Advances in ozone technology for preservation of grains and end products: Application techniques, control of microbial contaminants, mitigation of mycotoxins, impact on quality, and regulatory approvals

Abstract

Ozone has emerged as a promising technology for preserving stored grains and end products. Its efficiency as a biocide and the absence of residues make it an attractive alternative to traditional chemical methods of food preservation. This study reviews recent advancements in ozone application techniques, including continuous flow treatments, closed‐loop recirculation systems, and low‐pressure application systems, as well as their impact on product quality. The study also examines the mechanisms of ozone action, its half‐life in grain storage environments, and methods to ensure uniform gas distribution. The results of this study provide a foundation for understanding ozone reactions in various grain types and application systems, offering essential information for effectively sizing treatment systems, estimating ozone concentrations over time, and determining the quantity of products to be treated. A thorough comprehension of ozone behavior in porous environments, such as silos, and its stability under diverse environmental conditions is crucial for enhancing its applicability. While scientific evidence supports ozone's efficacy in controlling pests and microorganisms, further investigation is needed on its impact on the nutritional quality of grains and final products. Additionally, the review highlights the latest regulatory approvals for ozone use in the food industry, emphasizing the importance of compliance and safety. The findings underscore the need for continued technological development and economic analysis to evaluate the long‐term viability of ozone applications in agriculture.

1. INTRODUCTION

Cereals, pulses, and oilseeds, which constitute food grains, represent approximately 50% of global calorific intake. However, 20–40% of agricultural production is lost during storage due to the activity of microorganisms responsible for spoilage and pathogenicity, such as some bacteria and fungi (Ziuzina et al., 2021). Subsequent losses occur during threshing, drying, transport, storage, and processing (Kumar & Kalita, 2017). Insects can cause losses of 30–40% in production, while mycotoxins can reduce grain quality and yield by 25–50% (Ziuzina et al., 2021).

Despite the implementation of modern postharvest techniques, these losses persist, particularly in regions with limited infrastructure. The efficacy of advanced cultivation and storage practices in minimizing losses has been demonstrated; however, the implementation of these practices necessitates adequate inputs and the establishment of effective policies (Kitinoja et al., 2018). The employment of chemical fumigation, such as the use of phosphine, has been the subject of extensive research (Nath et al., 2011). Nevertheless, concerns regarding its detrimental effects on human and environmental health, arising from toxicity and residues in food, have led to limitations in its application (Paul et al., 2020). Furthermore, the continuous utilization of chemical fumigants has been demonstrated to encourage the development of microbial resistance, thereby necessitating escalating doses to maintain efficacy (Collins et al., 2005; Pimentel et al., 2008).

In this context, the increasing demand for safe pest control technologies and pesticide‐free food has driven the search for viable alternatives (Monica et al., 2024). Among these, ozone (O₃) has emerged as a promising tool, attracting significant research interest in recent years, particularly in the context of postharvest preservation of grains (Boopathy et al., 2022; Guzel‐Seydim et al., 2004). Its high reactivity enables the elimination of microorganisms responsible for spoilage, such as certain bacteria, fungi, and pathogens, thereby ensuring food quality (Epelle et al., 2023a; Fundo et al., 2018; Ziyaina & Rasco, 2021). Its application as a sanitizer has been regulated on a global scale and has been approved by the United States Food and Drug Administration (US FDA) in North America, influencing regulations in Australia, New Zealand, Japan, and Europe (FDA, 2001a). In Brazil, it is used to protect stored grain in an environmentally safe way (Mishra et al., 2019). In contrast to conventional pesticides, O₃ is known to degrade rapidly into molecular oxygen, thereby eliminating the necessity for removal steps and minimizing the environmental impact (Obadi et al., 2018a; Pandiselvam et al., 2017; Pandiselvam et al., 2020).

A substantial body of research has previously demonstrated the effectiveness of these treatments in the control of pests, the inactivation of fungal organisms, and the mitigation of mycotoxins (Silva et al., 2022; Uzoma et al., 2024; Brito et al., 2018; Gomes et al., 2020; Reinholds et al., 2016; Savi et al., 2020; Afsah‐Hejri et al., 2020). Furthermore, its capacity to degrade pesticide residues serves to expand its potential for utilization within the agri‐food industry (Avila et al., 2017; Freitas et al., 2017; Kaur et al., 2023). The increasing utilization of O₃ in cereal fumigation necessitates a detailed analysis of application strategies and their effects on product quality (Pandiselvam et al., 2017).

Several studies have investigated strategies to optimize the use of O₃ in the storage of grains and their derivatives (Bechlin et al., 2019; Granella et al., 2018; Mcdonough et al., 2011a; Silva et al., 2022; Uzoma et al., 2024). However, there is a necessity for additional research to be conducted into their mechanisms of interaction with foodstuffs and the factors that affect their efficiency.

A comprehensive understanding of the kinetics and fundamentals of O₃ reactions in diverse application systems for various types of grains and their byproducts is imperative for the effective sizing of treatment systems, estimation of O₃ concentration over time, and determination of the requisite quantity of products to be treated. Furthermore, a thorough comprehension of O₃ behavior in porous environments, such as silos, and its stability under diverse environmental conditions, is crucial for enhancing its applicability and effectiveness. However, the paucity and fragmentation of existing knowledge in this area necessitate the development of comprehensive reviews that integrate operational, physicochemical, and microbiological aspects of O₃ utilization in postharvest contexts.

This study reviews strategies for the application of O₃ to grains and end products, focusing on its impact on pest control, fungal inactivation, mycotoxin reduction, and overall food safety. The analysis encompasses the operating principles, distribution of O₃ in porous systems, and its stability on a large scale. The overarching objective of this approach is to optimize fumigation systems and size storage silos to maximize treatment efficiency. The subsequent sections of this study present case studies that illustrate various application strategies, highlighting their respective advantages and limitations. By addressing these elements in an integrated manner, this review contributes to a deeper understanding of O₃ technology and provides a scientific basis for its broader implementation in sustainable grain and end products management.

2. FUNDAMENTALS OF O₃ TECHNOLOGY

2.1. O₃ generation methods

O₃ is a powerful oxidant artificially produced by splitting diatomic oxygen molecules (O₂). This generation process is endothermic, requiring energy input, typically heat (Rakness, 2005). The primary methods for O₃ production include corona discharge (Figure 1), ultraviolet (UV) radiation (Figure 2), and electrolysis (Mottan et al., 2022; Rodríguez‐Peña et al., 2021; Ziyaina & Rasco, 2021).

FIGURE 1.

Method of generating ozone by corona discharge. Source: Goncalves (2009).

FIGURE 2.

Method of generating ozone using ultraviolet (UV) technology.

The corona discharge method generates O₃ by applying an intense electric charge to a dielectric surface, creating a high‐voltage electric field. This field induces the dissociation of oxygen molecules (O₂) into individual oxygen atoms (O), which then recombine with other O₂ molecules to form O₃. The efficiency of the process is influenced by several factors, including the applied voltage, the characteristics of the dielectric material, and the operating conditions such as pressure and temperature. This method is commonly used due to its effectiveness in generating O₃ at relatively low costs, while also being adaptable for various applications that require controlled O₃ production (Table 1) (Pandiselvam et al., 2017). The reactions involved are shown in Equations (1) and (2) below:

$${\mathrm{O}}_2+e^{-}\to 2\mathrm{O}$$ (1)

$$2\mathrm{O}+2{\mathrm{O}}_2\to 2{\mathrm{O}}_3$$ (2)

TABLE 1.

Comparison of ozone production methods.

Methods of O3 generation	Cost of O3 production	O₃ generation efficiency	Specific energy consumption/g O3 produced	Commercial application	O3 output flow	O3 concentration in the output feed by weight	References
Electrolysis	High	Moderate	–	Limited	Variable	–	Sousa et al. (2018); Okada et al. (2019); Rodríguez‐Peña et al. (2021).
UV irradiation	Bass	Low (1.94 g/kWh)	0.515 kWh/g O3 using 140–190 nm UV	Very limited	Variable	1.8 g/m3 0.14%	Harrison and Blazek (2000); Zoschke et al. (2014); Wardenier et al. (2019); Claus (2021)
Corona discharge	Moderate	High (55 g/kWh)	0.018 kWh/g O3 of dry air	Widely used	Constant	12–60 g/m3 0.1–4.8%	Harrison & Blazek (2000); Anuwat and Nuttee (2016); Cuong et al. (2019); Zylka (2020)

The efficiency of O₃ production depends on several factors, including the concentration of oxygen in the feed gas, the properties of the dielectric material, the discharge interval, and the frequency and voltage of the current used (Pandiselvam et al., 2017). When dry air is used as the feed gas, the O₃ concentration in the output typically ranges from 1 to 3%, whereas using high‐purity oxygen (93 ± 3%) can yield 3–6% O₃ (Brodowska et al., 2018; Pandiselvam et al., 2017).

The UV irradiation method generates O₃ by exposing oxygen molecules (O₂) to UV light within the 140–190 nm wavelength range. This UV radiation provides sufficient energy to dissociate the oxygen molecules through a process called photodissociation, where the absorbed photons break the O₂ bonds, resulting in the formation of individual oxygen atoms (O) (Zoschke et al., 2014). These atoms possess a high degree of reactivity and rapidly combine with other oxygen molecules to form O₃. The efficiency of this process is predominantly influenced by the wavelength of the UV radiation. Shorter wavelengths (closer to 140 nm) possess higher energy levels, thereby promoting a higher rate of dissociation and, consequently, O₃ production (Brodowska et al., 2018; Tapp & Rice, 2012).

An alternative method for generating O₃ is the electrolysis of water, a process that was introduced by Lynntech, Inc. (College Station, Texas) (Murphy et al., 1994). In this process, an electric current is passed through water, thereby splitting the water molecule (H₂O) into hydrogen (H₂) and oxygen (O₂) atoms. The oxygen is produced at the anode, while the hydrogen is generated at the cathode. However, while electrolysis produces oxygen, it does not directly form O₃. The generation of O₃ through electrolysis necessitates an additional mechanism, such as the application of a high‐energy electric field or the use of specialized electrodes that favor the conversion of O₂ into O₃. Therefore, the assertion that electrolysis of water directly generates O₃ is not entirely accurate. In order to generate O₃, it is necessary to implement additional steps to facilitate the transformation of O₂ into O₃ (Claus, 2021; Okada et al., 2019).

2.2. O₃ gas concentration measurement method

O₃ gas is a vital agent in the protection of stored grains, a fact that is firmly established by its proven efficacy in various studies (Parray et al., 2025). The concentration of O₃ is a crucial factor that directly impacts the effectiveness of fumigation systems used for grain preservation (Sivaranjani et al., 2021). Therefore, accurate measurement of O₃ concentration is essential for optimizing these systems and ensuring their successful application.

Several techniques are employed to measure O₃ concentration. Among these, the UV absorption method is widely recognized and standardized by the International Organization for Standardization (ISO13964, 1988). This technique relies on the absorption of UV light by O₃ molecules, providing a reliable and precise measure of O₃ concentration. Another established method is the iodometric technique, which involves the O₃ reaction with iodide ions, leading to a measurable color change that can be quantified spectrophotometrically (Rakness et al., 1996). This method is noted for its accuracy and sensitivity in various applications.

In recent years, metal oxide semiconductor (MOS) technology has gained prominence as an innovative approach to O₃ quantification (Pandiselvam et al., 2017; Peterson et al., 2017). MOS sensors detect O₃ by monitoring changes in the electrical resistance of metal oxide materials, offering real‐time measurement capabilities and ease of integration into monitoring systems. Each of these approaches has its distinct benefits and drawbacks. The selection of the most appropriate method depends on the specific requirements of the fumigation system and the environmental conditions under which O₃ is applied.

2.3. Benefits of using O₃

O₃, a gas that has gained significant popularity in nonthermal treatments (Ziyaina & Rasco, 2021), is widely acknowledged for its highly effective and dependable disinfectant properties, particularly in contexts involving stored grains. Its high oxidation potential (2.07 V) exceeds that of chlorine (1.36 V) (Blanco et al., 2021; Wen et al., 2020; Tripathi & Hussain, 2022; Xue et al., 2023), making it an effective agent for microbial control. In contrast to numerous conventional chemical disinfectants, O₃ decomposes into oxygen following application (Mishra et al., 2019), thereby eliminating the necessity for additional steps to remove residues from the food matrix.

A notable benefit of this approach is the capacity for on‐site O₃ generation, a feature that distinguishes it from conventional disinfection methods. O₃ can be produced on demand, thus eliminating the necessity for transportation and storage, which are typically required for chemical disinfectants (Epelle et al., 2023b; Sanchez et al., 2024; Ziyaina & Rasco, 2021). This feature enhances the efficiency of the disinfection process and reduces logistical challenges and associated costs. Moreover, the operational costs of ozonation systems are relatively low, as they primarily consume a limited amount of electricity. This cost effectiveness contributes to the economic viability of O₃‐based treatments, providing a financial advantage over other methods (Ziyaina & Rasco, 2021).

Furthermore, O₃ treatment is distinguished by its high adaptability, allowing for customization to diverse operational scales, ranging from small‐scale grain storage units to large‐scale industrial facilities (Epelle et al., 2023a; Sinha et al., 2014). The flexibility of O₃ systems facilitates their integration into a wide range of storage environments, thereby ensuring their versatility for both smallholder and commercial grain producers. Through the modulation of O₃ concentration and exposure duration, the system can be customized to address distinct preservation requirements, thereby ensuring a high degree of control over the treatment process (Ziyaina & Rasco, 2021).

Another key advantage of O₃ is its rapid action (Parray et al., 2025). O₃ exerts its effect with great rapidity, inactivating microbial contaminants and degrading harmful compounds within min of exposure (Afsah‐Hejri et al., 2020; Yudiastuti et al., 2021). This characteristic renders O₃ an optimal solution for grain preservation, where temporal constraints are frequently a pivotal consideration. The ability to rapidly eliminate microorganisms responsible for spoilage, including certain molds and bacteria, reduces the risk of spoilage, allowing producers to preserve high‐quality grains and extend their shelf life without significant delays in the storage process (Kaur et al., 2023; Parray et al., 2025; Savi et al., 2020).

