Efficacy of non-thermal technologies and sanitizer solutions on microbial load reduction and quality retention of strawberries

Journal of Food Engineering 108 (2012) 417–426

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Efficacy of non-thermal technologies and sanitizer solutions on microbial load reduction and quality retention of strawberries Elisabete M.C. Alexandre, Teresa R.S. Brandão, Cristina L.M. Silva ⇑ CBQF-Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

a r t i c l e

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Article history: Received 22 February 2011 Received in revised form 16 August 2011 Accepted 5 September 2011 Available online 10 September 2011 Keywords: Strawberries Microbial loads Quality Sanitizer solutions Non-thermal technologies Refrigeration

a b s t r a c t The effect of non-thermal technologies (ozone in aqueous solution, ultrasound and ultraviolet C radiation) and washings with chemical solutions (sodium hypochlorite and hydrogen peroxide) on safety and quality features of strawberries was studied. These treatments were applied before fruit storage at two different temperatures (4 and 15 °C). The overall impact on microbial loads (total mesophiles, and yeasts and moulds) and selected quality attributes (colour, firmness, pH, total anthocyanins and ascorbic acid content) was assessed. During storage under refrigerated temperature, washing with hydrogen peroxide solutions resulted in strawberries with lower microbial loads, when compared to the other treatments. However, it produced significant key quality attributes losses, such as colour and total anthocyanins content. The results presented show that ozone and ultrasound are promising alternatives to thermal treatments. The application of such technologies, before refrigerated storage of strawberries, allowed a satisfactory retention of all quality characteristics analysed, while being efficient in controlling microbial contamination. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The strawberry is an attractive fruit, with potential benefits to human health, due to its high vitamin C, anthocyanins and flavonols content, and high antioxidant activity (Odriozola-Serrano et al., 2010; Tiwari et al., 2009). However, strawberries are extremely perishable as a consequence of tenderness and susceptibility to mechanical damage, physiological deterioration, water loss and fungal spoilage (Fan et al., 2009). Storage under refrigerated conditions reduces fruit deterioration as chemical and biochemical reactions and microbial growth, which may reduce quality or shelf-life, slow down when temperature is reduced. To stabilize food products during storage, certain processes complementary to refrigeration can be used. Traditionally, several sanitizer agents, such as chlorine and hydrogen peroxide solutions, have been used to rinse fresh fruits, with the main objective of reducing microbial contamination, therefore extending product shelf life. The chlorination process usually consists on adding chlorine (Cl2) or sodium or calcium hypochlorite (NaOCl or Ca(OCl)2) to wash waters, at concentrations between 50 and 200 ppm and for a contact time of 1–2 min (Bachmann and Earles, 2000; Beuchat, 1998). Hydrogen peroxide (H2O2) is another well recognized germicidal agent, considered to be environmentally friendly due to

⇑ Corresponding author. Tel.: +351 22 5580058; fax: +351 22 5090351. E-mail address: [email protected] (C.L.M. Silva). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.09.002

its low toxicity. However, Sapers and Simmons (1998) reported that it may degrade the quality of some products by browning induction (in mushrooms and lettuce) or bleaching of anthocyanins (in strawberries and raspberries). The effect of using sanitizer agents on fruits has been assessed by several authors. Ukuku (2004) reported a 3 log unit reduction of aerobic mesophilic bacteria and Salmonella spp. in melons washed with hydrogen peroxide solutions (2.5% and 5%), and observed an improvement in the acceptability of fresh-cut melons dipped in such solutions. Ukuku (2006) reported for cantaloupes (using 200 ppm of hypochlorite solutions) log unit reductions of 2.7, 0.38, 2.7 and 1.8 for total bacterial, Pseudomonas spp., yeasts and moulds, and lactic acid bacteria counts, respectively. Non-thermal technologies, such as ozonation, ultrasonication and ultraviolet (UV) light radiation, can be applied to an extensive variety of food products to destroy microorganisms associated with spoilage and contamination (Cao et al., 2010; Hernández et al., 2010; Ölmez and Akbas, 2009). Ozone is considered one of the most powerful oxidizing agents. However, in aqueous solution ozone is more unstable than in gaseous phase. Its efficiency depends on the pH and temperature of the medium, and the balance between ozone concentration and organic matter content (Cullen et al., 2009; Sharma, 2005). Power ultrasound is defined as pressure waves, with a frequency of 20 kHz or more, which cause chemical and physical changes in biological structures (in a liquid medium) due to intracellular cavitation (Butz and Tauscher, 2002).

