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Neutral and acidic electrolysed water kept microbial quality and healthpromoting compounds of fresh-cut broccoli throughout shelf life

Javier Navarro-Rico, Francisco Artes-Hernandez, Perla A. Gomez, MarıaAngeles Nunez, Francisco Artes, Gines Benito Martınez-Hernandez

PII: S1466-8564(13)00174-4DOI: doi: 10.1016/j.ifset.2013.11.004Reference: INNFOO 1092

To appear in: Innovative Food Science and Emerging Technologies

Received date: 22 April 2013Accepted date: 6 November 2013

Please cite this article as: Navarro-Rico, J., Artes-Hernandez, F., Gomez, P.A.,Nunez, M.A., Artes, F. & Martınez-Hernandez, G.B., Neutral and acidic electrolysedwater kept microbial quality and health promoting compounds of fresh-cut broccolithroughout shelf life, Innovative Food Science and Emerging Technologies (2013), doi:10.1016/j.ifset.2013.11.004

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Neutral and acidic electrolysed water kept microbial quality and health promoting

compounds of fresh-cut broccoli throughout shelf life

Javier Navarro-Rico1,2

, Francisco Artés-Hernández1,2

, Perla A., Gómez2, María Ángeles

Núñez1, Francisco Artés

1,2 and Ginés Benito Martínez-Hernández*

1,2

1 Postharvest and Refrigeration Group. Department of Food Engineering. Universidad

Politécnica de Cartagena (UPCT). Paseo Alfonso XIII, 48. 30203 Cartagena, Murcia,

Spain.

2 Institute of Plant Biotechnology. UPCT. Campus Muralla del Mar. 30202 Cartagena,

Murcia, Spain.

* To whom correspondence should be addressed:

Tel: +34-968-325921; Fax: +34-968-325433.

E-mail: gpostref@upct.es Web site: www.upct.es/gpostref

Abstract

The effect of neutral (NEW) and acidic (AEW) electrolysed water (EW) sanitizing

treatments (both with 70 and 100 mg L-1

free chlorine) on the bioactive profile and

microbial quality of fresh-cut ‘Parthenon’ and kailan-hybrid broccoli throughout 19

days at 5 ºC were studied. Disinfection with 100 mg L−1

NaClO was used as control.

EW treatments, particularly NEW100, achieved the best microbial reductions after shelf

life, being a promising alternative to chlorine in both broccoli cvs. In addition, all EW-

treated samples, regardless of pH or free chlorine levels, showed up to 30 % higher total

phenolic (TP) contents than chlorine-disinfected ones, which reported values of 556.5

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(kailan-hybrid) and 444.0 mg gallic acid kg-1

fw (‘Parthenon’). The best correlation

between total antioxidant capacity (TAC) and TP was attained by the FRAP method

with an R2=0.65-0.68. Throughout shelf life, TP and FRAP-analysed TAC kept stable

values, or even slightly increased. On the processing day, the APX, GPX, CAT and

SOD activities of chlorine-treated samples were 105.6/115.4, 3783.2/6791.0,

359.0/433.0 and 798.0/1489.0 U g-1

protein for kailan-hybrid/’Parthenon’, respectively.

SOD and CAT activities of EW-treated samples were 13-46 % lower than those of the

NaOCl-disinfected samples. Myrosinase activity in kailan-hybrid was 1.5-fold higher

than in ‘Parthenon’. Generally, the myrosinase activity in EW-disinfected samples was

kept during shelf life, while in NaOCl-sanitised samples decreased. In general, both EW

treatments seem to be promising techniques for keeping good microbial quality in both

fresh-cut broccoli cvs. Furthermore, this alternative water sanitising technique showed

better bioactive compounds retention in broccoli.

Keywords: Minimally processed; ready-to-eat; phenolics; antioxidants; enzymes;

myrosinase.

1. Introduction

Kailan-hybrid is a new natural hybrid between kailan (Brassica oleracea, Alboglabra

group, also called kalian, Chinese broccoli or Chinese kale) and conventional broccoli

(B. oleracea, Italica group). This kailan-hybrid has a long slender stem and a mild sweet

taste with a completely edible portion being a suitable product for the minimal

processing or fresh-cut (FC) industry (Martínez-Hernández et al., 2013a). However, the

processing steps required good result in microbial proliferation and, currently in the FC

vegetables industry, washing with 50–150 mg L−1

NaClO is a sanitising method widely

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used (Artés et al, 2007). However, chlorine, among other disadvantages, may be

potentially harmful for humans and the environment (Hrudey, 2009). Thus, alternative

techniques such as water disinfection with electrolysed water (EW), both neutral (NEW)

and acidic (AEW) types seems to be useful for keeping microbial quality and to prevent

cross contamination in several FC horticultural products (Abadías et al., 2008; Artés et

al., 2009; Rico et al., 2008; Tomás-Callejas et al., 2011) and recently have been

described to be effective against moth proliferation (Jemni et al., 2014).

