EP4240161A1

EP4240161A1 - Biocidal composition

Info

Publication number: EP4240161A1
Application number: EP21806023.4A
Authority: EP
Inventors: Martin Richards; Nicholas James HARMER
Original assignee: Smarti Environmental Ltd
Current assignee: Smarti Environmental Ltd
Priority date: 2020-11-06
Filing date: 2021-11-04
Publication date: 2023-09-13
Also published as: WO2022096881A1; GB202017605D0

Abstract

The invention provides a biocidal composition comprising: — at least one protease selected from a serine protease, a metalloprotease and a cysteine protease; and — at least one lipase selected from a phospholipase, an esterase and a microbial lipase; and a carrier.

Description

Biocidal Composition

Technical Field of the Invention

The present invention relates to a biocidal composition and to its use in protecting surfaces from microorganisms such as coronaviruses.

Background to the Invention

It is known that coronaviruses such as Covid- 19 persist on surfaces such as metal, wood, plastic, ceramics and glass for between two and five days, meaning there is an increased risk of a person coming into contact and subsequently becoming infected by the virus. While coronaviruses are readily disrupted by cleaning solutions such as bleach, ethanol, or detergents, these solutions do not provide surfaces with protection after the agent has been wiped down. Accordingly, there is an urgent need to protect surfaces against coronaviruses and other microorganisms for extended periods where surface cleaning cannot meet the rate at which the surface is touched by different people.

In light of the above it is an object of embodiments of the present invention to provide a composition that is able to protect surfaces from viruses, particularly coronaviruses, for longer periods relative to existing cleaning solutions.

It is also an object of embodiments of the present invention to provide a composition that is, relative to existing cleaning solutions, more effective at eliminating or reducing coronavirus levels on surfaces.

It is another object of embodiments of the present invention to provide an improved cleaning solution which is economic.

Summary of the Invention

According to a first aspect of the invention there is provided a biocidal composition comprising:

— at least one protease selected from a serine protease, a metalloprotease and a cysteine protease; and

— at least one lipase selected from a phospholipase, esterase and a microbial lipase; and — a carrier.

The biocidal composition enables coronavirus levels on surfaces to be reduced by 99.9%. Moreover, it has been demonstrated that the biocidal composition is able to substantially eliminate coronaviruses from surfaces for a period of at least 16 hours after a single application of the biocidal composition to the surface. Therefore, the biocidal composition offers improved biocidal performance relative to existing cleaning solutions and only needs to be applied once daily as part of a regular cleaning routine. In particular, it has surprisingly been found that compositions comprising a mix of the claimed proteases and lipases exhibit improved coronavirus killing performance relative to compositions which comprise only one type of enzyme (protease or lipase). Without wishing to be bound by theory, it is believed that the improved performance is due to the presence of the specific proteases and lipases in the composition which are able to respectively hydrolyse proteins and ester bonds in the lipid bilayer which envelops and protects coronaviruses; whereas other types or combinations of proteases and lipases may not have such efficacy.

In some embodiments the serine protease may comprise a subtilisin or alkaline protease. In particular, the serine protease may comprise a subtilase. In some embodiments, the metalloprotease may be obtained from Bacillus subtilis. In some embodiments the phospholipase may comprise a pancreatic phospholipase.

The biocidal composition may comprise at least two proteases and at least one lipase. The biocidal composition may comprise a metalloprotease, a cysteine protease and a lipase. The biocidal composition may comprise a metalloprotease, a cysteine protease and at least one lipase independently selected from the group comprising: a phospholipase, an esterase, and a microbial lipase. In particular, the biocidal composition may comprise a metalloprotease, a cysteine protease and an esterase or microbial lipase. Biocidal compositions comprising this combination of enzymes are particularly effective at eliminating coronaviruses at surfaces for extended periods of time.

The biocidal composition may comprise a pair of enzymes for hydrolysing proteins and ester bonds in the virus’ lipid bilayer. In particular, it has been found that good biocidal activity can be obtained when the biocidal composition comprises one protease and one lipase. It will be appreciated that the composition may comprise other enzymes, such as fragrance enzymes, which do not play a role in breaking down the virus’ lipid bilayer.

In some embodiments, the biocidal composition may comprise a metalloprotease and an esterase or microbial lipase. By applying a biocidal composition comprising this combination of enzymes improved biocidal activity can be obtained over conventional cleaning solutions and over compositions that independently contain a metalloprotease or a lipase selected from esterase or microbial lipase.

In some embodiments, the biocidal composition may comprise a cysteine protease and a lipase. The biocidal composition may comprise a cysteine protease and a lipase independently selected from the group comprising: a phospholipase, an esterase, and a microbial lipase. In some embodiments the biocidal composition may comprise a cysteine protease and an esterase or microbial lipase. Relative to conventional cleaning solutions, improved biocidal performance is obtained when a biocidal composition comprising this combination of enzymes is applied to a surface.

In some embodiments the biocidal composition may comprise a serine protease and an esterase or microbial lipase. Improved biocidal activity was obtained over conventional cleaning solutions and over compositions which independently contained a serine protease and an esterase or microbial lipase.

In some embodiments the biocidal composition may comprise a metalloprotease and a phospholipase, preferably a pancreatic phospholipase. This combination of enzymes is effective at reducing coronavirus levels and exhibits improved performance relative to compositions comprising metalloproteases and phospholipases independently and relative to conventional cleaning solutions.

