Nonthermal plasma treatment of Aspergillus spp. spores on hazelnuts in an atmospheric pressure fluidized bed plasma system: Impact of process parameters and surveillance of the residual viability of spores

Accepted Manuscript Nonthermal plasma treatment of Aspergillus spp. spores on hazelnuts in an atmospheric pressure fluidized bed plasma system: Impact of process parameters and surveillance of the residual viability of spores Beyhan Gunaydin Dasan, Ismail Hakki Boyaci, Mehmet Mutlu PII:

S0260-8774(16)30351-X

DOI:

10.1016/j.jfoodeng.2016.09.028

Reference:

JFOE 8673

To appear in:

Journal of Food Engineering

Received Date: 19 February 2016 Revised Date:

26 August 2016

Accepted Date: 26 September 2016

Please cite this article as: Dasan, B.G., Boyaci, I.H., Mutlu, M., Nonthermal plasma treatment of Aspergillus spp. spores on hazelnuts in an atmospheric pressure fluidized bed plasma system: Impact of process parameters and surveillance of the residual viability of spores, Journal of Food Engineering (2016), doi: 10.1016/j.jfoodeng.2016.09.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT

1

Nonthermal plasma treatment of Aspergillus spp. spores on hazelnuts in

2

an atmospheric pressure fluidized bed plasma system: Impact of

3

process parameters and surveillance of the residual viability of spores

RI PT

4 5

Name of the Authors:

6

Beyhan Gunaydin Dasan1, Ismail Hakki Boyaci1, 2, Mehmet Mutlu3*

8

Affiliation of the authors:

9

1

SC

7

M AN U

Department of Food Engineering, Faculty of Engineering, Hacettepe

10

University, Beytepe, 06800 Ankara, Turkey

11

2

12

Turkey

13

3

14

Biomedical Engineering, Faculty of Engineering, TOBB University of

15

Economics and Technology, Sogutozu, 06560 Ankara, Turkey

Plasma Aided Biomedical Research Center (pabmed), Department of

TE D

16

Food Research Center, Hacettepe University, Beytepe, 06800 Ankara,

*Corresponding author:

18

Prof. Dr. Mehmet Mutlu

19

TOBB University of Economics and Technology

21 22

AC C

20

EP

17

Faculty of Engineering Department of Biomedical Engineering Sogutozu, Ankara, 06560, Turkey Phone: +90 312 292 42 68

23

Fax: +90 312 292 40 91

24

e-mail: [email protected]

1

ACCEPTED MANUSCRIPT

25

Abstract In this study, the impact of fluidized bed reactor diameters and plasma forming

27

gases on inactivation efficiency of the Atmospheric Pressure Fluidized Bed Plasma

28

(APFBP) system for aflatoxigenic spores of Aspergillus flavus and Aspergillus

29

parasiticus on hazelnuts were investigated. Hazelnuts were artificially contaminated

30

with A. flavus and A. parasiticus and then treated with dry air or nitrogen plasma for

31

up to 5 min in two different fluidizing bed reactors of APFBP system at various

32

plasma parameters. The decontamination effect of APFBP on Aspergillus spp.

33

spores increased with the applied reference voltage and the frequency. The killing

34

effect of plasma on the spores decreased as the diameter of the fluidized bed reactor

35

increased. The fungicidal effects on A. flavus (4.17 log) and A. parasiticus (4.09 log)

36

were found for air plasma treatment after 5 min. Due to the formation of active

37

plasma species in the presence of oxygen, the air plasma generated at APFBP

38

system was more effective than nitrogen plasma on decontamination of Aspergillus

39

spp. spores, as expected. The total inactivation of the natural background microbiota

40

of hazelnuts was obtained within maximum 2 min APFBP treatment. The

41

aflatoxigenic spores that remained on hazelnuts after plasma process were

42

considered as damaged cells, because they could not continue growing during

43

storage at 25 °C for 30 days. The damage caused by APFBP treatment on

44

Aspergillus spp. spore cells was demonstrated by using scanning electron

45

microscopy.

47 48

SC

M AN U

TE D

EP

AC C

46

RI PT

26

Keywords: Atmospheric pressure plasma, Fluidized bed reactor, Inactivation, Aspergillus flavus, Aspergillus parasiticus, Hazelnut

2

ACCEPTED MANUSCRIPT

49

1.

Introduction Food safety is a crucial element of food industry, and decontamination is an

51

essential step in many food processing industries. Due to more restrictive food laws

52

and higher quality expectations, food safety for agricultural products, which are under

53

the threat of toxigenic fungal infection, is gaining further importance. Filamentous

54

fungi are ubiquitous in nature, but they present a potential threat for humans,

55

especially those who are immunosuppressed, and for economically important plants

56

and animals. Fungi can contaminate foods at several stages of production, whenever

57

the conditions of temperature and humidity are favorable (Frisvad, 1991). Aflatoxins,

58

naturally produced by Aspergillus species (i.e. Aspergillus flavus and Aspergillus

59

parasiticus) during pre or post harvest of different crops, are the most toxic class of

60

mycotoxins (Iqbal et al., 2012; Iqbal and Asi, 2013). The most commonly produced

61

toxins are aflatoxin B1 (AFB1) and AFB2 (Smith, 1991). AFB1 was classified as a

62

group 1A carcinogen by the International Agency for Research on Cancer (IARC,

63

1993), and it has been associated with the development of human hepatic and extra

64

hepatic carcinogenesis (Iqbal et al., 2012). Human exposure to AFs can result

65

directly from the ingestion of contaminated foods, or indirectly through the

66

consumption of foods derived from animals previously exposed to AFs through their

67

feed (Hammami et al., 2014). The inhibition of aflatoxigenic fungal growth on food

68

and feed is necessary to reduce the potential risk to human and animal health.

70 71 72

SC

M AN U

TE D

EP

AC C

69

RI PT

50

The application of conventional thermal sterilization methods is limited since many

food products are sensitive to heat and moisture. With respect to the inactivation of Aspergillus spp., a variety of potential sterilization methods have already been researched, e.g. use of natural, biological or chemical agents (Guynot et al., 2005;

73

Magnusson et al., 2003; Rusul and Marth, 1987; Stiles et al., 2002), microwave

74

treatment (Basaran and Akhan, 2010; Czylkowski et al., 2013), gamma, UV and

75

electron beam irradiation (Assuncao et al., 2015; Aziz and Moussa, 2002; Refai et

3

ACCEPTED MANUSCRIPT

al., 1996; Trompeter et al., 2002). Due to problems concerning safety issues,

77

possible losses in the nutritional quality of treated commodities, and unsatisfactory

78

efficacy, poor consumer acceptance and cost implications; none of these methods

79

are efficient for reducing the fungal load.

RI PT

76

A promising nonthermal alternative to these methods is cold atmospheric pressure

81

plasma technology, which enables a microbial multi target inactivation on food

82

surface. Plasma, occasionally referred as the fourth state of matter, is a partially or

83

completely ionized gas and a reactive atmosphere where a variety of energetic

84

species (charged and excited particles, reactive neutrals and UV photons) are

85

formed mainly from collisions of energetic electrons with heavy particles (atoms,

86

molecules, ions). These plasma generated species can inactivate microorganisms

87

both individually and in their synergetic combinations, but the latter gives a more

88

effective outcome (Fridman, 2008; Moisan et al., 2002; von Keudell et al., 2010). The

89

working gas used and other process parameters determine the concentration of

90

these different reagents (Ehlbeck et al., 2011; Weltmann et al., 2008). Cold plasma

91

can be generated under atmospheric and low pressure conditions by using radio

92

frequency or microwave sources. Plasma applications performed under atmospheric

93

pressure have already antimicrobial effects at temperatures below 40 °C (Frohling et

94

al., 2012; Knorr et al., 2011).

EP

TE D

M AN U

SC

80

The use of plasma is well established in several fields such as surface

96

modification for variable applications (Biederman et al., 2001; Mutlu et al., 1998),

97 98 99

AC C

95

electrochemical sensors (Gunaydin et al., 2010), and preparation of functional surfaces e.g. amphoteric, bacterial anti-fouling (Akdogan and Mutlu, 2012; Cokeliler et al., 2007; Sen et al., 2012). Due to the nonthermal characteristic of non-equilibrium

100

plasma discharges (energetic electrons but cold heavy particles), these plasma

101

processes offer unique combination of high reactivity at moderate temperatures,

102

which is beneficial for the treatment of temperature sensitive substrates. In general,

103

plasma-generated species only act on the surface of substrates; therefore, the

4

ACCEPTED MANUSCRIPT

reduction of microbial contamination is limited to the outer surface, where most of the

105

contamination is located (Laca et al., 2006). However, bulk properties are not

106

affected. There are three primary mechanisms by which cold plasma inactivates

107

microorganisms: (1) direct chemical interaction of cells with reactive species and

108

charged particles; (2) UV damage of cellular components and membranes; (3) UV-

109

mediated DNA strand breakage. While one mode of action may be more

110

predominant than another in any given cold plasma system, the greatest sanitizing

111

efficacy will be achieved through multiple antimicrobial mechanisms (Laroussi et al.,

112

2004; Moisan et al., 2002; Niemira, 2012).