The inactivation mechanism for EW treatments proposed that oxidation-reduction

potential (ORP) could first affect and damage the redox state of glutathione disulfide–

glutathione couple, and then penetrate the outer and inner membranes of

microorganisms resulting in the bacteria necrosis (Liao et al., 2007). The main

advantage of EW is its safety. In contrast with the risks of using NaClO, such as skin

and membrane irritation and toxicity, EW is not corrosive to skin, mucous membranes,

or organic material. Additionally, EW is more eco-friendly than NaClO and is not

potentially harmful for human health (Huang et al., 2008). Among NEW and AEW,

NEW does not affect the surface colour and visual appearance of FC produce.

Furthermore, NEW is preferable to AEW, since a neutral pH is less aggressive for the

processing equipment. NEW showed great sanitising effects against natural microflora

and pathogenic bacteria in several FC vegetables, such as kailan-hybrid, carrots,

spinach, bell pepper, potato, cucumber, Japanese radish, lettuce and mizuna baby

leaves, lowering microbial counts from 0.6 to 2.6 log units (Abadías et al., 2008;

Martínez-Hernández et al., 2013b; Rico et al., 2008; Tomás-Callejas et al., 2011).

Sanitising treatments (such as EW), wounding, modified atmosphere packaging (MAP)

and chilled storage involved during the processing and shelf life of FC produce have

been regarded as abiotic stresses (Martínez-Hernández et al., 2013b; Jacobo-Velázquez

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et al., 2011). Such stresses may increase the accumulation of reactive oxygen species

(ROS) in the plant cells and, in order to mitigate the possible plant cell damage, the

enzymatic and non-enzymatic antioxidant systems are activated. Kailan-hybrid is rich in

polyphenols (Martínez-Hernández et al., 2013c), which have been widely reported as

non-enzymatic antioxidants. Other non-enzymatic antioxidants present in broccoli are

vitamins C and E, folic acid and carotenoids. Members of the enzymatic antioxidant

defence system include superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC

1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), guaiacol peroxidase (GPX; EC

1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2). The super-oxide radical (O2−) is

dismutated to H2O2 by SOD, and CAT, APX and GPX metabolize H2O2 to H2O (Kang

and Saltveit, 2001). Furthermore, kailan-hybrid is highly rich in glucosinolates

(biologically inactive) which are separated in different cell compartments from the

enzyme myrosinase (MYR; β-thioglucoside glucohydrolase; EC 3.2.3.1) which is

located in the myrosin cells. However, after tissue disruption (cutting, chewing, etc.)

glucosinolates come into contact with the MYR which causes rapid hydrolysis to form,

among others, chemopreventive compounds known as isothiocyanates. Several studies

have focused on the effects of postharvest treatments on the glucosinolates levels,

although there is very scarce information about postharvest treatments on the MYR

activity, which is crucial to get the chemopreventive benefits from the glucosinolates

(Halkier & Gershonzen, 2006; Jones et al., 2010).

However, there is no information on the effects of EW treatments on the levels of the

above mentioned bioactive compounds in FC products. In this way, the main aim of this

work was to study the effects of NEW and AEW as emergent sanitizers versus a control

washing with NaClO on the microbial quality of a new hybrid and a conventional

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broccoli cv. and their impact on their bioactive compounds profiles throughout shelf

life.

2. Materials and methods

2.1. Plant material

Kailan-hybrid broccoli (B. oleracea Italica group × Alboglabra group, cv. Bimi®) of 15

to 18 cm length and ‘Parthenon’ cv. (B. oleracea Italica group) were hand-harvested in

December from open air commercial cultivation in the southeast Mediterranean area of

Spain (Lorca, Murcia). The broccoli was grown according to integrated pest

management cultural practices. Immediately after harvesting, broccoli was selected in a

packinghouse for discarding heads and stems with yellowing, decay, cuts or bruises and

sound pieces were precooled with crushed ice and transported by car about 80 km to the

Technical University of Cartagena. Both kind of broccoli were then stored at 1 ºC and

90–95% RH until next day, when they were processed.

2.2. Sample preparation, minimal processing and storage conditions

Minimal processing was accomplished in a disinfected cold room at 8 ºC. Plant material

was inspected, selecting those free from defects and with similar visual appearance.

