteins, and their interactions on

protection of E. coli and Listeria monocytogenes against USW.

4.3 Materials and Methods

There is some repetition in materials and methods in this chapter since they were

the same as those in chapter 3.

4.3.1 Bacterial cultures and culture conditions

Escherichia coli ATCC 25922 was obtained from American Type Culture

Collection (Manassas, VA) and L. monocytogenes ATCC 19115 was obtained from Dr.

Stephen J. Knabel, Department of Food Science, Penn State University. The cultures

were activated by transferring a loopful of frozen culture into 10 ml Tryptic Soy Broth

(TSB; Becton Dickinson and Company, Sparks, MD; BD) and incubating the tube for 24

h at 37±2°C. The activated cultures were then streaked onto Tryptic Soy Agar (TSA; BD)

plates and incubated for 24 h at 37±2°C. Isolated colonies from plates were transferred

to TSA slants and incubated for 18 h at 37±2°C, then stored at 4°C until needed. The

cultures were periodically transferred to fresh TSA slants.

4.3.2 Preparation of sonication media

Simulated milk ultrafiltrate (SMUF) was prepared according to the method of

Jenness and Koops (1962; Appendix A.2). Milk ingredients were added to SMUF at the

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concentration present in milk (Table 4.1) with 1N potassium hydroxide (KOH) solution.

The pH of SMUF and SMUF with ingredients was adjusted to 6.6±0.1. The solutions

were filter sterilized by passing through 0.2 µm filters (Nalgene, Rochester, NY).

4.3.3 Preparation of culture for sonication

A loopful of E. coli or L. monocytogenes culture was transferred from the TSA

slant to 10 ml of TSB and incubated for 18 h at 37±2°C. The resulting culture was diluted

to ~107 CFU/ml in sterile phosphate buffer (pH 7.2; Appendix A) and 1 ml of the diluted

culture was added to 250 ml and 100 ml TSB for E. coli and L. monocytogenes,

respectively. For E. coli, the flasks were incubated for 24 h at 37±2°C in a shaking

incubator (Model 3597, Lab Line Instruments Inc., Melrose Park, IL) set at 200 rpm,

while for L. monocytogenes, the flasks were incubated under static conditions for 20 h at

37±2°C. The incubation times were determined by growth curves of bacteria performed

previously by plating and absorbance measurements at 600 nm (Appendix B.1 and B.2)

to achieve the mid-stationary phase cells of bacteria. Growth phase was determined by

absorbance at 600 nm for subsequent experiments. The mid-stationary phase cells were

added to 100 ml sonication medium to achieve a final concentration of ~107 CFU/ml. The

sonication medium inoculated with bacterial cells constituted a sonication sample.

4.3.4 Ultrasound treatment of sonication sample

Sonication samples were treated with 24 kHz pulse (80% pulse/sec) USW

(Model UP400S, Hielscher, Ringwood, NJ) with an amplitude of 100 µm and energy

density of 85 W/cm2, respectively. The probe was immersed in 70% alcohol for 15 min

and left to air dry prior to sonication of the sample in order to avoid any contamination of

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Table 4.1:Suspending media used for ultrasound treatment of E. coli and L. monocytogenes

Sonication media Description

SMUF Simulated milk ultrafiltrate SMUF + Lactose SMUF containing 50 gL-1 lactose SMUF + β lactoglobulin SMUF containing 3 gL-1 β-lactoglobulin SMUF + PhosCN SMUF containing 30 gL-1 phosphocasein SMUF + NaCN SMUF containing 3 gL-1 sodium caseinate SMUF + 3I SMUF containing lactose, β-lactoglobulin and sodium caseinate Water + 3I Water containing lactose, β-lactoglobulin and sodium caseinate SMUF + Suc SMUF containing sucrose 50,100 or 150 gL-1 SMUF + Glu SMUF containing glucose 50,100 or 150 gL-1 Skim milk Parmalat skim milk

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the sample during sonication. The probe was rinsed with 2 ml of sterile phosphate buffer

and plated onto TSA plates to confirm that the probe was not contaminated with

bacteria.

An aliquot of the sonication sample was withdrawn and placed into a 2 ml sterile

vial before starting the sonication; this was the 0 time sample. The sonication sample

was then transferred to a 250 ml reaction beaker (Cole Parmer, Vernon Hills, IL)

connected to a water bath (Model 9610, Fisher Scientific, Pittsburgh, PA) maintained at

5°C. The probe was inserted into the center of the beaker; immersed into the sonication

sample and the ultrasound was switched on. Aliquots (2 ml) were withdrawn and

transferred to 2 ml sterile vials at set time intervals during sonication. Temperatures of

the sonication sample were recorded with digital a thermocouple at various time intervals

throughout the sonication treatment. The temperature during sonication never exceeded

35°C which is a sublethal temperature for E. coli and L. monocytogenes (Mack and

Roberts, 2007, Romero et al., 2007).

4.3.5 Enumeration of bacteria and D values

The previously collected aliquots were serially diluted in sterile phosphate buffer

(pH 7.2) and surface plated onto duplicate TSA plates. The plates were incubated at

37±2°C and counted after 24 h for E. coli and 48 h for L. monocytogenes. The survival

curves were drawn by plotting Log10 CFU/ml against time. A trendline was drawn on the

straight line portion of survival curve and D values were calculated by taking the

negative inverse of slope of the trendline.

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4.3.6 Statistical analysis

All experiments were performed in triplicate and the D values from all three

replicates were used for data analysis. Data were analyzed using one way ANOVA

followed by Tukey’s pairwise comparison test at p = 0.05 using SPSS version 11 (SPSS

Inc. Chicago, IL).