SC

RI PT

104

Many studies on plasma inactivation are available and have been reviewed by

114

many researchers (Boudam et al., 2006; Lerouge et al., 2001; Moisan et al., 2001).

115

There are also studies on plasma decontamination of various kinds of food e.g.

116

bacon, pork, ham, apples, melons, mangos, lettuce, cheese, eggs and nuts (Misra et

117

al., 2011; Shama and Kong, 2012). Furthermore, recent studies on plasma treatment

118

of corn salad (Baier et al., 2013), fresh fruit and vegetables (Schnabel et al., 2014),

119

cheddar cheese (Yong et al., 2015), wheat grains (Butscher et al., 2015; Butscher et

120

al., 2016), herbs and spices (Hertwig et al., 2015a), and whole black pepper (Hertwig

121

et al., 2015b) have been published.

TE D

M AN U

113

To the best of our knowledge, there has been no available study focusing on the

123

atmospheric pressure plasma decontamination of aflatoxigenic fungal infection on

124

grain-like food products due to their high pathogenicity. Hazelnut was chosen as

126 127

AC C

125

EP

122

model food sample owing to the facts that they are susceptible to toxigenic fungal infection during postharvest operations, transport and storage, and that fungal contamination may result in mycotoxin production (Arrus et al., 2005; Williams et al.,

128

2004), cause health problems and financial burden especially during exportation.

129

Mycotoxins are produced at the end of the exponential phase or at the beginning of

130

the stationary phase of the mould growth, which corresponds to the fifth day for

131

Aspergillus, at optimal conditions of temperature (27-30 °C) and relative humidity

5

ACCEPTED MANUSCRIPT

132

(85%) to produce aflatoxins (Carjaval and Castillo, 2002). Rather than eliminating the

133

toxins after they have been produced, the goal should be the inactivation of fungus

134

before toxin production. The aim of this study is to investigate the feasibility of plasma inactivation of

136

aflatoxigenic spores on hazelnuts in two different fluidizing bed reactors and to study

137

the influence of process parameters on decontamination efficacy of atmospheric

138

pressure fluidized bed plasma (APFBP) system. The APFBP system allows for the

139

plasma treatment of a continuously flowing granular product and is, in principle,

140

scalable to industrial dimensions. In addition, multiple circulations of the product in

141

the system make it possible to realize an adequate treatment time. In some studies,

142

other researchers have only used a vibrating table that ensures homogeneous

143

treatment of granular particles (Butscher et al., 2016). In our study, granular food

144

sample was subjected to fluidization, which causes bouncing and rotation of

145

particles. This continuous movement and the avoidance of permanent contact points

146

with the plasma jet provide homogenous particle treatment. In our previous study, we

147

achieved significant reductions of aflatoxigenic fungi on hazelnuts in APFBP system

148

in one fluidizing reactor by applying air plasma. In this study, the impact of fluidized

149

bed reactor diameter and plasma forming gases on inactivation efficiency of the

150

APFBP system on aflatoxigenic fungi were investigated. And then, survival of the

151

spores after plasma treatment during a certain storage period, an aspect which is not

152

taken into consideration in most other studies on plasma decontamination of food

154 155

SC

M AN U

TE D

EP

AC C

153

RI PT

135

products, was studied.

2.

Materials and methods

2.1.

Hazelnut samples

156

Hazelnut was selected as model food sample in this study. Hazelnut samples

157

were purchased from a local supplier from the northeast region of Turkey as

6

ACCEPTED MANUSCRIPT

158

unroasted, and they retained their inner shells that were 9-11 mm in diameter,

159

0.59±0.07 g in weight, and 0.97±0.06 g/cm3 in density. Samples were stored at 4 °C.

160

2.2.

161

survivor microorganisms on sample surfaces

RI PT

Microbial procedure/Evaluation of the initial and plasma treated

Aflatoxin producing species of Aspergillus [A. flavus (ATCC 327) and A.

163

parasiticus (ATCC 1041)] were obtained from TÜBİTAK MAM Culture Collection,

164

Turkey, on slant agar media. These pure cultures were inoculated onto slants and

165

plates of Yeast Extract Glucose Chloramphenicol (YGC) Agar (Merck KGaA,

166

Darmstadt, Germany) and cultured for 5 days at 25-28 °C.

SC

162

A pre-decontamination process was applied to hazelnuts with 70% ethanol (Merck

168

KGaA, Darmstadt, Germany) in order to avoid the natural background microbiota,

169

and then artificial contamination procedure with A. flavus / A. parasiticus was carried

170

out, which was described in detail in our previous study (Dasan et al., 2016).

M AN U

167

The same microbiological procedure was applied to the samples both before the

172

plasma treatment to determine the initial fungal load on the surface and after the

173

plasma

174

decontamination process. Samples were dispersed into 0.85% sterile saline solution

175

containing 0.1% Tween 80, which ease the transfer of especially fungal spores from

176

the surface to the dilution solution by altering the surface energy (Aberkane et al.,

177

2002). After rinsing for 15 min, the resulting washing solution was serially diluted and

179 180

evaluate

the

surviving

microorganisms

during

the

EP

to

AC C

178

treatment

TE D

171

mold spore counts were determined by surface plating (100 µL) of appropriate aliquots in triplicate on YGC agar. YGC plates were incubated at 25-28 °C for 3-7 days and colonies were counted after the incubation period. The results were

181

expressed as log colony-forming units per gram (log cfu/g) and the detection limit of

182

this method was 10 cfu/g. Three samples were taken from each experiment having

183

been performed at varying parameters and evaluated in duplicate. For standard

184

difference calculation, first the duplicates were averaged, and then mean was

7

ACCEPTED MANUSCRIPT

calculated from three sample results. Then, the surviving fungal spore counts after

186

plasma process was compared to the mean of six control samples, which were

187

inoculated but untreated samples representing the initial microbial concentration. D-

188

values of the Aspergillus spp., which express the time required for the reduction by

189

one logarithmic unit, were determined for each specific plasma treatment condition,

190

where Nt is the final spore concentration and N0 is the initial value:

191

RI PT

185




ℎ     = −.  =  

(1)



2.3.

Experimental setup

SC

192

The atmospheric pressure plasma system (Plasmatreat GmbH, Steinhagen,

194

Germany) consists of a 3x400V-16A power generator, a plasma jet, a high voltage

195

transformer and a pressure supply control unit. In the atmospheric pressure plasma

196

jets, the active region of the discharge is blown by flowing auxiliary gas, which pulls

197

the particles outside the electrode area in propagating ionization waves and forms a

198

stream of active particles burning as a small jet. Local applicability and higher power

199

are coupled, and the generated plasma at atmospheric pressure directly gets into

200

contact with the treated surface. The atmospheric pressure plasma system could be

201

operated with dry air and nitrogen in the range of 5–10 kV electrode voltage and 18-

202

25 kHz frequencies (at a maximum power of 655 W) with a precursor gas flow of

203

1000-5000 L/h.

205 206 207

TE D

EP

Two fluidized bed reactors at two different diameters with the same L/D ratio of 3

AC C

204

M AN U

193

were designed to be compatible with APFBP system. These reactors were made from glass with a 49 mm diameter, 147 mm length for the first reactor and 65 mm diameter, 195 mm length for the second reactor with the same L/D ratio of 3. A

208

schematic representation of the atmospheric pressure fluidized bed plasma reactor

209

was given in Fig.1. Detailed information and demonstration of the designed APFBP

210

system was given in our previous study (Dasan et al., 2016).

8

ACCEPTED MANUSCRIPT

Hazelnuts that were artificially contaminated with A. flavus and A. parasiticus were

212

placed in the APFBP reactor in direct contact with plasma afterglow. The plasma was

213

ignited for the respective treatment time at variable plasma parameters [655 W-460

214

W] by using dry air and nitrogen as the plasma forming gas at 3000 L/h flow rate.

215

Reference trials only with airflow were performed under the same conditions without

216

generating plasma using the method given in detail by Dasan et al. (2016). It was

217

clearly seen that mold spores were strongly adhered to the hazelnut surface and the

218

pure gas flow had no effect on the inactivation of fungal spores. Following the

219

plasma treatment, the substrates were poured into sterile Erlenmeyer flasks and the

220

microbiological procedure was applied as described in previous section.

221

2.4.

M AN U

SC

RI PT

211

Decontamination of natural background flora

A homogeneous sampling of the hazelnut samples that represents the whole bulk

223

was performed to determine the natural background micro flora concentration, and

224

then the same microbiological surface washing procedure was applied as described

225

above. Total yeast-mold (YM) counts were determined by surface plating on YGC

226

agar, while total aerobic mesophilic bacteria (TAMB) counts were determined by

227

surface plating on Tryptic Soy Agar (TSA). YGC and TSA plates were incubated at

228

25-28 °C for 3-7 days and 35-37 °C for 2 days, resp ectively. Colonies were counted

229

after the incubation period and the results were expressed as log colony-forming

230

units per gram (log cfu/g).

