Accepted Manuscript

A combination treatment of ultrasound and ε-polylysine to improve microorganismsand storage quality of fresh-cut lettuce

Kai Fan, Min Zhang, Bhesh Bhandari, Fangjun Jiang

PII: S0023-6438(19)30655-3

DOI: https://doi.org/10.1016/j.lwt.2019.108315

Article Number: 108315

Reference: YFSTL 108315

To appear in: LWT - Food Science and Technology

Received Date: 25 February 2019

Revised Date: 23 June 2019

Accepted Date: 25 June 2019

Please cite this article as: Fan, K., Zhang, M., Bhandari, B., Jiang, F., A combination treatment ofultrasound and ε-polylysine to improve microorganisms and storage quality of fresh-cut lettuce, LWT -Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108315.

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A combination treatment of ultrasound and ε-polylysine to 1

improve microorganisms and storage quality of fresh-cut lettuce 2

3

Kai Fan a, b, Min Zhang a, c, * , Bhesh Bhandari d, Fangjun Jiang e 4

5

a State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 6

Wuxi, Jiangsu, China 7

b International Joint Laboratory on Food Safety, Jiangnan University, China 8

c Jiangsu Province Key Laboratory of Advanced Food Manufacturing Equipment and 9

Technology, Jiangnan University, China 10

d School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, 11

Australia 12

e Jiangsu First Farm Technology Co., Changzhou, Jiangsu, China 13

14

*Corresponding author: Professor Min Zhang, School of Food Science and 15

Technology, Jiangnan University, 214122 Wuxi, Jiangsu Province, China. 16

E-mail: min@jiangnan.edu.cn Fax: 0086-(0)510-8580797617

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Abstract: 18

A combination treatment of ultrasound and ε-polylysine to improve microorganisms 19

and storage quality of fresh-cut lettuce were investigated. Fresh-cut lettuce was 20

treated with ultrasound (20kHz, 17-29 W/L), ε-polylysine (0.1-0.6 g/L) and their 21

combination at 20 ºC for 10 min and then packaged as well as stored at 4 ºC for 12 d. 22

The results showed that 23 W/L ultrasound combined with 0.4 g/L ε-polylysine 23

treatment was superior to 23 W/L ultrasound or 0.4 g/L ε-polylysine treatment alone 24

in inhibiting the growth of microorganisms such as total number of colonies, mold 25

and yeast, and total coliform counts of fresh-cut lettuce during storage. Ultrasound 26

combined with ε-polylysine treatment reduced weight loss and total color difference 27

(∆E), declined POD and PPO activities, maintained the higher level of total phenolics 28

content, vitamin C and chlorophyll, as well as decreased water mobility and 29

respiration rate in fresh-cut lettuce during storage compared to control. Therefore, 30

these results demonstrated that ultrasound combined with ε-polylysine treatment was 31

helpful for inhibiting microorganisms and improving storage quality, and can be an 32

effective preservation method to extend shelf-life of fresh-cut lettuce. 33

Keywords: ε-Polylysine; Ultrasound; Microorganisms; Storage quality; Fresh-cut 34

lettuce 35

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1. Introduction 36

Fresh-cut lettuce is a very popular vegetable in human diet (Kim, Moon, Tou, 37

Mou, & Waterland, 2016). It’s vulnerability to microbial infection in postharvest 38

handling and processing leading to short shelf-life and poor quality. Washing is 39

necessary to decontaminate fresh-cut products. A suitable cleaning method for 40

fresh-cut vegetables will not only reduce the risk of diseases caused by pathogenic 41

microorganism contaminants, but also prolong shelf life (Sothornvit & 42

Kiatchanapaibul, 2009). The chlorine-containing disinfectants in fruits and vegetables 43

processing are unacceptable for affecting the environment and human health from 44

consumers’ concern (Gómez-López, Marín, Medina-Martínez, Gil, & Allende, 2013). 45

Therefore, it is necessary to identify safe washing ways to improve the safety of 46

fresh-cut lettuce. 47

ε-Polylysine (ε-PL) is a polypeptide which includes above 25 L-lysine linked by 48

amide bond at the ɛ position of the amino acid (Yoshida & Nagasawa, 2003). ε-PL is a 49

natural antibacterial agent acting also as detergent which is not only effective against 50

microorganisms such as Gram-positive and Gram-negative bacteria, yeast and mold, 51

but also heat stable, has good water solubility and is non-toxic to human (Li et al., 52

