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Comparison of sanitizing technologies on the quality appearance and antioxidant levels in onion slices
Food Control 22 (2011) 2052e2058
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Comparison of sanitizing technologies on the quality appearance and antioxidant levels in onion slices M.R. Pérez-Gregorio, C. González-Barreiro, R. Rial-Otero, J. Simal-Gándara* Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, Ourense Campus, University of Vigo, E-32004 Ourense, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2010 Received in revised form 25 May 2011 Accepted 31 May 2011
Background: A Portuguese landrace onion variety grown in Póvoa do Varzim (Northwest of Portugal) was selected for this study. It is a red onion known as “vermelha da Póvoa”. The effect of different chemicallyand irradiation-based disinfectant procedures of fresh-cut onions on their flavonoid content was compared. Amongst the chemical treatments the most used were selected: sodium hypochlorite, amukine, hydrogen peroxide, and sodium dichloroisocyanate. UV-C irradiation was also selected amongst all irradiation treatments. Results: The main effect contributing to the loss of flavonols in fresh-cut onion slices is their solubility in immersion water (17e23% both at 4 or 50 C). The acidification of sodium hypochlorite with sulphuric acid is also rendering losses (6e19%) vs. the use of citric acid. Anyway, in dry decontaminantion treatments, like UV-C irradiation, the natural levels of flavonoids in fresh-cut onion slices significantly increase at 35% for flavonols, and at 29% for anthocyanins. Conclusions: Onion flavonoids decreased by chemically disinfectant procedures of fresh-cut onion by solubility in immersion water. UV-C irradiation does not only maintain the initial levels of flavonoids but increases them; therefore, this is the recommended treatment. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Anthocyanins Chemical disinfectants UV-C irradiation Flavonols Fresh-cut onion slices
1. Introduction According to the United Fresh Produce Association, minimally processed vegetables are defined as any fruit or vegetable that has been altered physically from its original, but retains its status fresh. Because of consumer interest in the health benefits, convenience and value-added qualities of fresh produce, there is a growing need for convenience vegetables such as fresh-cut and ready-to-eat salads. It is then required that these products have optima hygienic, sensorial and nutritional properties. The most used systems for disinfectant are chemical and irradiation-based treatments. Generally, the chlorine-containing compounds group is considered first by those searching for antimicrobial activity, for being inexpensive and easy to use, and for have a broad spectrum of activity (Choi & Spacers, 1994; Delaquis, Stewart, Toivonen, & Moyls, 1999; Delaquis, Stewart, Cliff, Toivonen, & Moyls, 2000; Martínez, Sgroppo, Sánchez-Moreno, De Ancos, & Cano, 2005; Artés, Gómez, Aguayo, Escalona, & ArtésHernández, 2009). Hypochlorites are the most frequently used forms of chlorine, with sodium hypochlorite receiving broadest
* Corresponding author. E-mail address: [email protected] (J. Simal-Gándara). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.05.028
application. Chlorine can oxidize organic matter in foods or in water, and in the latter case by-products such haloforms and haloacetic acids, which are potentially carcinogenic and mutagenic, can be formed. In some European countries, sodium hypochlorite is not allowed for use in fresh-cut vegetables and might be possible that the UE banned its use by law for such applications (Ölmez & Kretzschmar, 2009). Thus, there is a need for alternative sanitizers to be used for the disinfection of fresh-cut vegetables. Hydrogen peroxide (Beerli, Vilas Boas, & Piccoli, 2004; Khadre & Yousef, 2001) or organic chlorinated products (sodium dichloroisocyanurate, potassium dichloroisocyanurate, dichloroisocyanuric acid and trichloroisocyanuric acid) are alternative sanitizing agents that gained interest in recent years (Beerli et al., 2004). Among the chemical methods for controlling post harvest diseases, UV-C irradiation appears to be one of the most attractive. UV radiation in the range of 250e260 nm is lethal to most microorganisms, including bacteria, viruses, protozoa, mycelial fungi, yeasts and algae. There are many publications with studies about the bactericide effects of different disinfectant agents, but only a few on potential losses of bioactive components such as polyphenols of fresh-cut fruits and vegetables (Fukumoto, Toivonen, & Delaquis, 2002). Onions are considered as a source of antioxidants including flavonols (quercetin derivatives) and also anthocyanins in the case of red bulbs (PérezGregorio, García-Falcón, Simal-Gándara, Rodrigues, & Almeida,
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2010; Pérez-Gregorio, García-Falcón, & Simal-Gándara, 2011; PérezGregorio, Regueiro, González-Barreiro, Rial-Otero, & Simal-Gándara, 2011; Rodrigues, Pérez-Gregorio, García-Falcón, Simal-Gándara, & Almeida, 2010 and 2011), but also mulberry fruits (Pérez-Gregorio, Regueiro, Alonso-González, Pastrana-Castro, & Simal-Gándara, 2011) and plant foods in general (Alén-Ruiz, Pérez-Gregorio, Martínez-Carballo, García-Falcón, & Simal-Gándara, 2008). During disinfection treatments of the bulbs, important chemical and biochemical reactions occur in onion tissue. Such reactions may have an impact on the flavonoids structure, resulting in changes of the bioavailability and activity of these compounds. The present work was undertaken in order to clarify the impact of regular disinfection treatments (sodium hypochlorite, or amukine, hydrogen peroxide, sodium dichloroisocyanate and UV-C irradiation) on the flavonols and anthocyanins content of onions, as well as the role of structural features on their degradation. 2. Experimental 2.1. Raw material A Portuguese landrace onion variety grown in Póvoa do Varzim (Northwest of Portugal), using standard cultural practices, was selected. It is a red onion known as “vermelha da Póvoa”. The onions were variable in size but with an average weight of 66.5 g and brown-red colour. 2.2. Sample randomization for fresh-cut onions and chemical disinfectant treatments Four types of chemical disinfectant were assayed (Sodium hypochlorite, Amukine (commercial product), hydrogen peroxide and sodium dichloroisocyanate). For each of the treatments realized with these disinfectants, 5 raw onions of average weight were selected. The reason for selecting onion bulbs with an average weight is to reduce variability in the measurements of flavonoids in onions. Since flavonoids in onions are not homogenously distributed in bulb scales, each bulb was split-up in quarters, and every quarter was divided into slices ranging from the center to the outer side and from top to bottom. The slices had an approximate thickness of 2.5 mm with a weight between 1.5 and 2.5 g depending on bulb size. To take an onion representative sample (Fig. 1), for each chemical treatment, 10 g were taken from the 5 different raw onions to have a total of 50 g. This operation was repeated 3 times. One 50 g portion was used for monitoring flavonoids directly fresh sample another portion (50 g) was submitted to a treatment of chemical disinfectant, and the latter was washed with distilled
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water, without disinfectant, in the same time-temperature conditions followed for any of the disinfectant procedures (control sample). Before analysis, each of the onion slices sets (50 g) was homogenized at 1200 rpm for 3 min, and splitted into three identical fractions of 15 g (n ¼ 3) to monitoring flavonoids. In general, all treatments were normalized by immersing the slices portions (50 g) under the chemical aqueous solution at a ratio of 8e10 L/kg (400 mL) in the established conditions of timetemperature. Afterwards, distilled water was used to wash-off all remaining chemicals residues in the bulb tissue. So the slices portions (50 g) were washed with 400 mL of water (4 C) during 1 min. In all disinfection treatments tested, not only the onion tissue was analyzed, but also the water used for disinfection. The conditions of chemical treatments, below described, were selected by literature (Adams, Hartley, & Cox, 1989; Baur, Klaiber, Wei, Hammes, & Carle, 2005; Beerli et al., 2004) to keep the onions in proper edible conditions for 7 days after processing. 2.2.1. Sodium hypochlorite Water containing 50e200 mg/L of chlorine with a contact time of 1e2 min is widely used to sanitize whole vegetables as well as fresh-cut produce on a commercial scale. In this assay, different concentrations of sodium hypochlorite were used (50 and 300 ppm) at a pH of 4.5e5.0 set-up with 2 acids, the organic citric acid (0.25 M) and the inorganic sulphuric acid (1 N). The concentrations of acid employed to adjust the pH were 0.5% and 3% for sulphuric acid, and 1% and 5% for citric acid, when hypochlorite concentrations were 50 and 300 ppm, respectively. With the purpose of finding differences between concentrations, a relatively high temperature of 50 C for 2 min was selected. For comparison with other decontaminant agents, a middle concentration of sodium hypochlorite (100 ppm) was selected, washing the onion samples in cold water (4 C) for 5 min (usual conditions). 2.2.2. Amukine Amukine (Angelini Farmacéutica, S.A.; Barcelona; Spain) is a commercial liquid disinfectant (1.15 g NaOCl in 100 mL) for fruit and vegetables. Following manufacture recommendations, 50 mL Amukine were poured in 2.5 L water, and the onion slices (50 g) were then immersed in the Amukine diluted solution (400 mL) for 5 min at 4 C. 2.2.3. Hydrogen peroxide Onion slices (50 g) were immersed in 400 mL of an aqueous solution of H2O2 (6%) for 5 min at 4 C. 2.2.4. Sodium dichloroisocyanate Onion slices (50 g) were immersed in 400 mL of an aqueous solution of C3Cl2N3NaO3$2H2O (100 ppm; 60% NaOCl) for 5 min at 4 C.
