EP4263778A2 - Elimination of coronaviridae (including sars-cov-2) viral contamination and recontamination with probiotic hygienic solutions - Google Patents

Elimination of coronaviridae (including sars-cov-2) viral contamination and recontamination with probiotic hygienic solutions

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Publication number
EP4263778A2
EP4263778A2 EP21886990.7A EP21886990A EP4263778A2 EP 4263778 A2 EP4263778 A2 EP 4263778A2 EP 21886990 A EP21886990 A EP 21886990A EP 4263778 A2 EP4263778 A2 EP 4263778A2
Authority
EP
European Patent Office
Prior art keywords
probiotic
coronaviridae
cov
test
bacteria
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21886990.7A
Other languages
German (de)
French (fr)
Inventor
Johannes VAN OVERVELD
Tim KEMLAND
Gjorgji JOVANOVSKI
Natalija ATANASOVA-PANCEVSKA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Investstone Doo Skopje
Original Assignee
Investstone Doo Skopje
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Investstone Doo Skopje filed Critical Investstone Doo Skopje
Publication of EP4263778A2 publication Critical patent/EP4263778A2/en
Pending legal-status Critical Current

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Classifications

    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/381—Microorganisms
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions

Definitions

  • the invention relates to probiotic hygiene solutions and methods for the use of probiotic non- pathogenic bacteria as an innovative solution for efficient cleaning, sanitation, disinfection and protection against contamination, elimination and re-contamination and of Coronaviridae (family of Corona viruses, including SARS-Cov-2, causing a global pandemic of Covid 19) with immediate, residual and prolonged efficacy applicable to hard surfaces, soft surfaces, human and animal skin, plants and the general environment as a natural-based product with numerous health and environmental benefits.
  • Coronaviridae family of Corona viruses, including SARS-Cov-2, causing a global pandemic of Covid 19
  • the invention is in the field of human needs, chemistry and procedures, which according to the International Patent Classification belongs to the:
  • Sub area preparations for medical and cosmetic use i.e. medicines containing probiotics and in the sub area of microbiology, i.e. measurement and testing of procedures involving microorganisms (bacteria).
  • the invention can be classified into the following classes: A61K 35/741 , A61L 2/18, C12Q 1/00, C12Q 1/02, C12Q 1/689, B08B 3/04, B05D 1/00.
  • probiotic hygienic solution is based on biological active substance and has the following new applications:
  • the invention is tested via probiotic hand spray, which is able to reduce and, in less than one minute, completely eliminate the growth of SARS-Cov-2 virus.
  • the product has also general antimicrobial properties and has been tested against other most common pathogenic microbial species namely: Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9037, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.
  • probiotic solution is able to reduce and, completely eliminate the growth of specific pathogenic microbial species namely Coronaviridae family of viruses, including the novel SARS-Cov-2.
  • the in vitro tests allowed the reduction in microbial count of the pathogens to be verified under controlled conditions, thus verifying the efficacy of tested Probiotic Hand spray to combat microorganisms where there is no risk of recontamination.
  • the results achieved confirms that the continuous and constant action of this probiotic-based solution is able to reduce and eliminate the proliferation of tested microorganisms.
  • this invention is effective and innovative product for environmental management.
  • Probiotics are ecological, easy to use and biodegradable. They render the skin, surfaces and the overall environment hygienically stable and are able to survive on and colonize non biological surfaces, combatting the proliferation of Coronaviridae viruses and other pathogens. In this study they were also found to perform well on different surfaces. Probiotics are therefore effective innovative products for sanitizing different environment and constitute a valid “green” alternative to the chemical disinfectants used up to now.
  • EP2556823 A review of the European patent application EP2556823 has established that there are cosmetic products that contain probiotic microorganisms as cleaning agents, but also contain chemical ingredients such as alkyl sulfate and alkyl polyglycol cholesterol sulphate salts. They are used to improve the skin barrier. Compared to the invention, it does not refer to cleaning or disinfecting the surface from viral contamination, i.e. SARS-Cov-2 (corona virus).
  • Russian patent number RU2699540 C2 relates to compositions containing probiotic bacteria and procedures for their use in the treatment of immune skin diseases.
  • Any surface or environment can be contaminated with a variety of microorganisms derived from various sources, some of them pathogen (harmful) to humans.
