Microbial response to some nonthermal physical technologies

Journal Pre-proof Microbial response to some nonthermal physical technologies Dan Wu, Fereidoun Forghani, Eric Banan-Mwine Daliri, Jiao Li, Xinyu Liao, Donghong Liu, Xingqian Ye, Shiguo Chen, Tian Ding PII:

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DOI:

https://doi.org/10.1016/j.tifs.2019.11.012

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Trends in Food Science & Technology

Received Date: 18 February 2019 Revised Date:

8 October 2019

Accepted Date: 10 November 2019

Please cite this article as: Wu, D., Forghani, F., Banan-Mwine Daliri, E., Li, J., Liao, X., Liu, D., Ye, X., Chen, S., Ding, T., Microbial response to some nonthermal physical technologies, Trends in Food Science & Technology (2019), doi: https://doi.org/10.1016/j.tifs.2019.11.012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Microbial Response to some Nonthermal Physical Technologies

Dan Wu a, Fereidoun Forghani b, Eric Banan-Mwine Daliri c, Jiao Li a, Xinyu Liao a, Donghong Liu a*, Xingqian Ye a, Shiguo Chen a, Tian Ding a*

*

Corresponding author. Donghong Liu, Tian Ding

Department of Food Science and Nutrition, Zhejiang University, Hangzhou, Zhejiang 310058, China Tel: 86-571-88982287, Fax: 86-571-88982287 E-mail address: [email protected] (D. Liu); [email protected] (T. Ding).

a

Department of Food Science and Nutrition, Zhejiang University, Hangzhou,

Zhejiang 310058, China

b

Department of Plant Pathology, University of Georgia, 2360 Rainwater Road, Tifton,

GA, 31793, USA

c

Department of Food Science and Biotechnology, Kangwon National University,

Chuncheon, 200-701, South Korea

2

1

Abstract

2

Background: In response to the worldwide interest in safe, nutritious and minimally

3

processed food products, innovative nonthermal processing for microbial inactivation

4

has been developed as one of the major growth sectors in the food industry. In

5

contrast to traditional thermal processing, nonthermal physical technologies have the

6

ability to inactivate microorganisms at lower temperatures and maintain the

7

organoleptic and nutritional qualities of food products. However, microbial cells can

8

also develop a range of strategies to adapt rapidly to environmental stimuli and to

9

survive under harsh conditions, posing a potential hazard to the food processing

10

industry.

11

Scope and approach: This review concluded the microbial response to nonthermal

12

technologies from the perspective of three states of microbes, sublethal cells, viable

13

but non-culturable cells, and apoptosis. This work describes the responses of

14

microorganisms to nonthermal physical technologies, mainly focusing on their

15

physiological modifications and genetic regulatory mechanisms.

16

Key Findings and Conclusion: Most nonthermal physical treatments are unable to

17

sterilize thoroughly, thereby resulting in suppressed or sublethally injured microbial

18

cells instead of killing them completely. This poses food safety hazards since

19

microorganisms may re-grow at post-processing stage as favorable conditions are

20

available again. Studying the mechanism of these responses on microorganisms may

21

help us to do better in anticipating possible risks during food processing and

22

preventing potential food safety incidents. 2

3

23

Keywords: Microbial response; Nonthermal physical technologies; Sublethal; VBNC;

24

Apoptosis

25

3

4

26 27

Introduction Thermal technologies have been widely used in food sterilization processing for a

28

long time. However, in response to the worldwide interest for safe, nutritious, and

29

minimally processed food products, innovative nonthermal processing for microbial

30

inactivation has been developed as one of the major growth sectors in the food

31

industry (Liao et al., 2018a). These emerging technologies are mainly reliant on

32

physical processes, including but not limited to high hydrostatic pressure, high-power

33

ultrasounds, pulsed electric fields, ultraviolet radiation, cold plasma, and high

34

pressure carbon dioxide. Several reviews have summarized their impact on microbial

35

reductions, cells morphology and composition, as well as food constituents (Ekezie,

36

Cheng, & Sun, 2018; Marx, Moody, & Bermúdezaguirre, 2011). For example, high

37

pressure processing (HPP) has been studied since 1881 and several reports have been

38

published on its application in food products to inactivate microorganisms

39

(Balasubramaniam, Martínezmonteagudo, & Gupta, 2015). Nowadays, HPP is

40

gradually becoming a commercial pasteurization method accepted by food

41

manufacturers and consumers due to the non-involvement of heat which helps to

42

maintain the flavor, texture, and nutritional quality of food products.

43

Many of these advanced nonthermal approaches have shown great superiority over

44

thermal treatments, such as killing microorganisms at lower temperatures and keeping

45

good conditions of organoleptic and nutritional qualities of food products. However,

46

there are still limitations. It has been demonstrated that environmental stress might

47

initiate a range of responsive strategies within the bacterial cells (Cabiscol, Tamarit, & 4

5

48

Ros, 2000; Ramona et al., 2012). Accordingly, not all of the bacterial pathogens

49

treated with nonthermal processing will be killed. In order to survive the stress from

50

the environment, a series of adjustments and control mechanisms play important roles

51

in microbial cells. Research studies have been performed to investigate how bacteria

52

rapidly adjust themselves into environmental stress (Schimel, Balser, & Wallenstein,

53

2007).

