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CALORIMETRIC AND MICROBIOLOGICAL EVALUATION OF BACTERIA
AFTER EXPOSURE TO FOOD PRESERVATION TREATMENTS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy in the Graduate School of
The Ohio State University
By
Jaesung Lee, M. S.
* * * * *
The Ohio State University 2004
Dissertation Committee: Approved By
Dr. Gönül Kaletunç, Adviser
Dr. Polly D. Courtney
Dr. Michael E. Mangino Adviser
Dr. Olli H. Tuovinen Food Science and Nutrition Graduate Program
ABSTRACT
Thermal and non-thermal food preservation treatments affect cellular components
of foodborne microorganisms that cause physiological changes in cells and eventually
death of bacteria. Differential scanning calorimetry (DSC) thermograms of whole
bacterial cells display thermally-induced transitions revealing the response of bacteria to
heat under linearly increasing temperature condition. Therefore, DSC of the whole
microbial cells can allow the detection in vivo of changes in their cellular components
including ribosomes, nucleic acids, proteins and cell envelopes. The main purpose of this
study was to evaluate the effects of physical and chemical treatments on microorganisms
based on the changes in thermal stability (Tm) of the cellular components and the total
apparent enthalpy (∆H) from the calorimetric data. To compare with DSC data, the
viability data from microbiological methods (plate counting) was also evaluated.
The viability and the change in the thermal stability of individual transitions of
Escherichia coli and Lactobacillus plantarum were evaluated after pre-heating in the
DSC to various temperatures. The fractional viability based on calorimetric data [(∆H-
∆Hf)/(∆H0-∆Hf)] and plate count data (N/N0) showed a linear relationship. Viability loss
and the irreversible changes in DSC thermograms of whole cells pre-treated in DSC to
various temperatures were highly correlated between 55 and 70oC. Comparison of DSC
ii
scans for isolated ribosomes showed that the thermal stability of ribosomes from E. coli
is greater than the thermal stability of L. plantarum ribosomes, consistent with the greater
thermal tolerance of E. coli observed from viability loss and DSC scans of whole cells.
The apparent enthalpy data obtained from DSC of E. coli cells were applied to a
theoretical formalism to predict the number of surviving microorganisms as a function of
linearly increasing temperature. The decimal reduction time (D) and thermal resistance
constant (z) values for E. coli determined from the calorimetric data were compared to
the corresponding values from plate count data obtained after heat treatment in the DSC
and after isothermal treatment to validate the new approach. The calculated D values
using both apparent enthalpy and viability data for cells heat treated in the DSC were
similar to the D values obtained from isothermal treatment. Temperatures for 1 through
10-log microbial population reductions, calculated from plate count and enthalpy data
were in agreement within 0.5-2.4oC at a 4oC min-1 heating rate.
The effect of chemical agents (acids, ethanol or NaCl) on the cellular components
and the survivability in subsequent heat treatment of E. coli was evaluated using DSC
and viable counting methods. The thermal stability for ribosomal subunits denaturation
decreased as concentration of the chemical agents increased. The apparent enthalpy also
decreased, mainly due to reduction of ribosomal subunit peak as the concentration
increased. The size and thermal stability of DNA transition were reduced by inorganic
(HCl) and organic (CH3COOH) acid treatments. The survival of cells received chemical
treatments was lower than that of non-treated cells after mild heat treatment (at 60, 62.5
and 65oC) indicating that the conformational changes in cellular components by the
chemical treatments may cause sensitization to heat.
iii
High hydrostatic pressure (HHP)-induced changes in cell structures of E. coli were
determined using DSC and electron microscopy (EM) to relate the structural changes to
viability of the cells. The reversibility of transition and the change in the thermal stability
of ribosome of E. coli are affected by 200 MPa and above pressures in HHP treatment.
The enthalpy and the thermal stability of the DNA melting transition were reduced by
HHP treatments above 300 MPa. The pressure-induced changes in ribosome and DNA
were also detected in thin sections under transmission electron microscopy. In EM study,
integrity of cell envelope was maintained in pressure- or heat-inactivated cells; however,
the leakage of cell wall or outer membrane substance and empty space between cell
envelope and inside structure were exclusively observed in pressure- inactivated cells.
The effects of HHP and nisin treatment alone and in combination on cellular
components and viability of Salmonella enterica subsp. enterica serova Enteritidis
(Salmonella Enteritidis) FDA and OSU 799 strains were evaluated by DSC and plate
counting in order to evaluate the relative resistance and to optimize the treatment
conditions. An 8-log cfu/ml reduction was observed after a pressure treatment at 500
MPa for FDA strain and 450 MPa for the OSU 799 strain. When nisin was added, a
similar reduction was obtained at 400 MPa for FDA strain and 350 MPa for the OSU 799
strain. The decrease in apparent enthalpy appeared to be mainly due to reduction in the
ribosome denaturation peak for the pressure alone and nisin-combination treatments.
DNA might be irreversibly damaged by the combination treatments. There is a linear
relationship in a logarithmic plot of fractional apparent enthalpy values [(∆H-∆Hf)/(∆H0-
∆Hf)] versus the fractional survivors from plate count data (N/N0) for treated cells.
iv
Dedicated to my parents and my daughters
v
ACKNOWLEDGMENTS
I thank Dr. Gönül Kaletunç, my adviser, for her guidance, encouragement, patience
and suggestions throughout this work. I also to express my gratitude to committee
members, Dr. Olli H. Tuovinen, Dr. Michael E. Mangino and Dr. Polly D. Courtney, for
their valuable criticism, suggestions and comments.
I wish to express appreciation to my colleagues, Kelley Yosik, Hyunjung Chung,
and U.C. Rakhith for their help. I thank the USDA laboratory for the instrumental
support to complete my research.
I am especially grateful to my parents and sisters for their concern, encouragement
and moral support throughout my education. Finally, this dissertation is dedicated to Jiae
Park, my wife, and Yunjung Lee and Yunmi Lee, my daughters, for their love, support,
and patience.
vi
VITA
January 5, 1964 ……………………………….
Born in Inchon, Korea
1994 …………………………………………... B.S., Microbiology, University of
Minnesota, Minneapolis, MN
1999 …………………………………………... M.S., Food Science, University of
Delaware, Newark, DE
1999-present ………………………………….. Graduate Research Associate, The Ohio
State University, Columbus, OH
PUBLICATIONS
Lee, J. and Kaletunç, G. 2002. Evaluation by differential scanning calorimetry of the heat inactivation of Escherichia coli and Lactobacillus plantarum. Appl. Environ. Microbiol. 68:5379-5386. Lee, J. and Kaletunç, G. 2002. Calorimetric determination of inactivation parameters of microorganisms. J. Appl. Microbiol. 93:178-189. Alpas, H., Lee, J., Bozoglu, F. and Kaletunç, G. 2003. Differential scanning calorimetry of pressure-resistant and pressure-sensitive strains of Staphylococcus aureus and Escherichia coli O157:H7. Int. J. Food Microbiol. 87:229-237. Kaletunç, G., Lee, J., Alpas, H. and Bozoglu, F. 2004. Evaluation of structural changes induced by high hydrostatic pressure in Leuconostoc mesenteroides. Appl. Environ. Microbiol. 70:1116-1122.
vii
PUBLISHED ABSTRACTS
Lee, J. and Kaletunç, G. Inactivation of Salmonella Enteritidis treated by a combination of high hydrostatic pressure (HHP) and Nisin: A calorimetric investigation. American Society of Microbiology General Meeting, Washington, DC, 2003. Lee, J. and Kaletunç, G. Calorimetric evaluation of the thermal stability of ribosomes isolated from Escherichia coli and Lactobacillus plantarum. Institute of Food Technology (IFT) Annual Meeting, Anaheim, CA, 2002. Lee, J., Alpas, H., Bozoglu, F. and Kaletunç, G. Studies on the effect of high hydrostatic pressure (HHP) on cell morphology of Leuconostoc mesenteroides with scanning electron microscopy (SEM). IFT Annual Meeting, Anaheim, CA, 2002. Lee, J. and Kaletunç, G. Calorimetric determination of microbial survival curve of Escherichia coli. IFT Annual Meeting, New Orleans, LA. 2001. Lee, J. and Kaletunç, G. Calorimetric evaluation of chemically stressed Escherichia coli. Ohio Valley IFT, Columbus, OH. 2001. Lee, J. and Kaletunç, G. Evaluation of the influence of environmental stresses on inactivation of microorganisms by differential scanning calorimetry. IFT Annual Meeting, Dallas, TX, 2000.
FIELDS OF STUDY
Major Field: Food Science and Nutrition
viii
TABLE OF CONTENTS Page ABSTRACT………………………………………………………………………… ii
DEDICATION……………………………………………………………………… v
ACKNOWLEDGMENTS…………………………………………………………. vi
VITA………………………………………………………………………………... vii
LIST OF TABLES………………………………………………………………….. xii
LIST OF FIGURES………………………………………………………………… xiii
INTRODUCTION…………………………………………………………………. 1
Chapters:
1. Literature Review………………………………………………………………... 3
Differential Scanning Calorimetry (DSC)………………………………………. 3 Principles of DSC………………………………………………………………. 3 Bacterial Thermal Analysis by Differential Scanning Calorimetry……………. 6
Thermal Processing Effect on Microorganisms…………………………………. 11 Effect on cell components……………………………………………………… 11 Mechanism of cell death……………………………………………………….. 16
Chemical Effect on Microorganisms……………………………………………. 18 Hurdle technology……………………………………………………………… 18 Effect of ethanol………………………………………………………………... 18 Effect of NaCl………………………………………………………………….. 19 Effect of acids…………………………………………………………………. 20
High hydrostatic pressure (HHP)……………………………………………….. 20 HHP technology for food preservation………………………………………… 21 Effects of HHP on microorganisms……………………………………………. 22 HHP in combination with other processing technologies……………………… 24 HHP in combination with bacteriocins………………………………………… 25
ix
2. Evaluation by differential scanning calorimetry of the heat inactivation of
Escherichia coli and Lactobacillus plantarum………………………………….
28
Abstracts………………………………………………………………………... 28 Introduction…………………………………………………………………….. 30 Materials and Methods…………………………………………………………. 33 Results………………………………………………………………………….. 39 Discussions…………………………………………………………………….. 55 References……………………………………………………………………… 63 3. Calorimetric determination of inactivation parameters of microorganisms ……. 66 Abstracts………………………………………………………………………... 66 Introduction…………………………………………………………………….. 68 Materials and Methods…………………………………………………………. 71 Theory………………………………………………………………………….. 75 Results………………………………………………………………………….. 80 Discussions…………………………………………………………………….. 86 References……………………………………………………………………… 102 4. Evaluation by differential scanning calorimetry of the effects of ethanol, NaCl,
acetic acid and pH on Escherichia coli …………………………………………
107 Abstracts………………………………………………………………………... 107 Introduction…………………………………………………………………….. 109 Materials and Methods…………………………………………………………. 111 Results………………………………………………………………………….. 115 Discussions…………………………………………………………………….. 125 References……………………………………………………………………… 131 5. Evaluation of viability and structural changes induced by high hydrostatic
pressure in Escherichia coli ……….……………………………………………
137 Abstracts………………………………………………………………………... 137 Introduction…………………………………………………………………….. 139 Materials and Methods…………………………………………………………. 141 Results………………………………………………………………………….. 146 Discussions…………………………………………………………………….. 159 References……………………………………………………………………… 167 6. Inactivation of Salmonella Enteritidis FDA by combination of high hydrostatic
pressure and nisin.……………………………………………………………….
172
x
Abstracts………………………………………………………………………... 172 Introduction…………………………………………………………………….. 174 Materials and Methods…………………………………………………………. 177 Results………………………………………………………………………….. 182 Discussions…………………………………………………………………….. 195 References……………………………………………………………………… 202 General Conclusions……………………………………………………….……….. 208 Bibliography…………………………………………………………………….….. 212 Appendix. Figures and Table of the evaluation of Salmonella Enteritidis inactivation after HHP treatment with different concentrations of nisin……………
225
xi
LIST OF TABLES
Table Page
1.1 The G+C content of each strain DNA and the temperature recorded for peak associated with the melting of putative DNA from whole cell DSC………….
9
1.2 Major transition temperatures in the thermograms of whole cells of E. coli….. 10 1.3 Survivors of four pathogens by pressurizing in the absence and presence of
bacteriocins……………………………………………………………………
27 2.1 Transition temperature and apparent enthalpy values for E. coli and L.
plantarum ribosomes after DSC in different pH………………………………
44 3.1 Viability and apparent enthalpy values for E. coli cells after pre-treatment in
the DSC……………………………………………………………………….
83 3.2 D and z values reported for E. coli from isothermal and non-isothermal heat
treatments……………………………………………………………………...
84 4.1 Effects of chemicals on viability and calorimetry of E. coli………………….. 116 5.1 Viability, apparent enthalpy values and transition temperatures of each peak
for E. coli cells after treatments…………………………………….…………
147 6.1 Viability and apparent enthalpy values for cells of Salmonella Enteritidis
strains after HHP treatments…………………………………………………..
186 Appendix. 1 Viability and apparent enthalpy values for cells of Salmonella
Enteritidis FDA after HHP treatments in combination with nisin…………….
231
xii
LIST OF FIGURES
Figure Page 1.1 Chamber of cylindrical type DSC …………………………………………….. 4 1.2 Diagram of the DSC chamber…………………………………………………. 4 1.3 Typical DSC curve of starch.………………………………………………….. 5 1.4 Scheme for the sequence of events leading to the death of microorganism
from heating…………………………………………………………………...
17 2.1 DSC thermogram of whole cells of E. coli ATCC 14948…………………….. 32 2.2 Experimental scheme of calorimetric and microbial analysis…………………. 38 2.3 Thermograms of whole cells of E. coli and L. plantarum obtained by DSC….. 40 2.4 Thermograms of isolated intact ribosomes of E. coli and L. plantarum
obtained by DSC………………………………………………………………
42 2.5 Thermograms of whole cells (A) and isolated intact ribosomes (B) of E. coli
(a) and L. plantarum (b) obtained by DSC after HEPES buffer (pH 7.5) wash.…………………………………………………………………………..
43 2.6 DSC thermogram of isolate ribosome of E. coli (a) and L. plantarum (b) at
different pH of phosphate buffer………………………………………………
45 2.7 Effect of heat pre-treatment on the thermogram of E. coli……………………. 48 2.8 Effect of heat pre-treatment on the thermogram of L. plantarum……………... 49 2.9 Viable counts and DSC thermograms of E. coli after heat pre-treatment at
60oC, 62.5oC, 64oC, 65oC and 70oC……………………………………….…..
52 2.10 Viable counts and DSC thermograms of L. plantarum after heat pre-treatment
at 55oC, 57.5oC, 60oC, 65oC and 70oC………………………………………...
53
xiii
2.11 Correlation between fractional apparent enthalpy and fractional viability for
E. coli and L. plantarum………………………………………………………
54 3.1 Experimental scheme of calorimetric and microbial analysis…………………. 79 3.2 Apparent specific heat capacity versus temperature curves of control and
heat-treated E. coli…………………………………………………………….
81 3.3 A typical DSC thermogram for whole cells of E. coli K12 after empty
crucible baseline subtraction…………………………………………………..
89 3.4 DSC thermogram for whole cells of E. coli K12 displaying curve base line
used to determine the apparent enthalpy value………………………………..
90 3.5 Temperature dependence of fractional survivor population determined from
plate count data after heat pre-treatment of E. coli cells in the DSC………….
92 3.6 Temperature dependence of fractional survivor population determined from
calorimetric data after heat pre-treatment of E. coli cells in the DSC………...
93 3.7 Comparison of D values calculated from the calorimetric and viability data
obtained under non-isothermal heat treatment in the DSC and D values obtained from isothermal heat treatment………………………………………
96 3.8 Comparison of D values calculated from the calorimetric and viability data
obtained under non-isothermal heat treatment in the DSC and D values obtained from isothermal heat treatment………………………………………
99 4.1 Experimental scheme of calorimetric and microbial analysis…………………. 114 4.2 DSC thermogram of E. coli pellet after ethanol treatment…………………….. 117 4.3 DSC thermogram of E. coli pellet after NaCl treatment………………………. 119 4.4 DSC thermogram of E. coli pellet after inorganic acid (HCl) treatment……… 120 4.5 DSC thermogram of E. coli pellet after organic acid (acetic acid) treatment…. 122 4.6 Survival of untreated and chemically treated E. coli after heat treatment under
linearly increasing temperature………………………………………………..
124 5.1 Experimental scheme of calorimetric, EM and microbial analysis……………. 145 5.2 Pressure dependence of fractional viability determined by plate count……….. 146
xiv
5.3 DSC thermograms of pellets of E. coli whole cell after HHP (35oC for 5 min) or heat (65oC for 6 min) treatments…………………………………………...
150
5.4 Pressure dependence of fractional apparent enthalpy determined by DSC…… 151 5.5 Correlation between fractional apparent enthalpy and fractional viability for
HHP treated E. coli……………………………………………………………
152 5.6 SEM micrograph of control (a), pressure-inactivated (b, at 700 MPa, 35oC, 5
min), and heat-inactivated (c, at 65oC for 6 min) E. coli cells.………………..
153 5.7 TEM micrographs of untreated (a), pressure-inactivated (b, at 700 MPa, 35oC,
5 min), and heat-inactivated (c, at 65oC for 6 min) E. coli cells.……………...
156 6.1 Experimental scheme of calorimetric and microbial analysis…………………. 181 6.2 Thermograms of whole cells of Salmonella Enteritidis OSU 799 and
Salmonella Enteritidis FDA obtained by DSC………………………………..
183 6.3 Pressure dependence of fractional viability of Salmonella Enteritidis strains
determined by plate count. The cells treated with or without nisin…………..
187 6.4 DSC thermograms of Salmonella Enteritidis FDA pellets after pressure alone
treatment (a) and pressure-nisin combination treatment (b)…………………..
190 6.5 DSC thermograms of Salmonella Enteritidis OSU 799 pellets after pressure
alone treatment (a) and pressure-nisin combination treatment (b………….….
191 6.6 Pressure dependence of fractional viability of Salmonella Enteritidis strains
determined by calorimetric data. The cells treated with or without nisin…….
192 6.7 Correlation between fractional apparent enthalpy and fractional viability for
Salmonella Enteritidis FDA after HHP treatment……………………………..
193 6.8 Correlation between fractional apparent enthalpy and fractional viability for
Salmonella Enteritidis OSU 799 after HHP treatment………………………..
194 Appendix.1 DSC thermograms of Salmonella Enteritidis FDA pellets after
combinations of pressure and nisin (200 IU/ml) treatments..…………………
226 Appendix.2 DSC thermograms of Salmonella Enteritidis FDA pellets after
combinations of pressure and nisin (400 IU/ml Nisaplin) treatments………...
227
xv
Appendix.3 DSC thermograms of Salmonella Enteritidis FDA pellets after combinations of pressure and nisin (600 IU/ml Nisaplin) treatments………...
228
Appendix.4 Pressure dependence of fractional viability of Salmonella Enteritidis
FDA determined by plate count……………………………………………….
229 Appendix.5 Pressure dependence of fractional viability of Salmonella Enteritidis
FDA determined by calorimetric data…………………………………………
230
xvi
INTRODUCTION
Thermal and non-thermal processing technologies are widely applied in the food
industry for the preservation of food materials. The main goals of food preservation
technologies are to inactivate the spoilage and pathogenic microorganisms to produce a
safe product with enhanced shelf life. An understanding of the mechanism of the
microbial inactivation by physical and chemical stress is vital to assess the foodborne
disease and spoilage risk associated with food processing. Inactivation of
microorganisms results from irreversible denaturation of cell walls, membranes,
ribosomes, nucleic acids, and proteins such as enzymes by application of chemical and
physical stresses. Therefore, the investigation of the patterns of above macromolecular
changes that induce cell death during given treatments will provide knowledge for
designing optimal food process conditions.
Differential scanning calorimetry (DSC) thermograms of whole bacterial cells
display thermally-induced transitions revealing the response of bacteria to heat under
linearly increasing temperature condition. A number of overlapping transitions with a
net endothermic effect are observed when microorganisms are heated (Miles et al., 1986;
Anderson et al., 1991; Mackey et al., 1991; Belliveau et al., 1992; Kaletunç, 2001). The
observed transition peaks correspond to the denaturation of cellular components. A peak
1
temperature corresponding to each transition represents the thermal stability of a cellular
component of bacteria. Mackey et al. (1991) investigated the origins of apparent
individual transitions on the thermogram of Escherichia coli. Individual peaks observed
in thermograms of whole cells of E. coli were assigned to cell components by comparing
the transition temperatures of isolated cell components with corresponding transitions in
whole cells. In addition, DSC measurement provides information about amount of heat
energy (apparent enthalpy, ∆H) associated with the transition. Therefore, DSC can be
utilized for thermal characterization of microorganisms before and after exposure to a
treatment to evaluate the impact of such treatment. Comparison of various final states
achieved under different treatment conditions starting from same initial state will allow
one to predict the effectiveness of various treatments for inactivation of bacteria.
My research focused on the investigation of changes in thermal properties of
cellular components after physical (heat, pressure) or chemical (acids, salt, ethanol and
nisin) treatment alone and in combination. Effectiveness of each treatment was evaluated
by calorimetric and microbiological data. Using the results of this study, a fundamental
understanding of the cause of those inactivation treatments of bacteria can be developed.
2
CHAPTER 1
LITERATURE REVIEW
Differential Scanning Calorimetry
Principles of DSC DSC is a thermal analysis technique that measures heat flow
difference between a sample and a reference as a function of temperature at a fixed
heating rate (Chowdhry and Cole, 1989; Hohne et al. 1996). DSC detects and monitors
thermally induced conformational transitions and phase transitions when components in
sample are heated. DSC data allow to determine transition temperature (Tm), heat
capacity (Cp), and heat of transition (∆H) (Chowdhry and Cole, 1989; Hohne et al., 1996;
Kaletunç, 2001).
There are two types in DSC: power compensated DSC and Heat flux DSC. In the
power compensated DSC, the sample and reference materials are held in a separate
chamber with its own heater. When a thermal event occurs in the sample, power or
energy is applied to or removed from chamber(s) to compensate for the energy change
(heat flow) occurring in the sample. The amount of power required to maintain the
3
system in equilibrium condition is proportional to the heat absorbed or released by the
sample. In a heat flux DSC (Calvet DSC 111, Setaram, Lyon, France), which was used
for this research, the sample and reference materials are heated in a single chamber (Fig.
1.1). There are two heating elements in the Calvet DSC sensor of the chamber, one on
top of the block, the other underneath. Compared with conventional heat flux DSC, in
which temperature is measured only through the bottom of the crucible by a
thermocouple, the Calvet DSC has greater accuracy and sensitivity on the temperature
measurement because the heat flux transducer containing 24 thermocouple wires located
inside a thermosated calorimetric block, fully surround each crucible. Such a design
allows to measure almost all the heat that is exchanged with the sample. Additional
advantage of using Calvet DSC is that it can be applied for larger amount of sample.
s
Heat element
Temperature probee
1
Crucibl
1
Figure 1.1. Chamber of DSC 114
Figure 1.2. Diagram of the DSC 11
When thermal event occurs in the sample crucible, a temperature differential (dT) is
created between the sample and reference area. Thermocouples surrounded in both
crucibles detect dT per time (Fig. 1.2). DSC program converts the detected dT to heat
flow (Q, J/sec) versus temperature (T, oC) using an equation;
LkAdTQ −=)(Heat flow
where k is thermal conductivity of crucible (Q/moC); A is area through which heat flows
(m2); and L is thickness of crucible (m). As a result, the changes in the sample that are
associated with absorption or evolution of heat cause a change in the differential heat
flow which is then recorded as a DSC curve (thermogram, Fig. 1.3).
Hea
t Flo
w
Figure
Tm
Endoderm
Exoderm
Temperature
5 1.3. Typical DSC curve (thermogram) of s
Tm
Tonset
tarch
The value of the Q is assessed to calculate heat capacity (Cp) by an equation;
mrQC p ×
−=
where r is heating rate (oC/sec) and m is weight of the sample (g). The area under the
peak is directly proportional to the heat or enthalpic change (∆H = Cp ∆T) and its
direction indicates whether the thermal event on a sample is endothermic (denaturation or
melting) or exothermic (crystallization or aggregation). The characteristic peak of such a
plot provides the transition temperature (thermal stability, Tm) at which the thermal event
is half-complete (Fig. 1.3).
Bacterial thermal analysis by differential scanning calorimetry Bacteria are
composed of cellular components such as cell envelope, ribosomes, nucleic acids, and
proteins. Since the basic structures of those macromolecular components are
biopolymers, the components in whole cells may go through conformational transitions
upon exposure to heating by DSC. In DSC, the transitions recorded as endothermic (heat
absorption) or exothermic (heat loss) peaks in the thermogram.
The first application of DSC on bacterial thermal analysis was the study on the
physical properties of biomembranes. Steim et al. (1969) studied the physical properties
of lipids in cell membranes of Mycoplasma laidlwii using DSC of their whole cells, cell
membranes, and extracted lipids. They reported that both isolated cell membranes and
extracted membrane lipids showed an endothermic transition around 40oC in DSC
6
thermograms. They claimed that the lipids in the membranes were as much stable as
extracted lipids because the enthalpies of lipid melting in both samples were not different.
However, they could not obtain distinguishable peaks in the DSC thermogram from pure
whole cell (Bach and Chapman, 1980).
The first successful DSC on whole cell was the study on heat inactivation and
spontaneous germination of bacterial spores. Maeda et al. (1974) observed that
germinated Bacillus megaterium spores had endothermic peaks at about 100oC and 130oC
in their DSC thermogram. For vegetative cells, Verrips and Kwast (1977) reported eight
unidentified endothermic peaks for whole cell DSC thermograms of Citrobacter freundii.
Maeda et al. (1974) believed that the first two peaks (at 50oC and 55oC) were related with
the thermal death because of the loss of viability occurring at the same range of
temperature.
Using DSC, Miles et al. (1986) studied heat resistance of bacterial species of nine
genera including Gram-negatives, Gram-positives and spore formers. Cells on agar
surfaces were collected into DSC pans and heated at rate of 10oC min-1 from 10 to 120oC.
Vegetative bacteria showed distinguished major peaks in the regions of 68-73, 77-84, 89-
99 and 105-110oC in DSC thermogram. The onset temperature of first peak, the largest
among the peaks, was the lowest (36-49oC) in psychrophilic bacteria (36oC for Vibrio
marinus, 42oC for Brochothrix thermoshacta, 47oC for Hafnia alvei and 49oC for
Pseudomonas fragi) and highest in thermophilic bacteria (67oC for Bacillus
stearothermophilus). Mesophilic bacteria such as Streptococcus faecalis and Escherichia
coli showed the onset temperature at range of 50 to 52oC. Therefore, Miles et al. (1986)
7
claimed that the onset temperature of the first thermal denaturation is strongly related
with thermal tolerance of studied organisms. Later studies showed that the onset
temperature is correlated with the maximum growth temperatures of bacteria (Lepock et
al., 1990; Mackey et al., 1993; Mohacsi-Farkas et al., 1994; Teixeira et al., 1997). Miles
et al. (1986) also identified that the peak in the range of 89-99oC in each organism was
associated with the melting of DNA because it was reversible and its transition
temperature (Tm) was within the expected range of the melting of bacterial DNA.
Using the melting temperatures of the putative DNA peaks from the whole cell
DSC of 58 bacteria strains, Mackey et al. (1988) observed the relationship between the
transition temperatures of the DNA peaks in the whole cell thermograms and the
literature values of mole fraction of guanine + cytosine (G + C) base pairs in isolated
DNA (Table 1.1). Table 1.1 shows that the transition temperature of the putative DNA
peak was higher in bacterial strains containing greater content of guanine + cytosine (G +
C) base pairs in their DNA. It has been known that the thermal stability for bacterial
DNA increases with the G + C content due to more extensive hydrogen bonding (Jay,
1996). Using the transition temperature value of the DNA peaks in the whole cell DSC,
Mackey and coworkers (1988) also developed the model further to predict the mole
fraction of G + C in bacterial DNA (XGC) as Tm = (41.0 x XGC) + 73.8. The value of XGC
in the equation for each organism closely agreed with the XGC values based on the
spectroscopically determined transition temperatures of isolated bacterial DNA (De Ley,
1970).
8
Bacterial Strain G + C content (mol%) Tm (oC)
Bacillus cereus ATCC 14579 35.7 88.6
Bacillus macerans ATCC 8244 52.2 95.8
Campylobacter coli NCTC 11366 32.3 86.2
Campylobacter jejuni NCTC 11351 31.6 83.8
Escherichia coli KL 16 51.6 94.3
Enterobacter agglomerans NCTC 9381 56.0 96.0
Lactobacillus bulgaricus NCDO 1489 50.0 91.7
Lactobacillus cremoris NCDO 543 42.0 91.0
Pseudomonas aeruginosa NCTC 10332 66.4 100.3
Table 1.1. The G + C content and the temperature recorded for peak associated with the melting of putative DNA from whole cell DSC (adapted from Mackey et al., 1988).
Mackey et al. (1991) investigated the identification of the origins of individual
transitions on the thermogram of E. coli NCTC 8164. For the whole cell DSC, the
pellets of the organism were heated from 5 to 130oC at 10oC min-1 heating rate.
Individual peaks observed in thermograms of whole cells of E. coli were assigned to cell
components by comparing the transition temperatures of isolated cell components with
corresponding transitions in whole cells (Table 1.2). Among the main thermogram peaks
obtained in the E. coli whole cells, the most prominent peaks (temperature range of
60~80oC) being associated with ribosome denaturation. The ribosomal denaturation by
the DSC was associated with the 30S and 50S ribosomal subunits in increasing order of
thermal stability.
9
Cell component Mean transition temperature (Tm, oC)
30S ribosomal subunit 62-64
50S and 70S ribosomal subunits 69-80
Transfer RNA (tRNA) 75-76
DNA and cell wall 95, 105
Cell envelope 118-125
Table 1.2. Major transition temperatures in the thermograms of whole cells of E. coli NTCT 8164 and corresponding cell components (Mackey et al., 1991). Since the ribosome-associated components of the DSC thermogram were identified,
many DSC studies on bacteria have focused on the relationship between ribosome
stability and thermal resistance (Anderson et al., 1991; Mackey et al., 1993; Mohacsi-
Farkas et al., 1994; Teixeira et al., 1997). In those studies, a DSC instrument was used to
apply heat to the bacteria for determining the reduction of cell numbers by plate count
method as well as for generating thermogram. The studies showed strong correlations
between the temperature of ribosome-associated DSC thermogram events and the
temperature at which thermal death of bacteria occurs.