2014; Liu, Pei, Han, Feng, & Li, 2015). In 2003, ε-PL was listed as FDA-permitted 53

additives in USA and is widely applied to food. Recently, some studies have reported 54

that ε-PL had a good antimicrobial effect against various pathogens in food (Hiraki et 55

al., 2003). According to Chang, Lu, Park, and Kang (2010), ε-PL showed an effective 56

inhibition against pathogens in roast beef slurry. Song et al. (2017) reported that ε-PL 57

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showed strong effectiveness in inhibiting microbial growth in fresh-cut carrot. 58

Ultrasound (US), as a novel and safe technology, is widely used for food 59

processing and preservation due to the cavitation effect in liquid media causing the 60

microbial inactivation (Aday, Temizkan, Büyükcan, & Caner, 2013; Cheng, Zhang, 61

Adhikari, & Islam, 2014; Islam, Zhang, & Adhikari, 2013; Lagnika, Zhang, & 62

Mothibe, 2013). Several studies have reported that ultrasound was applied for the 63

preservation of fresh-cut vegetables. Pinheiro, Alegria, Abreu, Gonçalves, and Silva 64

(2015) presented that tomato subjected to US treatment keep good storage quality. 65

Hashemi (2018) found that US treatment decreased bacterial count by 0.42-1.47 log 66

CFU/g in Mirabelle plum. Although many studies have reported that the ultrasound 67

treatment is beneficial to reduce microorganisms and prolong shelf-life of fresh 68

products, the effect of ultrasound treatment alone is limited. Therefore, ultrasound has 69

been combined with other hurdles such as the preservatives of organic acids, essential 70

oils, ozone and sanitizers in order to enhance its effectiveness. Sagong et al. (2011) 71

found that US combined with organic acids can effectively reduce pathogen load 72

without inducing the degradation of quality. Aday and Caner (2014) found that 73

ultrasound combined with ozone or chlorine dioxide for 5 min prolonged shelf-life of 74

strawberries up to 4 weeks. Millan-Sango, McElhatton, and Valdramidis (2015) found 75

that essential oils combined with ultrasound can be effective against Escherichia 76

strains. Previous researches have reported antimicrobial effect of individual US or 77

ε-PL on fresh-cut products. Nonetheless, there is limited information on the effect of 78

ultrasound combined with ε-polylysine on microorganisms and quality of fresh-cut 79

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vegetables during storage. The cavitation effect of US can promote the adsorption of 80

ε-PL on the cell membrane leading to the increase of cell membrane permeability, 81

which affect the synthesis of protein and nucleic acid, and then inhibit the growth of 82

microorganisms (Tan, Bo, Guo, Cui, & Jia, 2018). The synergistic effect of two 83

treatments can make the process further effective in producing safe food products. 84

Therefore, this study paid attention to evaluate a combination treatment of ultrasound 85

and ε-polylysine on microorganisms and quality of fresh-cut lettuce during storage. 86

2. Materials and methods 87

2.1 Raw materials 88

Lettuce was purchased at Auchan supermarket (Wuxi, China), and then stored at 89

4 ºC until use. The outer and core leaves were discarded, then cut into pieces of 3 cm2 90

by a stainless knife sterilized with 75% alcohol. The cut lettuce was fully mixed to 91

achieve uniformity and used in the below sections. 92

2.2 ε-Polylysine and ultrasound treatment 93

For ε-PL treatment, fresh-cut lettuce (50 g) was put into 2 L glass beaker with 1 L 94

different concentrations of ε-polylysine solution (0, 0.1, 0.2, 0.3, 0.4 and 0.6 g/L) 95

prepared using distilled water for 10 min at 20 ºC according to the temperature 96

decreased by Chen and Zhu (2011). After treatments, sterile filter paper was used to 97

wipe the sample surface for removing excess water and air-dried for 30 min in the 98

biosafety cabinet. 99

For US treatment, fresh-cut lettuce (50 g) was put into 2 L glass beaker with 1 L 100

distilled water at 20 ºC. A 15 mm diameter ultrasound probe (NingBo Scientz 101

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Biotechnology Co., China) immersed 2 cm into distilled water at 20 kHz. Schematic 102

diagram of ultrasound instrument is shown in Fig. 1. US treatment were conducted at 103

power densities of 17, 23 and 29 W/L (according to the method calculated by Ricce, 104

