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WO2022114970A1 - Antiviral food grade - Google Patents

Antiviral food grade Download PDF

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Publication number
WO2022114970A1
WO2022114970A1 PCT/OM2020/050011 OM2020050011W WO2022114970A1 WO 2022114970 A1 WO2022114970 A1 WO 2022114970A1 OM 2020050011 W OM2020050011 W OM 2020050011W WO 2022114970 A1 WO2022114970 A1 WO 2022114970A1
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WIPO (PCT)
Prior art keywords
present
composition
cells
food
antiviral
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PCT/OM2020/050011
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French (fr)
Inventor
Dr.Abduallh AL-MAHRUKI
Ung Eng Huan UNG ENG HUAN
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Industrial Innovation Center
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Publication of WO2022114970A1 publication Critical patent/WO2022114970A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids

Definitions

  • the present invention generally relates to an antiviral composition and particularly relates to aCOVID-19 antiviral composition.
  • the present invention more particularly relates to a food grade antiviral contact disinfectant composition which disinfects food surfaces as well as surfaces that come in contact with food and making it safer for human consumption.
  • Background Art Within 9 months, COVID-19 has infected about40 million people globally with a death toll of about 1.1 million at the time of writing this submission. Projections show that the USA alone may have around 400,000 deaths by February 2021.
  • COVID-19 is the 3rd major human Coronavirus epidemic after the SARS-CoV- 1 outbreak in 2003 (also known as SARS) and MERS-CoV outbreak in 2012. Although direct droplet transmission is the main route of transmission, faecal excretion, environmental contamination, and fomites might contribute also to viral transmission. Considering the evidence of faecal excretion, it may be possible that SARS-CoV-2 causing COVID-19 is also transmitted via the faecal-oral transmission route or from fomites being ingested orally as is common in feline Coronaviruses. There have been many studies exploring the possibility of faecal-oral transmission of COVID-19.
  • ACE2 and TMPRSS2 are also co-expressed in both upper epithelial and gland cells from oesophagus and absorptive enterocytes from ileum and colon and oral transmission is a possible route of SARS-CoV-2 transmission.
  • a disinfecting agent that can stop SARS-CoV-2 infectivity from food or food-contact surfaces.
  • this agent does not require rinsing after contact and also not distasteful.
  • EP 1331947 use of citric acid as an antimicrobial agent or enhancer or as an anticancer agent is provided.
  • Simple carboxylic acids in particular dicarboxylic acids such as citric acid shows an unexpected ability to enhance the antimicrobial power of a wide range of disinfectant and/or antibiotic agents. As little as 1 % citrate greatly enhances the ability of antibiotics to kill or inhibit a wide range of bacterial species including antibiotic resistant strains. While there are no claims against viruses in this patent, there is nonetheless use of citric acid in antiviral wet wipes that are being marketed.
  • US patent number US 9,980,497 provides an antimicrobial- antibiofilm compositions and methods of use thereof, which provides disinfectant composition comprising GRAS or “generally recognized as safe” substances and the composition may be suitably used in the disinfection of fruits, vegetables, meat products and food processing facilities.
  • US patent number US 6,617,290 provides preparation of concentrated sanitizing and cleaning preparation. The preparation has dual use for cleaning and sanitizing food surfaces as well as food contact and non-food contact surfaces using GRAS food additive ingredients and food ingredients.
  • US patent application number US 2016/0166498 provides oral care compositions comprising cannabinoids, preferably cannabidiol and/or cannabigerol.
  • the oral care composition disclosed in this application may be a tooth paste, a tooth powder, or a mouthwash solution with sodium citrate, citric acid being used as a preservative.
  • the prior art discloses the use of sodium bicarbonate as an abrasive agent, sodium chloride being used as a cavity prevention agent and sodium laureth sulphate as a surfactant.
  • Another product, Proxitane ® made by Solvay has a Cleaning in Place (CIP) and Food Contact Sanitizer disinfectant that uses Peracetic Acid (PAA).
  • the product is said to be effective at doses as low as 157 ppm (mg/L) leaving no harmful residues as it dissociates to water and vinegar.
  • the Proxitane 15:10 specifically has a USFDA approval for a Food Contact Surface disinfectant. But, there is a slightly vinegar like residual taste mentioned.
  • PAA is also a strong oxidizing agent and is unsuitable for use by laymen as it is meant to be a technical disinfectant at factory level.
  • a contact disinfecting antiviral solution for SARS-CoV-2 causing COVID-19 which is safe for human consumption, rapidly acts on the SARS-CoV-2 virus and not distasteful.
  • a composition that may be applied on food items to make them safe for consumption.
  • a food disinfectant which does not have vinegar like after taste and that is very simple for use by common people, and which is highly safe for being completely free of any oxidizing agents. 3.
  • the primary object of the present invention is to provide an antiviral food disinfectant composition that can be directly applied on food items before consumption.
  • Another object of the present invention is to provide an antiviral food disinfectant composition that is used in low concentration either as a spray or as an agent where food may be dipped in and does not pose any threat to health or as an irritant and not distasteful.
  • Yet another object of the present invention is to provide an antiviral food disinfectant composition that can be packaged in the form of a powder mixture in sachets for solubilization into a liquid form easily by non-technical common people as a spray.
  • an antiviral food disinfectant composition that is used for an immediate application at restaurant level or used by the diners themselves whereby the composition is surface sprayed onto the food surfaces for rapid killing of the SARS-CoV-2 virus within 15-60 seconds.
  • Yet another object of the present invention is to provide an antiviral food disinfectant composition that may be used as a bath or dip solution where various fruits, meats and vegetables may be treated with a short duration contact dip or bath to eliminate any surface contamination of SARS-CoV-2.
  • an antiviral food grade disinfectant composition comprises sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate.
  • the sodium citrate is present in a concentration of 19.99-24.99 g/L.
  • the sodium bicarbonate is present in a concentration of 6.99-14.99 g/L.
  • the sodium chloride is present in a concentration of 5.99-9.99 g/L.
  • the citric acid is present in a concentration of 2.99-8.99 g/L.
  • the glycine is present in a concentration of 0.99-1.99 g/L.
  • the sodium lauryl sulphate is present in a concentration of 0.099 – 0.499 g/L.
  • the pH of the composition is in the range of 2.99-6.99.
  • the composition is a powder.
  • the composition is a mixture of powdered ingredients, wherein the powdered ingredients comprises sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate.
  • the various embodiments of the present invention provide an antiviral food disinfectant composition.
  • the antiviral food disinfectant composition is in the form of a powder.
  • the powder is added to water to make a liquid composition that is fully dissolved without any precipitate.
  • the composition is safe for human consumption as all components are GRAS (Generally Regarded as Safe) and have been used in common processed foods for many decades.
  • the composition is sprayed on food items. The composition does not affect the taste of the food items.
  • an antiviral food grade disinfectant composition comprises sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate.
  • the sodium citrate is present in a concentration of 19.99-24.99 g/L.
  • citric acid is mentioned as the only active agent in wet wipes made by two US based companies against another virus i.e. Rhinovirus (without a specific test conducted on SARS-CoV-2).
  • citric acid is used to act together with sodium citrate at various ratios. Therefore, citric acid is added as an ingredient to balance out the aforementioned sodium citrate. Both citric acid and sodium citrate has been used in numerous process foods over many decades. According to an embodiment of the present invention, the sodium bicarbonate is present in a concentration of 6.99-14.99 g/L.
  • sodium bicarbonate is used to alter the pH further towards neutrality at the pH 6.19-6.99 range.
  • the water source that has its own unique composition determines the final pH but sodium bicarbonate is nonetheless useful in making the final solution more neutral such that food processors that prefer a more neutral final pH may be given a product closer to neutrality so that there is less chance of changes in coloration, taste or texture of their product due to pH fluctuations.
  • Sodium bicarbonate is being used as an acidulant for indigestion over many decades under the brand name ENO as well as others and is extremely regarded as safe for ingestion.
  • the sodium chloride is present in a concentration of 5.99-9.99 g/L.
  • Sodium chloride is used to adjust the taste from the citric acid and sodium citrate buffers that give a somewhat citrus like flavour with a slightly salty taste. Salt has been used as a source of sodium that is totally necessary for normal human nutrition.
  • the citric acid is present in a concentration of 2.99-8.99 g/L.
