Chapter 7

Analysis of Foodborne Bacteria by

Differential Scanning Calorimetry
Michael H. Tunick, John S. Novak, Darrell O. Bayles,
Jaesung Lee, and Gnl Kaletun

Introduction
C. perfringens and L. monocytogenes Analysis by DSC
Sample Preparations
C. perfringens Results
L. monocytogenes Results
Effect of Antibiotics on Bacteria
E. coli and Lactobacillus plantarum Analysis by DSC
Sample Preparations
E. coli and L. plantarum Results
Application of DSC for Evaluation of Food-Processing Treatments
Determination of Heat Inactivation Parameters of Bacteria from
Calorimetric Data
Determination of Efficacy of Nonthermal Treatments from
Calorimetric Data
Determination of Impact of Antimicrobials on Bacteria from
Calorimetric Data
Conclusions
References

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Introduction
The World Health Organization estimates that 325,000 hospitalizations
and 5000 deaths result from foodborne illness in the United States each
147

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year (WHO 2007). Tens of thousands of cases of foodborne illness in

the United States each year are the result of contamination by
Clostridium perfringens (Mead et al. 1999), a spore-forming anaerobe
that may initiate spore production in response to acidic conditions in
the gastrointestinal tract (Novak, Tunick, and Juneja 2001). Illness due
to Listeria monocytogenes is much less prevalent but far more serious,
leading to 500 deaths in the United States annually (Mead et al. 1999).
These and other foodborne pathogens can be inactivated by heat or
antibiotics, which alter the efficacy of protein synthesis in ribosomes.
Ribosomes, which are organelles found in the cytoplasm of all cells,
assemble amino acids into proteins by using the directions supplied by
messenger RNA molecules (Borman 2007). In bacteria, ribosomes
consist of a small 30S subunit and a large 50S subunit about twice the
size of the smaller subunit, which fit together to form the 70S ribosome. An Escherichia coli cell contains thousands of ribosomes, each
made up of three RNA components and over 50 proteins weighing
2.5 106 Da (Borman 2007). Stressing microorganisms at relatively
high or low temperatures, known as heat shocking or cold shocking,
decreases their thermal tolerance by impairing the 30S subunit
(Stephens and Jones 1993). This decreased thermal tolerance can be
measured by determining the microorganisms D60 value, which is the
length of time required for the viable population to decrease 10-fold
at 60 C.
About 35%40% of the mass of the ribosome consists of proteins,
which are analyzable by differential scanning calorimeter (DSC) if the
sample is sufficiently concentrated. Ribosomal proteins are similar to
many other proteins in that they are irreversibly denatured when heated,
producing an endothermal effect that disappears upon reheating. In
addition to ribosomes, bacterial cells contain other macromolecular
components, such as the cell envelope, nucleic acids, and proteins.
These components in whole cells go through conformational transitions upon exposure to heating in DSC. The transitions are recorded
as endothermic (heat absorption) or exothermic (heat release) peaks in
the thermogram. The area under the peak (enthalpy of transition, H)
and the thermal stability (transition temperature, Tm), of each cellular
component present on a typical DSC thermogram have been used to
characterize bacterial cells.
The first application of DSC on bacterial thermal analysis was the
study on the physical properties of biomembranes. The physical prop-

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149

erties of lipids in cell membranes of Mycoplasma laidlawii were investigated by Steim et al. (1969) with DSC, by using whole cells, cell
membranes, and extracted lipids. DSC thermograms of both isolated
cell membranes and extracted membrane lipids showed an endothermic transition around 40 C, suggesting extraction of lipids did not
change the stability. However, whole-cell thermogram did not exhibit
any distinguishable peaks (Bach and Chapman 1980).
The first successful DSC on whole cells was the study on heat inactivation and spontaneous germination of bacterial spores. Maeda and
colleagues (1974) observed that germinated Bacillus megaterium
spores had endothermic peaks at about 100 C and 130 C. For vegetative cells, Verrips and Kwast (1977) reported eight endothermic peaks
on the whole-cell thermogram of Citrobacter freundii.
It is necessary to obtain distinguishable and reproducible transitions
to identify the origin of the transitions and to examine their stability.
The resolution of peaks can be enhanced by increasing viable cell
density in the sample, by increasing sample size, and by improving the
sensitivity of DSC instrument. Recent studies showed that larger and
more distinguishable peaks can be obtained by using cell pellets instead
of cell suspensions and by using the cells at a late logarithmic growth
stage (Mackey et al. 1991; Lee and Kaletun 2002a,b).
This chapter focuses on the characterization of bacterial inactivation
by using DSC-relevant conditions on precooking, refrigerating, or
high-pressure processing of food to ensure its safety.

