May. 06, 2024
Ozone application is a non-thermal technology used in food preservation, which is a powerful oxidant agent used in water and air treatment specially in disinfection processes for agriculture and food industry. The objective of this revision work is to publicize ozone applications in the growing, harvest, and postharvest handling of fruit and vegetables (F & V) across México. Ozonated water by foliar spraying and irrigation were proved to be effective in the control of pathogens, bacteria, and bugs. The use of Ozone was effective to heighten quality parameters of F & V, such as color, flavor, and soluble solids in mango, sugarcane, citric fruits, and nopal, increasing shelf life of fresh products up to 15 days after harvesting. Several protocols mentioned to fulfill the requirements of the producer were developed by TRIO3. The methodology proposed and the designed equipment by the company suggest a wider approach of this green technology in agriculture.
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Ozone (O3), also called trioxygen, is a gaseous substance whose molecule is formed by three oxygen atoms linked with an angular geometry. The ozone is formed by applying on the oxygen molecule enough energy to divide it forming various molecular structures [1]. This homonuclear molecule is the oxidant agent most powerful used for water and air treatment specially in disinfection processes for agriculture and food industry. Ozone is friendly with the environment and classified as Generally Recognized as Safe (GRAS) according to the Food and Drug Administration (FDA) [2].
Ozone is a universal disinfectant that reacts with the contaminant agents. It suppresses bad color and undesirable odor at the same time, destroying molds, bacteria, virus and algae [2, 3]. The deodorant action of ozone is due to the oxidation of chemical compounds such as ketones, hydrocarbons, acids, sulfides and nitrogenated derivatives [4]. Ozone oxidizes the cell wall, breaking its membrane and attacking to the DNA and RNA constituents. For this reason, the microorganisms are unable to develop immunity to the action of ozone as they do against other chemical compounds [1, 4, 5]. The use of ozone as sanitizer is effective without compromising health of consumers [6]. Traditional sanitizers are cheaper than ozone, however, sometimes they are not appropriate when a new outbreak emerges or when the presence of new food pathogens is detected [7, 8].
Ozone as a non-thermal technology is used in food preservation, which helps to improve the organoleptic characteristics of food following good manufacture practices [9]. Ozone can be pumped into a postharvest cold room. In water applications, ozone is drawn into a low negative pressure water stream using a Venturi injection system (Mazzei Company, LLT). The excess of not mixed ozone must be captured and destroyed to avoid the corrosion and personal injury [2]. A useful method of destroying the remanent ozone is combining UV light and catalytic agents such as granulated activated carbon [10]. The aim of this review is to describe several memories in the application of ozone in agriculture. Adhered to this, every project mentioned was adjusted to the state regulations achieving the expectations of the producer.
AdvertisementSome of the variables affecting the postharvest conditions of raw materials are related with their metabolic processes. Respiration as a process in living cells moderates the release of energy through the breakdown of carbon chains and the formation of new compounds, necessary for the maintenance and generation of synthetic reactions after harvest [11]. A low respiration rate in fresh materials increase postharvest life of perishable fruits. Ethylene concentration acts as a maturation hormone due to its biological activity in ripening climacteric fruits. Therefore, ethylene concentration can be monitored using precise instrumental methods [12]. Fundo et al. (2018) monitored the quality parameters of Cantaloupe melon juice using 30 min O3 and 60 min O3 treatments. They observed a diminution in color, vitamin C, total carotenoids, and antioxidant activity; but an increase in total phenolics registered in ozonated juices.
One of the most relevant stages of growth and development in F & V is probably the ripening or maturity. During ripeness, the enzymatic changes promote softening, hydrolytic conversion of storage material-starch to free sugars, and pigmentation changes, especially in climacteric fruits [13]. Flavor changes are considered an important topic in the metabolic pathway of F & V depending on sugars, acids and volatile compounds changes during ripening. As the fruit ripens, there is an increase in the synthesis of their volatile compounds [14].
In order to assure a good maturity index of raw materials, some factors such as water loss, changes in size, and changes in shape or surface must be considered, particularly when a marketable size is reached. Therefore, raw materials that presents physical damage must be separated to prevent postharvest losses. Measurement of texture, either manually or instrumentally can help to detect the maturity index of the fruit. Paciulli et al. [15] measured texture to frozen vegetables such as asparagus stems, zucchini and green beans. They observed a softening effect on the vegetable structure probably due to the blanching treatment affecting the inner cell wall. Depolymerization and/or solubilization reactions of the tissue occurred as a consequence of thermal treatment. In addition, it is advisable to avoid the improper handling or biodeterioration by microorganisms, insects, rodents or birds [16].
AdvertisementThe harvesting of fresh F & V, its management during the freezing, packaging and processing involves the use of water. The presence of water on the agricultural products can increase the probability of contamination of plant pathogens and microorganisms of major concern in food safety. Inappropriate application of procedures related with prevention of contamination and disinfection of water can have severe consequences. Special attention on the microorganisms present in the water recirculation system must be taken because of a rapid reproduction and adaptability in adverse circumstances [16]. Ozone used for vegetable washing and disinfection either gas or dissolved in water has been effective in reducing several microorganisms such as Escherichia coli, Penicillium Italicum, and P. digitatum [17, 18]. In this way, damage and deterioration of the fruit can be prevented, and postharvest life increased [19]. Ozone is used to eliminate pests and insects without harming the food quality and the environment. Applied at proper doses, ozone diminishes the proliferation of bugs, and is an excellent choice to replace chemical products.
Feston et al. [20] used ozone and observed a reduction of bed bug (Cimex lectularius L.) in different life stages. They concluded that LC50 and LC90 values were higher in nymph and adults than the observed in eggs. The damage caused by insects it is not only restricted to what the vector physically damages and ingests, but also what is defecated over the plant. All these factors promote fungal development like Fusarium spp. and Aspergillus spp., and in most of the cases, they are responsible of promoting the development of mycotoxins [21].
Ozone applied in a foliar way allows the addition of nutrients in the plant and the elimination of pests and diseases. The effectiveness of this chemical agent in reducing microbial load of foods includes the presence of fungi, molds, spores, viruses, bacteria, eggs, and nymphs of insects as shown in Figure 1. Ozone has no unfavorable effects on the organoleptic characteristics of foods including textural and nutritional quality. Besides, ozone acts directly in the metabolism of the microorganism suppressing its development [19, 22]. During foliar spraying, plant diseases can be reduced due to the effect of nutrients and ozone added. Another benefit observed of using foliar spraying is that the dose of fertilizers applied in cultivars and the operating costs can be decreased. According to Ali [23] an appropriate foliar spraying improved the quality parameters of papaya using 2.5 ppm and 3.5 ppm ozone. Weight loss, firmness, ripening and soluble solids concentration were maintained and preserved for a longer storage period.
There are different ways to carry out an irrigation system: by gravity, sprinkling, dripping, micro sprinkling or sub-irrigation, and capillary diffusion as shown in Figure 2. The ozone irrigation system in cultivars allows the incorporation of nutrients and oxygen, the strengthening of roots, thickening of the tree trunk, and the increase of soluble solids and sugars. Ozonated water is effective to eliminate virus and bacteria and control in nopal-vegetable such as Fusarium spp. and Aspergillus spp. [24]. The nematodes present in their root are destroyed due to the sanitizing effect of ozonated water. The mature fruit (tuna or prickly pear fruit) can be sprayed with ozonated water to guarantee the innocuity and integrity of fruit [25].
Ozonated water irrigation provides oxygen to the root and the stream that flows in its path reaching the root of the plant. The inner membrane of viruses, bacteria, fungi, algae, spores, and other microorganisms are destroyed using ozone dissolved in water, and promotes strength and productivity in the plant preventing diseases. This technique is widely applied in fruit trees, vineyards and nopal-vegetable among others [26]. Table 1 shows uses of ozone in several materials and its benefits.
CommodityOzone treatmentEffects on qualityReferenceBroccoli0.04 μL L−1, 7 d, 10 °CDelay of metabolic process & oxidative reactions[2]Cucumber0.04 μL L−1, 17 d, 3 °CEnhancement of appearance.7±2.4
gL−1 for 30 & 60 minIncrease of polyphenols and vitamins.[5]Mango10 μL L−1, 3 d, 25 °CDelay of ripening, Improvement of quality characteristics.[7]Papaya2.5–3.5 ppmWeight loss reduction, higher firmness values.[8]Soil and water depositsOzone variable doseBactericidal action on responsible for food delay (P.aeruginosa)[6]Uses of ozone in several fresh produces and its benefits.
Several studies have been conducted to identify the vector disease in the plant as follows: leaf yellow curl virus in tomato [27], whitefly (Bemisia tabici) affects cotton [28], tropical fruits [29], and potato [30]. Ozonated water irrigation used in a regular way effectively eliminates plant disorders leaving no smell or trace [19]. Several researchers emphasize the effectiveness of ozone [30, 31, 32, 33, 34]. Contigiani et al. [35] applied ozonated water effectively in strawberries to reduce the risk of pathogen proliferation. They found significative differences (P > 0.05) in the fungal control (B. cinedea) using ozonated water for 5 min (2.73 mgL-1). Additionally, quality attributes were preserved. Ozone seems to be a very promising technology to reduce post-harvest losses.
AdvertisementMango (Ataulfo variety) cultivated in the tropical areas of south of Mexico has a supreme quality but achieves a fast state of maturity and decomposes easily due to a high microbiological load. Local growers apply higher doses of fertilizers and pesticides several times per year to avoid the infestation of mango tree, specifically whitefly and their larvae. Effective post-harvest techniques to avoid major economic losses must be employed.
The research team of Pérez-Nafarrate [36] designed processes using ozone gas and ozone water for disinfection and packaging of mango in an industrial plant in Guerrero, Mexico. The accomplishment of concise food safety regulations was according to studies realized by Tran et al. [37]. In order to preserve the physicochemical characteristics of mango fruit, the next premises were followed:
Ozone gas used in a regular basis (one dose every third day) should eliminate gradually the presence of pests.