  • a pathogen is the term used to describe an infectious microorganism or agent, such as a virus, bacterium, protozoan, prion, viroid or fungus; or this is an organism that causes disease.
  • viruses are submicroscopic infectious agent that replicates only inside the living cells of an organism. When not inside an infected cell or in the process of infecting a cell, viruses exist in the form of independent particles, or virions, consisting of genetic material (DNA or RNA) and a protein coat.
  • Probiotics are live microorganisms promoted with claims that they provide health and environmental benefits or in the case of this patent application as proven by the microbiological tests provided in this patent application, non-pathogen good bacteria safe for handling and use that promote biological dirt degradation and anti-microbial and antiviral disinfection and after-protection. Probiotics are even considered generally safe to consume.
  • the novel SARS-Cov-2 (Corona) virus that causes Covid-19 infections belongs to Coronaviridae family of viruses. Its particles (or “virions”) are spheres with diameters of approximately 0.125 microns (125 nm). The smallest particles are 0.06 microns, and the largest are 0.14 microns. Coronaviridae Viruses can infect all types of life forms: animals, plants and humans and can survive on hard and soft surfaces for up to 48 hours mainly due to the biofilm created by pathogen (bad) bacteria which virus is using as a host.
  • the invention is new break-through solution composed of probiotic non-pathogen bacteria as an innovative solution for efficient cleaning, sanitation, disinfection and protection against contamination, elimination and re-contamination with Coronaviridae (Corona-family of viruses, including SARS-Cov-2 virus that causes the pandemic of Covid 19) with immediate in less than 60 seconds and time-extended efficacy applicable on hard surfaces, soft surfaces, human and animal skin, plants and the general environment as a natural-based product with numerous health and ecological benefits. They offer hours or days of effective residual after-protection against Coronaviridae viruses after their application on abovementioned skin or surfaces.
  • the solution is aqueous active substance of Probiotic non-pathogen bacteria belonging to Class 1 EU / Generally Regarded As Safe and mixed with water - resulting in sufficient end-concentrations of viable probiotic bacteria. Therefore it provides non-toxic activity to skin or surfaces - due to absence of chemical components.
  • the application method of such innovative cleaning, sanitation, disinfection and protection solutions is to be made by means of manual mechanical application, mopping, rinsing, spraying or machine application by spraying trough nozzles in hand-disinfection machines, disinfection tunnels, spray cabins or any external human application by air- borne solution particles.
  • probiotic-based cleaning products employ the capability of living microorganisms to produce extracellular enzymes, such as cellulases, proteases and ureases. These enzymes can degrade high molecular weight compounds often associated with dirt and effectively mitigate unfavourable odours by further metabolism of intermediates from these processes.
  • surfactins produced by various Bacillus species have detergent-like properties and have been shown to exhibit antimicrobial properties by interacting with bio-membranes (Bartal et al., 2018).
  • Other microorganisms such as the lactic acid bacteria can inhibit growth by lowering the pH in its environment (Dural-Erem et al., 2019).
  • a synergy of various mechanisms could come into play when it comes to the cleaning effect of these probiotic-based products (Spbk et al., 2018).
  • Disk diffusion test A clear zone or ring is formed around the disk after incubation if the agent inhibits microbial growth.
  • Figure 2 Protocol for testing protection time: in order to assess how effectively probiotics were able to compete against above mentioned microbial strains in the absence of external factors linked to recontamination.
  • the two surfaces floor and desk, with area of 30*30 cm
  • Samples of each microorganism were taken daily over a five-days period and inoculated on agar medium. Then bacterial strains were incubated at 37°C for 18-24 hours, and fungal strains were incubated at room temperature, 3-5 days.
  • Figure 3 The product has also general antimicrobial properties and has been tested against other most common pathogenic microbial species namely: antimicrobial activity of probiotic based solution (original and diluted 1 :10 Handspray against Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9037, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.
  • probiotic based solution original and diluted 1 :10 Handspray against Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9037, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.
  • the first test is a PCR sampling test on a surface populated with SARS-Cov-2. According to the analytical rapport i.e. efficiency test of probiotic hand spray vis-a-vis Sars-Cov-2 performed by TLR International laboratories in Rotterdam is confirmed that after use of probiotic hand spray there is no detection of Coronaviridae / SARS-Cov-2.
