Open Veterinary Journal, (2024), Vol. 14(11): 2794-2805

Research Article

10.5455/OVJ.2024.v14.i11.8

Effectiveness of gaseous ozone on Arcobacter butzleri and bacterial loads on retailed meat sold at Iraqi Wasit markets

Manal Hadi Ghaffoori Kanaan*

Department of Nursing, Technical Institute of Suwaria, Middle Technical University, Baghdad, Iraq

*Corresponding Author: Manal Hadi Ghaffoori Kanaan. Department of Nursing, Technical Institute of Suwaria, Middle Technical University, Baghdad, Iraq. Email: manalhadi73 [at] yahoo.com

Submitted: 04/07/2024 Accepted: 22/10/2024 Published: 30/11/2024

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Abstract

Background: Ozone (O₃) is a promising alternative antibacterial agent that has recently been used in meat processing. The understanding of the appropriate functional settings of O₃ for addressing food safety problems is still insufficient.

Aim: The aim of this study was, therefore, to investigate the effects of exposure to O₃ on the bacteriological quality of retail meat inoculated with Arcobacter butzleri at refrigeration temperatures.

Methods: Chicken and cattle meat were inoculated with 7 log CFU/g of A. butzleri previously isolated from fresh and chilled retail meat. Using an O₃ generator (Ivation, Multipurpose Air Sterilizing, USA), the meat samples were treated with O₃ gas (0.5 ppm) for 20 and 40 minutes at temperatures ranging from 3°C to 7°C for 48 hours. Anaerobic and aerobic bacterial growth, as well as A. butzleri were assessed.

Results: The growth rates of A. butzleri, total aerobic bacteria, and anaerobic bacteria in chilled meat samples were all slowed by significant reduction by 0.75, 1.13, and 0.7 log CFU/g, respectively, when exposed to O₃ gas (p ≤ 0.05). Compared to control samples kept at 3°C–7°C for a further 24 hours, the decrease was greater with an additional exposure time of 20 minutes. In contrast to the samples that did not appear to be exposed to O₃, the total bacterial count of the samples exposed to O₃ showed a statistically significant reduction (p ≤ 0.01) compared to the samples that were not exposed to O₃. This decrease is important for the health of the population.

Conclusion: This study explored the use of O₃ experience as a potential antibacterial agent for meat and meat products stored in refrigerators. The results of this research may support further applications of O₃ exposure, such as the installation of an O₃ generator inside a refrigerator. However, further research is needed on O₃ levels and exposure durations.

Keywords: Arcobacter butzleri, Cattle meat, Chicken meat, Ozone, Storage conditions.

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Introduction

Meat is a valuable source of protein with high biological value, and the human body has become accustomed to eating meat over the course of evolution. Furthermore, the nutritional profile of meat is rich in vitamins, minerals, and other elements, and some of these nutrients are either more bioavailable or found exclusively in animal products (Font-i-Furnols, 2023). The Organization for Economic Co-operation and Development and the Food and Agriculture Organization of the United Nations forecast a 14% increase in meat consumption by 2030. The growing world population is the main reason for this increase in meat consumption (OECD/FAO, 2021).