4.4 Results and Discussion

4.4.1 Sonoprotective effect of milk components

Milk components were tested for their sonoprotective effect on E. coli and L.

monocytogenes upon sonication with 24 kHz USW (Table 4.1). It was previously

demonstrated that the presence of fat in milk does not affect the inactivation of E. coli

and L. monocytogenes by 24 kHz USW (Chapter 3). Hence, fat was not added to SMUF

at any point during this study. There were no significant differences in D values obtained

between SMUF and SMUF with NaCN or phosphocasein for either the organism

suggesting that casein is not protecting these organisms against USW (Figure 4.1). The

D value for E. coli in SMUF with β lactoglobulin was also not significantly different than

SMUF alone, while that for L. monocytogenes was significantly higher (p < 0.05) than

SMUF. The D values for E. coli (3.42 and 2.84 min) and L. monocytogenes (8.52 and

7.30min) inactivation were significantly higher (p < 0.05) in SMUF with lactose compared

to SMUF alone (Figure 4.1).

Caseins have been reported to protect more bacteria in micellar form than in

non-micellar form due to the presence of minerals in micellar casein (Black et al., 2007).

However, in this study, the presence of both micellar (phosphocasein) and non-micellar

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8.52

3.152.84

7.30

3.42

2.82

8.09

3.05

7.90 8.07

3.08

8.20

0

1

2

3

4

5

6

7

8

9

10

E. coli L. monocytogenes

D V

alue

(min

)

SMUF

SMUF +Lactose

SMUF + β lactoglobulin

SMUF + NaCN

SMUF + Phosphocasein

SMUF + 3I

D values with same superscript are not significantly different at p = 0.05

Figure 4.1: Inactivation of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 by 24kHz ultrasound waves in different sonication media

b

a

b ab ab ab

B

A A AB AB A

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(NaCN) casein in SMUF did not exert any protective effect on either organism (Figure

4.1). There was no significant difference between D values for inactivation in SMUF with

NaCN and SMUF with phosphocasein. This could be due to the disruption of casein

micelles by USW. Taylor and Richardson (1980) reported that the turbidity of milk, which

is an estimate of micelle size, vanished upon sonication of milk indicating that micellar

size was reduced upon sonication.

Escherichia coli and L. monocytogenes exhibited different inactivation behavior in

SMUF + β lactoglobulin solution. Proteins have been shown to have little effect on

protection of bacteria (Moats et al., 1971). This has been attributed to exclusion of

macromolecules by bacterial cell walls which depends upon molecular weight of protein

as well as bacterial species (Gerhardt and Judge, 1964). Moreover, the outer membrane

present in Gram-negative bacteria is known to be impermeable to large molecular weight

proteins (Pelczar et al., 1998). It is likely that β lactoglobulin was not able to pass

through the outer membrane of E. coli and confer protection to it. This indicates that

structures internal to the outer membrane could be the target of USW. However, in the

case of L. monocytogenes, β lactoglobulin might have been in contact with the target of

USW, thus protecting it.

Sugars have been known to protect bacteria either by decreasing the water

activity (aw) of the solution or by interaction of solute molecules with bacterial

biomolecules (Alvarez et al., 2003, Leslie et al., 1995, Sumner et al., 1991). In this study

while the water activity of SMUF decreased slightly from 0.990 to 0.987 upon addition of

lactose, the D value for inactivation of E. coli and L. monocytogenes increased

significantly as compared to SMUF (Figure 4.1). This suggests that aw of the sonication

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medium could not be the sole reason for increased resistance of bacteria to USW upon

addition of lactose. Sonoprotective effect of lactose might be dependent on specific

interactions of lactose with biological macromolecules (Fujii et al., 1996).

Microorganisms produce compatible solutes such as betaines or sugars to

maintain osmotic balance of cells in the presence of sugars in the environment. In

addition, these solutes also provide protection to bacteria against physicochemical

treatments such as heating, freezing, drying and high pressure (Welsh, 2000). The

increased resistance to physicochemical treatments has been attributed to the response

of microorganisms to osmotic stress and specific interactions of sugar molecules with

biomolecules such as phospholipids and proteins (Molina-Hoppner et al., 2004). The

phospholipids in bacterial membranes may undergo a transition from crystalline liquid to

gel phase accompanied by leakage of cells upon physical treatments such as freeze

drying thereby making cells more sensitive to the treatment. Sugars increase the stability

of cell membranes against such physicochemical treatments by lowering the phase

transition temperature (Tm) leading to delay in phase transition from liquid crystalline to

gel phase thus avoiding the leakage of the membranes (Leslie et al., 1995, Molina-

Hoppner et al., 2004). The increased stability of cell membranes has been attributed to

the attachment of the polar head group (phosphate group) of phospholipids layer with

the free –OH group of sugar (Crowe et al., 1987). Another plausible reason for protection

by lactose could be stabilization of proteins of bacterial cells by preferential exclusion of

the sugar from contact surface of the protein. Addition of sugar to protein solution is a

thermodynamically unfavorable phenomenon. To avoid this, the protein structure

stabilizes in the presence of sugars because the denaturation of the protein molecule will

lead to a larger contact surface between the protein and the solvent (Timasheff et al.,

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1975). Kilimann et al. (2006) also reported that sucrose protects Lactococcus lactis

against heat inactivation by stabilizing the secondary protein structure of bacteria.

Studies show that in the presence of sucrose and lactose in the environment, the

concentration of sucrose and lactose present in cell cytoplasm increased suggesting that

sugars were transported into the cells (Glaasker et al., 1998). Similar results were also

reported by Molina-Hoppner et al., (2004) for Lactococcus lactis. In this study, it is

possible that accumulation of compatible solute in the presence of lactose or

stabilization of membrane or proteins by lactose resulted in increased resistance of

bacteria to USW.