  • Citric acid has been used in multiple food preparations over many decades.
  • the glycine is present in a concentration of 0.99-1.99 g/L.
  • Glycine is a non-essential amino acid occurring in a wide range of foods.
  • the sodium lauryl sulphate is present in a concentration of 0.199 – 0.499 g/L.
  • Sodium Lauryl Sulphate (SLS) is an anionic surfactant naturally derived from coconut and/or palm kernel oil. SLS usually consists of a mixture of sodium alkyl sulphates, mainly the lauryl.
  • the pH of the composition may be within the range of 2.28-8.16.
  • the composition is a powder mixture.
  • SLS sodium laureth sulphate
  • SLS sodium laureth sulphate
  • the addition of SLS may vary from 0.099-0.499 g/L depending upon the sources and fineness and moisture content of the various component powder substances.
  • the composition is mixed in water to make a liquid.
  • the powders are mixed mechanically in any type of conventional powder mixers and tested using Endecott sieves to confirm that by comparing the granulometry of the samples taken after a manufacturer’s specified time in minutes of mixing, there is a close resemblance to 95% of the expected composite granulometry of all the individual ingredients.
  • the powders are sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate.
  • the sodium citrate is present in a concentration of 19.99-24.99 g/L.
  • the sodium bicarbonate is added in a concentration of 6.99-14.99 g/L.
  • the sodium chloride is added in a concentration of 5.99-9.99 g/L.
  • the citric acid is present in a concentration of 2.99-8.99 g/L.
  • the glycine is present in a concentration of 0.99-1.99 g/L.
  • the sodium lauryl sulphate is present in a concentration of 0.099 – 0.499 g/L.
  • Water used in the present invention ranges from filtered underground water, RO (reverse osmosis) water, distilled water, and drinking water that has been sterilized using UV or ozonation or by any other conventional means such that it complies to normal potable water standards.
  • the solubilization shall be done in a facility that complies with GMP and has HACCP in place with ISO standards pertaining to food ingredient manufacturing. As an added precaution, the incoming water for solubilization should pass through a final 0.22–0.25 ⁇ filter prior to the solubilization step.
  • the mixing is done in a sterilized non-reactive vessel that can be steel as in SS312, SS304 or any conventional superior grade commonly used in food processing plants such as SS316 and SS430.
  • a high-speed agitator guarantees a rapid and efficient solubilization to any temperature ranging from 8-45°C that is considered as most indoor facility temperatures in almost any country even during winter or peak summer conditions.
  • the samples are checked for the expected final pH that should range from pH 5.99-6.99 for food contact products where pH is desired to be more neutral so as to not cause any pH related discoloration of the food.
  • the solution should be clear and precipitates, colloids or any form or turbidity is a cause for rejection of the manufacturing lot. Any change in pH beyond the expected range is also a possible cause for rejection of that batch.
  • the disinfecting agent can also be sold in aluminium foil powder sachets in addition to an already solubilized liquid preparation. Any type of inert packaging is used for the bottles that may be any type of conventionally available plastic or glass material.
  • Spray-caps are fitted on the tops of the bottles for ease of application in homes and restaurants but in processing plants, solubilized powder may be used in bath-dips such that a 30-60 seconds minimum contact time is assured but where the food being treated is of convoluted or irregular surface, such as broccoli for example, the bath-dip duration may be extended for 1 – 5 minutes.
  • An antiviral food disinfectant composition which is packaged as a liquid under pressure in small metal cylinders with a nozzle that is engineered to emit a fine spray upon pressing. This is especially useful for an intranasal spray to reduce the number of SARS-CoV-2 virus within the nasal passage and easily used by non-technical common people. This may also reduce other airborne respiratory viruses such as any form of the Avian Influenza virus.
  • the antiviral food disinfectant composition is sprayed on food items, wherein the users are unable to easily distinguish between food that is treated and food that is left untreated.
  • the composition provides no offensive, bitter, strange, or repulsive off-taste that may affect its use as a no rinse contact disinfecting agent.
  • the formulation does not affect the morphology and structure of mammalian cells, which means that it has no negative effect on cell structure, growth, and appearance. As the agent will be in contact with the tongue, it is important that it does not affect the structure of the cells of the 2000-8000 taste buds on a normal tongue each of which having an average lifespan of 10 days.
  • EXPERIMENTAL DETAILS EXAMPLE 1 Preliminary Screening for Viral Reduction evidenced by Real Time-PCR Viral Inactivation Assay
  • CRFK cells in T-75 flask were washed twice with PBS.
  • Cells were harvested with trypsin-EDTA (1X) solution and incubated at 37°C with 5% CO 2 until the cells were detached from the flask.
  • Cells were then collected and centrifuged at 1500rpm for 5 min.
  • Cell pellet was resuspended in DMEM supplemented with 2% FBS. The number of cells were counted using a haemocytometer and seeded in 24-wells flat- bottom plate at a concentration of 5 x 10 4 cells/well.
  • FIG. 1 shows the percentage of viral reduction caused by formulation in various contact time, according to an embodiment of the present invention. With respect to FIG. 1, the formulations show 99.9999% to 100% reduction. There was seen a 6 log reduction, with preferably atleast 4-5 log reduction within 1 minute of contact time.
  • EXAMPLE 2 Role of pH in viral inactivation evidenced by TCID 50 quantification Virus Quantification – TCID 50 TCID 50 is defined as the dilution that is used to achieve infection of 50% of cell culture wells.
  • CRFK cells were seeded in 24-well plates at a cell density of 1.5 x 10 5 cells/well in 500 ⁇ L of DMEM supplemented with 2% FBS. The plate was incubated overnight at 37°C with 5% CO 2 . After 24 h of incubation, culture medium from the plate was removed and washed twice with PBS, followed by the addition of 500 ⁇ L of fresh medium into each well. A 10-fold serial dilutions of FIPV virus stock (10 1 to 10 6 ) was prepared in DMEM containing 2% FBS. Then, 100 ⁇ L of virus suspension from each respective dilution was added into the wells accordingly.
  • CRFK cells in T-75 flask were washed twice with PBS. Cells were harvested with trypsin-EDTA (1X) solution and incubated at 37°C with 5% CO 2 until the cells were detached from the flask.
  • Cells were then collected and centrifuged at 1500rpm for 5 min. Cell pellet was resuspended in DMEM supplemented with 2% FBS. The number of cells were counted using a haemocytometer and seeded in 24-wells flat- bottom plate at a concentration of 5 x 10 4 cells/well. After 24 h of incubation at 37°C with 5% CO 2 , culture medium was removed and replaced with 500 ⁇ L of fresh medium supplemented with 2% FBS. For formulation-FIPV sample preparation, three parts (20 ⁇ L per part) of undiluted formulation was mixed with three parts of virus (1.3 ⁇ L per part at MOI 0.1) at the contact time of one minute.
  • Controls in this study included cell control (cells in culture medium only), virus control (cells with virus only) and cytotoxic control (cells with formulation only). After 72 h of incubation, cells were observed under the inverted microscope for visible CPE and cytotoxicity by comparing with the virus and cytotoxic controls. Formulation that did not showed any sign of CPE and toxicity to the cells were proceed for viral quantification using TCID 50 . In brief, the plate was stored in -80°C overnight, followed by two cycle of freeze-thawing process. Samples from the plate were harvested through centrifugation at 4°C, 1500 rpm for 10 min.
  • FIG. 2 shows normal CRFK cells and FIPV-infected CRFK cells, wherein (a) shows normal cells, (b) and (c) shows FIPV-infected cells, according to an embodiment of the present invention.
  • pH variation between 2.28-8.16 does not affect the antiviral effect nor the cause any cytotoxicity
  • Table 3 Viral titre of the formulation-treated FIPV in CRFK cells.
  • FIG. 3 shows microscopic images of cytotoxic and formulation-treated FIPV on CRFK cells
  • FIG. 4 shows microscopic images of the virus control and cell control, according to an embodiment of the present invention.
  • no CPE (cytopathic effect) on the CRFK cells meaning antiviral efficacy is not affected by pH between 2.28-8.16.
  • TCID 50 is defined as the dilution that is used to achieve infection of 50% of cell culture wells.
  • the viral titre was estimated by Reed and Meunch (1938) method as shown in equation (2) below.
