Effect of slightly acidic electrolyzed water on natural Salmonella reduction and seed germination in the production of alfalfa sprouts

Journal Pre-proof Effect of slightly acidic electrolyzed water on natural Salmonella reduction and seed germination in the production of alfalfa sprouts Chunling Zhang, Zhiyi Zhao, Gaoji Yang, Yiqi Shi, Yuyu Zhang, Xiaodong Xia, Chao Shi PII:

S0740-0020(20)30003-4

DOI:

https://doi.org/10.1016/j.fm.2020.103414

Reference:

YFMIC 103414

To appear in:

Food Microbiology

Received Date: 23 May 2019 Revised Date:

2 January 2020

Accepted Date: 6 January 2020

Please cite this article as: Zhang, C., Zhao, Z., Yang, G., Shi, Y., Zhang, Y., Xia, X., Shi, C., Effect of slightly acidic electrolyzed water on natural Salmonella reduction and seed germination in the production of alfalfa sprouts, Food Microbiology, https://doi.org/10.1016/j.fm.2020.103414. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

1

Effect of slightly acidic electrolyzed water on natural Enterobacteriaceae

2

reduction and seed germination in the production of alfalfa sprouts

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Chunling Zhanga, Zhiyi Zhaoa, Gaoji Yanga, Yiqi Shia, Yuyu Zhanga, Xiaodong Xiaa,b*,

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Chao Shia*

6 7

a

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Shaanxi 712100, P.R. China

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b

College of Food Science and Engineering, Northwest A&F University, Yangling,

School of Food Science and Technology, National Engineering Research Center of

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Seafood, Dalian Polytechnic University, 1 Qinggongyuan, Ganjingzi District, Dalian,

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Liaoning, 116034 China.

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* Corresponding authors:

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Xiaodong Xia, Professor, College of Food Science and Engineering, Northwest A&F

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University, No.22 Xinong Road, Yangling, Shaanxi 712100, P.R. China.

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School of Food Science and Technology, National Engineering Research Center of

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Seafood, Dalian Polytechnic University, 1 Qinggongyuan, Ganjingzi District, Dalian,

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Liaoning, 116034 China.

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Phone: 86-29-87091391, E-mail: [email protected]

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Chao Shi, Ph.D., College of Food Science and Engineering, Northwest A&F

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university, No.22 Xinong Road, Yangling, Shaanxi 712100, P.R. China.

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Phone: 86-29-87092486, E-mail: [email protected] 1

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Abstract

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Microbial contamination of sprouts occurs easily because of the pathogens

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present on and in the seeds and the optimal conditions for bacteria growth provided

26

during the germination and sprouting processes. This study examined the effect of

27

using slightly acidic electrolyzed water (SAEW), a ‘generally recognized as safe’

28

(GRAS) disinfectant, in place of regular water in the production process of alfalfa

29

sprouts. In the experiment, SAEW with various available chlorine concentrations

30

(ACC, 25, 35, 45 mg/L) and different pH levels (5.0, 5.7 and 6.4) was used to soak

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seeds for different length of time (0.5 and 6 h), after which the variations in natural

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Enterobacteriaceae, water absorption and seed germination (germination rate, weight

33

and length of sprouts) were determined. The results showed that when the seeds were

34

soaked with SAEW, albeit with different ACC (25, 35 and 45 mg/L) and pH levels

35

(5.0, 5.7 and 6.4), a significant reduction of Enterobacteriaceae and no negative effect

36

on sprout quality was observed. The water absorption and germination rates were also

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not significantly adversely affected by SAEW soaking. These findings suggest that

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SAEW could be used to decontaminate natural Enterobacteriaceae in the production

39

of alfalfa sprouts, with no negative side effects on the alfalfa seeds.

40 41 42

Keywords: Slightly acidic electrolyzed water; alfalfa sprouts; Enterobacteriaceae;

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decontamination

44 2

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1. Introduction

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Edible sprouts are young seedlings, obtained from seeds germination, which has

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high nutritional value. The modern consumer trend is for natural, healthy and

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convenient food (Ma et al., 2019) and, as seed sprouts fall into this category, this fresh

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produce is increasingly in demand across the world. However, seed sprouts have been

50

involved in a range of outbreaks of foodborne illnesses induced by a number of

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pathogenic bacteria, especially Salmonella and Escherichia coli O157:H7, as well as

