Efficacy of sweep ultrasound on natural microbiota reduction and quality preservation of Chinese cabbage during storage

Journal Pre-Proof Efficacy of sweep ultrasound on natural microbiota reduction and quality preservation of Chinese cabbage during storage Evans Adingba Alenyorege, Haile Ma, Ishmael Ayim, Feng Lu, Cunshan Zhou PII: DOI: Reference:

S1350-4177(19)31017-X https://doi.org/10.1016/j.ultsonch.2019.104712 ULTSON 104712

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

30 June 2019 27 July 2019 30 July 2019

Please cite this article as: E.A. Alenyorege, H. Ma, I. Ayim, F. Lu, C. Zhou, Efficacy of sweep ultrasound on natural microbiota reduction and quality preservation of Chinese cabbage during storage, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.2019.104712

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Efficacy of sweep ultrasound on natural microbiota reduction and quality preservation of Chinese cabbage during storage

of Food and Biological Engineering, Jiangsu University, No. 301 Xuefu Road, Zhenjiang

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a School

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Evans Adingba Alenyorege a, b, Haile Ma a, Ishmael Ayim a, c, Feng Lu a, Cunshan Zhou a

212013, Jiangsu, P.R China

of Agriculture, University for Development Studies, Tamale, Ghana

c Faculty

of Applied Science, Kumasi Technical University, Kumasi, Ghana

Correspondence

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b Faculty

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School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu, China

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E-mails: [email protected] (H. Ma) [email protected] (E. A Alenyorege)

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JOURNAL PRE-PROOF Abstract In this study, the effect of sweep frequency ultrasound (SFUS), sodium hypochlorite (NaOCl) and their combinations (SFUS + NaOCl) in reducing and inhibiting natural microbiota as well as preserving quality of fresh-cut Chinese cabbage during storage (4 °C

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and 25 °C ) for up to 7 days was investigated. In effect, 40 kHz sweep frequency ultrasound

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in combination with 100 mg/L sodium hypochlorite resulted in maximum reduction and

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inhibition of mesophilic counts, yeast and molds and minimum chlorophyll depletion, weight

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loss and electrolyte leakage. However, colour and textural characteristics deteriorated. The combined treatment suppressed the activities of polyphenol oxidase and peroxidase and

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manifested its preservative effect after Fourier Transform near-infrared spectroscopy

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analysis. Synergistic reductions were recorded in most of the combined treatments though largely less than 1.0 log CFU/g. Specifically, the combined treatment significantly (P < 0.05) reduced mesophilic counts by an added 2.7 log CFU/g, yeasts and molds by an added 2.0 log

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CFU/g when compared to the individual treatments. During storage at 4 and 25 °C, washing with SFUS + NaOCl produced Chinese cabbage with lower microbial counts, in comparison

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with the individual treatments. However, post-treatment storage could not entirely inhibit microbial survival as populations increased during storage even at refrigeration

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temperature of 4 °C. The results demonstrate that ultrasound and sodium hypochlorite are

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promising hurdle alternatives for the reduction and inhibition of microorganisms, as well as prolonging the shelf life and retaining the quality characteristics of Chinese cabbage. Keywords: Sweep frequency ultrasound; microbial counts; quality; fresh-cut Chinese cabbage; chemical sanitizer

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JOURNAL PRE-PROOF 1. Introduction There has been an upsurge in the proliferation of fruits and vegetables with unknown microbiological history and quality due to changes in lifestyle and consumption patterns in recent times [1]. Consumption of fresh-cut vegetables has become increasingly popular

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because of their high sensory quality and convenience. However, fresh and fresh-cut

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vegetables have been progressively associated with outbreaks of foodborne illness [2].

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Additionally, the cut tissues of these produce discharge nutrients that support and sustain

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the growth of natural microbiota present on raw produce [3]. This including the fact that most of these vegetables are not exposed to some form of lethal conditioning makes them of

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exceptional concern for consumers [4]. Chinese cabbage which is one of the most important, widely grown and patronized vegetables in Eastern Asia is most often consumed raw or

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minimally processed. It can therefore, be touted as a possible high-risk transmission vehicle of microorganisms due to the relatively close distance between the edible portions and the

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soil.

Chlorine based washing of fresh-cut vegetables can result in lower counts of

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microorganisms compared to unwashed full vegetables [3]. Commercially available sodium hypochlorite (NaOCl) has been extensively accepted and used in fresh produce washing

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operations for microbial reduction [5]. Through hydrolysis, NaOCl can be converted into

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hypochlorous acid in its non-ionized form, making it an efficient antimicrobial agent due to its non-dissociated form permeating target cells through non-polar portions [6]. It has been established however that, washing with sanitizers alone has not been entirely successful in microbial reduction investigations [7,8], with chlorine sanitizers typically accomplishing between 1 and 2 log CFU/g reductions in fresh produce decontamination [9]. Hence, there is 3

JOURNAL PRE-PROOF a need to improve the efficacy of treatments to kill naturally occurring microbiota, thus increasing the shelf life and safety of fresh-cut Chinese cabbage. Power ultrasound (20 – 100 kHz) surface decontamination has remained a promising technique in microbial control studies involving fruits and vegetables [10,11]. Cavitation

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generated by the application of sound waves in wash solution is the main occurrence

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accountable for microorganism detachment and destruction in decontamination processes

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[12]. This is as a result of cell wall weakening as a consequence of localized heat and stress;