  • ...equation (2) CRFK cells were seeded in 24-well plates at a cell density of 1.5 x 10 5 cells/well in 500 ⁇ L of DMEM supplemented with 2% FBS. The plate was incubated overnight at 37°C with 5% CO 2 . After 24 h of incubation, culture medium from the plate was removed and washed twice with PBS, followed by the addition of 500 ⁇ L of fresh medium into each well.
  • a 10-fold serial dilutions of FIPV virus stock (10 1 to 10 6 ) was prepared in DMEM containing 2% FBS. Then, 100 ⁇ L of virus suspension from each respective dilution was added into the wells accordingly. Each dilution was repeated in triplicate. The plate was then gently tapped to ensure complete mixing of the virus. For cell control (cells and culture medium only), 100 ⁇ L of DMEM with 2% FBS was added instead of virus suspension. The plate was incubated for 72 h at 37°C in humidified 5% CO2 atmosphere. The cells were observed daily under inverted microscope for visible cytopathic effect (CPE) by comparing the morphology of virally infected-cells with the non-infected cells (cell control).
  • CPE visible cytopathic effect
  • CRFK cells in T-75 flask were washed twice with PBS. Cells were harvested with trypsin-EDTA (1X) solution and incubated at 37°C with 5% CO 2 until the cells were detached from the flask. Cells were then collected and centrifuged at 1500rpm for 5 min. Cell pellet was resuspended in DMEM supplemented with 2% FBS. The number of cells were counted using a haemocytometer and seeded in 24-wells flat- bottom plate at a concentration of 5 x 10 4 cells/well.
  • formulation-FIPV sample preparation After 24 h of incubation at 37°C with 5% CO 2 , culture medium was removed and replaced with 500 ⁇ L of fresh medium supplemented with 2% FBS.
  • three parts (20 ⁇ L per part) of undiluted formulation was mixed with three parts of clean solution (2.37 ⁇ L per part) and followed by three parts of virus (1.3 ⁇ L per part at MOI 0.1) at the contact time of 15, 30 and 60 seconds, respectively. Then, one part (23.67 ⁇ L per part) of the formulation-FIPV mixture was added into the cells accordingly. Same procedure was repeated for the study of dirty condition.
  • Controls in this study included cell control (cells in culture medium only), virus control (cells with virus only) and cytotoxic control (cells with formulation and clean/dirty solution only). After 72 h of incubation, cells were observed under the inverted microscope for visible CPE and cytotoxicity by comparing with the virus and cytotoxic controls. Viral quantification for the samples was proceeded using TCID 50 . In brief, the plate was stored in -80°C overnight, followed by two cycle of freeze-thawing process. Samples from the plate were harvested through centrifugation at 4°C, 1500 rpm for 10 min. Supernatant of each sample was collected and stored in -80°C prior to TCID 50 . The result was obtained from three independent experiments. FIG.
  • FIG. 5 shows are microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of 15 seconds, according to an embodiment of the present invention.
  • FIG. 5 it can be seen that the dirty conditions as demonstrated by using erythrocytes does not affect antiviral efficacy within 15 sec contact time.
  • FIG. 6 shows microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of 30 seconds, according to an embodiment of the present invention.
  • FIG. 6 it is seen that dirty conditions as demonstrated by using erythrocytes does not affect antiviral efficacy within 30 sec contact time.
  • FIG. 5 shows are microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of 15 seconds, according to an embodiment of the present invention.
  • FIG. 6 shows microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of
  • FIG. 7 shows microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of 60 seconds, according to an embodiment of the present invention.
  • the dirty conditions as demonstrated by using erythrocytes does not affect antiviral efficacy within 60 sec contact time.
  • FIG. 8 shows microscopic images of the virus control and cell control, according to an embodiment of the present invention. These controls are to show the validity of the experiment whereby uninfected cell control does not form CPE while the FIPV-infected virus control formed CPE.
  • Table 5 Viral titre of the formulation-treated FIPV in CRFK cells.
  • FIG. 9 shows TCID50 of formulation-treated FIPV in CRFK cells, according to an embodiment of the present invention.
  • FIG. 10A and FIG. 10B shows Transmission Electron Microscopic (TEM) images before and after contact with disinfecting agent, according to an embodiment of the present invention.
  • TEM Transmission Electron Microscopic
  • EXAMPLE 5 Virucidal effect of disinfectant upon SARS-CoV-2 in clean &dirty conditions Viral Kill Time Assay
  • the Food Disinfectant Spray was tested against SARS-CoV-2 in accordance with the European Standard EN14476:2013/FprA1:2015 protocol.
  • the product was tested undiluted under 2 different conditions; dirty condition (3.0 g/l BSA + 3 ml/l erythrocytes interfering substance) and clean conditions (0.3 g/l BSA interfering substance) at 15s, 30s, 60s contact time.
  • the test assay comprised of 100 ⁇ l of interfering substance, 100 ⁇ l of virus suspension at concentration of 5.42 x 10 5 TCID 50 /mL & 800 ⁇ l of Food Disinfectant Spray. After the specified contact time (15s, 30s and 60s), virucidal activity of the product was suppressed by adding DMEM+ 2% FBS and then the mixture was diluted in 10-fold dilution in ice cold media (DMEM+ 2% FBS). This diluted virus media was added to the Vero E6 cells to determine TCID 50 /mL. Virus controls for this test was distilled water in place of test product for both dirty and clean conditions. The cells were incubated for 72 hours till the CPE developed.
  • a mixture of paraformaldehyde and crystal violet were used to fix and stain the infected cells.
  • the virus titres were determined using the Spearman-Karber method and expressed as tissue culture infectious dose 50% (TCID50/ml).
  • the virucidal activity was determined by the difference of the logarithmic titre of the virus control minus the logarithmic titre of the test virus ( ⁇ log10 TCID50/ml).
  • a reduction in virus titre of 4 log 10 (corresponding to an inactivation of ⁇ 99.99%) was necessary for claiming virucidal activity of the product.
  • Product Suppression Assay The product suppression assay was performed to accurately determine the activity of the test product at the given contact time.
  • the activity was suppressed by adding cold DMEM+2% FBS, followed by serially diluting it 10-fold in cell culture medium. The suppression of product activity was assayed at 1 min exposure. Results from the suppression assay showed no difference in viral titres compared to controls (Table 6). This suggested that addition of cold media and serial dilution effectively suppresses the product activity, resulting in no reduction of the viral titres. This assay acts as a control to verify that the clean and dirty conditions tested alone without the XC-19 disinfectant had no effect on the viral titre reduction.
  • Table 6 Suppression of product activity *Undiluted mixture Virucidal Activity of Food Disinfectant Spray
  • the Food Disinfectant Spray was tested against SARS-CoV-2 in accordance with the European Standard EN14476:2013/FprA1:2015. Manifestation of virus cytopathic effects in cell culture were determined by comparing the study product-treated groups against that of the water -treated controls.
  • the Food Disinfectant Spray when tested neat achieved >4 log 10 reduction in viral titres when exposed for 15s, 30s and 60s under clean condition only(Table 7).
  • Table 7 Virucidal activity of Food Disinfectant Spray against SARS- CoV-2.
  • the virucidal efficacy of the food disinfectant spray was tested against SARS-CoV-2 in a suspension assay following the European Standard EN144762013/FprA1:2015 protocol.
  • the Food Disinfectant Spray when tested undiluted demonstrated potent and rapid virucidal activity of ⁇ 4 log 10 reduction of SARS-CoV-2 viral titre in 15 second in clean conditions.
  • the Food Disinfectant Spray hence, can kill 99.99% SARS-CoV-2 under clean condition that is defined here as the absence of RBC (Red Blood Cells or Human Erythrocytes).
  • RBC Red Blood Cells or Human Erythrocytes
  • FIG. 11B shows CRFK cell morphology before/after disinfectant contact, where FIG. 11A shows CRFK cells without contact with disinfectant, and FIG. 11B shows CRFK cells after contact with disinfectant for 72hours, according to an embodiment of the present invention. This shows the safety of disinfectant whereby it does not alter the cell morphology or causing cytotoxicity to the cell after 72 hours of exposure.
  • EXAMPLE 7 Taste Trial 1 and 2 STATISTICAL ANALYSIS OF TASTE TEST 1 A group of 10 random participants were selected to participate in a blind taste test with the aim to determine the perception of disinfectant taste in both raw and cooked food samples.
  • FIG. 12A shows the statistics of food Samples sprayed with disinfectant, according to an embodiment of the present invention.
  • Table 8 ANOVA: Two-Factor Without Replication With respect to table 8, it is seen that since the P-value (0.000022) is less than 0.05, it indicates that the results are statistically significant. Hence, there is strong evidence against the null hypothesis (the results are by chance). Overall, majority of the participants could not taste the disinfectant that was sprayed onto the food samples except for raw fish. Therefore, a second blind taste test using raw fish samples was conducted. STATISTICAL ANALYSIS OF TASTE TEST2 A group of 10 random participants were selected to participate in a blind taste test with the aim to determine the perception of disinfectant taste in 5 raw fish samples.
  • FIG. 12B shows the statistics of food Samples sprayed with disinfectant, according to an embodiment of the present invention. Table 9: ANOVA: Two-Factor Without Replication
  • the present invention comprises food grade additives that have long been used in the food industry. Also, the concentrations used are low and do not pose any threat to health or as an irritant.
  • the present invention allows for direct contact with the buccal cavity as a gargle or mouthwash (which is not recommended for products containing PAA or peracetic acid).
  • PAA and hydrogen peroxide releasing products may be used for mouthwashes
  • long term usage may cause side effects such as hypertrophy of the lingual papillae that may diminish the sense of taste.
  • COVID-19 can manifest initially as a sore throat in the early stages of infection and the present invention in the form of a gargle can reduce viral titre by rapid contact with the protein coat as well as the lipid bilayer of the Coronavirus causing its disruption.
  • SARS-CoV is killed after exposure to 65°C for 3 minutes.
  • not all food is subjected to heat before ingestion (e.g. fruits, salads, and raw fish such as salmon as in sushi and sashimi).

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Abstract

The present invention relates to an antiviral food grade disinfectant composition which is a combination of salts that is applied directly on food items to make them safe for consumption. The composition of the present invention is in the form of a powder or liquid, gargle or mouthwash or intranasal spray. The composition is safe for human consumption as all composition are GRAS (Generally Regarded as Safe). The composition is a mixture of salts present in unique concentration.English Drawings

Description

Description Title of Invention : ANTIVIRAL FOOD GRADE DISINFECTION COMPOSITION 1. Technical Field The present invention generally relates to an antiviral composition and particularly relates to aCOVID-19 antiviral composition. The present invention more particularly relates to a food grade antiviral contact disinfectant composition which disinfects food surfaces as well as surfaces that come in contact with food and making it safer for human consumption. 2. Background Art Within 9 months, COVID-19 has infected about40 million people globally with a death toll of about 1.1 million at the time of writing this submission. Projections show that the USA alone may have around 400,000 deaths by February 2021. COVID-19 is the 3rd major human Coronavirus epidemic after the SARS-CoV- 1 outbreak in 2003 (also known as SARS) and MERS-CoV outbreak in 2012. Although direct droplet transmission is the main route of transmission, faecal excretion, environmental contamination, and fomites might contribute also to viral transmission. Considering the evidence of faecal excretion, it may be possible that SARS-CoV-2 causing COVID-19 is also transmitted via the faecal-oral transmission route or from fomites being ingested orally as is common in feline Coronaviruses. There have been many studies exploring the possibility of faecal-oral transmission of COVID-19. Potential SARS-CoV-2 infection in the gastrointestinal tract has been discussed in regard to the expression of ACE2 and TMPRSS2 in the gut epithelium. There is evidence that the ACE2 receptors of the GI tract will allow for the Spike protein to attach, leading toa potential oral route of transmission of COVID-19. Bulk tissue RNA sequencing analysis indicated that digestive tract organs had higher ACE2 expression levels compared to the lung epithelia, and that the expression of ACE2 in the lung epithelia increased with age. Also, single-cell RNA- Seq results showed that the ACE2-positive-cell ratio in digestive tract organs was significantly higher compared to the lungs. Therefore, this may explain why 25% of COVID-19 patients in a study has GI-related symptomatic presentations. Since lung infections via respiratory droplets presents clinical signs of severe respiratory distress, researchers have not focused the same degree of attention to the GI tract where life-threatening presentations are less common. Nonetheless, there is evidence that suggests a mechanism for an oral transmission route. The results revealed that the expressions of mRNA and protein of the ACE2 gene are extremely high in the intestinal cells, especially in enterocytes. Therefore, the SARS-CoV-2 spike protein may enter the small intestinal enterocytes by binding to ACE2, which induces immune cells infiltration and abnormal intestinal function. One clinical consequence may be the inhibition of the absorption of dietary tryptophan in the small intestine causing changes of the intestinal microbiota resulting in susceptibility to colitis. ACE2 and TMPRSS2 are also co-expressed in both upper epithelial and gland cells from oesophagus and absorptive enterocytes from ileum and colon and oral transmission is a possible route of SARS-CoV-2 transmission. There is therefore a potential for a disinfecting agent that can stop SARS-CoV-2 infectivity from food or food-contact surfaces. There is an added utility if this agent does not require rinsing after contact and also not distasteful. In European patent application number EP 1331947, use of citric acid as an antimicrobial agent or enhancer or as an anticancer agent is provided. Simple carboxylic acids, in particular dicarboxylic acids such as citric acid shows an unexpected ability to enhance the antimicrobial power of a wide range of disinfectant and/or antibiotic agents. As little as 1 % citrate greatly enhances the ability of antibiotics to kill or inhibit a wide range of bacterial species including antibiotic resistant strains. While there are no claims against viruses in this patent, there is nonetheless use of citric acid in antiviral wet wipes that are being marketed. US patent number US 9,980,497 provides an antimicrobial- antibiofilm compositions and methods of use thereof, which provides disinfectant composition comprising GRAS or “generally recognized as safe” substances and the composition may be suitably used in the disinfection of fruits, vegetables, meat products and food processing facilities. The use of GRAS substances in this patent makes no claim for SARS-CoV-2 disinfection. US patent number US 6,617,290 provides preparation of concentrated sanitizing and cleaning preparation. The preparation has dual use for cleaning and sanitizing food surfaces as well as food contact and non-food contact surfaces using GRAS food additive ingredients and food ingredients. US patent application number US 2016/0166498 provides oral care compositions comprising cannabinoids, preferably cannabidiol and/or cannabigerol. The oral care composition disclosed in this application may be a tooth paste, a tooth powder, or a mouthwash solution with sodium citrate, citric acid being used as a preservative. The prior art discloses the use of sodium bicarbonate as an abrasive agent, sodium chloride being used as a cavity prevention agent and sodium laureth sulphate as a surfactant. Another product, Proxitane® made by Solvay, has a Cleaning in Place (CIP) and Food Contact Sanitizer disinfectant that uses Peracetic Acid (PAA). The product is said to be effective at doses as low as 157 ppm (mg/L) leaving no harmful residues as it dissociates to water and vinegar. The Proxitane 15:10 specifically has a USFDA approval for a Food Contact Surface disinfectant. But, there is a slightly vinegar like residual taste mentioned. PAA is also a strong oxidizing agent and is unsuitable for use by laymen as it is meant to be a technical disinfectant at factory level. In view of the foregoing, there is a need to develop a contact disinfecting antiviral solution for SARS-CoV-2 causing COVID-19, which is safe for human consumption, rapidly acts on the SARS-CoV-2 virus and not distasteful. There is a need to invent a composition that may be applied on food items to make them safe for consumption. Further, there is a need to develop a food disinfectant which does not have vinegar like after taste and that is very simple for use by common people, and which is highly safe for being completely free of any oxidizing agents. 