Control Measures of Pathogenic Microorganisms and Shelf-Life Extension of Fresh-Cut Vegetables

We investigated the combined effect of using slightly acidic electrolyzed water (SAEW), ultrasounds (US), and ultraviolet-C light-emitting diodes (UV-C LED; 275 nm) for decreasing pathogenic Escherichia coli and Staphylococcus aureus (SEA) in fresh-cut vegetables, including carrots, celery, paprika, and cabbage. Survival of pathogenic E. coli and SEA and quality properties of fresh-cut vegetables at 5 and 15 °C for 7 days were also investigated. When combined treatment (SAEW + US + UV-C LED) was applied to fresh-cut vegetables for 3 min, its microbial reduction effect was significantly higher (0.97~2.17 log CFU/g) than a single treatment (p < 0.05). Overall, the reduction effect was more significant for SEA than for pathogenic E. coli. At 5 °C, SAEW + US and SAEW + US + UV-C LED treatments reduced populations of pathogenic E. coli and SEA in all vegetables. At 15 °C, SAEW + US + UV-C LED treatment inhibited the growth of both pathogens in carrot and celery and extended the shelf life of fresh-cut vegetables by preventing color changes in all vegetables. Although the effects of treatments varied depending on the characteristics of the vegetables and pathogens, UV-C LED can be suggested as a new hurdle technology in fresh-cut vegetable industry.


Introduction
Recently, purchase rates for fresh-cut vegetables such as ready-to-eat (RTE) food and meal kits have increased [1,2]. RTE fresh-cut vegetables generally refers to salads and stickshaped and raw cut vegetables. They are generally consumed without washing or heating. They mainly include lettuce, cabbage, carrot, celery, cherry tomatoes, paprika, and so on [1][2][3][4]. However, due to non-thermal and minimal processing, contamination by pathogenic bacteria and viruses can be a risk factor during their production and distribution [5].
Foodborne illness outbreaks caused by pathogenic Escherichia coli and Staphylococcus aureus have been constantly occurring in Korea [6]. In the US and Europe, foodborne illness outbreaks at homes and restaurants due to consumption of fresh products contaminated by pathogenic E. coli O157:H7 and E. coli O104:H4 have been reported [7][8][9][10]. In addition, S. aureus and pathogenic E. coli have been consistently detected at high rates in agricultural products and RTE fresh-cut vegetables [11][12][13]. Recent research has also shown that some pathogens, including L. monocytogenes, E. coli O157:H7, Salmonella spp., S. aureus, B. cereus, and C. jejuni, have been isolated from various fruits and vegetables [14]. Therefore, appropriate measures to minimize contamination of foodborne pathogens and their effective control are required for RTE fresh-cut vegetables.
In general, sodium hypochlorite (NaClO) or a slightly acidic electrolyzed water (SAEW) is used in the washing and disinfection step as a critical control point (CCP) during the processing of fresh-cut vegetables [15,16]. Ultrasonic inactivation has been associated with damage to the cell wall and membrane of bacteria. However, it cannot be aureus suspensions (7 log CFU/g) and stirred for 2 min. Initial inoculation levels were approximately 5.5-6 log CFU/g. These inoculated samples were air-dried on a sterile stainless tray under a clean bench for 30 min.

Microbial Analysis
To investigate effect of each treatment for reducing the growth of pathogenic bacteria, viable cell count was measured using a spread plate culture method. Following disinfection treatment, 10 g of each treated sample was homogenized with 90 mL of sterile 0.1% peptone water using a stomacher (Interscience, Paris, France). The homogenate was serially diluted ten-fold with 0.1% peptone water and spread in duplicate onto selective media: eosin methylene blue agar (EMB, MBcell, Seoul, Korea) for pathogenic E. coli and Baird-Parker agar base (BPA, MBcell, Seoul, Korea) supplemented with egg yolk tellurite emulsion (MBcell, Seoul, Korea) for S. aureus. Tryptic soy agar (TSA, MBcell, Seoul, Korea), a nonselective medium, was used for both E. coli and S. aureus. Plates with selective and nonselective media were then incubated at 37 • C for 24~48 h. The number of colonies on each plate was then counted. Bacterial counts from duplicate plates were converted to log numbers.

