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Destruction of Escherichia coli O157:H7 in food and non-food systems by microwaves

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MICROFILMED 1996
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DESTRUCTION OF ESCHERICHIA COLI 0157:H7
IN FOOD AND NON-FOOD SYSTEMS BY MICROWAVES
A Thesis
Subm itted To T h e Faculty O f The G raduate School
O f The U niversity o f M innesota
By
Sophia Magdalen Czechowicz
In P artial F u lfillm e n t O f The R equirem snts
F o r The Degree O f
D octor O f Philosophy
Dr. E. A. Zottola, Advisor
July, 1996
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U N I V E R S I T Y OF M I N N E S O T A
This is to certify that I have examined this copy of a doctoral thesis by
50"PH1A
M A ttA L tN
CZECHOWIGZ
and have found that it is complete and satisfactory in all respects,
and that any and all revisions required by the final
examining committee have been made.
eDMlW JD A. Z OTTO L A
Name o f Faculty Adviser(s)
(LSigh> ___
0
GRADUATE SCHOOL
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ACKNOWLEDGEMENTS
I am sincerely thankful to many people who helped me in various way to finish
this project.
To my advisor, Dr. Zottola, fo r giving me the opportunity to work in his
laboratory.
I am grateful for his constant support, scientific freedom and sense of
humor.
To Dr. Hanson, Dr. Flickinger, and Dr. Busta for their interest in this project and
helpful suggestion.
To Dr. Schafer and Dr. Addis for serving on my committee.
To my husband Darek, my children, Agnieszka and Robert, and my parents-inlaw for their encouragement, love and dedication through my years in Graduate School.
To all my friends, for great support and friendship.
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ABSTRACT
The interactions between foods and microwaves result in uneven heating.
Therefore, it is difficult to predict traditional destruction parameters for bacteria in food
heated in the microwave oven. This research was developed to explore areas that
relate to microwave pasteurization of foods.
A consumer-sized microwave oven was modified to maintain a constant
temperature during the heating of liquid samples. This oven was used to determined
D-values for E. coli 0157.H7 at 57°C and 60°C.
These were compared to the
corresponding values obtained during conventional heating.
Statistical analysis
showed that there was no difference related to mode of heating if similar lethality rates
were delivered to the bacterial suspensions.
The D- and z-values were used to calculate pasteurization time for beef patties
inoculated with E. co li 0157:H7. With these data, F^bc was calculated and found to be
1 sec. Pasteurization was then defined to be a combination of the come-up time and
the hold time at this temperature.
Power levels in 5 different consumer microwave
ovens were determined and compared with the power levels in the modified oven. In
addition, thermographs that showed cold and hot spots in these ovens were developed.
Ground beef patties were microwave treated in each of the test ovens to achieve a
heat process equivalent to the selected F-value at 67.8°C.
Microbiological analysis
demonstrated that the calculated pasteurization times when achieved in the microwave
oven will destroy E. co li 0157:H7. Using this technique and knowing two major factors,
II
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power value and a thermograph o f the microwave oven, will allow the calculation of
equivalent heat processes fo r the destruction o f these bacteria in beef patties.
Microscopic examination of E. coli 0157:H7 cells obtained from microbial
colonies grown on solid agar surface and exposed to microwaves showed startling
results. Bacteria heated by microwaves and dry heat at 55°C revealed different cell
morphology. It appears from the TEM cross sections attained that microwave heated
bacteria showed damage occurring in the center of the cell as opposed to uneven
destruction of membrane or various areas within the cells that were conventionally
heated. Epifluorescence microscopy studies confirmed these results.
Ill
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS...................................................................................... I
ABSTRACT.......................................................................................................... II
TABLE OF CONTENTS......................................................................................IV
LIST OF FIGURES............................................................................................VIII
LIST OF TABLES................................................................................................XI
INTRODUCTION.................................................................................................. 1
LITERATURE REVIEW........................................................................................3
Microwave heating o f foods.................................................................................................. 4
Introduction........................................................................................................................... 5
Physical properties of microwave energy............................................................................... 5
Microwave heating at microscopic level.............................................................................6
Microwave - material interaction at molecular level............................................................ 7
Principles of the electromagnetic field (Maxwell's equation) and the laws of radiation
(Lambert’s law) as applied to predict heat distribution......................................................... 8
Rules that govern radiation............................................................................................ 8
Polarity of molecules................................................................................................... 11
Viscosity increases relaxation.......................................................................................11
Dielectric properties of mixtures.................................................................................... 12
Dielectric behavior of biological materials..................................................................... 13
Characteristics of the food system that relate to microwave thermoprocessing..................... 14
Dielectric properties of food - kj, k", penetration depth.......................................................15
Water content of foods and level of ions determine heating..............................................15
Geometry of sample........................................................................................................ 16
Physicothermal properties of the foods............................................................................. 17
Effect of environment surrounding heated foods.............................................................. 18
Microwave equipment determines distribution of electromagnetic field and uneven heating... 18
Conclusion.......................................................................................................................... 19
References......................................................................................................................... 20
Comparison o f the destruction of bacteria in foods during microwave heating and
conventional heating........................................................................................................... 27
Introduction......................................................................................................................... 28
Microwave heating versus conventional heating.................................................................. 28
IV
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Difference in mode of heating and distribution of temperature in processed samples........ 28
Modeling prediction of temperature distributions within a food during microwave heating
based on the dielectric properties of a food...................................................................... 30
Destruction of pathogenic and spoilage bacteria in foods by microwave heating................... 31
Microwave heating of meat products................................................................................31
Microwave cooking of other foods....................................................................................39
Microwave heating in food service systems...................................................................... 43
Interaction of microwaves with microorganisms................................................................... 44
Thermal and athermal effects of microwaves on distruction of bacteria.............................44
Kinetics of microbial destruction during microwave irradiation and conventional heating ....45
Heat resistance of bacteria heated at constant temperatures by both methods............... 45
Role of water during heating bacteria by microwaves.....................................................48
Biological effect of microwaves on microorganisms...........................................................49
Instrumentation influences progress in research on bacteria-microwave interactions..........50
Application of microwave technology and its future in food thermoprocessing....................... 51
Conclusion..........................................................................................................................53
References.........................................................................................................................53
EXPERIMENTAL PART..................................................................................... 70
Recovery o f thermally-stressed Escherichia coli 0157:H7 by media supplemented with
pyruvate................................................................................................................................71
Abstract..............................................................................................................................72
Introduction.........................................................................................................................73
Materials and Methods........................................................................................................ 75
Strain..............................................................................................................................75
Culture preparation.......................................................................................................... 75
Heating challenge........................................................................................................... 76
Recovery conditions........................................................................................................ 76
Statistical Analysis.......................................................................................................... 77
Results...............................................................................................................................77
Discussion..........................................................................................................................79
References.........................................................................................................................84
Inactivation rates of Escherichia coli 0157:H7 heated conventionally and by microwaves92
Abstract..............................................................................................................................93
Introduction.........................................................................................................................94
Materials and Methods........................................................................................................ 98
Bacterial strain................................................................................................................ 98
Microwave system maintaining constant temperature.......................................................99
Modification of microwave oven....................................................................................99
The temperature control loop...................................................................................... 100
Sample agitation loop.................................................................................................101
Temperature measuring system..................................................................................102
Sample holder and sample enclosure......................................................................... 102
Operation of the microwave system - setting proper parameters..................................... 103
Conventional heating in the water bath........................................................................... 104
Heating conditions......................................................................................................104
Lag correction factor of the Pyrex tubes...................................................................... 105
Heat inactivation studies during isothermal microwave heating and conventional heating 105
Lethality experiments.....................................................................................................107
V
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Evaluation of thermal destruction data - statistical analysis............................................. 108
Calculation of D-and z-values data............................................................................. 108
Regression analysis of survivor curve data.................................................................. 108
Statistical comparison of microwave and conventional heat treatments........................ 109
Results............................................................................................................................. 110
Maintaining a constant temperature in a microwave system............................................ 110
Water bath experiments.................................................................................................111
D and z-value data of E. coli 0157:H7 determined during microwave and conventional
heating.......................................................................................................................... 111
Statistical tests...............................................................................................................113
Lethality tests.................................................................................................................114
Discussion.........................................................................................................................116
Conclusions...................................................................................................................... 120
References....................................................................................................................... 120
Microwave pasteurization o f foods. Part A. A proposed method to determine
pasteurization time and testing its validation using ground beef patties as a model
system................................................................................................................................ 145
Abstract............................................................................................................................ 146
Introduction........................................................................................................................146
Materials and Methods.......................................................................................................149
Development of microwave pasteurization model............................................................ 149
Microwave heat destruction parameters for E. coli 0157:H7........................................ 149
Heat penetration studies.................................................................................................150
Microwave oven.........................................................................................................150
Beef patties................................................................................................................151
Temperature measuring system.................................................................................. 152
Heat penetration test...................................................................................................152
Microbiological studies...................................................................................................153
Test organisms...........................................................................................................153
Test organism - enumeration methods........................................................................154
Meat microflora...........................................................................................................155
Preparation of beef with test organism........................................................................ 155
Microwave pasteurization experiments - Inoculated pack studies..................................156
Results............................................................................................................................. 156
Calculation of pasteurization times during microwave heating - proposed method............ 156
Experimental microwave conditions Controlling and monitoring critical factors influencing
temperature distribution within microwave heated foods.................................................. 159
Thermal profiles.............................................................................................................161
Microbiological studies validating predicted pasteurization values................................... 163
Discussion........................................................................................................................ 163
Conclusion........................................................................................................................ 169
References....................................................................................................................... 170
Microwave pasteurization o f foods. PartB. Critical parameters of the process as
demonstrated during microwave pasteurization of beef patties fo r predicted
pasteurization time in various microwave ovens...............................................................190
Abstract............................................................................................................................ 191
Introduction....................................................................................................................... 192
Material and methods........................................................................................................194
Standard pasteurization value........................................................................................ 194
Microwave ovens used in study...................................................................................... 195
VI
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Power of microwave ovens............................................................................................195
Microwave pasteurization of patties - microbiological tests..............................................196
Thermographs of microwave oven.................................................................................197
Picture of meat patty in the oven cavity - location of the slowest heating zone within the
meat patty.....................................................................................................................197
Results............................................................................................................................ 198
Power of microwave ovens............................................................................................198
Temperature distribution - thermographs........................................................................ 198
Pictures of heated patties...............................................................................................199
Microbial studies correlated to the heating performance of the microwave ovens.............200
Readjusting of pasteurization time due to the power....................................................... 201
Discussion....................................................................................................................... 201
Conclusion....................................................................................................................... 206
References...................................................................................................................... 206
Effect of dry and microwave heating on the cell structure................................................218
Abstract........................................................................................................................... 219
Introduction...................................................................................................................... 219
Materials and Methods...................................................................................................... 221
Culture......................................................................................................................... 221
Heat treatment..............................................................................................................222
Microwave heating..................................................................................................... 222
Dry heat treatment..................................................................................................... 223
Microscopic examination of structural changes in heat treated cells................................223
Light microscopy........................................................................................................ 223
Transmission electron microscopy studies..................................................................224
Epifluorescence microscopy...................................................................................... 224
Results and discussion..................................................................................................... 226
Conclusions..................................................................................................................... 229
References...................................................................................................................... 230
CONCLUSIONS...............................................................................................239
VII
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LIST OF FIGURES
Recovery of thermally-stressed Escherichia coli 0157:H7 by media supplemented with
pyruvate................................................................................................................................71
Inactivation rates of Escherichia coli 0157:H7 heated conventionally and by microwaves92
Figure 1. Microwave system for providing constant temperature during microbial inactivation
studies.................................................................................................................................. 128
Figure. 2. Typical and modified microwave oven power supply circuits..................................129
Figure 3. Modified microwave oven for providing constant temperature during microbial
inactivation studies.............................................................................................................. 130
Figure 4. Cavity of modified microwave oven with sample and sample holder....................... 131
Figure 5. Thermal profiles of 20 mL water heated in A) Quasar microwave oven; B) modified
Quasar oven - temperature set up at 55°C and variac operated at 100% setting; C) modified
Quasar oven - temperature set up at 55°C and variac operated at 70% setting....................... 132
Figure 6. Outline of the procedure used to calculate lag correction factor for tubes used in
microbial thermal resistance studies...................................................................................... 133
Figure 7. Survivor curves for E. coli 0157:H7 in phosphate buffer heated at 57°C conventionally
( • - water bath) and by microwaves (■ - isothermal microwave system). Survivors were
recovered on Tryptic Soy Agar ( — ) spread plates and Plate Count agar with 1% sodium
pyruvate ( — ) spread plates. Three replicate experiments a, b, and c were performed
135
Figure 8. Survivor curves for E. coli 0157:H7 in phosphate buffer heated at 60°C conventionally
(• - water bath) and by microwaves (■ - isothermal microwave system). Survivors were
recovered on Tryptic Soy Agar ( — ) spread plates and Plate Count agar with 1% sodium
pyruvate ( — ) spread plates. Three replicate experiments a, b, and c were performed
136
Figure 9. Survivor curves for E. coli 0157:H7 in phosphate buffer heated at 60°C conventionally
(• * water bath) and by microwaves (■ • isothermal microwave system). Survivors were
recovered on Tryptic Soy Agar ( — ) spread plates and Plate Count agar with 1% sodium
pyruvate ( — ) spread plates. Three replicate experiments d, e, and f were performed
137
Figure 10. Thermal resistance curves of E. coli 0157:H7 heated conventionally and by
microwaves and enumerated using two different media Tryptic Soy Agar (TSA) and Plate Count
Agar with 1% sodium pyruvate (PCA + 1% NaPyr).................................................................139
Figure 11. Comparison of lethality delivered to E. coli 0157.H7 suspensions and number of
survivors recovered after heating samples at 60oC by microwaves (•) and in water bath (■).
Lethality ( j l - microwave, | - water bath) was calculated from time-temperature data by
VIII
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General Method. Two experiments were conducted - LRa and LRb. Numbers of survivors are
average values obtained from two replicate samples that were used per each heating tim e.... 142
Figure 12. Representative time-temperature profiles recorded during the survivor curve
experiments. E. coli 0157:H7 suspensions in phosphate buffer were heated: A) by microwaves
(isothermal microwave system, vials agitated), and B) conventionally (water bath; vials without
agitation). Two replicate vials (a, b) were heated for 1 min (heating time interval) during each
treatment at 60°C. Water bath heating intervals were 2 min longer because vials possessed 2min lag correction factors...................................................................................................... 144
Microwave pasteurization of foods. Part A. A proposed method to determine
pasteurization time and testing its validation using ground beef patties as a model
system................................................................................................................................145
Figure 1. Geometry of beef patties heated by microwaves. Temperature probe positions in
round and doughnut-shaped meat patties during heat penetration tests...................................182
Figure 2. Location of Luxtron temperature probes within the meat pattydetermined by
calculation of penetration depth of microwaves penetrating the meat......................................183
Figure 3. Location of beef patty in MW oven cavity.............................................................. 184
Figure 4. Flow diagram of the procedure used to determine pasteurization time for beef patties
inoculated with E. coli 0157:H7 microwave heated in standard microwave oven.....................185
Figure 5.
Formula used for calculation of end-point temperature during microwave
pasteurization of beef patties................................................................................................ 186
Figure 6. Heat penetration profiles of beef patty heated by microwaves at high power level.
Location of probes is described in the Figure 1. Pasteurization time was determined when the
lowest temperature within the meat reached minimum 67.8°C................................................187
Figure 7. Example of temperature values within round-shaped beef patties exposed to high MW
power during various trials to determine proper pasteurization time. Location of probes within
meat patty was described in Figure 1. Trial and error' approach was used to determine
pasteurization time . Pasteurization time was defined as a microwave heating time when the
lowest temperature in 30 consecutively heated beef patties was minimum or above 67.8°C.... 188
Figure 8. Influence of initial temperature of beef on the temperature development in round­
shaped beef patties fully pasteurized after 2 min 30 sec of microwave heating at high power
level. Location of probes within meat samples is shown in Figure 1....................................... 189
Microwave pasteurization of foods. PartB. Critical parameters of the process as
demonstrated during microwave pasteurization of beef patties for predicted
pasteurization time in various microwave ovens...............................................................190
Figure 1. Thermographs showing cold spots (light colors)and hotspots (darkcolors) on the
bottom of the cavity of microwave ovens used in this study.Thermal paper wasexposed to
microwaves for 5min at the high power setting...................................................................... 216
IX
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Figure 2. Distribution of cold and hot spots in microwave heated meat patties due to its location
in the oven cavity. A cold spot is located in the center of a hamburger when it is heated in the
center of the quasar microwave oven for A) 60 %, B)73%, and C) 87% of the total cooking time
respectively. The position of a cold spot in a hamburger changes when its heated D) on the left
side, E) in the front, and F) on the right side of the oven for 60% of the total cooking time
217
Effect of dry and microwave heating on the cell structure................................................218
Figure 1. Protocol of experiments designed to show the structure of £. coli 0157:H7 cell matrix
after heating by microwaves and dry heat at constant temperature conditions........................ 233
Figure 2. Morphology of E. coli 0157:H7 stained with crystal violet. Cell matrix was distributed
on the internal wall of the micro tube and then exposed to microwaves at 55°C. Arrows show
concentration of the stain at the both ends of the cells suggesting uneven distribution of
intracellular material in these microwave heated cells........................................................... 234
Figure 3. Cross section of E. coli 0157:H cells heated at 55°C for 10 min. Cell matix from a
single colony was distributed on the wall of micro tube and exposed to microwaves and dry heat.
TEM pictures show A) conventionally heated cells, B) microwave heated cells, and C) unheated
cells - control....................................................................................................................... 235
Figure 4. Thermal profiles of E. coli 0157:H7 cell matrix heated at 55°C by microwaves and dry
heat and used in A) TEM and B) epifluorescence microscopy studies.....................................236
Figure 5. Permeability of membranes of E. coli 0157:H7 heated by A) microwaves and B) dry
heat. Number of viable Ontact membranes - green) and nonviable (permeable fluorescent stains
- red) cells was determined with LIVE/DEAD® BacLight™ Bacterial Viability Kit (Molecular
Probes, Inc.)........................................................................................................................ 237
X
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LIST OF TABLES
Microwave heating of foods..................................................................................................4
Table 1. Comparison of plating methods. Population of E. coli 0157:H7 heated at 57° C and
recovered on Tryptic Soy Agar by spread and plate methods. Statistical analysis of results* 89
Table 2. Population of E. coli 0157:H7 after heating at S7°C and recovered on various media by
spread methods. Statistical analysis of results*........................................................................90
Table 3. Percent of enumerated E. coli 0157:H7 on media, unheated and heated at 57°C.
Numbers log N were compared to the population recovered on the most effective media - Plate
count agar+ 1% sodium pyruvate...........................................................................................91
Inactivation rates of Escherichia coli 0157:H7 heated conventionally and by microwaves92
Table 1. Calculation of the lag correction factor for vials used in E. coli 0157:H7 thermal
resistance studies. Screw-capped Pyrex tubes (10 x 130 mm) filled with 5-mL bacterial
suspension were heated in water bath at 57°C....................................................................... 134
Table 2. D-values and regression values for E. coli 0157:H7 heated by microwaves and in water
bath..................................................................................................................................... 138
Table 3. Comparison of the D- and z-values for E. coli 0157:H7 heated conventionally (water
bath) and by microwaves (isothermal microwave system) and enumerated on Tryptic Soy Agar
(TSA) and Plate Count Agar with 1% sodium pyruvate (PCA +1% NaPyr) spread plates
140
Table 4. O-values and 95% confidence interval for D-values determined during survivor curve
experiments for E. coli 0157:H7 heated by microwaves and in water bath..............................141
Table 5. Lethality delivered to E. co/i 0157:H7 suspensions during survivor curve experiments
earned out in microwave oven and water bath at 60°C. Temperature was monitored in each
heated vial. The lethality was calculated from the time-temperature data using General Method
(Tref = 60°C , z = 2.3°C). Two replicate vials (a, b) with E. coff 0157-.H7 in phosphate buffer were
heated per each time interval. Plate count agar with 1% sodium pyruvate was used as a
recovery media. Statistical difference between microwave and water bath treatment was
determined for each experiment............................................................................................143
Microwave pasteurization of foods. Part A. A proposed method to determine
pasteurization time and testing its validation using ground beef patties as a model
system..........................................................
145
Table 1. Microbial destruction kinetics of E. co/i 0157:H7 obtained in modified microwave oven
maintaining constant temperature......................................................................................... 178
Table 2. Calculation of the end-point temperature................................................................ 179
XI
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Table 3. Pasteurization time of beef patties determined from thermal profiles studies using
Quasar microwave oven. Pasteurization time is the cooking time to reach the 67.8°C
temperature in the coldest spot within the patty as found by trial and error approach. A total
number of 376 patties were used........................................................................................... 180
Table 4.
Survival of £. coli 0157.H7 and other mesophilic bacteria in beef patties after
microwave pasteurization in Quasar microwave oven. Initial average population of £. coli
0157:H7 was 1.2 x 104 CFU/g. Beef contained 3.8 x 104 CFU/g of indigenous bacteria
enumerated using Anaerobic Plate Count method.................................................................. 181
Microwave pasteurization of foods. PartB. Critical parameters of the process as
demonstrated during microwave pasteurization of beef patties for predicted
pasteurization time in various microwave ovens...............................................................190
Table 1. Types of consumer microwave ovens operating at 2450 MHz frequency used in
studies..................................................................................................................................211
Table 2. Power of MW ovens used in studies........................................................................ 212
Table 3. Survival of E. coli 0157:H7 and other microorganisms present in beef patties during
microwave pasteurization. Round and doughnut-shaped beef patties were processed in 6
different microwave ovens operating at high and medium power settings. Initial population of E.
coli 0157:H7 in all samples were 1.2 x 104 CFU/g and other microorganisms enumerated by
APC method 3.8 x 104 CFU/g................................................................................................213
Table 4. Recalculation of microwave heating time determined in standard oven (Quasar) for
microwave oven operating at different power level (Litton Industrial) to obtain equal
pasteurization values............................................................................................................ 214
Table 5. Microwave heating of foods - major factors that determined temperature level and
distribution in foods during microwave heating....................................................................... 215
Effect of dry and microwave heating on the cell structure................................................218
Table 1. Survival of £. coli 0157:H7 cell matrix heated by microwaves and dry heat in open
micro tubes at 55°C. Per cent of cells that survived treatment were determined with
epifluorescence microscopy using LIVE/DEAD® BacLight™ Bacterial Viability Kit....................238
XII
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INTRODUCTION
Application of microwave power in various science and engineering disciplines
is steadily growing. Microwave food processing that utilizes heat produced during
microwave irradiation is used with varying degree of success. Since the early 1940s,
the time of its initial discovery, attempts to utilize microwave technology in many
industrial systems such as drying, pasteurizing, tempering, baking have been met with
minimal success. An opposite trend occurs in the sale o f microwave oven models to
consumers. Microwave ovens have become a very successful product and are present
in 90% of U.S. households. Microwavable foods also have gained popularity, although
developers encountered difficulties sim ilar to those experienced by designers o f
microwave industrial systems. The number of new microwavable food products has
been decreasing since its peak in 1990; from 10% of all new product introductions in
1990 to 3% in 1993. The major problem is the uneven temperature distribution within
the heated foods that results from the varied characteristics of food components and
the uneven electric field formed within the microwave oven. Microbiological safety and
the quality of the food is unpredictable since it is difficult to determine time-temperature
profiles during microwave heating of products.
There is a significant number of reports on the destruction o f various bacteria in
food and nonfood systems during microwave heating.
Limited knowledge on the
mechanisms of microwaves-foods-bacteria interactions, microwave heating, lack of
microwave equipment that will uniformly heat a microbial sample at a desired
1
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temperature, and lack o f technology to continuously measure the temperature during
the heating process are major factors that contributed to unreliable results.
These factors and the difficulties associated with applying methods developed
for conventional thermoprocessing to calculate pasteurization or sterilization time during
microwave heating led to the intensification o f research on differences in the
mechanisms of heat formation during both processes.
2
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LITERATURE REVIEW
3
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Microwave heating of foods
4
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Introduction
Microwave heating can be a very attractive method o f thermoprocessing. Its'
advantage is a short come up time to reach the desire cooking temperature or high
temperature. The ability to achieve higher temperature inside the food rather then on
the surface can be a desirable attribute in the heating of some food systems. The
difficulty in use microwave heating as an alternative form o f thermoprocessing on a
wide scale is associated with a lack o f understanding of physical and engineering
aspects of this process. The objective of this review is to explain and summarize the
physical parameters and physical events occurring during interaction o f electromagnetic
field of microwaves with biological entities present in the food system.
Physical properties of microwave energy
Microwave heating is the heating o f a substance by electromagnetic energy
operating in the frequency range of 300 MHz to 300 GHz as defined by Risman (1991)
based on terminology published by International Electrotechnical Commission (IEC).
Materials that contain polar molecules, including biological samples, are heated when
exposed to microwaves.
Mechanism o f microwave heating is determined by
physicochemical properties of exposed materials and physical characteristics of
microwaves.
Microwaves are electromagnetic waves located in the electromagnetic spectrum
between infrared light and radiofrequencies.
The explanation fo r electromagnetic
waves phenomenon and the equations that govern it was given by Maxwell (1873), and
5
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is known as a electromagnetic wave theory (Thuery, 1992; Cumutte,
Accordingly,
electromagnetic waves propagate at different speeds
1980).
in various
environments and consist of electric and magnetic fields which are perpendicular to
each other. Magnetic and electric energy o f equal value is associated with each field.
In the microwave heating of foods, the alternating electric field is responsible for
heating.
The effect of the magnetic field on food has been ignored since food
molecules do not have magnetic properties, e. g., are not ferromagnetics (Cumutte,
1980; Buffier.1992). The current interest in research on the use of a magnetic field to
preserve foods should prove that oscillating magnetic fields at microwave frequency
does not affect bacteria (Pothakamury et al., 1993). The microwave frequencies of
2450 MHz are applied in the consumer microwave oven and some industrial microwave
systems; the latter works often at 915 MHz frequencies. The commonly used 2450
MHz frequency means that the electric field alternates a t 2.45 billion times per second.
Microwave heating at microscopic level
Charged and polar particles present in the microwave field attempt to align in it
and thus will be forced to oscillate at a 2.45 billion times per second rate to follow
charge rotation of the electric field.
mainly water and ions of salts)
Polar molecules and ions (in biological systems
during physical agitation collide with surrounding
particles. Adjacent particles expend their kinetic energy, agitate more intensely and
their temperature increases.
Following that, the collision of molecules with higher
energy with those o f lower energy occurs. Energy between molecules is exchanged as
during conventional heating (Buffler, 1992). Thus, mechanisms of microwave heating
consist o f two events; electromagnetic heating and heat transfer. For comparison of
6
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terms, in conventional heating the heat transfer progresses when molecules at a lower
temperature (lower energy) are brought to a higher energy level through collision with
other molecules (Mertens & Knorr, 1992). External temperature difference is a driving
force of conventional heating.
Microwave - m aterial interaction a t molecular level
The velocity and wavelength of electromagnetic waves traveling through a given
material depend on its electric and magnetic properties. At 2450 MHz frequency, the
wavelength can vary from 12.2 cm in the vacuum to 1.41 cm in water (Buffler, 1992).
Properties of electromagnetic radiation can be described in terms of wave theory and
corpuscular (particle-like) theory.
The corpuscular theory assumes that quantum
energy associated with electromagnetic waves is related to the frequency of radiation
as defined by the Planck's law (Cumutte, 1980). The energy of microwaves at 2450
MHz is approximately in the range of 1CT4 eV (Cleary, 1977). To cause changes within
the structure of any molecule, the quantum energy delivered has to be greater than the
activation energy of molecular bonds. The quantum of microwave energy is too low to
disrupt strong molecular bonds (covalent or ionization), weak hydrogen bonds which
require an activation energy of 0.08 to 0.2 eV, or even 2 eV-Van der Waals bonds
(Stuchly, 1978; Thuery, 1992).
Therefore, the direct effect of microwaves at the
molecular level have to be excluded. Cleary (1977) presents interesting descriptions of
forces imposed on biomolecules by the electric field and discusses possible
destabilization or destruction of macromolecular complexes irradiated by microwaves.
7
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Principles o f the electromagnetic field (Maxwell's equation) and the laws of
radiation (Lambert's law) as applied to predict heat distribution
Rules that govern radiation
Interactions of microwaves with materials is governed by the law of optics
(Copson, 1962; Lin, 1978; Mudgett, 1982; Buffler, 1992). Microwaves that impinge on
the surface are reflected, those that enter material are refracted and transmitted, and
some o f the microwave energy is absorbed by molecules which partially convert it to
heat. Dielectric properties of materials determine which event prevails. Understanding
of the aspects of reflection, absorption, refraction and transmission is essential in
designing microwave equipment, microwavable foods and establishing correct models
for predicting temperature distribution in heated samples. Particularly, reflection of the
microwaves becomes important to interpret uneven heat distribution. It may result in
formation o f standing waves and local overheating due to reflected waves occurring at
the interfaces in non-homogenous systems (e. g., oil-water emulsion) or the cylinder
shape o f heated samples. Lambert's law is used in models calculating the electric field
distribution and the heating pattern. It predicts an exponential decay of radiation with
increasing depth into the sample. This relationship is expressed by following equation:
P(z) = P0 e '2a
(1)
where P(z) - power as a function o f distance z into the sample, a - attenuation factor
(inverse of penetration depth), P0 - power transmitted into the sample.
8
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A modified form o f the Lambert's law, the Beer-Lambert equation describes the
optical transmission of waves and is applied to estimate intensity o f irradiation.
For
example, it is used in spectrophotometry to predict optical transmission o f UV or
infrared light.
Dielectric values and other terms assist in predicting heating o f materials. The
release of heat from material during microwave irradiation is caused by intermolecular
friction of polar molecules possessing high dipole moment.
polar characteristics are called dielectrics.
Materials that possess
They absorb microwave energy as
compared to insulators (e.g., Teflon) that mostly transmit waves or conductors (e.g.,
metals) which reflect microwaves.
Knowledge of dielectric properties values is
essential to the design of foods that will be heated uniformly during microwave
processing. Two major dielectric values describe interaction of foods with microwaves:
complex permittivity (x) and complex permeability (ft).
Complex permittivity (x) consists of two values: dielectric constant ( x ' ) and
dielectric loss ( x " ). The dielectric loss is the imaginary part of complex permittivity.
x = x '+ ix "
(2)
The x* and x" are the basic dielectric values that have practical meaning.
Dielectric constant, x', characterizes material ability to store electrical energy and
determines how much of the electrical energy w ill be absorbed by molecules, whereas
the remaining part of incident microwaves w ill be reflected and not participate in the
heating process. The second value, x", dielectric loss, describes material capacity to
thermalize
absorbed
energy and
measures
the
material's
ability
to
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convert
electromagnetic energy to heat. Metaxas and Meredith (1983) called it the effective
loss factor because it includes the effect of conductivity. Values of dielectric properties
change with temperature and frequency. In a sim ilar way, complex permeability (j i )
describes the magnetic properties of materials.
The magnetic values for biological
samples are equal to those in vacuum, confirming that absorbance of magnetic energy
in biological systems does not occur.
The other parameters important to predict heating profiles include: loss tangent,
penetration depth, thermal properties (thermal conductivity, heat capacity), and the
electric field distribution within the sample.
Loss tangent (tg 5 ), called dissipation
factor, expresses the ratio o f dielectric loss ( k " ) to the dielectric constant ( v * ) and
represents the energy loss.
Penetration depth is the depth within the sample at which microwave power has
decreased to 1/e or 36.8% of the power that entered the sample.
If the values of
dielectric constant x'a n d dielectric loss K'are known, the penetration depth (Rj) can be
calculated (Mudgett, 1986b; Datta, 1990).