Rojas, Miano, Siche, and Augusto (2016)) for 10 min with pulse way (10 s on : 5 s 105

off ) at 20 ºC using a cold water circulation connected to a thermostatic bath. The 106

increase of temperature was below 2 °C after 10 min of US treatment. Distilled water 107

treatment for 10 min was used as a control. After the experiments, excess water from 108

the sample surface was removed by sterile filter paper, and samples were put into the 109

biosafety cabinet to dry for 30 min. 110

For the combination of ultrasound and ε-polylysine treatment (US+ε-PL), the 111

optimum treatment conditions were first measured for ε-PL treatments and US 112

treatments alone and evaluated the combined effects of ε-PL and US treatments at 113

those optimum conditions. Fresh-cut lettuce samples (50 g) were put into 2 L glass 114

beaker filling with 1 L ε-PL solution at 20 ºC and subjected to ultrasound treatment. 115

Samples treated by distilled water for 10 min were used as a control. Then, samples 116

were taken out and wiped with sterile filter paper to remove the excess water from the 117

surface, and the biosafety cabinet was used to dry samples for 30 min. 118

All samples were packaged at atmospheric conditions with 5 % O2, 5 % CO2, 119

90 % N2, and stored under 4 ºC. Samples were tested every 3 days (0, 3, 6, 9 and 12 d). 120

Triplicates of the experiments were done. 121

2.3 Microorganisms determination 122

Total number of colonies (total bacterial count), mold and yeast, and total 123

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coliform counts of samples were tested as reported by Alexopoulos et al. (2013). Total 124

number of colonies (5.87 CFU/g), mold and yeast (4.09 CFU/g), and total coliform 125

counts (4.88 CFU/g) were observed in fresh samples without treatment. Samples (25 g) 126

were put into sterile physiological saline solution with 0.2% Tween 80 (225 mL), 127

homogenized for 2 min in a stomacher at 8000 rpm (SH-IIM, Anke Biotechnology 128

Co., Shanghai, China), and then, a series of 10 times dilutions in physiological saline 129

buffer was made. One mL of diluted sample were surface-plated on plate count agar 130

in petri dish, incubated at 37 ºC for 48 h for total number of colonies count, incubated 131

at 28 ºC for 96 h in Bangladeshi-red culture medium for mold and yeast counts (Cao 132

et al., 2010) and incubated at 37 ºC for 48 h in violet red bile glucose agar for total 133

coliform counts. Results were expressed as log CFU/g. 134

2.4 Quality parameters 135

2.4.1 Weight loss and total color difference 136

Weight loss was tested as reported by Meng, Zhang, and Adhikari (2012). The 137

mass of samples was weighted every 3 days. Results were expressed as %. 138

A colorimeter (CR-400, Konica Minolta, Japan) used to estimate the chromatic 139

characteristics of samples as L*, a* and b* according to Lu et al. (2010). Three 140

different samples were determined for each treatment and each sample was taken at 141

three positions to measure color. Total color difference (∆E) was determined by the 142

below equation. 143

144

where Lo*, ao

* and bo* were color of fresh-cut lettuce samples at 0 d after treatment. 145

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2.4.2 Respiratory rate 146

Respiratory rate was tested as presented by Wu, Zhang, and Wang (2012). 147

Samples (50 g) were put into a 2 L airtight container for 30 min at 4 ºC. Gas (5 mL) 148

was taken and analyzed using gas chromatograph (Shimadzu, Co., Japan). The set 149

parameters were used for detecting CO2 as follows: Detector temperature 80 ºC, 150

column temperature 50 ºC and flow rate 0.5 mL/s. Data results were expressed as mg 151

CO2/kg·h. 152

2.4.3 Peroxidases and polyphenoloxidase activities 153

Crude enzyme extract was prepared as presented by Fante and Noreña (2012). 154

Samples (1 g) were grinded with mortar adding 5 mL 0.05 mol/L phosphate buffer 155

(pH 7.0), and then centrifuged for 20 min at 3,500 g and 4 ºC. Supernatant was used 156

for the following analysis. 157

Peroxidases (POD) activity was tested according to Meng et al. (2012). The 158

experiment was conducted by mixing 2.2 mL 1 % guaiacol, 0.2 mL 1.5 % hydrogen 159

peroxide, and 0.5 mL crude enzyme extract. Absorbance was tested using the 160

spectrophotometer at 470 nm. 161

Polyphenoloxidase (PPO) activity tested as presented by Fang, Zhang, Sun, and 162

Sun (2007). Assay was conducted by mixing 3 mL 0.01 mol/L catechol and 0.5 mL 163

crude enzyme extract. Absorbance was measured at 420 nm. 164

One unit of enzyme activity was defined as the quantity of the enzyme able to 165

produce a 0.01 increase of the absorbance units per min, both for POD and PPO. 166

2.4.4 Total phenolics content 167

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Total phenolics content was tested as reported by Wang, Zhang, and Mujumdar 168

(2012). Samples (1 g) and 80% cold acetone (5 mL) were ground, then centrifuged for 169

