Journal Pre-proof

Inactivation of Listeria Monocytogenes at various growth temperatures by ultrasound

pretreatment and cold plasma

Yuanyuan Pan, Yan Zhang, Jun-Hu Cheng, Da-Wen Sun

PII: S0023-6438(19)30977-6
DOI: https://doi.org/10.1016/j.lwt.2019.108635
Reference: YFSTL 108635

To appear in: LWT - Food Science and Technology

Received Date: 2 May 2019

Revised Date: 6 August 2019
Accepted Date: 15 September 2019

Please cite this article as: Pan, Y., Zhang, Y., Cheng, J.-H., Sun, D.-W., Inactivation of Listeria
Monocytogenes at various growth temperatures by ultrasound pretreatment and cold plasma, LWT -
Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108635.

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 1 Inactivation of Listeria Monocytogenes at Various Growth Temperatures by

2 Ultrasound Pretreatment and Cold Plasma

4 Yuanyuan Pan a, b, c, Yan Zhang a, Jun-Hu Cheng a, b, c, Da-Wen Sun a, b, c, d∗

a
5 School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China
b
6 Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher

7 Education Mega Center, Guangzhou 510006, China

c
8 Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process

9 Control of Cold Chain Foods, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China
d
10 Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre,

11 University College Dublin, National University of Ireland, Belfield, Dublin 4, Ireland

12

13 Abstract

14 Effects of ultrasound pretreatments and dielectric barrier discharge (DBD) cold plasma on the

15 inactivation of Listeria monocytogenes (L. monocytogenes) under various growth temperatures were

16 investigated, and membrane fatty acid profiles of L. monocytogenes were also determined. Fatty

17 acids anteiso-C15:0, anteiso-C17:0 and C15:0 were the most abundant fatty acids at any given

18 growth temperature (10, 25, 37 and 42 °C). Principal components analysis (PCA) and redundancy

19 analysis (RDA) displayed that preconditioning growth temperatures induced noticeable

20 modifications in membrane fatty acid profile, which could be separated from each other and

21 classified roughly into four groups. In addition, growth temperature-mediated alterations in fatty acid

22 profile and membrane fluidity of L. monocytogenes were associated with cell viability, with a

23 significant increase in intracellular reactive oxygen species (ROS) levels (P < 0.01) and a significant

24 reduction in the resistance to DBD plasma exposure (P < 0.01). For inactivation kinetics, the

∗
Corresponding author. School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China.
Email: dawen.sun@ucd.ie, URLs: http://www.ucd.ie/refrig; http://www.ucd.ie/sun

1
25 sigmoidal-like model (RMSE10 °C = 0.037; RMSE25 °C = 0.055; RMSE37 °C = 0.073; RMSE42 °C =

26 0.192) with three phases (shoulder region, log-linear phase and tailing phase) was the most

27 appropriate model that could describe the inactivation curves of L. monocytogenes. Survival assays

28 and scanning electron microscopy (SEM) indicated that ultrasound pretreatments caused a

29 weakening effect on the membrane. In general, this study provided new insights on correlating

30 growth temperature-mediated alterations in fatty acid profile with intracellular ROS levels and

31 inactivation efficiency.

32

33 Keywords: DBD plasma, L. monocytogenes, membrane fluidity, kinetic models

34

35 1. Introduction

36 In the food industry, many of the sustained contamination, corruption, and safety issues related

37 to foods are caused by microorganism (Bourke et al., 2018), advanced alternative approaches such as

38 nonthermal techniques for microbial control are therefore needed to maintain the quality attributes of

39 foods. In the past decade, cold plasma has been rapidly developed as a novel nonthermal technique in

40 microbial decontamination, enzymes inactivation, and modifications of packaging materials, with

41 demonstrated effectiveness to extend the shelf life of foodstuffs (Bourke et al., 2017). However, the

42 efficacy of plasma-mediated inactivation of food-borne microorganism is affected by many factors,

43 including processing conditions (e.g., agent, associated process variables and exposure modes), food

44 matrix (e.g., solid or liquid) and microorganism (e.g., cell type, cell concentration, physiological

45 state, growth temperature) (Liao et al., 2017). Amongst them, temperature-induced environment

46 stress could affect the resistance of food-borne bacteria to nonthermal process, because

47 temperature-mediated variations in membrane fatty acid composition and its fluidity can seriously

48 affect the degree of fatty acid oxidation, the transmembrane diffusion of hydrogen peroxide (Bienert

49 et al., 2006), pH stress tolerance and the expression of critical virulence factor (Sun et al., 2012). For

2
50 example, Yusupov et al. (2017) found that lipid oxidation influenced the overall free energy barriers

51 of ROS into cells, and the oxidation of lipid tails induced a decrease of lipid order, thereby increasing

52 the bilayer fluidity as well as the permeability of the bilayer to ROS. Furthermore, Hong et al. (2014)

53 showed that cold atmospheric plasma treatment of phospholipid vesicles in eukaryotes was

54 conductive to the delivery of ROS into the vesicles with no compromise to membrane integrity.

55 Although growth temperature-induced changes in membrane fatty acid profile of microorganism

56 have been extensively studied, literature associated with its correlation with plasma-mediated

57 oxidative/acid stress are relatively limited. Therefore, it is necessary to have a deep research about

58 the correlation analysis between cultivated temperature-mediated variations in membrane fluidity of

59 food-borne bacteria and intracellular ROS levels.

60 Ultrasound, on the other hand, is another promising alternative with the ability to inactivate

61 microorganism by generating ultrasonic wave and acoustic cavitation mainly on microbial cell

62 envelopes, but some studies reported that only use ultrasound treatment can easily cause inefficient

63 inactivation (Li et al., 2017a). in particular, little is known about the synergistic effect of cold plasma

64 and ultrasound, especially, its effect on the translocation of ROS through cytoplasmic membrane into

65 cell interior to cause higher intracellular oxidative stress. In addition, Listeria monocytogenes (L.

66 monocytogenes), as a food-borne pathogenic bacteria, is capable of surviving in a wide range of pH

67 (4.1-9.0) and temperature (0-45 °C) and L. monocytogenes has already been set as zero tolerance

68 status in ready-to-eat foods by FDA, which can also easily cause infection to immunocompromised

69 people (Najjar et al., 2007). Therefore, gas chromatography-mass spectrometry (GC-MS),

70 fluorescence polarization, optical emission spectroscopy (OES), flow cytometry (FCM) combined

71 with fluorescent techniques, and scanning electron microscopy (SEM) were adopted to elucidate the

72 effects of ultrasound pretreatments and DBD cold plasma on the inactivation of L. monocytogenes

73 under various growth temperatures, and membrane fatty acid profiles as well as membrane fluidity of

74 L. monocytogenes were also examined. Fig. 1 shows the research strategy used in the current study

3
75 as compared with previously employed strategy published in literature. In general, this study could

76 provide new insights on correlating growth temperature-mediated alterations in fatty acid profile

77 with intracellular ROS levels and inactivation efficiency, suggesting the potential possibility to

78 improve or optimize the sterilization efficiency in real food systems by preconditioning different

79 growth temperatures or by ultrasound pretreatment.

80

81 2. Materials and methods

82 2.1. Strains and culture conditions

83 L. monocytogenes (ATCC19115) used in this study was provided by Guangzhou Microbial

84 Culture Center (GDMCC) (Guangzhou, China) and stored at - 80 °C. A loopful of isolated colony

85 was suspended to sterile Trypticase Soy-Yeast Extract Broth (TSB-YE) and grown at 10, 25, 37 and

86 42 °C until the required optical density at 600 nm was achieved. The corresponding growth curves

87 for L. monocytogenes at different growth temperatures are displayed in Fig. 2.

