Inactivation Method of Rotavirus by Multiple Treatment of Chemical Sanitizers and Gamma Irradiation on Food and Food Contact Surfaces

Abstract

The present invention relates to a combined treatment method of a chemical disinfectant and gamma ray irradiation for rotavirus inactivation. When the combined treatment method is applied to food or a food contact surface, it not only exhibits an excellent rotavirus inactivation effect but also shows a significant synergistic effect in rotavirus inactivation efficiency compared to each single treatment. Furthermore, by confirming through color and quality analysis of changes in food quality that there is no change in visual or textural appearance compared to before treatment, it can be usefully employed as a method that can be safely applied to fresh convenience foods and the food industry.

Description

Technology Field

[0001] The present invention relates to a method for effectively inactivating rotavirus on food and surfaces in contact with food through a combined treatment of a chemical disinfectant and gamma ray irradiation. Background Technology

[0003] Rotavirus (RV) is one of the leading causes of severe foodborne gastroenteritis and is particularly common in infants and young children. This places a significant health burden on developing countries. Despite the introduction of vaccines, approximately 128,500 children under the age of five died from rotavirus gastroenteritis in 2016, accounting for 28.8% of all diarrhea-related deaths in this age group worldwide (Troeger et al., 2018). Additionally, according to the Korea Centers for Disease Control and Prevention (KDCA), 73 cases of rotavirus infection were reported in schools and hospitals in Korea between 2021 and 2022 (KDCA, 2022). Individuals infected with rotavirus exhibit symptoms such as severe watery diarrhea, vomiting, fever, and abdominal pain (CDC, 2024).

[0004] Rotavirus is an unenveloped virus belonging to the family Reoviridae. The rotavirus genome consists of 11 double-stranded RNA segments and is surrounded by three layers of concentric proteins (capsids). It is generally classified into several serogroups (A–E, F, G), among which RV-A is the most common type infecting humans (Matthijnssens & Van Ranst, 2012; Wentzel et al., 2013). In particular, the Wa strain of RV-A, classified as G1P, is the most dominant and accounts for the majority of rotavirus infections worldwide (Shintani et al., 2012).

[0005] Rotavirus is primarily transmitted via the fecal-oral route and can be transmitted through close contact between people or contaminated environmental surfaces. Additionally, it exists stably in various environments and its rate of transmission varies greatly.

[0006] In addition, rotavirus can survive in sewage and may be transmitted to humans through crops irrigated with sewage (Badawy et al., 1985). The virus has been detected in various fresh vegetables, including celery, spinach, cherries, and strawberries in Mexico City (Parada-Fabian et al., 2016; Quiroz-Santiago et al., 2014), tomatoes in South Africa (van Zyl et al., 2006), sweet potatoes, carrots, radishes, and potatoes in Korea (Park et al., 2023), and lettuce in the United States (Aw et al., 2016), suggesting that enteric viruses, including rotavirus, can be transmitted through vegetables.

[0007] With growing interest in health, the consumption of fresh produce is on the rise. Fresh produce is generally consumed without the use of preservatives or antimicrobial agents, or after minimal processing. However, fresh produce has frequently been identified as a vector for the transmission of infectious microorganisms.

[0008] Contamination of agricultural products can occur before harvest when irrigated with contaminated water, and after harvest, it can lead to cross-contamination through contact with infected workers or contaminated equipment. While water is a widely used washing medium to remove soil and dust from the surfaces of fruits and vegetables, it is often insufficient to effectively remove viruses. Therefore, it is important to utilize appropriate and effective disinfectants to reduce the risk of viral infection in fresh vegetables.

[0009] Disinfectants generally inactivate viruses by damaging or degrading their capsid proteins and encapsulated genomes. Among these, chlorine-based disinfectants are widely used because they are inexpensive, highly effective, and provide residual bactericidal effects. Chlorine dioxide (ClO2) has been considered an alternative to sodium hypochlorite (NaOCl) because it does not react with humic substances to form halogenated disinfection byproducts. Furthermore, short treatment times using low concentrations of ClO2 are effective against various microbial pathogens over a wide pH range of 3 to 7.

[0010] Peracetic acid (PAA) is another powerful disinfectant with broad-spectrum antimicrobial activity, characterized by strong bactericidal ability and low pH dependence. Additionally, PAA is considered an environmentally friendly disinfectant because it decomposes into acetic acid, water, and oxygen, minimizing its impact on agricultural ecosystems. Previous studies have confirmed that PAA can effectively inhibit the proliferation of foodborne viruses when applied to various foods.

[0011] Food irradiation is a technology that processes and preserves food by exposing it to specific levels of ionizing radiation, such as gamma rays, electron beams, and X-rays. This method is generally considered safe and does not pose a significant risk to human health. The United Nations Food and Agriculture Organization (FAO), the International Atomic Energy Agency (IAEA), and the World Health Organization (WHO) have reported that irradiating food with radiation doses of less than 10 kGy does not cause toxicological or nutritional problems (Lung et al., 2015).

[0012] However, it is known that there is a correlation between the neutralization of reactive oxygen species (ROS) generated during the irradiation process and the reduction of antioxidant components such as vitamin C (De Figueiredo et al., 2014). Food irradiation is classified into low dose (maximum 1 kGy), medium dose (1–10 kGy), and high dose (10 kGy or more) depending on the radiation dose; this is an environmentally friendly method that does not use chemicals and does not cause pollution. Gamma rays are emitted during the negative beta decay of radioactive isotopes. Currently, Cobalt-60 and Cesium-137 are the gamma ray sources approved for use in food irradiation.