C. perfringens and L. monocytogenes Analysis by DSC

DSC was used to examine changes in temperatures of endothermal
effects of ribosomal proteins under cold- and heat-shocked conditions
to determine thermal tolerance of ribosomes in C. perfringens and
L. monocytogenes. In addition, L. monocytogenes cells were exposed
to several antibiotics that bind to ribosomes to mimic cold-shock
responses.
Sample Preparations
Enterotoxin-producing strains of C. perfringens were grown in fluid
thyoglycolate bacteriological medium. The ribosomes from C. perfrin-

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gens were isolated according to procedure of Novak, Tunick, and

Juneja (2001). Harvested vegetative cells were concentrated by centrifugation and resuspended in buffer consisting of 25 mM Tris (pH
7.5), 1 mM EDTA (pH 7.5), 5 mM -mercaptoethanol, 6 mM MgCl2,
and 30 mM NH4Cl. Cells were broken in a French pressure cell at
82.7 MPa, and DNase was added. Pellets of crude ribosomes were
produced by further centrifugation at 32,500 g.
Whole cells of L. monocytogenes were concentrated by centrifugation and resuspended in buffer consisting of 10 mM Tris (pH 7.5),
6 mM MgCl2, and 30 mM NH4Cl using the procedure of Bayles et al.
(2000). Investigations of antibiotic-treated cultures were performed
after exposing cells to antibiotics for 30 min at 37 C and then centrifuging and resuspending in buffer. Antibiotics used included chloramphenicol, erythromycin, kanamycin, puromycin, rifampin, streptomycin,
and tetracycline (Sigma Chemical Co., St. Louis, MO). Cold shocking
was performed by incubating at the specified temperature for 3 h.
C. perfringens cells and ribosomes were analyzed using a PerkinElmer DSC-7 equipped with an intercooler cooling accessory, and
DSC of L. monocytogenes cells was performed in a Perkin-Elmer Pyris
I with a liquid nitrogen cooler (Perkin-Elmer Corp., Norwalk, CT).
Samples weighing approximately 1220 mg were hermetically sealed
in volatile sample pans, and the appropriate Tris buffer was used as a
reference. After placing the pan in the instrument, C. perfringens
samples were cooled to 10 C and L. monocytogenes samples were
cooled to 0 C. After 2 min, samples were scanned to 100 C at 10 C/
min, and the baseline obtained from scanning the sample a second time
was subtracted, producing the final curve. At least three replicate
analyses of each sample were performed. Peak temperatures were
calculated by using the instruments software. Helium was used as the
flow gas in both instruments, which were regularly calibrated with an
indium standard.
Thermal tolerance studies and determination of D60 values were
conducted by dilution, submerged-coil heating, plating, and enumeration as described previously (Bayles et al. 2000).
C. perfringens Results
A DSC scan of ribosomes isolated from C. perfringens vegetative cells
is shown in Figure 7.1, curve A. There was an endothermal effect with

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151

Figure 7.1. DSC of C. perfringens vegetative cells. Curve A, isolated ribosomal

proteins; curve B, whole cells treated at 46 C for 60 min; curve C, whole cells treated
at 28 C for 60 min; curve D, technique for B followed by storage at 4 C for several
days; curve E, technique for C followed by storage at 4 C for several days.

a peak around 72 C, which corresponded to the 50S subunit and 70S

particle (Miles, Mackey, and Parsons 1986). A shoulder at 66 67 C
was due to the 30S subunit (Mackey et al. 1991). The peak and shoulder disappeared with a subsequent scan without a change in baseline,
proving that the ribosomal proteins were denatured by heat. The denaturation peaks of whole cells kept at 46 C (heat-shocked) and 28 C
(control) were several degrees higher (Figure 7.1, curves B and C).
The heat-shocked sample exhibited an increased resistance to heat,
indicating that the structure or conformation of the protein was altered
at elevated temperatures (Novak, Tunick, and Juneja 2001). Additional
endothermal effects were observed around 81 85 C, which have
been attributed to bacterial DNA (Miles, Mackey, and Parsons 1986).
Storage at 4 C for several days, mimicking refrigeration in a