232 233

EP

AC C

231

TE D

222

Plasma decontamination process at optimum parameters were applied to the

control hazelnut samples (without pre-decontamination process) at APFBP system. The surviving microbial counts on the samples after plasma treatment were

234

determined as described before. Three samples were taken from each experimental

235

run and evaluated in duplicate. Standard errors were calculated from three sample

236

results.

9

ACCEPTED MANUSCRIPT

237

2.5.

Survival of the Aspergillus spp. spore cells after plasma treatment /

238

Discrimination of the healthy and damaged spore cells Microbial cells exposed to different physical and chemical treatments suffer injury

240

that could be reversible in food materials during storage. The injured cells can repair

241

in a medium containing the necessary nutrients under optimum conditions (pH,

242

temperature) leading to outbreaks of foodborne disease and food spoilage. The

243

injured cells can form colonies on non-selective but not on selective agar. A suitable

244

medium was designed to show inhibitor effect on plasma treated and damaged

245

Aspergillus spp. spore cells, while allowing untreated and healthy ones to grow. A

246

similar method was applied to investigate the injury recovery of pathogenic bacteria

247

(S. aureus, L. monocytogenes) in high hydrostatic pressure treated milk during

248

storage in which varying supplements were used to compose selectivity in the

249

growing media for each tested pathogen (Bozoglu et al., 2004). From this point of

250

view, YGC agar medium was modified by adding varying concentrations of weak acid

251

preservatives (sodium benzoate (SB), potassium sorbate (PS)) which are known as

252

preservatives for fungal growth of common contaminants of bakery products

253

including isolates of Eurotium, Aspergillus and Penicillium (Marin et al., 2003; Marin

254

et al., 2002). The basic aim was to prove that the remainder fungal spore load on the

255

food samples after plasma process were also damaged cells, which could not

256

continue growing during storage. Distinguishing healthy Aspergillus spp. spore cells

258 259 260

SC

M AN U

TE D

EP

AC C

257

RI PT

239

from those, which were damaged after plasma process, was also a focus of this study. For this purpose, SB and PS were added to YGC agar composition at the required amount before autoclaving to obtain varying final concentrations (0.05- 2%). Molten media (15 mL) was poured into 9 cm sterile Petri plates. The pHs of the final

261

media were checked after sterilization with a PHM210 pH meter (Radiometer,

262

Denmark).

10

ACCEPTED MANUSCRIPT

For each isolate, an untreated spore suspension of ≈ 107 spores/mL was

264

prepared, and Petri plates were point-inoculated in the center. Plates were incubated

265

for 7 days at 25-28 °C. Colony diameters were recor ded, and visible growth period

266

was traced. The Petri plates were examined daily or when necessary, and the

267

diameter of the growing colonies were measured in two directions at right angles to

268

each other. The concentrations of preservatives added to YGC agar media for

269

modification were optimized by taking into consideration the inhibitor effects on

270

Aspergillus spp. Following this process, untreated spore suspensions of A. flavus

271

and A. parasiticus (≈ 107 spores/mL), as the control group, were inoculated on both

272

control and modified YGC agar media by surface plating (100 µL) of appropriate

273

aliquots in triplicate to determine the spore counts. At the same time, spore counts of

274

surviving Aspergillus spp. on hazelnut samples after plasma treatment at optimum

275

plasma parameter (655 W air plasma at D1: 49 mm reactor) were determined through

276

surface plating of appropriate aliquots in triplicate on the same control and modified

277

YGC agar media. Plates were incubated for 7 days at 25-28 °C.

278

2.6.

279

(SEM)

TE D

M AN U

SC

RI PT

263

Sample preparation procedure for Scanning Electron Microscope

Scanning electron micrographs were obtained from control and plasma treated

281

(655 W – 30 s) A. flavus and A. parasiticus spores. The suspensions consisting of

282

fungal hyphae and spores were fixed in 2.5% gluteraldehyde overnight at 4 °C,

284 285

AC C

283

EP

280

rinsed with 0.1 M sodium phosphate buffer (PBS), and then fixed with 1% osmium tetraoxide (OsO4) in 0.1 M PBS for 30 min. Following three rinses in 0.1 M PBS (10 min each) and two rinses with water (10 min each), fixed samples were dehydrated

286

in a graded series of ethanol concentrations (from 30% to 100%) for 15 min each.

287

The samples were fully dehydrated by applying three times 10 min washes in 100%

288

ethanol. 10 µL portions of the sample were placed on SEM stubs, which were air-

289

dried in desiccators before sputter coating with a thin layer of gold palladium. After

11

ACCEPTED MANUSCRIPT

coating, the samples were examined using a scanning electron microscope

291

(QUANTA 400F Field Emission SEM, METU Central Lab., Ankara Turkey) operated

292

at the high vacuum mode with an acceleration voltage of 20 kV (Dasan et al., 2016;

293

Guven et al., 2011).

294

2.7.

RI PT

290

Statistical analysis

All experiments were performed in triplicate. Each observation within

296

each replicate was determined in duplicate. The microbial counts for each

297

replicate were transformed to log CFU/g and treatments were assigned for

298

comparison. The statistical analysis was performed by one-way analysis of

299

variance (ANOVA) using SPSS 13.0 statistical package (SPSS Inc., Chicago,

300

USA). When significant differences were detected, the differences among the

301

mean values were determined by performing Duncan’s multiple comparison

302

tests at a confidence level of p < 0.05.

303

3.

304

3.1.

M AN U

SC

295

TE D

Results and discussion

Initial concentrations of Aspergillus spp.

Hazelnuts were artificially contaminated with A. flavus/A. parasiticus and

306

incubated for 18-24 h at 25-28 °C to enable mold spores to adhere to the sample

307

surfaces. The initial fungal load concentrations on the samples were given in Table 1.

309 310

AC C

308

EP

305

3.2.

Reduction of A. flavus and A. parasiticus in APFBP reactors via air

plasma

APFBP was applied at two different frequencies (25 kHz and 20 kHz) and varying

311

reference voltages (100% V, 90% V, 80% V) for 1 to 5 min by using dry air as the

312

precursor with a flow of 3000 L/h in two fluidizing reactors with different diameters

313

(D1: 49 mm and D2: 65 mm).

12

ACCEPTED MANUSCRIPT

314

3.2.1 First fluidized bed reactor (D1: 49 mm) The results of plasma inactivation of A. flavus and A. parasiticus spores on

316

hazelnut samples in the APFBP system was reported in our previous study in which

317

the first reactor (D1: 49 mm) was used (Dasan et al., 2016). In that study, air plasma

318

treatment at 25 kHz with the reference voltage of 100% V at atmospheric conditions

319

were the optimum parameter that ended up with the highest inactivation. This plasma

320

treatment inhibited A. flavus and A. parasiticus spores by 4.50±0.15 and 4.19±0.06

321

log (cfu/g) in 5 min.

322

3.2.2 Second fluidized bed reactor (D2: 65 mm)

M AN U

SC

RI PT

315

The same plasma decontamination process was carried out in the second

324

fluidizing reactor (D2: 65 mm) with a greater diameter to investigate the impact of

325

reactor dimensions on lethal effect of the plasma. At the end of 5 min APFBP

326

treatment at 25 kHz and 20 kHz with the reference voltages of 100% V, 90% V, and

327

80% V; A. flavus on hazelnuts was inhibited by 3.82±0.20, 3.45±0.15, 3.02±0.17 log

328

(cfu/g) and 3.22±0.20, 2.77±0.10, 2.58±0.10 log (cfu/g), respectively (Fig.2.). All

329

substrates presented in this study show a fungal spore reduction characteristic,

330

which is fast in the beginning and slows down after the fast initial phase because of

331

the spore distribution on the sample surface. A big proportion of the spores were

332

distributed over the outer side of the hazelnuts, and hence easily accessible for the

334 335 336

EP

AC C

333

TE D

323

plasma species. At the same time, some spores may have been hidden in the porous surface of the hazelnut where they could be shaded from the plasma species during the process. A major challenge in the plasma decontamination of grain-like products is presumed to result from their complex surface structure and geometry, where

337

microorganisms can be sheltered by the uneven surface, loose pieces of particle or

338

hidden deep inside the surface. The penetration limitation of the active species

339

formed during plasma process is the biggest restricting factor for the use of plasma

13

ACCEPTED MANUSCRIPT

340

technology in sterilization and decontamination treatments. For this reason,

341

decontamination efficiency of plasma process will be increased with the smoothness

342

of the applied surfaces. Parallel with the findings in our studies, Hertwig et al. (2015b) reported in their

344

study on plasma decontamination of whole black pepper that the inactivation

345

efficiency of direct plasma treatment is reduced since the surface structure of

346

peppercorns with cracks, grooves and pits shield microorganisms from active

347

species. In another study, plasma decontamination of rapeseeds to molecular sieves,