  • the second test EN 1500 disinfection standard test for hand-rub, EU standard is aimed to evaluate and compare the efficacy of probiotic-based hand soap and hand spray in reducing the microbial load present on artificially contaminated hands; and to compare the antimicrobial efficacy of the tested products with those of reference indicated in the methodology of EN1499 and EN1500, which used the non-antimicrobial soft soap and propan-2-ol 60% (v/v).
  • the EN 1499 and EN 1500 are a European Standard test methods that evaluates the efficacy of a hygienic handwash and handrub by measuring the number of viable bacteria remaining on the fingertips after contamination and handwash (handrub) exposure.
  • the EN 1499 standard is used to test antiseptic soaps and requires 12-15 subjects.
  • the EN 1500 is used to test antiseptic hand rubs and requires 18-22 subjects. Experimental contamination is with a non-pathogenic strain of Escherichia coli. Subjects are allotted randomly to two groups of approximately the same size, where one group applies the test formulation, and the other a reference antiseptic solution using a standardised procedure.
  • the mean reduction caused by the test product in the numbers of viable bacteria shall be significantly higher than that obtained with the non-antimicrobial soft soap.
  • the mean reduction in the numbers of viable bacteria shall not be significantly inferior to that with the reference alcohol-based hand rub (propan-2-ol 60% (v/v)).
  • the pre- and post-values recovered from the fingertips are evaluated against one another, resulting in a ratio called the reduction factor.
  • the reduction factor provides a quantitative measure of the antimicrobial efficacy. This is determined by comparing average counts of viable microorganism colony forming units of the pre-values to the average counts after exposure to the hygienic handrub.
  • Probiotic-based products hand soap and hand spray, were used in experiments for reducing the microbial load present on artificially contaminated hands, according to protocol EN1499 and EN1500.
  • biotechnology cleaning products contain non-pathogenic probiotic bacteria. These nonpathogenic bacteria are considered as a GRAS (generally recognized as safe) organism as they do not deemed to cause human diseases, with no impact on plants or animals. They also produce antimicrobial components active against many dangerous pathogenic bacteria. Due to their safety and activity on pathogens, biotechnology products have been used probiotic sanitation, demonstrating a significant reduction in microbial contamination with a stable effect over time.
  • GRAS generally recognized as safe
  • the EN1499 European Standard specifies a test method simulating practical conditions for establishing whether a product for hygienic handwash reduces the release of transient microbial flora on hands when used to wash the artificially contaminated hands of volunteers. In our study, we used 20 people for all tests. When tested in accordance with this test, the mean reduction of the release of the test organism Escherichia coli achieved by the hygienic handwash with the product under test shall be larger than that achieved by a specified reference hygienic handwash (non-antimicrobial soft soap).
  • the log reduction rate and % of reduction of the test microorganism after application of the probiotic-based hand soap and non-antimicrobial soft soap is shown in Table 1.
  • probiotic-based hand soap when applied in the manner described meet the requirement stipulated in EN 1499 i.e. the mean reduction of the release of test organisms achieved by the tested product shall be larger than that achieved by the reference product (non-antimicrobial soft soap) and therefore passes the European Standard EN 1499 for a hygienic handwash.
  • the E 1500 European Standard specifies a test method simulating practical conditions for establishing whether a product for hygienic handrub reduces the release of transient microbial flora on hands when rubbed onto the artificially contaminated hands of volunteers.
  • the mean reduction of the release of the test organism Escherichia coli achieved by the hygienic handrub with the product under test shall be at least not inferior to that achieved by a specified reference hygienic handrub (60 % volume concentration of propan-2-ol).
  • the log reduction rate and % of reduction of the test microorganism after application of the probiotic-based hand spray and propan-2-ol (60%) is shown in Table 2.
  • probiotic-based hand spray when applied and rubbed into the hands get reduction of about 99.9 % (log reduction of 3.697651187), higher than the reduction % of the reference product propan-2-ol 60% (w/v) (log reduction of 3.518963).
  • probiotic-based hand spray when applied in the manner described in the Standard meets the requirement stipulated in EN 1500- the mean reduction of the release of test organisms achieved by the test product is not significantly smaller than that achieved by the reference product (propan-2-ol 60% w/v) and therefore passes the European Standard EN 1500 for a hygienic hand rub.
