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Food Control

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Clove bud essential oil emulsion containing benzethonium chlorideinactivates Salmonella Typhimurium and Listeria monocytogenes on fresh-cutpak choi during modified atmosphere storage

Jun-Beom Park, Ji-Hoon Kang, Kyung Bin Song∗

Department of Food Science and Technology, Chungnam National University, Daejeon, 34134, Republic of Korea

A R T I C L E I N F O

Keywords:Benzethonium chlorideClove bud essential oilEssential oil emulsionFoodborne pathogenFresh-cut pak choi

A B S T R A C T

In this study, positively charged clove bud essential oil (CB)/benzethonium chloride (BEC) emulsion (CBB,0.02% CB + 0.002% BEC) was used to inactivate Salmonella Typhimurium and Listeria monocytogenes inoculatedon fresh-cut pak choi (FCPC). CBB treatment reduced the populations of S. Typhimurium and L. monocytogenesby 1.97 and 2.00 log CFU/g, respectively. The log reductions by CBB were greater than those by sodium hy-pochlorite at the same concentration (0.02%). Inactivation of the two pathogens on the FCPC surface wasconfirmed by scanning electron microscopy. In addition, after CBB treatment, the number of total aerobicbacteria on FCPC packaged with polyamide multi-layer films was significantly (p < 0.05) reduced duringmodified atmosphere (MA) storage (5% O2, 10% CO2). The degree of browning in FCPC was less for the CBB/MAgroup than for other treatment groups. Particularly, the CBB/MA group exhibited the highest sensory evaluationscores in terms of FCPC quality. Thus, CBB washing/MA storage can enhance microbial safety and extend theshelf life of FCPC.

1. Introduction

In recent years, consumer demand for fresh-cut vegetables (FCVs)has increased due to their health benefits (Fernández, Agüero, & Jagus,2018). Pak choi (Brassica campestris L. chinensis), a member of Brassi-caceae, is rich in various bioactive compounds beneficial to the humanbody and has gained much popularity (Harbaum-Piayda et al., 2010).With increasing consumption, FCVs are naturally exposed to microbialcontamination (Park, Kang, & Song, 2018). Several cases of foodbornepoisoning related to FCVs contaminated by Salmonella Typhimuriumand Listeria monocytogenes have been reported (Ma, Zhang, Bhandari, &Gao, 2017; Park et al., 2018). Thus, it is necessary to establish appro-priate washing treatments and postharvest technologies to reduce themicrobiological hazards and prolong the shelf life of FCVs (Chun &Song, 2013).

Chlorine-based sanitizers have been used for washing FCVs due totheir effectiveness and low cost (Kang, Park, Park, & Song, 2019).However, there is a possibility to generate harmful components duringthe washing step; hence, the use of chlorine-based sanitizers has beenlimited to a minimum (Pablos et al., 2018). Therefore, essential oils(EOs) with strong antimicrobial activities have been studied as an al-ternative to chlorine-based sanitizers for washing FCVs (Kang & Song,

2018). Among these, clove (Syzygium aromaticum) bud EO (CB) hasbeen used in the food industry (Ivanovic, Dimitrijevic-Brankovic, Misic,Ristic, & Zizovic, 2013). CB, whose main component is eugenol, couldimprove the microbiological safety of FCVs (Burt, 2004; Ivanovic et al.,2013). However, its use in washing FCVs is limited (0.01%–1%) due toits strong odor and low water solubility (Zhang, Critzer, Davidson, &Zhong, 2014). To improve the antimicrobial effect of EOs at low con-centrations, surfactant-containing EO emulsions have been studied as awashing solution (Prakash, Baskaran, Paramasivam, & Vadivel, 2018).Previously, we reported the antimicrobial effect of an EO emulsioncontaining a cationic surfactant [cetylpyridinium chloride (CPC) orbenzalkonium chloride (BAC)] against various pathogens on FCVs(Kang et al., 2019; Kang & Song, 2018; Park et al., 2018). Notably, thesecationic surfactant-based EO emulsions have an additive effect in con-trolling foodborne pathogens. Similar to CPC and BAC, benzethoniumchloride (BEC), used in this study as a cationic surfactant, is a qua-ternary ammonium compound (Komaiko & McClements, 2016). BECexhibits antibacterial activity by inducing morphological changesthrough electrostatic interactions with cell membrane (Park et al.,2018). Thus, CB/BEC emulsion (CBB) is expected to enhance the mi-crobial safety of fresh-cut pak choi (FCPC).