348

glass beads and glass helixes were compared and an improved inactivation

349

efficiency was obtained for glass substrates while molecular sieves showed even

350

lower reductions than rapeseeds (Schnabel et al., 2012b). Butscher et al. (2016) also

351

inactivated G. stearothermophilus endospores by 5 log on polypropylene (PP)

352

granules and 4 log on PP plates, but only 1 log CFU on wheat grains due to the

353

surface topography. Differences in inactivation efficiency were attributed to the

354

complexity of the surface structure. A further comparison of rapeseeds, radish, dill,

355

carrot, parsley, soft wheat and peppercorns also revealed a correlation between

356

surface topologies and inactivation kinetics (Schnabel et al., 2012a). On the other

357

hand, higher inactivation levels of Aspergillus spp. spores could be reached by a

358

lower initial concentration, which prevents stacking of spores on the surface of

359

samples. Stacking tend to decrease the efficiency of plasma inactivation since

360

covered microorganisms are shielded from the produced plasma species (Hury et al.,

362 363

SC

M AN U

TE D

EP

AC C

361

RI PT

343

1998; Moisan et al., 2002). The influence of inoculation level is an important aspect, and its impact should be further investigated. The linear velocity of the particles decreases in the fluidized bed when the gas

364

flow that provides the fluidizing process –which is higher than the minimum

365

fluidization velocity- is kept constant, while the diameter of the reactor increases. In

366

this case, the decontamination effect of plasma might be expected to be higher

367

because particles will move closer to the plasma jet as their linear velocity decrease.

14

ACCEPTED MANUSCRIPT

However, as seen through the results, the killing effect of plasma on the spores

369

decreased as the diameter of the fluidized bed reactor increased, which might be a

370

consequence of the increase in the reactor volume. Compared to the first reactor,

371

particles gain movement and fluidize in a larger volume, and the number of passing

372

of particles in front of the plasma jet might decrease as the diameter of the reactor

373

increases. On the basis of “collision theory” and “probability density function”

374

concept, having a larger diameter in reactor reduces the probability of the meeting of

375

the active species of plasma with hazelnut particles. Besides, the total density of the

376

active species generated during plasma process, which causes the killing process of

377

microorganisms, decreases as the reactor volume increases, and thus the lethal

378

affect also decreases.

M AN U

SC

RI PT

368

In the case of A. parasiticus, APFBP treatment at 25 kHz and 20 kHz with the

380

reference voltages of 100% V, 90% V, 80% V caused reductions by 3.75±0.08,

381

3.28±0.04, 2.94±0.08 log (cfu/g) and 3.12±0.10, 2.61±0.15, 2.34±0.08 log (cfu/g),

382

respectively (Fig.3.). Parallel with the results of the previous study, it was observed

383

that A. flavus spores were more susceptible to plasma decontamination process than

384

A. parasiticus spores. The killing effect of APFBP treatment on A. flavus spores were

385

found to be higher, thus a more considerable reduction was achieved when all other

386

parameters were kept constant. The definitive difference between the two species is

387

that A. flavus produces conidia which are rather variable in shape and size, have

388

relatively thin walls and range from smooth to moderately rough, the majority being

390 391 392 393 394

EP

AC C

389

TE D

379

finely rough. In contrast, conidia of A. parasiticus are spherical and have relatively thick, rough walls (Pitt and Hocking, 2009). For this reason, reactive species produced during plasma would easily react to relatively thin-walled and smoother conidia of A. flavus and a higher lethal effect is an expected finding. The statistical analyses of the decontamination results were given in the supplementary material.

15

ACCEPTED MANUSCRIPT

395

3.3.

Reduction of A. flavus and A. parasiticus in APFBP reactors via

396

nitrogen plasma The results achieved from the first part of the experiments in which air plasma was

398

applied on hazelnut samples showed that the maximum aflatoxigenic fungal spore

399

reduction was obtained at 100% reference voltage and 25 kHz frequencies with a

400

total power of 655 W was applied at both fluidized bed reactors (D1: 49 mm and D2:

401

65 mm). In the second part of the study, nitrogen was used as the plasma forming

402

gas to investigate the effect of gas composition on inactivation of Aspergillus spp.

403

spores during plasma process.

SC

APFBP was applied to hazelnuts at the optimum plasma parameter (655 W) for 1

M AN U

404

RI PT

397

405

to 5 min by using nitrogen as the precursor at both fluidizing bed reactors.

406

3.3.1 First fluidized bed reactor (D1: 49 mm)

At the end of 5 min nitrogen plasma treatment at optimum parameters to hazelnut

408

samples artificially contaminated with Aspergillus spp., 4.17±0.15 and 4.09±0.16 log

409

(cfu/g) reductions were achieved on A. flavus and A. parasiticus spores at D1: 49 mm

410

reactor (Fig.4.a).

TE D

407

It was observed that the air plasma generated at APFBP system was found to be

412

more effective than nitrogen plasma for decontamination of Aspergillus spp. spores.

413

Reactive atoms, charged particles, reactive oxygen species (ROSs), reactive

415 416 417

AC C

414

EP

411

nitrogen species (RONs) and UV photons, each of which contributes to inactivation of microorganisms, are formed when air is used as plasma process gas (MaiProchnow et al., 2014). However, a large part of the antimicrobial effect of atmospheric pressure plasma is attributed to the reactive oxygen and nitrogen

418

species (RONS) (Graves, 2012). Reactive oxygen species (atomic oxygen (O),

419

ozone (O3), and hydroxyl radicals (OH•)) generated during plasma in the presence of

420

oxygen are known to be the most important components that provide plasma

16

ACCEPTED MANUSCRIPT

inactivation, cell damage and cell death (Fridman et al., 2007; Moisan et al., 2001;

422

von Woedtke et al., 2013). Therefore, owing to oxygen content of air, the damage

423

caused by the plasma active species generated during plasma process on cells was

424

higher than nitrogen.

425

3.3.2 Second fluidized bed reactor (D2: 65 mm)

RI PT

421

Reductions of 3.70±0.12 and 3.57±0.11 log (cfu/g) were achieved at D2: 65 mm

427

reactor on A. flavus and A. parasiticus spores after nitrogen plasma treatment of 5

428

min in APFBP system (Fig.4.b).

SC

426

While the reductions on A. flavus and A. parasiticus achieved at D1: 49 mm

430

fluidized bed reactor was higher, A. flavus spores were more susceptible to plasma

431

decontamination process than A. parasiticus spores. These results confirm the

432

decontamination data obtained from APFBP reactors by using dry air as precursor.

M AN U

429

A closer look at the inactivation characteristics (see Figures 2-4) indicates

434

multiphasic behavior with a faster initial inactivation and a subsequent decrease in

435

inactivation rate. This phenomenon implies that not all microorganisms are equally

436

exposed to plasma treatment, and it is often explained by stacking of microorganisms

437

and the action of different inactivation mechanisms. High contamination levels can

438

result in stacking of microorganisms, where the topmost layer is easily accessible for

439

fast inactivation mechanisms (e.g. UV inactivation), while the covered layers are

440

shielded until the upper layers are eroded, a process which is considered to proceed

442 443

EP

AC C

441

TE D

433

more slowly (Hury et al., 1998; Moisan et al., 2002). Besides stacking of microorganisms, the distribution of microorganisms over the sample surfaces might also motivate multiphasic inactivation characteristics. While the majority of

444

microorganisms are spread over the outer surface and accessible to plasma-

445

generated species, a part of the microorganisms might be hidden inside the rough

446

structure where they are shielded from reactive species. The statistical analyses of

447

the decontamination results were given in the supplementary material.

17

ACCEPTED MANUSCRIPT

The temperature inside the fluidized bed reactors during the plasma process was

449

below 45 °C, which could be considered as acceptabl e for “nonthermal” treatment of

450

food materials. The temperature change on hazelnut surfaces during APFBP

451

treatment at varying parameters were given in our previous study in detail (Dasan et

452

al., 2016).

453

3.4.

RI PT

448

Inhibition of natural background flora

The microbial load of total aerobic mesophilic bacteria (TAMB) and yeast-mold

455

(YM) flora found on natural background microbiata of hazelnuts were 3.27±0.17 log

456

(cfu/g) and 3.45±0.03 log (cfu/g), respectively.

SC

454

Plasma decontamination process was applied to hazelnuts to inactivate the

458

natural background flora without pre-decontamination treatment with 70% ethanol

459

and artificial contamination with aflatoxigenic fungi. APFBP treatment was performed

460

at the optimum process parameters at which the maximum microbial reduction was

461

achieved in previous experiments, 655 W plasma power was applied for 1 to 5 min

462

by using dry air as the precursor at D1: 49 mm fluidized bed reactor.

TE D

M AN U

457

No viable cells of TAMB and YM flora on hazelnut samples were detected after 2

464

min of APFBP treatment applied at optimum parameters. A 3.27±0.17 log (cfu/g)

465

reduction was achieved on TAMB, while in the case of YM flora, a 3.45±0.03 log

466

(cfu/g) reduction was obtained after 2 min of plasma decontamination process on

467

hazelnuts (Fig.5.).

