THE EFFECTS OF GASEOUS OZONE TREATMENT ON THE SAFETY AND SHELF LIFE OF BERRIES

Maria Paola Previdi 1 , Rita Fiori 2 , Nicolo Dallacasa 3 , Gabriele Fortini 1 and Barbara Franceschini 1 *. 1. Stazione Sperimentale per l‟Industria delle Conserve Alimentari, SSICA, Viale F. Tanara, 31/A, 43121 Parma, Italy. 2. Via IX Luglio, 4243040 Varano dè Melegari (PR). 3. Via Ugo la Malfa 8, 43123 Parma. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Tests at high ozone gas concentrationswere carried outin a Plexiglas chamber equipped with a fan to homogeneously diffuse gaseous ozone. The ozone gas concentration was monitored and controlled using a chemical detector (Model Regulator 100 ppm, MET Company s.r.l., Bologna, Italy).
Tests at low ozone gas concentrations were carried out in a refrigeratorequipped with an ozone gas generator (Model 200 mg/h, Mosquito, MET Company s.r.l. Bologna Italy). For the control condition, an identical refrigerator without an ozone gas generator was used. Trials were carried out in high humidity (RH>90%) that was checked using a hygrometer (Model 174H, Testo Spa, Milano, Italy).

Microorganisms and sample inoculation:-
The following microorganisms were used for berry contamination:  Escherichia coli SSICA E21 from freshmeat  Escherichia coli ATCC 25922 from clinical isolate  Listeria innocua ATCC 33090 from cow brain  Listeriainnocua SSICA F1 from frozen mushrooms  ListeriainnocuaSSICA 8 from frozen peas The microorganisms were cultured at 37°C for 24 h in Brain Heart Infusion (BHI, Oxoid, Basingstoke, UK).
A mixture of L.innocua or E.coli strainswas prepared by combining 2 ml of each culture, washing by centrifugation at 4000 rpm for 10 minutes (Model Megafuge 11R, AhsiS.p.a., Milano, Italy) and suspending the mixture in a saline solution.
To inoculate the berry samples (30 g), 150 µl of the microorganism suspensionswere depositedas a droplet on the skin of the berries. The berries were left in a laminar flow cabinet for 1-2 hours in order to allow the microorganisms to attach. The concentration of both bacterial species ranged from 10 4 to 10 5 cfu/g. Ozone gas treatments:-Gaseous ozone treatments at room temperature (100 ppm):-To evaluate the effects of 100 ppmozone gas treatment on native microflora, samples of blackberries and blueberries (50 g) were transferred toaluminium-perforated baskets and placed in a Plexiglas chamber equipped with an ozone gas generator. The exposure times were 15, 30, 60 and 90 minutes. Tests were performed in triplicates at room temperature. In the evaluation test on pathogen survival, the same ozone gas concentration was tested against L. innocua and E.coli mixtures that were used to contaminate the 30 g berry samples. The samples were treated for 15 and 30 minutes.
Gaseous ozone treatments at low temperatures (0.3 ppm and 1 ppm):-Each sample of blackberries and blueberries (50 g)was placed in an open plastic basket and stored in the refrigerator with continuous ozone gas exposure. For comparison, other fruit samples were placed in an identical refrigerator without ozonegas exposure. At increasing exposure times, samples were collected from therefrigerators and analysed. Tests were carried out in triplicate under two different conditions: 0.3 ppm at 4°C for up to 20 days and 1 ppm at 8°C for up to 7 days. For the test on pathogen survival, treatments with 1 ppmozone gas at 8°C were also carried out on 30 g berry samples that were inoculated with L. innocua and E. coli mixtures. After 24, 48 and 72 h exposure times, three samples from each refrigerator were collected and counted.
Microbiological analysis:-Treated berries (30 g) were placed in a sterile-filtered stomacher bag and diluted to 1:10inbuffered peptone water (ISO -APT). Microbial counts were performed using a plate count technique on the Listeria Oxford Agar Base 18 (MOX) for L. innocua(37°C for 48 h), TBX medium for E. coli (44°C for 24 h), and Malt Extract Agar (MEA acidified to pH = 4.0 with citric acid 50% w/w) for yeast and mould (25°C for 5 days). All media were purchasedfrom Oxoid (Basingstoke, England).