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Ultraviolet radiation, in the wavelength range of 200–280 nm (UV-C), has a germicidal effect due to photochemical reactions that are induced in microorganisms (Giese and Darby, 2000). It is also a promising alternative to traditional sanitizers and thermal treatments. Some researchers have studied the impact of these technologies on fruit and vegetable quality and safety aspects, when applied before product storage under refrigerated conditions. Wang et al. (2004) used ozonated solutions to wash fresh-cut cilantro, prior to refrigerated storage. They reported a 1.0–1.5 log unit increase in total aerobic plate count during a storage period of 11 days at 0 °C. A small decrease in firmness was observed, while no influence in colour, off-odours, aroma and overall quality was detected. Beltran et al. (2005) used ozonated water and chlorine solutions to reduce the total mesophilic population on fresh-cut iceberg lettuce. Reductions of 1.6 and 2.1 log units were observed after ozonation and chlorine rinses, respectively. After 13 days of storage at 4 °C, the microbial loads increased but the treatments reduced final microbial counts by 1.8 (ozonated samples) and 2.7 (chlorine washed samples) log units when compared to control. Baka et al. (1999) verified shelf-life extension (with higher anthocyanins content and firmness) of refrigerated strawberries that had been previously UV-C irradiated (0.25 and 1.0 kJ m2). Moreover, the treatment allowed the control of decay caused by Botrytis cinerea. The main objective of this work was to assess the effect of two sanitizing agents (sodium hypochlorite prepared from a commercially available solution; and hydrogen peroxide at two different concentrations, 1% and 5% w/w) and three non-thermal technologies (UV-C radiation, ultrasonication and ozone in aqueous solution) on total mesophile and yeast and mould counts on strawberries throughout storage under refrigerated conditions. The impact on some quality factors, such as colour, firmness and pH was studied, since these characteristics are easily perceived by the consumer. Anthocyanins and ascorbic acid content were studied, since these are important indices of the health benefits of berries. 2. Materials and methods 2.1. Fruit samples Strawberries (Fragaria ananassa D. cv Camarosa) were obtained in a local market. 2.2. Microbial enumeration Endogenous total mesophiles and yeasts and moulds were quantified in strawberries. After each treatment and during storage, samples (20 g) were aseptically cut in small pieces and homogenized in a stomacher using 80 mL of Buffered Peptone Water (BPW; Lab M, Lancashire, UK) for 5 min. Decimal dilutions were carried out in BPW. Enumeration of total mesophiles was assessed, in duplicate, using Plate Count Agar (PCA; Lab M, Lancashire, UK). Samples were incubated at 30 °C for 3 days. Three replicates were carried out for each treatment and storage time. Yeast and mould enumeration was assessed, in duplicate, using Potato Dextrose Agar containing lactic acid (Lab M, Lancashire, UK). Samples were incubated at 30 °C for 5 days. Three replicates were carried out for each treatment and storage time. 2.3. Quality measurements 2.3.1. Colour Colour was measured with a hand held tristimulus colorimeter (Chroma Meter CR-400, Konica Minolta Sensing, Inc., Tokyo, Japan)

using the Hunter Lab scale. The total colour difference (TCD) parameter was considered for evaluation of colour changes. This parameter quantifies the overall colour difference of a given sample when compared to a reference one (L0, a0, b0), according to the expression (Drlange, 1994):

TCD ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ða  a0 Þ2 þ ðb  b0 Þ2 þ ðL  L0 Þ2

