kinetics and mechanism of bacterial inactivation by
TRANSCRIPT
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
ii
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
x
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.
1
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.
2
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)
3
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).
4
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
5
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)
6
• 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
7
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
8
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
9
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).
10
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
11
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
12
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).
13
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
14
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
15
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
16
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
17
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,
18
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
19
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
20
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
21
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
22
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)
23
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
24
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.
25
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
26
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.
27
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
28
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.
29
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
30
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
31
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).
32
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
33
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
34
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
35
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
36
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.
37
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CHAPTER 3
EFFECT OF SONICATION MEDIUM AND GROWTH PHASE ON
INACTIVATION OF E. coli AND L. monocytogenes BY ULTRASOUND WAVES
45
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.
46
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.
47
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
48
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.,
49
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.
50
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
51
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
52
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
53
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
54
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.
55
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.
56
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)
57
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
58
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
59
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
60
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
61
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.
62
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.
63
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
64
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.
65
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
66
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.
67
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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.
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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
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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-
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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
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continuous-flow ultrasonic treatment and conventional heating. J. Food Eng. 45:171-179.
70
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.
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1:211-218.
CHAPTER 4
SONOPROTECTIVE EFFECT OF MILK COMPONENTS ON E. coli
AND L. monocytogenes
72
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.
73
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
74
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
75
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
76
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
77
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
78
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.
79
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
80
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
81
(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
82
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.,
83
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
84
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
85
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
86
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
87
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
88
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
89
types of sugars at the same concentration was not evident in the concentration range
tested.
90
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
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Gerhardt, P. and J. A. Judge. 1964. Porosity of isolated cell walls of
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Kaur, J., D. A. Ledward, R. W. Park, and R. L. Robson. 1998. Factors affecting
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Lee, B. H., S. Kermasha, and B. E. Baker. 1989. Thermal, ultrasonic and
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66(9):1642-1649.
CHAPTER 5
MECHANISM OF INACTIVATION OF E. coli AND L.
monocytogenes BY ULTRASOUND WAVES
94
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
95
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.
96
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.
97
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
98
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
99
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
100
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
101
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
102
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.
103
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.
104
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
105
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.
106
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.
107
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).
108
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
109
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
110
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
111
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).
112
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
113
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).
114
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
115
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