52

Listeria monocytogenes and other Shiga-toxin-producing E. coli (STEC) (Chen et al.,

53

2018; Sadler-Reeves et al., 2016; Callejon et al., 2015; Crowe et al., 2015). The

54

European Food Safety Authority (EFSA, 2011) assessed risks from the consumption

55

of sprouted seeds worldwide and ascertained a further 43 outbreaks connected with a

56

variety of sprouted seeds (alfalfa, mung bean, soybean, cress, radish, clover, bean,

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fenugreek) consumption: 34 were due to Salmonella, 6 to STEC, and one each due to

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Yersinia enterocolitica, Bacillus cereus and L. monocytogenes. Salmonella and

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pathogenic E. coli are the bacterial pathogens most frequently found as the cause of

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outbreaks related to the contaminated sprouts (alfalfa, mung bean, cress, radish, clover,

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fenugreek) consumed, while implication of other bacterial pathogens (B. cereus, L.

62

monocytogenes, Y. enterocolitica, and Staphylococcus aureus) have rarely been

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reported. Among these, alfalfa sprout and mung bean sprout have been most often

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consumed and the most commonly involved products in outbreaks (EFSA, 2011).

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Jeter and Matthysse (2005) demonstrated that diarrheagenic E. coli strains, including

66

serovar O157:H7, were able to attach to alfalfa sprouts, whereas other strains did not. 3

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Alfalfa, which is a sprout commonly available worldwide, has reportedly been

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implicated in most of the Salmonella spp. (22/27) and E. coli (5/27) outbreaks from

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2000 to 2011 (Yang et al., 2013).

70

After the 1999 recommendation from the United States’ National Advisory

71

Committee on Microbiological Criteria for Foods (NACMCF, 1999) was promulgated,

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researchers have developed a substantial number of technologies on chemically-based

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treatments for seeds sanitizing, including acidified sodium chloride, organic acids,

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stabilized oxychloride complexes, ethanol, hydrogen peroxide, electrolyzed waters,

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ozone and natural antimicrobials (Pajak et al., 2014; Yang et al., 2013; Sikin et al.,

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2013). The primary sources of microbial contamination in seed sprouts are

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contaminated seeds and, therefore, disinfection of the seeds is the critical primary step

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in the safe production of sprouts. However, no chemically-based treatment has yet

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proven entirely effective, partly because of their limitations in contacting with

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microbes on and in the crevices of seeds, and also because of their stresses on seed

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germination or product quality (Sikin et al., 2013). Regulatory authorities have yet to

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recommend suitable alternative treatments, however, and 20,000 mg/L calcium

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hypochlorite solution remains the industry common intervention for reducing

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pathogens from seeds and sprouts (NACMCF, 1999), although chemical residues of

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this solution in sprouts are still unknown (Smith and Herges, 2018).

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Electrolyzed oxidizing water (EOW) has generally been recommended as a

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substitute sanitizer based on electrochemistry. Due to the strong bactericidal effects,

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environment-friendly attributes and ‘generally recognized as safe’ (GRAS) status, 4

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EOW has become an acclaimed alternative to harsh chemical sanitizers (Han et al.,

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2018; Huang et al., 2008). EOW is typically applied in the forms of acidic

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electrolyzed water (AEW) and slightly acidic electrolyzed water (SAEW). SAEW, at

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pH from 5.0 to 6.5, is generated by electrolyzing sodium chloride and/or hydrochloric

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acid solution in a chamber without a membrane. Previous studies have indicated that

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SAEW has no negative effect on seed germination, and on the contrary, may even

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contribute to improving the produce (radish sprout, germinated brown rice) because of

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its near-neutral pH (5.0~6.5), compared to the low pH (<3.0) of AEW (Zhang et al.,

97

2018; Zhang et al., 2016). Therefore, SAEW is more widely used in food disinfection.

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When it was used to decontaminate pea and mung bean seeds in the sprouts

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production, SAEW has been considered to be a useful way to eliminate pathogens

100

with little effect on seed germination (Zhang et al., 2019; Liu et al., 2011; Zhang et al.,

101

2011).

102

Seed germination is a most important phase in the growth cycle of plants, which

103

starts at water absorption of seeds and followed by the expansion of seeds embryos

104

(Hermann et al., 2007). Consequently, it is essential to consider the effects of

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disinfectants and their residues on the germination of seeds and ultimate food quality.