54

Pavlov and Ehrenberg have once come up with a subtle model in which bacteria

55

manipulate their own gene expression to quickly adapt to changes in the environment

56

(Pavlov & Ehrenberg, 2013). It has been pointed out that microbial response will

57

provide bacteria with an ability to concentrate on growth metabolism instead of

58

multiplying under environmental stress. These responses include 1) sublethally

59

injured state, defining a condition that bacteria are able to repair themselves against

60

the damages caused by external stimuli (Berney et al. 2007); 2) viable but

61

non-culturable (VBNC), a physiological state, in which the bacteria cannot form

62

colony on the solid media but still possess the capability of renewed metabolic activity,

63

namely, still alive (Oliver, 2000); 3) apoptosis, which is the spontaneous and orderly

64

death controlled by genes to maintain stability of intracellular mechanisms and

65

microbial population. These strategies inevitably pose a potential hazard to the food

66

processing industry.

67

Microorganisms respond to acid (Álvarez-Ordóñez, Prieto, Bernardo, Hill, & López,

68

2012), alkali (Giotis, Muthaiyan, Blair, Wilkinson, & McDowell, 2008), cold

69

(Stenfors Arnesen, Fagerlund, & Granum, 2008), heat (Ramos et al., 2001), antibiotics 5

6

70

(Nguyen et al., 2011), and oxidative stress (Touati et al., 2000) have been studied and

71

summarized previously. However, detailed descriptions on the responses of

72

microorganisms to nonthermal treatments are not available to date. In this review, we

73

provide a comprehensive overview of the responses of microorganisms under

74

nonthermal physical technologies. As such, the mechanisms of sublethally injured

75

state, VBNC state, and apoptosis induced by nonthermal physical techniques at the

76

molecular level have been discussed.

77 78

1. Sublethally injured state

79 80

Bacterial injury is briefly defined as the impact of one or more sublethal treatments

81

on a microorganism. When a microorganism is exposed to chemical or physical

82

processes which do not kill it, it may enter into sublethally injured state (Andrew &

83

Russell, 1984). To be more specific, it is a reversible condition where cells enter into a

84

growth stagnation phase due to cellular membrane alteration. After the stimulating

85

factor is removed, the sublethally injured cells can repair themselves and return to the

86

normal state (Hollaender, 1943; Jofré, Aymerich, Bover-Cid, & Garriga, 2010).

87

Sublethal injury of microbes includes structural damage such as some wall

88

components, cell membrane damage within the cells, and the expression of some

89

genes which incurs functional disorders that may be transient or permanent (Ray,

90

1986). As shown in Fig. 1, sublethal stress stimulates cell repair, and the response of

91

microorganisms to stress constitutes a potential hazard in the food processing industry 6

7

92

(Lado & Yousef, 2002).

93 94

1.1. Occurrence of microbial sublethally injured state

95 96

Most physical, chemical, and nutritional intervention strategies may produce

97

successive sublethal effects on pathogenic and spoilage microorganisms. Chemical

98

treatments refer to chemical sanitizers, including, but not limited to chlorine, iodine,

99

and quaternary and ammonium compounds (Ray, 1989); oxidative treatments,

100

including ozone, H2O2, bioactive antimicrobial peptides, and the lacto-peroxidase

101

system (Thanomsub, 2002); pH, including alkalis and acids (organic and inorganic);

102

and preservatives, including sorbate, benzoate, nitrate, and bacteriocins (Ray, 1978).

103

Chronic starvation, freezing, and thawing can also disrupt the metabolic system of

104

microorganism and consequently injure the cells.

105

With regards to physical treatments, they are generally classified into two

106

categories: thermal and nonthermal. The latter are mostly emerging technologies (i.e.,

107

high hydrostatic pressure, ultraviolet light, ultrasonication, pulsed electric field, high

108

pressure carbon dioxide, cold plasma, and others) with advantages such as little

109

negative effects on nutritional and organoleptic qualities (Liao et al., 2018a). These

110

factors are the driving forces for the increased attention and interest of consumers,

111

researchers and the food industry. Table 1 summarizes several nonthermal physical

112

technologies by which sublethally injured state may be induced and their occurrence

113

proportion. The proportion of sublethal injury stems from the count of sublethally 7

8

114

injured cells, i.e., the D-value difference between the number of colonies in the

115

non-selective medium and the selective medium (Wang, Dong, Yan, Xu, & Zhou,

116

2012). The proportion of sublethally injured cells may be found from research articles,

117

yet data that are not explicitly given can be calculated according to the following

118

equation: rate of sublethally injured cells % =

/

!"#

/

− %&%

/

%&%

!"#

× 100%

!"#

119

where CFU/mLselective is the counts in selective medium; and CFU/mLnon-selective is the

120

counts in non-selective medium (Ray, Hawkins, & Hackney, 1978). Among all

121

nonthermal processing technologies, the use of pulsed electric fields (PEF) to cause

122

microbial sublethally injury has been studied in details, both in number and depth.

123

Staphylococcus aureus, Listeria monocytogenes (both Gram-positive) and Escherichia

124

coli (Gram-negative) in milk were respectively treated with PEF to study sublethal

125

injury as well as inactivation kinetics (Zhao, Yang, Shen, Zhang, & Chen, 2013). In

126

the case of L. monocytogenes, electric field strengths from 15 to 30 kV/cm caused

127

sublethal rates ranging from 18.98 to 43.64%, correspondingly. For E. coli and S.