4.4.2 Sonoprotective effect of salts of milk

To test if the salts in milk protected bacteria from USW, E. coli and L.

monocytogenes were sonicated with 24 kHz USW in water + 3I (Table 4.1). Data

analysis showed that there was no significant difference in D values for E. coli (3.08 and

2.87 min) and L. monocytogenes (8.2 and 7.75 min) in SMUF + 3I and water + 3I,

respectively, suggesting that salts in SMUF do not exert sonoprotective effect on E. coli

and L. monocytogenes (Figure 4.2). Divalent cations present in milk have been reported

to increase heat resistance of bacteria by stabilizing their ribosomes (Manas et al.,

2001). These results indicate that the bacterial ribosomes might not be the potential

target of USW.

4.4.3 Synergistic or additive sonoprotective effect of milk components

To test if milk components interact in some way to exert an additive or synergistic

sonoprotective effect on bacteria, the three components (Table 4.1) were added to

SMUF and D values for inactivation of E. coli and L. monocytogenes were calculated. D

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3.08

8.2

2.87

7.75

0

1

2

3

4

5

6

7

8

9

E. coli L. monocytogenes

D V

alue

(min

)

SMUF + 3I

Water + 3I

D values with same superscript are not significantly different at p = 0.05

Figure 4.2: Inactivation of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 in SMUF + NaCN + lactose + β lactoglobulin and Water + NaCN + lactose + β lactoglobulin by 24 kHz ultrasound waves

a a

A A

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values for inactivation of E. coli and L. monocytogenes in SMUF + 3I were not

significantly different from SMUF + lactose suggesting that the milk components do not

interact to produce any additive or synergistic sonoprotective effect on these two

organisms (Figure 4.1). Moreover, the D values for E. coli (3.08 and 3.42 min) and L.

monocytogenes (8.61 and 8.52 min) were not significantly different in skim milk and

SMUF + lactose, respectively, suggesting that lactose present in milk is exerting a

protective effect on these organisms to sonication (Figure 4.3) .

4.4.4 Effect of different sugars on protection of E. coli

Three sugars, lactose, glucose or sucrose, were added to SMUF at a

concentration of 5, 10 or 15% to determine the effect of concentration and type of sugar

on inactivation of E. coli by USW. D values for inactivation by ultrasound treatments

increased significantly as the concentration of sucrose and glucose was increased

(Table 4.2). However, no significant change in D values was observed in case of lactose

in the range tested. Moreover, all three sugars showed significant increase in D values

when compared to SMUF alone (0%) suggesting that the sugars are exerting protective

effect on bacteria against sonication with 24 kHz USW (Table 4.2). The presence of

different sugars at the same concentration did not exhibit any significant differences in D

values. Since glucose is a monosaccharide while sucrose and lactose are disaccharides,

the number of sugar molecules of glucose would be twice the number of molecules of

sucrose and lactose at same sugar concentration. No differential effect of these sugars

at the same concentration suggests that number of molecules of sugar could not be

influencing the inactivation of bacteria. This could be due to the fact that the range tested

in the study was very narrow with concentrations less than 0.4 M. In most of the studies

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3.08

8.61

3.42

8.52

0

1

2

3

4

5

6

7

8

9

10

E. coli L. monocytogenes

D V

alue

(min

)

Skim Milk

SMUF + Lactose

a

a

A

A

D values with same superscript are not significantly different at p = 0.05

Figure 4.3: Comparison of D values for inactivation of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 in skim milk and SMUF + lactose upon sonication with 24 kHz USW

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D values± standard deviation within the same sugar type with the same lower case superscript are not significantly different at p = 0.05

D values ± standard deviation within the same sugar concentration with the same upper case superscript are not significantly different at p = 0.05

Table 4.2: Effect of sugar concentration and sugar type on inactivation of E. coli ATCC 25922 upon sonication with 24 kHz USW.

D Value (min) Sugar concentration (% w/v) Lactose Sucrose Glucose

0 2.84±0.17b 2.84±0.17b 2.84±0.17b 5 3.42±0.40aA 2.97±0.46bA 3.02±0.17bA

10 3.41±0.13aA 3.27±0.06abA 3.38±0.19abA

15 3.58±0.16aA 3.55±0.23aA 3.55±0.39aA

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for other physical treatments, the sugar concentrations of more than 0.5 M have been

used to differentiate their protective effects (Fujii et al., 1996, Kilimann et al., 2006).The

protection of bacteria by sugars has been reported to be dependent upon the

concentration and type of sugars. Resistance of Saccharomyces cerevisiae to high

pressure treatment increased with increase in concentration and number of equatorial

OH groups (Fujii et al., 1996). These results suggest that as the concentration of sugars

increases, the protection conferred to bacteria increases. However, it is not clear if the

type of sugar would affect the inactivation of bacteria by USW.

4.5 Conclusions

Sonoprotective effects of milk components on E. coli and L. monocytogenes

upon treatment with 24 kHz USW were investigated in this study. Presence of salts and

caseins in SMUF did not result in significant change in D values of ultrasound

inactivation for both E. coli and L. monocytogenes. However, the addition of lactose to

SMUF resulted in significant increase (p < 0.05) in D values of both the organisms.