Slightly Acidic Electrolyzed Water (SAEW) Treatment
Slightly acidic electrolyzed water (SAEW) at 30 ppm was produced using a SAEW generator (BC-120, Cosmic Round Korea Co., Seongnam, Korea) that basically consisted of a non-membrane electrolytic chamber with anode and cathode electrodes. The pH and available chlorine concentration of SAEW were measured with a pH meter (Orion-star pH-Bechtop, Thermo, USA) and a chlorine test paper (Toyo Roshi Kaisha, Ltd., Toyo, Japan), respectively, before SAEW was used to treat samples. The pH and available chlorine concentration of SAEW were 5.5 and 30 ppm, respectively.

Ultrasound (US) Treatment
Ultrasound (US) treatment was performed using a sterile bench-top ultrasonic cleaner (POWERSONIC 620, Hwashin Tech Co., Ltd., Gyeonggi-do, Korea) at a fixed frequency of 40 kHz and an ultrasonic power of 700 W. After an ultrasound device (rectangular tank, 500 × 300 × 150 mm) was filled with 4 L of tap water, a stainless container (280 × 215 × 140 mm) was placed in this device and filled with SAEW prior to experiment. Ten samples (100 g) were immersed in SAEW.

Combined Washing Treatment with SAEW and US
In order to investigate the optimal conditions of combined washing treatment with SAEW and US, the reduction effect of combined treatment was tested with carrot contaminated with EPEC alone under the following experimental conditions: time (0 and 3 min), the temperature of treatment solution (25 and 40 • C), and solution volume (10 and 20 times) with referring the previous works [21,29]. Carrots inoculated with EPEC were placed in a sterile stainless container filled with one or two liters of SAEW along with the ultrasound device. Reduction effects of EPEC in carrot under various conditions were compared as mentioned above. After treatments, samples were air-dried under a clean bench for 30 min. The most efficient reduction was observed for samples washed with SAEW for 3 min at ten times the sample volume in this preliminary work (Table 1). Thus, all fresh-cut vegetable samples (carrot, celery, paprika, and cabbage) were inoculated with pathogenic E. coli and S. aureus (SEA), respectively. They were treated in the same way. Table 1. Application conditions of single or combined treatments with SAEW, US, and UV-C LED applied for fresh-cut vegetables.

Treatments Application Conditions
NaClO Washing 1 with NaClO SAEW Washing with SAEW SAEW + US Washing with SAEW and US UV-C LED UV-C LED irradiation 2 SAEW + UV-C LED Washing with SAEW → UV-C LED irradiation SAEW + US + UV-C LED Washing with SAEW and US → UV-C LED irradiation 1 washing for 3 min with SAEW + US at 10 times volume of the sample at 25 • C. 2 Irradiation for 3 min and 6 cm distance between sample and UV-C LED source. NaClO, sodium hypochlorite solution (control, 100 ppm); SAEW, slightly acidic electrolyzed water (30 ppm); US, ultrasounds; UV-C LED, ultraviolet-C light-emitting diodes (275 nm).

Treatment with Ultraviolet-C Light-Emitting Diodes (UV-C LED)
Five ultraviolet-C light-emitting diodes (UV-C LED, 275 nm) modules (Seoulviosys Co., Ltd., Gyeonggi-do, Korea) were connected onto bar-type electronic printed circuit boards (PCBs) of 45 cm in length. Direct current voltage (500 mA) from a power supply (TY-1006, Tae Young Electronics Co., Ltd., Gyeonggi-do, Korea) was applied to PCBs. UV-C LED treatment was performed in a chamber (500 × 400 × 520 mm) equipped with two PCBs. The UV-C radiation intensity of UV-C LEDs was measured with a UV light meter (UVC-254SD, Lutron Electronics Co., Inc., Coopersburg, PA, USA) calibrated for a range of 240~390 nm. It was measured to be 23 µW/cm 2 .
To determine optimum irradiation conditions (processing time: 1 and 3 min; distance between sample and UV-C LED: 6 and 14 cm), effects of UV-C LED for reducing pathogenic bacteria were compared at each condition with two PCBs. The effect of microbial reduction was first tested for carrots and celeries contaminated with a three-strain cocktail of pathogenic E. coli. The superior performance of UV-C LED treatment was observed with an exposure time of 3 min and a distance between sample and UV-C LED source of 6 cm in this work ( Figure 1). Then, prewashed and dried fresh-cut vegetables (carrot, celery, paprika, and cabbage, 10 g each) were inoculated with the three-strain cocktail of pathogenic E. coli and S. aureus (SEA), respectively, and then transferred to polyethylene terephthalate (PET) containers. They were placed in the chamber. The distance between each sample and the UV-C LED source was 6 cm. Samples were then irradiated with UV-C LED for 3 min. The UV-C dose at this condition was 4.14 mJ/cm 2 .

