WO2021240315A1

WO2021240315A1 - Probiotic bacillus sanitiser

Info

Publication number: WO2021240315A1
Application number: PCT/IB2021/054415
Authority: WO
Inventors: Dylan John SETTER; Quinten Colin DICKERSON
Original assignee: Setter Dylan John; Dickerson Quinten Colin
Priority date: 2020-05-27
Filing date: 2021-05-21
Publication date: 2021-12-02

Abstract

The invention herein described refers to a probiotic Bacillus surface sanitiser comprising a sanitising agent, a probiotic Bacillus sp. and a surfactant, but excluding any stabiliser, as well as a process for manufacturing the probiotic Bacillus surface sanitiser. The invention further refers to a method of killing or inhibition of the growth of pathogenic or undesirable microorganisms on a surface by application of the probiotic Bacillus surface sanitiser of the invention.

Description

PROBIOTIC BACILLUS SANITISER

FIELD OF THE INVENTION

The invention herein described refers to a probiotic Bacillus surface sanitiser comprising a sanitiser, a probiotic Bacillus and a surfactant, but excluding any stabiliser, as well as a process for manufacturing the probiotic Bacillus surface sanitiser. The invention further refers to a method of killing or inhibition of the growth of pathogenic or undesirable microorganisms on a surface by application of the probiotic Bacillus surface sanitiser of the invention.

BACKGROUND OF THE INVENTION

Sanitising agents, disinfectants or biocides, including those for treating surfaces are typically comprised of chemicals or enzymes that are designed to kill or limit the growth of microorganisms including bacteria, fungi, viruses, algae and protozoa in order to prevent the harmful consequences of the presence and activity of such microorganisms. Such products may be used in medical settings as well as in industrial products or processes, in public spaces, or even in the home, but it is well known that conventional sanitisers, disinfectants and biocides often pose significant risk to both the natural environment and human and animal health by killing the beneficial bacteria as well as the pathogenic or undesirable bacteria, leaving the surface being treated unprotected from further accelerated growth of pathogenic or undesirable bacteria due to the beneficial bacteria also no longer being present. Manufacture and use of chemical sanitisers, disinfectants and biocides is strictly regulated due to the toxic and corrosive content, particularly in the food, medical, pharmaceutical, agriculture, cosmetic and water industries.

Sanitisers or disinfectants to inactivate or inhibit the growth of microorganisms are dependent on the contact time and the concentrations applied. However, due to increased resistance of microorganisms to conventionally used sanitisers and disinfectants, the contact time and concentration have needed to be increased over time to remain effective. Furthermore, certain species of bacteria can form spores or a biofilm to survive the effects of a sanitiser or disinfectant.

It would be useful to be able to provide a sanitising product with a natural component that is effective over an extended period but with either a reduced concentration of sanitiser/disinfectant or a reduction in the number of times that the product needs to be applied in order to maintain effectiveness, or both. Such a product could find application in many diverse settings, from the home to businesses, for public spaces including public transport, buildings and places, to medical, agricultural and industrial settings.

It would further be useful if such a sanitiser could reduce the unwanted microorganism content on surfaces without having a detrimental effect on the environment by replacing the unwanted pathogenic or undesirable microorganisms with beneficial and natural bacteria.

Furthermore it would be useful if such a product was able to act without causing damage to the surface on which it is used, such as corrosion, which is caused by frequent application of high concentrations of chemical sanitisers used at present.

In addition, it would be beneficial if the sanitiser was able to be simply manufactured without requiring the addition of stabilising chemicals, thereby to reduce the cost of production and simplifying the manufacturing process. SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a probiotic Bacillus sanitiser for killing or inhibiting the growth of microorganisms on a surface comprising or consisting of:

(i) as active ingredients, at least one species of Bacillus, at least one sanitising agent, and at least one surfactant; and

(ii) optionally water, and/or additives including one or more colourants, fragrances, effervescent compounds, fillers such as soda ash (sodium bicarbonate) or sodium chloride, degreasing agents, and cleaning agents if desired, wherein no stabilising chemicals are present.

The probiotic Bacillus sanitiser may further comprise one or more colourants, fragrances or effervescent compounds such as soda ash (sodium bicarbonate) if desired.

The Bacillus may be any one or more isolated GRAS (generally classified as safe) Bacillus species known to those skilled in the art, for example, B. subtilis, B. pumilus and B. megaterium, B. licheniformis, B. coagulans, B. mojavensis, or B. amyloliquefaciens as well as any other Bacillus species that may be discovered in future or are not currently utilized.