Gas monitoring was performed using an ozone analyzer (Model IN-2000, L2-LC, in USA Incorporated).
Ozone concentrations (1.5, 2.5, 3.5, 5 ppm) applied (96 h, 30 ± 3 °C) in the storage container should reduce the ethylene content generated as part of the fruit ripening process.
The most effective ozone dose (3.5 ppm) preserved the organoleptic characteristics of the fruit and delayed the ripening for 15 days. According to producers, optimal shelf life of mango is 10 days [37]. Soluble solids, color, flavor and empiric firmness were improved (data not published) and a sensory evaluation of the fruit was developed by the operators. More and Rao [38, 39] used a combination of UV-C irradiation (210–280 nm), guar gum (2.5%), and ozone water (400 mg-h-1) to preserve organoleptic characteristics of mangoes at 32 ± 3 °C after 19 days.
Compliance with good manufacturing practices diminish the fruit deterioration, and the economic profit increased. Harvesting of climacteric fruits, including mangoes, papaya, and banana; requires storage temperature and relative humidity similar to that observed in subtropical environments. According to Pérez-Nafarrate [36], key factors to conduct an ozonated water experiment consist on reliability and accuracy of the measurement devices, type and growth stage of the microorganism, ozone concentration and time of contact with the product.
AdvertisementThe nopal vegetable (Opuntia ficus-indica) grows in semi-arid zones around the world, it has a high dietary fiber content, and is usually consumed Mexico [40, 41]. Physical and morphological characteristics of nopal allow a quick adaptation to low precipitation and excessive heat (Figure 3). The nopal consist of two main parts: the edible part (84–90% water), and prickly pear (45–67% of reducing sugars) also called “tuna” [42]. Relative humidity is a crucial variable in nopal for development of pests and diseases. The most commonly microorganisms present in nopal are: cactus weevil Cactophagus spinulae (Gyllenhal), [43]; boll weevil (Cylindrocopturus biradiatus Champs), [40]; white worm (Lanifera cyclades Druce), zebra worn (Olycella nephelepsa Dyar), [44]; and cochineal (Dactylopius coccus costa), [45]. The most important include the bacterial stain (Bacterium sp.), rotting of the epidermis, anthracnose, prickly pear gold [46], the thickening of cladodes and the presence of bold among others [47]. The objective was to give added value to nopal-vegetable (raw and processed) using ozonated foliar spraying and ozonated water irrigation.
Due to the nature of nopal, good manufacture practices in soil and water are required to control outbreaks of diseases. The experimental plantation was located in Matehuala, San Luis Potosi; México. The producers gave light watering to nopal seedlings every 8 or 15 days during the dry months as shown in Figure 4, and the irrigation program was monitored periodically to establish the ozone generator requirements. Salinity, hardness, pH and sedimented solids were registered for 15 days before installing the irrigation system. The ozone solubility control included the measurement of pressure and temperature, concentration of carbonates, nitrates and metals [6]. Once the nopal “pencas” (thick and fleshy central nerve that has leaves) were cut, they were placed in recyclable plastic boxes preserving the thorns to avoid the oxidation reactions [48].
Ozonated water favored the organoleptic integrity of the nopal, reducing oxidation reactions and retaining green color in sliced raw nopal. Other goals achieved were the reduction in microbial load and the increase in shelf life (data not shown). The final product was packaged and sealed in plastic bags with 500 g and 1000 g. An investment proposal to install a rural cooperative company with capacity of processing 500 kg per day was presented to the cooperative.
Technical specifications and cost of a nopal sider, an immersion washing machine, a centrifugal dryer, and a vibrator dryer with cold were included in the investment project for $1,500,000.00 pesos.
Good manufacturing practices are essential to accomplish nopal processing with ozone. Water quality and soil requirements monitoring were key factors to improve the postharvest life of nopal opuntia. An innovative option for rural population centers was presented to detonate the local economy. Almost 50% of inhabitants in Mexico are distributed in rural areas, therefore ozone technology should be affordable to most of the population.
AdvertisementThe Huanglongbing (HLB) disease or “yellow dragon”, affects the worldwide production of citrus (lemon, tangerine, orange). Diaphorina citri (Kuwayama), the insect vector of HLB, is the main responsible as shown in Figure 5 [49, 50]. HLB is mainly distributed in Asia (Malaysia, Thailand and Vietnam) and Africa. Farmers invest up to 50 insecticide doses per year to control the vector and avoid the spread of the disease [51]. Symptoms of this plant disease include damage in tree and fruit, sparce yellow foliage, and stunting among others as shown in Figure 6 [52], shortening of the productive life of the plant, and detriment of final quality product [53].
Since 2010, HLB has been spread to the American continent especially those areas with frequent rainfall and template temperatures. HLB in Mexico is a serious threat to the 526 thousand hectares of citrus distributed in twenty-three Federal Entities. This in turn represents a production risk of 6.7 million tons per year with a value of more than 400 million dollars. Around 67,000 producers engaged to citric industry generated approximately 70,000 direct jobs and 250,000 indirect jobs, all of them endangered by HLB [54]. Specific research in Mexico to counteract this pandemic is urgently recommended.
In 2013, a collaboration protocol was signed between TRIO3 Food Technologies, the National Institute of Forestry, Agricultural and Livestock Research (INIFAP, for its acronym in spanish) and Los Limones Orchard, located in Tecoman; Colima (18°46′38.04′´ N, 103°48′56.74′´ W). Pérez-Nafarrate [55] evaluated the effect of ozone to eradicate the HLB disease in the productive region previously mentioned. Two main objectives were proposed: first, the early detection of infected trees in the growing season; and second to evaluate the damage spread by the vector in those trees with an advanced stage of the disease, and register its total loss [56]. Fifteen hectares planted with real lemon were micro-sprinkler irrigated with ozonated water, and nutrients were added to roots of trees every third day during six months. The sprinkler irrigation process was evaluated at the end of the experiment.
Ozone applied in Los Limones Orchard considerably reduced the population of larvae and adult vectors increasing the citrus production (data not shown). After ozonated water process concluded in lemon trees, other benefits were observed by farmers and technical staff including the improvement of the foliage coloration, and strengthening of trunk and root trees, which are very promising. Showler [57] used an organic mixture composed of corn meal, humic acid, molasses and fish oil to suppress greasy spot in infected trees. An increase in soluble solids content and a weight loss reduction in treated trees, as a result of the healing treatment during three years, provided similar results as ozone did. According to Perez-Nafarrate [55], the insecticide and plaguicide action of ozonated water proved to be faster than traditional methods.
HLB, like many other plant diseases, be diminished effectively with ozonated water. Gas concentration used, soil quality and the effectiveness of the irrigation system were the critical parameters to observe in the experiment. This clean and affordable technology is efficient to control HLB, but more research is still needed to eradicate this bacterium from Mexico and its surroundings.
AdvertisementSugarcane (Saccharum spp hybrids) is a tall perennial grass traditionally grown in tropical zones and used for obtaining sugar concentrates, molasses and other derivatives [58, 59]. Good manufacturing practices and biological control of diseases allow the correct development of this plant. It is common to find a considerable number of microorganisms in sugarcane such as bacteria, fungi and viruses, all these entities cause several diseases in the cultivar [60] as shown below:
Most common organisms found in sugarcane include: red rot (Colletotrichum falcatum) [61]; wilt (Fusarium sacchari) [62]; grassy shoot (Exitanius indicus) [63]; leaf scald (Xanthomonas albilineans) [64]; smut (Sporisorium scitamineum) [65, 66]; brown rust (Puccinia melanocephala) and orange rust (Puccinia kuchnii) [67].
The diseases of major economic importance in sugarcane are described as follows:
Mosaic disease. Is produced by Sugarcane mosaic virus and attacks young plants making areas appear on leaves pale green and yellowish within a normal green. The reeds become stunted causing production declines [68].
Eyespot. This name is derived from the powdery black mass of spores associated with this disease. The affected plants show a reddish elliptical lesion surrounded by a yellowish structure that varies in size. The spores of the fungus Ustilago scitaminea are transmitted by wind, rain, irrigation water, seeds or animals [69].
Rust. Produced by the fungus Puccinia melanocephala manifested by many elongated spots on the leaves, show no growth and the stem become thin. The yellowish spots look at the whole sheet [70].
Leaf scald. The causal organism Xanthomonas albineans is a bacterial disease first observed in Cuba in 1979. X. albineans causes sudden death of entire stems and seedlings affecting significantly sugarcane yields [64]. Leaf scald is also called in Latin America “gomosis”, a plant disease characterized by an abundant production of gum. Gomosis caused by Xanthomona axonopodis involves widespread dwarfism in plant [70].
Ratoon stunting disease. RSD caused by Leifsonia xyli spp. was observed in Cuba for the first time in 1953. Several stunted stems very thin are present within the plant and short internodes. Damaged stems increase as the number of cuts in the field is higher [71].
The ozonated water irrigation provides a greater contribution of oxygen to the root. Ozonated water is free of viruses, bacteria, fungi, algae, spores and any other microorganism. Growth is achieved faster than usual with more liveliness and strength as well as more productivity. This irrigation method is beneficial for plants, and is currently used in fruit trees, vineyards and crops in general. Ozonated water achieves the prevention of plant diseases such as leaf scald and rust reducing the microbiological load. Chemical products such as pesticides and fungicides are reduced improving the shelf life of fresh products [60].