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supermarket or by a consumer, caused the peaks of both the heatshocked and control samples to flatten and shift to lower temperatures
(Figure 7.1, curves D and E). The increased heat resistance of the heatshocked cells was lost, supporting the theory that this resistance is
transient (Heredia, Labb, and Garca-Alvarado 1998). Heat is uniformly distributed in a cell, resulting in damage to the most sensitive
molecules within it. The results suggest that conformational changes
in ribosomal proteins in response to temperature differences alter
protein synthesis in C. perfringens and that refrigeration will destroy
this organism in food. These conformational changes, which may
involve changing the shape and structure of the protein, are readily
discerned by evaluation of DSC scans.
L. monocytogenes Results
The DSC curve of L. monocytogenes cells (Figure 7.2A) exhibited
melting transitions at 67.5 0.4 C, corresponding to thermal denaturation of the 30S subunit, and at 73.4 0.1 C, corresponding to the
combined 50S subunit and 70S particle (Bayles et al. 2000). Cold
shocking the cells at 0 C for 3 h caused a shift in the 50S/70S peak
denaturation temperature to 72.1 0.5 C (Figure 7.2B). The position

Figure 7.2. DSC of L. monocytogenes cells. Curve A, control grown at 37 C; curve

B, grown at 37 C and cold shocked at 0 C for 3 h.

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153

of the 30S peak did not shift significantly. Similar results were observed
with a cold shock to 5 C. Peak shoulders observed around 81 C were
due to bacterial DNA (Miles, Mackey, and Parsons 1986), as with the
Clostridium samples. The results indicate that intracellular changes in
the ribosomes, such as an alteration in the association status of the 70S
particles, are correlated with changes in the thermal properties of L.
monocytogenes. The 30S and 50S subunits are more thermally labile
than the associated 70S particle, so any change that causes dissociation
of 70S would make the ribosome more sensitive to heat (Stephens and
Jones 1993).
Effect of Antibiotics on Bacteria
Certain antibiotics inhibit protein synthesis by selectively targeting
bacterial 70S ribosomes while leaving eukaryotic ribosomes unaffected (Weisblum and Davies 1968). The effects of seven antibiotics,
six active against the ribosome and one (rifampin) active against RNA
polymerase, were tested on the cells to determine whether the antibiotic treatment produced alterations in peak denaturation temperatures
corresponding to ribosomes or their subunits. Figure 7.3, curve A, is

Figure 7.3. DSC of L. monocytogenes cells treated with antibiotic. Curve A, control;
curve B, kanamycin-treated cells; curve C, tetracycline-treated cells.

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the DSC curve of a control similar to that in Figure 7.2, curve A, and
Figure 7.3, curve B, is the curve of cells treated with kanamycin. The
50S/70S peak shifted from 73.3 0.1 C to 72.1 0.7 C, a shift
that was similar to the temperature reduction in cells that had been cold
shocked (Figure 7.2). Treatment with tetracycline removed the 30S
transition that had been observed around 67 C (Figure 7.3, curve C).
Thus, DSC analysis showed evidence of structural changes in the
ribosomal protein. Treatment with chloramphenicol, erythromycin,
puromycin, rifampin, or streptomycin produced results that were
similar to those of the control.
Cells were also cold shocked from 37 to 0 C for 3 h and then
thermally challenged at 60 C to determine thermal tolerance. Previous
research revealed that the D60 value of L. monocytogenes is 75.6 s
(Miller, Bayles, and Eblen 2000). Kanamycin and tetracycline, which
measurably altered the DSC curves of L. monocytogenes cells, were
the antibiotics that caused reductions in thermal tolerance; chloramphenicol, erythromycin, puromycin, rifampin, and streptomycin did
not alter the D60 values. Compared with the controls, kanamycin and
tetracycline each reduced the D60 value by 20 s. These 26% reductions
were approximately the same as those observed following cold shocks
of 37 0 C (Miller, Bayles, and Eblen 2000) and 37 5 C. The antibiotic treatment data indicate that ribosomal changes have a significant
impact on the thermal resistance of L. monocytogenes. Cold shock and
certain antibiotics alter the state and modify the structure of ribosomes,
as reflected by changes in the DSC curves. The results are probably
due to disassociation of the 30S subunits, which are more thermally
labile and more effectively denatured by heat.
Similar results were observed in Dr. Kaletuns laboratory when
erythromycin-treated E. coli cells were analyzed by DSC (Figure 7.4).
E. coli cells suspended in HEPES buffer were treated with erythromycin for 40 min. With increasing concentration of erythromycin, it
appears that major ribosomal transition shifts to a higher temperature
in comparison with the thermogram of untreated cells. Furthermore,
the shape of the peak changes and becomes less broad. Erythromycin
is known to bind the 50S of bacterial ribosome, blocking the exit of
the growing peptide chain, thus inhibiting the translocation of peptide.
It can be speculated that treatment with erythromycin removes the 50S
transition from the 50S/70S peak observed in the control cell thermo-