The environments in which animals have been reared up and processed, along with the properties of the end products, in which it provides ideal conditions for the development and maintenance of spoilage and harmful bacteria, making meat and meat products vulnerable to microbiological contamination (Mohammed et al., 2022; Liu et al., 2023). The microbiological safety of meat has been a public health concern for many years (Kanaan et al., 2022a; Kanaan, 2024). Different species of bacteria that cause food poisoning have been detected in meat and meat products that cause food poisoning, including Shiga-toxin-producing Escherichia coli, Campylobacter, Salmonella species, Staphylococcus aureus, Acinetobacter baumannii, and Listeria monocytogenes (Kanaan and Al-Isawi, 2019; Xu et al., 2019; Kanaan and Mohammed, 2020; Kanaan et al., 2020, 2022b; Kanaan and Khashan, 2022; Liu et al., 2023; Kanaan, 2023). Moreover, chicken meat has been recognized as the primary source of foodborne outbreaks of Arcobacter butzleri (Oliveira et al., 2018; Shange et al., 2019). Infections caused by A. butzleri provide a considerable threat to human health, resulting in severe food poisoning and sepsis (Oliveira et al. 2023). A global epidemic of A. butzleri has been documented, although the incidence of human infection is limited (Chieffi et al., 2020; Jiménez-Guerra et al., 2020). The bacterium A. butzleri has been mentioned in several studies as one of the numerous Campylobacter-like bacteria in human feces (Jiménez-Guerra et al., 2020; Oliveira et al., 2023). Moreover, any obstacles to infection with this type of bacteria are compounded by the fact that the multidrug resistant Arcobacter spreads much more widely than Campylobacter (Son et al., 2007; Riesenberg et al., 2017). Various disinfectants and some common chemical agents such as chlorine, peracetic acid, hydrogen peroxide, butylated hydroxytoluene, and tetra-butylhydroquinone were mentioned by Xue et al. (2023), for treatment and preservation to remove bacterial contamination and improve the shelf life of meat quality. Numerous of research studies have examined and assessed many food processing technologies, encompassing both thermal and non-thermal processes (Jiang et al., 2018; Stadler et al., 2020; Chacha et al., 2021; Kanaan and Tarek, 2022).

Aqueous chlorine (0.5–1 ppm) is employed in the meat sector to diminish microbial load, mitigate cross-contamination risk, and maintain carcass safety. The efficacy of chlorine is contingent upon the amount employed. Nevertheless, several research indicates that chlorine by-products, including trihalomethanes, haloacetic acids, and chloramines, may possess carcinogenic properties (Gil et al., 2016; Kanaan and Abdullah, 2021; Kanaan et al., 2024a). Investigations are underway about alternatives to traditional cleaning chemicals and disinfectants that pose no risk to human and environmental health, yet possess the capability to diminish and eradicate the transmission of foodborne diseases (Kanaan et al., 2024a).

O₃ is classified as a Generally Recognized as Safe food processing additive that has minimal or no risk. It works considerably better for washing or sanitizing food than chemical sanitizers. O₃ is a more eco-friendly technique than other antimicrobial agents currently in use as it kills microbes without leaving harmful residues on food or equipment (Pandiselvam et al., 2020; Roobab et al., 2022). Megahed et al. (2018) discovered that temperature, pH, and O₃-oxidizable materials have a significant impact on O₃ degradation rate and half-life. Therefore, O₃ is useful in the meat processing industry when stored in cold, moist, and refrigerated environments (Bridges et al., 2018). O₃ is very effective against bacteria due to its significant oxidation potential; even at doses as low as 0.01 ppm, the condition remains dangerous to microbes (Megahed et al., 2018). In addition to bacteria, molds, viruses, and protozoa are also susceptible to the antibacterial properties of O₃ (Kanaan, 2018). Therefore, it has proposed O₃ as a strong substitute for conventional disinfectants (Megahed et al., 2018).

This study was conducted to investigate the efficacy of gaseous O₃ against bacterial growth on chicken and cattle meat inoculated with A. butzleri and to evaluate its effectiveness in preserving meat at refrigeration temperature.

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Materials and Methods

Study period and location

This study was conducted from October 2023 to January 2024, in Al-Suwaira, a city in Iraq’s Wasit governorate in the middle east.

