ozone is a gas that is naturally present in the stratosphere ; it is water - soluble and has a high oxidative power . the latter characteristic has been positively considered since ozone was discovered by van marum in 1783 and it has been used thanks to its bactericidal , fungicidal and virucidal activities . ozone toxicity towards cells is due to its decomposition to oxygen ; during this transformation , direct and indirect ( via hydroxyl radicals formation ) substrates oxidation and peroxidation take place , leading to the alteration of both the structure and the functionality of biomolecules ( khadre et al . , 2001 ; laisk et al . , 1989 ; sarti et al . , 2002 ; staehelin and hoigne , 1985 ) . in particular , it is used for disinfection of water ( bottled water , potable water networks and swimming pools ) , air , wood , several foodstuffs ( in particular fruits and vegetables ) and surfaces that are intended to be in direct contact with food . the application of ozone does not leave any residue in the environment or on treated substrates , thanks to its natural transformation into oxygen . it has been defined as a generally recognised as safe agent by the usa food and drug administration , but the toxicity towards workers must be taken into account , so safety measures must be carefully applied . the antimicrobial efficacy of ozone is influenced by environmental conditions , mainly temperature , humidity and ph values . the best microbicidal action is obtained in environments with low temperatures and presence of water or high relative humidity . sensitivity of bacteria to the action of ozone is variable : gram positive bacteria are more susceptible than gram negative bacteria , due to the different cell wall composition and the resulting sensitivity to lipid peroxidation , while bacterial spores are strongly resistant to the action of ozone ( khadre et al . , 2001 ; khadre and yousef , 2001 ; kim et al . , 1999 the mechanism of virus inactivation is not completely cleared , but it is known that higher ozone concentrations are required to exert a virucidal effect ( kim et al . , 1999 ) . as regards fungi , several authors evidenced the efficacy of gaseous ozone as inactivating agent against spores . since 1951 , moulds growth prevention on cheese surfaces during ripening was demonstrated with a dose equal to 1 ppm ( cnsa , 2010 ) . a study conducted on cheddar cheese in canada ( gibson et al . , 1960 ) showed that the exposure to an ozone concentration of 3 - 10 ppm resulted in moulds darkening on cheese surface , associated to a 96% reduction in air moulds concentration in ripening rooms . the application of lower ozone concentrations ( 0.2 - 0.3 ppm ) was sufficient to obtain a significant reduction of moulds , with no modification of sensory characteristics of cheese . during the 70s , these findings were confirmed , identifying a 10 ppm ozone concentration as efficient for spore inactivation ( cnsa , 2010 ) . the inhibitory activity of ozone against yeasts has been evidenced by several authors , both in vitro and on different foods . yeasts are naturally more resistant than bacteria to the action of ozone , thanks to their thicker cell wall , but they are clearly more susceptible than moulds ( kim et al . , 1999 ; moore et al . , 2000 ) . a different sensitivity has been observed among different yeast species ; for example candida spp . and saccharomyces spp . watanabe et al . , 2010 ) . considering the growing diffusion of ozone as disinfectant in food industries , this study aimed to evaluate the antimicrobial efficacy of gaseous ozone on potentially contaminating fungi and yeats in a meat deboning and processing plant , in real working conditions . the efficacy of gaseous ozone was tested in a deboning and processing industry in which vacuum packaged beef meats are produced . the plant is equipped with a centralised , medium frequency corona discharge ozone generator , with a production capacity of 2 - 20 g / h . our evaluation was performed in the deboning room , with a surface of 220 m and 4 m high . gaseous ozone was supplied by the refrigeration sock ducts for 4 h during the night ( from 11.00 p.m. to 03.00 a.m. ) ; ozone amount was settled to obtain a constant environmental concentration of 20 ppm , revealed by a fixed probe . with the aim of detecting the eventual presence of autochthonous moulds in the environment , the contamination of air was evaluated by keeping open petri plates with m2 ( composition : malt extract=20 g / l , nacl=50 g / l , agar=15 g / l ) culture media ( dragoni et al . , 1997 ) in different points of the room for 2 h before every test session . after the exposition , the plates were collected , transferred to the laboratory and then incubated at 25 and 35c for 7 - 10 days . for inactivation tests , three moulds strains , namely mucor racemosus , penicillium roqueforti and aspergillus niger , and one yeast strain , i.e. saccharomyces cerevisiae , from the collection of the laboratory of mycology , department of health , animal science and food safety , university of milan , italy , were selected , in view of their wide diffusion in meat production and storage plants ( dragoni et al . , 1997 ) . each strain was grown on m2 ( m. racemosus , p. roqueforti and s. cerevisiae ) or m5s5 ( a. niger ) petri plates , incubated at 30c for 2 days ( s. cerevisiae ) , 25c for 7 - 10 days then , a portion of mycelium from each strain was collected by sterile loop and inoculated onto petri plates with their specific culture . for s. cerevisiae , a suspension with a known concentration ( settled by optical density determination with spectrophotometer ) was prepared in a diluent solution ( nacl / triptone , 0.85% ) ; decimal dilutions were inoculated onto m2 petri plates . the inactivation of selected fungal strains , inoculated onto culture media and submitted to gaseous ozone was used as index to evaluate the efficacy of ozonation treatment . after the usual cleaning / disinfection procedures , plates were placed with opened covers , in different points of the deboning room . three positions ( identified as 1 , 2 and 3 ) were chosen , based on their distance from the refrigeration sock ducts , and three points at different heights ( identified as l=10 cm ; m=110 cm ; h=270 cm ) for each position , in order to evaluate the vertical stratification of ozone , as it is characterised by a high molecular weight and tends to accumulate in lower layers . after the overnight ozonation , the plates were collected , transported to the laboratory and