Combined Treatment with SAEW, US, and UV-C LED
To evaluate the combined effect, combined treatment with SAEW, US, and UV-C LED was carried out in this study using conditions shown in Table 1. As a positive control, 100 ppm of sodium hypochlorite (NaClO; Hanson Hygiene Co., Chungcheongnam-do, Korea) solution was used. This solution was prepared by diluting 4% NaClO solution using distilled water.

Quality Evaluation for Treated Vegetables during Storage at 5 and 15 • C after Packaging
To evaluate qualities of fresh-cut vegetables, carrot, celery, paprika, and cabbage were treated with SAEW + US or SAEW + US + UV-C LED irradiation. Treated samples were packaged with PET containers (Modenpack, Seoul, Korea) and then stored at 5 • C (storage temperature) and 15 • C (abused temperature) for 7 days.

Survival of Pathogenic E. coli and S. aureus on Fresh-Cut Vegetables after Combined Treatment
For fresh-cut vegetables inoculated with pathogenic E. coli and S. aureus, survival rates of these pathogens after treatments during storage were investigated and compared with those in the control group without any treatment. This experiment was repeated twice at different time.

Effects of Combined Treatments on Quality Parameters of Fresh-Cut Vegetables
To investigate effects of applied treatments on physical qualities of fresh-cut vegetables, moisture loss, instrumental color, and appearance observation were measured. Moisture loss was determined by taking the difference between the initial weight of treated fresh-cut vegetable and that obtained one at the end of each storage time using the following formula: (%)Moisture loss = (initial mass − final mass) ÷ initial mass × 100 (1) The color of each sample was measured using a colorimeter (Minolta CR-400, Osaka, Japan) every day for 7 days. The instrument was standardized against a white standardization plate before each measurement. All measurements were performed in triplicate. Color was described as coordinates: lightness (L*), redness (a*, ±red-green), and yellowness (b*, ±yellow-blue).

Statistical Analysis
Experiments for US, SAEW, and UV-C LED treatments were repeated twice with three replicates per treatment. All statistical analyses were conducted with the Statistical Analysis System SAS V 9.4 (SAS Institute Inc., Cary, NC, USA). The significance of differences among or between samples was determined by one-way ANOVA followed by Duncan's test for multiple range tests or t-test at p < 0.05.

Effects of Volume and Temperature of Combined Treatment with SAEW and US
First of all, reduction effects of combined treatments on EPEC in carrots were compared under various conditions (temperature 25 and 40 • C and volume of treatment solution 10 and 20 times) as described previously [21,29]. Microbial reduction effects of combined treatments were not significantly different regardless of temperature or volume of washing solution (p < 0.05) ( Table 2). Previous studies have reported that higher temperature of treatment solution (25~60 • C) is more effective for reducing pathogenic bacteria in freshcut carrot, potato, and bell pepper [21,29,30]. However, potatoes washed at 60 • C with US treatment have shown color changes [29]. Other studies have reported that dipping in electrolyzed water for 3 min has the best sanitizing effect [31][32][33]. The present study also showed that washing for 3 min with SAEW + US at 10 times the sample volume at 25 • C was the most effective treatment condition to reduce the population of EPEC in fresh-cut carrot. Ultrasonic inactivation has been associated with damage to the cell wall and membrane of bacteria. Ultrasound can enhance the inactivation of microorganisms in chemical solutions such as SAEW [20,34,35]. The volume of SAEW was a multiple of the sample volume. 2 The temperature of SAEW. 3 The data were obtained from a non-selective media. * Significant difference between the volume of SAEW was observed at each temperature (p < 0.05). ** Significant difference between the temperature of SAEW was observed at each volume (p < 0.05).