469 470 471

AC C

468

EP

463

The inactivation of natural background microbiota of hazelnuts within maximum 2

min of APFBP treatment demonstrates that this decontamination process is also effective on non-toxigenic and mixed flora found naturally on food samples.

3.5.

Determination of D-values of microorganisms

472

Experimental investigation of the kinetics of cell inactivation is paramount in

473

providing a reliable temporal measure of microbial destruction and one kinetics

18

ACCEPTED MANUSCRIPT

measurement parameter, which has been used extensively by researchers studying

475

sterilization by plasma, is what is referred to as the “D” value (Decimal value)

476

(Laroussi, 2002). D-value is a kinetic measurement parameter used for

477

characterizing the slope of survival curves and inactivation phases (Laroussi and

478

Leipold, 2004; Selcuk et al., 2008) which is expressed in the unit of time and

479

determined as the time for one log reduction. Exposure to plasma provides survival

480

pilots with multiphasic linear segments (Figs. 2-5). Therefore, D1, D2 and D3 values

481

were calculated from interpolated trend-lines by using the data between 0 and 1, 1-3

482

and 3-5 min treatment periods, respectively, to characterize the slope of each

483

segment, which refers to an inactivation phase (Table 2). As clearly seen from the

484

results, the first step of inactivation kinetics generally has the smallest D values,

485

while the second and third steps have larger ones. The fungal spores that were

486

distributed over the outer side of the hazelnuts were easily inactivated by plasma

487

species, while a part of them may have been sheltered in the uneven surface.

488

Therefore this could require a longer time for inactivation.

TE D

M AN U

SC

RI PT

474

Under plasma treatment, microorganisms undergo intense electron or ion

490

bombardments and their spore coatings or cell wall materials are eroded and

491

sometimes rupture that result in fatal outcomes (Moisan et al., 2001). To date, the

492

experimental work on germicidal effects of atmospheric pressure plasmas has shown

493

that D-value could vary considerably and survivor curves take different shapes

494

depending on the type of microorganism, the type of the medium supporting the

496 497

AC C

495

EP

489

microorganisms and the method of exposure (direct or remote). On inorganic substances D-values to inactivate a great variety of microorganisms ranged from 2 s to 5.5 min (Park et al., 2007). D-values of 1.1 min and 4.2 min were calculated from a

498

plasma treatment of 15 min for A. parasiticus when SF6 and air was used as process

499

gases in low-pressure plasma (Basaran et al., 2008).

19

ACCEPTED MANUSCRIPT

3.6.

Recovery of the Aspergillus spp. spore cells after plasma treatment /

501

Discrimination of the healthy and damaged Aspergillus spp. spore cells after

502

plasma treatment

503

Plasma could either compromise the integrity of the whole cell, leading to its death,

504

or could simply render the cell uncultivable but not necessarily dead (Laroussi and

505

Leipold, 2004). Therefore, the impact of storage on recovery of aflatoxigenic fungi on

506

the surface of hazelnut samples following the plasma treatment was investigated

507

after 30 days of storage at 25 °C. The results obta ined from these experiments had

508

been given in our previous study (Dasan et al., 2016). Table 3 shows the viable

509

spore counts of Aspergillus spp. which were evaluated both immediately and after 30

510

days of storage at 25 °C following the plasma treat ment. And also, hazelnuts, which

511

were artificially contaminated with Aspergillus spp. spores at a level close to the final

512

concentration on the surface after plasma treatment (≈ 1-2 log cfu/g) were evaluated

513

as control.

M AN U

SC

RI PT

500

According to the results, Aspergillus spp. spores continued to grow on the control

515

samples of hazelnuts under the storage conditions. In A. flavus and A. parasiticus

516

counts of control hazelnut samples, 115% and 76% increases were observed at the

517

end of the storage period. However, A. flavus and A. parasiticus counts on plasma

518

treated hazelnuts samples decreased by 5% and 10% after storage for 30 days. It

519

was observed that the damage caused by plasma process on Aspergillus spp.

521 522

EP

AC C

520

TE D

514

spores were not temporary; the detrimental effect occurred on the structure of spores could not be repaired during storage. Besides, contrary to what is expected, the viable spore counts decreased. Damaged spore cells could maintain their viability

523

immediately after plasma treatment; however, they could not protect their vitality

524

during storage period due to the damage caused in the course of plasma treatment.

525

Since hazelnut surface provides less optimal growing conditions for fungal spores

526

compared to growing medium, Aspergillus spp. spore counts on control samples

20

ACCEPTED MANUSCRIPT

after 30 days of storage were ≈ 4-5 log (cfu/g), while their concentrations could reach

528

to ≈ 7 log (cfu/g) at growing medium. As a result, the remaining fungal spore cells on

529

hazelnuts could preserve only their vitality in dormant state during the storage since

530

they were damaged. When inoculated on growing media at the end of storage, they

531

could form colonies on agar plate as they are living cells.

RI PT

527

In addition to this, modified YGC agar media were prepared by using weak acid

533

preservatives, which were known to prevent the fungal growth. They were used to

534

confirm that Aspergillus spp. spores remaining after plasma treatment could not grow

535

on hazelnut samples during storage as a consequence of the damage they were

536

exposed to during plasma treatment. The basic aim was to prove that the remaining

537

fungal spore load on the sample surfaces after plasma process were also damaged

538

cells that could not continue growing during storage, while Aspergillus spp. spores on

539

untreated samples as control group could grow at the same storage conditions. In

540

the presence of optimum concentration of preservative, distinguishing healthy

541

Aspergillus spp. spore cells from the ones damaged after plasma process was

542

aimed. For this purpose, modified YGC agar media were prepared by adding varying

543

concentrations of SB and PS. It was observed that PS showed greater inhibition of

544

Aspergillus spp. colonies on the basis of colony diameters compared to SB;

545

therefore, further experiments were performed by using varying concentrations of PS

546

in the modification of YGC agar medium. Potassium sorbate was also defined as the

547

most suitable weak-acid preservative for preventing fungal growth regardless of

549 550 551

M AN U

TE D

EP

AC C

548

SC

532

physical conditions such as pH and aw (Marin et al., 2002). The reduction percentage of colony diameter of point-inoculated untreated Aspergillus spp. on 7th day of incubation at 25-28 °C on PS modified YGC media in comparison with YGC (control) agar were given in Table 4.

552

Fig.6. illustrates the results of spore counts of untreated Aspergillus spp. spores

553

on PS modified and control YGC agar media. The untreated Aspergillus spp. spores

21

ACCEPTED MANUSCRIPT

could grow on YGC agar media modified with PS at concentrations varying between

555

0.25-1%, while the growth of both isolates were inadequate at 2% PS. At this point,

556

it’s obvious that the untreated Aspergillus spp. spores could grow in the presence of

557

1% PS, and PS did not show inhibitor effect on spores before this concentration. The

558

pH values of the media used were given in Table 5.

RI PT

554

Spore counts of surviving Aspergillus spp. on hazelnut samples after plasma

560

treatment of 5 min at optimum plasma parameter (655 W air plasma at D1: 49 mm

561

reactor) on the same growing media were given in Fig.7.

SC

559

As clearly seen from the results, no substantial difference was observed in the

563

growth of plasma treated Aspergillus spp. spores at 0.25- 0.50% PS modified and

564

control YGC agar media. However, the spore counts of plasma treated Aspergillus

565

spp. spores on hazelnut samples were much less at 1% PS modified YGC agar than

566

at control YGC agar. Also, no viable spore counts of plasma treated species were

567

observed on 2% PS modified YGC agar, but at the same time, this concentration of

568

preservative inhibited the growth of healthy control Aspergillus spp. spore cells

569

(Fig.6.). As a result, the presence of 1% PS in growing medium did not show any

570

inhibitor effect on untreated Aspergillus spp. spores, while the same concentration of

571

PS inhibited the growth of plasma treated ones. The harm given by plasma to the

572

structure of Aspergillus spp. spores on hazelnut surfaces after plasma treatment

573

inhibited their growth during storage time. The damaged cells could preserve their

574

vitality on the hazelnut samples during storage without increasing their concentration.

576

TE D

EP

AC C

575

M AN U

562

3.7.

Scanning Electron Microscopy (SEM) images

The effects of atmospheric plasma process on the structure of Aspergillus spp.

577

were demonstrated via scanning electron microscopy. Fig. 8 (a-d) shows the SEM

578

imaging of A. flavus and A. parasiticus spores before and after plasma treatment (at

579

655 W for 30s). The damage caused by plasma on the structure of conidial head of

580

A. flavus could be clearly seen in Fig. 8 (a) and (b). The metulae and phialide

22

ACCEPTED MANUSCRIPT

structures of A. flavus were completely destroyed and splintered because of the

582

damage caused by active species formed during plasma process. In Fig. 8 (c) and

583

(d), the untreated spores of A. parasiticus were observed as individually spread

584

spheres, whereas the integrity of the spore cells were broken after plasma process

585

and the cell contents were dispersed as clusters. SEM results reveal the damage

586

occurred after plasma process that destroyed the cell integrity and consequently the

587

death of the cells. The treated samples show clear indications of a strong erosion

588

mechanism, which causes considerable perforation and disintegration. Presumably,

589

this erosion phenomenon results from so-called chemical sputtering, a process

590

where defects are created by the impact of ions, and oxygen species interact with

591

these defects to form volatile compounds like CO, CO2 and H2O (Raballand et al.,

592

2008; Rauscher et al., 2010; Rossi et al., 2009).