Collection and processing of samples

Between October 2023 and January 2024, a total of 32 samples of chicken (n=16) and cattle (n=16) meat were randomly collected from various supermarkets and vendor shops. The samples were packed separately in a sterile plastic bag and sent to the laboratory with ice packs within 3 hours. From the time of collection until use, all samples were stored at a temperature of −20°C in a freezer. After defrosting in the refrigerator at a temperature of 4°C for 12 hours, the wrapped meat bags were allowed to cool to room temperature for 1 hour before being inoculated. To inoculate the samples with A. butzleri, around 20 g of the material was placed on a sterile petri dish (65 × 15 mm; FB0875711C, Fisherbrand ^TM, UK). Two separate plastic containers, one for the inoculated and one for the non-inoculated samples, were washed with 70% ethanol to eliminate the possibility of contamination and cross-contamination before and during treatment. This study utilized a 600 mg/hour O₃ generator (Ivation, Multipurpose Air Sterilising, USA) to produce O₃ gas (0.5 ppm) in an O₃ container, informed by previous research (Khudhir and Mahdi, 2017; Kanaan, 2018; Mohammed et al., 2022; Kanaan et al., 2024b) that employed the O₃ generator 600 mg/hour to generate aqueous O₃ at a concentration of 0.5 ppm over varying durations (20, 30, and 45 minutes). The O₃ generator was used according to the manufacturer’s instructions. This generator has 15-time grades, so it may conduct a variety of disinfection and sterilizing duties by selecting different durations. An automatic timer plug was used to program it for 20 and 40-minute operation times, respectively. The efficacy of O₃ gas (0.5 ppm) on A. butzleri and on the total bacterial population (log CFU/g) was carried out daily during this time.

Calculation of ozone (O₃) concentration

Ozone- CHEMets Visual Kit (K-7404 O₃ analysis kit, USA) (Supplementary Fig. 1) was used to measure the O₃ concentration (ppm). A plastic container with 1.5 liter of tap water was covered. The container was injected with an aeration stone via the lid cavity. Four contact periods (10, 15, 30, and 45 minutes) were selected. After each exposure, the tap water was changed and the container was cleaned with fresh tap water. Five drops of A-7400 activator were added to the empty sample cup, which was filled to the 25 ml mark with ozonated water, the CHE-Met ampoule tip was inserted into the cup, the tip was broken off and ingested by the water. The ampoule was inverted several times and left for 1 minute to allow the color to change. The ampoule was placed between the color standards until a high-range comparator indicated the best color. The maximum concentration was 0.5 ppm after 15, 30, and 45 minutes.

Bacterial strain preparation

The A. butzleri used in this study was obtained from a previous study (Kanaan, 2021) in which the bacterium was isolated from retail chicken and cattle meat sold in the markets of Wasit governorate in the middle east of Iraq. The bacterial strains were prepared by thawing them at 4°C and then resuscitating them on modified charcoal cefoperazone deoxycholate agar (mCCDA, Oxoid CM739, UK). They were then incubated at 30°C for 2 days. The pure colonies were cultured in Lauryl Tryptose Broth (Oxoid, CM0451, UK) (overnight at 30°C) and optical density was measured after 2 washing with buffered peptone water (BPW, Oxoid, CM0509, UK).

Microbiological assessment

For each type of sample containing both chicken and cattle meat, two different groups were formed. In the first group, an inoculum of A. butzleri at a concentration of 10⁷ CFU per milliliter was spread evenly on the surface of meat using sterile spatulas. The second group, on the other hand, received no inoculum for comparison. Also, each group was divided into two sections, the first one was placed in a plastic chamber (L × W × H=30 × 25 × 25 cm) with normal air (21% oxygen, 78% nitrogen, and 1% other gasses) and represented the control group. The other section was placed in an airtight O₃ chamber made of sturdy Tritan plastic with a lockable lid (L × W × H=30 × 25 × 25 cm), with an O₃ gas intake port to inject O₃ gas using an aeration stone (Diffuser) and disperse it uniformly throughout the air. An O₃ generator (600 mg/hour) was used to inject gas. The O₃ generator was fed with 1 liter per minute [flow rate (l pm)] of compressed air as feed gas. The gaseous O₃ (0.5 ppm) was diffused into the meat samples at two different exposure times (20 and 40 minutes) at a temperature of 3°C–7°C (supplementary Fig. 2). First, O₃ gas (0.5 ppm) was injected for 20 minutes at 3°C–7°C. The container was then kept at the same temperature for 24 hours before being compared with untreated samples that had also been stored at 3°C–7°C for 24 hours. In addition, the treated samples were exposed to O₃ gas (0.5 ppm) at 3°C–7°C for a further 20 minutes. The chamber containing the treated samples was kept at 3°C–7°C for a further 24 hours to compare the bacterial load of the treated and untreated samples (Fig. 1). During this period, the effect of O₃ gas (0.5 ppm) on A. butzleri and on the total bacterial population was constantly monitored. After every 24 hours, another meat sample was taken to assess the log CFU/g. The samples were weighed (1 g each), homogenized in a stomacher for 2 minutes, and then diluted with 9 ml of buffered peptone water. Plate Count Agar (Oxoid, CM0325B, UK) was used for the count of aerobes and anaerobes, and mCCDA-free agar was used for the total A. butzleri count. Incubation was carried out continuously at 30°C for 2 days. Dilutions were then performed according to the decimal system.