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micro-streaming induced shearing; and the creation of free species that cause cell wall disintegration [13–15]. Meanwhile, ultrasound as a standalone decontamination tool is also

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inadequate with numerous studies recommending a hurdle technology of combining

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ultrasound with chemical sanitizers to maximize microbial reduction [16–20]. Following the hurdle theory, ultrasound has been combined with other

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decontamination methodologies, including other non-thermal techniques and chemical disinfectants to yield synergistic reduction of microorganisms and preservation of food

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quality [8,21,22]. Ultrasound may reduce bacterial viability through weakening of cell wall and exposure of internal matter to the uptake of sanitizers without adversely affecting

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physicochemical and sensory attributes [6,23]. The present study therefore sought to

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investigate: (1) the reduction and synergistic effects of sweep frequency ultrasound (SFUS) in combination with sodium hypochlorite against natural microbiota (mesophilic counts and yeasts and molds) in Chinese cabbage, compared to only SFUS or NaOCl treatments. In addition, single and combined treatments that achieved maximum log-cycle reduction of natural microbiota were further applied to (2) determine the post-treatment survival of

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JOURNAL PRE-PROOF mesophilic counts (MC) and, yeasts and molds (YM) on Chinese cabbage during storage at 4 °C for 7 days and 25 °C for 4 days. Furthermore, the study sought (3) to establish the treatment effects during storage on total colour change, electrolyte leakage, texture (firmness), weight loss, chlorophyll content, enzyme activities and Fourier transform near-

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infrared (FT-NIR) spectroscopy.

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2. Materials and methods

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2.1 Vegetable preprocessing

Fresh loose-headed Chinese cabbage (Brassica rapa var. Chinensis L.) samples were

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procured from an irrigated farm in Zhenjiang, China immediately after harvesting, on the

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week of the experiment and maintained at 4 °C. Intact leaves were selected and used in the experiments based on appearance. Leaf samples for each experiment were air-dried for 10

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min and subsequently cut aseptically into 2 cm×3 cm pieces.

2.2 Application of sweep ultrasound and chemical sanitizer

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The sensitivity of naturally occurring microorganisms to multiple ultrasound

frequency washing treatments was assessed according to a previous investigations [24],

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with varying sweep ultrasound frequencies (28 ±, 33 ±, 40 ±, and 68 ± β kHz, where β = 2).

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Additionally, NaOCl (34 g/L free chlorine) at different concentrations of 20 – 100 mg/L (pH of 6.31) was prepared according to the description of Park et al. [8]. The triplicate experiments were designed as follows:

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JOURNAL PRE-PROOF i.

Control treatments: samples (30 g) were submerged in 300 mL of sterile distilled water in a beaker and taken through shaking at 190 rpm without any form of ultrasound and NaOCl treatments for 10 min.

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SFUS washing treatments: samples (30 g) were subjected to 10 min of various SFUS

For NaOCl washing treatment without ultrasound, 30 g portions of samples were

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iii.

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treatments in a beaker containing 300 mL of sterile distilled water without NaOCl.

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submerged in 300 mL of NaOCl solution in a beaker and agitated (190 rpm) in a

iv.

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detour shaker for 10 min at 25 ± 2 °C.

A procedure according to Park et al. [25] with some modifications was applied with

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regards to the combined simultaneous application of SFUS and NaOCl washing

SFUS for 10 min.

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activities. Thirty-gram portions were submerged in 300 mL of NaOCl and treated with

The synergistic effect (SE) was estimated by applying the following equation:

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SE = R1 ‒ (R2 + R3). Where, R1= microbial load decline due to the combined application of SFUS and NaOCl; R2 = microbial load decline due to the exclusive

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application of SFUS, and R3 = microbial load decline due to the exclusive use of NaOCl. Synergistic, antagonistic and null efficacies were identified according to the

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categorization of Bang et al. [21]. Figure 1 summarizes the process design of SFUS and

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NaOCl aided washing of fresh-cut Chinese cabbage leaves.

2.3 Quantification of microorganisms Precisely, 25 g samples were homogenized in 225 mL of 0.1 % sterile peptone water (PW) in stomacher bags (Inter-science Co. Ltd, SN, France) for 2 min. Appropriate ten-fold 6

JOURNAL PRE-PROOF dilutions were prepared in 0.1 % PW and aliquots (0.1 mL) plated on plate count agar (PCA) and incubated for 48 h at 35 °C for mesophilic counts. Subsequently, yeast and molds were determined using potato dextrose agar (PDA) and incubated for 7 days at 25 °C. All results were presented as log CFU/g. This plate count technique was according to the description of

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Chen and Zhu [26] with some modifications.

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2.4 Storage and physicochemical analysis

Storage survival of microorganisms and physicochemical quality characteristics of

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samples including colour attributes, electrolyte leakage, texture (firmness), chlorophyll content, weight loss, enzyme activity and FT-NIR spectroscopy were evaluated applying the

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treatments that were successful in achieving maximum reductions of natural microbiota

Storage of treated samples

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2.4.1

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after processing. Instruments used for the various experiments are listed in Table 1.

The effect of storage on the survival of natural microbiota on Chinese cabbage after

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the washing treatments was determined by keeping the samples in sterile bags at

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refrigeration temperature (4 °C) for 7 days and room temperature (25 °C) for 4 days.

