61 1. Introduction It is well known that disinfection represents one of the most critical processing steps influencing quality, safety and shelf-life of fresh-cut fruits and vegetables. In the fresh-cut industry, chlorine is generally used as a product sanitizer; however, there is a trend in eliminating this sub- stance due to the environmental and health risks associated with the formation of carcinogenic halogenated disinfec- tion by-products (Gil et al., 2009). Ozonation represents a sustainable technology able to improve the quality of the product; ozone is useful in diminishing the microbial load and the level of toxic or- ganic compounds. Furthermore, this gas has a stronger and more rapid antimicrobial action against spores, faecal and pathogenic microorganisms and viruses with respect to chlorine (Artés and Allende, 2005). The antimicrobial ef- fect of ozone and its relatively simple application, in addi- tion to its characteristic of not leaving any residue or form- ing carcinogenic trihalomethanes (such as chlorination), make this treatment suitable for various applications in the food industry (Gil et al., 2009; Alexopoulos et al., 2013). Moreover, products treated with ozone do not modify their sensory characteristics. After it gained GRAS (Generally Recognized As Safe) status in 1997, the use of this gas has been approved as a disinfectant or sanitizer in foods and food processing in Europe and in the US (Ölmez and Kretzschmar, 2009). The primary systems for ozone application include gas- eous phase storage and ozonated dips (Artés et al., 2009). When ozone is used as a gas, the exposure time is longer (1-4 h) than ozone dissolved in water (1-10 min) (Carletti et al., 2013). In recent years the use of this gas as a pretreatment to preserve fruit and vegetable quality has received great atten- tion for its antimicrobial activity against bacteria, fungi, vi- ruses, and bacterial and fungal spores. Several studies have focused on the effect of ozone treatment on the safety and quality of iceberg lettuce (Rico et al., 2006; Yuk et al., 2006; Hassenberg et al., 2007). In addition, various applications have been proposed concerned the sanitizing effect of ozone dissolved in water used for washing on the quality and safe- ty of minimally processed fruits and vegetables (Seydim et al., 2004; Zhang et al., 2004; Selma et al., 2008; Artes et al., 2009; Ölmez and Kretzschmar, 2009), but there is scant literature about ozone applied to fresh-cut iceberg lettuce (Beltran et al., 2005; Ölmez and Akbas, 2009). The aim of this work was to evaluate the effect of re- frigeration at 4°C, associated with ozonization treatment Application of ozone in fresh-cut iceberg lettuce refrigeration F. Galgano1(*), M.C. Caruso1, N. Condelli1, S. Stassano1, F. Favati 2 1 School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy. 2 Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134- Verona, Italy. Key words: environmental sustainability, fresh-cut vegetables, fresh vegetable quality, minimally fresh processed leaf vegetables, shelf-life. Abstract: Recently, technological innovations have been geared to supporting environmental sustainability, also in the fruit and vegetable industry. The application of ozone in the cold storage of fruits and vegetables is a sustainable tech- nology used to improve product quality and its antimicrobial effect, simple use, and characteristic of not leaving any residue, makes this treatment suitable for many applications in this field. The aim of this work was to evaluate the effect of refrigeration at 4°C, associated with ozonization treatment at a concentration of 0.2 ppm on the shelf life of fresh-cut lettuce, compared to a lettuce control stored only at 4°C. Lettuce quality throughout the storage period (7 days) was de- termined by means of color and microbiological indexes, such as total bacterial count, total coliforms, Escherichia coli, Pseudomonadaceae, yeasts and molds. The lettuce used in the experiment was found to have a low microbiological load. Microbiological results obtained at different storage times have shown that the use of ozone is effective in containing mi- crobial growth during chilling storage of the raw material compared to the refrigerated control. In particular, the positive effects of ozonation were appreciable