88

89 2.2. Extraction and analysis of membrane fatty acid

90 After incubation, these cultures harvested in desired phase were placed into sterilized centrifuge

91 tubes and centrifuged to obtain the pellets (2500 × g, 15 min, 4 °C). And then, the pellets were

92 washed and re-suspended twice in 0.01 M sterile phosphate buffered saline (PBS, pH 7.4) with final

93 concentrations of 107-108 CFU/mL.

94 The fatty acids in the cells from 40 mg of fresh pellets were saponified, methylated, extracted

95 and washed by following a standard protocol described in Sherlock (Sherlock 4.0 MIDI, Inc.,

96 Newark, DE, USA), and more details about this extracted method can be found in Wang et al. (2016).

97 Finally, two-thirds of the organic phase was transferred to a gas chromatograph vial for subsequent

98 GC-MS analysis.

99 The GC-MS analysis was conducted based on the method reported by Rogiers et al. (2017) with

4
100 some modifications. Briefly, the fingerprints analysis of fatty acid methyl esters was conducted by a

101 GCMS-QP2010 (Shimadzu Co., Tokyo, Japan) equipped with an InertCap 5MS/Sil capillary column

102 (30 × 0.25 mm ID, 0.25 µm film thickness) (GL Sciences Inc., Tokyo, Japan) using helium as the

103 carrier gas in a split mode (10:1). In addition, 10 µL of 10 mM methyl nonadecanoate (≥ 98.0%)

104 (Sigma Aldrich Co., St. Louis, Missouri, USA) was added to each sample (600 µL) as an internal

105 standard. More detailed process and parameters are described in the experiment of Rogiers et al.

106 (2017).

107

108 2.3. Analysis of membrane fluidity

109 Membrane fluidity can be measured as fluorescence polarization and its anisotropy value, which

110 indicates how a fluorescent probe inside the membrane reacts to polarized light (Royce et al., 2013).

111 For the measurement, 1 mL 2 µM TMA-DPH (Aladdin Reagent Co., Ltd., Shanghai, China) was

112 added to 1 mL harvested cells and incubated for 20-30 min in the dark at 37 °C, the suspended cells

113 were then centrifuged and washed by PBS to remove excessive TMA-DPH (8000 ×g, 10 min, 4 °C).

114 Bacterium suspensions transferred to sterile black Costar clear flat bottom 96-well plates (Corning)

115 were immediately used for fluorescence polarization (P) measurement, which was performed by

116 using a FlexStation 3.0 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at

117 excitation and emission wavelengths of 355 and 430 nm, respectively (Rudiger et al., 2001).

118 Fluorescence polarization from control incubations lacking the fluorescent probe was subtracted

119 from each sample. Fluorescence polarization and its anisotropy as well as microviscosity can be

120 described as follows:

121 P = (I − G ∙ I )/(I +G∙I ) (1)

122 r = (I − G ∙ I )/(I + 2G ∙ I ) = 2P/(3 − P) (2)

123 η = 2P/(0.46 − P) (3)

124 where P, r, η and G are the fluorescence polarization, anisotropy, microviscosity and instrument

5
125 correction factor, respectively. In addition, Ivv and Ivh represent the fluorescence intensities polarized

126 parallel and perpendicular relative to the direction of vertically polarized excitation beam,

127 respectively.

128

129 2.4. Plasma system configuration and ultrasound pretreatments

130 The DBD plasma system configuration (CTP-2000 K, Nanjing Suman Electronics Co., Ltd.,

131 Nanjing, China) used in this study consists of a high-frequency alternating current power source

132 (CTP-2000 K) and a DBD plasma reactor (DBD-50). The DBD plasma reactor consists of two steel

133 electrodes (Φ50 mm) and an upper quartz plate (Φ102 mm × 1 mm). The scheme diagram of the

134 chemical processes induced by DBD atmospheric cold plasma in gas-liquid interface is shown in Fig.

135 4a. For direct plasma treatment, pellets with 10 mL sterilized PBS were transferred to 60-mm Petri

136 dishes for plasma treatment at sterile laminar flow. In this case, the operating input voltage, input

137 power, frequency and the gap distance between the quartz plate and sample surface were fixed at 50

138 V, 1000 W, 10 kHz and 5 mm, respectively. Experiments were conducted at room temperature and

139 atmospheric pressure, using air as feeding gas.

140 The ultrasound treatment system (SB-600 DTY, Ningbo Scientz Biotechnology Co., Ltd.,

141 Ningbo, China) with a bath-type sonochemical reactor was applied to investigate the resistance of L.

142 monocytogenes to ultrasonic-assisted plasma treatment regime. An amount of 10 mL of the bacterial

143 suspension was transferred into a sterilized cylindrical glass tube. In order to avoid the interference

144 of thermal effects induced by ultrasound regime, the temperature of samples was maintained at

145 around 20 °C and the input power and frequency were fixed at 500 W and 40 kHz, respectively.

146 In addition, the ultrasound-plasma (UP) treatment regime referred to ultrasound pretreatments

147 for 0, 5, 10 and 15 min followed by plasma treatment for 2 min, while plasma (P) treatment regime

148 referred to only use plasma treatment for different durations (0, 1, 2, 3 and 4 min).

149

6
150 2.5. Optical diagnostics of air plasma

151 Optical emission spectroscopy was used for optical diagnostics of the reactive gas species

152 generated by DBD plasma in gas-phase (Wan et al., 2017). The emission spectra were measured

153 using a computer controlled HR2000+ spectrometer (Ocean Optics Inc., Florida, USA) with a

154 0.66-nm optical resolution and 1000-mm optical fibers. In addition, the fibers had a numerical

155 aperture of 0.22 and were optimized for use in the ultraviolet and visible portion of the spectra with a

156 wavelength range between 200 nm and 1100 nm, which were attached to an optical fiber cable with

157 1000 µm in diameter to transmit data to the spectrometer, while the distance between the optical fiber

158 and arc was controlled in 1 - 2 mm. The OES spectra were corrected for background noise and the

159 spectra were recorded every 1 s during the 4 min plasma treatment at 50 V. The data were analyzed

160 using OceanView Optics software (Ocean Optics Inc., Florida, USA). The peaks were identified by

161 comparing with the NIST Atomic Spectra Database (Xu et al., 2017).

162

163 2.6. Measurement of pH

164 A pH meter (PHSJ-4F, Shanghai INESA Co., Ltd., Shanghai, China) was used to determine the

165 pH values of treated and control samples. The samples (10 mL) were continually stirred until stable,

166 and pH was then measured at 25 °C.

167

168 2.7. Intracellular ROS level detection

169 As a cellular assay probe, 2’,7’-dichloro-dihydro-fluorescein diacetate (DCFH-DA)

170 (Sigma-Aldrich Trading Co. Ltd., Shanghai, China) has been widely applied to detect the levels of

171 intracellular ROS (Eruslanov and Kusmartsev, 2010). Intracellular esterases allow the conversion of

172 DCFH-DA into cell membrane-impermeable product DCFH, and this non-fluorescent DCFH can be

173 further oxidized by intracellular ROS into highly fluorescent 2’,7’-dichlorodihydrofluorescein (DCF).

174 The contents of DCF can be assumed to be proportional to the concentration of intracellular ROS

7
175 formation (Pan et al., 2019). Therefore, after treatment, 1 mL sample solution was incubated with

176 100 µL DCFH - DA (Sigma-Aldrich Trading Co. Ltd., Shanghai, China) at a final concentration of

177 20 µM in 0.01 M PBS for 30 min in the dark at 37 °C. The fluorescent analysis was performed by a

178 guava® easyCyte 6HT-2L flow cytometer (Merck Millipore Co., Billerica, MA, USA). Suspensions

179 were delivered at a relatively low flowrate of 400 - 600 cells/s. An amount of 200 µL sample was

180 transferred into a 96-well fluorescence microplate well and measured by FCM. The

181 dichloro-dihydro-fluorescein (DCF) emitted green fluorescence at 525 nm following excitation with

182 laser light at 488 nm. GuavaSoft (Version 2.7; Merck Millipore Co., Billerica, MA, USA) was used

183 to acquire the fluorescent events and quantify the percentage of the cells with positive staining. In

184 addition, in each sample, a minimum of 5,000 gated events was captured.