Modified atmosphere (MA) storage is used as a preservation method

https://doi.org/10.1016/j.foodcont.2019.01.001Received 11 October 2018; Received in revised form 2 January 2019; Accepted 3 January 2019

∗ Corresponding author.E-mail address: [email protected] (K.B. Song).

Food Control 100 (2019) 17–23

Available online 03 January 20190956-7135/ © 2019 Elsevier Ltd. All rights reserved.

T

to extend the shelf life and maintain the quality of FCVs during storageby modifying the gas composition inside the food package (Altieri,Genovese, Matera, Tauriello, & Di Renzo, 2018). Each FCV requires aspecific gas composition and it is necessary to use MA storage with theappropriate packaging materials (Oliveira et al., 2015). To date, therehave been no studies on washing FCVs using EO emulsions along withMA storage to extend their shelf life. Therefore, we examined the effectof CBB washing on the removal of pathogens and the quality changes inFCPC during MA storage with an appropriate gas composition andpackaging material.

2. Materials and methods

2.1. Bacterial strains and media

Each bacterial cocktail was prepared using strains of S.Typhimurium (KCTC 2421 and ATCC 14028) and L. monocytogenes(KCTC 13064 and ATCC 19115) (Kang et al., 2019). To determine thepopulation of the bacteria in the cocktail, standard plate countingmethod was used. All general and selective media used for culturingand enumeration of the bacteria were BD Difco brand purchased fromBecton, Dickinson and Company (Franklin Lakes, NJ, USA). The finalpopulation of each cocktail was approximately 107–108 CFU/mL.

2.2. Sample preparation

Pak choi (harvested in September 2018; 5 kg) was purchased from alocal market in Daejeon, Korea, sliced (2.5× 3.5 cm, 0.5 g), and usedwithin 24 h. Ultraviolet-C irradiation was performed on both surfacesfor 8min to reduce levels of the indigenous microflora and neither ofthe two pathogens were detected on the product following treatment.Standard plate counting method was applied to determine the popu-lation of the indigenous microflora or pathogens used in this study.Each bacterial cocktail (total 50 μL, 107–108 CFU/mL) was spot-in-oculated on the front side of FCPC surface, and the inoculated FCPC waskept on a clean bench for 40min to allow spotted inoculum to dry andgive cells time to firmly attach.

2.3. Washing treatment

Considering the maximum concentration of NaOCl that can be usedfor washing FCVs, the concentration of CB (eugenol; 76.8%, EcocationCo., Seongnam, Korea) was fixed at 0.02%. The concentration of BEC(Sigma-Aldrich Co., St. Louis, MO, USA) was fixed at 0.002%, at a ratioof 10:1 with CB, based on our previous report (Park et al., 2018). Allwashing solutions were prepared as described by Park et al. (2018).FCPC samples (2 g) were immersed in the prepared solutions (100mL)for 3min and kept on a clean bench for 50min to dry. For comparisonwith the CB/BEC emulsion (CBB) solution, NaOCl solution (0.02%) wasprepared and adjusted to pH 7.0 using HCl (1 N, Daejung Co., Siheuong,Korea) and used for FCPC washing as described above.

