**4. Discussion**

Commonly used methods for egg disinfection include chemical agents, but adverse effects of high temperature on, e.g., fatty acids, vitamins or cholesterol, are widely known. However, increased consumers' awareness forced the researchers to search for new methods, effective against microbes and safe for the environment and public health. New technologies such as RCI can reduce the use of disinfectants, but are not intended to completely replace them. The combined use of different physical and chemical methods results in increased microbiological safety of eggs. RCI technology, for maximum effectiveness, requires adequate working time, the right time of contact of active air with egg shells and the appropriate distance [26]. Such devices could be placed directly in laying hen houses or in egg stores or in hatching apparatuses. Then the working time of the technology could be sufficiently long. This technology is safe for humans and animals and is designed for continuous operation. Research conducted on the influence of RCI technology on hatchability of eggs, weight of chicks and occurrence of developmental defects did not show significant differences compared to the control variant without the use of this technology (unpublished data).

In the current study, we determined the effect of RCI and ozonation on the eggshells contaminated with *Salmonella* spp. Both methods significantly reduced the bacterial number of all tested serotypes, but their effectiveness varied depending on the variant used.

The initial bacterial contamination seems to play a crucial role in the efficacy of tested technology. However, our study shows that both methods are efficient even at high bacterial density. In low bacterial contamination level (103 CFU log/egg) and shorter time exposure (30 min) RCI-technology was more effective, suggesting its application as the disinfection method in egg processing plants. Soljour et al. [27] indicated that sodium carbonate, sodium hypochlorite and potassium hydroxide applied at the recommended concentrations eliminate *S. enteritidis* from eggshells contaminated with 10<sup>4</sup> or 10<sup>6</sup> CFU/mL of the bacterial suspension.

Moreover, temperature, organic solution and exposure time of RCI or ozone largely affect the decontamination power. Our previous research showed the lowest reduction of bacteria number for surfaces contaminated with meat and fish pulp before the action of RCI (24 h, 0.5 m, 20 ◦C). The reduction rate was equal to 0.89 log CFU × cm–2 for *Staphylococcus aureus*, 1.17 log CFU × cm–2 for *Listeria monocytogenes*, 1.43 log CFU × cm–2 for *S. enteritidis* and 1.61 log CFU × cm–2 for *Escherichia coli* O157:H7 [26]. The influence of poultry manure on the survival of bacteria was noticeable in these studies. Each time

their addition decreased the effectiveness of the tested methods and increased the survival rate of *Salmonella* spp. Furthermore, Bing et al. [28] showed that feces protect bacteria on eggshells against the bactericidal effects of other methods, e.g., UV-C radiation.

In most cases, the effectiveness of RCI was higher than that of ozonation, but usually, these differences were not statistically significant. Mannozzi et al. [22] indicated the reduction of bacteria (*E. coli*, *S. typhimurium* and *Listeria innocua*) from the surface of apple peel and spinach leaves after 90 min of exposure to RCI. For cantaloupe researchers obtained reductions of 94% and 88% for *E. coli* and *S. typhimurium*, respectively. The effectiveness of the RCI was influenced by the operating time of the device and the type of surface [22].

In our study, the treatment with ozone (10 g/h) reduced bacterial number on the eggshell. This number decreased with the exposure time extension, in both variants of the bacterial suspension density and at both temperatures. On the contrary, Braun et al. [14] have reported complete inactivation of *S. enteritidis* (contamination level of 102–104 CFU/g) on the eggshell after 120 min treatment with 1% ozone. In turn, Rodriguez-Romo and Yousef [29] applying ozonation for 10 min (15 lb/in2 [103.421 kPa], 4 to 8 ◦C) reduced up to 5.9 log CFU/g of *S. enteritidis*. The discrepancy between our research and the studies discussed above can result from the different strains, experimental conditions and ozone concentration used. A fundamental aspect of the use of ozone for egg disinfection is its safety. Wlazlo et al. [17] have demonstrated that ozone treatment reduced hatching of eggs and significantly increased egg mortality. This finding may indicate the negative impact of this gas on developing embryos. Some studies indicated that exposure of hen eggs to ozone deteriorated the nutritive characteristics of the eggs (low amount of yolk tocols, carotenoids, cholesterol and lipid oxidative status) and lowered eggshell breaking strength [30,31].

The conducted study has a several limitations that should be supplemented in further studies. In next experiments, it is worth considering other bacteria that may also be present on the shells of hens' eggs (e.g., *E. coli*, *Campylobacter* spp., *Staphylococcus* spp., *Streptococcus* spp., *Yersinia* spp. and *L. monocytogenes*). It is also worth considering shorter exposure times, e.g., a few minutes. If satisfactory efficiency were demonstrated, the technology could be applied at stages other than egg storage. Valuable information would also be provided by the study of hatching eggs, including the impact of RCI technology on their hatchability as well as the health and survival of chicks.