Arcobacter butzleri and total aerobic bacteria were counted by incubating the plates aerobically for 48 hours at 30°C and 24 hours at 37°C, respectively. To perform the enumeration of anaerobic bacteria, Oxoid™ AnaeroGen^™ 2.5 l Sachet (Oxoid, AN0025A, UK) was used in an anaerobic jar (Oxoid, AG25, UK) under anaerobic conditions. The plates were incubated at 37°C for a duration of 24 hours. The total count of aerobic and anaerobic bacteria, as well as Arcobacter butzleri, was determined using the standard plating procedure.

Statistical Analysis

The Statistical Analysis System (SAS, 2018) program, version 9, 6th edition (N.C., USA), was employed to ascertain the influence of diverse factors on the study parameters. In this research, the Least Significant Difference (LSD) test, part of the Analysis of Variance, was employed for mean comparisons (SAS, 2018).

Ethical approval

The Middle Technical University/Medical Ethics Committee/Ethics Committee Reference Number: MEC: 17 granted approval for this research on October 1, 2023. No humans or animals were employed in this investigation. Fresh meat samples were purchased from various supermarkets and vendor shops. All processes were carried out in compliance with the applicable norms and legislation.

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Results

In this study, the efficacy of gaseous O₃ against the bacterial load in meat preserved at a refrigerated temperature was investigated. The results (Tables 1 and 2) showed that the initial concentrations of A. butzleri in the inoculated chicken and cattle meat samples were 3.72 and 4.05 log CFU/g, respectively. When the inoculated chicken and cattle meat samples were exposed to gaseous O₃ (0.5 ppm) for 20 minutes, the number of A. butzleri decreased by 0.75 and 0.68 log CFU/g, respectively, after being kept at 3°C–7°C for 24 hours. Compared to samples not exposed to O₃, high counts of A. butzleri were detected. In addition, this study showed that the total count of aerobic and anaerobic bacteria decreased in chicken and beef meat samples treated with O₃ gas for 20 minutes during 24 hours of storage at 3°C–7°C compared to the untreated samples. The changes in chicken meat were also smaller: the number of aerobic and anaerobic bacteria decreased to only 1.13 log CFU/g and 0.7, respectively. The chicken meat was then kept at a refrigeration temperature of 3°C–7°C for a further day. The observations indicated a decrease in A. butzleri, aerobic and anaerobic bacteria (CFU/g) by 0.5, 0.32, and 0.29, respectively, compared to the samples used in the control group. Cattle meat showed a similar trend, with a reported lower aerobic bacterial count of 0.59 log CFU/g. Compared to the non-O₃-exposed samples, a statistically significant decrease (p ≤ 0.01) in bacterial counts was observed in the O₃-exposed samples. As shown in Tables 3 and 4, the non-inoculated meat samples showed no growth of A. butzleri (zero growth). Furthermore, after the non-inoculated samples were exposed to gaseous O₃ at 3°C–7°C for 48 hours, there was a decrease of 0.53 log CFU/g in aerobic bacteria and 0.98 log CFU/g in anaerobic bacteria. The total count number of aerobic and anaerobic bacteria decreased by 0.65 and 0.8 log CFU/g, respectively, in the non-inoculated cattle meat samples that were exposed to O₃ in the same way. These samples showed a similar trend (Fig. 2). The number of aerobic and anaerobic bacteria decreased significantly (p ≤ 0.01) in the samples that were exposed to O₃ compared with the samples that were not exposed to O₃ (Tables 3 and 4).