Small leaves of kailan-hybrid broccoli stems (when they were present) were eliminated

with a sharp knife. The kailan-hybrid broccoli was cut in about 15-cm-long spears and

the ‘Parthenon’ heads were cut into florets. All broccoli pieces were washed for 1 min

with tap water (4 ºC) in order to remove organic material. The EW treatment (5 ºC; pH

7 ± 0.1; ORP = +900 mV) for washing broccoli pieces consisted in two NEW (70 and

100 mg L−1

free Cl) and two AEW (70 and 100 mg L−1

free Cl). Both EW were

produced by an Envirolyte EL 400 device (Aquarioja, Madrid, Spain). Subsequently,

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the broccoli pieces were drained in a perforated basket for 1 min. Contact time was 2

min using a ratio 300-g plant material/5-L disinfectant (w/v). As control, a standard

industrial disinfection treatment with NaClO (100 mg L−1

; 5 ºC; pH 6.5 ± 0.1) for 2 min

was used with the same contact time, plant material/disinfectant ratio and rinsing

conditions than EW treatments. During all washing treatments continuous stirring of

plant material and liquid was carefully made by hand. Once drained, 120 g of broccoli

pieces were disposed in 1.5-L (kailan-hybrid) and 2-L (‘Parthenon’) rigid polypropylene

(PP) trays (12 x 17 cm) which were thermally sealed on the top with an antimist 30-

mm-thick bioriented PP film (Plásticos del Segura S.L., Murcia, Spain). Five replicates

of one tray per treatment and sampling time were prepared. The film permeability was

900 cm3 O2 m

-2 day

-1 atm

-1 and 1100 cm

3 CO2 m

-2 day

-1 atm

-1 at 23 ºC and 0% RH (data

from the supplier). In order to check appropriate MAP conditions, O2 and CO2 partial

pressures within packages were analysed according to Fernández-León et al. (2013)

using three replicates for each treatment and evaluation period. The product was stored

in darkness at 5 ºC (90-95 % RH). Analyses were conducted on the processing day and

after 5, 9, 14 and 19 days of shelf life.

2.3. Microbial analysis

To determine mesophilic, psychrophilic, enterobacteria and yeasts and moulds counts,

standard enumeration methods were used (Tomás-Callejas et al., 2011). All microbial

counts were reported as log10 colony forming units per gram (log CFU g-1

). Salmonella

spp., Listeria monocytogenes and generic Escherichia coli were monitored according to

the European legislation for FC vegetables (Regulation EC 1441/2007, 2007). All

analyses were made by triplicate.

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2.4. Total phenolics and antioxidant capacity

The total phenolics (TP) content was analysed according to Singleton and Rossi

(1965) method with slight modifications proposed by Martínez-Hernández et al.

(2013d). TP content was expressed in mg gallic acid equivalent per kg of fresh weight

(fw). Each one of the five replicates was analysed by triplicate.

The total antioxidant capacity (TAC) was determined by three methods: free radical

scavenging capacity (DPPH) (Brand-Williams, Cuvelier, & Berset, 1995), ferric

reducing antioxidant power (FRAP) (Benzie & Strain, 1999) and 2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Cano et al., 1998). The absorbance of

samples was measured using the same device as for TP content. TAC was expressed in

mg ascorbic acid equivalent antioxidant capacity per kg fw. Each of the five replicates

was analysed by triplicate.

2.5. Enzymatic activity

Antioxidant enzymes extractions and analyses were performed according to Martínez-

Hernández et al. (2013b) based on original methods from Kang and Saltveit (2001),

Chen and Asada (1989), Upadhyaya et al. (1985), Dhindsa et al. (1981) and Maehly and

Chance (1959). APX, GPX and CAT activities were estimated by the initial velocities

method from the linear portion of the curves. The estimated enzyme activity was

expressed per gram of protein, which was determined by the protein-dye binding

method (Bradford, 1976). One unit of activity (U) of APX and CAT was defined as a

decrease in absorbance of min−1

. One GPX U was defined as a decrease in absorbance

of s−1

. SOD activity was calculated per g protein and expressed as a percentage of the

control. Each of the five replicates was analysed by triplicate.

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The MYR extraction was made according to Guo et al. (2011) with slight modifications.

Briefly, 2.5 g of ground frozen sample were homogenised at 4 ºC in 10 mL of 100 mM

sodium phosphate buffer (pH 6.5) using an Ultraturrax. Subsequently, the homogenate

was centrifuged (15,000 × g; 20 min; 4 ºC) and the supernatant was used as the enzyme

extract for the MYR assay. Then, 60 μL of enzyme extract were mixed with 300 μL of

preheated (37 ºC for 5 min) reaction buffer [100 mM sodium phosphate buffer with 1

mM sinigrin (Sigma-Aldrich, Germany)]. The latter reaction mixture was incubated at

37 ºC for 15 min and the MYR activity was stopped in an oil bath at 100 ºC for 5 min.