In some embodiments the biocidal composition may comprise cysteine protease and a phospholipase, preferably a pancreatic phospholipase. This combination of enzymes is effective at reducing coronavirus levels and exhibits improved performance relative to compositions comprising cysteine proteases and phospholipases independently and relative to conventional cleaning solutions. In some embodiments the biocidal composition may comprise a serine protease and a phospholipase, preferably a pancreatic phospholipase. This combination of enzymes exhibits improved biocidal activity over compositions which independently contain a serine protease and a phospholipase. Improved biocidal activity is also observed relative to known cleaning solutions without enzymes.

Each protease and lipase may be independently present in the composition at a concentration of between 0.001 and 1 %wt., preferably between 0.001 and 0.5 %wt. and more preferably between 0.001 and 0.1 %wt. In some embodiments each enzyme is independently present in the composition at a concentration of between 0.001 %. and 0.5 %wt. In some embodiments, the protease may be present at a concentration of between 0.001 and 0.1 %wt. of the final composition; while the lipase may be present at a concentration of between 0.001 and 0.02 % wt, such as between 0.001 and 0.01 % wt. of the final composition.

In preferred embodiments the serine protease may be PROMOD 439L, the metalloprotease may be PROMOD 24L and/or the cysteine protease may be PROMOD 950L, all manufactured by Biocatalysts Limited, Unit 1, Cefn Coed, Parc Nantgarw, Cardiff, CF15 7QQ, Wales, UK.

In preferred embodiment, the phospholipase may be LIPOMOD 699L or LIPOMOD 833L and/or the esterase may be LIPOMOD 34MDP all manufactured by Biocatalysts Limited, Unit 1, Cefn Coed, Parc Nantgarw, Cardiff, CF15 7QQ, Wales, UK.

Particularly useful combinations, having the highest virucidal activity against coronaviruses are:

PROMOD 24L, PROMOD 950L and LIPOMOD 34MDP; PROMOD 24L and LIPOMOD 34MDP; PROMOD 950L and LIPOMOD 699L; PROMOD 24L and LIPOMOD 699L; PROMOD 24L, PROMOD 950L and LIPOMOD 699L; and PROMOD 950L and LIPOMOD 34MDP.

In preferred embodiments the composition may comprise one of the following combinations:

In some embodiments the composition may comprise a fragrance enzyme. When the biocidal composition comprises two proteases and one lipase, the composition may comprise 0.001 to 1 %wt. of metalloprotease, 0.001 to 1 %wt. of cysteine protease and 0.001 to 1 %wt. of esterase or microbial lipase. Suitably, the composition may comprise 0.001-0.5 %wt. of metalloprotease, 0.001-0.5 %wt. of cysteine protease and 0.001 - 0.5 %wt. of esterase or microbial lipase. The composition may comprise 0.001-0.05 %wt. of metalloprotease; 0.001-0.1 %wt. of cysteine protease; and 0.001-0.05 %wt. or 0.001-0.01 %wt. of esterase or microbial lipase. In this way, a biocidal composition can be obtained with robust efficacy at relatively low cost.

The biocidal composition may comprise a polymer derived from ethylene oxide. Suitably, the composition may comprise ethylene glycol. Without being bound by theory, it is believed that the presence of a polymer derived from ethylene oxide such as ethylene glycol may protect the enzymes in the carrier and/or on the surface to an extent that they are able to remain active and biocidally effective over extended periods of time. The biocidal composition may comprise at least one glycol ether of a C5-C15 alcohol, preferably of a CIO alcohol. The CIO alcohol may be independently selected from the group comprising: 1-decanol and 2-propylheptanol. The glycol ether may comprise at least one glycol ether independently selected from the group comprising: a polyethylene glycol-based glycol ether, a polypropylene glycol-based glycol ether, and a mixed polyethylene glycol and polypropylene glycol-based glycol ether. In a particular embodiment, the biocidal composition comprises a Deceth glycol ether, preferably Deceth-7.

The biocidal composition may comprise at least one isothiazolinone derivative. The isothiazolinone derivative may be independently selected from the group comprising: methylisothiazolinone, chloromethylisothiazolinone, benzisothiazolinone, octylisothiazolinone, dichlorooctylisothiazolinone, and butylbenzisothiazolinone. In preferred embodiments, the biocidal composition comprises benzisothiazolinone.

The carrier may comprise a liquid, preferably the liquid comprises a disinfectant. It has been shown that the addition of enzyme mixtures to a known cleaning solution improves the effectiveness of the cleaning solution in eliminating coronaviruses from a surface. The carrier may comprise an alcohol, suitably an ethanol- based alcohol. In particular, the alcohol may comprise 2-butoxy ethanol. The carrier may comprise an acid, suitably a sulfonic acid. In particular, the acid may comprise benzenesulfonic acid. The acid may comprise a benzenesulfonic acid alkyl derivative, preferably a benzenesulfonic acid CIO- 14 alkyl derivative. In some preferred embodiments, the carrier comprises an alcohol and an acid. Such a combination provides for effective delivery of the enzymes to a surface requiring disinfection to protect it for longer periods of time against microorganisms.

In preferred embodiments, the carrier comprises at least one detergent. The detergent may comprise one or more detergents independently selected from the group comprising: an anionic, cationic, zwitterionic, and non-ionic surfactant. In preferred embodiments, the detergent comprises an anionic detergent and/or a non-ionic detergent. A carrier comprising detergents, and in particular anionic and/or non-ionic detergents, is believed to loosen up the structure of viral particles allowing the enzymes in the composition better access to virus particles.