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Sprouts are usually consumed raw, and therefore, there tends to be an elevated level of

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risk associated with sprout consumption since no steps are taken to remove the

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pathogens that might be present. Ideally, disinfection should be performed throughout

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the sprout production process, including during seed soaking and germination. The

110

purpose of this work, therefore, is to investigate the effect of SAEW on natural 5

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Enterobacteriaceae reduction, as well as seed germination by using SAEW instead of

112

regular water throughout the production process.

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2. Materials and methods

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2.1 SAEW preparation

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Slightly acidic electrolyzed water (SAEW) was generated by electrolyzing 6%

116

hydrochloric acid solution using a SAEW generator (WaterGod HD-240L, Shanghai

117

Fu Qiang-Want Sanitary Accessories Ltd., China). The available chlorine

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concentration (ACC) was detected immediately after generation using a chlorine

119

tester (Chlorometer Duo, Palintest, UK). The oxidation-reduction potential (ORP) and

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pH values were determined using an ORP/pH meter (Five Easy Plus FE28, Mettler

121

Toledo, China) with an ORP electrode (FE510) and a pH electrode (FE438). Different

122

levels of ACC were obtained by adjusting the electrolytic and dilution parameters.

123

SAEW with different pH was slightly adjusted using 1 mol/L hydrochloric acid. In

124

this study, we used SAEW at ACC of 25~45 mg/L, pH of 5.0~6.4, ORP of 810~875

125

mV for disinfection effect assessment, and used tap water (TW) as a control.

126

2.2 Seed soaking and germination

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Alfalfa seeds (weight of 1.88 ± 0.01g/1000 seeds) were purchased from a local

128

seed supplier (Yongxin Technology Co., Ltd., Beijing, China). Seeds uniform in size

129

and shape were picked and soaked in various solutions at the ratio of 1:5 (m/v). After

130

soaking, the seeds were drained and placed on filter paper for 10 min to remove any

131

remaining water on their surface. The seeds were weighed using an electronic scale

132

(AL 204, Mettler Toledo, China) and water absorption rate was calculated as follows: 6

133 134

Water absorption rate (%) = (seeds weight after soaking- seeds weight before soaking)

135

/ seeds weight before soaking) ×100%

136

One hundred grains of alfalfa seeds were spread in a glass culture dish with two-

137

layer of sterile filter paper. Then the culture dish was placed in a biochemical

138

incubator (22°C; humidity 80%~90%). The seeds on the filter paper were kept moist

139

by spraying with corresponding treatment solutions, as described in Tables 1, 2 and 3,

140

respectively. Microbial analysis was carried out after seed soaking and

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pre-germination, following the method described in 2.4. Seeds were considered to

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have germinated when their coats ruptured, and the number of germinated seeds was

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counted when most sprouts were approximately 1 cm in length (about 2 days), after

144

which the rate of germination was calculated. For seed soaking test, each 25 g of

145

alfalfa seeds was immersed in SAEW with different soaking time, ACC, pH levels,

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and these three trials were conducted independently.

147

2.3 Seed sprouting

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Each 50 g of alfalfa seeds were soaked in 250 mL of SAEW for 6 h and then

149

drained and aseptically spread in single layers onto an EOW cleansed seedling tray

150

(54×27×6 cm), which was covered by 4 layers of sterilized cheese cloth. The trays

151

that filled with soaked seeds were stacked in between the two trays that with no seeds

152

filled, in order to blocking light and keep the suitable humidity. Seed-filled seedling

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trays in the stack were exchanged the sequence to ensure similar conditions. After

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germination for 40 hours, the trays filled with germinated seeds were then arranged by 7

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a single layer in climate chambers (22~25°C; relative humidity 80~90%) for

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sprouting. For each day, sufficient fresh SAEW was given and the sprouts were

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manually sprayed for 1 ~ 3 times, with no spraying on the last day (harvest day). All

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operations on seeds and sprouts were consistent during seed sprouting. When sprouts

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were 3 ~ 4 cm long, a weak LED light was given for 8 h daily until they were

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harvested on the 9th day. Microbial analysis was carried out at day 1, day 3, day 5, day

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7 and day 9, respectively, during the sprouting period.

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2.4 Microbiological analysis

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Selective growth medium was used in this study to detect the microbial

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contaminants of the alfalfa sprouts, and the standard plate count method was used for

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viable count enumeration. Ten grams of seeds and sprouts from each tray were

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selected randomly before water spraying and packed into a sterilized stomacher bag to

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detect the viable count of natural Enterobacteriaceae on the seeds and sprouts.

