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Chapter 7

Analysis of Foodborne Bacteria by Differential Scanning Calorimetry

Michael H. Tunick, John S. Novak, Darrell O. Bayles, Jaesung Lee, and Gönül Kaletunç

Introduction

147

C.

perfringens and L. monocytogenes Analysis by DSC

149

Sample Preparations

149

C.

perfringens Results

150

L.

monocytogenes Results

152

Effect of Antibiotics on Bacteria

153

E.

coli and Lactobacillus plantarum Analysis by DSC

155

Sample Preparations

156

E.

coli and L. plantarum Results

156

Application of DSC for Evaluation of Food-Processing Treatments

158

Determination of Heat Inactivation Parameters of Bacteria from Calorimetric Data

158

Determination of Efficacy of Nonthermal Treatments from Calorimetric Data

161

Determination of Impact of Antimicrobials on Bacteria from Calorimetric Data

163

Conclusions

164

References

164

Introduction

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

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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 ribo- some. An Escherichia coli cell contains thousands of ribosomes, each made up of three RNA components and over 50 proteins weighing 2.5 × 10 6 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 microorganism’s D 60 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 transi- tions 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, T m ), 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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erties of lipids in cell membranes of Mycoplasma laidlawii were inves- tigated 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 endother- mic 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 inac- tivation 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 vegeta- tive 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 Kaletunç 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 cen- trifugation and resuspended in buffer consisting of 25 mM Tris (pH 7.5), 1 mM EDTA (pH 7.5), 5 mM β-mercaptoethanol, 6 mM MgCl 2 , and 30 mM NH 4 Cl. 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 centrifuga- tion and resuspended in buffer consisting of 10 mM Tris (pH 7.5), 6 mM MgCl 2 , and 30 mM NH 4 Cl 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 centri- fuging and resuspending in buffer. Antibiotics used included chloram- phenicol, 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 Perkin- Elmer 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 12–20 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 D 60 values were conducted by dilution, submerged-coil heating, plating, and enumera- tion 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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Analysis of Foodborne Bacteria 151 Figure 7.1. DSC of C. perfringens vegetative cells. Curve A, isolated

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 shoul- der disappeared with a subsequent scan without a change in baseline, proving that the ribosomal proteins were denatured by heat. The dena- turation 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 heat- shocked and control samples to flatten and shift to lower temperatures (Figure 7.1, curves D and E). The increased heat resistance of the heat- shocked cells was lost, supporting the theory that this resistance is transient (Heredia, Labbé, and García-Alvarado 1998). Heat is uni- formly 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 denatur- ation 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

to 72.1 ° ± 0.5 °C (Figure 7.2B). The position Figure 7.2. DSC of L. monocytogenes

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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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 unaf- fected (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 antibi- otic treatment produced alterations in peak denaturation temperatures corresponding to ribosomes or their subunits. Figure 7.3, curve A, is

to ribosomes or their subunits. Figure 7.3, curve A, is Figure 7.3. DSC of L. monocytogenes

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 D 60 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; chloram- phenicol, erythromycin, puromycin, rifampin, and streptomycin did not alter the D 60 values. Compared with the controls, kanamycin and tetracycline each reduced the D 60 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 anti- biotic 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. Kaletunç’s laboratory when erythromycin-treated E. coli cells were analyzed by DSC (Figure 7.4). E. coli cells suspended in HEPES buffer were treated with erythromy- cin 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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Analysis of Foodborne Bacteria 155 Figure 7.4. Thermograms of whole cells of E. coli treated with

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

gram as one broad peak. The thermogram after a treatment at a 50 µ 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 stabil- ity 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 Kaletunç 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 corre-

sponding 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 × 10 9 cfu ml 1 for E. coli and 9.0 ± 0.1 × 10 8 cfu ml 1 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 min 1 . 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 nitro- gen 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 Kaletunç 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 a 2 , shows up at a higher tempera- ture in the E. coli thermogram (70 °C) in comparison with the L. plan- tarum 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. Kaletunç’s labora- tory for Gram-negative (Pseudomonas fluorescens) and Gram-positive (Staphylococcus aureus and Leuconostoc mesenteroides) bacteria, Lee and Kaletunç (2002a) suggested the origin of this peak is a cellular component of Gram-negative bacteria, more likely to be due to lipo- polysaccharide transition. Lee and Kaletunç (2002a) also evaluated the thermal stabilities and the reversibility of individual transitions by a second temperature scan