Fig. 1. A flow diagram for the study design.

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Discussion

Researchers have examined the effects of gaseous and liquid O₃ on many microorganisms, such as viruses, protozoa, bacterial and fungal spores, and even coronaviruses, on food (Xue et al., 2023). Consumers in Iraq could be at risk of infection with A. butzleri, due to the recent detection of this bacterium in both fresh and chilled retail meat sold in the country (Jasim et al., 2021; Kanaan, 2021). Therefore, this bacterium was introduced into meat samples to evaluate the effects of O₃ gas at temperatures between 3°C and 7°C over a 48-hour storage period.

Table 1. Effect of O₃ gas on the A. butzleri, total aerobic and anaerobic bacterial counts (log CFU/g) of inoculated chicken meat with A. butzleri during storage at 3°C–7°C.

Data in Tables (1 and 2) indicated that the exposure of meat samples infected with A. butzleri to O₃ gas (0.5 ppm) for 20 minutes at 3°C–7°C resulted in a strong reduction (p ≤ 0.01) of bacterial counts. Moreover, a significant decrease in aerobic and anaerobic counts in chicken meat was observed, with aerobic counts at 1.13 log CFU/g and the anaerobic counts at 0.7 log CFU/g. This study confirmed the findings of the previously mentioned studies (Cho et al., 2014; Muhlisin et al., 2016; Khudhir and Mahdi, 2017; Kanaan, 2018; Megahed et al., 2018; Megahed et al., 2020; Giménez et al., 2021). Balamatsia et al. (2007) and Capita et al. (2013), whose research included microbial counts by type of meat at various stages of the distribution chain, found that total bacterial loads in poultry meat at the start of the storage period ranged from 4.9 to 5.66 log CFU/g. In the study by Rani et al. (2023), the most negative result was reported, the mean ± SE was only 7.7 ± 0.17 log of total bacterial count CFU/cm² in beef during high capacity abatements. Therefore, the findings of this study were also of crucial importance for the improvement of public health and for sustainable improvement of quality and safety of food products.

Due to its high reactivity and lack of toxic residues, O₃ was actively studied for its bactericidal effect against foodborne pathogens. Cho et al. (2014) conducted some studies of this type and demonstrated that the growth of E. coli O157:H7 and some other anaerobic and aerobic bacteria was significantly inhibited (p ≤ 0.05) when ground beef samples were exposed to an O₃ concentration of 10 × 10^-6 kg O3/hour for 3 days at 4°C.

Muhlisin et al. (2016) also explored the bactericidal properties of O₃ in a gaseous state with regard to the total bacterial count of the chicken meat. Anaerobic and aerobic bacterial growth as well as coliform bacilli growth was inhibited, when chicken meat was treated with O₃ (temperature 4°C ± 1°C) at a concentration of 10 mg O₃/m³/hour over a period of 4 days.

In another study conducted by Khudhir and Mahdi (2017), there was a very significant effect of antimicrobial influence on microbial counts (reduction as much as 6 logs) after aqueous O₃ exposure of 0.5 parts per million for 20 minutes on bovine and ovine soft cheeses that were locally produced.