128

aureus, the sublethal rates were maximum (40.74% and 36.51%, respectively) at 25

129

kV/cm for 500 µs and 400 µs, respectively before decreasing. Under PEF stress, cell

130

membrane surfaces accumulate free charges which compress the cytoplasmic

131

membrane. This results in mechanical instability of cell permeabilization (cell injury).

132

Interestingly, more than 99.90% of sublethally injured E. coli cells were obtained

133

when McIlvaine buffer at pH 4 was added to PEF at 19 kV/cm for 400 µs (García et

134

al., 2005). It is likely that sublethally injured cells were promoted when PEF was 8

9

135

combined with pH treatment. In general, sublethality is more likely to occur in strains

136

that are more tolerant to PEF (García et al., 2005).

137

As another frequently used nonthermal processing technology, high-hydrostatic-

138

pressure (HHP) treatment can normally cause sublethally injured state. The literature

139

indicates that pressure size, time, and method, as well as the type of microorganism

140

will have various impacts on pasteurization (Benito, Ventoura, Casadei, Robinson, &

141

Mackey, 1999). Under the case of individual HHP treatment, the resistance of

142

endospore-forming microorganisms to growth inhibition can be observed (Ahn &

143

Balasubramaniam, 2007). A study reported that E. coli was 35.60% sublethally injured

144

by treating with 400 MPa HHP at 25℃ for 5 min. In contrast, the range of pressure

145

resistance was greatly reduced when the temperature was increased from 25 to 50℃

146

during the pressure treatment (Alpas et al., 1999). At 25℃, the viability loss of six E.

147

coli strains ranged from 2.8 to 5.64 log cycles, whereas more than 8 log cycles

148

viability loss was found at 50℃ under the same pressure conditions. This shows that,

149

although HHP is a nonthermal process, the sample temperature itself may influence its

150

microbial inactivation efficiency. In the light of numerous studies, three factors can be

151

concluded that influence the degree of sublethal injury caused by nonthermal

152

technology: 1) bacterial species; 2) treatment parameters (time, strength, temperature);

153

and 3) medium (components, pH).

154 155

1.2. Mechanism of sublethally injured state in microbial cells

156

9

10

157

Bacterial stress induced by processing conditions may result in decreased cell

158

function and/or protein denaturation (Lou, 1996). Table 2 outlines cellular sites

159

damaged by various sublethal nonthermal physical treatments.

160 161 162

1.2.1. Sublethally injured state and recovery mechanisms in microbial cells after PEF treatment

163

Zhao and colleagues (2014) investigated Saccharomyces cerevisiae sublethally

164

injured state induced by PEF treatment and reported that PEF caused reduction in

165

cytomembrane fluidity, increase in micro-viscosity, alteration in membrane lipid

166

composition (increase in saturated fatty acids to unsaturated fatty acid ratio) and

167

disruption of RNA. In addition, PEF caused alteration in cellular structures and

168

protein functions (Liu, Zeng, & Han, 2010), enzymes (Zhao, Yang, & Zhang, 2012),

169

and oxidation of lipids by free radicals produced during PEF treatment (Zhao et al.,

170

2012). Any of these alterations may eventually lead to sublethal injury.

171

Fig. 2 depicts a schematic representation of the cellular response mechanisms to

172

PEF treatment. Rivas et al. (2013) studied E. coli sublethally injured state and their

173

resuscitation processes induced by PEF at a molecular level. They found that

174

sublethally injured cells caused by PEF differentially expressed some structural

175

functional proteins (ompA, gmhA, CIpA, RS6, Dut, FtnA, TufB, ftsH, putA, atpA,

176

sdhA). Also, during resuscitation there was significant increase in the expression of

177

membrane proteins within sublethally injured cells indicating their close relationship

178

with resuscitation. OmpA is an outer membrane protein that is most damaged by PEF 10

11

179

and its level decreases upon PEF treatment (Torres et al., 2006). While in the process

180

of recovery from sublethally injury, OmpA increases significantly, and this suggests

181

the important role of OmpA in cell recovery. Besides, during PEF treatment, there are

182

some changes of protein level in sublethally injured E. coli cells. gmhA, a

183

phosphoheptose

184

lipopolysaccharide increases. Other proteins including ClpA, RS6, Dut, FtnA, also

185

increase. In addition, it has been shown that proteins regulated by a sigma factor,

186

RpoS (σ38), play significant roles in repairing sublethally injured E. coli cells caused

187

by PEF treatment.

isomerase,

involved

in

the

biosynthesis

of

cell

wall

188 189

1.2.2. Sublethally injured state and recovery mechanism in microbial cells after HHP

190

treatment

191

Kilimann and colleagues (2006) found a strong correlation between the generation

192

of sublethal cells and multidrug resistance (MDR) transport enzymes (LmrP) activity

193

in HHP (200 MPa, 5-50℃) treatment of Lacotoccous lactis, indicating that membrane

194

proteases were sensitive sites during HHP processing. In addition, they also pointed

195

out that the cellular living state could be well characterized by metabolic activity

196

rather than membrane integrity. Molina-Höppner and colleagues (2004) also proved

197

that the reversible or irreversible degeneration of cytoplasmic proteins induced by

198

HHP treatment caused the occurrence of sublethal state or death of cells. A study

199

carried out by Ulmer et al. (2003) showed that the activity of energy metabolism

200

index HorA which is closely related to the state of plasmalemma had impact on the 11

12

201

occurrence of sublethally injured cells. It is believed that HHP treatment has severe

202

influence on the transport enzyme system located on the plasmalemma, causing

203

sublethally injured state or death. Fig. 3 shows the schematic representation of cellular

204

mechanisms of response to HHP treatment.