[0013] Gamma rays have the potential to completely penetrate food and inactivate not only surface pathogens but also pathogens present within the food matrix. In particular, they are effective for fresh produce such as leafy vegetables, which are difficult to heat treat. According to a study by Bidawid et al. (2000), a gamma irradiation dose of 2.7–3.0 kGy is required to reduce the hepatitis A virus (HAV) population in fruits and vegetables by more than 90%.

[0014] Related prior art includes Korean published patent 10-2023-0165410 (Method for controlling norovirus by combined treatment with ultraviolet irradiation and PAA) and Korean published patent 10-2024-0157439 (Method for removing coronavirus by combined treatment with ultraviolet irradiation and ClO2), which disclose a method of combining chemical disinfectants and ultraviolet rays. However, no literature has been reported disclosing a combined treatment of chemical disinfectants and gamma ray irradiation targeting rotavirus.

[0015] However, current measures to ensure food safety are primarily optimized for inactivating bacterial growth, which may not be effective for controlling viruses that can persist on food or food contact surfaces (FCS) for several days without losing their infectivity. Furthermore, as mentioned above, chemical methods using chlorine-based compounds are not environmentally friendly, can generate environmentally toxic residues, and cause corrosion on the surfaces of applied equipment, processing equipment, or machinery; meanwhile, physical methods can alter the physical composition and flavor. Additionally, some physical methods, such as heat treatment, cannot be used on fresh convenience foods.

[0016] Accordingly, while searching for a method to inactivate rotavirus that can be safely used in the food industry, the inventors derived a combined treatment condition of a chemical disinfectant and gamma irradiation that can effectively inactivate rotavirus on food or food contact surfaces without degrading the quality of the food. Furthermore, the inventors completed the invention by confirming that the combined treatment method presented in this invention not only increases the inactivation efficiency of rotavirus compared to each single treatment, but also exhibits a significant synergistic effect when the two treatments are performed in combination. The problem to be solved

[0018] The present invention relates to a complex treatment method for rotavirus inactivation and a method for removing rotavirus using said method, which exhibits an excellent rotavirus inactivation effect and does not impair the hardness and quality of food, and can be applied to fresh convenience foods and the food industry as a safe method for inactivating rotavirus. means of solving the problem

[0020] In order to achieve the above objective,

[0021] The present invention provides a complex treatment method for rotavirus inactivation.

[0022] The present invention provides a method for removing rotavirus using a complex treatment method. Effects of the invention

[0024] When the combined treatment method of the chemical disinfectant and gamma ray irradiation of the present invention is applied to food or food contact surfaces, it not only exhibits an excellent rotavirus inactivation effect but also shows a significant synergistic effect in rotavirus inactivation efficiency compared to each single treatment and confirms that it does not impair the quality of food, so it can be applied to fresh convenience foods and the food industry as a safe method for inactivating rotavirus. Brief explanation of the drawing

[0026] Figure 1a shows the rotavirus inactivation effect of chlorine dioxide (ClO2) alone on rotavirus in a virus suspension. Figure 1b shows the rotavirus inactivation effect of peracetic acid (PAA) alone on rotavirus in a virus suspension. Figure 2a shows the rotavirus inactivation effect of chlorine dioxide (ClO2) alone on a food contact surface (stainless steel, SS). Figure 2b shows the rotavirus inactivation effect of peracetic acid (PAA) alone on a food contact surface (stainless steel, SS). Figure 3a shows the rotavirus inactivation effect of chlorine dioxide (ClO2) alone on a food surface (lettuce). Figure 3b shows the rotavirus inactivation effect of peracetic acid (PAA) alone on a food surface (lettuce). Figure 4a shows the rotavirus inactivation effect of gamma irradiation alone on rotavirus in a virus suspension. Figure 4b shows the rotavirus inactivation effect of gamma irradiation alone on a food surface (lettuce). Figure 5a shows the rotavirus inactivation effect of combined treatment with chlorine dioxide (ClO2) and gamma irradiation on a food surface (lettuce). Figure 5b shows the rotavirus inactivation effect of combined treatment with peracetic acid (PAA) and gamma irradiation on a food surface (lettuce). Specific details for implementing the invention

[0027] The present invention will be described in detail below.

[0028] The present invention provides a complex treatment method for rotavirus inactivation.

[0029] In the present invention, the composite treatment method

[0030] (i) a step of treating rotavirus (RV) with a chemical disinfectant; and

[0031] (ii) a step of irradiating the rotavirus treated with the chemical disinfectant with gamma rays; includes.

[0032] In the present invention, the chemical disinfectant is chlorine dioxide (ClO2) or peracetic acid (PAA).

[0033] In the present invention, the chlorine dioxide can be treated at a concentration of 5 to 50 ppm, preferably 20 to 40 ppm, and more preferably 30 ppm.

[0034] In the present invention, the peracetic acid can be treated at a concentration of 10 to 80 ppm, preferably 40 to 80 ppm, and more preferably 60 to 80 ppm.

[0035] In the present invention, the treatment time of the chemical disinfectant may be 1 to 5 minutes, preferably 1 to 3 minutes, more preferably 1 minute.