O₃ treatment represents a chemical‐free alternative, insofar as no harmful substances are introduced into the grains during the preservation process (Parray et al., 2025; Srivastava et al., 2021). This is of particular significance for organic and pesticide‐free grain producers, as it guarantees that the grains remain free from synthetic chemicals, thereby enhancing their appeal to environmentally conscious consumers. O₃’s natural decomposition into oxygen serves to reinforce its status as a safe and sustainable alternative to conventional chemical treatments (Obadi et al., 2018a; Pandiselvam et al., 2017; Pandiselvam et al., 2020).

2.4. Factors affecting the effectiveness of O₃ in grain preservation

The effectiveness of O₃ in grain preservation is shaped by a range of internal and external factors that critically influence its antimicrobial capabilities within storage environments (Table 2).

TABLE 2.

Extrinsic and intrinsic factors affecting the effectiveness of ozone in preserving grains.

Parameters	Factors
Extrinsic factors	Temperature; relative humidity; air flow rate; ozone treatment (application method, gas concentration, treatment time)
Intrinsic factors	Food product (grain type, surface characteristics, moisture content); microbial load (type of pests and their life stages, type of fungus, and microbial strain characteristics)

Source: Adapted from Pandiselvam et al. (2020); Swamy and Kasiviswanathan (2021); Kaur et al., 2023); Kaur et al. (2023).

2.4.1. Extrinsic factors

Temperature, relative humidity, and air flow rate

The efficacy and stability of O₃ are strongly influenced by environmental factors such as temperature, relative humidity, and air flow (Kaur et al., 2023; Premjit et al., 2022). These parameters directly affect the half‐life and reactivity of O₃, influencing its biocidal efficacy in preserving stored grains.

Studies suggest that high temperatures can enhance the effectiveness of O₃ in combating fungi (Wu et al., 2006). Nevertheless, the literature shows conflicting findings: in certain cases, higher temperatures accelerate the breakdown of O₃, reducing its effectiveness (Faroni et al., 2007; Sousa et al., 2006). The susceptibility of insects to O₃ also varies according to temperature. As an example, when  Sitophilus zeamais adults were exposed to 50 ppm for 24 h, mortality was under 20% at 20°C, but reached 100% at 40°C (Sousa et al., 2006). In contrast, for Tribolium castaneum, there was no clear linear relationship between temperature and mortality, suggesting that the specific physiology and metabolism of each species influences its resistance to oxidative stress (Pereira et al., 2008).

Temperatures above a certain threshold accelerate O₃’s conversion into reactive free radicals, which enhances its effectiveness against microorganisms and insects (Khadre et al., 2001). However, this also increases the rate of O₃ decomposition. To mitigate this, it is essential to provide a stable and controlled O₃ supply. Although O₃ concentration is relatively unaffected by temperature variations, changes in the biochemical and behavioral responses of organisms, such as shifts in respiration and mobility, can explain the differences in effectiveness (Hardin et al., 2010).

Aeration is a widely adopted method in grain storage to manage temperature and humidity conditions. This process involves the circulation of air through the grain mass, with the objective of eliminating excess heat and humidity. However, O₃ application has been demonstrated to be more efficacious in static air and under conditions of reduced temperature and humidity (McClurkin & Maier, 2010). Notwithstanding, the integration of ozonation with aeration has shown considerable promise by reducing the temperature within the intergranular space and thereby hindering the proliferation of pests.

The efficacy of this treatment method depends on a comprehensive understanding of O₃ kinetics and its transport dynamics within the grain mass. To establish effective fumigation protocols, it is vital to assess the time required for O₃ to saturate the intergranular spaces and the decomposition rate of the gas, which will inform the correct dosages and exposure times (Alencar et al., 2011; Silva et al., 2019).

O₃’s reactivity is largely influenced by the relative humidity present during application. Environments with higher humidity levels promote O₃ degradation, leading to the generation of hydroxyl radicals (·OH), which are potent oxidizers and enhance the antimicrobial effect (Cho et al., 2003). While O₃ demonstrates enhanced stability in arid environments, its antimicrobial efficacy is augmented under conditions of sufficient humidity due to the generation of these radicals (Epelle et al., 2023b; Ewell, 1946; Varga & Szigeti, 2016). To ensure the optimal efficacy of O₃, it is imperative to calibrate the air flow rate and relative humidity levels, thereby facilitating the gas’ penetration into the grain mass and maintaining sufficient reactivity to induce the generation of oxidizing radicals.

The specific air flow rate (m³ min⁻¹ t⁻¹) directly influences the distribution of O₃ and the contact time in the intergranular spaces. High flow rates promote faster distribution, but with a shorter residence time, which limits the biocidal action. On the other hand, lower flow rates increase contact time, favoring the inactivation of microorganisms, although they require more time to saturate the grain mass.

Silva et al. (2024) examined two distinct flow rates (0.15 and 1.00 m³ min⁻¹ t⁻¹) for 3.0 kg grain samples, exposed to an input concentration of 16.0 mg L⁻¹ for up to 48 h. At a flow rate of 0.15 m³ min⁻¹ t⁻¹, saturation occurred in 3.59 h, with consumption of 18.43 g of O₃, residence time of 6.72 min and saturation concentration of 8.59 mg L⁻¹. When the flow rate was set to 1.00 m³ min⁻¹ t⁻¹, saturation was achieved within 0.88 h, with a reduced residence time (1.12 min), elevated O₃ concentration (15.44 mg L⁻¹), and a notably higher consumption (138.24 g).

The results highlight the need to strike a balance between operational efficiency and cost effectiveness. Elevated flow rates expedite the process; however, they concomitantly augment O₃ consumption and expenses. Conversely, diminished flow rates engender greater economic efficiency but necessitate augmented exposure times. Consequently, the selection of the optimal flow rate must take into account a triad of factors: microbiological efficiency, uniform gas distribution, and economic viability, particularly in industrial applications.

O₃ treatment parameters

The application parameters of O₃ are of critical importance for the success of the treatment and the preservation of grain quality. The effectiveness of O₃ treatment in controlling insect pests, inhibiting the growth of specific microorganisms such as molds and spoilage bacteria, and preserving quality parameters in stored grains is strongly influenced by both the applied O₃ concentration and the duration of exposure (Sarron et al., 2021).The CT concept refers to the efficacy of O₃ treatment on a given organic or inorganic compound, where C represents the residual O₃ concentration (mg L⁻¹) and T the exposure time (min). The intensity of the ozonation process is thus expressed as CT (mg min⁻¹ L⁻¹), varying according to the target component and environmental conditions (Sarron et al., 2021). In many cases, a low O₃ concentration combined with a prolonged exposure time can yield effects similar to those of a high concentration applied over a shorter period, as long as the CT value remains constant (Sarron et al., 2021). However, it should be noted that this equivalence does not apply universally, as short‐term exposure to high doses has been shown to be more phytotoxic than longer exposures at lower concentrations (Souza & Pagliuso, 2009). Therefore, the effective application of ozonation requires a balance between O₃ concentration and an appropriate exposure duration (McDonough et al., 2011b).

A model for estimating insect mortality based on the CT concept was developed using the product of O₃ concentration (C) and exposure time (T) required to achieve the desired population control level. The CT values indicated that T. castaneum required either longer exposure or higher O₃ concentrations to achieve 100% mortality compared with Plodia interpunctella, with mean exposure values of 256,500 ppm‐min and 183,000 ppm‐min, respectively (McDonough et al., 2011b). In a separate study, Bonjour et al. (2011) investigated the effects of different O₃ concentrations on wheat grains for the management of Cryptolestes ferrugineus. The study observed that at 25 ppm, insect survival rates remained high after 4 days of exposure. Conversely, concentrations of 50 and 70 ppm resulted in reduced survival rates or complete adult mortality within the respective exposure periods.

Concurrently, Gad et al. (2021) conducted a study that evaluated the insecticidal activity of O₃ against all life stages of Callosobruchus maculatus (egg, larva, pupa, and adult) using a concentration of 600 ppm (1.2 mg L⁻¹) across six different exposure times (0.5, 1, 2, 3, 4, and 5 h). The results showed that longer exposure times significantly increased insect mortality. However, since the main goal of seed protectants is to ensure long‐term protection during storage, the reduction in insect progeny is a more relevant indicator of effectiveness than the initial mortality of adult insects present on the treated grains. The reduction in adult emergence from eggs, larvae, and pupae can be achieved either through extended O₃ exposure durations (Bonjour et al., 2011) or the application of a high concentration, such as 5 mg L⁻¹ (El‐Ghaffar et al., 2016).

2.4.2. Intrinsic factors

Grain characteristics

The intrinsic properties of the grain have a direct influence on the efficiency of O₃ treatment, determining its penetration capacity, reactivity, and impact on the preservation of the quality of the stored product (Silva et al., 2019). Among these factors, grain temperature plays a critical role in insect control and mycotoxin degradation (Kaur et al., 2023). Studies indicate that elevated temperatures enhance the efficacy of O₃ in degrading aflatoxins in groundnuts, reducing the exposure time necessary to achieve significant degradation. For instance, increasing the temperature from 25 to 75°C reduces the treatment time from 15 to 10 min, achieving 77 and 80% removal of aflatoxins AFB1 and AFG1, respectively (Proctor et al., 2004). This effect is attributed to the enhancement of chemical reactions induced by O₃ at higher temperatures, which accelerates the degradation of these toxins.

Another key factor influencing the efficiency of ozonization is the moisture content of the grain, which directly affects the stability of O₃ and its ability to target pests and microorganisms. Grains with a moisture content below 12% promote longer retention of the gas, as reduced water availability limits collateral reactions that could otherwise accelerate its degradation. On the other hand, high levels of moisture can intensify O₃ decomposition and promote the formation of oxidative byproducts such as free radicals, which can alter the biochemical structure of the grain and affect its nutritional quality. In this context, Ravi et al. (2015) evaluated the application of O₃ (1.43 mg L⁻¹) to rice grains with different moisture contents (11.4 and 14.2 g 100 g⁻¹ w.b.). The results showed that the half‐life of O₃ was longer in grains with 11.4 g 100 g⁻¹ w.b. compared with grains with 14.4 g 100 g⁻¹ w.b., with values of 13.80 and 11.61 min, respectively.

In addition to humidity, genetic variation between different cultivars of the same cereal species is an important factor to consider when applying O₃. The study by Souza et al. (2018) evaluated the effect of O₃ on different maize hybrids (AG 1051, Tropical Plus, GSS 41499, GSS 42072, and GSS 41243), all with an initial moisture content of 13.0 g 100 g⁻¹ w.b. The results showed significant differences in O₃ half‐life between the hybrids with values of 10.50, 6.30, 0.16, 0.80, and 3.80 min for AG 1051, Tropical Plus, GSS 41499, GSS 42072, and GSS 41243 respectively. The half‐life of O₃ is a fundamental parameter as it determines its persistence and stability within the grain mass, which directly influences its effectiveness in both pest control and microbial inactivation.

Therefore, when applying O₃ to stored grain, factors such as grain temperature, humidity, and genetic differences between varieties must be taken into account to ensure that the exposure conditions (time, concentration, and relative humidity) are adjusted to maximize the efficiency of microbiological decontamination without compromising the physical and chemical integrity of the grain.

Microbial load

The antimicrobial efficacy of O₃ is significantly influenced by multiple factors, including microorganism type, physiological state, population density, and environmental stress conditions (Giuliani et al., 2018; Premjit et al., 2022). Among these, the microbial load present on grains plays a fundamental role in determining O₃ treatment effectiveness. The microbiological composition of grains typically consists of bacteria, yeasts, and filamentous fungi, not all of which are pathogenic. Their susceptibility to oxidative stress varies depending on the species and developmental stage (Giuliani et al., 2018). For instance, the vegetative forms of Aspergillus flavus and Penicillium expansum exhibit heightened sensitivity to O₃‐induced oxidation, whereas Penicillium and Rhizopus require elevated O₃ concentrations or extended exposure times for effective inactivation (Afsah‐Hejri et al., 2020).

One of the most extensively studied fungal genera with regard to O₃ susceptibility is Fusarium. Numerous studies have confirmed the effectiveness of O₃ in inactivating Fusarium species (Piacentini et al., 2017; Savi et al., 2015). Research findings indicate that Fusarium graminearum was completely inhibited after 120 min of O₃ exposure, while A. flavus and Penicillium citrinum required longer exposure times of 160 and 180 min, respectively, to achieve growth inhibition (Savi et al., 2014; Savi et al., 2015). It is noteworthy that when working with reduced sample sizes, shorter exposure times were sufficient to inhibit fungal proliferation (Savi & Scussel, 2014), thus emphasizing the pivotal role of sample volume in O₃ efficiency.

In addition to the sensitivity of fungi to O₃, the volume of grains subjected to treatment is a critical determinant of its effectiveness. Trombete et al. (2017) demonstrated that ozonation was significantly more effective in small wheat samples stored in silos. The highest recorded inhibition rates, characterized by a 3‐log reduction in fungal populations, were observed in 2 kg wheat samples treated with high O₃ concentrations (60 mg/L) for 300 min. In a similar study, Granella et al. (2018) reported a significant reduction (92.86%) in total fungal counts following 45 min of air drying with O₃ at 50°C. These findings suggest that the optimization of O₃ treatment parameters, particularly exposure time and concentration, is essential for maximizing its antimicrobial efficacy.

The high susceptibility of Fusarium to O₃ has been further corroborated by previous investigations. Raila et al. (2006) and White et al. (2013) both identified Fusarium as the most sensitive fungal genus to O₃ treatment. However, variations in Aspergillus response across studies suggest that differences in O₃ concentration and application methods may influence treatment outcomes. These discrepancies underscore the necessity of standardizing O₃ treatment protocols to ensure consistent disinfection performance across different microbial species.