2.4. Scanning electron microscopy (SEM) analysis

For visualizing the morphological changes in both pathogens on theFCPC surface after the washing treatments, low-voltage field-emissionSEM (LVFE-SEM, Zeiss MERLIN, Oberkochen, Germany) was used asdescribed by Park et al. (2018). The FCPC was placed in potassiumphosphate buffer (PPB, 0.05M, pH 7.2) containing glutaraldehyde(2.5%, Sigma-Aldrich Co.) at 4 °C for 4 h to fix the microorganisms toFCPC, followed by washing with PPB. FCPC, which was dehydratedsequentially from low to high concentrations of ethanol (50%, 80%, and100%), and was air-dried on a clean bench and coated with platinum ona carbon tape for 30 s. LVFE-SEM photographs were captured at 5 kV at5000×magnification.

2.5. MA storage

Following the washing treatment, un-inoculated FCPC samples(control/normal air, control/MA, CBB washing/normal air, and CBBwashing/MA) were packed in polyamide multi-layer [polyamide/polyamide/polyethylene (PA/PA/FE)] film bags (N707, 25×30 cm,70 μm thickness, Barflex Co., Daejeon, Korea) with low gas perme-ability (60 cc/m·day·atm) and kept at 4 °C for 14 days. Normal airpackaging was performed without changing the gas composition in thepackaging films. For MA storage, the gas composition (5% O2, 10%CO2) recommended for Brassicaceae vegetables as described by Oliveiraet al. (2015) was injected into the packaging film using an MA packa-ging machine (MAP MIX 9001ME, Ringsted, Denmark) and im-mediately heat-sealed. The population of total aerobic bacteria (TAB)and the quality changes in FCPC were measured, and the changes in O2

and CO2 composition in the packaging films were analyzed in triplicateusing a gas analyzer (Checkpoint2, Ringsted, Denmark) during storage.

2.6. Microbiological analysis

Microbiological analysis was performed as described by Kang et al.(2019). Samples (1 g) and sterile peptone water (9mL) were placed in asterile bag and homogenized using a homogenizer (Mix2, AES Lab.,France) for 3min. The resulting homogenate (100 μL) was dilutedwhere applicable and 100 μL aliquots were spread onto plate countagar, Oxford medium base, and XLD agar for the enumeration of TAB, L.monocytogenes, and S. Typhimurium, respectively. Before counting thecolonies, all plates were incubated at 37 °C for 24–48 h (n=3).

2.7. Browning index and color changes during MA storage

To determine the degree of FCPC browning during MA storage, thebrowning index was determined as described by Chen et al. (2017).Color changes in FCPC by browning were measured five times using acolorimeter (Minolta Camera Co., Osaka, Japan) to examine the qualitychange in FCPC during MA storage. The parameters (a, redness; b,yellowness) were measured, and ΔE (total color difference) was calcu-lated.

△E= [(Lsample-Lcontrol)2 + (asample-acontrol)2 + (bsample-bcontrol)2]1/2

2.8. Total glucosinolate content (TGC)

TGC in FCPC samples was determined as described by Mawlong,Sujith Kumar, Gurung, Singh, and Singh (2017) with minor modifica-tions. Lyophilized FCPC powder (0.1 g) was added to 70% methanol(1.5 mL) and extracted overnight in a shaking incubator at 25 °C. Thesupernatant obtained after centrifugation (3000×g for 8min) was se-quentially reacted with distilled water (DW, 300 μL) and 2mM sodiumtetrachloropalladate (3 mL) for 1 h. Optical density was measured at425 nm with five replicates and the TGC was calculated.

2.9. Sensory evaluation

For sensory evaluation, 11 trained panelists (male: 5, female: 6, age:24–32) evaluated the appearance (AO), odor (OD), texture (TX), andoverall acceptability (OA) of the samples during storage. To examinethe organoleptic changes in FCPC samples during MA storage, panelistsevaluated the samples with scores ranging from 1 (very poor) to 5 (verygood) for at least three independent samples in the same treatment.

2.10. Statistical analysis

All experimental trials were repeated at least three times and results

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are expressed as mean ± standard deviation. Statistical significancewas analyzed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA)and Duncan's multiple range test (p < 0.05) was performed.