185

186 2.8. Survival assays

187 After treatments, several ten-fold dilution series with sterile saline solution (0.85% sodium

188 chloride) were used to achieve the optimum number of CFUs grown on TSA medium. Finally, all

189 plates were incubated at 37 °C from 24 to 48 h, and results were expressed in log CFU/mL.

190

191 2.9. Inactivation kinetic models

192 The kinetic models for microbial survival curves were analyzed by GInaFIT tool (Version 1.6),

193 which is a freeware tool to assess non-log-linear microbial survivor curves (Geeraerd et al., 2005).

194 Three models were adopted in the current study.

195

196 2.9.1. Weibull model

197 Weibull model is used to describe the kinetics of the survival curves of microorganisms by

198 assuming a non-linear relationship between the logarithm of survivor fraction and the treatment time

199 for a given input power, and is shown below:

8
200 ( )= ( ) − ( / ) (4)

201 where N represents the number of residual colony forming units after treatment, N0 is the initial

202 number of colony forming units, and t is the plasma exposure time (min), which is solely dependent

203 on the chosen microorganisms. In addition, δ is the necessary plasma exposure time (min) required

204 for the first log-reduction and ρ is a scale and shape parameter depicting concavity (ρ < 1) and

205 convexity (ρ > 1) as well as log-linearity (ρ = 1) of the survival curve.

206

207 2.9.2. Log-linear and shoulder model

208 In order to verify the shoulder effect of the initial resistance of cells to plasma-mediated stress,

209 the log-linear and shoulder model can be used according to Geeraerd et al. (2000):

210 ( )= ( )+ !" ∙ (# − ) ∙ ($) − %1 + $ '()* ∙(+, -.) − $ -'()* ∙. / (5)

211 where kmax is the specific inactivation rate (min-1), and S1 (min) is a parameter representing the

212 shoulder length.

213

214 2.9.3. Sigmoidal-like model

215 Sigmoidal-like model was used to describe the inactivation curves which exhibited a log-linear

216 behavior with shoulder and tailing effect, reading as follows.

( )

123
= ( )+ 0$ '()* ∙(+, -.) + × (1 − $ -'()* ∙. )5 − %1 + $ '()* ∙(+, -.) − $ -'()* ∙. /

217 (6)

218 where Nres (CFU/mL) is related to the residual population density.

219

220 2.9.4. Model performance evaluation

221 Root mean sum of squared error (RMSE) was used to describe the goodness of fit for linear or

9
222 nonlinear models by showing the difference between predicated and observed values (Buzrul and

223 Alpas, 2007). Usually, the best fit is obtained if RMSEs are in low values.

224

225 2.10. Scanning electron microscopy

226 For morphological observations by SEM, 1 mL re-suspended samples in PBS exposed to

227 ultrasonic-assisted plasma for different treatment times were selected. The glutaraldehyde solution

228 (2.5% in 0.01 M PBS, pH 7.4) was firstly employed to fix the treated samples at 4 °C overnight and

229 followed by triple rinses with 500 µL PBS (pH 7.4) (8000 × g, 5 min, 4 °C). The pellets were then

230 dehydrated in graded series of ethanol concentrations glutaraldehyde (30, 50, 70, 85, and 95%) for

231 15 min in each solution, and dehydrated twice in 100% ethanol for 20 min. It should be noted that

232 after each step, the samples needed to be centrifuged in a high speed refrigerated centrifuge (8000 ×

233 g, 5 min, 4 °C) (JW-3024HR, Anhui Jiaven Equipment Industry Co., Ltd., Heifei, China), and the

234 ethanol-dehydrated cells were replaced twice by 200 µL isoamyl acetate for 20-30 min, and 7-10 µL

235 cell samples were dropped on a silicon wafer for vacuum freeze-drying (SCIENTZ-18N, Ningbo

236 Xinzhi Bioscience Co., Inc., Ningbo, China). All cell samples were finally coated with gold (5 nm)

237 using ion sputter for approximately 2 min before SEM imaging. Cell imaging was performed with a

238 high-resolution Merlin SEM (Zeiss Merlin Field Emission SEM, Carl Zeiss NTS GmbH,

239 Oberkochen, Germany) at 5 kV.

240

241 2.11. Data analyses

242 2.11.1. Principal components analysis (PCA)

243 PCA summarizes a data set with many variables by creating a few new variables (called

244 principal components, PCs) containing most of the information (Lv and Yang, 2012). Therefore, PCA

245 was thus adopted to further overview clusters and identify the difference in eight identified fatty

246 acids, which was carried out by SIMCA-P software (Version 14.1, Umetrics, Umea, Sweden).

10
247 2.11.2 Redundancy analysis (RDA)

248 RDA is usually used to describe the response of taxa to environmental gradients (Shen et al.,

249 2014). Therefore, considering the environment effects (growth temperatures), the CANOCO

250 software (Version 4.5, Microcomputer Power, Ithaca, NY, USA) was adopted to perform RDA of the

251 correlation between membrane fatty acid composition and environmental factors. The cosine of the

252 angle between two arrows represents the approximated correlation between two corresponding

253 variables (Shen et al., 2014). Usually, an angle less/more than 90° represents a positive/negative

254 correlation, whereas an angle close to 90° means almost no correlation (Pan et al., 2016).

255

256 2.11. Statistical analysis

257 OriginLab 8.0 software (OriginLab Inc., Northampton, MA, USA), SPSS software (version 20.0)

258 (IBM Analytics, NY, USA), SIMCA-P software (version 14.1, Umetrics AB, Umea, Sweden) and

259 CANOCO software (version 4.5, Microcomputer Power, Ithaca, NY, USA) were adopted to perform

260 statistical analysis. One-way or multifactorial analysis of variance (ANOVA) was applied to evaluate

261 the antimicrobial effect and intracellular ROS levels. In addition, the differences were further

262 determined by the least significant difference test (LSD) at p < 0.05 level. Each experiment was

263 repeated in triplicate and averaged data were reported.

264

265 3. Results and discussion

266 3.1. Principal components analysis and redundancy analysis of membrane fatty acid profile

267 The effects of growth temperatures on membrane fatty acid profile of L. monocytogenes are

268 displayed in Table 1 and Fig. 3a. The results indicated that eight identified fatty acids were

269 specifically divided into straight chain fatty acids (SCFAs) (including C14:0, C15:0, C16:0 and

270 C18:0) and branched chain fatty acids (BCFAs) (including iso-C14:0, anteiso-C15:0, iso-C16:0 and

271 anteiso-C17:0), and there was a higher proportion of BCFAs at any given temperatures (> 78%). As

11
272 shown in Table 1, the most abundant fatty acids were anteiso-C15:0 and anteiso-C17:0 as well as

273 C15:0 at any given temperatures, and this pattern was also observed in most published papers

274 (Julotok et al., 2010; Juneja et al., 1998). In addition, at low temperature of 10 °C, the cells survived

275 with more abundant proportion of anteiso-C15:0 (60.29%) and BCFAs (83.83%). This result

276 indicated that anteiso-C15:0 with low phase transition temperature (Tm) (around -16.5 °C) was the

277 major regulator of BCFAs contents (Kaneda, 1991), and higher proportion of BCFAs in microbial

278 membrane might attribute to self-protection of L. monocytogenes to ensure sufficient membrane

279 fluidity for survival in low temperature perturbation (Zhu et al., 2005). Simultaneously, with an

280 increase of temperature from 10 °C to 42 °C, there was a concomitant increase in the proportion of

281 anteiso-C17:0 from 17.31% to 36.28% (P < 0.05) (Fig. 3a). These results confirmed the adaptation

282 mode of L. monocytogenes to low growth temperature by augmenting the amount of odd-numbered

283 fatty acids or shortening the length of fatty acid chain (Annous et al., 1997; Mastronicolis et al.,

284 2005). More interestingly, from the distribution of fatty acids in Fig. 3a, mild cultivation scenarios at

285 25 or 37 °C seemed to form more uniform fatty acids profile in microbial membrane than at low or

286 high growth temperature (10 or 42 °C). However, fatty acids C14:0 and iso-C14:0 were observed in

287 minor proportions, i.e., < 1.5%, and fatty acids C18:0 was found in the range from 1.07% to 2.33%

288 (Table 1).