  • biotechnology cleaning products containing probiotic bacteria was found effective in reducing the number of viable bacteria remaining on the fingertips after artificial contamination. They are eco-friendly; thus, they can reduce the exposure to chemical disinfectants. It is necessary to establish a new biosafety protocol which includes adequate disinfection by appropriate cleaning products. The information obtained from this study can be used to develop proper interventions to improve disinfection practices in all aspects of our lives. The only demand set by this method is that the frequency of disinfection is kept constant, so the probiotic bacteria (the good bacteria).

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Abstract

The invention relates to probiotic hygiene solutions composed of probiotic non-pathogenic bacteria as an innovative solution for efficient cleaning, sanitation, disinfection and protection against contamination, elimination and re-contamination with Coronaviridae (family of Corona viruses, including SARS-Cov-2, causing a global pandemic of Covid 19) with immediate and time-extended (hours' or days) effective residual after-protection applicable to hard surfaces, soft surfaces, human and animal skin, plants and the general environment as a natural- based product with numerous health and environmental benefits.

Description

Elimination of Coronaviridae (including SARS-Cov-2) viral contamination and recontamination with Probiotic hygienic solutions
Technical field to which the invention relates
The invention relates to probiotic hygiene solutions and methods for the use of probiotic non- pathogenic bacteria as an innovative solution for efficient cleaning, sanitation, disinfection and protection against contamination, elimination and re-contamination and of Coronaviridae (family of Corona viruses, including SARS-Cov-2, causing a global pandemic of Covid 19) with immediate, residual and prolonged efficacy applicable to hard surfaces, soft surfaces, human and animal skin, plants and the general environment as a natural-based product with numerous health and environmental benefits.
The invention is in the field of human needs, chemistry and procedures, which according to the International Patent Classification belongs to the:
Sub area of cleaning and applying liquids and other liquid materials to surfaces
Sub area preparations for medical and cosmetic use, i.e. medicines containing probiotics and in the sub area of microbiology, i.e. measurement and testing of procedures involving microorganisms (bacteria).
According to the International Patent Classification, the invention can be classified into the following classes: A61K 35/741 , A61L 2/18, C12Q 1/00, C12Q 1/02, C12Q 1/689, B08B 3/04, B05D 1/00.
Technical problem solved by the invention
It is hereby disclosed that probiotic hygienic solution, is based on biological active substance and has the following new applications:
- Effectively eliminate viruses from the Coronaviridae family in less than 60 seconds, including the novel SARS-Cov-2 (Corona virus) that causes Covid 19.
- Effective After-protection - they offer hours or days of effective protection against Coronaviridae viruses after their application on skin or surface. This means, once the surface is being treated (cleaned or sprayed), any contamination with Coronaviridae family of viruses from external source will not sustain and the virus will not survive.
- Non-toxic to skin or surfaces - due to absence of chemical components.
The invention is tested via probiotic hand spray, which is able to reduce and, in less than one minute, completely eliminate the growth of SARS-Cov-2 virus. The product has also general antimicrobial properties and has been tested against other most common pathogenic microbial species namely: Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9037, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.
In overall, our invention confirms that probiotic solution (hand spray) is able to reduce and, completely eliminate the growth of specific pathogenic microbial species namely Coronaviridae family of viruses, including the novel SARS-Cov-2.
The in vitro tests allowed the reduction in microbial count of the pathogens to be verified under controlled conditions, thus verifying the efficacy of tested Probiotic Hand spray to combat microorganisms where there is no risk of recontamination. The results achieved confirms that the continuous and constant action of this probiotic-based solution is able to reduce and eliminate the proliferation of tested microorganisms.
Based on the results obtained, this invention is effective and innovative product for environmental management. Probiotics are ecological, easy to use and biodegradable. They render the skin, surfaces and the overall environment hygienically stable and are able to survive on and colonize non biological surfaces, combatting the proliferation of Coronaviridae viruses and other pathogens. In this study they were also found to perform well on different surfaces. Probiotics are therefore effective innovative products for sanitizing different environment and constitute a valid “green” alternative to the chemical disinfectants used up to now.