Beyond species‐specific susceptibility, microbial population density has been demonstrated to play a crucial role in O₃ effectiveness. High microbial loads, measured in colony‐forming units per gram (CFU/g), can significantly reduce O₃ efficiency by providing a shielding effect and protecting the grain surfaces with organic matter and biofilms. Furthermore, genetic variability among microbial strains has been demonstrated to influence their resistance to oxidative stress, resulting in differential responses to O₃ treatment (Alexander et al., 2016; Frei, 2015).

The microbial susceptibility to O₃ is also influenced by physiological traits such as cell age and environmental adaptations. Wani et al. (2016) observed that Pseudomonas spp. cells from older colonies (7, 10, and 12 days) exhibited greater resistance to gaseous O₃ compared with younger colonies (2 and 4 days). In addition, refrigerated Pseudomonas sp. strains exhibited increased O₃ resistance in vitro. Furthermore, O₃ exposure has been demonstrated to induce bacterial aggregation, rendering Pseudomonas syringae noncultivable before complete viability loss (Sarron et al., 2013). Furthermore, the physical positioning of microorganisms has been demonstrated to impact O₃ penetration, with those embedded within surface irregularities being better protected than those directly exposed (Kim et al., 1999).

In line with these findings, initial bacterial load is another decisive factor influencing O₃’s antimicrobial potential. Feng et al. (2018) investigated the inactivation of Vibrio parahaemolyticus by O₃ and examined the relationship between initial cell density and sterilization efficiency. The experimental design entailed the mixture of bacterial cultures at varying concentrations (10⁶, 10⁸, and 10¹⁰ CFU/mL) with 99 mL of ozonated water (1.0 mg/mL). The results obtained demonstrated that an increase in bacterial density resulted in a decrease in sterilization efficiency, even when O₃ concentration and exposure time were constant. This finding indicates a direct correlation between microbial load and O₃’s inactivation capacity, as higher bacterial densities promote cell clustering, increasing O₃ consumption and reducing disinfection effectiveness (Megahed et al., 2020).

The findings, when considered as a whole, highlight the complexity of O₃‐based microbial inactivation, emphasizing the necessity of precisely controlling O₃ concentration and exposure duration to achieve optimal disinfection. It is imperative to comprehend the intricate relationship between microbial density, species‐specific resistance mechanisms, and environmental factors to optimize O₃ as a microbial control strategy. By addressing these variables, O₃ applications can be tailored to enhance its efficacy in diverse agricultural and food storage contexts.

2.5. O₃ movement/flow process in a porous medium

The application of O₃ gas to grains is usually carried out in a fixed bed. The grain mass remains static on a perforated plate above the gas injection point (Hardin et al., 2010). This arrangement is similar to conventional grain aeration and drying systems (Noyes et al., 2002). In these systems, when atmospheric air enters the plenum or aeration ducts, there is a decrease in velocity and an increase in static pressure. The pressure gradient created causes air to flow from the bottom to the top of the grain. The predominant transport mechanism in this process is forced convection, which is responsible for the transportation of gaseous O₃ through the grain mass (Noyes et al., 2002). However, it is also a consequence of gas diffusion and is limited by its reactivity with grains (Silva et al., 2019).

Dong et al. (2022) show that O₃ outflow in the intergranular space occurs in two different ways. First, O₃ has a high reaction rate with grains. Then, the intense reaction slows down as the different types of grains, including beans, reach the saturation stage. When the O₃ injection is stopped, the gas decomposes naturally into oxygen. The rate of reaction and decomposition of O₃ gas in the grain layer depends on the O₃ concentration, specific flow rate, physical properties, and grain moisture content (Sitoe et al., 2023; Tiwari et al., 2010). This behavior has been observed in rice (Assis‐Silva et al., 2019), beans (Sitoe et al., 2024a), peanuts (Alencar et al., 2011), and popcorn kernels (Silva et al., 2019).

When O₃ gas is applied by forced aeration, the flow regime is classified according to the surface velocity values of the gas in the intergranular space. The velocity values vary from operation to operation, depending on the specific flow rates applied (Olatunde et al., 2016). The flow regime is classified as laminar or turbulent (Nijemeisland & Dixon, 2001; Dumas et al., 2010; Lesage et al., 2004) based on the calculation of the Reynolds number for porous media (Equation 3) (Table 3). Below 110, the flow regime is considered to be laminar. For values greater than 110 and less than 280, the flow regime is considered transient. Turbulent flow regimes have a Reynolds number greater than 280 (Lesage et al., 2004).

TABLE 3.

Fundamental equations for ozone transport in porous media.

Equation	Parameters	Explanation/application	Definition of parameters
Equation (3)	$\mathrm{R}{\mathrm{e}}_{\mathrm{p}}=\frac{{\rho }_{\mathrm{g}}v{\overline{D}}_{\mathrm{P}}}{{\mu }_{\mathrm{g}}}$	Reynolds number for flow in porous media, which classifies the flow as laminar (Re < 110), transitional (110 < Re < 280), or turbulent (Re > 280).	${\rho }_{\mathrm{g}}$(kg m−3) is the density of the fluid; $v$(m s−1) Surface velocity; ${\overline{D}}_{\mathrm{P}}$ (m) average grain diameter; and ${\mu }_{\mathrm{g}}$ (kg m−1 s−1) is the dynamic viscosity of the gas
Equation (4)	${\epsilon }_{\mathrm{g}}\frac{\partial {\rho }_{\mathrm{g}}}{\partial t}+\nabla ·({\rho }_{\mathrm{g}}v)=0$	Continuity equation for gas phase, ensuring mass conservation	${\epsilon }_{\mathrm{g}}$ corresponds to the volumetric fraction of the gas; $(1-{\epsilon }_{\mathrm{g}})$ corresponds to the volumetric fraction of the solid matrix, that is, the grains that make up the porous medium; t (s) is time; ρ g (kg m−3) is the density of the gas; ρ s (kg m−3) is the density of the particle; ${\mu }_{\mathrm{g}}$(Pa s−1) is the dynamic viscosity of the fluid; $g$ (m s−2) is gravity; $v$ (m s−1) is the velocity vector; ${\omega }_{{\mathrm{O}}_3}^g$ is the mass fraction of ozone in intergranular air; ${\omega }_{{\mathrm{O}}_3}^{\mathrm{ss}}$ é a Mass fraction of ozone in solid matrix; ${\omega }_{{\mathrm{O}}_3}^{\mathrm{ss}}$ is the mass fraction of ozone on the surface of the solid matrix; F (N m−3) represents the resistive forces (viscous and drag) resulting from the interaction between the solid and fluid phases in the porous matrix; K (m−2) represents the permeability of the porous medium; I is the unit vector of velocity; A s (m−1) is the specific surface area of the porous medium consisting of rice grains and h m (m s−1) is the mass transfer coefficient; D s (m2 s−1) is the diffusivity of the ozone gas through the solid matrix of the porous medium and D g (m2 s−1) is the diffusivity of ozone gas in the fluid domain and in the intergranular space.
Equation (5)	$\frac{\partial }{\partial t}({\rho }_{\mathrm{g}}v)+{{\epsilon }_{\mathrm{g}}}^{-1}\nabla ·({\rho }_{\mathrm{g}}vv)=-{{\epsilon }_{\mathrm{g}}}^{-1}\nabla P_{\mathrm{g}}+{{\epsilon }_{\mathrm{g}}}^{-1}{\mu }_{\mathrm{g}}{\nabla }^{2}v-K^{-1}{\mu }_{\mathrm{g}}v-F{\epsilon }_{\mathrm{g}}{K}^{-1/2}{\rho }_{\mathrm{g}}(|v|·|v|)\mathrm{I}$	Navier–Stokes equation for gas flow in porous media, describing momentum conservation
Equation (6)	${\epsilon }_{\mathrm{g}}\frac{\partial }{\partial t}({\rho }_{\mathrm{g}}{\omega }_{{\mathrm{O}}_3}^g)+\nabla ·({\rho }_{\mathrm{g}}{\omega }_{{\mathrm{O}}_3}^{g}v)={\epsilon }_{\mathrm{g}}\nabla ·({\rho }_{\mathrm{g}}{D}_{\mathrm{g}}\nabla {\omega }_{{\mathrm{O}}_3}^g)-k_1{\epsilon }_{\mathrm{g}}{\rho }_{\mathrm{g}}{\omega }_{{\mathrm{O}}_3}^g-{\rho }_{\mathrm{g}}{h}_{\mathrm{m}}{A}_{\mathrm{s}}({\omega }_{{\mathrm{O}}_3}^g-{\omega }_{{\mathrm{O}}_3}^{\mathrm{ss}})$	Species transport equation for ozone in the gas phase, including diffusion and reaction with grain surface
Equation (7)	$(1-{\epsilon }_{\mathrm{g}})$ $\frac{\partial }{\partial t}({\rho }_{\mathrm{s}}{\omega }_{{\mathrm{O}}_3}^s)=(1-{\epsilon }_{\mathrm{g}})$ $\nabla ·({\rho }_{\mathrm{s}}{D}_{\mathrm{s}}\nabla {\omega }_{{\mathrm{O}}_3}^{\mathrm{s}})-k_2(1-{\epsilon }_{\mathrm{g}}){\rho }_{\mathrm{s}}{\omega }_{{\mathrm{O}}_3}^{\mathrm{s}}+{\rho }_{\mathrm{g}}{h}_{\mathrm{m}}{A}_{\mathrm{s}}({\omega }_{{\mathrm{O}}_3}^{\mathrm{g}}-{\omega }_{{\mathrm{O}}_3}^{\mathrm{ss}})$	Species transport equation for ozone in solid matrix, describing the interaction between ozone and grains

The correct characterization of the flow regime allows an appropriate choice of equations that allow the mathematical modelling of the O₃ gas flow in the intergranular space of the grain mass. Using the Navier–Stokes equations and the species transport equations, it is possible to quantify the contribution of each transport mechanism involved in the O₃ flow in the grain mass. In the case of a laminar flow problem, the governing equations are Navier–Stokes (Equations 4 and 5) (Kaviany, 1985) and species transport (Equations 6 and 7) (Elhalwagy and Straatman, 2017). For this set of equations, transient and isothermal flow regimes are considered. In addition, Equations (6) and (7) describe the transport of species assuming a nonequilibrium state between the solid phase of the porous matrix and the intergranular gas (air + O₃) (Table 3).

For the nonequilibrium state of the O₃ concentration between the solid and liquid phases, the decomposition rates are different. Thus, k₁ (s⁻¹) represents the O₃ decomposition rate in the intergranular space over time, while the parameter k₂ (s⁻¹) indicates the reaction rate constant of O₃ due to its interaction with the grains. The last right term in Equations (6) and (7) represents the driving force for mass transfer at the surface of the grains. Equation (4) describes the transport of O₃ from the liquid phase to the solid phase, and this term has a negative sign because it represents the decay of O₃ in the liquid phase. Equation (7) describes the transport of O₃ gas within the solid phase and this term has a positive sign because it represents the increase in O₃ concentration within the grains.

The set of equations presented (Equations 4, 5, 6, and 7) refers to the equations governing the laminar flow regime and the mass transfer between the O₃ gas and the grains. For the equation of a problem of application of O₃ gas in grains, where the flow regime is characterized as turbulent, only the equation of conservation of momentum is different and is presented in Equation (8) (Table 4). This equation (Equation 8) represents the conservation of momentum for turbulent flow in a porous medium. The second term on the right refers to the surface forces exerted on the fluid (air + O₃) due to fluctuations in the velocity vector caused by turbulence. In this term, τ (kg m⁻³) is the viscous stress tensor. The other terms are similar to those in Equation (4). In both cases, laminar or turbulent, the pressure drop imposed by the porous medium on the airflow is defined by (Equation 9) proposed by Hunter (1983) (Table 4).

TABLE 4.

Equations for ozone flow behavior in laminar and turbulent regimes.

Equation	Parameters	Explanation/application
Equation (8)	$\frac{\partial }{\partial t}({\epsilon }_{\mathrm{g}}{\rho }_{\mathrm{g}}v)+\nabla ·({\epsilon }_{\mathrm{g}}{\rho }_{\mathrm{g}}vv)=-\nabla P_{\mathrm{g}}+\nabla ·({\epsilon }_{\mathrm{g}}\tau )+{\epsilon }_{\mathrm{g}}{\rho }_{\mathrm{g}}g+{\epsilon }_{\mathrm{g}}F$	Conservation of momentum in turbulent flow; accounts for velocity fluctuations in the fluid
Equation (9)	ΔP g  = R $v$ + S $v^2$	Pressure drop in the porous medium, showing the relationship between pressure and velocity in both laminar and turbulent flows

Hunter (1983), based on the results obtained by Shedd (1953), calculated the coefficients R (Pa·s·m⁻²) and S (Pa·s²·m⁻³) for the most common types of grains. According to Hunter (1983), when air flows through a fixed bed formed by grains, the pressure gradient is proportional to the velocity (Equation 9). The pressure gradient is directly proportional to the velocity in laminar flow regimes and proportional to the square of the velocity in turbulent flow regimes. As shown in Equation (9), for low‐velocity values (0.005–0.1 m s⁻¹), which are commonly employed in aeration operations, the quadratic term has a minimal contribution to the pressure drop.

When O₃ is applied to grains at low pressure, the transport mechanisms involved are the same as those involved in transporting O₃ by forced air movement. The purpose of using O₃ gas at low pressures is to improve the distribution and interaction of the gas with the grains. The movement of O₃ into the grain mass within the treatment chamber depends on the convection and diffusion of the gas (Silva et al., 2022; Sitoe et al., 2023). Initially, the action of the forced convection of the gas is more important, as the pressure gradient within the treatment chamber is high. Soon after the stabilization of the pressure inside the hypobaric chamber and the interruption of the injection of O₃, the transport mechanism that will act will be purely diffusive. O₃ will migrate into the grain mass due to the concentration gradient. Simultaneously with this process, O₃ decomposition will occur due to its natural instability and the grain reaction (Sitoe et al., 2023).