2.4.2

Colour Parameters of surface colour were taken using a Chroma meter. The total colour

difference

(TCD)

was

estimated

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the

following

equation: TCD =

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[(L ∗ 1 ‒ L ∗ 0)2 + (a ∗ 1 ‒ a ∗ 0)2 + (b ∗ 1 ‒ b ∗ 0)2]

0.5

, where L*, a* and b* are brightness,

greenness and yellowness of the sample and subscript notations 1 and 0 representing

Weight loss determination

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2.4.3

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treated and untreated sample parameters respectively [24].

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Treated Chinese cabbage samples were weighed daily with a precision balance and

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the percentage loss in weight (WL) estimated during storage. The results were computed according to the following equation: 𝑊𝐿 = 100 [(𝑊0 ‒ 𝑊1) 𝑊0], where W0 = initial weight of

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sample (g) and W1 = final weight of sample (g) [27].

Texture

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Textural quality was evaluated by means of a penetration test using a texture analyzer

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according to the procedure outlined by Rajkumar et al. [28], using a 2500-gram weight cell and a 2 mm diameter aluminum probe. The penetration test was performed at a distance of 5 mm and pre-test-, test-, and post-test-speeds of 2 mm/s, 0.5 mm/s and 0.5 mm/s

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respectively. The highest puncture force (HPF) was assessed and used as the product

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firmness (N) depicting texture. Five independent measurements were performed for the respective treatments.

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JOURNAL PRE-PROOF 2.4.5

Electrolyte leakage rate Leakage of electrolytes was confirmed by plunging 5 g of sample into 100 mL of

deionized water according to the description of Huang et al. [12]. This was subsequently incubated at 23 °C with shaking at 100 rpm. Electrolyte leakage (EL) from fresh-cut Chinese

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cabbage was estimated according to the following equation: EL = 100[(𝐶60 ‒ 𝐶1)/𝑇𝑐], where C1 = electrical conductivity of the dip solution at 1 min, C60 = electrical conductivity of the dip

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solution at 60 min and Tc = total conductivity after cooling following 25 min of 121 °C

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Chlorophyll content determination

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2.4.6

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autoclaving.

Chlorophyll pigments were extracted by homogenizing 1 g samples in 50 mL of 80 % methanol for 2 min. The content of chlorophyll was determined at 653 and 666 nm using a

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spectrophotometer. The quantity of total chlorophyll present in the samples was estimated

Enzyme activity analysis

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2.4.7

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based on a previous study [29] and the results expressed as mg/100 g of fresh weight (FW).

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Polyphenol oxidase (PPO) and polyphenol peroxidase (POD) determinations were

carried out following the procedure of Zhou et al. [30] using catechol, guaiacol and hydrogen peroxide as substrates. Concisely, chopped Chinese cabbage samples (5 g) were homogenized in 0.05 M phosphate buffer (pH 6.5, 20 mL). The extracts were centrifuged at 10000 g for 30 min at 4 °C and the upper layer sampled as enzyme solution. 9

JOURNAL PRE-PROOF The PPO analysis entailed a mixture of 0.1 M phosphate buffer (3 mL), 1.0 mL of 0.1 M catechol and 0.5 mL of the enzyme extract solution. The variation in absorbance at 420 nm was spontaneously measured for 3 min at 25 °C. Similarly, POD activity was evaluated comparable to that of PPO, but with 1.0 mL 0.05 M guaiacol, 2.0 mL hydrogen peroxide (2 %

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(v/v)) and enzyme extract (0.2 mL). The absorbance was spontaneously measured at 470

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nm for 3 min at 25 °C. . A unit of PPO and POD activities was described as the change in the

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absorbance of 0.001 per minute. The enzyme activities were conducted in triplicates and

Fourier Transform near-infrared (FT-NIR) analysis

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2.4.8

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stated as U/g fresh weight (FW).

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Spectra acquisition and preprocessing was performed according to the description outlined by Yang et al. [31]. FT-NIR analysis was performed in transmission mode on fresh and treated samples stored for 0 and 7 days using a portable near-infrared

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spectrophotometer equipped with a light point, photosensitive fiber, spectrometer, specimen cell and a computer. Five-gram sample portions were placed in a standardized

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colorimetric sample holder and 3 spectra randomly taken. The average of the three spectra was obtained as light transmittance data. The transmittance (T) data were presented as

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absorbance (A) [A = log (1/T)] and the range of spectra was from 900 to 1700 nm,

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comprising 512 variables. The experiment was performed at 60 % humidity and 25 ± 2 °C temperature.

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JOURNAL PRE-PROOF 2.5 Statistical analysis Analysis of variance (ANOVA) using MINITAB version 17 software was performed on the data and the results presented as mean values ± standard deviation. Means of statistically significant triplicate analyses for the various parameters were compared via Tukey’s

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multiple comparison analysis considering a 5 % probability value.