Addition of whey protein β lactoglobulin to SMUF did not result in any significant change

in D value for E. coli while that for L. monocytogenes increased significantly. The D

values for E. coli and L. monocytogenes in SMUF + lactose were not significantly

different from that in SMUF with lactose, sodium caseinate and β lactoglobulin

suggesting that these milk components do not exert an additive or synergistic protective

effect on these bacteria. Moreover, D values for SMUF + lactose were not significantly

different from skim milk suggesting that among the components tested only lactose was

conferring protection to bacteria. The experiments with lactose, sucrose and glucose at

different concentrations (5-15% w/v) showed that as the concentration of sugars was

increased, the protection conferred to E. coli increased. However, the effect of different

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types of sugars at the same concentration was not evident in the concentration range

tested.

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4.6 References

Ahmed, N. M., D. E. Conner, and D. L. Huffmann. 1995. Heat-resistance of

Escherichia coli 0157:H7 in meat and poultry as affected by product composition. J Food

Sci 60(3):606-610.

Alvarez, I., P. Manas, F. J. Sala, and S. Condon. 2003. Inactivation of Salmonella

enterica serovar Enteritidis by ultrasonic waves under pressure at different water

activities. Appl Environ Microbiol 69(1):668-672.

Baumann, A. R., S. E. Martin, and H. Feng. 2005. Power ultrasound treatment of

Listeria monocytogenes in apple cider. J Food Prot 68(11):2333-2340.

Black, E. P., T. Huppertz, G. F. Fitzgerald, and A. L. Kelly. 2007. Baroprotection

of vegetative bacteria by milk constituents: A study of Listeria innocua. Int Dairy J

17(104-110):104.

Crowe, J.H., L. M. Crowe, J. F. Carpenter, and A. Wistrom, C. 1987 Stabilization

of dry phospholipid bilayers and proteins by sugars. Biochem J 242, 1-10.

Fujii, S., K. Obuchi, H. Iwahashi, T. Fujii, and Y. Komatsu. 1996. Saccharides

that protect yeast against hydrostatic pressure stress correlated to the mean number of

equatorial OH groups. Biosci Biotechnol Biochem 60(3):476-478.

Gerhardt, P. and J. A. Judge. 1964. Porosity of isolated cell walls of

Saccharomyces cerevisiae and Bacillus megaterium. J Bacteriol 87:945-951.

Glaasker, E., F. S. Tjan, P. F. Ter Steeg, W. N. Konings, and B. Poolman. 1998.

Physiological response of Lactobacillus plantarum to salt and nonelectrolyte stress. J

Bacteriol 180(17):4718-4723.

Hua, I. and J. E. Thompson. 2000. Inactivation of Escherichia coli by sonication

at discrete ultrasonic frequencies. Wat Res 34(15):3888-3893.

Jay, J. M., M. J. Loessner, and D. A. Golden. 2005. Modern Food Microbiology.

7th ed. Springer, New York

Jenness, R. and J. Koops. 1962. Preparation and properties of a salt solution

which simulates milk ultrafiltrate. Netherlands Milk Dairy J 16(3):153-164.

Kaur, J., D. A. Ledward, R. W. Park, and R. L. Robson. 1998. Factors affecting

the heat resistance of Escherichia coli O157:H7. Lett Appl Microbiol 26(4):325-330.

Kilimann, K. V., W. Doster, R. F. Vogel, C. Hartmann, and M. G. Ganzle. 2006.

Protection by sucrose against heat-induced lethal and sublethal injury of Lactococcus

lactis: an FT-IR study. Biochim Biophys Acta 1764(7):1188-1197.

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Lee, B. H., S. Kermasha, and B. E. Baker. 1989. Thermal, ultrasonic and

ultraviolet inactivation of Salmonella in thin films of aqueous media and chocolate. Food

Microbiol 6:143-152.

Leslie, S. B., E. Israeli, B. Lighthart, J. H. Crowe, and L. M. Crowe. 1995.

Trehalose and sucrose protect both membranes and proteins in intact bacteria during

drying. Appl Environ Microbiol 61(10):3592-3597.

Mack, S. Z. and J. S. Roberts. 2007. Ultrasound pasteurization: The effects of

temperature, soluble solids, organic acids and pH on the inactivation of Escherichia coli

ATCC 25922. Ultrason Sonochem 14:323-329.

Manas, P. and R. Pagan. 2005. Microbial inactivation by new technologies of

food preservation. J Appl Microbiol 98:1387-1399.

Manas, P., R. Pagan, J. Raso, F. J. Sala, and S. Condon. 2000. Inactivation of

Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg by

ultrasonic waves under pressure. J Food Prot 63(4):451-456.

Manas, P., R. Pagan, F. J. Sala, and S. Condon. 2001. Low molecular weight

milk whey components protect Salmonella senftenberg 775W against heat by a

mechanism involving divalent cations. J Appl Microbiol 91(5):871-877.

Moats, W. A., R. Dabbah, and V. M. Edwards. 1971. Survival of Salmonella

anatum heated in various media. Appl Microbiol 21(3):476-481.

Molina-Gutierrez, A., B. Rademacher, M. G. Ganzle, and R. F. Vogel. 2002.

Effect of sucrose and sodium chloride on the survival and metabolic activity of

Lactococcus lactis under high-pressure conditions Pages 295-302 in Trends in high

pressure bioscience and biotechnology Elsevier Science, Amsterdam, The Netherlands.

Molina-Hoppner, A., W. Doster, R. F. Vogel, and M. G. Ganzle. 2004. Protective

effect of sucrose and sodium chloride for Lactococcus lactis during sublethal and lethal

high-pressure treatments. Appl Environ Microbiol 70(4):2013-2020.

Pagan, R., P. Manas, J. Raso, and S. Condon. 1999. Bacterial resistance to

ultrasonic waves under pressure at nonlethal (manosonication) and lethal

(manothermosonication) temperatures. Appl Environ Microbiol 65(1):297-300.