Effects of Time and Distance between Sample and UV-C LED source
To determine optimum irradiation conditions such as application time (1 and 3 min) and distance between a sample and UV-C LED (6 and 14 cm), reduction effects of UV-C LED were compared with pathogenic E. coli cocktail strains in carrot and celery at different conditions. The highest reduction was observed for both carrot and celery after UV-C irradiation for 3 min with a distance of 6 cm ( Figure 1). Thus, this was confirmed as the optimal condition under the present UV-C irradiation dose (4.14 mJ/cm 2 ). Reduction levels of E. coli O157:H7, S. Typhimurium, and L. monocytogenes in culture media under UV-C (266~279 nm) dose of 1.67~3 mJ/cm 2 have been reported to be 3~6 log CFU/g in previous works [36].

Effects of Time and Distance between Sample and UV-C LED source
To determine optimum irradiation conditions such as application time (1 and 3 m and distance between a sample and UV-C LED (6 and 14 cm), reduction effects of UV LED were compared with pathogenic E. coli cocktail strains in carrot and celery at dif ent conditions. The highest reduction was observed for both carrot and celery after UV irradiation for 3 min with a distance of 6 cm ( Figure 1). Thus, this was confirmed as optimal condition under the present UV-C irradiation dose (4.14 mJ/cm 2 ). Reduction els of E. coli O157:H7, S. Typhimurium, and L. monocytogenes in culture media under U C (266~279 nm) dose of 1.67~3 mJ/cm 2 have been reported to be 3~6 log CFU/g in previ works [36].

Effect of Microbial Reduction in Combined Treatments with SAEW, US, and UV LED before Packaging
Finally, synergistic effects of combined treatments were evaluated to reduce pat genic E. coli and S. aureus (SEA) in carrot, celery, paprika, and cabbage at the optimal c dition of the present study, as shown in Table 3. Results revealed that the combined tr ment of 30 ppm SAEW + US (0.61~1.84 log CFU/g reduction) had a greater washing ef than a single treatment by 30 ppm SAEW (0.49~1.39 log CFU/g reduction) for both pat genic E. coli and S. aureus (SEA) in all vegetables. Additionally, application of UV-C L irradiation as a pre-packaging step after washing with SAEW or SAEW + US increa the reduction effect compared to UV-C LED irradiation only. Combined treatment o ppm SAEW + US + UV-C LED show higher reduction effect than 100 ppm NaClO solut alone, indicating that UV-C LED could be used as a new hurdle technology in the indus of fresh-cut vegetables. Regardless of the kind of pathogen, the highest reduction was served with SAEW + US + UV-C LED (0.97~2.17 log CFU/g) for all four vegetable samp

