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Article

Effect of Ozonized Water against Pathogenic Bacteria and Filamentous Fungi on Stainless Steel

by
Elettra Berni
1,*,
Chiara Moroni
2,
Massimo Cigarini
1,
Demetrio Brindani
3,
Claudia Catelani Cardoso
3 and
Davide Imperiale
3
1
Microbiology—Quality, Safety and Pre-Industrialization Area, SSICA, Stazione Sperimentale per l’Industria delle Conserve Alimentari—Fondazione di Ricerca, Viale F. Tanara, 31/A, 43121 Parma, Italy
2
Dipartimento di Scienze Chimiche, Della vita e Della Sostenibilità Ambientale, Parco Area delle Scienze, Università degli Studi di Parma, 31/A, 43124 Parma, Italy
3
Environment—Sustainability and Valorization Area, SSICA, Stazione Sperimentale per l’Industria delle Conserve Alimentari—Fondazione di Ricerca, Viale F. Tanara, 31/A, 43121 Parma, Italy
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(18), 8392; https://doi.org/10.3390/app14188392
Submission received: 9 August 2024 / Revised: 13 September 2024 / Accepted: 14 September 2024 / Published: 18 September 2024
(This article belongs to the Special Issue Innovative Technologies for Food Preservation and Processing)
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Versions Notes

Abstract

:
Ozone is a molecule that has gained increasing interest in recent years by food industries for sanitization of food-grade surfaces. Compared to chemical sanitizers such as chlorine, hydrogen peroxide, or peracetic acid, ozone shows undeniable advantages, such as the absence of by-products that should affect human health or the possibility of generating it when needed. Therefore, the aim of this paper was the assessment of the resistance to ozonized water of two pathogenic bacteria (Listeria monocytogenes, Salmonella) and of three airborne food-spoiling fungi (Aspergillus brasiliensis, Hyphopichia burtonii, and Penicillium nordicum) inoculated on stainless steel tiles and treated in static conditions with 1 to 6 mg L−1 (pathogens) or 8.5 mg L−1 (filamentous fungi). Ozonized water gave different results based on the tested microorganisms: pathogenic bacteria proved markedly more sensible to ozone than filamentous fungi, even if great differences were observed at inter- and intra-specific levels for both categories of microorganisms. Nevertheless, the non-linear inactivation kinetics of the studied strains made the calculation of a punctual F-value difficult, so in industrial practice, adequate tailoring of the treatments to be applied, based on the registered extrinsic factors and the industrial bio-burden, would be appropriate.

1. Introduction

The food industry is always looking for alternatives to traditional chemical sanitizers, which can guarantee the safety of foods, both maintaining their chemical–physical properties as well as their microbiological stability and reducing the water consumption rates in large-scale processes [1]. Ozone, either in its gaseous or in its liquid form, is a sanitizing agent that has gained interest in recent years for such applications since it can be generated when needed and does not form appreciable levels of by-products (mainly hydroperoxide molecules and unsaturated aldehydes, but also substances such as dibromo-acetonitrile, which is formed when bromide levels are over 1 ppm) [2]. However, it has one of the highest oxidation potentials [for instance, it proved to be 1 to 2 times stronger than chlorine and significantly stronger (>3000 times) than hypochlorous acid] [3]. As a comparison, the use of chlorine is associated with the production of carcinogenic by-products, including trihalomethanes and halo-acetic acids [4], whereas ozone eventually decomposes it into non-toxic molecular oxygen.
Aqueous ozone has been known for over a hundred years as a sanitizing agent. At the beginning, it was used for the sanitization of drinking water, whereas in the last decades its usage has been extended to a wide range of activities like swimming pool disinfection, food industry processing, clinical medical applications, and even industrial laundry operations [5,6]. Nowadays, food industries use aqueous ozone to reduce microbial loads on wastewater [7,8] and a wide range of foods such as fresh fruits [9,10,11], dried fruits [12], nuts [13], vegetables [14,15,16], and meats [17,18]. Its use concentrations are usually up to 10 ppm for application times varying from some minutes to one hour, alone or in combination with other sanitizing methods. Concerning this aspect, the UV-C light is the most common coupling [8,19], even if the use of aqueous ozone in combination with chlorine [20], organic acids [21,22], or hydrogen peroxide [7] has also been registered.
Similarly, its use as a sanitizing agent on different types of surfaces has been deeply investigated. The papers concerning this topic were referred to as food-grade steel [23,24,25,26,27], multi-laminated food packaging [28], polyethylene terephthalate [29], polystyrene [30], and polypropylene [31]. Naitou and Takahara [32] even tested the effectiveness of ozonized water up to 30 ppm in inactivating microbes in a food-packaging film sterilizing machine. Unfortunately, most of the works supply punctual data without assessing the inactivation kinetics of the studied microorganisms; only the paper by Shelobolina et al. [33] assessed the D-value of the pathogen Pseudomonas aeruginosa on glass, ceramics, and medical industry-related plastics. In addition to this, filamentous fungi were not included in the group of investigated microorganisms when aqueous ozone was tested on materials, even if they can contaminate working surfaces and machinery interiors as well as bacteria.
Therefore, the aim of this paper was the assessment of the resistance to aqueous ozone of two pathogenic bacteria, Listeria monocytogenes and Salmonella spp., and of two airborne food-spoiling fungi, Hyphopichia burtonii and Penicillium nordicum, inoculated on stainless steel (SS) and treated in static conditions. The inactivation kinetics of the mycetes were also compared with those of Aspergillus brasiliensis ATCC 16404, which already proved to withstand physical stresses such as UV-C irradiation [34] or chemical stresses such as the gaseous ozone itself [35] and had been adopted by the European Standards (EN) as a target microorganism for checking the effectiveness of chemical disinfectants and antiseptics against microbes [36,37,38].

2. Materials and Methods

2.1. Microbial Strains

The tested microorganisms were the ones used in previous tests performed with gaseous ozone on SS by the same authors [35].

2.1.1. Bacteria

Among bacteria, the following strains were selected for aqueous ozone treatments:
  • Listeria monocytogenes (Murray et al.) Pirie ATCC 7644, isolated from a source not specified by the ATCC website.
  • Listeria monocytogenes Scott A (Murray et al.) Pirie ATCC 49594, derived from an existing strain.
  • Salmonella enterica subsp. enterica serotype Senftenberg NCTC 9959 (=ATCC 43845 = Col Sal 385/57 = DSM 10062 = JT 493; 775/W), isolated from a source not specified by the ATCC website.
  • Salmonella enterica subsp. enterica serotype Typhimurium ATCC 14028 (=CIP 104115 = DSM 19587 = CDC 6516-60 = NCTC 12023) isolated from the pooled heart and liver tissue of four-week-old chickens.

2.1.2. Filamentous Fungi

Among filamentous fungi, the following strains were selected for aqueous ozone treatments:
  • Penicillium nordicum SSICA 1169, isolated from dry-cured meat production environments in Italy;
  • Penicillium nordicum SSICA B4798, isolated from a fermented meat product in Italy;
  • Hyphopichia burtonii SSICA 175717, isolated from spoiled sandwich bread in Italy;
  • Hyphopichia burtonii SSICA 251105, isolated from a spreadable fat used to cover dry-cured hams during seasoning in Italy;
  • Aspergillus brasiliensis ATCC 16404, isolated from blueberries in North Carolina (USA).

2.2. Preparation of the Microbial Suspensions

Microbial suspensions were prepared according to the protocols previously used by the same authors [35].

2.2.1. Bacteria

Bacterial suspensions were prepared from strains preserved at −20 °C in porous beads (Cryoinstant; WVR, Milan, Italy). One bead for each strain was added to 10 mL of Tryptic Soy Broth (TSB—Oxoid, Cambridge, UK) and incubated at 37 °C for 24 h. After incubation, 1 mL of bacterial suspension was centrifuged at 4100 rpm for 15 min, and the pellet was resuspended in 1 mL of a physiological salt solution (8.0 g L−1 sodium chloride) to obtain a concentration between 107 and 108 CFU/mL. The suspensions were discharged just after their use and freshly prepared each time.

2.2.2. Filamentous Fungi

Fungal suspensions were prepared from strains preserved at −20 °C in glycerol–water suspensions. Each strain was defrosted and separately inoculated on Malt Extract Agar (MEA; OXOID, Cambridge, UK) and incubated at 25 °C for up to seven days. Conidia and mycelium were harvested with a sterile loop in a 0.1% (v/v) Tween 80 aqueous solution, filtered through sterile glass wool, and counted on MEA supplemented with 0.01% chlortetracycline (Sigma-Aldrich, St. Louis, MO, USA), to obtain a concentration between 107 and 108 CFU/mL. The filtered conidial suspensions of A. brasiliensis or H. burtonii were stored at 2 °C up to 30 days, whereas that of P. brevicompactum was stored at −18 °C up to six months, in accordance with the different physiological characteristics of the selected strains.

2.3. Ozonized Water Treatments

The ozone generator used was a waterproof unit model with a generation capacity of 8 g/h (230 VAC, 50 Hz, 750 W) (MET Srl, Bologna, Italy). The generator was supplied with purified oxygen obtained from a Pressure Swing Adsorption (PSA) apparatus that separates oxygen from atmospheric air, providing the generator with a higher oxygen concentration in order to increase its efficiency. The ozone produced was then delivered at the bottom of a conical filter funnel Robu (Borosilicate Glass 3.3, diameter = 120 mm, capacity = 2 L) equipped with a sintered disc filter of porosity 3. The conical filter funnel was filled with sterile water that was ozonized due to the microbubbles produced by the ozone flow passing through the disc filter. In this way, stable ozonized water was generated. Its concentration was measured by means of an Ozone Vacu-vials® Kit (K-7423, CHEMetrics Inc., Midland, VA, USA). The spectrophotometric measurement and the determination of the ozone concentration in the water solution were performed using a V-2000 Photometer (CHEMetrics Inc., Midland, VA, USA). From the stock solution of ozonized water, solutions at the desired ozone concentrations were obtained by dilution. The final concentration of the diluted solution was measured again before starting the treatment.
SS tiles (15 × 20 × 1 mm) were utilized as germ carriers. They were previously sterilized at 121 °C for 15 min, inoculated with one microbial suspension at a time, and dried for one hour in sterile conditions under a laminar flow hood. Some of the inoculated tiles, marked as “positive controls,” were analyzed without being treated (see the correspondence with the “time 0” bars in each figure) to consent to a comparison with those that were treated. Treatments were carried out just after the drying of the inoculum in order to avoid the loss of viable cells (Figure 1).

2.3.1. Bacteria

Tests were carried out at 25 °C in ultrapure water added with ozone (1.0, 3.0, 6.0 ± 0.5 mg L−1) by inoculating tiles with a multi-layered (0.01 mL of the undiluted bacterial suspension) drop. The spotted tiles were positioned into screw-capped test tubes filled with 1.5 mL of ozonized water that was left to react for up to 9 min. Such a maximum treatment time was established based on preliminary tests showing that partial ozone degradation started after 10 min (unpublished data). After treatments, 1.5 mL of a 1.0% sodium thiosulfate (powder from Sigma-Aldrich, St. Louis, MO, USA) solution was added to each tube in order to neutralize the ozone, together with 0.5 g of sterile glass microbeads. Each tube was then vortexed for two minutes by an infrared apparatus (Starlab, Milan, Italy), the appropriate decimal dilutions being plated on Agar Listeria acc. to Ottaviani and Agosti (ALOA®, Biolife Italiana srl, Milan, Italy) and incubated at 37 °C for 48 h. Each combination was tested four times.

2.3.2. Filamentous Fungi

Tests were carried out at 25 °C in neutral or acidified ultrapure water [1% w/v citric acid (powder from Sigma-Aldrich, St. Louis, MO, USA)] in order to assess any potential synergistic effect of citric acid with dissolved ozone (final concentration 8.5 ± 0.5 mg L−1). The tiles were inoculated by applying either a multi-layered (0.01 mL of the undiluted suspension) or a single-layered (0.10 mL of the 1:50 diluted suspension) drop of each suspension. The experiment was carried out as it was for bacteria, even if in tests with acidified ozonized water, a 0.1% (v/v) Tween 80 aqueous solution was used as a neutralizer in place of sodium thiosulphate since it is known to react with citric acid-forming molecules with an inactivating activity against molds, such as SO2 and S. After treating and vortexing, the appropriate decimal dilutions were plated on MEA supplemented with 0.01% chlortetracycline, and the colonies were counted after incubation at 25 °C for five days. Each combination was tested four times.

2.4. Statistical Analysis

Microsoft® Excel 2013 (Microsoft, Redmond, WA, USA) was used to draw the inactivation curves based on the raw thermal reduction data that were changed into logarithmic mean values. The inactivation achieved, given as Logarithmic Count Reductions (LCR), was calculated for both bacteria and filamentous fungi based on the difference between the decimal logarithm of the initial average concentration of cells deposited and the decimal logarithm of the average concentration of cells surviving the treatment.
The tested strains showed different behavior in their inactivation curves, so log-linear models (followed or not by a tail) and Weibull models were used to determine the 1D-value (defined as the time required to bring the first 1-log reduction in the population of a given microorganism at a given temperature) of each strain at the corresponding aqueous ozone concentration. For such microorganisms, the calculation of the 1D values and the statistical analysis were performed using GInaFiT (Geeraerd and Van Impe Inactivation Model Fitting Tool, version 1.7), a freeware add-in tool for Microsoft® Excel (https://cit.kuleuven.be/biotec/software/GinaFit accessed on 11 March 2024). The goodness-of-fit for each elaboration was assessed using the root mean squared error (RMSE) and the regression coefficient (R2). In calculating the time needed for the first decimal reduction (1D-value), kmax was the parameter taken into consideration for log-linear models with tailing where the first logarithmic reduction occurred during the inactivation of the major, less resistant population. In this case, the 1D-value was calculated as 2.303/kmax [39]. On the contrary, delta (δ) was considered the 1D-value itself when Weibull-type models were applied [39,40].
Statistical significance between means within each treatment group was identified by applying an ANOVA multivariate analysis with the Tukey post hoc test. The software package SPSS v24 (IBM website, www.ibm.com/software/it/analytics/spss/downloads.html accessed on 5 September 2024) was used for this analysis.

3. Results and Discussion

The results obtained against pathogenic bacteria are reported in Table 1 and Figure 2. The ones against filamentous fungi are reported in Table 2 and Figure 3, with reference to the tests with ultrapure water, and in Table 3 and Figure 4, with reference to the tests with acidified ultrapure water. The best-fitting microbial survival models applied by GInaFiT were derived from the ones elaborated by different authors (see footnotes in each table for the corresponding bibliographical reference).

3.1. Bacteria

For the tested strains, a marked tailing was observed at all the tested ozone concentrations. This phenomenon was already observed by the same authors when sanitizing agents such as UV-C radiation [46] or the ozone itself [35] were used. Similarly, it has been registered by other authors and related to heterogeneous treatments, to the aggregation of microorganisms, to some intrinsic mechanism of bacterial physiology leading to resistant sub-populations [47,48,49]. Together with the tailing, a huge variability in the concentrations of the surviving microorganisms at each of the considered combinations was highlighted, albeit the high number of repetitions for each tested combination. This variability had not been registered in tests carried out on bacterial and fungal suspensions (unpublished data), so it was hypothesized that the multi-layering of the inoculum could have given protection to the lower layers of cells against the oxidative processes exerted by ozone, even considering that variations within the same train of tests have been already registered by other authors who tested aqueous ozone [6,25].
For all the considered strains, a progressive reduction in the surviving cells was observed, and the reduction proved more marked as the ozone concentration increased. When ozone concentrations increased from 1.0 to 6.0 mg L−1, the LCR moved from 2.4 to more than 3.9 for the tested S. senftemberg and from 1.1 to 2.0 for the tested S. typhimurium, the former showing a higher sensibility to aqueous ozone compared to the latter. Analogously, considering the same increase in ozone concentration, the LCR moved from 1.3 to 5.2 for the tested Listeria monocytogenes Scott A and from 2.5 to 5.3 for the tested Listeria monocytogenes ATCC 7644, the latter strain proving more sensible to the ozone applied in its liquid form.
A huge variability was observed considering the 1-D values of the different strains or species tested. At the lowest ozone concentration, the resistance of L. monocytogenes Scott A and S. typhimurium proved sensibly higher (from 6.5 to 9 times) than those registered for the other two tested strains, whereas at higher concentrations the calculated resistance values prove comparable.
The comparison with previous works on the same topic is not easy since literature data focused on biofilms rather than free cells inoculated on specific surfaces. The only paper concerning inoculated cells was that by Marino et al. [25], where cells of L. monocytogenes attached to SS were subjected to 2 to 3 LCR, yet in the first 2 min of treatment, 1 ppm of aqueous ozone in static conditions was applied. Differently, on biofilms, more papers are available. With regard to L. monocytogenes, ozonized water proved active at a concentration of 4 ppm for 3 min on cells attached to SS, whereas a four-fold increase in sanitizer was needed to destroy all biofilm cells [50]. On polystyrene, a reduction of about 0.9, 3.4, and 4.1 Log was reached when ozonized water was applied at 1.0, 2.0, 1 md, and 4.0 ppm, respectively [30]. With regard to Salmonella spp., the effectiveness of ozonized water seemed less marked, a reduction less than 0.8 CFU/cm2 being obtained on cells adhered to SS after 20 min of exposure [26].

3.2. Filamentous Fungi

On filamentous fungi, a concentration of ozone equal to 8.5 mg L−1 was applied since preliminary tests at the concentrations used against bacteria did not prove effective at all in inactivating fungal conidia.
The tested strains were not affected by aqueous ozone if inoculated as a multi-layer of cells, with the exception of H. burtonii strains that proved subjected to a partial inactivation when acidified ozonized water was used. Dissimilarly, on single layers of cells, a gradual decrease was observed for all strains belonging to P. nordicum or H. burtonii. A statistically significant difference within the same fungal species was observed comparing data obtained testing either not-acidified or acidified ozonated water. On multi-layered cells, differences were observed only for Hyphopichia strains at any treatment time, with a marked decrease in survivors registered when citric acid was added to ozonized water. Differently, on single-layered cells, the observed statistical differences seemed randomized: they were registered at 8 min for A. brasiliensis, at 6 min for P. nordicum 45, or from 4 to 8 min for P. nordicum 1169, and from 2 to 8 min for Hyphopichia strains.
When the multi-layered cells were tested in both liquid matrices, the A. brasiliensis and P. nordicum strains did not reach even 1.0 LCR after the longest treatment time. Conversely, for H. burtonii strains, the results differed when not-acidified and acidified ozonated water were tested: from 1.0 to 1.1 LCR after 10 min in the former case or more than 5.0 LCR after 6–10 min in the latter case were reached, depending on the isolate considered.
When the single-layered cells were tested, A. brasiliensis inactivation kinetics did not change compared to the multi-layered spatial arrangement. On the contrary, strain- and species-specificity was observed for the tested Hyphopichia and Penicillium strains. P. nordicum isolates underwent from 2.9 to 4.7+ LCR in not-acidified water and from 3.8 to 4.40 LCR in acidified water. Similarly, H. burtonii strains were subjected to a minimum of 3.4 LCR just after 6 min of treatment up to 5.0+ LCR after 10 min in not-acidified water and to 3.8–4.4 LCR in acidified water.
When compared with the other tested strains, Aspergillus brasiliensis showed the greatest resistance to the sanitizing agent applied whenever arranged in a multi-layer or in a single layer. It yet resulted in a point of reference when not-ionizing radiation such as the UVC light [34,46] or chemicals such as the ozone itself [35] have to be tested. This was probably due to the great amounts of melanin and melanin-like pigments in its outer cellular wall [51,52]. Furthermore, it must be taken into account that, like other filamentous fungi in the genus Aspergillus, it possesses good amounts of ROS-scavenging enzymes such as superoxide dismutases (SOD) and catalases (CAT) [53]. SOD proved critical to cell survival following ozone exposure by eliminating the superoxide radicals and thereby preventing the formation of hydroxyl radicals, whereas CAT proved necessary for protection against ozone exposure [54]. Therefore, the comprehension of melanin and of major ROS-scavenging enzymes could be responsible for its higher robustness against ozonized water.
With regard to the literature, few papers have dealt with the effect of ozonized water on filamentous fungi either in static or dynamic conditions, and none of them reported tests on metallic or plastic supports.
In static conditions, Hageskal et al. [55] tested filamentous fungi inoculated in ozonized water and registered a great variability in fungal response. All the tested strains (Fusarium solani, Trichoderma viride, and Aspergillus calidoustus) were reduced from one to three Logs after 10 min of treatment at 8 ppm, with the exception of P. spinulosum that did not prove affected by aqueous ozone. In agreement with our observations, [56] found that Aspergillus brasiliensis ATCC 16404 spores were still alive in freshly ozonized water at 1.5–3.0 ppm after 30 min, requiring longer times than those used for totally inactivating both [57] bacterial and yeast cells.
If dynamic fluxes were considered, the point of reference could be the paper by Beuchat et al. [58], where the D-values of two aflatoxigenic aspergilli (A. flavus and A. parasiticus) exposed to 1.74 ppm ozone were determined. D-values of A. flavus conidia were 1.72 and 1.54 min at pH 5.5 and 7.0, respectively, whereas D-values of A. parasiticus were 2.08 and 1.71 min, respectively. One of the most interesting ones is that by Restaino et al. [57], where the effect of the aqueous zone was tested on mycetes, and their strain of Aspergillus niger behaved similarly to the one used in this paper, its concentration being affected by less than one logarithmic reduction after 5 min, even if the ozone concentration tested by Restaino’s group was sensibly lower (less than 0.2 ppm).

4. Conclusions

Aqueous ozone has a Generally Regarded as Safe (GRAS) designation from the U.S. Food and Drug Administration (FDA, 2001), and it is allowed in many countries, including Japan, Australia, France, and Canada [59]. On the contrary, in Europe, no regulation exists about the use of ozonized water for agro-industrial purposes, with a recent EFSA report (European Union, 2021) indicating that “no conclusion could be drawn concerning the risk assessment to non-target species from potentially occurring by-products of the chemical interaction between ozone and substances occurring in water.” Nevertheless, the use of ozone in its liquid form for sanitization purposes is gaining an increasing interest in the food and feed industry due to its undeniable advantages compared with chemicals, such as the absence of by-products that should affect human health or the possibility to generate it when needed.
In our paper, ozonized water gave different results based on the tested microorganism, as observed yet for gaseous ozone [35]. Pathogenic bacteria proved markedly more sensible to ozone than filamentous fungi, even if great differences were observed at the inter- and intra-specific level for both categories of microorganisms, as in the case of L. monocytogenes and P. nordicum. For a better interpretation of the obtained results, it could be taken into consideration the European Standard EN 13697:2015+A1 [36], which establishes that any product with bactericidal and/or fungicidal activity used in food, industrial, domestic, and institutional areas should demonstrate at least a four-decimal log reduction for bacteria and at least a three-decimal log reduction for fungi. In this perspective, an appropriate number of log reductions were achieved only for three out of four pathogenic strains when higher concentrations of ozonized water were used or for some P. nordicum and H. burtonii at different treatment times, depending on their spatial arrangement of the conidia and the use of neutral or acidified pure water. Nevertheless, the non-linear inactivation kinetics of the studied strains made the calculation of a punctual F-value difficult, so in industrial practice, adequate tailoring of the treatments to be applied, based on the registered extrinsic factors and the industrial bio-burden, would be appropriate.

Author Contributions

E.B.: conceptualization (filamentous fungi), microbiological software, and writing; C.M.: execution of microbiological analyses and acquisition of data; M.C.: conceptualization (pathogens); D.B.: execution of chemical analyses and acquisition of data (ozone treatments); C.C.C.: conceptualization and writing (ozone treatments); D.I.: project supervision, conceptualization, statistical analysis, and review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 08392 g001
Figure 1. Graphical representation of the operations carried out to test the effectiveness of ozonized water against pathogenic bacteria and filamentous fungi. (Created with BioRender.com—https://biorender.com/; accessed on 12 April 2024).
Figure 1. Graphical representation of the operations carried out to test the effectiveness of ozonized water against pathogenic bacteria and filamentous fungi. (Created with BioRender.com—https://biorender.com/; accessed on 12 April 2024).
Applsci 14 08392 g001
Applsci 14 08392 g002
Figure 2. Behavior of Salmonella (a) and Listeria monocytogenes (b) strains treated with ozonized water (Lm = Listeria monocytogenes Pirie ATCC 7644; Ls = Listeria monocytogenes Scott A; Se = Salmonella enterica subsp. Enterica serotype Senftenberg NCTC 9959; St = Salmonella enterica subsp. Enterica serotype Typhimurium ATCC 14028). Vertical error bars indicate the standard deviation for mean values. Within each treatment group, different letters indicate significant differences between means at p < 0.05 (ANOVA and Tukey HSD post hoc test).
Figure 2. Behavior of Salmonella (a) and Listeria monocytogenes (b) strains treated with ozonized water (Lm = Listeria monocytogenes Pirie ATCC 7644; Ls = Listeria monocytogenes Scott A; Se = Salmonella enterica subsp. Enterica serotype Senftenberg NCTC 9959; St = Salmonella enterica subsp. Enterica serotype Typhimurium ATCC 14028). Vertical error bars indicate the standard deviation for mean values. Within each treatment group, different letters indicate significant differences between means at p < 0.05 (ANOVA and Tukey HSD post hoc test).
Applsci 14 08392 g002
Applsci 14 08392 g003
Figure 3. Behavior of filamentous fungi inoculated as a multi-layer (a) or a single-layer (b) and treated with 8.5 mg L−1 ozone in neutral ultrapure water. Vertical error bars indicate the standard deviation for mean values. Within each treatment group, different letters indicate significant differences between means at p < 0.05 (ANOVA and Tukey HSD post hoc test).
Figure 3. Behavior of filamentous fungi inoculated as a multi-layer (a) or a single-layer (b) and treated with 8.5 mg L−1 ozone in neutral ultrapure water. Vertical error bars indicate the standard deviation for mean values. Within each treatment group, different letters indicate significant differences between means at p < 0.05 (ANOVA and Tukey HSD post hoc test).
Applsci 14 08392 g003
Applsci 14 08392 g004
Figure 4. Behavior of filamentous fungi inoculated as a multi-layer (a) or a single-layer (b) and treated with 8.5 mg L−1 ozone in acidified ultrapure water. Vertical error bars indicate the standard deviation for mean values. Within each treatment group, different letters indicate significant differences between means at p < 0.05 (ANOVA and Tukey HSD post hoc test).
Figure 4. Behavior of filamentous fungi inoculated as a multi-layer (a) or a single-layer (b) and treated with 8.5 mg L−1 ozone in acidified ultrapure water. Vertical error bars indicate the standard deviation for mean values. Within each treatment group, different letters indicate significant differences between means at p < 0.05 (ANOVA and Tukey HSD post hoc test).
Applsci 14 08392 g004
Table 1. Inactivation parameters of the tested strains inoculated as a multi-layer on SS tiles and treated with different concentrations of aqueous ozone (Lm = Listeria monocytogenes ATCC 7644; Ls = Listeria monocytogenes Scott A; Se = Salmonella enterica subsp. enterica serotype Senftenberg NCTC 9959; St = Salmonella enterica subsp. enterica serotype Typhimurium ATCC 14028).
Table 1. Inactivation parameters of the tested strains inoculated as a multi-layer on SS tiles and treated with different concentrations of aqueous ozone (Lm = Listeria monocytogenes ATCC 7644; Ls = Listeria monocytogenes Scott A; Se = Salmonella enterica subsp. enterica serotype Senftenberg NCTC 9959; St = Salmonella enterica subsp. enterica serotype Typhimurium ATCC 14028).
Ozone Concentration (mg L−1)StrainInactivation CurveInactivation Model
ParameterRMSE
(-)		R2
(-)		1D-Value
(min)
1.0Lsδ0.14040.975.20[41]
3.0Kmax0.28670.960.44[42]
6.0Kmax0.26040.990.17[42]
1.0Lmδ0.19100.980.81[41]
3.0Kmax0.05201.000.27[42]
6.0Kmax0.17351.000.10[42]
1.0Seδ0.23900.970.82[41]
3.0δ0.17010.990.26[41]
6.0Kmax0.22170.990.22[42]
1.0Stδ0.03001.007.27[41]
3.0δ0.12410.981.70[41]
6.0δ0.09260.991.68[41]
Table 2. Inactivation parameters of the fungal strains inoculated as a multi-layer or a single-layer on SS tiles and treated with 8.5 mg L−1 ozone in neutral ultrapure water (Ab = Aspergillus brasiliensis ATCC 16404; Hb1 = Hyphopichia burtonii SSICA 175717; Hb2 = Hyphopichia burtonii SSICA 251105; Pn1 = Penicillium nordicum SSICA 1169; Pn2 = Penicillium nordicum SSICA B4798).
Table 2. Inactivation parameters of the fungal strains inoculated as a multi-layer or a single-layer on SS tiles and treated with 8.5 mg L−1 ozone in neutral ultrapure water (Ab = Aspergillus brasiliensis ATCC 16404; Hb1 = Hyphopichia burtonii SSICA 175717; Hb2 = Hyphopichia burtonii SSICA 251105; Pn1 = Penicillium nordicum SSICA 1169; Pn2 = Penicillium nordicum SSICA B4798).
InoculumStrainInactivation CurveInactivation Model
ParameterRMSE
(-)		R2
(-)		1D-Value
(min)
multi-layerAbndndndnd-
Hb1ndndndnd-
Hb2ndndndnd-
Pn1ndndndnd-
Pn2ndndndnd-
single-layerAbndndndnd-
Hb1Kmax1.19240.541.47[42]
Hb2δ0.30460.980.52[43]
Pn1Kmax0.40850.951.77[44]
Pn2Kmax0.35670.893.66[44]
Note. Nd: not determined since at least 1.0 LCR was not reached within the time considered.
Table 3. Inactivation parameters of the fungal strains inoculated as a multi-layer or a single-layer on SS tiles and treated with 8.5 mg L−1 ozone in acidified ultrapure water (Ab = Aspergillus brasiliensis ATCC 16404; Hb1 = Hyphopichia burtonii SSICA 175717; Hb2 = Hyphopichia burtonii SSICA 251105; Pn1 = Penicillium nordicum SSICA 1169; Pn2 = Penicillium nordicum SSICA B4798).
Table 3. Inactivation parameters of the fungal strains inoculated as a multi-layer or a single-layer on SS tiles and treated with 8.5 mg L−1 ozone in acidified ultrapure water (Ab = Aspergillus brasiliensis ATCC 16404; Hb1 = Hyphopichia burtonii SSICA 175717; Hb2 = Hyphopichia burtonii SSICA 251105; Pn1 = Penicillium nordicum SSICA 1169; Pn2 = Penicillium nordicum SSICA B4798).
InoculumStrainInactivation CurveInactivation Model
ParameterRMSE
(-)		R2
(-)		1D-Value
(min)
multi-layerAbndndndnd-
Hb1δ0.56130.960.48[43]
Hb2δ0.37240.973.86[43]
Pn1ndndndnd-
Pn2ndndndnd-
single-layerAbδ0.18420.925.00[41]
Hb1Kmax0.09491.000.18[45]
Hb2δ0.46580.910.09[41]
Pn1δ0.52240.891.62[41]
Pn2δ0.38750.944.89[41]
Note. Nd: not determined since the minimum value of 1.0 LCR was not reached within the time considered.
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Berni, E.; Moroni, C.; Cigarini, M.; Brindani, D.; Catelani Cardoso, C.; Imperiale, D. Effect of Ozonized Water against Pathogenic Bacteria and Filamentous Fungi on Stainless Steel. Appl. Sci. 2024, 14, 8392. https://doi.org/10.3390/app14188392

AMA Style

Berni E, Moroni C, Cigarini M, Brindani D, Catelani Cardoso C, Imperiale D. Effect of Ozonized Water against Pathogenic Bacteria and Filamentous Fungi on Stainless Steel. Applied Sciences. 2024; 14(18):8392. https://doi.org/10.3390/app14188392

Chicago/Turabian Style

Berni, Elettra, Chiara Moroni, Massimo Cigarini, Demetrio Brindani, Claudia Catelani Cardoso, and Davide Imperiale. 2024. "Effect of Ozonized Water against Pathogenic Bacteria and Filamentous Fungi on Stainless Steel" Applied Sciences 14, no. 18: 8392. https://doi.org/10.3390/app14188392

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