2.3. Sample randomization for fresh cut onions disinfected by UV-C irradiation
Fig. 1. Sampling procedure for comparison of representative treated and untreated subsamples.
Five bulbs were selected and cut into 2.5 mm slices. Two sets of 50 g each were obtained. Before analysis, one of the sets was irradiated with UV light. A dose in the range from 0.5 to 20 kJ/m2 leads to lethality by directly altering microbial DNA through dimmer formation. In this way, three replicates of 15 g onion slices were placed on a closed platform and were irradiated with 10 kJ/m2 at 4 C (UV lamp, wavelength at 254 nm, 2 tubes at 4 W; Afora-FVL-4LC, Afora, Barcelona, Spain). The lamp was placed 10 cm above the fresh-cut onion. Samples were rotated two times during the treatment to ensure uniform exposure. The other set (50 g) was used as control: three replicates of 15 g onion slices were kept at 4 C before analysis.
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2.4. Flavonoids extraction and determination by HPLC-DAD
The visual quality assessment included discoloration and glassy texture defects in the minimally processed onions.
The analytical treatment, which gave recovery figures higher than 92% with relative standard deviations lower than 4%, was based on a procedure previously reported by the authors (PérezGregorio et al., 2010; Pérez-Gregorio, García-Falcón, & SimalGándara, 2011; Pérez-Gregorio, Regueiro, González-Barreiro, Rial-Otero, & Simal-Gándara, 2011; Rodrigues et al., 2010 and 2011). A representative sample of edible portion (15 g) was homogenized and extracted by shacking with methanol:formic acid:water (MFW; 50:5:45; v:v:v) and stabilized with 2 g L1 of tertbutylhydroquinone. 50 mL of the final extract were then injected into the HPLC/DAD system (Thermo Separation Products). Quantification of single flavonols and anthocyanins was achieved by calibration curves obtained using pure quercetin and cyanidin 3-glucoside as standards, respectively. Results were then expressed in fresh weight (FW) onion, always referred to the weight at the starting of the experiment (about 15 g) with the intention to account result variations affected by the treatments. The main classes of flavonoids in this kind of onions are 11(Pérez-Gregorio et al., 2010; Pérez-Gregorio, García-Falcón & Simal-Gándara, 2011; Pérez-Gregorio, Regueiro, GonzálezBarreiro, Rial-Otero, & Simal-Gándara, 2011; Rodrigues et al., 2010 and 2011):
2.6. Statistical analysis Results are the means standard deviations from three replicates of independent experiments of the same treatment. Data were subjected to one-way analysis of variance with treatment as fixed factor. Whenever significant differences were detected at the 0.05 level, the means were separated by Duncan’s multiple range test in groups coded by a, b and c. All analyses were performed with the Statgraphics statistical software. 3. Results and discussion 3.1. Effect of concentration of sodium hypochlorite and temperature on onion flavonoids content Sodium hypochlorite is a powerful decontaminant with oxidizing properties very much used in the food industry, but it is necessary to take into consideration that its efficiency is pH-dependent. According to Adams et al. (1989), adjusting the pH of hypochlorite solutions from 9 to 4.5e5.0 with inorganic or organic acids produced a 1.5e4.0 fold increase in the disinfectant effect. Fresh-cut vegetables are usually washed in cold (4 C) chlorinated water before packaging. Hot water treatments are currently under investigation as a mean to reduce the extent of microbial contamination and improve quality retention in fruits and vegetables. There are many authors who reported the benefits of reduced microbial load and browning after brief dipping into water at 50 C due to a reduction in the activity of phenylalanine ammonia lyase (PAL) on minimally processed vegetables (Delaquis, Stewart, Toivonen & Moyls, 1999; Delaquis, Stewart, Cliff, Toivonen, & Moyls, 2000; and Fukumoto, Toivonen & Delaquis, 2002). In our work, changes in flavonoid content before washing at 50 C with sodium hypochlorite were assessed. Due to the hydrosolubility of flavonols there will have potential losses by migration into water. This was the reason for a control with just water: to distinguish between solubilization into water and oxidation of flavonols by the disinfectant agent selected. Flavonols and anthocyanins in onions slices were found to leach to water a 23% and 13%, respectively, at 50 C (Table 1). In our assay, no preferential leaching of any flavonol conjugate into the water occurred (24 and 23% for Q3,4dg and Q4g, respectively). Rodrigues, Pérez-Gregorio, GarcíaFalcón, and Simal-Gándara (2009) verified that boiling onions (100 C for 30 min) leaded to losses of quercetin glycosides, which
- Flavonols: two major (quercetin 3,4-diglucoside (Q3,4dg) and quercetin 4-glucoside (Q4g)) and five minor (quercetin 7,4diglucoside, isorhamnetin 3,4-diglucoside, quercetin 3glucoside, isorhamnetin 4-glucoside, and quercetin aglycon (Q)) - Anthocyanins (four): cyanidin 3-glucoside (C3g), cyanidin 3laminaribioside (C3lmb), cyanidin 3-(6”-malonylglucoside) (C3,6 mg) and cyanidin 3-(600 - malonyl-laminaribioside) (C3,6mlmb). It is important to note that the levels of these pigments in onions are much lower than those reached by the flavonols.
2.5. Quality evaluations Water loss assessment within each treatment was conducted by measuring the water volume at the end of the storage period due to mucilage exudation from the onion tissues, or the water condensation in the inner walls of the plastic cups. Visual appearance was determined based on the following subjective score on a 9-1 scale, with reference points of: 9, excellent; 7, good; 5, fair; 3, poor; and 1, unusable. A score of 6 was regarded as the limit of marketability.
Table 1 Losses % of flavonoids in onions by washing with water (control). (a) Flavonols. (b) Anthocyanins. (a) Q3,4dg
4 C
50 C
Fresh Control Fresh Control
Q4g
Q
[mg Q/kg FW]
%Change
[mg Q/kg FW]
142.6 18.1 119.1 8.1 117.1 8.5 89.0 9.1
Y16
191.6 160.8 126.4 96.8
Y24
25.1 11.4 6.3 13.6
Others
%Change
[mg Q/kg FW]
Y16
3.6 2.5 3.9 4.1
Y23
0.6 0.2 1.7 1.5
Total
%Change
[mg Q/kg FW]
Y29
23.0 17.6 29.4 21.4
[5
2.6 0.5 1.6 3.0
%Change
[mg Q/kg FW]
Y24
360.8 300.1 278.7 215.6
Y27
1.6 0.1 17.6 27.7
%Change Y17 Y23
(b) C3g [mg C3g/kg FW] 4 C 50 C
Fresh Control Fresh Control
1.3 0.9 0.7 0.4
C3,600 mg
C3lmb
0.2 0.0 0.2 0.2
%Change Y27 Y35
[mg C3g/kg FW] 0.54 0.3 0.4 0.4
0.1 0.0 0.1 0.1
%Change
[mg C3g/kg FW]
Y28
1.6 1.1 0.9 0.8
[10
0.2 0.0 0.1 0.1
C3,600 mlmb %Change
[mg C3g/kg FW]
Y33
0.9 0.7 0.6 0.6
Y15
0.1 0.0 0.1 0.1
Total %Change
[mg C3g/kg FW]
Y27
4.2 3.0 2.6 2.2
Y2
0.6 0.1 0.3 0.5
%Change Y29 Y13
*following manufacture recommendations on concentration (230 ppm). WWO, water-washed onion; CS, commercial solution (Amukine); HP, hydrogen peroxide; DS, dichloroisocyanuric sodium salt; SH, sodium hypochlorite; SA, sulphuric acid; CA, citric acid. aec Significantly different groups were coded with different letters.