ð1Þ

being the index ‘‘0’’ indicative of reference untreated samples. Thirty replicates were carried out for each treatment and storage time. 2.3.2. Firmness These analyses were conducted in a TA-XT2 plus texture analyser (Stable Micro Systems Ltd., Godalming, UK) equipped with a 5 kg force cell. Tests were performed in a compression mode (30% deformation, 1 mm s1 velocity) using a 36 mm diameter cylindrical probe. Firmness values were registered as the maximum force observed during compression of samples. Forty replicates were carried out for each treatment and storage time. 2.3.3. pH Samples were homogenized with a kitchen mixer (Braun, Warszawa, Poland) for 5 min. A pH meter (GLP 22 CRISON, Barcelona, Spain) was used for measurements. Six replicates were carried out for each treatment and storage time. 2.3.4. Anthocyanins Extraction of strawberry anthocyanins was performed by mixing 5 g of product (previously homogenized) with 15 mL of acetone (70% v/v) for 5 min. The solution was then filtered. The pH differential method was used to evaluate total anthocyanin content (Giusti and Wrolstad, 2001; Teow et al., 2007). Two buffers were used: potassium chloride (0.025 M, pH 1.0) and sodium acetate (0.4 M, pH 4.5). The pH was adjusted with concentrated hydrochloric acid. A volume of 200 lL of the extracted solution was mixed with 1.8 mL of each buffer, for 15 min. The absorbance of solutions was read at 510 and 700 nm (UV-1601, Shimadzu Co., Kyoto, Japan). Total anthocyanin content was expressed as the mass (mg) of pelargonidin 3-glycoside (predominant anthocyanin in strawberries) per 100 g of fresh weight (Cordenunsi et al., 2002; Gil et al., 1997). Four replicates were carried out for each treatment and storage time. 2.3.5. Ascorbic acid The methodology used for ascorbic acid (AA) determination was based on HPLC UV detection, using isoascorbic acid (IAA; Fluka) as an internal standard (Zapata and Dufour, 1992). The mobile phase was prepared with 13.61 g of potassium dihydrogen phosphate (Fluka), 3.64 g of cetrimide (Fluka) and 2 L of methanol (Merck)ultrapure water (5:95 v/v), filtered under vacuum using a 0.45 lm membrane (Millipore) and degassed for 15 min in a ultrasonic bath. AA buffers, as well as IAA solutions were prepared with methanol-ultrapure water (5:95 v/v). Samples were homogenised for 5 min with a kitchen mixer (Braun, Warszawa, Poland) and filtered with gauze. A volume of 5 mL of the filtrated was added to 1 mL of IAA (0.03 g/50 mL) in a volumetric flask. Each pH sample was adjusted to 2.45 with HCl, and methanol-ultra pure water (5/95 v/v) was added to reach a volume of 20 mL. The solution was centrifuged (Sorvall Instruments RC5C Du Pont, Delaware, USA) at 10,000 rpm, for 5 min at 4 °C. The mixture was maintained in a dark room for 37 min, and was further filtered (0.45 lm filter; Millipore) into a vial (Chromacol LTD). The HPLC system used consisted of a controller (Hercule Lite – Chromatography interface, Tokyo, Japan), a solvent pump (Jasco PU – 1580, Tokyo, Japan), an injector (Jasco AS – 1555, Tokyo, Japan)

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with a valve of 20 lL sample loop, a reversed phase column (Macherey–Nagel, Chromcart 100–10 Nucleosil, 250  4.6 mm) and a UV detector (Jasco UV – 1575, Tokyo, Japan). The wavelength detector was programmed to run at 262 nm for AA and IAA detection. Four replicates were carried out for each treatment and storage time.

(time  intensity) of the lamps were continuously measured using an UV digital photometer (DO 9721, Delta Ohm, Padova, Italy). The temperature was also monitored (25.2 ± 1.8 °C). 2.4.2. Ultrasonication A Sonorex Super RK 106 equipment with 5.6 L of water capacity (Bandelin Electronic, Berlin, Germany), operating at 35 kHz and 120 W, was used for ultrasonication treatments (Cao et al., 2010; Yang et al., 2008). The temperature of water used for sonication was 15 ± 2 °C. A 4 L volume of water was used to treat approximately 160 g of strawberries.

2.4. The treatments The contact time of all treatments was 2 min. 2.4.1. Ultraviolet-C radiation UV-C treatments were carried out in a chamber designed by the University of Algarve, Portugal. Samples were submitted to a bank of four germicidal UV lamps (TUV G30T8, 16 W, Phillips, Amsterdam, The Netherlands) with peak emission at 254 nm and an average intensity of 12.36 W m2. Flux intensity and exposure dose

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Refrigerated temperature

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Log (N)

2.4.3. Ozonation Ozone treatments were performed in a pilot plant. An ozone generator (OZ5, SPO3 – Sociedade Portuguesa de Ozono, Lda., Porto, Portugal) was interconnected to a container filled with deionised water

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Storage time (days) Fig. 1. (a) Impact of ozone (), ultrasound (j) and UV-C radiation (N), (b) NaOCl (h), H2O2 1% (e) and H2O2 5% (D) on total mesophiles on strawberries stored under refrigerated conditions and at room temperature; water-washed controls (s) and untreated (d) samples. The bars represent standard deviations.