In thermal study on Listeria monocytogenes by Anderson et al. (1991), cell
suspensions in pans were heated to 60oC in the DSC with different holding times and
10
removed, then survivors were counted by plate counting method. Anderson et al. (1991)
observed that the first major peak (ribosomal subunits) disappeared and viability
decreased by two orders of magnitude after 60oC for 5 min.
Mohacsi-Farkas et al. (1994) observed that the temperatures at which the loss of cell
viability started (55oC for E. coli, 52oC for Lactobacillus plantarum, and 58oC for L.
monocytogenes) matched with the transition temperatures of the first irreversible
endothermic peaks (ribosomal subunits). Similar results were reported for vegetative
cells of Bacillus stearothermophilus (Mackey et al., 1993) and Lactobacillus bulgaricus
(Teixeira et al., 1997). Those results suggest that ribosome damage is an important factor
in causing the loss of bacterial viability during heat treatment.
Thermal Processing Effect on Microorganisms
The effect on cell components Bacterial cells contain several targets for the action of
heat (Anderson et al., 1991; Russell, 2003). Therefore, it can be anticipated that the
extent of heat effect is related to the stability of macromolecules in cell wall, membrane
lipids, ribosomes, nucleic acids, and proteins.
Peptidoglycan represents the main component (50% of the weight) of the cell walls
of Gram-positive bacteria (Murray et al., 1965). The net-shaped structure of the
peptidoglycan layer may not be seriously affected by a mild heat treatment because it
contains polysaccharide chains cross-linked by tight peptide bridges to maintain the
11
stability of the bacterial shape (Hammond et al., 1984; Novak and Juneja, 2001).
Peptidoglycan also plays a major role in heat resistance of spores of Gram-positive
bacteria, such as Bacillus subtilis and Clostridium botulinum (Ellar, 1978; Popham et al.,
1996). However, the effect of peptidoglycan on the thermal stability of intracellular
structures in vegetative cells has not been clearly elucidated. The main structure of the
Gram-negative cell wall is the outer membrane. Lipopolysaccharide (LPS) is the
predominant component (~40% weight) of the outer membrane and the remainder is
made up from phospholipids and proteins. LPS is held in the outer membrane by
relatively weak cohesive forces (ionic and hydrophobic interactions) and can be
dissociated from the cell surface with mild heat (Tsuchido et al., 1985). LPS consisting
of lipid A, core, and O antigen, is heat stable because those three structures are covalently
linked to each other (Wright and Tipper, 1979). Thermal studies of isolated outer
membrane components showed that the denaturation of LPS required much higher
temperature (>120oC) than the cell death temperature, while that for outer membrane
protein was around 70oC (Rodriguez-Torres et al., 1993; Phale et al., 1998).
It has been suggested that the primary cause of cellular heat injury is the damage of
membrane lipoprotein complexes or proteins that confer integrity of the cytoplasmic cell
membrane (Bowler et al., 1973). The damage leads to the dissipation of the
transmembrane H+ gradient and a decrease in intracellular pH (Weitzel et al., 1987; Piper
et al., 1997). Heat-induced damage of the membrane can be detected by measuring the
amount leakage of intracellular substances such as ions, nucleotides, and amino acids
(Russel and Harries, 1967). However, there was no relationship between the rate of the
12
increase in leakage amounts and the loss of viability during mild heat (<58oC) treatment
in several studies, indicating that cytoplasmic membrane damage is not a major factor in
cell inactivation (Allwood and Russell, 1967; Russell and Harries, 1968). Mackey et al.
(1991) reported that the thermal transition of isolated membrane lipid of E. coli is the
range of 30 ~ 40oC in DSC. However, the membrane lipid transition was hardly detected
as a peak when whole bacterial cells were used for DSC (Anderson et al., 1991; Mackey
et al., 1991; Teixeira et al., 1997; Mohacsi-Farkas et al., 1999). The melting temperature
of the membrane lipids in thermophilic bacteria is proposed to be higher than that of
mesophilic bacteria since their membranes are rich in saturated fatty acids (Russell,
2003).
Ribosomes are large complexes of proteins and three rRNA (ribosomal ribonucleic
acid; 30S, 50S and 70S) subunits in prokaryotes. Ribosomes comprise a major part of the
bacterial cell, constituting 25% of the total cell mass. Approximately 65% of E. coli
ribosome consists of rRNA, with the rest consisting of ribosomal proteins. The
protein/RNA, RNA/RNA, and protein/protein interactions in the ribosomes stabilize
tertiary structures. These interactions in the ribosomal subunits can also be affected by
heat stress (Bonincontro et al., 1998). Heat inactivation of microorganisms was proposed
to be related to denaturation of ribosomal subunits, mainly 30S and 50S (Rosenthal and
Iandolo, 1970; Hurst, 1984). The loss of Mg2+, which stabilizes ribosomal subunits,
from membrane damage is the primary reason of thermal degradation of ribosome in
microorganisms (Hurst and Hughes, 1978; Hurst, 1984). After the loss of Mg, divalent
cation-inhibited nucleases become activated and catalyze the degradation of 30S and 50S
13
RNA by cleavage of phosphodiester bonds, leading to irreversible ribosomal unfolding
(Datta and Niyogi, 1976). Mackey et al. (1991) isolated subunits (30S and 50S) of E. coli
ribosomes and compared them with whole cells in DSC thermograms. The denaturation
of the ribosomal subunits occurred at 50~80oC range in both thermograms. The 50S and
70S subunits, which have more rigid structures, were more stable than 30S subunits
during heat treatments. In a recent study on isolated E. coli ribosome, Bonincontro et al.
(1998) reported the DSC profile of thermal degradation of 50S was identical to that of
70S.
Heat treatment affects both double stranded DNA (dsDNA) and single stranded
DNA (ssDNA). The dsDNA damage is induced by direct heat which breaks the
hydrogen bonds between base pairs of DNA while ssDNA damage is mainly due to the
cleavage activity from endonucleases after heating (Russell, 2003). It has been known
that the denaturation temperatures of DNA are strongly related to base composition (Pace
and Campbell, 1967; Mackey et al., 1988). There is an important contribution from
intracellular cation concentration, shifting DNA denaturation to higher temperatures
because negatively charged phosphate (PO43-) backbones of dsDNA interact with cations
(Kumar, 1995). It has been suggested that the heat denaturation of DNA might not be a
major factor of vegetative cells or spore death because the event is only partially
irreversible and requires higher temperature (85-100oC) than bacterial death (Verrips et
al., 1977; Mackey et al., 1988; Mohacsi-Farkas et al., 1999). The DNA melting
temperature increases due to stabilizing interactions with other intracellular molecules
14
such as cationic proteins and polyamines (Worcel and Burgi, 1972; Flink and Pettijohn,
1975).
Unlike DNA/DNA interactions, RNA usually exists as a single chain without a
complementary strand. RNA can hold back on itself to form double helical regions
(Saenger, 1984). The denaturation temperature of RNA is also strongly related with the
ratio of base pairs. Because of rotational freedom in the backbone of its non-base paired
regions, RNA can hold into tertiary structures involving irregular base pairing
(Gesteland, 1993). Among RNA structures, the thermal stability for tRNA (~79oC),
which has more complex tertiary structure, is higher than that for rRNA (~73oC) (Mackey
et al., 1991).
Thermal process leads denaturation and coagulation of bacteria proteins (Russell,
2003). Many of the thermal denaturations of proteins in microorganisms are irreversible
due to following aggregation and alterations of amino acid residues (Kurganov et al.,
1997). Thermal property of proteins in the cells largely depends on the presence of water
attached within groups or at surface of protein molecules having free charges and water
in the tertiary structure of protein (Earnshaw et al., 1995). It has been suggested that the
thermal resistance of cells is higher when the presence of the protein contacted water
level is low because more dipoles of the protein interact each other to stabilize the protein
complex (Warth, 1985). The environmental pH is also an important factor for the
thermal properties of cell proteins. The heat stability of proteins decreases if the pH
condition of heating environment is far below or above the isoelectric points of proteins
(Condon et al., 1992). In a recent spectrophotometric study on thermal stability of
15
bacterial protein, Boer and Koivula (2003) reported that the thermal stability (Tm, ~65oC)
of purified Trichoderma reesei enzyme (cellobiohydrolase), which has optimal thermal
stability at pH 5, decreased by >10oC when pH of heating environment was adjusted with
3.5 or 8.0.
Many proteins in bacterial cells have been proposed to be stable at higher
temperatures than those known to support viability of microbial cells due to hydrophobic
interaction and binding with other components (Daniel and Cowan, 2000). It is
hypothesized that thermally or non-thermally induced complete denaturation of protein
molecules may not lead to cell death if the corresponding gene is undamaged and the
energy and building bocks are supplied to reform that proteins. However, the irreversible
denaturation of proteins such as RNA polymerase, ribosomal proteins and some enzymes
which are involved in protein synthesis, should cause loss of cell viability (Davis, 1990).
Mechanism of cell death The death of microorganisms during thermal processing has
been known as a two-step process; reversible damage occurs initially and is increasingly
converted into lethal events that result in cell death (Jung, 1986; Bowler and Manning,
1994). Figure 1.4 shows a possible sequence of lethal events during thermal death of
microorganisms and indicates that the plasma membrane is the primary target. However,
all of macromolecular components such as cell wall, enzymes and proteins, nucleic acids
can be directly affected to some degree by high temperatures. Since those components
are important structures for cell viability, the study on the irreversible heat damages of
the components has been highly recommended (Earnshaw et al., 1995; Russell, 2003).
16
Thermal perturbation of plasma membrane
Inactivation of membrane proteins
Decreased order of lipid layer
Leakage of mono- and divalent ions
Failure of ion pump and nutrient transport
Loss of ion gradients
Calcium overload
Loss of coupling and inactivation of receptors
Disintegration of cell membrane
Activation of phospholipases, proteases and protein kinases
Breakdown of metabolic control and loss of cellular homeostasis
Cell death
Figure 1.4. A scheme for the possible sequence of events leading to the death of microorganism from heating (Bowler and Manning, 1994).
17
Effect of chemical agents on microorganisms
Hurdle technology Homeostasis is an important adaptation mechanism of
microorganisms that maintains the stability of internal environment of them against
changes in living external environment. Disturbing the homeostatic mechanisms has
been regarded as main goal of hurdle technology in which food treatments are combined
to produce shelf-stable, minimally processed foods that have maintained the nutritional
qualities with extended shelf-life (Leistner and Gorris, 1995; Leistner, 2000). It has been
believed that effectiveness of the hurdle technology on microbial inactivation can be
improved when cells are exposed to heat after injured with chemicals (Karatzas et al.,
2000; Leistner, 2000). The physiological conditions of bacterial cells are known to be
affected by ethanol (Salton, 1963; Ingram, 1986), NaCl (Gutierrez et al., 1995; Poirier et
al., 1998) and acids (Abee and Wouters, 1999; Brul and Coote, 1999), which are utilized
during food processing.
Effect of ethanol The cellular membrane is a semipermeable barrier for survival of
bacteria; however, it is also the primary target of ethanol damage (Ingram, 1986).
Replacement of water molecules with ethanol can disrupt the hydrophobic core by
weakening hydrophobic interactions which maintain membrane integrity (Ingram, 1990).
Leakage of intracellular components through damaged cell membranes and end-product
inhibition of enzymes in glycolysis are considered as the basic mechanisms of ethanol
inhibition in microorganisms (Salton, 1963; Ingram, 1986). The presence of high
18
concentration of ethanol in cells may change the dielectric properties of the intracellular
components; however, the result of these ethanol-induced changes on the cellular
structures and cell viability are not clearly defined. Most bacteria demonstrate a dose-
dependent inhibition of growth over a range of 1 to 10% (v/v) ethanol, and few organisms
such as ethanol producers and lactobacilli are viable at concentration above 10% (Ingram
and Buttke, 1984). The growth of E. coli strain is inhibited by ~5% of ethanol
concentration (Ingram, 1986).
Effect of NaCl Most food-borne bacteria are inhibited by ≥5% (w/v) of NaCl due to
plasmolysis in which water is drawn out of the cell and into the outside cell (Jay, 1996).
A rapid decrease in cell volume due to the loss of water from the cell was evidence for
the inactivation (Munns et al., 1983). Recent studies on E. coli indicated that cell death
observed in the presence of NaCl (>5% concentration, w/v) may be related to the toxic
effect of Na+ in the cell rather than a decrease in cell volume because solutes can be
accumulated in cells to maintain the internal osmotic pressure (Gutierrez et al., 1995;
Poirier et al., 1998; Shadbolt et al., 1999). E. coli is known to accumulate “compatible
solutes” such as betaine, trehalose, glycerol, sucrose, proline, mannitol, sorbitol and small
peptides, which can increase internal osmolarity against a hyperosmotic shock without
interfering functions of cellular enzymes. Poirier et al. (1998) hypothesized that the cell
volume decrease can also be limited due to counterbalance the external osmotic pressure
by increased Na+ and Cl- concentration inside of the cell. However, specific
macromolecular targets of the ionic stress of Na+ have not been identified.
19
Effect of Acids Because neutral pH is optimum for the growth and survival of most
microorganisms, low pH values have long been considered as important factors to
inactivate bacteria (Jay, 1996). Bacteria can regulate their intracellular pH (pHi) at a
value close to neutrality against low pH environment using the pH homeostasis system
(Hill et al., 1995). Under high acidic conditions (<pH 3), however, the penetration of H+
ions across the cell membrane is faster than the removal of the ions by the pH
homeostasis system. As a result, the cell ceases essential biochemical activities (Bearson
et al., 1998). When the pH of food or solution is lower than pKa of a weak organic acid,
undissociated organic acids readily pass through cellular membrane and enter the cell.
Once weak organic acids reach the inside of bacterial cells, the cells are inactivated by
the release of both charged anions and protons from organic molecule (Brul and Coote,
1999). The charged form of the organic acid intensively lowers intracellular pH by
interfering with metabolic and anabolic processes of the cell (Abee and Wouters, 1999).
Among weak organic acids, acetic acid (CH3COOH) and lactic acid (CH3CHOHCOOH)
are more effective in the inactivation of bacteria because they have relatively small
molecular weight and easily diffuse into the cell (Hisao & Siebert, 1999).
High Hydrostatic Pressure (HHP)
HHP technology for food preservation Similar to thermal treatment, the
conformational and phase transitions of macromolecules in sample are affected in HHP
20
treatment because of volume changes (Cheftel and Culioli, 1997). High pressure
treatment follows Le Chatalier’s principle in which a process associated with a reduction
in volume is favored by an increase in pressure and vice versa under equilibrium
(Earnshaw et al., 1995, Farkas and Hoover, 2000). HHP treatment also follows the
isostatic principle, in which pressure is transmitted in a uniform manner throughout the
sample (Cheftel, 1995). Therefore, the time required for reaching the pressure to all
components is instant and independent of the volume and shape of sample. This
principle represents a significant benefit of HHP, as compare to thermal treatment, where
a thermal gradient must be established in sample (Balny and Masson, 1993). Another
principle in HHP is that at constant temperature, elevated pressure levels increase the
degree of ordering of the molecules of a substance in sample. Thus, the melting point of
solids increases with the pressure (Heremans, 1992). Because of above advantages based
on its operation principles, high pressure processing technology of food is being
investigated as an alternative to thermal processing. This novel technology can be
potential to produce microbiologically safe food with enhanced quality, flavor and
textural properties in comparison to thermal processing (Mertens and Deplace, 1993;
Roberts and Hoover, 1996).
As early as 1889 Hite showed that pressures of 450 MPa or greater could eliminate
spoilage microorganisms and improve the storage quality of milk. Despite the early
imploring of the technology, the two main barriers that prevented rapid commercial
application of HHP were the lack of knowledge about the significant advantage of the
process over other existing preservation methods and difficulty in high pressure vessel
21
manufacture and operation (Earnshaw et al., 1995); however, both of these problem areas
have been resolved. The recent research in HHP technology has intensively been
studying in two areas. Researchers have been focusing on the development of kinetic
data and advanced knowledge of the mechanism of HHP effect on food systems.
Engineering fields have been trying to solve the problems on the temperature distribution
within pressure vessels and compressibility differences within complex food systems
(Knorr, 1993).
Effects of HHP on microorganisms The effectiveness of hydrostatic pasteurization has
been reported for several foodborne pathogens, namely Salmonella spp., Escherichia coli
O157:H7, Camplylobacter jejuni, Vibrio parahaemolyticus, Listeria monocytogenes and
Staphylococcus aureus (Metrick et al., 1989; Shigehisa et al., 1991; Styles et al., 1991;
Patterson et al., 1995; Gervilla et al., 1997; Kalchayanand et al., 1998; Alpas et al., 1999).
Studies revealed that cell viability decreases with increasing pressure and time (Metrick et
al., 1989; Robey et al., 2001). The effect of HHP on bacterial cells is enhanced when the
cells are pressurized at ≥35oC (Kalchayanand et al., 1998; Alpas et al., 2000).
Resistance to high pressure varies among strains of the same species. Various strains
on foodborne pathogens were observed to be relatively resistant to pressure in
comparison to other strains (Styles et al., 1991; Patterson et al., 1995; Hauben et al.,
1997; Alpas et al., 1999; Benito et al., 1999). The destruction of ≥ 8 log cycles of some
strains of Escherichia coli O157:H7 and Staphylococcus aureus in phosphate buffer was
achieved by pressurization for 15 min at 20oC at 700 MPa (Patterson et al, 1995).
22
However, strains of pressure-sensitive E. coli were reported to develop resistance to high
pressure by adaptation or mutation to survive at 800 MPa in the buffer (Hauben et al., 1997).
The composition of the pressurizing menstruum has a great impact on the effect of
HHP on microorganisms. Viability loss is lower in a food system than in phosphate buffer
(Metrick et al., 1989; Patterson et al, 1995). Proteins in culture medium or food were
reported to protect bacterial cells against pressure (Simpson and Gilmore, 1997; Park et al.,
2001). In addition, enriched media provide nutrients such as amino acids and vitamins to
pressure-injured cells to recover.
Cell inactivation by high pressure is strongly related with cell wall type and cellular
morphology. Gram-negative bacteria and rod-shaped cells showed more sensitivity to
pressure treatment than gram-positive bacteria and cocci-shaped cells (Styles et. al, 1991;
Cheftel, 1995; Ludwig and Schreck, 1997; Kalchayanand et al., 1998).
Studies have revealed that cell viability decreases with increasing pressure and time,
suggesting critical cellular activities have been irreversibly damaged (Hoover et al., 1989;
Metrick et al., 1989; Cheftel, 1995). The primary target of bacterial cells in HHP
treatment has been proposed to be the cytoplasmic membrane (Kalchayanand et al., 1998;
Farkas and Hoover, 2000). It was observed that bacterial cell viability is related with the
loss of the membrane integrity (Shigehisa et al., 1991) and the failure of active transport
system (Cheftel, 1995). The denaturation of enzymes, which includes the alteration of
molecular structures and change in active sites, has also been considered as a major factor
in pressure-induced cell injury (Suzuky and Suzuky, 1962; Mackey et al., 1995). Studies
using electron microscopy reported that the separation between the cell wall and
23
cytoplasmic membrane, and destruction of ribosomes were shown in dead cells of
microorganisms after HHP treatment (Mackey et al. 1994; Isaacs et al., 1995). However,
there is little information in literature about the nature of irreversible changes occurring
in cells, which leads to cell death as a result of HHP treatment.
HHP in combination with other processing technologies High hydrostatic pressure
(HHP) has been shown to inactivate spoilage and pathogenic bacteria without altering the
food quality and has been recognized as an alternative to thermal processing (Roberts and
Hoover, 1996; Mertens and Deplace, 1993; Knorr, 1993). However, excessive high
pressure necessary to obtain a desirable reduction of pathogens is expected to alter the
conformational structure of high molecular weight compounds such as starch and protein.
As a result, normal texture and color of many foods can be adversely changed at high
pressure. In fish meat, lipid oxidation occurred since the peroxide value of fish oil increased
with increasing pressure (Ohshima et al., 1992). HHP treated tomato juice gave rancid
flavor due to the oxidation of free fatty acids (Porretta et al., 1995). The lightness of skim
milk color was significantly decreased after HHP at 600 MPa due to disintegration of casein
micelles into small fragments (Mussa and Ramaswamy, 1997). The HHP treated cheeses
had higher moisture, salt contents than raw or pasteurized cheeses (Trujillo et al., 1999). In
addition, very high pressures the process may not be economical for commercial use due
to the high cost of equipment and increased metal fatigue which leads to high
maintenance costs (Hoover et al., 1989; Mertens and Deplace, 1993). Microbiological
problems to be addressed include sublethal damage and following recovery in HHP
24
inactivation of foodborne bacteria (Earnshaw, 1995; Patterson et al., 1995). The
problems can be significant issue to food industry when HHP is used as a single
preservation against food pathogens.
There have been studies employing the concept of “Hurdle technology” in which
HHP technique is combined with one or more suitable antimicrobial agent to produce
shelf-stable, minimally processed foods that have maintained the nutritional qualities
with extended shelf-life (Kalchayanand et al., 1998; Massachalck et al., 2001). Hurdle
technology has been applied to inactivate pathogenic bacteria by combining HHP with
CO2 (Hass et al., 1989), irradiation (Crawford et al., 1996; Paul et al., 1997), heat
(Patterson and Kilpatrick, 1998; Benito et al., 1999; Alpas et al., 2000), low pH (Alpas et
al., 2000), or lysozyme (Popper and Knorr, 1990; Masschalck et al., 2001).
HHP in combination with bacteriocins Some of antimicrobial peptides produced by
lactic acid bacteria, termed bacteriocins, have been used in foods as safe and natural
preservatives. In recent literature, there are studies employing combination of HHP
technique with bacteriocins (Kalchayanand et al., 1998; Ponce et al., 1998; Yuste et al.,
1998; Garcia-Graells et al., 1999; Masschalck et al., 2000, 2001). Kalchayanand and co-
workers (1998) reported a study based on application of combined HHP and bacteriocin
(mixture of pediocin and nisin) treatment on model systems involving various strains of
four pathogenic bacteria. The results in the Table 1.3 clearly indicate that bacteriocins
can provide an additional 1 to 5 log cycle reductions in bacterial populations in buffer
medium.
25
Nisin is an antibacterial peptide produced by certain strains of Lactococcus lactis.
Nisin was approved as a food preservative in over 50 countries including European
Economic Community (EEC) and by FDA in US (Delves-Broughton, 1990; Yuste et al.,
1998). Nisin is effective against Gram-positive bacteria but shows very little activity
against Gram-negative bacteria which have nisin-impermeable barrier (outer membrane)
in their cell envelope (Delves-Broughton, 1990; Massachalck et al., 2001). Recent
studies showed that damaged cells of Gram-negative bacteria may be sensitive to nisin
(Kordel and Sahl, 1986; Kalchayanand et al., 1992; Masschalck et al., 2000). Moderate
HHP treatment has been reported to cause a number of morphological changes in
bacterial cells including cell lengthening, separation of cell membrane from the cell wall,
and pore formation in the cell wall (Mackey et al., 1994; Cheftel, 1995). Antimicrobial
molecules such as nisin may penetrate the damaged outer membrane of Gram-negative
bacteria. In a combined HPP and nisin treatment, the primary target in bacterial cell was
proposed to be the cytoplasmic membrane (Kalchayanand et al., 1998). However, the
mechanism of the inactivation of bacteria either HHP or nisin is still not clearly known.
26
Log10 cfu/ml of survivors
Pathogens Unpressurized Pressurized Pressurized+bacteriocin
Staphylococcus aureus 582 8.8 4.0 <1.0
Listeria monocytogenes Scott A 8.9 5.1 <1.0
Salmonella typhimurium ATCC 14028 8.9 4.1 1.0
Escherichia coli O157:H7,932 7.9 3.4 2.1
Table 1.3. Survivors of four pathogens by pressurizing at 345 MPa for 10 min at 25oC in the absence and presence of bacteriocins (Kalchayanand et al., 1998).
27
CHAPTER 2
EVALUATION BY DIFFERENTIAL SCANNING CALORIMETRY OF THE HEAT
INACTIVATION OF ESCHEICHIA COLI AND LACTOBACILLUS PLANTARUM*
ABSTRACT
Differential scanning calorimetry (DSC) was used to evaluate the thermal stability and
reversibility after heat treatment of transitions associated with various cellular
components of Escherichia coli and Lactobacillus plantarum. The reversibility and the
change in the thermal stability of individual transitions were evaluated by a second
temperature scan after pre-heating in the DSC to various temperatures between 40 and
130oC. Viability of bacteria subsequent to a heat treatment between 55 and 70oC in the
DSC was determined by both plate count and calorimetric data. The fractional viability
based on calorimetric data and plate count data showed a linear relationship. Viability
loss and the irreversible change in DSC thermograms of pre-treated whole cells were
highly correlated between 55 and 70oC. Comparison of DSC scans for isolated
ribosomes showed that the thermal stability of ribosomes from E. coli is greater than the
28
thermal stability of L. plantarum ribosomes, consistent with the greater thermal tolerance
of E. coli observed from viability loss and DSC scans of whole cells.
Key Words: heat treatment, differential scanning calorimetry, ribosome denaturation, E.
coli, L. plantarum
* Adapted from Applied and Environmental Microbiology, 68:5379-5386 (2002).
29
INTRODUCTION
The main goal of thermal processing is to inactivate the spoilage and pathogenic
microorganisms to produce a safe product with enhanced shelf life. An understanding of
the mechanism of microbial inactivation by heat is potentially useful for optimizing heat
treatments in order to eliminate foodborne disease and spoilage risk associated with
common and emerging strains while avoiding over processing of the food material.
Thermal inactivation of microorganisms is associated with irreversible denaturation of
membranes, ribosomes, and nucleic acids. However, the patterns of macromolecular
changes that induce the cell death of microorganisms during heat treatment are still not
clearly known.
Differential scanning calorimetry (DSC) is a thermal analysis technique that detects,
monitors, and characterizes thermally-induced conformational transitions and phase
transitions as a function of temperature. A number of overlapping transitions with a net
endothermic effect are observed when microorganisms are heated (Miles et al., 1986;
Anderson et al., 1991; Mackey et al., 1991; Mohacsi-Farkas et al., 1999; Kaletunç, 2001).
The observed transition peaks correspond to the denaturation of cellular components.
Mackey et al. (1991) investigated the origins of apparent individual transitions on the
thermogram of E. coli. Individual peaks observed in thermograms of whole cells of E.
coli were assigned to cell components by comparing the transition temperatures of
isolated cell components with corresponding transitions in whole cells (Fig. 2.1). It is
believed that a strong relationship exists between thermal death of bacteria and the first
30
major peak in DSC thermograms (temperature range of 60~80oC) which is attributed to
ribosomal melting (Mackey et al., 1993; Teixeira et al., 1997). Several investigators have
shown correlations between the stability of ribosomes and cell viability for
Staphylococcus aureus (Allwood and Russel, 1967), Listeria monocytogenes (Stephens
and Jones, 1993), and Salmonella enterica serovar Typhimurium (Tolker-Nielsen and
Molin, 1996). Furthermore, a recent DSC investigation of pressure-treated E. coli NCTC
8164 demonstrated that lethality of cells and ribosome damage are closely related (Niven
et al., 1999). Irreversible denaturation of cellular DNA requires temperatures well above
the temperature of cell inactivation (Mackey et al., 1991). At temperatures that cause
ribosome denaturation, the DNA transition is reversible (Mohacsi-Farkas et al., 1999).
Previous DSC investigations of microorganisms employed scans to high
temperatures (at or above 100oC) resulting in inactivation of the microorganisms. Most
rescans did not display any peaks except for an endothermic transition attributable to
DNA (Miles et al., 1986; Anderson et al., 1991; Mackey et al., 1991; Mohacsi-Farkas et
al., 1999). Although DSC thermograms were compared to viability studies, no studies
examined the relationship between thermal stability differences in whole cells and in
isolated ribosomes or correlations between viability measures based on plate count and
calorimetric data. The objectives of this study include: i) comparison using calorimetry
of the thermal stability of two selected microorganisms, E. coli and L. plantarum, in
relation to the thermal stabilities of their ribosomes; ii) investigation of the reversibility
of individual transitions associated with various components of whole cells of E. coli and
L. plantarum, and iii) determination and comparison of the temperature dependence of
31
cell viability for a linearly increasing temperature protocol from plate counts and
calorimetric data.
20 40 60 80 100 120 140
a1
a2
a3 b
c d
a1, a2, a3 --- Ribosome subunits b --- DNA
c --- DNA and cell wall d --- G- bacterial cell wall
Probable components of peaks
Heat Flow 0.2 mW
Temperature (oC)
Figure 2.1. DSC thermogram of whole cells of E. coli ATCC 14948
32
MATERIALS AND METHODS
Source and preparation of organisms
E. coli ATCC 14948 and L. plantarum ATCC 10241 were obtained from the Culture
Collection, Department of Microbiology at the Ohio State University. A loopful of each
organism was revived in 10 ml Trypticase soy broth (Difco laboratories, Detroit, MI)
supplemented with 0.3 % (w/w) yeast extract for E. coli or MRS broth (Difco) for L.
plantarum and incubated at 37oC for 18 hours. Each culture was stored frozen (-80oC) in
30 % (v/v) sterile glycerol. A loopful of each stock culture was transferred to 10 ml
Trypticase soy or MRS broth and incubated 10 hrs at 37oC before use.
Each culture was inoculated (1 % v/v) into a broth containing Trypticase soy or MRS
broth. Cultures were incubated at 37oC. The growth phase was determined by measuring
absorbance (A640), using a Beckman Du-50 spectrophotometer, and matching appropriate
viable counts from a standard growth curve. The cells were grown to late exponential
growth phase, as determined from the growth curve. The final concentration of cells in
the medium was 1.3 ± 0.1 x 109 cfu ml-1 for E. coli and 9.0 ± 0.1 x 108 cfu ml-1 for L.
plantarum. Cells in the broth were harvested by centrifugation (Beckman J2-21
centrifuge) at 10 000 g for 10 min at 4oC. The supernatant was discarded and the pellets
were washed with sterile distilled water and centrifuged for a second time before
transferring into DSC crucibles.
