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In situ Determination of Membrane Fluidity of Endospores of 1
Clostridium spp. During Pressure-Assisted Thermal Processing in 2
Combination with Nisin or Reutericyclin 3
4
S. Hofstetter1,3), R. Winter2), L.M. McMullen1), M.G. Gänzle*1) 5
University of Alberta, Department of Agricultural, Food and Nutritional Science, 6
Edmonton, Alberta, Canada. 7
Running title: Heat and pressure effects on membranes of endospores 8
9
1) University of Alberta, Department of Agricultural, Food and Nutritional Science, 10
Edmonton, Alberta, Canada. 11
2) Technische Universität, Biophysikalische Chemie, Fakultät Chemie, Otto-Hahn Str. 6, 12
D-44227 Dortmund, Germany. 13
3) present address, Department of Civil and Environmental Engineering, University of 14
Alberta, Edmonton, Alberta, Canada. 15
*) corresponding author footnote: 16
Michael Gänzle, University of Alberta, Department of Agricultural, Food and Nutritional 17
Science, Edmonton, Canada, T6G 3P5, phone + 1 780 492 0774; fax + 1 780 492 4265; 18
e-mail mgaenzle@ualberta.ca 19
20
Copyright © 2013, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.03755-12 AEM Accepts, published online ahead of print on 18 January 2013
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Abstract (50 words) 21
This study determined membrane fluidity of clostridial endospores during 22
treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced 23
LAURDAN general polarization, corresponding to membrane fluidization. Pressure (200 24
MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on 25
heat- and pressure induced spore inactivation and membrane fluidity. 26
Keywords: Endospore, membrane, Clostridium, LAURDAN, PATS 27
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Pressure-assisted thermal sterilization (PATS) of food is an alternative to thermal 29
processing (1). PATS releases dipicolinic acid (DPA) from endospores, rehydrates the 30
core of endospores and allows for spore inactivation (2, 3). A combination of 200 to 800 31
MPa with 120°C eliminates resistant spores of C. botulinum (2, 4). At or above 120°C, 32
however, pressure may fail to accelerate thermal inactivation, or even exerts protective 33
effects (2, 4). Antimicrobials acting in concert with PATS may enhance inactivation of 34
endospores, thus ensuring product safety at reduced treatment intensity. Nisin and 35
reutericyclin exhibit antimicrobial activity against endospores (5, 6). Nisin is a pore-36
forming lantibiotic (6), reutericyclin is a proton-ionophore (7). Remarkably, nisin 37
enhances pressure-induced spore inactivation (8, 9), whereas reutericyclin had no effect 38
or even attenuated pressure-induced inactivation of bacterial endospores (10). 39
Endospores have multiple, distinct layers, that contribute to resistance and metabolic 40
dormancy (2, 11, 12). Dehydration of the spore core contributes to endospore resistance 41
(11). Endospores possess an outer membrane, a remnant of sporulation, and an inner 42
membrane separating the dehydrated core from the hydrated exterior (13, 14). Lipids of 43
the inner membrane are compressed and the surface area expands during germination 44
without lipid synthesis (15). Disruption of the inner membrane of endospores rehydrates 45
the core, and allows inactivation of endospores by antimicrobials (16, 17). An improved 46
understanding of the effect of pressure on endospore membranes will improve the control 47
of endospores by pressure, and facilitate selection of antimicrobials to support pressure-48
assisted sterilization; however, little is known about the behavior of membranes of 49
endospores during pressure processing. 50
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This study examined effects of pressure and temperature in combination with 51
nisin or reutericyclin on the membrane fluidity of endospores of Clostridium spp. Nisin 52
and reutericyclin were selected because they are both membrane active but differ with 53
respect to their mode of action, and their effect on spore survival after heat or pressure 54
treatments (6, 7, 10). Nisin and reutericyclin were applied at 16 and 6.4 mg L-1, 55
respectively, exceeding their minimum inhibitory concentration 16-fold. Experiments 56