593

4.

M AN U

SC

RI PT

581

Conclusion

This study shows the difference in the antimicrobial efficiency between

595

atmospheric pressure fluidizing bed plasma reactors with varying diameters by

596

means of using two different precursors as plasma forming gas on hazelnuts. The

597

focus of the study was the plasma inactivation of aflatoxigenic Aspergillus spp.

598

spores on model food sample at atmospheric pressure fluidized bed plasma reactors

599

flooded with air and nitrogen. Aspergillus spp. spores could be efficiently inactivated

600

on hazelnut samples. Yet, optimizing the plasma process conditions and decreasing

602 603

EP

AC C

601

TE D

594

the initial microbial load could improve the treatment efficiency. Although the mechanisms of plasma inactivation in our experiments are still not

fully clarified, further studies should focus on the question whether the

604

decontamination process can be improved by carefully tuning the gas mixture.

605

Reactive gases like oxygen could be added to increase the formation of reactive

606

oxygen species with high antimicrobial potential (Fridman, 2008). Furthermore, the

607

influence of moisture should be studied in detail to optimize the balance between

23

ACCEPTED MANUSCRIPT

608

quenching of the discharge and production of reactive species from water.

609

Presumably, chemical sputtering, where defects are created by the impact of ions

610

and attacked by oxygen species, may be responsible for spore inactivation. The results of this study demonstrate the potential of APFBP system designed for

612

the decontamination of granular, dry, heat sensitive food products on an

613

experimental scale as a post harvest sanitation process, which could be easily

614

incorporated into existing food production lines. Further research is needed to fully

615

understand and improve the application of cold atmospheric pressure plasma and

616

the product-plasma interactions as well as scale-up this technology to pilot scale.

SC

RI PT

611

Acknowledgement

618

This study was supported by The Scientific and Technological Research

EP

TE D

Council of Turkey; Project Number: 113O779.

AC C

619

M AN U

617

24

ACCEPTED MANUSCRIPT

620

References Aberkane, A., Cuenca-Estrella, M., Gomez-Lopez, A., Petrikkou, E., Mellado, E., Monzon, A., Rodriguez-Tudela, J.L., (2002). Comparative evaluation of two different methods of inoculum preparation for antifungal susceptibility testing of filamentous fungi. J Antimicrob Chemother 50(5), 719-722.

625 626 627

Akdogan, E., Mutlu, M., (2012). Generation of amphoteric surfaces via glowdischarge technique with single precursor and the behavior of bovine serum albumin at the surface. Colloids and Surfaces B-Biointerfaces 89, 289-294.

628 629 630

Arrus, K., Blank, G., Abramson, D., Clear, R., Holley, R.A., (2005). Aflatoxin production by Aspergillus flavus in Brazil nuts. Journal of Stored Products Research 41(5), 513-527.

631 632 633

Assuncao, E., Reis, T.A., Baquiao, A.C., Correa, B., (2015). Effects of Gamma and Electron Beam Radiation on Brazil Nuts Artificially Inoculated with Aspergillus flavus. Journal of Food Protection 78(7), 1397-1401.

634 635

Aziz, N.H., Moussa, L.A.A., (2002). Influence of gamma-radiation on mycotoxin producing moulds and mycotoxins in fruits. Food Control 13(4-5), 281-288.

636 637 638 639

Baier, M., Foerster, J., Schnabel, U., Knorr, D., Ehlbeck, J., Herppich, W.B., Schluter, O., (2013). Direct non-thermal plasma treatment for the sanitation of fresh corn salad leaves: Evaluation of physical and physiological effects and antimicrobial efficacy. Postharvest Biology and Technology 84, 81-87.

640 641 642

Basaran, P., Akhan, U., (2010). Microwave irradiation of hazelnuts for the control of aflatoxin producing Aspergillus parasiticus. Innovative Food Science & Emerging Technologies 11(1), 113-117.

643 644 645

Basaran, P., Basaran-Akgul, N., Oksuz, L., (2008). Elimination of Aspergillus parasiticus from nut surface with low pressure cold plasma (LPCP) treatment. Food Microbiology 25(4), 626-632.

646 647 648 649

Biederman, H., Boyaci, I.H., Bilkova, P., Slavinska, D., Mutlu, S., Zemek, J., Trchova, M., Klimovic, J., Mutlu, M., (2001). Characterization of glow-discharge-treated cellulose acetate membrane surfaces for single-layer enzyme electrode studies. Journal of Applied Polymer Science 81(6), 1341-1352.

650 651 652 653

Boudam, M.K., Moisan, M., Saoudi, B., Popovici, C., Gherardi, N., Massines, F., (2006). Bacterial spore inactivation by atmospheric-pressure plasmas in the presence or absence of UV photons as obtained with the same gas mixture. Journal of Physics D-Applied Physics 39(16), 3494-3507.

654 655 656

Bozoglu, F., Alpas, H., Kaletunc, G., (2004). Injury recovery of foodborne pathogens in high hydrostatic pressure treated milk during storage. Fems Immunology and Medical Microbiology 40(3), 243-247.

SC

M AN U

TE D

EP

AC C

657 658 659 660 661

RI PT

621 622 623 624

Butscher, D., Schlup, T., Roth, C., Muller-Fischer, N., Gantenbein-Demarchi, C., von Rohr, P.R., (2015). Inactivation of microorganisms on granular materials: Reduction of Bacillus amyloliquefaciens endospores on wheat grains in a low pressure plasma circulating fluidized bed reactor. Journal of Food Engineering 159, 48-56.

662 663 664

Butscher, D., Zimmermann, D., Schuppler, M., Rudolf von Rohr, P., (2016). Plasma inactivation of bacterial endospores on wheat grains and polymeric model substrates in a dielectric barrier discharge. Food Control 60, 636-645.

665 666

Carjaval, M., Castillo, P., (2002). Effects of Aflatoxins Contaminating Food on Human Health, Tropical Biology and Conservation Management. EOLSS pp. 1-6.

25

ACCEPTED MANUSCRIPT

Cokeliler, D., Caner, H., Zemek, J., Choukourov, A., Biederman, H., Mutlu, M., (2007). A plasma polymerization technique to overcome cerebrospinal fluid shunt infections. Biomedical Materials 2(1), 39-47.

670 671 672

Czylkowski, D., Hrycak, B., Jasinski, M., Dors, M., Mizeraczyk, J., (2013). Atmospheric pressure microwave microplasma microorganism deactivation. Surface & Coatings Technology 234, 114-119.

673 674 675 676

Dasan, B.G., Mutlu, M., Boyaci, I.H., (2016). Decontamination of Aspergillus flavus and Aspergillus parasiticus spores on hazelnuts via atmospheric pressure fluidized bed plasma reactor. International Journal of Food Microbiology 216, 50-59.

677 678 679 680

Ehlbeck, J., Schnabel, U., Polak, M., Winter, J., von Woedtke, T., Brandenburg, R., von dem Hagen, T., Weltmann, K.D., (2011). Low temperature atmospheric pressure plasma sources for microbial decontamination. Journal of Physics DApplied Physics 44(1), 013002.

681 682

Fridman, A., (2008). Plasma biology and plasma medicine, Plasma chemistry. Cambrigde University Press, pp. 848-913.

683 684 685 686

Fridman, G., Brooks, A.D., Balasubramanian, M., Fridman, A., Gutsol, A., Vasilets, V.N., Ayan, H., Friedman, G., (2007). Comparison of direct and indirect effects of non-thermal atmospheric-pressure plasma on bacteria. Plasma Processes and Polymers 4(4), 370-375.

687 688 689 690

Frisvad, J.C., Samson, R. A., (1991). Filamentous fungi in foods and feeds: ecology, spoilage and mycotoxins production, in: D. K. Arora, M., K. G., Marth, E. H. (Ed.), Handbook of applied mycology: foods and feeds. Marcel Dekker, New York, pp. 31-68.

691 692 693 694 695

Frohling, A., Baier, M., Ehlbeck, J., Knorr, D., Schluter, O., (2012). Atmospheric pressure plasma treatment of Listeria innocua and Escherichia coli at polysaccharide surfaces: Inactivation kinetics and flow cytometric characterization. Innovative Food Science & Emerging Technologies 13, 142150.

696 697 698

Graves, D.B., (2012). The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology. Journal of Physics D-Applied Physics 45(26), 263001 (42pp).