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Samples were mixed with 50 mL of sterilized 0.85% sodium chloride solution (pH:

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6.78±0.13) using a stomacher at the paddle speed of 10 times per second for 2 min

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(DH-11L, Ningbo Lawson Scientific Co., Ltd., China). One milliliter of obtained

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suspensions was taken and serially diluted using 0.85% sodium chloride. One hundred

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microliter of the proper dilution was spread onto sterilized Bismuth Sulfite Agar

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(Beijing Land Bridge Technology Co., Ltd.) and the petri dishes were then placed at

174

37

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forming unit per gram (log CFU/g) to express the surviving Enterobacteriaceae

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counts, and the detection limit was 1.7 log CFU/g in this study.

for 24 hours to determine the Enterobacteriaceae counts. We used log colony

8

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2.5 Properties of sprouts

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Length of sprouts: fifty plants were picked from each seedling tray to measure the

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sprout length (root excluded) using a dividing ruler (minimum scale is 0.5 mm). An

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average value was used to show length of the sprouts.

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Fresh weight of one hundred plants: 100 plants of sprout were selected randomly

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from each seedling tray to measure their fresh weight by an electronic scale

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(PTT-A2000, Fuzhou Huazhi Science Apparatus Co., Ltd., China).

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2.6 Statistical analysis

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Results presented in this manuscript were obtained from independently

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replicated experiments. Statistical analysis was carried out by SPSS (SPSS 24.0, Inc.,

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Chicago, IL). The data were presented as the mean ± SD (n=3) and differences were

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tested by one-way ANOVA. All tests were performed at a level of significance of

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0.05.

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3. Results and discussion

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3.1 Effect of different time treatments of SAEW on Enterobacteriaceae reduction in

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alfalfa seeds

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Enterobacteriaceae was at a non-detectable level after the seeds had been soaked

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in SAEW for 6 h, while Enterobacteriaceae at 2.94 log CFU/g was present after the

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seeds had been soaked in SAEW for 0.5 h. These results (Table 1) indicate that longer

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soaking in SAEW reached a significantly greater decrease of Enterobacteriaceae

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counts on the seeds, due to the antimicrobial activity of SAEW. In a previous study,

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seeds soaked in SAEW with ACC of 50~100 mg/L for 0.5 h reduced the microbiota 9

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counts on brown rice to a non-detectable level (<1.0 log CFU/g) (Zhang et al., 2018).

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However, this previous study differed from the present one in that the microbial

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detection was performed immediately after 0.5 h soaking, while here it was detected

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after 6 h with an additional 5.5 h tap water soaking. This suggests that inhibited

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bacteria could regrow and proliferate within a short time under appropriate conditions.

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Table 1. Enterobacteriaceae counts in alfalfa seeds soaked in SAEW for different

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soaking time Viable Enterobacteriaceae after soaking Treatments

206 207 208 209

Viable Enterobacteriaceae after

pre-germination (log CFU/g)

(log CFU/g)

SAEW+0.5 h

2.94±0.23 b

6.73±0.20 b

SAEW+6 h

ND c

6.25±0.1 c

Tap water+0.5 h

3.92±0.0 a

6.98±0.09 ab

Tap water+6 h

4.12±0.11 a

7.08±0.24 a

SAEW (ACC: 35 mg/L, pH: 6.0, ORP: 826 mV). Tap water (ACC: 0 mg/L, pH: 7.6, ORP: 502 mV). SAEW (tap water) + 0.5 h: seeds soaked in SAEW (tap water) for 0.5 h, and in tap water for additional 5.5 h. ND: Non-detection by plate counting. Data marked with non-common letters in the same column are significantly different (p<0.05).

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There was significantly different between the SAEW and tap water treatments

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(p<0.05), although, as can be seen, the population of Enterobacteriaceae on seeds

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soaked in tap water for 0.5 h followed by additional 5.5 h was only slightly lower than

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that of seeds soaked in tap water for 6 h. It could possibly be attributed to the water

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change removing a small quantity of the Enterobacteriaceae attached on the seeds.