Megahed et al. (2018) studied the efficacy of gaseous and aqueous O₃ in eliminating pathogens derived from dairy cow dung that had infected various farm surfaces under varying operating circumstances. The researchers found that 4 minutes of gaseous O₃ at 9 ppm inactivated 3.3 log of the pathogens detected in the manure. Additionally, they discovered that on rather rough surfaces, aqueous O₃ at 9 ppm for 2 minutes may be an effective way to eliminate manure-based pathogens to a safe level. In contrast, on smooth surfaces, it was determined to be 4 ppm. Furthermore, Kanaan (2018) found that after half an hour of exposure to 0.5 ppm O₃ in water, the bacterial count of S. aureus in infected chicken meat decreased by up to 2 logs.

Table 2. Effect of O₃ gas on the A. butzleri, total aerobic and anaerobic bacterial counts (log CFU/g) of inoculated cattle meat with A. butzleri during storage at 3°C–7°C.

Table 3. Effect of O₃ gas on the total aerobic and anaerobic bacterial counts (log CFU/g) of non-inoculated chicken meat with A. butzleri during storage at 3°C–7°C.

Table 4. Effect of O₃ gas on the total aerobic and anaerobic bacteria counts (log CFU/g) of non-inoculated cattle meat with A. butzleri during storage at 3°C–7°C.

Fig. 2. Log CFU/g reduction of bacterial counts in inoculated and non-inoculated samples after exposure to gaseous O₃.

Megahed et al. (2020) examined the microbial eradication capacity of aqueous mixtures of O₃ and lactic acid-O₃ (O₃-LA) under various operation settings on chicken meat infected with a high load of Salmonella. Further studies showed that aqueous O₃ (8 ppm) had a lethal effect of 1.6 log/cm² when administered topically, while it was 1.2 log/cm² and 1 when injected subcutaneously.

Based on the observation obtained by Giménez et al. (2021), it was found that increasing the O₃ concentration greatly reduced the quantity of mesophilic bacteria, lactic acid bacteria, Enterobacteriaceae, molds, and yeasts. In contrast, Botta et al. (2018) found in their study that the initial bacterial count did not change when beef samples were treated with aqueous O₃.

According to Xue et al. (2023), the inactivation of O₃ is a multiphase process that affects various cellular structures and content elements. O₃ primarily affects cell membranes, as it can filter intracellular material during stress (Botta et al., 2018). Sheng et al. (2018) noted that O₃ may be able to interfere with enzymes and prevent the growth and activity of bacteria. This could be the reason for the changes reported by Aponte et al. (2018), which are due to the permeability of the cell membrane. Another research showed that O₃ can cleave the DNA double bond by oxidation using of a highly reactive form of O₂ (O) (Botta et al., 2018).

There are a variety of internal and external variables that influence O₃ when applied to microbes. According to Crowe et al. (2012), Shu et al. (2021), Liu et al. (2022), and other authors, these include various different intrinsic microbiological factors such as the type of microorganisms, characteristic of food, the physiological conditions of the microbial cells, and the microorganisms that are naturally present or artificially introduced. Additionally, the intrinsic parameters of food include the type of food, surface properties, weight, and water activity (aW) (Xue et al., 2023). Brodowska et al. (2018), Chen et al. (2020), Liu et al. (2021), Bigi et al. (2021), and Onopiuk et al. (2021) have reported that the variables for O₃ therapy include temperature, contact time, O₃ concentration, and the application techniques were the most common stress factors. As a result, these variations may arise from differences between studies.

Tables 1 and 2 demonstrate that following preservation at 3°C–7°C for an additional 24 hours, an extension of the duration of exposure to 20 minutes resulted in a greater reduction compared with the controls.

Other studies also showed that with increased exposure to O₃, its efficiency against bacteria becomes higher. This was evident from the works of Kanaan (2018); Chen et al. (2020); Onopiuk et al. (2021); Aslam et al. (2021); and Kanaan et al. (2024b). However, this increase in effects seems to reach an asymptote beyond which further increases are not possible to achieve, probably due to structures within food or even due to non-viable microbial cells assumed to protect against the oxidative action of O₃ (Ayranci et al., 2020).