3. STRATEGIES FOR APPLYING O₃ TO GRAINS AND THEIR FINAL PRODUCTS

3.1. Ozonation in flux

Ozonation in flow is an effective strategy for applying O₃ to bulk‐stored grains. This method can be employed in a closed‐loop recirculation system during grain drying. The flow ozonation system comprises several components: (i) air compressor, (ii) refrigerated air dryer, (iii) compressed air tank, (iv) oxygen concentrator, (v) oxygen tank, (vi) O₃ generator, and (vii) silo (Figure 3). During ozonation, grains remain static on a perforated plate positioned above the gas injection point (Figure 3). This setup is akin to conventional grain aeration and drying systems, as Noyes et al. (2002) described.

FIGURE 3.

Schematic drawing of the ozonation components of flowing grains.

A gas generation system produces the O₃ used in the treatment, which is then pumped into the silo where it is diluted with the air stream to treat the grain. As the gas enters the plenum or aeration duct, the velocity decreases and the static pressure increases. This pressure gradient causes the gas to flow from the bottom to the top of the grain mass. The duration of the ozonation process can range from hours to days or even weeks, depending on factors such as silo size, product volume and the type of micro‐organisms being controlled. It is important to note that stainless steel silos (SS 304) are recommended for ozonation of bulk stored grain due to their corrosion resistance, as reported by Pandiselvam et al. (2017).

Scientific studies have demonstrated flow‐applied O₃’s efficacy in controlling S. zeamais in popcorn kernels (Silva et al., 2019). Mishra et al. (2019) evaluated the effect of ozonation on wheat grains. They found that the gas concentration of 2.5 mg L⁻¹ applied for 8 h caused the mortality of 97, 100, 99, and 100% of adults, pupae, larvae, and eggs of Rhyzopertha dominica, respectively. The study developed by Gadet al. (2021) observed that treatment with O₃ gas at a concentration of 2.0 mg L⁻¹ caused 100% mortality of Callosobruchus maculatus and C. chinensis 5 days after exposure to the gas. Other scientific investigations have demonstrated that O₃ gas applied in flow was effective in inactivating fungi in peanuts (Ferreira et al., 2021), nuts (Oliveira et al., 2020), rice (Savi et al., 2020; Luz et al., 2022), and corn (Ribeiro et al., 2022).

The ozonation of grains during the drying process was initially investigated by Granella et al. (2018). The authors applied O₃ during the wheat seed drying for total fungal inactivation. O₃ was produced in a generator with a 2000 mg/h capacity and applied at 30, 40, and 50°C temperatures for 15, 30, and 45 min. The drying air velocity was 0.5 ± 0.1 m s⁻¹. The drying chamber was made of a cylindrical column 0.45 m long and 0.15 m in diameter. The grains were placed on a perforated metal plate located at a height of 0.20 cm. The data obtained demonstrated that fungal reduction increased significantly with longer O₃ exposure and higher drying air temperatures. The maximum decrease in the total fungal count was 92.86%, with a reduction from 1.87 to 0.13 cfu/g when wheat seeds were treated with O₃ for 45 min and dried at an air temperature of 50°C. Uzoma et al. (2024) evaluated the effect of specific flow rate for the control of S. zeamais and on the inactivation of Aspergillus flavus during low‐temperature drying associated with the ozonation process in corn. Specific air flow rates of 0.50, 0.82, and 1.05 m 3 min⁻¹ t⁻¹ were adopted for drying. The concentration of O₃ at the intake was measured at 2.3 mg L⁻¹. The study's results pointed to the association of O₃ with low‐temperature drying as efficient in the total inactivation of A. flavus and control of S. zeamais for all specific flows.

Hardin et al. (2010) evaluated the process of ozonation of wheat in the silo with a gas recirculation system. In the study, O₃ was injected into three stainless steel containers with an internal volume of 18 m³ containing 13.600 kg of wheat. The concentration of O₃ applied ranged from 0.07 to 0.19 mg L⁻¹. The total time of gas injection was 26 days. Three O₃ injection velocities were adopted, in which for the first 17 days, the surface velocity of the gas was 0.020 m s⁻¹; for the subsequent 7 days, it was 0.026 m s⁻¹; and for the last 2 days, an acceleration of 0.036 m s⁻¹ was adopted. For the velocities of 0.020, 0.026, and 0.036 m s⁻¹, the residence time of O₃ gas in the grain mass was 58, 45, and 33 s, respectively. The recirculation system was efficient from the fifth day of ozonation, and higher surface velocities resulted in higher values of O₃ concentration above the grain layer.

The effectiveness of O₃ gas applied in flow for the control of insect pests and inactivation of fungi and other microorganisms depends on several factors, with greater emphasis on the moisture content of the grains, the concentration of the gas, the exposure time (Dong et al., 2022; Lemic et al., 2019; Mishra et al., 2019; Sunisha, 2019), size and shape of the ozonation prototype, and physical properties of grains (Pandiselvam et al., 2019a; Akbar, Medina and Magan, 2020; Sirohi et al., 2021; Sivaranjani et al., 2021). The thickness of the grain bed is also crucial when considering O₃ diffusion. The concentration of O₃ decreases from the bottom to the surface of the grains when the thickness of the bed increases, and the maximum concentration is observed at the bottom of the silo (Pandiselvam et al., 2017; Pandiselvam et al., 2018). Therefore, a diffusion channel must be provided in the ozonation silo to ensure the uniform distribution of the gas concentration throughout the grain mass and make the fumigation process efficient.

3.1.1. Advantages of the ozonation in flux system

The ozonation in the flow system, a process where O₃ is introduced into the grain mass as it moves through a system, offers several notable advantages for grain preservation. First, this approach ensures uniform distribution of O₃, providing continuous and effective treatment throughout the grain mass. This results in consistent control over microbial contaminants and specific pathogens, thereby enhancing overall grain safety (Silva et al., 2019). The integration of ozonation with existing drying and storage processes is particularly beneficial, as it optimizes the use of pre‐installed systems and minimizes the need for substantial new investments (Oliveira et al., 2020). Furthermore, the system excels in reducing microbial contamination by maintaining high O₃ concentrations, which are critical for suppressing spoilage fungi and insect pests (Pandiselvam et al., 2017). The ability to sustain a sanitary environment during storage is vital for maintaining grain quality.

Another significant advantage is the operational efficiency of the system. The capacity to precisely adjust parameters such as O₃ concentration and exposure time allows for effective management tailored to different grain types and volumes (Mishra et al., 2019). This adaptability ensures that the system can be used for a wide range of grains, enhancing its versatility. Additionally, the system is environmentally friendly and economically viable. O₃ naturally decomposes into molecular oxygen after treatment, which minimizes the risk of toxic residues and reduces the need for intensive posttreatment cleaning or waste disposal procedures (Zhu, 2018). The operational costs are relatively low, primarily involving electricity, which supports the economic sustainability of the system. Finally, the system's scalability and simplicity in operation, without the need for large volumes of O₃ or complex handling procedures, make it a practical and efficient solution for large‐scale grain preservation (Hardin et al., 2010).

3.2. Continuous‐flow ozonation

Figure 4 shows the components of the ozonation system on the screw conveyor. It includes a screw conveyor, an O₃ generator (OzoBlast), grain storage silos, and connecting hoses that link the generator's gas outlet to the injection points at the conveyor and silos (McDonough et al., 2011a).

FIGURE 4.

Schematic drawing of the grain ozonation components on the screw conveyor. (1) ozone generator (OzoBlast); (2) grain storage silos; (3) power generator; and (4) screw conveyor system. Fonte: McDonough et al. (2011a).

In the McDonough et al. (2011a) study, the authors applied O₃ gas to control T. castaneum and S. zeamais in corn grains. The screw conveyor was constructed of stainless steel with dimensions of 6.40 m in length and 0.102 m in diameter. A 22.7 kg hopper fed the system. During the study, the screw conveyor was set to a fixed inclination angle of 35°. The O₃ was produced by a generator from OzoBlast with a production capacity of 660 g. A 56‐kW diesel power source powered the generator. The O₃ gas was injected into the spiral conveyor, which was humidified and dried. Humidification was achieved by passing the gas through a 76 L volume tank containing 11 L of water. The O₃ concentration adopted in the study was 0.09 mg L⁻¹. The O₃ retention time inside the carrier was 1.80 min. A mass of 15.90 kg of grain took 6 min to pass through the system, which had a feed rate of 220 kg h⁻¹. With three passes of the grain mass through the system, 100% mortality of adult T. castaneum and S. zeamais was observed.

3.2.1. Advantages of continuous‐flow ozonation

The continuous‐flow ozonation system offers several distinct benefits for grain preservation. A primary advantage is its capability to ensure uniform O₃ distribution throughout the grain mass. The continuous movement of grains via the screw conveyor ensures consistent and effective O₃ application, thereby enhancing treatment efficiency (McDonough et al., 2011a). This uniform exposure improves the effectiveness of pest control and enhances the reduction of populations of harmful or contaminant microorganisms.

Additionally, the system enables precise regulation of O₃ concentration and exposure time, which is essential for optimizing disinfection efficacy, reducing resource waste, and ensuring efficient O₃ utilization (Silva et al., 2022). The controlled and continuous application of O₃ also reduces the risk of cross‐contamination between different grain batches, ensuring uniform quality of the final product (Ferreira et al., 2021).

The system's integration with existing grain transportation and storage facilities is another significant advantage. It can seamlessly adapt to established processes, providing an efficient solution without extensive modifications. Furthermore, the system's energy consumption is optimized, potentially reducing operational costs (McDonough et al., 2011a). Continuous O₃ application is also advantageous as it does not leave chemical residues on the grains, preserving product quality and minimizing environmental impacts associated with traditional chemical treatments. This makes the continuous flow ozonation system a sustainable and environmentally friendly option for grain preservation. In summary, the constant flow ozonation system enhances treatment efficiency and uniformity while offering a cost‐effective and ecologically safe solution for grain preservation (Ferreira et al., 2021; McDonough et al., 2011a).

3.3. Application of O₃ in flour

The application of O₃ to flour is an innovative technique designed to improve the safety and quality of flour products (Alexandre et al., 2017). This method involves exposing flour to O₃ gas, which can be implemented using either a batch system or a continuous‐flow system (Alexandre et al., 2019; Paes et al., 2017).

The ozonization system is composed of several critical components. The O₃ generator produces O₃ from oxygen, which must be at a concentration high enough to ensure effective treatment (Figure 5a). The O₃ delivery system, which includes piping and distribution mechanisms, is crucial for ensuring even O₃ distribution throughout the flour. The reaction chamber, where the flour is exposed to O₃, must be designed to facilitate thorough contact between the flour particles and the O₃ gas. Control systems regulate O₃ concentration, exposure time, and flow rate, maintaining optimal treatment conditions (Figure 5b). Last, the O₃ destruction system is essential for safely decomposing residual O₃ after treatment, mitigating potential safety risks (Figure 5c).

FIGURE 5.

Components of the ozone generation system, consisting of an oxygen cylinder and ozone generator (a); ozone concentration monitoring equipment (b); and residual ozone destroyer (c).

The ozonization process begins with preparing the flour, conditioned to a specific moisture level to optimize O₃ contact. During the O₃ exposure phase, the flour is treated in a controlled environment where O₃ concentration and exposure time are meticulously monitored. Following the treatment, the flour undergoes posttreatment ventilation to eliminate residual O₃ before packaging (Alexandre et al., 2017; Paes et al., 2017).

Typically, the flour treatment prototype includes a system that ensures the rotation of the flour during the ozonization process (Figure 6b). This rotation is achieved via a helical mixing system, which guarantees thorough and uniform movement of the flour, thereby ensuring comprehensive O₃ incorporation (Figure 6a) (Paes et al., 2017). The helical mixing system consists of a central shaft and two helices. The central shaft, connected to an induction motor, drives the rotation of the helical mixing system. The motor's frequency inverter allows for precise adjustment of the rotation speed, thereby providing tight control over the mixing process. This system effectively addresses the compact and dense nature of flour, facilitating thorough O₃ distribution (Paes et al., 2017). With such uniform distribution, the intended effects (such as insect control, dough quality enhancement, or inhibition of spoilage molds) can be consistently achieved, thereby ensuring the overall quality of the flour.

FIGURE 6.

Prototype for the ozonation of flour: (a) cylindrical roll, induction motor, and frequency inverter; (b) double helical mixing system. Source: Paes et al. (2017).

O₃ application in flour processing offers several advantages compared with traditional treatment methods. O₃ provides effective and uniform disinfection due to its strong oxidative properties. When applied through the ozonation system, O₃ reacts with the surface of the flour, efficiently reducing microbial loads, including populations of bacteria, filamentous fungi, and yeasts. This results in a substantial reduction of microbial contamination, thus enhancing the safety of the final product.

Several studies have confirmed the efficacy of O₃ in reducing microbial loads and degrading mycotoxins in flour and related products. For instance, Alexandre et al. (2017) evaluated the effect of O₃ on the degradation of deoxynivalenol (DON) in whole wheat flour and wet milling effluent. The study demonstrated that DON degradation increased with higher moisture content and prolonged exposure times, achieving a maximum reduction of approximately 80%. This highlights ozonation as a promising strategy for mitigating mycotoxin contamination in various food matrices. In wheat flour, Li et al. (2015) reported reductions in DON levels of up to 94% following O₃ treatment.

In another study, Alexandre et al. (2019) assessed the degradation kinetics of zearalenone (ZEN) in whole corn flour subjected to O₃ concentrations of 51.5 mg L⁻¹ for 20 and 60 min. The ozonation process resulted in ZEN reductions of 60.2 and 62.3%, respectively, depending on exposure time. These results reinforce the potential of O₃ as a viable alternative to conventional decontamination techniques in flour processing. By effectively decreasing specific chemical hazards and microbial contaminants, ozonation contributes to enhanced food safety and product quality.