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155

Figure 7.4. Thermograms of whole cells of E. coli treated with erythromycin, control
(thick dashes), 10 /ml erythromycin (thin dashes), 50 g/ml erythromycin (dots).

gram as one broad peak. The thermogram after a treatment at a 50 g/

ml erythromycin level therefore shows the endothermic transition
being shifted to a higher temperature because it belongs to denaturation
of 70S ribosomes, which is expected to have the highest thermal stability among the ribosomal subunits.

E. coli and Lactobacillus plantarum Analysis by DSC

When microorganisms are heated in DSC, thermograms exhibit a
number of overlapping transitions with a net endothermic effect (Miles,
Mackey, and Parsons 1986; Anderson et al. 1991; Mackey et al. 1991;
Mohacsi-Farkas et al. 1999; Lee and Kaletun 2002a). 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.

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Sample Preparations
E. coli cells were grown in trypticase soy broth, and Lactobacillus
plantarum cells were grown in MRS broth at 37 C to late exponential
growth phase. The final concentration of cells in the medium was
1.3 0.1 109 cfu ml1 for E. coli and 9.0 0.1 108 cfu ml1 for L.
plantarum. The cells were harvested by centrifugation at 10,000 g for
10 min at 4 C. The supernatant was discarded and the pellets were
washed with sterile distilled water and centrifuged for a second time
before transferring into DSC crucibles.
A differential scanning calorimeter (DSC 111, Setaram, Lyon,
France) was used to record thermograms of microorganisms heated at
a 3 C min1. All DSC measurements were conducted using fluid-tight,
stainless steel crucibles. For each DSC run, the reference crucible was
filled with distilled water equivalent to the water content of the sample.
After heating in the DSC, samples were cooled rapidly by liquid nitrogen and rescanned to evaluate the reversibility of transitions. DSC
thermograms were corrected for differences in the empty crucibles by
subtracting an empty crucible baseline.

E. coli and L. plantarum Results

DSC thermograms for E. coli and L. plantarum whole cells are shown
in Figure 7.5 (Lee and Kaletun 2002a). The peaks on the thermograms
correspond to the thermally induced transitions of cellular components.
Several differences exist between the DSC thermograms of E. coli and
L. plantarum. The major peak, peak a2, shows up at a higher temperature in the E. coli thermogram (70 C) in comparison with the L. plantarum thermogram (63 C). Another visible difference between the E.
coli and L. plantarum thermograms is a high-temperature endothermic
transition (peak d) observed only in the DSC thermogram of E. coli
whole cells. Based on the other DSC studies in Dr. Kaletuns laboratory for Gram-negative (Pseudomonas fluorescens) and Gram-positive
(Staphylococcus aureus and Leuconostoc mesenteroides) bacteria, Lee
and Kaletun (2002a) suggested the origin of this peak is a cellular
component of Gram-negative bacteria, more likely to be due to lipopolysaccharide transition.
Lee and Kaletun (2002a) also evaluated the thermal stabilities and
the reversibility of individual transitions by a second temperature scan