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The Enterobacteriaceae count increased during the seed pre-germination period,

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due to the presence of Enterobacteriaceae in seeds and the favorable conditions for

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Enterobacteriaceae growth in the pre-germination process. Even at the undetectable

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level (<1.7 log CFU/g) by SAEW soaking for 6 h, 6.25 logs of surviving 10

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Enterobacteriaceae were observed on alfalfa seeds after pre-germination. Although

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the pathogens attached on seeds are generally low, it can increase exponentially

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during seed germination to exceed the initial quantity, and microbial counts have been

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observed to reach about 7~8 log within 2 days (Splittstoesser, 1983). The bacteria on

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the dry seeds may have been dormant before soaking, with low level detected under

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SAEW treatment indicating that the bacteria was not completely eliminated or

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inhibited during soaking. Bacteria may be harbored and protected from disinfecting

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chemicals in the cracks, abrasions and crevices of the seeds (Taormina and Beuchat,

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1999). Furthermore, surviving bacteria can regrow and proliferate quickly to a high

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level under the warm, humid environment required for seed germination. They are

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affected not only by growth conditions, but also by their initial density prior to

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treatment.

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3.2 Effect of SAEW with different ACC on Enterobacteriaceae reduction in alfalfa

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seeds

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The results for decontaminating Enterobacteriaceae on alfalfa seeds by SAEW

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treatment with different ACC are listed in Table 2.

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Table 2. Variation of Enterobacteriaceae counts in alfalfa seeds by SAEW with

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different ACC Viable Enterobacteriaceae after soaking Treatments

Viable Enterobacteriaceae after

pre-germination (log CFU/g)

(log CFU/g)

SAEW 1 (ACC 25)

ND b

6.52±0.21 b

SAEW 2 (ACC 35)

ND b

6.28±0.27 bc

SAEW 3 (ACC 45)

ND b

6.04±0.11 c

3.94±0.20 a

7.09±0.25 a

Tap water

11

237 238 239

(ACC: 45 mg/L, pH: 6.0, ORP: 859 mV); Tap water (ACC: 0 mg/L, pH: 7.6, ORP: 502 mV). ND: Non-detection

240

The Enterobacteriaceae population was 3.94 log CFU/g on the seeds soaked in

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tap water, while it was at a non-detectable level on those soaked by SAEW with ACC

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of 25, 35 and 45, respectively. However, during an extra 40 h of pre-germination, the

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population of the Enterobacteriaceae in seeds grew by over 6 logs. The viable count

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of Enterobacteriaceae in seeds with the tap water treatment was significantly greater

245

than those with the SAEW treatments (p<0.05). SAEW with increasing ACC used in

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the process of seed soaking and pre-germination resulted in greater reductions in the

247

Enterobacteriaceae populations. This finding was in agreement with that in the

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previous research (Zhang et al., 2018). The results also indicated that bacteria could

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grow and multiply most rapidly in the sprouts production environment, which provide

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moist, warm conditions for bacteria growth. Moreover, only a small amount of SAEW

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applied during seeds pre-germination was not adequate to inhibit bacteria to grow and

252

proliferate.

SAEW 1 (ACC: 25 mg/L, pH: 6.0, ORP: 810 mV); SAEW 2 (ACC: 35 mg/L, pH: 6.0, ORP: 819 mV); SAEW 3

by plate counting. Data marked with non-common letters in the same column are significantly different (p<0.05).

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The contamination of final product was primarily affected by the pathogen load

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and seeds quality. One of the sources that introduce pathogens onto seeds is the use of

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fertilizers. Animal manure that is a cheap and practical solution on agricultural land

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commonly contains pathogens, such as Salmonella, E. coli and Campylobacter. (Guan

257

and Holley, 2003). Therefore, the application of animal manure may introduce the

258

pathogens into the soil, which then migrate and attach to the seeds and produce.

259

Furthermore, the bacterial cells residing in seed cracks and cavities or between the 12

260

seeds’ coats and cotyledons may support the survival of pathogenic bacteria, and

261

provide protection from chemical sanitation (Charkowski et al., 2001; Yang et al.,

262

2013). Therefore, effective disinfection will be the most important step to preventing

263

bacteria contamination in the seeds and the produce.

264

3.3 Effect of SAEW at different pH on Enterobacteriaceae reduction in alfalfa seeds

265

At a pH of 5.0~6.5, hypochlorous acid is primary form of ACC in SAEW, which

266

exhibits 80 times higher disinfection effect than that of hypochlorite under a similar

267

treatment conditions (Anonymous, 1997). However, the form of ACC may be affected

268

and differentiated by the pH of SAEW, even at the range of 5.0~6.5, which would

269

change the bactericidal activity. Therefore, in the present study, SAEW with different

270

pH levels (5.0, 5.7 and 6.4) was applied in seed soaking and the variations in the

271

viable populations of Enterobacteriaceae are shown in Table 3.