289 Fig. 3b shows the result of score plot of PCA using t1 vs t2 from relative peak areas of fatty acid

290 composition in GC-MS fingerprints of L. monocytogenes at various growth temperatures. The first

291 two components of the model explained 78.4% (R2X [1] = 0.473, R2X [2] = 0.311) of the variance

292 and predicted 67% of the variance (R2 = 0.987, Q2 = 0.67). As the parameters of the PCA model (R2

293 and Q2) were more than 50%, the analytical protocol employed in this study was sufficiently robust

294 for recognition analysis. As shown in Fig. 3b, the samples could be separated from each other and

295 classified roughly into four groups in the score plot using two principal components, which was

296 consistent with the actual situation. Additionally, the group cultivated at 10 °C was obviously

12
297 separated from other groups, implying that the fatty acid profile of 10 °C was significantly different

298 from the other three groups. Thus, PCA result in Fig. 3b indicated that preconditioning growth

299 temperatures induced noticeable modifications of membrane fatty acid profile during cultivation.

300 Fig. 3c shows the result of biplot of the RDA relating to the correlation between relative

301 abundance of fatty acid composition and environmental factors. The correlation coefficient of fatty

302 acids profile and environmental factors axis was 0.971, indicating that a linear combination could

303 well reflect the relationship between the fatty acids and temperatures. In addition, the first axis of the

304 RDA biplot explained 49.2%, while the second axis explained 45.7% of the variation in fatty acids

305 content. As shown in Fig. 3c, the fatty acids anteiso-C15:0 and C14:0 mainly located near the arrow

306 of low growth temperature (10 °C) were negatively correlated with temperature. On the contrary, the

307 fatty acids anteiso-C17:0 and C15:0 mainly located near the arrow of high growth temperature

308 (42 °C) were positively correlated with temperature. This may be related to the common assumptions

309 that the biosynthetic pathway of anteiso-C15:0 would compete with anteiso-C17:0 during low

310 temperature perturbation (Zhu et al., 2005).

311

312 3.2. Membrane fluidity

313 For microorganisms in plasma-mediated oxidative stress, biomembranes are one of the

314 preferential targets that are very susceptible to the attack of reactive species. Meanwhile,

315 biomembranes are the direct vehicles of reactive species into cells for further intracellular damage.

316 However, membrane fluidity is involved in many cell functions such as permeability, lateral motion

317 of membrane constituents and osmotic stability of cells, which directly affects the delivery of ROS

318 into cells (de la Haba et al., 2013; Hong et al., 2014). Therefore, in order to establish the relationship

319 between membrane fluidity and plasma resistance of L. monocytogenes, the development of

320 membrane fluidity was conducted in this study.

321 Fig. 2 also shows the effect of different growth temperatures on fluorescence polarization (P)

13
322 and anisotropy (r) as well as microviscosity (η) of cytomembrane of L. monocytogenes. With the

323 growth temperature increasing from 10 °C to 37 °C, a significant increase in P values and r values of

324 unadapted L. monocytogenes was observed (P < 0.05), corresponding to a decrease of membrane

325 fluidity (Fig. 3c). The decrease in membrane fluidity implies a more motion-restricted (more orderly)

326 environment at higher temperatures (Edgcomb et al., 2000). However, with the growth temperature

327 further increasing from 37 °C to 42 °C, a slight decline in P values (P > 0.05) and r values (P > 0.05)

328 was witnessed, that is, a higher membrane fluidity was displayed. Additionally, as depicted in Fig. 3d,

329 similar to anisotropy results, microviscosity (η), which was determined according to Eq. (3), showed

330 an obvious increase with elevated growth temperatures (10 °C to 37 °C). This trend implied that the

331 rigidity of membrane was increased with elevated growth temperatures. However, compared with

332 37 °C, η values declined from 0.66 to 0.59 at 42 °C, corresponding to a decreased in the rigidity of

333 membrane. These results were in agreement with the observation in Staphylococcus aureus (Wang et

334 al., 2016) and Salmonella typhimurium (Yun et al., 2016) when they were cultivated at different

335 temperatures. For example, Liu et al. (2017) reported the research results related to the change of

336 membranal fluorescence anisotropy of Escherichia coli after growing at different stages

337 (exponential/stationary stage) and temperatures (15, 20, 30, 37 and 45 °C). Regardless of growth

338 stage, a significant increase was witnessed in fluorescence anisotropy with increasing growth

339 temperatures. However, for the cells at stationary stage, a slight decline in fluorescence anisotropy

340 was also observed when the growth temperature was further increased from 37 °C to 42 °C. It has

341 been intensively studied that the modification of membrane fluidity was attributed to the Tm of their

342 respective fatty acid profile within membrane phospholipids at a given growth temperature (Kaneda,

343 1991). Therefore, as described above, the reason for the turning point of membrane fluidity and

344 rigidity may be associated with the phase transition of membrane lipids of L. monocytogenes

345 (Edgcomb et al., 2000).

346

14
347 3.3. Diagnosis of reactive gas species

348 Fig. 4b shows the main peaks of OES corresponding to the emissions of excited species

349 generated by DBD plasma in air at 50 V over the range of 200 to 1100 nm. The bulk of the observed

350 emission was in the near-UV region corresponding to the emissions of excited species of atomic

351 nitrogen, namely the second positive system N2(C-B) (around 316, 337, 353, 370, 375, 380, 400, 405

352 nm) and first negative system N2+(B-X) (around 357, 394, 434 nm), while there were nearly no

353 obvious strong peaks in the region of visible and near-infrared regions, which was in line with the

354 results of Wan et al. (2017). The major peak around 313 nm was also identified, which could be

355 assigned to the optical transition of CO (Connolly et al., 2013). In addition, a NO emission band

356 around 297 nm was also noted (Walsh et al., 2010). However, the peaks (e.g., 777 and 844 nm)

357 associated with optical transitions of O atom in air plasma was observed at extremely low intensities,

358 which was most likely related to the quenching of O I (5P) and O I (3P) (Walsh et al., 2010). These

359 results pointed to the fact that DBD plasma was a significant source of ample reactive nitrogen

360 species (RNS) and ROS, which could induce the oxidative stress and then caused the microbial

361 inactivation.

362

363 3.4. The development of pH

364 Fig. 4c shows the development of pH values in bacterium suspension subjected to DBD plasma

365 treatment after growth at different temperatures. There was a gradual decline in pH values when

366 plasma treatment time was increased from 0 to 3 min, and a sharp drop at relatively low pH values

367 (around 3.16) was then observed when plasma exposure time was extended to 4 min. These results

368 indicated that air plasma discharge could lead to the acidification of suspensions, which was

369 favorable for the penetration of ample reactive species into cell walls (Ma et al., 2016). When air was

370 used as the working gas, ample RNS (e.g., species nitric acid (HNO3), nitrous acid (HNO2)) were

371 generated in liquid matrixes, which could explain the corresponding decline in pH values (Tian et al.,

15
372 2015).