[0036] In the present invention, the gamma rays of step (ii) may be irradiated at a dose of 1.0 to 4.0 kGy, preferably 1.0 to 3.0 kGy, more preferably 1.0 to 2.0 kGy, and most preferably 1.0 to 1.5 kGy.

[0037] In the present invention, the rotavirus is generated on food or a food contact surface.

[0038] In the present invention, the food refers to vegetables, fish, or meat products distributed in a fresh state.

[0039] In the present invention, the vegetables include fresh vegetables that have not been physically or chemically processed and maintain their freshness, such as cut vegetables or chopped vegetables, and can be interpreted to include all unprocessed fruits or unprocessed agricultural products, grains, roots, stems, leaves, or fruits that can obtain an effect equivalent to that of fresh vegetables, in addition to such unprocessed vegetables.

[0040] In the present invention, the term "fish" refers to fish used as food, and in the present invention, it refers to aquatic products, which is a general term for edible animals and plants found in water, such as seas or rivers.

[0041] In the present invention, the meat refers to types of meat such as beef, pork, and chicken that are edible by humans, and the present invention includes all cut meat, packaged meat, raw milk, edible eggs, processed meat products, processed dairy products, and processed egg products.

[0042] In the present invention, the food contact surface may be tableware, utensils, packaging materials, cooking utensils or cooking facility equipment made of glass, metal, plastic, rubber, or paper, or surfaces thereof.

[0043] In the present invention, the surface in contact with the food is a surface of various materials that come into contact during food processing, and includes glass, stainless steel (SS), or plastic materials.

[0044] In the present invention, the stainless steel is a steel used in various fields ranging from building interior and exterior materials to kitchenware, industrial parts, automotive parts, and medical devices, and is an alloy made to prevent rusting by containing 12% or more of chromium. Various types of stainless steel products are also produced by adding other metals such as nickel, molybdenum, and nitrogen in addition to chromium, and as the added metal changes, the properties such as strength, ductility, heat resistance, and workability also change.

[0045] Polyethylene terephthalate (PET), polystyrene (PS), and polypropylene (PP) are representative plastic materials used for food containers.

[0046] The polyethylene terephthalate (PET) mentioned above is what we commonly refer to as PET bottles; it has high transparency and good thermal insulation. It is thermoplastic, lightweight, and odorless. Its excellent transparency makes it easy to check the freshness of food, and because the material is soft, it does not easily dent upon impact. However, it has the disadvantage of low heat resistance, so it cannot hold hot food.

[0047] The above polystyrene (PS) is a polymer synthesized by polymerizing styrene as a monomer. It is used for a wide variety of purposes, ranging from instant noodle containers to the exteriors of home appliances, and is also used in plastic model kits. Pure polystyrene is moderately hard, transparent, inexpensive, and has low reactivity, so it is used in the production of disposable Petri dishes instead of glass. While it has the advantage of being inexpensive and capable of mass production, it has the disadvantage of having low heat resistance and being weak against impact, making it difficult to hold hot food in many products made of this material.

[0048] The above polypropylene (PP) is produced by polymerizing propylene obtained from petroleum, like polyethylene, using a catalyst. It has a structure in which a methyl group (CH3) is attached to every other carbon in the polyethylene molecular chain, and it has a form with regularly arranged short branches. Polypropylene film has higher transparency and is slightly harder than polyethylene film. Its specific gravity is 0.92, making it the lightest among currently available plastics. Its melting temperature is also high at 135–160°C, and it has a wide range of applications. It has excellent heat resistance and is strong against impact, but it has the disadvantage of low transparency.

[0049] The present invention provides a method for removing rotavirus using a chemical disinfectant and a combined gamma ray irradiation treatment method.

[0050] In a specific embodiment of the present invention, the rotavirus inactivation effect by single treatment with a chemical disinfectant in rotavirus suspensions was confirmed. As a result, there was a significant difference between the reduction values ​​of rotavirus suspensions treated with 5, 10, and 15 ppm of ClO2 for 1 minute (0.74, 1.63, and 2.84, respectively) and 5 minutes (0.51, 1.58, and 3.22, respectively). It was confirmed that rotavirus was completely inactivated at a concentration of 20 ppm in both the 1-minute and 5-minute treatment groups. The detection limit was 1 log. 10It was PFU / mL (Fig. 1a). In the case of PAA, after treatment for 1 minute at concentrations of 20, 40, 60, 80, and 100 ppm, it showed decrease values ​​of 0.35, 0.41, 0.46, 0.54, and 1.79, respectively. When treated with PAA at a concentration of 120 ppm, the rotavirus titer was at the detection limit (1 log 10 It reached (PFU / mL). When PAA at 80 to 120 and 60 to 120 ppm was treated for 1 minute and 5 minutes, respectively, a significant difference (p<0.05) was observed (Fig. 1b).

[0051] As a result of confirming the rotavirus inactivation effect following disinfectant treatment (1 min and 5 min) on food contact surfaces (SS), with the exception of samples treated with 75 to 100 ppn of ClO2 for 1 min, the reduction values ​​of rotavirus increased significantly as the ClO2 concentration increased, reaching 0.10, 0.44, 1.00, 1.23, 2.38, and 4.15 after 1 min treatment, and 0.11, 0.59, 1.35, 2.31, and 4.08 after 5 min treatment (Fig. 2a). In the case of PPA, it increased significantly regardless of concentration and treatment time (Fig. 2b).