On July 4 2008 was signed a collaborative project between the National Institute of Agrarian Innovation (INIA, for its acronym in spanish) and the National Institute of Research of Sugarcane in Havana-Pinar del Río. Red ferritic soil was used to grow up a 9-month agamic seed from 5 varieties identified as follows: C323–68, C86–56, C86–456, C88–380, and C90–530. The experimental design (divided plots 5 x 6 x 2) included 180 buds from each variety with two replicates, 5 varieties and 6 treatments. Finally, sugarcane certified quality seeds (P. Vista Florida cv. 115–2014) previously treated with ozonated water were introduced among the producers as shown in Figure 7.
The exposure time in ozonated water was 10, 20, and 30 min respectively. The treatments included a hot water-chemical treatment, a biological one, and a sample control [52]. The measurements were carried out one and six months after sowing. The main objective was to evaluate the effect of ozonated water on sugarcane seed growing. The viability and growth of agamic seeds of sugarcane used was also evaluated. The ozone equipment used is shown in Figure 8.
An irrigation and sprinkler system with ozonated water to promote the growth of sugarcane is shown in Figures 9 and 10. The fungicide was reduced progressively to control and eliminate fungus and pests as the amount of ozonated water increased. Previously, 90 buds were submerged in ozonated water during 24 h. Thirty buds per variety were immersed in ozonated water (1 ppm) for 10, 20, and 30 min respectively.
Thirty buds per variety were soaked on water for 24 h without applying any treatment.
Thirty buds per variety were submerged 24 h in water, then immersed in hot water at 51 °C for one hour. Immediately they were treated with propiconazole (Tilt EC 250) at 0.4% (w/w) for 15 min. This time was considered enough to eliminate leaf scald and red rot diseases.
This treatment started on July 4 2008 with the immersion in water of 30 sugarcane buds per variety for 24 h, and then submerged for 30 min in a water solution of Nemacid (EDOCA, Amealco, Querétaro) at 2% (w/w). This organic insecticide is used in the control and inhibition of nemathodes. Quivican experimental station was used to plant the sprouted seeds treated with Nemacid. After 30 days, the sprouting count was carried out and analyzed in the laboratory. All the experiments were made by duplicate.
All the treatments showed significant differences (p < 0.0001). Best results obtained on seeds immersed in ozonated water for different time intervals are shown in Figure 11. The sprouted seeds treated with 10 min-ozonated water immersion reached the highest germination level: 10 seeds in total. The sample control seeds and seeds treated with 30 min-ozonated water reached an acceptable germination rate: 9 and 8 respectively. Nemacid and hot water samples presented the lowest germination rate: 7 in total. Using ozonated water did not affect the viability of the seed. The experimental ozonated water immersion system proposed to producers of sugarcane reduced drastically eyespot and leaf scald. This treatment was beneficial for agamic seed viability showing a different response in every treatment, especially that with 10-min immersion time.
AdvertisementOzone has demonstrated be useful for management of postharvest of climacteric fruits such as mangoes and papayas. This ecotechnology is cost effective and is very promising to use in developing countries of Latin América. Using ozone watering in Nopal opuntia crops, proved to be very effective to control diseases and increase plant health. An adequate ozone irrigation system prevents growth of bacteria and bugs in citrus trees, and other fruits, and is an alternative to reduce HLB infestation. Sugarcane and other important crops growing in tropical areas increase yield using ozonated water, and an early diagnose of diseases reduce losses and increase the economic benefit in productive areas. TRIO3 acknowledges all the producers and scientific investigators involved in the protocols developed during these last 10 years.
AdvertisementThanks to TRIO3 Food Technology support and also thanks to Superior Technological Institute Zacatecas Norte (ITSZN-TecNM).
AdvertisementThe authors declare no conflict of interest.
The dairy field has considerable economic relevance in the agri-food system, but also has the need to develop new ‘green’ supply chain actions to ensure that sustainable products are in line with consumer requirements. In recent years, the dairy farming industry has generally improved in terms of equipment and product performance, but innovation must be linked to traditional product specifications. During cheese ripening, the storage areas and the direct contact of the cheese with the wood must be carefully managed because the proliferation of contaminating microorganisms, parasites, and insects increases significantly and product quality quickly declines, notably from a sensory level. The use of ozone (as gas or as ozonated water) can be effective for sanitizing air, water, and surfaces in contact with food, and its use can also be extended to the treatment of waste and process water. Ozone is easily generated and is eco-sustainable as it tends to disappear in a short time, leaving no residues of ozone. However, its oxidation potential can lead to the peroxidation of cheese polyunsaturated fatty acids. In this review we intend to investigate the use of ozone in the dairy sector, selecting the studies that have been most relevant over the last years.
The aim of this review is to give an overview of the current state of knowledge on several manufacturing and storage processes of the dairy chain, highlighting the potential and limiting the use of gaseous or ozonated water against the pathogenic and spoilage microflora, parasites, and insects, such as molds and mites. Specifically, we intend to start from the raw material of the dairy supply chain, the milk, up to the different final products, including their quality and sensory parameters, passing through the sanitization of the working surface areas and equipment and through the treatment of wastewater. The process flow diagram of the dairy industry is shown below ( ).
Ozonation is a clean technique with antimicrobial power due to its oxidation potential able to reduce the microorganisms, limiting the production of enzymes, but the effectiveness of ozone can be affected by the temperature, pH, humidity, and the amount of organic matter around the cells [ 41 ].
Product losses also occur due to infestation problems, such as mites, which infest the ripening rooms through the wooden boards used to mature the cheeses, with consequences ranging from product weight loss to the waste of the entire cheese. The mites are very small, hardly visible to the naked eye, and are practically ubiquitous in the dairy industry and, more generally, in the food sectors in which a product maturation phase is foreseen [ 36 ]. They are present in the wooden shelves used in the maturing rooms and spread through the clothes of the operators, or they can be transported by air; dirty environments favor their proliferation [ 36 , 37 , 38 ]. Therefore, cleaning and disinfection are key operations for food safety, but they produce a high environmental impact due to water use and wastewater. In the care areas, chemicals such as chlorine, quaternary ammonium compounds, etc., are used. Health and environmental concerns with chemical use on food products or food contact facilities are supporting the need for alternative sanitation technologies. In this sense, the potential utility of ozone lies in the fact that ozone is a stronger oxidant compound than chlorine, and it has been shown to be effective over a wider spectrum of microorganisms. Furthermore, ozone reacts with some organic compounds in food matrices, and the possible by-products are aldehydes, ketones, or carboxylic acids, which do not pose a threat to human health [ 39 ].
The transport of bulk milk from the dairy to the processing plant is critical, as well as the various stages of storage and processing, because it could significantly alter the milk microbiota due to contamination or the growth of microflora [ 30 , 31 , 32 , 33 , 34 , 35 ].
The many varieties of cheese found are characterized by different flavors, textures, and shapes, and are produced using milk, rennet, salt, and lactic ferments. The microbiota, made up of lactic acid bacteria (LAB) [ 23 ], contributes significantly to the quality and safety of the final product, even to the nutritional composition [ 24 ], developing desirable characteristics and flavor compounds, and it inhibits the growth of spoilage and pathogenic bacteria in cheese [ 25 ]. Moreover, their variation leads to obtaining a different quality of the product and, for this reason, some studies were conducted to isolate microbial strains that were obtained from the best natural fermentations [ 26 ]. Examples of starter LAB dominant during the fermentation period are Lactococcus and Streptococcus [ 25 ], while during the maturation process the dominant species are Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis, Enterobacter faecium, and Lactobacillus plantarum, qualitatively appreciated by consumers thanks to flavor intensity and texture, as well as overall acceptability [ 27 , 28 , 29 ]. Since the inoculation of starter cultures has been shown by several studies to have a better impact on the overall quality of the cheese, there are commercial starter cultures with which to inoculate cheeses [ 26 ].
Milk and dairy products offer highly nutritious food for microorganisms to multiply, so they can produce spoilage. The microbial contamination depends on the raw material quality, the conditions under which the products were produced, and the temperature and duration of their storage [ 20 , 21 ], resulting in product decay due to contamination by spoilage microorganisms, bringing the product to be unsaleable or greatly depreciated. In the case of pathogenic microorganisms, the product is not marketable, as it is harmful to human health. The control of contamination by spoilage bacteria, like Pseudomonas spp., and pathogenic bacteria, such as Listeria monocytogenes, and Salmonella spp., is a big challenge for the dairy industry [ 22 ].
The cleaning of equipment was once completely manual in the dairy sector, using brushes and detergent solutions, and it involved dismantling the equipment to access every surface. This approach was labor- and time-intensive and often quite counterproductive, since contamination can be reintroduced to the system by imperfectly cleaned equipment. These issues were solved by the introduction of CIP (Cleaning In Place) programs, which adopted automated cleaning systems in various parts of the processing plant to achieve the necessary cleaning and sanitation results [ 19 ]. However, in sanitation systems, it is advisable to try to lower the environmental impact of the various operations.
The amount of chlorine required for sanitization applications depends on the concentration in water of the species to be oxidized, organic substances, micro-organisms, ferrous ions and manganese, nitrites, and hydrogen sulfide [ 16 ]. The legislative framework regulating the quality of water for human consumption has recently been updated with EU directive 2020/2184 of 21 December 2021 [ 17 ], which replaces the 98/83 directive [ 18 ]. The new directive provides for the monitoring of some by-products of chlorine disinfection, such as chlorates and haloacetic acids five (HAA5-monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, monobromoacetic acid, and dibromoacetic acid).
The treatment of wastewater with ozone allows for the production of effluents at a level that can be discharged into natural aquatic systems [ 13 , 14 ]. The common practice for the sanitation of water is the use of chlorine gas (Cl 2 ), chlorine dioxide (ClO 2 ), or hypochlorite (ClO − ) due to the bactericidal and oxidative properties of chlorine. Ozone is a viable alternative for sanitization concerning other traditional sanitizers, such as chlorine, for over-chemical residues and potential environmental impacts that consumers care about [ 15 ].