30 min at 3,500 g and 4 ºC. The absorbance was recorded at 765 nm after 90min using 170

the spectrophotometer (UV-2600, Precision Science Instrument, Shanghai, China). 171

2.4.5 Vitamin C and chlorophyll contents 172

Vitamin C content was determined according to Yang, Cao, Cai, and Zheng 173

(2011). Samples (10 g) were ground with 10 mL 3 % metaphosphoric acid with 174

volume made up to 100 mL, and then centrifuged for 20 min at 3,500 g. Supernatant 175

(10 mL) was titrated against standard 2,6-dichloroindophenol. 176

Chlorophyll content were tested as presented by An, Zhang, Lu, and Zhang (2006) 177

with slight modifications. Samples (1 g) were mixed with 10 mL 80 % acetone and 178

centrifuged (3,500 g, 20 ºC) for 20 min. Supernatant was determined for absorbance 179

at 645 nm (A645) and 663 nm (A663). Chlorophyll content was calculated by the 180

equation: 181

2.4.6 LF-NMR 182

A low-field nuclear magnetic resonance (LF-NMR) analyzer (Niumag Electric 183

Corporation, China) with 22.6 MHz was used to test water status of fresh-cut lettuce 184

(Xin, Zhang, & Adhikari, 2013). Sample (1±0.05 g) was placed in glass tube, then put 185

into the magnet chamber at 32 ºC. Carre-Purcelle-Meiboome-Gill (CPMG) sequence 186

was used to test transverse relaxation time (T2). 187

2.5 Statistical analysis 188

One-way analysis of variance (ANOVA) was performed by SPSS 19 (SPSS Inc., 189

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Chicago, USA). Duncan’s test was used to determine significant difference at 95 % of 190

confidence (P < 0.05). 191

3. Results and discussion 192

3.1 Effect of ε-PL treatment on microorganisms 193

Effect of different ε-PL concentration treatments on total number of colonies, 194

mold and yeast, and total coliforms counts in fresh-cut lettuce during 12 days of 195

storage are shown in Fig. 2A, B and C. ε-PL treatment significantly decreased (P < 196

0.05) initial microbial numbers. An increase in ε-PL concentration elevated the 197

inhibition effect of microorganisms at 12 d of storage period. The ε-PL can penetrate 198

microbial cells causing the interaction of ε-PL with nucleic acids to prevent nucleic 199

acids transcription (Song et al., 2017). As ε-PL concentration raised from 0.1 to 0.4 200

g/L, there were significant differences (P < 0.05) in inhibiting total number of 201

colonies, mold and yeast and total coliform counts during storage. There were no 202

significant differences (P > 0.05) on the reduction of total number of colonies, mold 203

and yeast and total coliform counts between 0.4 and 0.6 g/L at the storage period. Li 204

et al. (2014) reported that 0.5%-1.5% ε-PL treated chilled pork. They found that 205

1.25% ε-PL can effectively inhibit microbial growth. Therefore, 0.4 g/L ε-PL was 206

selected to study individual and combined effects. 207

3.2 Effect of ultrasound treatment on microorganisms 208

As can be seen from Fig. 3A, B and C. US treatment significantly decreased (P < 209

0.05) the initial microbial numbers. In comparison to control, as US power increased 210

from 17 to 29 W/L, total number of colonies, mold and yeast and total coliform 211

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counts decreased. US can bring the cavitation phenomenon, which generate 212

mechanical and chemical energies to inactivate microorganisms (Brilhante São José & 213

Dantas Vanetti, 2012). Gani et al. (2016) reported that ultrasound treatment decreased 214

microbial number of strawberry at the storage period. US treatment at 23 and 29 W/L 215

had no significant differences (P > 0.05) in inhibiting the growth of total number of 216

colonies, mold and yeast and total coliform counts at 12 d storage period. Similarly, 217

66.64-145.74 W/L US treatment of cherry tomato, in which 106.19 W/L US 218

effectively reduced spoilage microorganisms (Wang et al., 2015). Therefore, 23 W/L 219

US treatment was selected as the treatment condition to study its individual and 220

combined effects on quality during storage. 221

3.3 Effect of ultrasound combined with ε-PL treatment on microorganisms 222

As can be seen from Fig. 4A, B and C, compared to control, US (23 W/L), ε-PL 223

(0.4 g/L) and US (23 W/L) combined with ε-PL (0.4 g/L) treatments decreased initial 224

microbial numbers in fresh-cut lettuce by 0.85, 1.39 and 2.21 log CFU/g for total 225

number of colonies, 0.55, 0.8 and 1.35 log CFU/g for mold and yeast, 0.83, 1.14 and 226