205

Inhibitory studies that assessed the recovery mechanisms of sublethally injured E.

206

coli cell found that repair of cytomembrane damage was energy-dependent and

207

required RNA and protein synthesis, whereas repair of outer membrane damage

208

required no energy, RNA or protein synthesis (Chilton, Isaacs, Manas, & Mackey,

209

2001). Also, it has been found that the activation of RpoS (the alternative sigma

210

subunit of RNA polymerase) induced transcription of a set of over 50 genes related to

211

stress survival when bacteria entered the stationary growth stage (Huisman, Siegele,

212

Zambrano, & Kolter, 1996). In line with this, Robey et al. (2001) showed that lack of

213

stationary phase inducible sigma factor RpoS led to decreased resistance to HHP

214

treatment in E. coli O157: H7 cells.

215 216

1.2.3 Sublethally injured state and recovery mechanism in microbial cells after High

217

pressure carbon dioxide treatment

218

The effects of High pressure carbon dioxide (HPCD) on microorganisms have

219

been ascribed to the interaction of anaerobic conditions, acidification, pressure, and

220

high CO2 concentration. In the analyses of cells subjected to HPCD at moderate

221

values of operational pressure and temperature conditions, CO2 is known to be used

222

for carbonation, from which the main effects are replacement of the oxygen and 12

13

223

reduction of the pH in the medium, inhibiting enzymatic reactions and microbial

224

growth (Bonnaillie & Tomasula, 2015). In a recent study, a metabolic inhibitor was

225

added during the resuscitation process to study the repair of sublethal E. coli cells

226

induced by HPCD treatment. It was found that resuscitation required production of

227

energy, protein and RNA, but was not dependent on peptidoglycan synthesis. It was

228

also found that Mg and Ca cations were needed during the resuscitation process (Bi et

229

al., 2015). In addition, results obtained suggested that transient response in E. coli

230

O157:H7 happened during the formation of sublethally injured cells, such as

231

decreased metabolic activity, repressed cell division and enhanced survival ability. A

232

hypothetical inactivation mechanism of the above conditions has been simplified in a

233

series of steps schematically depicted in Fig. 4.

234 235

2. Viable but non-culturable state

236 237

The viable but non-culturable (VBNC) state is a survival strategy adopted by

238

many microorganisms when exposed to extreme environmental stress. They exhibit a

239

pattern similar to dormancy (Oliver, 2005), in which bacteria cannot form a colony in

240

standard medium but can retain their metabolic activity and express toxic proteins

241

(Fakruddin, Mannan, &Andrews, 2013; Oliver 2010; Pinto, Santos, & Chambel,

242

2015). Fig. 5 shows a schematic view of the cells in VBNC state growth as influenced

243

by post-stress conditions and culture media. VBNC cells generally exhibit very low

244

levels of metabolic activity, but are again culturable, once under resuscitation 13

14

245

conditions (Ding et al., 2017). Many studies suggested that VBNC state was a

246

self-protection strategy for some bacteria, which made the least of cellular energetic

247

requirements as well as enhanced the resistance to environmental stress (Zhao, Zhong,

248

Wei, Lin, & Ding, 2017). Due to the typical survival characteristics, VBNC cells have

249

the ability to evade routine microbiological detection methods, while still posing a

250

potential risk of food safety (Ding et al., 2017). At present, it is known that more than

251

60 kinds of bacteria can enter the VBNC state, most of which (accounting for more

252

than 75%) are pathogenic bacteria, including L. monocytogenes, Salmonella spp.,

253

Vibrio spp., enteropathogenic and enterohemorrhagic E. coli, etc. The finding of

254

VBNC state in Brettanomyces cells revealed that in addition to bacteria, fungi would

255

also adopt this survival strategy under adverse circumstances (Agnolucci, 2010).

256 257

2.1. The occurrence of VBNC cells

258 259

Cells enter the VBNC state in response to environmental stress or sterilization

260

process which are normally known to be bactericidal, including such treatments as

261

pasteurization of milk (Gunasekera et al., 2002) and chlorination of wastewater

262

(Oliver et al., 2005). Ultraviolet light (Serpaggi, 2012), TiO2 photocatalysis (Kacem,

263

2016), and plasma treatment (Doležalová, Prukner, Lukeš, & Šimek, 2016) have also

264

been demonstrated to induce the formation of VBNC microbial cells. Zhao and

265

colleagues (2013) reported that VBNC E. coli O157:H7 could be induced after

266

high-pressure CO2 (HPCD) treatment at 5 MPa in 25, 31, 34, and 37℃, with nearly an 14

15

267

8 log reduction in 40, 30, 28, and 25 min, respectively. Many studies have been

268

carried out on other nonthermal physical technologies that may result in VBNC

269

formation. Some of them are summarized in Table 3. The VBNC percentages not

270

explicitly given were calculated according to the following equation: Percentage of VBNC cells % =