_____________
Ri "
7Z {2 k ' ) 1/2 {[1 + ( K " / f C ' ) : I 1'2 - 1 }1,2
(3)
where wavelength ( Xo) in free space at 2450 MHz equals 12.24 cm.
The knowledge o f thermal properties is essential to determine the material's
ability to distribute the heat released by rotating polar molecules by convectionconduction mode. The dielectric properties values change when the temperature of the
10
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material and microwave frequency change. The electric field allows us to calculate the
power absorption according to the equation:
Pv = 5.56 x 104 x f £" E2
(5)
where Pv - power absorbed per unit volume (W/cm3), f - frequency, s" - relative
loss factor and E - electric field (V/cm) (Goldblith, 1966).
Polarity of molecules
Materials can be effected by electric field if they contain charged molecules
such as permanent dipoles (e.g., water molecule) or ions (e.g., Na+, C l - ). In the static
electric field they will undergo either dipole redistribution or charge redistribution
(Metaxas & Meredith, 1983).
Molecules that consist o f permanent dipoles resulting
from asymmetric charge distribution are known as a polar dielectric. In the alternating
electric field of low frequency, polar molecules are able to follow charge changes of the
field and the heat is not released. At higher microwave frequencies, the rotation of
dipoles can not keep up with the changes of the field and lags behind the applied field.
The relaxation of the polar molecules occurs and heat dissipates into the environment.
Dielectric loss value increases when relaxation increases.
Viscosity increases relaxation
Relaxation (lag time) relates to frictional forces in the medium according to
Debye's theory (Metaxas & Meredith, 1983).
In turn, high frictional forces are
associated with the viscosity of samples. In an electric field, charged polar molecules
can experience high frictional forces due to the high frictional coefficient of the
11
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molecules. This proportional relationship between the frictional force and the frictional
coefficient is expressed by Stoke's law (Alberty, 1987).
Generally, the greater the
viscosity of the sample, the larger the frictional forces and the greatest relaxation. The
frictional factor and viscosity relate to the size and the shape of molecules, and to the
strength o f bonds between rotating polar molecule and neighboring molecule layers.
The importance of the above factors on the dielectric properties o f materials has been
recognized. Modification to the Deby's theory has been presented (Blythe, 1979). The
equation which calculates the dipolar moment was improved by adding a parameter
that accounts for the intermolecular interactions (Thuery, 1992).
These theoretical
terms have become important to understand the difference in behavior of the bound
versus mobile water, or water in the gaseous state during microwave heating. Water
mobility is a technique-dependent term. The mobility of water can be characterized by
the dielectric properties o f the system but measurements are different when determined
by ESR and NMR as reported by Tsoubeli (1994).
Rotations of ion salts and many
other polar molecules such as proteins or hydrocolloids are limited by their
intermolecular bounds and the viscosity of the system (Decareau, 1986).
Dielectric properties of mixtures
Aqueous solutions of dissolved salts have different dielectric properties than
that o f pure water (Mudgett et al., 1971; Mudgett 1986a, 1986b, 1990).
adverse factors account for these changes.
alternating
electric field which
Two major
First is the migration o f ions in an
increases dielectric properties.
However, this
electrophoretic migration depends on how freely ions can move in the electric field.
The second factor, which lowers the dielectric constant as compared to pure water, is
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the restrained rotation o f water molecules. W ater molecules are bound to ions through
hydrophilic interaction (electrostatic forces) and their movement is restricted (Mudgett,
1990; Decareau, 1985).
Dielectric properties of water are greatly modified by ions
which is particularly significant for dielectric properties o f biological samples including
bacteria and various food materials. Dielectric properties of aqueous mixtures consist
of components that do not interact with each other and these values can be predicted
using established models such the Hasted-Debye model or the Fricke model as
described by Mudgett (Decareau, 1985). Mixtures with strongly interacting components
possess dielectric values different from predicted values, therefore they can only be
established experimentally.
Strong hydrogen bonding between hydroxyl groups of
alcohols or sugars and water molecules affect the relaxation time of mixtures (Copson,
1962; Mudgett, 1986a, 1986b). These interactions explain unique properties o f some
ingredients during microwave cooking.
Starches, honey and other hydrocolloids
possess moisture retention properties and are used to replace ingredients that turn
tough and rubbery when microwaved (Zylema et al., 1985; Kevin, 1994).
Dielectric behavior of biological materials
Dielectric properties of simple biological structures such as amino acids and
small proteins were studied over a range o f frequencies and summarized by Grant et
al., (1978). In the frequency near 1 GHz, the level of hydration of molecules accounted
for major differences of dielectric values. The water molecules that are unable to rotate
at frequencies near 1GHz were defined as bound water. Amino acids have significantly
higher relaxation frequencies than proteins. On the contrary, Thuery (1992) claimed
that in the aqueous solution of proteins, the protein molecules do not contribute to
13
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permittivity o f the sample regardless of the microwave frequency. The protein molecule
with its layer of bound water is too large for its dipole alignm ent Reports are available
on the study of the behavior of large-structure macromolecules o f proteins, lipoproteins,
DNA, viruses, cells and membranes in an electric field a t microwave frequency but the
results are highly controversial (Grant et al., 1978; Thuery, 1992; Baranski & Czerski,
1976). The influence of size, shape, and hydration o f biological macromolecules on
their dielectric properties was underlined in the initial research. Interestingly, dielectric
properties of bacteria were studied and found to be related to the cytofogical structure
of the bacterial cells (Marquis & Carstensen, 1973). The effect o f the reflection of the
microwaves in heterogeneous biological systems and foods was recognized as a major
factor contributing
to
uneven
heating
(Lin,
1978;
Stuchly
&
Hamid,
1972).
Understanding the complex interaction becomes critical in order to predict the amount
of thermal energy released from foods during microwave processing.
Characteristics of the food system that relate to microwave
thermoprocessing
The progress in application of microwave heating is related to the enormous
amount of knowledge accumulated from practical observations by food product
developers and engineers designing microwave processing equipment.
Transfer of
microwave irradiation principles led to application of theoretical terms to microwave
cooking.
Dielectric properties o f ingredients became basic parameters used in
exploring the microwave capacity o f many foods (Kent, 1994).
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Dielectric properties of food- k ',
a t"
penetration depth
The dielectric properties are essential values applied in microwave technology.
They determine the functionality o f any food ingredient used to formulate microwavable
foods and affect process time to achieve desired textural quality and microbiological
safety.
The importance of dielectric values of foods in microwave processing was
stressed in early research by several workers (Risman & Ohlsson , 1973; Nelson,
1973).
Dielectric properties have been established fo r many foods (Kent, 1987;
Bengtsson & Risman, 1971; Tsoubeli, 1994) but more studies are needed in this area.
Dielectric properties change dramatically with frequency, temperature, density o f food,
water content, salt content and state (frozen vs. liquid), and these data are particularly
needed to design microwavable foods.
Heterogeneous systems may have unusual
values, and their kf and k " cannot be predicted based on composition. The example is
a milk system where dielectric properties cannot be predicted by applying Debye model
(Mudgett et al., 1971).
Availability of proper technology to measure the dielectric
properties becomes critical (Barringer, 1994; Engelder & Buffler, 1991).
Water content of foods and level of ions determine heating
W ater is a major polar molecule in food systems. The mobility of water in the
structure of material rather than the total amount o f water affects the dielectric
properties of foods. Mobile water includes free water and water that may be partially
kinetically constrained as opposed to bound water. Although there is a lack of strict
definitions of these terms, generally mobile water is related to the water activity
(Engelder & Buffler, 1991).
Thus, mobile water affects growth of microorganisms.
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Dielectric properties can relate to the moisture content of foods. They may reflect the
interactions between molecules or monitor the alteration in the system as a result o f
denaturation, gellation, freezing and other processing changes. Tsoubeli (1994) and
Miller et al. (1991) reported that binding o f water by proteins, starches and
carbohydrates and the interactions between them as they occur during baking or
cooking resulted in changes of dielectric properties. Nelson et al. (1991) reported that
permittivities (dielectric constant and dielectric loss) of starch and other hydrocolloids
such as carrageenan increased with increased moisture content and temperature
Geometry of sample
Lack of uniform heating is dictated mostly by reflections and refractions at the
boundary between dissimilar materials such as foods and air. Microwaves that entered
food can be reflected from surfaces, scattered or concentrated inside the sample that
may result in formation o f cold and hot spots within the sample. For a food of regular
shape, the focusing of heating in the center can be predicted by calculating the
penetration depth. The temperature map o f irregularly shaped foods can be completely
unexpected, and the slowest heating point is not located at the center as in
conventional heating. This is a common mistake occurring in the reports on microbial
lethality o f microwave heated foods.
Ohlsson and Risman (1978) showed heating
concentration in the central parts of cylinder and spheres.
They demonstrated,
experimentally using infrared thermography and mathematically by calculation of
electric field based on dielectric properties o f foods, the relation between the diameter
of food sample and the center focusing o f microwaves.
pronounced in the sphere than in the cylinder.
The effect was more
Foods cooked in the cylindrical
16
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containers had a center temperature above the boiling point, whereas sides were
undercooked. Center focusing of microwaves in the smaller diameter containers (11
cm vs. 17cm) with oil and water was reported by Prosetya and Datta (1991).
Additionally, standing wave pattern may develop as was shown by Ohlsson (1990) in
the slabs of meat.
Prosetya and Datta (1991) demonstrated formation of standing
waves in liquids heated in cylindrical containers o f specific diameter.
The standing
waves are responsible fo r a high heating rate in the specific position (anti-nodes) and
lack o f heat at other locations (nodes). This explains several problems encountered
during microwaving of food such as bubbling in the center of liquids (soups, water,
coffee) and formation of steam leading to explosion in solid foods (eggs, potatoes)
(Schiffmann, 1992b).
To avoid center focusing the penetration depth must be
calculated.
The edge-heating effect is another factor that accounts for uneven power
density patterns particularly in large loads.
It is caused by scattering waves a t the
comers and along the edges and boundary between air and dielectric food component
(Ohlsson, 1990).
Physicathermal properties of the foods
Radiation energy that is delivered to a solid food sample is dissipated through
heat conduction.
Unsteady-state heat conduction process takes place until the
temperature equilibrates. The rate of heat transfer via conduction is determined by the
thermal properties of materials, heat capacity, thermal conductivity, and heat transfer
17
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coefficient. Two mechanical properties, density and viscosity, are equally important
(Metaxas & Meredith, 1983; Buffler & Stanford, 1991; Barringer, 1994).
Effect of environment surrounding heated foods
Unlike conventional heating, the temperature in the cavity o f the microwave
oven is lower than at the surface of the heated food.
The heat and mass transfer
occurs not only within the sample but also between the surface and outside. Surface
convection depends on the air temperature and velocity (Datta, 1990).
Moisture
gradient between the sample and the air causes evaporation of w ater from the surface
of the sample.
Evaporative losses can be prevented by heating foods wrapped or
packaged in the containers.
Radiative heat transfer occurs in the enclosed sample.
Loss of heat to the walls and the bottom of the oven cavity is observed. Evaporation is
responsible fo r low temperature and survival of bacteria on the surface o f microwave
heated foods. Lentz (1980) demonstrated that evaporative losses are accounted for
lower microwave power uptake from an 1% saline solution load than an equal water
load.
Microwave equipment determines distribution of electromagnetic field and
uneven heating
Electromagnetic waves emitted by magnetron and delivered to the oven cavity
are reflected when they impinge on the walls of the cavity.
This secondary
electromagnetic field interferes with the primary field and creates a complicated pattern
of various density fields. Scattering of electromagnetic waves leads to the formation of
a unique electrical field. Each microwave oven has a cold and a hot spot within the
18
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cavity that adds to uneven heat distribution within the sample. The role of multiport
feeds, stirrers and turntables installed in consumer microwave oven is to improve
uniformity of electrical field (Risman et al., 1987). To minimize the effect o f reflected
waves and in order to heat the sample in a uniform electromagnetic field, the
transmission line method is utilized in some studies. It involves placing the sample in a
waveguide, a rectangular tube of the conducting metal (Hung, 1980).
Diversity in
models of microwave ovens (output power, cavity size, with or without turntable) makes
a microwave cooking oven specific (Geriing, 1987). Microwave power delivered to the
cavity varies depending on the methods of its determination. Therefore, the power
output of a particular oven model may vary due to the method selected by
manufacturer.
Recently, a standard method that determines power output was
proposed (Buffler, 1992). The power o f the ovens used in research should be retested
by applying standard procedures before carrying out any experiments.
Conclusions
Microwave thermoprocessing of foods is a complex phenomenon.
The
mechanism o f microwave heating at the macroscopic level consists of electromagnetic
heating and heat transfer within the food.
Electromagnetic heating results from the
inability of polar molecules to rotate at the frequencies dictated by the alternating
electric field. Heat transfer under these conditions are well known physical concepts.
However, application of these concepts to explain and predict a temperature
distribution within heated food systems is very difficult.
The rate of microwave heating is determined by many factors including:
19
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• physical properties o f microwaves (frequency and power of the electric field
and the radiation of the microwaves - reflection, refraction, transmission and
absorption)
•
heated
properties of foods (dielectric properties ■ water, ion content; geometry o f
sample,
sample
size,
initial
temperature,
mechanical
and
thermal
characteristics, packaging)
•
microwave system (geometry and power o f MW oven, environment in the
microwave oven cavity - temperature and humidity).
It is essential to understand and incorporate these factors into a microwave
process for pasteurization or cooking o f designing food products during production o f
such foods.
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Biotechnol. Progress. 9:481-487.
Welt, B. A., C. H. Tong and J. L. Rossen. 1992. An apparatus for providing
constant and homogeneous temperatures in low viscosity liquids during microwave
heating. Microwave World. 13:9-13.
Zylema, B. J., J. A. Grider, J. Gordon and E. A. Davis.
1985. Model wheat
starch systems heated by microwave irradiation and conduction with equalized heating
times. Cereal Chem. 62:47-45.
26
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Comparison of the destruction of bacteria in foods during
microwave heating and conventional heating
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Introduction
Attempts have been made to use microwave thermoprocessing to pasteurize or
sterilize biological systems including foods.
The difficulty in doing this is related to
predicting temperature distribution during the exposure o f food to microwaves.
Deficiencies in instrumentation and lack of adequate methodology to predict
pasteurization times has slowed down the application of microwave technology for
these purposes.
This review summarizes research that has been reported on the application of
microwaves to inactivate microorganisms present in foods.
Studies that aimed to
elucidate which microwave heating parameters are critical to determine microwave
pasteurization or sterilization times are presented. Progress in development of proper
methodology for microwave thermoprocessing as they relate to the advances in
mathematical modeling and instrumentation is discussed.
Reports o f research on
interaction of microwaves with microorganisms have been reviewed.
Microwave heating versus conventional heating
Difference in mode of heating and distribution of temperature in processed
samples
Short come-up time and non-uniformity of heating are two major factors that
make microwave heating different from conventional thermoprocessing.
Difficulty in
identifying the coldest area within microwaved does not permit to calculate heat
processing time in a simple manner (Datta and Hu, 1992). Mathematical modeling and
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experimental setup have been used to determine the coolest temperature necessary to
estimate the slowest inactivation rate of bacteria. Datta et al. (1992) showed that in
cylindrical containers containing 500 mL o f water, the slowest heating point was located
at the bottom o f the sample.
Stratification of axial temperatures occurred, whereas
transient temperature profiles were almost linear.
The difference in mass and heat
transfer modes are the major factors affecting the quality and the temperature
distribution in foods heated conventionally and by microwaves.
Mass transport in
porous materials heated by convection is capillary-driven compared to temperaturedriven that occurs in microwave heating (Wei et al., 1985).
Several studies showed the extensive variability of temperatures in food
products heated by microwaves. Temperature increased with the increase of time and
microwave power. The location of the slowest heating point appears to be difficult to
predict since so many factors are involved in temperature distribution within samples.
Ramaswamy and Pillet-W ill (1992) observed differences of 50°C and greater during
heating of starch-based products on square-sized trays.
Lin and Sawyer (1988)
recommended using beef patties o f doughnut shape because they possess more even
heat distribution than patties of circular shape. The microwaves are reflected at the
boundaries forming the center of the doughnut-shaped hamburger, therefore the
distance that microwaves penetrate is reduced. The number of cold areas within the
meat is minimized in hamburger of doughnut-shape.
Datta and Hu (1992) pointed out that as the heating time increases the thermal
distribution also spreads out with time.
Some areas (e.g. comers, edges) undergo
unnecessary thermal treatm ent resulting in deterioration of quality. In contrast to that,
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the increase of temperature during the initial stage (come-up time) is really short The
partial solution to that problem is discontinuation o f microwave irradiation once
sterilization temperature is reached. The thermal properties of foods play an important
role. Sawyer (1985) proposed using the term “the post-processing rise of temperature".
Modeling prediction of temperature distributions within a food during microwave
heating based on die dielectric properties of a food
The mathematical models that were developed were based either on Maxwell's
equation or Lambert's law to calculate the electric field generated within a sample
(Ayappa et al., 1991a, 1991b, 1992; Wei et al., 1985; Datta et al., 1992). By applying
the thermal properties o f foods and the external cooking conditions, the temperature
pattern can be determined (Buffler, 1992).
The calculation based on both models
requires sophisticated mathematical procedures and involves extensive computer
programming. Significant progress has been made in research related to prediction of
microwave power distribution and temperature during microwave heating.
Predicted
temperatures based on both models are not in full agreement with the experimental
data.
Both equations used in calculation have limitations: Lambert's law assumes a
simple, exponential decay of power with a distance that does not include reflections
while Maxwell's equation incorporates reflections (Barringer, 1994).
Progress in
application of microwave technology is closely related to the advances in mathematical
analysis of microwave heating, particularly in development of mathematical formulas
that allow the prediction o f transient temperature profiles of foods during microwave
exposure.
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Destruction of pathogenic and spoilage bacteria in foods by microwave
heating
Microwave heating of meat products
As the popularity of microwave ovens increased during the 1970s, proper
cooking guidelines were requested by consumers. The safety of microwave cooked
meals, with meat as major component became an issue (Woodbum et al., 1962;
Goldblith, 1966).
In the report by Woodbum et al. (1962), the effectiveness of microwaves on the
destruction of salmonellae and staphylococci in chicken products was studied.
Samples (200 g) were cooked in plastic bags and, after cooking, the temperature of the
product was measured.
Heating fo r 120 sec was found satisfactory to reduce the
number o f bacteria to a safe level, but 90-sec cooking did not deliver required lethality
to inactivate pathogens.
Bengtsson et al. (1970) reported on the pasteurization o f cured hams by
microwave heating at 60 MHz and 2450 MHz to the final temperature of 66-68°C. After
treatment at both frequencies, the number of bacteria was ten times higher when
compared to controls that underwent conventional hot water treatment at 69°C. Shorter
surface heat treatment (low temperature) was responsible for bacterial survival.
Similar results were obtained by Chen et al. (1973) during the cooking of
chicken parts in hot water (88°C, 20 min) and cooking by microwave heating (2450
MHz, 1600 W, 6 min).
The number of mesophilic and psychrophilic bacteria that
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survived the microwave treatment was ten times or more higher than the number of
bacteria found in water-cooked chicken breasts.
Failure to uniformly expose all
surfaces of chicken pieces to microwaves was the major reason of higher microbial
counts.
Clostridium perfringens (10s cell/g), spores or vegetative cells were eliminated
from precooked chicken samples during microwave heating up to 84°C in studies
earned out by Craven and Lillard (1974).
Considerable variations in internal
temperature were noticed even though the pieces were the same weight and
microwave oven working at a 2 kW power setting was equipped with a turntable.
Crespo and Ockerman (1977) attempted to compare effectiveness o f microwave
cooking with low temperature oven cooking (149°F) and high temperature oven cooking
(232°F). The ground beef samples (250 g) containing natural spoilage microflora were
heated in conventional oven and microwave oven to the internal temperature of 34°C,
61°C and 75°C. After treatment, 50-g samples were removed from the center o f the
heated sample and the logarithmic reduction o f bacteria was determined.
The
microwave heating was least effective in destruction of the spoilage bacteria to a 3.61
log reduction at the 75°C final temperature. Greater reduction o f microorganisms was
observed during high temperature oven cooking (5.24 log reduction at a 75°C final
temperature) than at low temperature oven cooking (4.32 log reduction at a 75°C final
temperature).
Although the end-point cooking temperature was identical, different
lethality levels were observed within samples as they were determined by the
temperature values obtained during the entire process and the distribution of
temperature within samples. The shape o f the sample, power of the microwave oven,
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and other factors that determine temperature pattern in microwave heated foods were
not monitored. The attem pt to minimize the variability during microwave cooking was
presented in another study by Crespo et al. (1977). The objective was to compare
destruction of Streptococcus faecalis, Pseudomonas putrefadens and Lactobacillus
plantarum during microwave and conventional heating at 176°C. Sterile, 50-g samples
of ground beef, were inoculated with bacteria and heated in conventional oven and
microwave oven by both methods until the specific internal temperature was reached.
The shape of the heated samples were uniform and the position of meat patty in the
oven cavity was controlled. Conventional cooking was more destructive for S. faecalis
than L. plantarum. The authors stated that differences in sensitivities to the heating
methods were observed between microorganisms. Both heating methods were equally
effective in decreasing the level of P. putrefadens.
In earlier studies, the major
problem was associated w ith instrumental limitation to monitor the temperature, which
was solved later by application o f fiber-optic temperature probes.
Baldwin et al. (1971) determined the time adequate fo r destruction of two strains
of Salmonella typhimurium distributed on the surface o f 270-g fish samples.
Both
strains survived 195 sec o f microwave cooking at 2450 MHz, but the power level o f the
oven was not reported.
There was a concern about even temperature distribution
within the sample; therefore, an experiment was carried out to observe the temperature
rise in fish and fish products. The time required to reach 55°C in the center of the
samples varied from 49 sec to 390 sec. Baldwin (1983) pointed out that the speed and
eveness of heating are influenced by the mass and composition of a food and the
features of the microwave oven.
Payton and Baldwin, (1985) carried out a study to
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compare the properties of top round steaks after cooking by microwave-convection,
forced-air convection and conventional ovens.
Although less cooking time was
required and less total energy was used during microwave-convection heating, the
evaporation and drip losses were greater than those observed in other cooking
procedures.
The process of transferring information and principles applied by microwave
engineers to food developers, food processors and microbiologists has progressed. As
a result of accumulated information from various microbiological studies on the
microbiological safety of microwave heated foods, the National Livestock and Meat
Board (Starrack, 1980) recommended a procedure that should be followed during
microwave cooking. A number of factors should be controlled to improve uniformity of
microwave heating including: 1) keep constant the geometric shape o f the sample, 2)
cover the sample to reduce evaporative losses; 3) make a hollow in the center of the
sample to avoid center overheating; and 4) allow standing time fo r conductive heat
transfer. This recommendation also included practical suggestions that are important
to minimize, but not eliminate, problems associated with the transfer of heat during
microwave cooking.
Harrison (1980) listed factors that affected quality attributes of
cooked meats which included initial temperature, density and homogeneity of meat,
shape and quantity of meat piece, utensils, post-oven temperature rise and distribution
of energy in the oven cavity.
Cunningham (1980) investigated if a short microwave treatm ent in the
microwave oven would decrease the number of psychrotrophic bacteria on chicken
parts and subsequently increase their shelf life during refrigerated storage. Reduction
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of psychrotrophs by 1 to 2 logs was observed on raw poultry parts and chicken skin
after 20 to 40 sec microwave treatm ent
The weight and size of samples were not
monitored and the power of microwave irradiation was not specified.
Inoculated
cultures of these bacteria in 10 mL nutrient broth was tested at various time exposures.
The temperature at the end of heating was measured. A fter 5 to 20 sec o f microwave
heating, the number o f bacteria in the broth decreased substantially or was totally
reduced if a final temperature 60°C was achieved.
Fruin and Guthertz (1982) recognized the dilemma of uneven heating
distribution when they assayed for the survival o f pathogens in meatloaf cooked by
microwave oven, conventional oven and slow cookers.
The destruction rates of
Escherichia coii, Clostridium perfringens and Streptococcus faecalis were related to the
temperature variations rather than to method of cooking.
The slow cooker and the
conventional oven were more efficient in reducing pathogens compared to microwave
oven. The temperature after cooking was measured at nine different locations. It was
found that the cumulative time-temperature relationship during microwave cooking was
not as effective as during conventional cooking as the rapid increase of the
temperature would suggest.
Sawyer et al. (1984) conducted studies to demonstrate problems associated
with microwave cooking. They tested the difference in the internal temperature and the
effect on the survival of Staphylococcus aureus and Salmonella senftenberg in meats
heated enclosed in plastic wrap and without it. They found that wrapping products did
not have a statistically significant effect but slightly improved microbial quality when
mean microbial numbers were considered.
Duration and extent of post-processing
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temperature rise in foods including
meat entrees
after conventional cooking
(conventional hot air, 160°C) and microwave cooking (oven - 2450 MHz, 645 W - half
and full pow er) were compared by Sawyer (1985). Temperature distribution in the beef
patty of doughnut shape, heated in the microwave oven fo r 90 sec, ranged from
approximately 80°C to 93°C. A fter an additional 41-sec standing time, the temperature
increased slightly at a few locations yet a highly uniform temperature pattern was
observed. Factors that contributed to the temperature variation included: 1) wrapping;
2) wattage output; 3) time of processing; 4) microwave wavelength; 5) product
composition and; 6) size and shape. Lin and Sawyer (1988) suggested establishing a
new term, “the exposed microwave dose”, that would assist in predicting microbial
quality in foods processed by microwaves.
The microwave dose (watts x minutes
processed / gram of food) correlated highly to the percent of survival o f S. aureus and
E. co li in the beef loaves and to the end-point temperature o f the beef loaves.
The low effectiveness of microwave cooking versus conventional cooking for the
destruction of aerobic bacteria and C. perfringens in ground beef patties was
demonstrated by Wright-Rudolph et al. (1986). A rigorous protocol was followed to
better control microwave heating. Ground beef patties, 120-g in weight and of uniform
shape, were processed by both heating methods to the same internal temperatures
measured after 1-min standing time and the location between center and edge.
Reduction in log numbers of C. perfingens during microwave cooking was 0.75 to 1.48 ,
whereas fo r conventional cooking the log reduction was 3.06 to 3.51.
Aleixo et al. (1985) evaluated the microbiological safety o f cooking guidelines to
the end-point temperature of 76.6°C during microwave heating o f whole turkey and
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cook-in-bag heating inoculated with S. typhimurium, S. aureus and C. perfringens. This
procedure did not completely eliminate pathogens from meat in the microwave heated
turkey, but cooking-in-bag increased destruction of microorganisms.
A series of studies was conducted to test efficacy of microwave cooking for
inactivating Trichinella spiralis larvae from pork roasts and chops (Zimmermann &
Beach, 1982; Zimmermann, 1984; Carlin et al., 1983). It was found that the cooking
procedure used during conventional heating was not satisfactory during microwave
processing to the same final temperature of 76.7°C although various adjustments were
made to improve heat distribution in the meat.
Schnepf and Barbeau (1989) attempted to demonstrate the reliability of cooking
recommendations issued by different authorities [i.e., U.S. Department of Agriculture
(71°C), Food and Drug Administration (74°C) and American Home Economics
Association (85°C)]. Fresh whole roasting chicken inoculated with S. typhimurium were
cooked to four different internal temperatures (min. 74°C, max. 85°C) in microwave,
microwave-convection and conventional electric oven.
Results showed that cooking
poultry to a temperature of 85°C does not ensure complete destruction of S.
typhimurium possibly present in the meat.
Huang et al. (1993) earned out a study on the destruction of Listeria
monocytogenes and Aeromonas hydmphila during microwave cooking. Catfish fillets
inoculated with these bacteria were cooked, covered and uncovered with polyvinylidene
chloride film, to internal temperatures o f 55°C, 60°C and 70°C. The temperature was
monitored at several locations and the F-value was calculated fo r these points during
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individual temperature treatments.
It was found that microwave heating of fillet,
covered with film, to the final temperature o f 70°C eliminated both pathogens.
Microwave cooking o f these samples to the final temperature o f 60°C did not eliminate
a subpopulation o f L. monocytogenes.
The effect of different microwave power levels on the survival o f L
monocytogenes
in
inoculated
shrimp
samples
(100
g) was
investigated
by
Gundavarapu et al. (1995). A mathematical model was used to predict cooking times
based on established by authors the D-values of L. monocytogenes. These thermal
inactivation rates (D-values) for L. monocytogenes in shrimp were determined during
thermal resistance studies conducted in a water bath.
The cooking time that was
predicted by the model, was not effective in inactivating this pathogen when exposed to
microwave heating.
No viable bacteria were detected when shrimp were cooked for
120% of the predicted time.
Hollywood et al. (1991b) cooked minced beef, containing 107 colonies/g of
contaminating organisms, by microwave and conventional oven methods following the
procedures recommended fo r heating roast beef to obtain rare, medium and well-done
states. External and internal temperatures during heating o f 500-g meat samples were
monitored. Standing time of 30 min was allowed fo r microwave-cooked samples after
they reached the final end-point temperature.
Only the samples cooked in the
conventional oven to the well-done state resulted in the absence of microflora. In all of
the samples heated in the microwave oven and the conventional oven, samples heated
to the rare and medium states bacteria were detected, including L monocytogenes.
One of the most accurate studies carried out to compare efficacy of meat
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pasteurization by microwave and conventional heating, where attention was paid to
small temperature differences of heated samples, was reported by Coote et al. (1991).
The conventional heating process simulated temperatures obtained during microwave
cooking. L monocytogenes organisms were inoculated on the chicken skin samples or
in the culture broth.
Samples were heated in a thermocouple block calibrator
connected to a temperature programmer based on mean temperatures achieved during
the microwave heating process o f whole chickens fo r 28 min. The lethality delivered to
the samples caused a 6-log reduction of bacteria and was comparable to the
recommended cooking parameters of 70°C for 2 min.
The uneven temperature
distribution is responsible for survival of bacteria in microwave heated foods.
Microwave cooking of other foods
The destruction o f S. typhimurium and E. coli in soups (tomato, vegetable and
broth) during microwave cooking was studied by Culkin and Fung (1975).
Single
serving portions (200 mL) inoculated with 107 bacteria/mL were exposed to 915 MHz
microwaves. The power of the emitted microwaves was not specified. Soups were
heated in modified graduated-cylinder containers which affected the distribution of the
temperature during heating. Temperature within the sample was carefully controlled
using temperature-sensitive paper strips since fiberoptic thermometry was not available
at the time.
At any given heating time, the temperature o f the middle region was
warmest; at the bottom intermediate, and coolest at the top. Based on the mechanism
of microwave heating, the high temperature in the middle region could be explained as
the result of focusing of the microwave energy in the center through reflection and
standing waves effects. Evaporative losses and transfer o f heat to the environment is
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often responsible for the low temperature at the surfaces.
However, the authors
suggested that heat alone is inadequate to fully explain the lethality effect on
microorganisms. They support the hypotheses on the involvement of the non-thermal
portion o f the microwave field in the microbial destruction process (Fung and
Cunningham, 1980).
A few researchers earned out studies to establish parameters fo r pasteurization
of milk by microwave heating. Chiu et al. (1984) examined the possibility of extending
the shelf life of pasteurized milk by consumer use of their household microwave oven.
A milk portion (200 mL) stored for 8 to 11 days increased the number of psychotrophic
bacteria to a level approximately 106 per mL. Microwave heating the milk sample for
120 sec to the final temperature o f 60°C reduced the number of bacteria to a nondetectable level.
Sim ilar studies on the in-home pasteurization processing of milk were conducted
by Thompson and Thompson (1990).
The native microflora of raw goat milk was
successfully reduced (6-log reduction) by heating 2 to 3 L of milk in a microwave oven
at a 450 W power level. When the temperature of the probe immersed in the milk
showed 65°C, the "temperature hold " feature was used to maintain the 65°C for an
additional 30 min.