About 192.5 × 106 m 3 of dairy wastewater (DWW) are generated per year in EU-27 countries, 49% of which is to produce cheese, while 19%, 18%, and 13% are required to produce drinking milk, acidified milk, and butterfat products, respectively. Germany, France, Italy, Poland, Spain, and the Netherlands produced more than 73% of DWW [ 12 ].
According to the 2022 Industry Sheet drawn up by Ismea, the dairy chain has strong economic importance in the national agri-food system. However, weaknesses in the supply chain has resulted in insufficient diffusion and implementation of measures to combat climate change that can be transformed into opportunities, such as the creation of sustainable products that respond to consumer needs [ 7 ]. In the dairy sector, several problems involve huge costs, economic losses, and excessive product waste. For the disinfection of the premises and equipment, a large amount of water and steam at high temperatures and chlorine-based chemicals is used. For example, ultra-high-temperature (UHT)-processed milk uses a lot of chemicals (alkaline cleaners, such as NaOH, and HNO 3 ) for cleaning [ 8 ]. This results in further problems and costs for the disposal of wastewater, both for the sanitizers used, and for the production residues. Dairy wastewater (DWW) contains high levels of mineral and organic compounds, which can collect in soil and water, affecting serious environmental pollution [ 9 ]. The huge amounts of wastewater with high organic content must be disposed of and, conventionally, they are purified by physical–chemical and biological methods. Some authors reported a generation of 1–10 L of wastewater per liter of processed milk [ 10 , 11 ].
Dairies in Germany accounted for the highest share of EU production of all main fresh and manufactured dairy products, including drinking milk (19.3% of the EU total), butter (21.0%), cheese (22.9%), and acidified milk products (23.7%) [ 5 ]. In 2021, Germany was the largest producer of cheese globally, with 2,461,334 tons. France was the second largest producer of cheese, recording a value of 1,716,120 tons, while Italy was the third largest producer of cheese globally, recording a value of 1,197,390 [ 6 ] ( ).
Cheese is a product of great importance in the dairy sector, notably in the western States, and for this reason, today, it has a globalized origin, ranging from Europe to the USA [ 2 ]. The COVID-19 pandemic has led to a global increase in food consumption, including cheese. The global cheese market was valued at USD 72.26 billion in 2020, with a production volume of 21.69 million metric tons compared to 20.98 million metric tons in 2019. The global cheese market is expected to grow from $192.81 billion in 2021 to $211.02 billion in 2022 at a compound annual growth rate (CAGR) of 9.4%. The market is expected to grow to $289.57 billion by 2026 at a compound annual growth rate (CAGR) of 8.2% [ 3 ]. The world’s largest cheese producer in 2021 is Europe (EU-27) with 10 million tons, followed by 6 million tons in the USA [ 4 ] ( ).
The environmental impact from the production of food is a focus of interest at the global level due to its relationship with climate change and competition with existing natural and energy resources [ 1 ].
In summary, it turns out that molds are more resistant than yeasts which, in turn, are more resistant than viruses and bacteria and, among these, the most susceptible are Gram-negatives. Moreover, both bacterial and fungal spores are more resistant to the antibacterial action of ozone. Efficacy is also interesting in detoxification: mycotoxins are completely degraded or modified so that their bioactivity is compromised. Moreover, it has been seen that treatment with ozone can degrade biofilms [ 55 ]. It has been seen that the inactivation mechanisms of viruses, although so far less studied, differ from those of bacteria: viruses, in fact, do not undergo cellular lysis, but the inactivation of specific viral receptors which allow them to form cells gami with the wall of the cell to be invaded, thus allowing the virus to enter it. The result is the arrest of the viral reproduction mechanism already at the level of cellular invasion. The spectrum of action, depending, among other things, on the exposure time and applied concentration, is quite broad [ 37 ].
Finch et al. [ 70 ] determined concentration and exposure time to obtain the inactivation of E. coli; Morandi et al. [ 71 ] positively evaluated its efficiency for the control of Listeria monocytogenes on some cheeses. Brodowska et al. [ 72 ] showed that vegetative cells are much more susceptible to treatment than sporogens. In the study, Bacillus cereus, Bacillus subtilis, Bacillus pumilus, Escherichia coli, Pseudomonas fluorescens, Eupenicillium cinnamopurpureum, and Aspergillus niger were examined, and the results showed that longer treatments were required precisely for the inactivation of the microorganisms belonging to the genus Bacillus. Moore et al. [ 73 ] reported the efficacy of ozone against various bacteria of food interest, underlining that in the presence of biofilm this is limited; Farooq et al. [ 74 ] focused on the study of the inactivation of Candida parapsilosis. Recently, ozone has also been shown to be effective against Aspergillus fumigatus, a difficult fungus to inactivate using other methods [ 75 ]. The researchers demonstrated the effectiveness of ozone in completely removing the microbiota on the tested swatches (20 ppm for 4 min) in a test ozone chamber.
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Other conditions being equal, the efficiency of ozone treatment varies depending on the different susceptibility of the target microorganisms. There are numerous studies that demonstrate its efficiency against both Gram-negative, less resistant, and Gram-positive bacteria, as well as against viruses, yeasts and molds, protozoam and parasites, including mites [ 37 ].
Ozone acts in two different ways: a direct way, in which the ozone molecule itself reacts with the organic or inorganic substance and an indirect way, in which the radicals, mainly the hydroxyl radical, are obtained following the decomposition of ozone to react with organic matter. In direct reactions, ozone acts as a dipole, with electrophilic and nucleophilic properties: its action is very selective. In the case of addition reaction to the double bond (mostly in reference to unsaturated fatty acids), a compound called ozonoid is formed, which in turn degenerates in proton solution (for example in water) into aldehyde, ketone, or zwitterion; the latter degenerates into hydrogen peroxide and carboxyl residues. As far as indirect reactions are concerned, three phases can be identified: activation, propagation, and termination. In the first phase, in the presence of an activator, such as the hydroperoxide radical (HO 2 • ), the decomposition of ozone accelerates: at a pH higher than the value of 4.8 (corresponding to the pKa of this radical) the radical forms the anion superoxide, which triggers the chain propagation by reacting with the ozone to form the hydroxyl radical, which in turn reacts with the ozone, determining the formation of the HO 2 radical, which will trigger the process again. In general, indirect oxidation can be applied to many organic pollutants, while direct oxidation can be applied to many inorganic pollutants, and it is no coincidence that its first and main uses concern water treatment [ 45 , 69 ]. Ozone acts at different levels and the mechanisms of inactivation of bacterial cells are many. As far as the cell walls and membranes are concerned, it oxidizes various components and, in particular, the unsaturated fatty acids, the enzymes present at the membrane level, the glycoproteins, and glycolipids, leading to the modification of the permeability of the cell membrane and finally to the cell lysis. The bacterial spores are protected from the action of the ozone thanks to the bark which itself, in the first instance, undergoes the oxidizing action; cellular enzymes are inactivated by O 3 in a very effective and less selective way compared to other disinfectants, such as chlorine [ 59 ].
Due to its high oxidation potential and its ability to diffuse across biological membranes, ozone oxidizes the cellular components of the bacterial cell wall and, once it enters the cells, it oxidizes all essential components: enzymes, proteins, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA). The result of this process is the destruction of the cell. The mechanism of action is, therefore, different from that of other disinfectants: chlorine, for example, penetrates the cell by diffusion and, once inside, it affects the various enzymes [ 45 ].
The way ozone acts against microorganisms is a complex process. Ozone can attack various cell membrane constituents-cell walls, the cytoplasm, endospore coats, virus capsids, and viral envelopes [ 54 , 67 ]. The double bonds of unsaturated fatty acids are particularly susceptible to ozone [ 56 ]. The effective antimicrobial properties of the molecule are its high oxidation potential and its ability to spread through biological membranes [ 68 ].
An interesting study on the penetration capacity of ozone for the disinfection of textile materials shows that, for effective decontamination, it is important to use ozone concentrations to allow for reaching hard-to-reach regions of different types of garments, considering the distance between the garments used [ 66 ].
In this sense, the results of the work of Shynkaryk et al. [ 65 ] have demonstrated that such changes due to ozone are limited to the product surface only, without penetration into the observed mass of material.
However, an important factor to consider is the ability of ozone to penetrate food, which can affect the quality of the product. Therefore, a careful balance must be maintained between food safety and the maintenance of nutritional aspects [ 43 ].
Therefore, it has become even more important to sanitize these products immediately after their harvest. Ozone could be used in this phase, as it appears to have a strong virucidal activity and is capable of inactivating viruses and bacteria [ 42 ].
Additionally, due to the recent Coronavirus epidemic (COVID-19), whose transmission occurs through droplets, more attention is being given to fruit and vegetables that are bought for household consumption.
A final significant use is its use in fruit and vegetable production, including applications for the disinfection of the water used during the sanitization of products, both in the storage and conservation phases, by acting as an antimicrobial inside the storage rooms [ 61 , 62 , 63 , 64 ].
In poultry farming, it is used to sanitize the environment from pathogenic microorganisms, to eliminate ammonia fumes and for deodorization, as well as to improve the oxygenation of the environment, reduce the risk of cross-contamination, and improve the hygienic–sanitary conditions of the feed. Ozone is also used in the preservation of fish and meat to reduce the microbial load in storage cold rooms, and it avoids contamination of products with bad smells; however, it is advisable to keep the concentration of ozone under control, as it can be a vehicle for an increase in lipid peroxidation, which causes the deterioration of the final product [ 59 ]. Additionally, for storing cereals and grains in silos, it is used as a fumigant to inhibit the formation of molds (with consequent mitigation of the risk of mycotoxin release), but also for insects that can infest stored cereals [ 60 ].