272

Table 3. Enterobacteriaceae counts in alfalfa seeds soaked in SAEW at different pH

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levels Viable Enterobacteriaceae after soaking Treatments

274 275 276

Viable Enterobacteriaceae after

pre-germination (log CFU/g)

(log CFU/g)

SAEW 1 (pH 5.0)

ND b

6.09±0.17 c

SAEW 2 (pH 5.7)

ND b

6.48±0.16 b

SAEW 3 (pH 6.4)

ND b

6.45±0.07 b

Tap water

4.22±0.13 a

7.23±0.19 a

SAEW 1 (ACC: 45 mg/L, pH: 5.0, ORP: 875 mV); SAEW 2 (ACC: 45 mg/L, pH: 5.7, ORP: 863 mV); SAEW 3 (ACC: 45 mg/L, pH: 6.4, ORP: 856 mV); Tap water (ACC: 0 mg/L, pH: 7.6, ORP: 502 mV). ND: Non-detection by plate counting. Data marked with non-common letters in the same column are significantly different (p<0.05).

277

The surviving Enterobacteriaceae on alfalfa seeds by SAEW treatment with pH

278

of 5.0, 5.7, and 6.4 was at a non-detectable level. Tap water treated-seeds resulting in 13

279

4.22 log CFU/g of viable Enterobacteriaceae was great higher than that of SAEW

280

treated seeds. As can be seen, the SAEW significantly decreased the population of

281

Enterobacteriaceae during the seeds’ pre-germination stage, in comparison to the

282

treatment of tap water, and SAEW at pH of 5.0 showed the highest bactericidal

283

activity. Research performed on the antimicrobial effect of EOW with pH from 2.5 to

284

6.5 indicated that there was no great difference among EOWs with pH of 5.5~6.5

285

(Zhang et al., 2016). Furthermore, the previous studies have demonstrated that the

286

form of ACC in SAEW variated with the pH change, which is mainly associated with

287

the bactericidal activity of EOW (Xiong et al., 2014; Xiong et al., 2012).

288

3.4 Effect of SAEW soaking on the water absorption of alfalfa seeds

289

Seed germination is a most important phase in the growth cycle of plants, which

290

starts at water absorption of seeds and followed by the expansion of seed embryos

291

(Hermann et al., 2007). Therefore, the water absorption of alfalfa seeds soaked in

292

SAEW with different treatments was analyzed in this study. The results are shown in

293

Fig. 1.

294

SAEW treatments for different time periods, with different ACC, and at different

295

pH all showed no significant influence on the water absorption of seeds, compared

296

with tap water-treated seeds (p>0.05). The water absorption for seeds soaked with

297

SAEW for 0.5 h and 6 h, and with tap water for 0.5 h, and 6 h, was 59%, 66%, 54%

298

and 63%, respectively. The seeds were first soaked for 0.5 h, followed by an

299

additional 5.5 h, resulting in a slightly lower water absorption (Fig. 1A). The reason

300

for this could be that the water change during soaking may have damaged the seed 14

301

coat cells and, hence, affected the water absorption. Water absorption rate was at the

302

range of 54% to 66%, 62% to 68%, 57% to 69%, by SAEW treatment with different

303

treatment time, ACC, pH, and tap water, respectively, and no significant difference

304

were observed among treatments in the same group. The result is in agreement with

305

that in previous publication, in which SAEW, AEW with different ACC (50~150

306

mg/L) applied in brown rice soaking for 30~60 min made no significant difference to

307

its water absorption (Zhang et al., 2018).

308

Water absorption of seeds during soaking is generally influenced by their

309

composition and structure, their age, water quality, temperature and the duration of

310

soaking (Vishwakarma et al., 2013). The kinetics of water absorption in alfalfa seeds

311

should be studied in further study.

312

3.5 Effect of SAEW on seed germination

313

Seed germination is a critical factor for sprout quality. To clarify the effect of

314

SAEW on alfalfa seed germination, we investigated the change in the rate of

315

germination depending on different soaking times, ACC and pH levels in the seed

316

soaking process.

317

As shown in Fig. 2, seeds germination rate was at the range of 88% to 92% (Fig.