Pelczar, M. J., E. C. S. Chan, and N. R. Krieg. 1998. Microbiology. Fifth ed.

McGraw-Hill, New Delhi.

Piyasena, P., E. Mohareb, and R. C. McKellar. 2003. Inactivation of microbes

using ultrasound: A review. Int J Food Microbiol 87(3):207-216.

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Romero, E., H. Feng, and S. E. Martin. 2007. Inactivation of Shigella boydii 18

IDPH and Listeria monocytogenes Scott A with power ultrasound at different acoustic

energy densities and temperatures. J Food Sci 72(4):M103-M107.

Sumner, S. S., T. M. Sandros, M. C. Harmon, V. N. Scott, and D. T. Bernard.

1991. Heat resistance of Salmonella typhimurium and Listeria monocytogenes in

sucrose solutions of various water activities. J Food Sci 56(6):1741-1743.

Taylor, M. J. and T. Richardson. 1980. Antioxidant activity of skim milk: effect of

sonication. J Dairy Sci 63(11):1938-1942.

Timasheff, S. N., J. C. Lee, E. P. Pitts, and N. Tweedy. 1975. The interaction of

tubulin and other protein structure-stabilizing solvents J Colloid Interface Sci 55(3):658-

663.

Valero, M., N. Recrosio, D. Saura, N. Munoz, N. Marti, and V. Lizama. 2007.

Effects of ultrasonic treatments in orange juice processing. J Food Eng 80(509-516):509.

Wan, J., R. Mawson, M. Ashokkumar, and K. Ronacher. 2005. Emerging

processing technologies for functional foods. Australian J Dairy Technol 60(2):167-169.

Welsh, D. T. 2000. Ecological significance of compatible solute accumulation by

micro-organisms: from single cells to global climate. FEMS Microbiol Rev 24(3):263-290.

Wrigley, D. M. and N. G. Llorca. 1992. Decrease of Salmonella typhimurium in

skim milk and eggs by heat and ultrasonic wave treatment. J Food Prot 55(9):678-680.

Zenker, M., V. Heinz, and D. Knorr. 2003. Application of ultrasound-assisted

thermal processing for preservation and quality retention of liquid foods. J Food Prot

66(9):1642-1649.

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CHAPTER 5

MECHANISM OF INACTIVATION OF E. coli AND L.

monocytogenes BY ULTRASOUND WAVES

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5.1 Abstract

Scanning electron microscopy (SEM) and spectrophotometry of ultrasound

treated cells of E. coli and L. monocytogenes was performed to investigate the

mechanism of inactivation by ultrasound waves (USW). SEM images of ultrasound

treated cells of E. coli and L. monocytogenes showed that the cells were disintegrated

by USW. Cell debris was visible in E. coli sample suggesting that ultrasound waves

broke the cells into pieces. However, L. monocytogenes were broken into halves and no

cell debris was visible in the sample. Ultrasound treated samples of E. coli and L.

monocytogenes also showed a significant (p < 0.05) increase in absorbance at 260nm,

suggesting that nucleic acids of bacteria was leaking out of the bacterial cells. SEM

images and spectrophotometry data suggest that USW cause physical damage to cell

membranes of E. coli and L. monocytogenes leading to their cell death.

5.2 Introduction

Ultrasound waves (USW) are sound waves with frequencies above 20 kHz,

which is above the threshold frequency for human hearing (Piyasena et al., 2003). In a

liquid medium, they travel as longitudinal waves with alternate compression and

rarefaction regions characterized by positive and negative pressure zones, respectively

(Suslick, 1988). Microbial inactivation by ultrasound waves is attributed to cavitation,

which is a process of formation, growth and collapse of bubbles in a liquid medium

(Raso et al., 1999, Schebra et al., 1991). As the ultrasound waves pass through a liquid

medium, continuous pressure changes lead to cavitation. Cavitation bubbles form in the

rarefaction region of the sound wave due to negative pressure and grow in size in the

compression region, a positive pressure region. The bubbles grow to a maximum

unstable size over many alternating compression and rarefaction cycles, beyond which

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they are unable to sustain themselves and finally collapse. This collapse results in

radiation of shock waves from the site of collapse. The collapse also generates high

local temperatures leading to the formation of free radicals in the medium (Atchley and

Crum, 1988, Frizzell, 1988, Suslick, 1990). The free radicals and shock waves produced

by ultrasound waves are thought to be involved in the inactivation of microorganisms

(El'piner, 1964, Joyce et al., 2003). However, the exact reason for the lethality of

ultrasound is not yet completely understood.

The objective of this study was to investigate the mechanism of inactivation of E.

coli and L. monocytogenes by USW using spectrophotometry and scanning electron

microscopy.

5.3 Materials and Methods

Culture preparation and ultrasound treatment was performed by the methods as

cited in chapter 4.

5.3.1 Measurement of cellular leakage upon ultrasound treatment

Leakage of cytoplasmic content was determined by measuring absorbance at

260 nm using a spectrophotometer (Model ND 1000, Nanodrop, Wilmington, DE). Cells

of E. coli and L. monocytogenes were grown to mid stationary-phase and inoculated into

SMUF to achieve a final concentration of 107 cells/ml. The bacteria were then sonicated

with 24 kHz ultrasound waves. The total sonication time for E. coli and L.

monocytogenes was 20 and 40 min, respectively. The sonicated sample was then

filtered through 0.2 µm filter and absorbance of filtrate was measured at 260 nm.

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5.3.2 Scanning electron microscopy (SEM)

Cells exposed to USW were harvested by centrifugation at 2000 rpm for 10 min

at 4°C. The supernatant was discarded and the pellet was resuspended in 2 ml SMUF.