State of the Art
So far, there are disinfection products on the market that eliminate Coronaviridae viruses based only on chemical active components (alcohol, peroxide, bleach etc.) that offer only elimination of the viruses, but not the after-protection. This means, the surface or skin will be free from the viral contamination only after the solution dries out but not after few hours or days. All these chemical products also have allergenic and toxic properties for the people and the environment, frequently causing skin irritations, itching and other unwanted consequences.
World health organization (WHO) in their Guide for local production of handrub formulations, recommends two types of formulations for immediate disinfection offering no antiviral afterprotection:
1.Chemical active formula - Final concentrations: Ethanol 80% (v/v), Glycerol 1.45% (v/v), Hydrogen peroxide 0.125%(v/v), and
2. Chemical active formula - Final concentrations: Isopropyl alcohol 75 % (v/v), Glycerol 1.45% (v/v) and Hydrogen peroxide 0.125% (v/v)
In the current state of the art in accordance with database searches: Google patent, European Patent Office, USPTO, WIPO, and market search, there are known patents relating to inventions containing probiotics, procedures for their obtaining and use in the treatment of patients who have COVID 19, but used for the treatment of allergic diseases. Then patents relating to cleansing agents containing probiotics, which are used to improve the skin barrier.
According to search of the international patent classification codes reveals that there are only procedures and compositions for detecting suitable bacteria.
A review of the European patent application EP2556823 has established that there are cosmetic products that contain probiotic microorganisms as cleaning agents, but also contain chemical ingredients such as alkyl sulfate and alkyl polyglycol cholesterol sulphate salts. They are used to improve the skin barrier. Compared to the invention, it does not refer to cleaning or disinfecting the surface from viral contamination, i.e. SARS-Cov-2 (corona virus). Russian patent number RU2699540 C2 relates to compositions containing probiotic bacteria and procedures for their use in the treatment of immune skin diseases.
In the current state of the art, there are no patents for cleaning hard surfaces, soft surfaces, skin of humans and animals, plants, which are containing probiotics and can eliminate the corona virus. Additionally, the invention has a residual (time-extended) effectiveness that can last for hours and days.
Description of the invention
Any surface or environment can be contaminated with a variety of microorganisms derived from various sources, some of them pathogen (harmful) to humans.
A pathogen is the term used to describe an infectious microorganism or agent, such as a virus, bacterium, protozoan, prion, viroid or fungus; or this is an organism that causes disease.
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. When not inside an infected cell or in the process of infecting a cell, viruses exist in the form of independent particles, or virions, consisting of genetic material (DNA or RNA) and a protein coat.
Probiotics are live microorganisms promoted with claims that they provide health and environmental benefits or in the case of this patent application as proven by the microbiological tests provided in this patent application, non-pathogen good bacteria safe for handling and use that promote biological dirt degradation and anti-microbial and antiviral disinfection and after-protection. Probiotics are even considered generally safe to consume. The novel SARS-Cov-2 (Corona) virus that causes Covid-19 infections belongs to Coronaviridae family of viruses. Its particles (or “virions”) are spheres with diameters of approximately 0.125 microns (125 nm). The smallest particles are 0.06 microns, and the largest are 0.14 microns. Coronaviridae Viruses can infect all types of life forms: animals, plants and humans and can survive on hard and soft surfaces for up to 48 hours mainly due to the biofilm created by pathogen (bad) bacteria which virus is using as a host.
In 2020, the outbreak of Covid 19 viral pathogen caused 38.450.000 infections and 1.092.252 deaths worldwide (statistic until 15 October 2020, the day of writing the patent fiche).
The invention is new break-through solution composed of probiotic non-pathogen bacteria as an innovative solution for efficient cleaning, sanitation, disinfection and protection against contamination, elimination and re-contamination with Coronaviridae (Corona-family of viruses, including SARS-Cov-2 virus that causes the pandemic of Covid 19) with immediate in less than 60 seconds and time-extended efficacy applicable on hard surfaces, soft surfaces, human and animal skin, plants and the general environment as a natural-based product with numerous health and ecological benefits. They offer hours or days of effective residual after-protection against Coronaviridae viruses after their application on abovementioned skin or surfaces. This means, once the surface is being treated (cleaned or sprayed), any contamination with Coronaviridae family of viruses from external source will not sustain and the virus will not survive. The solution is aqueous active substance of Probiotic non-pathogen bacteria belonging to Class 1 EU / Generally Regarded As Safe and mixed with water - resulting in sufficient end-concentrations of viable probiotic bacteria. Therefore it provides non-toxic activity to skin or surfaces - due to absence of chemical components.