The sanitising agent may be any one or more chemical sanitiser(s) known to those skilled in the art, for example chlorine, sodium dichloroisocyanurate (SDIC), chlorine dioxide, hypochlorous acid, hydrogen peroxide, sodium hypochorite, quaternary ammonium, alcohol such as ethanol or isopropyl alcohol or any derivative of the above. In a preferred embodiment of the invention, the chemical sanitiser is SDIC or quaternary ammonium.

Alternatively, the sanitiser may be a natural sanitiser known to those skilled in the art, such as comprising one or more essential oils or compounds including enzymes and/or flavonoids from antiseptic plants, such as eucalyptus, neem, mint, rosemary, tea tree, witch-hazel, citrus peel, lactic acid and the like. The surfactant may be any one or more cationic and/or one or more non-ionic surfactant(s) known to those skilled in the art. Cationic surfactants are those where the polar group carries a positive charge, for example dimethyl distearyl ammonium chloride or sodium dodecylbenzene sulfonate. Non-ionic surfactants have a polar group that cannot be ionized in an aqueous solution, a fatty acid chain forming a hydrophobic component and a hydrophilic component containing non ionizable atoms of oxygen, nitrogen or sulphur. Examples include derivatives of polyoxyethylene (POE) or polyoxypropylene (POP), sugar esters and alkanolamides.

The pH of the probiotic sanitiser may be between about 4.5 to about 9.5, or from about 5 to 9, or about 3.5 to 10.5, or about 8 to 9, or about 7 to 10, or about 9.5 to 10.5. A buffering agent known to those skilled in the art may be added to obtain the desired pH.

The probiotic Bacillus sanitiser may be in a liquid or a dry (solid) form. The liquid form may be formulated as a stock solution for further dilution by a user or distributor, or may be formulated ready to use. The solid form may be any solid formulation including a granular or powder formulation which can be mixed with water to the desired liquid concentration by a user or distributor. In particular, the solid form may further comprise a filler including soda ash (sodium carbonate) or sodium chloride or other known fillers. A filler is a particulate material that is added to the probiotic Bacillus sanitiser to bulk it up and make the final product cheaper, but may also have beneficial properties.

The probiotic sanitiser may further comprise degreasing components including soda ash (sodium carbonate), sodium tripolyphosphate, tetrapotassium pyrophosphate, sodium metasilicate anhydrous and sodium dodecylbenzene sulfonate.

In one possible embodiment of the invention, the probiotic Bacillus sanitiser is formulated as a liquid product and comprises per 1 ,000 L:

Bacillus sp. at between about 6,00E+05 to about at least 0,0001% - 30%

1 ,00E+11 colony forming units (CFU) depending on the CFU of the Bacillis sp. used sanitising agent

10ppm - 1500ppm cationic and/or non-ionic surfactants 5% - 15% water 40% - 80% In an alternative possible embodiment of the invention, the probiotic Bacillus sanitiser is formulated as a dry product and comprises per 1 ,000 g:

In a further alternative possible embodiment of the invention, the probiotic sanitiser is formulated as a chemical degreasing probiotic Bacillus sanitiser and comprises per 1,000 g:

Bacillus sp. at between about 6,00E+05 to about at least 0,0001% - 30%

1 ,00E+11 CFU depending on the CFU of

According to a further aspect of the invention, there is provided a simplified process for manufacturing a probiotic Bacillus sanitiser of the invention, comprising or consisting of a step of blending:

(ii) optionally water and/or additives including one or more colourants, fragrances, effervescent compounds, fillers such as soda ash (sodium bicarbonate) or sodium chloride, degreasing agents, and cleaning agents if desired, where no stabiliser is added. The Bacillus may be any one or more isolated GRAS (generally classified as safe) Bacillus species known to those skilled in the art, for example, B. subtilis, B. pumilus and B. megaterium, B. licheniformis, B. coagulans, B. mojavensis, or B. amyloliquefaciens as well as any other Bacillus species that may be discovered in future or are not currently utilized.

Alternatively, the sanitising agent may be a natural sanitiser known to those skilled in the art, such as comprising one or more essential oils or compounds including enzymes and/or flavonoids from antiseptic plants, such as eucalyptus, neem, mint, rosemary, tea tree, witch-hazel, citrus peel or lactic acid and the like.