373

374 3.5. Intracellular ROS levels

375 In order to assess the oxidative stress within the cells after ultrasonic-assisted plasma exposure,

376 FCM combined with fluorescent techniques was adopted to measure the abundance of intracellular

377 ROS and RNS. The percentage of DCFH-DA stained cells is correlated with the abundance of

378 intracellular ROS. The histogram representing DCF fluorescent signals of L. monocytogenes cells

379 cultivated at 10 °C is displayed in Fig. 5a. For the negative control group without adding DCFH-DA

380 at 10 °C, the DCF fluorescent signals of cells were weak (0%), which was the prerequisite for the

381 following assay. Without ultrasound pretreatment, the percentage of DCFH-DA stained cells

382 measured by FCM was remarkably elevated from 4.73% to 95.81% as a function of treatment time

383 from 0 min to 4 min (P < 0.05), which exhibited a plasma treatment time-dependent behavior.

384 Usually, during aerobic metabolism, the constantly generated intracellular ROS played a vital

385 protective and functional role in the immune system, which explained the reason why the percentage

386 of DCFH-DA stained cells of control group (P-0 min) was 4.73% (Eruslanov and Kusmartsev, 2010).

387 On the other hand, by taking the assistance of ultrasound pretreatment into consideration, the

388 percentage of DCFH-DA stained cells was significantly (P < 0.05) increased during the first UP-5

389 min treatment (36.04% to 48.40%), and then a sudden successive descent was observed when the

390 ultrasonic-assisted pretreatment time was extended to 10 min (43.15%) and 15 min (42.34%). In

391 agreement, a similar result was reported by Liao et al. (2018), who assessed the bactericidal effect of

392 nonthermal plasma assisted ultrasound treatment on Staphylococcus aureus. Therefore, a hypothesis

393 could be proposed from the current study that short-time ultrasonic-assisted (UP-5 min) plasma

394 pretreatment created pores in membrane to facilitate the penetration of ROS into cells, however, cells

395 rupture would happen when ultrasonic-assisted pretreatment time was prolonged to 10 min or 15 min,

396 which ultimately caused the leakage of intracellular ROS. The following assay of morphological

16
397 observations could further support this hypothesis.

398 In order to further assess the effect of growth temperatures on the abundance of intracellular

399 ROS, L. monocytogenes grown at different temperatures was treated by ultrasonic-assisted plasma,

400 and results are shown in Fig. 5b. On the whole, after exposure to plasma treatments for different

401 times (0, 1, 2, 3 and 4 min), the results based on multifactorial ANOVA revealed that significant

402 difference (P < 0.01) in intracellular ROS level was only observed between the L. monocytogenes

403 cells cultivated at 10 °C and the other temperatures (25, 37 and 42 °C). This result was consistent

404 with results from PCA based on membrane fatty acid composition, showing that cells grown at 10 °C

405 was prominent and different from other groups. In addition, an interesting phenomenon was

406 observed, showing that pronounced difference in initial endogenous ROS level appeared to be

407 associated with the aerobic metabolism of L. monocytogenes. To be more specific, for the control

408 group (P-0 min), the percentage of DCF-stained L. monocytogenes cells was increased from 4.73%

409 to 7.72% when growth temperature was increased from 10 °C to 42 °C. This implied that the aerobic

410 metabolism of L. monocytogenes was improved at higher temperatures (42 °C). According to

411 previous results reported by Schulte (2015), the generation of intracellular ROS by aerobic

412 metabolism was increased with environmental temperature, but intracellular ROS also had the

413 potential to damage cellular macromolecules such as lipids, proteins and DNA, which is not

414 conducive to the survival of living tissue. Perhaps this is the reason why L. monocytogenes cultivated

415 at high temperatures with higher endogenous ROS level was more sensitive to plasma treatments. On

416 the other hand, at other growth temperatures (25, 37 and 42 °C), a trend similar to that at 10 °C was

417 observed when samples were exposed to plasma treatments (0, 1, 2, 3 and 4 min) alone and to

418 ultrasonic-assisted plasma pretreatments for different times (0, 5, 10, 15 min). However, compared

419 with the L. monocytogenes cells cultivated at 10 °C (36.04%), plasma treatment for 2 min induced an

420 up to 2.02-fold (72.66%), 2.38-fold (85.63%) and 2.53-fold (91.10%) augment of the percentage of

421 DCFH-DA stained cells at 25 °C, 37 °C and 42 °C, respectively. Under the same plasma exposure

17
422 condition, these results could explain that L. monocytogenes cells cultivated at higher temperatures

423 appeared to be more prone to the penetration of DBD plasma-mediated ROS into cells interior.

424 Xiong et al. (2011) reported that cold plasma-mediated ROS could penetrate 15 µm thick

425 biofilms and effectively deactivated the Porphyromonas gingivalis with no damage to the biofilm

426 structures of cells, and it was also confirmed that the length of fatty acids, the ratio of unsaturated

427 versus saturated fatty acids indeed affected the membrane permeability and transmembrane diffusion

428 of ROS (Bienert et al., 2006; Sousa-Lopes et al., 2004). Additionally, molecular dynamics

429 simulations were conducted by Cordeiro (2014) to determine the distribution, mobility and

430 permeation of ROS at phospholipid bilayers, indicating that there was no significant energy barrier

431 for the permeation of hydrophobic species such as molecular oxygen (O2) and singlet oxygen (1O2),

432 which were mainly accumulated at the membrane interior. However, hydrophilic ROS, such as

433 hydrogen peroxide (H2O2) and hydroxyl (HO) as well as hydroperoxyl (HO2), were involved in

434 long-lived H-bonds interactions with lipid carbonylester groups, which led to severe impairment of

435 their lateral mobility along the membrane surface. From the analysis of membrane fatty acid profile

436 in the current study, L. monocytogenes cells, inoculated at low temperatures with more abundant

437 short-chain fatty acids (anteiso-C15:0), were more prone to interacting with hydrophilic ROS,

438 impairing the permeation efficiency of ROS into cells interior. Thus, the abundant proportion of

439 long-chain fatty acids (anteiso-C17:0) after inoculation at high temperatures was responsible for the

440 high intracellular ROS levels of L. monocytogenes cells after plasma treatment.

441 The results from intracellular ROS level and membrane fluidity as well as membrane fatty acid

442 profile analysis showed that growth temperatures had remarkable effects on the plasma-mediated

443 accumulated abundance of intracellular ROS that ultimately contributed to fragmenting nuclear DNA

444 or other cellular macromolecules (Ma et al., 2013). However, more investigations are still needed to

445 have a better understanding of the distribution, penetration and interaction mechanism of

446 plasma-mediated ROS from the bulk liquid phase to membrane interior at a molecular level

18
447 especially in real food systems.

448

449 3.6. Survival assays and its relationship with membrane fatty acid profile

450 The effect of different growth temperatures on the resistance of L. monocytogenes was

451 determined by the average number of CFUs grown on TSA medium after exposure to plasma only

452 (Fig. 6a) and to ultrasonic-assisted (Fig. 6b) pretreatments for different periods. On the whole, after

453 exposure to plasma treatment for 0, 1, 2, 3 and 4 min, the results based on multifactorial ANOVA

454 revealed that there were significant differences (P < 0.01) in different growth temperatures (10, 25,

455 37 and 42 °C), except between 25 °C and 37 °C. However, Fernandez et al. (2013) reported that the

456 growth temperatures in the range from 20 °C to 45 °C did not significantly affect (P > 0.05) the

457 resistance of Salmonella enterica serovar Typhimurium to cold atmospheric plasma inactivation,

458 probably due to the different optimal survival temperature ranges and strains used in their study. As

459 shown in Fig. 6a, for short time treatment of 1 min, the reduction in the number of L. monocytogenes

460 cultivated at different temperatures was limited (< 0.4 log), but the inactivation degree of L.