[29 5.48 0.60 [24 3.08 0.13 [ 33 2.33 0.04 [35 486.0 56.1 [32 [42 202.4 50.6
252.9 73.3
e 4.23 0.64 e 2.49 0.09 e 1.75 0.07 e 360.8 46.4 e 191.6 25.1 e 142.6 18.1
Fresh onion (Control) UV-C radiation (254 nm 60 min) (c) UV-C physical treatment Physical Treatment 10 kJ/m2)
e Y2a Y6a Y2a Y0a Y3a
1.3 0.1 1.2 0.1 1.3 0.0 1.2 0.1 0.9 0.1 1.5 0.0
e Y2 [2 Y4 Y24 [19
1.7 0.0 1.6 0.0 1.7 0.1 1.7 0.2 1.2 0.1 1.8 0.1
e Y2 Y2 Y1 Y30 [3
3.0 0.1 2.9 0.1 3.0 0.1 2.9 0.2 2.2 0.1 3.3 0.1
e Y2 Y0 Y2 Y28 [10
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(b) Comparison of different chemical treatments at conventional disinfection conditions (100 ppm-4 C-5 min) Control (WWO) 119.1 8.1 e 160.8 11.4 e 300.1 19.9 154.4 21.6 Y4a 295.4 28.3 SH CA 119.1 14.3 Y0a a a 152.8 19.8 Y5 282.7 35.2 SA 112.0 11.1 Y6 DS 117.9 8.3 Y1a 156.0 10.9 Y3a 293.5 15.0 160.8 6.4 Y0a 300.1 20.9 HP 119.1 3.6 Y0a 155.5 2.8 Y3a 291.3 24.7 CS* 116.3 1.2 Y2a
e Y12 Y8 [2 Y25 0.4 0.1 0.3 0.2 0.1 2.2 1.9 2.0 1.7 2.3 e Y12 Y8 [2 Y21 1.4 0.16 1.2 0.1 1.2 1.4 1.1 e Y12 Y8 [3 Y18 0.1 0.0 0.0 0.1 0.0 0.87 0.7 0.8 0.9 0.7 e Y7a Y4a Y7a Y19c 27.7 16.0 22.8 19.4 11.4 215.6 200.6 207.2 201.1 173.7 e Y10b Y7a,b Y3a Y 20c
[mg C3 g/kg FW] %Change
Total
[mg Q/kg FW] %Change %Change
acids (50 C-2 min) e 96.8 13.6 Y4a 94.2 3.5 a 77.5 10.8 Y1 86.9 12.2 Y10b 89.9 5.7 Y17c
Total
[mg C3 g/kg FW] %Change
Acylated
[mg Q/kg FW] [mg Q/kg FW]
[mg C3 g/kg FW]
Glycosilated Q4g Q3,4dg
%Change
Major Flavonoids (Anthocyanins)
(a) Treatments with sodium hypochlorite and different Control (WWO) 89.0 9.1 SH CA 50 ppm 85.1 3.4 300 ppm 87.9 9.7 SA 50 ppm 80.1 6.3 300 ppm 73.9 3.6
Chlorine is a common efficient disinfectant agent but there is the risk of undesirable by-products upon reaction with organic matter and this may lead to new regulatory restrictions in the future (Artés et al., 2009; Ölmez & Kretzschmar, 2009). Because of this, it is important to assay other disinfectants such as hydrogen peroxide or sodium dichloroisocyanure. It is well known that hydrogen peroxide is a strong bactericide agent (affecting also to spores) by the generation of oxidant cytotoxic species such as hydroxyl radicals (Khadre & Yousef, 2001). It has been proved its efficiency increasing the shelf-life of convenience vegetables (Artés
Major Flavonoids (Flavonols)
3.2. Comparison of the different disinfectant treatments at usual operation conditions
Table 2 Losses % of flavonoids in onions by different disinfectant treatments.
leached to the boiling water without being degraded, at 37% and 29% for Q3,4dg and Q4g, respectively. Makris and Rossiter (2000) and Rodrigues et al. (2009) also found that leaching of the diglucoside derivative was favored. Flavonols content was affected by the acid used to fix the pH of the sanitation solution (Fig. 2). Because of the powerful oxidizing capacity of sulphuric acid, when this acid was used to adjust the pH more flavonol losses occurred. Flavonol content did not significantly change with the wash treatment when citric acid was used for the acidification of the sanitization solution. This treatment produces losses of overall a 4 % and 7 % at 50 ppm and 300 ppm of sodium hypochlorite, respectively, compared with a distilled water washing control. When the acid used to fix the pH of the sanitation solution was sulphuric acid, losses of 7 and 19% at 50 and 300 ppm of sodium hypochlorite, respectively, were produced (Table 2a). Results in Table 2a also suggest that Q4g is more susceptible to oxidation than Q3,4dg. The difference in the stability is, respectively, attributed to the presence and absence of the hydroxyl group at the C-3 position. The faster oxidation of Q4g, which is catalyzed by onion peroxidize, indicates that the hydroxyl group of the C-3 position is attacked by the enzyme (Hirota, Shimoda, & Takahama, 1998). Anthocyanins also experienced losses of 0%e25% at 50 and 300 ppm when sulphuric acid was used for the acidification and about 10% when citric acid was used (Table 2a). We found no articles comparing the different oxidizing capacity of acids used to set pH. It was previously shown the effect of the presence of hypochlorite in wash water, but without any specification of the acid agent used. Choi and Sapers (1994) studied the effects of washing with sodium hypochlorite on the phenolics compounds of mushrooms; they found that washing with 50 ppm of sodium hypochlorite at room temperature caused the disappearance of phenolics and the formation of their oxidation products. Instead, Klaiber, Baur, Wolf, Hammes, and Carle (2005) found that the use of chlorinated water (100 mg/L) to wash shredded carrots had negligible influence on the phenolics content. Martínez et al. (2005) reported the impact of minimal processing on onion quercetin. They did not find any difference between washing the onion with tap water or with sodium hypochlorite (100 ppm, 1 min). On the other hand, Odriozola-Serrano, Soliva-Fortuny, and Marti cn-Belloso (2008) investigated the effect of minimal processing on bioactive compounds and colour attributes of fresh-cut tomatoes. This study shows than phenolic content was not significantly affected by sanitization for 2 min in chlorinated water (0.2 mg free chlorine/L) at 4 C. Other authors (Hirota et al., 1998) establish that when scales of onion were boiled in tap water, Q4g degraded rapidly. It is known that tap water contains Feþ3 released from steel pipe and chlorine added for sterilization. Therefore, they examined the effects of these oxidants on the degradation of flavonols during boiling, and found that neither chlorine nor Feþ3 is important to enhance the degradation of flavonols.
%Change
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A
B
Fig. 2. Effect of acid and hypochlorite concentration on the levels of the main flavonoids in onion slices (A) flavonols; B) anthocyanins) after a treatment at 50 C for 2 min, a to c: significantly different groups were coded with different letters.
et al., 2009; Beerli et al., 2004; Ölmez & Kretzschmar, 2009). It is a GRAS (Generally Recognized As Safe) substance; in USA, however, the CFR (the Code of Federal Regulation) limits the concentration to be used in some products as bleaching or antimicrobial agent (Ölmez & Kretzschmar, 2009). As previously indicated, sodium dichloroisocyanurate, potassium dichloroisocyanurate, dichloroisocyanuric acid and trichloroisocyanuric acid are other common chlorine-based disinfectants. Most reports indicate that these compounds are somewhat more effective, albeit slower acting, than hypochlorites (Beerli et al., 2004). In our work, different disinfectants were selected and their effect was assayed on onion flavonoid content under the same conditions (100 ppm - 4 C for 5 min). These sanitation conditions were selected as the most usual conditions (Beerli et al., 2004). According to Fig. 3, it can be confirmed that there are no significant losses of flavonols by oxidation effects due to the decontaminant agent and that the only decrease of their levels is by solubilisation into water (Table 1, and Fig. 3). The 29% of anthocyanins have moved into water versus the 17% of flavonols. As shown in Table 1 the percentage of flavonols migration to water is greater in more
drastic conditions. Therefore, the migration at 50 C for 2 min of immersion is higher than at 4 C for 5 min of exposure (23% vs. 17%). Most losses were produced in flavonols when the acid employed to keep pH around 6.0 in the sodium hypochlorite solution was the sulphuric acid (6%). If we focus in anthocyanins treated with sodium hypochlorite and sulphuric acid we can observe that the total content of anthocyanins is unaffected by the treatment. Dichloroisocyanuric acid sodium salt treatment, instead, showed no effect in anthocyanins. Most losses in anthocyanins (28%) were produced with the hydrogen peroxide treatment, may be due to their blanching effect. 3.3. Effect of UV-C treatment on onion flavonoids content The damage inflicted by UV-C probably involves specific target molecules and a dose in the range from 0.5 to 20 kJ/m2 leads to lethality by directly altering microbial DNA through dimmer formation. In addition, no known toxic or significant non-toxic byproducts are formed during the UV-C treatment (Keyser et al., 2008).