Statistical analysis:-
Microbial counts were transformed to logarithms before means and standard deviations were computed, and counts were reported as Log cfu/g. The statistical elaboration of the results was performed using SPPSS 13.0.

Results and Discussion:-
The microbial population on berries mainly consists of yeast and mould, so the effects of gaseous ozone on these microorganisms were tested.
Yeast and mould populations resulted in floating, especially on blackberries. Sometimes a high mould concentration (10 5 cfu/g) together with a low yeast concentration (it was not possible to enumerate the yeasts because of overgrowth of MEA plates by moulds) was found. At other times, the latter microorganisms were predominantly expressed. In blueberries, both fungi were detected at concentrations between 10 3 and 10 4 cfu/g.
Microbial concentrations were correlated with the ripeness and integrity of the fruit. In fact, lower consistency, bruising and mechanical damage increase microbial proliferation (EFSA 2014).

Gaseous ozone treatments at room temperature (100 ppm):-
The results of these trials were carried out with a high ozone gas concentration, as shown in Table 1. Treatment with a 100ppm ozone gas concentration for 15 minutes resulted in less than 1Log reduction for yeast and mould, on both blueberries and blackberries. When the treatment timewas increasedto up to 30 minutes, a 1.5 Log reductionwas observed for mould on both berries and for yeast on blueberries.
A further increase in treatment time (60 min) did not significantly affect microbial population reduction. A 90minute treatment time resulted in a2Log reduction in mould on both berries, while no significant differences were observed for yeast on blueberries compared to the 30 minutes treatments.
The results indicated that a 30-minutes treatment improves the microbiological quality of fresh blueberries and blackberries, a treatment time suitable for production requirements.

Gaseous ozone treatments at low temperature (0.3 ppm and 1 ppm):-
The results of trials with low ozone gas concentrations are shown in Figures 1 and 2.
For the trials carried out at 4°C with 0.3ppm of ozone gas, the continuous ozone gas exposure did not significantly affect fungi concentration within twenty days of storage in both berries. A slight increase in the mould concentration in blueberries stored without ozone gas was observed, while the yeast concentration did not significantly change. Barth et al. (1995) reported that a continuous exposure of blackberries to 0.3-ppm ozone at 2°C for 12 days inhibited fungal development, while 20% of control fruits showed decay. Palou et al. (2002) observed that continuous exposure to ozone at a concentration of 0.3 ppm for 4 weeks at 5°C, inhibited the growth of mould on "Elegant Lady" peaches; however, aerial growth and sporulation resumed in ambient atmospheres.
In the present study, the trials carried out at 8°C with 1ppm ozone gas exposure to both berries, did not change the fungi concentration after 7 days of storage. The slight variations in fungi population observed during storage were probably due to different contamination rates of fruits, as seen by the high standard deviation values.
Pathogen inactivation:-Trials were carried out using a mixture of three L. innocua strains (gram-positive bacteria) and another mixture of two E. coli strains (gram-negative bacteria);

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L.innocuawas chosen as a non-pathogenic indicator of L. monocytogenes due to its phylogenetic proximity (Fan et al. 2007;Fairchild and Foegeding 1993).
According to the Regulation EC No.2073/2005 on microbiological criteria for foodstuffs, berries are considered 'ready-to-eat" do not allow the growth of L. monocytogenes(due to their low pH). Thus, its concentration during the shelf life of berries must be less than 100 cfu/g. E. coli is commonly present in faecal material and is widely used as a hygiene indicator. The hygienic microbial criteria for E.coli in "ready-to-eat" pre-cut fruits and vegetables are between 100 and 1000 cfu/g (Regulation EC No. 2073/2005).