461 monocytogenes was the highest (0.37 log) at 37 °C, corresponding to the implication that L.

462 monocytogenes cultivated at 37 °C with lower membrane fluidity was more sensitive to plasma

463 exposure. However, as treatment time progressed, the inactivation efficiency of L. monocytogenes

464 was significantly enhanced (P < 0.01), but L. monocytogenes cultivated at around 42 °C was found to

465 be the most sensitive growth temperature range to plasma exposure. For example, L. monocytogenes

466 cultivated at 10, 25 and 37 °C yielded 2.50, 3.16 and 3.29 log-reduction of viability for 4 min plasma

467 treatment, respectively, and the maximum log-reduction of 3.78 log was at 42 °C. Therefore, a

468 hypothesis could be proposed that when L. monocytogenes survived in relatively mild oxidative

469 stress, i.e., plasma exposure for 1 min, L. monocytogenes cultivated at 42 °C with higher endogenous

470 ROS levels was conducive to its transient adaptation to initial mild oxidative stress (De Abrew

471 Abeysundara et al., 2016). Furthermore, with the progress of plasma treatment times, the advantage

19
472 of oxidative stress adaptation was weakened, while the impact of long-chain fatty acids (such as

473 anteiso-C17:0) gradually dominated.

474 On the other hand, as depicted in Fig. 6b, for cells grown at 37 °C, plasma only treatment for 2

475 min resulted in 1.59 log-reduction of L. monocytogenes viability, while ultrasound pretreatment for 5

476 min followed by plasma treatment for 2 min was enough to achieve 1.88 log-reductions of viability.

477 Upon prolonged ultrasonic-assisted pretreatment time for 15 min, the inactivation level was

478 increased to 2.14-fold. Similar inactivation effectiveness was also observed at other growth

479 temperatures (10, 25 and 42 °C). In addition, the effect of growth temperatures on bacterial

480 resistance to ultrasonic-assisted plasma treatment was consistent with plasma only treatments.

481 However, some researchers believed that bacterial cells inoculated at low cultured temperatures

482 exhibited low stress resistances to thermal or some nonthermal processing techniques (Wang et al.,

483 2016). For example, Álvarez et al. (2002) reported that L. monocytogenes was less resistant to pulsed

484 electric fields if strain was grown at low temperatures. Likewise, Juneja et al. (1998) showed the

485 effect of growth temperatures on heat resistance of L. monocytogenes and indicated that the D value

486 (the decimal log-reduction time required at a given heating temperature) was significantly increased

487 with increasing temperatures (10, 19, or 37 °C) when the pH of the growth medium was 7, whereas

488 D value was significantly decreased (P < 0.05) with increasing growth temperature at pH 5.4.

489 However, literature available to the effect of growth temperatures on plasma resistance was scarce

490 except a report by Fernandez et al. (2013), who found that the inactivation efficiency of Salmonella

491 enterica serovar Typhimurium was independent on growth temperatures of 20, 25, 37 and 45 °C. In

492 the present study, with increase in growth temperatures, growth temperature-mediated alterations in

493 fatty acid profile were accompanied by a decline in resistance to cold plasma. It is possible that

494 plasma-mediated low pH (around 3.16) and different inactivation mechanisms can account for the

495 different inactivation trends among these processing techniques.

496 As mentioned above, the length of fatty acids indeed affected the membrane permeability and

20
497 the transmembrane diffusion of ROS (Bienert et al., 2006), while the intracellular accumulation

498 levels of exogenous ROS had a high positive correlation with the inactivation efficiency (Liao et al.,

499 2018). Therefore, growth temperature-mediated alterations in membrane fatty acid profile of L.

500 monocytogenes were associated with its viability and resistance to DBD plasma exposure.

501

502 3.7. Inactivation kinetics

503 It is important to have a reliable mathematical model that can accurately describe the

504 inactivation kinetics of microbial population to establish appropriate plasma processing conditions

505 (Fröhling et al., 2012). The results of the statistical analysis on kinetics changes of L. monocytogenes

506 cells subjected to DBD plasma are listed in Table 2, and the associated available fitting curves are

507 shown in Fig. 6. Many kinetic models are available with Weibull model as the predominant model

508 for plasma-mediated inactivation (Fröhling et al., 2012), however, as indicated in Table 2, the

509 Weibull model in the current study provided some details about shape parameter (ρ), but the fitting

510 was not adequate because there was a shoulder or tail effect in plasma-mediated inactivation.

511 Therefore, based on RMSE values, the sigmoidal-like model was the most appropriate model that

512 described the inactivation curves. In the sigmoidal-like model, all parameters have a clear

513 biological/graphical meaning and three phases (shoulder, log-linear phase and tailing region) are easy

514 to recognize (Geeraerd et al., 2005). The initial counts of L. monocytogenes inoculated at 10, 25, 37

515 and 42 °C before plasma treatment were 6.50 ± 0.102 log, 7.35 ± 0.052 log, 8.06 ± 0.040 log and

516 7.27 ± 0.023 log, respectively, and the shoulder length (S1) parameters of L. monocytogenes resistant

517 to initial plasma exposure were 2.389 ± 0.028 min, 2.097 ± 0.078 min, 1.016 ± 0.233 min and 1.926

518 ± 0.104 min, respectively. Usually, inactivation data exhibiting a very short shoulder phase should

519 suggest that the initial threshold of bacterial resistance is relatively easy to overcome (Perni et al.,

520 2006). Therefore, contrarily to the above, the current result further emphasized that L.

521 monocytogenes inoculated at low temperature (10 °C) with high membrane fluidity had a strong

21
522 initial resistance to plasma treatment (Shivaji and Prakash, 2010). In addition, the tailing phase is

523 usually a manifestation of a heterogeneous population, some of which are more resistant than others,

524 or shows some physical phenomena such as cell aggregation, where those cells at the exterior of a

525 clump can effectively protect those at the interior (Perni et al., 2006). In the current study, the

526 residual population density (Nres) of L. monocytogenes inoculated at different temperatures after

527 plasma treatment was applied to evaluate the tailing effect, and the result is shown in Table 2.

528 In fact, this sigmoidal-like model has been applied to survival curves, such as the acid tolerance

529 response of Salmonella enterica (Greenacre et al., 2003), a mild thermal inactivation of L.

530 monocytogenes (Geeraerd et al., 2000), and cold plasma-mediated inactivation kinetics of Listeria

531 innocua and Escherichia coli (Fröhling et al., 2012). However, literature about sigmoidal-like model

532 for plasma-mediated bacterial inactivation is still limited, and the current study should provide new

533 insights about plasma-mediated bacterial inactivation kinetics.

534

535 3.8. Morphological observations

536 SEM analysis was utilized to observe the morphological change of L. monocytogenes cells

537 grown at 25 °C by ultrasound-assisted plasma treatment (Fig. 7). Smooth and intact short rods were

538 observed in L. monocytogenes samples before treatment (Fig. 7a), whereas the morphology of treated

539 cells was changed after the ultrasound-assisted plasma exposure. In details, after plasma only

540 exposure for 2 min, most of the cells exhibited rough surfaces and were shrunken with discrete

541 ridges (red arrows) (Fig. 7b). Prolonging plasma exposure time to 4 min led to more obviously

542 profound shrinkage and deformation but with no rupture (Fig. 7d). Similar observations were

543 reported in earlier work regarding plasma-mediated inactivation mechanism of Staphylococcus

544 aureus (Han et al., 2015). Therefore, the major target of plasma-mediated inactivation of L.