01234 563748 9844: − ;42;<3748 9844: × 100% 01234 9844 =1=;43261>

271 272

2.2. VBNC mechanism in microbial cells

273 274

At present, most of the researches on the VBNC state focus on biological

275

characteristics, while the mechanism of how VBNC E. coli adjusts itself still remains

276

unclear. Some researchers have studied the genes and proteins involved in the VBNC

277

cells. Continued expression of the major stress factor gene, rpoS, was observed for as

278

long as 14 days by Smith and Oliver (2006a) which is in accordance with the findings

279

of Boaretti et al. (2003), reporting that rpoS was involved in the persistence of E. coli

280

in the VBNC state. Yaron & Matthews (2002) reported that a variety of genes

281

including mobA, rfbE, stx1 and those for 16S rRNA synthesis were expressed in

282

non-culturable E. coli O157:H7 cells. Pai and colleagues (2000) also found

283

continuous expression of antigen 85B in Mycobacterium tuberculosis and Gunasekera

284

et al. (2002) reported the expression of gfp gene in VBNC cells of E. coli and

285

Pseudomonas putida following pasteurization.

286

MurG is a glycosyltransferase that has been shown by Signoretto and colleagues

287

(2002) to be required for the late assembly of peptidoglycan in Enterococcus faecalis 15

16

288

cells entering the VBNC state. Zhu, Plikaytis, and Shinnick (2003) reported that the

289

Rpf protein of Micrococcus luteus can promote the resuscitation of VBNC cells.

290

Similar studies have been summarized in Table 4. Smith and Oliver (2006a) found

291

that the expression level of katG, a peroxidase gene in VBNC Vibrio vulnificus, was

292

down-regulated. Masmoudi, Denis, and Maalej (2010) demonstrated that the

293

reduction of S. aureus culturability was positively correlated with the reduction of

294

peroxidase activity, when the relationship between catalase activity and S. aureus

295

culturability was studied. Thus, they concluded that H2O2 may be involved in

296

inducing VBNC state in bacteria.

297 298

3. Apoptosis

299 300

Under a low dose of environmental stress, the cells can adapt and become more

301

resistant to continuous exposure (Collinson & Dawes, 1992). At higher doses, cell

302

death occurs as apoptosis, but at extreme doses it manifests as necrosis. Apoptosis is a

303

voluntary death process, which involves activation, expression and regulation of a

304

subset of genes, in order to adapt to environmental stress. A typical series of

305

morphological modifications happen during apoptosis, including chromatin

306

condensation (Haupt, Barak, & Oren, 1996), DNA and nuclear fragmentation, etc.

307

Cells that are unable to repair the damaged DNA would resort to apoptosis, while

308

those with misrepaired DNA would survive with accumulated mutations. The

309

dynamic balance of apoptosis and proliferation plays an irreplaceable role in 16

17

310

maintaining the stability of cell population under environmental stress.

311 312

3.1. Occurrence of apoptosis induced by nonthermal physical technology

313 314

Apoptosis is an active, signal-dependent process that can be induced by many

315

factors, which can be divided into physical, biological and chemical factors. Physical

316

factors include radiation (such as UV, gamma rays, etc.) and moderate temperature

317

stimulation (such as heat shock, cold shock). Chemical and biological factors include

318

reactive oxygen species (ROS) groups, molecular, cytotoxic, DNA and protein

319

synthesis inhibitors, physiology disorders, etc. (Wen-yi, 2010). Table 5 summarizes

320

briefly the induction of apoptosis in microorganisms by a number of nonthermal

321

physical techniques known to date. One of the common DNA damaging agents,

322

ultraviolet irradiation, which was investigated for S. cerevisiae apoptosis by Del

323

Carratore et al. (2002). According to their study, the quantification of apoptotic cells

324

measured by flow cytometric analysis (FACS) revealed that UV irradiation actually

325

caused a dose-dependent increase in apoptosis within S. cerevisiae cells. The

326

Dielectric Barrier Discharge Plasma treatment, which would cause oxidative stress,

327

was also performed on S. cerevisiae to explore the occurrence of apoptosis (Chen et

328

al., 2010). In this study, cell cycle alterations were observed as an indicator to

329

apoptosis.

330 331

3.2. The mechanism of apoptosis induced by nonthermal physical technology 17

18

332 333

Although there is still lack of sufficient research on the mechanism of apoptosis,

334

the process so far is defined to include: accepting apoptosis signal, regulating the

335

intermolecular interaction, activation of proteolytic enzyme (Caspase), and entering

336

into the continuous reaction process (Carmona-Gutierrez & Madeo, 2009; Balzan,

337

2004; Egbe, 2017). The signal of apoptosis, which has been expounded in last

338

paragraph, can trigger apoptosis by activating the death receptor or other pathways

339

(Clarke, 1990). Generally, apoptosis is related to the generation of ROS in cells across

340

a wide range of organisms including yeast. In many cases, apoptosis occurs in

341

unicellular organisms as an altruistic response to severe oxidative damage (Galluzzi et

342

al., 2012). Later, cells develop mechanisms to produce ROS as a regulator of

343

apoptosis (Perrone, Tan, & Dawes, 2008). Numerous studies have shown that many

344

external stimulations (such as physical and chemical factors) can induce cells to

345

produce ROS, and they respond according to intracellular ROS levels (Uren et al.,

346

2000). At low concentrations of ROS, cells can adapt to the environment by

347

increasing tolerance (Collinson & Dawes, 1992). At lower concentrations, cells

348

activate their antioxidant systems and delay cell division. In contrast, at higher ROS

349

concentrations, cells undergo apoptosis or necrosis (Flattery-O’Brien & Dawes, 1998;

350

Gasch et al., 2000).