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A
B
Fig. 3. Comparison of the effects of different disinfectant systems on the levels of the main flavonoids in onion slices A) flavonols; B) anthocyanins. SH-Sodium Hypochlorite; CACitric Acid; SA- Sulphuric Acid; HP-Hydrogen peroxide; DS-Dichloroisocyanuric Acid Sodium Salt; CS-Commercial Solution; a to c: significantly different groups were coded with different letters.
In our work, when the treatment used was the UV-C radiation, flavonol increased at 35%, and anthocyanins increased at 29%. Quercetin diglucosides increase much more than glucosides (42% vs. 32%) and glycosilated anthocyanins than acylated forms (33% vs. 24%) (Table 2c). Post harvest irradiation with UV light has been proposed as an appropriate method to increase the health value of different fruits and vegetables by inducing phenylpropanoid metabolism (Higashio, Hirokane, Sato, & Tokuda, 2005; Hagen et al., 2007; Mahdavian, Ghorbanli, & Kalantari, 2008). Hagen et al. (2007) found that posthaverst irradiation with VisþUV-B treatment enhanced the sum of flavonoids and total phenols in the peel of shade-grown apples. Mahdavian et al. (2008) also verified that contents of flavonoids, rutin and UV-absorbing compounds of pepper plants increased rapidly in response to UV-B and UV-C radiation. Higashio et al. (2005) verified that the irradiation of onion slices increased the quercetin content, reaching the duplication of their levels. The best visual quality results were obtained when sliced onions were treated with UV-C irradiation (score 8, excellent/good, in a 9-1 scale), but also a good score was obtained
for any of the chemical disinfectant treatments (score 7, good, in a 9-1 scale). 4. Conclusions This paper addresses some recent results obtained with alternative sanitizers on fresh-cut onion commodities. The main effect contributing to the loss of flavonols in fresh-cut onion slices is their solubility in immersion water (17e23% both at 4 or 50 C). There are no any further losses by oxidation due to the use of decontaminant chemical agents such as sodium hypochlorite, amukine, and hydrogen peroxide or sodium dichloroisocyanate systems. Only in the case of high levels of sodium hypochlorite (300 ppm) treated with sulphuric acid at temperatures of 50 C there is a clear trend for oxidation losses of the main flavonols (at a 15%). It is for such a reason that, in the case of sodium hypochlorite, concentrations of 100 ppm are recommended together with the use of organic acids for acidification purposes at refrigeration temperatures (4 C). Most losses occur for anthocyanins when the treatment employed was
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that based on hydrogen peroxide (28%). Anyway, in-dry decontaminant treatments like UV-C irradiation the natural levels of flavonoids in fresh-cut onion slices increase, being then superior to any of the chemically-based procedures. The combination of well designed integrated production, handling, processing and distribution chains for fresh-cut produces is crucial for achieving the high quality and safety demanded by consumers. References Adams, M., Hartley, A., & Cox, L. (1989). Factors affecting the efficacy of washing procedures used in the production of prepared salads. Food Microbiology, 6, 69e77. Alén-Ruiz, F., Pérez-Gregorio, M. R., Martínez-Carballo, E., García-Falcón, M. S., & Simal-Gándara, J. (2008). Influence of polyphenols on colour and antioxidant value in plant foods. Electronic Journal of Environmental, Agricultural and Food Chemistry, 7(8), 3171e3176. Artés, F., Gómez, P., Aguayo, E., Escalona, V., & Artés-Hernández, F. (2009). Sustainable sanitation techniques for keeping quality and safety of fresh-cut plant commodities. Postharvest Biology and Technology, 51, 287e296. Baur, S., Klaiber, R., Wei, H., Hammes, W. P., & Carle, R. (2005). Effect of temperature and chlorination of pre-washing water on shelf-life and physiological properties of ready-to-use iceberg lettuce. Innovative Food Science and Emerging Technologies, 6(2), 171e182. Beerli, K. M. C., Vilas Boas, E. V.de B., & Piccoli, R. H. (2004). Effect of sanitizer on the microbiol, physical and physical-chemical characteristics of fresh-cut onions (Allium cepa L.).Ciência e Agrotecnología. Lavras, 28, 107e112. Choi, S. W., & Sapers, G. M. (1994). Effects of washing on polyphenols and polyphenol oxidase in commercial mushrooms (Agaricus bisporus). Journal of Agricultural and Food Chemistry, 42(10), 2286e2290. Delaquis, P. J., Stewart, S., Cliff, M., Toivonen, P. M., & Moyls, A. L. (2000). Sensory quality of ready-to-eat lettuce washed in warm, chlorinated water. Journal of Food Quality, 23, 553e563. Delaquis, P. J., Stewart, S., Toivonen, P. M. A., & Moyls, A. L. (1999). Effect of warm, chlorinated water on the microbial flora of shredded iceberg lettuce. Food Research International, 32, 7e14. Fukumoto, L. R., Toivonen Peter, M. A., & Delaquis Pascal, J. (2002). Effect of wash water temperature and chlorination on phenolic metabolism and browning of stored iceberg lettuce photosyntetic and vascular tissues. Journal of Agricultural and Food Chemistry, 50(16), 4503e4511. Hagen, S. F., Borge, G. I. A., Bengtsson, G. B., Bilger, W., Berge, A., Haffner, K., et al. (2007). Phenolic contents and other health and sensory related properties of apple fruit (Malus domestica Borkh., cv. Aroma): effect of postharvest UV-B irradiation. Postharvest Biology and Technology, 45(1), 1e10. Higashio, H., Hirokane, H., Sato, F., & Tokuda, S. (2005). Uragami A Effect of UV irradiation after the harvest on the content of flavonoid in vegetables. Acta Horticulture, 682, 1007e1012. Hirota, S., Shimoda, T., & Takahama, U. (1998). Tissue and spatial distribution of flavonol and peroxidase in onion bulbs and stablilty of flavonol glucosides
during boiling of the scales. Journal of Agricultural and Food Chemistry, 46, 3497e3502. } ller, I. A., Cilliers, F. P., Nel, W., & Gouws, P. (2008). AUltraviolet Keyser, M., Mu radiation as a non-thermal treatment for the inactivation of microorganisms in fruit juice. Innovative Food Science and Emerging Technologies, 9, 348e354. Khadre, M. A., & Yousef, A. E. (2001). Sporicidal action of ozone and hydrogen peroxide: a comparative study. International Journal of Food Microbiology, 71, 131e138. Klaiber, R. G., Baur, S., Wolf, G., Hammes, W. P., & Carle, R. (2005). Quality of minimally processed carrots as affected by warm water washing and chlorination. Innovative Food Science and Emerging Technologies, 6(3), 351e362. Mahdavian, K., Ghorbanli, Kh, & Kalantari, M. (2008). The effects of Ultraviolet radiation on the contents of Chlorophyll, flavonoid, anthocyanin and Proline in Capsicum annuum L. Turkish Journal of Botany, 32, 25e33. Makris, D. P., & Rossiter, J. T. (2000). Heat - induced, metal e catalyzed oxidative degradation of quercetin and rutin (quercetin 3-o-rhamnosylglucoside) in aqueous model systems. Journal of Agricultural and Food Chemistry, 48(9), 3830e3838. Martínez, J. A., Sgroppo, S., Sánchez-Moreno, C., De Ancos, B., & Cano, M. P. (2005). Effects of processing and storage of fresh-cut onion on quercetin. In F. Mencarelli, & P. Tonutti (Eds.), Proceedings of the Fifth International Postharvest Symposium. Acta Horticulturae, Vol. 682. ISHS. Ölmez, H., & Kretzschmar, U. (2009). Potential alternative disinfection methods for organic fresh-cut industry for minimizing water consumption and environmental impact. Journal of Food Science and Technology, 42, 686e693. Odriozola-Serrano, I., Soliva-Fortuny, R., & Marti cn-Belloso, O. (2008). Effect of minimal processing on bioactive compounds and color attributes of fresh-cut tomatoes. Journal of Food Science and Technology, 41(2), 217e226. Pérez-Gregorio, R. M., García-Falcón, M. S., & Simal-Gándara, J. (2011). Flavonoids changes in fresh-cut onions during storage in different packaging systems. Food Chemistry, 124(2), 652e658. Pérez-Gregorio, R. M., García-Falcón, M. S., Simal-Gándara, J., Rodrigues, A. S., & Almeida, D. P. F. (2010). Identification and quantification of flavonoids in traditional cultivars of red and white onions at Harvest. Journal of Food Composition and Analysis, 23, 592e598. Pérez-Gregorio, R. M., Regueiro, J., Alonso-González, E., Pastrana-Castro, L. M., & Simal-Gándara, J. (2011). Influence of alcoholic fermentation process on antioxidant activity and phenolic levels from mulberries (Morus nigra L.). LWT e Food Science and Technology, 44, 1793e1801. Pérez-Gregorio, R. M., Regueiro, J., González-Barreiro, C., Rial-Otero, R., & SimalGándara, J. (2011). Changes in antioxidant flavonoids during freeze-drying of red onions and subsequent storage. Food Control, 22, 1108e1113. Rodrigues, A. S., Pérez-Gregorio, M. R., García-Falcón, M. S., & Simal-Gándara, J. (2009). Effect of curing and cooking on flavonols and anthocyanins in traditional varieties of onion bulbs. Food Research International, 42(9), 1331e1336. Rodrigues, A. S., Pérez-Gregorio, R. M., García-Falcón, M. S., Simal-Gándara, J., & Almeida, D. P. F. (2011). Effect of meteorological conditions on antioxidant flavonoids in Portuguese cultivars of white and red onions. Food Chemistry, 124(1), 303e308. Rodrigues, A. S., Pérez-Gregorio, M. R., García-Falcón, M. S., Simal-Gándara, J., & Almeida, D. P. F. (2010). Effect of post-harvest practices on flavonoid content of red and white onion cultivars. Food Control, 21(6), 878e884.