In some preferred embodiments, the carrier comprises at least one glycol ether non-ionic detergent. The glycol ether non-ionic detergent may comprise at least one glycol ether independently selected from the group comprising: a polyethylene glycol (PEG)-based glycol ether, a polypropylene glycol-based glycol ether, and a mixed polyethylene glycol and polypropylene glycol-based glycol ether. The glycol ether non- ionic detergent preferably comprises a PEG-based glycol ether. The glycol ether non- ionic detergent may comprise a glycol ether of a C5-C25 alcohol, preferably of a C8- C20 alcohol, more preferably of a C9-C15 alcohol, and most preferably of a C10-C13 alcohol. In some preferred embodiments, the carrier comprises a glycol ether of a CIO and/or C13 alcohol. The CIO alcohol may be independently selected from the group comprising: 1-decanol and 2-propylheptanol. The C13 alcohol may preferably comprise 1 -tridecanol. The glycol ether may comprise between 2-25 alkylene glycol units, more preferably between 3-20, 4-18, 5-16, 6-14, 7-12, or most preferably between 7-10 repeat units. In preferred embodiments, the carrier comprises a glycol ether with 7 or 10 alkylene glycol repeat units.

In preferred embodiments, the carrier comprises a PEG-based glycol ether comprising a C5-C15 alkoxy group, C7-C14, C8-C14, C9-C13, or C10-C13 alkoxy group; and a PEG chain comprising between 3-15 PEG units, more preferably 5-10 PEG units, or most preferably 7-10 PEG units.

In one preferred embodiment, the carrier comprises a decyl glycol ether, preferably PEG-7 decyl ether.

In a particularly preferred embodiment, the carrier comprises a tridecyl glycol ether, preferably PEG- 10 tridecyl ether.

In some embodiments, the carrier comprises at least one alkyl anionic detergent or salts thereof, preferably an alkyl sulfate or phosphate or salts thereof, and most preferably an alkyl sulfate or salts thereof. The carrier may comprise a C5-C25 alkyl sulfate, preferably a C6-C20, C7-C18, C8-C16, C9-C14, C10-C13, more preferably a C10-C12 alkyl sulfate, or most preferably lauryl sulfate, or salts thereof.

In some particularly preferred embodiments, the carrier comprises an anionic detergent and a non-ionic detergent.

In preferred embodiments, the carrier comprises an anionic detergent and a PEG-based glycol ether non-ionic detergent. The PEG-based glycol ether is preferably as described above. The PEG-based glycol ether may comprise a C5-C15 alkoxy group, C7-C14, C8-C14, C9-C13, or C10-C13 alkoxy group; and a PEG chain comprising between 3-15 PEG units, more preferably 5-10 PEG units, or most preferably 7-10 PEG units.

In preferred embodiments, the carrier comprises a non-ionic detergent and an alkyl anionic detergent or a salt thereof. The alkyl anionic detergent is preferably as described above. The alkyl anionic detergent preferably comprises an alkyl sulfate or a salt thereof, preferably a C5-C25, C8-C16, or C10-C13 alkyl sulfate. Most preferably, the alkyl anionic detergent comprises a lauryl sulfate or a salt thereof.

In preferred embodiments, the carrier comprises at least one PEG-based glycol ether non-ionic detergent and an alkyl anionic detergent or salt thereof. The carrier may comprise a PEG-based glycol ether with a C5-C15 alkoxy group and 3-15 PEG units, or a PEG-based glycol ether with a C8-C14 alkoxy group and 5-10 PEG units, or a PEG-based glycol ether with a C10-C13 alkoxy group and 7-10 PEG units; and an alkyl sulfate or salt thereof, preferably a C5-C25 alkyl sulfate or salt thereof. The carrier may comprise a PEG-based glycol ether with a C5-C15 alkoxy group and 3-15 PEG units, or a PEG-based glycol ether with a C8-C14 alkoxy group and 5-10 PEG units, or a PEG-based glycol ether with a C10-C13 alkoxy group and 7- 10 PEG units; and a C8-C16 alkyl sulfate or salt thereof.

The carrier may comprise a PEG-based glycol ether with a C5-C15 alkoxy group and 3-15 PEG units, or a PEG-based glycol ether with a C8-C14 alkoxy group and 5-10 PEG units, or a PEG-based glycol ether with a C10-C13 alkoxy group and 7- 10 PEG units; and a C10-C13 alkyl sulfate or salt thereof, preferably a lauryl sulfate or salt thereof.

In a particularly preferred embodiment, the carrier comprises PEG- 10 tridecyl ether and sodium lauryl sulfate.

Salts of the alkyl anionic detergent as described in the above statements may comprise an ammonium cation or a metal cation. The metal cation may be independently selected from the group comprising: ammonium, sodium, potassium, lithium, and copper.

In preferred embodiments, the carrier comprises at least one film-forming agent. The film- forming agent may be independently selected from the group comprising: a polyvinylpyrrolidone, an acrylate, an acrylamide, a styrene, and combinations, blends or copolymers thereof. In preferred embodiments, the film-forming agent may be independently selected from: an acrylate, a styrene, and combinations, blends, or copolymers thereof. In preferred embodiments, the film- forming agent comprises a film-forming polymer. The film-forming polymer may preferably comprise a polyacrylate and/or an acrylate copolymer. In some particularly preferred embodiments, the film-forming polymer comprises an acrylate-styrene copolymer. When applied to a surface, such film-forming agents enable the enzymes and other actives in the biocidal composition to contact the surface for longer periods of time, enabling protection of the surface from microorganisms, particularly viruses, for > 16 hours.