318

2A), 90% to 92% (Fig. 2B), 92% to 98% (Fig. 2C) by SAEW treatment with different

319

soaking time, ACC, pH, and tap water, respectively. There a great increase in the rate

320

of seed germination was observed at pH 5.7 of the SAEW treatment, exceeding 6%,

321

compared with the tap water treatment (Fig. 2C). This suggests that the seed

322

germination is affected by pH (Shoemaker and Carlson, 1990). Previous studies 15

323

showed that the germination rate of seed in different species are differently influenced

324

by ACC and pH. Deska and Jankowska (2011) reported that a range of pH from 5.5 to

325

6.5 was appropriate for the seeds germination such as Festuca pratensis, Dactylis

326

glomerata, Trifolium repens, and Medicago sativa seeds (Deska and Jankowska,

327

2011). Zhang et al. (2016) reported that a near neutral pH slightly enhanced radish

328

seed germination, while a lower ACC (15 mg/L) achieved a slightly higher

329

germination rate, and a higher ACC (40 mg/L) reached a bit lower rate of the seed

330

germination. However, the germination rate of mung bean reduced with elevated ACC

331

of SAEW (20~80 mg/L) (Zhang et al., 2011). The higher germination rate of alfalfa

332

seed was observed when the medium at pH 7.0, with significantly lower germination

333

rate at pH 5.0 (Mandic et al., 2012). Different kinds of seeds need different optimal

334

pH level and require a chemical stimulus to trigger seed germination (Joel et al.,

335

2012); hence, an in-depth study is needed to elucidate the impact mechanism of

336

SAEW applied in seed soaking for seed germination.

337

3.5 Population variation of Enterobacteriaceae on alfalfa sprouts treated with SAEW

338

during seed sprouting

339

SAEW at ACC levels of 25, 35, 45 mg/L were applied in the production process

340

of alfalfa sprouts in place of tap water, and the variation of surviving

341

Enterobacteriaceae counts is presented in Fig. 3.

342

The initial counts of Enterobacteriaceae (day 1) in seeds was 7.61, 7.29 and 7.05

343

log CFU/g, respectively, treated by SAEW at ACC of 25, 35 and 45 mg/L. Although

344

the initial counts of Enterobacteriaceae ranged from 7.05~7.61 log CFU/g (day 1), 16

345

the Enterobacteriaceae counts decreased under SAEW treatment to 5.55~6.64 log

346

CFU/g (day 9), with a reduction of 0.97~1.50 log. The viable counts of

347

Enterobacteriaceae in alfalfa sprouts was observed significantly decreased from day 1

348

to day 9 (p<0.05). However, a slightly increased count of Enterobacteriaceae was

349

obtained on the seed sprouts from day 7 (5.27~7.08 log CFU/g) to day 9 (5.55~7.24

350

log CFU/g), possibly because watering was reduced on the day of harvest to reduce

351

the humidity of the sprouts. Bacteria can grow and reproduce at any time in the sprout

352

production process and, thus, frequent watering and disinfection is required to

353

maintain the low level of bacteria in the sprouts. The tap water treatment also resulted

354

in a slight reduction of Enterobacteriaceae during the sprouting process, showing that

355

regular watering 1~3 times per day during seed sprouting does remove a small

356

amount of Enterobacteriaceae from the sprouts. In various studies, it reported that the

357

microbial loads in seeds were between 3.0 and 6.0 logs, with population that were 2.0

358

to 3.0 logs more in sprouts (Ren et al., 2009; Martinez-Villaluenga et al., 2008).

359

Therefore, increasing quantity and frequency of water supply will result in greater

360

bacterial reduction, however, the appropriate humidity for seed sprouting should also

361

be considered.

362

The significance of any products’ contamination is dependent mainly on the load

363

of pathogen’s infection and its surviving population in the period of storage. The

364

factors influencing the growth of pathogens was as temperature, humidity, gaseous

365

component, and available nutrient (Yang et al., 2013). Here, alfalfa seed was firstly

366

soaked in SAEW and then placed in warm and moist environment for germination. 17

367

These conditions are favorable for bacteria to multiply, in which bacteria may reach

368

significantly high levels if some counts remain on the seeds due to incomplete

369

disinfection (Splittstoesser, 1983; Zhang et al., 2018). The germination stage is a

370

principle source of microbial contamination in sprouts as microbe that present in

371

seeds could be internalized during seed sprouting (Yang et al., 2013). Other potential

372

sources of contamination, such as the irrigation water, fertilizer type, soil quality and

373

the production conditions may contaminate seeds and sprouts during sprouts

374

production (Guan and Holley, 2003; Pachepsky et al., 2011). Therefore,

375

contamination elimination should be executed throughout of the production cycle.