employed Clostridium sporogenes ATCC 7955 and Clostridium beijerinckii ATCC 8260, 57
which were previously characterized with respect to spore inactivation and DPA release 58
by heat and pressure (10). Membrane fluidity changes and phase transitions were 59
assessed with the fluorescent dye LAURDAN. The LAURDAN general polarization 60
(GP) values indicate membrane fluidity of bacterial cells and endospores (18, 19). 61
LAURDAN was previously used to characterize the response of bacterial membranes to 62
high pressure, nisin, and reutericyclin (20, 21). Fourier-transform infrared spectroscopy 63
(FT-IR) scans of cells complemented information on membranes (21, 22). Details on the 64
experimental protocol used for in situ assessment of membrane fluidity are provided as 65
online supplementary material. 66
Analysis of spore membranes with FT-IR spectroscopy of endospores. FT-IR 67
spectroscopy of clostridial endospores examined changes in the hydrocarbon chain region 68
of membranes during heating to 90°C. The symmetrical CH2-stretching mode at ∼2850 69
cm-1 was analyzed (22, 23). FT-IR spectra were recorded and analyzed after drying of 70
spores on a CaF2 window as described (24). During treatment, spores of both Clostridium 71
spp. exhibited a shift at wavenumber 2849 cm-1 to 2853 - 2854 cm-1 (Figure 1 and data 72
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not shown), indicating a membrane phase transition from a gel phase to the liquid-73
crystalline phase. 74
In situ GP measurements of endospores. GP values of LAURDAN-labeled endospores 75
(19) exposed to nisin or reutericyclin at 90°C or 90°C/200 MPa were measured in situ to 76
assess membrane fluidity during treatments (25). Samples were sealed in quartz vials, 77
placed in a custom pressure vessel, and heated at ambient pressure or after compression 78
to 200 MPa. Heating to 90°C decreased GP values, corresponding to a phase transition 79
from the gel phase to the liquid-crystalline phase (Figure 2 A and C). The GP values of 80
the samples treated at 90°C did not return to initial levels upon cooling (Figure 2, A and 81
C). Higher GP values were maintained in presence of nisin when compared to control 82
samples and samples with reutericyclin. Heating to 90°C at 200 MPa also decreased GP 83
values for both Clostridium spp. but GP values remained high and indicative of a gel-84
phase membrane (Figure 2 B and D). 85
Effect of heat, pressure, and antimicrobials on spore membrane properties. 86
FT-IR and LAURDAN measurements confirm a gel state of inner membranes of 87
endospores (15, 19). A phase shift toward a liquid-crystalline phase was observed after 88
heating, in keeping with the effects of heat and pressure on model membranes (26). 89
Changes in the GP during heating of LAURDAN labeled endospores persisted after 90
decompression and overnight storage, suggesting that treatment at 90°C altered the 91
ordered state of the inner membrane of endospores. This effect may relate to the heat 92
activation of spore germination (27). In situ GP measurements of endospores heated to 93
90°C at 200 MPa highlight the antagonistic effect of pressure on fluidization of 94
membranes. A high degree of order, consistent with gel-state membranes, was maintained 95
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for all treatments of both Clostridium spp. Nisin and reutericyclin exerted divergent 96
effects on inner membrane fluidity. Nisin forms tetrameric pores (6) and increased 97
resistance to membrane fluidization by treatment at 90°C, thereby introducing order to a 98
membrane. Reutericyclin preferentially interacts with the hydrophobic interior of lipid 99
bilayers, and increases the membrane fluidity in L. reuteri (20). Reutericyclin 100
counteracted the ordering effect of pressure, but not to an extent that caused a deviation 101
from a gel-state membrane during treatment at 200 MPa. 102
Correlation of membrane properties to spore inactivation. GP values of LAURDAN-103
labeled endospores, exposed to nisin or reutericyclin at 90°C or 90°C/600 MPa, were 104
measured ex situ to relate membrane properties to spore inactivation. Spore preparation, 105