Natural Organic Matters (NOM) are micro-organic pollutants that can affect the efficacy of drinking water treatment processes and the safety of drinking water. Advanced removal of water NOM by PEB (Pre-ozonation, Enhanced coagulation, and Bio-augmented Granular Activated Carbon) was studied. The results showed that the pre-ozonation treatment with 0.5 mg of O 3 per mg of TOC (Total Organic Carbon) resulted in a TOC removal of 87%, with a concentration of 0.8 mg L −1 in the finished water, lower than the standard limit for chlorination treatment (2 mg L −1 ) with no formation of bromate as a carcinogenic byproduct [ 58 ].
Today, ozone is currently used in water for food sanitization, but also for swimming pools, showers, irrigation systems, and water purification; it is used in the environment in the gaseous phase, mainly for the elimination of volatile molecules (connected to the development of odors) and toxins [ 57 ].
To prevent microbial contamination in several industrial fields, disinfectants are used, such as quaternary ammonium salts, iodine, acids, and chlorine-based compounds, compounds which, although particularly effective in disinfection, release toxic substances into the environment. UV radiation can also be used (to which problems of release of radioactive substances can be associated, which accounts for more marginal use in the food sector) in addition to heat treatment [ 56 ]. The use of hot water or steam is a very effective system, but excessive heat can damage the equipment, and the associated energy cost is by no means negligible. For these reasons, ozone presents itself as a valid alternative.
In the food industry, chlorine and hydrogen peroxide-based sanitizers are the most used. Although their antimicrobial action is extremely effective, Khadre et al. [ 54 ] have shown that they release residues that can have a potentially toxic effect; hence the need to look for alternatives that can be just as effective, but without leading to the formation of residues. Therefore, the introduction of ozone as a sanitizer in the food sector must be seen in this context. Being characterized by a strong redox potential, ozone has excellent capacity as a disinfectant, but, having a short half-life, it decomposes rapidly into non-toxic products and therefore leaves no residues that could have an impact on the environment, much less on human health. This is also clearly correlated to a reduction in costs for the company, who will not have to deal with the disposal of residues of chemical substances used for sanitization. Furthermore, being generated on-site and on-demand, both the environmental and economic impact is further reduced, in fact, there is no need for transport and much less storage (as otherwise happens for conventional products). Finally, the costs to be incurred for the operation of ozonation systems are also low, as they require relatively low levels of electricity to operate [ 55 ].
The legislation related to ozone must also stop dealing with issues related to safety in the workplace as the effects related to oxidation make this molecule potentially toxic, in a logic that remains dependent on the concentration and exposure time. The human olfactory perceptibility threshold for ozone is very low (0.02–0.04 ppm in air); for exposures to concentrations of 0.1 ppm in air, irritation to the eyes, nose, and throat can be generated; one-hour exposures to increasing concentrations of 2, 4, and 15 ppm, respectively, lead to the appearance of symptoms, irritant, and finally toxic effects, while higher concentrations can have lethal effects on humans [ 44 ]. The Occupational Safety and Health Administration (OSHA) has approved, as the concentration limit for ozone gas, 0.1 ppm with an exposure time of 8 h at the workplace in the food industry [ 51 ]. Advice on the treatment of air with ozone in cheese-maturing environments from the National Committee for Food Safety of the Italian Ministry of Health (CNSA) states that operators must not be exposed to more than 0.1 ppm ozone in 8 h or more than 0.3 ppm twice a day for 15 min [ 37 ]. However, ozone treatments with high concentrations can be undertaken with no operators or during the weekly closing days [ 52 , 53 ].
Disinfectants such as ozone can be manufactured, placed on the market, and used in the European Union exclusively on the basis of an authorization issued under Regulation 528/2012 [ 49 ] and/or under the national legislation currently in force in each Member State. There are also directives on some specific uses of ozone, such as the directive 2003/40/EC [ 50 ] that defines the conditions for using ozone-enriched air for separating compounds of iron, manganese, sulfur, and arsenic from natural mineral waters or spring waters, and the labeling requirements for waters which have undergone such treatment.
Since 2001, the FDA (Food and Drug Administration) has recognized the use of ozone as an antimicrobial agent in the gaseous phase or in aqueous solution in food production processes, defining this as GRAS (Generally Recognized As Safe) element, i.e., a food additive considered safe for human health [ 48 ].
Having a strong pro-oxidant effect, ozone in contact with some materials could lose effectiveness, but also generate corrosion. The compatibility of this molecule obviously depends, in a directly proportional manner, on its concentration; if this remains as dry gas in a range of 2% by weight, it is compatible with most plastic materials [ 46 ] used in the food industry: PTFE, PVDF, PVC, ECTFE–Polytetrafluoroethylene, Polyvinylidene fluoride, Polyvinyl chloride, and Ethylene Chlorotrifluoroethylene. 316L and 304L stainless steel proved to be more resistant to ozone than to chlorine [ 47 ]; natural rubber is instead very susceptible, while silicone tolerates short periods of exposure well, but if exposed for long periods it oxidizes.
Ozone is not a radical species, but it is a very reactive molecule: in water it decomposes very quickly, through mechanisms giving rise to a series of reactive ROS (Reactive Oxygen Species), including the superoxide radical (O 2 • − ), hydroxyl radical (OH•), and hydrogen peroxide (H 2 O 2 ), which, in turn, react with the biological macromolecules, causing modifications of the structure and therefore of the functions. Its reactivity depends on the electronic configuration of the molecule: one of the terminal oxygen atoms is characterized by a lack of electrons and therefore by an electrophilic behavior, the other by a negative charge, which gives the molecule a nucleophilic character. It is an unstable molecule characterized by a very short half-life, greater in the gaseous state than in the aqueous solution; its decomposition is so rapid in the aqueous phase of food that its antimicrobial action takes place mainly on the surface [ 44 ]. The stability of the molecule is influenced, among other things, by the purity of the water: it has been seen observed at 20 °C that 50% of the ozone decomposes in twenty minutes if in the presence of distilled or tap water, a percentage which is reduced to 10% if double-distilled water is used. At pH 7, as the temperature increases from 15 °C to 30 °C, the half-life decreases from 30 to 12 min; moreover, the half-life in water is much shorter than in air. Taking, for example, the temperature of 20 °C, the half-life is three days in the air and only twenty minutes in water at pH 7 [ 45 ].
Connected to the antimicrobial power of ozone molecule is the oxidation potential of well −2.07 V; higher than that of chlorine (−1.36 V) and hydrogen peroxide (−1.78 V), and lower than fluorine (which has the highest redox potential with −2.87 V), hydroxyl radical, persulphate ion, and atomic oxygen. In nature it is present in the ozonosphere, part of the stratosphere, and is concentrated at about 25 km from sea level, and its concentration in the atmosphere is about 0.04 ppm (1 ppm = 1.96 mg m −3 ), which remains constant thanks to the dynamic balance existing between the formation and photolysis processes. The formation is due to UV radiation with a wavelength of 242 nm which dissociates molecular oxygen into atomic oxygen [ 37 ].
In industrial practice, ozone generators are of three different types: corona generator, electrochemical, and UV radiation. The most used is the ozone generator through the corona effect as compared to the other methods. Three methods are used for the determination of ozone: physical, based on the measurement of some ozone properties, such as absorbance in the visible or infrared range; physical–chemical: it measures the effect of the reaction of ozone with certain reagents including chemiluminescence and heat reaction; chemical: it measures the number of reaction products between ozone and a specific reagent (an example is the indigo colorimetric method) or the reduction of the molecular weight of a polymer the ozone adds to the double bonds causing the discoloration, which is then measured with the spectrophotometer as a change in absorbance [ 44 ].
Ozone is soluble in aqueous solution (from 0 to 30 °C its solubility is thirteen times higher than that of oxygen molecule), in a logic that is inversely proportional to the temperature (solubility decreasing as the temperature of the water increases) [ 37 ].
The inactivation mechanism against microorganisms through ozone can be obtained in two ways, oxidizing amino acids and sulfhydryl groups of peptides, proteins, and enzymes to produce small peptides at the time of ozone exposure, or oxidizing the poly-unsaturated fatty acids, with the formation of acid peroxides [ 42 , 43 ].
Ozone (from the Greek ozein, which has an odor) is a molecule made up of three oxygen atoms arranged in space with a trigonal planar geometry with a bond angle of 116.8° and a bond length of 1.278 Å. Ozone has a molecular weight of 48 g mol −1 at 1 atm of pressure, a boiling point of −111.9 °C (beyond which it appears as a dark blue liquid), and a melting point of −192.7 °C. At room temperature and normal pressure, ozone is present in the gaseous state and is characterized by a pungent odor (the fresh and clean smell, typical of the air after a storm, is due precisely to the ozone that has just been generated in the atmosphere) and bluish in color if generated from dry air [ 37 ].
Some phases within the dairy industry are particularly critical from a microbiological point of view. Examples in the production of cheese are the thermization or pasteurization of milk which must be sufficient to inactivate all human pathogenic bacteria, as well as reduce the endogenous microbial load [76]; the syneresis which may be more or less marked, influencing the aw (water activity index) and therefore the possibility of development of the various microorganisms; the salting phase which determines a change in the microbiological habitat and, therefore, also the variation of the microbial ecosystem (from the population of starter lactic acid bacteria, SLAB, to that of non-starter bacteria, NSLAB); stewing, during which the optimal environment is recreated between temperature and relative humidity for the proliferation of the microorganisms responsible for the acidification of the “pasta”; finally, the maturation phase is a fundamental one in which the NSLAB microorganisms will be responsible for the changes that will occur in the cheese and which will influence the consistency, the sensory properties of the product, and its shelf-life. For the maturation of some aged cheeses, a further criticality must be considered: the presence of mites, arthropods that tend to settle in the wooden boards on which the cheeses are placed to age, on which the parasites feed, causing serious economic losses to the companies [77].