376

3.6 Effect of SAEW on biological quality of alfalfa sprouts

377

It has been proved that SAEW used in the process of seed sprouts production

378

could eliminate the bacterial contamination on seeds and sprouts to a relatively low

379

level, however, the quality of sprouts treated with SAEW is also important.

380

Table 4. The biological quality of alfalfa sprouts after SAEW treatment

381

ACC (mg/L)

Fresh weight of 100 plants (g)

Sprouts length (cm)

25

3.56±0.14 a

5.47±0.09 a

35

3.43±0.13 a

5.30±0.08 bc

45

3.48±0.14 a

5.31±0.04 bc

Tap water

3.42±0.09 a

5.36±0.07 ab

Data marked with non-common letters in the same column are significantly different (p<0.05).

382

As shown in Table 4, the weight of fresh alfalfa sprouts was 3.56, 3.43, 3.48 and

383

3.42 g/100 plants by SAEW treatments at ACC of 25, 35, 45 mg/L, and tap water,

384

respectively. There was no significantly different in fresh weight of the sprouts under

385

these different treatments. SAEW at 25 mg/L available chlorine concentration

18

386

obtained a slight increase in the length of the sprouts, while ACC at 35 and 45 mg/L

387

resulted in a slight decrease, compared with tap water treatment.

388

Published literature on EOW-treated mung beans found that SAEW with ACC of

389

10~30 mg/L promoted their growth, resulting in longer radicles and hypocotyls,

390

compared with tap water treatment. Moreover, near neutral pH in the EOW was found

391

to be optimal for the growth of mung, resulting in a longer length of sprout (Liu et al.,

392

2011). Short time (30~60 min) soaking by SAEW with ACC from 50 to 150 mg/L

393

achieved an obvious improvement in the length of germinated brown rice (Zhang et

394

al., 2018). In this present study, we used SAEW with different ACC at the pH of 6.0

395

to produce alfalfa sprouts and observed no negative impact on the length of sprouts.

396

4. Conclusions

397

Low concentration (25~45 mg/L) of SAEW was used to soak alfalfa seeds and

398

water the sprouts during their production. The results show that SAEW decontaminate

399

the natural Enterobacteriaceae on seeds with maximum 2.52 log CFU/g reduction

400

during soaking, with no adverse effect on the water absorption and germination, and

401

even slightly improved. SAEW applied in the production process reduced the

402

Enterobacteriaceae on sprouts with 0.73~1.81 log CFU/g reduction, in comparison to

403

tap water, while having no negative effect on the sprouts’ quality. With incomplete

404

decontamination effect by low concentration SAEW, therefore, higher concentration

405

SAEW, or SAEW combine with other approaches could be a good option in the

406

production of alfalfa sprouts.

407

Acknowledgment 19

408

This study was funded by NSFC (the national Natural Science Foundation of

409

China, No.31801658, 31772048 and 31801659), the China Postdoctoral Science

410

Foundation Funded Project (No.2018M633587), and the Fundamental Research

411

Funds for the Central Universities (No.2452017146 and 2452017228).

412

Disclosure statement

413

The authors have no conflicts of interest to declare.

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Fig. 1 Effect of SAEW on water absorption of seeds with different treatments. Data labeled with non-common letters on the bar are significantly different (p<0.05).

Fig. 2 Effect of SAEW on the germination rate of seeds with different treatments. Data labeled with non-common letters on the bar are significantly different (p<0.05).

Fig. 3. Variation of Salmonella on alfalfa sprouts treated with SAEW during seed sprouting. Data point and error bars represent mean and standard deviation (n=3). Data labeled with non-common letters on the same line are significantly different (p<0.05).

Highlights


SAEW was applied instead of tap water in the production of alfalfa sprout




SAEW effectively reduced the natural Salmonella during seed soaking and sprouting




No adverse effect was found on water absorption and germination of seeds by SAEW




The fresh weight and sprout length was not negatively affected by SAEW




SAEW is a promising method in the production of seed sprouts

Disclosure statement No authors have any financial or professional conflict of interest to disclose pertaining to the research described in this manuscript.