3. Results and discussion

3.1. Effects of CB, BEC, or CBB washing on FCPC

This study demonstrated the antimicrobial activity of CBB treatmentand proposes the use of CBB as a novel agent for washing FCVs. Fig. 1

shows the log reductions in pathogen numbers after various washingtreatments. Water washing reduced the populations of S. Typhimuriumand L. monocytogenes by 0.29 and 0.37 log CFU/g, respectively, bydetaching the pathogens from the FCPC surface. Using water forwashing FCVs is not effective for removing foodborne pathogens;hence, other novel washing agents are needed (Poimenidou et al.,2016). Single treatment of CB or BEC decreased the populations of S.Typhimurium by 0.94 and 0.97 log CFU/g, respectively, and of L.monocytogenes by 1.01 and 0.95 log CFU/g, respectively, compared tothe control. In addition, similar to our previous studies (Kang et al.,2019; Kang & Song, 2018; Park et al., 2018), CBB washing showed anadditive effect of each single treatment. CBB treatment reduced thepopulations of the two pathogens by 1.97 and 2.00 log CFU/g, re-spectively, suggesting that CBB washing can be an effective sanitizer forimproving microbial safety against pathogens on FCPC. CB and BECpossess strong antimicrobial properties due to eugenol, which can da-mage the cell membrane and increase the permeability of cell mem-brane, and electrostatic interactions with the bacterial cell membrane,respectively (Chen et al., 2017; Park et al., 2018). When EOs areemulsified by surfactants, the wettability applied to the food surfaceincreases, resulting in an increase in the antibacterial activities of EOs(Bhargava, Conti, da Rocha, & Zhang, 2015). In particular, the anti-bacterial activity of EO emulsions depends on the type of surfactantused (Kang et al., 2019). The antimicrobial activity of EO emulsioncontaining Tween 80 (T80, non-ionic surfactant) was reported to be lessthan that of EO alone because of hydrogen bond formation between T80and EO (El-Sayed, Chizzola, Ramadan, & Edris, 2017; Shaaban & Edris,2015). In contrast, BEC, a cationic surfactant used in this study, de-monstrated an additive effect against both pathogens tested here byadding its antibacterial activity, while maintaining the antimicrobialactivity of CB. Thus, the cationic surfactant BEC, can be considered as auseful surfactant for the preparation of an EO emulsion for washingFCVs (Kang et al., 2019). In addition, it should be noted that the logreductions by CBB treatment against both pathogens on FCPC weresignificantly (p < 0.05) greater than those by sodium hypochlorite(0.02%) treatment, suggesting that CBB is a novel washing agent that

Fig. 1. Changes in the populations of L. monocytogenes and SalmonellaTyphimurium inoculated on fresh-cut pak choi following various washingtreatments. Error bars represent standard deviations. A−DAny means in thesame bacteria with different letters are significantly different (p < 0.05) byDuncan's multiple range test.■, Listeria monocytogenes; □, SalmonellaTyphimurium. CB; clove bud essential oil 0.02%, BEC; benzethonium chloride0.002%, CBB; clove bud essential oil 0.02% + benzethonium chloride 0.002%,SH; sodium hypochlorite 0.02%, NaOCl, pH 7.0.

Fig. 2. Scanning electron micrographs of pathogens on fresh-cut pak choi surface (magnification, 5000× ).(A) L. monocytogenes, (B) L. monocytogenes after CBBtreatment, (C) S.Typhimurium, (D) S. Typhimurium after CBB treatment.

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can replace chlorine-based sanitizers such as NaOCl.