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3. Results and discussion

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3.1 Efficacy of sweep ultrasound and chemical sanitizer on reduction of Chinese cabbage

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natural microbiota

As a single treatment, the control merely achieved less than 0.5 log CFU/g reductions

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of both mesophilic counts (MC), and yeasts and molds (YM) as compared to the other treatments. Therefore, data from the control treatments involving water washing only were

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not shown and the treatment left out in subsequent investigations. The natural microbiota of vegetables contributes to the loss of quality during storage

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[6]. Hence, produce sanitation is recommended to reduce the natural microbiota for shelf life extension and safety of raw or minimally processed vegetables [23]. The natural microbiota

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of fresh greens consists mainly of mesophilic aerobic bacteria as well as yeasts and molds,

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and their reductions were studied with washing treatments including SFUS, NaOCl or SFUS + NaOCl. The initial MC and YM on the samples before washing treatments were 7.93 ± 0.28 log CFU/g and 6.64 ± 0.32 log CFU/g respectively. Reduction counts were significantly (P < 0.05) different for MC in the range of 0.41 – 1.13 log CFU/g for NaOCl; 0.24 – 1.83 log CFU/g for SFUS; and 0.63 – 4.51 log CFU/g for the combined treatments (Fig. 2). Similarly, 11

JOURNAL PRE-PROOF significant reductions were recorded for YM in the range of 0.39 – 1.07 CFU/g for NaOCl; 0.15 – 2.26 log CFU/g for SFUS; and 0.55 – 4.28 CFU/g for the combined treatments (Fig. 2). Despite the effectiveness of ultrasound technology and chemical sanitizers in produce decontamination, the results indicated that their individual applications (Fig. 2 A and B) have

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not achieved satisfactory reductions compared to their combined treatments (Fig. 2 C, D, E

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and F). Similar trend of results were reported, indicating that the single application of

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ultrasound and sanitizers was inadequate in reducing microorganisms in fresh produce,

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whereas combined treatments achieved maximum reductions [1,6,22–24,32]. The synergistic effect of multi-frequency ultrasound and chemical sanitizers brought about 1.0

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log CFU/g additional decrease in Salmonella Typhimurium on lettuce compared to their respective treatments [33]. In the current study, microbial reduction reached the highest

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(4.51 log CFU/g) level when samples were subjected to a combined washing treatment involving sweep ultrasound of 40 kHz plus 100 mg/L of sodium hypochlorite as shown in

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Figure 2E. Despite the significant reduction in microbial populations with individual washing treatments, the combined washing treatment however achieved an additional 2.68 log

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reduction (4.51 - 1.83 = 2.68 log CFU/g) of MC and a 2.02 log reduction (4.28 - 2.26 = 2.02 log CFU/g) of YM. Similarly, Francisco et al. [22] reported that 40 kHz ultrasound treatment

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improved the antimicrobial efficacy of NaOCl, leading to the removal of 1.5 log cycles relative

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to the single NaOCl treatment that achieved a 0.9 log cycle reduction of aerobic mesophilic bacteria on Eruca sativa. The bactericidal effect of the sanitizer was possibly augmented after the cavitation inflicted exposure of microorganism content. The intense pressure gradient created via ultrasound cavitation may have facilitated the penetration of NaOCl through the outer thinner peptidoglycan layer and delicate cell casing [34], improving the effectiveness 12

JOURNAL PRE-PROOF of the sanitizer. Cavitation efficacy have been stated to be heightened with the simultaneous use of a range of frequencies with possible strong antimicrobial effects [35,36]. The 40 kHz sweep frequency applied swept from 38 to 42 kHz around the central frequency (40 kHz) at a constant speed without interruption. Similarly, reduction of bacteria on alfalfa was largely

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improved by a joint simultaneous application of ultrasound (38.5 – 40.5 kHz) and chemical

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sanitizers [17].

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The current study was aimed at overcoming the obvious limitations of both

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ultrasound and NaOCl as separate washing treatments by harnessing their individual disinfection capacities. Their combination was intended to increase the disinfection

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efficiency through a multifaceted damage approach [37]. The synergy as a result of the

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capacity of acoustic cavitation to generate free radicals adding to its energy input expedites the infiltration of hypochlorous acid into bacterial cells and promotes the mechanism that augments the removal and disintegration of microorganisms [22]. A similar conclusion was

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reached after the application of ultrasound (40 kHz, 360 W) expedited the infiltration of acetic and gibberellic acids in Asparagus officinalis L., synergistically inhibiting naturally

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available microbiota [32]. Ultrasound combined with sodium hypochlorite is effective for the

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reduction of naturally occurring microorganisms in fresh-cut Chinese cabbage.

3.2 Interaction efficacy of ultrasound and NaOCl washing treatments on Chinese cabbage natural microbiota To evaluate the single and combined effects of SFUS and NaOCl in reducing natural microbiota on Chinese cabbage, it is necessary to understand whether the individual

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JOURNAL PRE-PROOF washing treatments achieved equal level of reduction as achieved by their combinations, or if their combined treatments exceeded the reduction achieved by the individual treatments. Significant (P < 0.05) positive synergistic effects were observed aside few antagonistic effects as shown in Table 2. Various antagonistic effects were recorded for some combined

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treatments in the range of –0.02 to –0.76 and –0.01 to –1.33 for MC and YM respectively. The

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most probable explanation for the detection of unexpected antagonistic effects could be that,

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microorganism cells were highly sensitive and susceptible to the cavitation effect of

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ultrasound as a standalone treatment. This could further advance the uptake of NaOCl as a supplementary treatment and may serve as a credible elucidation for the numerous

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synergistic effects observed. Synergistic effects were observed in almost all combined treatments even though most were less than 1.0 log CFU/g. Similarly, the interaction effect

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of power ultrasound and chemical disinfectants have been reported in bok choy [38]; lettuce [25,37]; arugula [22]; bell pepper [39]; raw laver [40]; green asparagus [32] and watercress

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[41] decontamination studies.