545 monocytogenes (G+) was cellular macromolecules. However, ultrasound pretreatment for 15 min

546 followed by plasma treatment for 2 min not only caused the shrinkage and deformation, but also

22
547 considerably higher number of collapsed cells (Fig. 7c). This result confirmed the preceding

548 hypothesis that prolonging ultrasonic-assisted pretreatment time to 15 min followed by 2 min plasma

549 treatment caused the leakage of intracellular ROS. According to Li et al. (2017a and 2017b), after

550 ultrasound treatment, only partial pore formation and disruption of morphological changes in

551 Staphylococcus aureus were observed. The current results indicated that ultrasound pretreatment

552 caused a weakening effect, which then increased the susceptibility of L. monocytogenes cells to the

553 subsequent cold plasma treatment.

554

555 4. Conclusions

556 In the current study, effects of growth temperatures on membrane fatty acid profile of L.

557 monocytogenes were firstly examined. Their correlation with oxidative stress induced by ultrasound

558 pretreatments and DBD cold plasma treatments were then investigated. Fatty acids anteiso-C15:0,

559 anteiso-C17:0 and C15:0 were the most abundant fatty acids at any growth temperatures (10, 25, 37

560 and 42 °C), while the cells survived in low temperature perturbation (10 °C) with more abundant

561 proportion of anteiso-C15:0 (60.29%) and BCFAs (83.83%). Both PCA and RDA based on relative

562 peak areas of fatty acid composition in GC-MS fingerprints of L. monocytogenes at various growth

563 temperatures displayed that preconditioning growth temperatures induced noticeable modifications

564 of membrane fatty acid profile during cultivation. In addition, growth temperature-mediated

565 alterations in fatty acid profile and membrane fluidity of L. monocytogenes were associated with its

566 viability to DBD plasma exposure and accompanied by significant augment in intracellular ROS

567 levels (P < 0.01) and significant decline in resistance with increase in growth temperatures (P <

568 0.01). With respect to inactivation kinetic models, the sigmoidal-like model (RMSE10 °C = 0.037;

569 RMSE25 °C = 0.055; RMSE37 °C = 0.073; RMSE42 °C = 0.192) with three phases (shoulder region,

570 log-linear phase and tailing phase) was the most appropriate model to describe the inactivation

571 curves of L. monocytogenes. The results obtained from survival assays and SEM indicated that

23
572 ultrasound pretreatment caused a weakening effect, which could then increase the susceptibility of L.

573 monocytogenes cells to the subsequent cold plasma treatment. In general, the current study provided

574 new insights on correlating temperature-mediated alterations in fatty acid profile and membrane

575 fluidity with intracellular ROS levels and inactivation efficiency, and suggested the potential

576 possibility to improve or optimize the sterilization efficiency in real food systems by preconditioning

577 growth temperatures at low or high temperatures or by ultrasound pretreatment. However, more

578 investigations are still needed to have a better understanding of the distribution, penetration and

579 interaction mechanism of plasma-mediated ROS from the bulk liquid phase to membrane interior at a

580 molecular level especially in real food systems.

581

582 Acknowledgements

583 This study was funded by the Fishery Development Project of Guangdong Province, China (2019B9).

584 This research was also supported by Natural Science Foundation of Guangdong Province

585 (2017A030310558), the S&T Project of Guangzhou (201804010469), the S&T Project of

586 Guangdong Province (2018D1002, 2017B020207002), the Agricultural Development and Rural

587 Work of Guangdong Province (2017LM4173), the Pearl River S&T Nova Program of Guangzhou

588 (201610010104), the International and Hong Kong – Macau - Taiwan Collaborative Innovation

589 Platform of Guangdong Province on Intelligent Food Quality Control and Process Technology &

590 Equipment (2015KGJHZ001), the Guangdong Provincial R & D Centre for the Modern Agricultural

591 Industry on Non-destructive Detection and Intensive Processing of Agricultural Products, the

592 Common Technical Innovation Team of Guangdong Province on Preservation and Logistics of

593 Agricultural Products (2016LM2154) and the Innovation Centre of Guangdong Province for Modern

594 Agricultural Science and Technology on Intelligent Sensing and Precision Control of Agricultural

595 Product Qualities.

596

24
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717 Archives of Microbiology, 192, 85-95.

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720 Letters, 578, 152-156.

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725 Physicochemical Properties and Biological Effects of Water Activated by Non-thermal Plasma

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747 Zhu, K., Ding, X., Julotok, M., & Wilkinson, B. J. (2005). Exogenous isoleucine and fatty acid

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31
750 Table 1. The effect of different growth temperatures on membrane fatty acid profile of L. monocytogenes cells
751
Relative proportion (%) at different growth temperature (°C)
Fatty acid profile Tm (°C) (Kaneda, 1991)
10 25 37 42

C14:0 1.08 ± 0.17ef 1.10 ± 0.17de 0.85 ± 0.24f 0.92 ± 0.12f 24

Iso-C14:0 0.43 ± 0.025f 0.55 ± 0.17e 0.55 ± 0.13f 0.44 ± 0.08f 6.5

C15:0 10.98 ± 0.53c 16.80 ± 1.75c 13.84 ± 0.25c 14.75 ± 0.48c 34.2

Anteiso-C15:0 60.29 ± 1.58a 50.38 ± 1.49a 41.83 ± 0.13a 38.98 ± 0.69a -16.5

C16:0 1.78 ± 0.19ef 3.03 ± 0.23d 2.72 ± 0.20e 1.45 ± 0.06ef 41.5

Iso-C16:0 5.79 ±0.25d 2.48 ± 0.58de 4.07 ± 0.40d 5.05 ± 0.30d 22

Anteiso-C17:0 17.31 ± 0.30b 24.59 ± 0.76b 34.32 ± 0.10b 36.28 ± 0.32b 7.6

C18:0 2.33 ± 0.09e 1.07 ± 0.33de 1.83 ± 0.31e 2.1 ± 0.30e 54.8

SCFAs 16.17 ± 0.99 22.00 ± 1.47 19.24 ± 0.16 19.21 ± 0.25

BCFAs 83.83 ± 1.00 78.00 ± 1.47 80.77 ± 0.15 80.75 ± 0.31

BCFAs/SCFAs 5.18 3.55 4.20 4.20

752 Note: Tm: phase transition temperature (°C); Values in the same column with different letters were significantly different (P < 0.05).

32
753 Table 2. Results of the statistical analysis on kinetics changes of L. monocytogenes cells in resistance to DBD plasma
754
Temperature (oC) Model Constants Log10 N0 (CFU/mL) RMSE R2

Weibull model δ= 4.742 ± 0.096 min, ρ = 1.761 ± 0.067 6.50 ± 0.102 0.175 0.998
10 Log-linear and shoulder model Kmax = 1.020 ± 0.010 min-1, S1 =2.344 ± 0.042 min 6.50 ± 0.102 0.052 0.999
-1
Sigmoidal-like model Nres = 2361.41 ± 1008.01 CFU/mL, Kmax = 1.068 ± 0.024 min , S1 =2.389 ± 0.028 min 6.50 ± 0.102 0.037 0.999

Weibull model δ= 3.313 ± 0.415 min, ρ = 1.460 ± 0.269 7.53 ± 0.052 0.584 0.947
-1
25 Log-linear and shoulder model Kmax = 1.324 ± 0.159 min , S1 =1.736 ± 0.435 min 7.53 ± 0.052 0.500 0.971
Sigmoidal-like model Nres =22879.54 ± 3972.95 CFU/mL, Kmax = 1.650 ± 0.056 min-1, S1 =2.097 ± 0.078 min 7.53 ± 0.052 0.055 0.999