1.88 log CFU/g for total coliform counts, respectively. US and ε-PL treatments all 227

reduced the initial microbial numbers of fresh-cut lettuce, and their combination had a 228

certain synergistic effect. Compared with the literature, Sagong et al. (2011) presented 229

that ultrasound combined with organic acid significantly reduced pathogen compared 230

to their individual treatments. The microbial numbers of fresh-cut lettuce subjected to 231

US treatment, ε-PL and US combined with ε-PL exhibited a growth with the 232

prolongation of storage time. Microbial numbers in fresh-cut lettuce subjected to US, 233

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ε-PL and their combined treatments were obviously lower (P < 0.05) compared to 234

control. US showed 0.75-1.11 log CFU/g reduction, ε-PL showed 1.04-1.59 log 235

CFU/g reduction and the combined effects of US and ε-PL showed 1.52-2.57 log 236

CFU/g reduction for total number of colonies, mold and yeast, and total coliform 237

counts at 12 d of storage. This suggested that US combined with ε-PL treatment can 238

effectively reduce the microbial numbers and relatively inhibited microbial growth 239

during storage. This may be due to the fact that US promoted the adsorption of ε-PL 240

on the cell membrane to inhibit microbial growth (Cao et al., 2010). Ge, Liu, Ding, 241

Xu, and Zhong (2013) also obtained that individual and combined treatments of US 242

and slightly acidic electrolyzed water inhibited the microbial number growth of 243

strawberry during storage, and the microbial numbers of treated strawberry were 244

lower than that of control. 245

3.4 Effect of ultrasound combined with ε-PL treatment on weight loss and total color 246

difference 247

Weight loss reflects the loss of water in fruits and vegetables during storage 248

(Jiang, 2013; Qian et al., 2018). Fig. 5A shows that weight loss of all treated samples 249

gradually raised as the storage time prolonged. To compare with control, weight loss 250

of fresh-cut lettuce treated by US, ε-PL and US combined with ε-PL significantly (P < 251

0.05) reduced. US combined with ε-PL treated samples had the lowest weight loss of 252

1.42 % at 12 d. This could be because US combined with ε-PL treatment preserved 253

hydrogen bonding between water molecules and macromolecules to reduce 254

respiratory metabolism of samples leading to decrease in water loss (Li et al., 2017). 255

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To compare with literature, Feng, Zhang, Adhikari, and Guo (2018) also reported that 256

combination of US and controlled atmosphere effectively decreased water loss. Hence, 257

the results suggest that US combined with ε-PL can reduce the moisture loss in 258

fresh-cut lettuce. 259

Total color difference is a critical quality indicator (Engmann, Ma, Tchabo, & Ma, 260

2015). As can be seen from Fig. 5B, ∆E of fresh-cut lettuce subjected to control, US, 261

ε-PL and US combined with ε-PL treatments increased as the storage time prolonged. 262

The ∆E of treated samples was significantly (P < 0.05) lower in comparison to control. 263

∆E of US combined with ε-PL treatment was the lowest. This could be because US 264

combined with ε-PL treatment retarded the degradation rate of pigments such as 265

chlorophyll (Feng et al., 2018). Aday and Caner (2014) also reported that US 266

combined with ozone and chlorine dioxide treatments greatly inhibited the color 267

changes. 268

3.5 Effect of ultrasound combined with ε-PL treatment on peroxidases activity, 269

polyphenoloxidase activity and total phenolics content 270

Browning related peroxidases (POD) and polyphenoloxidase (PPO) activities 271

affect the preservation of fresh-cut products (Li et al., 2017). Fig. 6 shows the effect 272

of US combined with ε-PL treatment on POD and PPO activities in fresh-cut lettuce at 273

12 d of storage period. In Fig. 6A, it is seen that POD activity increased first and then 274

decreased. POD activity for US and combination of US and ε-PL treatments 275

significantly decreased (P < 0.05) in comparison to control during the storage period. 276

However, control and ε-PL treatment had no significant differences (P > 0.05) in POD 277

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activity. At 6 d of storage, POD activity reached a maximum value of 304.5 U/kg·s for 278

control and 287.67 U/kg·s for ε-PL treatment, respectively. At 9 d of storage, POD 279

activity of fresh-cut lettuce reached the maximum value of 267 U/kg·s for US 280

treatment and 256.33 U/kg·s for US combined with ε-PL treatment, respectively. POD 281

activity for US combined with ε-PL treatment was lower compared to other treatments 282

at 12 d. This could be because free radicals produced by mechanical and chemical 283

effects of US can react with the amino acids of the enzyme structure inhibiting POD 284

activity (Wang et al., 2015). Thus, this suggested that US combined with ε-PL can 285

effectively inhibit POD activity. Wei and Ye (2011) also found that POD activity in 286

asparagus subjected to US assisted with 6-benzylaminopurinetreatment declined 287

during storage. 288

Fig. 6B shows that PPO activity increased first and then decreased during storage. 289