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3.3 Post-treatment storage survival of natural microbiota on Chinese cabbage The survival of MC and YM on fresh-cut Chinese cabbage after treatment with SFUS

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(40 kHz), NaOCl (100 mg/L) or their combination for 10 min followed by refrigeration (4 °C)

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and room temperature (25 °C) storage for up to a week was investigated and the results presented in Fig. 3. The SFUS, NaOCl and their combination (SFUS +NaOCl) reduced the initial populations of MC on the samples immediately after treatment (0 d) by 1.54, 1.23 and 4.69 log CFU/g respectively (Fig. 3 (A and C)). However, no significant differences (P > 0.05) existed amongst the survivals on the Chinese cabbage samples on day 0 and after day 1, 2 14

JOURNAL PRE-PROOF and 3 of storage (4 °C). Possibly, this could be deduced as a stability in microorganism survival during storage, indicating that a stationary phase of growth has been attained [42,43]. Reduction dynamics of MC revealed a comparable trend with that of YM, where SFUS (40 kHz), NaOCl (100 mg/L) and their combination reduced YM by 2.26, 0.94 and 4.4 log

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CFU/g respectively immediately after treatments (0 d) (Fig. 3 (B and D)).

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However, the MC and YM of all treated samples irrespective of the treatment type

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revealed a prompt growth as storage time increased. Post-treatment storage controlled the

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level of microbial counts on the Chinese cabbage samples in the first three days of storage (4 °C) regardless of the washing treatment. Conversely, a survival analysis at day 7 revealed an

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increase in the initial MC and YM summing up to 7.71, 7.98 and 4.5 log CFU/g and 5.63, 6.8 and 3.32 log CFU/g after SFUS, NaOCl and their combined treatments respectively (Fig. 3 (A

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and B)). At day 7, the observed increase in both MC and YM could be due to sample impairment, the presence of oxygen, or the existence of moistness and nutrients on sample

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surfaces capable of supporting microbial survival and development [43]. Generally, the increase in natural microbiota after 7 days of storage (4 °C) was < 1.5 log CFU/g for all

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treatments. The differences among treatments were however not significant (P > 0.05). Populations of MC and YM on Chinese cabbage, subjected to washing treatments and

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4 days of storage at room temperature (25 °C) are shown in Fig. 3 (C and D). Populations

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increased as storage time increased irrespective of treatment. At 25 °C, microbial counts on the Chinese cabbage samples increased significantly (P < 0.05) after day 1 of storage regardless of the washing method. Differences were significant (P < 0.05) amongst the survivals of MC and YM on the Chinese cabbage after 2, 3, and 4 days of storage. Additionally, the counts on the cut leaves treated with SFUS, NaOCl and SFUS + NaOCl increased by 1.57, 15

JOURNAL PRE-PROOF 1.73 and 0.83 log CFU/g for MC and 1.79, 1.38 and 0.92 log CFU/g for YM respectively at day 4 of storage. Samples washed with the combined treatment (SFUS + NaOCl) had the lowest MC and YM survivals at the end of storage for both temperature conditions. Similar results were

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reported by Salgado et al. [43] who indicated that lettuce treated with a combined treatment

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of chlorine and ultrasound presented the lowest aerobic plate counts at the end of storage

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with considerably varied results compared to other treatments. Comparable results were

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observed by Wu et al. [38], who specified that 180 W ultrasonication coupled with aqueous ClO2 could reduce microbial load and prolong the storability of Brassica chinensis. In the

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present investigation, microbial survival increased more quickly on Chinese cabbage stored at 25 °C compared to that stored at 4 °C. Refrigeration storage better inhibits microbial

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growth than room temperature storage that may cause damaging effects on the food material

3.4 Colour

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and promote microbial survival [42].

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Colour is influenced by the chemical, biochemical, and physical processes which occur

during growth and postharvest handling stages [44]. Colour change analysis results are

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shown in Fig 4A. For colour variations, the SFUS and SFUS + NaOCl treatments were more

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noticeable than the NaOCl treatment, probably due to cavitation-induced degradation. The SFUS and SFUS + NaOCl samples did not differ in total colour during day 1, 2 and 3 of storage and were among the best in colour. At the completion of storage, and for all washing treatments, colour change was visibly different (1.5 < TCD < 3) according to the classification of Pathare et al. [44]. Total colour difference values ≥ 5.0 signifies easily detectable colour 16

JOURNAL PRE-PROOF alterations [23]. The TCD values were however within acceptable ranges, which is key for the preservation of the fresh characteristics of the vegetable. This is imperative such that microbial reduction may occur devoid of alterations in the appearance of the product. Ultrasound treatments possibly destabilized the vegetable tissues and by this, the colour

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stability during storage declined through the breakdown of colour constituents [45].

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3.5 Weight loss

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Undesirable physiological changes including weight loss in vegetables can be minimized via refrigeration storage [46] . The influence of the washing treatments and

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storage at 4 °C on weight loss of fresh-cut Chinese cabbage was studied and the results presented in Fig. 4B. As shown in the figure, weight loss of treated Chinese cabbage samples

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gradually increased during the storage period. A gradual (P > 0.05) rise in weight loss was observed during storage at day 1, 2, and 3 regardless of the treatment. However, a significant

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(P < 0.05) increase in weight loss was observed from the fourth to the last day of storage, at which the lowest weight loss of 1.02 % was recorded for SFUS + NaOCl washing treatment,

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and 1.57 % and 1.33 % for SFUS and NaOCl treatments respectively. Comparable effects were also reported by Wang and Fan [32], who established that ultrasound in combination with

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acetic acid and gibberellin acid treatment recorded the smallest weight loss in green

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asparagus during storage citing the ability of ultrasound to protect the hydrogen bonds between water molecules as the reason for this occurrence.