The culture thus obtained was filtered through 0.2 µm filters mounted on a filter carrier

and immersed in a mixture of 25% glutaraldehyde in 0.2M cacodylate buffer (CB) and

16% paraformaldehyde and 0.4 g sucrose for 1 h at room temperature. The filters were

fixed with 1% osmium tetraoxide for 1 h at room temperature followed by three washes

with 0.1M CB. The filters were then dehydrated in a gradient of ethanol (50, 70, 85, 95

and 100%) with 5 min immersion for each alcohol concentration. The filters were critical

point dried in PolaronE 3000 critical point dryer (Fissons, Middlebury, CT) using carbon

dioxide. Dried fillers were placed on aluminum stubs and coated with 10 nm gold-

palladium using a SCD050 sputter coater (BAL-TEC Middlebury, CT). The samples were

then examined using a JSM 5400 scanning electron microscope (JEOL, Peabody, MA)

in the electron microscopy facility at Penn State University, PA at 20 kV of accelerating

voltage.

5.4 Results and Discussion

5.4.1 Cellular leakage upon ultrasound treatment

A significant increase (p < 0.05) in absorbance at 260 nm was observed in

sonicated samples of E. coli and L. monocytogenes as compared to control samples

(Figure 5.1) suggesting that there was leakage in cells of both the organisms upon

ultrasound treatment (Mendonca et al., 1994). This indicates that cell membrane may be

the target of inactivation by USW.

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0

0.1

0.2

0.3

0.4

0.5

E. coli L. monocytogenes

Abs

orba

nce

260

nm

Control Ultrasound treatment

Values with same superscript are not significantly different at p = 0.05

Figure 5.1: Change in 260 nm absorbance of E. coli ATCC 25922 and L. monocytogenes ATCC 19115 sample upon treatment with 24 kHz USW

a

b

A

B

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5.4.2 Changes in cell morphology by USW

SEM images of sonicated E. coli and L. monocytogenes cells show that USW

caused mechanical damage to the cells. Escherichia coli cells were broken into pieces

and cell debris was visible throughout the field (Figure 5.2 B). Some E. coli cells showed

an intermediate stage where they were not disrupted but the cell walls were roughened

and discontinued as compared to control E. coli. In some cells of E. coli, invaginations

were also present and the cell membrane or cell wall seemed ruptured at some places

(Figures 5.2 C and D). This shows that the cell wall and cell membrane was damaged

upon sonication of E. coli. Listeria monocytogenes cells also exhibited physical damage

caused by USW (Figure 5.3 B, C and D). The cells were broken into halves and cell

debris was not visible in sonicated samples of L. monocytogenes as it was in case of E.

coli. These images suggest that cell wall and cell membrane were the target of

ultrasound waves.

Cell death involving cell membranes could either be due to pore formation in cell

membrane or mechanical disruption of the cell membrane by the shock waves produced

during cavitation. It is even possible that the pore formation could have lead to

mechanical disruption of the cells. It has been shown that small sized pores are created

upon treatment with PEF. These pores are small enough not to allow the passage of

molecules responsible for osmotic balance but they allow entrance of water molecules

leading to an increase in the volume of the cell. The inflow of water into cells leads to

increase in turgor pressure and hence bursting of the cells resulting in their cell death

(Tsong, 1991). In this study, the SEM images of treated samples did not show any

significant increase in volume of the cells indicating that pore formation might not be

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Figure 5.2: Scanning Electron microscopy images of E. coli ATCC 25922 upon treatment with 24 kHz USW in SMUF; Control E. coli ATCC 25922(A), Treated E. coli ATCC 25922 (B, C, D)

A

DC

B

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Figure 5.3: Scanning Electron microscopy images of L. monocytogenes ATCC 19115 upon treatment with 24 kHz USW in SMUF; Control L. monocytogenes ATCC 19115(A), Treated L. monocytogenes ATCC 19115(B, C, D)

DC

A B

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involved in the destruction of cells by USW. But, the presence of invaginations and

discontinuities in the membrane show that the membrane was physically damaged by

USW. This is also supported by the fact that no sublethal injury was observed during

sonication of E. coli and L. monocytogenes (Figures 3.4 and 3.5). The cell membrane

controls the cell’s metabolic activities by maintaining an osmotic balance between the

cell and its surrounding environment. Any damage to the cell membrane could lead to

impairment of this balance leading to cell death (Pelczar et al., 1998). Physical damage

to cell membrane has been demonstrated by leakage of cytoplasmic material

(Mendonca et al., 1994) from cell. In this study, it was observed that cytoplasmic content

leaked into the cytoplasm upon treatment of bacterial cells with USW as suggested by

increase in absorbance at 260nm (Figure 5.1). The UV absorbance data and SEM

images suggest that the possible mechanism of inactivation of these bacteria by USW is

the physical damage to the cell membrane of bacteria.

In addition, the SEM images of ultrasound treated cells of E. coli showing cell

debris could also explain the tailing phenomenon observed during inactivation of E. coli

(Figure 3.3). Both stationary-phase and log-phase cells of E. coli exhibited tailing upon

sonication with 24 kHz USW (Figure 3.3 and Appendix B; B.7). However, L.

monocytogenes did not show any tailing during inactivation by USW. Based on the

electron microscopy observations it is postulated that live cells of E. coli might have

become entrapped in the debris of dead cells and were protected from mechanical

damage caused by USW, leading to tailing of inactivation curve. However, L.

monocytogenes, which did not show any debris in the sonicated sample, exhibited first

order inactivation kinetics. This suggests that presence of cell debris in the sample might

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be involved in the protection of E. coli from ultrasound treatment leading to tailing in

inactivation curves.