Weibull model δ= 2.024 ± 0.482 min, ρ = 0.896 ± 0.179 8.06 ± 0.040 0.688 0.934
-1
37 Log-linear and shoulder model Kmax = 0.951 ± 0.181 min , S1 =-0.377 ± 1.279 min 8.06 ± 0.040 0.661 0.928
-1
Sigmoidal-like model Nres = 51255.38 ± 2920.61 CFU/mL, Kmax = 1.495 ± 0.037 min , S1 =1.016 ± 0.233 min 8.06 ± 0.040 0.073 0.999

Weibull model δ= 2.421 ± 0.460 min, ρ = 1.265 ± 0.295 7.27 ± 0.023 0.951 0.925
-1
42 Log-linear and shoulder model Kmax = 1.595 ± 0.303 min , S1 =1.255 ± 0.580 min 7.27 ± 0.023 0.973 0.949
-1
Sigmoidal-like model Nres = 4365.88 ± 1130.06 CFU/mL, Kmax = 2.362 ± 0.131 min , S1 =1.926 ± 0.104 min 7.27 ± 0.023 0.192 0.998
755 Note: δ: time for the first log cycle reduction (min); ρ: the scale and shape parameter; Kmax: specific inactivation rate (min-1); S1: shoulder length (min); Log10 N0 (CFU/mL): predicted
756 logarithm of initial count; Nres (CFU/mL): the residual population density; RMSE: the root mean square error.

33
757 Lists of figures:

758 Fig. 1. (a) Research strategy used in the current study as compared with (b) previously employed

759 strategy published in literature.

760 Fig. 2. Growth curves of L. monocytogenes at various temperatures, (a) 10 °C, (b) 25, 37 and 42 °C;

761 (c) the fluorescence polarization (P) and anisotropy (r), and (d) microviscosity (η) values of

762 cytomembrane of L. monocytogenes at different growth temperatures. OD 600 nm = optical

763 density at 600 nm.

764 Fig. 3. (a) The effect of growth temperatures on the relative proportion of membrane fatty acid

765 profile in L. monocytogenes; (b) score plot of PCA using t1 vs t2 from relative peak areas of

766 fatty acid composition in GC-MS fingerprints of L. monocytogenes at various growth

767 temperatures; (c) biplot of RDA showed the correlation between relative abundance of fatty

768 acid composition and growth temperatures. Values for the same component with different

769 letters were significantly different (P < 0.05).

770 Fig. 4. (a) The scheme diagram of chemical processes induced by DBD atmospheric cold plasma in

771 gas-liquid interface; (b) the main peaks in OES corresponding to the emissions of excited

772 species generated by DBD plasma in air at 50 V over the range of 200 to 1100 nm; (c) the

773 effect of growth temperatures on the development of pH values in bacterium suspension

774 subjected to ultrasonic-assisted plasma treatment. UP involved different ultrasound

775 pretreatment times followed by subsequent 2 min plasma treatment, while P referred to single

776 plasma treatment for different times.

777 Fig. 5. (a) Histogram represented the DCF fluorescent signals of L. monocytogenes cells grown at

778 10 °C after exposure to ultrasonic-assisted plasma treatment; (b) the effect of growth

779 temperatures on the percentage of DCF-stained L. monocytogenes cells subjected to

780 ultrasonic-assisted plasma treatment. UP involved different ultrasound pretreatment times

781 followed by subsequent 2 min plasma treatment, while P referred to single plasma treatment

34
782 for different times.

783 Fig. 6. The effect of different growth temperatures on the resistance of L. monocytogenes which was

784 determined by the average number of CFUs grown on TSA medium after exposure to a range

785 of (a) single plasma treatment time and (b) ultrasonic-assisted plasma patterns; the effect of

786 different growth temperatures on inactivation kinetic models of L. monocytogenes subjected to

787 DBD plasma treatment at a given power: (c) Weibull model; (d) log-linear and shoulder model;

788 (e) sigmoidal-like model. UP involved different ultrasound pretreatment times followed by

789 subsequent 2 min plasma treatment, while P referred to single plasma treatment for different

790 times.

791 Fig. 7. SEM images of L. monocytogenes grown at 25 °C after exposure to different treatment

792 patterns, (a) untreated cells; (b) singe plasma treatment for 2 min; (c) 15 min ultrasound

793 pretreatment followed by 2 min plasma treatment; (d) single plasma treatment for 4 min.

35
794
795 Fig. 1. (a) Research strategy used in the current study as compared with (b) previously employed strategy published in
796 literature.

36
797

798
799 Fig. 2. Growth curves of L. monocytogenes at various temperatures, (a) 10 °C, (b) 25, 37 and 42 °C; (c) the fluorescence

800 polarization (P) and anisotropy (r), and (d) microviscosity (η) values of cytomembrane of L. monocytogenes at different

801 growth temperatures. OD 600 nm = optical density at 600 nm.

37
802
803 Fig. 3. (a) The effect of growth temperatures on the relative proportion of membrane fatty acid profile in L.

804 monocytogenes; (b) score plot of PCA using t1 vs t2 from relative peak areas of fatty acid composition in GC-MS

805 fingerprints of L. monocytogenes at various growth temperatures; (c) biplot of RDA showed the correlation between

806 relative abundance of fatty acid composition and growth temperatures. Values for the same component with different

807 letters were significantly different (P < 0.05).

38
808
39
809 Fig. 4. (a) The scheme diagram of chemical processes induced by DBD atmospheric cold plasma in gas-liquid interface; (b) the main peaks in OES corresponding to the emissions of

810 excited species generated by DBD plasma in air at 50 V over the range of 200 to 1100 nm; (c) the effect of growth temperatures on the development of pH values in bacterium

811 suspension subjected to ultrasonic-assisted plasma treatment. UP involved different ultrasound pretreatment times followed by subsequent 2 min plasma treatment, while P referred to

812 single plasma treatment for different times.

40
813
814 Fig. 5. (a) Histogram represented the DCF fluorescent signals of L. monocytogenes cells grown at 10 °C after exposure to ultrasonic-assisted plasma treatment; (b) the effect of growth

815 temperatures on the percentage of DCF-stained L. monocytogenes cells subjected to ultrasonic-assisted plasma treatment. UP involved different ultrasound pretreatment times

816 followed by subsequent 2 min plasma treatment, while P referred to single plasma treatment for different times.

41
817
818
819 Fig. 6. The effect of different growth temperatures on the resistance of L. monocytogenes which was determined by the average number of CFUs grown on TSA medium after

820 exposure to a range of (a) single plasma treatment time and (b) ultrasonic-assisted plasma patterns; the effect of different growth temperatures on inactivation kinetic models of L.

821 monocytogenes subjected to DBD plasma treatment at a given power: (c) Weibull model; (d) log-linear and shoulder model; (e) sigmoidal-like model. UP involved different ultrasound

822 pretreatment times followed by subsequent 2 min plasma treatment, while P referred to single plasma treatment for different times.

42
823

824
825 Fig. 7. SEM images of L. monocytogenes grown at 25 °C after exposure to different treatment patterns, (a) untreated cells;

826 (b) singe plasma treatment for 2 min; (c) 15 min ultrasound pretreatment followed by 2 min plasma treatment; (d) single

827 plasma treatment for 4 min.

43
Highlights

• Prior growth temperature induced a noticeable alteration in fatty acid

profile

• Anteiso-C15:0 was the most abundant membrane fatty acids at low

temperature

• Elevating growth temperature was accompanied by a significant

decline in viability

• Ultrasound pretreatment increased the susceptibility to plasma

treatment

• The inactivation kinetics was adequately described by sigmoidal-like

model
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: