The genus Pseudomonas includes bacteria that are able to cause several alterations in milk and dairy products. Specifically, Pseudomonas spp. are responsible for undesirable odors and flavors as well as unusual pigments of foods [61]. These microorganisms are ubiquitous; therefore, they are usually isolated at different production stages in the dairy environment. Species more commonly isolated from dairy plants are P. fluorescens, P. koreensis, P. marginalis, P. rhodesiae, P.fragi, P. putida, P. entomophila, P. mendocina, and P. aeruginosa [62]. Even if these bacteria are sensitive to thermal treatments commonly used in dairy processing, thermostable enzymes, such as proteases and lipases, could persist after treatments causing spoilage in finished products [33]. Several studies demonstrated that Pseudomonas isolates from milk, dairy products and dairy processing environments are able to form biofilm. In this regard, a recent study highlighted the relationship between biofilm formation abilities and the production of blue pigment of P. fluorescens dairy-related strains [63].

Several studies demonstrated how microorganisms belonging to this genus are generally susceptible to ozone exposure even when they are attached to common surfaces or organized in biofilms. As early as 1993, Greene et al. [94] showed that ozonated (0.5 ppm) water treatment (10 min exposure) was effective in reducing the loads (~4 Log10) of common psychrotrophic spoilage bacteria, including P. fluorescens and Alcaligenes faecalis, on stainless steel surfaces, meanwhile highlighting that the effect of this technology was better performing than the commercial chlorinated sanitizers used in high concentration (100 ppm). Similarly, Dosti et al. [95] reported the effectiveness of ozone treatment (0.6 ppm for 10 min) on P. fluorescens (ATCC 948), P. fragi (ATCC 4973), P. putida (ATCC 795), Enterobacter aerogenes (ATCC 35028), E. cloacae (ATCC 35030) and B. licheniformis (ATCC 14580) on stainless steel coupons. The sensitivity of P. fluorescens at ozone treatments was also shown by Marino et al. [8], which demonstrated the effectiveness of ozonated water (0.5 ppm) applied in static as well as dynamic conditions on biofilms. The authors also reported that ozone in gaseous form (20 ppm) led to a reduction of 5.51 Log CFU/cm² after 60 min treatment. Khadre and Yousef [53] studied the effect of ozone on bacterial biofilms and dried films of B. subtilis spores and P. fluorescens in a multilaminated aseptic food packaging material and stainless steel. Ozone inactivated P. fluorescens in biofilms more effectively on stainless steel than on the multilaminated packaging material. Shelobolina et al. [96] studied the effect of dissolved ozone (2, 5 and 7 ppm for 10 and 20 min) on P. aeruginosa biofilm grown on glass. The regression equation, used to analyze the effect of ozone, highlighted that biofilm inactivation was correlated to the concentration and the contact time (predicted D-values: 11.1, 5.7 and 2.2 min at 2, 5 and 7 ppm, respectively). The same authors studied the inactivation of biofilms on various surfaces by dissolved ozone (5 ppm for 20 min). The outcomes emphasized that biofilms grown on ceramics were more difficult to inactivate than those grown on plastic materials. Ozone can also be effective on Pseudomonas biofilm in combination with other technologies. For example, ozone water in combination with a hydrogen peroxide solution was effective on P. fluorescens biofilm. In this regard, a sequential treatment with 1.0 and 1.7 mg/L of ozone followed by 0.8 and 1.1% of hydrogen peroxide showed synergistic disinfection effects [97].

Among spore-forming bacteria, the genus Bacillus is of high importance, since it includes bacteria that can cause spoilage in milk and dairy products, as well as foodborne pathogens. Bacillus species are ubiquitous, Gram-positive, motile, and rod-shaped bacteria, characterized by high versatility and adaptability to different environmental conditions and can survive during the different stages of processing and manufacturing of dairy products [64]. The most common species found in dairy environments are B. licheniformis, B. cereus, B. subtilis, B. thuringiensis, B. weihenstephanensis, B. mycoides, B. sporothermodurans, and B. megaterium [64,65,66]. Bacillus are able to adhere and persist on different surfaces; in addition, they can form other biofilm types, including bundles in the liquid phase and pellicles at the air-liquid interface [67,68]. Additionally, Bacillus can form heat-resistant spores that can survive after the routinary pasteurization processes.

Different studies have emphasized how ozone is effective on biofilms formed by dairy-related Bacillus. A recent researchdemonstrated the efficacy of gaseous ozone treatment (45 ± 2 ppm) on B. cereus biofilms formed on stainless steel and polypropylene [98]. Another study evaluated the effect of ozonated water on B. cereus biofilms grown on dairy processing membranes and highlighted an average reduction of 1.0 Log CFU/cm² for treated membranes [99]. The efficacy of ozone, in combination with cleaning in place reagent (NaOH), was shown on biofilms formed by B. subtilis and B. amyloliquefaciens on stainless steel. Higher inactivation of biofilms (60 and 120″) was obtained with 1.4 ppm of ozone coupled with 1% NaOH as compared to NaOH alone, which required 240 s to completely remove the film from the stainless steel coupons [100].

This genus comprises one of the most studied bacteria worldwide, that is L. monocytogenes. L. monocytogenes is a Gram-positive foodborne pathogen. This pathogen, when organized in established biofilms, can persist over a long period of time on surfaces and food processing environments, thus representing a potential cause of repeated contaminations of the finished products [74,75,76]. Detection of biofilm forming and persistent L. monocytogenes strains in the dairy environment was reported in several studies [45,46]. It has been demonstrated that adhesion capacity and biofilm formation abilities differ among several L. monocytogenes strains. This strain variability seems to be linked with the presence of specific genes and/or accessory genetic elements, such as phages, plasmids and stress survival islets [26,77].

Several experiments have been performed so far on L. monocytogenes biofilm, highlighting that high ozone levels and long exposure times are needed to achieve an effect against biofilms formed by this bacterium. Korany et al. [101] reported that ozonated water treatment (1 min at 1.0, 2.0 and 4.0 ppm) resulted in ∼0.9, 3.4 and 4.1 log reduction of L. monocytogenes single strain biofilm on polystyrene, but the effect was lower with multi-strain biofilms and in the presence of organic matter. Robbins et al. [102] obtained a complete elimination of attached L. monocytogenes Scott A and 10403S strains cells after exposure to 4 ppm of ozone. Nicholas et al. [103] reported a mean reduction of 3.41 Log10 CFU/cm² for stainless steel-attached L. monocytogenes cells after 1 h treatment at 45 ppm of gaseous ozone, but the same strains organized in biofilm were significantly more resistant after a treatment with ozone gas at 45 ppm for 1 h. Harada et al. [104] demonstrated the efficiency of gaseous ozone (45 ppm) as a dry sanitizing method on L. monocytogenes. The authors observed a reduction of sessile cells below the limit of detection (1.7 Log CFU/cm²) in 5 min on polypropylene, while a reduction of 3.4 Log CFU/cm² was observed in stainless steel. A recent experiment conducted on dairy- and meat-related L. monocytogenes showed that ozone gas in high concentrations (50 ppm for 6 h) caused a significant decrease of the biofilm biomass for 59% of the strains tested, but only a slight reduction of live cells in the formed biofilm was observed [26]. De Candia et al. [21] demonstrated the efficacy of cold gaseous ozone treatments at low concentrations in the eradication of L. monocytogenes from different food contact surfaces (glass, polypropylene, stain-less steel, expanded polystyrene). A continuous ozone flow (1.07 mg m⁻³) after 24 or 48 h of cold incubation resulted in the inactivation of 11 strains, while with higher inoculum levels (9 log CFU coupon⁻¹) the best inactivation rate was detected after 48 h of treatment at 3.21 mg m⁻³ of ozone on stainless steel and expanded polystyrene. Baumann et al. [105] tested ozone (concentrations of 0.25, 0.5 and 1.0 ppm) in combination with power ultrasound cycled through 250 mL of a potassium phosphate buffer containing L. monocytogenes biofilm chips for 30 or 60 s. Reductions obtained with the combined treatments were significantly (p < 0.05) higher than each treatment alone. No recoverable cells were detected (reduction = 7.31 Log CFU/mL) after 60 s of the combined treatment when ozone was used at a concentration of 0.5 ppm.

S. aureus is deemed as an important pathogen detectable in dairy products. Several studies showed how this bacterium can form biofilm in the dairy environment. Lee et al. [78] studied the biofilm production abilities of strains isolated from milking parlor environments on dairy farms in Brazil. Around 45% of S. aureus pulsotypes were able to form biofilms in at least one assay, suggesting their possible persistence in milking environments. Biofilm forming abilities were demonstrated also for dairy-related S. aureus isolated from Switzerland and Italy, including methicillin-resistant S. aureus (MRSA) [79], and for strains isolated from food-contact surfaces in dairy industries in Mexico [80].

S. aureus is generally sensitive to ozone exposure. Cabo et al. demonstrated that the application of 1 μg/g of ozonized water allowed 99% inactivation in 2 min of S. aureus CECT4459 biofilm on polypropylene [106]. Shao et al. [107] studied the effect of ozone water on mature S. aureus and Salmonella spp. biofilm, detecting less than 0.8 Log cfu/cm² of cells reduction in biofilm exposed to ozonized water for 20 min. In the study performed by Marino et al. [8], S. aureus was highly responsive to aqueous ozone treatments at dynamic conditions, while exposure to gaseous ozone at high concentrations (20 ppm) resulted in a reduction of 4.72 Log CFU/cm² of S. aureus biofilm. A recent study investigated the effect of ozonated oils with concentrations ranging from 0.53 to 17 mg/g on Methicillin-resistant S. aureus (MRSA) biofilm; most strains were inhibited at concentrations of 4.24 mg/g. Additionally, ozonated oils showed ability in removing adherent cells and high capacity in the eradication of 24 h biofilms [108].

The genus Salmonella includes well known pathogenic bacteria which can be found in different types of foods. Historically, several Salmonella outbreaks were linked to the consumption of dairy products, especially raw milk products [81,82,83]. Bacteria of this genus, indeed, can persist in fresh and fermented dairy products for their adaptation to an acid environment. Leyer and Johnson [84] demonstrated that acid-adapted S. typhimurium cells had increased resistance to organic acids usually present in cheese, such as lactic, propionic, and acetic acid. In a study of Kessel et al. [85], Salmonella was isolated from 36 of 75 PCR-positive bulk tank milk samples and 105 of 174 PCR-positive milk filter samples. Lamas et al. [86] proved that milk residues are a source of nutrients for S. enterica biofilm formation on stainless steel and that the biofilm forming abilities of this bacterium are strongly related to oxygen levels.

Few studies exist about the effectiveness of ozone treatments against biofilms formed by bacteria belonging to this genus. Shao et al. [107] reported a reduction less than 0.8 Log cfu/cm² of S. aureus and Salmonella spp. biofilm after exposure to ozonized water for 20 min. In another study [109], the effect of malic acid and ozone against S. typhimurium biofilm on different food contact surfaces (PVC pipes, polyethylene, plastic, and fresh produce) was explored. The mutual effect of malic acid with ozone resulted in a reduction of biofilm formation on plastic bags and PVC pipes. In microtiter plates, reductions in biofilm formation were observed after 20 h and 40 h treatments.