33
Calorimetry of whole cells
Pellets of whole cells were transferred into the empty sample crucible and were
weighed (56 ± 0.3 mg wet weight). The dry material content of the pellets was
determined by freeze drying (Freezone 4.5, Freeze dry system, Model 77510, Labconco,
Missouri) as 19 ± 0.3 % for E. coli and 20 ± 0.5 % for L. plantarum on a wet basis. The
standard deviations were calculated based on twelve freeze dried pellets for each
bacterium.
A differential scanning calorimeter (DSC 111, Setaram, Lyon, France) was used to
record thermograms of microorganisms heated at a 3oC min-1. All DSC measurements
were conducted using fluid-tight, stainless steel crucibles. For each DSC run, the
reference crucible was filled with ~45 µl (~80 % of sample wt) of distilled water. A DSC
run was performed with unsealed, empty sample and reference crucibles to record an
empty crucible baseline. Crucibles were sealed using aluminum o-rings and were
refrigerated at 4oC prior to DSC runs. The sample and reference crucibles were placed in
the DSC and equilibrated at 1oC using a liquid nitrogen cooling system. After heating in
the DSC, samples were cooled rapidly by liquid nitrogen and rescanned to ascertain the
reversibility of thermograms. Samples were reweighed after DSC measurements to
check for loss of mass during heating. Thermograms of samples showing signs of
leakage were discarded.
34
Heat pre-treatment in the DSC of whole cell pellets
Heat pre-treatment was performed in the DSC. Throughout the text an unheated
sample will be referred to as an untreated sample. The pellet was sealed in the sample
crucible, heated to the pre-treatment temperature and maintained at the pre-treatment
temperature for 60 seconds, followed by rapid cooling to 1oC. The sample was rescanned
from 1 to 130oC at 3oC min-1 to assess the reversibility of thermally-induced transitions in
bacterial cells. The reversibility of the transitions was evaluated by performing partial
scans between 40 and 130oC 5oC intervals. Additional pre-treatment runs were
conducted at 57.5oC for L. plantarum and at 57.5, 62.5, and 64oC for E. coli due to sharp
decreases in viability observed over the temperature range of 50-70oC.
Measurement of cell viability after heat pre-treatment
Heat pre-treatment prior to viability measurements was conducted in the DSC as
described in section 3. The crucible containing a pellet was capped (not sealed) using an
aluminum ring and screw cap. The reference crucible was filled with distilled water (~80
% of sample wt). The crucibles were refrigerated (4oC) until use. Pellets in crucibles
were heated to pre-treatment temperatures between 50 and 70oC as specified in the
previous section at a 3oC min-1 heating rate in the DSC. After rapid cooling, a portion
(40 or 50 mg) of heat treated pellet from the sample crucible was transferred using a
sterile loop to a (1.5 ml) sterile polyethylene tube. Sterile peptone water was added to
make a final volume of 1 ml with 1/25 or 1/20 (w/v) ratio. After careful suspension in
the tube, the cells were serially diluted and plated into Trypticase soy agar or MRS agar
35
to determine viable counts. After 36 hours incubation at 37oC, viable counts of each
sample were obtained by calculation of the dilution ratio. The level of the lowest
detection was 2.5 x 101 or 2 x 101 cfu g-1 in pellet. An untreated sample was used as a
control.
Preparation and calorimetry of intact ribosomes
The protocol described by Mackey et al. (1991) with modification of buffer solutions
was applied to prepare the intact ribosomes for both bacteria. The cell pellets obtained by
centrifugation of 3.5 l of late exponential phase cultures were washed and resuspended in
20 mM HEPES buffer at pH 7.5, containing 6 mM MgCl2 and 50 mM NH4Cl. The cell
suspension was disrupted by passing twice or three times through a previously cooled
French press (AMINco SLM Instruments, Inc. Urbana, IL). Deoxyribonuclease (RNase
free) (Sigma) was added (0.4 mg ml-1) and the material was centrifuged (Beckman L85-
55M Ultracentrifuge) at 32 500 g for 30 min. The supernatant (cell-free extract) was
centrifuged at 150 000 g for 3.5 h to obtain a pellet of crude intact ribosomes. The water
content of the ribosome pellet was determined to be 65.7 % for E. coli ribosome and 64.9
% for L. plantarum ribosome on a wet basis. Pellets of intact ribosomes were placed in
the DSC sample crucible. The reference crucible was filled with HEPES buffer equal to
the amount of buffer in the sample. The crucibles were heated from 1 to 140oC at a 4oC
min-1 in the DSC.
36
Calorimetry of intact ribosomes in different pH conditions
After freeze drying, dried pellets were weighed (~2 mg) and transferred into DSC
crucible. The pellets were mixed with ~36 mg of 50 mM potassium phosphate buffer
(pH 6, 5, 4 or 3, Fisher Chemicals, Fair Lawn, NJ). The reference crucible was filled
with potassium phosphate buffer equal to the amount of buffer in the sample. The
crucibles were heated from 1 to 140oC at a 4oC min-1 in the DSC.
Data analysis
DSC thermograms were corrected for differences in the empty crucibles by
subtracting an empty crucible baseline. Total heats corresponding to the envelope of
endothermic peaks (enthalpy, J g-1) between approximately 45-130oC for E. coli and 45-
110oC for L. plantarum were determined by integrating the temperature vs. heat flow
curve using software provided by the instrument manufacturers. A curved baseline using
three-temperature points was utilized to calculate the apparent enthalpy of both whole
cells and the intact ribosomes. Use of a curved baseline which takes into account the
apparent heat capacity change before and after the transition(s) of interest is explained in
Chapter 3. Peak temperatures for the thermally induced transitions were also determined.
37
e
Growth of the cells to the end of exponential growth stag
Centrifugation to obtain cell pellets Intact ribosome isolation DSC38
Plate counting
Calorimetric curvedata
MicrobiologicalViable count data
t
Analysis of the effects of heat treatmenHeat treatment in DSC
Figure 2.2. Experimental scheme of calorimetric and microbial analysis
RESULTS
Thermograms of E. coli and L. plantarum whole cells
Figure 2.3 shows the DSC thermograms for untreated E. coli and L. plantarum
pellets. The major peak in the DSC thermograms of both bacteria was observed over a
temperature range of 40 to 80oC. Several differences exist between the DSC
thermograms of E. coli and L. plantarum. The first peak, a1, (Tm, 56 oC), which is
proposed to be the denaturation of the smallest ribosomal subunit (30S) in E. coli
(Mackey et al., 1991), is not observed in the thermogram of L. plantarum as a separate
peak or shoulder. The major peak, peak a2, appears at a higher temperature in the E. coli
thermogram (70oC) in comparison to the L. plantarum thermogram (63oC). A peak (peak
b) similar to the peak reported by Mackey et al. (1991) as the melting of DNA in E. coli
exists, although at slightly different temperatures, in thermograms of both E. coli (94oC)
and L. plantarum (93oC). Similarly, peak c, a peak suggested by Mackey et al. (1991) to
be related to denaturation of DNA with a cell wall component appears at 102.5oC in E.
coli thermogram and at 100oC in L. plantarum thermogram. Figure 2.3 also shows that
peak d (Tm, 118oC) which appears in the E. coli thermogram, is absent from the L.
plantarum thermogram. Also apparent from Figure 2.3 is a difference in apparent heat
capacity of the live and inactivated cells (difference between the baseline before and after
the transition) of about 0.6 J g-1 K-1 for both organisms.
39
20 40 60 80 100 120
a
dc
ba
a1
3
2
Heat Flow
0.2 mW
Temperature (oC)
Figure 2.3. Thermograms of whole cells of E. coli ( ▬ ) and L. plantarum ( ••• ) obtained by DSC (1 to 150oC with 3oC min-1 heating rate).
40
Thermograms of isolated ribosomes
Intact ribosomes from both bacteria were isolated and DSC thermograms of
ribosomes suspended in HEPES buffer at pH 7.5 were collected and compared with those
of whole cells. Two endothermic transitions were observed for E. coli ribosomes (Fig.
2.4). The L. plantarum ribosome thermogram displayed an endothermic peak with a
shoulder on the ascending side of the peak. Comparison of denaturation peaks for
ribosomes suspended in HEPES buffer at pH 7.5 showed that the peak temperature of the
transition was higher for E. coli ribosome (74.3oC) than for L. plantarum ribosome
(70.7oC), indicating higher thermal stability. The area of the peak which corresponds to
the enthalpy of ribosome denaturation also was different for each ribosome. E. coli
ribosomes display a higher enthalpy of denaturation (26.6 J g-1 dry ribosome) than L.
plantarum ribosomes (23.5 J g-1 dry ribosome). A heat capacity change as a result of
ribosome denaturation (difference between the baseline before and after the transition) is
observed for both E. coli (0.4 J g-1 K-1) and L. plantarum (0.33 J g-1 K-1).
DSC thermograms of intact ribosomes are compared to those of whole cells (Figs.
2.5a and 2.5b, thermograms A and B). For E. coli, the transition temperature (75oC) of
the major peak in the thermogram of the whole cell pellet washed in HEPES buffer
coincides with the transition temperature (74.3oC) of the ribosome denaturation peak of
the isolated intact ribosomes suspended in HEPES buffer (Fig. 2.5a). However, for L.
plantarum, while a single peak is observed for ribosomes suspended in HEPES buffer at
pH 7.5, the whole cell thermogram shows dual transition temperatures (63.5 and 68.2oC).
41
1
2
3
4
5
6
20 40 60 80 100 120 140
App
aren
t hea
t cap
acity
(J/g
o C)
Temperature (oC)
Figure 2.4. Thermograms of isolated intact ribosomes of E. coli ( ▬ ) and L. plantarum ( ••• ) obtained by DSC (1 to 140oC with 4oC min-1 heating rate).
42
20 40 60 80 100 120 140
A
B
0.5 mWHeat Flow
(a)
Temperature (oC)
20 40 60 80 100 120 140
A
B
Heat Flow0.5 mW
(b)
Temperature (oC)
Figure 2.5. Thermograms of whole cells (A) and isolated intact ribosomes (B) of
E. coli (a) and L. plantarum (b) obtained by DSC after HEPES buffer (pH 7.5) wash.
43
Thermograms of isolated ribosomes in acidic condition
The effect of low pH (levels 6 to 3) on the thermal characteristics of isolated E. coli
and L. plantarum ribosomes was evaluated using DSC (Table 2.1, Figs. 2.6a,b).
Decrease in transition peak of their ribosomes was accompanied with increase in acidic
heating condition. Both peak temperature and enthalpy value (J/g) of the transition are
higher in E. coli ribosome thermogram than in L. plantarum ribosome thermogram at any
pH level (Table 2.1). Figure 2.6b shows that the size of the peak (enthalpy) of the L.
plantarum ribosome was apparently decreased (~13%) at pH 5 while that of E coli
ribosome remained same (Fig. 2.6a). The thermogram of L. plantarum ribosomes
suspended in the buffer at pH 4 (Fig. 2.6b) displays a profile (transition temperatures,
61.8 and 67.4oC) which strongly resembles the major peak in the thermogram of L.
plantarum whole cells (Fig. 2.5b thermogram A).
Isolated E. coli ribosome Isolated L. plantarum ribosomeBuffer pH Transition
temperature (oC) Enthalpy (J g-1
dry wt) Transition
temperature (oC) Enthalpy (J g-1
dry wt)
6.0 70.6 28.52 69.8 17.87
5.0 67.6 28.51 66.1 15.51
4.0 65.3 23.27 61.8, 67.4 14.57
3.0 61.0 11.86 59.8 9.75
Table 2.1. Transition temperature and apparent enthalpy values for E. coli and L. plantarum ribosomes after DSC in different pH.
44
20 40 60 80 100 120 140
Heat Flow 0.2 mW
DSC in pH 3 buffer
DSC in pH 4 buffer
DSC in pH 5 buffer
DSC in pH 6 buffer
(a)
Temperature (oC)
20 40 60 80 100 120 140
Heat Flow 0.2 mW
DSC in pH 3 buffer
DSC in pH 4 buffer
DSC in pH 5 buffer
DSC in pH 6 buffer
(b)
Temperature (oC)
Figure 2.6. DSC thermogram of isolate ribosome of E. coli (a) and L. plantarum (b)
at different pH of phosphate buffer.
45
Effect of heat pre-treatment on the DSC profiles of E. coli and L. plantarum
Pre-treatment of bacteria at various temperatures was performed in the DSC with a
partial scan to a pre-defined temperature. The reversibility of transitions after pre-
treatment was evaluated with a full second DSC scan (1 to 130oC) following the partial
scan. Thermograms displaying changes in major conformational transitions of both
organisms as a function of heat treatment are shown in Figures 2.7 and 2.8. In general,
pre-treatments below 70oC resulted in significant changes in both the temperature and the
area of the major peak observed in the thermograms of both bacteria indicating
irreversible effect of pre-treatment. It is apparent from Figures 2.7 and 2.8 that the major
peak in the DSC thermogram is obliterated after a pre-treatment temperature to 85oC for
E. coli (Fig. 2.7 thermogram F) and 75oC for L. plantarum (Fig. 2.8 thermogram F). Pre-
treatment resulted in changes in the shapes of existing peaks as well as in the appearance
of new peaks. Thermograms of un-treated bacteria are composed of several overlapping
transitions. A new peak, which was previously partially obscured, may seem to appear if
the overlapping transition disappears due to the heat treatment. For E. coli, peak a3
which is only partially visible due to the overlapping peak a2 in the control and 50-65oC
pre-treatment thermograms becomes visible after pre-treatment at 70oC (Fig. 2.7).
In Figures 2.7 and 2.8, only the full scans following pre-treatment which resulted in
major irreversible changes are displayed. For E. coli, peak b, which is associated mainly
with DNA denaturation (Mackey et al., 1991), was reversible after heat treatment at
115oC. For L. plantarum, peak b shifted to a lower temperature following a heat
treatment at 95 oC (Fig. 2.8 thermogram G) and was not reversible after the cell pellet
46
was pre-heated to 100oC (Fig. 2.8 thermogram H). After a partial scan to 115oC, the
transition due to an outer-cell wall component of E. coli (peak d) was absent. No
evidence of native cellular components was observed in the thermogram of the 130oC
heat-treated E. coli pellet (Fig. 2.7 thermogram H). However, for L. plantarum there was
no evidence of native cellular components detected in thermograms of pellets heat-treated
at 100oC.
47
40 60 80 100 120 140
a
a
a
1
2
3 b
c dA
H
G
F
E
D
C
B
Heat Flow0.2 mW
Temperature (oC)
Figure 2.7. Effect of heat pre-treatment on the thermogram of E. coli. Control (A), pre-treatment temperatures: 50oC (B), 60oC (C), 65oC (D), 70oC (E), 85oC (F), 115oC (G), 130oC (H). Thermograms are offset for clarity.
48
40 60 80 100 120 140
a
a
2
3 b
A
H
G
F
E
D
C
B
Heat Flow0.2 mW
c
Temperature (oC)
Figure 2.8. Effect of heat pre-treatment on the thermogram of L. plantarum. Control (A), pre-treatment temperatures: 50oC (B), 55oC (C), 60oC (D), 65oC (E) 75oC (F), 95oC (G), 100oC (H). Thermograms are offset for clarity.
49
Comparison of DSC thermograms and viability of E. coli and L. plantarum after heat pre-treatment DSC thermograms of pellets of each microorganism were compared to each control
thermogram after heat-treatment at different temperatures (Figs. 2.9 and 2.10). The area
under the curve of the second scan (apparent enthalpy, J g-1) was evaluated by
integration. Noticeable reductions in the apparent enthalpy value occurred with heat-
treatment up to 65oC (~53 %) for E. coli and up to 60oC (~58 %) for L. plantarum. The
viability of each microorganism treated in DSC under conditions identical to the
corresponding DSC experiment was determined and plotted on Figures 2.9 and 2.10. For
E. coli, the viable cell counts of the culture pellet displayed a slight change with heat
treatment up to 60oC. Heat treatment of E. coli to higher temperature resulted in 6-log10
unit reductions for 65oC and 7-log10 unit reductions for 70oC treatments. These
reductions were accompanied by a decrease in the area of the peak (a2) corresponding to
the denaturation of ribosomes in the thermogram (Fig. 2.9 thermograms E and F). For L.
plantarum, irreversible denaturation of ribosomes was observed in the thermogram
following a 57.5oC heat treatment (Fig. 2.10 thermogram C) with a viability loss of 2.3-
log10 units.
As described in Chapter 3, by assuming that the apparent enthalpy is proportional to
number of viable cells after correction for residual apparent enthalpy, the fractional
viability can be defined as the reduced apparent enthalpy, [(∆H-∆Hf)/(∆H0-∆Hf)], where
∆H is the apparent enthalpy after a pre-treatment, ∆Hf is the residual apparent enthalpy
after treatment resulting in no viability and ∆H0 is the apparent enthalpy of untreated
50
cells. Fractional viability values calculated from calorimetric data [(∆H-∆Hf)/(∆H0-∆Hf)]
and plate count data (N/N0) are plotted in Figure 2.11. A linear relationship between the
reduced apparent enthalpy and the fraction of survivors is observed, except for the points
corresponding to high temperature treatment.
51
2
4
6
8
10
12
30 40 50 60 70 80 90
A
A
B
C
C
B
ED
F
E
Heat Flow0.2 mW
a1
a 2
a3
F
Log
(CFU
/g)
Temperature (oC)
Figure 2.9. Viable counts (--∙--) and DSC thermograms of E. coli for control (A) and after heat pre-treatment at 60oC (B), 62.5oC (C), 64oC (D, thermogram not shown), 65oC (E) and 70oC (F). Thermograms are offset for clarity.
52
2
4
6
8
10
12
30 40 50 60 70 80 90
A
A
B
B
C
D
D
E
E
F
F
Heat Flow
0.2 mW
2
3
C
a
a
Log
(CFU
/g)
Temperature (oC)
Figure 2.10. Viable counts (--∙--) and DSC thermograms of L. plantarum for control (A) and after heat pre-treatment at 55oC (B), 57.5oC (C), 60oC (D), 65oC (E) and 70oC (F). Thermograms are offset for clarity.
53
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1
N /N 0
[(∆H
-∆H
f)/(∆
H0-∆
Hf)]
Figure 2.11. Correlation between fractional apparent enthalpy and fractional viability for E. coli (o) and L. plantarum (+).
54
DISCUSSION
This study aims to assess bacterial resistance to heat treatment by i. comparing the
thermal resistance of bacteria and the thermal stability of isolated intact ribosomes; ii.
evaluating the reversibility of thermal transitions of various cellular components
following heat treatment; and iii. developing a relationship between fractional viability
calculated from plate count data and calorimetric data.
The primary low temperature features (40~80oC) of whole cell DSC profiles of
bacteria are believed to correspond to the thermal unfolding of ribosomes (Mackey et al.,
1991). Using ribosomes isolated from E. coli, Mackey et al. (1991) showed that an
endotherm with three overlapping peaks appearing between 47 and 85oC for E. coli
whole cells is associated with ribosome denaturation. In present study, although E. coli
whole cells display a ribosomal denaturation endotherm consisting of three peaks, two
peaks (a2 and a3) are observed for ribosomal denaturation in L. plantarum thermogram
(Fig. 2.3). It has been reported that the 30S ribosomal subunit is less thermally stable
than the larger ribosomal subunit (Mackey et al., 1991; Stephens and Jones, 1993;
Bonincontro et al., 1998) suggesting peak a1 may be attributed to denaturation of the 30S
ribosomal subunit. It is also apparent from Figure 2.3 that the peaks a2 and a3 are shifted
to lower temperatures in comparison to the corresponding peaks for E. coli. The lower
peak temperatures of peaks a2 and a3 of the L. plantarum thermogram suggest that the
relative stabilities of L. plantarum ribosomes are lower than those of E. coli ribosomes.
The pH of the L. plantarum medium is reduced due to lactic acid production during L.
plantarum growth. It is possible that the increased acidity in the medium of L. plantarum
55
may influence the stability of the ribosomal subunits directly or indirectly. Another
factor that may influence ribosome stability is altered intracellular cation concentrations,
in particular Mg2+is required for ribosome integrity (Hurst, 1984). A loss of the peak (a1)
is observed in the thermogram of acid-treated E. coli (Chapter 4. Figs. 4.4 and 4.5).
Mohacsi-Farkas et al. (1994) reported that the ribosomal denaturation peak of L.
plantarum shifted to lower temperatures as the pH of the suspending medium decreased
below 5. Furthermore, their results show that while a low temperature (Tm, 57oC)
endothermic transition appears when cells are suspended in buffer at pH 6.8 and 5, this
transition is not observed on the thermograms for whole cells suspended in buffer at pH
4.6 and lower. Because the pH of the growth medium for L. plantarum was measured to
be 4.4 in the present study, our results are in agreement with the previous data showing
an absence of peak a1 and lower transition temperatures for peaks a2 and a3 induced by
low pH. Ribosomes also were reported to be destabilized by loss of Mg2+ from cells
(Hurst and Hughes, 1978; Rheinberger et al., 1988; Anderson et al., 1991). The lack of a
visible peak, a1, in the L. plantarum thermogram may indicate denaturation of the 30S
ribosomal subunit as a result of Mg2+ loss (Tomlins and Ordal, 1976; Hurst, 1984).
Alternatively, peak a1 may be present but obscured by the other ribosomal peaks a2 and
a3 because their transition are shifted to lower temperatures.
The comparison of ribosomal denaturation for each bacterium at pH 7.5 shows that
both the transition temperature and the apparent enthalpy for E. coli are higher than those
for L. plantarum indicating a higher thermal stability and a greater energy requirement to
disrupt the structure of the E. coli ribosome (Fig. 2.4). Similar behavior is observed for
56
whole cells suggesting that both the thermal stability and the energy required to inactivate
the bacterial cells are higher for E. coli.
E. coli ribosomes show similar thermal stabilities when in whole cells (75.1oC) and
when isolated (74.3oC) in terms of transition temperatures of corresponding peaks.
However, for L. plantarum the transition shapes and the thermal stabilities of whole cells
washed with HEPES buffer at pH 7.5 and of isolated ribosomes are similar only when the
ribosomes are suspended in potassium phosphate buffer at pH 4. Although an effect on
ribosomes is indicated in both cases, the strong resemblance in shape of observed peaks
in whole cells and isolated ribosomes may have different causes and must be explored
further. The higher thermal stability of isolated L. plantarum ribosomes when suspended
in HEPES buffer at pH 7.5 containing 6mM MgCl2 (Fig. 2.5b thermogram B) in
comparison with isolated ribosomes suspended in potassium phosphate buffer without
Mg2+ at pH 4 (Fig. 2.6b) may be attributed to stabilization of ribosomes by magnesium
ions in vitro (Noll and Noll, 1976). Similar behavior also was observed with E. coli
whole cells where the transition temperature of the major peak was 70oC following a
water wash but 75.1oC following a HEPES buffer wash. Anderson et al. (1991) note that
the ionic composition and concentration of the buffer affect the thermal stability and
shape of the ribosomal denaturation peak which in turn may affect the thermal resistance
of bacteria.
In the whole cell thermograms, there are likely more transitions than are observable
as discrete peaks. Some of these transitions may occur within the same temperature
range and be obscured by the larger ribosome denaturation peaks. Comparison of
57
thermograms of ribosomal denaturation and whole cells (Figs. 2.7 and 2.8) shows that the
difference in heat capacity between the native and denatured states, as shown by the
difference in pre- and post-transition baselines, is 1.5 times greater for whole cells. A
positive heat capacity is typically observed for denaturation of proteins. A typical
globular protein of ~15 kDa, the change in the heat capacity is on the order of 0.4-0.67 J
g-1 K-1 (Gomez et al., 1995). Given the larger heat capacity observed for whole cells in
comparison to denaturation of ribosomes, it is probable that other cellular components
contribute to the endothermic transitions attributed to the denaturation of ribosomes.
Anderson et al. (1991) indicate that the small number of peaks observed in whole cell
thermograms can be due to a larger number of transitions including protein unfolding and
denaturation.
Another visible difference between the E. coli and L. plantarum thermograms is a
high temperature endothermic transition (peak d) only observed in the DSC thermogram
of E. coli whole cells. Mackey et al. (1991) observed a peak corresponding to peak d in
the thermogram of the cell envelope fraction and proposed this peak to be the result of
cell envelope denaturation. These investigators hypothesized that a cell wall associated
thermo-stable protein may account for the appearance of this peak. Other DSC studies in
our laboratory showed that the peak was observed in thermograms of Pseudomonas
fluorescens but was absent from the thermograms of Staphylococcus aureus and
Leuconostoc mesenteroides (unpublished results) suggesting the origin of this peak is a
cellular component of Gram-negative bacteria. In most Gram-negative bacteria the outer-
cell wall layer exists as a true unit membrane. The outer-cell wall membrane contains
58
lipid, phospholipid, polysaccharide, and protein. The lipid and polysaccharide form a
specific lipopolysaccharide (LPS) layer. Rodriguez-Torres et al. (1993) using DSC
reported that LPS show endothermic transitions above 120oC, with the specific
temperature depending on the linkage type. Reversibility studies demonstrate that this
peak in E. coli is denatured by heat treatment above 110oC.
The transition temperatures associated with DNA denaturation are not considerably
different for both microorganisms, with that for L. plantarum is being slightly lower
(93oC) than that for E. coli (94oC). Although the thermal stabilities of the DNA for both
microorganisms are similar, their reversibility subsequent to heat treatment differs
significantly. There is no indication of a DNA peak in the thermogram of the L.
plantarum pellet pre-heated to 100oC (Fig. 2.8 thermogram H). The peak is preserved in
the thermogram of the E. coli pellet heated up to 125oC, although the apparent enthalpy is
reduced and peak is shifted to lower temperatures as the heat pre-treatment temperature is
increased. The change in energy required for denaturation of DNA subsequent to heat
treatment indicates partial refolding upon cooling or folding to a different state (Cantor
and Schimmel, 1980). Furthermore, the appearance of a previously obscured, reversible
peak (Tm at 88.4oC) in the thermogram of 95oC pre-heated L. plantarum may be due to
partial reversibility of denatured DNA (Fig. 2.8 thermogram G). Mackey et al. (1988)
showed that there is a strong correlation between guanine + cytosine (G + C) content of
DNA and the Tm of the putative DNA peak determined from a DSC scan of whole cells.
The G + C content of L. plantarum (44~46 mol %) (Kandler and Weiss, 1986) is lower
than that of E. coli (51.6 mol %) (Mackey et al., 1988). Using the empirical relation
59
between G + C content and Tm reported by Mackey et al. (1988) gives a predicted DNA
transition temperature of 92.4-93oC for L. plantarum and 94.8oC for E. coli. Mackey et
al. (1988) also reported 94.3oC DNA transition temperature determined using DSC for E.
coli. The experimental values for DNA melting in this study are in close agreement with
the literature data, including the expectation of a lower DNA peak temperature for L.
plantarum.
DSC curves can be exploited further to determine the fractional viability of
microorganisms based on calorimetric data as described in Chapter 3. For both E. coli
and L. plantarum, Figures 2.9 and 2.10 reveal that as the severity of the heat treatment
increases, the observed peak temperature of ribosomal denaturation increases, implying
sequential damage to the ribosomal subunits and/or the existence of a range of thermal
resistance in the microorganism population. It is apparent that a loss of viability of cells
of both organisms occurs when the microorganisms are subjected to heat pre-treatment in
the range of 50~70oC. The viability loss is related to the apparent enthalpy change of
ribosomal subunits monitored by DSC because preheating to 55~70oC affected the peaks
associated with ribosome subunits but had no apparent influence on the thermally-
induced transitions of other cellular structures. Both the putative ribosomal peaks in the
thermogram and cell viability of L. plantarum were noticeably reduced by pre-heating
from 55oC to 70oC (Fig. 2.10). A similar pattern was observed in the DSC profiles of E.
coli, although the reductions in ribosomal peaks and cell viability occurred at higher
temperature (Fig. 2.9). However, as discussed in Chapter 3, the peak area corresponding
to only the ribosome transition within the whole cell thermogram can not be determined
60
accurately because the baseline is not well defined due to overlapping transitions. Instead
the total peak area corresponding to the total apparent enthalpy must be used. With
increasing treatment temperature, the total apparent enthalpy (between approximately 50-
130oC for E. coli and 50-110oC for L. plantarum) decreases gradually compared to the
peak area for the untreated control. It is apparent from Figures 2.7 and 2.8 that residual
transitions remain even after the cells are inactivated implying that the total area under
the thermogram includes contributions related to both cell death and additional
macromolecular transitions. After subtracting the contributions due to enthalpy
associated with inactive cells, a reduced apparent enthalpy value can be defined to
determine the fraction of survivors in terms of calorimetric data. A plot of reduced
apparent enthalpy versus the fractional survivors from plate count data (Fig. 2.11) gives a
linear relationship. As I have shown in Chapter 3, these data can be interpreted in terms
of D and z values of microorganisms which are subjected to linearly increasing
temperature. In this Figure, the points close to a viability value 1 represent low
temperature treatment, while the points close to 0 represent high temperature treatments.
It is apparent that for both microorganisms at very low temperature viability calculated
from both plate count and calorimetric data are in close agreement. However, as the
treatment temperature increases a disparity appears between the plate count and
calorimetric data for both microorganisms. The disparity between the viability data
derived from the two methods is larger for L. plantarum than for E. coli. As the
temperature of the treatment increases further the disparity decreases. It is expected that
as the temperature of the heat treatment increases the number of injured microorganisms
61
will increase. The injured microorganisms die during a complete DSC scan following
the partial scan without having a chance to repair. However, the plate count method
provides favorable conditions for injured cells to recover. We speculate that the disparity
between viabilities calculated from calorimetric data and from plate count data at
intermediate treatment temperatures may be due to the microorganisms injured during
pre-treatment. L. plantarum may have a greater tendency toward injury than E. coli. This
speculation may also explain the lag period typically observed in the semi-log survival
curves of microorganisms and needs to be explored further.
In this study, the patterns of the temperature induced changes in ribosomes, cell
envelope components, and DNA of E. coli (Gram-negative) and L. plantarum (Gram-
positive) bacteria are compared by DSC. The results indicate that more intensive heat
treatment is needed to inactivate E. coli in comparison to L. plantarum. Mohacsi-Farkas
and co-workers (1999) also reported a higher heat-inactivation temperature for E. coli in
comparison to L. plantarum. The thermal tolerance of microorganisms may depend on
the growth conditions as well as cell structure. Both the thermal stability and enthalpy of
ribosome denaturation are influenced by low pH in vitro and in vivo for E. coli and L.
plantarum. The calorimetric evaluation of the thermal stability and enthalpy change of
isolated ribosomes as a function of pH in present study shows that the values for E. coli
are higher than those for L. plantarum indicating a higher thermal stability and a greater
energy requirement to disrupt the structure of the E. coli ribosome at low pH conditions.