3. Summary of Invention Thus, the primary object of the present invention is to provide an antiviral food disinfectant composition that can be directly applied on food items before consumption. Another object of the present invention is to provide an antiviral food disinfectant composition that is used in low concentration either as a spray or as an agent where food may be dipped in and does not pose any threat to health or as an irritant and not distasteful. Yet another object of the present invention is to provide an antiviral food disinfectant composition that can be packaged in the form of a powder mixture in sachets for solubilization into a liquid form easily by non-technical common people as a spray. Yet another object of the present invention is to provide an antiviral food disinfectant composition that cannot be distinguished between treated and untreated food samples which means that there are not distinctly offensive or bitter off- tastes. Yet another object of the present invention is to provide an antiviral food disinfectant commercial formulation that does not adversely affect the cell morphology of a mammalian cell which has the indication that it will not also adversely affect the morphology of taste receptor cells on the tongue. Yet another object of the present invention is to provide an antiviral food disinfectant composition in liquid form, wherein the liquid form is in the form of a gargle or mouthwash in order to reduce SARS-CoV-2 upon contact within the buccal cavity of any person. Yet another object of the present invention is to provide an antiviral food disinfectant composition that is able to reduce 99.99% of SARS-CoV-2 upon a 15-30 seconds of contact time. Yet another object of the present invention is to provide an antiviral food disinfectant composition that is packaged as a liquid under pressure in small metal cylinders with a nozzle that is engineered to emit a fine spray upon pressing. Yet another object of the present invention is to provide an antiviral food disinfectant composition that is effective for COVID-19 and is used not only on food surfaces but on any surfaces that come into contact with food such as plates and cutlery made of plastic, ceramic, steel and so on and the surfaces of food processing equipment or food storage containers that is made of plastic, steel or any hard non-porous commonly used material. Yet another object of the present invention is to provide an antiviral food disinfectant composition that is used for an immediate application at restaurant level or used by the diners themselves whereby the composition is surface sprayed onto the food surfaces for rapid killing of the SARS-CoV-2 virus within 15-60 seconds. Yet another object of the present invention is to provide an antiviral food disinfectant composition that may be used as a bath or dip solution where various fruits, meats and vegetables may be treated with a short duration contact dip or bath to eliminate any surface contamination of SARS-CoV-2. According to the embodiments of the present invention, an antiviral food grade disinfectant composition comprises sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate. According to an embodiment of the present invention, the sodium citrate is present in a concentration of 19.99-24.99 g/L. According to an embodiment of the present invention, the sodium bicarbonate is present in a concentration of 6.99-14.99 g/L. According to an embodiment of the present invention, the sodium chloride is present in a concentration of 5.99-9.99 g/L. According to an embodiment of the present invention, the citric acid is present in a concentration of 2.99-8.99 g/L. According to an embodiment of the present invention, the glycine is present in a concentration of 0.99-1.99 g/L. According to an embodiment of the present invention, the sodium lauryl sulphate is present in a concentration of 0.099 – 0.499 g/L. According to an embodiment of the present invention, the pH of the composition is in the range of 2.99-6.99. According to an embodiment of the present invention, the composition is a powder. The composition is a mixture of powdered ingredients, wherein the powdered ingredients comprises sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate. These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications. 4. Technical Problem 5. Solution to Problem 6. Advantageous Effects of Invention 7. Brief Description of Drawings 8. Fig.1 9. [Fig.1] Description of Embodiments The present invention will now be described in detail with reference to the accompanying drawings. The various embodiments of the present invention provide an antiviral food disinfectant composition. The antiviral food disinfectant composition is in the form of a powder. The powder is added to water to make a liquid composition that is fully dissolved without any precipitate. The composition is safe for human consumption as all components are GRAS (Generally Regarded as Safe) and have been used in common processed foods for many decades. The composition is sprayed on food items. The composition does not affect the taste of the food items. The composition does not even affect the morphology or structure of mammalian cells when the composition comes in contact with tongue surface or any other surface of an organ. According to an embodiment of the present invention, an antiviral food grade disinfectant composition comprises sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate. According to an embodiment of the present invention, the sodium citrate is present in a concentration of 19.99-24.99 g/L. In the US EPA list of approved SARS-CoV-2 surface disinfectants, citric acid is mentioned as the only active agent in wet wipes made by two US based companies against another virus i.e. Rhinovirus (without a specific test conducted on SARS-CoV-2). The details are in the following USEPA link https://www.epa.gov/pesticide-registration/list-n- disinfectants-use-against-sars-cov-2-covid-19. In the manufacture of physiological buffers between the pH of 3.0 – 6.2, citric acid is used to act together with sodium citrate at various ratios. Therefore, citric acid is added as an ingredient to balance out the aforementioned sodium citrate. Both citric acid and sodium citrate has been used in numerous process foods over many decades. According to an embodiment of the present invention, the sodium bicarbonate is present in a concentration of 6.99-14.99 g/L. In the event that a higher pH formulation is required beyond pH 3.0-6.2 for the sodium citrate- citric acid buffer ratio, sodium bicarbonate is used to alter the pH further towards neutrality at the pH 6.19-6.99 range. Ultimately, the water source that has its own unique composition determines the final pH but sodium bicarbonate is nonetheless useful in making the final solution more neutral such that food processors that prefer a more neutral final pH may be given a product closer to neutrality so that there is less chance of changes in coloration, taste or texture of their product due to pH fluctuations. Sodium bicarbonate is being used as an acidulant for indigestion over many decades under the brand name ENO as well as others and is extremely regarded as safe for ingestion. According to an embodiment of the present invention, the sodium chloride is present in a concentration of 5.99-9.99 g/L. Sodium chloride is used to adjust the taste from the citric acid and sodium citrate buffers that give a somewhat citrus like flavour with a slightly salty taste. Salt has been used as a source of sodium that is totally necessary for normal human nutrition. According to an embodiment of the present invention, the citric acid is present in a concentration of 2.99-8.99 g/L. Two surface contact wet wipe products in the USA that have been registered with an Emerging Viral Pathogen Claim on the 14th May 2020 with the US EPA. Their claim is based on Rhinovirus and have no validations based on SARS-CoV-2 causing COVID-19. One comes under the trade name of Freak while the other under Wexford Disinfection wipes. See the following link https://www.epa.gov/pesticide-registration/list-n- disinfectants-use-against-sars-cov-2-covid-19. Citric acid has been used in multiple food preparations over many decades. According to an embodiment of the present invention, the glycine is present in a concentration of 0.99-1.99 g/L. Glycine is a non-essential amino acid occurring in a wide range of foods. It is recognized as GRAS with the specific safety number as GRAS 3287 and is recognized by FEMA (Flavour and Extract Manufacturers Association) as a human food and drink flavour enhancer (https://www.femaflavor.org/flavor-library/glycine). According to an embodiment of the present invention, the sodium lauryl sulphate is present in a concentration of 0.199 – 0.499 g/L. Sodium Lauryl Sulphate (SLS) is an anionic surfactant naturally derived from coconut and/or palm kernel oil. SLS usually consists of a mixture of sodium alkyl sulphates, mainly the lauryl. SLS lowers surface tension of aqueous solutions and is used as fat emulsifier, wetting agent, and detergent in cosmetics, pharmaceuticals, and toothpastes. Sodium dodecyl sulphate, appearing as its synonym sodium lauryl sulphate (SLS), is considered a generally recognized as safe (GRAS) ingredient for food use according to the USFDA (21 CFR 172.822.) According to an embodiment of the present invention, the pH of the composition may be within the range of 2.28-8.16. Our data indicates that there is very significant antiviral activity that is maintained from pH 2.28-8.16 when virus quantification of FCoV based on TCID50 is carried out using Crandell Reese Feline Kidney (CRFK) cells seeded with an FCoV suspension treated with various formulations of the aforementioned disinfectant solution based on a 60 seconds contact time. For two trial formulations, where the virus control was 1.9 x 109 the titre dropped to 1.02 x 103 and 3.03 x 103 respectively at a MOI of 0.1. According to an embodiment of the present invention, the composition is a powder mixture. In mixing of the various powder components, SLS or sodium laureth sulphate is added as an anti- caking agent and to assist in powder flow during mixing to give a better evenness of the mixture. The addition of SLS may vary from 0.099-0.499 g/L depending upon the sources and fineness and moisture content of the various component powder substances. According to an embodiment of the present invention, the composition is mixed in water to make a liquid. The powders are mixed mechanically in any type of conventional powder mixers and tested using Endecott sieves to confirm that by comparing the granulometry of the samples taken after a manufacturer’s specified time in minutes of mixing, there is a close resemblance to 95% of the expected composite granulometry of all the individual ingredients. The time will differ according to the mixing device and can range from below 1 minute to about 5 minutes for most modern mixers. According to an embodiment of the present invention, the powders are sodium citrate, sodium bicarbonate, sodium chloride, citric acid, glycine, and sodium lauryl sulphate. According to an embodiment of the present invention, the sodium citrate is present in a concentration of 19.99-24.99 g/L. The sodium bicarbonate is added in a concentration of 6.99-14.99 g/L. The sodium chloride is added in a concentration of 5.99-9.99 g/L. The citric acid is present in a concentration of 2.99-8.99 g/L. The glycine is present in a concentration of 0.99-1.99 g/L. The sodium lauryl sulphate is present in a concentration of 0.099 – 0.499 g/L. Once an even mix has been obtained, the composite powder is solubilized