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Analysis of Foodborne Bacteria 157 Figure 7.5. Thermograms of whole cells of E. coli (dashes) and

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 min 1 heating rate). From Lee and Kaletunç

(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 H f )/(H 0 H f )] and plate count data defined as (N/N 0 ) 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 ribo- somes, 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 ther- mograms. 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 microorgan- isms 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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is only partially irreversible and requires a higher temperature (85– 100 °C) than bacterial death (Lee and Kaletunç 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 bac- terial cells exhibit differences in thermally induced transitions, reveal- ing 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 Kaletunç 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 calo- rimetric data.

Determination of Heat Inactivation Parameters of Bacteria from Calorimetric Data

The efficacy of a given treatment for inactivation of foodborne patho- genic and spoilage microorganisms depends on the inactivation kinetics of a target microorganism. In general, bacterial inactivation is consid- ered as a first-order kinetics process. Therefore, the bacterial inactiva- tion 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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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 deter- mine 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 Kaletunç (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 tran- sitions associated with the bacterial cells surviving after the preheat. Peak areas (apparent enthalpies, H, J g 1 ) corresponding to the con- tributions of survivors were determined from the apparent heat capac- ity 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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–0.75 –1.00 –1.25 –1.50 –1.75 –2.00 –2.25 10 20 30 40 50 60 70 80
–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, N 0 is the initial number of viable cells, r is the heating rate, and D e is the D value at an arbitrary temperature T e . The value N/N 0 represents the fraction of survivors as a result of heat treatment. Lee and Kaletunç (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 Kaletunç (2002b) obtained an equation that enables one to obtain kinetic param- eters 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 quali- tative 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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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 non–spore-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; Kaletunç et al. 2004). The com- parison 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 sensi- tivities 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.

 

Fractional

 

reduction in

 

Apparent

apparent

enthalpy

enthalpy

 

Log reduction in viability log 10 (N/N 0 )

(J/g wet

(

H 0

Viable cells

Bacteria

weight)

H)/ ∆∆ H 0

(cfu/ml)

S.

aureus 485

4.0

1.6 × 10 9

Control

 

S.

aureus 485

2.7

0.32

5.0 × 10 6

2.5

345

MPa

S.

aureus 765

3.8

2.0 × 10 9

Control

 

S.

aureus 765

2.4

0.37

1.6 × 10 6

3.1

345

MPa

E.

coli

3.7

2.0 × 10 9

O157:H7

933

Control

 

E.

coli

2.8

0.24

2.0 × 10 7

2.0

O157:H7

933

275

MPa

E.

coli

3.7

1.3 × 10 9

O157:H7

931

Control

 

E.

coli

2.7

0.27

6.3 × 10 6

2.3

O157:H7

931

275 MPa

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Whereas S. aureus 765 had a relatively higher resistance to thermal treatment in comparison with S. aureus 485, S. aureus 485 was deter- mined to be more resistant to pressure than S. aureus 765. This infor- mation 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 com- monly 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 Kaletunç (2005) investigated the influence of organic (acetic acid) and inorganic (hydrochloric acid) acids, ethanol, or NaCl treat- ment 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 thermo- grams 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. mono- cytogenes 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 treat- ments, thereby providing insight into the mechanism of bacterial inac- tivation. 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.

References

Adams , M. R. , O ’ Brien , P. J. and Taylor, G. T. , 1989 . Effect of ethanol content of beer on the heat resistance of a spoilage Lactobacillus. J Appl Bacteriol,

66:491–495.

Alpas, H., Kalchayanand, N., Bozoglu, F., Sikes, A., Dunne, C.P. and Ray, B., 1999. Variation in resistance to hydrostatic pressure among strains of food-borne patho- gens. Appl Environ Microbiol, 65(9):4248–4251. Alpas, H., Lee, J., Bozoglu, F. and Kaletunç, G. 2003. Differential scanning calorim- etry of pressure-resistant and pressure-sensitive strains of Staphylococcus aureus and Escherichia coli O157:H7. Int J Food Microbiol, 87:229–237.

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