treatments, and GP measurements were performed as described (10, 19). Treatment of 106
spores at 90°C for 8 min reduced viable spore counts by less than 1 log cfu mL-1 and 107
spores released less than 5% DPA (10) but GP values of clostridial endospores were 108
lowered (Figure 3). Reutericyclin reduced GP values when compared to controls but high 109
GP-values were maintained in presence of nisin. Treatment of clostridial endospores at 110
600 MPa/90°C for 8 min reduced viable spore counts by more than 3 log cfu mL-1 and 111
released of more than 95% DPA for all treatments (10) but did not affect GP values 112
(Figure 3). 113
Spores thus retained DPA and remained viable after heating, indicating that the core 114
remained dehydrated despite the heat-induced membrane phase transition to the liquid-115
crystalline phase. Heating in presence of nisin reduced spore counts by 90% (10) but also 116
mitigated thermal effects on membrane fluidity (this study). Conversely, 90°C/600 MPa 117
treatments released DPA from clostridial endospores, and inactivated a majority of the 118
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spore population (10), but did not severely alter membrane rigidity (this study). The 119
presence of nisin during treatment at 90°C/600 MPa did not affect spore membranes (this 120
study) but accelerated endospores inactivation (10). Addition of reutericyclin resulted in 121
more fluid membranes during and after high pressure thermal processing (this study) but 122
did not consistently enhance spore inactivation (10). Taken together, these findings 123
suggest that pressure-mediated spore inactivation and release of DPA does not require 124
disturbance of the highly ordered state of endospore membranes. 125
In conclusion, high pressure counteracts the fluidizing effects of heat on the inner 126
membrane of endospores. The antimicrobials nisin and reutericyclin exert opposite 127
effects on the membrane fluidity of endospores. Reutericyclin increased disorder within 128
membranes during thermal and combined thermal and high pressure treatments. Nisin 129
facilitates a return to a highly ordered membrane state following high pressure, thermal 130
processing. These findings demonstrate that endospores can be inactivated by heat and 131
pressure without altering the highly ordered state of the membrane. Moreover, the 132
divergent effect of reutericyclin and nisin on spore membrane fluidity provides a 133
rationale for their divergent effects on heat- and pressure induced spore inactivation and 134
DPA release. 135
Acknowledgments 136
We would like to thank Kim Sørensen of Chr. Hansen HS for the gift of Chrisin. 137
We would also like to thank Yong Zhai and Shobhna Kapoor for their technical 138
assistance and expertise. This research was supported by the Natural Sciences and 139
Engineering Research Council of Canada, and the Alberta Livestock and Meat Agency. 140
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Michael Gänzle acknowledges financial support from the Canada Research Chairs 141
Program. 142
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Figure Legends. 145
Figure 1. Second derivative Fourier-transform infrared spectroscopy spectra of Laurdan-146
labeled endospores of C. sporogenes heated to 90°C. Samples were heated in the absence 147
of antimicrobials (A), in presence of 16 mg L-1 nisin (B), or in presence of 6.4 mg L-1 148
reutericyclin (C). Measurements were taken every 10 min as samples were heated at a 149
rate of 5°C /10 min. The graph highlights the area corresponding to the CH2 150
asymmetrical stretching absorbance; (i) and (ii) denoting the wavenumbers corresponding 151
to gel state and liquid crystalline membranes, respectively. Comparable results were 152
obtained with C. beijerinckii (data not shown). 153
Figure 2. Generalized polarization of Laurdan-labeled endospores of C. sporogenes (A 154
and B) and C. beijerinckii (C and D) standardized to OD600 0.5 and treated at 90°C (A 155
and C) or 90 ˚C and 200 MPa (B and D). Samples were treated in the presence of 6.4 mg 156
L-1 reutericyclin (red lines), 16 mg L-1 nisin (blue lines), or in the absence of 157
antimicrobials (black lines). Measurements were taken every 12 s during treatments. 158