New microwave ovens are equipped with options to control
temperature during heating.
Knutson et al. (1988) simulated, in a microwave oven, the heat treatm ent given
milk in HTST (high-temperature short-time, 71.7°C fo r 15 sec) and LTLT (lowtemperature long-time, 62.8°C for 30 min) pasteurization. Steamed milk, without initial
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microflora, was inoculated with S. typhimurium, E. co li and Pseudomonas fluorescens.
Volumes of 76 mL were heated to the required pasteurization temperatures in a
microwave oven but such treatments failed to inactivate all bacteria. Sim ilar results
were obtained when two different volumes of milk (1/2 and 3/4 quarts) inoculated with
Streptococcus faecalis were heated in a microwave oven to the final temperature in the
range 62.8°C to 71.7°C. The number of bacteria killed in these treatments was lower
than when these same samples were heated in the water bath.
The non-uniform
distribution of heat in the mass o f milk was found to be responsible for the differences.
Several other food
systems were tested
using
microwave heating
in
pasteurization processes. Spite (1984) determined the survival of pathogens in frozen
foods with controlled content o f carbohydrates (mashed potatoes used as a base),
lipids, proteins and moisture or in a commercially prepared food.
Various time-
temperature configurations were applied during microwave heating of foods ranging
from 3 to 5 min at a power level of 1500 W. Bacteria inoculated in the food before
experiments at a concentration of 10s to 107/g were not totally eliminated during
heating. Therefore, the author suggested that cooking instructions should be changed
by manufacturers. Another study earned out by Hollywood et al. (1991b) led to similar
conclusions.
Pathogenic microorganisms survived microwave heating of frozen
convenience foods. The procedure recommended by manufacturers was found to be
inadequate. Microwave reheating of convenience meals was the objective o f a study
by Deader and Lacey (1990). Poor heat penetration was shown in the foods containing
a higher salt content.
Although the relationship between ion content and the heat
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penetration depth is a well described phenomenon in microwave heating, it was
reported as a result of artificial microwave heating.
Application of microwave irradiation to pasteurize dry foods was suggested by
Bookwalter et al. (1982). Enriched com-soy blends were successfully processed in a
continuous microwave tunnel (2450 MHz, 60 kW) to destroy salmonellae. When timetemperature microwave exposure parameters were optimized, a 3-log reduction was
obtained without significant change in the quality of the product.
A series of studies to determine various factors affecting efficiency of
microwave heating of foods were earned out by Heddleson et al. (1991, 1993, 1994).
The relationship between temperature achieved, composition of the aqueous food
system, particularly salt content and microbial destruction, was studied. The presence
of salts changed the dielectric properties of the samples and the amount o f heat and
heat distribution within the samples which affected the inactivation rates o f Salmonella
during heating.
Power level, post-heating holding time, heating medium volume,
heating food sample in container versus without container and container shape all play
a role in the destruction o f bacteria in foods heated by microwaves.
The safety of chilled retailed products reheated in microwave ovens was studied
by researchers at Campden Food and Drink Research Association.
W alker et al.
(1989) found that the recommended microwave cooking instructions were not
satisfactory for 3 of 8 food products inoculated with 105 to 106 CFU/g o f L
monocytogenes. The follow-up studies by George et al. (1991) revealed the influence
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of thermal, electrical and physical properties on the quality o f products heated by
microwaves.
Microwave heating in food service systems
Microbial safety of the foods prepared in the cook/chill foodservice systems that
use microwave ovens was reported. Dahl et al. (1981a,b) determined how microwave
processing in cook/chill systems affected
the survival of a radiation-resistant
Streptococcus faecium and S. aureus and the quantity of sublethally injured aerobic
bacteria present in cooked beef loafs and cooked potatoes and beans, in both reports
it was found that an increase in the microwave heating time during a reheating process
decreased the proportion of viable or injured bacteria. Processing o f foods above 74°C
during the reheating stage should be carried out to ensure safety. S. faecium did not
show a special ability to survive microwave heating. In studies reported by Cremer and
Chipley (1980) on the microbiological safety of scrambled eggs in a hospital
foodservice system, a similar recommendation of cooking above 74°C was presented.
To obtain this temperature, heating time should be correlated with the output power of
the microwave oven, sample weight and
initial temperature
of the
sample.
Microbiological quality of foods produced by the cook/chill foodservice system after
reheating by conduction, convection and microwave radiation was presented by
Sawyer et al. (1983). The position of the plate in the oven, location o f the lowest
temperature in the reheated food sample, and the amount of moisture were listed as
major factors responsible for inadequacy of microwave processing in 83% of the
situations observed.
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Interaction of microwaves with microorganisms
Thermal and athermal effects of microwaves on destruction of bacteria
Since the time when microwave irradiation was applied to food processing, the
safety of this process has been questioned.
Certain unexplained phenomenon that
occurred during various microbial destruction tests, especially during poorly controlled
studies, were attributed to nontherma! effects o f microwaves on bacteria.
Many
researchers believed in the existence of effects associated with radiation portion o f the
microwaves. Microwave energy at 2450 MHz frequency does not alter strong molecular
bonds, thus new, possibly toxic compounds, cannot be formed.
However, theories
have been proposed and experiments were earned out to prove that selective
absorption of radiation energy leads to biological effects. They may result in altering
physiology, metabolic activity or, together with heat, lead to lethality to bacterial cells.
Similar changes were expected to occur in human or animal tissues. The safety of the
electromagnetic fields issue created a new research area combining the elements of
medical field and biophysics. This controversy did not play an important role in the
development o f microwave thermoprocessing in the food industry other than to
encourage conducting more research. Meticulous microwave heating studies, where
more attention was paid to details, allowed a better understanding of the principles of
microwave heating. One of the latest issues discussed is that bacteria suspended in
media may absorb more microwave energy than media alone. This may result from
higher intercellular conductivity of bacteria compared to the media, thus higher than
expected lethality. To disapprove this claim, the internal temperature of the bacteria
should be measured and compared to the media temperature (Mudgett, 1989). This
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would be a rather difficult measurement to carry o u t Sastry and Palaniappan (1991),
reported that calculations based on heat transfer principles reject this claim.
As Baldwin (1983) pointed out, the topic of athermal effects o f microwaves will
"continue to stimulate interest o f researchers, but is doubtful that such effect will ever
be demonstrated to have practical application in the food industry."
Kinetics of microbial destruction during microwave irradiation and conventional
heating
Heat resistance of bacteria heated at constant temperatures by both
methods.
Several studies were earned out to determine the heat resistance of bacteria in
aqueous solutions during microwave heating and to compare these values with
corresponding values obtained during conventional heating.
The difficulties arising
from the lack of constant temperature were solved by designing special equipment or
by conducting appropriate mathematical calculations.
Goldblith and W ang (1967) reported that the degree of inactivation o f E. coli
and Bacillus subtilis spores by microwave energy was identical to that by conventional
heating. A small volume (0.025 mL) of bacteria suspended in phosphate buffer was
enclosed in capillary tubes.
Temperature profiles during conventional heating were
recorded. To simulate sim ilar time-temperature conditions during microwave heating,
ice was added to the w ater with submerged capillaries. Cautious techniques during
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heating by both methods resulted in obtaining sim ilar inactivation rates of tested
bacteria.
Lechowicz et al. (1969) designed a microwave system that allowed them to
conduct survivor curve experiments at 52°C and 55°C during exposure to 2450 MHz
microwaves. The experimental setup consisted o f a cooling system surrounding the
vial containing the bacterial suspension.
The reduction rates o f S. faecalis and
Saccharomyces cerevisiae suspensions heated in a w ater bath and by microwave were
not different.
A report by Carroll and Lopez (1969) indicated no killing effect of the radiofrequency energy (60 mc/sec) per se on bacteria was observed.
Radio-frequency
evaporation apparatus was constructed to eliminate the heat effect on irradiated
bacteria including S. cerevisiae, E. coli and B. subtilis suspended in buffer at different
pH values. On the contrary, Dreyfuss and Chipley (1980) and Chipley (1980) claimed
that microwaves affect metabolic activity of Staphylococcus aureus in a manner that
cannot be explained by thermal effect alone. They found increased specific activity of
several enzymes from cell lysates and walls of microwave-heated cells compared to
conventionally heated enzymes.
Studies carried out by Khalil and Villota (1985, 1988, 1989) supported the
existence of athermal effects on bacteria heated by microwaves. The extent of injury of
S. aureus heated by microwaves and conventionally was studied. Microwave-heated
cells possessed greater membrane damage, slower ability to regain enterotoxin
production, and greater lesion of the 23S RNA. Microwave-heated samples were kept
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at a constant temperature through a cooling jacke t but the temperature during heating
was not reported.
Spores o f Bacillus stearothermophilus heated by microwaves
exhibited more lethality than spores heated conventionally.
Time-temperature profiles of microbial suspensions heated in uniformed
microwave field (waveguide connected to magnetron) are included in the report of
Rosenberg and Sinell (1990). As a result of small differences in the heating profiles,
the lethalities delivered to the microbial samples varied.
This explains the small
differences between the D-values for S. typhimurium, S. aureus and E. coli.
The kinetics o f E. coli destruction by microwave irradiation were studied by
Fujikawa et al. (1992). Inactivation profiles fo r microwave exposure and conventional
heating did not show remarkable differences.
Research earned out by W elt et al. (1994) produced convincing data only on the
thermal effects of microwaves on bacterial spores.
Three techniques were used to
study inactivation kinetics of Clostridium sporogenes spores.
For the first method, a
especially designed kinetics vessel working at a constant temperature was used. In the
two other methods, a heated suspension was continuously cooled by pumping the
suspension outside o f the microwave oven cavity.
The data presented from
inactivation studies by conventional heating and microwave heating strongly showed
lack of any difference in the sensitivity of bacteria to both heating modes.
Similar
conclusions were reached in enzymatic inactivation studies conducted by Janhgen et
al. (1990).
47
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Role of water during heating bacteria by microwaves
Efficiency of microwaves to inactivate bacteria is related to the water content of
bacteria.
Very often the difficulty in pasteurizing food is caused by the dry food
environment and the low am ount o f cellular water. Vela and Wu (1979) showed that
samples of soil could not be decontaminated from bacteria, actinomycetes, fungi and
bacteriophages without the presence of water. Lyophilized microorganisms were not
affected by 2450 MHz microwave energy at 240 kW power at various exposure times.
The amount of moisture in soil determined the rate of the microbial destruction.
Rosenberg and Sinell (1989) reported that the viability of lyophilized E. coli was not
changed after exposure for 30 min to 1037 W microwave power at 34°C and 2000 W
power at 45°C.
The inactivation rate o f dry spores of B. subtilis subsp. niger during microwave
exposure and conventional heating was the subject o f research earned out by Wayland
et al. (1977).
Measurable differences in the destruction rates were found when
comparing heat alone to microwaves. The author suggested that in future experiments
improvements in the experimental procedure should be done to substantiate a clear
indication of non-thermal effects.
Jeng et al. (1987) carried out research on the mechanisms of microwave
sterilization during destruction o f bacteria in a dry state. Spores of 8. subtilis subsp.
niger were dried to a 33% water content in a relative humidity chamber. Destruction of
spores was done in the dry oven and microwave oven using identical time-temperature
profiles studied. Equal D-values were obtained at three different temperatures by both
methods.
48
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Biological effect of microwaves on microorganisms
There is an enormous amount o f literature on the biological effects of
microwaves at a wide range of frequencies including 2450 MHz (Hileman, 1993). The
objective
of many studies was
to
observe any
metabolic changes
of the
microorganisms resulting from exposure to microwave fields. Although many prove a
lack o f any influence on the cell physiology and are of academic interest, however, it is
im portant to mention the areas that were already explored.
Growth of microorganisms in an electromagnetic field (70, 75, 42 GHz),
particularly S. cerevisiae, was found not to be different than growth in normal conditions
(Dardalhon et al., 1979; Furia et al., 1986) as opposed to the results o f earlier reports
(Webb & Booth, 1969; Grundler & Keilmann, 1983).
When bacteria were subjected to MW fields, changes in bacterial membrane or
enzymes were not observed in studies earned out by Kazbekov and Vyacheslavov,
(1987), unlike the reports of Pickard and Rosenbaum (1978), and Saffer and Profenno
(1989, 1992).
Controversial theories and data were reported regarding the interaction of
microwaves with the genetic materials of microbial cells that would lead to mutogenic
changes (Leonard et al., 1983; Blevins et al., 1980; Hamnerius et al., 1985). Theory on
the resonant microwave absorption of selected DNA molecules was proposed by
(Edwards et al., 1984; Gabriel et al., 1989). However, other studies showed lack of
specific changes o f cell genetic material as a result of exposure to microwave field
49
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Meltz et al., 1989; Sagripanti et al. 1987; Batbyamba & Drasil, 1988; Maleev et al.,
1987).
Lack of understanding of microwave heating and problems on uneven heat
distribution in the samples lead to the incorrect conclusion that microwaves interact with
amino adds or anti-infective factors (Quan et al., 1992; Lubec et al., 1989; SigmanG rante ta l., 1992)
Instrumentation influences progress in research on bacteria-microwave
interactions
Reliable microbial inactivation data can be produced only during carefully
controlled microwave heating with accurate temperature measurements. Availability of
sophisticated instrumentation that incorporates the newest technology allows us to
make progress in the application of microwave heating.
The biggest obstade is to
accurately control temperature in the heated sample. Metal thermocouples could not
be used to measure temperature because they interfered with the electromagnetic field
and caused discharges, unless other modifications were made (van de Voort et al.,
1987). Fiberoptic temperature probes (Berek & Wickersheim, 1988) were introduced
which could be calibrated to the temperature standards and capable o f sending
frequent temperature data without interrupting the microwave heating. Infrared imaging
was applied successfully to continuous temperature measurement (Geodeken et al.,
1991; Ohlsson & Risman, 1978). MRI (magnetic resonance imaging) is a promising
new technology that could be used (Sun et al., 1993). The key to studying microbial
kinetics is a constant temperature during microwave heating. Microwave ovens were
50
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developed to work at a preset temperature by adding a feedback temperature system
(Ramaswamy et al., 1991; W elt et al., 1992, 1993).
Development of advances in
general numerical methods and applications of the supercomputer led to successful
computational studies. Models based on Maxwell's equation or Lambert’s law were
developed that predicted the temperature profiles fo r specific food systems during
microwave heating (Ayappa et al., 1992; Jia & Jolly, 1992).
A simple method was
suggested by Holyoak et al. (1993) to determine the safety of microwave cooking.
Alginate beads with immobilized bacteria of known heat resistance could be used to
estimate the lethality delivered to the microwave-heated foods. Uniform distribution of
beads within the foods resulted in multipoint measurements to determine the effect of
lethal temperature.
Application of microwave technology and its future in food
thermoprocessing
Utilization of microwave energy in industrial sterilization processes was initiated
by researchers from U.S. Army Natick Laboratory. Kenyon et al. (1971) and Ayoub et
al. (1974) described the continuous microwave sterilization process of m eat Portions
o f meat packed in flexible pouches were first preheated to 93°C, then exposed to
microwave energy to the final temperature of 121°C followed by holding in water to
achieve the desired F-value. However, inoculated-packed studies showed that specific
areas in the pouches did not receive the adequate sterilization value and that
methodology was discontinued (Decareau, 1985).
51
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Development o f microwave sterilization/pasteurization technology has been
continued and complete microwave sterilization or pasteurization systems are available
for food processors (Schlegel, 1992).
The TW Kutteris Omac system conducts a
sterilization process based on the product's properties including dielectric parameters,
size, weight, homogeneity and the moisture content. It consists of 1.9 kW magnetrons
with a frequency of 2450 MHz and a programmable logic controller which allows for
entering parameters such as power, air temperature and travel speed of the product
(Harifinger, 1992). A number of microwave pasteurization processes were installed in
industrial settings and some of the microwave thermoprocesses produced high quality
products. An extensive review on that topic is presented in several excellent reports by
Goldblith (1966), Decareau (1985), Knutson et al. (1987), Rosenberg & Bogl (1987),
Thuery (1992) and Heddleson & Doores (1994).
Applications o f microwaves to sterilize laboratory materials, products such as
parenteral solutions and medical devices were tested (Latimer & Matsen, 1977; Rohrer
& Bulard, 1985; Lohman & Manique, 1986; Cowan & Allen, 1985, Diaz-Cinco &
Martinelli, 1991, Najdovski et al., 1991). It was demonstrated that the reduction of the
number of spores was small and variable due to the moisture level in the bioload. A
reliable sterilization or disinfection process could not be carried out.
The use of
microwaves to melt agar-based microbiological media was found convenient and a
time-saving method (Fung & Lin, 1984)
Growing applications of microwaves and microwave technology in nonfood
areas will influence progress in microwave thermoprocessing as the transfer of
technology proceeds (Young & Jolly, 1990).
There have been attempts to apply
52
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microwaves in other agriculture related areas such as weed control. The innovative
microwave systems recently developed in analytical chemistry (Kingston & Jassie,
1988) and material science (Landry & Barron, 1993) show that microwave energy has a
future that has not been fully explored.
Conclusions
A number of studies were carried out to show the lethal effect of microwaves on
bacteria. Difficulties occurred during the process when quantifying parameters that are
responsible for the amount o f lethality delivered to the food system. Inconsistent and
confusing results have been obtained during many studies that were earned out to
determine safe end-point microwave cooking guidelines.
As research progresses it
becomes evident that the nature of microwave heating is very complex and many
factors contribute to uneven heat distribution in foods. It is these factors that determine
the effectiveness of the pasteurization or sterilization process. When factors such as
microwave power, microwave oven size, shape, initial temperature and the dielectric
properties of foods are controlled it is possible to predict proper pasteurization
methods. The focus of current research is to establish the appropriate methodology to
assure
proper
pasteurization
of
foods
and
biologicals
during
microwave
thermoprocessing.
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EXPERIMENTAL PART
70
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Recovery of thermally-stressed Escherichia coli 0157:H7 by
media supplemented with pyruvate
71
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Abstract
Unheated and heat-stressed (57°C, 50 min and 60 min) cells of Escherichia coli
0157:H7, were enumerated using three media supplemented with 1% sodium pyruvate
(NaPyr): plate count agar (PCA), tryptic soy agar (TSA) and phenol red sorbitol agar
(PhRSA) using the spread plate method. Medium recovering the greatest numbers of
severely heated E. coli 0157:H7 was PCA with 1% NaPyr. Recovery on this medium of
the heat stressed E. coli 0157:H7 was significantly higher (p<0.05) than the two other
media with pyruvate: 16.3% (50 min heating) and 0.55% (60 min heating) o f the total
population was recovered with TSA + 1 % NaPyr when compared to those numbers
found on PCA + 1% NaPyr. The PhRSA + 1% NaPyr ability to recover heat-stressed £.
coli 0157:H7 was sim ilar to TSA + 1% NaPyr.
Using PhRSA + 1% NaPyr media,
12.9% (50 min heating) and 0.61% (60 min heating) of the total population were
recovered when compared with the cells enumerated on PCA + 1% NaPyr. Recovery
of the heat stressed cells using the spread plate method was greater than using pour
plate method.
Recovery was significantly higher (p<0.05) on the spread plates for
highly stressed E. coli 0157:H7(1.2 log) heated for 60 min than on the pour plates.
Overall, the populations on the TSA spread and pour plates were low compared with
the same heat-stressed cells recovered on media containing pyruvate. Enumeration of
unheated bacteria was not affected by either the type o f medium or the plating method.
72
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Introduction
Successful elimination of E. coli 0157.H7, a recognized foodbome pathogen,
from food systems during heat processing can be achieved by applying heat under
adequate
conditions o f time
and
temperature.
Thus,
the
thermoresistance
characteristics of E. coli 0157:H 7,that is, the decimal reduction time (D-value) and the
time-temperature coefficient (z-value) have to be established and used during food
preparation to avoid foodbome infection from this microorganism.
In microbial
thermoresistance studies, several factors have to be considered which may potentially
affect the reliability of the results. These are selection o f heating system, growth stage
and growth environment o f microorganisms used in the study, heating menstrum, and
recovery conditions (Andrews, 1989). However, as suggested by the research that has
been conducted on microbial injury, the most important is a choice o f media that could
provide optimal conditions to repair cellular damages o f heated microorganisms (Ray,
1989).
A medium is needed that will give the highest number of CFU/ml from the
population that has survived the heat treatment (H urst
1977; Speck, 1984).
Enumerating a severely heat-stressed subpopulation is important in assessing the true
lethality of the tested microbial culture.
Sublethal heating causes damage to components of microbial cells at the
molecular level (Andrews, 1989; Gould, 1989; Hurst, 1977; Ray, 1989). The most often
reported are an increase o f cell surface hydrophobicity, loss o f components from outer
and cytoplasmic membranes, inactivation of cell enzymes, degradation of rRNA and
breaks in single DNA strands which can lead to mutation.
Subsequently, there is a
73
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substantial loss of cell viability. Since metabolism is impaired, heat-damaged bacteria
can express different physiological and nutritional requirements.
Assessing the true lethality of a microbial population in a food system has been
a significant problem and directly affects food product safety (Ray, 1989). Approaches
that have been used to recover heat injured bacteria include: i) use o f general growth
media in place of selective media, ii) add compounds to the growth media that support
the
recovery,
and
iii)
eliminate
additional
stresses
during
the
detection
of
microorganisms. Heat-damaged cells are very sensitive to chemicals such as bile salts,
NaCI and deoxycholate. These may be present in selective media used to enumerate
these microorganisms. Thus, nonselective media such as PCA or TSA should be used
in heat resistance studies. Specific compounds that facilitate the repair process such
as amino acids, protein digest, yeast extract ATP, sodium pyruvate, and inorganic
compounds including K2 HPO4 , Mg+Z may be added to these media (Hurst, 1984; Ray,
1989).
Recovery conditions for stressed bacteria might be different than those
satisfying undamaged bacteria (e.g. low tolerance to fluctuation of temperature or pH
and longer incubation time). The purpose o f this research was to i) determine optimal
method of recovering heat-stressed E. coli 0157:H7 using TSA, PCA and PhRSA with
or without 1% sodium pyruvate, ii) investigate whether recovery with these media can
be maximized by adding pyruvate, and iii) establish if there is a difference in recovery
of heat-stressed cells using either the spread plate or the pour plate method.
74
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Materials and Methods
Strain
A culture of E. coli 0157:H7 strain 933 was obtained from the FDA
(Minneapolis, MN) and was used in these studies. The strain was preserved fo r future
experiments by freezing (-50°C) the culture in a 10%(vol/vol) mixture o f glycerol in
Tryptic soy broth (TSB) (Difco; Detroit, Ml).
Culture preparation
Cell suspensions utilized in heating tests were propagated in TSB for 24 hr at
37°C prior to each experim ent
The culture was inoculated by the transfer o f two
loopfuls o f frozen or fresh cell suspension to 20 mL o f TSB. It was maintained by
consecutive daily transfers fo r two weeks. After this period, a new series of culture was
initiated from the frozen stock. The first transferred culture was never used in heating
tests, since it contained freeze-damaged cells.
Growth curve experiments were
performed to assure that the 24-hr cell suspension was in the stationary phase. In this
time span, the concentration o f cells at the time of harvest was 109 CFU/mL. Twenty ml
of culture were pelleted by centrifugation at 17,000 x g for 20 min. The pellet was
cleaned in Butterfield's phosphate buffer (pH = 7.2) (Speck, 1984), pelleted again and
diluted with buffer to the initial volume.
The working cell suspension with the
concentration of 107 CFU/mL was prepared by transferring 1 mL of washed cells into
99 mL of phosphate buffer. Five-ml portions were transferred to screw-capped Pyrex
tubes (10 x 130 mm) which were kept in ice-water bath prior to the heating
75
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experiments. The tubes were protected against leakage during heating by using caps
with liners made of silicone septa (gas-chromatography type). This cap successfully
prevented leakage of the water from the bath into the submerged tubes during the
heating process.
Heating challenge
Cell suspensions were heated at 57°C in the temperature-controlled water bath
equipped with a Haake E2 (Haake Buchler Instruments Inc., Saddle Brook, NJ.) heating
element.
Tubes were totally immersed in water and secured to prevent them from
floating to the surface. The bacterial suspension was subjected to a total of 50 or 60
min of heating. Immediately after heating, tubes were transferred to an ice-water bath.
Five independent experiments were earned out and three replicate tubes were used per
each heating time. Heating time intervals were selected after conducting a series o f
preliminary experiments that showed appropriate time spans to be used in order to
obtain a population of highly heat-stressed microorganisms.
Recovery conditions
Four types of recovery media were used: Tryptic soy agar (TSA), Tryptic soy
agar +1% sodium pyruvate (TSA + 1% NaPyr), Plate count agar +1% sodium pyruvate
(PCA + 1% NaPyr) and Phenol red sorbitol agar + 1 % sodium pyruvate (PhRSA +
1%NaPyr). The efficiency o f two plating techniques, pour plates and spread plates,
were tested using TSA media. Spread plates were used with three other media - TSA
+ 1% NaPyr, PCA + 1% NaPyr, PhRSA + 1%NaPyr. Media powders TSA, PCA, PhRA
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and Sorbitol were purchased from Difco Laboratories, (Detroit, Ml.). Sodium pyruvate
was obtained from Sigma Chemical Co. (S t Louis, MO.). All plates were prepared a
day before use and stored at 4°C. Prior to inoculation with the heated suspension of E.
coli 0157:H 7 the plates were warmed to room temperature. The same lots of media
were used throughout the experiments.
Butterfield's phosphate buffer was used to
dilute heated suspensions. One-tenth ml of diluted sample was transferred into Petri
plates with appropriate media and inoculum was spread on the surface of agar with
individually-sterilized L-rods. For pour plates, TSA liquid agar was tempered to 45°C
before being used. All plates were incubated at 37°C and counted after approximately
78 hr.
Statistical Analysis
An analysis of variance (Anova) was performed using Excel software (Microsoft
Corp., Redmond, WA.) to determine if results were statistically different (Devore and
Peck, 1989).
Results
Efficiency of plating techniques using TSA showed that the spread plate
technique rather than pour plate method was more effective in the recovery of heatstressed microbial subpopulations.
Significantly more (>1.2 log) CFU/mL were
recovered for 60-min heated bacteria on spread plates compared to pour plates (Table
1).
As Table 2 illustrates, the population of unheated E. coli 0157:H7 was
approximately 107 CFU/mL, irrespective of the recovery media (p < 0.05). When the
77
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cell suspension was exposed to a 50 min heat treatment at 57°C, there was
approximately a 2-log reduction in numbers.
type of media used.
Exact numbers obtained depended on
PCA + 1% NaPyr resulted in recovery of significantly higher
numbers of heated E. coli 0157:H7 than other media (p < 0.05). When the cells were
heated for 50 min, the PhRSA + 1% NaPyr enumerated 16.3%, and the TSA + 1%
NaPyr enumerated 12.9% of the total population observed on PCA + 1% NaPyr (Table
3).
The difference in numbers of CFU/mL counted on TSA + 1%NaPyr, PhRA +
1%NaPyr and spread and pour plates with TSA was noticed, but differences were not
statistically significant at the p < 0.05. For highly stressed cells heated at 57°C for 60
min, the differences between the efficiency of three media with pyruvate were more
pronounced (Table 2). The PCA + 1%NaPyr continued to be an excellent recovery
media as was observed for E. coli 0157:H7 heated fo r 50 min. The number of bacteria
enumerated with this media was 105 CFU/mL compared to other media which gave 102
or lower CFU/mL.
On media with added pyruvate enumeration of severely heated cells was
greater than on media without pyruvate, but the type of media to which pyruvate was
added played an important role. Pyruvate incorporated into PCA is more effective than
when added to TSA or PhRSA. The last two media with pyruvate supported growth of
the same number o f viable cells since the CFU/mL recovered with both media were not
significantly different (Tables 2 and 3). The number of unheated E. coli 0157:H7 on
each medium was similar (p<0.05) and was not influenced by media type, presence of
pyruvate or type of plating method used.
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Discussion
Results of this research and other studies indicate that pyruvate enhances
recovery of heat-stressed cells.
Sodium pyruvate was recognized as an important supplement to the media
since the report of Baird-Parker and Davenport (1965). They showed that recovery o f
heated and dried Staphylococcus aureus cells was the greatest on media containing
sodium pyruvate and blood.
It was concluded that heating or drying processes
destroyed the cells' ability to protect itself against toxic oxygen compounds. Damaged
S. aureus cells lacked catalase, thus pyruvate and catalase in the blood added to the
agar became powerful decomposers of hydrogen peroxide.
Baird-Parker agar, as
tested by many groups (Flower et al., 1977; Hurst et. al, 1976) appeared to be the
superior recovery media fo r S. aureus and the presence of sodium pyruvate
significantly contributed to the recovery of stressed cells. Researchers (Brewer et al.,
1977; Flow eret al., 11977; Martin et al., 1976) that reported on recovery of S. aureus
partially elucidated the role of pyruvate and catalase during the repair process of
stressed cells. These observations encouraged others to continue research on the role
o f catalase in the recovery o f injured microorganisms. New media supplemented with
pyruvate were formulated. Flowers et al. (1977) found that incorporation of catalase
into media increased the number of recovered cells (heated, reduced water activity, or
freeze-dried) up to 1,100-fold. In the report by Brewer e t al. (1977), it was shown that
pyruvate was effective in degradation of hydrogen peroxide.
When the enzyme
catalase was added to the media, increased numbers (MPN) were observed for heat-
79
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stressed and nonheat-stressed bacteria present in ground beef samples. The addition
of 10% NaCI to liquid medium did not change the observation. Based on the study of
Martin et al. (1976) the positive impact o f pyruvate and catalase supplements on the
recovery of physically and chemically injured bacteria was extended to Gram-negative
species: Salmonella typhimurium, Pseudomonas fluorescens and E. coli.
One of the first events
occurring
during
the
thermal
inactivation
of
microorganisms is the loss of enzymes that protect the cells against destructive oxygen
species and lig h t Therefore, in order to resuscitate an injured cell, it is imperative that
the media possess an oxygen scavenging property. Media supplemented with catalase
will protect injured cells against oxygen radicals. Pyruvate has been claimed to play a
similar role to catalase.
Creating anaerobic conditions in the absence of light,
minimizes the production of undesirable oxygen species and improves the recovery
process. Mossel and van Netten (1984) reported that incubation o f stressed cells in a
liquid medium is superior to solid agar media.
The advantage o f recovering the
stressed cells in an anaerobic environment was demonstrated in studies presented by
Andrews (1989) and Hurst (1984). An effective repair procedure based on the agar
double layer developed by Ray and Speck (1978) certainly allowed the recovery of
bacteria in the low p 0 2 condition.
Other methods include using the commercially
produced enzyme 'Oxyrase' which converts 0 2 to H20 2 thereby reduces the oxygen
tension
of
medium
and
consequently
establishes
anaerobic
conditions
microorganisms (Fung, 1992).
80
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for
Two structurally different types of catalase enzymes have been identified in E.
c o li: catalase HPI (encoded by katG gene) and catalase HPII (product of katE gene)
(Loewen, 1984). The reason for which the bacterial cell produces two sim ilar enzymes
is that they protect the cell in different circumstances (Hengge-Aronis, 1993). Catalase
HPI is a part of cell adaptive system (oxidative stress-induced regulon) functioning
during the growth stage, whereas catalase HPII production is induced during entry into
the stationery phase. The level of catalase HPI is related to the p 0 2 growth conditions.
The more oxygenic the environment, the higher the amount o f hydrogen peroxide
produced, and the more catalase HPI is needed to degrade it Therefore, accumulated
H20 2 triggers transcription of catalase HPI and regulates its amount (Christman et
al.,1985). Additionally, due to cell protective needs, the cellular location o f these two
enzymes is different. HPI is present in the periplasm and inhibits undesired transfer of
H20 2 into the cytoplasm. In turn, catalase HPII, located in cytoplasm, is able to protect
cell genetic material during oxidative stress in the stationery phase. HPII belongs to the
group of starvation inducible proteins as described by Jenkins et al. (1988).