after the third day of storage. Furthermore, the ozone treatment did not affect the color of the product. (*) Corresponding author: fernanda.galgano@unibas.it Received for publication 26 September 2014 Accepted for publication 9 March 2015 Adv. Hort. Sci., 2015 29(2-3): 61-64 62 Adv. Hort. Sci., 2015 29(2-3) 61-64: at a concentration of 0.2 ppm on the shelf life of fresh- cut lettuce, compared to a lettuce control stored only for 7 days at 4°C. 2. Materials and Methods Sampling and ozone treatment Iceberg lettuce (Lactuca sativa L.) was obtained from Società Agricola ”Rago” (Battipaglia, SA, Italy) and, im- mediately after harvesting, it was stored at 4°C and sub- mitted to a continuous ozone exposure of 0.2 ppm. A con- trol sample was stored at 4°C without ozone treatment. Ozone gas was generated using a corona discharge ozone generator (Model OXY MET TWIN, MET, San Lazzaro di Savena, BO, Italy) from purified oxygen gas. Functioning of this equipment takes place through sensors placed in- side the storage compartment/chamber that reveal the ex- act concentration present in the refrigeration cell and it is reported on the display of the instrument. The tests lasted up to 7 days and the ozonated and non-ozonated product was sampled at 1, 2, 3, 5 and 7 days for analyses. Colorimetric analysis Color was measured on the lettuce surface using a Mi- nolta Chroma meter CR-300 (Minolta Camera Co. Ltd., Osaka, Japan), with a D 65 illuminant, using CIELAB L*, a*, b* values. The data reported are the average of a set of four determinations carried out on the lower and upper surface of two leaves of lettuce. Microbiological analysis At each treatment time, 9 g of ozonated or control prod- uct were placed in a stomacher bag containing 81 ml of Tryptone Water (Liofilchem, Teramo, Italia) and homog- enized for 2 min. Samples were then serially diluted in tripton water and surface plated in duplicate on Compact- Dry EC (PBI International, Milano, Italy) for the recovery of surviving total coliforms and Escherichia coli, on Com- pact-Dry YM (PBI International, Milano, Italy) for yeasts and moulds and on Cetrimide Agar (Liofilchem, Teramo, Italy) for the enumeration of Pseudomonas aeruginosa and Pseudomonas spp. Total mesophilic bacteria were enumerated on Plate Count Agar (Liofilchem, Teramo, Italy) using the pour plate technique. All the inoculated media were incubated at 30°C for 48 h. Statistical analysis In order to study the effect of ozone treatment and storage time on fresh-cut iceberg lettuce quality charac- teristics, data were processed by two-way analysis of vari- ance (ANOVA). Moreover, a Student test was performed to compare the mean values between the samples treated with ozone and control samples for each time of storage. All statistical procedures were computed using the statisti- cal package SYSTAT for Windows (ver. 10, 2003) (Systat Software, Chicago, USA). 3. Results and Discussion Ozone can be used to disinfect fruit and vegetables by dissolving it in the water used for washing (Gil et al., 2009), or by keeping a constant ozone concentration, through the use of an appropriate generator, during storage of the product in a cooling room at 4°C. In this paper we evaluated the effect of cold storage at 4°C, accompanied by ozonization at a concentration of 0.2 ppm on the shelf life of the fresh-cut lettuce, compared to a control subjected only to cold storage. Preliminary tests showed that the use of ozone at low concentrations, such as 0.2 ppm, did not determine sensory alterations; for this reason, the effect of treatment on both perceptible sensory parameters, such as color stability, and microbiological parameters, for safety of the product, has been evaluated. A two-way analysis (time, treatment) showed a significant effect of both factors on the colorimetric parameters (p= 0.000). In particular, total bacterial count and coliforms in- creased throughout the storage period, being significantly lower for ozonated samples than control samples. There- fore, the microbiological results obtained at different stor- age times show that the use of ozone is effective in con- taining microbial growth during storage in a cooling room of raw material compared to the untreated control. In par- ticular, the positive effects of ozonation were appreciable after