699 700 701 702

Gunaydin, B., Sir, N., Kavlak, S., Guner, A., Mutlu, M., (2010). A New Approach for the Electrochemical Detection of Phenolic Compounds. Part I: Modification of Graphite Surface by Plasma Polymerization Technique and Characterization by Raman Spectroscopy. Food and Bioprocess Technology 3(3), 473-479.

706 707 708 709 710

SC

M AN U

TE D

EP

AC C

703 704 705

RI PT

667 668 669

Guven, B., Basaran-Akgul, N., Temur, E., Tamer, U., Boyaci, I.H., (2011). SERSbased sandwich immunoassay using antibody coated magnetic nanoparticles for Escherichia coli enumeration. Analyst 136(4), 740-748. Guynot, M.E., Marin, S., Sanchis, V., Ramos, A.J., (2005). An attempt to optimize potassium sorbate use to preserve low pH (4.5-5.5) intermediate moisture bakery products by modelling Eurotium spp., Aspergillus spp. and Penicillium corylophilum growth. International Journal of Food Microbiology 101(2), 169177.

711 712 713

Hammami, W., Fiori, S., Al Thani, R., Kali, N.A., Balmas, V., Migheli, Q., Jaoua, S., (2014). Fungal and aflatoxin contamination of marketed spices. Food Control 37, 177-181.

714 715

Hertwig, C., Reineke, K., Ehlbeck, J., Erdoğdu, B., Rauh, C., Schlüter, O., (2015a). Impact of remote plasma treatment on natural microbial load and quality

26

ACCEPTED MANUSCRIPT

parameters of selected herbs and spices. Journal of Food Engineering 167, Part A, 12-17.

718 719 720

Hertwig, C., Reineke, K., Ehlbeck, J., Knorr, D., Schlüter, O., (2015b). Decontamination of whole black pepper using different cold atmospheric pressure plasma applications. Food Control 55, 221-229.

721 722 723

Hury, Vidal, Desor, Pelletier, Lagarde, (1998). A parametric study of the destruction efficiency of Bacillus spores in low pressure oxygen-based plasmas. Letters in Applied Microbiology 26(6), 417-421.

724 725 726 727

IARC, (1993). Some naturally occurring substances: food items and constituents, heterocyclic amines and mycotoxins, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. International Agency for Research on Cancer, Lyon, France.

728 729 730

Iqbal, S., Asi, M., Ariño, A., Akram, N., Zuber, M., (2012). Aflatoxin contamination in different fractions of rice from Pakistan and estimation of dietary intakes. Mycotoxin Research 28(3), 175-180.

731 732

Iqbal, S.Z., Asi, M.R., (2013). Assessment of aflatoxin M-1 in milk and milk products from Punjab, Pakistan. Food Control 30(1), 235-239.

733 734 735

Knorr, D., Froehling, A., Jaeger, H., Reineke, K., Schlueter, O., Schoessler, K., (2011). Emerging Technologies in Food Processing. Annual Review of Food Science and Technology 2, 203-235.

736 737 738

Laca, A., Mousia, Z., Diaz, M., Webb, C., Pandiella, S.S., (2006). Distribution of microbial contamination within cereal grains. Journal of Food Engineering 72(4), 332-338.

739 740 741

Laroussi, M., (2002). Nonthermal decontamination of biological media by atmospheric-pressure plasmas: Review, analysis, and prospects. Ieee Transactions on Plasma Science 30(4), 1409-1415.

742 743 744 745

Laroussi, M., Leipold, F., (2004). Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. International Journal of Mass Spectrometry 233(1-3), 81-86.

746 747 748

Laroussi, M., Lu, X., Kolobov, V., Arslanbekov, R., (2004). Power consideration in the pulsed dielectric barrier discharge at atmospheric pressure. Journal of Applied Physics 96(5), 3028-3030.

749 750 751

Lerouge, S., Wertheimer, M.R., Yahia, L.H., (2001). Plasma Sterilization: A Review of Parameters, Mechanisms, and Limitations. Plasmas and Polymers 6(3), 175188.

755 756 757

SC

M AN U

TE D

EP

AC C

752 753 754

RI PT

716 717

Magnusson, J., Strom, K., Roos, S., Sjogren, J., Schnurer, J., (2003). Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. Fems Microbiology Letters 219(1), 129-135. Mai-Prochnow, A., Murphy, A.B., McLean, K.M., Kong, M.G., Ostrikov, K., (2014). Atmospheric pressure plasmas: Infection control and bacterial responses. International Journal of Antimicrobial Agents 43(6), 508-517.

758 759 760 761

Marin, S., Abellana, M., Rubinat, M., Sanchis, V., Ramos, A.J., (2003). Efficacy of sorbates on the control of the growth of Eurotium species in bakery products with near neutral pH. International Journal of Food Microbiology 87(3), 251258.

762 763

Marin, S., Guynot, M.E., Neira, P., Bernado, M., Sanchis, V., Ramos, A.J., (2002). Risk assessment of the use of sub-optimal levels of weak-acid preservatives in

27

ACCEPTED MANUSCRIPT

the control of mould growth on bakery products. International Journal of Food Microbiology 79(3), 203-211.

766 767 768

Misra, N.N., Tiwari, B.K., Raghavarao, K.S.M.S., Cullen, P.J., (2011). Nonthermal Plasma Inactivation of Food-Borne Pathogens. Food Engineering Reviews 3(34), 159-170.

769 770 771

Moisan, M., Barbeau, J., Crevier, M.C., Pelletier, J., Philip, N., Saoudi, B., (2002). Plasma sterilization. Methods mechanisms. Pure and Applied Chemistry 74(3), 349-358.

772 773 774 775

Moisan, M., Barbeau, J., Moreau, S., Pelletier, J., Tabrizian, M., Yahia, L.H., (2001). Low-temperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms. International Journal of Pharmaceutics 226(1-2), 1-21.

776 777 778

Mutlu, M., Mutlu, S., Boyaci, I.H., Alp, B., Piskin, E., (1998). High-linearity glucose enzyme electrodes for food industries: Preparation by a plasma polymerization technique. Polymers in Sensors 690, 57-65.

779 780 781

Niemira, B.A., (2012). Cold Plasma Reduction of Salmonella and Escherichia coli O157:H7 on Almonds Using Ambient Pressure Gases. Journal of Food Science 77(3), M171-M175.

782 783 784 785

Park, B.J., Takatori, K., Lee, M.H., Han, D.W., Woo, Y.I., Son, H.J., Kim, J.K., Chung, K.H., Hyun, S.O., Park, J.C., (2007). Escherichia coli sterilization and lipopolysaccharide inactivation using microwave-induced argon plasma at atmospheric pressure. Surface & Coatings Technology 201(9-11), 5738-5741.

786 787

Pitt, J.I., Hocking, A.D., (2009). Aspergillus and Related Teleomorphs, Fungi and Food Spoilage, third ed. Springer, New York, pp. 305-311.

788 789 790 791

Raballand, V., Benedikt, J., Wunderlich, J., von Keudell, A., (2008). Inactivation of Bacillus atrophaeus and of Aspergillus niger using beams of argon ions, of oxygen molecules and of oxygen atoms. Journal of Physics D-Applied Physics 41(11), 115207 (8pp).

792 793 794

Rauscher, H., Kylian, O., Benedikt, J., von Keudell, A., Rossi, F., (2010). Elimination of Biological Contaminations from Surfaces by Plasma Discharges: Chemical Sputtering. Chemphyschem 11(7), 1382-1389.

795 796 797

Refai, M.K., Aziz, N.H., ElFar, F., Hassan, A.A., (1996). Detection of ochratoxin produced by A-ochraceus in feedstuffs and its control by gamma radiation. Applied Radiation and Isotopes 47(7), 617-621.

798 799 800

Rossi, F., Kylian, O., Rauscher, H., Hasiwa, M., Gilliland, D., (2009). Low pressure plasma discharges for the sterilization and decontamination of surfaces. New Journal of Physics 11, 115017 (33pp).

SC

M AN U

TE D

EP

AC C

801 802 803 804

RI PT

764 765

Rusul, G., Marth, E.H., (1987). Growth and Aflatoxin Production by AspergillusParasiticus Nrrl-2999 in the Presence of Potassium Benzoate or Potassium Sorbate and at Different Initial Ph Values. Journal of Food Protection 50(10), 820-825.

805 806 807 808

Schnabel, U., Niquet, R., Krohmann, U., Polak, M., Schlüter, O., Weltmann, K.-D., Ehlbeck, J., (2012a). Decontamination of Microbiologically Contaminated Seeds by Microwave Driven Discharge Processed Gas. Journal of Agricultural Science and Applications 1(4), 99-105.

809 810

Schnabel, U., Niquet, R., Krohmann, U., Winter, J., Schlüter, O., Weltmann, K.D., Ehlbeck, J., (2012b). Decontamination of Microbiologically Contaminated

28

ACCEPTED MANUSCRIPT

Specimen by Direct and Indirect Plasma Treatment. Plasma Processes and Polymers 9(6), 569-575.