In searching for optimal recovery media, pyruvate and catalase are becoming
important supplements for the recovery of stressed cells of various species.
It was
shown that pyruvate increased the number o f cells recovered after freezing and heating
with E. coli (McDonald et al., 1983), E. coli 0157:H 7 (Line et al., 1991), heat-stressed
and uninjured Shigella flexneri (Smith and Dell, 1990), heat-stressed Salmonella
senftenberg (Raymen et al., 1978), S. aureus (Hurst et al., 1976), Clostridium
perfringens (Hood et al., 1990) and fungi isolated from foods (Koburger, 1986).
81
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A
concentration of 1% is commonly used, but the am ount of pyruvate recommended as a
media supplement was as low as 0.02% (Lee and Hartman, 1989).
In our studies we found that pyruvate and the type of medium are important
factors in recovery process. TSA + 1%NaPyr resulted in enumeration of 10 times the
CFU/mL that TSA, but the biggest difference was with PCA.
Synergistic effects o f
pyruvate and PCA components allowed recovery of four more logs of severely heated
E. coli 0157:H7 compared to commonly used TSA pour plates. After comparing the
ingredients o f PCA and TSA (Table 3) it can be concluded that the presence of glucose
and yeast extract in PCA may be as important as incorporation of pyruvate. The repair
process of heat-damaged bacteria may require energy source components such as
glucose and additional growth factors which are available in the yeast extract agar. It is
known that glucose enhances recovery o f stressed cells (Draughon and Nelson, 1981).
Two other media, TSA + 1%NaPyr and PhRSA + 1%NaPyr, lack these compounds.
Unheated bacteria are not affected by these factors.
Although pyruvate is recognized as a oxygen radical scavenger in many reports,
the mechanism of this reaction has not been clearly explained. It would be of interest
to study the potential role of pyruvate as an energy generating compound that is
utilized by unheated and heat-injured bacteria under aerobic and anaerobic conditions.
This hypothesis is supported by the fact that E. coli possesses a specific pyruvate
transport system through which pyruvate can enter the cell (Lang et al., 1987). Studies
by Heinmets et al. (1954) indicated a possible involvement of pyruvate as a substrate
fo r energy production, but Baird-Parker and Davenport (1965) did not support that
82
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theory. D'Aoust (1978) studied the effect of pyruvate and other intermediates o f the
TCA
cycle
(e.g.
citrate,
succinate,
phosphoenolpyruvate) on
recovery
of
S.
typhimurium. Under the conditions that they used, pyruvate supported the recovery of
70% of the initial population but media with lactate appeared to be better and
recovered 100% of the population.
Tomlins et al. (1971) determined the effect of
thermal injury on TCA cycle enzymes of S. aureus heated at 52°C and S. typhimurium
heated at 48°C.
They reported heat denaturation of oxaloglutarate dehydrogenase
enzyme which caused the loss of metabolic activity of S. aureus cells, but there was no
apparent enzyme inactivation in heat-injured S. typhimurium.
Another conclusion that was reached in our studies is that the spread plate
technique resulted in better recovery of more severely stressed bacteria than did the
pour plate method.
It can be speculated that the exposure of injured cell to the
tempered agar at a temperature of 45°C decreased the chance of cell recovery. This
short and mild heat exposure did not affect unstressed bacteria. A sim ilar conclusion
was reached by Mossel and van Netten (1984).
Clark (1967) and Ray and Speck
(1973) reported that the spread plate technique is better than the pour plate technique
in recovery of injured bacteria. This observation shows how demanding the stressed
microorganisms are and how important the formulation of optimal recovery condition is
to determine the number of viable cells after heat treatment.
83
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1988.
Starvation-induced cross
protection against heat or H20 2 challenge in Escherichia coli. J. Bacteriol. 170:39103914.
Koburger, J. A. 1986. Effect of pyruvate of fungi from foods. J. Food Prot.
49:231-232.
Lang, V. J., C. Leystra-Lantz. and R. A. Cook. 1987. Characterization o f the
specific pyruvate transport system in Escherichia coli K-12. J. Bacteriol. 169:380-385.
Lee, R. M. and P. A. Hartman. 1989. Optimal pyruvate concentration for the
recovery of Coliforms from food and water. J. Food Prot. 52:119-121..
Line, J. E., A. R. Fain, A. B. Moran, L. M. Martin, R. V. Lechowich, J. M.
Carosella and W . L. Brown. 1991. Lethality of heat to Escherichia coli 0157.H7: Dvalue and z-value determinations in ground beef. J. Food Prot. 54:762-766.
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Loewen, P. C.
1984.
Isolation of catalase-deficient Escherichia coli mutants
and genetic mapping of KatE, a locus that affects catalase activity.
J. Bacteriol.
157:622-626.
Martin, S. E., R. S. Flowers and Z. J. Ordal.
1976.
Catalase: its effect on
microbial enumeration. Appl. Environ. Microbiol. 32:731-734.
McDonald, L. C., C. R. Hackney and B. Ray.
1983.
Enhanced recovery of
injured Escherichia coli by compounds that degrade hydrogen peroxide or block its
formation. Appl. Environ. Microbiol. 45:360-365.
Mossel, D. A. and A. P. van Netten. 1984. Harmful effects of selective media
on stressed micro-organisms: nature and remedies, p. 329-371. In M. H. E. Andrew
and A. D. Russell (ed.) The Revival of Injured Microbes. Academic Press. New York.
Ray, B. 1989. Enumeration o f injured indicator bacteria from foods, p. 9-54. In
B. Ray (ed.) Injured Index and Pathogenic Bacteria: Occurrence and Detection in
Foods, W ater and Feeds. CRC Press, Inc. Boca Raton, Florida.
Ray, B. and M. L. Speck.
1973. Enumeration of Escherichia coli in frozen
samples after recovery from injury. Appl. Environ. Microbiol. 25:499-503.
Ray, B. and M. L. Speck.
1978.
Plating procedure for the enumeration of
coliforms from dairy products. Appl. Environ. Microbiol. 35:820-822.
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Rayman, M. K., B. Aris and H. B. El Derea. 1978. The effect o f compounds
which degrade hydrogen peroxide on the enumeration of heat-stressed cells of
Salmonella senftenberg. Can. J. Microbiol. 24:883-885.
Smith, J. L. and B. J. Dell. 1990. Capability o f selective media to detect heatinjured Shigella flexneri. J. Food P ro t 45:360-365.
Speck M. L. (ed.)
1984.
Compendium of Methods for the Microbiological
Examination of Foods. American Public Health Association. Washington, D. C.
Tomlins, R. I., M. D. Pierson and Z. J. Ordal. 1971. Effect o f thermal injury on
TCA cycle enzymes o f Staphylococcus aureus MF 31 and Salmonella typhimurium
7136. Can. J. Microbiol. 17:759-765.
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 1. Comparison of plating methods. Population of E. coli 0157:H7 heated at 57° C and recovered on Tryptic Soy Agar by
spread and plate methods. Statistical analysis of results0.
Log of E. coli 0157:H7 cells ± 95% confidence interval
Media - plating methods
0 min heatingb
0 min heating®
0 min heatingd
A) TSA - spread plates
7.04 ±0.17
4.16 ±0.41
2.37 ± 0.58
B) TSA - pour plates
7.11 ±0.06
3.87 ± 0.39
1.17 ±0.55
a - Single-factor ANOVA analysis. The F, F critical and p-values with alpha = 0.05 were calculated. When p<0.05, there is
statistical difference between media
b - (AxB) p = 0.144 - no significant difference between plating methods
c - (AxB) p = 0.085 - no significant difference between plating methods
d - (AxB) p = 0.007 - significant difference between plating methods
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Table 2. Population of E. coli 0157.H7 after heating at 57°C and recovered on various media by spread methods. Statistical
analysis of results".
Media with pyruvate
(spread plates)
Log of E. coli 0157:H7 cells ± 95% confidence interval
0 min heatingb
50 min heatingc,d
60 min heating6’1
A) PCA + 1% NaPyr
7.00 ±0 .1 0
5.21 ±0 .3 3 °
5.04 ± 0.47
B) TSA + 1% NaPyr
7.07 ± 0.06
4.43 ± 0.24
2.78 ± 0.34
C) PhRSA + 1% NaPyr
7.01 ±0.10
4.32 ± 0.25
2.83 ± 0.36
* - Single-factor ANOVA analysis. The F, F critical and p-values with alpha = 0.05 were calculated. When p<0.05, there is
statistical difference between media
b - (AxBxC) p = 0.427 - no significant difference between plating methods
0 - (AxB) p = 0.00025 - significant difference between plating methods
d - (BxC) p = 0.693 - no significant difference between plating methods
e - (AxB) p = 22 x 10'7 - significant difference between plating methods
f
' (BxC) p = 0.829 - no significant difference between plating methods
90
Table 3. Percent o f enumerated E. coli 0157:H7 on media, unheated and heated at
57°C. Numbers log N were compared to the population recovered on the most effective
media - Plate count agar + 1% sodium pyruvate.
Medium*
Percent recoveryb
0 min (unheated)
60 min at 57°C
60 min at 57°C
Comparison of olatina methods
TSA - spread pi.
110.11
8.73
0.21
T S A -p o u r pi.
131.20
4.48
0.01
P C A+1% NaPyr. - spread pi.
100.00
100.00
100.00
TSA + 1% NaPyr. - spread pi.
118.32
16.34
0.55
PhRSA + 1% NaPyr. - spread pi.
103.50
12.88
0.61
Media with Dvruvate
* - Media ingredients. Per liter of water, media are comprised of: PCA (5 g
tryptone, 2.5 g yeast extract, 1 g glucose), TSA (15 g tryptone, 5 g soytone, 5 g NaCI),
PhRSA (5 g NaCI, 10 g proteose peptone, 1 g beef extract, 10 g sorbitol, 25 mg phenol
red)
b - Percent o f recovered bacteria was calculated as follows: CFU/mL on medium
was divided by CFU/mL on PCA + 1% NaPyr and multiplied by 100%.
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Inactivation rates of Escherichia coli 0157:H7 heated
conventionally and by microwaves
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Abstract
The inactivation rates of Esherichia coli 0157.H7 heated conventionally and by
microwaves during isothermal conditions were compared. A microwave system that
maintains constant temperature conditions was constructed for this study.
Survivor
curve experiments fo r E. coli 0157:H7 suspended in phosphate buffer were carried out
at 57°C and 60°C. Two parallel experiments were performed, one in the microwave
system and one in a water bath, to test the difference between both modes o f heating
in relation to the kill rate of bacteria. Regression analysis of the survival data were
performed for the individual tests and they revealed that the D57C values for the MW
heated suspensions were higher than for the conventionally heated suspensions and
average Dsrc values were 19.77 min and 11.96 min respectively. In contrast the D«>c
were higher for the conventionally heated E. coli 0157:H7 suspension than fo r the
microwaved suspensions, and these average O-values were 2.49 min and 1.93 min
respectively. A parallel model with a categorical predictors compared regression lines
from the microwave and the water bath experiment, and analyzed data fo r a treatment
effect. This statistical calculation revealed that in 13 out of 18 individual experiments
no significant difference was observed between the microwave and the water bath
heating. Additional experiments were carried out to seek more conclusive results. Two
survivor tests at 60°C were performed during which thermal profiles o f the heated
samples were collected. The lethality delivered to individual vials was calculated from
the time-temperature data and compared to the number of survivors recovered from
these samples. It was found that the difference in lethality delivered to each sample of
E. coli 0157:H7 suspensions can be attributed to the low number o f survivors rather
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than the difference between treatments. As the thermal profiles showed, the lethality
varied between the samples not only these heated in the microwave system but also in
the water bath.
Unless microbial suspensions w ill receive an equal amount o f the
lethality during the microwave and the conventional heating no claim can be made
about the difference between these two heating modes in relation to the destruction of
bacteria.
Introduction
A growing application o f microwaves in the food industry and popularity of
microwave
ovens
among
individual
consumers
raises
the
question
of
the
microbiological safety o f microwave heated foods (Spite, 1984; Knutson et al., 1988;
Schnepf and Barbeau, 1989; Dealier and Lacey, 1990; Hollywood et al., 1991; Quan et
al., 1992). In respond to consumers' and food developers’ needs a significant amount
of research on the use o f microwaves for microbial control has been earned out over
recent years.
However, a methodology that enables prediction of appropriate
processing parameters is still under development (Ayappa et al., 1992; Nelson and
Labuza 1992; Holyoak et al., 1993; Huang et al., 1993; Gundavarapu et al., 1995) The
difficulty in working with microwaves resulted in many o f the reports to be either
inconclusive or unable to predict appropriate processing which was summarized by
Heddleson and Doores (1994), Rosenberg and Bogl (1987) and Knutson et al. (1987).
Information on the resistance o f bacteria exposed to microwaves is needed in order to
develop a method for predicting the parameters of microwave thermoprocesing.
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The effect of microwaves on bacteria has been a subject o f interest conducted
over the last few decades.
Many experimental studies (Goldblith and Wang, 1967;
Lechowicz et al., 1969; Dreyfuss and Chipley; 1980; Rosenberg and Sinell, 1989;
Fujikawa et al., 1992; W elt et al., 1994) and theoretical discussion (Frohlich, 1975;
Pickard and Rosenbaum, 1978; Edwards et al., 1984) have been published to elucidate
the mechanism of microbial destruction and growth of bacteria exposed to microwave
irradiation.
The major factors that contributed to this perceived problem were: a)
complicated scientific laws that govern microwave radiation as they combine knowledge
from many areas including optics, electromagnetic theory, heat and mass transfer
(Metaxas and Meredith, 1983; Mudgett, 1986; Datta, 1990); b) lack o f understanding of
the biophysical aspects of microwave-bacteria interactions (Thuery, 1992), c) lack of
understanding the engineering principles of microwave heating by researchers,
especially microbiologists (Stanford, 1990); and d) the lack of appropriate technology to
monitor or control critical parameters during microwave heating (W elt et al., 1993).
Nevertheless, significant progress has been accomplished.
The possibility of
forming toxic compounds has been excluded, because an energy of 2450 MMz
frequency does not alter strong molecular bonds. It is too low to form new chemical
bonds and consequently new molecules (Stuchly, 1978; Thuery, 1992). The problem
whether the electric field
(Sastry and Palaniappan,
1991) or magnetic field
(Pothakamury et al., 1993) impairs metabolism of bacteria has been addressed. No
strong evidence has been found to support this claim.
Any additional influence of the
electric or the magnetic fields on the bacteria other than those causing an increase of
temperature has been neglected.
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There is general agreement that the death o f bacteria subjected to microwave
irradiation is caused exclusively by thermal effects, and the death o f the microbial cell is
a function of the temperature (Heddleson and Doores, 1994; W elt et al., 1994).
The theory that is discussed currently is that there is an enhanced microbial kill
under microwave heating as a result of selective absorption of microwaves (Mudgett,
1989).
Some researchers claim that a temperature gradient is created within the
microbial cell due to various dielectric properties of cell constituents (Khalil and Villota,
1988,1989). Selective absorption of microwaves could lead to localized heat formation
and possibly higher temperatures within the cell than the temperature of the
environment surrounding the cell (media, food system).
Thus, the sequence of the
thermal events occurring within the cell may not be the same as in conventional
heating. This question is not o f practical importance as long as the killing rate or the
lethality of bacteria is equal in both processes. Moreover, if the temperature within the
cells are higher than surrounding medium, then bacteria are killed faster as compared
to a conventional treatment where heat is transferred from the medium to the cells.
This could possibly have an effect on the process of recovery of the injured cells, since
the site of injury could be different in microwave heated cells than cells heated
conventionally.
Sastry and Palaniappan (1991) reported that calculations based on heat
transfer principles reject this claim.
Additionally, the reports of (Kahail and Villota,
1988, 1989) suggest that the selective absorption of microwaves by microbial cells
failed to demonstrate an isothermal condition of the microwave heating. A constant
temperature
observed
on
the
time-temperature
profiles
of
heated
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microbial
suspensions during exposure to microwave are the fundamental data that have to be
reported to support such experimental conclusions.
Before these controversial and interesting problems w ill be fully resolved the
only method to determine the D and z-values for bacteria, and using these values in the
calculation of the pasteurization time, is to conduct the survivor curve study during
isothermal microwave heating. The heat inactivation parameters of the pathogen of
interest can be established using a constant temperature microwave system, rather
than assuming that microbial destruction parameters from microwave and conventional
heating modes are equal.
There is a technology available now to conduct such a
study. Recently, W elt et al., (1992) introduced a suitable apparatus capable of heating
liquids at constant temperature.
There were excellent studies on reaction kinetics of biological systems during
microwave heating where temperature profiles were collected during heating and
compared to inactivation rates.
However, these studies dealt mostly with enzymes
(Jahngen et al., 1990, W e lt e t al., 1993) or spores (Jeng et al., 1987; W elt et al.,
1994).
Research is needed that would show clearly that the resistance o f bacteria
during microwave heating is a function of the temperature in the heated environment
created during this process, and the 0 and z-values are not different as in conventional
heating at this same temperature.
Therefore, the objective o f this research was to compare the inactivation kinetics
of E. coli 0157:H7 in phosphate buffer during microwave exposure with corresponding
values obtained during conventional heating. An effort was made to subject bacteria to
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an equal amount o f heat as measured by the time and the temperature parameters. To
achieve these objectives the following steps were incorporated into this study: a)
constructing a microwave system that would maintain constant temperature; b)
establishing the 0 and z-values obtained during microwave and water bath treatments;
c) performing a statistical test to compare conventional and microwave heat treatments;
d) analyzing the thermal profiles of the heated samples and relate these data to the
number of survivors;, and e) studying injury of the bacteria during microwave and
conventional heating by using two different recovery media.
Materials and Methods
Bacterial strain
The organism used in this study was E. coli 0157:H7 strain 933, obtained from
the Food and Drug Administration Laboratory (Minneapolis, MN) and maintained in 10%
(vol/vol) mixture of glycerol in tryptic soy broth [(TSB) (Difco; Detroit, Ml)] at -60°C.
Cells were grown to the stationary phase (approx. 109 CFU/mL) in TSB at 37°C fo r 24
hrs. They were propagated by daily transfer o f one loopful of the fresh cell suspension
to 10 mL TSB.
The cells were harvested by centrifugation (17,000 x g, 20 min),
washed and resuspended to the original volume in Butterfield’s buffer (pH = 7.2)
(Speck, 1984). The cell suspension used in the heating test was prepared by mixing 10
mL of washed cells with 990 mL of Butterfield's buffer (Sigma Chemical Co., SL Louis,
MO).
Twenty-mL and five-mL aliquots (approx. concentration 107 CFU/mL) were
transferred to EPA tubes (28 x 95 mm) and Pyrex tubes (10 x 130 mm) that were used
in microwave and water bath heating tests respectively.
The tubes with the E. coli
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0157.H7 suspensions were kept in an ice-water bath prior to the survivor curve
experiments.
Initial counts in the suspensions and the number o f cells that survived
microwave and conventional heating were determined by spread plating. Two types of
recovery media were used: tryptic soy agar [(TSA) (Difco; Detroit, Ml)] and plate count
agar (Difco; Detroit, Ml) supplemented with 1% sodium pyruvate [(PCA + 1 % NaPyr)
(Sigma Chemical Co., MO)]. The latter medium was found to be an excellent recovery
medium fo r heat-stressed E. coli 0157:H7 as previously reported. Samples o f the cell
suspension (heated or control) were serially diluted in Butterfield’s phosphate buffer.
One-tenth mL o f the diluted sample was transferred into each o f two duplicate plates
with appropriate media. The same lot of media was used throughout the course of this
study. Plates were incubated at 37°C and counted after approximately 78 hrs, the time
necessary to recover the heat-injured E. coli 0157:H7 cells.
Microwave system maintaining constant temperature
E. coli 0157:H7 inactivation kinetics studies during were performed microwave
heating under constant temperature conditions.
W elt e t al. (1992) described an
apparatus that permits the maintenance of uniform temperature of liquid samples
during microwave irradiation. This design was adapted for the purpose of this study.
Modification of microwave oven
A Quasar microwave oven (model MQS 1403, Quasar Co., Elk Grove, IL) ) was
selected fo r this study. The Quasar oven had a uniform heating pattern inside the oven
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cavity (Buffler, 1992; Lentz, 1993).
It did not have a turntable; therefore fiberoptic
temperature probes could be used. This model had a large cavity (1.4 c u .ft) and was
rated at 900W power output by manufacturer. The oven power circuit was modified to
deliver a continuous variable power as is presented in Figure 2.
A filam ent power
transformer, a variable transformer [(variac) (model 05F1303; Staco Energy Product
Co., Dayton, OH)] and a current panel meter were incorporated into the circuit. The
variac possessed 0 to 100 settings fo r manual power reduction.
The current meter
reflected the power delivered by a magnetron to the oven cavity.
The constructed microwave system consisted o f a modified microwave oven
and two additional loops: a feedback temperature control loop and a sample agitation
loop. The assembled microwave system is shown in Figures 1 and 3.
The temperature control loop
The feedback temperature control loop consisted of a mechanical power relay,
a computer and temperature-sensing equipm ent
The relay had a 120-ohm coil
resistance and was switched on by 12V DC source. The miniature power relay was
connected to an IBM computer called the oven power computer. A computer program
was developed fo r the control of the temperature by Lentz (1993).
The set-point
temperature was entered into the computer software menu prior to the experiment.
The computer sent the TTL (transistor - transistor logic) signal to the optocoupler which
turned the mechanical relay on. The magnetron was pulsed on/off when the sample
temperature was below or above the desired value.
Occasionally, the liquid
temperature reached higher than the set-point level because of an overheating
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phenomenon. This was strongly observed when the magnetron operated at full power.
To minimize this effect, the variac setting was changed manually from 100 to
approximately 70 for the 55 to 60°C temperature range used in this study. A t the lower
setting, the transformer possessed a lower output voltage, the magnetron functioned at
the reduced power, and consequently the sample was heated gradually.
This
prevented overheating of sample and caused a small fluctuation of temperature of the
heated suspension. The power delivered to the oven cavity was read on the 0 to 500
mA miliammeter ( O m A - O W power; 300 mA - full power).
Sample agitation loop
This loop was built into the microwave system to ensure a uniform temperature
throughout the sample during the microwave exposure. The sample agitation loop
consisted of a stepper motor, the second computer (called stepper motor computer)
and a signal booster circuit.
This computer was equipped with a digital I/O board
[(model PCDIO 24-P, XT/AT compatible) (Industrial Computer Sources, San Diego,
CA)]. The computer was connected to the signal booster circuit through the cable and
the screw termination [(model CAB50A-6)(lndustrial Computer Sources, San Diego,
CA)]. A small stepper motor was install on the side of the back wall of the oven by a
standard laboratory stand with a clamp. The shaft of the stepper motor was mounted in
the nylon rod that connected it with the sample holder placed in the oven cavity. A hole
was drilled in the back wall o f the oven to accommodate the coupling rod. A computer
program was written (Lentz, 1993) to operate the stepper motor.
The appropriate
agitation parameters were found to assure that the suspension was well mixed during
exposure to microwaves.
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Temperature measuring system
The temperature measuring system consisted of a Luxtron [(Model 750)(Luxtron
Corp.; Santa Barabara, CA)] unit connected to the power oven computer through the
RS-232 cable. The Luxtron temperature-sensing unit was equipped with four fiberoptic
temperature probes that were calibrated with an accurate thermometer (readings
traceable to the NBS temperature standards).
The calibration of the Luxtron
temperature probes was carried out prior to each survivor curve experim ent
The
fiberoptic probes were cleaned between runs with 400ppm quaternary ammonium
prepared in 70% ethyl alcohol.
Sample holder and sample enclosure
The sample holder and sample enclosure were designed to accommodate a
glass vial with the E. coli 0157:H7 suspension in the oven cavity. The sample holder
was an L-shaped, structural support made o f nylon. The top o f its vertical bar was
attached to the coupling rod connecting the holder with the shaft o f the stepper motor.
The horizontal bar of the holder supported the bottom of the glass vial. Two Velcro
strips tightly attached the vial to the side of the vertical bar. This support system that
stabilized the vial during agitation is shown in Figure 4. The 20-mL liquid sample was
enclosed in an EPA type (28 x 95 mm) glass vial. The EPA tube had a 30-mL total
capacity and was closed with a screw cap.
The fiberoptic temperature probe was
inserted in the center of the liquid sample. A hole (2-mm diameter) was drilled in the
phenolic cap of the vial. The caps contained liners which were cut out from the gas
chromatography septa [(214495)(Dionex Corp.; Sunyvale, CA)]. The liners protected
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the sample from leakage and evaporation. This same septum, but without reducing the
size, was inserted in the center of the heated liquid in the horizontal position.
The
septum formed a support for the temperature probe inside the vial and ensured that the
temperature was controlled at this same location fo r various samples. Several holes
were cut out in each septum to allow for free circulation o f the liquid in the vial.
Operation o f the microwave system - setting proper parameters
A series o f tests were performed to establish proper agitation parameters, variac
operation conditions and the experimental protocol fo r heating o f the cell suspensions.
The vials with 20 mL of w ater were kept in the ice w ater until they were placed inside
the oven cavity.
The Luxtron probe was inserted in the center o f the liquid. The vial
was placed in the sample holder inside the oven. The agitation system was turned on
several seconds before the heating to obtain a uniform temperature gradient within the
sample. The set-point temperature was entered into the computer program and the
microwave heating was initiated. The Luxtron probes monitored the temperature within
the sample every 1 sec. It was noticed that w ater reached the desired temperature of
55°C after 15 sec o f heating. When the temperature was above 55°C, a signal was
sent to the computer and the magnetron was turn off. Operation of the magnetron was
initiated when the temperature value decreased to 55°C o r below. Figure 5 shows the
temperature fluctuation observed and the initial overheating o f the sample.
To
eliminate these problems, a series of tests were conducted at a reduced microwave
power by changing the transformer setting from 100 to a lower value. The stability of
the temperature was achieved at a power setting o f 70.
In the initial stage of the
microwave exposure (14 to 15 sec), the magnetron operated at full power (100) in order
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to minimize a come-up time and quickly bring the temperature of the sample to the
desired level. Immediately, the power was changed by switching the variac setting from
100 to 70. Optimal agitation of the sample was maintained by rotating the vial from the
left horizontal position to the right horizontal position, allowing fo r 180° turns. Figure 5
presents a comparison o f temperature profiles obtained fo r 20 mL of water heated in
the Quasar oven during three operating modes: a) the regular mode; b) the
temperature control mode at full power; and c) at the power reduced to 70% after initial
15 sec of come-up time at 100 setting. After heating, the computer was turned off
which terminated the operation o f magnetron. The vial was quickly removed from the
oven cavity and immersed in ice water.
These parameters were followed during survivor curve experiments.
Conventional heating in the water bath
Heating conditions
Conventional heating was earned out in the temperature-controlled, circulated
water bath equipped with Haake E2 (Hakke Buchler Instruments Inc., Saddle Brook,
NJ). The temperature of the water in the water bath fluctuated within ± 2°C from the
desired 57°C and 60°C levels. The microbial suspension was heated in screw-capped
Pyrex tubes (10 x130 mm). The tubes containing 5-mL cell suspensions were totally
submerged in the water.
To protect tubes against leakage, special cap liners were
made from silicone septa (gas-chromatography type). Leakage tests were conducted.
The vials with 5 mL of water were placed in the water bath containing water mixed with
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green food coloring. This cap successfully prevented leakage o f the green water into
the tubes. The tubes were heated individually during survivor curve tests.
Lag correction factor of the Pyrex tubes
Lag correction factor was determined for the 10 x 130 mm Pyrex vials with 5 mL
of water to account for the time necessary for the liquid to come-up to the desired 57°C
temperature.
The heat penetration test was earned out using the Luxtron probes
placed inside the tube in the slowest heating zone.
In the convection heat transfer,
this point is located a small distance from the bottom of the vial - approximately 1/10 of
the liquid height.
The vial was transferred from the ice water to the water bath
maintained at 57°C. Vials were submerged in water and the temperature was recorded
every 1 sec until water in the vial achieved a constant 57°C. Four replicate tests were
conducted and the data are reported in Table 1. The General Method was used to
calculate lethality rates assuming a z-value of 5°C as an appropriate z-value fo r this
process (Pflug, 1988). The protocol o f the calculation process to determine the lag
correction factor is outlined in Figure 6. The lag correction factor fo r the Pyrex vials (10
x 130 mm) used in the water bath experiments was found to be 2 min. Thus, each
heating time used in the survivor curves experiment carried out in the water bath was
increased for 2 min.
Heat inactivation studies during isothermal microwave heating and conventional
heating
Heat destruction tests fo r E. coli 0157:H7 were designed as a comparison study
between microwave and conventional heating.
All steps in these parallel experiments
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were identical, except the heat treatments. Preparation o f the vials fo r the microwave
and the water bath test, the heating process, the plating of controls and heated
suspensions on the agar plates were performed on the same day.
Survivor curve
experiments fo r E. coli 0157:H7 were conducted at two temperatures:57°C and 60°C.
In order to choose appropriate heating times and temperatures, a preliminary study was
conducted. The following heating times were selected:
57°C test - 8 min, 18 min, 28 min, 38 min, 48 min, 58 min
60°C test -1 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min
Three experiments were performed at 57°C and six experiments at 60°C. Two replicate
vials were used per each heating time. Serially-diluted samples from each vial were
plated, in duplicate, on two types of agar media, the TSA and the PCA + 1 % NaPyr.
The spread plate method was used. Each microwave heating test consisted of 14 to
15 sec come-up time (100 variac setting) and heating time listed above when the
temperature of 57°C or 60°C was reached (70 variac setting). The variac setting was
changed manually during each heating test after 14 to 15 sec.
Each water bath
heating test was 2 min longer than the desired heating time because of the additional
2-min lag correction factor. The test tubes before and immediately after the heating
were cooled in the ice water.
Plates from both experiments were incubated at the
same conditions as described previously.
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Lethality experiments
The lethality studies consisted of the survivor curve tests during which heat
penetration data fo r the heated suspensions were obtained. The survivor curve tests
were performed in the microwave system and the w ater bath as described previously.
The only difference was that the temperature probes were inserted into heated vials.
The Luxtron probes monitored the temperature within the heated vial during the 2-min
come-up time, appropriate heating time and a 2-min cool-down time. Temperature data
were recorded every 1 sec. The vials used in the water bath studies were modified for
the lethality experiments. In order to place the fibroptic probe within the liquid, a hole
was drilled in the cap, and the liner was punctured to insert the temperature probe. The
tip of the probe was placed in the required distance from the bottom of the vial. To
record the temperature in the microwave system during the cooling stage, the computer
was not terminated after the heating but, instead, the power setting on the variac was
reduced to zero. The sample with the temperature probe was removed from the cavity
and transferred to the ice water.
During the lethality test, the temperature of the w ater in the water bath and the
temperature of the air in the microwave oven cavity was monitored with the Luxtron
probes to follow any unusual changes in these heating environments.
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Evaluation o f thermal destruction data - statistical analysis
C alculation o f D-and z-values data
The survival data were analyzed following a procedure described by Stumbo,
1973 and Pflug and Holcomb, 1991. The D-value is the time required to inactivate 90%
of the microbial population.
This value was determined from the slope (negative
reciprocal of the slope) o f the best fit line of the survivor curve. The survivor curve was
obtained by plotting the log of survivors versus the heating time. The z-value is the
degrees of temperature change needed to bring about a tenfold reduction in the Dvalue. Similarly, as the D-value, the z-value was calculated from the slopes (negative
reciprocal of the slope) of the decimal reduction curve established by plotting log D as a
function of temperature.
Regression analysis of survivor curve data
The D-value was calculated for each survivor curve test performed in the water
bath and the microwave oven by using linear regression analysis (Devore and Peck,
1986). The initial number of E. coli 0157:H7 cells in the heated suspensions was not
included in this analyses.
The correlation coefficient (r2) was reported which
characterized the linear correlation of the log of the survivors as a function of the
heating time.
The average values of the D-values at the 57°C and 60°C for both
treatments and two types of recovery media were calculated.
analyses were conducted to determine the z-values.
Similar regression
The logs of the individual D-
values were plotted against 57°C and 60°C. The regression analyses were carried out
and four sets of z-values were calculated, corresponding to the two types o f heat
108
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treatment and the two recovery media. The regression analyses were conducted with
the help of regression function, a part of statistical package o f the Excel software
(Microsoft Corp., Richmond, WA).
Statistical comparison of microwave and conventional heat treatments
To determine if there was a significant difference between microwave and the
water bath treatment, the parallel regression model was used (Weisberg, 1985; Martin,
1996).
Y = p0 + P-i A! + p2 A2 + ps S + e
(1)
where p0, Pi, p2 are intercepts for treatm ent conditions, A1 and A2 are categorical
predictors (variable: 0 - no treatment, 1 - treatment), S is the additive variable, p8 is
slope of the additive, e - error.
This model compares regression lines representing both treatments.
The
procedure involves carrying out regression analysis by combining data from both
survivor curve tests which were obtained during microwave and water bath
experiments. The P-value was calculated for the interaction between both treatments.
There is no statistical difference between the microwave and the conventional heating if
the P-value is above 0.05.
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Results
Maintaining a constant temperature in a microwave system
The microwave system that was constructed for E. coli 0157:H 7 inactivation
studies is presented in Figures 1 and 3. A constant temperature was maintained by
modifying the electric circuit of the Quasar oven (Figure 2).
Uniform temperature
distribution within the 20 mL of E. coli 0157:H7 in Butterfield’s buffer was accomplished
by rotation of the vial through the action of the stepper motor. Appropriate agitation
parameters were determined during trial runs at temperatures set at 57°C and 60°C. A
Luxtron probe was inserted into the center of the liquid as shown in Figure 4 which
stayed immersed in the liquid during rotations.
A series o f heating tests were performed using 20 mL of water to observe if the
liquid in the vials was heated at a constant temperature. First, a come-up time to reach
the temperature in the experimental range of 55 to 60°C was determined by heating the
vial in the Quasar oven operating at full power mode. This temperature was achieved
during 15 to 18 sec as the thermal profile (A) in the Figure 5 presents. This time was
not included in the performed heating time of the sample during survivor curve tests
and possibly added more lethality to the heated population of cells.
The feedback
temperature loop cycled the oven’s magnetron o ff and on, but the stability o f the
temperature during the heating time was accomplished by manually changing the
power on the variac immediately after the desired temperature was recorded on the
computer.
The difference between these two types of heating conditions are
demonstrated in Figure 5 (profiles B and C). Profile C shows that excellent isothermal
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conditions were maintained during the heating te s t
The only problem was making
manual adjustments of the power at the appropriate fraction of the second to avoid
overheating of the sample. The liquid was well secured within the vial and no leakage
was observed during rotations.
Water bath experiments
In contrast to the microwave test, the conventional heating of £. coli 0157:H7 in
the water bath was a simple task but required establishing a lag correction factor for the
vials heated with suspension. The procedure of this experiment is described in Figure
6. This procedure was followed fo r four tubes tested, and the lag correction factor for
10 x 130 mm tubes was found to be 2 min (Table 1).
D and z-value data o f E coli 01S7:H7 determined during microwave and
conventional heating
Thermal resistance studies for E. coli 0157:H 7 in phosphate buffer were
performed at two temperatures, 57°C and 60°C, using two recovery media, TSA and
PCA + 1% NaPyr. Survivor curve experiments were carried out in the water bath and
parallel tests were performed in the microwave system on the same day, using the
same working suspension of cells. Studies were limited to two replicate vials at each
heating interval because samples had to be heated and plated within one day.
To
assure the accuracy of the results, 6 heating time spans were used for the 57°C test
and 8 spans for 60°C. Stationary-phase cells were heated during the study because
they are more resistant than cells from the logarithmic stage.
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The individual survivor curves at these temperature are presented in Figures 7,
8, and 9. It appears that the experiments conducted at 57°C were controlled better
than experiments a t 60°C since the shapes o f the curves show even, gradual decline
especially those obtained for bacteria heated in the water bath.
This confirms the
observation that during the heat resistance studies, the shorter the heating time the
more error is introduced to the survival data.
More variability was observed for
microwave-heated samples than for the water bath samples.
A sim ilar trend was
observed during experiments 60a, 60b and 60c when E. coli 0157:H 7 was exposed to
microwaves at the set-up temperature of 60°C (Figure 8).
The D-values were calculated from the data presented using regression
analysis and results are shown in Table 2. The initial number o f cells was not included
in the calculation o f the regression line. The D-values were determined from the slope
of the regression line. The summary of the D-value data are presented in the Table 2.
These values show that E. coli 0157:H7 is less resistant to conventional heating than
to microwaves at 57°C but not 60°C. Only statistical analysis can determine if this
relation is valid. The average D-value for E. coli 0157.H7 exposed to microwaves was
19.77 min and 1.05 min heated at 57°C and 60°C respectively when TSA was used as
a recovery media.
From these D-values, the z-values were estimated for four
experimental conditions (combination of treatm ent and recovery media), as reported in
Figure 10 and Table 3. The z-values ranged from 2.32°C to 3.29°C and the smallest zvalue was obtained fo r microwave heating when cells were enumerated on TSA media.
The PCA + 1% NaPyr demonstrated its excellent ability to recover higher
number of heat-stressed cells as compared to the TSA. However, this test using two
112
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recovery media failed to show clear differences between recovery of conventionally
heated or microwaved cells. There were no distinctive trends showing that PCA + 1%
NaPyr had a better ability to recover cells from either water bath or microwave
treatm ent
Statistical tests
Statistical analyses of survival data were performed during calculation o f the
regression lines (Devore and Peck, 1986) to show the relation between the log of the
survivors and the heating time. The linearity of the survival data is demonstrated by the
correlation coefficient (r2 value) (National Canners Association, 1968). These values
rather than confidence intervals, are more appropriate (Pflug and Holcomb. 1991) to
express distribution of the log of survivors as a the function of the heating tim e. The
regression analysis revealed that t2 values ranged from 0.96 to 0.85 for the microwave
treatments and 0.99 to 0.55 for water bath treatments.
The most im portant statistical analyses that were earned out aimed to determine
whether there was a significant difference between the heat resistance o f E. coli
0157.H7 during microwave and conventional heating.
The comparison of the
regression lines representing both treatments is the appropriate statistical method
according to Martin (1996) and Weisberg (1985). A regression model was established
that included two indicator variables (dummy variables) representing microwave and
water bath treatments.
Regression analyses for this new model were earned out to
demonstrate the treatm ent effect on two regression lines (two D-values). The results of
the analyses of 18 survivor cun/e experiments are presented in Table 4.
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The
significance level P-value was 0.05. It appears from the P-value data that 6 out of a
total 18 experiments showed a significant difference between both treatments. Thus in
most of the experiment (12 out o f 18) no difference was observed between the
conventional and the microwave heating.
The results o f the test were not fully
conclusive; therefore, additional experiments were earned out.
Lethality tests
A claim can be made that bacteria express different levels o f resistance during
microwave and conventional heating only if isothermal conditions occur during these
heatings.
Thus thermal profiles of heated samples should be identical during both
survivor curve experiments.
Because the results of our test were not definitive, two
additional experiments at 60°C were earned out during which time temperature data
were collected fo r each heated vial. A total of 56 thermal profiles were collected. The
problem of presenting the data in a simple manner was solved by applying the lethality
concept.
The time-temperature data allowed the calculation o f the lethality delivered to
the heated sample during each survivor curve test.
The procedure to determine
lethality is based on the General Method (Stumbo, 1973) According to this method the
lethality is a sum of lethal rates that are calculated from the following equation;
L = 10 T'Trste
(2)
where T is a temperature that changes with time during heat penetration tests, T ^ is a
reference temperature, a constant temperature maintained during heat penetration
114
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tests (60°C in this study), z is a temperature coefficient (2.3°C determined previously in
this study).
Figure 11 presents the results o f two survivor curve experiments: LRa and LRb.
For both replicate samples heated at the specific time interval, the lethality values were
determined. They appear as two bars fo r each heating tim e on the graph. The log of
survivors recovered from heated samples in also presented on this same graph.
Analyses of these data suggest that more heat was delivered to the E. coli 0157.H7
suspension than desired.
However, when the suspension was exposed to an
excessive amount of heat during microwave heating as compared to a sample heated
in the water bath, then a smaller number of organisms were recovered from the
microwave sample.
This can be observed for the 3.5-min and 4-min sample in
experiment LRA and 2-min sample in experiment LRb.
As data in Table 5 shows,
substantial differences in lethal values were found for E. co li 0157:H7 suspensions
heated in two modes.
Nevertheless, the parallel test showed that there was no
difference in the D-values determined during both treatments although larger variability
of the lethality values were observed. The statement can be made that the differences
in the temperature of heated suspensions during microwave and conventional heat
treatment are responsible for the variability of the thermal resistance data. Figure 12
shows typical time-temperature profiles of the two samples heated in the microwave
system and the water bath. The curve representing the temperature in the microwaved
sample demonstrates the difficulty in obtaining 60°C during the come-up time.
The
overheating phenomenon as was explained earlier did o ccu r. Although the sample was
exposed to 2 to 3°C higher temperature only for a few seconds, but this contributed
115
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significantly to the total lethality o f the process. This small increase in temperature is
particularly disadvantageous fo r bacteria with small z-value characteristics.
The z-
values of 2.3°C used in this experiment showed that the lethality values increased
dramatically when the temperature data above the 60°C range were added to the total
lethality time.
Discussion
The microwave system constructed during this study was a suitable apparatus
to maintain constant temperature conditions.
The thermal profiles o f the samples
heated during preliminary experiments showed that a constant temperature was
observed within the agitated vials and a short come-up time was achieved. However,
detailed comparison of heat penetration profiles demonstrated that initial overheating
led to differences in D-values fo r E. coli 0157:H7. Nevertheless, this is the best system
available now for such a comparison study to determine the difference in thermal
resistance of bacteria heated by microwaves and conventionally (Welt et al., 1992,
1993).
Early research on microwave destruction of bacteria reports on other innovative
methods that were used to simulate heating patterns observed in conventionally heated
samples. However, the technology was not advanced enough at that time that would
allow for the design of satisfactory systems. Researchers were forced to use metal
thermocouples.
Computerized electric circuits to maintain constant temperature or
mixing of the samples were not part o f the designs. Carroll and Lopez (1969) built a
radio-frequency electronic evaporator which was used to study microbial inactivation
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rates which then were compared to the water bath treatments.
thermal damage to the
microwaved microorganisms would
evaporation of the suspension
at 20°C under vacuum.
They assumed that
be eliminated
by
Lechowicz et al., (1969)
modified a microwave oven by placing a glass condenser inside the oven and
circulating kerosene through the jacket to cool the microbial sample during exposure to
microwaves. A metal thermocouple measured the temperature inside the sample when
the magnetron pulsed on and o ff to maintain isothermal microwave heating. Although
the design of a system did not allow for a perfect isothermal condition, the overall
lethality effect on bacteria was sim ilar as in conventionally heated suspensions.
A
similar approach was used in a recent years in the experimental design used by Khail
and Villota (1988).
Cold kerosene was circulated around heated vials placed in a
regular microwave oven to keep the sample at a constant temperature but thermal
profiles were not reported.
In many reports, comparison studies were earned out using the approach that
the temperatures developed in the microwave oven were simulated during conventional
heating.
A few of these reports presented controversial results and researchers
claimed there was a difference between microwave and conventional heated bacteria
(Dreyfuss and Chipley, 1980). However, the comparison studies showed satisfactory
results when more sophisticated technology was applied to the experimental design.
For example, Coote et al. (1991) used a programmable heating block fo r conventional
heating of Listeria monocytogenes to simulate temperature profiles obtained in
microwave heated samples.
Similarly, no difference was found in the thermal
destruction characteristic o f E. co li 0157:H7 when meticulous heat penetration studies
117
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were conducted in the regular microwave oven and then replicate tests were performed
in the water bath at the same rate of temperature increase in samples (Fujikawa, 1992).
In the above discussed studies, thermal profiles were reported adding credibility to the
data and underlining the error margin between the temperature developed in a
microwaved and water bath sample.
The studies carried out on destruction of spores by microwaves and
conventional heat spores by W elt et al., (1994) and Jeng et al., (1987) demonstrated a
similar pattern o f inactivation during both treatments. In both systems programmable
temperature control circuits were employed and the time-temperature data were
monitored.
The best method to demonstrate the accuracy of those experiments was to
present thermal profiles data.
lethality during both treatments.
The better approach is to determine and compare
In our study, time-temperature curves (Figure 12)
show that small fluctuations of temperature occurred. However, these small changes
can cause substantial kill o f bacteria if the temperature coefficient expressed by the zvalue is small. Lethality calculated from the time-temperature profiles is the only value
that demonstrates realistically the amount of the kill delivered to the bacteria.
The
lethality calculated for the particular sample expresses the equivalent time at the
temperature o f the experiment delivered to the sample. Figures 11 and Table 5 show
that lethality can explain unusual differences between a sample heated conventionally
and by microwaves.
This is the first report showing the importance of calculating
lethality in comparison studies. This is particularly important when bacteria are used as
a test organism since they are more heat sensitive than spores.
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Appropriate statistical analyses have to be carried out to compare lethality of the
data. Since the thermal resistance characteristics are calculated employing regression
analysis only statistical models that compare regression lines should be used
(Weisberg. 1985). A parallel model with categorical predictors was applied in our study
and showed that in 6 out of 18 experiments there was a significant difference between
the microwave kill rates and the kill rates obtained during conventional heating (Table
5). This difference can be explained based on the variability in lethality time delivered
to the heated vials.
Unusually high lethality was also delivered to the water bath samples, although
the lag correction factor was calculated and included in the survivor curve tests. These
differences can be explained by uneven circulation o f water in the water bath or a
temperature gradient created in the microbial suspension caused by lack of agitation
and convection heat transfer.
Other methods could be used to perform thermal
resistance studies such as using Thermal Death Time vials (National Canners
Association, 1984) or capillaries but all of these methods of determining D and z-values
have advantages and disadvantages such as uneven heat transfer that creates
localized overheating, difficulties in removal of suspension, and variability between
capillaries such as glass thickness (Perkin et al., 1977, W escott et al., 1995).
The calculated D-values for E. coli 0157:H7 were higher than the values
reported by Doyle and Schoeni (1988) (45 sec at 60°C) and Line et al., (1991) (4.1 to
7.4 min at 57°C). However, the experimental conditions of this study were different.
The cell suspensions in these reports were heated in ground beef, a different strain of
E. coli 0157:H7 was used and survivors were enumerated on different media. Ahmed
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et al. (1995 ) reported the D-values for E. coli 0157:H7 heated in meat and poultry at
various levels of fa t (0.55 to 0.38 min at 60°C) which appears to be smaller than the
values established in our study fo r the E. coli 0 1 57:H7 suspended in buffer.
Conclusions
Results of this study show that a lack of isothermal temperature conditions
during the survivor curve experiment is responsible fo r variability in the number of
bacteria recovered from heated samples rather than the difference between microwave
and conventional heating.
This was shown clearly in lethality tests where the
temperature inside the bacterial suspensions was monitored.
Although statistical
analyses showed no difference between microwave and water bath heated samples
during these tests, the lethality delivered to the microbial suspension varied and were
not delivered at the expected level.
Thus, problems of selective absorption o f microwaves cannot be discussed
unless bacteria are exposed to this same lethality level.
Perhaps a new microwave
system could be built to eliminate the manual switching of power during the survivor
test and minimize the overheating of samples.
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Spite, G. T.
1984.
Microwave-inactivation of bacterial pathogens in various
controlled frozen food compositions and in a commercially available frozen food
product. J. Food Prot. 7:458-462.
Stanford, M. S. 1990. Microwave oven characteristics and implications for food
safety in product development. Microwave World. 11 (3):9-11.
126
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Stuchly, S. S.
1978.
Radiowaves and microwaves - basic definitions and
concepts, p. 23-56. In: S. S. Stuchly (ed.), Microwave bioeffects and radiation safety.
The International Microwave Power Institute, Edmonton.
Stumbo, C. R.
1973
Thermobacteriology in food processing, p.143-151.
Academic Press, New York.
Thuery, J.
1992.
Microwaves: industrial, scientific, and medical applications,
p.3-106. Artech House, Boston.
Weisberg, S. 1985. Applied linear regression, p. 177-184. John W iley & Sons,
New York.
Welt, B. A., C. H. Tong and J. L. Rossen. 1992. An apparatus fo r providing
constant and homogeneous temperatures in low viscosity liquids during microwave
heating. Microwave World. 13:9-13.
Welt, B. A., J. A. Steet, C. H. Tong, J. L. Rossen and D. B. Lund.
1993.
Utilization of microwaves in the study of reaction kinetics in liquid and semisolid media.
Biotechnol. Progress. 9:481-487.
Welt, B. A., C. H. Tong, J. L. Rossen and D. B. Lund.
1994.
Effect of
microwave radiation on inactivation of Clostridium sporogenes (PA 3679) spores. Appl.
Environ. Microbiol. 60:482-488.
Wescott, G. G., T. M. Fairchild, P. M. Foegeding. 1995. Bacillus cereus and
Bacillus stearothermophilus spore inactivation in batch and continuous flow system. J.
FoodSci. 60:446-410.
127
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Variac
Sample
Stepper motor
MW oven
Luxtron
Signal booster circuit
Computers
mm&ma
Figure 1. Microwave system for providing constant temperature during microbial inactivation studies.
128
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
POWB? SUPPLY
TRANSFORMER
BASIC MW OVBV FOWB? SUPPLY CIRCUIT
120 VAC
MAGNETRON
MODIFIED MW Q V frl POWB? SUPPLY. CIRCUIT
TRANSFORMER
DOOR
INTERLOCKS
TRANSFORMS?
OVB4 CONTROLLER
120 VAC
POWER ADJ
Figure. 2. Typical and modified microwave oven power supply circuits.
129
F igure 3. M odified m icrow ave oven fo r providing co n sta n t tem perature d u rin g
m icro b ia l inactivation stu d ies.
130
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4. C avity o f m odified m icrow ave oven w ith sam ple and sam ple holder.
131
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
140_
0
120
£ 100-
1
I
60
80 ‘ '
I
402 0
-
0
10
20
30
time (sec)
40
60
60
70 T
60 -
u
o
3
(0
I
I
io ■■
0
60
120
180
240
180
240
time (sec)
70 T
60-
o so-
so -
0
60
120
time (sec)
Figure 5. Thermal profiles of 20 mL water heated in A) Quasar microwave oven; B)
modified Quasar oven - temperature set up at 55°C and variac operated at 100%
setting; C) modified Quasar oven - temperature set up at 55°C and variac operated at
70% setting.
132
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 6. Outline of the procedure used to calculate lag correction factor fo r tubes
used in microbial thermal resistance studies.
A) Lag correction factor (LCF)
LCF is used in the thermal resistance studies, and it corrects for the lag in the
transfer of heat to and from a sample.
B) Calculation process
• Method of Ball (Ball and Olson, 1957)
• General Method (Stumbo, 1973; Pflug, 1988)
C) Procedure to calculate time LCF(tt.cF) based on General Method
The tj_cF is the difference between the heating time - th and the sterilization value - F(Ti):
t LCF = th -
F(Ti)
F(Ti) = 2L x At
where: At - time interval between temperature measurements,
L - lethal rate
D) Lethal rate
L - lethal rate calculated from equation:
L = lo TTref/z
where: T - temperature that changes with time during heat penetration tests,
Tref - reference temperature; constant temperature maintained during
heat penetration tests,
z - temperature coefficient
133
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Table 1.
Calculation of the lag correction factor for vials used in E. coli 0157.H7
thermal resistance studies. Screw-capped Pyrex tubes (10 x 130 mm) filled with 5-mL
bacterial suspension were heated in water bath at 57°C.
Time (t)
(s)
Temp. (T) in four replicate tubes (°C)
Lethal rate (L) (sec at 57°C/sec at T)
where T ^ = 57°C, z = 5°C
tube 1
tube 2
tube 3
tube 4
tube 1
0
4.4
4.8
0.0
Z1
10
4.2
4.7
0.4
2.7
20
3.8
4.6
0.5
2.6
30
13.3
9.5
11.1
5.0
0.000
0.000
0.000
0.000
40
27.4
20.3
25.9
15.7
0.000
tube 2
tube 3
tube 4
50
31.4
25.7
28.3
21.0
60
37.6
33.4
34.4
29.1
0.000
0.000
70
43.1
39.7
40.6
36.7
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
80
47.3
45.4
45.4
42.5
0.011
0.004
0.005
0.001
90
50.3
49
49.0
46.9
0.046
0.021
0.027
0.008
100
52.4
51.5
51.6
49.9
0.120
0.066
0.086
0.034
110
53.7
53.2
53.3
52.1
0.219
0.145
0.188
0.090
120
54.8
54.5
54.4
53.7
0.363
0.263
0.318
0.186
130
55.5
55.3
55.2
54.7
0.501
0.380
0.466
0.306
140
55.9
55.8
58.8
55.5
0.603
0.479
2.432.
0.439
150
56.3
56.4
56.1
56.1
0.724
0.631
0.701
0.562
160
56.5
56.7
56.4
56.4
0.794
0.724
0.787
0.664
170
56.6
56.7
56.6
56.7
0.832
0.724
0.855
0.752
180
56.7
57
56.7
57.0
0.871
0.832
0.900
0.863
190
56.8
57
56.8
57.1
0.912
0.832
0.959
0.908
200
57
57.2
56.9
57.0
1.000
0.912
0.982
0.879
210
57
57.5
56.9
57.2
1.000
1.047
0.995
0.973
220
57
57.3
57.0
57.2
1.000
0.955
1.042
0.959
230
57.1
57.5
56.9
57.3
1.047
1.047
1.019
1.014
EL =
10.05
9.06
11.76
8.64
EL x At = EL x 10 (sec) =
100.5
90.6
117.6
86.4
2.32
1.87
2.39
LCF (min) ==230 (sec) - EL X At = 2.16
Lag Cor. Factor (average value) (min) -
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.001
2.0 min*
* data from 4 additional experiments were included
134
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0.000
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Exp. 67a
6
Exp. 67b
Exp. 67c
6
-
•\
6z
$
6
• •
6
■■
z
4
"
3 ■•
3 -
3 •-
2
2-
2
1
-
1 ■■
• •
0
20
40
time (min)
Figure 7.
■ ■
60
0
20
40
time (min)
60
0
20
40
60
time (min)
Survivor curves for E. coli 0157.H7 in phosphate buffer heated at 57°C conventionally ( • - water bath) and by
microwaves (■ - isothermal microwave system). Survivors were recovered on Tryptic Soy Agar ( — ) spread plates and Plate
Count agar with 1% sodium pyruvate ( — ) spread plates. Three replicate experiments a, b, and c were performed.
135
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
8
8
8
Exp. 60a
Exp. 60b
Exp. 60c
7
7
7
6
6
6
•~ -S
4
6
6
4
4
3
3
2
2
1
1
0
0
0
1
2
3
time (min)
Figure 8.
4
6
0
0
1
2
3
time (min)
4
6
0
1
2
3
4
6
time (min)
Survivor curves for E. coli 0157:H7 in phosphate buffer heated at 60°C conventionally ( • - water bath) and by
microwaves (■ - isothermal microwave system). Survivors were recovered on Tryptic Soy Agar ( — ) spread plates and Plate
Count agar with 1% sodium pyruvate ( — ) spread plates. Three replicate experiments a, b, and c were performed.
136
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Exp. 60d
Exp. 60e
6
5
Exp. 60(
6-
• •
5-
z
6
z
-•
z
g, 4 - -
9
3 -
3 -
3 ■■
2-
2-
2
- ■
1
0
1
2
3
time (min)
Figure 9.
4
6
0
1
2
3
time (min)
4
S
0
1
2
3
4
0
time (min)
Survivor curves for E. coli 0157:H 7 in phosphate buffer heated at 60°C conventionally ( • - water bath) and by
microwaves (■ - isothermal microwave system). Survivors were recovered on Tryptic Soy Agar ( — ) spread plates and Plate
Count agar with 1% sodium pyruvate ( — ) spread plates. Three replicate experiments d, e, and f were performed.
137
Table 2. D-values and regression values for E. coli 0157:H7 heated by microwaves
and in water bath.
Temp.
Media
Exp.
Conventional heating
D - value
(min)
57°C
57a
TSA
57b
57c
Avg. D-value =
PCA + 1%
NaPyr
57a
57b
57c
Corr. coeff.
(r2)
0.94
10.12
13.94
11.83
0.94
0.95
11.96
60a
TSA
60b
60c
60d
60e
60f
Avg. D-value =
PCA + 1%
NaPyr
60a
60b
60c
60d
60e
60f
D - value
(min)
18.68
19.64
20.98
Corr. coeff.
(r2)
0.83
0.81
0.85
19.77
0.94
17.68
23.31
19.95
0.93
0.94
Avg. D-value = 20.31
60°C
Microwave heating
21.83
36.79
32.19
0.88
0.55
0.92
30.27
0.92
0.96
1.11
1.37
1.14
0.97
1.17
0.95
0.96
0.95
0.92
0.93
0.83
0.76
0.98
1.76
0.97
1.00
0.91
0.92
0.97
0.93
0.97
0.99
1.05
1.12
0.96
2.44
3.05
2.26
2.04
2.37
2.77
0.85
0.91
0.87
0.84
0.94
Avg. D-value = 2.49
1.65
1.50
1.86
2.74
1.40
2.41
0.84
0.97
0.96
0.85
0.84
0.93
1.93
138
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
z-value (water bath, TSA)
z-value (microwave, TSA)
100
100
©
3
(8
>
Q
3
:>
*5
D
88
0.1
0.1
temperature (C)
temperature (C)
z-value (water bath,
PCA+1%NaPyr)
z-value (microwave,
PCA+1%NaPyr)
100
100
«
a
m
>
a
•i
3
"5
>
Q
56
58
60
56
temperature (C)
58
60
temperature (C)
Figure 10. Thermal resistance curves of E. coli 0157:H7 heated conventionally and by
microwaves and enumerated using two different media Tryptic Soy Agar (TSA) and
Plate Count Agar with 1% sodium pyruvate (PCA + 1% NaPyr).
139
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 3. Comparison o f the D- and z-values for E. coli 0157:H 7 heated conventionally
(water bath) and by microwaves (isothermal microwave system) and enumerated on
Tryptic Soy Agar (TSA) and Plate Count Agar with 1% sodium pyruvate (PCA + 1%
NaPyr) spread plates.
Treatment
microwave
conventional
Media
Mean D-value (min)
z-value (°C)
57°C
60°C
TSA
19.77
1.05
2.32
PCA + 1% NaPyr
30.27
1.93
2.50
TSA
11.96
1.12
2.92
PCA + 1% NaPyr
20.31
2.49
3.29
140
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 4. D-values and 95% confidence interval for D-values determined during survivor curve experiments for E. coli 0157:H7
heated by microwaves and in water bath.
Temp.
57°C
Media
TSA
Exp.
57a
57b
57c
PCA + 1% NaPyr
57a
57b
57c
60°C
TSA
60a
60b
60c
60d
60e
60f
PCA + 1% NaPyr
60a
60b
60c
60d
60e
6Of .
•
D - value (min) -
Statistical comparison of treatments*
water bath
microwave
p-value
results
10.12
13.94
11.83
17.68
23.31
19.95
18.68
19.64
20.98
21.83
36.79
32.19
0.020
0.185
0.034
0.276
0.261
0.042
-
0.96
1.11
1.37
1.14
0.97
1.17
2.44
3.05
2.26
2.04
2.37
2.77
0.83
0.76
0.98
1.76
0.97
1.00
1.65
1.50
1.86
2.74
1.40
2.41
0.455
0.056
0.334
0.002
0.803
0.221
0.110
0.002
0.208
0.328
0.012
0.312
+
+
+
+
-
+
+
-
-
+
+
+
-
+
+
-
+
Statistical comparison of D-values for microwave and water bath treatments was carried out. Parallel model with categorical predictors was used
(Weisberg, 1985). There is significant difference (-) between microwave and water bath treatments if p < 05 and no difference (+) if p > 0.05
141
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
12 T
Exp.LRa
2
2.6
12 T
t 8
3
3.6
4
Exp.LRb
2
2.6
Heating time (min)
T8
3
3.6
4
Heating time (min)
Figure 11. Comparison of lethality delivered to E. coli 0157:H7 suspensions and number of survivors recovered after heating
samples at 60oC by microwaves (•) and in water bath (■). Lethality ( « - microwave, |
- water bath) was calculated from time-
temperature data by General Method. Two experiments were conducted * LRa and LRb. Numbers of survivors are average values
obtained from two replicate samples that were used per each heating time.
142
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Table 5. Lethality delivered to E. coli 0157:H7 suspensions during survivor curve experiments carried out in microwave oven and
water bath at 60°C. Temperature was monitored in each heated vial. The lethality was calculated from the time-temperature data
using General Method (Tret = 60°C , z = 2.3°C). Two replicate vials (a, b) with E. coli 0157:H7 in phosphate buffer were heated per
each time interval.
Plate count agar with 1% sodium pyruvate was used as a recovery media.
Statistical difference between
microwave and water bath treatment was determined for each experiment.
Exp.
LRa
Treatment
Microwave
D-value
Statistics*
(min)
p-value
1.80
0.2315
Water bath
2.81
Lethality delivered to replicate samples at the heating intervals (sec at 60°C)
1 min
2 min
2.5 min
3 min
3.5 min
4 min
4.5 min
5 min
vial a,b (sec)
147,1033
176, 221
181,155
297, 481
507,268
4770,310
322,322
348,346
Avg. (min)
9.80
3.31
2.80
6.48
6.46
5.17
5.37
5.78
102,85
177,175
258, 231
265,222
257, 221
287, 267
468,274
2.93
4.08
4.06
3.98
4.62
6.18
vial a,b (sec)
Avg. (min)
LRb
Microwave
7.33
vial a.b (sec)
0.6242
Water bath
5.54
Avg. (min)
vial a,b (sec)
Avg. (min)
64, 41
0.86
1.56
ND, 76
682, N D
172, N D
319,219
220, 218
470, 224
287, 324
439,498
1.27
11.37
2.87
4.48
3.65
5.78
5.09
7.81
212,163
311,286
384,300
356,229
386, 410
305,345
353,382
3.13
4.98
5.70
4.88
6.63
5.42
6.13
149,82
1.93
•
* - There is no sig n ifica n t difference between m icrow ave and conventional heating if p > 0.05
•
N D - not deterim ed
143
A - microwave treatment
70 T
p.
60 O 50-
0
5 40 -
■*
**iil
j
-M1a
■
1-7
1 20 • ■
10
0
r,,rr,v.'•ii.
-M 1b
Hi.,,,
"'■ -.M r.
'""ill.
Ir
60
120
180
240
tim e(s)
B - water bath treatment
(■■■■••■••I1
o
• V
0
£30--
—
W3a
—
W3b
W
" •r,"•N,,
- -
K.
■im.
60
120
180
240
time(s)
Figure 12. Representative time-temperature profiles recorded during the survivor curve
experiments.
E. coli 0157:H7 suspensions in phosphate buffer were heated: A) by
microwaves (isothermal microwave system, vials agitated), and B) conventionally (water
bath; vials without agitation). Two replicate vials (a, b) were heated fo r 1 min (heating
time interval) during each treatment at 60°C. Water bath heating intervals were 2 min
longer because vials possessed 2-min lag correction factors.