[0052] As a result of confirming the rotavirus inactivation effect of disinfectant treatment (1 min and 5 min) on food (lettuce), in the case of ClO2, the reduction values ​​of rotavirus increased as the concentration increased (at 1 min treatment: 0.17, 1.01, 1.17, 1.40, and 1.77; at 5 min treatment: 0.66, 0.96, 1.32, 1.49, and 1.79). However, there was no significant difference between the groups treated at concentrations of 20 and 40 ppm for 1 min (Fig. 3a). In the case of PAA, after 1 minute of treatment, decrease values ​​of 0.09, 0.27, 0.53, and 0.64 were observed at concentrations of 20, 40, 60, and 80 ppm, respectively, and after 5 minutes of treatment, decrease values ​​of 0.31, 0.39, 0.66, and 0.75 were observed at the same concentrations. No significant difference was observed according to treatment time (Fig. 3b).

[0053] As a result of confirming the rotavirus inactivation effect following disinfectant treatment (1 min and 5 min) on food contact surfaces (SS), with the exception of samples treated with 75 to 100 ppn of ClO2 for 1 min, the reduction values ​​of rotavirus increased significantly as the ClO2 concentration increased, reaching 0.10, 0.44, 1.00, 1.23, 2.38, and 4.15 after 1 min treatment, and 0.11, 0.59, 1.35, 2.31, and 4.08 after 5 min treatment (Fig. 2a). In the case of PPA, it increased significantly regardless of concentration and treatment time (Fig. 2b).

[0054] As a result of confirming the rotavirus inactivation effect of disinfectant treatment (1 min and 5 min) on food (lettuce), in the case of ClO2, the reduction values ​​of rotavirus increased as the concentration increased (at 1 min treatment: 0.17, 1.01, 1.17, 1.40, and 1.77; at 5 min treatment: 0.66, 0.96, 1.32, 1.49, and 1.79). However, there was no significant difference between the groups treated at concentrations of 20 and 40 ppm for 1 min (Fig. 3a). In the case of PAA, after 1 minute of treatment, decrease values ​​of 0.09, 0.27, 0.53, and 0.64 were observed at concentrations of 20, 40, 60, and 80 ppm, respectively, and after 5 minutes of treatment, decrease values ​​of 0.31, 0.39, 0.66, and 0.75 were observed at the same concentrations. No significant difference was observed according to treatment time (Fig. 3b).

[0055] As a result of confirming the rotavirus inactivation effect of gamma irradiation in suspensions, the initial virus titer of the untreated sample was 6.77log 10 It was PFU / mL. The rotavirus titer was 5.40 log after gamma irradiation at doses of 1, 3, 5, 7, and 10 kGy, respectively. 10 PFU / mL (1.33log 10 PFU / mL decrease), 4.79 log 10PFU / mL (1.94log 10 PFU / mL decrease), 4.11 log 10 PFU / mL (2.62log 10 PFU / mL decrease), 3.14 log 10 PFU / mL (3.59log 10 PFU / mL decrease), 2.06 log 10 PFU / mL (4.67log 10 As shown by a decrease in PFU / mL, rotavirus titer significantly decreased as the gamma irradiation dose increased (Fig. 4a).

[0056] As a result of confirming the rotavirus inactivation effect of gamma irradiation (1.0, 1.5, 2.0, 2.5, 3.0, 4.0 kGy) on food (lettuce), the rotavirus titer was 3.97 log with increasing gamma irradiation dose, respectively. 10 PFU / mL (0.30log 10 PFU / mL decrease), 2.92 log 10 PFU / mL (1.35log 10 PFU / mL decrease), 2.27 log 10 PFU / mL (2log 10 PFU / mL decrease), 1.80 log 10 PFU / mL (2.47log 10 It was shown as a decrease in PFU / mL). At 4 kGy, the U.S. FDA gamma irradiation standard for agricultural products, the rotavirus titer was at the detection limit (1.5 log). 10 It reached (PFU / mL). The reduction value of rotavirus in lettuce increased with increasing gamma irradiation dose, but while there was no difference in the average rotavirus titer observed at gamma irradiation doses of 1.0 to 1.5 kGy, a significant difference was confirmed at 1.5 to 4.0 kGy (Fig. 4b).

[0057] As a result of confirming the rotavirus inactivation effect of combined treatment with the chemical disinfectant ClO2 and gamma irradiation on food (lettuce), the reduction values ​​in rotavirus titers at a gamma irradiation dose of 1 kGy according to ClO2 concentrations (10, 20, 30, 40, and 50 ppm) were 0.65, 1.07, 1.65, 1.80, and 2.00 log, respectively. 10 It was expressed in PFU / mL. At a gamma irradiation dose of 1.5 kGy, the rotavirus reduction values ​​according to ClO2 concentrations (10, 20, 30, 40, and 50 ppm) were 0.80, 1.25, 1.85, 2.00, and 2.40 log, respectively. 10 It was found to be in PFU / mL (Fig. 5a and Table 1). Under the combined treatment conditions of ClO2 and gamma irradiation, it was confirmed that 1.5 kGy gamma irradiation after 30 ppm ClO2 treatment showed the highest synergistic effect.