100ppmozone gas treatment:-
The efficacy of 100ppm gaseous ozone treatment on reducing contamination from theL.innocua and E.colimixture was comparable to that observed on the native microflora in berries.
A 15-minutes treatment resulted in a slight reduction of both L.innocuaand E.coliinoculation on blackberries and blueberries. A 30-minutes treatment resulted inmore than one logarithmic decrease (ranged from 1.22 to 1.85 log reductions) on both fruits ( Table 2).
In a previous study (Previdi et al.2011) it was observed that ozone gas dissolved in sanitizing water was more effective against inoculated cells than native microorganisms. Native microbiotaare mainly comprisedof fungi and eukaryotic cellsthat provide defence mechanisms, such as the production of waxy cuticles, biofilm formation. These cells can be protectedby cracks or cuts on fruits. The artificially inoculated bacteria, (prokaryotic and much more simple cells), that underwent to ozone gas exposure for 1-2 hours after inoculation, could presumably have lower chances to stick to a substrate; in the present study, gaseous ozone treatment resulted in the same degree of microbial inactivation.
The decimal reductions achieved in the present study were lower than those reported by Bermúdez-Aguirre et al. (2013), where more than 2Log reductions were obtained on tomatoes artificially inoculated with E. coli and treated for 3 minutes with 5 ppm of ozone gas. However, the same treatment on carrots and lettuce achieved less than 1Log reduction compared to the aforementioned microorganism.
In literature it wasreported that the higher resistance observed in Gram-negative bacteria could be due to a high phospholipid content, which results in increased cell wall rigidity, and provides extra protection for the cell membrane against ozone gas. Despite the presence of a peptidoglycan layer in L. monocytogenes cell walls, this Gram-positive bacterium proved to be very susceptible to ozone gastreatment (Alwiet al. 2014).

ppmozone gas treatments:-
The efficacy of 1ppmgaseous ozone in reducing the population of L. innocuaand E.coli inoculated on berries that were stored at 8°C are shown in Figures 3 and 4.
Within three days of storage, ozone gas treatment reduced L. innocua population by 3logarithms in blackberries and 1.88 logarithms in blueberries compared to time zero. Additionally, 1.88 and 1.29 logarithm reductions in blackberries and blueberries, respectively, were observed, compared to the inoculated, untreated samples. Concha-Meyer et al. (2014) studied the survival of L.monocytogenes in fresh blueberries stored at 4°C with 4ppmozone gas and at 12°C with 2.5-ppm of ozone gas: the ozone gas treatments achieved 3 and 2 logarithm reductions, respectively, when compared with air treatment.
In the present study, we observed that storage for several days in the refrigerated condition in the presence of 1 ppm of ozone gas, maintained good sanitizing action against Listeria.

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Exposure to 1 ppm of gaseous ozone at 8°C on the inoculated fruits with E. coliresulted in no significant differences between treated and untreated fruits after three days of storage ( Figure 4).

Conclusions:-
A critical point for the microbiological safetyin the processing line of ready-to-eat vegetables is the washing step. Several studies have investigated the antimicrobial activity of different compounds and have indicated that a log reduction of more than 2.0of naturally present microorganisms is rarely achieved (Tomasoniet al. 2001;Guerzoniet al. 1996;Gras et al. 1994). Therefore, the observations from the present study are in agreement with the previousstudies that used gaseous ozone as a sanitizer: a 100ppm ozone concentration for 30 minutes resulted in a 1.5 Log reduction in mould on both types of berries and in yeast on blueberries. Similar results were observed forL. innocua and E.coliinoculation of both types of berries.
Storage for three days in refrigerated condition in the presence of 1 ppm of ozone gas resultedin the sanitizationofL.innocua(3Log reduction in blackberries and 1.88Log reduction in blueberries).
Furthermore, because berries are generally not washed and are stored and transported under refrigerated conditions, ozone gastreatment could improve the microbiological quality of fresh blueberries and blackberries.