PPO activity for US combined with ε-PL treatment significantly decreased (P < 0.05) 290

compared to control at the storage period. At storage for 6 d, PPO activity reached 291

maximum value of 297.16 U/kg·s for control, 277.5 U U/kg·s for ε-PL treatment, 292

252.83 U/kg·s for US treatment and 234.67 U/kg·s for US combined ε-PL treatment, 293

respectively. PPO activity for US combined with ε-PL treatment was lower in 294

comparison to other treatments. This could be because US produced the mechanical 295

and chemical effects inhibiting PPO activity (Li et al., 2017). Thus, US combined 296

with ε-PL had a positive effect in inhibition of PPO activity. Lagnika, Zhang, 297

Nsoratindana, and Bashari (2014) obtained a similar result with white mushroom 298

when treated with US combined with citric acid. 299

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Fig. 6C shows that total phenolics content increased first and then decreased at 12 300

d of storage period. At 6 d, total phenolics content for control and ε-PL treatment 301

reached maximum value. At 9 d, total phenolics content for US and US combined 302

with ε-PL treatment reached maximum value. Total phenolics content for US 303

combined with ε-PL treatment significantly higher (P < 0.05) compared to control at 304

12 d. An increase in phenolic content could be attributed to the reason that US 305

combined with ε-PL treatment caused release of bound phenolics leading to maintain 306

a high total phenolics content during storage. As reported by Yang et al. (2011), the 307

combination treatment of ultrasound and salicylic acid can decay incidence of peach 308

fruit. 309

3.6 Effect of ultrasound combined with ε-PL treatment on vitamin C and chlorophyll 310

contents 311

Vitamin C and chlorophyll are key nutritional indicator in vegetables (Miller & 312

Riceevans, 1997). Fig. 7 shows effect of US combined with ε-PL treatment on vitamin 313

C and chlorophyll contents in fresh-cut lettuce at the storage period. As can be seen 314

from Fig. 7A, vitamin C content decreased as the storage time prolonged. To compare 315

with control, vitamin C content for US combined with ε-PL treatment significantly 316

reduced (P < 0.05) the loss of vitamin C during storage. At 12 d, vitamin C content 317

decreased by 56.48 % for control, 51.34 % for ε-PL treatment, 48.9 % for US 318

treatment and 44.01 % for US combined with ε-PL treatment. Thus, vitamin C content 319

for US and US combined with ε-PL treatments was higher in comparison to control 320

and ε-PL treatment at 12 d. This attributed to cavitation effect of ultrasound leading to 321

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elimination of dissolved oxygen and microbial inactivation (Bhat, Kamaruddin, 322

Min-Tze, & Karim, 2011). As presented by Chen and Zhu (2011), US combined with 323

aqueous chlorine dioxide maintained high vitamin C content at the storage period. 324

As can be seen from Fig. 7B, chlorophyll content decreased in fresh-cut lettuce at 325

the storage period. Chlorophyll content for US combined with ε-PL treatment was a 326

significantly higher (P < 0.05) in comparison to control at the same storage period. 327

Control and ε-PL treatment had significant differences in chlorophyll content after 328

storage for 6 d (P < 0.05). At 12 d, US and US combined with ε-PL treatments 329

maintained higher chlorophyll content in comparison to control and ε-PL treatment 330

during storage. This may be due to the fact that mechanical and chemical effects of 331

ultrasound inhibited reaction of enzyme and substrates to retard chlorophyll 332

degradation rate (Wei & Ye, 2011). Thus, US combined with ε-PL treatment 333

effectively maintained the chlorophyll content in the product. Wei and Ye (2011) also 334

reported that US assisted with 6-benzylaminopurine can maintain chlorophyll content 335

of asparagus. 336

3.7 Water status and respiration rate of fresh-cut lettuce 337

Fig. 8 presents water status of fresh-cut lettuce by LF-NMR. The amplitude 338

versus T2 plot can be divided into three relaxation peaks (T21, T22 and T23). Relaxation 339

time of three peaks were centered at 0.01-1 ms (T21), 4-20 ms (T22) and 80-1000 ms 340

(T23), respectively. T21 represents the water status of bound water. T22 indicates the 341

water status of immobilized water. T23 reveals the water status of free water. 342