3.4. Application of O₃ in packaged products (low‐pressure)

Low‐pressure storage involves the design and construction of specialized grain storage structures in which the internal pressure is reduced in a controlled manner. This technology represents a significant advance in grain storage, as it allows the physical environment to be precisely modified, generally through the application of vacuum and the creation of low‐oxygen (O₂) atmospheres (Guru et al., 2022). In this context, the application of O₃ in low‐pressure systems has emerged as a promising strategy for the treatment of packaged grains, maximizing the preservation and decontamination of stored products (Sitoe et al., 2023). The scheme used for the ozonation of grains in a closed system is shown in Figure 7. The scheme consists of an oxygen concentrator (1), O₃ generator (2), vacuum pump (3), hypobaric chamber (4), O₃ concentration quantification system (5), and vacuum gauge (5). For closed‐system ozonation, the packaged grains are inserted into the hypobaric chamber, which is then carefully closed. At the subsequent, the chamber's internal pressure is reduced by a vacuum pump.  O₃ is injected into the chamber, and the flow is interrupted when the internal pressure reaches the value of 1000 hPa (Silva et al., 2022). In this grain treatment system, the oxygen concentration inside the chamber directly correlates with the internal chamber pressure and injection time (Sitoe et al., 2023).

FIGURE 7.

Schematic drawing of the low‐pressure grain ozonation components.

3.4.1. Advantages of the low‐pressure ozonation system

The application of O₃ at low pressure offers several advantages compared with conventional flow ozonation systems. In low‐pressure systems, O₃ interacts differently with the product due to the controlled environment of the hypobaric chamber (Silva et al., 2022). In this context, every volume of oxygen is efficiently converted into O₃, which reacts with the product inside the chamber. This process results in a more uniform and controlled ozonation than flow ozonation, where O₃ may not be evenly distributed across the product (Oliveira et al., 2020; Silva et al., 2019).

Moreover, the low‐pressure ozonation system enhances occupational safety. The applications are conducted within a hermetically sealed chamber, minimizing the risk of O₃ exposure to operators and preventing the generation of residual O₃ that could pose health risks (Sitoe et al., 2023). The enclosed nature of the system ensures that O₃ is fully utilized during the treatment process, reducing the potential for O₃ leakage and improving overall safety throughout the ozonation process.

4. EFFECT OF O₃ ON THE CONTROL OF MICROORGANISMS IN GRAINS AND THEIR FINAL PRODUCTS

4.1. Effect of O₃ on pest insect control

O₃ has been shown to be highly effective in controlling insects found in grains and their end products due to its powerful oxidative properties (Boopathy et al., 2022; Xinyi et al., 2017). The mechanism by which O₃ acts against insects involves several key processes. First, O₃ oxidizes the lipids within the cellular membranes of insects, leading to the loss of membrane integrity and the disruption of cellular functions. Furthermore, O₃ has been shown to oxidize essential proteins involved in the metabolism and reproduction of insects, thereby impairing their biological functions (Bi et al., 2022; Boopathy et al., 2022). Additionally, O₃ has been demonstrated to penetrate insects’ respiratory systems, oxidizing critical components and interfering with cellular respiration (Boopathy et al., 2022). This disruption is particularly detrimental for insects, which depend on an efficient respiratory system for survival (Bi et al., 2022; Boopathy et al., 2022). Furthermore, O₃ has been demonstrated to induce oxidative stress in insects by generating reactive oxygen species (ROS), which in turn cause further cellular damage (Bi et al., 2022; Ghazawy et al., 2021). The combined effects of these factors ultimately result in insect mortality, thus establishing O₃ as a highly effective tool for the control of pests in grains and their final products. Tables 5 and 6 present detailed results on the effects of O₃ on different types of grain and flour, highlighting its effectiveness in reducing insect contamination.

TABLE 5.

Insect mortality as a function of concentration and exposure time to ozone gas during fumigation of different types of grain, including beans.

Ozonation system	Grains	Insects	Ozone concentration	Exposure time	Mortality	References
Ozonation in flux	Wheat	Adults of R. dominica	0.42 and 0.84 mg L−1	66.65 and 41.81 h	99%	Subramanyam et al. (2017)
		Adults of T. castaneum, Oryzaephilus surinamensis, S. zeamais	200 ppm	12 h assessed for 5 days	100%—Sitophilus spp and O. Surinamensis. 90%—T. castaneum	Xinyi et al. (2017)
		Adults of Sitophilus granarius	25 mg L−1 50 mg L−1 75 mg L−1 150 mg L−1 300 mg L−1	10 min 20 min 30 min 60 min 120 min	20.7% 17.0% 13.8% 44.2% 97.8%	Lemic et al. (2019)
		Adults of Trogoderma granarium	1200 ppmv	2 h	100%	Mahmoud et al. (2023)
	Cowpeas	Adults of C. maculatus	500 ppmv	274.40 min	100%	Pandiselvam et al. (2019b)
		Pupa of C. maculatus	500 ppmv	1.816,54 min	100%
		Adults of C. maculates and Callosobruchus Chinensis L	0.25, 0.5, 1.0, 1.5, and 2 mg L−1	1, 3, 5, and 7 days	C. maculates—100% at 1.0, 1.5, and 2.0 g/m3 for 7 days C. Chinensis—100% at all concentration.	Gad et al. (2021)
		Adults of C. maculatus	1.61 mg L−1	43.65 min	95%	Ramos et al. (2023)
		Adults of C. maculatus	500, 1.500, 2.500, and 3.500 µg L−1	1.698.00, 331.20, 157.07, and 84.40 min	95%	Abreu et al. (2022)
	Chickpea	Eggs and adults of C. maculatus	1000 ppm	5 days	100 %	Nickhil et al. (2021)
	Rice	Adults of R. dominica	500 mg/kg	540 min	100%	Sunisha (2019)
		Eggs and adults of S. oryzae	1.0–2.0 mg L−1	2–6 h	Eggs: 46.55–99.89% Adult: 44.13–99.02%	Srivastava et al. (2021)
	Barley	Adults of R. dominica e T. castaneum	700 ppm	1440 min	97%	Dong et al. (2022)
	Wheat	Penicillium spp. And Tilletia spp.	0, 10, 50, and 100 ppm	1 and 6 h each	82 and 92% for total fungal count and Tilletia spp. incidence, respectively	Sanchez et al. (2025)
Low‐pressure ozonation	Popcorn kernels	Adults of S. zeamais	13.0 mg L−1	–	94.45%	Silva et al. (2022)
	Common beans	Egg and larvae of Zabrotes subfasciatus	61.37 mg L−1	10 injections until the pressure inside the chamber reached 1000 hPa	100%	Sitoe et al. (2024a)

TABLE 6.

Insect mortality as a function of concentration and exposure time to ozone gas during the flour fumigation process.

Ozonation system	Flour	Insects	Ozone concentration	Exposure time		Mortality	References
Ozonation in flux	Wheat flour	Eggs, larvae, and adults of R. dominica, T. castaneum	0.428 mg L−1	0, 2, 4, and 6 h	Eggs—41.5; 64.7 and 100% Larvae—23.3; 54.4 and 100% Adults—7.3; 56.0 and 100%		Abdelfattah et al. (2023)
		Eggs, larvae, and pupae of Corcyra cephalonica	1.0, 3.0, and 5.0 mg L−1	0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 h	Eggs—100.0% Larvae—89.1% Pupae—96.2% With 5.0 g/m3 and 5.0 h		Gad et al. (2025)
		Eggs, larvae, pupae and adults of T. castaneum	30, 35, 40, and 50 mg L−1	3 days	Eggs—80.06% Larvae—85.6% Pupae—76.7% Adults—82.2%		Baume et al. (2024)
	Organic wholemeal flour	Eggs of P. interpunctella and Adults of T. castaneum and Lasioderma serricorne	0–300 ppm	8–96 h	>80% for all stages		Ingegno and Tavella (2022).

4.2. Effect of O₃ on the inactivation of fungi and degradation of mycotoxins

O₃ exhibits substantial antifungal activity, effectively inhibiting the growth of filamentous fungi and contributing to the degradation of mycotoxins in grains and their processed products. The mechanisms by which O₃ acts on fungal structures involve two primary oxidative processes (Sivaranjani et al., 2021). First, O₃ reacts with sulfhydryl groups and amino acid residues in microbial proteins and enzymes, altering their structural conformation and impairing essential biological functions. Second, O₃ oxidizes polyunsaturated fatty acids in the lipid bilayer of fungal membranes, compromising membrane integrity. These oxidative disruptions lead to increased membrane permeability, leakage of intracellular components, and ultimately cell lysis (Afsah‐Hejri et al., 2020; Brodowska et al., 2018; Pandiselvam et al., 2017).

In addition to its effects on fungal cells, O₃ has also demonstrated efficacy in the chemical degradation of mycotoxins (toxic secondary metabolites synthesized by certain fungal species). Studies indicate that O₃ can cleave specific molecular bonds in these compounds, converting them into less toxic or inactive derivatives (Chen et al., 2014; Conte et al., 2020). The efficiency of fungal growth inhibition and mycotoxin degradation by O₃ depends on multiple factors, including O₃ concentration, exposure time, and the physicochemical properties of the substrate undergoing treatment (Afsah‐Hejri et al., 2020). Application of O₃ in a controlled environment allows for the optimization of these parameters, enhancing treatment efficacy and contributing to the microbiological and chemical safety of grain‐based products. Tables 7 and 8 provide detailed data on the impact of O₃ on fungal load reduction and mycotoxin degradation across different types of grain and flour.

TABLE 7.

Effect of ozone on fungal decontamination and mycotoxin degradation in different types of grains, including beans.

Ozonation system	Fungus and mycotoxin	Grains	Contaminants	Ozone concentration	Exposure time	Impact of ozone application	References
Ozonation in flux	Fungus	Wheat and corn	A. parasiticus e A. flavus	40 ppm	30 min	99.9%	Scussel et al. (2016)
		Rice	Penicillium spp. and Aspergillus spp.	5.00 mg L−1	13.97 min	3.8 logs cycle (100%) reduction	Santos et al. (2016)
		Popcorn kernels	Aspergillus flavus	16.0 mg L−1	6 h	80.0%	Silva et al. (2024)
		Corn	Aspergillus spp. Penicillium spp.	2.14 mg L−1	50 h	78.5% 98.0%	Brito et al. (2018)
			Aspergillus spp. Fusarium spp.	60 mg L−1	480 min	99.74% 99.94%	Porto et al. (2019)
		Wheat	Aspergillus spp Fusarium spp	60 mg L−1	300 min	99.9%	Trombete et al. (2017)
		Rice	Aspergillus spp. Penicillium spp.	10.13 mg/L	60 h	100%	Santos et al. (2016)
	Mycotoxin	Corn	Aflatoxina G1 (AFG1), aflatoxina B1 (AFB1), aflatoxina G2 (AFG2), and aflatoxina B2 (AFB2)	60 mg/L	480 min	54.6, 57.0, 36.1, and 30.0% decline, respectively	Porto et al. (2019)
		Corn	Zearalenona (ZEN) and Ochratoxina A (OTA)	100 mg/L	180 min	ZEN—86.0% reduction OTA—64.2% reduction	Qi et al. (2016)
		Wheat	Deoxynivalenol (DON)	75 mg L−1	90 min	53.48%	Wang et al. (2016)
			Total aflatoxin and DON	60 mg L−1	300 min	64.3 and 48.0%	Trombete et al. (2017)
		Peanuts	AFB1	50	60 h	89%	Qi et al. (2016)
		Durum wheat	DON	32.5 g/h 48 g/h 32.5 g/h	12 h 12 h 16 h	31–48%	Piemontese et al. (2018)
		Scabbed wheat	DON	10 g m−3	30 s	94%	Li et al. (2019)
Low‐pressure ozonation	Fungus	Common beans	A. flavus	61.37 mg L−1	1, 4, 7, and 10 injections	0; 0; 30, and 70%	Sitoe et al. (2024a)

TABLE 8.

Effect of ozone on mycotoxin degradation in different types of flour.

Ozonation system	Flour	Contaminants	Ozone concentration (mg L−1)	Exposure time	Impact of ozone application	References
Ozonation in flux	Corn flour	AFB1, AFB2, AFG1, and total aflatoxin	75	60 min	78.76, 70.73, 72.09, and 77.29%	Luo et al. (2014)
		AFB1, AFB2, AFG1, and AFG2	75	60 min	79, 71, and 72%	Wang et al. (2016)
	Wheat flour	DON	100	1 h	78%	Mohammadi‐Kouchesfahani et al. (2015)
	Wheat bran	DON	62	4 h	32%	Santos‐Alexandre et al. (2018)
		ZEN			61%
	Whole maize flour	ZEN	51.5	60 min	62.3%	Alexandre et al. (2019)
	Wheat flour	DON	60	1 h	33, 33%	Zhuang et al. (2020)

5. REACTION KINETICS OF O₃ GAS IN GRAINS AND THEIR FINAL PRODUCTS

The kinetics of O₃ gas reactions in grains and their final products involve complex interactions that are influenced by several factors, including O₃ concentration, exposure time, and the physical and chemical properties of the grains and final products (Alexandre et al., 2019; Paes et al., 2017). O₃ is a potent oxidizing agent that reacts with contaminants such as pesticides, mycotoxins, and microbial pathogens (e.g., fungi and bacteria) in grains and their processed products, leading to their oxidation and inactivation (Alexandre et al., 2019; Sitoe et al., 2023).

Upon exposure, O₃ gas diffuses into the interstitial spaces of the grains and final products, where it comes into contact with target compounds. The reaction rate is governed by the concentration of O₃ and the availability of reactive sites (Alexandre et al., 2019; Paes et al., 2017). Increased concentrations of O₃ gas and extended exposure durations generally enhance the efficacy of microbial inactivation and mycotoxin degradation during decontamination processes. The surface area and porosity of the grains and final products also play critical roles in determining the diffusion rate and overall effectiveness of O₃ treatment (Afsah‐Hejri et al., 2020; Alexandre et al., 2019; Kaur et al., 2023; Paes et al., 2017).