In preferred embodiments, the carrier comprises at least one detergent and at least one film-forming agent, preferably as described above. The carrier may preferably comprise an anionic detergent and/or a non-ionic detergent, and at least one filmforming agent, preferably as described above. In preferred embodiments, the carrier comprises an anionic detergent, a non-ionic detergent, and at least one film-forming agent. In particularly preferred embodiments, the carrier comprises at least one alkyl anionic detergent or a salt thereof, preferably an alkyl sulfate or salt thereof as described above; at least one glycol ether non-ionic detergent, preferably a PEG-based glycol ether as described above; and at least one film-forming agent, preferably a film-forming polymer as described above, and more preferably a polyacrylate and/or an acrylate copolymer. In an especially preferred embodiment, the carrier comprises a lauryl sulfate or a salt thereof, PEG- 10 tridecyl ether, and an acrylate- styrene copolymer.

In some preferred embodiments, the biocidal composition comprises at least two proteases and at least one lipase, and a carrier comprising an alcohol and an acid, preferably an ethanol-based alcohol and a sulfonic acid. In some preferred embodiments, the biocidal composition comprises a metalloprotease, a cysteine protease and a lipase, preferably an esterase or microbial lipase; and the composition also comprises a carrier comprising an alcohol and an acid, preferably an ethanol-based alcohol and a sulfonic acid.

In some preferred embodiments, the biocidal composition comprises a cysteine protease and a lipase, preferably an esterase or microbial lipase; and the composition also comprises a carrier comprising an alcohol and an acid, preferably an ethanol-based alcohol and a sulfonic acid.

In particularly preferred embodiments, the biocidal composition comprises at least two proteases and at least one lipase; and a carrier comprising at least one PEG glycol ether and at least one alkyl sulfate anionic detergent, preferably as described in statements above, and most preferably PEG- 10 tridecyl ether and sodium dodecyl sulfate. In some preferred embodiments, the biocidal composition comprises a metalloprotease, a cysteine protease and a lipase, preferably an esterase or microbial lipase; and the composition also comprises a carrier comprising at least one PEG glycol ether and at least one alkyl sulfate anionic detergent, preferably as described in statements above, and most preferably PEG- 10 tridecyl ether and sodium dodecyl sulfate. In particularly preferred embodiments, the biocidal composition comprises a cysteine protease and a lipase, preferably an esterase or microbial lipase; and the composition also comprises a carrier comprising at least one PEG glycol ether and at least one alkyl sulfate anionic detergent, preferably as described in statements above, and most preferably PEG- 10 tridecyl ether and sodium dodecyl sulfate.

According to a second aspect of the invention there is provided a method of protecting a surface from microorganisms, the method comprising the steps of:

— providing a biocidal composition according to the first aspect of the invention; and

— applying the biocidal composition on the surface to be protected.

The method according to the second aspect of the invention may, as appropriate, include any or all of the features described in relation to the biocidal composition of the first aspect of the invention.

The step of providing the biocidal composition may comprise the step of mixing a carrier with at least one protease selected from a serine protease, a metalloprotease and a cysteine protease, and at least one lipase selected from a phospholipase, an esterase and a microbial lipase. The or each protease and lipase may be as defined and described above for the first aspect of the invention.

The biocidal composition may be applied onto glass, metal, ceramic, wooden plastic or textile surfaces. It has been found that the biocidal composition is able to substantially eliminate coronaviruses on most surfaces, including glass, metal, ceramic, wooden plastic or textile surfaces. The textile surface may comprise cotton.

The biocidal composition may be applied onto the surface by spraying, wiping, dipping or by immersing the surface in the biocidal composition. In this way, the biocidal composition can be applied onto a variety of surfaces easily and quickly.

According to a third aspect of the invention there is provided a product that comprises the biocidal composition according to the first aspect of the invention. The product according to the third aspect of the invention may be include, as appropriate, any or all of the features described in relation to the biocidal composition of the first aspect of the invention.

The product may comprise a solid support soaked or treated with the biocidal composition. In particular, the solid support may comprise a fabric sheet such as a wipe or cloth. The fabric may be formed from natural or synthetic fibres.

According to a fourth aspect of the invention there is provided a use of an enzyme mixture as a biocide, the mixture comprising:

— at least one lipase selected from a phospholipase, an esterase and a microbial lipase.

The use of the mixture according to the fourth aspect of the invention, may include, as appropriate, any or all of the features described in relation to the first, second and third aspects of the invention. The use may include using a composition of the first, second or third aspects of the invention.

In some embodiments the mixture may be used as a virucide. In particular, the mixture may be used as a virucide against coronaviruses. For example, the mixture can be used as a virucide against Covid- 19, SARS-COV and MERS-COV. In some embodiments, the mixture may be used as a virucide against an enveloped virus. The mixture may be used as a virucide against Influenza. In some embodiments, the mixture may be used as a virucide against a non-enveloped virus. The mixture may be used as a virucide against Norovirus.

According to another aspect of the invention there is provided use of a protease to inhibit, kill or disable a coronavirus.

The protease may be a metalloprotease, serine protease or cysteine protease, or any combination thereof, and may be described above for the other aspects of the invention. In some embodiments the protease may be a metalloprotease, which has been found to be particularly effective at killing coronaviruses. The protease may be selected from PROMOD 439L (serine protease, specifically a subtilase), PROMOD 24L (metalloprotease from Bacillus subtilis') or PROMOD 950L (cysteine protease) or any combination thereof. The protease may be present in a carrier. The protease may be applied to the coronavirus in a carrier at a concentration of between 0.001 and 0.5 %wt.