Table 1 presents T23 and free water proportion (A23) in the vacuole. T23 values of 343

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all lettuce samples increased with the prolongation of storage time. T23 of US 344

combined with ε-PL treatment was significantly smaller (P < 0.05) in comparison to 345

control during storage. As seen from Table 1, A23 of all treated samples decreased as 346

storage time prolonged. A23 of US combined with ε-PL treatment was significantly 347

higher (P < 0.05) in comparison to other treatments at the storage period. This could 348

be because issue cell in fresh-cut lettuce had low permeability of tonoplast membrane 349

and metabolic activity, and US combined with ε-PL treatment decreased the 350

respiration rate (Fundo et al., 2016). Feng et al. (2018) reported similar results with 351

changes of water status in cucumber by US treatment. 352

As can be seen from Fig. 9, respiration rate increased first and then decreased 353

during storage. At 3 d, respiration rate reached maximum value. Fresh-cut lettuce 354

treated by US combined with ε-PL was significantly (P < 0.05) lower level of CO2 355

production compared to other treatment at the end of storage. Chen and Zhu (2011) 356

presented that US treatment can effectively retard respiration rate. This demonstrated 357

that US combined with ε-PL treatment can decrease water mobility and slow down 358

the respiration rate. 359

4. Conclusion 360

The study indicated that ε-PL, ultrasound and ultrasound combined with ε-PL 361

treatments can effectively inhibit the microorganisms growth and improve quality of 362

fresh-cut lettuce at the storage period. Ultrasound combined with ε-PL treatment can 363

be preferred over ε-PL and ultrasound alone treatments for the storage of fresh-cut 364

lettuce. Ultrasound combined with ε-PL treatment significantly decreased total 365

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number of colonies, mold and yeast, and total coliform counts. Ultrasound combined 366

with ε-PL treatment had a better effect on the reduction of weight loss and change of 367

color, the inhibition of POD and PPO activities, and the maintenance of total 368

phenolics content, vitamin C and chlorophyll contents as compared to ε-PL and 369

ultrasound alone treatments. Ultrasound combined with ε-PL treatment can also 370

decrease water mobility as indicated by LF-NMR and respiration rate. In overall 371

summary, the results exhibited that the ultrasound combined with ε-PL treatment can 372

prolong preservation period of fresh-cut lettuce. This could also be a promising result 373

that can be applied to other vegetables. 374

375

Acknowledgments 376

We acknowledge the financial support from National Key R&D Program of 377

China (No. 2018YFD0700303), Postgraduate Research & Practice Innovation 378

Program of Jiangsu Province (No. KYCX19_1802), Jiangsu Province (China) Key 379

Project in Agriculture (Contract No. BE2015310217), Jiangsu Province Key 380

Laboratory Project of Advanced Food Manufacturing Equipment and Technology (No. 381

FMZ201803), National First-class Discipline Program of Food Science and 382

Technology (No. JUFSTR20180205), all of which enabled us to carry out this study.383

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Figure and Table captions: 542

Fig. 1 Schematic diagram of ultrasound treatment set-up 543

Fig. 2 Total number of colonies (A), mold and yeast (B) and total coliform counts (C) 544

of fresh-cut lettuce at different ε-PL concentrations treatments 545

Fig. 3 Total number of colonies (A), mold and yeast (B) and total coliform counts (C) 546

of fresh-cut lettuce at different ultrasound powers treatments 547

Fig. 4 Effect of ultrasound combined with ε-PL treatments on total number of 548

colonies (A), mold and yeast (B) and total coliform counts (C) of fresh-cut lettuce 549

Fig. 5 Effect of ultrasound combined with ε-PL treatments on weight loss (A) and 550

total color difference (B) of fresh-cut lettuce 551

Fig. 6 Effect of ultrasound combined with ε-PL treatments on peroxidases activity (A), 552

polyphenoloxidase activity (B) and total phenolics content (C) of fresh-cut lettuce 553

Fig. 7 Effect of ultrasound combined with ε-PL treatments on vitamin C content (A) 554

and chlorophyll content (B) of fresh-cut lettuce 555

Fig. 8 Distribution of transverse relaxation times (T2) in fresh-cut lettuce 556

Fig. 9 Effect of ultrasound combined with ε-PL treatments on respiratory rate of 557

fresh-cut lettuce 558

Table 1 Transverse relaxation time (T23) and free water proportion (A23) of fresh-cut 559

lettuce during storage560

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561

562

Fig. 1 Schematic diagram of ultrasound treatment set-up: 1. ultrasound generator, 2. 563

beaker; 3. ultrasonic transducer, 4. ultrasound probe, 5. thermocouple, 6. distilled 564

water, 7. samples, 8. data logger565

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569

Fig. 2 Total number of colonies (A), mold and yeast (B) and total coliform counts (C) 570

of fresh-cut lettuce at different ε-PL concentrations treatments 571

Values are the mean of three replicates and error bars stand for standard deviation. 572