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JOURNAL PRE-PROOF 3.6 Textural analysis Firmness is a major determinant of texture that is mostly used as a measure of quality during storage of fruits and vegetables. Due to respiration and transpiration, firmness of fresh products reduces steadily throughout storage, affecting quality and permitting

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microbial contamination [47]. The plot of firmness as affected by storage time and washing

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treatments presented a decline in firmness with increase in storage time (Fig. 5A). The initial

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firmness of fresh–cut Chinese cabbage was 5.23 ± 0.13 N, and after 7 days of storage at 4 °C,

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all SFUS + NaOCl treated samples showed an enhanced forfeiture of firmness (2.34 ± 0.04 N). The single treatments of SFUS and NaOCl provided more stability to samples during storage

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compared to the combined treatment (SFUS + NaOCl) that led to rapid softening. The application of ultrasound, which results in cell wall weakening, could possibly have

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undesirably affected the sample firmness and initiated softening in leaves compared to the treatment without ultrasound. Ultrasound induced softening in vegetables has been

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investigated [43,48] and associated to the influence of pectin methylesterase and polygalacturonase. These enzymes are accountable for solubilization and depolymerization

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of cell wall components. Hence, the cavitation effect of ultrasound may have influenced these

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enzyme activities and triggered cell wall depletion.

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3.7 Electrolyte leakage Fluctuations in electrolyte leakage (EL) rates in Chinese cabbage as influenced by

washing treatments and storage are shown in Fig. 5B. The level of cell membrane damage caused by stress may be simply measured through estimations of electrolyte leakage from tissues [49]. During storage, samples treated with only NaOCl had the lowest EL compared 18

JOURNAL PRE-PROOF with other treatments involving ultrasound. It is plausible to conclude that cavitation induced stress and micro-streaming activities brought about tissue structural alterations including cell wall and protopectin collapse, which resulted in increased ionic movement [50]. Wu et al. [38] detected the continuous increase in EL of Chinese cabbage (bok choy)

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during the advancement of storage and after ultrasound treatments, suggesting a persistent

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forfeiture of cell membrane strength. In the current study, Chinese cabbage samples exposed

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to SFUS + NaOCl treatment presented minimal additional increments in membrane

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perviousness, signifying a delay in tissue damage that may promote extension of shelf life.

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3.8 Chlorophyll content

Chlorophyll content values of Chinese cabbage used as a measure of the green colour

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loss (senescence) during storage are presented in Fig. 5C. Per an initial chlorophyll content of 8.69 ± 0.23 mg/100g FW, extended storage resulted in a gradual decrease in chlorophyll

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content regardless of the treatment type. However, aside the NaOCl treated samples, differences among SFUS and SFUS + NaOCl treated samples were not significant (P > 0.05)

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on the 1st and 2nd days of storage. Chlorophyll decrease in Chinese cabbage samples exposed to SFUS + NaOCl treatments was low compared to the single treatments. At the 7th

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day of storage, 11 % of chlorophyll was lost under the combined treatment (SFUS + NaOCl)

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compared to 18 and 26 % under SFUS and NaOCl single treatments respectively, probably due to the ability of ultrasound to inhibit increasing levels of ethylene, a precursor of senescence [46]. Since chlorophyll is a major pigment in plants, it can be concluded that the enzymatic degradation of pigments was inhibited to some level by ultrasonication [47].

19

JOURNAL PRE-PROOF 3.9 Enzyme activity The effect of different washing treatments on enzyme activity of Chinese cabbage during storage at 4 °C is shown in Fig. 6. The results presented in Fig. 6(A) demonstrated that, at day 0 maximum PPO activity of 8.3 U/g FW was recorded. After 7 days of storage, the

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PPO activity of the SFUS and NaOCl individually treated samples were 5.3 and 5.7 U/g FW,

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decreasing the activity by 1.57 – and 1.46 –folds respectively. However, with the application

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of the combined treatment, the synergistic inhibition effect of SFUS + NaOCl on PPO activity

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became predominant especially after day 4 of storage, leading to a 2.31–fold reduction at the end of storage. Similar results have been indicated by Wu et al. [38], where the PPO activity

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of bok choy after a combined treatment of aqueous chlorine dioxide and ultrasound treatment was comparatively smaller compared to other treatments. On one hand, the POD

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activity of Chinese cabbage increased minimally on the early days of storage up to the 3rd day where it started to decline up to day 6 with a slight increase at day 7 as shown in Fig. 6B. A

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comparable trend was observed where the POD activity of treated asparagus under storage increased swiftly and reached the highest values, then declined gradually afterwards [32].

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The combine treatment (SFUS +NaOCl) significantly (P < 0.05) minimized the increase in POD activity after day 3 of storage, reducing it from 35.1 U/g FW to 28.5 U/g FW at the end

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of storage. As presented in Figure 6, the minimum PPO and POD activities were observed in

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the SFUS + NaOCl treated samples and the variations were uniform than in the other treatments during storage. It was established that application of ultrasound largely inhibited the activities of PPO and POD at the initial stages of storage, therefore significantly delaying cut-surface browning and tissue senescence.

