1

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

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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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