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Comparison of sanitizing technologies on the quality appearance and antioxidant levels in onion slices M.R. Pérez-Gregorio, C. González-Barreiro, R. Rial-Otero, J. Simal-Gándara* Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, Ourense Campus, University of Vigo, E-32004 Ourense, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2010 Received in revised form 25 May 2011 Accepted 31 May 2011
Background: A Portuguese landrace onion variety grown in Póvoa do Varzim (Northwest of Portugal) was selected for this study. It is a red onion known as “vermelha da Póvoa”. The effect of different chemicallyand irradiation-based disinfectant procedures of fresh-cut onions on their flavonoid content was compared. Amongst the chemical treatments the most used were selected: sodium hypochlorite, amukine, hydrogen peroxide, and sodium dichloroisocyanate. UV-C irradiation was also selected amongst all irradiation treatments. Results: The main effect contributing to the loss of flavonols in fresh-cut onion slices is their solubility in immersion water (17e23% both at 4 or 50 C). The acidification of sodium hypochlorite with sulphuric acid is also rendering losses (6e19%) vs. the use of citric acid. Anyway, in dry decontaminantion treatments, like UV-C irradiation, the natural levels of flavonoids in fresh-cut onion slices significantly increase at 35% for flavonols, and at 29% for anthocyanins. Conclusions: Onion flavonoids decreased by chemically disinfectant procedures of fresh-cut onion by solubility in immersion water. UV-C irradiation does not only maintain the initial levels of flavonoids but increases them; therefore, this is the recommended treatment. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Anthocyanins Chemical disinfectants UV-C irradiation Flavonols Fresh-cut onion slices
1. Introduction According to the United Fresh Produce Association, minimally processed vegetables are defined as any fruit or vegetable that has been altered physically from its original, but retains its status fresh. Because of consumer interest in the health benefits, convenience and value-added qualities of fresh produce, there is a growing need for convenience vegetables such as fresh-cut and ready-to-eat salads. It is then required that these products have optima hygienic, sensorial and nutritional properties. The most used systems for disinfectant are chemical and irradiation-based treatments. Generally, the chlorine-containing compounds group is considered first by those searching for antimicrobial activity, for being inexpensive and easy to use, and for have a broad spectrum of activity (Choi & Spacers, 1994; Delaquis, Stewart, Toivonen, & Moyls, 1999; Delaquis, Stewart, Cliff, Toivonen, & Moyls, 2000; Martínez, Sgroppo, Sánchez-Moreno, De Ancos, & Cano, 2005; Artés, Gómez, Aguayo, Escalona, & ArtésHernández, 2009). Hypochlorites are the most frequently used forms of chlorine, with sodium hypochlorite receiving broadest
* Corresponding author. E-mail address: [email protected] (J. Simal-Gándara). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.05.028
application. Chlorine can oxidize organic matter in foods or in water, and in the latter case by-products such haloforms and haloacetic acids, which are potentially carcinogenic and mutagenic, can be formed. In some European countries, sodium hypochlorite is not allowed for use in fresh-cut vegetables and might be possible that the UE banned its use by law for such applications (Ölmez & Kretzschmar, 2009). Thus, there is a need for alternative sanitizers to be used for the disinfection of fresh-cut vegetables. Hydrogen peroxide (Beerli, Vilas Boas, & Piccoli, 2004; Khadre & Yousef, 2001) or organic chlorinated products (sodium dichloroisocyanurate, potassium dichloroisocyanurate, dichloroisocyanuric acid and trichloroisocyanuric acid) are alternative sanitizing agents that gained interest in recent years (Beerli et al., 2004). Among the chemical methods for controlling post harvest diseases, UV-C irradiation appears to be one of the most attractive. UV radiation in the range of 250e260 nm is lethal to most microorganisms, including bacteria, viruses, protozoa, mycelial fungi, yeasts and algae. There are many publications with studies about the bactericide effects of different disinfectant agents, but only a few on potential losses of bioactive components such as polyphenols of fresh-cut fruits and vegetables (Fukumoto, Toivonen, & Delaquis, 2002). Onions are considered as a source of antioxidants including flavonols (quercetin derivatives) and also anthocyanins in the case of red bulbs (PérezGregorio, García-Falcón, Simal-Gándara, Rodrigues, & Almeida,
M.R. Pérez-Gregorio et al. / Food Control 22 (2011) 2052e2058
2010; Pérez-Gregorio, García-Falcón, & Simal-Gándara, 2011; PérezGregorio, Regueiro, González-Barreiro, Rial-Otero, & Simal-Gándara, 2011; Rodrigues, Pérez-Gregorio, García-Falcón, Simal-Gándara, & Almeida, 2010 and 2011), but also mulberry fruits (Pérez-Gregorio, Regueiro, Alonso-González, Pastrana-Castro, & Simal-Gándara, 2011) and plant foods in general (Alén-Ruiz, Pérez-Gregorio, Martínez-Carballo, García-Falcón, & Simal-Gándara, 2008). During disinfection treatments of the bulbs, important chemical and biochemical reactions occur in onion tissue. Such reactions may have an impact on the flavonoids structure, resulting in changes of the bioavailability and activity of these compounds. The present work was undertaken in order to clarify the impact of regular disinfection treatments (sodium hypochlorite, or amukine, hydrogen peroxide, sodium dichloroisocyanate and UV-C irradiation) on the flavonols and anthocyanins content of onions, as well as the role of structural features on their degradation. 2. Experimental 2.1. Raw material A Portuguese landrace onion variety grown in Póvoa do Varzim (Northwest of Portugal), using standard cultural practices, was selected. It is a red onion known as “vermelha da Póvoa”. The onions were variable in size but with an average weight of 66.5 g and brown-red colour. 2.2. Sample randomization for fresh-cut onions and chemical disinfectant treatments Four types of chemical disinfectant were assayed (Sodium hypochlorite, Amukine (commercial product), hydrogen peroxide and sodium dichloroisocyanate). For each of the treatments realized with these disinfectants, 5 raw onions of average weight were selected. The reason for selecting onion bulbs with an average weight is to reduce variability in the measurements of flavonoids in onions. Since flavonoids in onions are not homogenously distributed in bulb scales, each bulb was split-up in quarters, and every quarter was divided into slices ranging from the center to the outer side and from top to bottom. The slices had an approximate thickness of 2.5 mm with a weight between 1.5 and 2.5 g depending on bulb size. To take an onion representative sample (Fig. 1), for each chemical treatment, 10 g were taken from the 5 different raw onions to have a total of 50 g. This operation was repeated 3 times. One 50 g portion was used for monitoring flavonoids directly fresh sample another portion (50 g) was submitted to a treatment of chemical disinfectant, and the latter was washed with distilled