using water. Water used in the present invention ranges from filtered underground water, RO (reverse osmosis) water, distilled water, and drinking water that has been sterilized using UV or ozonation or by any other conventional means such that it complies to normal potable water standards. The solubilization shall be done in a facility that complies with GMP and has HACCP in place with ISO standards pertaining to food ingredient manufacturing. As an added precaution, the incoming water for solubilization should pass through a final 0.22–0.25µ filter prior to the solubilization step. Mixing with the sodium bicarbonate will release a certain amount of carbon dioxide gas and the mixing vessel if completely sealed should possess a pressure release valve such that the liquid is no longer releasing gas during the filling step as this may compromise some cap seals which may result in leakage during transportation. The mixing is done in a sterilized non-reactive vessel that can be steel as in SS312, SS304 or any conventional superior grade commonly used in food processing plants such as SS316 and SS430. The use of a high-speed agitator guarantees a rapid and efficient solubilization to any temperature ranging from 8-45°C that is considered as most indoor facility temperatures in almost any country even during winter or peak summer conditions. After solubilization is achieved, the samples are checked for the expected final pH that should range from pH 5.99-6.99 for food contact products where pH is desired to be more neutral so as to not cause any pH related discoloration of the food. The solution should be clear and precipitates, colloids or any form or turbidity is a cause for rejection of the manufacturing lot. Any change in pH beyond the expected range is also a possible cause for rejection of that batch. The disinfecting agent can also be sold in aluminium foil powder sachets in addition to an already solubilized liquid preparation. Any type of inert packaging is used for the bottles that may be any type of conventionally available plastic or glass material. Spray-caps are fitted on the tops of the bottles for ease of application in homes and restaurants but in processing plants, solubilized powder may be used in bath-dips such that a 30-60 seconds minimum contact time is assured but where the food being treated is of convoluted or irregular surface, such as broccoli for example, the bath-dip duration may be extended for 1 – 5 minutes. An antiviral food disinfectant composition which is packaged as a liquid under pressure in small metal cylinders with a nozzle that is engineered to emit a fine spray upon pressing. This is especially useful for an intranasal spray to reduce the number of SARS-CoV-2 virus within the nasal passage and easily used by non-technical common people. This may also reduce other airborne respiratory viruses such as any form of the Avian Influenza virus. This is especially useful as such viruses act in upregulation of the TMPRSS2 within respiratory epithelia that will also act in synergy to increase the ease of infectivity of SARS-CoV-2 during the Flu Season experienced in many countries especially within the Northern Hemisphere. This may also be of significant utility for medical personnel coming into close contact with those infected with SARS-CoV-2. There is also a potential use as an intranasal spray that is packed within slightly pressurised aluminium canisters of various convenient sizes. These are typically activated by depressing the top nozzle resulting in the release of a fine liquid aerosol that may be inhaled to a certain extent into the upper respiratory system. According to an embodiment of the present invention, the antiviral food disinfectant composition is sprayed on food items, wherein the users are unable to easily distinguish between food that is treated and food that is left untreated. The composition provides no offensive, bitter, strange, or repulsive off-taste that may affect its use as a no rinse contact disinfecting agent. According to an embodiment of the present invention, the formulation does not affect the morphology and structure of mammalian cells, which means that it has no negative effect on cell structure, growth, and appearance. As the agent will be in contact with the tongue, it is important that it does not affect the structure of the cells of the 2000-8000 taste buds on a normal tongue each of which having an average lifespan of 10 days. EXPERIMENTAL DETAILS EXAMPLE 1 Preliminary Screening for Viral Reduction evidenced by Real Time-PCR Viral Inactivation Assay Prior to the experiment, CRFK cells in T-75 flask were washed twice with PBS. Cells were harvested with trypsin-EDTA (1X) solution and incubated at 37°C with 5% CO2 until the cells were detached from the flask. Cells were then collected and centrifuged at 1500rpm for 5 min. Cell pellet was resuspended in DMEM supplemented with 2% FBS. The number of cells were counted using a haemocytometer and seeded in 24-wells flat- bottom plate at a concentration of 5 x 104 cells/well. After 24 h of incubation at 37°C with 5% CO2, culture medium was removed and replaced with 500µL of fresh medium supplemented with 2% FBS. For formulation-FIPV sample preparation, three parts (20µL per part) of undiluted formulation was mixed with three parts of virus (1.3µL per part at MOI 0.1) at the contact time of 1 min, 5 min, 10 min, and 30 min, respectively. Then, one part (21.3µL per part) of the formulation-FIPV mixture from different contact time was added into the cells accordingly. Controls in this study included cell control (cells in culture medium only), virus control (cells with virus only) and cytotoxic control (cells with formulation only). After 72 h of incubation, cells were observed under the inverted microscope for
Figure imgf000015_0001
Figure imgf000016_0001
visible CPE and cytotoxicity by comparing with the virus and cytotoxic controls. Formulation that did not showed any sign of CPE and toxicity to the cells were proceed for viral quantification. In brief, the plate was stored in -80°C overnight, followed by two cycle of freeze-thawing process. Samples from the plate were harvested through centrifugation at 4°C, 1500 rpm for 10 min. Supernatant of each sample was collected and stored in -80°C prior to viral RNA extraction and real time RT-PCR (Table 1). The result was obtained from three independent experiments. Table 1: FIPV primers and real time RT-PCR protocol. Table 2: The efficacy of formulation in viral copy number at various contact time.
Figure imgf000017_0001
*N/A means not available FIG. 1 shows the percentage of viral reduction caused by formulation in various contact time, according to an embodiment of the present invention. With respect to FIG. 1, the formulations show 99.9999% to 100% reduction. There was seen a 6 log reduction, with preferably atleast 4-5 log reduction within 1 minute of contact time. EXAMPLE 2 Role of pH in viral inactivation evidenced by TCID50 quantification Virus Quantification – TCID50 TCID50 is defined as the dilution that is used to achieve infection of 50% of cell culture wells. The viral titre was estimated by Reed and Meunch (1938) method as shown in equation (1) below:
Figure imgf000018_0001
…equation (1) CRFK cells were seeded in 24-well plates at a cell density of 1.5 x 105 cells/well in 500 µL of DMEM supplemented with 2% FBS. The plate was incubated overnight at 37°C with 5% CO2. After 24 h of incubation, culture medium from the plate was removed and washed twice with PBS, followed by the addition of 500 µL of fresh medium into each well. A 10-fold serial dilutions of FIPV virus stock (101 to 106) was prepared in DMEM containing 2% FBS. Then, 100 µL of virus suspension from each respective dilution was added into the wells accordingly. The plate was gently tapped to ensure complete mixing of the virus. For cell control (cells and culture medium only), 100 µL of DMEM with 2% FBS was added instead of virus suspension. The plate was incubated for 72 h at 37°C in humidified 5% CO2 atmosphere. The cells were then observed daily under inverted microscope for visible cytopathic effect (CPE) by comparing the morphology of virally infected cells with the non-infected cells (cell control). Viral Inactivation Assay Prior to the experiment, CRFK cells in T-75 flask were washed twice with PBS. Cells were harvested with trypsin-EDTA (1X) solution and incubated at 37°C with 5% CO2 until the cells were detached from the flask. Cells were then collected and centrifuged at 1500rpm for 5 min. Cell pellet was resuspended in DMEM supplemented with 2% FBS. The number of cells were counted using a haemocytometer and seeded in 24-wells flat- bottom plate at a concentration of 5 x 104 cells/well. After 24 h of incubation at 37°C with 5% CO2, culture medium was removed and replaced with 500µL of fresh medium supplemented with 2% FBS. For formulation-FIPV sample preparation, three parts (20µL per part) of undiluted formulation was mixed with three parts of virus (1.3µL per part at MOI 0.1) at the contact time of one minute. Then, one part (21.3µL per part) of the formulation- FIPV mixture was added into the cells accordingly. Controls in this study included cell control (cells in culture medium only), virus control (cells with virus only) and cytotoxic control (cells with formulation only). After 72 h of incubation, cells were observed under the inverted microscope for visible CPE and cytotoxicity by comparing with the virus and cytotoxic controls. Formulation that did not showed any sign of CPE and toxicity to the cells were proceed for viral quantification using TCID50. In brief, the plate was stored in -80°C overnight, followed by two cycle of freeze-thawing process. Samples from the plate were harvested through centrifugation at 4°C, 1500 rpm for 10 min. Supernatant of each sample was collected and stored in -80°C prior to TCID50. The result was obtained from three independent experiments. FIG. 2 shows normal CRFK cells and FIPV-infected CRFK cells, wherein (a) shows normal cells, (b) and (c) shows FIPV-infected cells, according to an embodiment of the present invention. With respect to FIG. 2, it is seen that pH variation between 2.28-8.16 does not affect the antiviral effect nor the cause any cytotoxicity Table 3: Viral titre of the formulation-treated FIPV in CRFK cells.