The protease may be applied to the coronavirus as a spray, for example or may be applied as a liquid or aerosol.

The protease may be applied to a surface, which may be as defined and described above for other aspects of the invention. The protease may be applied to the surface to kill, inhibit or disable coronavirus on the surface and/or which may contact the surface post-application of the protease.

It has surprisingly been found that proteases are effective at killing and inhibiting coronaviruses on surfaces, and that application of proteases on surfaces provides long-lasting prophylactic effect against coronaviruses (at least 16 hours).

In some embodiments the use may comprise two or more proteases, independently selected from a metalloprotease, serine protease or cysteine protease.

In another aspect of the invention there is provided use of at least one protease and at least one lipase to inhibit, kill or disable a coronavirus.

The or each protease and lipase may be as described and defined hereinabove for the first aspect of the invention.

The protease may be selected from PROMOD 439L (serine protease, specifically a subtilase), PROMOD 24L (metalloprotease from Bacillus subtilis) or PROMOD 950L (cysteine protease) or any combination thereof. The protease may be present in a carrier. The protease may be applied to the coronavirus in a carrier at a concentration of between 0.001 and 0.1 %wt. The lipase may be a phospholipase, esterase or microbial lipase. The phospholipase may be LIPOMOD 699L or LIPOMOD 833L and/or the esterase may be LIPOMOD 34MDP all manufactured by Biocatalysts Limited, Unit 1, Cefn Coed, Parc Nantgarw, Cardiff, CF157QQ, Wales, UK. The lipase may be applied to the coronavirus in a carrier at a concentration of between 0.001 and 0.5 %wt. The protease(s) and lipase(s) may be applied to the coronavirus in the same or separate compositions. In some embodiments the protease(s) and lipase(s) may be applied in a liquid composition, which may be applied as a spray or aerosol. The protease and lipase may be applied to a surface, which may be as defined and described above for other aspects of the invention. The protease and lipase may be applied to the surface to kill, inhibit or disable coronavirus on the surface and/or which may contact the surface post-application of the protease.

Detailed Description of the Invention

In order that the invention may be more clearly understood one or more embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 shows the results of a visual test for determining the activity of protease enzymes when dried on a surface;

Figure 2 shows the results of a quantitative test for determining the activity of protease enzymes when dried on a surface;

Figure 3 shows the results of a visual test for determining the activity of lipase enzymes when dried on a surface;

Figure 4 shows the results of tests for determining the effectiveness of individual proteases and lipases in reducing coronavirus levels;

Figure 5 shows the results of tests for determining the effectiveness of compositions comprising proteases and lipases in reducing coronavirus levels;

Figure 6 shows the results of enzyme concentration optimisation experiments;

Figure 7 shows the results of experiments for determining the effectiveness of a composition for reducing coronavirus levels on surface up to sixteen hours; and Figure 8 shows the results of a test for determining the effect of the carrier on the virucidal activity of a composition containing protease and lipase enzymes.

Enzymes and Chemicals

The Proteases and lipases described in the examples below were acquired from Biocatalysts Ltd. Three proteases (A: PROMOD 439L (serine protease, specifically a subtilase); B: PROMOD 24L (metalloprotease from Bacillus subtilis, 1.1 %wt. enzyme concentration); C: PROMOD 950L (cysteine protease, 3.4 %wt. enzyme concentration)) and three lipases (D: LIPOMOD 699L (phospholipase); E: LIPOMOD 833L (phospholipase); F: LIPOMOD 34MDP (microbial lipase/esterase, 76 %wt. solid enzyme, prepared as a 1% aqueous solution in PBS) were tested. Standard substrates used were 4-nitrophenyl propionate (Merck #N3377) and Anorogenic chymotrypsin substrate II (Merck # 230914-25MG). SteriKleen and SteriKleen concentrate were provided by Smarti Environmental Ltd, UK. Other suitable standards include 4- Methylumbelliferyl butyrate (Santa Cruz Biotechnology #sc-206912A).

Clinical Samples

A clinical sample of feline infectious peritonitis (hereinafter “FIP”) was acquired from Veterinary Pathology Group. This sample was of feline faeces from an infected animal. Part of this sample was used directly. For most tests, a suspension of one part feline faeces in nine parts sterile PBS was made and thoroughly mixed before each use.

Enzyme Assays

Enzyme F was supplied as a powder and was prepared to 10 mg/mL in PBS. Enzymes A-F were mixed one part in 20 with either PBS, SteriKleen, or. A 250 pL sample was dried onto glass (laboratory cover slide) or plastic (lid of Starlab TipOne box). Enzymes were left to dry onto the surface and were incubated at 4 °C, room temperature (20-22 °C) or 37 °C as appropriate. For protease samples, chymotrypsin substrate II was added to the dried enzyme. For visual tests, 25 pL of 1 mM substrate in 4% v/v methanol/PBS was added. For quantitative tests in microplates, 200 pL of 1 mM substrate in 4% v/v methanol/PBS was added to a 96 well plate (Greiner #655001). Visual tests were imaged under illumination with UV light. Quantitative tests were performed in an M200 Pro plate reader (Tecan) with excitation 380 nm, emission 460 nm. A sample from enzyme A incubated at 4 °C in PBS was chosen to optimise gain. For lipase tests, 250 pL 1 mM 4-nitrophenyl propionate in 4% v/v methanol/PBS was added to the dried enzyme. The results were imaged under white light.