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157

Figure 7.5. Thermograms of whole cells of E. coli (dashes) and L. plantarum (dots)
obtained by DSC (1 to 150 C with 3 C min1 heating rate). From Lee and Kaletun
(2002a).

after preheating in the DSC to various temperatures between 40 C and

130 C. They correlated with calorimetric data viability of bacteria
subsequent to a heat treatment between 55 C and 70 C in the DSC.
The fractional viability based on calorimetric data defined as the
reduced apparent enthalpy [(H Hf)/(H0 Hf)] and plate count
data defined as (N/N0) show a linear relationship. Viability loss and the
irreversible change in DSC thermograms of pretreated whole cells are
highly correlated between 55 C and 70 C. Comparison of DSC scans
for isolated ribosomes shows 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 denaturation of
the ribosomal subunits occurred at the 50 80 C range in both thermograms. The result indicated that the ribosomal denaturation by the
DSC was associated with the 30S and 50S ribosomal subunits in
increasing order of thermal stability. This study demonstrated that
calorimetric data can be used to evaluate the viabilities of microorganisms exposed to thermal treatments. Furthermore, the relative thermal
stabilities of different organisms to heat treatment can be compared.
The calorimetric data also show that the heat denaturation of DNA
might not be a major factor of vegetative cells death because the event

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Calorimetry in Food Processing

is only partially irreversible and requires a higher temperature (85

100 C) than bacterial death (Lee and Kaletun 2002a).

Application of DSC for Evaluation of Food-Processing

Treatments
Food preservation treatments are used to inactivate microorganisms
and to enhance the shelf life of food products. The food industry uses
thermal processing as the main technology for food preservation.
However, alternative thermal processes, nonthermal processes, and
processes using mild heating in conjunction with antimicrobial agents
also have been used to preserve nutritional and textural qualities of
food materials. Preservation treatments affect cellular components of
foodborne microorganisms, resulting in physiological changes in cells
and eventually the death of bacteria. DSC thermograms of whole bacterial cells exhibit differences in thermally induced transitions, revealing the response of bacteria to heat. Thus, DSC technique allows one
to monitor and to detect the impact of thermal treatment on cellular
components of bacterial cells, including ribosomal subunits, nucleic
acids, and cell wall components. The differences in ribosomal thermal
stabilities of various bacteria are shown to be related to the thermal
tolerances of bacterial cells to heat (Lee and Kaletun 2002a; Mackey
et al. 1993; Miles, Mackey, and Parsons 1986). In this section, we will
focus on the quantitative evaluation of cell viability from calorimetric
data and the evaluation of impact of nonthermal treatments using calorimetric data.
Determination of Heat Inactivation Parameters of Bacteria from
Calorimetric Data
The efficacy of a given treatment for inactivation of foodborne pathogenic and spoilage microorganisms depends on the inactivation kinetics
of a target microorganism. In general, bacterial inactivation is considered as a first-order kinetics process. Therefore, the bacterial inactivation kinetics can be described by the D value (the time needed to reduce
the population by 1 log) and z value (temperature change required for a
1-log reduction in D value. The D and z values are determined under
isothermal conditions. However, in industrial applications, processing

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159

temperature is reached over a period of time during that a significant

reduction of microbial population may occur as the temperature rises
(Peleg 1999). Therefore, it is important to determine the D and z values
under conditions similar to those used in processing.
There are several studies in the literature modeling microorganism
inactivation during increasing temperature protocols (Reichart 1979;
Thompson et al. 1979a,b; Van Impe et al. 1992). DSC is ideally suited
to achieve heat treatment under controlled conditions of linearly
increasing temperature. Some investigators have used DSC to determine the thermally induced transitions and to evaluate the relationship
between the stability of cellular components and cell injury or death
(Miles, Mackey, and Parsons 1986; Mackey et al. 1988, 1991, 1993).
An equation describing the rate of microorganism inactivation as a
function of linearly increasing temperature was used to determine the
temperature at which the maximum death rate occurred for vegetative
cells (Miles, Mackey, and Parsons 1986) and to predict the number of
surviving microorganisms as a function of temperature at a constant
heating rate (Miles and Mackey 1994). 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.
Lee and Kaletun (2002b) used a novel approach to obtain the
kinetic parameters of E. coli K12 inactivation using calorimetric data.
E. coli pellets were preheated in the DSC to preset temperatures, were
cooled immediately by liquid nitrogen, equilibrated at 1 C, and were
rescanned to 140 C. The rescan contained the thermally induced transitions associated with the bacterial cells surviving after the preheat.
Peak areas (apparent enthalpies, H, J g1) corresponding to the contributions of survivors were determined from the apparent heat capacity versus temperature profile by integrating the area under the curve
(Figure 7.6).
Miles and Mackey (1994) derived a mathematical model describing
the number of surviving cells under linear heating conditions (Equation
7.1).