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JOURNAL PRE-PROOF 3.10

FT-NIR assessment The FT-NIR spectroscopy is a nondestructive analytical technique applied in food

safety and quality regulations based on the chemical structure of the food item, describing the C–H, N–H and O–H groups [27,51]. The transmission mode can identify both outer and

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inner qualities of fruits and vegetables compared with reflectance measurements that

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mainly determines surface and outer surface qualities [52]. The transmission mode spectra

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of the fresh untreated control and treated Chinese cabbage samples at day 0 and 7 of storage

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are presented in Fig. 7. Generally, spectral shapes were the same irrespective of treatment and storage time, even though observable differences existed among spectra in absorbance

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units. However, the spectra of some treatments were markedly overlapped because of storage time. The spectra of the Chinese cabbage were largely even across the full spectral

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region of 900 nm (11111 cm-1) to 1700 nm (5882 cm-1), and had three descending peaks positioned around 925, 1250 and 1375 nm. Nicolai et al. [53] indicated that peaks between

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970 and 1450 are associated to the O–H of water content. From Fig. 7, the highest absorbance peaks of the samples appeared in the untreated (fresh) samples at the commencement of

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storage whereas the least was observed for the NaOCl at the end of storage. The results indicated that SFUS + NaOCl treatments were beneficial in the 7 days preservation of

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freshness followed by the SFUS treatment. The SFUS + NaOCl treatment plausibly reduced

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the rate of respiration, which led to a decrease in carbohydrate metabolism as well as inactivating enzymes accountable for depolymerization of cell wall [50].

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JOURNAL PRE-PROOF 4. Conclusion The application of ultrasound in combination with sodium hypochlorite was an efficient method of reducing microbial load in Chinese cabbage, and possibly in other green leafy vegetables. The combined treatments presented synergistic effects on the initial

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reduction of natural microbiota and inhibition of their survival on Chinese cabbage during

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storage. The results confirmed the effectiveness of the combined treatments of sweep

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ultrasound at 40 kHz and 100 mg/L of sodium hypochlorite compared to their individual

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treatments, with the combination achieving > 4.0 log cycle reduction in both mesophilic counts and yeasts and molds. The combined treatment extended the storability of Chinese

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cabbage while preserving some physical and chemical quality attributes during storage. The combination of sweep frequency ultrasound and chemical sanitizers should inspire further

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investigations from a hurdle perspective for the reduction and inhibition of other microorganisms on fresh produce, together with the evaluation of treatments on food quality

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attributes. These supplementary investigations could result in a more complete understanding of this hurdle technique and its feasibility for posterior domestic and

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industrial applications.

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Acknowledgements

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This research was supported by the National Key Research & Development Plan

[2017YFD0400901]; the Special Fund for the Transformation of Scientific and Technological Achievements in Jiangsu Province [BA2016169]; and the Jiangsu Agricultural Science and Technology Innovation Fund [CX(18)3038].

22

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Figure captions Fig. 1. Procedure flow illustration for sweep frequency ultrasound and sodium hypochlorite handling of fresh-cut Chinese cabbage.

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Fig. 2. Reduction effect of sweep frequency ultrasound, sodium hypochlorite and their

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combinations on natural microbiota in Chinese cabbage. Vertical error bars represent the

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standard deviation (n=3) SFUS: only sweep frequency ultrasound treatment; NaOCl: only

ultrasound and sodium hypochlorite treatment.

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sodium hypochlorite treatment; and SFUS + NaOCl: combination of sweep frequency

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Fig. 3. Survival of mesophilic counts (A and C), and yeasts and molds (B and D) on Chinese cabbage after washing with SFUS, NaOCl or SFUS + NaOCl and storage at 4 °C and 25 °C for

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up to 7 days. Vertical error bars represent the standard deviation (n=3). SFUS: only 40 ± 2 kHz sweep frequency ultrasound treatment; NaOCl: only 100 mg/L sodium hypochlorite

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treatment; and SFUS + NaOCl: combination of 40 ± 2 kHz sweep frequency ultrasound and 100 mg/L sodium hypochlorite treatment.

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Fig. 4. Effect of washing treatments on (A) total colour difference and (B) weight loss of Chinese cabbage during storage at 4 °C for 7 days. The same letter on bars means that

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differences between the samples after Tukey comparison test are not significant at 5 %

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probability. Vertical error bars represent the standard deviation (n=3). SFUS: only 40 ± 2 kHz sweep frequency ultrasound treatment; NaOCl: only 100 mg/L sodium hypochlorite treatment; and SFUS + NaOCl: combination of 40 ± 2 kHz sweep frequency ultrasound and 100 mg/L sodium hypochlorite treatment.

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JOURNAL PRE-PROOF Fig. 5. Effect of washing treatments on (A) firmness, (B) electrolyte leakage, and (C) chlorophyll content of Chinese cabbage during storage at 4 °C for 7 days. Vertical error bars represent the standard deviation (n=3). SFUS: only 40 ± 2 kHz sweep frequency ultrasound treatment; NaOCl: only 100 mg/L sodium hypochlorite treatment; and SFUS + NaOCl:

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combination of 40 ± 2 kHz sweep frequency ultrasound and 100 mg/L sodium hypochlorite

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treatment.