DNA and RNA Preparation

A 143 base pair fragment of FIP DNA (bases 12,605 to 12,747 of FIP accession number KC461237) was prepared by IDT using the method of Desmerets et al. (2013) Veterinary Research, 44, 71. (Doi: 10.1186/1297-9716-44-71). RNA was prepared from this using T7 RNA polymerase (NEB #M0251) following the manufacturer’s recommendations, and the parent DNA was removed using DNase I (NEB #MO3O3). RNA was prepared from samples using the QIAamp Viral RNA Mini kit (Qiagen #52906) following the manufacturer’s recommendations.

RT-qPCR Conditions

RT-qPCR was performed on a Rotor-Gene Q qPCR machine (Qiagen). The Luna Universal One-Step RT-qPCR kit (NEB #E3005) was used following the manufacturer’s recommendations, except that a final volume of 10 pL was used and all quantities reduced in proportion. 1 pL of RNA preparation was added to each sample. The cycling conditions were:

10 min at 55 °C (reverse transcription step); 1 min at 95 °C; 45 cycles of: 10 s at 95 °C then 20 s at 60 °C; and finally a melt curve from 60 °C to 95 °C at 5 s / °C.

For each experiment, a triplicate standard curve of RNA prepared as above was used, diluting in 10-fold steps from an initial 10 pg. All data were analysed using Graphpad v. 8.0.2.

Testing of Enzymes Against Coronaviruses

For initial tests, 11.3 pL enzyme, 214 pL SteriKleen and 25 pL FIP sample were mixed and incubated at room temperature for 10 minutes. RNA was prepared as described above and the RNA concentration determined. For testing of pairs of enzymes, 5.63 pL of each enzyme, 214 pL SteriKleen and 25 pL FIP were mixed and incubated at room temperature for 10 minutes. RNA was prepared as described above and the RNA concentration determined.

To determine optimal concentrations of enzymes B, C and F (Example 4 below), a Design of Experiments approach was calculated using Matlab v.2020a. Enzymes were mixed at the determined concentrations in SteriKleen and 225 |iL was added to 25 |iL FIP sample. RNA was prepared and the concentrated determined as described above. Following the data processing, a model was determined using Matlab v. 2020b using the following equation:

LoglORNA = B] + &[C] + c[F] + [B][C] + e[B][F] +/[C] [F] + g[B]² + /z[C]² + z[F]² + j

The final concentrations selected were chosen to give the minimal surviving FIP at the 95^th percentile of the model.

Surface Testing of Mixtures

The surfaces used were: plastic - the lid of a standard SBS microplate and glass - cover slides. A mixture of 0.125% (v/v) enzyme B (corresponding to a final concentration of 0.001375 %wt. in the composition, 0.125% (v/v) enzyme C (corresponding to a final concentration of 0.00425 %wt. in the composition) and 0.25% of a 1% (w/v) solution of enzyme F in PBS (corresponding to a final concentration of 0.0019 %wt. in the composition) in SteriKleen was used. 250 pL of the mixture was pipetted onto the surface and allowed to dry. After incubation at room temperature for four or sixteen hours, 25 pL of FIP suspension was added to the surface. The sample was incubated for ten minutes at room temperature. Viral samples were recovered from plastic and glass substrates by pipetting 200 pL PBS onto the surface and pipetting up and down six times. The PBS was stored in a plastic tube. RNA was prepared as above from all samples, and the RNA concentration determined as described above.

Example 1: Enzyme Activity after Drying on Surfaces

Experiments were undertaken to establish whether proteases remain active after being dried onto surfaces. This was tested by drying proteases A, B and C (one part in 20) in either a neutral buffer or SteriKleen onto a glass surface. Immediately after drying (Figure 1(A)) or after four hours (Figure 1(B)) incubation at 37 °C, a Anorogenic chymotrypsin substrate was added to the glass. After ten minutes, the release of Auorophore was measured by imaging the slides under illumination by UV light. Figures 1A and IB show that there is no apparent loss of activity in proteases A and C, whilst little activity was observed for protease B. The results also show no notable difference in activity between samples which were incubated with SteriKleen or buffer, indicating that SteriKleen does not cause any loss of enzyme activity. Similar tests were undertaken to test whether proteases A, B and C could retain their activity on surfaces over an extended period of time. Enzyme samples (one part in 20) in buffer or SteriKleen concentrate were dried onto wells in a plastic 96 well plate. The plates were incubated at 4 °C, room temperature or at 37 °C for 72 hours. Fluorogenic chymotrypsin substrates were then added to each well, and after 10 minutes the release of fluorophore was measured using a plate reader (excitation 380 nm, emission 460 nm). As best shown in Figure 2, there was no loss in observed activity for proteases A and C after 72 hours, irrespective of whether the proteases were incubated with buffer or SteriKleen, while protease B again exhibited relatively little activity by comparison.

Experiments were also undertaken to establish whether lipases remain active after being dried onto surfaces. This was achieved by drying lipases D, E and F (one part in 20) in either a neutral buffer or SteriKleen onto glass cover slides. Immediately after drying (A) or after four hours (B) incubation at 37 °C, pNP-propionate was added to the glass. After ten minutes, the release of fluorophore was measured by imaging the slides in white light. As best shown in Figure 3, only lipase F showed significant activity with a standard substrate and that there was no loss of activity over four hours at 37 °C in SteriKleen.