Effect of Microbial Reduction in Combined Treatments with SAEW, US, and UV-C LED before Packaging
Finally, synergistic effects of combined treatments were evaluated to reduce pathogenic E. coli and S. aureus (SEA) in carrot, celery, paprika, and cabbage at the optimal condition of the present study, as shown in Table 3. Results revealed that the combined treatment of 30 ppm SAEW + US (0.61~1.84 log CFU/g reduction) had a greater washing effect than a single treatment by 30 ppm SAEW (0.49~1.39 log CFU/g reduction) for both pathogenic E. coli and S. aureus (SEA) in all vegetables. Additionally, application of UV-C LED irradiation as a pre-packaging step after washing with SAEW or SAEW + US increased the reduction effect compared to UV-C LED irradiation only. Combined treatment of 30 ppm SAEW + US + UV-C LED show higher reduction effect than 100 ppm NaClO solution alone, indicating that UV-C LED could be used as a new hurdle technology in the industry of fresh-cut vegetables. Regardless of the kind of pathogen, the highest reduction was observed with SAEW + US + UV-C LED (0.97~2.17 log CFU/g) for all four vegetable samples. Combined washing with SAEW and US has been reported to be effective for microbial decontamination and shelf life extension of various vegetables [21,[37][38][39] and kashk [40]. Reduction levels of E. coli O157:H7 have been observed to be 1.3~3.3 log CFU/g in Chinese cabbage, lettuce, sesame leaf, spinach, and kale after washing with SAEW and US [37,39]. Combined washing with SAEW and US for kashk has also shown a reduction effect of 1.7 log CFU/g for E. coli O157:H7 and 1.9 log CFU/g for S. aureus [40]. Maximum reduction effect of washing with SAEW and US for B. cereus biofilms has been reported to be 4 log CFU/g on leafy green vegetables [38]. In our study, a combined washing treatment with 30 ppm SAEW and US showed similar reduction levels as those shown in other studies.
Recent studies have reported reduction effects of UV-C LED irradiation (254~280 nm wavelength) on apple juice and apple [26,27]. Jiang et al. [28] have also investigated the effect of combined treatment with SAEW and UV-C LED for inactivating Salmonella Typhimurium and E. coli O157:H7 on coriander. Their study also used 4 UV-C doses of 8~432 mJ/cm 2 along with SAEW washing. The reduction level of pathogens was 1.50~2.42 log CFU/g, similar to our results with UV-C LED irradiation (275 nm) at a dose of 4.14 mJ/cm 2 .
Green et al. [41] have compared effects for inactivating E. coli O157:H7 surrogate using a low-pressure mercury (LPM) lamp (253.7 nm) and UV-C LED (275 nm) at UV-C dose 0~20 mJ/cm 2 . UV-C LED irradiation for the same period of time resulted in a larger log reduction (~5 log CFU/g) than the LPM lamp, since a warm-up time was not required for UV-C LED irradiation compared to the LPM lamp. Reduction levels of S. aureus on lettuce and strawberry were also observed to be 1.21 and 0.5 log CFU/g, respectively, after applying UV-C LPM lamp irradiation (254 nm) for 45 min (540 mJ/cm 2 ) [42]. These results indicate that UV-C LED could be efficiently used with a shorter time and a less consumption of energy than UV-C LPM lamp.

Effect of the Kind of Pathogen and Vegetables on Microbial Reduction with SAEW, US, and UV-C LED
In the present study, a more effective reduction was observed for SEA (0.50~2.17 log CFU/g) than for pathogenic E. coli (0.41~1.59 log CFU/g) in all vegetables except cabbage (Table 3). Generally, Gram-positive bacteria are recognized as more resistant than Gramnegative bacteria, since they have thicker cell walls that can protect against physical and chemical treatments [34,43]. Pathogenic E. coli (0.30~1.27 log CFU/g) showed greater reduction than SEA (0.34~0.97 log CFU/g) in cabbage of this study. No significant difference in inactivation level between Gram-positive and Gram-negative bacteria after treatment with ultrasound has been reported [44]. On the other hand, Crook et al. [45] have reported that S. aureus is the most sensitive bacterium among seven pathogens (L. monocytogenes, E. coli, Serratia marcescens, Salmonella Senftenberg, Yersinia enterocolitica, Aeromonas hydrophila, and S. aureus) to UV-C irradiation.
Regarding the kind of vegetable, all treatments were more effective for pathogens in carrot and celery than in paprika and cabbage. When combined treatment of US + SAEW + UV-C LED was applied, the highest reduction levels for pathogenic E. coli and SEA were measured to be 1.48 and 2.17 log CFU/g, respectively, in carrot, and 1.59 and 2.02 log CFU/g, respectively, in celery (Table 3, Figure 2). A significantly greater reduction with SAEW + US was observed for pathogenic E. coli and SEA in carrot, while a more effective UV-C LED treatment was observed for both pathogens in celery (p < 0.05). Additional UV-C LED irradiation enhanced decontamination effects for carrot and celery. However, these reinforced effects were not significantly different between carrot and celery (p > 0.05). Surface characteristic of fresh produce was an important factor affecting inactivation effects of physicochemical treatments such as SAEW, US, and UV-C LED in most studies. Inactivation rates were higher for less hydrophobic fruits with smoother surfaces (apple, pears, and cherry tomato) than for those with rougher surfaces (cantaloupe, strawberry, and raspberry) [20,46]. reinforced effects were not significantly different between carrot and celery (p > 0.05). face characteristic of fresh produce was an important factor affecting inactivation ef of physicochemical treatments such as SAEW, US, and UV-C LED in most studies. In vation rates were higher for less hydrophobic fruits with smoother surfaces (apple, pe and cherry tomato) than for those with rougher surfaces (cantaloupe, strawberry, raspberry) [20,46]. The surface of celery itself is smoother than that of carrot. However, due to a lo regular groove shapes, a more significant reduction effect of SAEW + US was obser for carrots than for celeries during this study's washing step. However, a better disin tion effect was observed for celeries than for carrots with UV-C LED irradiation. In present study, a relatively lower microbial reduction effect was observed for paprika cabbage.
Moreover, a similar reduction effect in pathogenic E. coli and SEA was notice cabbage and paprika.
Since the effect of disinfection treatment was affected by various factors such as face properties of vegetables, adherence characteristics of pathogen on the surface of ferent vegetables, additional hurdle technology might be needed during processing distribution of various kinds of fresh-cut vegetables.