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Fig. 6. Effect of washing treatments on (A) PPO and (B) POD activities of Chinese cabbage

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during storage at 4 °C for 7 days. Vertical error bars represent the standard deviation (n=3). SFUS: only 40 ± 2 kHz sweep frequency ultrasound treatment; NaOCl: only 100 mg/L sodium

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hypochlorite treatment; and SFUS + NaOCl: combination of 40 ± 2 kHz sweep frequency

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ultrasound and 100 mg/L sodium hypochlorite treatment.

Fig. 7. Absorbance spectra of Chinese cabbage taken in transmission mode at the onset and

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at the end of storage. Fresh: untreated; SFUS: only 40 ± 2 kHz sweep frequency ultrasound treatment; NaOCl: only 100 mg/L sodium hypochlorite treatment; and SFUS + NaOCl:

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combination of 40 ± 2 kHz sweep frequency ultrasound and 100 mg/L sodium hypochlorite

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3

4

Storage time (d)

JO

U

0.0

36

5

6

7

JOURNAL PRE-PROOF Figure 5

28

6.0

A

B

SFUS NaOCl SFUS + NaOCl

5.5

SFUS NaOCl SFUS + NaOCl

26

3.5 3.0

F

24

22

O

4.0

20

18

2.5

16 2

3

4

5

6

0

7

1

2

3

4

Storage time (d)

E-

Storage time (d)

9.0

SFUS NaOCl SFUS + NaOCl

C

PR

8.5

8.0

7.5

7.0

AL

Chlorophyll content (mg/100g FW)

1

6.5

6.0

R N

0

PR

2.0

0

U

Firmness (N)

4.5

O

Electrolyte leakage (%)

5.0

1

2

3

4

Storage time (d)

37

5

6

7

5

6

7

JOURNAL PRE-PROOF Figure 6

9

A

SFUS NaOCl SFUS + NaOCl

F

7

O

6

5

O

PPO activity (U/g FW)

8

PR

4

3 0

1

2

3

4

5

6

7

E-

Storage time (d)

PR

40

B

38

34

AL

POD activity (U/g FW)

36

SFUS NaOCl SFUS + NaOCl

32

R N

30 28

0

1

2

3

4

Storage time (d)

JO

U

26

38

5

6

7

JOURNAL PRE-PROOF Figure 7

3.54 3.52

F

3.50

O O

3.46

SFUS (day 0) SFUS (day 7) NaOCl (day 7) SFUS + NaOCl (day 7) SFUS + NaOCl (day 0) NaOCl (day 0) Fresh (day 0) Fresh (day 7)

3.42

E-

3.40

PR

3.44

3.38 3.36 900

1000

PR

Absorbance units

3.48

1100

1200

1300

1400

1500

1600

1700

R N

AL

Wavelength (nm)

U

Table 1. List of analytical instruments Instrument and specifications

Ultrasonication

Sweep frequency ultrasound (Fanbo-Biological Industrial Co.,

JO

Analysis

Surface colour

Ltd., Wuxi, China). CR-400 Minolta Chroma Meter (Minolta Corporation, Osaka, Japan).

39

JOURNAL PRE-PROOF Texture

TA-XT2 Texture Analyzer (Stable Micro Systems, Ltd., Surrey, UK).

Electrical conductivity

DDS-11A Conductivity Meter (Shanghai, China).

Chlorophyll content and UV-VIS spectrophotometer (Purkinje General Instrument Co., Ltd. Shanghai, China).

Weight loss

Precision balance (PRACTUM124-1CN Sartorius Scientific Instrument Co., Ltd. Beijing, China). Antaris

II

NIR

spectrophotometer

JO

U

R N

AL

PR

E-

PR

Company, Waltham, MA, USA).

40

(Thermo-Electron

O

FT-NIR

O

F

enzyme activity

JOURNAL PRE-PROOF Table 2. Synergistic effect of sweep ultrasound and sodium hypochlorite washing treatments on the level of natural microbiota in Chinese cabbage Natural microbiota

SFUS (kHz)

a Synergistic

effect (SE) (log CFU/g)

NaOCl concentration (mg/L) 80

100

28 ± 2

-0.02

0.00

-0.04

-0.04

+0.18

33 ± 2

+0.19

+0.27

+0.27

+0.42

+0.52

40 ± 2

-0.76

+0.12

+0.62

+0.81

+1.55

68 ± 2

+0.32

+1.08

+1.64

+1.78

+2.34

28 ± 2

+0.01

-0.01

-0.06

+0.22

33 ± 2

+0.15

+0.07

-0.01

+0.39

+0.44

40 ± 2

-1.33

-0.60

+0.04

+0.16

+0.95

68 ± 2

-0.53

+0.14

+0.78

+0.81

+1.44

O

O

F

60

-0.05

PR

Yeasts and molds

40

E-

Mesophilic counts

20

= R1 – (R2 + R3): R1= microbial load reduction because of the combined application of SFUS and NaOCl; R2 = microbial load decline from the exclusive application of SFUS, and R3 = microbial load decline from the exclusive use of NaOCl. +SE =synergistic effect, – SE = antagonistic effect and 0.00 = null effect.

U

R N

AL

PR

a SE

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JOURNAL PRE-PROOF

Highlights Impact of ultrasound and sanitizer on quality of Chinese cabbage was investigated



Combined treatments were effective in reducing microbial load than single treatments



Sweep ultrasound plus NaOCl presented synergistic effects on microbiota reduction



Combined treatments subdued microbial survival and prolonged shelf life of samples



Combined treatments produced better-quality characteristics than single treatments

U

R N

AL

PR

E-

PR

O

O

F



42