Example 2: Capability of Individual Enzymes to Reduce Coronavirus Levels.

Feline infectious peritonitis (FIP) samples were added to an equal volume of PBS, SteriKleen, or SteriKleen supplemented with 5% enzymes (A-F). After 10 minutes RNA was prepared from the mix and the number of surviving virus particles enumerated by RT-qPCR. As best shown in Figure 4 enzymes B and F showed a strong effect while enzymes A, C, D and E showed a smaller but adequate apparent effect in reducing coronavirus levels.

Example 3: Capability of Pairs of Enzymes to Reduce Coronavirus Levels

Enzymes were tested in pairs to establish whether any enzyme combinations could lead to an enhanced coronavirus killing effect. FIP samples were added to an equal volume of PBS, SteriKleen, or SteriKleen supplemented with 2.5% each of two enzymes. After 10 minutes RNA was prepared from the mix and the number of surviving virus particles enumerated by RT-qPCR. Figure 5 is a graph showing the results of the enzyme pair tests. It can be seen that all combinations involving enzyme B gave good results, as expected from the individual enzyme results. However, enzyme C showed strong synergy with enzymes D and F and both combinations were effective in reducing corona virus levels and exhibited improved performance relative to enzymes C, D and F alone.

Example 4: Enzyme Concentration Optimisation Test

FIP samples were added to an equal volume of PBS, or SteriKleen supplemented with 0.125% each of proteases B and C, 0.25% of lipase F, or 0.125 % each of proteases B and C and 0.25 % of lipase F. After 10 minutes RNA was prepared from the mix and the number of surviving virus particles enumerated by RT-qPCR. As best shown in Figure 6, enzyme mixtures comprising 0.125% of proteases B and C, and 0.25% of lipase F show robust efficacy in that coronavirus levels could be reduced to less than 0.1%, even at low enzyme concentrations. Since only low enzyme concentrations are needed to obtain a good coronavirus killing effect, this has the added benefit that compositions comprising enzymes B, C and F are likely to be economic.

Example 5: 16 Hour Test

The composition comprising SteriKleen, 0.125 % each of proteases B and C and 0.25 % of lipase F was tested to establish whether the composition could reduce and keep coronavirus levels low for up to 16 hours when applied onto plastic and glass surfaces. 250 pL of enzyme/SteriKleen mix was applied to a surface and allowed to dry. After sixteen hours, FIP samples (25 pL) were added to the surface. After 10 minutes the FIP was recovered from the surface, RNA was prepared from the mix and the number of surviving virus particles enumerated by RT-qPCR.

Example 6: Quantitative suspension test for evaluation of virucidal activity against Influenza

A composition comprising an aqueous solution containing a Cirrus- 17 carrier (sodium lauryl sulfate, PEG- 10 tridecyl ether, and acrylate-styrene copolymer) and 0.022% of protease B, 0.068% of protease C and 0.0077 % of lipase F was tested for its activity against both Influenza and Norovirus. Samples of the composition were either left neat or diluted by 50% in distilled water and were independently added to test suspensions of Influenza Virus H1N1 (VR-1638) and Murine Norovirus strain S99 Berlin in solutions of clean 0.3 g/L bovine albumin interfering substance. The mixtures were maintained at 20°C ± 1°C for a contact time of 5 mins ± 10 s. At the end of this contact time, aliquots were taken; the virucidal action in these portions was immediately suppressed by a validated method (dilutions of the sample in ice-cold cell maintenance medium). The dilutions were transferred into cell culture units either using monolayer or cell suspension. Infectivity tests were done either by plaque test or quantal tests. After incubation, the titres of infectivity were calculated according to Spearman and Kaber or by plaque counting. Reduction of virus infectivity was calculated from differences of log virus titres before (virus control) and after treatment with the products.

Results for Influenza H1N1 are shown in Tables 1-3 below. As displayed in Tables 1-3, the composition achieved a greater than 4-log reduction against Influenza HINT. The product suppression control also showed a <0.5-log reduction in viral titre. The results also showed a 4-log difference between the cytotoxicity level and viral titre. Accordingly, the composition was shown to be greatly effective against Influenza H1N1.

Results for Murine Norovirus are shown in Tables 4-6 below. As displayed in Tables 4-6, the composition also achieved a high 3.92-log reduction when neat and 2.67-log reduction when at 50% dilution against Murine Norovirus, indicating good efficacy against the virus.

Table 1 Table 2

Table 3 Table 4

Table 5

Table 6

Example 7: Test to evaluate the effect of the carrier on biocidal performance

FIP samples were independently added to equal volumes of SteriKleen; SteriKleen supplemented with 0.011% of protease B, 0.034% of protease C, and 0.0038% of lipase F; an aqueous solution containing the same mixture of enzymes (same concentration as previous sample) and also a Cirrus- 17 carrier at a concentration 2-times that employed in Example 6 above; an aqueous solution containing the same mixture of enzymes (same concentration as previous sample) and also a Cirrus- 17 carrier at the same concentration as that employed in Example 6; an aqueous solution containing the same mixture of enzymes (same concentration as previous sample) and also a Cirrus- 17 carrier at a concentration 0.5-times that employed in Example 6; and an aqueous solution containing the same mixture of enzymes (same concentration as previous sample) and also a Cirrus- 17 carrier at a concentration 0.25-times that employed in Example 6. After 5 minutes, RNA was prepared from the mixtures and the number of surviving virus particles enumerated by RT-qPCR. Figure 8 is a graph showing the results of the tests.