351

Madeo et al. (1999) found that the expression of human apoptotic gene Bax in yeast

352

cells led to the production of large amounts of oxygen free radicals, provoking the

353

apoptotic reaction. However, heterologous expression of bcl-2 can enhance the 18

19

354

tolerance of yeast cells to H2O2 and extend the cell survival time (Chae et al., 2003;

355

Longo, Ellerby, Bredesen, Valentine, & Gralla, 1997). The main genes and proteins

356

that function to regulate apoptosis were listed in the Table 6.

357 358

4. Cross response induced by multiple nonthermal technologies

359 360

As discussed above, the response of microorganisms induced by individual

361

nonthermal physical technology is an issue which cannot be neglected from food

362

safety point of view. Therefore, it is necessary to explore whether hurdle technology

363

will provide a synergistic effect on antimicrobial activity. The response induced by

364

individual nonthermal techniques has been well investigated as previously discussed,

365

whereas there is limited knowledge available concerning the combination of different

366

treatments to achieve cross response within microbes.

367

Liao et al. (2018b) assessed the physiological changes in S. aureus cells treated by

368

single and combined application of ultrasound and nonthermal plasma (NTP). They

369

explored the physiological variations in lethal and sublethally injured S. aureus cells

370

induced by individual ultrasound, NTP, ultrasound-NTP (UP) and NTP-ultrasound

371

(PU) treatments. NTP is a mixture of various active species including free radicals,

372

charged particles, ultraviolet photons, etc. (Li, 2016; Scholtz, Pazlarova, Souskova,

373

Khun, & Julak, 2015). The results showed that the intracellular ROS level of S. aureus

374

produced during the individual ultrasound and NTP treatment were time-dependent.

375

When ultrasound treatment was exerted on S. aureus, low ROS level, low H2O2 19

20

376

concentration, high superoxide dismutase (SOD) and catalase (CAT) activity were

377

detected. Subsequently, NTP treatment was added, and it was observed that ROS level

378

and H2O2 concentration were greatly increased while SOD and CAT activity

379

decreased. This might be explained by the oxidative response of S. aureus provoked

380

by primary ultrasound treatment, and therefore, slightly increased its resistance to the

381

subsequent NTP stress. Conversely, initial NTP aided the provision of enough ROS

382

dissolved in the medium, and the subsequent ultrasound helped to inject ROS into S.

383

aureus cells. This accelerated the rate of death in microbes, as well as decreasing the

384

sublethally injured cells produced, which resulted from the reaction between ROS and

385

intracellular biomolecules. Fig. 6 schematically represents the mechanism of UP and

386

PU hurdle treatments on S. aureus. The yellow stars mean ROS (reactive oxygen

387

species) produced by nonthermal plasma. Several other studies have also partly

388

mentioned cross response, which are partly summarized in Table 7.

389 390

5. Concluding remarks

391 392

Nonthermal physical treatments have been regarded as novel processing methods

393

minimizing the negative impact of conventional treatments on food nutrition and

394

organoleptic quality during processing. However, due to different kinds of responses

395

of microorganisms to environmental stress, nonthermal physical processing seems to

396

show some deficiencies and risks during food processing. Currently, most nonthermal

397

physical treatments are unable to sterilize thoroughly, thereby resulting in suppressed 20

21

398

or sublethally injured microbial cells instead of killing them completely. This poses

399

food safety hazards since microorganisms may re-grow at post-processing stage as

400

favorable conditions are available again. Nonetheless, as research on microbial

401

responses deepens, we will become more rigorous in our control of food safety

402

hazards. Studying the mechanism of these responses on microorganisms may help us

403

to do better in anticipating possible risks during food processing and preventing

404

potential food safety incidents. Clearly, more studies should be undertaken on the

405

response mechanism of microorganisms under nonthermal physical environmental

406

stress. Hence, the expression of microbial oxidative stress systems, cell repair systems

407

and resistance regulation systems after nonthermal physical treatments should be

408

further studied at the molecular and genomic levels in future.

409 410

Acknowledgments

411 412 413

This study was supported by the National Natural Science Foundation of China (grant 31772079).

414 415

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Figure Legends

864 865

Fig. 1 Microbial stress, injury, adaption, and resistance to processing

866

Fig. 2 The representation of cellular mechanisms of response to PEF treatment

867

Fig. 3 The representation of cellular mechanisms of response to HHP treatment

868

Fig. 4 Cellular mechanisms of response to HPCD in E. coli

869

Fig. 5 The VBNC state of cells under extreme conditions

870

Fig. 6 Mechanisms of ultrasound-NTP and NTP-ultrasound hurdle treatments on S.

871

aureus

43

44

872 873

Table 1 Nonthermal physical technologies that can cause sublethal injury in microorganisms.