A fundamental element for the control of microbial loads is the disinfection of systems, instruments, and rooms: the microorganisms that can colonize environments are in fact many and companies carry out cleaning operations every day using water vapor at high temperature and pressure and chemical products that are chlorine-based; these are costly operations from both an economic and an environmental point of view. Furthermore, wastewater is rich in organic matter, an element that makes it difficult and expensive to dispose of, as well as representing an excellent growth medium for microflora.
The genus Pseudomonas includes spoilage bacteria, responsible for undesirable odors and flavors or unusual pigments of foods [78], often isolated at several stages of the dairy production environment (P. fluorescens, P. koreensis, P. marginalis, P. rhodesiae, P.fragi, P. putida, P. entomophila, P. mendocina, and P. aeruginosa) [79].
The genus Listeria comprises L. monocytogenes, a Gram-positive foodborne pathogen that can form biofilms and persist for a long period on surfaces and food processing environments, being able to cause frequent contaminations of the finished products [80,81,82].
The genus Salmonella comprises pathogenic bacteria found in various types of foods, like fresh and fermented dairy products, adapting to an acid environment [83,84,85,86].
Ozone is used upstream of the dairy supply chain, starting from animal husbandry: it is an alternative to traditional methods, which involve the use of hot water and chemical products, for the sanitization of the pipes that carry the milk from the rooms of milking to collection tanks. Using ozone reduces costs (in reference to the purchase of disinfectants and the use of hot water) and the environmental impact (as it does not release residues); moreover, the effectiveness of ozonized water in the disinfection of animals, surfaces and tools has been seen. In livestock farms, the ozone can be used for the sanitization of the air in the stables: at low concentrations, it contributes to the elimination of bad odors and any pathogens present in the air [26]. Ogata and Nagahata [87] studied its potential in the treatment of bovine mastitis, a problem that afflicts farms, causing important economic losses: according to the researchers, as many as 60% of treated mastitis heals without the use of antibiotics (6–30 mg of ozone).
Hot water with chemicals is usually used for cleaning and disinfection processes, generating large energy and chemicals consumptions. Many studies evaluated the use of ozonated water and gas to clean and sanitize equipment and different surfaces in dairy locations [55], ( ).
Guzel-Seydim et al. [88] evaluated the use of ozone for the treatment of stainless-steel surfaces to remove milk residues, demonstrating its greater effectiveness compared to the traditional treatment with hot water at 40 °C. 15 min treatments using either warm water (40 °C) or ozonated cold water (10 °C) were conducted. The results show that Chemical Oxygen Demand (C.O.D.) values are reduced by 84% (ozone treatment) against 51% (hot water treatment). Furthermore, many researchers have evaluated the effectiveness of ozone against microorganisms that can colonize metal surfaces.
Greene et al. [89] studied the effect of ozonated deionized water against psychrophilic contaminating microflora (Pseudomonas fluorescens and Alcaligenes faecalis) on stainless steel plates, showing that a 10-min exposure with a concentration of 0.5 ppm decreased the microbial growth by more than 4 log10. For the experimentation, stainless steel plates were incubated in UHT pasteurized milk and inoculated with pure cultures of Pseudomonas fluorescens (ATCC 949) or Alcaligenes faecalis (ATCC 337). Since these are metal surfaces, the corrosive power of ozone must always be considered. The use of ozonated water is recommended, instead of hot water and chlorine, when the surfaces of the milk processing equipment are not damaged. Recently, the potential of disinfection effectiveness of single and synergistic ozone (10 ppm for 15 min) and UVC (1 cm or 15.56 mW cm−2 for 15 min) treatment for the sterilization of bacteria and fungi (Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus fumigatus) was studied on different material surfaces (stainless steel, polymethyl methacrylate, copper, surgical facemask, denim, and a cotton-polyester fabric) [90].
In the work of Dosti et al. [91], fresh 24-h bacterial cultures were treated with ozone (0.6 ppm for 1 min and 10 min), chlorine (100 ppm for 2 min), or heat (77 ± 1 °C for 5 min), showing its effectiveness against food spoilage microorganism in synthetic broth. The bacterial biofilm on the metal coupons was significantly reduced by ozone and chlorine, but with no significant difference between ozone and chlorine, except for P. putida (ozone was more effective than chlorine).
Greene et al. [47] observed that 0.4–0.5 ppm of ozone, pulsed into water at 21–23 °C for 20 min per day over a 7-day period, caused a certain degree of weight loss of all materials tested (i.e., aluminum, copper, stainless steel, and carbon steel), but only weight loss for carbon steel was significant. Therefore, special attention is required when the treatment is used for dairy chilling water systems with copper or carbon steel parts.
Megahed et al. [92,93] studied the effect of gaseous-ozone and aqueous-ozone (from 1 to 10 ppm) in commonly used devices of the dairy industry (plastic, nylon, rubber, and wood) contaminated by cattle manure-based pathogens. It was observed that aqueous-ozone treatment at a concentration of 4 ppm or greater reduced bacterial load below detectable limits within 2 min of exposure predominantly on the plastic surface, while other surfaces were largely decontaminated after 4 min of treatment. Gaseous O3 cannot be an alternative to aqueous O3 in reducing the manure-based pathogens to a safe level, particularly in complex environments. The results obtained are in line with different previous studies [94].
It is important to consider the contamination of surfaces by spoilage bacteria, like Pseudomonas spp. and pathogenic bacteria, like Listeria monocytogenes and Salmonella spp., that lead to recurrent food contamination, with problems related to the shelf life and safety of dairy products.
Biofilm development in food processing facilities is prevented by using common chemical sanitizers, but their use has some disadvantages, such as damaging environmental impacts and harmful consequences for human health [52]. The use of ozone could be a promising technique to prevent spoilage or pathogenic bacteria contamination [22].
Shelobolina et al. [95] studied the effect of dissolved ozone (5 ppm for 20 min) on Pseudomonas biofilm on various surfaces, and they observed that ozone can be effective. Biofilms on plastic materials showed inactivation effects, such as for glass, while biofilms grown on ceramics were more difficult to inactivate. Therefore, it is important to use non-porous materials in industrial and clinical settings. It has also been demonstrated that ozone can have an antimicrobial effect in association with other technologies: for example, ozone water and hydrogen peroxide solution were effective on P. fluorescens biofilm.
A sequential treatment with 1.0 and 1.7 mg L−1 of ozone, followed by 0.8 and 1.1% of hydrogen peroxide, showed synergistic disinfection effects [96].
In a study of a cheese production plant, the gaseous ozonation of 2 ppm was provided during a weekend for 15 min (first treatment) and for 120 min (second treatment), when staff were not present. Testing for L. monocytogenes was carried out for a total of 360 environmental samples, over a period of 12 months, in 15 areas before and 15 areas after ozonation: there was a significant reduction in L. monocytogenes isolations from 15.0% in pre-zoning samples to 1.67% in post-ozonation samples in all areas, to include the ozonation regime in the hygiene-health program. No negative effects of ozonation treatment were noted on the surfaces and equipment [97].
Instead, Shao et al. [98] studied the effect of ozone water on mature S. aureus and Salmonella spp. biofilm on stainless steel surfaces, using acidic electrolyzed water, ozone water, or ultrasound (40 kHz) alone, and combinations of ultrasound and disinfectants. They detected less than 0.8 Log CFU (colony-forming units) cm−2 of cells reduction in biofilm exposed to ozonized water (16 mg L−1) for 20 min. Only in a few studies was the efficacy of ozone explored against biofilms formed by bacteria belonging to the Salmonella genus [99].
Indoor air in the dairy industry constitutes a source or a vehicle of microbial contamination, causing food safety and product shelf-life problems. Masotti et al. [100] monitored the air microbial load in the dairy plant and evaluated the impact of air disinfection through ozonation or chemical aerosolization by hydrogen peroxide. Ozonated air had a constant flow rate of 40 L min−1, and it generated 1.5 g ozone per hour (11–12 p.m. and 1–3 a.m. from Friday to Sunday). Hydrogen peroxide aerosolization was realized producing particles in the range of 5–15 μm at a concentration of 5–15% for 16 and 20 min. Both techniques were effective against airborne microorganisms.
In the treatment of raw milk, the use of ozone is debated: in fact, in contrast to the traditional thermal pasteurization process, ozone treatment minimizes the loss of the nutritional properties of milk, but it does not always show a total inactivating capacity on microorganisms [101] ( ).
Torlak and Sert [102] studied ozone treatment to eliminate Cronobacter sakazaki (responsible for fatal infections in infants) from milk powder. They treated both whole and skimmed milk with different results: for the skimmed milk, the initial levels of Cronobacter were reduced by 2.71 and 3.28 log, following an exposure of 120 min at the concentration of 2.8 mg L−1 and 5.3 mg L−1, and the whole milk sample showed a smaller logarithmic reduction.
Rojek et al. [103] used pressurized ozone (5–35 mg L−1 for 5–25 min) to safeguard skim milk by diminishing its microbial masses, and this treatment looked foremost to decrease the number of psychrotrophs by more than 99%.
Sheelamary and Muthukumar [104] totally wiped out Listeria monocytogenes from both crude and marked milk tests through gaseous ozonation, with a mean viable count of 5.5 and 5.7 log10 CFU mL−1, respectively. The results showed complete removal of Listeria monocytogenes after 15 min of treatments with a controlled flow rate of 0.5 m L−1 of oxygen (0.2 g h−1 of ozone).
Cavalcante et al. [101] evaluated in crude milk that ozone gas at 1.5 mg L−1 for 15 min was found to decrease bacterial as Listeria monocytogenes, Enterobacteriaceae, and Staphylococcus, and contagious checks by up to 1 log10 cycle.
A study by Alsager et al. [105] analyzed the decomposition of various antibiotic compounds in milk samples by the ozonation process. They observed that increasing concentration of gas ozone (75 mg L−1) caused a 95% reduction in antibiotics in milk samples.