144
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Microwave pasteurization of foods. Part A. A proposed method
to determine pasteurization time and testing its validation using
ground beef patties as a model system
145
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Abstract
A method fo r determination of pasteurization time during microwave heating
was established.
The procedure is based on a modification of the method used in
conventional thermoprocessing which determines F-process time at the specific
constant temperature.
The proposed methodology involves calculating an end-point
temperature for non-isothermal conditions occurring during microwave heating. Ground
beef patties (3.8 x 104 CFU/g - aerobic plate count) inoculated with Escherichia coli
0157:H7 (1.2 x 104 CFU/g) were used as a model system. The reference values (D
and z-value) were determined during a constant temperature microwave heating test.
Heat penetration data were used to determine pasteurization time (defined as come-up
time and 1-sec lethal process time).
The end-point temperature calculated fo r this
process was found to be 67.8°C. The lethality equal, 12 D, was delivered within 1 sec
and during this time isothermal conditions were assumed. The pasteurization values
were found to be: at the medium power (463W) 4 min and 3 min 20 sec, and at the
high power (808W) 2 min 30 sec and 2 min 10 sec fo r round and doughnut-shaped
patties respectively.
Microbiological testing confirmed validity o f the predicted
pasteurization times.
Introduction
The potential fo r using microwave technology in thermoprocessing of foods has
been recognized since the discovery o f microwave heating (Decareau,
1985).
Microwave heating, as compared to the conventional form of heating, has several
advantages. Short come-up time and short process time to reach a high temperature in
146
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
a clean environment makes this process very attractive. The disadvantages that slow
down an application of microwave thermoprocessing fo r consumer and industrial needs
are the difficulties to predict the temperature distribution and the location o f cold spots
within the heated foods.
A noticeable increase in the use of microwave heating in the preparation o f
foods has been achieved in the last decade when microwave ovens became available
to most consumers. Popularity of these microwave ovens raised the question about
microbial safety o f microwave heated foods. (Knutson e t al., 1988, Hollywood et al.,
1991, Schnepf and Barbeau, 1989)
Research on the application of microwave heating as an alternative method fo r
pasteurization of foods has been reported for almost 40 years (Decareau, 1985).
Microwave energy has been considered for pasteurization and sterilization o f many
foods including m eat juices, milk as discussed by Rosenberg and Bogl (1987), Sieber
et al., (1996), Decareau (1994), and Mudgett (1990).
evaluated for application in industrial systems.
Microwave processes were
In the last decade, consumer
microwave ovens have been used to study a variety o f microwave heat processes.
Using microwaves as a mild heat treatment to extend the storage life of vacuumpacked (sous-vide) refrigerated foods has been explored recently (Simpson et al.,
1995; Paterson et al., 1995), as the consumer demands minimally processed foods
with fewer preservatives.
A great deal of information exists in the literature with regard to the individual
conditions of microwave heating effective in decreasing populations of spoilage and
pathogenic bacteria which occur in foods heated in consumer microwave ovens.
147
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Reports revealed common problems with repeatability o f experiments and timetemperature measurements.
These studies have been summarized in reviews by
Knutson et al. (1987) and Heddleson and Doores (1994). There is a lack of guidelines
on how to carry out a microwave heating process. A standard methodology is needed
that would enable the processors to predict the destruction o f microorganisms during
the microwave pasteurization (Stanford, 1990).
Spatial distribution of low- and high-temperature areas within a food subjected
to microwave irradiation is dictated by many factors, such as dielectric properties
specific to the microwave-food interaction (Mudgett, 1990; Buffler and Stanford, 1991).
Attempts to use the conventional thermoprocessing method to determine the lethal
effect of microwave treatment were not very successful because of heat distribution
problems (Huang et al., 1993). However, non-isothermal conditions occurring during
microwave heat treatment are not uncommon in food thermoprocessing. Most of the
heat operations in industrial settings deal with unsteady-state heat transfer.
In
successful technologies, standard methods are modified by incorporating additional
parameters to predict lethality of the thermal treatments in a real situation. An example
is the UHT pasteurization of milk that required development o f the Equivalent Point
Method (EPM) to determine reaction kinetics during a continuous flow (Swartzel, 1984).
The presence of E. coli 0157:H7 in ground beef and its survival in cooked
hamburgers became a serious problem for consumers, the fast food industry and beef
producers in last decade. Research is needed on proper preservation methods that
would elim inate this pathogen from meat products ( Padhye and Doyle, 1992).
148
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The goal of this research was to determine a method that would aid in the
prediction of pasteurization times during microwave heating of foods in a consumer
microwave oven. The first objective was to establish a simple procedure to determine
the pasteurization time for beef patties inoculated with E. coli 0157.H7 used as a
model food system and document its validity.
The second objective was to
demonstrate that, although many parameters characterize the microwave heating,
these pasteurization values can be predicted based on a few critical factors.
Materials and Methods
Development o f microwave pasteurization model
Microwave heat destruction parameters for E. coli 0157:H7
E. coli 0157:H 7 was selected to test the proposed microwave pasteurization
model. Parameters o f microbial destruction kinetics, D-values (time to inactivate 90%
of population) and z-values (degrees of temperature needed to bring about a ten-fold
reduction in D-values) were generated during constant temperature microwave heating.
The isothermal microwave conditions were created by constructing a microwave system
that consisted o f a modified microwave oven connected to two computerized loops: an
agitation loop and a temperature control loop (W elt et al., 1992). Methodology of £.
coli 0157:H7 inactivation studies performed in this system were described in the
previous chapter. These values were used to determine an end-point temperature of
the microwave pasteurization process. The D and z-values used in model calculations
are presented in Table 1.
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Heat penetration studies
Microwave oven
Microwave heating was performed in a Quasar microwave oven model MQS
1403W (Quasar, Elk Grove Village, IL) working at 2450 MHz. This model can operate
a t 5 power levels: high, high-medium, medium, medium-low and low-power setting.
Microwaves enter the oven cavity through the rotating wave guide placed under the
ceramic floor.
This oven is equipped with a fan, thus hot air is replaced by room-
temperature air during heating.
This particular model does not have a turn table;
therefore Luxtron temperature probes could be used. Probes were connected to the
Luxtron data acquisition system (Model 750; Luxtron, Santa Clara, CA) through a small
opening drilled in the side wall o f the oven cavity.
A microwave leakage test was
performed to assure safety of the oven. The oven cavity has the following dimensions:
1011/16”(H) x 165/ 16b(W ) x 133/16”(D) and 1.4 f t 3 total capacity.
To assure consistent
conditions during experiments the oven was never used “cold”. Before starting a new
series o f tests, the microwave cavity was warmed up by heating a Pyrex beaker with 2
L of water (room temperature) fo r 5 min at high power as suggested by Buffler (1992)
The Quasar microwave oven has been rated by the m anufacturer at the 900W
maximum power as determined by the IEC-705-88 test (IEC.1988). Calibration tests
were carried out to determine the specific power associated with each power level. The
power output of the Quasar microwave oven was tested at the high, medium and lowpower setting using an established but fairly simple measurement technique, the IMPI
2-L method as described by B uffler (1993). The IMPI 2-L procedure involved heating
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two 1 L-Pyrex beakers filled with 1 L of water (20°C) in the oven fo r 2 min and 2 sec.
Measurement of the initial and the final temperature of water allowed for a calculation
of the heat absorbed in the process due to the oven power output
Cold and hot spots occurring within the oven cavity were identified with a
mapping technique that will be described in the next chapter.
Beef patties
Ground beef patties were obtained from the Meat Science Laboratory at the
Food Science Department University o f Minnesota. They were stored in the freezer
(-10°C, 5-6 mo.) until studies were conducted. One batch of meat was used throughout
the study to minimize any inconsistency o f the results.
Meat was analyzed fo r fat
(17.0%) and moisture content (61.6%) using AOAC (1984) procedure. To prepare 10
patties o f uniform consistency fo r each microbial or heat penetration test, 15 patties
(1/4 lb. each) were thawed at room temperature for about 1 hr and then mixed fo r 1 min
(2-speed) using a KitchenAid mixer (model K5-A, speed 1-10) (Hobart Corp., Troy, OH).
Appropriate mixing parameters were found by adding a green food coloring to the meat
that indicated homogeneous mix.
Ground meat portions, 110 g (± 0.5 g), were formed into round or doughnut­
shaped patties that corresponded to a typical Vi-lb. hamburger.
A Tupperware®
hamburger mold was used to assure consistency of shape during the course of this
study. The dimensions of the patties are presented in the Figure 1. For the doughnut­
shaped patties a hole was made in the center of each patty with a custom-made
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stainless steel cylindrical knife (1n diameter).
The doughnut-shaped patties were
thicker than the round-shaped patties to maintain the same weight.
Initial temperature o f beef patties was maintained constant in the range of 4 to
8°C (Figure 2). Each patty prepared from the thawed meat was placed in a polystyrene
Petri dish (150 x 15 mm) and stored at 4°C fo r 1 to 3 hrs prior to the microwave heating
tests.
Temperature measuring system
A Luxtron fiberoptic temperature probes system, Model 750 (Luxtron Co., Santa
Barbara, CA), was used to measure the temperature o f the meat during the microwave
heating. The probes were calibrated on each day of experimentation using an accurate
thermometer calibrated previously to NBS temperature standards. The Luxtron data
acquisition unit was connected to a PC computer.
During the experiments, the
temperature data were sent continuously to the computer and then were recorded for
future analysis.
Heat penetration test
The pasteurization time was determined at high, medium and low-power level
for the round and the doughnut-shaped patties. The pasteurization time was defined
as the time during which the lowest temperature within the patty, 67.8°C, was reached
and maintained fo r a minimum of 1 sec (previous chapter).
To determine the
pasteurization temperature a “trial and error” approach was used. The pasteurization
value was selected when a minimum of 30 patties heated fo r that specific process time
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achieved the lowest internal temperature of 67.8°C or higher.
Pasteurization times
were determined for beef patties at high, medium and low-power setting.
To assure the reliability of the pasteurization data, the heat penetration tests
were conducted for meat patties placed in the cold spot o f each oven. A Petri plate
with a beef patty was removed from the refrigerator, the lid was removed and placed
open side down in the center o f the oven cavity. The Petri dish containing the patty
was placed on the top of the lid (Figure 3). Meat patties were heated individually. Four
Luxtron temperature probes monitored the temperature during heat penetration studies
as shown in Figure 1. The temperature values were recorded every 1 sec at four
locations within the cold spot Data was sent to the computer connected to the Luxtron
data acquisition unit every 1 sec.
Microbiological studies
Test organism s
The organism used in the inoculated pack study was E. coli 0157:H7 (strain
933, obtained from FDA, Minneapolis). The culture was grown in Tryptic soy broth
(TSB) at 37°C for 18 to 24 hr. Original culture was kept frozen in TSB + 10% glycerol.
The fresh culture was inoculated into the TSB and maintained for a 3-to 4-week period
by daily transfer (1 loop to 10 mL of new TSB followed by incubation at 37°C for 18 to
24 hrs). To prepare the cell suspension that was added to the meat, a 0.1 mL aliquot
of a stationary-phase culture was mixed with 100 mL of peptone water.
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Test organism - enumeration methods
The concentration of E. coli 0157:H7 cells in the peptone water added to the
meat was determined. Three samples were serially diluted in peptone, spread-plated in
duplicate on Plate Count Agar (Difco, Detroit, Ml) supplemented with 1% sodium
pyruvate (Sigma, St. Louis, MO) (PCA + 1% NaPyr). These plates were incubated for
24 hr at 37°C. Data obtained from this study were used to estimate the concentration
of E. coli 0157.H 7 per 1 g of meat (14 mL of peptone water with cells was added to 15
meat patties).
The presence of E. coli 0157:H7 cells in meat slurries after pasteurization was
assayed using Dynabeads® anti-E. coli 0157 ( Dynal, Oslo, Norway).
The
manufacturer's instructions were followed. A 25-g portion of the meat slurry was added
to 225 mL o f pre-enrichment broth (buffered peptone water).
After mixing, a 1-mL
sample was pipetted into Eppendorf vials containing 20 pL of magnetic polystyrene
beads coated with E. coli 0157 antibodies. E. coli 0157:H7 cells present in the meat
were allowed to contact antibodies during gentle agitation of the vials at room
temperature fo r 30 min. Beads with attached E. coli 0157:H7 cells were removed from
the liquid by a magnetic separation, washed and transferred onto the surface of two
plates containing: Sorbitol MacConkey Agar (SMAC) (Difco, Detroit, MO) and Sorbitol
MacConkey Agar supplemented with antibiotic Cefixime (Dynal, Oslo, Nonway) and
Potassium Tellurate (Sigma, St. Louis, Ml). These selective compounds were added to
the medium according to Dynabeads® manufacturer’s suggestions. The detection level
of the Dynabeads® anti-E.coli 0157 method is 100 cells per mL o f enriched culture.
Occurrence o f colonies on both plates suggested survival of E. coli 0157:H7 during
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microwave treatment of patties.
The meat, before inoculation, was also tested for
presence o f E. coli 0157:H 7 (control).
Meat microflora
The number o f mesophilic microorganisms present in the meat before and after
the microwave heating was also determined by the Aerobic Plate Count method (APC).
A portion of the m eat slurry was diluted in peptone water.
A 0.1-mL aliquot was
spread-plated on the surface o f PCA + 1% sodium pyruvate. This medium was found
to be excellent for the recovery of the heat-injured E. coli 0157:H7 cells (previous
chapter). These plates were incubated at 30°C fo r 24 hr. The incubation time was
extended to 4 d for an enumeration of heat-injured bacteria in pasteurized m eat
Preparation o f beef with test organism
One m illiliter of E. coli 0157:H7 at approximately 106 CFU/mL, was added to
thawed and uniformly-mixed ground beef prepared with the KitchenAid mixer. After
adding the cell suspension, the process of mixing was continued for an additional 30
sec at number 1-speed setting. The round and the doughnut-shaped patties (110 g ±
0.5 g) of even shape were formed using the hamburger mold as described before.
These patties inoculated with E. coli 0157:H7 were refrigerated to reach a 4 to 8°C
temperature.
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Microwave pasteurization experiments - inoculated pack studies
The meat patties inoculated with E. coli 0157:H7 were heated in this same
manner as those samples used in heat penetration studies. The round and doughnut­
shaped patties were processed in the Quasar microwave oven.
One meat patty was
positioned in the open Petri dish in the center of each oven. A fter heating, each patty
was quickly transferred from the Petri plate into a sterile strainer bag with a plastic
mesh insert (Tekmar, Cincinnati, OH) filled with a 110 ml of cold peptone water. These
mixture was homogenized for 2 min in the Stomacher (A. J. Steward, London, U.K.).
Small portions of the meat slurry were taken for APC determination and Dynabead®
anti-E.coli 0157 tests. Ten beef patties were used to validate each pasteurization time.
Presence of E. coli 0157:H7, even in one out o f ten patties, would invalidate the
predicted pasteurization time.
Results
The first stage of this study involved the establishment o f the methodology to
determine the microwave pasteurization parameters fo r foods in a consumer oven. The
experimental part of the described methodology included the heat penetration studies
and the microbiological tests.
Calculation o f pasteurization rimes during microwave hearing - proposed method
The proposed method allows for the calculation o f the pasteurization time for a
microwave thermoprocessing during non-isothermal conditions. The approach is based
on the classical methodology to calculate F-process time fo r conventionally heated
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foods (Stumbo, 1973; Pflug and Holcomb, 1991).
Two major modifications were
incorporated into the process: a) the D and z-values, generated during the constant
temperature microwave heating, were used to calculate F-value as opposed to
conventional heating; b) the F-value process delivered at the end-point temperature
was 1 sec. Figure 4 outlines five critical steps of the protocol used, and the results
obtained using these techniques are as follows.
Reaction kinetics for E. coli 0157:H7 heated by microwaves during constant
temperature conditions were determined.
Generally, it has been assumed that
inactivation o f bacteria by microwaves, and similarly enzymes, is a temperature
dependent process, but recent controversial reports suggest that this issue is still not
resolved (Tajchakavit and Ramaswamy, 1995; W elt et al., 1994).
However, a
technology exists, to establish the D and z-values fo r bacteria inactivated by
microwaves at a constant temperature.
conducting
constant-temperature
isothermal microwave heating.
W elt et al. (1992) described a system for
microbial
destruction
kinetics
test
during
an
A similar microwave system was constructed in our
laboratory as was described in the previous chapter. Survival curve experiments were
earned out for the E. coli 0157:H7 suspended in phosphate buffer at 60°C and 57°C.
The D and z-values obtained are presented in Table 1.
The end-point temperature was calculated by following the procedure outlined in
Figure 5. The Bigelow formula derived from the first order reaction kinetics equation
(Pflug and Holcomb, 1991; Stumbo, 1973) was applied to calculate the end-point
temperature. This temperature was determined fo r the four different D and z-value sets
assuming that the F-value effective process will last 1 sec and will perform the 12 D
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reduction o f bacteria.
One second is the time interval between temperature
measurements monitored by Luxtron and it can be assumed that during 1 sec an
isothermal condition w ill occur in the food system. W hen the destruction parameters
fo r E. coli 0157:H7 (T ,* , z-value, F ^ - 12 D), and the F-value (1 sec) were incorporated
into the Bigelow formula (Table 2), four values o f the end point-temperature were
calculated. The highest value, 67.8°C, was selected as the end-point temperature for
the process.
The pasteurization times were determined during heat penetration study
experiments (Table 2).
For the experimental microwave heating conditions, the
pasteurization time was defined as the come-up time to reach the end-point
temperature, 67°C plus 1 sec of the process at the minimum temperature 67.8°C
(Figure 5).
Time-temperature profiles were obtained in the coldest area within the
heated meat. The challenge was to determine the coldest spot within the meat heated
by microwaves. A temperature probe was located in the cold spot of the beef patty
placed in the cold area of the oven.
The pasteurization time was determined after
analysis of the highest number of patties that would represent a large sample. Using a
statistical approach, a large sample consists of at least 30 observations (Devore and
Peck, 1986).
The microbiological testing of the predicted pasteurization times were earned
out in a typical microwave oven. Ground beef patties inoculated with E. coli 0157:H7
were pasteurized fo r the predicted time in the Quasar microwave oven.
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These pasteurization times were validated in other models of microwave ovens.
Simple parameters that characterize microwave ovens were compared: a power output
and a location of cold spot in the oven cavity.
Experimental microwave conditions. Controlling and monitoring critical factors
influencing temperature distribution within microwave heated foods
Heat penetration studies have to be conducted within the area heated at the
slowest rate where there is the highest probability fo r survival of pathogenic
microorganisms. Microwave heating is complex, and there are a number of parameters
which determine the location of the cold spot within food.
To determine the pasteurization times for the beef patties meticulous heat
penetration studies were conducted.
To minimize internal temperature gradient,
precise attention was given to maintain a number of parameters constant. In this study,
as compared to other investigators, we recognized that some parameters are more
detrimental then others, that few of them can be controlled, and other changes must be
monitored.
Heat penetration studies were conducted using a new Quasar microwave oven
which was selected as a typical household microwave oven available on the market. In
this report it will be referred to as a standard microwave oven.
This model was
recommended by Buffler (1992), W elt at al. (1993) and Lentz (personal communication,
1993) because o f a highly uniform heating pattern.
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The location o f the beef patties within the oven cavity was not changed during
heat penetration studies (Figure 3). A series of experiments were earned out to identify
that area.
For the heat penetration studies, the meat sample was situated in the
coldest spot of the Quasar microwave oven. Even temperature distribution occurred
within that area. The meat patties were heated in the Petri dish placed on the top of
Petri lid. This setup protected meat against rapid overheating occurring due to the
contact with the hot floor of the cavity. Plastic Petri plates were used instead o f glass
since dielectric loss o f polystyrene is lower than glass (Kingston and Jassie, 1988).
The glass container heats up slowly in the electromagnetic field and transfers heat into
food unlike the polystyrene plate. The Luxtron temperature probes were calibrated on
each day of the experiment to minimize experimental errors.
An important factor that effects the variability of heat penetration data is
maintaining a consistent geometry of the heated patties.
In this study this was
accomplished by using a standardized hamburger mold. An initial temperature o f the
meat patties as well as meat moisture and fat content influences the dielectric
parameters of the meat, thus the heating process. These parameters were established
(17.0% fat and 61.6% moisture) and did not change during the course of study. Beef
patties were stored at 4°C prior to the heat penetration test and the initial temperature
was measured before starting the oven. Meat used in the preparation of the patties
came from a thoroughly mixed single batch of ground meat using consistent mixing
parameters on each day of experiment.
The next step in the process was the identification of the size of the cold area
within the heated round and doughnut-shaped meat patties, and finding an appropriate
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location for temperature probes.
Observations o f the pink spots within the
undercooked meat were helpful in this determination.
Another factor that was
im portant in these tests was a microwave penetration depth (Figure 2).
This
characteristic was calculated by using dielectric properties as reported by Buffler
(1992), from which penetration depth was calculated. The penetration depth is a depth
at which microwave power has decreased to 36.8% o f its original power. This value
allows the prediction of the cold zone which may occur within the area located 2.4 cm
away from the sample edge fo r the round-shaped patties. The process of calculation of
penetration depth and application to determine the slowest heating area is presented in
Figure 2.
The location o f the temperature probes and the geometry of the round and the
doughnut-shaped patties are presented in the Figure 1.
placed close to the surface (1/4 of sample height).
The tip of the probe was
The coldest temperature was
observed at the surface as a result o f evaporation. The temperature o f the air in the
oven was monitored during the experiments and appeared to be consistent between
the runs, thus evaporation losses did not vary between tests.
Thermal profiles
The pasteurization times determined during the heat penetration studies in the
Quasar oven were 2 min 10 sec, 3 min 20 sec and 17 min fo r the doughnut-shaped
patties and 2 min 30 sec, 4 min and 23 min for the round-shaped patties at high,
medium and low-power settings respectively (Table 3).
As stated previously, the
pasteurization time is defined as the process time to reach the internal, end-point
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temperature at the coldest spot within the ground beef patty (Figure 6). This time was
selected when a minimum of 30 meat patties achieved a temperature of 67.7°C or
above when heating was terminated.
Doughnut-shaped patties heated faster than
round-shaped patties of the same weight (110 g) (Scarrack, 1980).
Figure 7 explains the trial and error approach used to find the proper
pasteurization times.
For this particular test, the round-shaped beef patties were
exposed to high power level for a selected short time. When the lowest temperature at
one of the locations within at least one beef patty was below 67.8°C during this time
interval, a new trial was initiated with time extended for 10 sec. This procedure was
repeated until the final temperature in 30 patties at four different locations within the
meat was in the range above 67.8°C. A total o f 376 beef patties were microwave
heated in the Quasar oven to establish the proper pasteurization time fo r two different
shapes of the beef patties at three power levels.
The initial temperature of food influences its dielectric properties, and an
amount of heat generated in the sample when placed in electromagnetic field. In these
studies, as can be concluded from temperature data o f 30 samples (Figure 8), the
temperature of the beef ranged from 4°C to 8°C.
This four °C variation of initial
temperature did not affect, in a significant way, the final temperature o f heated patties.
The temperature variations occurred between patties exposed to microwaves
for the same time. A large disparity of temperatures (up to 30°C) occurred within a
small area of the meat.
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The beef patties heated at the high and the medium power level possessed
good texture. It was observed that a low amount o f moisture was lost. In contrast,
meat exposed to microwaves at the low power level had a dry surface.
Microbiological studies validating predicted pasteurization values
Inoculated pack studies were conducted only at high and medium power
settings. The low power was not used because o f the long pasteurization time. Results
of microbiological tests are presented in Table 4.
heating pasteurization parameters were accurate.
They indicate that the predicted
Ten patties of each shape
inoculated with E. coli 0157:H7 to the level of 1.2 x 104 CFU/g were heated in the
Quasar microwave oven at both power settings. In all 10 patties, E. coli 015:H7 did not
survive microwave heating. However, other aerobic bacteria were found in pasteurized
meat. The initial number of bacteria associated with the raw meat was 3.8 x 104 CFU/g
as determined by the APC method. Although these bacteria were not fully destroyed,
but because pasteurization targets only pathogens this fact is not of importance.
Discussion
Microbiological safety of microwave processed foods has been the subject of
many studies. Since early investigations, it was understood that lack o f uniformity of
the microwave field resulted in wide ranges of internal temperatures in foods processed
in microwave ovens. However, defined methodology was not proposed to correct the
problem. Attention was focused on details o f the microbiological test (Chen et at.,
1973; Fruin and Guthertz, 1982) rather than the physical environment of microwave
heating. Researchers did not include evaluation o f physical parameters of microwave
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heating (intensity of microwave field and composition o f foods in relation to amount of
power deposited in the load) in their investigations. These studies were focused on
one o f the following objectives:
1. Heating to the end-point temperature (doneness) - Craven and Lillard, 1974;
Crespo and Ockerman, 1977.
2. Heating for the specific process time and compare reduction of bacteria to
results obtained during heating for this same time conventionally - Baldwin
et al., 1971; Fruin and Guthertz, 1982.
Unsatisfactory results led to some changes in methodology and the following
approaches were suggested:
a) wrapping food - Sawyer et al., 1984
b) adding post processing time - Zimmerman and Beach, 1982
c) using multiple temperature measurements - Sawyer, 1985
d) measuring the power o f the oven by the method of Compton - Zimmerman,
1984
e) placing food at different heights in the microwave oven - Crespo et al., 1977
f)
testing various shapes of loads - W right-Rudolf et al., 1986
g) using several ovens - Zimmerman and Beach, 1982
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h) selecting combinations of power and time - Carlin et al., 1982
i)
using doneness indicators - Nelson and Labuza, 1992.
Knowledge about the complexity o f microwave technology has increased
(Thuery, 1992).
Successful measurement of lethality delivered to foods involves
applying concepts o f food engineering and electrical engineering, and microbiological
tests are rather simple.
Analysis o f microwave pasteurization processes are not
different than conventional treatments since they both the involve inoculation of the
food with the test microorganism and its recovery from the heat-processed food. W hat
is different and challenging is determination of pasteurization times by including
dielectric properties of foods and characteristics of oven performance.
W ith the new and better models of microwave ovens it is possible that the trial
and error approach can be replaced with analytical approaches to experimental design
and measurement
Mathematical modeling is promising and a high correlation was
found in some models between predicted and experimental heat profiles (Barringer,
1994; Ohlsson, 1990).
Numerical methods are extremely complicated because they
involve solving the Maxwell or the Lambert equation, the heat transfer and the mass
transfer altogether, and do not as yet have practical applications (Ayappa e t al., 1992).
As the home microwave market expands, there is a tremendous need to
educate the consumers and the food product developers about the principles of
microwave cooking (Mudgett, 1989). Microwave processing is thermal in nature, but
the mechanisms of microwave heating are different than conventional heating since
they consist of two processes: electromagnetic heating and heat transfer. Difficulty in
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predicting temperature distribution in foods does not allow for the use of conventional
methods to determine pasteurization times.
We attempted to establish a simple
procedure, developed on a scientific basis, similar to that used in conventional heating
and test its practical applications.
The concept used in this study is not new since many investigations have been
focused on the end-point approach to microwave heating (Crespo et al., 1977). Few
researchers applied the end-point temperature values in their experiments suggested
by regulatory agencies (Schnepf and Barbeau, 1989).
Others calculated end-point
temperature for the specific food-bacteria system. They performed appropriate
destruction studies to obtain D and z-values for selected indicator bacteria (Huang et
al., 1993, Gundavarapu, e ta l., 1995).
What is novel in the proposed method is the approach to the heat penetration
studies. Meticulous tests were conducted to obtain reliable heat penetration profiles in
the undercooked areas of the patties. These cold spots were determined based on: a)
the dielectric properties of meat used to calculate the penetration depth; b) an
evaluation of oven performance through the power output test and the thermographs at
the bottom of the oven cavity.
Dielectric properties are the most significant food variables unique to microwave
heating (Mudgett, 1986). They describe how a food material interacts with microwaves
that penetrate food.
These properties can not be ignored in the designing o f a
microwave heating process (Buffler and Stanford, 1991). The process of calculation of
the penetration depth by incorporating dielectric properties is shown in the Figure 2.
The dielectric values fo r particular foods are available, although more data are needed
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(Sun et al., 1995 ). Tables with values were compiled by Kent (1987) and Bengtsson
and Risman (1971). The methodology to obtain dielectric values has been improved as
new equipment has been introduced (Engelder and Buffler, 1991).
Calculation of
penetration depth should be the first task when designing a microwave process. This
can be accomplished by either a simple calculation task as presented in Figure 2 or by
using food maps (Buffler and Stanford, 1991).
The initial temperature o f the food should be taken under consideration since
the dielectric properties change with the temperature.
In our studies the initial
temperature did not influence dramatically the final temperatures. For some foods the
dielectric constant and the dielectric loss change significantly when the temperature
changes from 5 to 20°C but not fo r raw meat
(e”
is approximately 18 at 20°C and 16 at
5°C) (Mudgett, 1986; Buffler, 1992).
Oven performance has to be tested by measuring its power output with
standard methods. A map o f temperature distribution was created to select the cold
area (conservative approach) within the oven for the heat penetration test.
Calculated pasteurization times are valid only for a sample of identical size and
the geometry. This is not surprising since in a conventional thermoprocessing specific
sterilization values are readjusted according to a can size (Ball & Olson, 1957). To
make this data practical, the hamburger of typical size (1/4 lb.) and the shape was
prepared. A standard hamburger mold (Tupperware®) was used. This enabled us to
prepare meat patties of consistent geometry throughout the course of the study. Tested
patties were fairly flat (1”) thus spatial heat distribution across the height of the sample
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has been assumed to be constant. Formation o f standing waves that occur in very
small loads of cylinder shape (< 0.5 cm) was excluded (Zhang, 1995).
Evaporative
losses do occur in an open enclosure (Datta, 1990), and the top surface was expected
to be the coldest If food is heated without enclosure, the conditions inside the oven
(air velocity and temperature) are detrimental as they dictate the rate of evaporation.
This causes a cold surface and a high probability fo r bacteria to survive. Therefore, we
chose an open system to work with, i.e., less favorable conditions under which more
bacteria are expected to survive. Additionally, the presence of bacteria in a whole beef
patty was evaluated as opposed to the small sample taken from the specific area o f the
heated food (Crespo and Ockerman, 1977).
In the suggested method additional safety factors were incorporated. First, a 12
D process instead o f 5 D was incorporated into method, and second, come-up time
delivers additional lethality to the process.
become an issue.
Therefore, overprocessing o f meat may
However, as long as texture and sensory quality are acceptable,
there is no reason why this pasteurization time should not be used either by consumers
or the fast-food industry. We performed informal sensory tests where the texture of
microwave cooked beef patties was positively scored.
Lack of crust was the only
negative characteritics described by a few members of sensory panel. This finding is
with agreement of Davies et al., (1993) who reported that microwave cooked
beefburgers were not only consumer-acceptable, but additionally they reported that
beefburgers prepared by microwaves is mutagen-free in contrast to a traditionally fried
burgers.
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The validity of the method proposed to calculate microwave pasteurization time
was confirmed. No E. coli 0157:H7 survived the process as shown in inoculation pack
studies (Table 4). A wide range of temperature differences tests were observed during
replicate trials (Figures 6, 7 and 8). This partially may be caused by the heterogeneity
o f the meat structure. Ground beef structure consists o f muscle tissue and fat. Meat
reflection phenomena take place at the interfaces o f fa t and meat, thus localized
underheating and superheating areas may be responsible for variation in temperature
distribution. Food compactness and amount of air incorporated into ground beef adds
to this reflection problem.
Knutson et al., (1988) Heddleson et al., (1994) and Coote et al., (1991) stated
that pasteurization of foods will be feasible if the following parameters will be consistent
throughout treatments: size and initial temperature of load, composition of food,
wattage and power of oven, type o f container and time of microwave exposure,
manipulation o f food after heating (post-treatment time and temperature, mixing). We
demonstrated how to overcome these difficulties. In this study we showed that the
focus of microwave heating lethality studies should be on physical parameters of
heating rather than on microbiologial tests which are fairly standard.