5.5 Conclusions

The SEM images suggest that ultrasound waves cause physical damage to cell

membrane of E. coli and L. monocytogenes leading to their death. Escherichia coli cells

were disintegrated into pieces by ultrasound waves and cell debris was visible in the

field. However, L. monocytogenes were broken into halves and no cell debris was visible

in the sample. Ultrasound treated samples of E. coli and L. monocytogenes showed a

significant (p < 0.05) increase in absorbance at 260 nm, suggesting that cell membrane

was the target of USW. Moreover, cell debris generated upon disintegration of E. coli

might be involved in protection of live cells of E. coli leading to tailing phenomenon.

5.6 Acknowledgements

I would like to thank SEM facility staff for helping me with sample preparation and

training with scanning electron microscope.

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5.7 References

Atchley, A. A. and L. A. Crum. 1988. Acoustic cavitation and bubble dynamics.

Pages 1-64 in Ultrasound: Its chemical, physical and biological effects. VCH Publishers

Inc., New York.

El'piner, I. E. 1964. Ultrasound Physical, Chemical and Biological Effects.

Consultants Bureau Enterprises Inc, New York

Frizzell, L. A. 1988. Biological effects of acoustic cavitation, Pages 287-301 in

Ultrasound: Its chemical, physical and biological effects. K. S. Suslick, ed. VCH

Publishers, New York.

Joyce, E., S. S. Phull, J. P. Lorimer, and T. J. Mason. 2003. The development

and evaluation of ultrasound for the treatment of bacterial suspensions. A study of

frequency, power and sonication time on cultured Bacillus species. Ultrason Sonochem

10(6):315-318.

Mendonca, A. F., T. L. Amoroso, and S. J. Knabel. 1994. Destruction of gram-

negative food-borne pathogens by high pH involves disruption of the cytoplasmic

membrane. Appl Environ Microbiol 60(11):4009-4014.

Pelczar, M. J., E. C. S. Chan, and N. R. Krieg. 1998. Microbiology. Fifth ed.

McGraw-Hill, New Delhi.

Piyasena, P., E. Mohareb, and R. C. McKellar. 2003. Inactivation of microbes

using ultrasound: A review. Int J Food Microbiol 87(3):207-216.

Raso, J., P. Manas, R. Pagan, and F. J. Sala. 1999. Influence of different factors

on the output power transferred into medium by ultrasound. Ultrason Sonochem

5(4):157-162.

Schebra, G., R. M. Weigel, and W. D. O’Brien. 1991. Quantitative assessment of

the germicidal efficacy of ultrasonic energy. Appl Environ Microbiol 57(7):2079-2084.

Suslick, K. S. 1988. Homogenous sonochemistry. Pages 123-159 in

Ultrasound:Its chemical, physical and biological effects. K. S. Suslick, ed. VCH

Publishers, New York.

Suslick, K. S. 1990. Sonochemistry. Science 247(4949):1439-1445.

Tsong, T. Y. 1991. Electroporation of cell membranes. Biophys J 60(2):297-306.

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CHAPTER 6 CONCLUSIONS AND FUTURE RESEARCH

6.1 Conclusions

Ultrasound technology is one of the emerging alternative technologies for food

processing. The overall goal of this thesis was to evaluate the use of ultrasound

technology for pasteurization using milk as a model system. First, the effects of

sonication medium, ultrasound frequency and bacterial growth phase on inactivation of

E. coli and L. monocytogenes by ultrasound waves were evaluated. The rate of

inactivation of E. coli and L. monocytogenes was significantly slower in whole and skim

milk as compared to phosphate buffer. Escherichia coli exhibited non log-linear

inactivation kinetics with tailing, whereas L. monocytogenes showed log-linear

inactivation kinetics throughout. Maximum inactivation of E. coli was observed with 24

kHz ultrasound waves (USW), while high frequency USW (650, 765 and 850 kHz) were

ineffective in killing E. coli. No injury was observed upon sonication of E. coli and L.

monocytogenes with 24 kHz USW indicating that ultrasound inactivation follows all or

none principle of inactivation. Log-phase cells of both bacteria were more sensitive to

ultrasound treatment than stationary phase cells.

Since the D values for both bacteria in whole and skim milk were significantly

higher than in phosphate buffer, the sonoprotective effect of milk components on E. coli

and L. monocytogenes upon treatment with 24 kHz USW were investigated in the

second part of this study. Presence of salts and caseins in Simulated Milk Ultrafiltrate

(SMUF) did not significantly increase (p < 0.05) D values for both E. coli and L.

monocytogenes; however, the addition of lactose to SMUF did. Addition of whey protein

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β lactoglobulin to SMUF did not result in any significant change in D value for E. coli

while that for L. monocytogenes increased significantly (p < 0.05). The D values for E.

coli and L. monocytogenes SMUF + lactose were not different from SMUF containing

lactose, sodium caseinate and β lactoglobulin suggesting that these components do not

exert an additive or synergistic protective effect on these bacteria. Moreover, D values

for SMUF + lactose were not significantly different from skim milk suggesting that among

the components tested, lactose was conferring maximum protection to bacteria. The

experiments examining the effects of lactose, sucrose and glucose at different

concentrations (5 to 15% w/v) showed that as the concentration of sugars was

increased, the protection conferred to E. coli increased. However, no significant

differences in D values were observed upon sonication in different sugars at the same

concentartion.

Significant increase (p < 0.05) in 260 nm absorbance of sonicated samples of E.

coli and L. monocytogenes suggested that cell membrane might be the target of USW.

Scanning electron microscopy images of the USW treated E. coli and L. monocytogenes

cells showed physical damage to the cell wall and cell membrane, suggesting that

membrane disruption is the mode of inactivation of bacteria by USW.