The absorbance by the incubated reaction mixture was read at 227 nm. The MYR

activity was determined using a standard curve of sinigrin (0.02-2.0 mg mL-1

). One

MYR U was defined as 1 nM sinigrin transformed of min-1

. Each of the five replicates

was analysed by triplicate.

2.6. Statistical analysis

The experiment was a 5×5 two-factor (treatment × storage time) design subjected to an

analysis of variance using Statgraphics Plus (version 5.1) software. When differences

among treatments were significant, the mean values were compared by the least

significant difference multiple range test (LSD, p < 0.05).

3. Theory

Several sanitising alternatives to NaOCl have been studied in the last few years. EW

treatments (neutral and acidic) seem to be a promising option. In that way, the

disinfecting potential of these treatments has been studied in several FC vegetables,

unless the effects on many bioactive compounds have not been studied. Concretely, the

high ORP of EW treatments may act as an abiotic stress inducing defence responses in

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the plant cells such as increases of important bioactive compounds such as phenolics or

antioxidant enzymes. In that way, the EW treatment of FC broccoli may enhance their

bioactive compounds giving to the consumer a healthier FC product which meet the

microbial quality and safety aspects being a sanitising alternative to the conventionally

used NaOCl.

4. Results and discussion

4.1. Gas composition

The gas partial pressure changes of both kailan-hybrid and ‘Parthenon’ broccoli within

MAP packages are shown in Figure 1. The equilibrium gas partial pressures were

reached after 5 days at 5 ºC for all treatments being 9-13 kPa O2 + 10-15 kPa CO2 and 6-

10 kPa O2 +10-14 kPa CO2 for kailan-hybrid and ‘Parthenon’, respectively, without

noticeable differences among them. Similar results were found by Torrieri et al. (2010)

and Martínez-Hernández et al. (2013a) for both MAP-stored broccoli cvs. This gas

composition was similar to that recommended for broccoli florets by Gorny (2001),

being these high CO2 and low O2 atmospheres excellent coadjutants to low temperature

and high RH, slowing down the senescence rate and extending the shelf life of the FC

broccoli.

The similar atmosphere reached around the samples to both NaOCl- and EW-treated

samples indicates that no stresses were produced owed to different gas compositions.

The latter finding limits the microbial and bioactive changes of EW-treated samples,

compared to chlorine, to the used sanitizing treatment itself.

4.2. Microbial analysis

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The initial mesophilic, psycrophilic, enterobacteria and yeast and moulds counts were

5.5/4.6, 5.0/4.0, 3.5/4.8 and 8.0/4.6 log CFU g-1

for kailan-hybrid/‘Parthenon’,

respectively (Figure 2). These differences between microbial counts could be explained

by the natural contamination of the plant material from the field, which was generally

higher for the kailan-hybrid than for the ‘Parthenon’. The mesophilic counts were

higher than those reported by Martínez-Hernández et al. (2013a) for the same broccoli

cv. However, higher mesophilic counts (6 log CFU g-1

) were reported by Brackett

(1989) and Olarte et al. (2009) in other conventional broccoli cvs. These relatively high

counts of enterobacteria could be due to any punctual inadvertent faecal contamination

of irrigation water. However, and accomplishing the EU legislation for FC vegetables

(Regulation EC 1441/2007, 2007), the plant material safety was confirmed after no

detection of Salmonella spp., L. monocytogenes and E. coli. Generally, all sanitizing

treatments achieved better microbial reductions (for the four microbial groups studied)

in kailan-hybrid than in ‘Parthenon’, probably owed to a better washing efficiency due

to the less compacted form of kailan-hybrid.

Attending to mesophilic counts on the processing day, NEW100 induced the highest

decrease around 1.7-fold for ‘Parthenon’ regarding to levels of the non-sanitized

product. The remaining treatments induced significant (p<0.05) mesophilic decreases

around 1.2-1.4-fold in ‘Parthenon’. AEW and NEW-treated kailan-hybrid achieved

around 2.2-2.4 and 1.7-1.8-fold significant (p<0.05) mesophilic reductions, respectively,

compared to ‘Parthenon’ results. However, NaOCl-treated kailan-hybrid broccoli

achieved the highest mesophilic reduction with 3.2-fold. This fact could be probably

due to a better efficacy of NaOCl than EW in plant material with high initial mesophilic

loads. The hereby found mesophilic decreases (p<0.05) were higher than those reported

in EW disinfected FC iceberg lettuce and four seasons salad (iceberg lettuce, carrot and

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cabbage) by Abadías et al. (2008) and in FC mizuna baby leaves (Tomás Callejas et al.,

2011).

On the processing day, the highest reduction of the psycrophilic microorganisms was

obtained after NEW100 treatment with 4.0 and 1.3-fold for kailan-hybrid and

‘Parthenon’, respectively. Meanwhile, the remaining treatments induced significant

(p<0.05) psycrophilic reductions around 2.2-3.6 and 1.0-1.3 for kailan-hybrid and

‘Parthenon’, respectively. The psycrophilic counts reached in kailan-hybrid after EW

treatments were higher than those recently reported (Martínez-Hernández et al., 2013b).