The genus Clostridium includes Gram-positive spore-forming anaerobes bacteria that can induce spoilage of dairy products by gas production arising from the fermentation of acetate, lactate, and butyrate [69,70]. C. tyrobutyricum is considered the species most frequently involved in cheese spoilage, as it is the causative agent of the so called “late blowing defect”, though C. sporogenes, C. beijerinckii, C. tyrobutyricum, and C. butyricum can also cause cheese alterations [69,70]. Additionally, this genus includes pathogenic bacteria, such as C. botulinum and C. perfringens. A recent study showed that dairy-related C. perfringens isolates were able to form biofilm at different temperatures (4, 25, and 35 °C) [71]. The genus Cronobacter comprises C. sakazakii, a relevant foodborne pathogen included in the food safety criteria for infant foods in the Regulation EC 2073/05 and amendments [110]. This bacterium is characterized by high adaptability to the dairy environment. This aspect was emphasized by Oh et al. [72], which studied the biofilm-forming abilities of 72 strains on plastic surfaces, as well as the influence of the artificial growth medium and infant milk formula (IMF). The diversity and biofilm forming abilities of Cronobacter isolated in New Zealand were investigated by Gupta et al. [73], which showed that adherence characteristics are related with nutrients and temperature. The genus Escherichia includes relevant foodborne pathogens associated with dairy products, such as the enterohemorrhagic E. coli O157:H7. Several studies demonstrated that this bacterium could survive in different types of dairy products, and E. coli O157:H7 outbreaks frequently occurred after consumption of unpasteurized cheese [87,88,89,90]. Sharma and Anand analyzed biofilms of pasteurization lines in a commercial plant and in an experimental dairy plant, revealing the presence of E. coli in both cases [91]. The high biofilm forming abilities of different Shiga toxin–producing E. coli (STEC) and the strong tolerance to common sanitizers led to concerns regarding the colonization of surfaces and the resultant downstream food contamination [92,93].

To the best of the knowledge, no relevant studies describing the effect of gaseous ozone against biofilms of these bacteria have been conducted, while the effect of ozone on the vegetative and spore forms is well documented. Foegeding [111] studied the effect of ozone on spores of C. perfringens NCTC 8798 and C. botulinum 12885A strains, highlighting how ozone was an effective sporicide, especially at acidic pH values. The action of ozone improved the efficiency of cooking temperatures (from 45 to 75 °C) against C. perfringens on beef surfaces [112]. Significant reductions of vegetative cells (from 5.59 ± 0.17 to 4.09 ± 0.72 and 3.50 ± 0.90 log CFU/g after treatments with aqueous ozone at 5 ppm and heating at 45 and 55 °C) were reported. Spores, indeed, were reduced from 2.94 ± 0.37 log spores/g to 2.07 ± 0.38 log spores/g and 1.70 ± 0.37 log spores/g after the treatments with 5 ppm of aqueous ozone and heating at 55 and 75 °C, respectively. Gaseous ozone was effective in the inactivation of Cronobacter in milk powders. A continuous stream of ozone led to a reduction of 2.71 and 3.28 log after 120 min at 2.8 and 5.3 mg/L⁻¹, respectively [113]. With regards to Escherichia, a study carried out in 2010 did not reveal any significative effect of ozone (2 mg/L) in removing E. coli and L. monocytogenes cells in biofilms on lettuce surfaces [114]. However, in the study of de Oliveira Souza et al. [115], ozonated water (35 and 45 mg/L⁻¹ for 0, 5, 15, and 25 min) was effective in inactivating E. coli O157:H7, while reductions of 1.5 log cycles were detected in lactose-free homogenized skim milk, indicating the influence of the substrate on the antimicrobial efficiency of ozone. Effectiveness of aqueous ozone treatment (5 mg/L) on Shiga toxin-producing E. coli inoculated in alfalfa seeds was demonstrated by Mohammad et al. [116], who reported mean log reductions of 1.5 ± 0.4, 1.6 ± 0.4, 2.1 ± 0.5 after 10, 15, and 20 min, respectively.