Survival of Pathogenic E. coli and S. aureus on Fresh-Cut Vegetables after Com bined Treatment
After combined treatment with SAEW + US + UV-C LED, survival characteristic pathogenic E. coli and enterotoxin A-producing S. aureus (SEA) inoculated on fresh carrot, celery, cabbage, and paprika were investigated after storage at 5 °C (storage perature) and 15 °C (abused temperature) for 7 days. At 5 °C, the combined treatm decreased both pathogens' populations to the lowest level (1.5 logs CFU/g) in all veg bles (data not shown). At 15 °C, the growth of pathogenic E. coli was inhibited in carrot and celery treated with SAEW + US or SAEW + US + UV-C LED during sto ( Figure 3A). The population of E. coli treated with SAEW + US + UV-C LED was incre up to 7.7 log CFU/g in cabbage, which was lower than the population in the non-tre sample (8.8 log CFU/g). The growth of E. coli was not also inhibited in paprika tre with SAEW + US or SAEW + US + UV-C LED.
On the other hand, the combined treatment decreased SEA populations to the low level (1.5 log CFU/g) in carrots and celeries after storage at 15 °C for 7 days (Figure The surface of celery itself is smoother than that of carrot. However, due to a lot of regular groove shapes, a more significant reduction effect of SAEW + US was observed for carrots than for celeries during this study's washing step. However, a better disinfection effect was observed for celeries than for carrots with UV-C LED irradiation. In the present study, a relatively lower microbial reduction effect was observed for paprika and cabbage.
Moreover, a similar reduction effect in pathogenic E. coli and SEA was noticed in cabbage and paprika.
Since the effect of disinfection treatment was affected by various factors such as surface properties of vegetables, adherence characteristics of pathogen on the surface of different vegetables, additional hurdle technology might be needed during processing and distribution of various kinds of fresh-cut vegetables. After combined treatment with SAEW + US + UV-C LED, survival characteristics of pathogenic E. coli and enterotoxin A-producing S. aureus (SEA) inoculated on fresh-cut carrot, celery, cabbage, and paprika were investigated after storage at 5 • C (storage temperature) and 15 • C (abused temperature) for 7 days. At 5 • C, the combined treatment decreased both pathogens' populations to the lowest level (1.5 logs CFU/g) in all vegetables (data not shown). At 15 • C, the growth of pathogenic E. coli was inhibited in only carrot and celery treated with SAEW + US or SAEW + US + UV-C LED during storage ( Figure 3A). The population of E. coli treated with SAEW + US + UV-C LED was increased up to 7.7 log CFU/g in cabbage, which was lower than the population in the non-treated sample (8.8 log CFU/g). The growth of E. coli was not also inhibited in paprika treated with SAEW + US or SAEW + US + UV-C LED.