The application method of such innovative cleaning, sanitation, disinfection and protection solutions is to be made by means of manual mechanical application, mopping, rinsing, spraying or machine application by spraying trough nozzles in hand-disinfection machines, disinfection tunnels, spray cabins or any external human application by air- borne solution particles.
The mechanism of action is based on that the probiotic-based product contains good bacteria (Class 1 EU classification, non-pathogen) that break up biofilm, reduce the ability for pathogenic bacteria to attach to a surface again, and create a protective shield against pathogens so the biofilm can’t survive while the probiotics remain active. Probiotic-based cleaning products employ the capability of living microorganisms to produce extracellular enzymes, such as cellulases, proteases and ureases. These enzymes can degrade high molecular weight compounds often associated with dirt and effectively mitigate unfavourable odours by further metabolism of intermediates from these processes. It employs sporeforming bacteria that compete pre-existing pathogens for nutrients and outgrow (Dural-Erem et al., 2019; Spok et al., 2018). While the main mechanism is considered to be based on competitive antagonism or exclusion, the ability to produce and release antimicrobial compounds also play a significant role in inhibiting growth of microbial cells or causing death (D'Accolti et al., 2019; Piewngam et al., 2018; Spok et al., 2018). Multiple members of the genus Bacillus are well recognized for the capability to produce multiple compounds with antimicrobial properties (JezewskaFrqckowiak et al., 2018). For example, surfactins produced by various Bacillus species, have detergent-like properties and have been shown to exhibit antimicrobial properties by interacting with bio-membranes (Bartal et al., 2018). Other microorganisms such as the lactic acid bacteria can inhibit growth by lowering the pH in its environment (Dural-Erem et al., 2019). Apparently, a synergy of various mechanisms could come into play when it comes to the cleaning effect of these probiotic-based products (Spbk et al., 2018).
Brief description of the Figures
Figure 1: Disk diffusion test: A clear zone or ring is formed around the disk after incubation if the agent inhibits microbial growth.
Figure 2: Protocol for testing protection time: in order to assess how effectively probiotics were able to compete against above mentioned microbial strains in the absence of external factors linked to recontamination. During this test the two surfaces (floor and desk, with area of 30*30 cm) were first contaminated using sterile swabs soaked with the microbial solutions with the initial concentration of approximately 1.5*10® CFU/mL, and then sanitized with spraying of tested probiotic-based cleaner. Samples of each microorganism were taken daily over a five-days period and inoculated on agar medium. Then bacterial strains were incubated at 37°C for 18-24 hours, and fungal strains were incubated at room temperature, 3-5 days.
Figure 3: The product has also general antimicrobial properties and has been tested against other most common pathogenic microbial species namely: antimicrobial activity of probiotic based solution (original and diluted 1 :10 Handspray against Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9037, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.
Several tests have been performed to prove the effectiveness of the probiotic solution:
- Efficiency Test of Probiotic handspray vis-a-vis SARS-Cov-2
- EN 1500 disinfection standard test for hand-rub, Ell standard The first test is a PCR sampling test on a surface populated with SARS-Cov-2. According to the analytical rapport i.e. efficiency test of probiotic hand spray vis-a-vis Sars-Cov-2 performed by TLR International laboratories in Rotterdam is confirmed that after use of probiotic hand spray there is no detection of Coronaviridae / SARS-Cov-2.
The second test EN 1500 disinfection standard test for hand-rub, EU standard is aimed to evaluate and compare the efficacy of probiotic-based hand soap and hand spray in reducing the microbial load present on artificially contaminated hands; and to compare the antimicrobial efficacy of the tested products with those of reference indicated in the methodology of EN1499 and EN1500, which used the non-antimicrobial soft soap and propan-2-ol 60% (v/v).
The EN 1499 and EN 1500 are a European Standard test methods that evaluates the efficacy of a hygienic handwash and handrub by measuring the number of viable bacteria remaining on the fingertips after contamination and handwash (handrub) exposure.