813 814 815 816

Schnabel, U., Niquet, R., Schlüter, O., Gniffke, H., Ehlbeck, J., (2014). Decontamination and Sensory Properties of Microbiologically Contaminated Fresh Fruits and Vegetables by Microwave Plasma Processed Air (PPA). Journal of Food Processing and Preservation 39(6),653-662.

817 818 819

Selcuk, M., Oksuz, L., Basaran, P., (2008). Decontamination of grains and legumes infected with Aspergillus spp. and Penicillum spp. by cold plasma treatment. Bioresource Technology 99(11), 5104-5109.

820 821 822 823

Sen, Y., Bagci, U., Gulec, H.A., Mutlu, M., (2012). Modification of Food-Contacting Surfaces by Plasma Polymerization Technique: Reducing the Biofouling of Microorganisms on Stainless Steel Surface. Food and Bioprocess Technology 5(1), 166-175.

824 825 826 827 828

Shama, G., Kong, M.G., (2012). Prospects for treating foods with cold atmospheric gas plasmas, in: Machala, Z., Hensel, K., Akishev, Y. (Ed.), Plasma for BioDecontamination, Medicine and Food Security. NATO Sicence for Peace and Security Series A: Chemistry and Biology. Springer, The Netherlands, pp. 433443.

829 830 831

Smith, J.E., Ross, I. C. , (1991). The toxigenic Aspergillus, in: Smith, J.E., Henderson, R. S. (Ed.), Mycotoxins and animal foods. CRC Press, London, pp. 31-60.

832 833 834

Stiles, J., Penkar, S., Plockova, N., Chumchalova, J., Bullerman, L.B., (2002). Antifungal activity of sodium acetate and Lactobacillus rhamnosus. Journal of Food Protection 65(7), 1188-1191.

835 836 837 838

Trompeter, F.J., Neff, W.J., Franken, O., Heise, M., Neiger, M., Liu, S.H., Pietsch, G.J., Saveljew, A.B., (2002). Reduction of Bacillus Subtilis and Aspergillus Niger spores using nonthermal atmospheric gas discharges. Ieee Transactions on Plasma Science 30(4), 1416-1423.

839 840 841 842 843 844

von Keudell, A., Awakowicz, P., Benedikt, J., Raballand, V., Yanguas-Gil, A., Opretzka, J., Flotgen, C., Reuter, R., Byelykh, L., Halfmann, H., Stapelmann, K., Denis, B., Wunderlich, J., Muranyi, P., Rossi, F., Kylian, O., Hasiwa, N., Ruiz, A., Rauscher, H., Sirghi, L., Comoy, E., Dehen, C., Challier, L., Deslys, J.P., (2010). Inactivation of Bacteria and Biomolecules by Low-Pressure Plasma Discharges. Plasma Processes and Polymers 7(3-4), 327-352.

845 846

von Woedtke, T., Reuter, S., Masur, K., Weltmann, K.D., (2013). Plasmas for medicine. Physics Reports-Review Section of Physics Letters 530(4), 291-320.

851 852 853 854 855 856 857 858

SC

M AN U

TE D

EP

AC C

847 848 849 850

RI PT

811 812

Weltmann, K.D., Brandenburg, R., von Woedtke, T., Ehlbeck, J., Foest, R., Stieber, M., Kindel, E., (2008). Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pressure plasma jets (APPJs). Journal of Physics DApplied Physics 41(19), 194008 (6pp). Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., Aggarwal, D., (2004). Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. American Journal of Clinical Nutrition 80(5), 1106-1122. Yong, H.I., Kim, H.J., Park, S., Kim, K., Choe, W., Yoo, S.J., Jo, C., (2015). Pathogen inactivation and quality changes in sliced cheddar cheese treated using flexible thin-layer dielectric barrier discharge plasma. Food Research International 69, 57-63.

859 29

ACCEPTED MANUSCRIPT

RI PT

Table 1

A. parasiticus

cfu/g

4.68±0.31*106

5.76±0.92*106

log (cfu/g)

6.67±0.03

6.76±0.08

AC C

EP

TE D

M AN U

A. flavus

SC

Initial fungal spore concentrations on sample surface

ACCEPTED MANUSCRIPT

RI PT

Table 2 D-values of A. flavus and A. parasiticus at varying process parameters applied to hazelnut samples

Air D1: 49 mm

D- values (min)

A. parasiticus

D1

D2

D3

D1

D2: 65 mm

A. flavus

M AN U

A. flavus

SC

Precursor

A. parasiticus

D2

D3

D1

D2

D3

D1

D2

D3

100% V 90% V 80% V

0.52 0.77 0.88

2.08 1.74 2.61

1.25 1.67 1.33

0.50 0.59 0.66

2.46 2.41 2.38

1.46 2.10 2.46

0.47 0.52 0.52

3.21 4.78 4.76

1.87 1.80 2.90

0.45 0.49 0.54

3.27 2.73 3.49

2.12 3.91 3.88

20 kHz

100% V 90% V 80% V

0.80 0.86 1.65

2.66 2.47 2.58

1.33 1.82 1.65

0.58 0.66 1.07

2.69 2.54 2.09

1.97 3.43 2.70

0.72 0.80 1.16

2.80 2.72 2.26

1.80 2.55 2.39

0.52 0.60 0.80

2.60 3.77 4.00

4.76 4.76 3.39

0.36

4.30

4.36

0.36

4.74

5.06

Precursor 0.35

3.90

2.45

0.36

EP

100% V

AC C

25 kHz

TE D

25 kHz

4.70

Nitrogen 2.29

ACCEPTED MANUSCRIPT

RI PT

Table 3 Aspergillus spp. spore counts of control and plasma treated hazelnuts before and after storage Control

After plasma

After plasma

(0. day)

(30. day)

(0. day)

(30. day)

A. flavus

2.52±0.23

5.41±0.14

2.17±0.15

2.07±0.09

A. parasiticus

2.50±0.12

4.39±0.04

2.57±0.06

2.31±0.11

M AN U

TE D EP AC C

SC

Control

log (cfu/g)

ACCEPTED MANUSCRIPT

RI PT

Table 4

agar YGC

YGC

YGC

reduction %

+ 0.25% PS

+ 0.50% PS

+ 1% PS

A. flavus

20%

30%

51%

A. parasiticus

18%

29%

51%

AC C

EP

TE D

M AN U

Colony diameter

SC

Reduction percentage of colony diameters on 7th day of incubation at 25-28 °C on PS modified YGC m edia in comparison with YGC (control)

ACCEPTED MANUSCRIPT

pH measurements of growing media YGC +

YGC +

(control)

0.25% PS

0.50% PS

6.48

6.54

6.58

AC C

EP

TE D

M AN U

pH (at 41 °C)

YGC

YGC +

YGC +

1% PS

2% PS

6.65

6.81

SC

Growing media

RI PT

Table 5

ACCEPTED MANUSCRIPT

1

FIGURE CAPTIONS

2 3

Fig.1. The schematic representation of APFBP reactor

RI PT

4 Fig.2. Logarithmic survival curves of A. flavus spores on hazelnuts at (a) 25

6

kHz and (b) 20 kHz 80% V, 90% V, 100% V at second fluidizing bed reactor

7

(D2: 65 mm) by using air as precursor.

SC

5

8

Fig.3. Logarithmic survival curves of A. parasiticus spores on hazelnuts at (a)

10

25 kHz and (b) 20 kHz 80% V, 90% V, 100% V at second fluidizing bed

11

reactor (D2: 65 mm) by using air as precursor.

M AN U

9

12

Fig.4. Logarithmic survival curves of A. flavus and A. parasiticus spores on

14

hazelnuts at 655 W by using nitrogen as precursor in (a) D1: 49 mm (b) D2: 65

15

mm fluidized bed reactor.

TE D

13

16

Fig.5. Logarithmic survival curve of natural background flora of hazelnuts after

18

APFBP treatment at 655 W in D1: 49 mm fluidized bed reactor by using air as

19

precursor.

21 22 23 24

AC C

20

EP

17

Fig.6. Viable spore counts of untreated A. flavus and A. parasiticus on control and PS modified YGC agar media. Different letters indicate significant (p <

0.05) differences between means.

ACCEPTED MANUSCRIPT

25

Fig.7. Viable spore counts of A. flavus and A. parasiticus on hazelnut samples

26

after plasma treatment on control and PS modified YGC agar media. Different

27

letters indicate significant (p < 0.05) differences between means.

RI PT

28 Fig.8. Scanning electron micrographs of control (without plasma treatment)

30

(a) A. flavus (c) A. parasiticus; after plasma treatment (b) A. flavus (d) A.

31

parasiticus.

AC C

EP

TE D

M AN U

SC

29

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights:

− A. flavus and A. parasiticus spores were significantly inhibited on

RI PT

hazelnuts. − The effect of process gas type, reactor diameter on inactivation was investigated.

SC

− The remaining aflatoxigenic spores on hazelnuts lost their viability for growing.

M AN U

− The damage on Aspergillus spp. spore cells was demonstrated by

AC C

EP

TE D

using SEM.