Quality Evaluation of Treated
On the other hand, the combined treatment decreased SEA populations to the lowest level (1.5 log CFU/g) in carrots and celeries after storage at 15 • C for 7 days ( Figure 3B). Similar results were found after storage at 5 • C. The populations of SEA decreased in cabbage treated with SAEW + US or SAEW + US + UV-C LED, while SEA population was not changed in the non-treated cabbage during 7 days of storage at 15 • C. On the other hand, the populations of SEA were not changed in paprika during 7 days of storage at 15 • C, regardless of treatment, indicating that SEA in paprika was the least affected by SAEW + US or SAEW + US + UV-C LED. Overall, the growth control was more effective for SEA in all vegetables than for pathogenic E. coli based on reduction effects of treatments (Table 3, Figure 3). A more rapid reduction in SEA in carrots and celeries than in cabbage and paprika during storage at 15 • C was noticed. of SAEW and ultrasound had no significant effect on the contents of total soluble solids, vitamin C, or total titratable acidity in both strawberries and cherry tomatoes [20]. Total phenol and ascorbic acid contents of UV-C-treated yellow bell pepper remained constant during refrigerated storage [56]. These results showed that UV-C is an effective non-chemical treatment to maintain the qualities of fruits and vegetables.  Previous studies have reported the survival of foodborne pathogens in vegetables after different treatments [19,32]. Total bacterium counts on lettuce and button mushroom applied combined treatment with SAEW and US are lower than those without treatments after 6-8 days of storage at 4 and 10 • C [19,32]. Jiang et al. [28] have also studied the reduction effect of combined treatment with SAEW and UV-LED on Salmonella Typhimurium and E. coli O157:H7 in coriander after 6 days of storage at 4 • C. Among tested samples, SAEW + UV-LED-treated samples showed the lowest levels of Salmonella Typhimurium and E. coli O157:H7. Combined treatment of SAEW, US, and UV-C LED show synergistic effects due to the impact of chlorine compounds, cavitation, and ultraviolet-C, causing structural damage of cell membrane efficiently [17,26,47,48]. A synergistic effect was also observed for pathogenic bacteria on vegetables treated with SAEW + US + UV-C LED in the present study, indicating that such combined technology might be effective in extending the shelf life of fresh-cut vegetables. The most effective method of elimination Shiga toxin (verotoxin)-producing E coli (STEC) from foods is to introduce a bactericidal treatment, such as heating or gamma irradiation [49,50]. The present results show that UV-C LED treatment with washing can replace the gamma irradiation in the fresh-cut vegetable industry. However, the retail market's storage temperature must not be abused, since the growth of pathogenic E. coli was not prevented by such treatment at 15 • C.
Overall, the growth of pathogens in carrot and celery was controlled better than in cabbage and paprika at 5 and 15 • C. Antimicrobial activities of water-soluble polysaccharides in carrot and sedanolide (aroma component) in celery might explain such results [51,52]. Consequently, it can be concluded that combined treatment may extend the shelf life of fresh-cut vegetables, although the microbial reduction effect may vary depending on the characteristics of vegetables.