incubated as described previously . for each selected strain , the same inoculation procedures were performed by petri plates used as control ; after the inoculum process , such plates were refrigerated at + 2c up to collect all exposed test plates , then they were incubated in the same conditions . a total of 6 analytical sessions were performed , during different working weeks ; for each session , the test was performed twice . the inhibitory activity of ozone was evaluated as follows : for moulds , any slow growth , or its absence , was evidenced as difference in mycelium diameter in treated plates when compared with controls . considering s. cerevisiae , the logarithmic decrease of microbial count in treated plates compared with controls was determined . these data were submitted to a mixed anova by sas / stat package version 8.0 ( sas inst . position and height of sampling points were identified as fixed factors , while analytical session was considered as random factor . the evaluation of the concentration of autochthonous moulds in the environment revealed a mean count of 5.6 cfu / plate ; moulds belonged to the genera aspergillus , penicillium , alternaria and mucor ; this presence was expected as these genera are extremely common and are widespread in food production environments . the night distribution of gaseous ozone did not exert any detectable inhibitory action against the selected mould strains in the usual production conditions . an optimal moulds growth on the treated plates was observed , if compared with the control plates in all the analytical sessions . no difference was revealed among the petri plates placed in different positions or at different heights . unlike moulds , an evident microbicide effect against s. cerevisiae was observed in all the analytical sessions ( table 1 ) ; the statistical analysis revealed a highly significant ( p<0.01 ) decrease in treated plates against the control . in 96.3% of the replicates a fall in the counts > 2 log was detected with a mean value of 2.8 log ( standard deviation=0.6 ) . the inhibitory action was evidenced in all the selected points of the room ( figure 1 ) ; no significant difference was detected among the three positions ( 1 - 2 : p=0.91 ; 1 - 3 : p=0.72 ; 2 - 3 : p=0.81 ) , or the different heights ( h - m : p=0.91 ; h - l : p=0.64 ; m - l : p=0.57 ) , revealing the uniform distribution of gaseous ozone within the treated environment . thus , we can suppose that the vertical stratification of ozone , due to its high molecular weight , could be contrasted by constant air flow supplied by the sock ducts placed near the ceiling . although the antimicrobial efficacy of ozone has been evidenced by several authors , various studies have already shown a lack of this activity against moulds . ( 1999 ) reported an increase in botrytis cinerea prevalence on strawberries treated with 0.35 ppm of ozone for 4 days ; the development of botrytis cinerea , mucor piriformis and rhizopus stolonifer on grapes was not reduced by a 0.3 ppm ozone treatment for 7 days ( palou et al . , 2002 ) and similar results were obtained with similar tests on apples , blueberries , cranberries and melons ( rice et al . , 1982 ) . these differences in experimental results can be ascribed to the different methodological approaches and to the variable functionality of different ozone generators and distribution systems ( de alencar et al . , 2012 ; kim et al . , 1999 , 2003 furthermore , it has to be considered the critical role played by environmental conditions , such as humidity and temperature , on antimicrobial efficacy of ozone , as it is known that also little variations in these parameters can significantly modify its activity . the natural moulds resistance , due to their cellular constituents , must also be considered in evaluating ozone application ; the structure of mould cell wall , unlike bacterial cells , is characterised by a very low lipid content ( < 5% ) and it is consequently less susceptible to oxidising agents ( dragoni et al . , 1997 ) . in many cases , 2011 ) observed that the dose needed to remove penicillium digitatum , penicillium italicum and botrytis cinerea from table grapes was also dangerous for workers . italian legislation allows a maximum exposition dose of 0.1 ppm of ozone within a 8 h working period , or a 0.3 ppm dose for max 15 min , twice a day ( lgs . , many authors indicated a higher sensitivity to ozone treatments than moulds ( naitoh and shiga , 1982 ; kim et al . , 1999 ) , but evident evident variability in ozone susceptibility has been observed among different yeast species ( kim et al . , 1999 ) . the efficacy of ozonisation assessed in our study towards saccharomyces cerevisiae confirmed the results of previous studies and it indicated a marked susceptibility of saccharomyces spp . to such treatments ( guzzon et al . the results obtained confirm the absolute need for an accurate definition of the treatment conditions with ozone in order to maximise its antimicrobial effect in real working situations , considering the treatment parameters ( ozone dose and supply modality ) in combination with environmental parameters , such as temperature , humidity and air flow .
ozone is already known as effective food / environmental disinfection agent , thanks to its oxidative action towards microbial cell components . 
 bactericidal effect of ozone is well documented , while data concerning its inhibitory activity towards fungi are still ambiguous . 
 our study aims to evaluate the antimicrobial activity of gaseous ozone towards potentially contaminant fungi in a meat production plant in real working conditions . 
 m2 and m5s5 plates were inoculated with aspergillus niger , penicillium roqueforti , mucor racemosus , saccharomyces cerevisiae strains and positioned in a deboning room , where gaseous ozone was dispensed throughout the night ( until a maximum concentration of 20 ppm ) . 
 nine different points were chosen , based on height and distance from the ozone dispenser . 
 after the treatment , the presence of strains growth was evaluated . 
 gaseous ozone did not show any inhibitory activity against mould strains , as optimum growth during all the trials was observed . 
 an appreciable and constant microbicidal effect against s. cerevisiae was evidenced , with a mean value of 2.8 log reduction . 
 our results suggest the importance of the definition of environmental and technical use conditions in order to optimise the antimicrobial efficacy of ozone in real working situations in food industries .