(7.1)

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Calorimetry in Food Processing

0.75
1.00
1.25
1.50
1.75
2.00
2.25
10

20

30

40

50

60

70

80

90 100 110 120 130

Figure 7.6. DSC thermogram for whole cells of E. coli K12 displaying curve baseline
used to determine the apparent enthalpy value. From Alpas et al. (2003).

where N is the number of survivors at time t, N0 is the initial number

of viable cells, r is the heating rate, and De is the D value at an arbitrary
temperature Te. The value N/N0 represents the fraction of survivors as
a result of heat treatment.
Lee and Kaletun (2002b), assuming that H is proportional to the
number of survivors, wrote Equation 7.2 to describe the fraction of
surviving cells in terms of the DSC observable:

(7.2)

By substituting Equation 7.2 into Equation 7.1, Lee and Kaletun

(2002b) obtained an equation that enables one to obtain kinetic parameters of bacterial inactivation from calorimetric data.

(7.3)

This novel approach demonstrated that calorimetric data obtained

with linearly rising temperature in DSC can be used not only for qualitative evaluation of bacterial inactivation kinetics but also quantitative
evaluation. The D and z values for E. coli K12 determined from the
calorimetric data and the corresponding values from plate count data

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161

obtained after heat treatment in the DSC and after isothermal treatment
displayed close agreement. This approach provides reproducible and
accurate results in a short time compared with the plate count technique
because the DSC approach eliminates the incubation time normally
used for plating, which might take 2 days or more.
Determination of Efficacy of Nonthermal Treatments from
Calorimetric Data
There is a growing interest in using techniques alternative to thermal
processing for food preservation to enhance safety and shelf life of
perishable foods (Hoover et al. 1989; Knorr 1993). Among nonthermal
treatment processes, high hydrostatic pressure (HHP) appears to be the
most promising technology. HHP processing has the advantage over
conventional heat treatments in that, while this technique is effective
in inactivation of nonspore-forming microorganisms, substantial food
quality retention can be retained by avoiding the destruction of small
molecular compounds such as vitamins.
It is reported that cell death increases as the level of the pressure
applied increases, implying that critical cellular activities or processes
have been irreversibly damaged (Hoover et al. 1989; Cheftel 1995).
However, the pressure tolerance varies among the species of bacteria
and even among the various strains of the same species (Styles et al.
1991; Patterson et al. 1995; Hauben et al. 1997; Alpas et al. 1999;
Benito et al. 1999).
Although DSC is a thermal analysis technique, it has been applied
to evaluate the impact of HHP processing on inactivation of bacteria
by comparing the pre- and postprocess thermograms (Niven, Miles,
and Mackey 1999; Alpas et al. 2003; Kaletun et al. 2004). The comparison of various final states as a function of various physical and
chemical factors, starting from the same initial state, makes it possible
to use DSC to predict the effectiveness of methods to inactivate
microorganisms.
Niven and colleagues (1999) demonstrated by DSC studies that cell
death due to high-pressure treatment may also be related to irreversible
ribosomal damage. Alpas et al. (2003) confirmed quantitatively that
cell viability decreases as the extent of ribosomal denaturation assessed
by calorimetry increases. The ribosomal denaturation was evaluated
by comparing the total apparent enthalpy of the control and pressure-

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treated cells and was related to the log reduction in viability.

Furthermore, they demonstrated quantitatively that the relative sensitivities to high hydrostatic pressure treatment of bacterial strains from
E. coli O157:H7 and S. aureus can be assessed from calorimetric data
(Table 7.1). The results showed that pressure and thermal tolerances
of bacteria can be different as can be the mechanism of denaturation.
Table 7.1. Apparent enthalpy and viability data for untreated control and
pressure-treated cells.