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water, without disinfectant, in the same time-temperature conditions followed for any of the disinfectant procedures (control sample). Before analysis, each of the onion slices sets (50 g) was homogenized at 1200 rpm for 3 min, and splitted into three identical fractions of 15 g (n ¼ 3) to monitoring flavonoids. In general, all treatments were normalized by immersing the slices portions (50 g) under the chemical aqueous solution at a ratio of 8e10 L/kg (400 mL) in the established conditions of timetemperature. Afterwards, distilled water was used to wash-off all remaining chemicals residues in the bulb tissue. So the slices portions (50 g) were washed with 400 mL of water (4 C) during 1 min. In all disinfection treatments tested, not only the onion tissue was analyzed, but also the water used for disinfection. The conditions of chemical treatments, below described, were selected by literature (Adams, Hartley, & Cox, 1989; Baur, Klaiber, Wei, Hammes, & Carle, 2005; Beerli et al., 2004) to keep the onions in proper edible conditions for 7 days after processing. 2.2.1. Sodium hypochlorite Water containing 50e200 mg/L of chlorine with a contact time of 1e2 min is widely used to sanitize whole vegetables as well as fresh-cut produce on a commercial scale. In this assay, different concentrations of sodium hypochlorite were used (50 and 300 ppm) at a pH of 4.5e5.0 set-up with 2 acids, the organic citric acid (0.25 M) and the inorganic sulphuric acid (1 N). The concentrations of acid employed to adjust the pH were 0.5% and 3% for sulphuric acid, and 1% and 5% for citric acid, when hypochlorite concentrations were 50 and 300 ppm, respectively. With the purpose of finding differences between concentrations, a relatively high temperature of 50 C for 2 min was selected. For comparison with other decontaminant agents, a middle concentration of sodium hypochlorite (100 ppm) was selected, washing the onion samples in cold water (4 C) for 5 min (usual conditions). 2.2.2. Amukine Amukine (Angelini Farmacéutica, S.A.; Barcelona; Spain) is a commercial liquid disinfectant (1.15 g NaOCl in 100 mL) for fruit and vegetables. Following manufacture recommendations, 50 mL Amukine were poured in 2.5 L water, and the onion slices (50 g) were then immersed in the Amukine diluted solution (400 mL) for 5 min at 4 C. 2.2.3. Hydrogen peroxide Onion slices (50 g) were immersed in 400 mL of an aqueous solution of H2O2 (6%) for 5 min at 4 C. 2.2.4. Sodium dichloroisocyanate Onion slices (50 g) were immersed in 400 mL of an aqueous solution of C3Cl2N3NaO3$2H2O (100 ppm; 60% NaOCl) for 5 min at 4 C.
2.3. Sample randomization for fresh cut onions disinfected by UV-C irradiation
Fig. 1. Sampling procedure for comparison of representative treated and untreated subsamples.
Five bulbs were selected and cut into 2.5 mm slices. Two sets of 50 g each were obtained. Before analysis, one of the sets was irradiated with UV light. A dose in the range from 0.5 to 20 kJ/m2 leads to lethality by directly altering microbial DNA through dimmer formation. In this way, three replicates of 15 g onion slices were placed on a closed platform and were irradiated with 10 kJ/m2 at 4 C (UV lamp, wavelength at 254 nm, 2 tubes at 4 W; Afora-FVL-4LC, Afora, Barcelona, Spain). The lamp was placed 10 cm above the fresh-cut onion. Samples were rotated two times during the treatment to ensure uniform exposure. The other set (50 g) was used as control: three replicates of 15 g onion slices were kept at 4 C before analysis.
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2.4. Flavonoids extraction and determination by HPLC-DAD
The visual quality assessment included discoloration and glassy texture defects in the minimally processed onions.
The analytical treatment, which gave recovery figures higher than 92% with relative standard deviations lower than 4%, was based on a procedure previously reported by the authors (PérezGregorio et al., 2010; Pérez-Gregorio, García-Falcón, & SimalGándara, 2011; Pérez-Gregorio, Regueiro, González-Barreiro, Rial-Otero, & Simal-Gándara, 2011; Rodrigues et al., 2010 and 2011). A representative sample of edible portion (15 g) was homogenized and extracted by shacking with methanol:formic acid:water (MFW; 50:5:45; v:v:v) and stabilized with 2 g L1 of tertbutylhydroquinone. 50 mL of the final extract were then injected into the HPLC/DAD system (Thermo Separation Products). Quantification of single flavonols and anthocyanins was achieved by calibration curves obtained using pure quercetin and cyanidin 3-glucoside as standards, respectively. Results were then expressed in fresh weight (FW) onion, always referred to the weight at the starting of the experiment (about 15 g) with the intention to account result variations affected by the treatments. The main classes of flavonoids in this kind of onions are 11(Pérez-Gregorio et al., 2010; Pérez-Gregorio, García-Falcón & Simal-Gándara, 2011; Pérez-Gregorio, Regueiro, GonzálezBarreiro, Rial-Otero, & Simal-Gándara, 2011; Rodrigues et al., 2010 and 2011):
2.6. Statistical analysis Results are the means standard deviations from three replicates of independent experiments of the same treatment. Data were subjected to one-way analysis of variance with treatment as fixed factor. Whenever significant differences were detected at the 0.05 level, the means were separated by Duncan’s multiple range test in groups coded by a, b and c. All analyses were performed with the Statgraphics statistical software. 3. Results and discussion 3.1. Effect of concentration of sodium hypochlorite and temperature on onion flavonoids content Sodium hypochlorite is a powerful decontaminant with oxidizing properties very much used in the food industry, but it is necessary to take into consideration that its efficiency is pH-dependent. According to Adams et al. (1989), adjusting the pH of hypochlorite solutions from 9 to 4.5e5.0 with inorganic or organic acids produced a 1.5e4.0 fold increase in the disinfectant effect. Fresh-cut vegetables are usually washed in cold (4 C) chlorinated water before packaging. Hot water treatments are currently under investigation as a mean to reduce the extent of microbial contamination and improve quality retention in fruits and vegetables. There are many authors who reported the benefits of reduced microbial load and browning after brief dipping into water at 50 C due to a reduction in the activity of phenylalanine ammonia lyase (PAL) on minimally processed vegetables (Delaquis, Stewart, Toivonen & Moyls, 1999; Delaquis, Stewart, Cliff, Toivonen, & Moyls, 2000; and Fukumoto, Toivonen & Delaquis, 2002). In our work, changes in flavonoid content before washing at 50 C with sodium hypochlorite were assessed. Due to the hydrosolubility of flavonols there will have potential losses by migration into water. This was the reason for a control with just water: to distinguish between solubilization into water and oxidation of flavonols by the disinfectant agent selected. Flavonols and anthocyanins in onions slices were found to leach to water a 23% and 13%, respectively, at 50 C (Table 1). In our assay, no preferential leaching of any flavonol conjugate into the water occurred (24 and 23% for Q3,4dg and Q4g, respectively). Rodrigues, Pérez-Gregorio, GarcíaFalcón, and Simal-Gándara (2009) verified that boiling onions (100 C for 30 min) leaded to losses of quercetin glycosides, which
- Flavonols: two major (quercetin 3,4-diglucoside (Q3,4dg) and quercetin 4-glucoside (Q4g)) and five minor (quercetin 7,4diglucoside, isorhamnetin 3,4-diglucoside, quercetin 3glucoside, isorhamnetin 4-glucoside, and quercetin aglycon (Q)) - Anthocyanins (four): cyanidin 3-glucoside (C3g), cyanidin 3laminaribioside (C3lmb), cyanidin 3-(6”-malonylglucoside) (C3,6 mg) and cyanidin 3-(600 - malonyl-laminaribioside) (C3,6mlmb). It is important to note that the levels of these pigments in onions are much lower than those reached by the flavonols.
2.5. Quality evaluations Water loss assessment within each treatment was conducted by measuring the water volume at the end of the storage period due to mucilage exudation from the onion tissues, or the water condensation in the inner walls of the plastic cups. Visual appearance was determined based on the following subjective score on a 9-1 scale, with reference points of: 9, excellent; 7, good; 5, fair; 3, poor; and 1, unusable. A score of 6 was regarded as the limit of marketability.