On the processing day, Enterobacteria counts in ‘Parthenon’ greatly decreased (around

3.6-fold) after NEW100 treatment while the remaining treatments only achieved

reductions ranging from 2.0 to 2.6-fold, without significant (p<0.05) differences among

them. However, and similarly to mesophilic results, the NaOCl washing of kailan-

hybrid induced the highest psycrophilic counts reduction of 3.5-fold over initial levels,

showing the rest of treatments decreases around 0.9 to 1.7-fold. Generally, all

treatments reduced the yeast and moulds counts around 1.8-2.0 and 1.0-1.3-fold for

kailan-hybrid and ‘Parthenon’, respectively, without significant (p<0.05) differences

among them.

In general, microbial counts significantly (p<0.05) increased 1 log CFU after 19 days at

5ºC regarding to values on the processing day. Although in some initial cases NaOCl

was more effective than EW treatments, after 19 days of shelf life NaOCl-treated

samples reached the highest mesophilic and enterobacteria growth with 1.5 to 2.5-fold.

In this way, EW treatments, particularly NEW100, can be regarded as a promising

alternative to conventional NaClO disinfection in both broccoli cvs. providing a good

microbial quality after 19 days of shelf life.

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4.3. Total phenolic content

On the processing day, the TP contents after NaOCl washing were 556.5 and 444.0 mg

gallic acid kg-1

fw for kailan-hybrid and ‘Parthenon’, respectively (Figure 3). Generally,

EW treatments showed higher significant (p<0.05) TP contents than NaOCl for both

broccoli cvs. Among treatments, AEW-treated samples achieved the highest TP content

with levels 16-30 % higher (p<0.05) than NaOCl-disinfected broccoli. In this way, EW

sanitation can be regarded as an abiotic stress that may induce a TP content

enhancement as it has been similarly described in other vegetables after several

sanitation treatments such as UV-C radiation, superatmospheric oxygen packaging,

wounding, etc. (Jacobo-Velázquez et al., 2011; Martínez-Hernández et al., 2013b).

Contrary to our results, no TP increases were early found after EW treatments in kailan-

hybrid (Martínez-Hernández et al., 2013b) and mizuna baby leaves (Tomás-Callejas et

al., 2011).

The general trend of the phenolic content throughout 19 days at 5 °C was to remain

quite stable for kailan-hybrid (except control and NEW100 that showed significant

(p<0.05) decreases of 10 and 16 %, respectively) and to significantly (p<0.05) increase

(except AEW100) up to 30 % for ‘Parthenon’ compared to the correspondent levels on

the processing day. The observed phenolics accumulation in FC ‘Parthenon’ throughout

shelf life may be promoted by the phenylalanine ammonia lyase (PAL) activity. This

enzyme may be activated by abiotic stresses, such as the wounding produced during the

minimal processing, which was more intense for ‘Parthenon’, synthesizing the main

phenolic compounds and new polyphenolic substances (Cisneros-Zevallos, 2003). The

diverse free chlorine levels used did not induced significant (p<0.05) TP differences

among them.

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4.4. Total antioxidant capacity

The TAC of kailan-hybrid and ‘Parthenon’ have been early reported to be highly

correlated to the TP content (Fernández-León et al., 2013; Martínez-Hernández et al.,

2013b, 2013e; Soengas et al., 2011). According to this, the correlation rates between

TAC –analyzed by FRAP, ABTS and DPPH methods– and TP content of kailan-hybrid

and ‘Parthenon’ are shown in Figure 4. The FRAP and ABTS methods showed the

highest correlations with regression factors (R2) of 0.68 and 0.54, respectively, for

kailan-hybrid; and 0.65 and 0.43, respectively, for ‘Parthenon’. However, DPPH only

showed a R2 of 0.14 and 0.30 for kailan-hybrid and ‘Parthenon’, respectively. The low

correlation rate between DPPH and total phenolics may be explained since broccoli

carotenoids have the maximum absorption in the same wavelength range than that used

for the DPPH determination. Consequently, and according to the DPPH method

procedure, the higher the carotenoids content of the plant material, the lower the TAC

determined by the DPPH method. It has been reported that broccoli florets have higher

carotenoid concentration than stems (Zhang & Hamauzu, 2004). In that way, the high

carotenoid content of the edible part of ‘Parthenon’ (practically just florets) compared to

the kailan-hybrid edible part (floret plus stems) may explain the lower TP vs TAC

(DPPH) correlation of ‘Parthenon’.