This report
suggests how to improve current methodology to determine reasonable data.
Conclusions
The methodology exists to determine pasteurization time for beef patties heated
by microwave ovens.
Heat penetration studies that include consistent geometry of
169
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heated meat sample, location of cold spot in the oven and meat, are in agreement with
microbiological studies.
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Survival of
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177
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Table 1.
Microbial destruction kinetics of E. coli 0157.H7 obtained in modified
microwave oven maintaining constant temperature.
Temperature
Media
D-value (min)
z-value (°C)
57°C
PCA +1% NaPyr
30.27
2.50
57°C
TSA
19.77
2.32
60°C
PCA +1% NaPyr
1.93
2.50
60°C
TSA
1.05
2.32
178
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Table 2. Calculation of the end-point temperature
D-value (min)
z-value (°C)
End-point T(°C)
30.27
2.5
67.84
19.77
2.3
66.63
1.93
2.5
67.85
1.05
2.3
66.68
Final temperature selected = 67.8°C
179
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Table 3. Pasteurization time of beef patties determined from thermal profiles studies
using Quasar microwave oven. Pasteurization tim e is the cooking time to reach the
67.8°C temperature in the coldest spot within the patty as found by trial and error
approach. A total number o f 376 patties were used.
Power setting in Quasar oven
Power (W)1)
Regular patties
Doughnut patties
High
808
2 min 30 sec
2 min 10 sec
Medium
463
4 min
3 min 20 sec
Low
105
23 min
17 min
11power determined by IMPI-2 L method (Buffler, 1992)
180
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Table 4. Survival of E. coli 0157:H7 and other mesophilic bacteria in beef patties after
microwave pasteurization in Quasar microwave oven. Initial average population of E.
coli 0157:H7 was 1.2 x 104 CFU/g.
Beef contained 3.8 x 104 CFU/g of indigenous
bacteria enumerated using Anaerobic Plate Count method.
Bacteria present in beef after
pasteurization
Beef patty
Round shape
Doughnut shape
MW power
E. coli 0 1 5 7 :H 7 1)
A PC 2’
High
-
+
Medium
-
+
High
-
+
Medium
-
+
1) + survivors; - no survivors determined by immunomagnetic separation with
Dynabeads® anti-E. coli 0157.
21+ survivors; - no survivors of mesophilic bacteria associated with meat
determined with Plate Count Agar +1% sodium pyruvate spread plate method.
181
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 1. Geometry of beef patties heated by microwaves, Temperature probe positions in round and doughnut-shaped meat
patties during heat penetration tests.
,Temperature probes
Temperature probes
(location: 1/4H, 1/3 R from center)
MW penetration distance (2.4 cm)
>
;
iiiiillP?
2r=2.5cm
.
W
W*3
mrnmm »-
182
-»
......... f
Figure 2. Location o f Luxtron temperature probes within the meat patty determined by
calculation of penetration depth of microwaves penetrating the m eat
a) penetration depth-dp
•
dp = X (s')'12/
•
for the following parameters: X - MW wavelenght in free-space -1 2 .2 cm,
dielectric constant - 50.8; s " - dielectric loss -1 6 (Buffler, 1992)
dp = 0.865 cm
•
Penetration depth is a depth at which the microwave power has decreased to
36.8% of its original power that has entered the sample. Thus, microwave power will
stop to penetrate sample at the 2.4 cm distance from the surface (100% reduction
of microwave power).
2 7 is”
s’ -
b) cold spot in the beef patty formed as a result of microwaves not fully
penetrating meat
Round shaped beef patty
Doughnut shaped beef patty
cold spots
183
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Figure 3. Location of beef patty in MW oven cavity.
Luxtron
temDerature Drobes
Location of rotating wave guide underse ramie oven floor
Beef patty in open container (Petri plate)
Isolation layer to minimize conventional
heat transfer (Petri plate lid)
184
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Figure 4. Flow diagram of the procedure used to determine pasteurization time for beef
patties inoculated with E. coli 0157:H 7 microwave heated in standard microwave oven.
Determine a D and z-value fo r E. c o li 0157:H7 during
constant tem perature m icrowave heating
I
Calculate an end-point tem perature fo r 12 D process
(assuming that F-value a t the end-point temperature is 1 sec)
i
•
Find a pasteurization tim e (come-up tim e + 1 sec)
based on analysis o f heating profiles
of at least 30 microwave heated patties.
C onsider:
• location of the cold spot in microwave oven cavity
• wattage at the selected power setting
• penetration depth of microwaves within patty
location of the temperature probes in the cold spot of the patties
I
Test accuracy o f pasteurization tim e
(in standard MW oven - Quasar model)
Inoculation pack studies
(patties spiked with E. coli 0157:H7)
I
Test accuracy o f pasteurization tim e in other models o f m icrowave ovens
Com pare:
• location of the cold spot in microwave oven cavities
• wattage at the selected power setting
185
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Figure 5.
Formula used for calculation o f end-point temperature during microwave
pasteurization o f beef patties.
Equation:
1
Assumptions:
•
•
12D process
F-process time (12D) = 1 sec
End-point Temperature
Calculated as follows:
e-g.
Fref = 12 D = 12 x 30.27 min
Tref = 57°C (Tab. 1)
z = 2.5°C
F = 1 sec
T=?
186
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Figure 6. Heat penetration profiles of beef patty heated by microwaves at high power
level.
Location of probes is described in the Figure 1.
Pasteurization time was
determined when the lowest temperature within the meat reached minimum 67.8°C.
100 T
i
90 J-
—■— tem. prob. 1
—»— tem. prob. 3
—•— tem. prob. 2
—
tem. prob. 4
1
End-point temp. = 67.8 C
E
50 t
£
40
i
IF(67.fl C)= 1 sec
Pasteurization time
= come-up time + 1:
= 2 min 30 sec
time(sec)
187
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 7. Example of temperature values within round-shaped beef patties exposed to high MW power during various trials to
determine proper pasteurization time. Location of probes within meat patty was described in Figure 1. "Trial and error" approach
was used to determine pasteurization time . Pasteurization time was defined as a microwave heating time when the lowest
temperature in 30 consecutively heated beef patties was minimum or above 67.8°C.
110
100
o
d>
3
F nri-noinl tpm naratnra = R7 RV.
E
&
E
Trial 1 - 9 min
CM
Trial 1 - 9 min 10 car
Trial 1 - 9 min 90 car
CM
188
Trial 1 - 9 min ^0 cor
110 ♦
100
♦
l.
A»:
l*
90
" ■
4
| 4
80 T
o
S
O.
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*
,
•
A
t
a
*A
" '
i
■
•
A
♦
•
■
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!
V
13
£03
60
C
50
£
3
2
q.
40
E
03
h30
■ 2 min 30 sec - probe 1
□ 0 sec - probe 1
A 2 min 30 sec - probe 2
A 0 sec-probe 2
• 2 min 30 sec - probe 3
O 0 sec - probe 3
♦ 2 min 30 sec - probe 4
O 0sec-probe4
20
10
- I — I— I— I— I— I— I -
01
2 3 4 5 6 7 8
9 101112131415161718192021222324252627282930
Number of beef patty
Figure 8. Influence of initial temperature of beef on the temperature development in
round-shaped beef patties fully pasteurized after 2 min 30 sec of microwave heating at
high power level. Location of probes within meat samples is shown in Figure 1.
189
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Microwave pasteurization of foods. Part B. Critical parameters
of the process as demonstrated during microwave pasteurization
of beef patties for predicted pasteurization time in various
microwave ovens
190
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Abstract
A method established to predict pasteurization times during microwave heating
was tested using four consumer size microwave ovens (Sharp, Kenmore, General
Electric, Litton Minutemaster) and one Litton Commercial oven. Ground beef patties
inoculated with Escherichia coli 0157:H7 were exposed to microwaves for a standard
pasteurization time of 2 min 10 sec and 2min 30 sec at a high power level, and at the
medium power level for 4 min and 3 min 20 sec for doughnut and round-shaped patties
respectively.
The pasteurized patties were tested for the presence of E. coli 0157:H7 and
mesophilic bacteria present originally in the m eat To characterize physical factors
associated with the microwave pasteurization process, the power of the oven and hot
and cold spots at the bottom of the oven cavity were determined. The analysis of this
microbiological data revealed that only the Sharp oven delivered enough lethality to
inactivate £. coli 0157:H7 in the round and the doughnut-shaped patties at both power
levels.
The Kenmore oven was the least efficient in pasteurizing meat as E. coli
0157:H7 was recovered from the samples which were cooked in 4 different
combinations of the shape and the power output. An ability to prepare microbiologically
safe hamburgers in the General Electric oven and the Litton Minutemaster oven
occurred only for certain shapes and power output.
Mesophilic bacteria present
originally in the meat survived in the most o f the treatment conditions.
Two major
factors: the power of microwave oven and the uneven heating pattern on the bottom of
the oven cavity were related to the survival of the pathogen in the pasteurized meat.
191
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Simple methods were suggested to test the performance of microwave ovens.
Recalculation of the pasteurization time for the specific microwave oven based on
microwave energy balance was successful as proved by the microbiological studies
carried out fo r the Litton Commercial oven.
introduction
Microwave pasteurization of foods has become an attractive process fo r food
preservation. A drawback of the microwave heating is a lack of a method to determine
the lethality of the process. Methods are needed that would allow us to include the
most important variables of the process and to predict the outcome. A great deal of
information exists, on the physiochemical aspects o f food-microwaves interactions
(Mudgett, 1986 b; Thuery, 1992). Factors were identified that determine an amount of
heat generated during the process.
Attempts were made to determine a heat
distribution pattern within the food (Ohlsson, 1990; Datta, 1990; Barringer, 1994).
There is minimal knowledge on which factors are the most critical during microwave
pasteurization.
The quality o f the research on the safety of microwave heated foods is related
to the understanding o f engineering aspects of microwave heating. Conflicting results
were reported in the literature due to the lack of a proper methodology that would
address the characteristics o f microwave heating. Several reviews are available on this
topic such as a recent review by Heddleson and Doores (1994). Different studies have
shown (Carlin et al., 1982, Knutson et al., 1987; Heddleson et al., 1994) a lack of
192
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consistency of the suggested pasteurization parameters, particularly when the old
generation o f microwave ovens were used.
Simple standard methods have to be established to validate the performance of
microwave ovens, although progress has been made in recent years (Buffler, 1992;
Buffler and McNutt, 1994; Schiffmann, 1990; Buffler, 1990; Schiffmann, 1992). Some
manufacturers adapted standard methods to measure the power of the microwave
ovens (Risman, 1993; Buffler, 1991). Innovations in oven design such as turntables,
programmable power levels and humidity sensors make microwave cooking more
predictable (Mudgett, 1990). Progress in using microwaves in the preservation of foods
is related to the developments in microwave oven technology. New microwave oven
design could improve the safety of microwave heated foods.
The engineering
modifications of microwave ovens might resolve the apparent differences such as the
power output difference o r the uniformity of electromagnetic field. This would enhance
the application of microwave ovens by consumers and on the industrial scale.
However, as Geriing (1987) stated, new innovative solutions exist but the cost of
microwave ovens would increase.
A microwave pasteurization technology could have immediate application in a
fast-food industry and a food-service industry if the microbial safety problem can be
solved through the proper monitoring of the microwave heating conditions (Sawyer et
al., 1983; Dahl et al., 1981).
Effectiveness of microwave pasteurization is heavily
dependent on the consistent performance parameters o f the microwave pasteurization
equipment (Stanford, 1990).
193
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This research is focused on identification of the most important parameters that
would allow us to distinguish between two different microwave heating conditions that
lead to the unexpected survival of pathogens in microwave heated foods.
In the
previous chapter a method was established to predict microwave pasteurization time for
beef patties which was proven to produce pathogen-free beef patties in the standard
microwave oven. In this report a validity o f standard pasteurization time will be tested
in five other microwave ovens that represent a variety of the oven designs present on
the market.
Material and methods
Standard pasteurization value
A new method to determine the pasteurization time for the ground beef patties
was established.
The approach is based on the procedure used in conventional
thermoprocessing. The F-process time method (Stumbo, 1973) was adapted to nonisothermal microwave heating conditions.
A protocol of the method involves a
calculation of the end-point temperature using E. coli 0157:H7 destruction data
determined during an isothermal microwave heating.
resulted in determining the pasteurization times.
Heat penetration experiments
A description of the process was
presented in the previous chapter.
The following pasteurization times should be used fo r 110g-patties: for the
round shaped patties 2 min 30 sec and 4 min; for the doughnut-shaped patties 2 min
10 sec and 3 min 20 sec respectively at the high and the medium power output.
194
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Microwave ovens used in study
The standard pasteurization times were determined in Quasar microwave oven
which has been described previously. Five other microwave ovens were used in the
study including Sharp, Kenmore, General Electric, Litton Minutemaster and Litton
Commercial models. They represent different designs of microwave ovens used by
consumers and a variety of microwave cooking conditions.
New generation models
are equipped with programmable power settings as compared to adjustable power
settings (Table 1). The Sharp microwave oven possesses a turntable in contrast to four
other models.
Power o f microwave ovens
The power of the microwave ovens used in the studies was determined with a
IMP - 2 L test as described by Buffler (1992). A load of two 1-L beakers (Pyrex), each
containing 1 L of water at approximately 20°C, was placed in the microwave oven
cavity. The oven was turned on fo r 2 min and 2 sec at the tested power level. The
beakers were removed from the oven, the final temperature of water was measured
and the power was calculated using the heat balance formula:
P = 70(ATi + AT2)/2
(1)
where A L and AT2 are temperature differences between the initial and the final
water temperature in beaker 1 and beaker 2. Three replicate tests were performed for
each power setting (Table 2).
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Microwave pasteurization of patties - microbiological tests
A detailed description o f the methodology was presented in the previous
chapter. Ground beef was mixed thoroughly with E. coli 0157.H7 culture grown fo r 18
to 24 hr at 37°C. The initial number of E. coli 0157:H 7 in the peptone suspension that
was added to the meat was enumerated using a spread-plate method. The recovery
media was Plate Count Agar with 1% sodium pyruvate. From this data, a concentration
o f bacteria in meat was calculated. A number o f mesophilic microorganisms present
originally in meat was enumerated using this same media except that plates were
incubated at 30°C for 24 hrs.
These plates were kept for an additional 3 d in the
incubator to recover the heat injured bacteria.
Patties containing E. coli 0157:H7 were refrigerated fo r 1 to 3 hrs until
pasteurization treatments were carried out in the particular microwave oven. Samples
were subjected to microwaves at the high and the medium power level as listed in
Table 2. One patty was placed in the center of the microwave oven cavity in an open
Petri plate (150 mm x 10 mm).
Immediately after heating, the cooked meat was
transferred to a stomacher bag filled with 110 mL peptone (4°C).
By adding cold
peptone water to the meat the heating process was terminated. A meat slurry (1:1 ratio
o f meat and peptone water) was obtained after stomaching the bag content. Samples
o f this meat slurry were used to enumerate E coli 0157:H 7 and other meat bacteria that
survived microwave pasteurization. A Dynabeads® anti-E. coli 0157 method was used
to test fo r the presence o f the pathogen in the beef.
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Thermographs of microwave oven
A thermal paper (high sensitivity fax paper, 216 mm wide, in rolls, Perfect Print
Co) was used to identify cold and hot spots occurring within the oven cavity of each
microwave oven. A simple, one-dimensional mapping technique was used by placing a
layer of the paper on the bottom o f the oven cavity. A total area o f the bottom surface
was covered to obtain the thermograph.
This thermal paper was exposed to
microwaves fo r 5 min at the high power setting in each oven.
Black color on the
thermograph represents the hot areas in the oven. The thermographs were scanned
and their size was reduced using ScanJet II (v. 2.0, Hewllet Packard, Paulo Alto, CA)
and Adobe Photoshop (v. 3.0, Adobe System Inc., Mountain View, CA) software.
Picture o f m eat patty in the oven cavity - location o f the slowest heating zone
within the m eat patty
A careful observation of the change of the meat tissue color that occurred
during the heating helped to locate the coldest spot within the meat patty.
Meat
samples were heated individually at the high power for 1 min 30 sec, 1 min 50 sec, 2
min 10 sec and 2 min 30 sec (pasteurization time) at the various locations within the
Quasar oven cavity - center, front, left and right side from center. The meat patties
were immediately removed from the cavity and pictures were taken. The cold spots
observed on the pictures were correlated with thermograph of the Quasar oven.
197
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Results
Power of microwave ovens
Microwave ovens were selected with different maximum power output and the
mode o f setting power to test a variety of cooking conditions. The proposed method to
estimate the pasteurization times fo r the beef patties processed by microwaves may
have practical application.
The protocol of this method is valid if the pasteurization
times, when used in various microwave ovens, will result in E. coli 0157:H7-free
cooked meat. The results of the IMPI - 2 L tests that were earned out to find a real
power output at the high and the medium level are shown in the Table 2. The Quasar
oven was rated by the m anufacturer at 900W and performed at 808W. The IEC power
measurement test was used by the manufacturer.
Buffler (1992) states that both
methods give different results. The IEC measurement is 100 to 150W higher than the
2-liter measurement which is in an agreement with our observations. The power o f the
Litton Commercial oven was approximately 2.5 times higher that the standard Quasar
oven. The power output o f the four ovens (Sharp, Kenmore, General Electric, Litton
Minutemaster) at the high setting was similar, 600W -power range. Bigger differences
were noted for these same ovens at the medium setting (up to 120W difference
between ovens).
Temperature distribution - thermographs
The thermographs o f the bottom cavity of the oven are shown in Figure 1.
These spots are caused by the uneven distribution of the electromagnetic field. The
more intensive is the dark color, the higher focusing of the microwave energy occurred
198
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in that region. Both Litton ovens, which represent the old oven designs, possessed a
very uneven heating pattern, particularly the Litton Minutemaster.
The Sharp oven,
which belongs to the new generation o f microwave ovens, is equipped with a turntable
and has an even distribution of the microwave power.
The difference between the
thermographs of these three ovens shows that the quality of the cooking can be
improved by installing turntables. The Kenmore and the General Electric models had a
similar temperature pattern with few er hot areas. The thermograph o f the Quasar oven
showed very intensive power distribution within the cavity, with the cold spot located in
the center where a rotating waveguide is mounted.
The Quasar oven had a high
amount of power delivered to the cavity since almost the whole area o f the Quasar
thermograph is covered by the black color. This explains why the power output of this
oven is a 100W or more as compared to the power output of the four other consumer
ovens.
Pictures o f heated patties
One beef patty was placed in the Quasar oven and heated at the high power
level fo r a short time at a few different locations. The microwave exposure time was
increased slowly for the next heated sample to observe the heating pattern that
occurred within the m eat Pictures o f these partially cooked patties were taken and are
presented in Figure 2. The picture a, b, c of the patties heated in the center show
clearly that the coldest area within the meat (pink color o f undercooked meat) occurs in
the center. Placing samples on the left, front or right side of the oven causes a shifting
o f the cold spot within the patty due to uneven electromagnetic field distribution at this
location in the oven as demonstrated in the picture d, e, f. This recorded pattern of the
199
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heating reveals that the location of cold areas within the patty strongly depends on the
distribution of the microwave field within the microwave oven cavity.
Microbial studies correlated to the heating performance o f the microwave ovens
To test whether the suggested pasteurization values can be used in other
microwave heating environments, five microwave ovens were used in addition to the
standard Quasar oven. The results of inoculated pack studies are presented in the
Table 3.
The Sharp microwave oven delivered enough microwave energy to inactivate E.
coli 0157:H7 as did the Quasar oven during this same heating time.
The Litton
Minutemaster and the General Electric microwave ovens were not effective in killing E.
coli 0157:H7 in the beef patties during the predicted pasteurization times.
A
comparison of the microwave power levels between these two ovens explains that
difference.
The Litton Minutemaster operates at higher energy fo r the high power
setting than does the General Electric microwave oven. An opposite trend occurred at
the medium power; therefore the General Electric microwave oven pasteurized all 10
beef patties. It appears that the timebase (% time “off” to the total time “o ff and “on”
when oven operates in pulsing mode) (Geriing, 1987) for these two ovens is different.
Processing in the Kenmore oven did not produce safe, pathogen-free hamburgers
although its power output was higher than in the two previous ovens.
thermographs fo r these ovens are presented in Figure 1.
Analysis of
They partially explain
problems of uneven heating occurring in the centers o f these ovens.
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Readjusting of pasteurization time due to the power
The power measurements in the Litton Commercial oven showed that this oven
operates at a much higher power level as compared to other ovens. Therefore, using
these same pasteurization
parameters was not appropriate.
For this oven,
recalculation o f the pasteurization time was done based on the energy needed to
achieve a successful treatm ent Table 4 shows that the new pasteurization values are
61 sec and 101 sec fo r the round patties and 53 sec and 85 sec fo r doughnut-patties at
high and medium power respectively. Those meat patties heated fo r the above
pasteurization times in the Litton Commercial oven were free from pathogens and
indigenous meat flora and still possessed a good texture. Thus, this method allows for
adjustments of pasteurization values when the power o f microwave oven is known.
Discussion
Microwave heating of foods is a very complex phenomenon that is determined
by many variables (Mudgett, 1986 a; Metaxas and Meredith, 1983, Decareau, 1985).
As compared to a conventional heating, the heat source is not external but results from
imposing an electromagnetic field on polar molecules of foods. The conventional heat
transfer is only a part of the microwave heating process (Datta, 1990).
Physical
properties of the microwave field (frequency and power) and rules of optics (reflection,
refraction, transmission and absorption) govern the effectiveness o f microwaves to
create the
thermal effects in the foods (Mudgett, 1982).
Properties of the foods,
including the dielectric properties (determined by water and ion content), characterize
an interaction of the food molecules with the electromagnetic field and they change
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with the temperature (Buffler and Stanford, 1991).
The penetration depth of
microwaves within the food is related to its geometry and size (Ohlsson and Risman,
1978).
Heat and mass transfer is influenced by packaging and the physiochemica!
characteristics of the system subjected to microwaves. The design of the microwave
oven is responsible for the unique pattern o f the electromagnetic field in the oven cavity
(Ohlsson, 1990). The physical environment in the oven cavity (air temperature and
velocity) influences the rate of heat and mass transfer. A summary o f listed factors,
which in some cases overlap each other, are presented in Table 5. The high number of
variables explains why difficulties were experienced in many studies to establish a
method predicting the lethality o f the microwave pasteurization (Huang et al., 1993;
Gundavarapu et al., 1995). However, by categorizing them according to their
importance on the lethality effect, better microbiological studies can be conducted with
the focus on its critical elements.
During the course of this investigation, the microbiological studies of the beef
patties cooked in various ovens showed that, even though the food variables are
eliminated (constant mass, size, temperature), a great variation in the oven
performance resulted in a different lethality delivered to bacteria.
Safety factors
implemented in model (12D, lethality during come-up time) were not enough to
compensate fo r variability o f the thermal effects produced by the different models o f
microwave ovens. It was proved, through the power measurements, thermographs and
pictures o f beef patties cooked gradually in cold and hot spots o f the Quasar oven, that
survival of bacteria is due to the variability in the microwave oven performance. Results
of the power test and the thermographs characterize ovens used in this study, and they
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are presented in the Table 2 and the Rgure 1. Pictures of the patties, taken gradually
during heating, show clearly that the cold spot within patties are in this same location
where the cold spots occur within the oven. Therefore, a power distribution in the oven
and the power output are the most important parameters of the microwave
pasteurization.
There is a great variation of microwave ovens on the market (Davidson, 1991).
Expensive models have either a turntable o r a stirrer that mixes microwaves entering
the oven and creates a uniform pattern. Less expensive models have an uneven heat
pattern thus more hot and cold spots. Microwaves usually enter the cavity from the
side but in the Quasar oven microwaves enter the cavity from the bottom of the ceramic
floor through the special rotating wave guide o f a unique design (Buffler, 1992). Ovens
show a wide variation in the total power and special distribution of power in oven cavity
(Buffler, 1991). After years of using microwave oven its power output decreases. A
magnetron, a major part that produce microwaves, looses its ability to generate
microwaves. Aging process of magnetron accelerates when microwave oven operates
with small loads or empty cavity.
Microwaves which are not absorbed by food or
dummy load build into the oven return to magnetron and cause significant damage of
magnetron.
One of the disturbing aspects of microwave technology is the fact that the ovens
on the market do not have standard power output (Mudgett, 1990). A regulation is
needed in this area, but there has been a long debate in the industry regarding which
methods should be used to rate the power o f ovens (Buffler, 1992). There are few
methods available to test the power output which may produce different results
203
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(Risman, 1993; Buffler, 1992). They use this same basic concept which is heating a
container of water in the oven for a specific time or to the required temperature and
calculating the power from the energy balance equation.
The power measurement
strictly depends on the size o f the load, volume and geometry of the water load. Two
tests are widely used, a 1-L test established by IEC (International Electrotechnical
Commission) and a 2-L test procedure promoted by the International Microwave Power
Institute (IMPI). Since the IEC test produces higher power values it has been used the
most often by oven manufacturers as in the case o f Quasar oven. However, Geriing
(1987) explains that the maximum power which can be delivered by a magnetron to the
oven cavity is in the range o f 750W. This value is simply dictated by a regulatory
voltage in outlet (120 Volts, 15 Amperes).
Lethality delivered to the bacteria in food exposed to the microwave field relates
to the thermal effects occurring in the food. The thermal performance of the oven is a
combination of the oven heating pattern and the geometry of the food.
The
pasteurization times determined using the proposed method are valid fo r the specific
size and geometry o f beef patties when pasteurization is performed in the standard
oven.
When a different oven is used, the power output and the distribution of the
electromagnetic field changes, and the pasteurization values do not produce a
pathogen-free beef patty. A solution to this problem is readjusting the pasteurization
time. Our simple tests showed that the pasteurization times can be adapted to the new
power values. But at this same time, the size and geometry of the heated food remains
this same.
Changes of food would required performing a new penetration test.
A
power measurement test is easy to perform and allows a comparison of a variety of
204
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microwave models. At this same time a thermograph has to be made (5-min test) to
assure that the new oven does not have unusual problems associated with heat
distribution pattern.
Results presented in this study show a model-to-model variation between
microwave ovens. There are also oven-to-oven variations among these same models
as a result of lot-to-lot variation of parts of the oven.
The example is a difference
between individual capacitors which shows that even fo r identical models power can
range from 575W to 675W (Geriing, 1987).
Oven design-to-oven design variations
include a timebase - a time between pulses during on-off cycles to achieve lower power
setting in the oven.
This phenomenon was noticed among the ovens used in this
study. For the Kenmore and the Sharp ovens a similar power output was measured at
the high power setting (672W vs. 636W) but different for the setting 60 (484W vs.
405W).
There are other factors that influence the thermal effect o f the oven (Buffler,
1992) such as a magnetron - age and type and changes in power supplies (5% voltage
drop will cause drop of power in 850W oven to 350W).
This also influenced the
performance of ovens used in this study and reflected on the variability o f temperature
during heating of beef patties.
Lentz (1993) and Geriing (1987) pointed out that many innovative features can
be added to existing oven designs but this would increased the cost o f ovens on the
market which is very price competitive. Unfortunately, this could eliminate the improved
or new models o f microwave ovens from the market which was the case with few
excellent designs in the past.
205
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The difficulty in determining the safety o f microwave heated foods is a
combination o f a lack of understanding of the principles o f the microwave heating as
well as the variability in the performance observed among microwave ovens.
The
technology of microwave heating and the knowledge about the microwave science is
still under development.
Conclusions
Differences between the microwave oven power and the power distribution are
major factors responsible for a lack of safety o f microwaveable foods.
Standard
pasteurization times can be used for heating hamburgers in other microwave ovens. A
comparison of different oven performance, their power output at the specific power
setting and their thermographs have to be carried out.
Both these factors can be
established using simple methods.
References
Barringer, S. A.
1994.
microwaved food systems.
Experimental and predictive heating rates of
Ph.D. thesis, p. 14-17.
University of Minnesota,
Minneapolis.
Buffler, C. R. and M. A. Stanford.
1991.
Effect of dielectric and thermal
properties on the microwave heating o f foods. Microwave World. 12:15-23.
Buffler, C. R.
1990.
An analysis of power data for the establishment of a
microwave oven standard. Microwave World. 11(3):10-14.
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Buffler, C. R.
1991.
Microwave power measurement: impact on food
processors. Microwave World. 12(1): 19-24.
Buffler, C. R.
1992.
Microwave cooking and processing, p.47-68.
Van
Nostrand Reinhold, New York
Buffler, C. R. and R. M cNutt
1994.
Efficiency improvement techniques for
microwave ovens. Microwave World. 15(1):16-19.
Carlin, F., W. Zimmermann and A. Sundberg.
1982.
Destruction of trichina
larvae in beef-pork loaves cooked in microwave ovens. J. Food Sci. 47:1096-1099,
1118.
Dahl, C. A., M. E. Matthews and E. H. Marth. 1981b. Survival o f Streptococcus
faecium in beef loaf and potatoes after microwave-heating in a simulated cook-chill
foodservice system. J. Food P rot 44:128-133.
Datta, A. K.
1990.
Heat and mass transfer in the microwave processing of
foods. Chem. Eng. Progress. 6:47-53.
Davidson, H. L. 1991. Microwave oven repair, p. 292-340. TAB Books, Blue
Ridge Summit.
Decareau, R. V.
1985.
Microwaves in food processing industry, p. 1-57.
Academic Press, Inc., Orlando, Florida.
Geriing, J. E. 1987. Microwave oven pow er a technical review. J. Microwave
Power Electromag. Energy. 22:199-207.
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Gundavarapu, S., Y. C. Hung, R. E. Brackett and P. Mallikarjunan.
1995.
Evaluation of microbiological safety of shrimp cooked in a microwave oven. J. Food
Prot. 58:742-747.
Heddleson, R. A., S. Doores and R. C. Anantheswaran.
1994.
Parameters
affecting destruction of Salmonella ssp. by microwave heating. J. Food So. 59:447451.
Heddleson, R. D. and S. Doores. 1994. Factors affecting microwave heating of
foods and microwave induced destruction of foodbome pathogens: a review. J. Food
P rot 57:1025-1037.
Huang, Y. W., C. K. Leung, M. A. Harrison and K. W. Gates. 1993. Fate of
Listeria monocytogenes and Aeromonas hydrophila on catfish fillets cooked in
microwave oven. J. Food S d. 58:519-521.
Knutson, K. M., E. H. Marth and M. K. Wagner. 1987. Microwave heating of
foods. Lebensmitt.-Wiss. und-Technol. 20:101-110.
Lentz, R. R. 1993. Personal communication.
Metaxas, A. C. and R. J. Meredith. 1983. Industrial microwave heating, p.1103. Peter Peregrinus Ltd., London.
Mudgett, R. E. 1982. Electrical properties of foods in microwave processing.
Food Technol. 36(2):109-115.
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Mudgett, R. E.
1986a.
Microwave properties and heating characteristics of
foods. FoodTechnol. 40(6):84-86.
Mudgett, R. E. 1986b. Electrical properties o f foods, p.329-390. In: M. A. Rao
and S. S. H. Rizvi (eds.), Engineering properties of foods. Marcel Dekker, New York.
Mudgett, R. E. 1990. Development in microwave food processing, p. 359-404.
In: H. G. Schwartzberg and M. A. Rao (eds.), Biotechnology and food process
engineering. Marcel Dekker, Inc., New York.
Ohlsson, T. and P. O. Risman. 1978. Temperature distribution of microwave
heating - spheres and cylinders. J. Microwave Power. 13:303-309.
Ohlsson, T. 1990. Temperature distribution in microwave heating -influence of
oven and food related factors, p. 231-241. In: W. E. L. Spiess and H. Schubert (eds.)
Engineering and food. Elsevier Applied Science, New York.