the third day of storage, as shown by the trend of the total bacterial count and total coliforms of the chilled and ozonized product compared to the refrigerated only prod- uct (Fig. 1 and 2). In particular, ozone was able to inhibit the proliferation of total coliforms, which were not detect- able even after 7 days of storage in the ozonized samples, while in control samples these microorganisms reached 1.6 x 103 cfu/g at the same storage time. The growth of colonies on the plates inoculated for the detection of other microorganisms was sporadic and not significant for both the experimental theses considered. In the literature a re- duction of 3-4 log units of Escherichia coli O157: H7 in- oculated in lettuce and carrots, by sequential washes with aqueous solutions of Cl 2 O/ozonated water, has been re- ported (Singh et al., 2002). Various experiments carried out by treating vegetables with ozonated water have shown the ability of ozone to reduce the growth of different mi- croorganisms, including pathogens, and also to reduce en- zymatic activities, including enzymatic browning (Artes et al., 2009). Alexopoulos et al. (2013) have highlighted the importance of keeping the concentration of ozone constant during treatment by continuous generation. In fact, the im- mersion of vegetables in water presaturated with ozone determined the reduction of 0.5 log of CBT after 15 min, whereas the immersion for the same time in continuously ozonated water involved the reduction of 2 log of CBT, showing in this latter case a greater effectiveness of ozon- ization with respect to chlorination. In fact, the reduction from 1.5 to 2.5 log units of the microflora of the lettuce was obtained with ozonated water with 1.5-3 ppm; this ef- fect was comparable to the use of 100 ppm of chlorine 63 Galgano et al., Application of ozone in fresh-cut iceberg lettuce refrigeration (Ölmez and Kretzschma, 2009). Furthermore, ozone treatment did not adversely affect product color (Fig. 3 and 4), as also confirmed by two- way analysis of variance; in fact, a non significant effect of ozone treatment and storage time was recorded for the col- orimetric indexes. It is well known that ozone is a strong oxidizing agent, which can induce damage to the structure and may influence the color of the products depending on the time of contact, the concentrations applied, and the type of product (Horvitz and Cantalejo, 2014; Olmez et al., 2009). However, in the present study, at concentrations of 0.2 ppm of gaseous ozone on lettuce stored at 4°C, no difference was detected relative to colorimetric parameters L *, a *, and b * between the control and the product treat- ed with ozone has (Fig. 5 and 6). 4. Conclusions The microbiological results obtained at different stor- age times have shown that the use of ozone is effective in containing microbial growth during storage of the raw material compared to the untreated control. In particular, the positive effects of ozonation were appreciable after the third day of storage. Moreover, ozone treatment did not affect the color of the product. In order to better assess the Fig. 1 - Evolution of total mesophilic bacteria during storage at 4°C. For each storage time, data followed by different letters are signifi- cantly different (t- Student test at p<0.05). Fig. 2 - Evolution of total coliforms during storage at 4°C. For each storage time, data followed by different letters are significantly different (t- Student test at p<0.05). Fig. 3 - Lettuce not treated with ozone after 3 days of storage at 4°C. Fig. 4 - Lettuce treated with ozone after 3 days of storage at 4°C. Fig. 5 - L*, a* and b* values determined on the upper leaf surface. 64 Adv. Hort. Sci., 2015 29(2-3) 61-64: treatment effect of ozonation on lettuce, studies consider- ing material with a higher degree of microbial contamina- tion, or inoculated with microorganisms that have not been detected even in the thesis used as a control in this experi- ment, should be undertaken. Acknowledgements The authors would like to thank Società Agricola ”Rago” for supplying the lettuce samples. The work reported in this paper was presented at the “POSTHARVEST2014 Reducing Postharvest Losses to Feed the World Congress” held in Barletta, Italy, on 22-23 May 2014. 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