3.2. SEM analysis

The LVFE-SEM image visually shows how the pathogens are influ-enced by CBB treatment on the FCPC surface (Fig. 2). In the control, alarge number of pathogens were uniformly distributed on the FCPCsurface. In contrast, the populations of each pathogen treated with CBBwas lower than in their respective control due to the detachment of cellsfrom the FCPC surface upon CBB washing. Furthermore, the mor-phology of each pathogen in the untreated sample remained intact,whereas those exposed to CBB washing displayed altered morphologiesdue to membrane damage. Using SEM images, we could visually con-firm that CBB causes morphological changes in microorganisms. It hasbeen reported that eugenol, the main component of CB, can damage thebacterial membrane by serial mechanisms that alter morphology, in-duce leakage of cell constituents, and increase permeability (Calo,Crandall, O'Bryan, & Ricke, 2015). In addition, L. monocytogenes and S.Typhimurium have cell membranes with a negative charge from tei-choic acid and lipopolysaccharide, respectively (Nazzaro, Fratianni, DeMartino, Coppola, & De Feo, 2013). Thus, electrostatic attraction be-tween positively charged BEC and negatively charged membranes ofthese pathogens changes the membrane properties and damages the cellmembranes (del Carmen Velázquez, Barbini, Escudero, Estrada, & deGuzmán, 2009).

3.3. Gas composition in packaging films and microbial analysis of FCPCduring MA storage

After the washing treatments (control and CBB washing) on FCPC,the change in the gas composition in the head space of each samplepackaged with PA/PA/PE films was analyzed (Fig. 3). The O2 content inthe normal air storage samples (control/normal air and CBB washing/normal air) was decreased from 21% to 9.7–9.8%, whereas the CO2

content increased from 0.03% to 4.9–5% during the 14-day storageperiod. In contrast, during MA storage (control/MA and CBB washing/MA), the O2 content decreased from 5% to 1.0–1.2% and the CO2

content increased from 10% to 11.8–11.9%. The optimal MA storagegas composition for Brassicaceae vegetables, such as pak choi, was re-ported to be 2–7.5% O2 and 6–15% CO2 (Oliveira et al., 2015). The gascomposition in the packaging is determined by the gas permeability ofthe packaging material and respiration rate (RR) of the samples (Costaet al., 2011). In the present study, the RR of FCPC affected the gascomposition within samples packaged with PA/PA/PE film due to itslow gas permeability. Therefore, PA/PA/PE packaging material is sui-table for MA storage of FCPC. Similarly, Yassoralipour, Bakar, AbdulRahman, and Abu Bakar (2016) reported that barramundi packaged inpolyamide film showed longer shelf life compared to other packagingmaterials.

Table 1 shows that the differences in the gas composition withinpackaging films during various treatments (control/normal air, control/MA, CBB washing/normal air, and CBB washing/MA) affected thechanges in the population of TAB on FCPC during MA storage. Thenumber of TAB for the control/normal air samples was 5.87 log CFU/gon day 0 and slightly decreased to 5.79 log CFU/g on day 14. For thecontrol/MA group, the population of TAB decreased from 5.76 to 5.20log CFU/g during storage. It has been reported that low O2 and highCO2 concentration under modified gas composition increases the lagphase and decreases the logarithmic phase of aerobic bacterial growth(Chun & Song, 2013; Horev et al., 2012). However, it should be notedthat MA storage in this study showed only a slight impact on bacterialnumbers. In contrast, due to the antimicrobial activities of CBB, thepopulation of TAB on day 0 was 3.88 log CFU/g for the CBB washing/normal air, which reduced by 2.00 log CFU/g compared with thecontrol, and showed 3.75 log CFU/g on day 14. Similarly, in case ofCBB washing/MA group, the population of TAB was reduced from 3.91to 3.22 log CFU/g during storage. These results indicate that washingwith CBB is more important for ensuring microbiological safety of FCPCthan storage condition. The shelf life of FCV is usually 5–7 days(Stranieri & Baldi, 2017); hence, it is difficult to improve the micro-biological safety of FCVs for more than 7 days under normal gas com-position (Stranieri & Baldi, 2017). Therefore, CBB washing treatmentwith MA storage can be useful to enhance the microbiological safety ofFCPC.