6.2 Future Research

This study demonstrated that ultrasound technology has the potential to be used

in the dairy industry for pasteurization of milk. To facilitate its application in the food

industry, scale-up feasibility studies for ultrasound technology need to be conducted.

Also, the impact of ultrasonication on the sensory and nutritional attributes of milk needs

to be studied.

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During the course of this research, several interesting questions arose which

need to be investigated to develop a mechanistic understanding of bacterial inactivation

by ultrasound waves.

1. It was found that sugars such as lactose, sucrose and glucose exhibit a

sonoprotective effect on the bacteria. The exact mechanism of this

behavior of sugar molecules is not clear yet and needs to be elucidated.

This could potentially help the food processors to better design ultrasound

processing operations for various food products.

2. This study provided some indications towards the mode and sites of

action of ultrasound waves on bacterial cells. Scanning electron

microscopy images suggested that USW potentially disrupt the bacterial

cell wall and cell membrane. However, further research needs to be

conducted to determine the exact target of USW at molecular level.

3. Bacterial inactivation could be mathematically modeled to help food

processors predict sonication times for food products. Advanced

mathematical models could be developed to include product parameters

such as viscosity, density, temperature of sonication and flow rates to

facilitate continuous ultrasound processing of food products.

4. Inactivation kinetics of E. coli exhibited tailing upon sonication, which is

another poorly understood phenomenon. While it has been reported by

several authors, it has not been understood clearly. Understanding of the

reason behind tailing would help food processors to develop processes

for various food products.

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APPENDIX A COMPOSITION OF BUFFERS

A.1 Preparation of phosphate buffer

A.1.1 Stock phosphate solution

Monobasic potassium phosphate (34 g; Chemical Corp. St. Louis, USA) was

dissolved in 500 ml of deionized water and pH was adjusted to 7.2 ± 0.2 with sodium

hydroxide (NaOH). The final volume of salt solution was made to 1 L by adding

deionized water. The solution was sterilized at 121°C for 15 min and then stored at 4°C

until needed (Davis and Hickey, 2004).

A.1.2 Phosphate dilution water (Class O)

Stock phosphate solution (1.25 ml) was added to deionized water and volume

was made to 1 L. The solution was dispensed (9 ml in tubes or as required) and

sterilized at 121°C for 15 min (Davis and Hickey, 2004).

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A.2 Simulated Milk Ultrafiltrate (SMUF)

Each salt was weighed out and ground in a mortar. The salts were mixed

thoroughly and stored in an air tight container at room temperature. A portion of this

mixture (7.59 g) was dissolved in 990 ml of deionized water and the pH of the solution

was adjusted to 6.6 with 1N KOH solution. Deionized water was added to bring it to 1 L

(Jenness and Koops, 1962).

References

Davis, G. L. and P. J. Hickey. 2004. Media and dilution water preparation. Pages

93-100 in Standard methods for the examination of dairy products, 17th ed. H. M. Wehr

and J. F. Frank, ed. APHA, Washington, DC.

Jenness, R. and J. Koops. 1962. Preparation and properties of a salt solution

which simulates milk ultrafiltrate. Netherlands Milk Dairy J 16(3):153-164.

Table A.1: Composition of dry blended mix

Salt Weight used (g)

KH2PO4 15.80

K3 citrate.H2O 5.08

Na3 citrate.5H2O 21.2

K2SO4 1.8

CaCl2.2H2O 13.2

Mg3 citrate.H2O 5.02

K2CO3 3.00 KCl 10.78

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4

5

6

7

8

9

10

0 10 20 30 40 50 60 70Time (h)

Log 1

0CFU

/ml

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

OD

600

nm

Log10CFU/ml

OD600 nm

APPENDIX B ADDITIONAL EXPERIMENTAL RESULTS

Figure B.1: Growth curve of E. coli (ATCC 25922) in Tryptic Soy Broth (TSB) incubated in a shaking incubator set at 37°C and 200 rpm

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4

5

6

7

8

9

10

0 10 20 30 40 50 60Time (hr)

Log 1

0CFU

/ml

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

OD

600

nm

Log10CFUs/ml

OD600 nm

Figure B.2: Growth curve of L. monocytogenes (ATCC 19115) in Tryptic Soy Broth (TSB) incubated in a incubator set at 37°C

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0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8 9 10 11 12Time (min)

Log 1

0 CFU

/ml

TSA VRBA

Figure B.3: Survival curves of mid stationary-phase cells of E. coli ATCC 25922 on TSA (-■-), a non-selective and VRBA (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in phosphate buffer (pH 7.0).

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0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16 18 20 22Time (min)

Log 1

0 CFU

/ml

TSA VRBA

Figure B.4: Survival curves of mid stationary-phase cells of E. coli ATCC 25922 on TSA (-■-), a non-selective and VRBA (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in skim milk

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0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30 35 40Time (min)

Log 1

0 CFU

/ml

TSA MOX

Figure B.5: Survival curves of mid stationary-phase cells of L. monocytogenes ATCC 19115 on TSA (-■-), a non-selective and MOX (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in phosphate buffer (pH 7.0).

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0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30 35 40Time (min)

Log 1

0 CFU

/ml

TSA MOX

Figure B.6: Survival curves of mid stationary-phase cells of L. monocytogenes ATCC 19115 on TSA (-■-), a non-selective and MOX (-∆-), a selective medium upon sonication with 24 kHz ultrasound waves in skim milk

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0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14Time (min)

Log 1

0 CFU

/ml

Figure B.7: Survival curve of mid-log phase cells of E. coli ATCC 25922 by 24 kHz USW in phosphate buffer