Risman, P.O.
1993.
Microwave oven loads fo r power measurements.
Microwave World. 14(1):14-19.
Sawyer, C. A., Y. M. Naidu and S. Thompson. 1983. Cook/chill food-service
systems: microbiological quality and end-point temperature o f beef loaf, peas and
potatoes after reheating by conduction, convection and microwave radiation. J. Food
Prot. 46:1036-1043.
Schiffmann, R. F.
1990.
Problems in standardizing microwave oven
performance. Microwave World. 11(3):20-24.
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Schiffmann, R. F. 1992. Major problems in heating foods in microwave ovens.
Microwave W orld. 13:21-23.
Stanford, M. S. 1990. Microwave oven characteristics and implications for food
safety in product developm ent Microwave W orld. 11(3):9-11.
Thuery, J.
1992. Microwaves: industrial, scientific, and medical applications,
p.3-106. Artech House, Boston.
210
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Table 1. Types o f consumer microwave ovens operating at 2450 MHz frequency used
in studies.
Application
Microwave oven
Brand
Power settings
Model
Standard oven
Quasar
MQS 1403
5 programmable levels
Other ovens tested
Sharp
Carousel II
10 programmable levels
Kenmore
88762
adjustable levels
General Electric
JET 91 OV1
3 power levels
Litton
Minutemaster 425
adjustable levels
Litton
Commercial Oven
System 70/80
2 power levels
211
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Table 2. Power of MW ovens used in studies.
Microwave oven type
Power measured1) ( W ) - Oven power setting
High
Medium
Quasar
808
(high)
463
(medium)
Sharp
672
(100)
484
(60)
General Electric
608
(high)
382
(medium)
Kenmore
636
(60 - bake)
405
(100 - max)
Litton Minutemaster
655
(high)
364
(simmer)
Litton Commercial Oven
1983
(high)
1098
(half)
1) power determined with IMPI 2 - liter test (Buffler, 1992)
212
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 3. Survival of E. coli 0157:H7 and other microorganisms present in beef patties during microwave pasteurization.
Round and doughnut-shaped beef patties were processed in 6 different microwave ovens operating at high and medium
power settings.
Initial population of E. coli 0157:H7 in all samples were 1.2 x 104 CFU/g and other microorganisms
enumerated by APC method 3.8 x 104 CFU/g.
Beef patties
Power/
Past, time
Round shape
High / 150 sec
Medium / 240 sec
Doughnut shape
High /1 5 0 sec
Medium / 240 sec
Ovens
Bacteria tested
Gen. Elect
Sharp
Kenmore
Litton Minut.
j)
+2>
+
-
a p c 3)
+
+
+
+
E. coli 0157: H7
-
+
+
APC
-
+
+
E. coli 0157.H7
-
+
-
APC
+
+
+
+
E. coli 0157:H7
-
+
+
+
APC
+
+
+
+
E. coli 0 1 57:H7
1) - no survivors ( in 10 patties tested)
2) + survivors (in at least one out of 10 patties tested)
3) - APC - Mesophilic bacteria (30°C, 4 days), detection level 102 CFU/g or higher
213
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Table 4. Recalculation of microwave heating time determined in standard oven (Quasar) for microwave oven operating at
different power level (Litton Industrial) to obtain equal pasteurization values.
Quasar Oven
Power (W)
Litton Industrial Oven
Energy1* (J)
MW heat, time(s)
Power (W)
MW heat, time (s)
Microbial survival
E. coli 0157:H7
A. Round patties
High power
808
High power
150
121,170
Medium power
463
1,983
61
Half power
240
111,312
1,098
101
B. D oughnut patties
High power
808
High power
130
105,014
Medium power
463
532)
1,983
Half power
200
92,760
85
1,098
1) - Energy = Power (Quasar) x Microwave Heating Time (Quasar)
2) - Microwave Heating Time (Litton) = Energy / Power (Litton)
214
Indigenous flora
Table 5. Microwave heating o f foods - major factors that determined temperature level
and distribution in foods during microwave heating.
Physical properties of microwaves
Properties of food system
Microwave equipment
•
EM (electromagnetic) field of
microwaves
- frequency
-pow er
•
radiation o f microwaves
- reflection
- refraction
- transmission
- absorption
•
dielectric properties
- k, k"
- penetration depth
•
water, ion content
•
geometry, size
•
initial temperature
•
physiochemical characteristics
- thermal
- mechanical
•
packaging
•
microwave oven
- geometry
- power output
•
environment in
cavity
- temperature
- humidity
microwave
215
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oven
Figure 1. Thermographs showing cold spots (light colors) and hot spots (dark colors) on
the bottom of the cavity o f microwave ovens used in this study.
Thermal paper w as
exposed to microwaves fo r 5 min at the high power setting.
A) Quasar MW Oven
C) Kenmore MW Oven
D) General Electric MW Oven
E) Litton - Minutemaster MW Oven
F) Litton - Industrial MW Oven
216
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Figure. 2 Distribution o f cold and hot spots in microwave heated meat patties due to its
location in the oven cavity. A cold spot is located in the center of a hamburger when it is
heated in the center of the Quasar microwave oven for A) 60%, B) 73% and C) 87% o f the
total cooking time respectively. The position of a cold spot in a hamburger changes w hen
its heated D) on the left side E) in the front and F) on the right side of the oven for 60% o f
the total cooking time.
Thermograph of Quasar microwave oven; gold spots - light colors, hot spots - dark colors.
urn
217
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Effect of dry and microwave heating on the cell structure
218
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Abstract
The mechanism of heat destruction of bacterial cell material caused by
microwaves versus conventional heating is not well understood.
This study was
initiated to describe approaches that can be used to determine structural changes that
occur as the result of the microwave heating o f bacteria. The extent of cell damage
that resulted from microwave treatment was compared to the changes in corresponding
samples exposed to dry heat during similar time-temperature treatments. A non-liquid
environment was used to carry out this comparison study. Cell material was transferred
from a single colony grown on agar to a micro tube. The colony was spread on the
inside surface o f this tube.
Then the tube was exposed to microwaves in the
microwave system maintaining 55°C (in the cell matrix environment) for 10 min.
A
replicate micro tube with the cell matter was exposed to dry heat at 55°C fo r 10 min.
Microscopic techniques used included bright-field microscopy, transmission electron
microscopy, and epifluorescence microscopy with propidium iodide and SYTO® stains.
These methods were used to compare the structure o f the cells after both heat
treatments.
This preliminary study indicated that in the microwave heated cells,
physical lesions occurred in different areas of the cell as opposed to the cells that were
heated conventionally. A more systematic study using combinations of heating time
and temperature should be conducted to confirm this observation.
Introduction
In recent years interest has been reported concerning the microwave treatment
for sample preparation in several techniques that are used to study biological samples.
219
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A microwave fixation of samples for electron and light microscopy will reduce
significantly the time of these assays. Microwave fixation has been promoted as a new,
efficient tool in microscopy especially when new microwave ovens were constructed to
accommodate specific needs o f the sample preparation (Login and Dvorak, 1994).
Excellent results have been obtained when microwave irradiation was used fo r
immunohistochemistry (Vitarelli et al.,1995). The microwave fixation process has been
claimed to improve antigenicity and to preserve ultrastructural details of the cells. A
new procedure for fixing Northern and Southern blots in the microwave oven was
reported (Angeletti et al., 1995). And finally, microwave heating has been discovered
to be a simple and an efficient method to extract genetic material from cells (Bollet et
al., 1991; Dawson and Harris, 1995; Wang e ta l., 1995)
In all these studies better results were obtained when samples were heated in
microwave ovens as opposed to conventional heat treatments. The higher temperature
that can be achieved during microwave heating o f the samples could be responsible for
the observed effects. However, it may be that the difference in structural changes of
the heated cells during microwave and conventional treatments resulted from a
different sequence of the destruction of proteins during microwave and conventional
heating.
The possibility o f developing a higher temperature inside the cell was
discussed (Khalil and Villota, 1988; Mudgett, 1989), but because of the small size o f a
cell it is thought that it is unlikely to occur (Sastry and Palaniappan, 1991). However, it
has been known that formation of standing waves and internal focusing of energy does
occur in small spheres and cylinders (Ohlsson and Risman, 1978). The question is, is
220
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this phenomenon responsible for heat formation in small spheres and cylinders such as
bacterial cells.
To date no studies have been reported that had examined the damage caused
in bacteria during exposure to microwave and conventional heat processed at similar
temperatures.
To explore this, a study has to be conducted when bacteria are not
suspended in liquids since the extraction of DNA from the bacterial cell can be obtained
by direct exposure of cell surface to microwaves.
In this study we attempted to explore the extent of cell damage caused in E coli
0157:H7 cells when the cells were exposed to similar temperature conditions during
microwave and conventional heat treatments. Cell injury of conventionally heated cells,
has been studied extensively in the past (Hurst, 1977). Our research was not intended
to carry out a systematic study of the cell damage that results from microwave
irradiation and compare it with injured cells after conventional heating. The purpose of
our study was to initiate research in the area o f cell damage upon microwave heating.
We explored various microscopic techniques that could be used to analyze the extent
of cell destruction resuling from these treatments.
Materials and Methods
C ulture
E. co li 0157:H7 (strain 933) was obtained from the
FDA
laboratory
(Minneapolis, MN). Cells were cultured a t 37°C in 10 m l. Tripticase soy broth [(TSB)
(Difco, Ditroit, Ml)]. To obtain the cell matrix used in the study, the E. co li 0157:H7
221
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suspension was streaked onto the surface of Plate count agar with 1% sodium
pyruvate. Plates were incubated fo r 24 hrs until single bacterial colonies grew on the
agar surface. Sterile Q-tips were used to pick the cell matrix from a single colony. The
bacteria were brushed o ff onto the internal wall of sterile 39 x 10-mm micro tube
(Sarstedt, Inc., Newton, NC) and a thin layer of cells was formed. Immediately after,
the cells were exposed to either microwaves or dry heat
Heat treatment
Microwave heating
The E. coli 0157:H 7 cell matrix, deposited on the wall o f the micro tube, was
exposed to microwave heating.
A microwave system maintaining a constant
temperature was used to create an isothermal condition. The structure and operation
o f the system was described previously. A Luxtron temperature probe (Luxtron, Santa
Barabara, CA) was inserted into the tube through a small hole cut in the cap. The tip of
the probe was placed into the cell layer. This was accomplished by locating the probe
close to the tube wall at approximately a 45° angle (Figure 1). Teflon tape was used to
secure the probe in the cap o f the micro tube. The E coli 0157.H7 cells were exposed
to microwaves for 10 min at 55°C.
This temperature value was selected after
preliminary tests showed that the maximum temperature o f 55°C could be maintained in
the micro tube. Samples prepared for the electron microscopy study were heated in
the closed tubes, whereas samples prepared for epifluorescence microscopy viewing
were not closed when heated. The temperature was monitored during heating of the
samples. One sample was used per each heating.
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Dry heat treatment
E. coli 0157:H7 cells were spread on the internal surface o f the micro tube and
exposed to dry heat. Each tube was placed in a well of a dry heat block (Lab-Line
Instruments, Inc., Melrose Park, IL) previously adjusted to 55°C. A temperature probe,
which monitored the temperature during 10 min-heat treatments, was inserted inside
the cell matrix.
Microscopic examination of structural changes in heat treated cells
Light microscopy
Dry cells were removed from the vial by adding 1 mL o f filter-sterilized water (0.2
(im-pore size filter) to each vial.
Five microiiters o f bacterial suspension were
transferred onto the glass slide. Cells were fixed to the glass surface by drying the
suspension at room temperature.
Heat fixation of the slide was not done to avoid
changes in cell structure that could result from additional exposure to h e a t The sample
was stained with crystal violet fo r 1 min and thoroughly washed.
Light microscopy
examination of the stained bacteria was made using an oil immersion technique at
1250X magnification.
A bright-field capability of a Zeiss Photomicroscope III (Carl
Zeiss, Oberkochen, Germany) equipped with Optronix VI-470 color CCD camera was
used to capture images o f the bacteria. The digital images were acquired with the help
of Image - Pro Plus software (v. 1.2 for Windows, Media Cybernetics, Silver Spring,
MD). Images of stained cells were processed and figures were compiled using Adobe
Photoshop™ (v. 3.0, Adobe Systems, Mountain View, CA).
223
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Transmission electron microscopy studies
The procedure described by Beveridge et al., (1994) was followed with some
modifications. The heat-treated cells and the unheated cells (control) were fixed in a
formaldehyde-glutaraldehyde mixture [(content 6% glutaraldehyde (15 mL), 8%
paraformaldehyde (2.5 mL), water (2.5 mL), 0.2 M cacodylate buffer in 15% sucrose
(20 mL), 1% ruthenium red (0.4mL)]. The cells were post-fixed in 1% osmium tetroxide
prepared in 0.1 M cacodylate buffer and then dehydrated in acetone solutions (25%,
50%, 75%, 95%, and 100%). After the dehydration series, the cells were embedded in
Quitol resin.
Ultra-thin sections were prepared with a microtome (model MT -7000,
RMC) using a diamond knife. The sections were stained for 1 hr in 1% uranil acetate
and then fo r 5 min in 2.6% lead citrate. Transmission electron microscopy (TEM) was
performed with a Philips 300 Transmission Electron Microscope located at the Electron
Microscopy Laboratory, University of Minnesota.
Epifluorescence microscopy
The degree of membrane damage caused by microwaves and dry heat was
determined using fluorescent dyes. The LIVE/DEAD BacLight Viability Kit (Molecular
Probes, Inc., Eugene, OR) consists of a mixture of nucleic acid stains; propidium iodide
- red nucleic acid stain and SYTO 9® - green fluorescent nucleic acid stain. Generally,
these stains are used to determine alive and dead cells based on their difference in
ability to penetrate membranes. Cells with intact membranes stain fluorescent green
and those with damaged membranes stain fluorescent red. The dry cells, from the
microwave and dry heat treatments, were suspended in 1 mL of filter-sterilized water
224
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(0.2 pm filter). The bacterial suspension was filtered onto a black, 13-mm diameter,
0.2-|im pore-size polycarbonate filte r (Poretics, Corp., Livermore, CA). The filte r and
drain disc-support filter was housed in Swinney filte r holder attached to a plastic syringe
with Luer-Lok tip that was used in the filtration. The filte r with bacteria and disc were
placed on a microscopic slide. One hundred microliters of dye suspension was layered
on the surface of the filter. The dye suspension was prepared by adding 1.5 nL of
each dye to 100 mL o f filter-sterilized water.
The glass slide with the filte r was
enclosed in a dark box (to avoid exposure to light) fo r 15 min at room temperature so
the dyes could penetrate the membranes of the heated cells. The filter and drain disc
were removed from the box and the excess dyes were drained onto the surface of
chromatography paper.
The filte r with disc was placed on a new glass microscope
slide. A drop of mounting oil was applied on the filte r with bacteria and then a cover
slip was placed on the top. The cover slip was sealed with epoxy resin to prevent
spilling of the mounting
oil.
The samples were examined with the Zeiss
Photomicroscope III which was described previously.
Microscope fluorescence
capabilities were obtained by using a 100W mercury lamp and a set of optical filters
(PB450-490 exciter, FT510 and LP520 barrier filters).
Sections of the filter were
examined and images were captured with the CCD camera. Green and red bacteria
were counted in a minimum of 20 fields, following a procedure described by Pettipher
(1986), Kepner and Pratt (1994), Tortorello and Gendel (1993), and Tortorello and
Steward (1994).
225
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Results and discussion
The experimental design presented in Figure 1 was aimed to explore the effect
of microwave heating on bacterial cells that were not suspended in a liquid medium. In
this condition, heat is not transferred to the bacteria from the outside environment. It is
produced as a result o f energy dissipated by water ions and other polar molecules that
occur within the cell. As the microwave irradiation proceeds in time, water evaporates
from the cell, the temperature in the environment surrounding the cell increases, and
the rate of the heat transfer from the cell to the environment decreases. In contrast,
during dry heating when the cell is exposed to hot air, heat is transferred from the
outside environment into the cells. These two different directions o f the heat flow may
effect the extent o f damage caused to the bacteria during heating.
We decided to
examine this process using microscopic techniques.
It was our intent to create similar conditions to those suggesting DNA extraction
from cells using the microwave oven (Dawson and Harris, 1995; W ang et al., 1995). In
our system the temperature was controlled and was constant during microwave heating
of the micro tubes. A thin layer of cells was formed on the internal wall of the tube.
The tip of the Luxtron temperature probe (diameter approximately 1mm) was placed
within the cell matrix. W e attempted to create similar temperatures outside the cells
during both conventional and microwave heating.
The morphology o f microwave and conventionally treated cells were compared
with bright-field light microscopy. The oil-immersion technique in combination with the
optical capability of the microscope allowed us to obtain 1250X magnification.
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Observation o f cells stained with crystal violet revealed a concentration o f intracellular
material at the poles of the cells that were exposed to microwaves. This phenomenon
occurred to a lesser extent in conventionally heated cells. Figure 2 shows a picture of a
microscopic field with microwave heated cells. A higher concentration o f dye is noticed
at the end of the cells, suggesting uneven distribution of cellular constituents upon
microwave heating.
Electron microscopy methods were explored to look at cross sections of cells
exposed to both treatments and to compare the structure o f the heated cells with the
unheated control.
Figure 3 indicates that cells heated by microwaves and dry heat
show different effects on ceil wall morphology. Physical lesions occurred in different
areas of the microwave heated cells when compared to the cells that were heated
conventionally. Extensive membrane disruption occurred in cells exposed to dry heat
but the membrane in microwave heated cells was not disrupted.
These cells were
heated at 55°C fo r 10 min by microwaves and dry heat and both treatm ent are
comparable in terms of tim e temperature profiles which are presented in Figure 4
(graph A).
An epifluorescence microscopy technique was used to investigate the effect of
both heat treatments on permeability of ceil membranes. Detection of fluorescent dyes
as they penetrate microbial cells has become a rapid and convenient method for
quantitative assessment o f the viability of bacteria (McFeters et al., 1995; Pettipher,
1986; Lloyd and Hayes, 1995). Epifluorescence microscopy is a real tim e technique
and measures the condition of certain cell constituents as they react with various
fiuorogenic compounds. This method has been proposed to use in the detection of
227
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pathogens in a food system (Pettipher, 1986).
Good results were obtained fo r the
enumeration of E. coli 0157:H7 in beef, milk and juice (Tortorello and Gendel, 1993;
Tortoello and Steward, 1994). However, the disadvantage of this method is that the
intensity of fluorescence, and thus concentration o f bacteria in a food sample, is
affected by the heat treatment to which bacteria were exposed during processing (Back
and Kroll, 1991). Therefore, we found this an excellent method to show the difference
between cells that were exposed to microwave and dry heat conditions. If the extent of
the heat damage in the membrane o f treated bacteria vary due to these two heating
methods, then these differences could be detected with epifluorescence microscopy
using various fluorescent dyes. Appropriate for these techniques are fluorescent stains
that bind to nucleic adds such as acridine orange, SYTO® or stains DAPI, propidium
iodide which mode of action is based on dye exdusion prindples (Haugland, 1994;
Lloyd and Hayes, 1995)). The LIVE/DEAD® BacLight™ Bacterial Viability Kit was used
in this study since it contained a mixture of two fluorescent stains, SYTO 9® and
propidium iodide. According to the manufacturer of the assay kit, these stains differ in
their ability to penetrate bacterial cells. SYTO 9® labels all the bacteria in a population,
those with intact and damaged membranes.
Propidium iodide (red) penetrates only
bacteria with damaged membranes, competing with SYTO 9® (green) when both dyes
are present.
Figure 5 shows the bacteria with the intact membranes (green) and
damaged membranes (red) in the populations exposed to microwave and dry heat
treatments.
Pictures A and B represent typical microscopic fields observed for the
bacteria exposed to microwaves and dry heat respectively. It appears that the number
of bacteria with intact membranes is higher in microwave treated population (22.7%)
when compared to conventionally heated bacteria (5.44%) (Table 1). Cells of E. coli
228
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0157:H 7 were microwaved in open micro tubes to minimize the heat flow to the cell
from the environment surrounding the cells.
The thermal profiles indicate that
microwave cells were exposed to lower temperatures at the early stage o f the heating
which maybe responsible for better survival o f cells. Nevertheless, these results are in
agreement with the observation of TEM cross sections o f the cells heated by these two
methods (Figure 3).
Different techniques could be used in future studies to determine the degree of
inactivation o f cell constituents caused by microwaves and dry heat such as respiratory
activity of bacteria which can be measured by using terazolium salts (Betts et al., 1989;
Schaule et al., 1993) and p- galactosidase activity which can be measured using flow
cytometry (Alvarez, 1993).
Future research on the effect of microwaves on bacterial cells should cover
areas yet unexplored such as exposure o f dry bacteria (with various levels of water
content) to microwaves and the study o f morphological or physiological changes
caused to bacterial cells due to this treatm ent
It could be a fascinating subject of
research to investigate the effect of sublethal microwave heating on bacteria by
studying the expression of a stress protein in bacteria or the release of phage from
lysogenic cells.
Conclusions
Results obtained here indicate that the structure of the bacterial cell when
heated under these conditions may be different than when exposed to sim ilar timetemperature condition during microwave and dry heat treatments.
However, more
229
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extensive studies are needed that would include various combination of temperature
and heating time.
References
Alvarez, A. M., A Ibanez and R. Rotger.
1993.
(5-galactosidase activity in
bacteria measured by flow cytometry. Biotechniques. 15:974-976.
Angeletti, B.. E. Battiloro, E. Pascale and E. Dambrosio. 1995. Southern and
Northern blot fixing by microwave oven. Nucleic A dd Res. 23:879-880.
Back, J. P. and R. G. Kroll.
1991. The differential fluorescence of bacteria
stained with acridine orange and the effect o f h e a t J. Appl. Bacteriol. 71:51-58.
Betts, R. P., P. Banks and J. G. Banks.
1989. Rapid enumeration o f viable
micro-organisms by staining and direct microscopy. Lett. Appl. Microbiol. 9:199-202.
Beveridge, T. J., T. J. Popkin and R. M. Cole. 1994. P. 42-64. In: P. Gerhard,
G. E. Murray, W. A. Woods and N. R. Krieg (eds.). Methods for general and molecular
bacteriology. American Sodety for Microbiology, Washington, D. C.
Bollet, C., M. J. Gevaudan, X. de Lamballerie, C. Zandotti and P. de Micco.
1991. A simple method for the isolation of chromosomal DNA from Gram positive or
a d d -fa st bacteria. Nudeic Add Res. 19:1995.
Dawson, E. P. and J. R. Hams.
1995.
Rapid PCR sample preparation for
multiple amplifications. J. NIH Res. 7:64.
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Haugland P. 1994. Molecular probes. 5th ed. Molecular Probes, Inc., Eugene.
Hurst, A. 1977. Bacterial injury: a review. Can. J. Microbiol. 23:935:944.
Kepner, R. L. and J. R. Pratt.
1994.
Use of fluorochromes for direct
enumeration o f total bacteria in environmental samples: past and present. Microbiol.
Rev. 58:603-:615.
Khalil, H. and R. Villota.
1988. Comparative study on injury and recovery of
Staphylococcus aureus using microwaves and conventional heating. J. Food Protect.
51:181-186.
Lloyd, D. and A. J.
Hayes.
1995.
Vigour, vitality and viability of
microorganisms. FEMS Microbiol. Lett. 133:1-7.
Login, G. R. and A. M. Dvorak.
1994.
Methods o f microwave fixation for
microscopy. Progr. Histochem. Cytochem. 27:72-94.
McFeters, G. A., F. P. Yu, B. H. Pyle and P.S. Stew art
1995. Physiological
assessment of bacteria using fluorochromes. J. Microbiol. Methods. 21:1-13.
Mudgett, R. E.
1989.
Microwave food processing.
Food Technol. 43:117-
126.
Ohlsson, T. and P. O. Risman. 1978. Temperature distribution of microwave
heating - spheres and cylinders. J. Microwave Power. 13:303-309.
Pettipher, G. L.
1986.
Review, the direct epifluorescent filte r technique.
Food Technol. 21:535-546.
231
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J.
Sastry, K. S. and S. Palaniappan. 1991. The temperature difference between a
microorganism and a liquid medium during microwave heating.
J. Food Process.
Preserv. 15:225-230.
Schaule, G., H.C. Fleming and H. F. Ridgway.
1993.
Use o f S-cyano-2,3-
ditolylterazolium chloride for quantifying planktonic and sessile respiring bacteria in
drinking water. Appl. Environ. Microbiol. 59:3850-3857.
Tortorello, M. L. and D. S. Steward. 1994. Antibody-direct epifluorescent filter
technique for rapid, direct enumeration o f Escherichia coli 0157:H7 in beef.
Appl.
Environ. Microbiol. 60:3553-3559.
Tortorello, M. L. and S. M. Gendel. 1993. Fluorescent antibodies applied to
direct epifluorescent filte r technique fo r microscopic enumeration of Escherichia coli
0157:H7 in milk and juice. J. Food Protect 56:672-677.
Vitarelli, E., G. Sippelli, G. Tuccari and G. Barresi. 1995. The use o f microwave
irradation for immunohistochemistry - a new methodological proposal.
Histol.
Histopathol. 10:35-38.
Wang, B., M. Merva, M. V. Williams and D. B. Weiner.
1995.
Large-scale
preparation of plasmid DNA by microwave lysis. Biotechniques. 18:554-555.
232
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 1. Protocol of experiments designed to show the structure of E. coli 0157:H7 cell matrix after heating by microwaves and
dry heat at constant temperature conditions.
Temp, probe
o
\
V
Microwave system • 55°C
Structural changes of E. coli 0157:H7 cell matrix
observed with:
/
V
\
Temp, probe
Dry heat block - 55°C
233
•
light microscopy
•
TEM microscopy - vials heated closed
•
epifluorescence microscopy ■vials heated open
Figure 2. M orphology of E. c o li 0 1 5 7 :H 7 sta in ed with crystal vio le t. C ell m atrix
w as d istrib u te d on the internal w all of the m icro tube and then exposed to
m icrow aves at 55°C. Arrow s show co n cen tra tio n of the stain at th e both ends o f
the ce ll suggesting
uneven
d istrib u tio n
o f in tra ce llu la r
m aterial
m icrow ave heated ce lls.
%
L
234
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
in these
Reproduced with permission of the
Figure 3. Cross section of E. coli 0157:H 7 cells heated as at 55°C for 10 min. Cell matrix from a single colony
was distributed on the wall of micro tube and exposed to microwaves and dry heat. TEM pictures show A)
conventionally heated cells, B) microwave heated cells, and C) unheated cells - control.
A)
B)
C)
Dry bath
§• 20 ■■
Microwaves
10- ■
60
120
H
180
1---------- (— — I---------- 1---------- h
240
300
360
420
480
640
600
640
600
time (sec)
i6 k
40-
u
S
3
nw
30-
20
/
Microwaves
- '
Dry bath
0
80
120
180
240
300
360
420
480
660
time (sec)
Figure 4. Thermal profiles of E. co li 0 1 57:H7 cell matrix heated at 55°C by microwaves
and dry heat and used in A) TEM and B) epifluorescence microscopy studies.
236
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Figure 5. Permeability of membranes of E. coli 0157:H 7 heated at by A) microwaves and
B) dry heat. Number of viable (intact membrane - green) and nonviable (permeable to
fluorescent stains - red) cells was determined with LIVE/DEAD BacLight* kit (Molecular
Probes, Inc.).
A)
237
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Table 1. Survival of E. co li 0157:H7 cell matrix heated by microwaves and dry heat in
open micro tubes at 55°C. Per cent of cells that survived treatment were determined
with epifluorescence microscopy using LIVE/DEAD® SacLight™ Bacterial Viability Kit.
Treatment
Viable / total population enumerated
% (green cells)/(green cell +red cells)
Standard deviation
Microwaves
22.70
4.56
Dry heat
5.44
3.07
238
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CONCLUSIONS
This study was intended to develop a better understanding o f the destruction of
microbial pathogens by microwave energy using E. coli 0157:H 7 as a test organism.
Three areas of research were explored and the major findings are presented below.
The detailed summary of each research topic covered in this thesis was given in the
conclusion section at the end o f each chapter.
1. Thermoresistance o f E. coli 0 1 57:H7 exposed to microwaves.
This is the first time that D-values were determined fo r microbial cells heated
exclusively by microwaves at constant temperature conditions. A microwave system
was constructed and used to determine the thermoresistance of E. coli 0157:H7
suspended in phosphate buffer during isothermal microwave heating. The D-values for
E. coli 0157:H7 exposed to microwaves were:
Ds7c
- 19.8 and
Deoc
- 1.12 min. For
conventionally heated bacteria, the heat resistance values were Ds7c - 11.96 min and
Deoc - 1-12 min.
The z-values were calculated from the above data and were
established as 2.32°C and 2.92°C for microwave and conventionally heated bacteria
respectively. These results were obtained when Tryptic soy agar was used as a plating
medium. Higher D-values resulted when heated cell suspensions were plated on Plate
count agar supplemented with 1% sodium pyruvate. This medium was observed to be
excellent for recovery of heat-stressed £. coli 0157:H7 cells followed by Tryptic soy
agar with 1% sodium pyruvate and Phenol red sorbitol agar with 1% sodium pyruvate.
The spread-plate method yielded higher CFU/mL than pour plating. Statistical analysis
o f the D-values for microwave and conventional heat treatment were carried out using a
239
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parallel model with categorical predictors. Calculated P-values showed that in 12 out of
18 individual experiments no significant differences were observed between microwave
and water bath heating. Additional lethality experiments were carried out to seek more
conclusive results. The lethality delivered to the individual bacterial suspensions were
calculated from the time- temperature data.
It was found that a lack of isothermal
conditions during the survivor curve experiments contributed to different lethality rates
to the heated samples. Thus, variable D-values were obtained not as a result of the
difference between a microwave and conventional heating.
2. Microwave pasteurization of foods.
A method to calculate microwave pasteurization time was established.
The
procedure involved calculation of the end-point temperature fo r the non-isothermal
conditions occurring during microwave heating. Ground beef patties inoculated with E.
coli 0157:H7 were used as a model system. Heat penetration studies were conducted
for beef patties pasteurized in a standard microwave oven.
Time-temperature data
from the meat patties heated at high and medium power allowed fo r the determination
of time necessary to pasteurize meat.
Pasteurization times found fo r round-shaped
beef patties were longer than fo r doughnut-shaped beef patties. Respectively, at the
high power, pasteurization was predicted to be achieved after 2 min 30 sec and 2 min
and 10 sec, and at the medium power output the pasteurization values were 4 min and
3 min 20 sec. An inoculation pack study with E. coli 0157:H7 added to meat confirmed
the validity of the predicted pasteurization times. Additional tests were carried out to
determine application of pasteurization times in five other microwave ovens. Two major
factors, the power of the microwave oven and an uneven heating pattern on the bottom
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of the oven cavity, were related to the survival o f pathogen. Simple methods were
suggested to test the performance of the microwave ovens.
Recalculation o f the
pasteurization time for a specific microwave oven, based on microwave energy, was
successful as proven by microbiological studies.
3. Effect of dry and microwave heating on the cell structure.
A study was initiated to determine the extent o f cell damage that resulted from
microwave treatment as compared to the changes in corresponding samples exposed
to dry heat
The observations were carried out fo r the cells heated in a non-liquid
environment during similar time-temperature conditions.
The cell matrix o f E. coli
0157:H7 obtained from a microbial colony grown on the agar was placed in a micro
tube and exposed to microwaves and dry h e at
Microscopic examination o f heated
cells using bright-field, TEM and epifluorescence microscopy revealed that cells from
both treatments had different morphology.
This preliminary study indicated that in
microwave heated cells, physical lesions occurred in different areas of the cells than in
cell heated conventionally.
However, more systematic studies are needed, using
combination o f time and temperature, to confirm this observation.
241
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