Effects of Combined Treatments on Quality Parameters of Fresh-Cut Vegetables
To evaluate the effects of various treatments on quality parameters of fresh-cut carrot, celery, cabbage, and paprika, moisture loss (Figure 4), appearance ( Figure 5), and instrumental color (Table 4) were measured during storage at 5 and 15 • C for 7 days. Figure 4 shows changes in moisture in samples treated with SAEW + US or SAEW + US + UV-C LED and stored for 7 days at 5 • C compared to non-treated samples.
The lowest moisture loss with SAEW + US + UV-C LED treatment was noticed for celery (1.4%) and cabbage (2.5%) after storage for 7 days. Such moisture loss was lower than that with SAEW + US or non-treated samples (3.7~9.0%). However, changes in moisture in carrot ( Figure 4a) and paprika ( Figure 4d) were observed to have similar levels at all conditions. A similar tendency of moisture loss was noticed at 15 • C (data not shown). Generally, a loss of moisture greater than 5% would cause a reduction in the retail value of vegetables and fruits [53]. The threshold level of moisture loss is 10% for ending the shelf life of fresh produce [54]. Regardless of treatment, less than 5% of moisture loss was observed for carrot and paprika. Less than 5% of moisture loss was also observed for celery and cabbage treated with SAEW + US or SAEW + US + UV-C LED in the present study. Other studies have also reported that moisture losses in tomatoes treated with the US and UV-C [55] and yellow bell pepper treated with UV-C irradiation [56] are lower than those in non-treated samples. Treatments with the US and various sanitization agents did not affect purple cabbage moisture during storage [57]. It is also reported that the combination of SAEW and ultrasound had no significant effect on the contents of total soluble solids, vitamin C, or total titratable acidity in both strawberries and cherry tomatoes [20]. Total phenol and ascorbic acid contents of UV-C-treated yellow bell pepper remained constant during refrigerated storage [56]. These results showed that UV-C is an effective non-chemical treatment to maintain the qualities of fruits and vegetables. Appearance changes in fresh-cut vegetables stored at 15 °C are shown in After 7 days of storage, conspicuous appearance changes in non-treated celery and paprika were identified at an abused temperature of 15 °C compared t treated with SAEW + US + UV-C LED. However, appearance changes were n for non-treated samples and samples treated with a combination of methods aft at 5 °C for 7 days (data not shown). Overall, significant changes in values of L*, were not observed for samples treated with SAEW + US + UV-C LED after 7 da age at 15 °C compared to SAEW + US or non-treated samples (p > 0.05) ( Table 4) visual inspection for sliced button mushrooms, application of SAEW and US t delayed browning after 8 days of storage at 5 °C [19]. Other previous works [56 also reported that L*, a*, and b* values were not affected for purple cabbage, to  Appearance changes in fresh-cut vegetables stored at 15 • C are shown in Figure 5. After 7 days of storage, conspicuous appearance changes in non-treated celery, cabbage, and paprika were identified at an abused temperature of 15 • C compared to samples treated with SAEW + US + UV-C LED. However, appearance changes were not noticed for non-treated samples and samples treated with a combination of methods after storage at 5 • C for 7 days (data not shown). Overall, significant changes in values of L*, a*, and b* were not observed for samples treated with SAEW + US + UV-C LED after 7 days of storage at 15 • C compared to SAEW + US or non-treated samples (p > 0.05) ( Table 4). Based on visual inspection for sliced button mushrooms, application of SAEW and US treatments delayed browning after 8 days of storage at 5 • C [19]. Other previous works [56][57][58] have also reported that L*, a*, and b* values were not affected for purple cabbage, tomato, and yellow bell pepper treated with the US, chemical sanitizer, and UV-C technologies during storage at 8~12 • C. The present study also proved that combined treatment of SAEW + US + UV-C LED prevented the color change in fresh-cut vegetables and could be applied to maintain the quality and safety of fresh-cut vegetables.

Conclusions
In the present study, combined treatment with 30 ppm SAEW + US + UV-C LED before packaging was more effective in reducing microbial contamination than every single treatment. Combined treatment with SAEW, US, and UV-C LED further reduced populations of pathogenic E. coli and enterotoxin A-producing S. aureus (SEA) in fresh-cut vegetables. Combined treatments (SAEW + US or SAEW + US + UV-C LED) reduced populations of pathogenic E. coli and SEA to the lowest levels in all vegetables after storage at 5 • C for 7 days. The growth of pathogenic E. coli and SEA was more effectively inhibited in carrot and celery than in cabbage and paprika treated with SAEW + US + UV-C LED during storage at an abused temperature of 15 • C. Overall, the growth control by combined treatment was more effective for SEA than for pathogenic E. coli. It was also noticed that combined treatment with SAEW + US + UV-C LED prevented moisture loss, color, and appearance change during storage at 5 and 15 • C. These results indicate that combined treatment of SAEW, US, and UV-C LED can be applied as a useful technology to extend the shelf life of fresh-cut vegetables. However, its effect may vary depending on the characteristics of vegetables. Especially, UV-C LED can be suggested as a new hurdle technology in the fresh-cut vegetable industry. Data Availability Statement: We did not report any additional data for this study.