We have demonstrated a correlation between viability calculated from calorimetric
data and from plate count data. Calorimetric data provide unique information by direct
62
measurement of the energy required to inactivate microorganisms. Evaluating and
quantifying differences in thermograms of whole cells and isolated components, permits
ranking of the relative thermal stabilities of the various cellular components and
identification of those most susceptible to thermal disruption.
REFERENCES
Allwood, M. C., and Russel, A. D. 1967. Mechanism of thermal injury in Staphylococcus aureus. Appl. Microbiol. 15:1266-1269. Anderson, W. A., Hedges, N. D., Jones, M. V. and Cole, M. B. 1991. Thermal inactivation of Listeria monocytogenes studied in differential scanning calorimetry. J. Gen. Microbiol. 137:1419-1424. Bonincontro, A., Cinelli, S., Mengoni, M., Onori, G., Risuleo, G. and Santucci, A. 1998. Differential stability of E. coli ribosomal particles and free RNA towards thermal degradation studied by microcalorimetry. Biophys. Chem. 75:97-103. Cantor, C. R. and Schimmel, P. R. 1980. Biophysical Chemistry Part III. The behavior of biological macromolecules, p. 1222-1223. W.H. Freeman and Company, San Francisco. Gomez, J., Hilser, V. J., Xie, D. and Freire, E. 1995. The heat capacity of proteins. Proteins: Structure, Function, and Genetics 22:404-412. Hurst, A. 1984. Reversible heat damage, p. 303-318. In A. Hurst, and A. Nasim (ed.), Repairable Lesions in Microorganisms. Academic Press, London.
63
Hurst, A., and Hughes, A. 1978. Stability of ribosomes of Staphylococcus aureus S6 sublethally heated in different buffers. J. Bacteriol. 133:564-568. Kaletunç, G. 2001. Thermal analysis of bacteria using differential scanning calorimetry, p. 227-235. In F. Bozoglu, T. Deak, and B. Ray (ed.), Novel Process and Control Technologies in the Food Industry. IOS press, Amsterdam. Kandler, O. and Weiss, D. 1986. Regular, nonsporing, gram-positive rods, p. 1208-1234. In Peter H. A. Sneath (ed.), Bergey's manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore, Md. Lee, J. and Kaletunç, G. 2002. Calorimetric determination of inactivation parameters of microorganisms. J. Appl. Microbiol. 93:178-189. Mackey, B. M., Parsons, S. E., Miles, C. A. and Owen, R. J. 1988. The relationship between base composition of bacterial DNA and its intracellular melting temperature as determined by differential scanning calorimetry. J. Gen. Microbiol. 134:1185-1195. Mackey, B. M., Miles, C. A., Parsons, S. E. and Seymour, D. A. 1991. Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry. J. Gen. Microbiol. 137:2361-2374. Mackey, B. M., Miles, C. A., Seymour, D. A. and Parsons, S. E. 1993. Thermal denaturation and loss of viability in Escherichia coli and Bacillus stearothermophilus. Lett. Appl. Microbiol. 16:56-58. Miles, C. A., Mackey, B. M. and Parsons, S. E. 1986. Differential Scanning Calorimetry of Bacteria. J. Gen. Microbiol. 132:939-952. Mohacsi-Farkas, Cs., Farkas, J., Meszaros, L., Reichart, O. and Andrassy, E. 1999. Thermal denaturation of bacterial cells examined by differential scanning calorimetry. J. Therm. Anal. Calor. 57:409-414.
64
Mohacsi-Farkas, Cs., Farkas, J. and Simon, A. 1994. Thermal denaturation of bacterial cells examined by differential scanning calorimetry. Acta Aliment. 23:157-168. Niven, G. W., Miles, C. A. and Mackey, B. M. 1999. The effect of hydrostatic pressure on ribosome conformation in Escherichia coli: an in vivo study using differential scanning calorimetry. Microbiology 145:419-425. Noll, M. and Noll, H. 1976. Structural dynamics of bacterial ribosomes. V. Magnesium-dependent dissociation of tight couples into subunits: measurement of dissociation constants and exchange rates. J. Mol. Biol. 105:111-130. Rheinberger, H., Geigenmuller, U., Wedde, M. and Neirhaus, K. H. 1988. Parameters for preparation of E. coli ribosomes and ribosomal sub-units active in tRNA binding. Method. Enzymol. 164:658-662. Rodriguez-Torres, A., Ramos-Sanchez, M. C., Orduna-Domingo, A., Martin-Gil, F. J. and Martin-Gil, J. 1993. Differential scanning calorimetry investigations on LPS and free lipids A of the bacterial cell wall. Res. Microbiol. 144(9):729-740. Stephens, P. J., and Jones, M. V. 1993. Reduced ribosomal thermal denaturation in Listeria monocytogenes following osmotic and heat shocks. FEMS Microbiol. Lett. 106:177-182. Teixeira, P., Castro, H., Mohacsi-Farkas, C. and Kirby, R. 1997. Identification of sites of injury in Lactobacillus bulgaricus during heat stress. J. Appl. Microbiol. 83 (2):219-226. Tolker-Nielsen, T. and Molin, S. 1996. Role of ribosome degradation in the death of heat-stressed Salmonella typhimurium. FEMS Microbiol. Lett. 142:155-160. Tomlins, R. H. and Ordal, Z. J. 1976. Thermal injury and inactivation in vegetative bacteria, p. 153-190. In F. A. Skinner, and W. B. Hugo (ed.), Inhibition and inactivation of vegetative microbes. Academic Press, London.
65
CHAPTER 3
CALORIMETRIC DETERMINATION OF INACTIVATION PARAMETERS OF
MICROORGANISMS*
ABSTRACT
This study aimed to apply differential scanning calorimetry (DSC) to evaluate the thermal
inactivation kinetics of bacteria. The apparent enthalpy (∆H) of Escherichia coli cells
was evaluated by a temperature scan in a DSC after thermal pre-treatment in the
calorimeter to various temperatures between 56 and 80oC. Conventional semi-
logarithmic survival curve analysis was combined with a linearly increasing temperature
protocol. Calorimetrically determined D and z values were compared to those obtained
from plate count data collected under isothermal conditions to validate the new approach.
The calculated D values using both apparent enthalpy and viability data for cells heat
treated in the DSC were similar to the D values obtained from isothermal treatment.
Temperatures for 1 through 10-log microbial population reductions, calculated from plate
count and enthalpy data were in agreement within 0.5-2.4oC at a 4oC min-1 heating rate.
66
This novel calorimetric method provides an approach to obtain accurate and reproducible
kinetic parameters for inactivation. The calorimetric method here described is time
efficient and is conducted under conditions similar to food processing conditions.
Key Words: thermal inactivation, differential scanning calorimetry, linearly increasing
temperature, apparent enthalpy, kinetic parameter
* Adapted from Journal of Applied Microbiology, 93:178-189 (2002).
67
INTRODUCTION The processing temperature and time necessary to produce a safe product are
determined using the D value (the time needed to reduce the population by one log) and z
value (temperature change required for a one log reduction in D value) for a target
microorganism. In general, D and z values for microorganisms are calculated from semi-
logarithmic survival curves produced as a function of time under isothermal conditions at
several temperatures. The thermal processing design is based on the thermal resistance of
target bacteria which is described by D and z values determined under isothermal
conditions. However, in industrial applications, processing temperature cannot be
reached instantly but requires a “come up time” in which a significant reduction of
microbial population may occur as the temperature rises (Peleg, 1999). Furthermore, the
reaction rate and D value are affected by the temperature and the rate of heat transfer
(Teixeira, 1992). Therefore, it is important to determine the D and z values under
conditions similar to those used in processing.
Kinetic parameters for chemical and biochemical reactions can be evaluated
analytically using differential and integral methods from data collected under non-
isothermal conditions (Deindoerfer and Humphrey, 1959; Rhim et al., 1989; Nunes et al.,
1991). There are several studies in the literature that model microorganism inactivation
during increasing temperature protocols (Reichart, 1979; Thompson et al., 1979a,b; Van
Impe et al., 1992). Reichart (1979) calculated the D and z values for Saccharomyces
cerevisae, Escherichia coli, and Bacillus stearothermophilus using the linear portion of
the survival curve generated as a function of rising temperature with decreasing heating
68
rate. Reichart (1977) reported that similar D and z values were obtained from analysis of
viable count data produced under isothermal and increasing temperature conditions. On
the other hand, Thompson and co-workers (1979a,b), from their investigation on the
effect of heating rate on the inactivation of Clostridium perfringens and Salmonella
typhimurium reported that the D value for C. perfringens at 60oC increased from 5.3 min
to 20 min when the heating rate increased from 0oC h-1 (isothermal treatment) to 15oC h-1,
while the D value for S. typhimurium at 50oC did not change significantly when the
heating rate changed from 6oC h-1 to 12.5oC h-1. In all cases, they report D values higher
than the D50 obtained with isothermal treatment.
Some investigators have used differential scanning calorimetry to achieve heat
treatment under controlled conditions of linearly increasing temperature and to determine
the thermally-induced transitions with the ultimate purpose of evaluating the relationship
between the stability of cellular components and cell injury or death (Miles et al., 1986;
Mackey et al., 1988; Lepock et al., 1990; Mackey et al., 1991; Belliveau et al., 1992;
Mackey et al., 1993). An equation describing the rate of microorganism inactivation as a
function of linearly increasing temperature was derived by Miles et al. (1986) and was
used to determine the temperature at which the maximum death rate occurred for
vegetative cells (Miles et al. 1986) and spores (Belliveau et al. 1992). Miles and Mackey
(1994) developed the model further to predict the number of surviving microorganisms as
a function of temperature at a constant heating rate. These investigators used the
resulting equation to predict the survival of Listeria monocytogenes heated to different
final temperatures in minced beef and compared the predictions with experimental
69
results. They also calculated the temperatures required to reduce viability by 7D at 0.1,
1, and 10oC min-1 heating rate using the published D and z values determined under
isothermal conditions. The results demonstrated that the temperatures required to
inactivate L. monocytogenes increased with the heating rate. Miles and Mackey (1994)
stated that the derived equation can also be used to calculate the D and z values under
linearly increasing temperature protocols.
Although several investigators determined the viable counts of microorganisms
following heat treatment in the DSC, no D and z values were reported. In this study, the
mathematical model proposed by Miles and Mackey (1994) was utilized to determine the
D and z values using both the viable count and calorimetric data obtained with linearly
rising temperature in DSC. The D and z values for E. coli K12 determined from the
calorimetric data were compared to the corresponding values from plate count data
obtained after heat treatment in the DSC and after isothermal treatment. The close
agreement between the calorimetrically determined microorganism inactivation kinetic
parameters and those of determined from plate count data demonstrates the advantage of
this unique approach in obtaining reproducible and accurate results in a short time.
70
MATERIALS AND METHODS
Source and preparation of organisms
E. coli K12 was obtained from the Culture Collection, Department of Microbiology
at the Ohio State University. A loopful of organism was revived in 10 ml Trypticase soy
broth (Difco laboratories, Detroit, MI) supplemented with 0.3 % (w/w) yeast extract
(Difco laboratories, Detroit, MI) (TSBYE) and incubated at 37oC for 18 hours. The
culture was stored frozen (-80oC) in 30 % (v/v) sterile glycerol. A loopful of the stock
culture was transferred to 10 ml TSBYE and incubated 10 hrs at 37oC before use.
A growth curve for E. coli was generated from viable count data to determine the
time required to reach the late exponential growth phase. Culture was inoculated (1 %
v/v) into TSBYE and incubated at 37oC until reaching late exponential growth phase,
when the final concentration of cells in the medium was 1.0 ± 0.1 x 109 cfu ml-1. After
reaching the late exponential phase, the growth medium was centrifuged (Beckman J2-
21 centrifuge) at 10 000 g for 10 min at 4oC to separate the cells as pellets. The pellet
was washed with 150 ml of sterile distilled water before transfer into DSC crucibles. A
differential scanning calorimeter (DSC 111, Setaram, Lyon, France) was used for
collection of all thermograms. A portion (~100 mg) of the pellet was transferred into a
tared (1.5 ml) polyethylene tube, weighed, freeze dried (Freezone 4.5, Labconco Freeze
Dry System), and reweighed to determine the percentage of dry matter in the pellet. The
amount of moisture in the E. coli pellet (wt/wt) used in the DSC experiments was
determined to be 83 ± 0.3 % by freeze drying.
71
Apparent enthalpy value of E. coli K12 from DSC
A DSC thermogram with empty stainless steel sample and reference crucibles was
collected to measure the empty crucible baseline. Temperature calibration was
confirmed using an indium sample in the stainless steel crucible at a constant heating
rate of 4oC min-1. Pellets of cells were carefully transferred into the sample crucible and
weighed (70 ± 0.3 mg wet weight). When the reference crucible is left empty an artifact
due to the heat capacity imbalance between crucibles is observed at the initiation of
temperature scanning. A known quantity of water, similar in mass to the moisture in
sample, was placed in the reference crucible to eliminate the artifact. The reference
crucible was filled with 58 ± 0.2 mg (~83 % of sample wt) of distilled water. Both
crucibles were sealed using aluminum o-rings. The sealed crucibles were refrigerated
(4oC) until used for DSC. The sample and reference crucibles were placed in the DSC
and equilibrated at 1oC. Pellets were pre-heated in the DSC to 56.7, 58.7, 60.7, 62.7,
64.7, 66.7, 68.7, 70.7, 75.7, or 80.7oC with 4oC min-1 heating rate. After pre-heating in
the DSC, samples were cooled immediately by liquid nitrogen, equilibrated at 1oC, and
rescanned to 140oC at 4oC min-1. Samples were reweighed after DSC measurements to
check for loss of mass during heating. Thermograms of samples showing signs of
leakage were not used. A control scan was recorded by heating the pellets from 1oC to
140oC at 4oC min-1. DSC thermograms of samples were corrected for differences in the
empty crucibles by subtraction of an empty crucible baseline. Peak areas (apparent
enthalpies, J g-1) corresponding to the contributions of survivors were determined from
the apparent heat capacity vs. temperature graph using software provided by the
72
instrument manufacturer. Apparent enthalpy values were also corrected for the amount
of dry matter in the pellets based on the freeze-dryer results. Because the moisture
content of the pellet was not known prior to the DSC measurement, the reference
crucible was filled with water equal to 83 % of the sample weight. When the moisture
content was determined, the difference between the sample moisture content and the
amount of water in the reference crucible was calculated. The enthalpy contributions of
excess water, based on the measured difference in water content, was calculated by
integrating the equation describing the specific heat of water as a function of
temperature over the temperature limits used to calculate the apparent enthalpy. The
apparent enthalpy value was corrected by adding (subtracting) the enthalpy contribution
of the water if the moisture content of the sample was lower (greater) than the amount of
water in the reference crucible.
The number of E. coli K12 survivors after DSC
Weighed pellets of cells (~70 mg wet weight) were transferred into sterile DSC
crucibles using sterile loops. A stainless steel cap, an aluminum o-ring and a screw cap
was placed in each crucible without sealing. The reference crucible was filled with
distilled water (~83 % of sample wt). The capped crucibles were kept under
refrigeration (4oC) until used for DSC. Pellets in crucibles were heat-treated to 56.7,
58.7, 60.7, 62.7, 64.7, 66.7, 68.7, 70.7oC at 4oC min-1 heating rate using the DSC
instrument. After cooling, a portion (50 mg) of the heated pellet from each crucible was
transferred to a (1.5 ml) sterile polyethylene tube using a sterile loop. Sterile peptone
73
water was added to make a final volume of 1 ml with 1/20 (w/v) ratio. After careful
suspension in the tube, the cells were serially diluted and plated onto Trypticase soy agar
to determine viable counts. After 36 hours incubation at 37oC, viable counts of each
sample were obtained by calculation of dilution ratios. The level of the lowest detection
was 2 x 101 cfu g-1 in pellet.
The number of E. coli K12 survivors after isothermal heat treatment
Weighted cell pellets (70 mg wet weight) were transferred into thin-walled
polypropylene reaction tubes containing smug-fitting snap caps. The tubes were
submerged in a temperature-controlled water bath stabilized at 56, 58, 60, 62, or 64oC.
The temperature was continuously monitored by a thermocouple placed in the water
bath next to the tubes. The temperature variance was ~±0.02oC during treatment. The
tubes were removed at time intervals; 10, 20, 30, 40 min for 56oC, 2, 5, 10, 20, 30 40
min for 58oC, 1, 2, 4, 5, 8, 11, 14 min for 60oC, 1, 2, 3, 4 min for 62oC, 0.5, 1, 1.5, 2 min
for 64oC, and were cooled in an ice-water bath. The cells in the tube were serially
diluted and plated onto Trypticase soy agar to determine viable counts using the
procedure described above.
DSC data analysis
DSC thermograms were corrected for differences in the empty crucibles by
subtracting an empty crucible baseline. Total heats corresponding to the envelope of
endothermic peaks (enthalpy, J g-1) between approximately 40 and 130oC were
74
determined by integrating the temperature vs. heat flow curve using software provided
by the instrument manufacturer. A curved baseline taking into account the variation in
heat capacity before and after the transition passing through three designated points on
the thermogram was used to calculate the apparent enthalpy of whole cells. Data points
at three temperatures were selected to determine the baselines for all DSC curves as
shown in Figure 3.4. The initial temperature point was on the pre-transition baseline
(40oC). The final point was on the post-transition baseline (130oC). An intermediate
point was selected at a temperature below the onset of the final peak which corresponds
to transitions in the cell envelope (108oC).
THEORY
Miles and Mackey (1994) developed a model to predict the viability of
microorganisms which have subjected to linearly increasing temperature. Their
derivation combining a first order inactivation model for microorganisms with a linearly
increasing temperature protocol was adapted to analyze the calorimetric data as described
below.
According to first order inactivation kinetics, the ratio of survivors as a function of
treatment time at a constant temperature can be described as;
75
Dt
NN
−=0
10log (1)
where, N is the number of survivors at time t and N0 is the initial number of viable cells.
The z value can be calculated from the D values determined at least at two temperatures
as the slope of a line of log D vs. temperature;
z
TTDD e
e
−=10log (2)
where, T is the temperature and De is the D value at an arbitrary temperature Te.
When the temperature increases at a constant linear heating rate, r; from an initial
temperature T0:
rtTT += 0 (3)
where, T0 is the initial temperature
the number of survivors is given by an equation, derived by Miles et al. 1986:
⎭⎬⎫
⎩⎨⎧
⎥⎦
⎤⎢⎣
⎡⎟⎠
⎞⎜⎝
⎛ −−⎟
⎠
⎞⎜⎝
⎛ −−=
zTT
zTT
rDz
NN ee
e
)(303.2exp
)(303.2expexp 0
0
(4)
When
20 >−zTT
,
76
equation 4 reduces to
⎥⎦
⎤⎢⎣
⎡⎟⎠⎞
⎜⎝⎛ −−
≈z
TTrDz
NN e
e
)(303.2expexp
0
(5)
Equation 5 is converted into linear form by taking the logarithm of both sides twice,
ee
TzrD
zTzN
N 303.2ln303.2lnln0
−+=⎥⎦
⎤⎢⎣
⎡⎟⎟⎠
⎞⎜⎜⎝
⎛− (6)
slope intercept
The slope (2.303/z) of ln[-ln(N/N0)] vs. T curve is used to calculate a z value. The z value
and the intercept from the graph are used to calculate a De value for the microorganism at
a given heating rate, r. Values for D at any temperature are obtained from equation 2.
The value N/N0 represents the fraction of survivors as a result of heat treatment. The
apparent enthalpy, ∆H, is the area under the DSC thermogram, ∆H = ∫ Cp dT.
Assuming that ∆H is proportional to the number of survivors after correction for the
residual area observed for killed cells and macromolecular transitions, ∆Hf, we can write
an expression for the fraction of survivors in terms of the DSC observable;
f
f
HHHH
NN
∆−∆
∆−∆≈
00
(7)
77
Substitution of equation 7 into equation 6 yields;
eef
f TzrD
zTzHH
HH 303.2ln303.2lnln0
−+=⎥⎥⎦
⎤
⎢⎢⎣
⎡⎟⎟⎠
⎞⎜⎜⎝
⎛
∆−∆
∆−∆− (8)
By analogy to the viability data, the z value is determined from the slope (2.303/z) of
ln[-ln(∆H-∆Hf)/(∆H0-∆Hf)] vs. T (Eqn. 8 and Fig. 5). Once De is determined from the
intercept, D at any temperature can be obtained from equation 2.
The temperature for any given log reduction in survivors, n, is calculated by rearranging
equation 6 as;
⎥⎦
⎤⎢⎣
⎡+⎟
⎠⎞
⎜⎝⎛+= )303.2ln(ln
303.2n
zrDzTT e
e (9)
where n = -log N/N0
78
e
C
Growth of the E. coli cells to the end of exponential stag
Centrifugation to obtain cell pellets
Heat-treatment in DSCi
g g
a a
Apparent enthalpydata
s
79
Heat-treatment in sothermal condition
DS
Plate countinViable count dat
Applied to the equation
e
Calorimetric D and z value Isothermal D and z valuViable count dat
Plate countin
Figure 3.1. Experimental scheme of calorimetric and microbial analysis
RESULTS
Heat treatment of E. coli with linearly rising temperature in DSC
DSC thermograms displaying the thermally-induced transitions of E. coli K12
control cells and the cells immediately subsequent to thermal pre-treatment are shown in
Figure 3.2. The pre-treatment was accomplished by partial scanning to temperatures
between 56.7 to 80.7oC followed by rapid cooling to the initial temperature. The
scanning rate was 4oC min-1 for both partial and subsequent complete scans. It is
apparent from Figure 3.2 that as the final temperature of the partial scan increases, the
shape and size of some transitions in the second scan change compared to the control
scan; while, other transitions remain unchanged. These observations indicate irreversible
and reversible changes in cellular components as a result of thermal pre-treatment. An
increase in pre-heating temperature results in a decrease in the area of the transition peak
(apparent enthalpy, ∆H, J g-1) observed in the 50~85oC region, an indication of
irreversible changes. The onset temperature of the first major transition also was
determined from the thermograms. A closer examination of Figure 3.2 reveals that the
onset temperature of the transition increases up to 70.7oC as the temperature of the pre-
treatment increases to 70.7oC. Further increases up to 80.7oC in the pre-treatment
temperature do not result in an increase in the onset temperature although the apparent
enthalpy calculated continues to decrease.
The DSC thermograms of E. coli K12 also exhibit a significant difference between
the apparent specific heat capacities of the live and inactivated cells. These heat capacity
differences are apparent as differences between the pre- and post transition baselines.
80
Figure 3.2. Apparent specific heat capacity versus temperature curves of control and heat-treated E. coli. Curves are displayed by 0.1 Jg-1 K-1 for clarity.
20 40 60 80 100 120 140
Temperature (oC)
control
56.7 o C pre-heated
58.7 o C pre-heated
60.7 o C pre-heated
62.7 o C pre-heated
64.7 o C pre-heated
70.7 o C pre-heated
68.7 o C pre-heated
66.7 o C pre-heated
80.7 o C pre-heated
75.7 o C pre-heated
0.5 J g -1 K -1
81
Evaluation of D and z values of E. coli K12 from viable counts data after a heat
After a partial scan in the DSC at 4 C min to the temperature specified in Table
3.1, the E. coli pellets were cooled rapidly and the pellet was removed from the crucible.
The number of survivors was determined using plate counting. If the assumption of first
order inactivation kinetics is valid, according to equation 6 ln[-ln(N/N0)] should be a
linear function of temperature. It is apparent from Figure 3.5 that over the temperature
range of 58.7 to 68.7 C a linear relationship exists between ln[-ln(N/N0)] and temperature
with regression coefficient r2 = 0.974. The number of survivors after heating to 56.7oC
with 4 C min in the DSC was equal to the initial number of viable cells, while no
survivors were detected when the cells were heated to 70.7 C. The D value at 60 C and z
value were calculated from the intercept and the slope of the line displayed in Figure 4
and were found to be 5.9 min and 3.8 C, respectively.
Evaluation of D and z values of E. coli K12 from calorimetric data after a heat
Following a partial scan to the temperature specified in Table 3.1 and rapid cooling
to 1 C, the E. coli pellets were scanned to monitor the thermally-induced transitions
associated with the bacterial cells surviving the partial scan. The DSC thermograms were
normalized to yield the apparent specific heat capacity (J g K ) as a function of
temperature. The area under the curve of the second scan (apparent enthalpies, J g ) was
evaluated by integrating the apparent heat capacity vs. temperature curve. A curved
baseline using three-temperature points was utilized to calculate the apparent enthalpy.
treatment to different final temperatures in DSC
o -1
o
o -1
o o
o
treatment to different final temperatures in DSC
o
-1 -1
-1
82
Treatment temperature (oC) *Viable counts (cfu g-1) †Apparent enthalpy (J g-1)
Control 3.6 x 1011 4.32
56.7 3.6 x 1011 4.28
58.7 3.4 x 1011 4.19
60.7 2.6 x 1011 3.89
62.7 2.1 x 1011 3.60
64.7 8.2 x 109 3.15
66.7 7.9 x 105 2.78
68.7 3.0 x 102 2.70
70.7 <2.0 x 101 2.67
*Plate count data after heat treatment in the DSC. †Calorimetric data after heat treatment in the DSC.
Table 3.1. Viability and apparent enthalpy values for E. coli K12 cell pellets after pre-treatment in the DSC.
83
D value (min) z value (oC)
Heating medium for E. coli
Temperature
(oC)
Isothermal
Non-
isothermal
Isothermal
Non-
isothermal
Reference
Nutrient broth 56 4.5 4.9 Chambers et al. (1957)
Ringer solution 55 4.0 Lemcke and White (1959)
Sucrose solution (0.99 aw) 57.2 1.2 Goepfert et al (1970)
Tryptic soy broth 52 25.6 Stiles et al. (1973)
Milk solution (10 %) Milk solution (51 %)
58 58
1.40 13.5
4.67.9
Dega et al. (1972)
Glucose solution (0.5 %)
55 56
57.2 60
3.54
0.29
6.7
3.52 1.6
0.27
3.68
3.58
Reichart (1979)
Cell pellets
58 60 62 64
5.90 1.80 0.50 0.24
4.23 Present work
84
Table 3.2. D and z values reported for E. coli from isothermal and non-isothermal heat treatments.
A representative curve and baseline for the E. coli control pellet heated from 1 to 140oC
are displayed in Figure 3.4. The apparent enthalpy value was found to be 4.32 J g-1 wet
weight. Because this sample was not subjected to a partial scan, the measured apparent
enthalpy corresponds to the ∆H0 value. The peak areas of heat-treated E. coli pellets (∆H)
were determined using the same analysis procedure applied to the second scan. The
apparent enthalpy values were corrected to account for moisture content differences
between the sample and reference crucibles. The apparent enthalpy of maximally treated
cells, ∆Hf, was calculated from the second scan after a partial scan to 70.7oC. The criteria
for selecting 70.7oC pre-treatment to calculate ∆Hf are discussed later in discussion
section. As expected from Equation 8, the ln[-ln(∆H-∆Hf)/(∆H0-∆Hf)] vs. temperature
graph produced a straight line (r2 = 0.988) from which the D value at 60oC and the z value
were calculated to be 6.1 min and 5.2oC, respectively (Fig. 3.6).
Evaluation of D and z values of E. coli K12 from viable counts data after isothermal heat treatment The number of survivors in E. coli pellets subjected to isothermal heat treatment at
58, 60, 62, and 64oC were determined. D values for each isothermal treatment were
determined from the slope of ln(N/N0) vs. time plots (Table 3.2). According to Equation
2, log10(D/D58) values were plotted against temperature and the z value was calculated to
be 4.23 oC from isothermal treatment studies.
85
DISCUSSION
DSC thermograms of microorganisms display several endothermic or net
endothermic transitions which may be a combination of exothermic and endothermic
events (Verrip and Kwast, 1977; Miles et al., 1986; Lepock et al., 1990; Anderson et al.,
1991; Mackey et al., 1991; Kaletunç, 2001). Several main peaks of the E. coli
thermogram can be assigned to thermally induced transitions of particular cellular
components by comparison to the transition temperatures of isolated cell components
(Mackey et al., 1991). It has been reported that a strong relationship exists between
thermal death and the peaks observed at 50~85oC for vegetative microorganisms (Miles
et al., 1986; Teixeira et al., 1997; Niven et al., 1999). Mackey et al. (1991) and Niven et
al. (1999) proposed that the temperature region over which the major reduction due to
pre-heat treatment in the area under the peaks is observed in the DSC thermogram is the
result of denaturation of the main ribosomal subunit. To calculate the enthalpy associated
with a certain peak, the baselines in the temperature regions well below and well above
the transition region should be apparent. In the DSC thermograms of microorganisms,
the existence of several overlapping transitions resulting from various cellular
components does not allow one to evaluate the enthalpy of the individual transitions. Pre
and post-transition baselines can be readily defined to determine total area under the
thermal transition curve. Therefore, we have measured the total apparent enthalpy of all
transitions. Miles et al. (1986) used a similar approach to determine the total enthalpy of
denaturation for a number of bacteria and reported that there was a significant difference
86
among bacteria, with enthalpy of denaturation values based on the dry matter content
ranging from 9 to 20 J g-1 dry matter. The total enthalpy of denaturation for the E. coli
K12 used in this study is 23 J g-1 dry matter.
A significant portion of the difference observed between the apparent specific heat
capacities of the live and inactivated cells occurs between the baselines prior to and after
the major peak which is attributed to disruption of the ribosome structure. As the
preheating temperature increases, the amount of inactivated cells during preheating
increases resulting in a visible reduction in the apparent heat capacity difference between
the pre- and post transition baselines in the rescan thermogram (Fig. 3.2). A similar
observation was reported by Miles et al. (1986) as the rescan thermogram displayed
slightly higher specific heat capacities at low temperatures compared to the initial scan.
Denaturation of proteins typically is accompanied by a positive heat capacity change.
For a typical globular protein of ~15 kDa, the change in the heat capacity is on the order
of 1.5-2.5 kcal K-1 mol-1 (0.4-0.67 J g-1 K-1) and may have a profound effect on the
overall energetics (Gomez et al. 1995). The apparent heat capacity change for E. coli
pellets upon inactivation is determined to be of the same order of magnitude, 0.6 J g-1 K-1
(Fig. 3.2).