The TAC values determined by FRAP, ABTS and DPPH for NaOCl-disinfected

samples on the processing day were 480.5±112.8, 904.0±65.7 and 114.0±12.2 mg

ascorbic acid equivalents kg-1

fw, for kalian-hybrid, respectively, and 268.6±38.5,

962.5±44.7 and 102.3±20.6 mg ascorbic acid equivalents kg-1

fw, for ‘Parthenon’,

respectively (supplementary data). Similar results have been early reported for kailan-

hybrid by DPPH and FRAP methods (Martínez-Hernández et al., 2013e). According to

this, the ABTS method showed the highest TAC levels while DPPH registered the

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lowest. There were no significant (p<0.05) TAC differences between NaOCl and all the

EW treatments for both broccoli cvs. on the processing day for any of the TAC methods

used.

Attending to TAC behaviour throughout shelf life, a general TAC increment around

1.5-2.1-fold was found for the ABTS and DPPH-analyzed samples, regardless of the

treatment. According to these findings, the EW treatments attained the same TAC

increases during storage as NaOCl, which shows how the EW-treated samples reached

the same TAC as NaOCl. Furthermore, there was no significant (p<0.05) TAC

differences related to either pH or free chlorine level of the EW treatments. This implies

that the best EW treatment selected, in order to attain the best microbial sanitation, will

not have any special impact on the TAC of both broccoli cvs. However, TAC of

samples determined by the FRAP method did not show any significant (p<0.05) change

during shelf life. The last fact may be owed to a decrease of the carotenoids content

during storage due to their degradation, as it has been reported on FC broccoli and kale

chilling stored (De Azevedo & Rodríguez-Amaya, 2005; Hussein et al., 2000).

Consequently, this carotenoid reduction during storage may interfere with the TAC

measured by FRAP as it has been above described.

4.5. Enzymatic activity

The activities of the APX, GPX, CAT, SOD and MYR enzymes were studied for

NaOCl and NEW100-treated FC broccoli samples (Table 2). This treatment was

selected since NEW100 was the most effective sanitising treatment and, as reported by

Tomás-Callejas et al. (2011), CAT and SOD enzymatic activities were not influenced

by different free chlorine levels of AEW and NEW treatments.

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On the processing day, the APX, GPX, CAT and SOD activities of NaOCl-treated

samples were 105.6/115.4, 3783.2/6791.0, 359.0/433.0 and 798.0/1489.0 U g-1

protein

for kailan-hybrid/’Parthenon’, respectively. As it can be observed, the antioxidant

enzymes activities of NaOCl-treated ‘Parthenon’ samples were 1.1-1.9-fold higher than

those of kailan-hybrid. Wounding has been described as an abiotic stress in several

fruits and vegetables, enhancing the activity of their antioxidant enzymes as a result of a

metabolic defence response (Cisneros-Zevallos, 2003; Jacobo-Velázquez et al., 2011).

In that way, since ‘Parthenon’ suffered a more intense processing due to the florets

cutting, it might lead to the greater enzymatic activity found compared to kailan-hybrid

which processing was less intense. Generally, APX and GPX activities after EW

treatment did not show significant (p<0.05) changes compared to NaOCl-treated

samples for both broccoli cvs. However, SOD and CAT activities significantly (p<0.05)

decreased after EW washing around 13-37 and 40-46 %, respectively, comparing to

NaOCl. EW has shown strong and stable SOD- and CAT-like activities because of

extremely high level of dissolved molecular hydrogen produced in EW during

electrolysis of water (Shirahata et al., 1997). According to that, the SOD- and CAT-like

activities of EW could greatly contribute to the scavenging of those ROS produced

throughout the shelf life of the FC product. As a result of the strong SOD- and CAT-like

activities of EW-treated samples, the activities of SOD and CAT might have been

diminished as it has been shown.

Generally, GPX and CAT activities decreased progressively after processing day once

the ROS generated during processing were being eliminated. Thus, minimum levels

with 45-60 % (NaOCl) and 20-40 % (EW) lower values, compared to their respective

levels on the processing day, were reached after 5 (GPX) and 9-14 days at 5 ºC (CAT).

The latter results show how EW-treated samples newly registered significantly (p<0.05)

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lower activities for those enzymes due to the scavenging-like properties of this

sanitising treatment as it was explained before. However, throughout storage, ROS were

being generated and accumulated, leading to an activity increase of the latter antioxidant

enzymes until the end of shelf life. Similarly, other abiotic stresses such as UV-C

radiation or salicylic acid have shown GPX increases during storage in conventional

broccoli, peach and mushrooms (Jiang et al., 2010; Lemoine et al., 2010; Tareen et al.,

2012). As it was early found in kailan-hybrid (Martínez-Hernández et al., 2013b), the

CAT activity enhancement throughout shelf life was retarded, since this enzyme has

affinity for H2O2 at mM ranges, contrary to the µM ranges where peroxidases act. APX

activity was also significantly (p<0.05) enhanced throughout storage with values 1.2-1.5

and 1.6-2.0-fold higher for ‘Parthenon’ and kailan-hybrid, respectively, compared to

initial levels, regardless of the treatment. These differences in substrate affinity might be

explained since APX is responsible for finely modulating ROS to serve as signalling

molecules, and CAT is in charge of removing ROS when toxic levels are attained

(Jacobo-Velázquez et al., 2011). Throughout shelf life, SOD activities did not show

significant differences.