[0058] As a result of confirming the rotavirus inactivation effect by combined treatment with the chemical disinfectant PAA and gamma irradiation on food (lettuce), the reduction values ​​of rotavirus titers at a gamma irradiation dose of 1 kGy were found to be 0.29, 0.38, 0.63, and 1.20 log10 PFU / mL depending on the PAA concentration (20, 40, 60, and 80 ppm), respectively. At a gamma irradiation dose of 1.5 kGy, the reduction values ​​of rotavirus titers at a gamma irradiation dose of 1.5 kGy were found to be 0.33, 0.78, 1.15, and 1.65 log10 PFU / mL depending on the PAA concentration (20, 40, 60, and 80 ppm), respectively, confirming a significant reduction (Fig. 5b and Table 2). Under the combined treatment conditions of PAA and gamma irradiation, it was confirmed that 1.5 kGy gamma irradiation after treatment with 80 ppm PAA showed the highest synergistic effect.

[0059] As a result of evaluating the physicochemical effects on food after combined treatment with chemical disinfectants and gamma irradiation, the hardness (g / cm²) of samples between the control group and the treatment group was compared. 2No significant differences were observed in the values ​​of ) and color (L*, a*, and b*) (Table 3). The overall color difference (△E*) in all samples was less than 1.2. It was confirmed that the quality characteristics of lettuce were not affected even at maximum concentrations and doses under all treatment conditions. Through this, it can be seen that the method of rotavirus inactivation by combined treatment of a chemical disinfectant and gamma irradiation according to the present invention can be very usefully utilized in the food industry.

[0060] The present invention will be explained in detail below through examples.

[0061] However, the following examples are merely specific illustrations of the present invention in one aspect, and the present invention is not limited to the following examples.

[0063] <Example 1> Experimental Preparation and Analysis Method

[0064] Host cells and viruses

[0065] The MA-104 cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). MA-104 cells were cultured in Dulbecco minimal medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, USA). Cells were cultured in a 5% CO2 incubator at 37°C for 75 cm². 2 The cells were dispensed into tissue culture flasks, and once the cells reached approximately 100% density, the cells in the flasks were subcultured every 3–4 days. First, the cell culture medium was aspirated, and then rinsed twice with Dulbecco Phosphate Buffered Saline (DPBS; Sigma-Aldrich). The flasks were incubated in a 5% CO2 incubator for 4 minutes, followed by centrifugation at 300 lb at 25°C for 5 minutes. The cell pellet was suspended in fresh FBS medium and 75 cm 2 It was inoculated into a flask and cultured.

[0066] Rotavirus (RV(Wa)) was obtained from ATCC. 75 cm 2 When the MA-104 cell monolayer in the flask reached 90-100% confluence, the growth medium was aspirated and removed, and the cells were washed with DPBS (pH 7.3). 3 mL of RV suspension was dispensed into the cells in the flask to infect them, and the virus was adsorbed by incubating for 90 minutes in a humidified incubator at 37°C with 5% CO2. Then, 7 mL of maintenance medium (DMEM, 44 mM sodium bicarbonate, 1% penicillin-streptomycin) was added to the flask, and the cells were incubated for 5 to 7 days in a 5% CO2 incubator at 37°C. When the cytopathic effect (CPE) reached 90% or higher, the virus-infected culture was subjected to three freeze-thaw cycles to induce cell lysis and release viral particles. The contents of the flask were centrifuged at 4000 lb for 10 minutes at 4°C, and the supernatant was filtered through a 0.2 μm pore size filter (Sartorius Stedim Biotech, Gottingen, Germany). The suspension was stored in a freezer at -80°C before use.

[0068] Preparation of food and food contact surfaces

[0069] Stainless steel (SS) was used as the food contact material. Specimens with a diameter of 1 cm were rinsed with distilled water and then immersed in 70% ethanol for at least one hour to remove residues. The specimens were dried on Whatman No. 4 filter paper, sterilized in an autoclave at 121°C for 15 minutes, and then placed in a drying oven until needed. To prepare the inoculum, a 160 μL soil load was prepared, consisting of 25 μL of 5% bovine serum albumin (BSA), 35 μL of 5% yeast extract, and 100 μL of 0.4% mucin. This was mixed with 340 μL of virus suspension (approx. 6–7 log 10A virus solution (stock) was prepared immediately before the experiment by mixing with PFU / mL. Subsequently, 50 μL of the prepared virus solution (stock) was applied to the center of the specimen and dried for 1 hour.

[0070] Fresh lettuce was purchased from a local market in Anseong, South Korea. The sample was washed at least three times with running tap water and distilled water to remove impurities and measured 2 x 2 x 0.2 cm. 3 Cut into squares. Each sample was dried in a laminar flow hood for 20 minutes and then exposed to UV light on both the front and back sides. Then, 100 μL of rotavirus (RV) suspension (approx. 6–7 log 10 After inoculating the sample with PFU / mL, it was placed on a clean bench for 1 hour (at 20-25℃) to allow the virus particles to dry and adhere.

[0072] Virus recovery and plaque analysis (Plaque assay)

[0073] 1X DMEM was used to neutralize the disinfectant, and 3% beef extract was used as the virus recovery solution. Samples that had undergone chemical or physical treatment were placed in a 50 mL conical tube with 2 mL of recovery solution, vortexed for 2 minutes, and then centrifuged at 5000 Hg for 10 minutes at 4°C. Subsequently, the supernatant was serially filtered using a 0.2 μm filter. The titer of each virus suspension was measured via plaque assay.