In the absence of a heat capacity change over the transition, the enthalpy
calculation is carried out by constructing a linear baseline connecting the segments of the
thermogram before and after the peak and by evaluating the area between the peak and
the baseline. The construction of the baseline is critical for accurate evaluation of the
enthalpy value for a first order transition coupled with a heat capacity change as typically
87
observed in thermograms of microorganisms. Upon rescanning of the samples which
were heated to 140oC, we observed a superimposition of baselines between the two scans
above 130oC (Fig. 3.3); implying that the heat capacity of the heat damaged cells is
independent of any thermal treatment. Therefore, a curved baseline was used between
the segment of the thermogram prior to the major thermally-induced transition (~40oC)
and the segment of the thermogram after the last peak (~130oC) (Fig. 3.4). The total peak
area was determined from the first scan of the control sample. For heat treated samples,
the second scan after a partial scan to pre-treatment temperature was used to calculate the
total apparent enthalpy. The heat flow data after correction for the difference between
the empty crucibles was used to calculate the total apparent enthalpy. The resultant
enthalpy value is further corrected for the difference in the amount of water between the
sample and reference crucibles.
A close examination of Figure 3.2 reveals that the peaks attributed to the ribosomal
subunits in the DSC thermogram (observed between 50 and 85oC) disappeared when the
E. coli pellet was heated to 80.7oC, while the thermal transitions associated with other
cellular components appear to be unaffected by pre-heating to the same temperature. The
absence of viability revealed in the plate counts of cells preheated to 70.7oC in the DSC
suggests that the total area under the thermogram peaks includes contributions directly
related to the cell death as well as contributions due to additional macromolecular
transitions. Macromolecular changes may include conformational changes and phase
transitions that manifest themselves as a combination of overlapping endothermic and/or
exothermic events resulting in a net endothermic profile. Residual peak area which
88
Hea
t flo
w (m
W)
Temperature (oC)
Figure 3.3. A typical DSC thermogram for whole cells of E. coli K12 after empty crucible baseline subtraction. initial scan (a), rescan after cooling (b).
89
Hea
t flo
w (m
W)
Temperature (oC)
Figure 3.4. DSC thermogram for whole cells of E. coli K12 displaying curve base line used to determine the apparent enthalpy value.
90
corresponds to apparent enthalpy changes associated with macromolecular transitions is
observed even after the cells are completely inactivated. The residual apparent enthalpy
(∆Hf) determined from samples pre-treated at 70.7oC was subtracted from the total
apparent enthalpy calculated from each DSC rescan to compensate for the contributions
of inactive cells. After pre-treatment to 70.7oC no viability was detected in the plate
count data. The analysis of each rescan thermogram showed that the onset temperature
of the major peak increases with increasing pre-treatment temperature up to 70.7oC and is
unchanged for pre-treatment temperatures of 70.7, 75.7, and 80.7oC. Furthermore, the
continuous decrease in apparent enthalpy stabilizes around 70.7oC (Table 3.1), but
continues to decrease to 1.86 J g-1 at 75.7oC and to 1.69 J g-1 at 80.7oC. Consequently,
the pre-treatment temperature used to calculate the residual apparent enthalpy (∆Hf) can
be determined using either plate count data or calorimetric data. Therefore, the fractional
enthalpy change associated with pretreatment is used to estimate the reduction in the
viable cell population. It is apparent from equation 7 that the fractional enthalpy
calculation depends on the choice of ∆Hf. Thus it is important to determine the value of
(∆Hf) carefully.
The corrected apparent enthalpies (∆H) of cells determined from second scans after
an initial scan to various temperatures in the DSC together with ∆H0 and ∆Hf values were
used to construct a fractional survivor versus temperature graph according to equation 8
(Fig. 3.6). Similar to the DSC based plate count data (Fig. 3.5), a linear relationship
(r2=0.988) is observed between the reduced apparent enthalpy [(∆H-∆Hf)/(∆H0-∆Hf)] and
the temperature. It is apparent that the assumption of equation 7, in which the fractional
91
-4
-3
-2
-1
0
1
2
3
4
55 60 65 70
Temperature (oC)
ln[-
ln(N
/N0
)]
Figure 3.5. Temperature dependence of fractional survivor population determined from plate count data after heat pre-treatment of E. coli cells in the DSC.
92
-3
-2
-1
0
1
2
3
55 60 65 70
Temperature (oC)
ln[-
ln((∆H
-∆H
f)/(∆
H0
-∆H
f)]
Figure 3.6. Temperature dependence of fractional survivor population determined from calorimetric data after heat pre-treatment of E. coli cells in the DSC.
93
survivor population from plate count viability and apparent enthalpy data are
proportional, is valid.
Microbial survival curves typically are parameterized in terms of D and z values,
where D represents the time required to reduce the population by one log unit and z
represents the temperature change required to reduce D by one log value. There are a
number of D and z values for E. coli, reported in the literature (Table 3.2). It is apparent
that even the D and z values determined from isothermal treatment are influenced by the
medium in which the bacteria are suspended. Furthermore, thermal resistance may
depend on the strain of bacteria. In this study, E. coli pellets with 1011 cfu mg-1 bacterial
concentration were prepared in order to produce a measurable and reproducible heat flow
signal in the DSC for calorimetric analysis of bacterial death. E. coli pellets of 70 mg are
used for comparison of D and z values obtained from both plate count and apparent
enthalpy data of microorganisms treated in the DSC under a 4oC min-1 heating rate.
Isothermal heat treatments in a water bath were applied to approximately 70 mg E. coli
pellets in order to be able to compare the D and z values from isothermal and
nonisothermal temperature protocols. For comparison, our D and z values calculated
under isothermal treatment conditions are included in Table 3.2. Although of the same
order of magnitude, D values calculated in this study are clearly higher than the values
reported in the literature. This could be due to a higher equilibration time required for the
pellet to reach a constant temperature compared to a microorganism sample in a capillary
tube or to differences between our strain and those for which D and z values are reported
in the literature. Very high cell densities, such as in the pellet in this study, are associated
94
with increased heat resistance of spore-forming and nonsporing microorganisms (Hansen
and Riemann, 1963; Beaman et al., 1981; Jay, 1996). Also, it is known that the values of
D and z depend on the composition of the medium, and the washing solution, and the
stage of growth (Strange and Shon, 1964; Hoffman et al., 1966; Tomlins and Ordal,
1976). It should be emphasized that the purpose of the study is to compare the D and z
values of the E. coli samples prepared from the same strain, with same microorganism
concentration, and amount of sample using different heating strategies and also to
demonstrate that calorimetric data can be utilized to evaluate the thermal resistance of
microorganisms.
Comparison of D values in Figure 3.7 reveals that D values obtained from data
collected under linearly increasing temperature protocols were higher than those obtained
under isothermal conditions. Several factors may lead to such results. The D values
determined from isothermal kinetic data may be lower than the actual values, because at
all of the isothermal treatment temperatures (58, 60, 62, 64oC) an initial lag time
unaccounted for in the standard model was observed. Because the number of points from
the log-linear portion of the survival curve were much larger compared to the number of
points in the initial lag time portion, calculated D values from the fitted data were
dominated by the linear portion of the survival curve which would be expected to lead to
an underestimation of D values compared to the actual ones.
If the heating rate is higher than the time necessary for the samples to reach
thermal equilibrium a thermal gradient may be established in the sample. The thermal
gradient is a function of sample dimensions, thermal diffusivity, and the heating rate.
95
0
2
4
6
8
10
55 60 65 70 75
Temperature(oC)
D v
alue
(min
)
Figure 3.7. Comparison of D values calculated from the calorimetric and viability data obtained under non-isothermal heat treatment in the DSC and D values obtained from isothermal heat treatment. Calorimetric data ( ), Viability data ( ), Isothermal data ( ).
96
Depending on the magnitude of the temperature gradient, a distribution of active, injured,
and inactive cells may be present in the pellet at any time. Miles and Mackey (1994)
calculated a 3oC thermal gradient between the surface and the center of a cylindrical
container of 0.01m radius filled with Listeria monocytogenes inoculated beef with a
thermal diffusivity of 1.4 x 10-7 m2 s-1. The DSC crucibles used in this study were
cylindrical shape with a radius of 0.0025 m. The pellets had approximately 83 ± 0.3 %
by weight water. Assuming a thermal diffusivity of 1.3 x 10-7 m2 s-1, similar to that of a
starch gel with 82 % moisture (Andrieu et al., 1989), the temperature gradient between
the center and the surface of the crucible calculated to be 0.8oC. If such a temperature
correction is applied to the data in Figure 3.7, both of the curves describing D values
obtained under linearly increasing temperature conditions approach the D values obtained
under isothermal conditions. This correction becomes more significant in the steep part
of each curve. A close examination of Figures 3.7 and 3.8 reveals that even an order of
magnitude change in D value at low temperatures (Fig. 3.7) results in a reduction in
microbial population of less than 0.5 log units (Fig. 3.8). At higher temperatures, where
significant microbial population reduction occurs, the differences among the D values
obtained by isothermal plate count, nonisothermal plate count, and nonisothermal
calorimetric data are insignificant.
The D values obtained using linearly increasing temperature protocols may be
influenced by heat transfer limitations as well as by stress adaptation of microorganisms
as a result of sublethal heat treatments during constant heating rate schemes. If slow
heating rates are employed, temperature adaptation may occur during the heating process
97
which may lead to an increase in the thermal tolerance of microorganisms and higher D
values compared to those obtained under isothermal conditions. The increase in heat
resistance under linearly increasing temperature protocols was reported to be dependent
on the heating rate (Tsuchido et al., 1982; Mackey and Derrick, 1987). The study on
Salmonella Typhimurium by Mackey and Derrick (1987) reveals that microorganism
survival under isothermal conditions subsequent to a 0.6oC min-1 heating rate was greater
in comparison to survival after heating at 10oC min-1 heating rate. The impact of
sublethal heat treatment on the thermotolerance of food pathogens has been studied
extensively (Bunning et al., 1990; Farber and Brown, 1990; Linton et al., 1992; Murano
and Pierson, 1992). Linton et al. (1992) reported that a 10 min heat treatment of L.
monocytogenes at 48oC, increased the D value at 55oC, by more than two-fold. However,
at heating rates typical of domestic or commercial cooking practices (0.5-5.0oC min-1),
the sample temperature is expected to increase 5oC or more in 10 minutes. Miles and
Mackey’s study (1994) showed that the temperatures required to reduce viable numbers
of L. monocytogenes by 7D increased as the heating rate increased from 0.1 to 10oC min-
1. A similar temperature increase of 5oC was observed when the heating rate increased
from 0.1 to 1oC min-1 and from 1 to 10oC min-1. Because heat adaptation of a
microorganism is more likely to occur at the slowest heating rate of 0.1oC min-1, it is
expected that a smaller increase in sample temperature required to kill 7D when the
heating rate is increased from 0.1 to 1oC min-1 in comparison to a heating rate increase
from 1 to 10oC min-1. Heat adaptation may become a greater concern for isothermal
98
0
1
2
3
4
5
6
7
8
9
10
55 60 65 70 75Temperature (oC)
n, L
og r
educ
tion
Figure 3.8. Predicted log reductions in cell population as a function of temperature using a 4 oC min-1 heating rate (equation 9) using apparent enthalpy data ( ), viability data ( ), and isothermal data for holding time of 5 sec ( ).
99
treatments, because as the sample temperature approaches the set temperature, the driving
force decreases causing an increase in the time required to reach the set temperature. The
temperatures required to reduce the number of viable E. coli by 0.1 through 10 log units
in the DSC employing a constant heating rate of 4oC min-1 are predicted using equation 9
(Fig. 3.8). The difference between the predicted temperatures using plate count and
enthalpy data varies between 0.5-2.4oC over the 10-log unit reduction in survivor
population. For comparison, corresponding curves derived from the isothermal data for
5 sec exposure time is also displayed in Figure 3.8. The calculation of the temperatures
for isothermal treatment was carried out using the following equation,
⎟⎟⎠
⎞⎜⎜⎝
⎛−=
ee nD
tzTT 10log (10)
where t is the time in minutes.
The plots in Figure 3.8 suggest that the log population reduction of the E. coli cells
treated at isothermal conditions with a 5 sec holding time is equivalent to the log
reduction of cells calculated from the enthalpy data obtained with a linearly increasing
temperature treatment at 4oC min-1.
DSC provides information on the thermal and thermodynamic stability of materials.
The thermal analysis of both spores (Belliveau et al. 1992) and vegetative cells (Miles et
al. 1986) demonstrated a clear relationship between the onset of cellular component
denaturation observed in the DSC and the thermal resistance of the organisms. The
maximum death rate of the organisms occurred above the onset of thermal denaturation
while the maximum growth temperatures were below the onset of thermal denaturation.
100
It is also apparent from our results on E. coli that the onset of thermal denaturation of the
control samples is around 58oC. The plate count results for E. coli after partial scanning
to various temperatures with 4oC min-1 scanning rate clearly shows that a decrease in
viable counts occurs after a partial scan to 58.7oC (Table 3.1).
Although a first order rate equation is generally employed to describe the survival
curve of bacteria and to evaluate the processing parameters necessary to inactivate
microorganisms, it has been demonstrated that survival curves may show deviations from
the log-linear model displaying an initial lag time, a tail after a linear semi-logarithmic
survival curve, or other nonlinear semi-logarithmic behavior (Miles et al. 1994; Peleg,
2000). Calorimetric data can be fitted to mathematical models other than linear semi-
logarithmic models to determine the inactivation parameters of microorganisms.
This study focused on the utilization of calorimetric data to evaluate thermal
inactivation of bacteria under a linearly increasing temperature protocol. The basis for
thermodynamic study of microorganism inactivation is that the relevant initial and final
states can be defined and the energetic differences between these states can be measured
using calorimetric instrumentation. The amount of thermal energy (apparent
enthalpy, ∆H) associated with denaturation of cellular components before and after
application of heat treatment with linearly rising temperature using DSC was related to
the number of viable cells of E. coli K12 and was used to calculate the fraction of
surviving cells. An equation based on first order inactivation kinetics is used to calculate
D and z values from the fraction of surviving cells exposed to heat treatment using a
linearly increasing temperature protocol. The results suggest that the apparent enthalpy
101
data obtained from DSC can be used to evaluate D and z values, as well as, the linear
temperature rise necessary to reduce a microbial population by a chosen factor. While
this calorimetric approach requires careful data collection and analysis, the significantly
shorter time required coupled with comparable accuracy, make this method competitive
with the plate count technique.
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Teixeira, P., Castro, H., Mohacsi-Farkas, C. and Kirby, R. 1997. Identification of sites of injury in Lactobacillus bulgaricus during heat stress. J. Appl. Microbiol. 83(2):219-226. Thompson, D.R., Willardsen, R.R., Busta, F.F. and Allen, C.E. 1979a. Clostridium perfringens population dynamics during constant and rising temperatures in beef. J. Food Sci. 44:646-651.
Thompson, W.S., Busta, F.F., Thompson, D.R. and Allen, C.E. 1979b. Inactivation of salmonellae in autoclaved ground beef exposed to constantly rising temperatures. J. Food Prot. 42:410-415. Tomlins, R.I. and Ordal, Z.J. 1976. Thermal injury and inactivation in vegetative bacteria. In Inhibition and Inactivation of Vegetative Microbes ed. Skinner, F.A. and Hugo, W.B. pp.153-190. New York: Academic Press. Tsuchido, T., Hayashi, M., Takano, M. and Shibasaki, I. 1982. Alteration of thermal resistance of microorganisms in a non-isothermal heating process. J. Antibacterial Antifungal Agents 10:105-109. Van Impe, J.F., Nicolai, B.M., Martens, T., De Baerdemaeker, J. and Vandewalle, J. 1992. Dynamic mathematical model to predict microbial growth and inactivation during food processing. Appl. Environ. Microbiol. 58:2901-2909. Verrips, C.T. and Kwast, R.H. 1977. Heat resistance of Citrobacter freundii in media with various water activities. Eur. J. Appl. Microbiol. 4:225-231.
106
CHAPTER 4
EVALUATION BY DIFFERENTIAL SCANNING CALORIMETRY OF THE
EFFECTS OF ETHANOL, NaCl, ACETIC ACID AND pH ON ESCHERICHIA COLI
ABSTRACT
The influence of chemical (acids, ethanol or NaCl) treatment on the cellular components
of Escherichia coli was evaluated using differential scanning calorimetry (DSC). Cell
viability was assessed using plate count. DSC thermograms showed that the transition of
ribosomal subunits in the cells was affected by mild chemical treatments where less than
0.4 log reduction of cell viability occurred. The thermal stability (Tm) for ribosomal
subunits denaturation decreases as chemical agent concentration increases. The total
apparent enthalpy (∆H) also decreases, mainly due to reduction of ribosomal subunit
peak as the concentration increases. Unlike in ethanol and NaCl treatments, the transition
of DNA was irreversibly affected after the treatment with inorganic (HCl) or organic
(CH3COOH) acid. The number of surviving cells received chemical treatments was
lower than that of non-treated cells after mild heat treatment (at 60, 62.5 and 65oC)
107
indicating the conformational changes in cellular components by chemical treatments
may have sensitized bacteria to heat. Greater heat sensitivity for acid-treated cells might
be due to the chemically-induced irreversible damages on DNA as well as the damages
on ribosomal subunits.
Key Words: chemical treatment, differential scanning calorimetry, thermal stability,
ribosome, Escherichia coli
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INTRODUCTION
Thermal processing is the main choice of food preservation for inactivation of
pathogenic and spoilage bacteria in order to produce a safe product with enhanced shelf
life. High temperatures employed during thermal processing adversely affect texture,
flavor, nutrient value of food products. Therefore, mild heating in conjunction with
antimicrobial agents have been utilized to preserve the nutritional qualities while
maintaining extended shelf-life. This approach is known as hurdle technology (Abee and
Wouters, 1999). Hurdle technology is based on the reduced thermal resistance observed
for bacteria treated with chemical or physical means prior to or during heat treatment
(Karatzas et al., 2000; Leistner, 2000). The most commonly employed hurdles to reduce
the intensity of heat treatment include controlling water activity (aw), acidity, and use of
preservatives (Cameron et al., 1980; Adams et al., 1989; Membre, 1997; Casadei et al.,
2001). The physiological conditions of bacterial cells such as pH, aw, and ionic
interactions, which maintain metabolic reactions and homeostasis, are shown to be
affected by ethanol (Ingram and Buttke, 1984; Ingram, 1986), salt (Csonka, 1989; Poirier
et al., 1998) and acids (Abee and Wouters, 1999; Brul and Coote, 1999).
Differential scanning calorimetry (DSC) has been used to characterize the
conformational transitions of cellular components of bacteria during heat treatment
(Mackey et al., 1991; Kaletunç, 2001). Thermal stability of a cellular component can be
evaluated from the peak temperature of the corresponding transition from DSC
thermograms of whole cells (Miles et al., 1986; Anderson et al., 1991; Mackey et al.,
109
1991; Bellivieau et al., 1992; Kaletunç, 2001; Lee and Kaletunç, 2002b). DSC has been
demonstrated to evaluate the effect of high hydrostatic pressure on bacteria by comparing
the DSC thermograms of pressure-treated cells with thermograms of untreated cells
(Niven et al., 1999; Alpas et al., 2003; Kaletunç et al., 2004). It has also been used to
investigate the survival of bacterial cells after heat treatment under linearly increasing
temperature using DSC instrument (Chapters 2 and 3).
To prevent the repair of the microbial homeostasis after food processing, chemical
hurdles of choice should target the various cellular components such as membrane,
nucleic acids, and proteins (Leistner, 1992; Leistner and Gorris, 1995). Therefore, to
achieve the optimal design of the hurdle technology, the investigations of the effect of
chemicals on the cellular components need to be investigated to elucidate the irreversible
changes in macromolecular components occurring of cells, which leads to cell injury and
death as a result of chemical treatments.
In the present study, the influence of acids, ethanol or NaCl treatment on the
cellular components of E. coli and the viability of the chemically treated cells during
subsequent heat treatment were evaluated using DSC. Plate count method was performed
to determine the effect of treatments on the viability of the E. coli.
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MATERIALS AND METHODS
Source and maintenance of organism
E. coli ATCC 14948 was obtained from the Culture Collection, Department of
Microbiology at the Ohio State University. A loopful of the organism was revived in 10
ml Trypticase soy broth supplemented with 0.3% (w/w) yeast extract (TSBYE) and
incubated at 37oC for 18 hours. The culture was stored frozen (-80oC) in 30% (v/v)
sterile glycerol. A loopful of this stock culture was transferred in 10 ml TSBYE and
incubated 10 hrs at 37oC before use.
DSC profiles of E. coli after chemical treatments
Revived E. coli culture was inoculated (1% v/v) into TSBYE. Cultures were
incubated at 37oC until the end of exponential growth phase, when the final concentration
of cells in a medium was 1.0 ± 0.1 x 109 cfu ml-1. E. coli cells in the broth was treated by
adding 6, 10, 12 and 15% (vol/vol) ethanol (95%, Pharmco Inc., Brookfield, CT), 6 and
10% (wt/wt) sodium chloride (NaCl, Sigma), pH 3.0 and 4.0 using hydrochloric acid
(36%, wt/vol; Fisher Scientific), or 0.2, 0.5, 1.0 and 2.0% acetic acid (glacial, Fisher
Scientific). After 1 h of treatment at 37oC, a portion (1 ml) of the treated and untreated
(control) cultures were pour plated into Trypticase soy agar to determine viable cell
counts in media. Remained cultures were centrifuged at 10 000 g for 10 min at 4oC.
Supernatants were discarded. Pellets were washed with 100 ml of sterile distilled water
and centrifuged again before transferring into DSC crucibles.
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Calorimetry
A differential scanning calorimeter (DSC 111, Setaram, Lyon, France) was used to
record the thermograms of the untreated and treated E. coli cells. All DSC measurements
were conducted using fluid-tight, stainless steel crucibles. A DSC run was performed
with unsealed-empty sample and reference crucibles to measure the instrument baseline.
Pellets of cells were weighed (56 ± 0.3 mg wet weight) and carefully transferred into the
sample crucible. The water content of the pellets was determined using freeze dryer
(Freezone 4.5, Freeze dry system, Model 77510, Labconco, MO) as 80 % on wet basis.
For each DSC run, reference crucible was filled with ~45 µl (~80 % of sample wt) of
pure distilled water. Both crucibles were sealed using aluminum rings and covered with
screw caps. The sealed crucibles were refrigerated at 4oC until used for DSC. The
sample and reference crucibles were placed in the DSC and equilibrated at 1oC using
liquid nitrogen cooling system.
Samples were heated in the DSC at 3oC min-1 from 1 to 150oC. After heating,
samples were rapidly cooled by liquid nitrogen and rescanned to observe the reversibility
of thermograms. Samples were re-weighed after measurements to check for loss of mass
during heating and the result of samples showing signs of leakage were discarded.
Viability after heat treatment in DSC following chemical treatment
Weighed cell pellets (~70 mg wet weight) of chemically-treated cultures and control
were carefully transferred into sterile DSC empty sample crucibles using sterile loops.
112
Each crucible was capped (not sealed) using aluminum ring and screwed cap. Empty
reference crucible was filled with ~56 µl (~80% of sample wt) of pure distilled water.
The capped crucibles were kept in refrigerator (4oC) until used for DSC. Pellets in
crucible were heat-treated to 60, 62.5, 65oC with 3oC/min heating rate using DSC
instrument. After cooling, a portion (50 mg) of the heated pellet from each crucible was
transferred to a (1.5 ml) sterile polyethylene tube using a sterile loop. Sterile peptone
water was added to make a final volume of 1 ml with 1/20 (w/v) ratio. After careful
suspension in the tube, the cells were serially diluted and plated onto Trypticase soy agar
to determine viable counts. After 36 hours incubation at 37oC, viable counts of each
sample were obtained by calculation of dilution ratios. The level of the lowest detection
was 2 x 101 cfu g-1 in pellet.
DSC data analysis
DSC thermograms were corrected for differences in the empty crucibles by
subtracting an empty crucible baseline. Total heats corresponding to the endothermic
peaks of whole cells (enthalpy, J g-1) between approximately 40 and 130oC were
determined by integrating the temperature vs. heat flow curve using software provided by
the instrument manufacture. A curved baseline taking into account the variation in heat
capacity before and after the transition passing through three designated points on the
thermogram was used to calculate the apparent enthalpy of whole cells. Data points at
three temperatures were selected to determine the baselines for all DSC curves. The
initial temperature point was on the pre-transition baseline (40oC). The mid-point was
113
selected at a temperature below the onset of the final peak which corresponds to
transitions in the cell envelope (~108oC). The final point was on the post-transition
baseline (130oC).
Growth of E. coli cells to the end of exponential growth stage
Chemical shock by adding acids, ethanol or NaCl for 1 h Plate counting forviabilitya
DSC for calorimetric datCentrifugation to obtain cell pellets
114
Analysis of the survivability of chemically- treated E. coli
cells in heat treatment
Analysis of the effects ofchemicals on E. coli cells
s
Figure 4.1. Experimental scheme of calorimetric and microbial analysiHeat-treatment in DSC
Plate counting
RESULTS
Effect of ethanol on thermal transitions and viability of E. coli
Figure 4.2 shows the DSC thermogram for untreated (A) and ethanol treated (B, C, D,
and E) E. coli pellets. Individual endothermic peaks (a-d) were shown to be associated
with components such as ribosomal subunits (a1, a2 and a3), DNA (b), DNA with cell wall
(c), and outer membrane of Gram-negative organisms (d) (Mackey et al, 1991; Lee and
Kaletunç, 2002b). Thermograms in Figure 4.2 show that ribosomal subunits, which are
composed of RNA and ribosomal proteins, in the E. coli cells were affected by ethanol
treatment, while other transitions remain same. The onset temperature and thermal
stability for ribosomal subunits denaturation decreased as ethanol concentration increase
(Table 4.1). The total apparent enthalpy also decreased, mainly due to reduction of
ribosomal subunit peak as ethanol concentration increased. After treatment with 12%
ethanol, the transition temperature and enthalpy of the peaks due to DNA (peak b) and
DNA with cell wall component (peak c) were markedly decreased. However, ethanol
concentration did not affect the plate count data (<1 log reduction) up to 12% ethanol
addition. The decrease in transition temperature and enthalpy associated with outer
membrane component (peak d) was apparent after 15% ethanol treatment (Table 4.1).
115
Transition temperature (Tm,, oC) Enthalpy (J/g) Concentration in broth (v/v)
Viability [log (N/N0)]
Total Apparent enthalpy (∆H, J/g)
Onset temperature
(oC) Peak a2 Peak a3 Peak b Peak c Peak d Peak b+c Peak d
Non-treatment - 4.19 54.6 69.8 77.9 93.8 102.4 117.7 0.47 0.19
Ethanol
6% 0.01 4.12 47.1 69.4 76.7 93.2 101.5 117.6 0.47 0.19
10% 0.09 3.80 43.6 67.5 75.7 93.2 101.9 117.5 0.43 0.19
12% 0.35 3.76 42.2 64.0 - 93.3 101.3 116.7 0.41 0.11
15% 4.79 2.85 36.4 62.4 - 92.8 100.9 115.9 0.38 0.11
NaCl 6% 0.24 4.21 46.6 67.2 76.6 93.6 101.3 117.4 0.43 0.17
10% 1.15 4.20 46.1 67.3 76.7 94.7 101.4 117.7 0.44 0.16
HCl pH 4.0 0.15 3.20 37.8 65.7 - 100.3 117.6 0.32 0.15
pH 3.0 0.22 2.61 33.2 61.1 - 93.9 116.2 0.39 0.14
Acetic acid 0.2% (pH 4.9) 0.09 4.15 42.7 67.8 71.7 - 93.9 102.4 117.7 0.39 0.13
0.5% (pH 4.2) 0.21 3.89 38.7 66.1 70.6 - 101.6 117.6 0.29 0.14
1.0% (pH 3.9) 0.38 3.01 38.3 60.2 68.2 - 97.9 117.7 0.36 0.10
2.0% (pH 2.8) 5.31 0.82 - - - 89.8 116.8 0.28 0.12
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Table 4.1. Effects of chemicals on viability and DSC transitions of E. coli.
20 40 60 80 100 120 140
a2
a1
a3 bc d
A
B
C
Heat flow
0.4 mW
D
E
Temperature (oC)
Figure 4.2. DSC thermogram of E. coli pellet after ethanol treatment. Control (A), ethanol concentration in the treatment: 6% (B), 10% (C), 12% (D), 15% (E). Thermograms are offset for clarity.
117
Effect of salt on thermal transitions and viability of E. coli
Sodium chloride (NaCl) was added to assess the effect of salt on E. coli cells in
growth medium. Except for the transitions in ribosomal subunits, no apparent differences
of major peaks were found between thermograms of control and salt-added cells (Fig.
4.3). Salt treatments resulted in absence of peak a1 and changes in transition
temperatures of peak a2 and a3 while total apparent enthalpy remain unchanged (Fig. 4.3
and Table 4.1). After 6% NaCl (wt/wt) treatment, the transition temperatures of
ribosomal peaks were lowered by 2oC for peak a2 and 1oC for peak a3. In addition,
decrease in onset temperature (~8oC) of the peak a2 is shown in thermograms of the
treated cells. NaCl treatment of cells to higher concentration (10%) resulted in ~1-log10
unit reduction (Table 4.1); however, except for increase in transition temperature (~1oC)
of DNA peak (peak b), the increase in NaCl concentration to 10% did not affect the
transitions of cellular components in DSC thermograms of the salt-treated cells (Fig. 4.3
thermograms B and C, Table 4.1).
Effect of HCl on thermal transitions and viability of E. coli
HCl was used to adjust the pH of environment to 4 and 3. The thermograms (B and
C) in Figure 4.4 show that HCl affected all of the cellular components in DSC
thermogram at both pH 4 and 3. The substantial decrease in onset temperature and
ribosomal transition (a2) temperature of the cells were observed as pH of the medium
decreased. Specifically, the temperature of a2 decreased 4oC after pH 4 treatment and
9oC after pH 3 treatment.
118
20 40 60 80 100 120 140
a2
a1
a3b
c d
A
B
C
Heat flow
0.2 mW
Temperature (oC)
Figure 4.3. DSC thermogram of E. coli pellet after NaCl treatment. Control (A), NaCl concentration in the treatment: 6% (B), 10% (C). Thermograms are offset for clarity.