Table 1 Losses % of flavonoids in onions by washing with water (control). (a) Flavonols. (b) Anthocyanins. (a) Q3,4dg
4 C
50 C
Fresh Control Fresh Control
Q4g
Q
[mg Q/kg FW]
%Change
[mg Q/kg FW]
142.6 18.1 119.1 8.1 117.1 8.5 89.0 9.1
Y16
191.6 160.8 126.4 96.8
Y24
25.1 11.4 6.3 13.6
Others
%Change
[mg Q/kg FW]
Y16
3.6 2.5 3.9 4.1
Y23
0.6 0.2 1.7 1.5
Total
%Change
[mg Q/kg FW]
Y29
23.0 17.6 29.4 21.4
[5
2.6 0.5 1.6 3.0
%Change
[mg Q/kg FW]
Y24
360.8 300.1 278.7 215.6
Y27
1.6 0.1 17.6 27.7
%Change Y17 Y23
(b) C3g [mg C3g/kg FW] 4 C 50 C
Fresh Control Fresh Control
1.3 0.9 0.7 0.4
C3,600 mg
C3lmb
0.2 0.0 0.2 0.2
%Change Y27 Y35
[mg C3g/kg FW] 0.54 0.3 0.4 0.4
0.1 0.0 0.1 0.1
%Change
[mg C3g/kg FW]
Y28
1.6 1.1 0.9 0.8
[10
0.2 0.0 0.1 0.1
C3,600 mlmb %Change
[mg C3g/kg FW]
Y33
0.9 0.7 0.6 0.6
Y15
0.1 0.0 0.1 0.1
Total %Change
[mg C3g/kg FW]
Y27
4.2 3.0 2.6 2.2
Y2
0.6 0.1 0.3 0.5
%Change Y29 Y13
*following manufacture recommendations on concentration (230 ppm). WWO, water-washed onion; CS, commercial solution (Amukine); HP, hydrogen peroxide; DS, dichloroisocyanuric sodium salt; SH, sodium hypochlorite; SA, sulphuric acid; CA, citric acid. aec Significantly different groups were coded with different letters.
[29 5.48 0.60 [24 3.08 0.13 [ 33 2.33 0.04 [35 486.0 56.1 [32 [42 202.4 50.6
252.9 73.3
e 4.23 0.64 e 2.49 0.09 e 1.75 0.07 e 360.8 46.4 e 191.6 25.1 e 142.6 18.1
Fresh onion (Control) UV-C radiation (254 nm 60 min) (c) UV-C physical treatment Physical Treatment 10 kJ/m2)
e Y2a Y6a Y2a Y0a Y3a
1.3 0.1 1.2 0.1 1.3 0.0 1.2 0.1 0.9 0.1 1.5 0.0
e Y2 [2 Y4 Y24 [19
1.7 0.0 1.6 0.0 1.7 0.1 1.7 0.2 1.2 0.1 1.8 0.1
e Y2 Y2 Y1 Y30 [3
3.0 0.1 2.9 0.1 3.0 0.1 2.9 0.2 2.2 0.1 3.3 0.1
e Y2 Y0 Y2 Y28 [10
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(b) Comparison of different chemical treatments at conventional disinfection conditions (100 ppm-4 C-5 min) Control (WWO) 119.1 8.1 e 160.8 11.4 e 300.1 19.9 154.4 21.6 Y4a 295.4 28.3 SH CA 119.1 14.3 Y0a a a 152.8 19.8 Y5 282.7 35.2 SA 112.0 11.1 Y6 DS 117.9 8.3 Y1a 156.0 10.9 Y3a 293.5 15.0 160.8 6.4 Y0a 300.1 20.9 HP 119.1 3.6 Y0a 155.5 2.8 Y3a 291.3 24.7 CS* 116.3 1.2 Y2a
e Y12 Y8 [2 Y25 0.4 0.1 0.3 0.2 0.1 2.2 1.9 2.0 1.7 2.3 e Y12 Y8 [2 Y21 1.4 0.16 1.2 0.1 1.2 1.4 1.1 e Y12 Y8 [3 Y18 0.1 0.0 0.0 0.1 0.0 0.87 0.7 0.8 0.9 0.7 e Y7a Y4a Y7a Y19c 27.7 16.0 22.8 19.4 11.4 215.6 200.6 207.2 201.1 173.7 e Y10b Y7a,b Y3a Y 20c
[mg C3 g/kg FW] %Change
Total
[mg Q/kg FW] %Change %Change
acids (50 C-2 min) e 96.8 13.6 Y4a 94.2 3.5 a 77.5 10.8 Y1 86.9 12.2 Y10b 89.9 5.7 Y17c
Total
[mg C3 g/kg FW] %Change
Acylated
[mg Q/kg FW] [mg Q/kg FW]
[mg C3 g/kg FW]
Glycosilated Q4g Q3,4dg
%Change
Major Flavonoids (Anthocyanins)
(a) Treatments with sodium hypochlorite and different Control (WWO) 89.0 9.1 SH CA 50 ppm 85.1 3.4 300 ppm 87.9 9.7 SA 50 ppm 80.1 6.3 300 ppm 73.9 3.6
Chlorine is a common efficient disinfectant agent but there is the risk of undesirable by-products upon reaction with organic matter and this may lead to new regulatory restrictions in the future (Artés et al., 2009; Ölmez & Kretzschmar, 2009). Because of this, it is important to assay other disinfectants such as hydrogen peroxide or sodium dichloroisocyanure. It is well known that hydrogen peroxide is a strong bactericide agent (affecting also to spores) by the generation of oxidant cytotoxic species such as hydroxyl radicals (Khadre & Yousef, 2001). It has been proved its efficiency increasing the shelf-life of convenience vegetables (Artés
Major Flavonoids (Flavonols)
3.2. Comparison of the different disinfectant treatments at usual operation conditions
Table 2 Losses % of flavonoids in onions by different disinfectant treatments.
leached to the boiling water without being degraded, at 37% and 29% for Q3,4dg and Q4g, respectively. Makris and Rossiter (2000) and Rodrigues et al. (2009) also found that leaching of the diglucoside derivative was favored. Flavonols content was affected by the acid used to fix the pH of the sanitation solution (Fig. 2). Because of the powerful oxidizing capacity of sulphuric acid, when this acid was used to adjust the pH more flavonol losses occurred. Flavonol content did not significantly change with the wash treatment when citric acid was used for the acidification of the sanitization solution. This treatment produces losses of overall a 4 % and 7 % at 50 ppm and 300 ppm of sodium hypochlorite, respectively, compared with a distilled water washing control. When the acid used to fix the pH of the sanitation solution was sulphuric acid, losses of 7 and 19% at 50 and 300 ppm of sodium hypochlorite, respectively, were produced (Table 2a). Results in Table 2a also suggest that Q4g is more susceptible to oxidation than Q3,4dg. The difference in the stability is, respectively, attributed to the presence and absence of the hydroxyl group at the C-3 position. The faster oxidation of Q4g, which is catalyzed by onion peroxidize, indicates that the hydroxyl group of the C-3 position is attacked by the enzyme (Hirota, Shimoda, & Takahama, 1998). Anthocyanins also experienced losses of 0%e25% at 50 and 300 ppm when sulphuric acid was used for the acidification and about 10% when citric acid was used (Table 2a). We found no articles comparing the different oxidizing capacity of acids used to set pH. It was previously shown the effect of the presence of hypochlorite in wash water, but without any specification of the acid agent used. Choi and Sapers (1994) studied the effects of washing with sodium hypochlorite on the phenolics compounds of mushrooms; they found that washing with 50 ppm of sodium hypochlorite at room temperature caused the disappearance of phenolics and the formation of their oxidation products. Instead, Klaiber, Baur, Wolf, Hammes, and Carle (2005) found that the use of chlorinated water (100 mg/L) to wash shredded carrots had negligible influence on the phenolics content. Martínez et al. (2005) reported the impact of minimal processing on onion quercetin. They did not find any difference between washing the onion with tap water or with sodium hypochlorite (100 ppm, 1 min). On the other hand, Odriozola-Serrano, Soliva-Fortuny, and Marti cn-Belloso (2008) investigated the effect of minimal processing on bioactive compounds and colour attributes of fresh-cut tomatoes. This study shows than phenolic content was not significantly affected by sanitization for 2 min in chlorinated water (0.2 mg free chlorine/L) at 4 C. Other authors (Hirota et al., 1998) establish that when scales of onion were boiled in tap water, Q4g degraded rapidly. It is known that tap water contains Feþ3 released from steel pipe and chlorine added for sterilization. Therefore, they examined the effects of these oxidants on the degradation of flavonols during boiling, and found that neither chlorine nor Feþ3 is important to enhance the degradation of flavonols.