[0074] MA-104 cells were seeded into 12-well cell culture plates and cultured for 2 days in a 5% CO2 incubator (37°C) with 1.5 mL of cell maintenance medium to increase cell density to 90%. Subsequently, diluted rotavirus (RV) was infecting each well, followed by incubation in a 5% CO2 incubator at 37°C. After incubating for 90 minutes for virus adsorption, 1.5 mL of medium mixed with 2% agarose and 2X DMEM was added to each well, and the cells were cultured for 5 days until a cytopathic effect (CPE) appeared. Afterward, 1 mL of 3.7% formaldehyde solution was added to each cell for fixation; after 4 hours, the formaldehyde was removed using tap water, and each well was stained with 1% crystal violet solution for at least 1 hour to determine the Plaque-Forming Unit (PFU) titer; Plaque-forming units were measured, and the reduction rate of rotavirus (log 10 The reduction was calculated as follows:

[0075] RV log 10 reduction = log 10 (Control group RV titer) - log 10 (RV titer after treatment)

[0077] Quality measurement

[0078] The color of each food sample treated with a combination of disinfectant and gamma rays was analyzed using a colorimeter (UltraScan PRO) and measured at a minimum of six points. The average values ​​of the measured colors were expressed as brightness (L*: + is bright, - is dark), redness (a*: + is red, - is green), and yellowness (b*: + is yellow, - is blue) according to CIE standards. In addition, the total color difference (△E) was calculated using the formula △E = √(△L² + △a² + △b²). The colorimeter was standardized prior to use, and the reference values ​​were L*: 99.95, a*: -0.14, and b*: 0.06.

[0079] The texture of the food samples was measured using a TA-XT Express texture analyzer (TAHDi / 500), and texture profile analysis (TPA test) was performed by compressing twice. The experimental conditions were set using an SMS P / 2N probe, with a pre-test speed of 1.0 mm / s, a test speed of 1.0 mm / s, a post-test speed of 5.0 mm / s, and a trigger force of 5.0 g. Texture analysis was performed using Exponent Lite Express software (version 4.0.8.0).

[0081] Statistical analysis

[0082] All experiments for single or combined treatments were performed using triplicate samples. Data analysis was conducted using IBM SPSS Statistics version 26.0 (IBM Corp., Chicago, IL, USA). Viral titers were calculated using the logarithmic function (log 10 Values ​​were expressed as mean ± standard deviation in PFU / mL. Statistical significance among treatment variables was assessed using Duncan's multiple-range test at a P < 0.05 level following a one-way analysis of variance (ANOVA).

[0084] <Example 2> Confirmation of Rotavirus Inactivation Effect by Single Treatment with Chemical Disinfectant

[0085] The chemical disinfectants used were chlorine dioxide (ClO2, effective chlorine concentration 0.1%) and peracetic acid (PAA), which are approved by the Ministry of Food and Drug Safety (MFDS) of Korea and the Food and Drug Administration (FDA) of the United States. Chlorine dioxide (ClO2) was applied at 5, 10, 15, and 20 ppm for the suspension experiment, at 25, 50, 75, 100, 200, and 300 ppm for the food contact surface experiment on stainless steel (SS), and at 10, 20, 30, 40, and 50 ppm for the food experiment on lettuce. Peracetic acid (PAA, initial concentration 10%) was treated at concentrations of 20, 40, 60, 80, 100, and 120 ppm for suspension experiments, 100, 500, 1000, 1500, 2000, and 2500 ppm on stainless steel (SS) for food contact surface experiments, and 20, 40, 60, and 80 ppm on lettuce for food experiments. The disinfectant was diluted using sterile hard water.

[0086] The rotavirus inactivation effect in suspensions was verified using the revised ASTM E1052 method. Specifically, 100 µl of rotavirus stock (approx. 6 log 10 800 µl of disinfectant and 100 µl of BSA as an interfering agent were mixed for 1 or 5 minutes. After treatment, 100 µl of the virus-disinfectant mixture was directly diluted in 900 µl of neutralizing agent (1×DMEM, 44 mM sodium bicarbonate, 1% penicillin-streptomycin). The control group (0 ppm) was evaluated using hard water as the diluent for the disinfectant.

[0087] As a result, 5, 10, and 15 ppm of ClO for 1 minute (0.74, 1.63, and 2.84, respectively) and 5 minutes (0.51, 1.58, and 3.22, respectively) 2-There was a significant difference in the reduction values ​​of the rotavirus suspensions treated with [method]. It was confirmed that rotavirus was completely inactivated at a concentration of 20 ppm in both the 1-minute and 5-minute treatment groups. The detection limit was 1 log. 10 It was PFU / mL (Fig. 1a).

[0088] In the case of PAA, after treatment for 1 minute at concentrations of 20, 40, 60, 80, and 100 ppm, decrease values ​​of 0.35, 0.41, 0.46, 0.54, and 1.79 were observed. When treated with PAA at a concentration of 120 ppm, the rotavirus titer was at the detection limit (1 log 10 It reached (PFU / mL). When PAA at 80 to 120 and 60 to 120 ppm was treated for 1 minute and 5 minutes, respectively, a significant difference (p<0.05) was observed (Fig. 1b).

[0089] The rotavirus inactivation effect on food contact surfaces (SS) and food (lettuce) was verified using the revised ASTM E1053 method. Specifically, 50 µl of rotavirus stock (approx. 6 log 10 PFU / mL) was mixed with 100 µL of disinfectant for 1 or 5 minutes. After treatment, 100 µL of the virus-disinfectant mixture was directly diluted in 900 µL of neutralizing agent. Samples inoculated in the food test were immersed in the diluted disinfectant solution for 1 or 5 minutes, and then all samples were immediately transferred to 2 mL of recovery solution.