MYR activity was found 1.5-fold higher in kailan-hybrid than in ‘Parthenon’ for

NaOCl-treated samples. The distribution of myrosin cells are usually considered to be

random and very different according to the plant material studied. There is no

information regarding to MYR localization in conventional broccoli neither in kailan-

hybrid, being most of the published studies performed on the Brassica species B. napus,

S. alba and A. thaliana. According to this, since the stem proportion in kailan-hybrid is

higher than in ‘Parthenon’ florets a high myrosin cells proportion could be located in

the stems of these Brassicas, concretely in the xylem, phloem or some cortical cells as it

was reported for B. napus (Andreasson et al., 2001; Höglund et al., 1991). Generally,

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the MYR activity of EW-treated samples did not show significant (p<0.05) changes

throughout storage. However, a marked significant (p<0.05) reduction of MYR activity

was found for NaOCl-treated samples after 5 days at 5 ºC, reaching in the last day of

shelf life activity levels 42 % (kailan-hybrid) and 61 % (‘Parthenon’) lower than the

respective values on the processing day.

5. Conclusions

Both studied EW treatments (NEW and AEW) seem to be promising water sanitation

alternatives to the commonly used chlorine in the fresh-cut vegetables industry. Its use

preserved a good microbial quality in fresh-cut broccoli during 19 days at 5ºC.

Generally, both EW treatments increased the bioactive compounds, regardless of pH

and free chlorine levels studied, achieving 16-30 % higher total phenolics content than

NaOCl-disinfected broccoli. Among the total antioxidant capacity methods studied,

FRAP showed the better correlations to total phenolic content. Generally, EW

treatments attained the same antioxidant enzyme activities than NaOCl samples.

Conclusively, among all EW treatments, NEW100 could be selected as a promising

water sanitising treatment for kailan-hybrid and conventional ‘Parthenon’ broccoli,

which additionally kept and, in some cases even enhanced, health-promoting properties.

ACKNOWLEDGMENTS

The authors are grateful to Sakata Seeds Ibérica S.L.U. and to Campo de Lorca S.C.L.

for providing financial support and plant material, respectively. Thanks are also due to

Fundación Séneca de la Region de Murcia for a grant to G.B. Martínez-Hernández and

to the Institute of Plant Biotechnology of UPCT for providing some facilities.

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FIGURE LEGENDS

Figure 1. Gas changes within packages of fresh-cut kailan-hybrid (A) and ‘Parthenon’

broccoli (B) washed under several disinfection treatments and stored up to 19 days at 4

ºC (n = 5). Average (CO2 and O2) SD values=1.0 (both A and B). O2 solid lines; CO2

dashed lines. Balanced with N2.

Figure 2. Mesophilic (A), psychrophilic (B), enterobacteria (C) and yeast and moulds

(D) counts (log CFU g−1

) of fresh-cut kailan-hybrid (A) and ‘Parthenon’ broccoli (B)

washed under several disinfection treatments and stored up to 19 days at 4 ºC (n = 5 ±

SD). N-S: non-sanitised samples.

Figure 3. Total phenolics content of fresh-cut kailan-hybrid (A) and ‘Parthenon’

broccoli (B) washed under several disinfection treatments and stored up to 19 days at 4

ºC (n = 5 ± SD).

Figure 4. Correlations between total phenolics content and total antioxidant capacity of

fresh-cut kailan-hybrid (A) and ‘Parthenon’ broccoli (B) washed under several

disinfection treatments.

Table 1. Enzymatic activities (APX, GPX, SOD, CAT and MYR) of fresh-cut kailan-

hybrid (K) and ‘Parthenon’ broccoli (P) washed under several disinfection treatments

and stored up to 19 days at 4 ºC (n = 5 ± SD). Different capital letter among each row

denotes significant difference (p < 0.05). Different lower case letter within each column

for the same broccoli cv. denotes significant difference (p < 0.05).

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Supplementary data a. Total antioxidant capacity of fresh-cut kailan-hybrid washed

under several disinfection treatments and stored up to 19 days at 4 ºC (n = 5 ± SD).

Different capital letter among each row denotes significant difference (p < 0.05).