Figure imgf000019_0001
Figure imgf000020_0001
With respect to table 3, the quantification of virus titer is done by calculating the number of wells for each dilution that showed CPE through the observation of morphology difference between normal and FIPV-infected CRFK cells. FIG. 3 shows microscopic images of cytotoxic and formulation-treated FIPV on CRFK cells, while FIG. 4 shows microscopic images of the virus control and cell control, according to an embodiment of the present invention. With respect to FIG. 3 and FIG. 4, no CPE (cytopathic effect) on the CRFK cells meaning antiviral efficacy is not affected by pH between 2.28-8.16. EXAMPLE 3 Effect of clean & dirty conditions on viral inactivation using the FCoV model Virus Quantification – TCID50 TCID50 is defined as the dilution that is used to achieve infection of 50% of cell culture wells. The viral titre was estimated by Reed and Meunch (1938) method as shown in equation (2) below.
Figure imgf000021_0001
…equation (2) CRFK cells were seeded in 24-well plates at a cell density of 1.5 x 105 cells/well in 500µL of DMEM supplemented with 2% FBS. The plate was incubated overnight at 37°C with 5% CO2. After 24 h of incubation, culture medium from the plate was removed and washed twice with PBS, followed by the addition of 500µL of fresh medium into each well. A 10-fold serial dilutions of FIPV virus stock (101 to 106) was prepared in DMEM containing 2% FBS. Then, 100µL of virus suspension from each respective dilution was added into the wells accordingly. Each dilution was repeated in triplicate. The plate was then gently tapped to ensure complete mixing of the virus. For cell control (cells and culture medium only), 100µL of DMEM with 2% FBS was added instead of virus suspension. The plate was incubated for 72 h at 37°C in humidified 5% CO2 atmosphere. The cells were observed daily under inverted microscope for visible cytopathic effect (CPE) by comparing the morphology of virally infected-cells with the non-infected cells (cell control). After the incubation, culture media from plate was discarded and cells were stained with 300µL of naphthalene black. The plate was further incubated for 1 hour at 37°C with 5% CO2 and followed by the removal naphthalene black. Then, plate was washed twice with distilled water and blotted dry to calculate the number of wells for each dilution that showed CPE. Clean and Dirty Solution Preparation As shown in Table 4, the clean and dirty solutions were prepared accordingly in distilled water and further sterilized using a 0.22 µm cellulose acetate syringe filter. For dirty condition, blood was added after the solution was filtered. The solutions were stored in 4ºC pending assay. Table 4: The composition of clean and dirty solutions.
Figure imgf000022_0001
Viral Inactivation Assay Prior to the experiment, CRFK cells in T-75 flask were washed twice with PBS. Cells were harvested with trypsin-EDTA (1X) solution and incubated at 37°C with 5% CO2 until the cells were detached from the flask. Cells were then collected and centrifuged at 1500rpm for 5 min. Cell pellet was resuspended in DMEM supplemented with 2% FBS. The number of cells were counted using a haemocytometer and seeded in 24-wells flat- bottom plate at a concentration of 5 x 104 cells/well. After 24 h of incubation at 37°C with 5% CO2, culture medium was removed and replaced with 500µL of fresh medium supplemented with 2% FBS. For clean condition of formulation-FIPV sample preparation, three parts (20µL per part) of undiluted formulation was mixed with three parts of clean solution (2.37µL per part) and followed by three parts of virus (1.3µL per part at MOI 0.1) at the contact time of 15, 30 and 60 seconds, respectively. Then, one part (23.67µL per part) of the formulation-FIPV mixture was added into the cells accordingly. Same procedure was repeated for the study of dirty condition. Controls in this study included cell control (cells in culture medium only), virus control (cells with virus only) and cytotoxic control (cells with formulation and clean/dirty solution only). After 72 h of incubation, cells were observed under the inverted microscope for visible CPE and cytotoxicity by comparing with the virus and cytotoxic controls. Viral quantification for the samples was proceeded using TCID50. In brief, the plate was stored in -80°C overnight, followed by two cycle of freeze-thawing process. Samples from the plate were harvested through centrifugation at 4°C, 1500 rpm for 10 min. Supernatant of each sample was collected and stored in -80°C prior to TCID50. The result was obtained from three independent experiments. FIG. 5 shows are microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of 15 seconds, according to an embodiment of the present invention. With respect to FIG. 5, it can be seen that the dirty conditions as demonstrated by using erythrocytes does not affect antiviral efficacy within 15 sec contact time. FIG. 6 shows microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of 30 seconds, according to an embodiment of the present invention. With respect to FIG. 6, it is seen that dirty conditions as demonstrated by using erythrocytes does not affect antiviral efficacy within 30 sec contact time. FIG. 7 shows microscopic images of cytotoxic and formulation-treated FIPV in CRFK cells under clean and dirty conditions at the contact time of 60 seconds, according to an embodiment of the present invention. With respect to FIG. 7, the dirty conditions as demonstrated by using erythrocytes does not affect antiviral efficacy within 60 sec contact time. FIG. 8 shows microscopic images of the virus control and cell control, according to an embodiment of the present invention. These controls are to show the validity of the experiment whereby uninfected cell control does not form CPE while the FIPV-infected virus control formed CPE. Table 5: Viral titre of the formulation-treated FIPV in CRFK cells.
Figure imgf000024_0001
FIG. 9 shows TCID50 of formulation-treated FIPV in CRFK cells, according to an embodiment of the present invention. EXAMPLE 4 Viral disruption evidenced by TEM imagery after 15 seconds contact time FIG. 10A and FIG. 10B shows Transmission Electron Microscopic (TEM) images before and after contact with disinfecting agent, according to an embodiment of the present invention. With respect to FIG. 10A, FCoV image (bar is 10 nm) prior to treatment with the disinfecting agent can be seen. With respect to FIG. 10B, remnants of FCoV coat protein after 15 seconds contact with the disinfecting agent can be seen ruptured. EXAMPLE 5 Virucidal effect of disinfectant upon SARS-CoV-2 in clean &dirty conditions Viral Kill Time Assay The Food Disinfectant Spray was tested against SARS-CoV-2 in accordance with the European Standard EN14476:2013/FprA1:2015 protocol. The product was tested undiluted under 2 different conditions; dirty condition (3.0 g/l BSA + 3 ml/l erythrocytes interfering substance) and clean conditions (0.3 g/l BSA interfering substance) at 15s, 30s, 60s contact time. The test assay comprised of 100 µl of interfering substance, 100 µl of virus suspension at concentration of 5.42 x 105 TCID50/mL & 800 µl of Food Disinfectant Spray. After the specified contact time (15s, 30s and 60s), virucidal activity of the product was suppressed by adding DMEM+ 2% FBS and then the mixture was diluted in 10-fold dilution in ice cold media (DMEM+ 2% FBS). This diluted virus media was added to the Vero E6 cells to determine TCID50/mL. Virus controls for this test was distilled water in place of test product for both dirty and clean conditions. The cells were incubated for 72 hours till the CPE developed. A mixture of paraformaldehyde and crystal violet were used to fix and stain the infected cells. The virus titres were determined using the Spearman-Karber method and expressed as tissue culture infectious dose 50% (TCID50/ml). The virucidal activity was determined by the difference of the logarithmic titre of the virus control minus the logarithmic titre of the test virus (Δ log10 TCID50/ml). A reduction in virus titre of 4 log10 (corresponding to an inactivation of ≥ 99.99%) was necessary for claiming virucidal activity of the product. Product Suppression Assay The product suppression assay was performed to accurately determine the activity of the test product at the given contact time. The activity was suppressed by adding cold DMEM+2% FBS, followed by serially diluting it 10-fold in cell culture medium. The suppression of product activity was assayed at 1 min exposure. Results from the suppression assay showed no difference in viral titres compared to controls (Table 6). This suggested that addition of cold media and serial dilution effectively suppresses the product activity, resulting in no reduction of the viral titres. This assay acts as a control to verify that the clean and dirty conditions tested alone without the XC-19 disinfectant had no effect on the viral titre reduction. Table 6: Suppression of product activity
Figure imgf000026_0001
*Undiluted mixture Virucidal Activity of Food Disinfectant Spray The Food Disinfectant Spray was tested against SARS-CoV-2 in accordance with the European Standard EN14476:2013/FprA1:2015. Manifestation of virus cytopathic effects in cell culture were determined by comparing the study product-treated groups against that of the water -treated controls. The SARS-CoV-2 titre in the control-treated samples under clean and dirty conditions, respectively, were at 5.42 x 105 TCID50/ml. The Food Disinfectant Spray when tested neat achieved >4 log10 reduction in viral titres when exposed for 15s, 30s and 60s under clean condition only(Table 7). Table 7: Virucidal activity of Food Disinfectant Spray against SARS- CoV-2.