This was confirmed by Liu et al. [106]: antibiotic residues in pasteurized milk reach tolerance levels by O3 in a vortex reactor (2.16, 4.54, and 6.12 mg h−1), and no antimicrobial activity was detected in the milk treated. The results showed that post-ozonation of 400 mL of 5.52 μM various antibiotics for 20–40 min, their residue concentrations (50.8–84.1 μg L−1) satisfy the relevant maximum residue limits. Therefore, antibiotic residues in milk arrive at tolerance levels by O3 in a vortex reactor. Increasing the input O3 concentration, O3 flow rate, and temperature can accelerate the degradation and shorten the time to meet the relevant MRLs.
de Oliveira Souza et al. [107] studied the effect of gaseous ozone on the inactivation of Escherichia coli O157:H7 inoculated on an organic substrate and the efficacy of ozonated water in controlling the pathogen. It was inoculated in milk with different compositions: unhomogenized whole milk (UHWM), homogenized whole milk (HWM), skim milk (SM), lactose-free skim milk (LFSM), and lactose-free whole milk (LFWM), which were sterilized by boiling in the laboratory to guarantee the elimination of microorganisms and cooled before inoculation. Milk was utilized only as a substrate, contemplating the differences in fat and lactose contents. The same procedures were also conducted in distilled water for comparison. Escherichia coli O157:H7 was inoculated in different ozonated water treatments (35 and 45 mg L−1 for 0, 5, 15, and 25 min). In a second experiment, the water was ozonated at 45 mg L−1 for 15 min. E. coli O157:H7 was exposed for 5 min to the ozonated water immediately after the treatment, and after storage for 0.5, 1.0, 1.5, 3.0, and 24 h at 8 °C. The results showed that in lactose-free homogenized skim milk, was observed a reduction of 1.5 log cycles for ozonation periods of 25 min at the concentrations tested. Overall, ozonated water was effective in all treatments, but the efficiency of ozone is influenced by the composition of the organic substrates. The refrigerated ozonated water put in storage for up to 24 h was effective to control E. coli O157:H7.
The degradation of mycotoxins and aflatoxins is also possible through the use of ozone [108]. As reported by Ismail et al. [109] when milk samples were exposed to gas ozone (80 mg min−1 for 5 min), and aflatoxins were reduced by 50%.
Khoori et al. [110] studied the synergistic effect of ozonation (9.99 mg min−1), UV light radiation (4.99 J cm−2), and pulsed electric field processes (13.15 µs) on the decrease of aflatoxin in probiotic milk. Data showed an effect in reducing the levels of aflatoxins (total and M1) in acidophilus milk samples. Additionally, the bacterial viability was reduced through these methods, with a level of Lactobacillus acidophilus in the final product of 106 CFU g−1.
Sert and Mercan [111] studied the effect of ozone treatment (OT) against milk concentrate (MC) and whey concentrate (WC), evaluating the physicochemical and textural properties and the effect on the microbial, antibiotic, and aflatoxin content. The ozone was applied to MC and WC samples for 0 (control), 5, 10, 15, 30, and 60 min. The treatment was performed as previously described [112], with some changes. Ozone treatment was conducted at 20 ± 2 °C. An ozone generator with a capacity of 3.5 g ozone h−1 was used. The results showed that OT (60 min) reached 18.9 and 9.9% degradation of the aflatoxin M1 in MC and WC, respectively. It caused the degradation of β–lactam and tetracycline in concentrates. The volume mean diameter of MC was increased by the ozone, while for WC it was decreased. In both MC and WC samples, the ozone treatment was successful in decreasing the microbial load. For MC samples, the coliform decreased from 3.19 ± 0.04 log CFU g−1 at time 0 to 2.37 ± 0.06 log CFU g−1 at time 60. For WC samples this decreased from 2.79 ± 0.06 log CFU g−1 to 2.04 ± 0.06 log CFU g−1. For MC samples, Enterobacteriaceae decreased from 3.33 ± 0.04 log CFU g−1 at time 0 to 2.60 ± 0.05 log CFU g−1 at time 60. For WC samples, this decreased from 3.17 ± 0.05 log CFU g−1 to 2.54 ± 0.06 log CFU g−1. For MC samples, the staphylococci decreased from 2.65 ± 0.08 log CFU g−1 at time 0 to 1.89 ± 0.06 log CFU g−1 at time 60. For WC samples, this decreased from 2.52 ± 0.03 log CFU g−1 to non-detectable. For MC samples, the total mesophilic aerobic bacteria decreased from 3.39 ± 0.08 log CFU g−1 at time 0 to 2.93 ± 0.08 log CFU g−1 at time 60. For WC samples, this decreased from 2.93 ± 0.06 log CFU g−1 to 2.35 ± 0.07 log CFU g−1. For MC samples, the yeast and mold decreased from 2.75 ± 0.07 log CFU g−1 at time 0 to non-detectable at time 60. For WC samples from 2.36 ± 0.06 log CFU g−1 to non-detectable ( ).
Open in a separate windowIn the dairy industry, water is used in a wide range of uses, generating significant volumes of wastewater rich in organic matter. It is currently purified with physicochemical and biological methods. Many studies have evaluated the possibility of using ozone for their treatment ( ), with the aim of recovering and reusing water [113,114,115,116].
Làszlò et al. [117] confirmed the ability of ozone (30 mg dm−3) to reduce the content of organic pollutants in wastewater at 25 °C for 5 min; thanks to its microflocculation effect, in fact, the effectiveness of the following nanofiltration phase increases and, consequently, the reduction of COD is performed; in addition, there is a 40% increase in the biodegradability of nanofiltration residues.
Additionally, dos Santos Pereira et al. [118] investigated the degradation of organic matter in a synthetic dairy wastewater. In this study, dairy wastewater has a chemical oxygen demand of 2.3 g L−1, and they treated this wastewater through an oxidation process using ozonation combined with hydrogen peroxide (30% w/w, [H2O2] = 9.007 mol L−1 and density of 1.1 g mL−1) and catalyzed by manganese (Mn2+) in alkaline conditions (MnSO4.H2O, 98% of purity). It was observed that the optimal condition for the ozonation catalyzed by manganese at alkaline medium (chemical oxygen demand removal of 69.4%) can be obtained at pH 10.2 and Mn2+ concentration of 1.71 g L−1, with COD removals above 60%.
In the work of Li et al. [119], it was observed that Mn-Fe-Ce/γ-Al2O3 can be used for catalytic ozonation of dairy farm wastewater, with efficient treatment results. The biodegradability of wastewater was drastically improved after 20 min of reaction time with Mn-Fe-Ce/γ-Al2O3 catalyst, which has a dosage of 12.5 mg·L−1·min−1 of ozone. The pH value was 9 and the catalyst dosage was 15 g·L−1. The mechanism involves the conversion of refractory color-developing organics into small-molecule organics that can be biodegraded. The results showed that COD removal ratio of dairy farming wastewater can reach 48.9%. The BOD/COD increased from 0.21 to 0.54. Therefore, it has been observed that as the catalyst dosage increases (0–25 g/L), the chemical oxygen demand decreases.
Recently, Zhu et al. [120] studied the effect of ozone on undiluted dairy farm liquid digestate that contains high levels of organic matters, chromaticity, and total ammonia nitrogen (TAN), resulting in inhibition of microalgal growth. Ozone greatly promoted microalgal growth, but it did not clearly reduce pollutants. After cascade pretreatment, TAN, total nitrogen (TN), COD, and chromaticity were reduced by 80.2%, 75.4%, 20.6%, and 75.8%, respectively.
In the work of Chen et al. [121], dairy wastewater was pre-treated with ultrasound (US 200 W), ozone (4.2 mg O3 L−1), and US combined with ozone (US/ozone) to study the fate of enteric indicator bacteria and antibiotic resistance genes (ARGs), and anaerobic digestion (AD). All pre-treatments were performed for 10 min, 20 min, and 30 min, respectively. US/ozone pre-treatment was effective in the inactivation of enteric indicator bacteria. Total coliforms and enterococci were reduced by 99% and 92% after 30 min US/ozone pre-treatment. Together, pre-treatment and AD clearly inhibited the enrichment of ARGs in relative abundance.
Chang et al. [122] designed a continuous type O3 treatment system (43.26, 87.40, and 132.46 mg L−1) to eliminate pathogens such as Salmonella Typhimurium and Escherichia coli O157: H7 in liquid dairy waste, and they observed that the ozone treatment reduced the amount of E. coli O157: H7 and of S. typhimurium, and the reductions increased with the exposure time, particularly at 87.40 or 132.46 mg·L−1. These results were confirmed by Choi et al. [123].
Many studies demonstrate the effectiveness of ozone in preventing the growth of mold in maturing environments against cheese mites ( ).
Gibson et al. [124] demonstrated the bacteriostatic effect by applying two different concentrations of ozone in the ripening phase of Cheddar cheese, verifying its effectiveness both at concentrations of 3–10 ppm for 30 days and at those of 0.2–0.3 ppm for 63 days with a difference of 6 percentage points in favor of the highest concentrations. Gabriel ‘yants’ et al. [125] confirmed these observations: the application of ozone under refrigerated conditions on Russian and Swiss-type cheese prevented mold growth for four months without damaging the sensory properties and chemical composition, while growth was observed on the control sample already after one month of storage. They stored the cheeses under refrigeration (2–4 °C, 85–90% RH) with or without ozonation of the air in the room. Periodic treatments were undertaken with concentrations of 2.5–3.5 ppm of gaseous ozone for 4 h at 2- to 3-day intervals, preventing mold growth on packaging materials for up to 4 months.