The result also showed that the bacterial inhibition rate was significantly lower when an additional day of cooling was applied. Such an effect seems to be related to the limitations of O₃ exposure, as the O₃ exposure could only remove superficial bacterial contamination.

Tables 3 and 4, and Figure 2 confirm that, with time, the counts of total aerobic and anaerobic bacteria were lower in the case of inoculated samples than in the case of non-inoculated samples. Furthermore, the study demonstrated that exposure to O₃ could in some way inhibit or retard the germs’ growth in a cold environment. According to this perspective, O₃ treatment acts as an antimicrobial agent for meat products kept or stored at a cold temperature to eliminate surface bacterial infection. Cárdenas et al. (2011) previously studied the application of gaseous O₃ treatment on bacterial counts on meats. Total and E. coli bacterial counts decreased by 0.5 log to attain 0 log, while the count of L. monocytogenes, injected at 10² CFU/g tissue under cold storage conditions, fell below the limit of detection. Another study conducted by Muhlisin et al. (2016) discovered that gaseous O₃ applied to the beef samples did not destroy the internal form of E. coli O157:H7, but only eliminated the external contamination by this bacterium. A further piece of data supporting the effectiveness of O₃ treatment during immersion chilling in improving the microbiological protection of beef comes from the finding of Kalchayanand et al. (2019) found that aqueous O₃ sprays cool significantly decreased the prevalence of E. coli O157:H7 on raw beef surfaces by 1.46 log. Studies that have used O₃ to reduce bacterial contamination in meat have generally shown that the surface area of ozonated meat is the primary target of O₃’s antibacterial activity. The most frequent and common cause of contamination in meat is surface contamination from pathogenic microorganisms. This is an alarming situation for both health and food safety. In addition to that, O₃ has been suggested as a more appropriate antibacterial agent that can improve the shelf life of meat and meat products stored in the refrigerator.

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Conclusion

From this investigation, it was found that gaseous O₃ of 0.5 ppm was effective in inhibiting the growth of A. butzleri in meat samples infected with A. butzleri for the first and second days of storage. In addition, gaseous O₃ caused a significant reduction of bacterial populations including both aerobic and anaerobic bacteria, and greater efficacy was demonstrated for the treatment as the time of the exposure period at the same dosage was increased. In terms of public health, these results are important. The cost-effective and effective antibacterial O₃-preserving treatments for food products have a number of advantages over others. Application of O₃ is more efficient and energy saving in the food industry because O₃ can be dissolved in much colder water, and without chemical residues, waste disposal and final rinsing are cheaper. This method improves food safety, quality, and shelf life, decreasing decomposition and spoiling losses and saving money. Additional research using different exposure times and various O₃ concentrations is necessary to elucidate the impact of O₃ on spoilage microorganisms and meat borne pathogens to substantiate its use and scalability in industrial environments.

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Acknowledgments

The author thanks the Technical Institute of Suwaria for letting her use its microbiology lab. The author would like to express her gratitude to Assist. Prof. Dr. Zuhair Al-Chalabi for his guidance and support. She would like to extend her gratitude to Prof. Dr. Nasr Noori Al-Anbari, College of Agriculture Engineering Sciences, University of Baghdad, for his statistical analysis support. She is also very grateful to Dr. Morteza Saki, Ahvaz Jundishapur University of Medical Sciences for his assistance in editing the English language.

Conflict of interest

According to what the author has claimed, there are no conflicting interests.

Funding

No funding or contributions supported this research.

Author’s Contributions

MHGK was in charge of everything involved in this work, from the research planning to the final revisions of the publication. The study’s design, sample collection, laboratory work, analysis, and writing are all included. The author evaluated and agreed to the final version of the manuscript before and during the submission of her paper.

Data availability

All data are provided in the manuscript.

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Supplementary Materials

Supplementary Fig. 1. Ozone- CHEMets Visual Kit (K-7404 ozone analysis kit).

Supplementary Fig. 2. The diagram of the gaseous O₃ chamber.