%Change
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A
B
Fig. 2. Effect of acid and hypochlorite concentration on the levels of the main flavonoids in onion slices (A) flavonols; B) anthocyanins) after a treatment at 50 C for 2 min, a to c: significantly different groups were coded with different letters.
et al., 2009; Beerli et al., 2004; Ölmez & Kretzschmar, 2009). It is a GRAS (Generally Recognized As Safe) substance; in USA, however, the CFR (the Code of Federal Regulation) limits the concentration to be used in some products as bleaching or antimicrobial agent (Ölmez & Kretzschmar, 2009). As previously indicated, sodium dichloroisocyanurate, potassium dichloroisocyanurate, dichloroisocyanuric acid and trichloroisocyanuric acid are other common chlorine-based disinfectants. Most reports indicate that these compounds are somewhat more effective, albeit slower acting, than hypochlorites (Beerli et al., 2004). In our work, different disinfectants were selected and their effect was assayed on onion flavonoid content under the same conditions (100 ppm - 4 C for 5 min). These sanitation conditions were selected as the most usual conditions (Beerli et al., 2004). According to Fig. 3, it can be confirmed that there are no significant losses of flavonols by oxidation effects due to the decontaminant agent and that the only decrease of their levels is by solubilisation into water (Table 1, and Fig. 3). The 29% of anthocyanins have moved into water versus the 17% of flavonols. As shown in Table 1 the percentage of flavonols migration to water is greater in more
drastic conditions. Therefore, the migration at 50 C for 2 min of immersion is higher than at 4 C for 5 min of exposure (23% vs. 17%). Most losses were produced in flavonols when the acid employed to keep pH around 6.0 in the sodium hypochlorite solution was the sulphuric acid (6%). If we focus in anthocyanins treated with sodium hypochlorite and sulphuric acid we can observe that the total content of anthocyanins is unaffected by the treatment. Dichloroisocyanuric acid sodium salt treatment, instead, showed no effect in anthocyanins. Most losses in anthocyanins (28%) were produced with the hydrogen peroxide treatment, may be due to their blanching effect. 3.3. Effect of UV-C treatment on onion flavonoids content The damage inflicted by UV-C probably involves specific target molecules and a dose in the range from 0.5 to 20 kJ/m2 leads to lethality by directly altering microbial DNA through dimmer formation. In addition, no known toxic or significant non-toxic byproducts are formed during the UV-C treatment (Keyser et al., 2008).
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A
B
Fig. 3. Comparison of the effects of different disinfectant systems on the levels of the main flavonoids in onion slices A) flavonols; B) anthocyanins. SH-Sodium Hypochlorite; CACitric Acid; SA- Sulphuric Acid; HP-Hydrogen peroxide; DS-Dichloroisocyanuric Acid Sodium Salt; CS-Commercial Solution; a to c: significantly different groups were coded with different letters.
In our work, when the treatment used was the UV-C radiation, flavonol increased at 35%, and anthocyanins increased at 29%. Quercetin diglucosides increase much more than glucosides (42% vs. 32%) and glycosilated anthocyanins than acylated forms (33% vs. 24%) (Table 2c). Post harvest irradiation with UV light has been proposed as an appropriate method to increase the health value of different fruits and vegetables by inducing phenylpropanoid metabolism (Higashio, Hirokane, Sato, & Tokuda, 2005; Hagen et al., 2007; Mahdavian, Ghorbanli, & Kalantari, 2008). Hagen et al. (2007) found that posthaverst irradiation with VisþUV-B treatment enhanced the sum of flavonoids and total phenols in the peel of shade-grown apples. Mahdavian et al. (2008) also verified that contents of flavonoids, rutin and UV-absorbing compounds of pepper plants increased rapidly in response to UV-B and UV-C radiation. Higashio et al. (2005) verified that the irradiation of onion slices increased the quercetin content, reaching the duplication of their levels. The best visual quality results were obtained when sliced onions were treated with UV-C irradiation (score 8, excellent/good, in a 9-1 scale), but also a good score was obtained
for any of the chemical disinfectant treatments (score 7, good, in a 9-1 scale). 4. Conclusions This paper addresses some recent results obtained with alternative sanitizers on fresh-cut onion commodities. The main effect contributing to the loss of flavonols in fresh-cut onion slices is their solubility in immersion water (17e23% both at 4 or 50 C). There are no any further losses by oxidation due to the use of decontaminant chemical agents such as sodium hypochlorite, amukine, and hydrogen peroxide or sodium dichloroisocyanate systems. Only in the case of high levels of sodium hypochlorite (300 ppm) treated with sulphuric acid at temperatures of 50 C there is a clear trend for oxidation losses of the main flavonols (at a 15%). It is for such a reason that, in the case of sodium hypochlorite, concentrations of 100 ppm are recommended together with the use of organic acids for acidification purposes at refrigeration temperatures (4 C). Most losses occur for anthocyanins when the treatment employed was
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that based on hydrogen peroxide (28%). Anyway, in-dry decontaminant treatments like UV-C irradiation the natural levels of flavonoids in fresh-cut onion slices increase, being then superior to any of the chemically-based procedures. The combination of well designed integrated production, handling, processing and distribution chains for fresh-cut produces is crucial for achieving the high quality and safety demanded by consumers. References Adams, M., Hartley, A., & Cox, L. (1989). Factors affecting the efficacy of washing procedures used in the production of prepared salads. Food Microbiology, 6, 69e77. Alén-Ruiz, F., Pérez-Gregorio, M. R., Martínez-Carballo, E., García-Falcón, M. S., & Simal-Gándara, J. (2008). Influence of polyphenols on colour and antioxidant value in plant foods. Electronic Journal of Environmental, Agricultural and Food Chemistry, 7(8), 3171e3176. Artés, F., Gómez, P., Aguayo, E., Escalona, V., & Artés-Hernández, F. (2009). Sustainable sanitation techniques for keeping quality and safety of fresh-cut plant commodities. Postharvest Biology and Technology, 51, 287e296. Baur, S., Klaiber, R., Wei, H., Hammes, W. P., & Carle, R. (2005). Effect of temperature and chlorination of pre-washing water on shelf-life and physiological properties of ready-to-use iceberg lettuce. Innovative Food Science and Emerging Technologies, 6(2), 171e182. Beerli, K. M. C., Vilas Boas, E. V.de B., & Piccoli, R. H. (2004). Effect of sanitizer on the microbiol, physical and physical-chemical characteristics of fresh-cut onions (Allium cepa L.).Ciência e Agrotecnología. Lavras, 28, 107e112. Choi, S. W., & Sapers, G. M. (1994). Effects of washing on polyphenols and polyphenol oxidase in commercial mushrooms (Agaricus bisporus). Journal of Agricultural and Food Chemistry, 42(10), 2286e2290. Delaquis, P. J., Stewart, S., Cliff, M., Toivonen, P. M., & Moyls, A. L. (2000). Sensory quality of ready-to-eat lettuce washed in warm, chlorinated water. Journal of Food Quality, 23, 553e563. Delaquis, P. J., Stewart, S., Toivonen, P. M. A., & Moyls, A. L. (1999). Effect of warm, chlorinated water on the microbial flora of shredded iceberg lettuce. Food Research International, 32, 7e14. Fukumoto, L. R., Toivonen Peter, M. A., & Delaquis Pascal, J. (2002). Effect of wash water temperature and chlorination on phenolic metabolism and browning of stored iceberg lettuce photosyntetic and vascular tissues. Journal of Agricultural and Food Chemistry, 50(16), 4503e4511. Hagen, S. F., Borge, G. I. A., Bengtsson, G. B., Bilger, W., Berge, A., Haffner, K., et al. (2007). Phenolic contents and other health and sensory related properties of apple fruit (Malus domestica Borkh., cv. Aroma): effect of postharvest UV-B irradiation. Postharvest Biology and Technology, 45(1), 1e10. Higashio, H., Hirokane, H., Sato, F., & Tokuda, S. (2005). Uragami A Effect of UV irradiation after the harvest on the content of flavonoid in vegetables. Acta Horticulture, 682, 1007e1012. Hirota, S., Shimoda, T., & Takahama, U. (1998). Tissue and spatial distribution of flavonol and peroxidase in onion bulbs and stablilty of flavonol glucosides
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