3.4. Changes in the quality of FCPC during MA storage

Table 2 demonstrates the color changes in FCPC during MA storage.There were no differences in the L parameter (data not shown) duringMA storage. In contrast, the redness (a) and yellowness (b) increasedwith the browning index of FCPC (Fig. 4). Among the treatment groups,the color change and browning index were the least in the CBBwashing/MA during storage. These results suggest that CBB washing/MA is effective in inhibiting the browning of FCPC. Browning is one ofthe problems related with quality deterioration in the FCV industry.Chen et al. (2017) reported that eugenol inhibits various enzymes in-volved in the browning of FCV. In addition, low concentrations of O2

under MA storage inhibits the browning of FCV by retarding the ac-tivities of polyphenol oxidases (Luna, Tudela, Tomás-Barberán, & Gil,2016). Thus, MA storage can maintain the sensorial qualities of FCV byreducing the self-degradative properties, thereby extending the shelflife of FCV.

Table 3 shows the changes in the TGC in FCPC during MA storage.The TGC decreased during storage and there was not much differenceamong treatments. It was thus confirmed that CBB treatment and MAstorage did not affect the TGC in FCPC. Glucosinolates are the majorsecondary bioactive substances of Brassicaceae, which are broken downby myrosinase during storage after harvest (Harbaum-Piayda et al.,

Fig. 3. Changes in air composition (O2 and CO2)inside the polyamide multilayer package films offresh-cut pak choi during MA storage. Error bars re-present standard deviations.(A) O2, (B) CO2. ●,control; ○, control/MA storage; ▼, CBB washing;△, CBB washing/MA storage.

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2010).The results of sensory evaluation on FCPC during MA storage are

shown in Fig. 5. For the control/normal air packaging, low scores wereobtained in all categories (AP, appearance; OD, odor; TX, texture; OA.overall acceptability) compared to other groups. In the case of CBBwashing/normal air packaging, high scores in all categories were ob-tained until day 7, suggesting that CBB washing maintained the qualityof FCPC. However, on day 14, the quality was not maintained due to theshelf-life characteristics (5–7 days) of FCV. Rather, due to the MAcondition, the control/MA packaging exhibited better scores than theCBB washing/normal air packaging. Overall, the CBB washing/MApackaging exhibited the highest score in all categories during MA sto-rage. The sensory evaluation data revealed the highest scores for CBBwashing/MA with the lowest changes in browning and moisture lossamong the treatments. Thus, these results suggest that CBB washingwith MA storage is a suitable method to prevent browning of FCVs. Inaddition, although high concentrations of EO can negatively affect thequality of FCPC, the appropriate concentration (0.02%) of EO main-tained the quality of the FCPC during storage. It is also apparent thatMA storage with low permeability packaging materials is necessary toimprove the shelf life of FCPC.

4. Conclusions

CBB washing exhibited an additive effect against pathogens onFCPC without affecting the color and total glucosinolate content duringstorage. CBB treatment is thus a good substitute for chlorine-based sa-nitizers for FCPC washing as it is more effective than sodium hypo-chlorite treatment. In addition, MA storage (5% O2, 10% CO2) usingpolyamide multi-layer packaging films with low gas permeability

extended the shelf life of FCPC. Therefore, CBB treatment/MA storage isa suitable treatment alternative to enhance the microbiological safetyand shelf life of FCPC.

Declarations of interest

None.

Table 1Changes in the population of total aerobic bacteria on fresh-cut pak choi treated with clove bud essential oil emulsion during MA storage.(log CFU/g).

Treatment Storage time (day)

0 4 7 14

Control/air 5.87 ± 0.09Aa 5.60 ± 0.02Ab 5.69 ± 0.04Ab 5.79 ± 0.04Aa

Control/MA 5.76 ± 0.10Aa 5.36 ± 0.05Bb 5.13 ± 0.08Bc 5.20 ± 0.09Bc

CBB/air 3.88 ± 0.13Ba 3.84 ± 0.06Ca 3.76 ± 0.08Cab 3.75 ± 0.02Cb

CBB/MA 3.91 ± 0.11Ba 3.70 ± 0.01Db 3.39 ± 0.09Dc 3.22 ± 0.07Dd

Means ± SD, n=3.A−D Any means in the same column followed by different letters are significantly different (p < 0.05) by Duncan's multiple range test.a-d Any means in the same row followed by different letters are significantly (p < 0.05) different.