[0090] As a result of confirming the rotavirus inactivation effect following disinfectant treatment (1 min and 5 min) on food contact surfaces (SS), excluding samples treated with 75 to 100 ppm of ClO2 for 1 min, the reduction values ​​of rotavirus increased significantly as the ClO2 concentration increased, reaching 0.10, 0.44, 1.00, 1.23, 2.38, and 4.15 after 1 min treatment, and 0.11, 0.59, 1.35, 2.31, and 4.08 after 5 min treatment (Fig. 2a). In the case of PPA, it increased significantly regardless of concentration and treatment time (Fig. 2b).

[0091] As a result of confirming the rotavirus inactivation effect of disinfectant treatment (1 min and 5 min) on food (lettuce), in the case of ClO2, the reduction values ​​of rotavirus increased as the concentration increased (at 1 min treatment: 0.17, 1.01, 1.17, 1.40, and 1.77; at 5 min treatment: 0.66, 0.96, 1.32, 1.49, and 1.79). However, there was no significant difference between the groups treated at concentrations of 20 and 40 ppm for 1 min (Fig. 3a). In the case of PAA, after 1 minute of treatment, decrease values ​​of 0.09, 0.27, 0.53, and 0.64 were observed at concentrations of 20, 40, 60, and 80 ppm, respectively, and after 5 minutes of treatment, decrease values ​​of 0.31, 0.39, 0.66, and 0.75 were observed at the same concentrations. No significant difference was observed according to treatment time (Fig. 3b).

[0093] <Example 3> Confirmation of Rotavirus Inactivation Effect by Single Gamma Irradiation Treatment

[0094] Gamma ray irradiation was performed at the Korea Atomic Energy Research Institute using a Cobalt-60 gamma ray irradiator (Point source, ACEL, IR-79, MDS Nordion). The source intensity was approximately 11.1 PBq at a dose rate of 10 kGy / h.

[0095] The rotavirus inactivation effect of gamma irradiation on suspensions and food (lettuce) was confirmed. For the suspensions, 1 mL of each rotavirus suspension was irradiated into a 1.5 mL Eppen tube at doses of 1, 3, 5, 7, and 10 kGy, and then the treated suspension was directly diluted 10-fold for plaque analysis. For the food, gamma irradiation was performed at doses of 1, 1.5, 2, 2.5, 3, and 4 kGy, and then the samples were transferred to a 2 mL recovery solution.

[0096] As a result of confirming the rotavirus inactivation effect of gamma irradiation in suspensions, the initial virus titer of the untreated sample was 6.77log 10 It was PFU / mL. The rotavirus titer was 5.40 log after gamma irradiation at doses of 1, 3, 5, 7, and 10 kGy, respectively. 10 PFU / mL (1.33log 10 PFU / mL decrease), 4.79 log 10 PFU / mL (1.94log 10 PFU / mL decrease), 4.11 log 10 PFU / mL (2.62log 10 PFU / mL decrease), 3.14 log 10 PFU / mL (3.59log 10 PFU / mL decrease), 2.06 log 10 PFU / mL (4.67log 10 As shown by a decrease in PFU / mL, rotavirus titer significantly decreased as the gamma irradiation dose increased (Fig. 4a).

[0097] As a result of confirming the rotavirus inactivation effect of gamma irradiation (1.0, 1.5, 2.0, 2.5, 3.0, 4.0 kGy) on food (lettuce), the rotavirus titer was 3.97 log with increasing gamma irradiation dose, respectively. 10 PFU / mL (0.30log 10 PFU / mL decrease), 2.92 log 10 PFU / mL (1.35log 10PFU / mL decrease), 2.27 log 10 PFU / mL (2log 10 PFU / mL decrease), 1.80 log 10 PFU / mL (2.47log 10 It was shown as a decrease in PFU / mL). At 4 kGy, the U.S. FDA gamma irradiation standard for agricultural products, the rotavirus titer was at the detection limit (1.5 log). 10 It reached (PFU / mL). The reduction value of rotavirus in lettuce increased with increasing gamma irradiation dose, but while there was no difference in the average rotavirus titer observed at gamma irradiation doses of 1.0 to 1.5 kGy, a significant difference was confirmed at 1.5 to 4.0 kGy (Fig. 4b).

[0099] <Example 4> Confirmation of Rotavirus Inactivation Effect by Combined Treatment of Chemical Disinfectant and Gamma Ray Irradiation

[0100] The rotavirus inactivation effect of combined treatment with chemical disinfectants and gamma irradiation in food was confirmed.

[0101] Rotavirus suspension on lettuce (approx. 6 log 10 The samples were inoculated with PFU / mL. Lettuce samples were immersed in ClO2 solutions (10, 20, 30, 40, 50 ppm) or PAA solutions (20, 40, 60, 80 ppm) at various concentrations. Each disinfectant was applied for 1 minute, followed by treatment with a neutralizing agent. After a rest period (3 hours) following disinfectant treatment, gamma rays were sequentially irradiated at doses of 1 and 1.5 kGy. After the combined treatment, the samples were transferred to 2 mL of recovery solution. The samples were shaken for 2 minutes and then centrifuged for 10 minutes to recover rotavirus. The efficacy of each treatment was evaluated by calculating the reduction in rotavirus titer. The synergistic effect of the combined treatment was expressed using the following formula.