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(approximate 30 L), forming a closed circuit ring apparatus. The ratio between mass of the samples and volume of ozonated water was approximately 80 g/30 L. Ozone was continuously incorporated by bubbling in the water (15 ± 2 °C), and the aqueous ozone concentration was measured by potential difference (Redox probe; SZ 275, B and C Electronics, Carnate, Italy), attaining 0.3 ppm. This concentration was confirmed using an ozone determination kit for O3 in water (25180–50 Ozone AccuVac colour disc kit, HACH Lange GmbH, Düsseldorf, Germany).

Table 1 Values of pH, firmness, and total anthocyanins and ascorbic acid content for fresh (untreated) strawberries.

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3.55 ± 0.24 8.63 ± 1.41 27.62 ± 0.92 46.77 ± 1.29

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pH Firmness (N) Total anthocyanins (mg/100 g) Ascorbic acid (mg/100 g)

commercial bleach solution of 1.15 mg/mL – AMUKINAÒ, FarmaLepori) and (ii) hydrogen peroxide (H2O2; 1% and 5% w/w, Riedelde Haën) at 15 ± 2 °C. A 4 L volume of sanitizer solution was used to treat approximately 160 g of strawberries.

2.4.4. Sanitizer solutions Samples were immersed in the following sanitizer solutions: (i) sodium hypochlorite (NaOCl; 200 lg/mL; prepared from a

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Storage time (days) Fig. 2. (a) Impact of ozone (), ultrasound (j) and UV-C radiation (N), (b) NaOCl (h), H2O2 1% (e) and H2O2 5% (D) on yeasts and moulds on strawberries stored under refrigerated conditions and at room temperature; water-washed controls (s) and untreated (d) samples. The bars represent standard deviations.

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both conditions and analyzed in terms of microbial populations and quality attributes for a total of 13 days.

2.4.5. Controls Additional experiments using untreated samples and water washing (deionised water at 15 ± 2 °C) were performed as controls. A 4 L volume of water was used to treat approximately 160 g of strawberries.

2.6. Data analysis The microbiological data were analyzed in terms of log(N/N0), where N is the microorganism load at a given time, and N0 corresponds to the initial microbial load of untreated samples. Data on quality attributes (colour, texture, pH, and anthocyanins and ascorbic acid content) were normalized in relation to values obtained for untreated samples. Results were compared by analyses of variance (two-way ANOVA, testing treatment and storage time effects) using SPSSÒ 17.0 for WindowsÒ (SPSS Inc., Chicago, USA). Tukey’s test was performed for paired means comparison (Walpole and Myers, 1993).

2.5. Storage conditions After each treatment, samples were separated into two different batches: one was kept at room temperature (15 ± 2 °C) and the other was stored under refrigerated conditions (4 ± 1 °C) in a conventional refrigerator (Balay 3FCB 1416, Pamplona, Spain). Temperatures were monitored using a data logger (Testo 174, New Jersey, USA). Samples were collected throughout storage time at

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Storage time (days) Fig. 3. (a) Impact of ozone (), ultrasound (j) and UV-C radiation (N), (b) NaOCl (h), H2O2 1% (e) and H2O2 5% (D) on total anthocyanins in strawberries stored under refrigerated conditions and at room temperature; water-washed controls (s) and untreated (d) samples. The bars represent standard deviations.

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resulted in strawberries with lower microbial loads, when compared to the results obtained with the remaining treatments. Among the technologies (Fig. 1a), ozonated water washing was the most effective. On average, a 1.21 ± 0.33 log unit reductions occurred when samples were washed with aqueous ozone solutions. At the end of refrigerated storage, samples pre-treated with ozone presented lower microbial loads than samples ultrasonicated or treated with UV-C radiation. The impact of ozone was greater when samples were maintained at room temperature. Throughout refrigerated storage, strawberries pre-washed with all sanitizer solutions and with ozone presented lower microbial loads (statistically significant at p < 0.05) than untreated, ultrasonicated, UV-C irradiated or water-washed samples. As expected, when samples were maintained at room temperature, mesophiles grew at a higher rate. Nevertheless, samples

3. Results and discussion 3.1. Microbial loads The effects of the technologies and sanitizer solutions on total mesophiles and yeasts and moulds (before treatments) are shown in Figs. 1 and 2. The impacts of refrigerated storage for 12 days and storage at room temperature for 4 days can also be visualised. For total mesophiles it was observed that untreated and water-washed samples presented the highest microbial loads during storage at both temperatures. Strawberries washed with hydrogen peroxide solutions (before storage) had the highest total mesophiles reduction: 2.26 ± 0.38 and 1.59 ± 0.41 log unit reductions, for H2O2 at 5% and 1%, respectively (Fig. 1b). During storage at refrigerated temperature, these two washing treatments

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Storage time (days) Fig. 4. (a) Impact of ozone (), ultrasound (j) and UV-C radiation (N), (b) NaOCl (h), H2O2 1% (e) and H2O2 5% (D) on colour of strawberries stored under refrigerated conditions and at room temperature; water-washed controls (s) and untreated (d) samples. The bars represent standard deviations.