Different lower case letter within each column denotes significant difference (p < 0.05).

Supplementary data b. Total antioxidant capacity of fresh-cut broccoli ‘Parthenon’

washed under several disinfection treatments and stored up to 19 days at 4 ºC (n = 5 ±

SD). Different capital letter among each row denotes significant difference (p < 0.05).

Different lower case letter within each column denotes significant difference (p < 0.05).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Table 1

K NaOCl 105.6 ±8.3) 111.8 ±10.7) 146.8 ±21.4) 142.3 ±33.2) 132.3 ± 28.2)

APX NEW100 104.4 ± 4.5) 115.8 ± 18.4), 124.7 ±13.2) 159.4 ±12.2) 159.2 ± 15.5)

P NaOCl 115.4 ±5.1) 107.6 ±12.5) 261.8 ±20.0) 193.8 ± 9.7) 180.8 ± 20.0)

NEW100 119.7 ±4.5) 160.0 ± 10.9) 146.6 ± 19.1) 209.1 ±22.9) 231.7 ±12.8)

K NaOCl 3783.2 ± 307.8® 2097.6 ±347.1) 2739.0 ± 184.0) 4111.0 ±498.7) 5829.4 ± 174.8)

GPX NEW100 6144.2 ±471.3) 7516.0 ±587.6) 3735.6 ±88.2) 4885.0 ±495.3) 7553.4 ±699.5)

P NaOCl 6791.0 ± 592.2® 2996.1 ±338.9) 4689.9 ±335.7) 5719.3 ±713.3) 9327.4 ± 1237.0)

NEW100 7244.4 ± 796.5) 4129.6 ±785.9) 6695.4 ± 193.6) 9939.9 ± 698.9) 8541.9 ±798.3)

K NaOCl 359.0 ±45.4;) 225.5 ± 20.9) 161.1 ± 17.0)d 142.2 ±6.7) 179.6 ± 18.5)

NEW100 227.6 ± 14.6® 415.8 ±42.8) 216.9 ±25.5) 156.5 ± 18.2) 184.1 ±22.3) CAT P NaOCl 433.0 ±18.3) 269.4 ±22.6) 211.4 ±30.0) 280.6 ±32.3) 312.0 ±17.5)

NEW100 374.8 ± 15.1)a 391.5 ± 12.4) 302.1 ±27.2) 340.5 ± 23.7) 362.8 ±38.1)

K NaOCl 798.8 ±38.3) 891.8 ± 20.3)d 784.2 ± 93.9) 933.8 ±63.9) 818.6 ± 98.5)d

SOD NEW100 1337.9 ± 167.9® 1329.9 ±186.7) 1283.2 ± 155.5)c 1276.3 ± 111.3)d 1198.6 ± 91.1)d

P NaOCl 1489.0 ± 107.2) 1087.3 ± 176.6) 1031.9 ±243.9)c 955.1 ±128.2) 837.5 ± 150.3)

NEW100 811.5 ± 78.2® 919.7 ± 98.7) 899.9 ± 75.2)t 982.6 ±125.4) 943.6 ±31.0)

K NaOCl 16.5 ± 1.9) 16.7 ± 1.1) 13.7 ± 0.9) 9.5 ± 0.4) 12.4 ±2.3)

MYR NEW100 10.5 ± 0.7) 11.7 ± 1.0) 11.0 ± 0.9)t 11.5 ± 1.0)„ 10.5 ± 1.4)

P

NaOCl 10.8 ± 1.9) 10.8 ± 1.3) 7.2 ±0.2) 4.2 ±0.3) 10.0 ± 1.0)

NEW100 15.7 ± 1.6) 10.0 ± 0.7) 5.5 ± 1.9) 8.3 ± 1.6) 7.7 ± 1.5)

Days at 5°C

Processing day ____________ 5 _________________ 9 ________________ 14 _________________ 19

Enzymes (U g'1 protein)

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NEUTRAL AND ACIDIC ELECTROLYSED WATER KEPT MICROBIAL QUALITY AND HEALTH

PROMOTING COMPOUNDS OF FRESH-CUT BROCCOLI THROUGHOUT SHELF LIFE

Javier Navarro-Rico, Francisco Artés-Hernández, Perla A., Gómez, María Ángeles Núñez,

Francisco Artés and Ginés Benito Martínez-Hernández*

Research Highlights

Effect of electrolyzed water (EW) sanitation was studied on two broccoli cvs.

Neutral EW (100 ppm free Cl) achieved the lowest microbial counts after shelf life.

EW-samples showed up to 30% higher total phenolics content than NaOCl-treated

ones.

Total phenolics/antioxidant capacity was preserved up to 19 days at 5 ºC.

Antioxidant enzyme activities were kept after EW treatments.