Kinetic studies have shown that the degradation of contaminants follows a pseudo‐first‐order reaction, where the reaction rate depends on the concentration of the contaminant and the presence of O₃ (Paes et al., 2017; Silva et al., 2019). The reaction kinetics can be described by mathematical models that account for the dynamic interactions between O₃ and the various constituents of grains and final products. These models help optimize the treatment conditions to achieve maximum decontamination efficiency while minimizing potential adverse effects on the quality of the treated products (Wright, 2004). Understanding the reaction kinetics of O₃ gas is crucial for optimizing fumigation systems, predicting O₃ distribution in porous media, and determining the gas’ half‐life in storage systems (Silva et al., 2019; Pandiselvam & Thirupathi, 2015; Pandiselvam et al., 2017). The interaction between O₃ and grain mass occurs in two distinct phases. O₃ is rapidly degraded due to its intense reaction with the grain mass (Afsah‐Hejri et al., 2020; Kaur et al., 2023). This rapid degradation leads to the formation of molecules responsible for O₃ decomposition, which, once reaching saturation, results in reduced degradation and allows the gas to move freely in the intergranular spaces (Pandiselvam et al., 2017).

During the first phase, O₃ reacts quickly with easily accessible sites on the grains, causing a significant drop in concentration. This phase is characterized by a high initial reaction rate that slows as O₃ interacts with the grain mass (Pandiselvam et al., 2017). Once the easily reactive sites are occupied, the remaining O₃ diffuses more slowly through the grain mass, resulting in a lower reaction rate. This behavior has been observed across various grains, including corn (Silva et al., 2019), wheat (Hardin et al., 2010), peanuts (Alencar et al., 2011), and beans (Abreu et al., 2022). For example, in bean grains subjected to low‐pressure O₃ treatment, initial O₃ concentrations were estimated at 17.0 and 27.9 mg L⁻¹ for one and 10 injections, respectively. Similarly, concentrations within the package were 11.9 and 21.2 mg L⁻¹ for one and 10 injections, respectively. The increased concentration observed with multiple injections, approximately 1.8 times higher than with a single infusion, supports the hypothesis that saturation occurs during the O₃ injection process, leading to higher gas concentrations.

Several factors influence the reaction kinetics of O₃ gas. Grain moisture is a critical parameter, as higher moisture content enhances the reaction rate due to the increased availability of reactive sites (Pandiselvam & Thirupathi, 2015). Grain type also plays a significant role; different types of grains have varying surface areas and chemical compositions that affect O₃ interaction (Souza et al., 2018). Additionally, the initial concentration of O₃ applied impacts the reaction kinetics; higher concentrations can lead to more rapid degradation but may also result in higher residual O₃ levels (Krstović et al., 2021; Silva et al., 2019).

Understanding these factors is essential when selecting an ozonation strategy. Rapid O₃ decomposition as it moves through the intergranular space presents a significant challenge when choosing an effective ozonation system for grains (Pandiselvam et al. 2017). This challenge necessitates careful consideration of factors such as O₃ concentration, grain moisture, and grain type to optimize the efficacy of the ozonation process. Table 9 illustrates that the half‐life of O₃ varies based on these parameters, emphasizing the need for tailored approaches to different grain types and storage conditions.

TABLE 9.

Kinetic parameters of ozone in different grains and gas application strategies.

Ozonation system	Grain	Moisture content of grains (g 100 g−1 w.b.)	Chamber pressure (hPa)	Ozone concentration (mg L−1)	$t_{\mathrm{sat}}$ (min)	t 1/2 (Min)	References
Ozonation in flux	Rice	11.4	–	1.43	119	13.80	Ravi et al. (2015)
		14.2	–		144	11.61
	Corn (AG 1051)**	13.0	–	1.28	6.5	10.50	Souza et al. (2018)
	Corn (Tropical Plus)*		–		52.4	6.30
	Corn (GSS 41499)*		–		163.9	0.16
	Corn (GSS 42072)*		–		119.8	0.80
	Corn (GSS 41243)*		–		88.4	3.80
	Popcorn kernels	11.2	–	0.50	600	5.20	Silva et al. (2019)
			–	2.20	186	6.22
			–	4.50	126	5.56
	Chestnut	4.40	–	2.42	–	3.96	Oliveira et al. (2020)
			–	4.38	–	6.20
			–	8.88	–	6.59
			–	13.24	–	7.01
Low‐pressure ozonation	Common beans	13.94	500	32.10	–	52.91	Sitoe et al. (2023)
	Cowpea	10.61			–	29.37
	Corn	12.32			–	34.48
	Paddy rice	13.04			–	21.53
	Polished rice	12.60			–	17.86
	Popcorn kernels	12.95			–	38.30
	Popcorn kernels	12.2	250	5.00	–	22.72	Silva et al. (2022)
				6.70	–	31.36
				8.50	–	32.09
				13.00	–	49.86

*, sweet corn hybrids; **, common corn hybrid; t _1/2, half‐life of ozone gas; $t_{\mathrm{sat}}$, saturation time.

Understanding the reaction kinetics of O₃ gas in grains and their final products is the key to designing effective ozonation protocols. By tailoring the O₃ concentration, exposure time, and environmental conditions, you have the potential to significantly enhance the safety and quality of grain products. This approach ensures that the grain products meet the required standards for human consumption and commercial use, instilling hope for a safer and healthier future.

6. EFFECT OF O₃ ON GRAIN QUALITY

Several factors, including physicochemical, nutritional, technological, and sensory properties, contribute to determining grain quality, which in turn impacts consumer acceptance (Baker et al., 2022; Melovic et al., 2020). In this context, O₃ treatment has emerged as a promising solution for extending the shelf life and improving the quality of stored grains. The key findings regarding the impact of O₃ on grain quality are summarized in Table 10.

TABLE 10.

Quality of ozonated grains in different gas application systems.

Ozonation system	Grain	Ozone concentration (mg L−1)	Effect of ozone on grain quality	References
Ozonation in flux	Popcorn kernels	0.50	Expansion volume; moisture content (∼); electrical conductivity (∼)	Silva et al. (2019)
		2.20	Expansion volume (↓); moisture content (∼); electrical conductivity (↑)
		4.50	Expansion volume (↓); moisture content (∼) electrical conductivity (↑)
	Chestnut	2.42	Moisture content (∼); free fatty acids (∼); iodine content of crude oil (∼); lipid profile of crude oil (∼)	Oliveira et al. (2020)
		4.38	Moisture content (∼); free fatty acids (∼); iodine content of crude oil (∼); lipid profile of crude oil (∼)
		8.88	Moisture content (↓); free fatty acids (∼); iodine content of crude oil (∼); lipid profile of crude oil (∼)
		13.24	Moisture content (∼); free fatty acids (∼); iodine content of crude oil (∼); lipid profile of crude oil (∼)
	Wheat	2.50	Moisture content (↓); protein (↓); fat (∼); fiber (∼); ash (∼); carbohydrates (∼); iron (∼); copper (↓); magnesium (∼); manganese (∼); potassium (∼); phosphorus (∼); zinc (↓); calcium (∼); selenium (↓); hardness (↑)	Mishra et al. (2019)
	Cowpea	2.00	Seed germination (∼); protein content (↓); fats (↓); carbohydrates (↓); moisture content (↓); fibers (↑); ash (↑); total phenolic content (↓), total flavonoids (↓); tannins (↓)	Gad et al. (2021)
	Wheat	0.19	Grain moisture content (∼); grain weight (∼)	Hardin et al. (2010)
	Corn	2.30	Electrical conductivity (∼); seed germination percentage (↓); luminosity (L*) (↑); hue angle (h*) (↑); chroma (C*) (↑)	Uzoma et al. (2024)
Low‐pressure ozonation	Common Beans	32.10	Moisture content (∼); color (∼); cooking time (∼)	Sitoe et al. (2023)
	Cowpea		Moisture content (∼); color (∼); cooking time (∼)
	Corn		Moisture content (∼); color (∼)
	Paddy rice		Moisture content (∼); color (∼)
	Polished rice		Moisture content (∼); color (∼)
	Popcorn kernels		Moisture content (∼); color (∼); expansion volume (∼); flake volume (∼)
	Popcorn kernels	5.00	Expansion volume (∼); moisture content (∼); electrical conductivity (∼)	Silva et al. (2022)
		6.70	Expansion volume (∼); moisture content (∼); electrical conductivity (∼)
		8.50	Expansion volume (∼); moisture content (∼); electrical conductivity (∼)
		13.00	Expansion volume (∼); moisture content (∼); electrical conductivity (∼)
	Common beans	61.37	Electrical conductivity (↑); dough of a thousand grains (↑); percentage (%) of whole grains after cooking (∼); total soluble solids in bean cooking broth (%) (∼); cooked bean length‐width ratio (mm) (∼), and volume of cooked beans (mm3) (∼)	Sitoe et al. (2024a)
		61.37	Minerals (∼); moisture content (∼); protein (∼); lipids (∼); carbohydrates (∼); total titratable acidity (↑); luminosity (L∗) (∼); color saturation or chroma (C∗) (∼); color hue (h∗) (∼); electrical conductivity (↑); water absorption capacity (%) (∼); hydration coefficient (%) (∼); hydration capacity (g grain−1) (∼); percentage of tough‐skin beans (%) (∼); cooking time (min) (∼); phenolic compounds (mg GAE g−1) (↓); antioxidant capacity (µmol of Trolox g−1) (∼)	Sitoe et al. (2024b)

*(↑), increases; (↓), decreases; (∼), no change.

6.1. Physico‐chemical properties

The physical and chemical properties of grains and their derivatives are crucial for their preservation, safety, and industrial applications. Modifications to these parameters through oxidative treatments, such as ozonation, have been shown to improve product stability, provided the operational conditions are strictly controlled.

O₃ treatment has been found to significantly reduce the pH, moisture content, and water activity of various cereals and their products (Çatal & Ibanoglu, 2012; Li et al., 2012; Li et al., 2013; Qi et al., 2016). The observed decrease in pH is likely associated with O₃‐induced oxidative changes in the functional properties of the product constituents (Lee et al., 2017). Furthermore, the interaction between O₃ and the grain matrix leads to the oxidation of free water, decreasing its availability and improving the prospects for long‐term storage.

While high O₃ concentrations are effective for sanitization, they can exacerbate these processes, resulting in discoloration, surface oxidation, textural changes, and the formation of undesirable odors (Xue et al., 2023). According to Oliveira et al. (2020), exposing chestnuts to O₃ in a continuous flow system for 240 min led to a decrease in moisture content from 4.31 to 3.54 g 100 g⁻¹, a change associated with the lower relative humidity in the gas mixture.

Grains treated with low‐pressure O₃ systems exhibited further significant alterations, including elevated electrical conductivity, higher titratable acidity, and changes in antioxidant activity, as detailed in Table 10. O₃ exposure was found to raise electrical conductivity in a concentration‐ and time‐dependent manner, as evidenced by studies on cowpea grains (Abreu et al., 2022) and popcorn kernels (Silva et al., 2022). This rise in electrical conductivity indicates damage to the cell membranes, which are essential for maintaining cell integrity and regulating internal processes. The oxidative action of O₃ leads to membrane damage, resulting in the release of ions, sugars, and other intracellular substances, thereby enhancing electrical conductivity (Abreu et al., 2022). Cellular degradation, as indicated by the observed changes, has the potential to impair the viability and quality of the grains, ultimately influencing their germination and long‐term storage capabilities. Therefore, the rise in electrical conductivity is viewed as a negative outcome, reflecting structural damage that could negatively affect grain quality (Fessel et al., 2006).

Color, a key physicochemical characteristic influencing product acceptance, can also be altered by O₃ treatment. Increases in lightness (L*) and decreases in yellowness (b*) are attributed to the oxidation of carotenoid pigments, particularly the breaking of conjugated double bonds (Çatal & Ibanoglu, 2012; Lee et al., 2017; Mei et al., 2016; Qi et al., 2016). These changes result in a brighter, more homogeneous color, which is typically desired in processed products.

The findings highlight that O₃ treatment can successfully modify the physicochemical characteristics of grains, enhancing their stability and preservation. However, it is crucial to apply the treatment with precision to minimize any detrimental effects on grain quality.

6.2. Nutritional properties

The nutritional profile of grains plays a crucial role in determining their suitability for human consumption and industrial use. Understanding how O₃ treatment influences the levels of macronutrients such as proteins, lipids, and carbohydrates is essential for determining its impact on the overall nutritional value of grains after processing. This assessment ensures that the beneficial components remain intact while undergoing ozonation. The effects of O₃ on the nutritional content of grains are influenced by factors such as the amount of O₃ used and the duration of exposure. Research on wheat has shown that when O₃ is applied under controlled conditions, it does not significantly alter the protein, phytate, or lipid content, though enzyme activities, such as alpha‐amylase, may be reduced (Gozé et al., 2016; Savi et al., 2014). However, extended exposure to O₃ can result in the deterioration of more sensitive components. For instance, Obadi et al. (2016) reported a decrease in sulfhydryl groups and changes in the gluten and glutenin subunits of isolated wheat proteins after exposure to O₃ (5 g/h) for up to 1 h.

Wang et al. (2016) reported that free fatty acid concentrations in wheat samples remained similar, whether treated with O₃ or not, even after 90 min of exposure to 75 mg/L O₃. This suggests that the O₃ treatment did not result in lipid degradation or the onset of oxidative rancidity. In addition, the total amino acid content in the treated samples remained unchanged, indicating that the O₃ treatment did not have a significant impact on nitrogen compounds under these conditions.

The structure of starch, the primary carbohydrate in grains, can be altered by the oxidative action of O₃. According to Chan et al. (2011), corn starch experienced a 22.6% reduction in molecular mass after 10 min of exposure to O₃ at a rate of 8 mL/s, suggesting a partial breakdown of its polymer chains. In contrast, Çatal and Ibanoglu (2012) reported an increase in the size of starch granules after 1 h of ozonation, a result they attributed to swelling induced by surface modifications and enhanced water uptake.

Thus, while O₃ may cause structural changes in the nutritional components of grains, its application under controlled conditions does not significantly compromise nutritional value, ensuring both technological viability and safety for consumption.