The EN 1499 standard is used to test antiseptic soaps and requires 12-15 subjects. The EN 1500 is used to test antiseptic hand rubs and requires 18-22 subjects. Experimental contamination is with a non-pathogenic strain of Escherichia coli. Subjects are allotted randomly to two groups of approximately the same size, where one group applies the test formulation, and the other a reference antiseptic solution using a standardised procedure. According to the EN 1499, the mean reduction caused by the test product in the numbers of viable bacteria shall be significantly higher than that obtained with the non-antimicrobial soft soap. To meet the EN 1500 requirement, the mean reduction in the numbers of viable bacteria shall not be significantly inferior to that with the reference alcohol-based hand rub (propan-2-ol 60% (v/v)).
The pre- and post-values recovered from the fingertips are evaluated against one another, resulting in a ratio called the reduction factor. The reduction factor provides a quantitative measure of the antimicrobial efficacy. This is determined by comparing average counts of viable microorganism colony forming units of the pre-values to the average counts after exposure to the hygienic handrub.
Probiotic-based products, hand soap and hand spray, were used in experiments for reducing the microbial load present on artificially contaminated hands, according to protocol EN1499 and EN1500.
These biotechnology cleaning products contain non-pathogenic probiotic bacteria. These nonpathogenic bacteria are considered as a GRAS (generally recognized as safe) organism as they do not deemed to cause human diseases, with no impact on plants or animals. They also produce antimicrobial components active against many dangerous pathogenic bacteria. Due to their safety and activity on pathogens, biotechnology products have been used probiotic sanitation, demonstrating a significant reduction in microbial contamination with a stable effect over time.
Beside basic EN 1499 and EN 1500 protocols, we applied additional hybrid tests based on EN 1499 and EN 1500. In all additional tests we made mixed and extended EN protocol (with different test substances and different contact time, up to 120 min).
The EN1499 European Standard specifies a test method simulating practical conditions for establishing whether a product for hygienic handwash reduces the release of transient microbial flora on hands when used to wash the artificially contaminated hands of volunteers. In our study, we used 20 people for all tests. When tested in accordance with this test, the mean reduction of the release of the test organism Escherichia coli achieved by the hygienic handwash with the product under test shall be larger than that achieved by a specified reference hygienic handwash (non-antimicrobial soft soap). The log reduction rate and % of reduction of the test microorganism after application of the probiotic-based hand soap and non-antimicrobial soft soap is shown in Table 1.
Table 1 - Results after EN1499
According to the results, probiotic-based hand soap when applied in the manner described meet the requirement stipulated in EN 1499 i.e. the mean reduction of the release of test organisms achieved by the tested product shall be larger than that achieved by the reference product (non-antimicrobial soft soap) and therefore passes the European Standard EN 1499 for a hygienic handwash.
The E 1500 European Standard specifies a test method simulating practical conditions for establishing whether a product for hygienic handrub reduces the release of transient microbial flora on hands when rubbed onto the artificially contaminated hands of volunteers. When tested in accordance with this test, the mean reduction of the release of the test organism Escherichia coli achieved by the hygienic handrub with the product under test shall be at least not inferior to that achieved by a specified reference hygienic handrub (60 % volume concentration of propan-2-ol). The log reduction rate and % of reduction of the test microorganism after application of the probiotic-based hand spray and propan-2-ol (60%) is shown in Table 2.
Table 2 - Results after EN1500
The above results showed that probiotic-based hand spray when applied and rubbed into the hands get reduction of about 99.9 % (log reduction of 3.697651187), higher than the reduction % of the reference product propan-2-ol 60% (w/v) (log reduction of 3.518963). So, probiotic-based hand spray when applied in the manner described in the Standard meets the requirement stipulated in EN 1500- the mean reduction of the release of test organisms achieved by the test product is not significantly smaller than that achieved by the reference product (propan-2-ol 60% w/v) and therefore passes the European Standard EN 1500 for a hygienic hand rub.