Different lowercase letters at the same storage time express the significant differences 573

(P < 0.05) 574

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577

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578

Fig. 3 Total number of colonies (A), mold and yeast (B) and total coliform counts (C) 579

of fresh-cut lettuce at different ultrasound powers treatments 580

Values are the mean of three replicates and error bars stand for standard deviation. 581

Different lowercase letters at the same storage time express the significant differences 582

(P < 0.05) 583

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584

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587

Fig. 4 Effect of ultrasound combined with ε-PL treatments on total number of 588

colonies (A), mold and yeast (B) and total coliform counts (C) of fresh-cut lettuce 589

Values are the mean of three replicates and error bars stand for standard deviation. 590

Different lowercase letters at the same storage time express the significant differences 591

(P < 0.05) 592

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593

594

Fig. 5 Effect of ultrasound combined with ε-PL treatments on weight loss (A) and 595

total color difference (B) of fresh-cut lettuce 596

Values are the mean of three replicates and error bars stand for standard deviation. 597

Different lowercase letters at the same storage time express the significant differences 598

(P < 0.05) 599

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603

Fig. 6 Effect of ultrasound combined with ε-PL treatments on peroxidases activity (A), 604

polyphenoloxidase activity (B) and total phenolics content (C) of fresh-cut lettuce 605

Values are the mean of three replicates and error bars stand for standard deviation. 606

Different lowercase letters at the same storage time express the significant differences 607

(P < 0.05) 608

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609

610

Fig. 7 Effect of ultrasound combined with ε-PL treatments on vitamin C content (A) 611

and chlorophyll content (B) of fresh-cut lettuce 612

Values are the mean of three replicates and error bars stand for standard deviation. 613

Different lowercase letters at the same storage time express the significant differences 614

(P < 0.05) 615

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616

617

Fig. 8 Distribution of transverse relaxation times (T2) in fresh-cut lettuce. Three 618

relaxation peaks: T21, bound water; T22, immobilized water; T23, free water 619

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620

621

Fig. 9 Effect of ultrasound combined with ε-PL treatments on respiratory rate of 622

fresh-cut lettuce 623

Values are the mean of three replicates and error bars stand for standard deviation. 624

Different lowercase letters at the same storage time express the significant differences 625

(P < 0.05) 626

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Table 1 Transverse relaxation time (T23) and free water proportion (A23) of fresh-cut lettuce during storage 627

Storage Time (d) Control ε-PL US US+ε-PL

T23 (ms) 0 219.64 ± 10.86a 219.64 ± 10.86a 219.64 ± 10.86a 219.64 ± 10.86a

3 289.94 ± 14.81a 252.35 ± 28.53ab 270.49 ± 13.07ab 235.43 ± 22.22b

6 302.15 ± 14.96a 284.42 ± 22.92a 311.27 ± 19.28ab 247.15 ±17.08b

9 355.26 ±21.39a 309.75 ± 18.20ab 346.53 ± 22.29a 261.29 ± 16.47b

12 388.37 ± 22.89a 330.28 ± 15.05b 372.16 ± 18.49ab 272.57 ± 13.84c

A23 (%) 0 96.31 ± 1.51a 96.31 ±1.51a 96.31 ±1.51a 96.31 ±1.51a

3 90.89 ± 0.63c 93.74 ± 0.52ab 92.98 ± 0.69b 94.17 ± 0.66a

6 88.65 ± 0.46d 91.58 ± 0.41c 89.17 ± 0.58b 92.34 ± 0.24a

9 86.78 ± 0.55c 89.83 ± 0.59b 87.84 ± 0.59c 91.12 ± 0.08a

12 84.54 ± 0.38d 87.22 ± 0.16b 85.25 ± 0.18c 89.56 ± 0.33a

Values are mean ± standard deviation of three replicates.Different lowercase letters in the same row express the significant differences (P < 0.05) 628

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Highlights

• Ultrasound combined with ε-polylysine treatment inhibited microorganisms.

• Ultrasound combined with ε-polylysine treatment maintained quality

attributes.

• Ultrasound combined with ε-polylysine treatment decreased water mobility.

• Ultrasound combined with ε-polylysine treatment extend shelf life of lettuce.

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Conflicts of interest

There are no conflicts to declare.