Microorganism

Nonthermal Technology

Parameters

Sublethal Cell Proportion

References

E. coli

Pulsed Electric Field

25 kV/ cm for 500 µs

40.74%

Zhao et al. (2013)

High Hydrostatic Pressure

25°C, 5 min, 400 MPa

35.60%

Kong et al. (2018)

20°C, 15 min, 350 MPa

100.00%

Munoz, de ANCOS, CANO (2007)

High Pressure Carbon Dioxide

5 MPa, 25°C, 50 min

>99.99%

Bi et al. (2015)

L. monocytogenes

Pulsed Electric Field Pulsed-plasma Gas Discharge

18.98~43.64% 46.80%

Zhao et al. (2013) Rowan et al. (2008)

Listeria innocua

High Hydrostatic Pressure

15~30 kV/ cm at 200µs 4°C, 24 s, pulse energy 3.7J, the charging voltage from pulse 23.5 KV, pulse frequency 124 pps, gas flow rate 10 L/min 400 MPa at 20°C for 10 min

100.00%

Sokołowska et al. (2014)

44

and

45

S. aureus

Pulsed Electric Field

36 KV/cm and 45 KV/cm

about 90%

Picart, Dumay, and Cheftel (2002)

High Hydrostatic Pressure

25°C for 10 min at 345 MPa

100%

Alpas, Kalchayanand, Bozoglu, and Ray (2000)

Pulsed Electric Field

25 kV/ cm for 400 µs

36.51%

Zhao et al. (2013)

High Hydrostatic Pressure

25°C for 10 min at 345 MPa

45.00%

Alpas, Kalchayanand, Bozoglu, and Ray (2000)

Dielectric Barrier Discharge Atmospheric Cold Plasma

60 w, 4 mm, 30 s

96.30%

Liao et al. (2017)

Nonthermal Plasma

40 w, 5 mm, 2 min 40 w, 5 mm, 5 min 40 w, 5 mm, 10 min

18.72 % 47.52 % 78.12%

Liao et al. (2018a)

negligible

Li et al. (2017)

12.25~20.00% 2.20~2.50%

Jofré, Aymerich, Bover-Cid, and Garriga (2010)

negligible

Zhou et al. (2007)

98.30%

Yuk and Geveke (2011)

Sonication Salmonella enterica

High Hydrostatic Pressure

400 MPa for 10 min at 15ºC 600 MPa for 10 min at 15ºC

Sonication Lactobacillus plantarum

Supercritical Carbon Dioxide

10% CO2 at 38°C

874

45

46

875 876

Table 2 Sites of cellular injury after exposure to various nonthermal sublethal treatments.

Sublethal treatment

Cell wall

Membrane (cell leakage)

Proteins

RNA (ribosomes)

DNA

References

Gamma radiation

√

√

√

?

√

Mackey and Derrick (1982)

HHP

√

√

Pulsed white light

√

√

PEF

√

HPCD

√

Somolinos, García, Pagán and Mackey (2008) √

Wuytack et al. (2003) Wuytack et al. (2003)

√ √

√

877

46

Bi et al. (2015)

47

878 879

Table 3 Nonthermal physical technologies that can induce microbial cells into VBNC state.

Microorganism

Nonthermal Technology

Medium and Parameters

Percentage of VBNC cells

References

E. coli O157:H7

High Pressure Carbon Dioxide

0.85% NaCl solution (pH 7.0), 5 MPa, 25°C, 20 min

99.00

Zhao, Bi, Hao, and Liao (2013)

0.85% NaCl solution (pH 7.0), 5 MPa, 25°C, 30 min

31.60

0.85% NaCl solution (pH 7.0), 5 MPa, 25°C, 40 min

9.90

3% Neutral Electrolyzed Water

56.00

25% Neutral Electrolyzed Water

<10.00

50% and 100% Electrolyzed Water

0.00

E. coli O157:H7 505B

E. coli

Neutral Electrolyzed Water

Ultraviolet Light

Neutral

Water, 15 W, 254 nm, 280 93.44 µW/cm2, 100 mJ/cm2

47

Han, Hung, and Wang (2018)

Zhang, Ye, Lin, Lv, and Yu (2015)

48

Salmonella Enteritidis PT 30

Yersinia enterocolitica strain 729

Salmonella Typhimurium

Atmospheric pressure plasma jet

15 min 30 min 45 min

55.00 35.00 12.00

Dolezalova and Lukes (2015)

Neutral Electrolyzed Water

3% Neutral Electrolyzed Water

68.31

Han, Hung, and Wang (2018)

25% Neutral Electrolyzed Water

<10.00

50% and 100% Electrolyzed Water

0.00

Neutral Electrolyzed Water

Thermosonication

Neutral

3% Neutral Electrolyzed Water

58.00

25% Neutral Electrolyzed Water

<10.00

50% and 100% Electrolyzed Water

0.00

53°C, 30 min, 380 W

48

Neutral

1.26

Han, Hung, and Wang (2018)

Cooper, Fridman, Fridman, and Joshi (2010)

49

52°C, 50 min, 380 W

880

49

10.00

50

881 882

Table 4 Functional genes and proteins regulated in VBNC.