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of 11 days at 0 °C. Beltran et al. (2005) used also ozonated water to reduce total mesophilic population on fresh-cut iceberg lettuce. Microbial loads increased approximately 4 log units after 13 days of storage at 4 °C. However, ozone treatment allowed reductions of 1.8 log units when compared with control samples.

pre-treated with ozone and H2O2 at 5% presented lower microbial counts (statistically different from the remaining treatments; p < 0.05). In relation to yeasts and moulds (Fig. 2), it was observed that untreated and water-washed samples presented the highest microbial loads throughout refrigerated storage; all remaining treatments were significantly different from those. However, among all treatments, hydrogen peroxide (at 5%) was the most efficient in reducing initial contamination of strawberries, in terms of yeasts and moulds with on average, a reduction of 2.61 ± 0.30 logunits observed. If strawberries were kept at room temperature, the antimicrobial effects of hydrogen peroxide solutions and ozone treatments were equivalent. They reduces the level of microbial contamination, and differed significantly (p < 0.05) from waterwashed and untreated samples. Wang et al. (2004) used ozonated solutions to wash fresh-cut cilantro, prior to refrigerated storage. They reported a 1.0–1.5 log unit increase in total aerobic plate count during a storage period

a) Firmness (normalized values)

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3.2. Quality factors Values of total anthocyanins, pH, firmness and ascorbic acid for fresh (untreated) strawberries are presented in Table 1. 3.2.1. Anthocyanins content Throughout storage, strawberries treated with non-thermal technologies (ozone, UV-C radiation and ultrasound) presented higher anthocyanins content than the samples washed with chemical solutions (hydrogen peroxide or sodium hypochlorite) (Fig. 3). Results showed that under refrigerated storage the anthocyanins content was better retained if strawberries were previously treated with ozone. These results were significantly higher than the ones

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Storage time (days) Fig. 5. (a) Impact of ozone (), ultrasound (j) and UV-C radiation (N), (b) NaOCl (h), H2O2 1% (e) and H2O2 5% (D) on firmness of strawberries stored under refrigerated conditions and at room temperature; water-washed controls (s) and untreated (d) samples. The bars represent standard deviations.

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Temperature has a considerable influence on anthocyanins degradation. If strawberries were kept at room temperature, anthocyanins content decreased rapidly. For all sanitizer solutions, and after 6 days of storage at room temperature, strawberries lost more than 90% of their initial anthocyanins content. However, if samples had been ultrasonicated, ozonated, UV-C radiated or waterwashed, losses were 44%, 69%, 84% and 95%, respectively. These values were significantly different (p < 0.05).

obtained by using ultrasonication or UV-C radiation. Untreated and water-washed samples had the lowest anthocyanins content. After 13 days of storage at 4 ± 1 °C, ozonated strawberries preserved on average 82% of anthocyanins (when compared to fresh samples), while untreated and water-washed samples only retained 55%. In relation to the sanitizer solutions (Fig. 3b), hydrogen peroxide at 5% had the greatest negative impact on anthocyanins content. Samples dipped in this solution lost approximately 38% of anthocyanins (in relation to fresh strawberries and before storage). Furthermore, and throughout refrigerated storage, samples washed in 5% H2O2 differed significantly (p < 0.05) from all the others. For this case, and after 13 days of storage, 88% of anthocyanins degraded. For the remaining treatments (controls, NaOCl and 1% H2O2) anthocyanins content was significantly higher.

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3.2.2. Colour For total colour changes, and as observed for anthocyanins results, the non-thermal technologies were less severe than chemical solutions (Fig. 4). Ultrasonicated and ozonated samples (Fig. 4a) did not differ throughout refrigeration (p < 0.05) and were the ones with better colour retention. In terms of TCD, those samples were different from controls and from UV-C treated strawberries.