119
20 40 60 80 100 120 140
a2
a1
a3 b
c d
A
B
C
Heat flow
0.2 mW
Temperature (oC)
Figure 4.4. DSC thermogram of E. coli pellet after inorganic acid (HCl) treatment. Control (A), pH in the treatment: pH 4 (B), pH 3 (C). Thermograms are offset for clarity.
120
The acid treatment resulted in the absence of peak b in thermogram B and shift of
peak c in thermogram C (Fig. 4.4 and Table 4.1). In addition, marked reductions in the
total apparent enthalpy occurred after the acid treatment at pH 4 (~24%) and at pH 3
(~38%). Although noticeable changes in peaks for various cellular components were
observed, loss of viability prior to DSC was minimal among control, pH 4- and pH 3-
treated cultures (<0.3 log reductions) (Table 4.1).
Effect of acetic acid on thermal transitions and viability of E. coli
Acetic acid (CH3COOH) was used to evaluate the effect of organic acid on E. coli.
Peak a2 appeared to include two overlapping endothermic transitions for acetic acid
treated cells (Fig. 4.5). The transition temperature for a2 peak decreased as acetic acid
concentration increased (Table 4.1). The absence of peak b in thermogram C and the
shifts of peak c in thermograms D,E were observed after acetic acid treatments (Fig 4.5).
Total apparent enthalpy decreased with increasing acetic acid concentration. The
viability loss of acetic acid treated cells prior to DSC measurement did not differ from
control cells until concentration of acetic acid was 1% (<0.4 log unit reduction of
viability) in the medium. When the medium acidity was dropped to pH 2.8 by 2% acetic
acid concentration, the viable count of the culture declined 5-log cycles (Table 4.1). The
transition due to ribosomal subunits of cells was absent after treatment with 2% acetic
acid (Fig. 4.5 Thermogram E).
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20 40 60 80 100 120 140
a2
a1
a3 b
c d
A
B
C
Heat flow
0.4 mW
D
E
Temperature (oC)
Figure 4.5. DSC thermogram of E. coli pellet after organic acid (acetic acid) treatment. Control (A), acetic acid concentration (v/v) in the treatment: 0.2% (B), 0.5% (C), 1.0% (D) and 2.0% (E). Thermograms are offset for clarity.
122
Effect of heat treatment on viability of chemically treated E. coli cells
E. coli cultures exposed to 12% ethanol, 6% NaCl, pH 4, pH 3, 0.5% acetic acid,
and 1% acetic acid treatments for 1 hr were heat treated in DSC to 60, 62.5 and 65oC.
Chemical treatments alone caused 0.2 to 0.4 log reductions in viability of E. coli cells.
Figure 5 compares viability of E. coli cells after chemical and heat treatment with the
cells after heat treatment alone. Application of chemical stresses such as HCl (pH 3) and
acetic acid (1%) prior to heat treatment markedly (log 3.7 for HCl and 3.8 for acetic acid)
reduced the viability of E. coli cells at the lowest heat treatment temperature of 60oC
compared to that of untreated (0.1 log) and ethanol (1.0 log) or NaCl (0.5 log) treated
cells. For 1.0% acetic acid treated cells, ~8 log reduction after 62.5oC treatment was
achieved. Viable cells were not observed after 65oC treatment for cells treated with 1%
acetic acid or by HCl at pH 3. After heat treatment in DSC at 65oC, a log reduction of
7.9 for 0.5% acetic acid treatment, 6.5 for HCl treatment at pH 4, 6.0 for 12% ethanol
treatment, and 5.7 for 6% salt treatment were observed while the control cells decreased
4.7 log units.
123
2
3
4
5
6
7
8
9
10
11
12
13
Before DSC 60 62.5 65
Control
Ethanol 12%
Salt 6%
pH4 usingHCl
pH3 usingHCl
Acetic acid0.5%
Acetic acid1.0%
Log
(cfu
/g) i
n pe
llet
Temperature (oC)
Figure 4.6. Survival of untreated and chemically treated E. coli after heat treatment under linearly increasing temperature.
124
DISCUSSION
The studies on effect of chemicals on bacteria have been focused on the
cytoplasmic membrane with minimal effort on the influence of chemical treatment on
intracellular structures such as ribosomes, nucleic acids and proteins. Modifications of
cell permeability and leakage of ions, failure of proton motive force in which ATP is
synthesized, and inhibition of membrane-associated enzyme activity were considered as
potential damages to bacteria due to interaction of chemicals with the membrane (Weitzel
et al., 1987; Maillard, 2002).
Chemicals, such as ethylenediamine tetraacetic acid and hydrogen peroxide (H2O2),
were reported to chelate metal ions associated with ribosomes leading to dissociation of
larger ribosomal subunits to smaller subunits or disintegration of each subunit (Nakamura
and Tamaoki, 1968). Except for above chelators and specific antibiotics such as
erythromycin (on 50S subunit) and tetracycline (on 30S subunit), ribosomal subunits
have not been considered as major target sites for most chemicals (Hugo, 1999; Maillard,
2002). However, the marked reductions in size and transition temperature of ribosomal
subunit transitions in DSC thermograms of ethanol (15%), HCl (<pH 4), and acetic acid
(>0.5%) treated E. coli cells in this study indicate ribosomal structure was irreversibly
altered by high concentrations of these chemicals. The disappearance of first peak (a1),
which is proposed to be the denaturation of 30S ribosomal subunit in E. coli (Mackey et
al., 1991), in thermogram of all of chemically treated E. coli whole cells in present DSC
study indicates the smaller and flexible ribosomal subunit might be very susceptible
125
component for chemical treatment. In Chapter 2, I discussed that ribosome thermal
stability is altered by low pH condition during cell growth as well as loss of intracellular
metals like Mg2+ ions during heat treatment.
The pH of inside cell was reported to be lower during chemical treatment due to
accumulation of excessive H+ ions in the cell (Maillard, 2002). Chemicals containing
hydrocarbons such as ethanol and acetic acid can induce membrane damage leads to the
disintegration of proton motive force which synthesizes ATP to maintain H+ level of
microbial cell (Quintas et al., 2000; Minamino et al., 2003). In addition, a recent study
showed that monovalent ions in NaCl generate the dissipation of the proton motive force
in bacterial membrane (Minahk and Morero, 2003). Therefore, the effect of chemical
treatments on ribosomal subunits in present study might be result from the interaction of
chemicals with ribosomal subunits and/or the interaction of chemicals with cytoplasmic
membrane. The occurrences of above interactions by a chemical might be concentration
dependent.
DSC thermograms in Figure 1 show that the shape and size of ribosome transitions
(peaks a1,2,3) changed after various levels of ethanol treatments; while, other transitions
remain unchanged. There were minimal changes in viability and the apparent enthalpy
(J/g) in DSC thermograms of the treated E. coli cells up to 12% ethanol concentration,
while those values were considerably reduced in 15% ethanol treated cells (Table 4.1). In
the studies of E. coli (Chapters 2 and 3), heat-treatment under linearly increasing
temperature slightly decreased the apparent enthalpy value of the whole cell DSC until
the temperature reached to a certain level where a log reduction in viable counts
126
occurred. Ethanol is known to have a similar effect to heat stress in initiating response
mechanisms by the microbial membrane (Piper, 1995; Casadei et al., 2001). Both
stresses cause induction of membrane-bound heat shock proteins which enhance the
proton efflux to help homeostasis of the increased membrane permeability that result
from the stresses (McElhaney, 1985; Piper, 1995). Therefore, the cell viability might be
protected by above response mechanism in the presence of ethanol concentration up to
12%. Because ethanol generally penetrate cytoplasmic membrane via lipid-bilayer
portion, the increase in the length of the fatty acid chains in the membrane of
microorganisms, which provide a more fortified hydrophobic barrier than normal fatty
acid chain, are also considered defense mechanism against ethanol (Ingram and Buttke,
1984; Broadbent and Lin, 1999). However, since the above result was observed after
growth of cells in the presence of ethanol, further study on the change in fatty acid
composition in the same condition as this study will be needed to verify the occurrence of
the mechanism. In present study, E. coli cells in the end of logarithmic growth phase
were treated by ethanol for 1 hour prior to DSC.
DSC thermograms show that there is no apparent change in transition of cellular
components of E. coli with the exception of ribosomal subunits after NaCl treatments
(Fig. 4.3 and Table 4.1). The concentrations of NaCl higher than 10% did not cause
further reduction of the ribosomal transition temperature and area in E. coli cells in spite
of decreased viability (Table 4.1). High concentration of intracellular cations has been
reported to protect DNA from denaturation at high temperatures because removal of
counterions (positively charged mono- or di-valent ions), which interact with negatively
127
charged phosphate backbones of the double stranded DNA, is increasingly unfavorable
due to surrounded bulk cations (Manning et al., 1978; Record et al., 1978). Therefore,
slight increase in the transition temperature of peak c in the thermogram of 10% NaCl
treated cells indicating the thermal stability of DNA structure might be enhanced by bulk
Na+ ions which surround the DNA.
E. coli cells are known to accumulate “compatible solutes” such as betaine and
trehalose which can increase internal osmotic pressure against a hyperosmotic shock
without interfering functions of cell components (Gutierrez et al., 1995; Abee and
Wouters, 1999). In a study on NaCl sensitivity of E. coli, Nakamura et al. (1992)
reported that the addition of NaCl to suspensions of mutant cells that do not have sodium
efflux system results in the loss of viability while wild-type cells show resistance.
Therefore, it is possible that E. coli cell inactivation at the higher concentration of NaCl
may be mainly due to leakage of more Na+ ions into the cell and inhibition of certain
regulatory pathway by interacting with key enzymes and proteins as well as with ions. It
is reported that excessive Na+ cause the failure of respiratory electron transport system
which is important factor for viability of cell (Allakhverdiev et al., 1999). One log unit
viability loss observed with addition of 10% NaCl (w/v) did not cause any change in the
total apparent enthalpy requirement for the cell death in subsequent heat treatment in
DSC in spite of thermal stability loss of 2oC in ribosomal subunits. The result may
indicate that NaCl-induced changes may be entropy driven rather than enthalpy driven.
Figure 4.4 and 4.5 reveals that the peaks attributed to the ribosomal subunits in the
DSC thermogram greatly shifted to lower temperature when the E. coli was subjected to
128
HCl or acetic acid treatment. The considerable reduction of total area under the
thermogram peaks (apparent enthalpy) of the HCl and acetic acid treated cells includes
the absence or size reduction of peaks associated with DNA transition (peaks b and c,
which remained unchanged after ethanol or salt treatment) as well as the decrease in area
associated with ribosomal subunit transitions. The absence of peak b in the thermograms
of Figures 4.4B and 4.5C possibly indicates that double stranded DNA structure was
affected by HCl (pH 4) or 0.5% acetic acid (pH 4.2) treatment. Exposure of bacterial
cells to acidic pH was known to induce DNA damage due to the reduction of divalent
ions such as Mg2+ and Ca2+ which stabilize DNA molecules (Hickey and Hirshfield,
1990; Juenja et al., 1995). Peak c, proposed to be due to denaturations of cell wall and
part of DNA (Mackey et al., 1991), becomes apparent due to the obliteration of peak b
which obscured peak c in the thermogram of untreated E. coli cells. The peak with 94oC
in Figure 4.4C and the peaks with 98oC (in Fig. 4.5D) or 90oC (in Fig. 4.5E) might
indicate that the thermal stability of the component(s) related in peak c was (were)
reduced after acid treatments. The possible reasons of the difference in thermal stability
of two peaks were from the following differences between two DNAs: the structure of
DNA (chromosome or plasmid), interaction with components such as cationic proteins
and polyamines in the cell (Worcel and Burgi, 1972; Flink and Pettijohn, 1975).
The survivability of the chemically treated cells during heat treatments was much
lower than those of untreated cells (Fig. 4.6). The result suggests that there is
relationship between reduction in the ribosomal subunits transition in DSC thermogram
and increase in heat sensitivity of chemically treated cells. The result also agrees with the
129
findings of previous DSC studies that heat resistance of bacteria is strongly related to the
onset temperature and the thermal stability of the main ribosomal subunit peak (Miles et
al., 1986; Mohacsi-Farkas et al., 1994; Lee and Kaletunç, 2002b). In present study, DSC
data show the reductions in the onset temperatures (by 8~12oC) and the transition
temperatures (peaks a2, by 3~10oC) of major ribosomal subunit occurred in the
thermograms of chemically treated cells that were used in heat treatments (Table 4.1).
Among chemically treated cells, the onset temperature and thermal stability of ribosome
were much lower in HCl- and acetic acid-treated cells than in ethanol- and NaCl-treated
cells. In addition, the reduction of total apparent enthalpy was greater in those acid
treated cells due to greater size reductions in ribosomal and DNA transitions of their
thermograms. Above results might be correlated to higher thermal sensitivity of acid
treated cells in subsequent heat treatment. Among acid treated cells, the highest
inactivation rate during the heat treatment up to 65oC for 1% acetic acid treated cells, as
judged by the steepest slope of the plots in Figure 4.6, indicates that the hurdle effect of
acetic acid is greater than that of HCl.
In conclusion, DSC thermograms for E. coli revealed conformational changes in
cellular components after chemical treatments. Mild treatments affect the thermal
stability of ribosomal subunits in the cell, thereby increasing the sensitivity of bacteria to
heat treatment. The heat sensitivity is greater for acid-treated cells because more cellular
components were irreversibly affected after the treatments. These hurdle effects should
be considered when current thermal processing technologies are modified.
130
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Leistner, L. 1992. Food preservation by combined methods. Food Res. Int. 25:151-158. Leistner, L. 2000. Basic aspects of food preservation by hurdle technology. Int. J. Food. Microbiol. 55:181-186. Leistner, L. and Gorris, L.G.M. 1995. Food preservation and hurdle technology. Trends Food Sci. Technol. 6:41-46. Mackey, B.M., Miles, C.A., Parsons, S.E., and Seymour, D.A. 1991. Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry. J. Gen. Microbiol. 137:2361-2374. Maillard, J,-Y. 2002. Bacterial target sites for biocide action. J. Appl. Microbiol. Sym. Suppl. 92:16S-27S. Manning, G.S. 1978. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q Rev Biophys. 11:179-246. McElhaney, R.N. 1985. The effect of membrane lipids on permeability and transport in prokaryotes. In Structure and properties of cell membranes ed. Benga, G. p. 20. vol. 2. CRC Press, Boca Raton, FL. Membre, J.-M., Majchrzak, V. and Jolly, I. 1997. Effects of temperature, pH, glucose and citric acid on the inactivation of Salmonella typhimurium in reduced calorie mayonnaise. J. Food Prot. 60:1497-1501. Miles, C.A., Mackey, B.M. and Parsons, S.E. 1986. Differential scanning calorimetry of bacteria. J. Gen. Microbiol. 132:939-952. Minahk, C.J. and Morero, R.D. 2003. Inhibition of entercin CRL35 antibiotic activity by mono- and divalent ions. Lett. Appl. Microbiol. 37:374-379.
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Minamino, T., Imae, Y., Oosawa, F., Kobayashi, Y. and Oosawa, K. 2003. Effect of intracellular pH on rotational speed of bacterial flagellar motors. J. Bacteriol. 185:1190-1194. Mohacsi-Farkas, Cs., Farkas, J. and Simon, A. 1994. Thermal denaturation of bacterial cells examined by differential scanning calorimetry. Acta Aliment. 23:157-168. Munns, R., Greenway, H., Setter, T.L. and Kuo, J. 1983. Tugor pressure, volumetric elastic modulus, osmotic volume and ultrastructure of Chlorella emersonii grown at high and low external NaCl. J. Exp. Bot. 34:144-155. Nakamura, H., Hase, A. and Funatsuki, K. 1992. Biological actions of acridines: Salt sensitivity of Escherichia coli which is determined by the acrA gene. Memo. Konan Univ. Sci. Ser. 39:213-223. Nakamura, K. and Tamaoki, T. 1968. Reversible dissociation of Escherichia coli ribosomes by hydrogen peroxide. Biochim. Biophys. Acta 161:368-376. Niven, G.W., Miles, C.A. and Mackey, B.M. 1999. The effect of hydrostatic pressure on ribosome conformation in Escherichia coli: an in vivo study using differential scanning calorimetry. Microbiology 145:419-425. Poirier, I., Marechal, P.-A., Evrard. C. and Gervais, P. 1998. Escherichia coli and Lactobacillus plantarum responses to osmotic stress. Appl. Microbiol. Biotechnol. 50:704-709. Piper, P.W. 1995. The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap. FEMS Microbiol. Lett. 134:121-127. Quintas, C., Lima-Costa, E. and Loureiro-Dias, M.C. 2000. The effect of ethanol on the plasma membrane permeability of spoilage yeasts. Food Technol. Biotechnol. 38:47-51. Record, M. Th., Jr. 1975. Effects of Na+ and Mg++ ions on the helix-coil transition of DNA. Biopolymers 14:2137-2158.
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Record, M. Th., Jr., Anderson, C.F. and Lohman, T.M. 1978. Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity. Q Rev Biophys. 11:103-178. Shadbolt, C.T., Ross, T. and McMeekin, T.A. 1999. Nonthermal death of Escherichia coli. Int. J. Food. Microbiol. 49:129-138. Salton, M.R.J. 1963. the relationship between the nature of the cell wall and the Gram strain. J. Gen. Microbiol. 30:233-235. Weitzel, G., Pilatus, U. and Rensing, L. 1987. The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast. Exp. Cell Res. 170:64-79. Worcel, A. and Burgi, E. 1972. On the structure of the folded chromosome of Escherichia coli. J. Mol. Biol. 71:127-147.
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CHAPTER 5
EVALUATION OF VIABILITY AND STRUCTURAL CHANGES INDUCED BY
HIGH HYDROSTATIC PRESSURE IN ESCHEICHIA COLI
ABSTRACT
The effect of high hydrostatic pressure (HHP) on the cell structures of Escherichia coli
was determined using differential scanning calorimetry (DSC) and electron microscopy
(EM). The changes in the structures were compared with viability. The cells were
pressurized between 200 and 700 MPa at 35oC for 5 min. DSC studies of whole cells
showed a decrease in apparent enthalpy above 200 MPa pressure treatments. The major
contribution to enthalpy decrease was due to reduction in the transition attributed to the
denaturation of ribosomes. The enthalpy and the thermal stability of the DNA transition
were affected by HHP treatments above 300 MPa. Linear relationship between the
fractional viability based on calorimetric data and plate count data was obtained. In EM
studies, integrity of cell envelope was maintained in pressure- or heat-inactivated cells;
however, the leakage of cell wall substances and the formation of empty space between
cell envelope and internal structure were observed in pressure- inactivated cells.
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Key Words: high hydrostatic pressure, differential scanning calorimetry, electron
microscopy, Escherichia coli, inactivation
138
INTRODUCTION
Because the interest among consumers in natural or minimally processed foods is
increasing, the inactivation of pathogenic and spoilage microorganisms using a treatment
alternative to thermal processing is under consideration by food industry. High
hydrostatic pressure (HHP) treatment has the potential to produce the microbiologically
safe food without impairing the nutrient content of a food (Mertens and Deplace, 1993;
Roberts and Hoover, 1996). As early as 1889 Hite showed that pressures of 450 MPa or
greater could eliminate spoilage microorganisms and improve the preservation of milk.
The effectiveness of hydrostatic pasteurization on the destruction of several foodborne
pathogens such as Salmonella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus,
Listeria monocytogenes and Staphylococcus aureus has been reported (Metrick et al., 1989;
Styles et al, 1991; Patterson et al, 1995; Kalchayanand et al, 1998; Alpas et al, 1999). Cell
viability decreases with increasing pressure, time, and temperature suggesting critical
cellular activities have been irreversibly damaged (Hoover et al., 1989; Metrick et al.,
1989; Alpas et al., 2000; Robey et al., 2001).
In HHP treatment, the primary target in bacterial cell is proposed to be the
cytoplasmic membrane (Kalchayanand et al., 1998; Farkas and Hoover, 2000). Studies
showed that bacterial cell viability is related to the loss of the membrane integrity
(Shigehisa et al., 1991; Casadei et al., 2002). The denaturation of membrane bound
ATPases, which includes the alteration of molecular structures and change in active sites,
has also been considered as a major factor in pressure-induced cell inactivation (Suzuky and
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Suzuky, 1962; Mackey et al., 1995; Wouters et al., 1998). Studies using electron
microscopy (EM) reported that the empty space between the cell envelope and inner cell
body, compacted amorphous clusters in ribosome or protein region and fibril formation in
DNA region were observed in transmission electron microscopy (TEM) sections on
pressure inactivated Salmonella Thompson (Mackey et al. 1994) and Lactobacillus
viridescens (Park et al., 2001). In a scanning electron microscopy (SEM) study on
Pseudomonas fluorescens, Lopez-Caballero et al. (2002) reported that pressure induced
rough and wrinkled cell surface is related to the destruction of cell wall which also leads
the leakage of intra cellular materials.
Differential scanning calorimetry (DSC) has been used to detect thermally induced
conformational transitions in bacterial cells and spores (Miles et al., 1986; Mackey et al.,
1991; Belliveau et al., 1992; Mohacsi-Farkas et al., 1999; Lee and Kaletunç, 2002a,b).
Peak temperatures of observed endothermic transitions, in DSC thermograms, correspond
to the thermal stabilities of cellular components of microorganism (Mackey et al., 1991;
Kaletunç, 2001). In addition, DSC measurement provides information about amount of
energy (enthalpy, ∆H) associated with the transition. DSC also has been utilized to
evaluate the effects of treatments other than heat such as pH (Mohacsi-Farkas et al.,
1994) and pressure (Niven et al., 1999; Alpas et al., 2003) by comparing the thermograms
of cells before and after treatment. Niven and coworkers (1999) investigated the effect of
high hydrostatic pressures (HHP) on ribosome conformation in whole E. coli NCTC 8164
cells using DSC. They observed reduction in ribosome-associated enthalpy was
correlated with loss of cell viability due to pressure treatment between 50 to 250 MPa.
140
The effect of maximum pressure treatment (250 MPa), however, was not enough to
inactivate (less than a log reduction) the E. coli cells in their study. Furthermore, the
information in literature about the irreversible changes of specific macromolecular
components occurring in cells, which leads to cell death as a result of HHP treatment is
limited.
The objectives of this study were (1) to investigate the stability of cellular
components of pressure treated E. coli using DSC, (2) to determine the fraction of
survivors as a function of HHP (up to 700 MPa) using plate count and calorimetric data,
and (3) to compare the changes in cellular components of pressure-inactivated cells with
that of heat-inactivated cells using the DSC and the electron microscopy.
MATERIALS AND METHODS
Preparation of organisms for pressurization
E. coli K12 was obtained from the Culture Collection, Department of
Microbiology at the Ohio State University. A loopful of organism was revived in 10 ml
Trypticase soy broth (Difco laboratories, Detroit, MI) supplemented with 0.3 % (w/w)
yeast extract (Difco laboratories, Detroit, MI) (TSBYE) and incubated at 37oC for 18
hours. The culture was stored frozen (-80 oC) in 30 % (v/v) sterile glycerol. A loopful of
the stock culture was transferred to 10 ml TSBYE and incubated 10 hrs at 37oC before
141
use. Revived E. coli K12 culture was inoculated (1% v/v) into TSBYE. Cultures are
incubated at 37oC until the late exponential growth phase, when the final concentration of
cells in a medium was 1.0 ± 0.1 x 109 cfu ml-1. Cell suspensions (200 ml) were removed
from the incubator and placed in 3-mil-thick sterile polyethylene stomacher bag (Fisher
scientific, Pittsburgh, PA) (10 x 15 cm). Air was removed from the bag prior to heat
sealing. The bag was placed inside a second stomacher bags (Fisher scientific, Pittsburgh,
PA) (18 x 30 cm) to prevent contamination of the high-pressure unit if the primary package
was to fail and heat sealed under vacuum. Prepared bags were kept at 4oC prior to
pressurization. Control samples were prepared for plate count, SEM, TEM, and DSC
analysis. One of the control samples was placed in hot water bath (65oC) until no viable
cells detected. The cellular morphology and components of cells in this heat-inactivated
broth or pellet was compared with that of pressure-inactivated cells using the electron
microscopy or DSC.
High Hydrostatic Pressure Processing
A High Pressure Processing Unit (ABB Quintus Food Processor QFP-6 Cold
Isostatic Press, Columbus, Ohio) was used for pressure treatments. The hydrostatic
pressurization unit was capable of operating up to 900 MPa (8,874 atm). A
water/propylene glycol (Houghton-Safe 620-TY, Houghton Int., Inc., Valley Forge, Pa)
mixture (1:1, vol/vol) was used as the pressure transmitting fluid. The liquid can be
heated to the desired temperature prior to pressurization by an electric heating system
around the chamber. The rate of pressure increase was about 400 MPa per min. The
142
pressure level, time, and temperature of pressurization were controlled and maintained
during the pressurization cycle.
The bags containing cell suspensions were pressurized at 100, 200, 300, 400, 500,
600 or 700 MPa pressure for 5 min at 35oC. Duplicate samples were prepared at each
treatment level. After HHP treatment, a portion (1 ml) of the pressured and untreated
(control) cultures in the bags was serially diluted in 0.1 % sterile peptone solution and
pour plated into Trypticase soy agar (TSA) to determine viable cell counts. Remained
cell suspensions were used for preparation of SEM and TEM samples, and for DSC
analysis.
Heat Inactivation
One of the bags containing control culture was placed in hot water bath (65oC) until no
viable cells detected (6 min). The cellular morphology and components of cells in this
heat-inactivated broth or pellet was compared with that of pressure-inactivated cells using
the electron microscopy or DSC.
Calorimetry
Cells in the pressure-treated and untreated broth were centrifuged (Beckman J2-21
centrifuge, Palo Alto, CA) at 10 000 g for 10 min at 4oC. Supernatants were discarded.
Pellets were washed with 100 ml of sterile distilled water before transferring into DSC
crucibles. A DSC run was performed with empty sample and reference crucibles to
measure the empty crucible baseline. Pellets of cells were weighed and carefully
143
transferred into the sample crucible. The dry material content of the pellets was
determined by freeze drying (Freezone 4.5, Freeze dry system, Model 77510, Labconco,
Missouri). For each DSC run, the reference crucible was filled with distilled water equal
to the amount of water in sample. The sealed crucibles were refrigerated at 4oC until
used for DSC. The sample and reference crucibles were placed in the DSC and
equilibrated at 1oC using liquid nitrogen cooling system. Samples were heated in the
DSC instrument at 4oC/min from 1 to 150oC. DSC thermograms of samples were
corrected for differences in the empty crucibles by subtraction of an empty crucible
baseline. Peak areas (apparent enthalpies, J g-1) corresponding to the contributions of
survivors were determined from the apparent heat capacity vs. temperature graph using
software provided by the instrument manufacturer.
EM preparation
Cells in the pressure-treated, heat-inactivated, and control broth were centrifuged at
10 000 g for 10 min at 4oC to separate the cells as pellets. Pellets were washed with 150
ml of sterile distilled water. A 1 mm3 pellet was transferred to a sterile vial and
resuspended in 1 ml of 0.1 M phosphate buffer at pH 7.4. The cells were fixed on the
membrane (0.45 µm pore size) by passing with 10 ml 3% glutaraldehyde in 0.1M
phosphate buffer (pH 7.4) through the filter. Fixative was left in contact with the cells
overnight at 4oC. The cells for SEM observation were washed with buffer and post-fixed
for 1 hour in 1% osmium tetroxide in phosphate buffer. Filters were rinsed with buffer,
dehydrated with a serial concentrations of ethanol, and then dried on a critical point drier.
144
The dried cells were coated with gold-palladium and examined using a Philips XL-30
SEM at 30 KV (FEI Inc., Oregon). Fixed cells for TEM observation were centrifuged
and the pellet embedded in 2% agar. Agar was cut into 1mm3 pieces and post-fixed for 1
hour in 1% osmium tetroxide in phosphate buffer. Samples were rinsed in distilled water
and in-block stained for 1 hour in 1% aqueous uranyl acetate. After dehydration with a
serial concentrations of ethanol, cells in agar were transferred to propylene oxide and
infiltrated and embedded in Spurr’s resin (Ted Pella Co., Redding, CA). The samples
were sliced (70 nm) with an ultramicrotome and stained with Reynold’s lead citrate
(Reynolds, 1963) prior to observing by a Philips CM-12 TEM at 60KV (FEI Inc.,
Oregon).
e
Growth of E. coli to the late exponential stag
High Hydrostatic Pressure (HHP)processingcal a M y
Plate countingfor viability DSC fororimetric dat
Centrifugation to obtain cell pellets
145
Heat inactivation usingwater bath
Electron icroscop
Determination of the effects of HHP on E. coli
Figure 5.1. Experimental scheme of calorimetric, EM and microbial analysis
RESULTS
Survivability of E. coli after pressure treatment
Inactivation of E. coli K12 by HHP in TSBY broth for 5 min at 35oC was
determined by microbial counts (Fig. 5.2). The plate counts were immediately done after
the application of pressure. The viability of E. coli was not affected by pressure up to
200 MPa (Table 5.1 and Fig. 5.2). The number of survivors decreased drastically when
pressure increased above 300 MPa. The inactivation was considered complete when no
colony was observed in TSA after incubation at 37oC for 36 hours. The complete
inactivation occurred after 600 MPa and above treatments (Fig. 5.2).
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
0 100 200 300 400 500 600 700
Log
(N/N
0)
)
Figure 5.2. Pressure dependence
Pressure (MPa
of fractional viability determined by plate count.
146
0.1 MPa 200 MPa 300 MPa 400 MPa 500 MPa 600 MPa 700 MPaWater bath (65oC for 6
min)
CFU/ml 1.4 x 109 1.1 x 109 1.6 x 107 2.0 x 105 9.4 x 103 <1 x 101 <1 <1
Apparent Enthalpy(J/g)
3.83 3.64 2.99 2.60 1.93 1.76 1.75
1.69
Tm (oC) a1 a2 b c d
55.0 71.9 92.4 103.4 118.2
70.5, 74.2 91.6
102.1 118.2
55.7
67.3, 70.0 89.2 101.9 118.0
60.4, 67.2 -
100.9 117.6
61.7, 72.7 -
100.8 118.6
66.8
100.4 117.3
70.2
100.6 117.4
63.8, 71.9 85.5 102.1 118.2
147
Table 5.1. Viability, apparent enthalpy values and transition temperature (Tm) of each peak for E. coli cells after treatments.