Values are the average of 10 measurements. Dotted lines indicate treatment conditions as 159
follows: Panels A and C. i, Heating to 90°C; ii, hold at 90°C; iii, cooling to 4°C and 160
refrigerated storage; iv, re-scan after overnight storage at -20°C. Panels B and D. i, 161
compression to 200 MPa; ii, heating to 90°C; iii, hold at 200 MPa, 90°C; iv, cooling to 162
4°C and hold at 4°C, 200 MPa; v, measurements following decompression and overnight 163
storage at -20°C. 164
Figure 3. General polarization of Laurdan-labeled endospores of C. sporogenes 165
(triangles) and C. beijerinckii (circles) standardized to OD600 0.5 and treated at 90°C (A) 166
or 90°C and 600 MPa (B). Samples were treated in the presence of 6.4 mg L-1 167
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reutericyclin (grey), 16 mg L-1 nisin (white), or in the absence of antimicrobials (black). 168
A treatment time of 0 min corresponds to placement of samples into the 90°C bath and 169
immediate withdrawal (Panel A), or compression to 600 MPa at 90°C, followed by 170
immediate decompression to 0.1 MPa and cooling (Panel B). GP values of C. sporogenes 171
and C. beijerinckii after treatment at 600 MPa and 90°C with nisin or in the absence of 172
were essentially identical; consequently, white and black symbols in Panel B overlap. 173
Measurements were taken after holding samples overnight at 4°C. Values are the average 174
± standard deviations of 3 measurements. 175
176
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Figure 1. Second derivative Fourier-transform infrared spectroscopy spectra of Laurdan-labeled 177 endospores of C. sporogenes heated to 90°C. Samples were heated in the absence of antimicrobials (A), in 178 presence of 16 mg L-1 nisin (B), or in presence of 6.4 mg L-1 reutericyclin (C). Measurements were taken 179 every 10 min as samples were heated to 90°C at a rate of 5°C /10 min. The graph highlights the area 180 corresponding to the CH2 asymmetrical stretching absorbance; (i) and (ii) denoting the wavenumbers 181 corresponding to gel state and liquid crystalline membranes, respectively. Comparable results were 182 obtained with C. beijerinckii (data not shown). 183
2840 2845 2850 2855 2860
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Figure 2. Hofstetter et al. Generalized polarization of Laurdan-labeled endospores of C. 185 sporogenes (A and B) and C. beijerinckii (C and D) standardized to OD600 0.5 and treated 186 at 90 ˚C (A and C) or 90 ˚C and 200 MPa (B and D). Samples were treated in the 187 presence of 6.4 mg L-1 reutericyclin (red lines), 16 mg L-1 nisin (blue lines), or in the 188 absence of antimicrobials (black lines). Measurements were taken every 12 s during 189 treatments. Values are the average of 10 measurements. Dotted lines indicate treatment 190 conditions as follows: Panels A and C. i, Heating to 90˚C; ii, hold at 90˚C; iii. iii, 191 cooling to 4˚C and refrigerated storage; iv, re-scan after overnight storage at -20˚C. 192 Panels B and D. i, compression to 200 MPa; ii, heating to 90˚C; iii, hold at 200 MPa, 193 90˚C; iv, cooling to 4˚C and hold at 4°C, 200 MPa; v, measurements following 194 decompression and overnight storage at -20˚C. 195
196
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Figure 3. Hofstetter et al. 197
198
Figure 3. General polarization of Laurdan-labeled endospores of C. sporogenes 199
(triangles) and C. beijerinckii (circles) standardized to OD600 0.5 and treated at 90 ˚C (A) 200
or 90 ˚C and 600 MPa (B). Samples were treated in the presence of 6.4 mg L-1 201
reutericyclin (grey), 16 mg L-1 nisin (white), or in the absence of antimicrobials (black). 202
A treatment time of 0 min corresponds to placement of samples into the 90°C bath and 203
immediate withdrawal (Panel A), or compression to 600 MPa, followed by immediate 204
decompression to 0.1 MPa and cooling (Panel B). Measurements were taken after holding 205
samples overnight at 4˚C. Values are the average ± standard deviations of 3 206
measurements. Data on viable spore counts was obtained using the same strains and 207
identical treatment conditions (10). 208
209
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