Bacteria
S. aureus 485
Control
S. aureus 485
345 MPa
S. aureus 765
Control
S. aureus 765
345 MPa
E. coli
O157:H7
933
Control
E. coli
O157:H7
933
275 MPa
E. coli
O157:H7
931
Control
E. coli
O157:H7
931
275 MPa

Apparent
enthalpy
(J/g wet
weight)

Fractional
reduction in
apparent
enthalpy
(H0
H)/H0

4.0
2.7

0.32

3.8
2.4

0.37

3.7

2.8

0.24

3.7

2.7

From: Alpas et al. 2003

0.27

Viable cells
(cfu/ml)

Log reduction
in viability
log10(N/N0)

1.6 109

5.0 106

2.5

2.0 109

1.6 106

3.1

2.0 109

2.0 107

2.0

1.3 109

6.3 106

2.3

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163

Whereas S. aureus 765 had a relatively higher resistance to thermal

treatment in comparison with S. aureus 485, S. aureus 485 was determined to be more resistant to pressure than S. aureus 765. This information can be used in the design of processes specific to targeting
certain cellular components by using different physical stresses.
Determination of Impact of Antimicrobials on Bacteria from
Calorimetric Data
Hurdle technology, which involves mild heating in conjunction with
antimicrobial agents, has been used by the food industry to preserve
nutritional and textural qualities of food while maintaining its extended
shelf life (Leistner 2000). Acids, salt, and ethanol are the most commonly employed preservatives used to reduce the intensity of the heat
treatment (Cameron, Leonard, and Barret 1980; Adams et al. 1989;
Casadei et al. 2001). The effectiveness of hurdle technology can be
enhanced if hurdles target different cellular components, thereby
reducing the tolerance of bacteria to heat treatment and preventing
cellular repair mechanisms during the storage of the food product. DSC
can be used to monitor changes in cellular components induced by
chemical agents in vivo by comparing the thermograms of bacteria
before and after treatment.
Lee and Kaletun (2005) investigated the influence of organic (acetic
acid) and inorganic (hydrochloric acid) acids, ethanol, or NaCl treatment on the cellular components of E. coli by using calorimetry and
compared the calorimetric data with viability results obtained by the
plate count method. All chemical treatments resulted in shifting of
ribosomal denaturation transition to a lower temperature, an indication
of the increasing sensitivity of the bacteria prior to heat treatment. The
comparison of the DSC thermograms of control cells with the thermograms of ethanol or acetic acid-treated cells showed, in addition to
thermal stability decrease, a major reduction in size of the ribosomal
subunit transition peak, which can be interpreted as the lower energy
requirement for denaturation of ribosomes. The observed changes in
the DSC profiles were irreversible and were associated with the loss
of viability assessed by a plate count method. The decrease in thermal
tolerance of the bacterial cell to heat treatment was chemical-specific
and a function of the chemical concentration. The heat sensitivity of
bacterial cells following an acid treatment was observed to be greater

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than in the cells treated with ethanol and salt. Differences also were
observed in the DSC profiles of bacterial cells treated with organic or
inorganic acid, suggesting that the mechanism of reduced thermal
tolerance of bacterial cells by these acids may be different. For design
of hurdle technology application in food processing, DSC studies in
vivo provide valuable information relevant to the effectiveness of
hurdles.
Conclusions
DSC is a valuable tool when investigating the effect of physical or
chemical treatments applied during food preservation on inactivation
of bacteria. Among the cellular components in a bacterial cell, the
damage to ribosomal proteins due to thermal, nonthermal, chemical,
or antibiotic treatments appears to be related to loss of cell viability.
DSC scans show that protein synthesis in C. perfringens and L. monocytogenes ribosomes is more efficiently destroyed during heating when
conformational changes and disassociation of the 30S subunits are
induced by temperature shocks. DSC thermograms display information
about the cellular components affected by various preservation treatments, thereby providing insight into the mechanism of bacterial inactivation. Furthermore, the calorimetric data can be analyzed to obtain
quantitative information about bacterial inactivation, including thermal
stability, thermal energy required for bacterial inactivation, and the
kinetic parameters of inactivation. Calorimetric data can be used to
optimize the processing conditions of food preservation in a rational
manner.
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