Comparison of DSC thermograms and viability of E. coli after pressure treatment
The shapes and temperatures of endothermic transitions observed in DSC
thermograms were different after pressure treatment for 5 min at 35oC in comparison
with thermograms of untreated cell pellets (Fig. 5.3). The fractional viability based on
apparent enthalpy data [(∆H-∆Hf)/(∆H0-∆Hf)], where ∆H is the apparent enthalpy after a
HHP treatment, ∆Hf is the residual apparent enthalpy after treatment resulting in no
viability (at 700 MPa) and ∆H0 is the apparent enthalpy of untreated cells, was plotted
versus pressure (Fig. 5.4). The slope of plots in Figure 5.4 is very steep at the pressure
range between 200 and 600 MPa where cells were exponentially inactivated in the plate
count data (Fig. 5.2). Natural logarithm of fractional viability based on the apparent
enthalpy data and plate count data (N/N0) are plotted in Figure 5.5. A linear relationship
(r2 = 0.91) between the reduced apparent enthalpy and the fraction of survivors is
observed. The decrease in apparent enthalpy values in Table 5.1 is strongly related with
the reduction of ribosomal subunit peaks (a1 and a2) in thermograms in Figure 5.3 as
pressure increases. Two endothermic transitions are observed in the temperature range of
major ribosomal subunit denaturation (70~75oC) in the thermogram of 200 MPa treated
cells (Fig. 5.3 thermogram B). Significant reductions in the apparent enthalpy value
(~22%) and the plate count value (~2 log10 unit) occurred with HHP treatment at 300
MPa (Table 5.1). The noticeable reduction of peak b, which is similar to the peak
identified by Mackey et al. (1991) as the melting of DNA, occurred in thermograms of
cells treated at 300 MPa and above pressures. Peak c became more apparent after the
HHP treatments. The area of the peak d which corresponds to the enthalpy of a
148
component in outer membrane denaturation was decreased (~30%) after 400 MPa and
above HPP levels.
149
20 40 60 80 100 120
A
B
E
G
F
C
D
H
0.5 mW
a1
a2
b c d
Temperature (oC)
Figure 5.3. DSC thermograms of pellets of E. coli whole cell after HHP (35oC for 5 min) or heat (65oC for 6 min) treatments. Control (A), HHP treatment levels: 200 MPa (B), 300 MPa (C), 400 MPa (D), 500 MPa (E), 600 MPa (F), 700 MPa (G), and heat treatment (H). Thermograms are offset for clarity.
150
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 100 200 300 400 500 600 700
[(∆H
-∆H
f)/(∆
H0-
∆Hf)]
Pressure (MPa)
Figure 5.4. Pressure dependence of fractional apparent enthalpy determined by DSC.
151
0
1
2
3
4
5
6
0 5 10 15 20 25
-ln[
(∆H
-∆H
f)/(∆
H0-
∆Hf)]
)
Figure 5.5. Correlation between fractionalHHP treated E. coli.
1
-ln(N/N0
apparent enthalpy and fractional viability for
52
SEM study on the cellular components after treatments
The shape and the surface appearance of inactivated E. coli cells were evaluated
from SEM photomicrographs obtained at 25,000X magnification after pressure treatment
at 700 MPa for 5 min at 35oC or heat treatment at 65oC for 6 min (Fig. 5.6). Before
treatment, the surface of cells in SEM micrograph was clean and smooth (Fig. 5.6a). The
appearances of cell surfaces became rough and wrinkled when the cells were inactivated
at 700 MPa (Fig. 5.6b). Many cells have protruding parts on their surfaces were also
observed in Figure 5.6b. Like pressure-inactivated cells, the surfaces of heat inactivated
cells became rough and cracked; however, no protruding formation were found on their
surfaces (Fig. 5.6c). The cell length of heat-inactivated cells appeared to be longer than
that of controls and pressure-inactivated cells.
(a)
153
(b)
Protruding parts
(c)
Figure 5.6. SEM micrograph of control (a), pressure-inactivated (b, at 700 MPa, 35oC, 5 min), and heat-inactivated (c, at 65oC for 6 min) E. coli cells.
154
TEM study on the cellular components after treatments
The morphology of intracellular structures and cell envelope of inactivated E. coli
cells were examined from TEM photomicrographs obtained at 70,000X magnification
after pressure treatment at 700 MPa for 5 min at 35oC or heat treatment at 65oC for 6 min
(Fig. 5.7). The internal appearance of untreated cells in TEM micrograph was
characterized by uniform density distribution of ribosomes and central electron
transparent region of DNA (Fig. 5.7a). When the cells were inactivated by 700 MPa
pressure, compacted peripheral dark regions and fibrous central area are shown in
internal bodies (Fig. 5.7b). Similar appearances are shown in internal structures of heat
inactivated (at 65oC for 6 min) cells (Fig. 5.7c). Empty spaces were observed between
internal bodies and cell envelope in the pressure inactivated cells while those were absent
in the heat inactivated cells (Fig. 5.7b,c). Most of pressure or heat inactivated cells
maintained integrity of membranes in their cell envelope.
155
(a)
DNA
Cell envelope
Ribosomes
Figure 5.7. TEM micrographs of untreated (a), pressure-inactivated (b, at 700 MPa, 35oC, 5 min), and heat-inactivated (c, at 65oC for 6 min) E. coli cells. Bar = 1.0 µm.
156
(b)
DNA
Ribosomes
Cell envelope
157
DNA
Cell envelope
(c)
Ribosomes
158
DISCUSSION
This study aims to assess the effect of HHP on viability and structure of E. coli cells
by: i. evaluating the relationship between the conformational changes in cellular
components and cell viability using plate count and DSC data; ii. comparing the
morphological and conformational changes in the components of pressure inactivated
cells with that of heat inactivated cells using electron microscopy and DSC. Preliminary
study showed that HHP treatment of 100 MPa did not affect either the viability of the
culture or transitions of cellular components in whole cell thermogram. Therefore, the
effects of 200 MPa and above treatments on the organism were evaluated in present
study.
DSC shows the changes in conformational transitions of cellular components and
apparent enthalpy of E. coli cell pellets after treated at each level of HHP (Fig. 5.3). The
ribosomal subunits are the cellular component most affected by the 200 MPa HHP
treatment (Fig. 5.3 thermogram B). Two endothermic transitions are observed in peak a2
which is proposed to be the denaturation of the main ribosomal subunit (Mackey et al.,
1991). Since a large and broad peak in the thermogram of bacterial whole cells has been
considered as the sum of several overlapped transitions, the appearance of the two peaks
possibly indicates that one of those transitions was shifted to lower temperature after the
HHP treatment while the other transition(s) was/were not affected or reversible. In the
DSC study on isolated 50S and 70S ribosomal subunits of E. coli, Bonincontro et al.
(1998) reported that the two peaks found in a ribosomal subunit is accounted as a
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complex unfolding process on interactions such as protein/RNA, protein/protein and
RNA/RNA within the subunit (Bonincontro et al., 1998). It has been known that
alteration of tertiary structure of proteins occurs at 200 MPa pressure level (Vidugiris et
al., 1995), while denaturation of RNA structure requires much higher level of pressure
(Smelt et al., 1998). Since ribosomal subunits are composed of RNA and protein, the
pressure-induced conformational change of ribosomal proteins may have important role
in the formation of two peaks in thermogram by altering protein/protein and protein/RNA
interactions in ribosome structures. However, there were no noticeable changes in the
apparent enthalpy and plate counts after 200 MPa in Table 5.1, indicating that the initial
conformational change in ribosomal subunits did not affect the cell viability.
The reductions in the apparent enthalpy value (~22%) and viable count (~2 log10
unit) of E. coli cells occurred after HHP at 300 MPa (Table 5.1). The temperature and
size of peak a2, which is associated with the denaturation of larger and more rigid
ribosomal subunits, were also lowered. However, peak a1 (probably 30S subunit), which
had been only partially visible due to the overlapping peak a2 in the control, became
visible (Fig. 5.3 thermogram C). It has been reported that the volume reduction of
ribosome molecule occurs during HHP treatment as results of hydration changes around
ribosome and conformational changes which are followed by denaturation (Balny and
Masson, 1993; Alpas et al., 2003). Among ribosomal subunits of bacteria, larger subunit
is believed to have greater tendency to decrease its volume in HHP treatment (Alpas et
al., 2003). Therefore, the decrease in the size of peak a2 and the appearance of peak a1 in
the thermogram of 300 MPa pressure-treated cells indicate the volume reduction of larger
160
ribosomal subunit (probably 50S) associated in peak a2 is greater than that of smaller
ribosomal subunit (probably 30S).
The peak in the range of 90-100oC in DSC of whole bacterial cells was associated
with the melting of double strand DNA due to the thermally-induced breakage of
hydrogen bonds between base pairs of two single strand DNA structures (Miles et al.,
1986, Mackey et al., 1988). The effect of HHP on thermal stability of DNA double helix
has commonly been studied by measuring the change in the ultraviolet adsorption as a
function of temperature after pressurization of isolated DNA in salt solution. Studies
showed that the base pairing of DNA duplex are stabilized by high pressure at least up to
200~270 MPa (Héden et al., 1964; Kumar, 1995; Lin and Macgregor Jr., 1996).
However, in a recent DSC study, a decrease in thermal stability of DNA duplex in
Leuconostoc mesenteroides was observed after HHP treatment at 250 MPa and above
pressures (Kaletunç et al., 2004). Noticeable size reduction of peak b in thermograms of
cells treated at 300 MPa and above pressures in present study indicates that dissociation
of double stranded DNA, which induces irreversible change of the DNA transition,
possibly occurred in the pressure treatments. Since little information about the HHP level
which leads denaturation of the DNA double helix in bacteria cell is available in
literature, further study on the detection of change in DNA structures in vivo after HHP
treatment will be needed to confirm the result of this study. The appearance of peak c, a
peak known to be also related to denaturation of DNA with a cell wall component
(Mackey et al., 1991) became apparent due to the reduction of peak b which partially
obscured the peak c in the thermogram of untreated E. coli cells (Fig. 5.3 thermogram C).
161
Figure 5.3 shows that the area of the peak d, which corresponds to the denaturation
enthalpy of components in outer membrane, was decreased (~30%) after 400 MPa and
above HHP treatments. In protein content analysis study of Salmonella Typhimurium
using electrophoresis SDS-PAGE, Ritz et al. (2000) observed that most of major outer
membrane proteins of the bacteria disappeared after 350 MPa and above HHP treatments.
The irreversible denaturation or forced out from the membrane has been considered as
reason for the loss of the membrane proteins by HHP (Casadei et al., 2002). However,
since thermal stabilities of the E. coli outer membrane proteins are ~70oC (Phale et al.,
1998), and these proteins are not directly interacted with thermally stable components in
outer membrane, it is doubted that pressure effect on the outer membrane proteins made
the change in peak d in the thermograms. Therefore, it is possible that the reduction of
the peak d in the thermograms is mainly related with the conformational change in other
outer membrane component such as LPS by higher levels (>400 MPa) of HHP
treatments. LPS is predominant component (~40% weight) in outer membrane of E. coli
and hypothesized to be associated with the endothermic transition (peak d) (Lee and
Kaletunç, 2002b).
The plots in Figure 5.2 and 5.4 suggest that the fraction of apparent enthalpy [(∆H-
∆Hf)/(∆H0-∆Hf)] from DSC data is closely related to the log of fractional viability [log
(N/N0)] from plate count data subsequent to pressure treatments. No slope changes up to
200 MPa treatment in both figures indicates that although it affected the peak associated
with major ribosome subunit (Fig. 5.3), HPP treatment at 200 MPa had no great influence
on either apparent enthalpy or plate count data. The steep slopes of the plots after 300
162
MPa treatment in the Figures 5.2 and 5.4 suggest that the reductions in the apparent
enthalpy and plate count data were accompanied by a decrease in the area of the peak (a2)
corresponding to the alterations of larger ribosomal subunits (Fig. 5.3) at 300 MPa and
above pressure levels. The size reduction of one of the DNA transition (peak b) due to
possible pressure-induced damages on DNA duplex attributed the reduction of apparent
enthalpy and might cause more cell viability loss on plate count result.
A plot of the fractional apparent enthalpy versus the fractional survivors from plate
count data (Fig. 5.5) gives a linear relationship by taking the logarithm of both sides.
However, in the pressure treatment range from 300 to 500 MPa, where the cell population
was exponentially decreased in plate count method (Fig. 5.2), disparities appear between
the calorimetric data and plate count data. The result indicates that the decrease in the
fraction of plate count data against the decrease in the fraction values of apparent
enthalpy data are relatively higher in the HHP treatment levels between 300 and 500 MPa
than that in other pressure levels (200, 600 MPa).
Many adhering cells covered with protruding substances were observed in Figure
5.6b indicating components of cell envelope released during the HHP. The observed
whole cell population in our SEM and TEM studies seemed to maintain its shape without
cell rupture or breakage at 700 MPa pressure inactivation. The property of the
smoothness of Gram-negative bacterial cell surface is mainly contributed by O
polysaccharide (O antigen) in LPS structure (Todar, 2002). Therefore, we assume that
the denaturation or the detachment from the cell of LPS by either pressure or heat
inactivation might play an important role in the formation of the roughness on the surface
163
of cells. However, it is still possible that the protruding substances also contain
intracellular components that leaked out via membranes which can lose their permeability
during high pressure treatment.
The dark compacted regions in TEM micrographs of pressure-inactivated cells have
been thought to be residual ribosomes and proteins in cytoplasm after denaturation
(Mackey et al., 1994). Because no clear evidence of cell membrane damages in TEM of
the pressure-inactivated cells (Fig. 5.7b), ribosomes and proteins might remain in the cell
and contribute the transitions in DSC thermogram. However, DSC thermogram G in
Figure 5.3 shows that peak associated with ribosome denaturation almost disappeared
after cells were inactivated by pressure while the peripheral dark region are clearly shown
in TEM of the cells. It is suggested that pressure induced denaturation and later
aggregation of ribosome result in the reduction of ribosomal peak in DSC thermogram
(Kaletunç et al., 2004). Therefore, the disappearance of ribosomal transition in the
thermogram G possibly indicates that both denaturation of residual ribosome
(endothermic event) and aggregation of denatured ribosome (exothermal event) occurred
during DSC scan to reduce ribosomal transition in the thermogram of 700 MPa treated
cells. Comparison of thermograms of pressure inactivated cells and heat inactivated cells
(Fig. 5.3. Thermogram G and H) shows that two peaks (63.5 and 68.2oC) remained in the
ribosome transition for heat inactivated cells although the apparent enthalpy for both cells
were similar. The result may suggest the effect of HHP on ribosome structure is greater
than that of heat treatment when lethal level of each treatment is applied.
164
The condensed fibril formation in central regions of both pressure and heat
inactivated cells possibly indicates that DNA structures were affected by both treatments.
Similar formations were shown in HHP (>500 MPa) treated cells of Lactobacillus
viridescens (Park et al., 2001) and Salmonella Thompson (Mackey et al., 1994).
Thermogram G and H in Figure 5.3 show that one of the DNA transition (peak b) was
irreversible after both treatments indicating the denaturation of DNA resulted in the
formation of fibrils in the TEM of the treated E. coli cells. In the comparison of peak c,
which is also associated with DNA transition, the thermal stability (Tm) of the peak in the
thermogram of heat inactivated cells is 1.5oC higher than that of HHP inactivated cells.
The result implies that the effect on DNA is also greater in HHP inactivation.
The cause of the appearances of empty spaces during HHP (Fig. 5.7b) has still been
controversial. It was thought to be the reversible invagination of cytoplasmic membrane,
which increases its surface area due to pressure-induced phase changes in lipid bilayer,
causes the spaces in Salmonella Thompson (Mackey et al., 1994). On the other hand,
Park et al. (2001) claimed that the separation processes of deformed cell wall structures
from the internal cell bodies cause the spaces in Lactobacillus viridescens. Because the
internal bodies of the pressurized cells in Figure 5.7b seem to be physically squeezed, the
cause of the empty spaces on E. coli in this study might be similar to the idea in Mackey
et al. (1994). Therefore, it is possible that the cause of empty spaces in pressurized
bacteria cells depends on their cell wall types (Gram-positive or Gram-negative). Most
pressurized cells retained the membrane line in cell envelope region in our TEM section,
indicating that there was no apparent evidence of cell envelope rupture shown after HHP.
165
Mackey et al. (1994) stated that the invaginated membrane return to original position
when pressure is released.
In conclusion, DSC study shows that the reversibility of transition and the change in
the thermal stability of ribosome of E. coli were affected by 200 MPa and above
pressures in HHP treatment. The enthalpy and the thermal stability of the DNA melting
transition were affected by HHP treatments above 300 MPa. The morphological changes
in structures of ribosome and DNA are also shown in TEM section. There is a close
relationship of fractional viability based on calorimetric data and plate count data. In
EM study, no rupture of cell envelope is shown in pressure- or heat- inactivated E. coli
cells. However, leakage of cell wall or outer membrane substance and empty space
between cell envelope and inside structure are exclusively observed in pressure-
inactivated cells. Comparison of the cell components by DSC combined with EM prior
and after HHP inactivation allows us to evaluate the changes resulting from HHP
processing which coincide with inactivation of microorganisms.
166
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Lopez-Caballero, M.E., Carballo, J., Solas, M.T. and Jimenez-Colmenero, F. 2002. Responses of Pseudomonas fluorescens to combined high pressure/temperature treatments. Eur. Food. Res. Technol. 214:511-515. Mackey, B.M., Forestiere, K. and Issacs, N. 1995. Factors affecting the resistance of Listeria monocytogenes to high hydrostatic pressure. Food Biotechnol. 9:1-11. Mackey, B.M., Forestiere, K., Issacs, N.S., Stenning, R., Brooker, B. 1994. The effect of high hydrostatic pressure on Salmonella Thompson and Listeria monocytogenes examined by electron microscopy. Lett. Appl. Microbiol. 19:429-432. Mackey, B.M., Miles, C.A., Parsons, S.E. and Seymour, D.A. 1991. Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry. J. Gen. Microbiol. 137:2361-2374. Mertens, B. and Deplace, G. 1993. Engineering aspects of high pressure technology in the food industry. Food Technol. 47(6):164-169. Metrick, C., Hoover, D.G. and Farkas, D.F. 1989. Effects of hydrostatic pressure on heat resistant and heat sensitive strains of Salmonella. J. Food Sci. 54:1547-1549. Miles, C.A., Mackey, B.M. and Parsons, S.E. 1986. Differential scanning calorimetry of bacteria. J. Gen. Microbiol. 132: 939-952. Mohacsi-Farkas, Cs., Farkas, J., Meszaros, L., Reichart, O. and Andrassy, E. 1999. Thermal denaturation of bacterial cells examined by differential scanning calorimetry. J. Therm. Anal. Calor. 57:409-414. Mohacsi-Farkas, Cs., Farkas, J. and Simon, A. 1994. Thermal denaturation of bacterial cells examined by differential scanning calorimetry. Acta Aliment. 23:157-168. Niven, G.W., Miles, C.A. and Mackey, B.M. 1999. The effect of hydrostatic pressure on ribosome conformation in Escherichia coli: an in vivo study using differential scanning calorimetry. Microbiology 145, 419-425.
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CHAPTER 6
INACTIVATION OF SALMONELLA ENTERITIDIS STRAINS BY COMBINATION
OF HIGH HYDROSTATIC PRESSURE AND NISIN
ABSTRACT
The effects of high hydrostatic pressure (HHP) and nisin treatment alone and in
combination on cellular components and viability of two Salmonella enterica subsp.
enterica serova Enteritidis (Salmonella Enteritidis) strains were evaluated by differential
scanning calorimetry (DSC) and plate counting in order to evaluate the relative resistance
and optimize the treatment conditions. Salmonella Enteritidis FDA and OSU 799 strains
were subjected to HHP (0.1- 550 MPa for 10 min at 25oC) alone and in combination with
nisin (200 IU/ml Nisaplin®) in culture broth (TSBY). HHP (up to 200 MPa) or the nisin
alone did not affect the viability and cellular components of either strain. An 8-log
cfu/ml reduction was observed after a pressure treatment at 500 MPa for FDA strain and
450 MPa for the OSU 799 strain. When nisin was added, a similar reduction was
obtained at 400 MPa for FDA strain and 350 MPa for the OSU 799 strain. The decrease
172
in apparent enthalpy appeared to be mainly due to reduction in the ribosome denaturation
peak for both pressure alone and pressure-nisin combination treatments. DNA might be
irreversibly damaged by the combination treatments. A linear relationship in a
logarithmic plot of fractional apparent enthalpy values [(∆H-∆Hf)/(∆H0-∆Hf)] versus the
fractional survivors from plate count data (N/N0) for treated cells indicates the apparent
enthalpy data obtained from DSC can be used to evaluate pressure levels necessary to
reduce a microbial population in the presence of nisin and provide information about
viability in shorter time with comparable accuracy to plate count.
Key Words: high hydrostatic pressure, nisin, differential scanning calorimetry,
Salmonella Enteritidis.
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INTRODUCTION
High hydrostatic pressure (HHP) has been shown to inactivate spoilage and
pathogenic bacteria while preserving the quality parameters of food products. HHP has
been recognized as a most likely alternative to thermal processing among emerging food
preservation technologies (Roberts and Hoover, 1996; Mertens and Deplace, 1993;
Knorr, 1993). Inactivation of pressure-resistant pathogens needs very high pressure
levels (>600 MPa) at which texture and color of many foods may be adversely altered
(Porretta et al., 1995; Mussa and Ramaswamy, 1997; Trujillo et al., 1999). Furthermore,
the initial cost and wear of equipment increase at pressure levels above 600 MPa, leading
to high maintenance costs or short equipment life (Hoover et. al., 1989; Mertens and
Deplace, 1993). If moderately high pressure treatments (up to 400 MPa) are applied,
sublethal injury and recovery during storage occur for pressure resistant pathogens
(Earnshaw, 1995; Patterson et al., 1995). Therefore, a processing protocol combining
moderately high pressures with chemical or physical preservation methods can provide
safe food without high processing cost.
The concept of “Hurdle technology” has been applied to inactivate pathogenic
bacteria by combining HHP with low pH (Alpas et al., 2000), heat (Patterson and
Kilpatrick, 1998; Benito et al., 1999; Alpas et al., 2000), lysozyme (Masschalck et al.,
2001), CO2 (Hass et al., 1989) or antimicrobial peptides (Kalchayanand et al., 1998;
Yuste et al., 1998; Garcia-Graells et al., 1999; Masschalck et al., 2000; Massachalck et
al., 2001). Most antimicrobial peptides produced by bacteria are bactericidal to Gram-
174
positive bacteria and to sublethally stressed Gram-negative bacteria (Ray et al., 2001).
Kalchayanand et al. (1998) reported that the mixture of pediocin and nisin provided 1 to 5
log unit reductions above the additive effects of bacteriocin and HHP alone in
Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica serovar
Typhimurium (S. Typhimurium) and Escherichia coli O157:H7. Among bacteriocins,
nisin, produced by some strains of Lactococcus lactis, is known to inactivate Gram-
positive bacteria by binding to the cytoplasmic membrane and forming pores which leads
to leakage of intracellular molecules and metabolites (Sahl and Bierbaum, 1998). Its
ineffectiveness against Gram-negative bacteria is attributed to the nisin-impermeable
outer membrane which prevents nisin reaching the cytoplasmic membrane (Kordel and
Sahl, 1986; Delves-Broughton, 1990). Recent studies show that HHP treated Gram-
negative bacterial cells may allow penetration of nisin through the outer membrane
(Masschalck et al., 2000; Masschalck et al., 2001; Ray et al., 2001). Garcia-Graells et al.
(1999) described that pressurization of E. coli in the presence of nisin (400 IU/ml)
decreased the survivability by an additional 3 logs in skim milk at 550 MPa. An
additional 1 to 2 log unit reductions in Pseudomonas fluorescens, E. coli O157:H7, and a
Salmonella sp. were achieved by nisin (100 IU/ml) under moderate (<300 MPa) HHP
treatment (Masschalck et al., 2001). Although the primary target for nisin and HHP in
bacterial cell is proposed to be the cytoplasmic membrane (Kalchayanand et al., 1998;
Masschalck et al., 2001), the exact mechanism of the inactivation of bacteria by HHP or
nisin is still not clearly known.
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DSC thermograms of whole bacterial cells display endothermic transitions
associated with the cellular components of bacteria to heat under linearly increasing
temperature condition. The peak temperature corresponding to each transition represents
the thermal stability of a cellular component of bacteria (Miles et al., 1986; Anderson et
al., 1991; Mackey et al., 1991; Belliveau et al., 1992; Kaletunç, 2001; Lee and Kaletunç
2002b). In addition, DSC measurement provides amount of heat energy (apparent
enthalpy, ∆H) associated with the transition. The apparent enthalpy can be used to
calculate fractional survivors following heat treatment (Lee and Kaletunç, 2002a,b).
DSC has also been utilized for characterization of changes in cellular components of
bacteria by recording scans before and after exposure to a high hydrostatic pressure, a
physical stress (Niven et al, 1999; Alpas et al., 2003; Kaletunç et al., 2004). Niven et al.
(1999) reported reductions in ribosome associated transitions for Escherichia coli NCTC
8164 cells as a function of pressure between 50-250 MPa. Alpas et al. (2003) used
apparent enthalpy data to evaluate the relative high pressure resistance of two bacterial
strains from E. coli O157:H7 and S. aureus.
Development of an understanding for inactivation of Gram-negative bacteria by
pressure-nisin treatment is necessary for optimization of HHP processing conditions to
inactivate Gram-negative bacteria using nisin. The objective of this study was to evaluate
the effect nisin and HHP alone and in combination on cellular components of Salmonella
Enteritidis (Gram-negative food-borne pathogen) using DSC. The fraction of survivors
as a function of treatment was determined using plate count and calorimetric data.
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MATERIALS AND METHODS
Bacterial strains
Salmonella Enteritidis FDA was obtained from the Food Microbiology Laboratory,
University of Wyoming. Salmonella Enteritidis OSU 799 was obtained from the Culture
Collection Center at the Ohio State University. Previous study has shown FDA strain to
be strongly resistant to high pressure among Salmonella Enteritidis strains (Alpas et al.,
1999). An isolated pure colony on of each organism grown on XLD agar plate was
suspended in 10 ml Trypticase soy broth supplemented with 0.6 % (w/w) yeast extract
(TSBY) and incubated at 37oC for 18 hours. Culture was stored frozen (-80oC) in 30 %
(v/v) sterile glycerol. A loopful of stock culture was transferred to 10 ml Trypticase soy
broth and incubated 10 hrs at 37oC before use.
Preparation of organisms for pressurization
Each culture was inoculated (1 % v/v) into a broth containing TSBY and incubated
at 37oC until reaching early stationary growth phase. The growth phase was determined
using viable counts. The growth was used in the subsequent studies to obtain cells at
early stationary phase for pressure treatment. After reaching to early stationary phase,
culture was removed from the incubator and placed (150 ml) in 3-mil-thick sterile
stomacher bags (Fisher scientific, Ottawa, Canada). Nisaplin® (Aplin and Barrett,
Milwaukee, USA) contains 2.5% nisin, was added to the bags containing grown culture
to obtain concentration of Nisaplin (0 and 200 IU/ml). After heat sealed, the bag was
177
then placed inside a second bag filled with chlorine solution (20% v/v) and heat sealed to
prevent contamination of the high-pressure unit if the primary package failure is to occur.
Air was removed from all of the bags as much as possible. Prepared bags were vacuum
sealed in sterile plastic bags prior to pressurization.
High Hydrostatic Pressure Processing
Pressurization of bacteria culture was carried out using a High Pressure Processing
Unit (ABB Quintus Food Processor QFP-6 Cold Isostatic Press, Columbus, Ohio). The
hydrostatic pressurization unit is capable of operating at 900 MPa. The pressure chamber
was filled with 50% (v/v) aqueous glycol solution. The temperature of the liquid was
controlled by an electric heating system around the chamber. The rate of pressure
increase was about 400 MPa per min. The pressure level, time, and temperature of
pressurization were controlled. All the processing parameters were maintained during
the pressurization cycle.
Various pressures (150, 200, 250, 300, 350, 400, 450, 500 and 550 MPa) were
applied for 10 min at 25oC. After HHP treatment, a portion (1 ml) of the pressured and
untreated (control) cultures in the bags were serially diluted in 0.1 % sterile peptone
solution and pour plated into Trypticase soy agar to determine viable cell counts. Cells in
the pressure-treated and untreated broth were centrifuged at 10,000 g for 10 min at 4oC
(Beckman J2-21 centrifuge). Supernatants were discarded. Pellets were washed with
100 ml of sterile distilled water before transferring into DSC crucibles for calorimetry.
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Calorimetry of whole cell
A differential scanning calorimeter (DSC 111, Setaram, Lyon, France) was used to
record the thermograms of the cells heated at a 4oC min-1 after HHP treatment. All DSC
measurements were conducted using fluid-tight, stainless steel crucibles. A DSC run was
performed with empty sample and reference crucibles to assure a reproducible empty
crucible baseline. Pellets of whole cells were transferred into the empty sample crucible
and were weighed (70 ± 0.5 mg wet weight). The dry material content of the pellets was
determined by freeze drying (Freezone 4.5, Freeze dry system, Model 77510, Labconco,
Missouri) as 18 ± 0.2 % on a wet basis. The standard deviation was calculated based on
four freeze dried pellets for Salmonella Enteritidis FDA. For each DSC run, reference
crucible was filled with water (~57 mg) equal to the amount of moisture in sample.
Crucibles were sealed using aluminum o-rings and were refrigerated at 4oC prior to DSC
runs. The sample and reference crucibles were placed in the DSC and equilibrated at 1oC
using liquid nitrogen cooling system. Samples were reweighed after DSC measurements
to check for the loss of mass during heating. Thermograms of samples showing signs of
leakage were discarded. DSC thermograms of samples were corrected for differences in
the empty crucibles by subtraction of an empty crucible baseline. Peak areas (apparent
enthalpies, J g-1) corresponding to the contributions of survivors were determined from
the apparent heat capacity vs. temperature graph using software provided by the
instrument manufacturer. A curved baseline using three-temperature points was
developed between the segment of the thermogram prior to the first thermally-induced
179
transition (~30oC) and the segment of the thermogram at a temperature below the onset of
the final peak (~110oC).