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Storage time (days) Fig. 6. (a) Impact of ozone (), ultrasound (j) and UV-C radiation (N), (b) NaOCl (h), H2O2 1% (e) and H2O2 5% (D) on ascorbic acid content of strawberries stored under refrigerated conditions and at room temperature; water-washed controls (s) and untreated (d) samples. The bars represent standard deviations.

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The selection of a chemical solution for maximum colour preservation during refrigerated storage was not evident (Fig. 4b). At the end of storage, and for all sanitizers, large differences (according to the classification of (Drlange, 1994) were observed in treated fruit colour, when compared to fresh ones. The temperature effect on colour alterations was high. At room temperature, samples suffered considerable changes in colour. Nevertheless, and as observed for refrigerated samples, ultrasound and ozone were the treatments that provided better results. After 6 days at room temperature, strawberries presented very great colour differences, independently of the treatment applied. 3.2.3. Firmness Fruit firmness was better retained throughout all refrigerated storage if samples had been previously ozonated and ultrasonicated (Fig. 5a). These treatments were statistically equivalent, but differed from controls (p < 0.05). In relation to sanitizers (Fig. 5b), no distinct differences were detected among them; moreover, the results for all solutions were similar to those for water-washed fruit. By the last day of refrigerated storage, strawberries that had been pre-treated with ozone, ultrasound and UV-C radiation lost 31%, 46% and 50% of firmness, respectively; water-washed samples lost 62% (when compared to fresh products). At room temperature, firmness degraded faster. However, and as observed for refrigerated conditions, samples that underwent ozone and ultrasound treatments maintained firmness better throughout storage. After 6 days at room temperature, ozonated, ultrasonicated and UV-C radiated samples lost 24%, 21% and 40% of firmness, respectively, while 67% of losses were observed for water-washed strawberries. Concerning chemical solutions, NaOCl and 1% H2O2 were equivalent, but different from all the other washing treatments, leading to better firmness retention throughout the storage period. 3.2.4. pH Fruit pH averaged 3.88 ± 0.08 (fresh samples). After treatments, no significant changes were detected. At both temperatures, the storage effect was not evident. 3.2.5. Ascorbic acid In relation to ascorbic acid content (Fig. 6), refrigerated samples, which had been previously treated with all technologies and sanitizers presented higher ascorbic acid content than untreated or water-washed controls. This was more evident after 9 days of storage. After this period, samples that had been pre-treated with ozone, ultrasound, UV-C radiation, or washed with all chemical solutions, did not differ significantly from each other. They lost on average 68% of the initial ascorbic acid content (p < 0.05). This value is significantly different from controls, in which losses of 91% were observed. However, at the end of the refrigerated storage period (13 days), only a residual content of ascorbic acid (lower than 2% of the initial content) was detected in strawberries that had been pre-treated with all sanitizers, ultrasound and UV-C radiation (as well as controls). The exception was ozonated strawberries, which retained 8% of initial ascorbic acid. If samples treated with ozone, ultrasound and UV-C were maintained at room temperature, they presented higher ascorbic acid content than those washed with chemical solutions (and controls). However, the degradation of ascorbic acid is rapid, and after 4 days it was only detected in ozonated and ultrasonicated strawberries (averaging 16% of the initial content). 4. Conclusions In terms of microbial loads (total mesophiles, and yeasts and moulds), the highest impact was observed in refrigerated

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strawberries previously washed with hydrogen peroxide solutions (at both concentrations). However, if samples were kept at room temperature, ozone provided better results. Although the efficacy of hydrogen peroxide solutions was proven in terms of reducing microbial contamination on strawberries, important quality attributes such as colour and total anthocyanins content suffered negative impacts. The application of ozone and ultrasound treatments, before storage of strawberries under refrigerated conditions, was efficient in controlling growth of microbial contamination and attained a product with satisfactory quality retention. Overall it can be concluded that ozone and ultrasound are promising alternatives to chemical rinses. The efficacy of UV-C radiation and sodium hypochlorite solutions was not relevant.

Acknowledgements The authors acknowledge the financial support through Programa Operacional Agricultura e Desenvolvimento Rural – Projecto AGRO n°822 (Novas Tecnologias de Processamento de Hortofrutículas Congelados – EMERCON). Authors E.M.C Alexandre and T.R.S. Brandão would like to thank Fundação para a Ciência e a Tecnologia (Grants SFRH/BD/16042/2004 and SFRH/BPD/11580/2002, respectively).

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