Genes and proteins

Microorganism

References

mobA, rfbE, stx1 gfp murG vvhA wza and wzb tufA Rpos katG gene ppGpp

E. coli O157:H7 E. coli & Pseudomonas putida Helicobacter pylori V. vulnificus V. vulnificus V. vulnificus V. vulnificus & S. Typhimurium V. vulnificus E. coli

Yaron and Matthews (2002). Gunasekera et al. (2002) Bates, Adams, and Oliver (2003) Smith and Oliver (2006a)

EnvZ

E. coli

Outer membrane protein W (OmpW)

E. coli

Magnusson, Farewell, and Nyström (2005) Darcan, Ozkanca, Idil, and Flint (2009) Muela et al. (2008)

883

50

51

884 885

Table 5 Nonthermal physical technologies which can induce apoptosis.

Microorganism

Nonthermal Technology

Conditions

Percentage apoptosis

S. cerevisiae

Dielectric Discharge Plasma

1 min 2 min 3 min 4 min 5 min 90 J/m2 120 J/m2 500 J/m2

15.00 35.00 39.00 40.00 43.00 6.50 18.60

Nanosecond duration pulsed electric field

30kV/cm, 250ns 30kV/cm, 500ns 30kV/cm, 750ns

21.00 28.00 28.00

Novickij et al. (2019)

Ultrasound

25.5 W/cm2, 5 min 25.5 W/cm2, 15min 25.5 W/cm2, 25min

3.15 5.82 12.77

Li et al. (2018)

255 W/cm2, 5 min 255 W/cm2, 15min 255 W/cm2, 25min

5.62 13.24 20.1

30kV/cm, 250ns 30kV/cm, 500ns 30kV/cm, 750ns

31.00 29.00 34.00

Barrier

Ultraviolet irradiation

E. coli O157:H7

C. lusitaniae

Nanosecond duration pulsed electric field

of

Reference Chen, Bai, (2010)

and

Del Carratore (2002)

et

undetectable

51

Novickij et al. (2019)

Xiu

al.

52

C. guilliermondii

Nanosecond duration pulsed electric field

30kV/cm, 250ns 30kV/cm, 500ns 30kV/cm, 750ns

23.00 30.00 46.00

886

52

Novickij et al. (2019)

53

887 888

Table 6 Functional genes and proteins regulated in apoptosis.

Functions in regulating apoptosis

Detailed working process

Genes and proteins

Found in Microorganism

References

The key to initiating apoptosis Inhibition of apoptosis

Once activated by the signal pathway, degrading the proteins in cells, making cells die irreversibly By inhibiting the permeability of apoptotic protein Bax and Bak to mitochondrial membrane, blocking the apoptosis Activated by apoptotic stimulators and bound to the mitochondrial outer membrane, inducing cytochrome C enter the cytoplasm through the mitochondrial membrane, binding and activating the cytosolic cohesive proteins in the cytoplasm, providing the binding site for the initial apoptotic protease and promoting the subsequent apoptotic cascade reaction Translocating to the nucleus resulting in DNA fragmentation under conditions that trigger apoptosis Acting as a control protein of cell cycle G1 to mediates apoptosis

Caspases

Yeast

Bcl-2

Yeast

Riedl and Shi (2004) Zha et al. (1996)

Bak/Bax

S. cerevisiae Schizosaccharomyces pombe

Aif1p

Yeast

p53

S. cerevisiae Schizosaccharomyces pombe

Induction of apoptosis

889

53

and

and

Cheng (1997)

et

al.

Riedl and Shi (2004) Fröhlich and Madeo (2000)

54

890 891

Table 7 The cross response within microbial cells induced by hurdle techniques.

Microorganism

Preceding treatment

Response

Following treatment

Response

Reference

S. aureus

ultrasound

activated oxidative response occurred

nonthermal plasma

Liao et al. (2018b)

S. aureus

nonthermal plasma

ROS produced

Ultrasound

E. coli

ultrasound

cell membranes thinning, localized heating, and free radicals produced

plasma (submerged system without aeration )

oxidative response scavenge ROS produced by NTP ROS injected into microbial cells by ultrasonic waves electric discharges take place inside the bubbles generated by cavitation

892

54

Chen, Lee, Chen, Chen, and Chang (2009)

55

893 894

Fig. 1. Microbial stress, injury, adaption, and resistance to processing.

55

56

895 896

Fig. 2. The representation of cellular mechanisms of response to PEF treatment.

897

56

57

898 899

Fig. 3. The representation of cellular mechanisms of response to HHP treatment.

900

57

58

901 902 903 904

Fig. 4. Cellular mechanisms of response to HPCD in E. coli (1) Solubilization of pressurized CO2 in the external liquid phase. (2) Structural changes in the cell membrane (3) Intracellular pH (pHi) decrease. (4) Key enzyme inactivation and cellular metabolism inhibition. (5) Direct inhibitory effect of molecular CO2 and HCO3- on metabolism. (6) Disorder of the intracellular electrolyte balance. (7)Removal of vital constituents from cells and cell membranes.

905 906

58

59

907 908 909

Fig. 5. The VBNC state of cells upon exposure to extreme conditions.

59

60

910

911 912 913 914

Fig. 6. Mechanisms of ultrasound-NTP and NTP-ultrasound hurdle treatments on S. aureus.

60

Sublethal cells, VBNC, and apoptosis may happen due to nonthermal treatment; These responses could lead to potential risk to food safety; Studying microbial mechanisms helps to anticipate possible risks during food processing.