Statistical Analysis
Viability after the experiments was analyzed using MINITAB statistical program
(Minitab Inc., State College, PA). The means from each variables (pressure level and
concentration of nisin) were determined by two-way analysis to compare the effect of
variables on treatment. Differences in inactivation between pressure only treated cells
and pressure-nisin combination treated cells were compared using t-test. The level of
significance was set for P<0.05.
180
Growth of the Salmonella strains to the end of exponential stagephase
Plate countinga
DSC for calorimetric datt
Analysis of the effect of HHP
or Nisin alone treatment
181
Combination treatment by HHPand nisin
Plate counting
Analysis of the effect of the combination treatmen
Treatment by HHP or nisin
Centrifugation to obtain cell pellets
Figure 6.1. Experimental scheme of calorimetric and microbial analysis
RESULTS
Thermograms of Salmonella Enteritidis whole cells
Figure 6.2 shows the DSC thermogram for untreated pellet of Salmonella
Enteritidis FDA (A) and OSU 799 (B) cells. Several overlapping endothermic peaks (a,
b, c and d) appear in the thermograms. The transition temperature (Tm) of first major
peak, a, which was identified as denaturation of ribosome in bacteria (Mackey et al.,
1991; Stephens and Jones, 1993; Lee and Kaletunç, 2002b), appears at a higher
temperature in the FDA strain thermogram (71.5oC) in comparison to OSU 799 strain
thermogram (69.5oC). Other peaks, which have been proposed to be thermally induced
transitions of DNA (b, Tm 93oC) and DNA together with cell wall components (c, Tm
100oC), are similar for the two strains. Figure 6.2 also shows that the transition of the
denaturation of outer membrane components of Gram-negative bacteria (Mackey et al.,
1991) appears to be at least two overlapping peaks (d, Tm 125 and 132.3oC ) in the FDA
strain thermogram, while it appears as one broad peak (Tm 119oC) in the OSU 799
thermogram. The total apparent enthalpy values for both untreated cells were 4.0 ± 0.4
J/g wet cell.
182
20 40 60 80 100 120
a
bc d
Heat Flow0.5 mW
Temperature (oC)
Figure 6.2. Thermograms of whole cells of Salmonella Enteritidis OSU Salmonella Enteritidis FDA (B) obtained by DSC (1 to 150oC with 4oC mrate).
183
A
B
140
799 (A) and in-1 heating
Evaluation of inactivation Salmonella Enteritidis from plate count data after HHP treatment in combination with nisin Pressures at 200, 250, 275, 300 or 350 MPa and Nisaplin at 200, 400 or 600 IU/ml
were used to inactivate Salmonella Enteritidis FDA culture (Appendix Figs. 1,2 and 3).
Nisin alone did not affect the viability of the culture at Nisaplin concentration levels of
200-600 IU/ml. The reduction of viability based on plate counting and apparent enthalpy
data was greater for nisin-pressure combination than for pressure alone (Appendix Figs. 4
and 5). Viability of cells were not significantly different (P>0.05) with respect to nisin
concentration at each pressure treatment level as assessed by both calorimetric and plate
count (Appendix Table 1). Therefore, 200 IU/ml Nisaplin was applied to cultures of two
Salmonella Enteritidis strains to use minimum amount of antimicrobial agent.
Table 6.1 gives the viability of both strains treated by nisin and pressure alone and
in combination. Nisin alone did not affect the viability of either strain. Table 6.1 also
shows that the combination treatment was more effective in comparison with pressure
alone treatment for both strains. At 200 MPa, the combined treatment of pressure and
nisin caused ~ 1 log unit decrease in cell viabilities of both strains, while the pressure
alone did not affect viabilities. At 300 MPa and above pressure, addition of nisin caused
between 2 and 3 log units additional inactivation for both strains.
Fractional viability values from plate count data (N/N0) was plotted against pressure
for two strains, which were pressurized with or without nisin (Fig. 6.3). The OSU 799
strain was more sensitive than FDA strain for both pressure alone and nisin-pressure
combination treatment (P<0.05). An 8 log unit reduction was observed after a pressure
treatment at 500 MPa for FDA strain and 450 MPa for the OSU strain. When nisin was
184
added, 8 log unit reduction was obtained at 400 MPa for FDA strain and 350 MPa for the
OSU strain, where the difference in log survivals between pressure alone and nisin-
combination treatments was greatest (Fig. 6.3). The difference of log survivals between
two strains in pressure alone treatment was greatest (3 log) at 450 MPa, while the
difference was greatest (~2.5 log) at 350 MPa in pressure-nisin combination treatment.
The difference of log survivals between the cells of two strains treated by a given
pressure level (except for 300 MPa level) was greater for pressure-nisin combination
treated cells than for pressure-alone treated cells (Fig. 6.3).
185
Viable counts (cfu/ml) Apparent enthalpy (J/g)
S. Enteritidis OSU S. Enteritidis FDA S. Enteritidis OSU S. Enteritidis FDAPressure
(MPa) 0 Nisaplin 200 IU/ml
Nisaplin 0 Nisaplin 200 IU/ml
Nisaplin 0 Nisaplin 200 IU/ml
Nisaplin 0 Nisaplin 200 IU/ml
Nisaplin 0.1 1.7 x 109 1.4 x 109 1.3 x 109 1.2 x 109 3.99 3.91 3.96 3.91
150 1.7 x 109 5.6 x 108
3.80 3.40
200 1.3 x 109 8.2 x 107 1.2 x 109 3.0 x 108 3.35 2.88 3.65 2.92
250 1.4 x 108 1.4 x 106 4.8 x 108 1.4 x 107 3.06 2.60 3.62 2.81
300 3.6 x 105 1.8 x 103 1.3 x 107 2.1 x 104 2.55 2.27 3.00 2.31
350 4.9 x 103 < 1.0 x 101 6.5 x 104 1.2 x 102 2.11 1.89 2.82 2.12
400 5.0 x 101 1.5 x 104 < 1.0 x 101 1.76 1.75 2.46 1.75
450 < 1.0 x 101 1.9 x 103 1.74 2.18
500 < 1.0 x 101 1.74 1.65
550 1.73
186
Table 6.1. Viability and apparent enthalpy values for cells of Salmonella Enteritidis strains after HHP treatments.
0.000000001
0.00000001
0.0000001
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
0 100 200 300 400 500
N/N
0
Figure 6.3. Pressure dependence of fracdetermined by plate count. The cells trstrain ♦). The cells treated with 200 IU/m
Pressure (MPa)
tional viability of Salmonella Enteritidis strains eated without nisin (FDA strain ▲, OSU 799 l nisin (FDA strain ∆, OSU 799 strain ◊).
187
Evaluation of inactivation Salmonella Enteritidis from apparent enthalpy data after HHP treatment in combination with nisin The effect of the pressure-nisin combination treatment on two strains was evaluated
by comparing apparent enthalpies of pressure-nisin treated cells with those of pressure
alone treated cells (Table. 6.1). No apparent differences found between thermograms of
control and the nisin alone treated cells for both strains (Figs 6.4a,b Thermogram A and
Figs 6.5a,b thermogram A). Similar to results of plate count method, the reduction of
apparent enthalpy was greater for combined nisin-pressure treatment for both strains.
The decrease in apparent enthalpy (∆H) appeared to be mainly due to reduction in the
ribosome denaturation peak, a, for both pressure alone and nisin-combination treatments
(Figs. 6.4a and 6.5a). As pressure increased, the greater decrease in the apparent
enthalpy of the pressure-nisin treated cells was observed due to the greater size reduction
of peak c as well as the greater decrease in the ribosome peak for both strains (Figs. 6.4b
and 6.5b).
Initial thermograms reveal an overlapping b (Tm 93oC) and c (Tm 100oC) peaks.
Peak, c, disappeared from thermograms revealing the endotherm b at 200 MPa and above
pressure treated cells on both strains (Figs. 6.4a and 6.5a). However, the peak c remained
(Tm 103oC) in the thermograms up to 300 MPa when nisin was added. Substantial
reduction of the size of the peak b was observed after a pressure treatment at 400 MPa for
FDA strain and 350 MPa for the OSU strain. When nisin was added, the peak b of FDA
strain seemed to be disappeared after 350 MPa treatment for FDA strain while an
endothermic transition still remained in the peak b area after 400 MPa for OSU strain.
188
The fractional viability based on apparent enthalpy data [(∆H-∆Hf)/(∆H0-∆Hf)] was
plotted against pressure for two strains, which were pressurized with or without nisin
(Fig. 6.6). The apparent enthalpy (∆H) and the residual apparent enthalpy (∆Hf) are
obtained after treatment resulting in no viability and ∆H0 is the apparent enthalpy of
untreated cells. A residual apparent enthalpy (∆Hf) was reached at 500 MPa for FDA
strain and 450 MPa for OSU strain in the pressure treatment. The ∆Hf was obtained at
lower pressure levels when nisin was added, the value was reached at 400 MPa for FDA
strain and 350 MPa for OSU strain. The slope of plots in Figure 6.6 became much
steeper after 250 MPa alone and nisin combination treatments for FDA strain and after
150 MPa for OSU strain. About 80% reduction of the enthalpy fraction was observed
after a pressure treatment at 450 MPa for FDA strain and 350 MPa for the OSU strain.
When nisin was added, the similar reduction was obtained at 350 MPa for FDA strain and
300 MPa for the OSU strain.
Natural logarithm of fractional viability values calculated from calorimetric data
[(∆H-∆Hf)/(∆H0-∆Hf)] and plate count data (N/N0) are plotted in Figure 6.7 and 6.8. The
correlation coefficients of all four curves were greater than 0.93. The difference of the
slope of the lines between pressure only and pressure-nisin combination treated cells are
shown in the graph of FDA strain, while very similar slope of those are observed in the
graph of OSU strain.
189
(a) (b)
20 40 60 80 100 120 140
0.4 mWIa
dcb
A
G
F
E
D
C
B
I
H
20 40 60 80 100 120 140
a
dcb
A
G
F
E
D
C
B
0.4 mWI
Temperature (oC) Temperature (oC)
Figure 6.4. DSC thermograms of Salmonella Enteritidis FDA pellets after pressure alone treatment (a) or pressure-nisin combination treatment (b). Control (A), 200 MPa (B), 250 MPa (C), 275 MPa (D), 300 MPa (E), 350 MPa (F), 400 MPa (G), 450 MPa (H) and 500 MPa (I). Thermograms are offset for clarity.
190
(a) (b)
20 40 60 80 100 120 140
0.4 mWIa
dcb
A
G
F
E
D
C
B
I
H
20 40 60 80 100 120 140
0.4 mWIa
dcb
A
F
E
D
C
B
G
Temperature (oC) Temperature (oC)
Figure 6.5. DSC thermograms of Salmonella Enteritidis OSU 799 pellets after pressure alone treatment (a) or pressure-nisin combination treatment (b). Control (A), 150 MPa (B), 200 MPa (C), 250 MPa (D), 300 MPa (E), 350 MPa (F), 400 MPa (G), 450 MPa (H) and 500 MPa (I). Thermograms are offset for clarity.
191
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 100 200 300 400 500
[(∆H
-∆H
f)/(∆
H0-∆
Hf)]
Figure 6.6. Pressure dependence of fradetermined by calorimetric data. The cestrain ♦). The cells treated with 200 IU
Pressure (MPa)
ctional viability of Salmonella Enteritidis strains lls treated without nisin (FDA strain ▲, OSU 799
/ml nisin (FDA strain ∆, OSU 799 strain ◊).
192
y = 0.0888x + 0.0584R2 = 0.9323
y = 0.1014x + 0.236R2 = 0.9398
0
0.5
1
1.5
2
2.5
0 5 10 15 20
-ln[
(∆H
-∆H
f)/(∆
H0-∆
Hf)]
-ln(N/N0)
Figure 6.7. Correlation between fractional apparent enthalpy and fractional viability for Salmonella Enteritidis FDA after HHP treatment. The cells treated without nisin (♦). The cells treated with 200 IU/ml nisin (◊).
193
y = 0.1225x + 0.133R2 = 0.9644
y = 0.1151x + 0.1865R2 = 0.9505
0
0.5
1
1.5
2
2.5
0 5 10 15 20
-ln[
(∆H
-∆H
f)/(∆
H0-∆
Hf)]
-ln(N/N0)
Figure 6.8. Correlation between fractional apparent enthalpy and fractional viability for Salmonella Enteritidis OSU 799 after HHP treatment. The cells treated without nisin (♦). The cells treated with 200 IU/ml nisin (◊).
194
DISCUSSION
For successful commercialization of HHP, a pressure processing protocol which will
employ the combination of low to intermediate pressure with antimicrobial agents and will
achieve a high level of destruction of foodborne pathogens must be developed
(Kalchayanand et al., 1998; Masschalck et al., 2001). Nisin has been approved as a food
preservative world wide. Therefore, commercially available nisin, Nisaplin, was used in
present study to evaluate the effect of pressure-nisin combination treatment on
inactivation of foodborne bacteria. Although the extent of bacterial death is directly
proportional to the pressure level, resistance to high pressure varies among the species and
among strains of the same species (Alpas et al., 1999; Robey et al., 2001; Alpas et al., 2003).
Therefore, the differences in pressure sensitivities of two different strains of Salmonella
Enteritidis were evaluated after HHP treatment with or without nisin.
The denaturation of ribosome has been considered as an important factor in death of
bacteria by both heat (Mackey et al., 1993; Mohacsi-Farkas et al., 1999; Lee and
Kaletunç, 2002b) and HHP treatments (Niven et al., 1999; Alpas et al., 2003). This study
confirms the findings of previous DSC studies that the change in the peak areas (apparent
enthalpy, ∆H) associated with denaturation of ribosome are correlated to loss of viability
in microorganisms as a function of pressure (Niven et al., 1999; Alpas et al., 2003). The
greater survivability of Salmonella Enteritidis FDA strain observed from plate counting
data (Fig. 6.3) and apparent enthalpy data (Fig. 6.6) subsequent to HHP treatment
indicates that the FDA strain have a relatively higher resistance to the pressure treatment
195
in comparison to Salmonella Enteritidis OSU 799 strain. Comparison of thermal
stabilities of ribosomal subunits for two control strains showed higher (~2oC) thermal
stability for the FDA strain. Thermal stability of ribosome has been shown to be related
to thermal resistance of bacterial cells (Mackey et al., 1993; Lee and Kaletunç, 2002b).
Thermal inactivation study on E. coli and Lactobacillus plantarum showed that the
greater thermal stability of ribosome in E. coli coincided with the greater resistance of E.
coli to heat treatment (Chapter 2). However, a recent study (Alpas et al., 2003) on
comparison of pressure sensitivities of two strains of Staphylococcus aureus
demonstrated that there is no clear relationship between the thermal stability of ribosome
and pressure resistance of bacteria. Alpas et al. (2003) observed that while S. aureus 765
has a higher thermal stability of ribosome than S. aureus 485, S. aureus 485 is more
resistant to pressure than S. aureus 765. Therefore, the relationship between the thermal
stability of ribosome and pressure resistance of bacterial strains may vary with the
different organisms.
DSC thermograms of two Salmonella Enteritidis strains also showed two
overlapping endotherms as peak d for FDA strain while a single endotherm occurred as
peak for OSU 799 strain. The endotherms had a temperature range of 115 to 130oC for
both strains are described as the denaturations of outer membrane components of Gram-
negative bacteria (Mackey et al., 1991; Lee and Kaletunç, 2002b).
The plate count results (Table 6.1) indicate that nisin itself is ineffective against
Salmonella Enteritidis strains. The finding agrees with the theory that nisin alone
treatment is ineffective against Gram-negative bacteria due to nisin-impermeable barrier
196
(outer membrane) in their cell wall (Kordel and Sahl, 1986; Delves-Broughton, 1990).
However, the susceptibility of the bacterial cells to nisin increased with pressure level
increased. It has been suggested that HHP causes alterations of structures in the outer
membrane of the cells thereby facilitating penetration of small water soluble proteins
such as nisin and lysozyme into the Gram-negative cells via permeabilized outer
membrane (Hauben et al., 1996; Kalchayanand et al. 1998). The pressure-induced
alteration of outer membrane components was confirmed by the leakage of a periplasmic
enzyme at 110 MPa and above treatments (Hauben et al., 1996) and the dissociation of
metal ions such as Ca2+ and Mg2+, which are involved in stabilization of outer membrane,
at 220 MPa and above treatments on E. coli (Hauben et al., 1997). However, there were
no apparent changes in the transitions of the outer membrane components (peak d)
detected in the thermograms of both strains after the pressure-nisin treatments (Figs. 6.4a
and 6.5b) indicating pressure-induced conformational change of the outer membrane
might be reversible due to immediate recovery after the release of pressure or nisin into
the cells. Hauben et al. (1996) reported that pressure-mediated change in permeability of
the outer membrane was not permanent at least up to 320 MPa pressure levels on E. coli
cells, and the cells regained their resistance to nisin immediately upon relief of pressure.
Another possible explanation for no change in the outer membrane transition in the
thermograms is that the energy requirements for the thermal events in outer membrane
components were not great enough to change the size of the transition.
The comparison of DSC data for pressurized (≥ 200 MPa) cells shows that the lack
of endotherm b, which is proposed to be transition of DNA denaturation, in the
197
thermograms of pressurized cells in the presence of nisin indicates that DNA might be
irreversibly damaged by the penetrated nisin in combination with the moderate HHP.
The absence of peak b and the reduction of ribosomal peaks (Figs. 6.4 and 6.5)
significantly (P<0.05) decreased apparent enthalpy (∆H) for pressure-nisin combination
treated cells than that for pressure alone treated cells. The difference of ∆H was greatest
at 250 MPa for FDA strain and 200 MPa for OSU 799 strain where a decrease in ∆H of
the pressure-nisin treated cells occurred due to the disappearance of the DNA peak (Fig
6.6). On the other hand, the difference of N/N0 increased as pressure increased and was
greatest at 400 MPa for FDA strain and 350 MPa for OUS 799 strain where tails on the
survival curves started (Fig. 6.3). Above results suggest the effectiveness of nisin
addition on the reduction of apparent enthalpy is higher at the low pressure treatment
levels while that on viability loss from plate count method is higher at the high pressure
treatment levels. These findings should be considered when apparent enthalpy data is
used to determine viability of bacteria after the pressure-nisin combination treatment.
The pressure levels less than 200 MPa enhance the stability of synthetic double
stranded DNA when the thermal stability of the DNA is higher than 50oC (Dubins et al.,
2001). Since the thermal stability (Tm) of bacterial DNA is generally 95oC, we may
expect that there is no damage on DNA structures during moderate HHP treatment.
Because little information available in literature, it is difficult to explain about the
mechanism of pressure-nisin combination treatment which irreversibly changes transition
of DNA (peak b) in DSC thermograms after 200 MPa for FDA strain and 150 MPa for
OSU strain (Figs. 6.4b and 6.5b). Possible hypothesis can be the additional dropping of
198
intracellular pH (pHi) by addition of nisin in the HHP treatment. Wouters et al. (1998)
reported that pHi of L. plantarum dropped from 7 to 5.5 after the cells pressurized at 250
MPa for 10 min while external pH was maintained at pH 5.5 during the pressurization. It
is known that HHP treatment drops pHi by damaging membrane bound ATPase which
regulates the efflux of protons (Cheftel, 1995). There is an evidence that pHi can also be
lowered by nisin treatment. In the study of the interaction between Listeria
monocytogenes and nisin, Budde and Jakobsen (2000) observed that pHi the cell
decreased from 7.9 to 5.5 in the nisin treatment (500 IU/ml, Nisaplin) for 12 min while
external pH was maintained at pH 5.5. It has been reported that the proton motive force
can be collapsed due to the dissipation of the membrane potential and the pH gradient
after nisin-induced pore formation in cytoplasmic membrane (Bruno et al., 1992; Chung
et al., 2000). DNA structure tends to be denatured in acidic conditions due to elimination
of hydrogen bonding between single strands (Cooper, 1997). Therefore, it is possible that
the damage of cytoplasmic membrane, which leads to affecting DNA by lowering pHi, is
greater when nisin is applied in HHP treatment. Further study on the measurement of pHi
of cells treated with pressure-nisin combination might be helpful to confirm above
hypothesis. In the comparison of DNA transitions in the thermograms of two strains,
there were no noticeable transition temperature and size differences between the
transitions of both DNA before and after treatment (peak b, Figs. 6.2, 6.4, and 6.5). The
result suggests that the thermal stabilities and reversibility following to pressure or/and
nisin treatments of the DNA for both strains are similar.
199
The plots in Figure 6.3 and 6.6 suggest that the fraction of apparent enthalpy [(∆H-
∆Hf)/(∆H0-∆Hf)] from DSC data is correlated to the log of fractional viability (N/N0) from
plate count data subsequent to pressure-nisin treatments for both strains. Previous
research showed that the apparent enthalpy is related to the fractional survivors after a
thermal treatment (Chapters 2 and 3). A linear relationship in the plots of [(∆H-
∆Hf)/(∆H0-∆Hf)] versus (N/N0) (Fig. 6.7 and 6.8) is observed after taking natural
logarithm. The slope of the lines from the fractions derived from the two data is lower
for pressure only treated cells than for pressure-nisin combination treated cells on FDA
strain (Fig. 6.7). The result indicates that the fraction values of apparent enthalpy data
against the fraction of plate count data for pressure only treated cells are relatively higher
than those for pressure-nisin combination treated cells. The plots of pressure only treated
cells in Figure 6.7 shows that the disparity between the apparent enthalpy data and plate
count data is greatest at 350 MPa treatment. At 350 MPa treatment, the fraction of
viability greatly dropped from 300 MPa treatment (Fig. 6.3) while the fraction of
apparent enthalpy was not much decreased from 300 MPa treatment (Fig. 6.6) for
pressure only treated cells. Thermograms in Figure 6.4 show that the main reason for
less change in the fraction of apparent enthalpy for pressure only treated cells is due to
reversibility of DNA transition (peak b). Unlike thermograms from three other treated
cells, in which the size of DNA transition is gradually decreased as the treatment pressure
increased, the DNA transition in the thermogram from pressure only treated cells of FDA
strain did not change up to 350 MPa treatment.
200
Moderate pressures (200 to 400 MPa) increased the susceptibility of both relatively
pressure-resistant and -sensitive Salmonella Enteritidis strains to nisin, although onset of
the susceptibility occurred in lower pressure level for the pressure-sensitive strain. The
pressure might cause alterations in the outer membrane of the cells thereby facilitating
penetration of nisin into the cell. Comparison of various final states achieved under
different treatment conditions starting from same initial state using DSC data allowed us
to investigate the effectiveness of combination treatment of HHP with the antimicrobial
agent for inactivation of bacteria. The apparent enthalpy data obtained from DSC can be
used to evaluate pressure levels necessary to reduce a microbial population in the
presence of nisin and provide information about viability in significantly shorter time
with comparable accuracy to plate count. Furthermore, DSC helps to identify changes in
cellular components as a function of treatment conditions. DNA might be irreversibly
damaged by the combination treatments. The finding of this combination treatment can
allow the rational design of HHP processing protocols for manufacture of
microbiologically safe and minimally processed food products.
201
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GENERAL CONCLUSIONS
The main goal of this study was to evaluate the effectiveness of physical and
chemical food preservation treatments for inactivation of bacteria using DSC. The
treatments studied included heat (linearly increasing temperature), chemical agents (HCl,
acetic acid, ethanol, NaCl, and nisin), and HHP. DSC of the whole bacterial cells
allowed us the detection of treatment induced changes in their cellular components
including ribosomes, nucleic acids, and cell wall. The changes in transition of ribosomal
subunits and the loss of cell viability appear to be related.
Since thermal processing has been the main choice of food preservation for
inactivation of bacteria in order to produce a safe product, the evaluation of thermal
sensitivity is important. The lower onset and peak temperatures of ribosomal transitions
of the L. plantarum thermogram indicate that the thermal stabilities of L. plantarum
ribosomes are lower than those of E. coli ribosomes. The viability loss was related to the
apparent enthalpy change for both organisms and L. plantarum cells were more sensitive
to heat treatment. The findings suggest heat resistance of bacteria is related to the onset
temperature and the thermal stability of the ribosomal subunit transition.
The amount of thermal energy (apparent enthalpy, ∆H) associated with denaturation
of cellular components due to heat treatment applied in DSC under linearly increasing
208
temperature conditions was found to be related to the number of viable cells of E. coli.
Similar D and z values were calculated from plate count data and calorimetric data,
indicating that apparent enthalpy obtained from calorimetric data can be used to
determine the kinetic inactivation parameters. Use of DSC data to determine kinetic
parameters can be accomplished in shorter time in comparison with plate count method
because this approach eliminates incubation time of plates thereby saving more than two
days to produce results.
Mild chemical treatment on E. coli cells resulted in a decrease in the onset and peak
temperature of ribosome transition. The transition of DNA was irreversibly affected after
HCl (pH≤4) and acetic acid (≥0.5%) treatments. Reduced survivability of chemically
treated cells in subsequent heat treatment in comparison with untreated cells indicates
mild chemical treatments affect the thermal stability of ribosomal subunits in the cell,
thereby increasing the sensitivity of bacteria to heat treatment. The thermal sensitivity
was greater for acid-treated cells due to both ribosome and DNA structures were
irreversibly affected after the treatments. The result can support the “hurdle technology”
concept, in which mild heating in conjunction with chemical agents have been utilized to
reduce processing requirements.
The effect of high hydrostatic pressure treatment, a potential non-thermal food
treatment, on E. coli was evaluated using DSC. The decrease in apparent enthalpy with
increasing pressure was mainly due to the reduction of ribosome and DNA peak size.
Comparison of the transmission electron micrographs indicated that the structures of
ribosome and DNA were damaged during HHP treatment. A close relationship of
209
fractional viability based on calorimetric data and plate count data indicates apparent
enthalpy data can be used to predict the viability of target bacteria during HHP
inactivation.
Salmonella Enteritidis is a Gram-negative bacterium, which has nisin-impermeable
barrier (outer membrane). Therefore, nisin treatment alone is not expected to inactivate
Salmonella Enteritidis. Recent HHP studies on Gram-negative bacteria showed that high
pressure combined with nisin increases the inactivation of bacteria in comparison with
HHP alone. Present study was performed to evaluate the changes in cellular components
using DSC and viability of relatively pressure-resistant and -sensitive Salmonella
Enteritidis strains after HHP treatment with and without nisin. Addition of nisin at
pressures of 200 to 400 MPa caused additional viability loss and change in DNA
transition for both strains, indicating the pressures might cause alterations in the outer
membrane of the cells thereby facilitating penetration of nisin into the cell membrane.
Addition of nisin also reduced the difference of pressure sensitivity between two strains.
There were close relationships of fractional viability based on calorimetric data and plate
count data for both strains suggest the apparent enthalpy data obtained from DSC can be
used to evaluate pressure levels necessary to reduce a microbial population in the
presence of nisin and provide information about viability.
Overall, DSC helps to identify changes in cellular components of bacteria as a
function of treatment conditions. The effects of thermal and non-thermal treatments can
be evaluated by comparing the corresponding thermograms of DSC before and after
treatment. The apparent enthalpy data obtained from DSC can be used to determine
210
viability and the kinetic inactivation parameters for bacteria. The findings of this study
help to develop the design of food processing protocols for manufacture of
microbiologically safe and minimally processed food products.
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APPENDIX
Figures and Table of the evaluation of Salmonella Enteritidis inactivation after HHP
treatment with different concentrations of nisin
225
20 40 60 80 100 120 140
0.2 mWI
350 MPa
300 MPa
275 MPa
250MPa
200 MPa
Control
Temperature (oC)
Figure Appendix.1. DSC thermograms of Salmonella Enteritidis FDA pellets after combinations of pressure and nisin (200 IU/ml Nisaplin) treatments.
226
20 40 60 80 100 120 140
0.2 mWI
350 MPa
300 MPa
275 MPa
250MPa
200 MPa
Control
Temperature (oC)
Figure Appendix.2. DSC thermograms of Salmonella Enteritidis FDA pellets after combinations of pressure and nisin (400 IU/ml Nisaplin) treatments.
227
20 40 60 80 100 120 140
0.2 mWI
300 MPa
275 MPa
250MPa
200 MPa
Control
Temperature (oC)
Figure Appendix.3. DSC thermograms of Salmonella Enteritidis FDA pellets after combinations of pressure and nisin (600 IU/ml Nisaplin) treatments.
228
0.000000001
0.00000001
0.0000001
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
0 100 200 300 400 500
0 Nisin
200 IU/mlNisaplin
400 IU/mlNisaplin
600 IU/mlNisaplin
N/N
0
Figure Appendix.4. Pressure dependeFDA determined by plate count.
Pressure (MPa)
nce of fractional viability of Salmonella Enteritidis
229
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 100 200 300 400 500
0 Nisin
200 IU/mlNisaplin
400 IU/mlNisaplin
600 IU/mlNisaplin
[(∆H
-∆H
f)/(∆
H0-∆
Hf)]
Figure Appendix.5. Pressure deFDA determined by calorimetric
Pressure (MPa)
pendence of fractional viability of Salmonella Enteritidis data.
230
Nisaplin (IU/ml)
0 200 400 600
Pressure (MPa) N1 ∆H2 N ∆H N ∆H N ∆H
0 1.3 x 109 3.96 1.2 x 109 3.91 1.2 x 109 3.88 1.1 x 109 3.83
200 1.2 x 109 3.65 3.0 x 108 2.92 1.4 x 108 2.79 1.3 x 108 2.64
250 4.8 x 108 3.62 1.4 x 107 2.81 5.3 x 106 2.76 5.7 x 106 2.63
275 8.4 x 107 3.15 1.2 x 106 2.61 1.0 x 106 2.58 1.0 x 106 2.58
300 1.3 x 107 3.00 2.1 x 104 2.22 1.5 x 104 2.16 5.6 x 103 2.10
350 6.5 x 104 2.82 1.2 x 102 2.12 <1 x 101 1.75 1.3 x 109 4.65
400 1.5 x 104 2.46 <1 x 101 1.75 1.3 x 109 4.65 1.3 x 109 4.65
450 1.9 x 103 2.18 <1 x 101 4.65 1.3 x 109 4.65 1.3 x 109 4.65
500 <1 x 101 1.65 <1 x 101 4.65 1.3 x 109 4.65 1.3 x 109 4.65 1 Viable counts (cfu/ml) in culture 2 Apparent enthalpy of cell pellet DSC
Table Appendix.1. Viability and apparent enthalpy values for cells of Salmonella Enteritidis FDA after HHP treatments in combination with nisin.
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