In order to see the performance of the combined effect of our two products, we conducted this hybrid test with simultaneously application of probiotic-based hand spray and hand soap (in that order) on previously artificially contaminated hands. After that, we evaluated the number of bacterial cells on both hands in all volunteers immediately, after 5 minutes of action, after 30 minutes, 60 minutes and after 120 minutes. In the meantime, the volunteers freely moved and worked usual things. Table 3: Results after hybrid test 1 (EN1500+ EN1499)
The results in table 3 showed significantly lowering the number of tested bacterium with combined use of products immediately and after 5 and 30 minutes, but further on it was observed slowly increasing of number of test microorganism. It was expecting result, because volunteers continued to work, and this way the number of bacteria continued to raise. Anyway, the number of test microorganism after 120 minutes was still low (7.17*10^ CFU mH) compare to initial number of bacterial suspension (2.2*10® CFU mH). This is due to the fact that probiotic bacteria from these two products had prolonged action against test microorganism, so the number of the test microorganism was low after 2 hours of application of the tested products. In case of using alcoholic disinfectant, an important disadvantage is the unspecific action of these agents, killing both good and bad micro-organisms. This results in a surface (hands) free of micro-organisms, making it readily available to any remaining pathogen in order to bloom within a very limited timeframe. Hence, this type of disinfection results in a fast reduction of the number of microorganisms of which the effect is very short and unstable. But, when we use probiotic-based products, during the cleaning procedure a layer of probiotic bacteria is placed on the treated surface, immediately occupying the ‘field’ by good bacteria. They will consume all remaining food sources, leaving nothing behind for potential pathogenic invaders looking for space and food.
Also, when the probiotic products are applied to hands, a competition for space, food and moisture is established. This immediately results in the fact that pathogenic bacteria, by means of quorum sensing, will inform each other about these unfavourable conditions, leading to a dormant phase and their subsequent removal. There are some data who said that it was estimate we have around 1,500 bacteria living on each square centimetre of skin on our hands According to WHO (2009) it was found that total bacterial counts on the hands have ranged from 3.9 * 10 to 4.6 * 10 CFU/cm .
Also, we wanted to see the activity of probiotic-based hand spray, when we use and immediately after that we use non-antimicrobial soft soap. So, we made a hybrid test with these two components, and the mean numbers of E. coli after 5, 30, 60 and 120 minutes are given in Table 4.
Table 4: Results after hybrid test 2 (EN1500+ non-antimicrobial soft soap) mean number of 20 volunteers/ both hands
| mean mean CFU ml" mean CFU ml" mean CFU ml" mean CFU ml" mean CFU ml"
I CFU ml"1 1 E. coli after 1 E. coli after 1 E. coli after 1 E. coli after 1 E. coli after ] j E. coli- probiotic- probiotic- probiotic- probiotic- probiotic-
I prevalues based hand based hand based hand based hand based hand !
( spray+non- spray+ non- spray+ non- spray+ non- spray+ non-
■ antimicrobial antimicrobial antimicrobial antimicrobial antimicrobial i soft soap- soft soap- soft soap- soft soap- soft soap -
■ postvalues postvalues postvalues postvalues postvalues i after 5 min after 30 min after 60 min after 120 min J
As we expected, the number of test microorganism E.coli was reduced slightly because of the use of non-antimicrobial soft soap. Namely, simultaneously application of probioticbased hand spray and non-antimicrobial soft soap will lead to washing out the active probiotic bacteria from our product, and that will be enable other bacteria to settle on hands of volunteers.
The application of biotechnology cleaning products containing probiotic bacteria was found effective in reducing the number of viable bacteria remaining on the fingertips after artificial contamination. They are eco-friendly; thus, they can reduce the exposure to chemical disinfectants. It is necessary to establish a new biosafety protocol which includes adequate disinfection by appropriate cleaning products. The information obtained from this study can be used to develop proper interventions to improve disinfection practices in all aspects of our lives. The only demand set by this method is that the frequency of disinfection is kept constant, so the probiotic bacteria (the good bacteria).

Claims

PATENT CLAIM
1. The probiotic hygienic solution composed of probiotic non-pathogen bacteria as an innovative solution for efficient cleaning, sanitation, disinfection and protection against contamination, elimination and re-contamination with Coronaviridae (Corona-family of viruses, including SARS-Cov-2 virus that causes the pandemic of Covid 19) which is characterized with immediate and time-extended (hours or days) effective residual afterprotection applicable on hard surfaces, soft surfaces, human and animal skin, plants and the general environment.
EP21886990.7A 2020-11-02 2021-11-01 Elimination of coronaviridae (including sars-cov-2) viral contamination and recontamination with probiotic hygienic solutions Pending EP4263778A2 (en)

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