6.3. Technological and functional properties

Technological and functional properties are crucial in determining how grains perform during processing and directly influence the quality of the final products. O₃ has been studied as a tool to modify these properties without leaving chemical residues. The oxidizing action of O₃ can interfere with the protein network in grains, promoting changes in properties such as elasticity, viscosity, and gas retention. Additionally, O₃ treatment affects properties such as solubility and thermal stability of proteins (Zhu, 2018). Gozé et al. (2017) observed a decrease in the solubility of prolamins after ozonation, suggesting the formation of cross‐links and loss of functionality.

Despite potential adverse effects, evidence indicates that the controlled application of O₃ can preserve the technological quality of grains. Abreu et al. (2022) showed that cowpea grains maintained critical qualities, such as cooking time and water content, even after exposure to a broad range of O₃ concentrations (0.5–3.5 g/m³) and long treatment durations (up to 1698 min). This highlights the necessity for stringent control over treatment parameters. Additionally, changes in starch structure, like those reported by Chan et al. (2011) and Çatal and Ibanoglu (2012), affect properties such as gelatinization, retrogradation, and water absorption, which influence the grains’ performance in industrial processing. Therefore, O₃ treatment may hold promise for maintaining or enhancing technological properties of grains, provided the conditions are carefully managed.

6.4. Sensory attributes

Ozonation can significantly influence sensory traits such as color, aroma, and texture, which are key determinants of product acceptance. The clarification of color is frequently observed postozonation, resulting in grains with a brighter, more appealing look, beneficial for many industrial processes (Lee et al., 2017; Li et al., 2012; Qi et al., 2016). However, high O₃ concentrations can lead to alterations in taste and smell, which necessitate careful management of both O₃ levels and exposure time (Xue et al., 2023). Therefore, although O₃ can enhance visual attributes, its application must be meticulously controlled to maintain the sensory qualities of the grains and ensure their acceptance. Proper regulation of the treatment conditions is vital to avoid any unwanted changes in sensory attributes.

7. EFFECT OF O₃ ON THE QUALITY ATTRIBUTES OF FLOUR AND ITS END PRODUCTS

The impact of O₃ on the quality parameters of grain‐derived food products has been thoroughly investigated, as summarized in Table 11. O₃ treatment of flour and its derivatives has been extensively researched for its potential to improve microbiological safety and enhance food quality. Several studies have rigorously examined the effect of O₃ on various quality aspects, including physicochemical, rheological, sensory, and microbiological properties. These studies have contributed significantly to understanding both the advantages and the limitations of applying O₃ to flour and its final products.

TABLE 11.

Quality attributes of various food products made from grains and their final products treated with ozone.

Ozone application technology	Foods	Ozone concentration	Effect of ozone on grain quality	References
Ozonation in flux	Buckwheat‐based noodles	2.4 g/h	Dough strength and elasticity (↑); sensory acceptability of fresh noodles during storage (∼); (↑) shelf life of noodles to 96 h	Hu et al. (2020)
	Buckwheat noodles	5 g/h	Microbiological count and extended shelf life from 2 to 5 days (↓); cooking loss and water absorption (↓); hardness and tensile strength (↑)	Bai et al. (2021)
	Wheat flour bread	5 L/min	Bread volume, color, and textural characteristics (↑); bread shelf life (↑); longer exposure time (↓) bread quality and shelf life	Obadi et al. (2018a, 2018b)
	Steamed bread	5 mg/L	Wet gluten content and whiteness of wheat flour (↑); binding properties (↑); positive effects on quality scores, volume/weight, height, crust color, crust structure, external appearance, and internal structure of bread (↑)	Mei et al. (2016)
	Whole wheat flour	5 g/h	Binding properties (peak viscosity, trough, breakdown, final viscosity, and setback) (↓); water and oil absorption capacities, swelling power, and solubility (↑); altered starch and protein structures	Obadi et al. (2018b)
	Wheat flour	6 g/h	Dough strength of wheat flour (↑); maximum viscosity of the flour (↑); insoluble protein polymer content of flour (↑); starch molecular weight (↑)	Zhang et al. (2021)
	Wheat flour	0.428 g/m3 (200 ppm)	Protein, fat, fiber, ash, and carbohydrate content of wheat flour and bread (); dough strength (↑); development time and stability time (↑); water absorption (WA) of the flour (↓); sensory parameters (∼)	Abdelfattah et al. (2023)
	Chinese steamed bread	5 mg/L, 3.3 L/min) for up to 2 h	The texture analysis showed that ozone treatment (↑) the chewiness of the bread. Increasing the treatment time up to 1 h (↑) the hardness and elasticity of the bread. Further increasing the treatment time (↓) these values. Sensory analysis showed that ozone treatment for 1 h provided the highest overall sensory acceptance of the bread, while further increasing the treatment time (↓) the sensory quality.	Mei et al. (2016)
	High ratio cake	0.06 L/min for up to 40 min	pH (↓); cake batter viscosity (↑). Increasing the ozone treatment time to up to 35 min (↑) the cake volume. Further increasing the treatment time to 40 min (↓) the cake volume. Cake hardness (↓); cohesion and elasticity (↑)	Chittrakorn et al. (2014)
	High ratio cake	20 mg/sL for up to 30 min	Increasing the ozone treatment time (↓) L* and (↑) a* of the cake crust, having little effect on the cake height and texture	Sui et al. (2016)

*(↑), increases; (↓), decreases; (∼), no change.

7.1. Physico‐chemical properties

O₃ treatment has been shown to induce a variety of physicochemical alterations in flour, primarily through the catalysis of oxidative processes within the composition of the food matrix. These modifications directly affect the functional characteristics of the material, such as its rheological properties and performance during baking (Obadi et al., 2018a).

Regarding lipids, the effects of O₃ vary depending on the form of the product (grain or flour) and the intensity of the treatment. Research suggests that the lipids in whole grains are generally stable, owing to the protective nature of the grain's physical structure (Dubois et al., 2006; Wang et al., 2016). In contrast, studies on flour suggest that lipids are more vulnerable to oxidative degradation. After ozonation, an increase in the acidity index, changes in fatty acid composition, an increase in peroxide and p‐anisidine levels, and an increase in the presence of volatile compounds such as hexanal and heptanal have been observed (Qi et al., 2016; Sandhu et al., 2011). The oxidation of lipids in flour likely leads to the formation of these volatile compounds, which are associated with oxidative rancidity, thus impacting the sensory qualities of the flour and its derived products. Although lipid oxidation may adversely affect the flavor and aroma of flour due to the generation of volatile compounds, it can simultaneously improve the microbiological stability of the product as a result of the pH decrease.

O₃ has also been shown to affect the activity of the flour's endogenous enzymes. The α‐amylase activity significantly decreased, as reflected by the increase in falling time observed during rheological testing (Ding et al., 2015; Mei et al., 2016; Trombete et al., 2016). This reduction can impact how the flour behaves during dough formation, thus affecting the texture of the final product. Alongside this, a reduction in polyphenol oxidase (PPO) activity was noted, which could lead to less browning in the end product (Li et al., 2012). The reduction in PPO activity could be beneficial for preventing undesirable discoloration in flour‐based products.

Regarding phenolic compounds, O₃ treatment has been shown to promote a slight reduction in the tannin content of sorghum flour due to oxidation (Yan et al., 2012). In contrast, other research indicates that compounds like ferulic acid in wheat grains remain largely unaffected by ozonation (Dubois et al., 2006). This resistance to oxidation may be attributed to the physical barrier provided by the grain or flour matrix, which limits O₃’s access to polyphenolic compounds. Thus, the physical structure of the flour matrix plays an essential role in determining the susceptibility of phenolic compounds to oxidation.

The oxidative process induced by O₃ on starch generates carbonyl and carboxyl groups, leading to the acidification of the flour matrix and a consequent reduction in its pH (Lee et al., 2017; Marston et al., 2015; Sui et al., 2016). The intensity of this reduction depends on the dose and duration of exposure to the gas. A study observed a significant decrease in the pH of a Korean flour from 5.7 to 4.7 after O₃ exposure, indicating the potential of O₃ to chemically modify the constituents of flour (Lee et al., 2017). The acidification of the medium induced by O₃ could influence the flour's reactivity, especially in processes such as dough fermentation, where pH is critical for yeast activity.

Although these physicochemical changes may be subtle, they directly influence the technological and functional properties of the flour, thereby affecting the quality and sensory attributes of the resulting products. The impact of O₃ on these properties emphasizes the need for careful control over treatment conditions (O₃ concentration, exposure time) to ensure desired outcomes, particularly regarding the preservation of the flour's functional attributes and sensory appeal in final products.

7.2. Rheological properties

7.2.1. Dough rheology

O₃ treatment has been shown to be a promising tool for modulating the rheological properties of flours, particularly with regard to gluten‐forming proteins. A considerable body of research has focused on the effects of ozonation on dough rheology using various instruments, such as the farinograph, mixograph, extensograph, alveograph, and Mixolab (Li et al., 2015; Mei et al., 2016; Violleau et al., 2012).

Farinographic analysis has shown that O₃ treatment can extend the dough development time and improve its stability (Li et al., 2015). However, preliminary studies reported that for soft and medium wheat flours, consistency remained unaltered (Naito, 1990). Based on the extensograph, O₃ concentrations from 0.5 to 50 ppm enhanced the dough's resistance to extension, while reducing extensibility at concentrations between 0.05 and 50 ppm for soft flour and from 5 to 50 ppm for medium flour (Naito, 1990).

Alveographic analysis indicated that O₃ treatment improved the W and P/L parameters, which are linked to increased dough toughness (Trombete et al., 2016; Violleau et al., 2012). However, it is essential to note that prolonged or excessive O₃ treatment may impair dough strength, pointing to the necessity of defining safe O₃ application thresholds (Violleau et al., 2012).

In the mixograph, an increase in O₃ treatment time led to an increase in peak time and the width of the cake flour mixing curve, with minimal interference on peak height (Sui et al., 2016). The Mixolab analyses indicated that ozonation, particularly at moderate concentrations, increased the development time, thermal stability, and torque at C3, associated with starch gelatinization. However, values of C2, reflecting protein stability, were reduced. Furthermore, the findings of studies conducted by Li et al. (2012), Chittrakorn et al. (2014), and Mei et al. (2016) suggested that moderate ozonation enhanced development time, thermal stability, and torque at C3, while concurrently reducing C2 values and the C3 and C5‐C4 values, which are linked to starch retrogradation.

These changes can be attributed to the formation of disulfide bridges, the insolubility of glutenin polymers, and the occurrence of cross‐links between proteins. These phenomena lead to reorganizations in the gluten network, thereby improving dough stability and positively affecting its mechanical strength (Violleau et al., 2012). Overapplication of O₃ can cause the oxidation of amino acids and the depolymerization of proteins, which could impair the dough's structural integrity and reduce its quality.

Consequently, it can be concluded that O₃ treatment, when applied in a controlled manner, can significantly enhance the rheological performance of flours, benefiting the processing of breads and pasta. However, identifying the optimal concentration and exposure time is key to preventing undesirable effects and ensuring the intended technological quality.

7.2.2. Pasting properties

The pasting properties of flours, which are closely linked to the structure and behavior of starch during heating, are also impacted by O₃ treatment. Studies have demonstrated that O₃ can modify the structure of starch and the activity of associated enzymes, such as α‐amylase, resulting in changes in viscosimetric parameters during the gelatinization process (Lee et al., 2017; Mei et al., 2016; Sandhu et al., 2011; Sui et al., 2016).

For instance, Ding et al. (2015) observed an increase in the viscosity of waxy rice flour after ozonation, attributed to the inactivation of α‐amylase. Similarly, Sui et al. (2016) reported an increase in the peak and final viscosity of soft wheat flour. Sandhu et al. (2011) and Lee et al. (2017) also observed that O₃ treatment led to an increase in indentation viscosity and a greater degree of breakage during the pasting of flours.

Mei et al. (2016) found that increasing O₃ exposure time (up to 2 h) resulted in a significant decrease in viscosity properties, including peak, break, and indentation viscosities. Marston et al. (2015) reported that prolonged ozonation in sorghum flour led to increased peak and final viscosities, with a reduction in break viscosity. These results highlight that the impact of O₃ on pasting properties is dependent on both the intensity and duration of the treatment.

In general, when applied under controlled conditions, O₃ treatment improves the viscosity of flour during the pasting process, thereby enhancing its technological properties. However, excessive exposure can compromise the structural stability of starch and other flour constituents, which may negatively affect pasting properties. Additionally, oxidized proteins can influence the flour's viscosimetric behavior, contributing to the final rheological profile.

7.3. Sensory attributes

Sensory attributes, including color, flavor, aroma, and texture, are crucial for consumer acceptance of food products and are significantly influenced by ozonation processes. Color, in particular, plays a key role in assessing the quality of products. Studies have demonstrated the ability of O₃ treatment to alter the color of flours and bakery products, with outcomes varying depending on the specific conditions of the treatment. Sui et al. (2016) found a marked increase in the brightness (L*) of wheat flour, from 93 to 97, following O₃ treatment. Similar effects were noted in other flours, such as corn and sorghum, where O₃ enhanced whiteness and reduced the yellow hue (Li et al., 2012; Mei et al., 2016; Qi et al., 2016; Wang et al., 2016). The oxidation of pigments, such as polyphenols and carotenoids present in grains, is believed to be the primary cause of this change (Marston et al., 2015). In addition, Sandhu et al. (2011) found that O₃ treatment improved the clarity of bread color by decreasing the yellow hue, which was attributed to the oxidation of wheat carotenoids.

When it comes to texture, ozonation has been shown to improve other sensory qualities such as softness and volume in breads and pastas (Chittrakorn et al., 2014). For instance, Mei et al. (2016) used medium‐hard flour treated with O₃ to make Chinese steamed bread (CSB). Texture analysis indicated that CSB made with O₃‐treated flour (5 mg/L, 3.3 L/min, up to 2 h) exhibited higher chewiness compared with bread made with untreated flour. Furthermore, O₃ treatment for up to 1 h improved the bread's hardness and elasticity, whereas extending the treatment beyond this duration led to a reduction in these attributes. Sensory evaluations revealed that the bread treated with O₃ for 1 h received the highest acceptance, while longer treatment durations, especially 2 h, negatively impacted the sensory quality, particularly elasticity. According to these findings, O₃ treatment impacts the flour's proteins and starches, with moderate exposure enhancing protein quality, but prolonged exposure causing their degradation.