Figure imgf000027_0001
The virucidal efficacy of the food disinfectant spray was tested against SARS-CoV-2 in a suspension assay following the European Standard EN144762013/FprA1:2015 protocol. The Food Disinfectant Spray when tested undiluted demonstrated potent and rapid virucidal activity of ≥4 log10 reduction of SARS-CoV-2 viral titre in 15 second in clean conditions. The Food Disinfectant Spray, hence, can kill 99.99% SARS-CoV-2 under clean condition that is defined here as the absence of RBC (Red Blood Cells or Human Erythrocytes). EXAMPLE 6 FIG. 11A and FIG. 11B shows CRFK cell morphology before/after disinfectant contact, where FIG. 11A shows CRFK cells without contact with disinfectant, and FIG. 11B shows CRFK cells after contact with disinfectant for 72hours, according to an embodiment of the present invention. This shows the safety of disinfectant whereby it does not alter the cell morphology or causing cytotoxicity to the cell after 72 hours of exposure. EXAMPLE 7 Taste Trial 1 and 2 STATISTICAL ANALYSIS OF TASTE TEST 1 A group of 10 random participants were selected to participate in a blind taste test with the aim to determine the perception of disinfectant taste in both raw and cooked food samples. FIG. 12A shows the statistics of food Samples sprayed with disinfectant, according to an embodiment of the present invention. Table 8: ANOVA: Two-Factor Without Replication
Figure imgf000028_0001
With respect to table 8, it is seen that since the P-value (0.000022) is less than 0.05, it indicates that the results are statistically significant. Hence, there is strong evidence against the null hypothesis (the results are by chance). Overall, majority of the participants could not taste the disinfectant that was sprayed onto the food samples except for raw fish. Therefore, a second blind taste test using raw fish samples was conducted. STATISTICAL ANALYSIS OF TASTE TEST2 A group of 10 random participants were selected to participate in a blind taste test with the aim to determine the perception of disinfectant taste in 5 raw fish samples. FIG. 12B shows the statistics of food Samples sprayed with disinfectant, according to an embodiment of the present invention. Table 9: ANOVA: Two-Factor Without Replication
Figure imgf000029_0001
With respect to Table 9, it can be seen that since the P-value (0.008636) is less than 0.05, it indicates that the results are statistically significant. Hence, there is strong evidence against the null hypothesis (the results are by chance). Overall, majority of the participants could not taste the disinfectant that was sprayed onto the raw fish samples. Several participants who perceived the presence of disinfectant in the food samples stated that the taste difference between that and the control (no disinfectant) was not obvious. Thus, the present invention comprises food grade additives that have long been used in the food industry. Also, the concentrations used are low and do not pose any threat to health or as an irritant. The present invention allows for direct contact with the buccal cavity as a gargle or mouthwash (which is not recommended for products containing PAA or peracetic acid). While PAA and hydrogen peroxide releasing products may be used for mouthwashes, long term usage may cause side effects such as hypertrophy of the lingual papillae that may diminish the sense of taste. COVID-19 can manifest initially as a sore throat in the early stages of infection and the present invention in the form of a gargle can reduce viral titre by rapid contact with the protein coat as well as the lipid bilayer of the Coronavirus causing its disruption. SARS-CoV is killed after exposure to 65°C for 3 minutes. However, not all food is subjected to heat before ingestion (e.g. fruits, salads, and raw fish such as salmon as in sushi and sashimi). There is therefore an immediate application where at the restaurant level or even executed by the diners themselves whereby the invention may be surface sprayed onto the food surfaces for rapid killing of the virus within 15-60 seconds. As will be readily apparent to those skilled in the art, the present invention may easily be produced in other specific forms without departing from its essential characteristics. The present embodiments is, therefore, to be considered as merely illustrative and not restrictive, the scope of the invention being indicated by the claims rather than the foregoing description, and all changes which come within therefore intended to be embraced therein.

Claims

Claims 1. An antiviral food grade disinfectant composition, comprises: sodium citrate; sodium bicarbonate; sodium chloride; citric acid; glycine; and sodium lauryl sulphate.
2. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the sodium citrate is present in a concentration of 19.99-24.99 g/L.
3. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the sodium bicarbonate is present in a concentration of 6.99-14.99 g/L.
4. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the sodium chloride is present in a concentration of 5.99-9.99 g/L.
5. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the citric acid is present in a concentration of 2.99-8.99 g/L.
6. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the glycine is present in a concentration of 0.99-1.99 g/L.
7. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the sodium lauryl sulphate is present in a concentration of 0.199 – 0.499 g/L.
8. The antiviral food grade disinfectant composition as claimed in claim 1, wherein a pH of the composition is in the range of 3.99-6.99, wherein a preferable range is 2.30-8.20.
9. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the composition is a powder.
10. The antiviral food grade disinfectant composition as claimed in claim 1, wherein the composition is mixed in water to make a liquid.
11. A method of manufacturing an antiviral food grade disinfectant composition comprises: mixing a predetermined amount of sodium citrate; a predetermined amount of sodium bicarbonate; a predetermined amount of sodium chloride; a predetermined amount of citric acid; a predetermined amount of glycine; and a predetermined amount of sodium lauryl sulphate to form a mixture; dissolving the mixture in water to form a solution.
12. The method as claimed in claim 11, wherein the predetermined amount of sodium citrate is 19.99-24.99 g/L.
13. The method as claimed in claim 11, wherein the predetermined amount of sodium bicarbonate is 6.99-14.99 g/L.
14. The method as claimed in claim 11, wherein the predetermined amount of sodium chloride is 5.99-9.99 g/L.
15. The method as claimed in claim 11, wherein the predetermined amount of citric acid is 2.99-8.99 g/L.
16. The method as claimed in claim 1, wherein the predetermined amount of glycine is 0.99-1.99 g/L.
17. The method as claimed in claim 11, wherein the predetermined amount of sodium lauryl sulphate is 0.199 – 0.499 g/L.
18. The method as claimed in claim 11, wherein a pH of the mixture is in the range of 3.99-6.99, wherein a preferable range is 2.30 – 8.20.
19. The method as claimed in claim 11, wherein the water should be passed through a 22-25-micron filter before using for dissolution.
20. The method as claimed in claim 11, wherein the dissolution is done at a temperature between 8-45°C.
PCT/OM2020/050011 2020-11-24 2020-11-29 Antiviral food grade WO2022114970A1 (en)

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Citations (6)

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WO2003028455A1 (en) * 2001-10-04 2003-04-10 Bioacumen Pty Limited Compositions for the treatment and prevention of plant pathogens
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