Table 2Changes in color of fresh-cut pak choi during MA storage.

Color parameter Treatment Storage time (day)

0 4 7 14

a Control/air −6.03 ± 1.04Ab −5.99 ± 1.03Ab −4.83 ± 0.99Ab −2.93 ± 0.57Aa

Control/MA −5.96 ± 0.99Ab −5.91 ± 1.00Ab −5.70 ± 0.59Aab −4.68 ± 0.47Ba

CBB/air −5.95 ± 0.75Ab −5.92 ± 0.77Ab −5.75 ± 0.77Ab −4.75 ± 0.35Ba

CBB/MA −6.03 ± 1.22Aa −5.96 ± 0.85Aa −5.83 ± 0.85Aa −5.63 ± 0.47Ca

b Control/air 9.23 ± 1.22Ac 9.37 ± 1.08Ac 11.42 ± 0.50Ab 13.11 ± 0.68Aa

Control/MA 9.29 ± 1.24Aa 9.35 ± 1.26Aa 9.81 ± 0.30BCa 10.45 ± 0.40Ba

CBB/air 9.20 ± 1.23Aa 9.33 ± 1.28Aa 9.94 ± 0.41Ba 10.46 ± 0.38Ba

CBB/MA 9.22 ± 1.40Aa 9.24 ± 1.34Aa 9.37 ± 0.38Ca 9.74 ± 0.50Ca

ΔE Control/air – 1.02 ± 0.35Aa 3.16 ± 0.34Ab 5.50 ± 0.34Aa

Control/MA 0.65 ± 0.18Ac 0.90 ± 0.09Ac 1.38 ± 0.36Bb 2.56 ± 0.40Ba

CBB/air 0.65 ± 0.35Ac 0.83 ± 0.20Ac 1.58 ± 0.22Bb 2.63 ± 0.20Ba

CBB/MA 0.77 ± 0.32Ac 0.83 ± 0.11Abc 1.06 ± 0.11Cb 1.60 ± 0.27Ca

Means ± SD, n=5.A−C Any means in the same column followed by different letters are significantly different (p < 0.05) by Duncan's multiple range test.a-c Any means in the same row followed by different letters are significantly (p < 0.05) different.

Fig. 4. Changes in browning index of fresh-cut pak choi during MA storage.Error bars represent standard deviations.●, control; ○, control/MA storage; ▼,CBB washing; △, CBB washing/MA storage.

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Acknowledgements

This research did not receive any specific grant from fundingagencies in the public, commercial, or not-for-profit sectors.

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Table 3Changes in total glucosinolate content of fresh-cut pak choi during MA storage. (μmol/g).

Treatment Storage time (day)

0 4 7 14

Control/air 37.46 ± 1.86Aa 38.56 ± 1.50Aa 31.99 ± 0.92Ab 27.64 ± 0.64Ac

Control/MA 37.49 ± 1.77Aa 37.72 ± 1.40Aa 32.76 ± 0.79Ab 27.15 ± 0.68Ac

CBB/air 37.03 ± 1.88Aa 38.39 ± 0.39Aa 32.07 ± 0.55Ab 27.43 ± 0.83Ac

CBB/MA 37.13 ± 1.08Aa 37.79 ± 0.96Aa 32.54 ± 0.61Ab 27.05 ± 1.23Ac

Means ± SD, n=5.A Any means in the same column followed by different letters are significantly different (p < 0.05) by Duncan's multiple range test.a-c Any means in the same row followed by different letters are significantly (p < 0.05) different.

Fig. 5. Sensory evaluation of fresh-cut pak choi during MA storage.(A) control, (B) control/MA storage, (C) CBB washing, (D) CBB washing/MA storage. AP;appearance, OD; odor, TX; texture, OA; overall acceptability.

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