It should be noted that excessive O₃ levels or prolonged treatment can alter the sensory qualities, particularly taste and texture, of the products, negatively impacting their overall quality (Obadi et al., 2018b). Nevertheless, when properly controlled, ozonation is an efficient and environmentally friendly technology that enhances the quality of grain‐based products without compromising their sensory attributes such as flavor, aroma, and color.

7.4. Microbiological aspects and shelf life

The antimicrobial properties of O₃ are a key factor supporting its use in the food industry. O₃ treatment has been proven to reduce microbial contamination in flour and other food products, without leaving harmful residues, thereby improving food safety and extending shelf life (Li et al., 2015). This antimicrobial effect also contributes to maintaining the microbiological quality of flours and derived products during storage (Sandhu et al., 2011), reducing the need for artificial preservatives and aligning with more sustainable production practices.

Controlled O₃ application can effectively inactivate both pathogenic and spoilage microorganisms, while preserving the quality of the final product. Successful production of fresh and semi‐dry buckwheat noodles has been achieved using O₃‐treated wheat flour (5 g/h, 5 L/min, for up to 1 h) (Bai et al., 2017; Li et al., 2012; Li et al., 2013). O₃‐treated flour significantly reduced the bacterial load in fresh noodles during storage for up to 10 days. Specifically, a treatment with 2.21 mg/L O₃ reduced the microbial concentration in buckwheat noodles by 1.8 log 10 CFU/g (Bai et al., 2017). The results underscore the potential of O₃ as a chemical‐free and sustainable alternative to traditional preservation methods, delivering effective microbial control and contributing to better food safety.

8. REGULATORY APPROVALS FOR THE USE OF O₃ IN FOOD TECHNOLOGY

Since 1939, O₃ has been successfully used to inhibit the growth of molds and yeasts on fruit, with its applications gradually expanding to the preservation of a wide range of foods (Brodowska et al., 2018; Pandiselvam et al., 2019a). However, prior to mid‐1997, the use of O₃ in food processing and treatment in the United States was limited, and commercial applications had not yet been widely implemented. At that time, the direct use of O₃ in contact with food was not allowed, as the US FDA had not approved O₃ as a safe substance. The US FDA classified any substance in contact with food as a food additive, which required specific approval through regulations. A major challenge in the approval process for a Food Additive Petition was the absence of specific guidance on the minimum O₃ exposure required for efficacy, and the maximum exposure that could potentially harm the food. In this context, an expert panel convened by the Energy Power Research Institute in 1997 classified O₃ as “Generally Recognized as Safe” for use in food (Xue et al., 2023). On June 26, 2001, the US FDA formally recognized this status, allowing the use of O₃ in food treatment, storage, and processing (FDA, 2001b). This regulatory approval paved the way for integrating O₃ into postharvest treatment and storage practices, enhancing food safety and quality (Brodowska et al., 2018; Qi et al., 2017; Schneider et al., 2016).

In the European Union, the use of gaseous O₃ for food decontamination remains a subject of debate. The European Food Safety Authority (EFSA) is currently evaluating the use of O₃ in food. In 2012, EFSA recommended the use of O₃, alongside other disinfectants, for the decontamination of leafy vegetables and red fruits, based on risks associated with pathogens in non‐animal food sources, such as Salmonella and norovirus (EFSA, 2012). However, in a more recent EFSA document, it was stated that gaseous O₃ should not be approved as a basic substance for plant protection. In contrast, ozonated water is considered potentially suitable for approval (EFSA, 2021). In Italy, the Ministry of Health has approved the use of gaseous O₃ for decontaminating cheese ripening rooms, though direct contact of O₃ with cheese is prohibited (Bigi et al., 2021). In Japan, O₃ has been used to treat various food crops (Naito & Takahara, 2006; Panebianco et al., 2022). O₃ and related treatments have also been approved as processing aids in several countries, including Australia, New Zealand, Russia, Armenia, Belarus, Kazakhstan, and Kyrgyzstan (Baggio et al., 2020; Marino et al., 2018). In Brazil, the Ministry of Agriculture, Livestock, and Supply sanctioned the use of O₃ for disinfection of organic products in 2011, as outlined in Annex IV of Normative Instruction No. 18 (Coelho et al., 2015). Despite increasing global acceptance, regulations governing O₃’s use in food preservation still vary across regions. Therefore, the grain industry must remain vigilant regarding local legislation and ensure compliance with national guidelines when employing O₃ in food treatment and storage processes.

9. IMPLICATIONS OF O₃ ON OPERATOR HEALTH

O₃, extensively utilized in various industrial applications and postharvest processing of grains due to its oxidative and antimicrobial properties, can pose health risks to humans. As an extremely potent oxidant, O₃ can react with biological tissues (Silveyra et al., 2020; Wang et al., 2019), raising significant concerns regarding the safety of its use in environments where the gas is directly applied to agricultural products, such as grains and flour.

To mitigate these risks, the US FDA has established stringent guidelines for O₃ exposure. The maximum permissible concentration of O₃ for human exposure is set at 0.05 ppm over 8 h (Wang et al., 2019). This limit is based on studies demonstrating the adverse effects of O₃ on health, including irritation and damage to the mucous membranes of the eyes and respiratory system. Prolonged exposure or concentrations exceeding this threshold can result in a range of symptoms, such as headaches, dizziness, a burning sensation in the eyes and throat, and persistent coughing (Nuvolone et al., 2018; Wang et al., 2019).

Individuals with pre‐existing respiratory conditions, such as asthma, emphysema, and chronic bronchitis, are particularly susceptible to the adverse effects of O₃, which can significantly exacerbate the symptoms of these diseases (Silveyra et al., 2020). Despite these risks, O₃ has a distinctively irritating odor that can be detected at concentrations as low as 0.02–0.05 ppm (by volume), allowing for early identification of potentially hazardous exposure (EPA, 1999; Nazaroff & Weschler, 2022; Salonen et al., 2018).

The Occupational Safety and Health Administration sets occupational exposure limits for O₃. These include a Short‐Term Exposure Limit of 0.3 ppm (0.6 mg/m³) for 15‐min periods and a time‐weighted average (TWA) of 0.1 ppm for an 8‐h workday. According to the American Conference of Governmental and Industrial Hygienists, the TWA is set at 0.1 ppm for light activities and 0.2 ppm for activities lasting up to 2 h (Pandiselvam et al., 2017).

A pungent and unpleasant odor characterizes a concentration of 1 ppm of O₃, which can irritate the eyes and throat (Suslow, 2004). Prolonged exposure to O₃ concentrations above 4 ppm can be fatal. The O₃ concentration considered immediately dangerous to life and health is 5 ppm. This is the maximum level for which approved respirators are effective; concentrations above this level pose a significant health risk (Pandiselvam et al., 2017).

Since its introduction into industrial applications over a century ago, O₃ has been widely used without reported fatalities due to poisoning. However, exposure risks remain a critical concern, especially in processes requiring high O₃ concentrations, such as grain disinfection and flour production (Sitoe et al., 2023). To ensure worker safety, preventive measures, including continuous monitoring of O₃ levels, effective ventilation systems, air filtration technologies, and the use of personal protective equipment, are essential to minimize exposure and maintain a safe working environment.

In conclusion, while O₃ offers substantial benefits in grain preservation and other postharvest applications, operators and industries must strictly adhere to safety guidelines to avoid potential health hazards. Implementing safe handling practices and complying with regulations established by health authorities are crucial to ensuring that O₃ use remains effective and safe.

10. ENVIRONMENTAL IMPACT AND SUSTAINABILITY OF O₃ USE

O₃ offers significant environmental advantages as an alternative to traditional chemical pesticides. Unlike conventional pesticides, O₃ leaves no chemical residues on grains or in the environment, thereby eliminating the risks of soil and water contamination (Sitoe et al., 2023). This is particularly important as consumer demand for chemical‐free food products increases, driven by health and environmental concerns (Sitoe et al., 2024a; Ziyaina and Rasco, 2021).

Additionally, O₃, being produced from oxygen, an abundant and renewable resource, and rapidly decomposing back into oxygen after application, without generating any toxic residues, underscores its eco‐friendly nature and safety (Silva et al., 2022; Sitoe et al., 2023; Sitoe et al., 2024a). This natural cycle not only reduces the chemical load on agricultural ecosystems but also promotes biodiversity and soil health. The ability to generate O₃ on‐site also reduces the need for transportation and storage of chemicals, thereby lowering the carbon footprint associated with grain storage operations (Ziyaina and Rasco, 2021).

However, O₃ generation requires electricity, and the energy efficiency of O₃ generators is a crucial factor to consider to ensure the sustainability of O₃ use. Technological advancements are being made to improve the efficiency of O₃ generators and reduce energy consumption, making O₃ applications even more environmentally friendly (Pandiselvam et al., 2017).

The adoption of O₃ in agricultural practices can also be integrated with other sustainable management strategies, such as precision agriculture and integrated pest management, to maximize environmental benefits. It is crucial that ongoing research and development in O₃ application technologies prioritize the minimization of environmental impact, reinforcing the commitment to sustainability in the agricultural industry.

11. CHALLENGES AND FUTURE PERSPECTIVES

The use of O₃ gas to protect stored grains offers significant advantages, such as its effectiveness in disinfection and absence of residues, making it an attractive alternative to conventional pesticides (Mishra et al., 2019; Zhu, 2018). However, substantial challenges associated with its application need to be addressed to maximize its effectiveness and feasibility in grain storage.

One of the primary challenges is the high reactivity of O₃, which leads to rapid gas degradation after application. This characteristic limits its persistence and efficacy, requiring on‐site O₃ generation and potentially increasing operational costs (Pandiselvam et al., 2015; Silva et al., 2019). The need to generate O₃ at the time of application means it cannot be stored, representing an additional challenge for practical use.

Furthermore, the porous and heterogeneous structure of grains complicates the distribution and penetration of O₃ within grain masses. The effectiveness of O₃ may be compromised by difficulties in reaching all areas of the grains and by variations in gas penetration, which can result in uneven and less effective treatment (Pandiselvam et al., 2017; Sitoe et al., 2024a). This necessitates strict control of application conditions and the implementation of systems that ensure uniform O₃ distribution.

The choice of O₃ concentration and exposure time is critical to the treatment's success. Inadequate concentrations or exposure times may not effectively eliminate microorganisms or negatively impact grain quality (Kaur et al., 2023; Mishra et al., 2019). Therefore, optimizing these parameters to balance treatment efficacy with grain quality preservation is necessary.

Developing new technologies and improvements in application systems are essential to address these challenges. Ongoing research into O₃ reaction kinetics, implementation of advanced ozonation methods, and adaptation of application technologies can enhance the efficiency and feasibility of O₃ as a solution for grain preservation. A deeper understanding of the factors influencing O₃ distribution and reaction within grains will enable the development of more effective and practical strategies for its use in the agricultural sector (Pandiselvam et al., 2017; Silva et al., 2019). Thus, despite the current difficulties, the prospects for O₃ gas use in stored grain protection are promising. Advances in technology and scientific knowledge could overcome existing challenges and enable more efficient and effective application of O₃, contributing to food safety and sustainability in grain storage.

12. CONCLUSIONS

The use of O₃ gas for the preservation of stored grains and their final products represents a promising alternative to traditional pest control and preservation methods. O₃ offers substantial advantages, including its effectiveness in eliminating microorganisms and its ability to avoid chemical residues, which contributes significantly to food safety and environmental sustainability. The evidence consistently demonstrates that O₃ is effective in disinfecting, controlling pests, fungi, and mycotoxins, making it a highly attractive solution for preserving grains and their derivatives.

However, the practical application of O₃ in grain preservation presents certain challenges. The high reactivity of O₃ leads to its rapid degradation, which limits its persistence and effectiveness. This necessitates on‐site O₃ generation, which, while addressing the degradation issue, can also increase operational costs. Furthermore, the porous and heterogeneous structure of grains complicates the uniform distribution and penetration of O₃ within grain masses, potentially leading to inconsistent treatment. Ensuring the right concentration and exposure time is crucial to maintaining treatment efficacy without compromising the quality of the grains.

To overcome these challenges, ongoing research and development are crucial. Advancements in O₃ reaction kinetics, coupled with innovations in ozonation methods and application technologies, are essential for enhancing the efficiency and practicality of O₃‐based preservation systems. Additionally, understanding the factors influencing O₃ distribution and reaction within grain matrices will enable the design of more effective and targeted strategies for its use in the agricultural sector.

Moreover, a comprehensive economic analysis is vital for assessing the long‐term feasibility of adopting O₃ as a preservation method. This includes not only the initial investment but also the potential indirect benefits such as improved health and safety, consumer preference, and sustainability. While challenges remain, the outlook for O₃ use in grain preservation is promising. Advances in technology and scientific knowledge hold the potential to overcome current limitations, facilitating more efficient O₃ application. These developments will contribute to enhanced food safety, greater sustainability, and improved efficiency in agricultural practices.

AUTHOR CONTRIBUTIONS

Eugénio da Piedade Edmundo Sitoe: Conceptualization; investigation; writing—original draft; methodology; validation; visualization; writing—review and editing; software; formal analysis; data curation; supervision; resources. Flaviana Coelho Pacheco: Investigation; writing—original draft; methodology; visualization; writing—review and editing; formal analysis; software; data curation; validation. Florentina Domingos Chilala: Investigation; funding acquisition; writing—original draft; methodology; validation; visualization; writing—review and editing; formal analysis; software; data curation.

FUNDING INFORMATION

This study did not obtain any targeted funding from public, commercial, or nonprofit organizations.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Sitoe, E. P. E. , Pacheco, F. C. , & Chilala, F. D. (2025). Advances in ozone technology for preservation of grains and end products: Application techniques, control of microbial contaminants, mitigation of mycotoxins, impact on quality, and regulatory approvals. Comprehensive Reviews in Food Science and Food Safety, 24, e70173. 10.1111/1541-4337.70173