The results demonstrate that a composition containing both a suitable carrier and enzymes is required for the best virucidal performance. Whilst “carrier (0.25X) + enzymes” displayed some virucidal effect relative to the SteriKleen control, increasing the Cirrus- 17 carrier concentration was found to have a major improvement on performance. This supports the reasoning that the mixture of anionic and non-ionic detergents in the carrier loosen up the structure of viral particles allowing the enzymes in the composition better access to them.

Summary

The results of the above tests demonstrate that the addition of proteases and lipases to a SteriKleen cleaning solution make it capable of breaking down and killing coronaviruses. The results also show good activity of compositions containing proteases and lipases and a carrier comprising detergents, particularly a combination of an anionic detergent and a non-ionic detergent, and preferably also a film-forming polymer, against enveloped and non-enveloped viruses, including Influenza and Norovirus. Enzyme mixtures comprising metalloproteases, cysteine proteases and esterases or microbial lipases were particularly effective, but very good biocidal activity was also observed when SteriKleen was supplemented with one protease and one lipase. In particular, enzyme mixtures comprising a metalloprotease and a phospholipase; a cysteine protease and a phospholipase or a cysteine protease and an esterase or microbial lipase exhibited very good biocidal performance. It has also been demonstrated that robust efficacy in eliminating coronaviruses at surfaces for up to sixteen hours even when the concentration of enzymes in the composition is low. Therefore, the biocidal compositions offer a cost-effective solution for protecting the surfaces from microorganisms such as coronaviruses over extended periods of time.

The one or more embodiments are described above by way of example only. Many variations are possible without departing from the scope of protection afforded by the appended claims.

Claims

24 CLAIMS

1. A biocidal composition comprising:

— at least one lipase selected from a phospholipase, an esterase and a microbial lipase; and

— a carrier.

2. A biocidal composition according to claim 1, wherein the composition comprises at least two proteases and at least one lipase.

3. A biocidal composition according to claim 1 or 2, wherein the composition comprises metalloprotease and cysteine protease.

4. A biocidal composition according to claim 3, wherein the composition comprises metalloprotease, cysteine protease and esterase or microbial lipase.

5. A biocidal composition according to claim 1, wherein the protease comprises metalloprotease and the lipase comprises esterase or microbial lipase.

6. A biocidal composition according to claim 1, wherein the protease comprises cysteine protease and the lipase comprises esterase or microbial lipase.

7. A biocidal composition according to claim 1, wherein the protease comprises serine protease and the lipase comprises esterase or microbial lipase.

8. A biocidal composition according to claim 1, wherein the protease comprises metalloprotease and the lipase comprises phospholipase.

9. A biocidal composition according to claim 1, wherein the protease comprises cysteine protease and the lipase comprises phospholipase.

10. A biocidal composition according to claim 1, wherein the protease comprises serine protease and the lipase comprises a phospholipase.

11. A biocidal composition according to claims 1, 2, 8, 9 or 10, wherein the phospholipase comprises a pancreatic phospholipase. A biocidal composition according to any preceding claim, wherein the composition comprises ethylene glycol. A biocidal composition according to any preceding claim, wherein the carrier comprises a liquid, preferably a liquid disinfectant. A biocidal composition according to any preceding claim, wherein the carrier comprises an alcohol and an acid, preferably an ethanol-based alcohol and a sulfonic acid. A biocidal composition according to any preceding claim, wherein the carrier comprises an anionic detergent, preferably comprising an alkyl sulfate or a salt thereof. A biocidal composition according to any preceding claim, wherein the carrier comprises a non-ionic detergent, preferably comprising a PEG glycol ether. A biocidal composition according to any preceding claim, wherein the carrier comprises at least one film-forming polymer, preferably a polyacrylate and/or acrylate copolymer. A method of protecting a surface from microorganisms, the method comprising the steps of:

— providing a biocidal composition according to any preceding claim; and

— applying the biocidal composition on the surface to be protected. A method according to claim 18, wherein the step of providing the biocidal composition comprises mixing a carrier with at least one protease selected from a serine protease, a metalloprotease and a cysteine protease, and at least one lipase selected from a phospholipase, an esterase and a microbial lipase. A method according to claim 18 or claim 19, wherein the biocidal composition is applied onto a glass, metal, plastic, wood, ceramic or textile surface. A method according to any of claims 18 to 20, wherein the biocidal composition is applied onto the surface by spraying, wiping, dipping or by immersing the surface in the biocidal composition. A product comprising the biocidal composition according to any of claims 1 to 17. A product according to claim 22, wherein the product comprises a solid support soaked or treated with the biocidal composition. A product according to claim 23, wherein the solid support comprises a fabric such as a wipe or cloth. Use of an enzyme mixture as a biocide, the mixture comprising: a) at least one protease selected from a serine protease, a metalloprotease and a cysteine protease; and b) at least one lipase selected from a phospholipase, esterase and a microbial lipase. Use of the enzyme mixture according to claim 25 as a virucide. Use according to claim 26, wherein the enzyme mixture is used as a virucide against coronaviruses. Use according to claim 27, wherein the mixture is used as a virucide against Covid-19. Use according to claim 26, wherein the mixture is used as a virucide against Influenza. Use according to claim 26, wherein the mixture is used as a virucide against

Norovirus. Use of a protease and optionally a lipase to inhibit, kill or disable a coronavirus. Use according to claim 31 wherein the protease is selected from the group of a metalloprotease, a serine protease, a cysteine protease or any combination thereof.