Other studies verify the effectiveness of ozone against the bacterial population: Morandi et al. [71] applied 4 ppm of gaseous ozone for 8 min at different stages of maturation and showed the effectiveness in controlling Listeria monocytogenes (artificially surface-inoculated up to 103 CFU g−1) on Ricotta Salata di Pecora (below 10 CFU g−1) and, limited to the first days of maturation on Gorgonzola PDO (Protected Designation of Origin) and Taleggio PDO, the effect was a complete elimination; Cavalcante et al. [126] proposed treatments with ozonated water (2 mg L−1 for 1–2 min) for washing Minas Frescal cheese during storage, highlighting, once again, the ability to reduce the initial microbial load (by approximately 2 log10 cycles) without damaging the sensory and physical–chemical properties of the product. However, the treatment did not show efficacy in controlling the growth rate of the surviving microflora.
Serra et al. [127] have shown the effectiveness of ozone treatment against the fungal spores that colonize the maturing rooms, but they have also shown that the treatment may not eliminate the molds present on the surface of the cheeses. They studied the effect of ozone to reduce molds in a cheese-ripening room (3500 m3) with a temperature of 5 ± 1 °C and a relative humidity of >80%. The efficacy of this treatment was evaluated in air and on surfaces through sampling on a weekly basis over a period of 3 months. The results demonstrated that ozonation decreased the viable airborne mold load, but not on surfaces, resulting in a 10-fold reduction compared with the level observed for the control. The mold genera usually isolated in the air were Penicillium, Cladosporium, and Aspergillus (89.9% of the mold isolates). They used 8 g h−1 as the rate of ozone generation.
In their study, Guzzon et al. [128] explored the microbiota of the red–brown defect in smear-ripened cheese and how to prevent it using different cleaning systems. Red–brown pigmentation can occasionally form in this type of cheese, such as Fontina, during the ripening process due to an over-development of the typical microbiota present on the rind. The microbiota of the shelf had a role in this defect in cheese. Different systems were tested for cleaning the wooden shelves (WS): washing with hot water and ozone treatment. The results showed that Actinobacteria, dominant on the WS, indicated to be responsible for the red–brown pigmentation; they were also in traces in the defected samples. Galactomyces and Debaryomyces were the main species for the yeast population, with Debaryomyces as the most dominant species on the shelves used during the maturation of the red–brown defective cheese. Even if the hot water treatment decreased the microbial load of shelves, only the use of ozone guaranteed a total elimination of yeast and bacteria, with no red–brown defect on the cheese rind. The ozone treatment (OT) was done using ozone in two different forms: gaseous (dry) or water-dissolved (wet). Therefore, washing plus ozone treatment ensures that the WS will be safe for the next ripening process. After the study, some trained experts reported that the wheels ripened on shelves cleaned by wet ozone treatment did not have the red–brown defect and no difference in cheese color, taste, and texture.
In the study of Alexopoulos et al. [129], an ozone stream (2.5–3 ppm) for 0, 10, 30, and 60 s was used on the surface of freshly filled yogurt cups (240 g) before storage for the development of the curd (24 h) to prevent cross-contamination from spoilage airborne microorganisms. Additionally, the brine solution was bubbled with ozone at different times and applied for the ripening of white (feta type-400 g) cheese. Ozone gas was supplied for 0, 10, 20, and 30 min to the brine, where the cheese sample was then left for ripening for a period of 2 months. Products were monitored for microbial load and tested for their sensorial characteristics. In ozonated yogurt samples, data showed a reduction in mold counts of about 0.6 Log CFU g−1 (25.1%) by the end of the monitoring period against the control samples. In white cheese matured with ozonated brine (1.3 mg L−1 O3, NaCl 5%), the treatment for two months decreased some of the mold load without offering advantages over the use of traditional brine (NaCl 7%). However, some alterations in the sensorial quality were observed, perhaps due to the organic load of the brine that soon neutralizes ozone. For the cheese samples ripened in 60 min ozonated brine, there was a reduction in the attribute of flavor, according to the global acceptance. Therefore, factors of time and concentration of ozone are essential, and they must be configured.
The efficacy of ozone is non-selective towards harmful or useful bacteria. It becomes challenging when lactic acid bacteria (LAB—one of the most significant groups of probiotic organisms), commonly used in fermented dairy products, is destroyed by the application of ozone. In this work, LAB was present in all yogurt samples in an adequate number (S. thermophillus 9.1 ± 0.5 Log CFU g−1 and L. delbrueckii ssp. bulgaricus 8.8 ± 0.6 Log CFU g−1). Although a drastic decline was observed after 50 days of storage by the end of the monitoring time, nevertheless no statistical differences were observed between the control and the ozonated samples. Therefore, ozone did not affect good microflora, maintaining the functional character of the yogurt, with LAB values higher than 7 Log CFU g−1 for a period of 70 days.
Segat et al. [130] used ozone treatment to reduce the microbial spoilage load on “mozzarella” cheese, concluding that the cooling water treatment (15 °C) with ozone (2 mg L−1) improves the microbiological qualities, positively affecting the shelf-life of the products. The results showed that “mozzarella” cheese samples that were cooled in water and pretreated were characterized by low microbial counts as compared to control samples cooled with non-ozonated water, following 21 d of storage (by 3.58 and 6.09 log10 CFU g−1 lower total plate counts and Pseudomonas spp. counts, respectively, compared to control samples).
Sert et al. [112] showed the effect of ozone on butter samples in their work, considering that the treatment can be applied to produce butter as a non-thermal technology. Raw cream was ozone-treated for 5 (OT-5), 15 (OT-15), 30 (OT-30), and 60 (OT-60) min. The ozone application was conducted at 8 ± 2 °C and, after the treatment, the churning was carried out at 4 ± 2 °C. Then, the samples were packaged and stored at 4 °C for 24 h. The ozone treatment increased the firmness, consistency, and fat particle size values of samples. Instead, it decreased the color parameter b value, so the yellowness of butter. OT-60 caused 2.01 log reduction in Staphylococci, and over 15 min, the ozone completely inactivated Salmonella and yeast-mold, while coliform was not detected in OT-30 and OT-60. The results showed a higher microbiological quality in butter from raw cream with ozone treatment. Instead, OT decreased the oxidative stability of butter and up to 15 min increased the spreadability characteristic of the sample.
Panebianco et al. [131] studied the effect of ozone on Listeria monocytogenes contamination and the resident microbiota on the Gorgonzola cheese rind. Concentrations of 2 and 4 ppm for 10 min were used against L. monocytogenes and resident microbiota of cheese rind samples stored at 4 °C for 63 days. Results showed that in ozonized rinds the final loads of L. monocytogenes were ~1 log CFU g−1 higher than controls. No significant differences were found for the other microbial determinations and the resident microbiota between ozonated and control samples.
Recently, Tabla and Roa [132] studied the effect of gaseous ozone in soft cheese ripening, in particular its effect on the rind microorganisms, evaluating the sensorial quality. The experiment was conducted at different production scales. On a pilot plant scale, the samples were inoculated with Mucor plumbeus, Kluyveromyces marxianus, and Pseudomonas fluorescens, while on an industrial scale they evaluated the effect of ozone on naturally present microorganisms. For the experiment at pilot plant scale, cheeses were surface-inoculated after salting to a final concentration of 105 spores g−1 of rind for Mucor plumbeus and 107 CFU g−1 of rind for Kluyveromyces marxianus and Pseudomonas fluorescens. The ripening was conducted in 8 m3 ripening rooms for 30 days with gaseous ozone at concentrations of 0, 2, and 6 mg m−3. For the experiment at dairy plant scale, a total of 30 cheeses with 15 days of ripening were subjected to maturation with gas ozone at concentrations of 2 mg m−3 for 45 days. The results showed that gaseous ozone treatments had a significant fungistatic effect on cheese surface molds for pilot and industrial scale, but its effect on yeast growth was only observed at pilot plant scale, and for Pseudomonas ssp., the ozone concentrations used did not affect it. For the sensory quality of the cheese, its typicality was not influenced by gas ozone, and the ozonized cheeses had significantly higher scores than the control samples for the appearance of the rind and color, preventing cheese rind discoloration.
In ripened cheeses, an important problem is the presence of mites that live and grow on the rind of the product. Some studies have shown that the most represented species in the dairy industry are Acarus siro, Tyrophagus casei, T. longior, T. palmarum, and T. putrescentiae [36,37,38]. The ripening environment is optimal for the proliferation of mites, above all by the high relative humidity and temperatures that hover around 12–13 °C; it has, in fact, been seen that at temperatures of °C these parasites do not survive, but there is also an excessive prolongation of the ripening times which makes them inapplicable [133], and in most cases the presence of mites in environments of maturation is associated with important economic losses. The intake of mites can cause the appearance of symptoms ranging from dermatitis to allergic rhinitis and asthma, up to gastro-intestinal problems. It has been seen that water at a temperature higher than 71.1 °C [36] eliminates the mites in a few seconds; moreover, there are several effective chemical treatments, and the most used substances are organophosphate compounds. However, chemical products leave residues and, in some cases, such as that of methyl bromide, they have been banned, as they have harmful effects on human health. For this reason, it is essential that research identifies alternative control methods. Therefore, the interest in the methods of sanitizing the maturing environments with ozone is growing. The effectiveness of ozone in killing mites has been ascertained thanks to a few studies, even in dairy environments. For example, Pirani [59] verified the effectiveness of ozone treatment for the control of mite infestations on matured speck products.
In a new study, Pecorino cheese samples were treated for 150 days overnight with gaseous ozone (200 and 300 ppb, 12 °C, and 85% R.H. for 8 h per day). It was observed that, starting from 25 days of storage, 200 ppb of ozone reduced the growth of mites and significantly reduced bacteria, molds, and yeast count, starting from 75 days of storage. Furthermore, it was observed that no significant differences were shown between the control samples and ozone treatment at 200 ppb for the centesimal composition of Pecorino cheese. However, treatments with ozone 300 ppb contained microbiological and mite growth but did not have the same positive impact on some aspects of overall quality [134].
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