[0103] Synergistic effect = A - (B + C)

[0104] A: log obtained by combined treatment with ClO2 or PAA and gamma irradiation10 PFU / mL reduction

[0105] B: log obtained by treating with ClO2 or PAA alone 10 PFU / mL reduction

[0106] C: log obtained by treating with gamma ray irradiation alone 10 PFU / mL reduction

[0107] Positive values ​​indicate a synergistic effect, while negative or zero values ​​mean there is no synergistic effect.

[0109] According to the single gamma irradiation treatment of <Example 3>, the decrease in rotavirus titer was 1 log at a gamma irradiation dose of 2 kGy. 10 As levels exceeded PFU / mL, the gamma irradiation dose was adjusted to 1.0 and 1.5 kGy for combined treatment with chemical disinfectants. As a result of confirming the rotavirus inactivation effect of combined treatment with the chemical disinfectant ClO2 and gamma irradiation on food (lettuce), the reduction in rotavirus titer at a gamma irradiation dose of 1 kGy varied according to ClO2 concentrations (10, 20, 30, 40, and 50 ppm), with values ​​of 0.65, 1.07, 1.65, 1.80, and 2.00 log, respectively. 10 It was expressed in PFU / mL. At a gamma irradiation dose of 1.5 kGy, the rotavirus reduction values ​​according to ClO2 concentrations (10, 20, 30, 40, and 50 ppm) were 0.80, 1.25, 1.85, 2.00, and 2.40 log, respectively. 10 It was found to be in PFU / mL (Fig. 5a and Table 1). Under the combined treatment conditions of ClO2 and gamma irradiation, it was confirmed that 1.5 kGy gamma irradiation after 30 ppm ClO2 treatment showed the highest synergistic effect.

[0110]

[0111] As a result of confirming the rotavirus inactivation effect by combined treatment with the chemical disinfectant PAA and gamma irradiation on food (lettuce), the reduction values ​​of rotavirus titers at a gamma irradiation dose of 1 kGy were found to be 0.29, 0.38, 0.63, and 1.20 log10 PFU / mL depending on the PAA concentration (20, 40, 60, and 80 ppm), respectively. At a gamma irradiation dose of 1.5 kGy, the reduction values ​​of rotavirus titers at a gamma irradiation dose of 1.5 kGy were found to be 0.33, 0.78, 1.15, and 1.65 log10 PFU / mL depending on the PAA concentration (20, 40, 60, and 80 ppm), respectively, confirming a significant reduction (Fig. 5b and Table 2). Under the combined treatment conditions of PAA and gamma irradiation, it was confirmed that 1.5 kGy gamma irradiation after treatment with 80 ppm PAA showed the highest synergistic effect.

[0112]

[0114] <Example 5> Measurement of food quality after combined treatment with chemical disinfectant and gamma irradiation

[0115] After treating food with a combination of chemical disinfectants (ClO2 and PAA) and gamma irradiation, changes in texture and color were measured to evaluate the physicochemical effects on the food. As a result, the hardness (g / cm²) between the control and treatment group samples was compared. 2 No significant differences were observed in the values ​​of ) and color (L*, a*, and b*) (Table 3). The overall color difference (△E*) in all samples was less than 1.2. It was confirmed that the quality characteristics of lettuce were not affected even at maximum concentrations and doses under all treatment conditions. Through this, it can be seen that the method of rotavirus inactivation by combined treatment of a chemical disinfectant and gamma irradiation according to the present invention can be very usefully utilized in the food industry.

[0116]

[0117]

Claims

Claim 1 A combined treatment method for rotavirus inactivation, comprising: (i) a step of treating rotavirus (RV) with a chemical disinfectant; and (ii) a step of irradiating the rotavirus treated with the chemical disinfectant with gamma rays. Claim 2 A composite treatment method according to claim 1, characterized in that the chemical disinfectant of step (i) is chlorine dioxide or peracetic acid. Claim 3 A composite treatment method according to paragraph 2, characterized in that the chlorine dioxide is treated at a concentration of 5 to 50 ppm. Claim 4 A composite treatment method according to claim 2, characterized in that the peracetic acid is treated at a concentration of 10 to 80 ppm. Claim 5 A composite treatment method according to claim 1, characterized in that the treatment time of the chemical disinfectant in step (i) is 1 to 5 minutes. Claim 6 A composite treatment method according to claim 1, characterized in that the gamma rays in step (ii) are irradiated at a dose of 1.0 to 4.0 kGy. Claim 7 A complex treatment method according to claim 1, characterized in that the rotavirus is generated in food. Claim 8 A composite processing method according to claim 7, characterized in that the food is a vegetable, fish, or meat product distributed in a fresh state. Claim 9 A composite treatment method according to claim 1, characterized in that the rotavirus is generated on a surface in contact with food. Claim 10 A composite processing method according to claim 9, characterized in that the food contact surface is tableware, utensils, packaging material, cooking utensils, or cooking facility equipment made of glass, metal, plastic, rubber, paper, or a composite material of the above. Claim 11 A composite treatment method according to claim 1, characterized in that the composite treatment method does not cause a change in color or texture when treated with food. Claim 12 A method for removing rotavirus using the combined treatment method of claim 1.

Images