The surfactant may be any one or more cationic or non-ionic surfactant known to those skilled in the art. Cationic surfactants are those where the polar group carries a positive charge, for example dimethyl distearyl ammonium chloride or sodium dodecylbenzene sulfonate. Non-ionic surfactants have a polar group that cannot be ionized in an aqueous solution, a fatty acid chain forming a hydrophobic component and a hydrophilic component containing non ionizable atoms of oxygen, nitrogen or sulphur. Examples include derivatives of polyoxyethylene (POE) or polyoxypropylene (POP), sugar esters and alkanolamides.

The pH of the probiotic sanitiser may between about 4.5 to about 9.5, or from about 5 to 9, or about 3.5 to 10.5 or about 8 to 9. In one embodiment of the invention, the pH is between about 7 to 10. In an alternative embodiment of the invention, the pH is between about 9.5 and 10.5. A pH adjusting agent known to those skilled in the art may be added to obtain the desired pH, including but not limited to potassium hydroxide, sodium hydroxide, glycolic acid. The process may be for manufacturing of a liquid probiotic Bacillus sanitiser formulation and may comprise providing water to a mixing tank followed by addition of each of the active components and optionally one or more additives, and mixing until blended before packaging.

The process may be for manufacturing of a granular or powder dry (or solid) probiotic Bacillus sanitiser formulation and may comprise providing each of the active components in dry form and optionally one or more additives in dry form to a mixer, and mixing until blended. The process may further comprise forming the solid sanitiser into a tablet by stamping the solid formulation into a mould before packaging.

According to a further aspect of the invention there is provided a method of killing or inhibition of the growth of one or more pathogenic or undesirable microorganisms on a surface by application of the probiotic Bacillus sanitiser of the invention. The microorganism(s) may be one or more bacteria, one or more virus(es), one or more fungus(i), one or more protozoa or one or more algae. In particular the microorganism may be one or more bacteria or one or more fungi.

The method may comprise dilution of a liquid formulation of the probiotic sanitiser of the invention to a desired concentration prior to use. For example, the dilution factor may be 1 :10 parts probiotic Bacillus sanitisenwater.

Application may comprise spraying the probiotic sanitiser of the invention at the desired concentration onto the surface and wiping the surface followed by allowing the surface to dry naturally. Alternatively, application may comprise providing a bucket containing the probiotic Bacillus sanitiser of the invention at the desired concentration, and application of the probiotic Bacillus sanitiser by means of a mop or cloth dipped into the bucket and then used to wipe or mop the surface, followed by natural drying of the surface.

In an alternative embodiment of the invention, the method may comprise dissolving the dry probiotic Bacillus sanitiser of the invention in water to obtain a liquid formulation. The liquid formulation may be further diluted to a desired concentration prior to use or may be mixed at the desired concentration without requiring further dilution. For example, 50g of dry probiotic Bacillus sanitiser may be mixed with 10L of water, followed by stirring until completely dissolved, and use within 3 hours of mixing, or if stored in an air tight container, use within 12 hours.

DETAILED DESCRIPTION OF THE INVENTION

The current invention relates to a probiotic Bacillus surface sanitiser for killing or inhibiting the growth of microorganisms on a surface comprising at least one species of Bacillus, at least one sanitiser, and at least one surfactant, wherein no stabilising chemicals are present. The invention further provides a process for manufacturing the probiotic Bacillus sanitiser. The invention further refers to a method of killing or inhibition of the growth of pathogenic or undesirable microorganisms on a surface by application of the probiotic Bacillus sanitiser of the invention.

Throughout this specification the terms “sanitiser”, “sanitising agent”, “disinfectant” and “biocide” are used interchangeably and mean a compound or composition that is active to inhibit the growth of a microorganisms or to kill a microorganism.

Currently available surface disinfectants or sanitisers are commonly formulated either as chemical or as natural biocides or sanitisers. The use of probiotic bacteria in combination with a disinfectant or sanitiser is rare, and when combined, requires the use of stabilisers to ensure that the product remains stable prior to use. Some examples of stabilisers that have been used in the past, but are not required for use in the formulation of the invention include: polyethylene glycol, or another humectant, such as glycerine, glycerol, an ethylene oxide polymer, 1 ,2-dihydroxypropane, 1,2- propanediol, methyl glycol, or trimethyl glycol, or sodium benzoate, nitrate, nitrite, sodium nitrate, benzoic acid, suphure dioxide, benzalkonium chloride, benzyle alcohol, paraben, tartaric, acetic or citric acid, or isothiazolinone. The required addition of such compounds adds to the cost and complexity of the product and the manufacturing process.

Disinfectants or sanitisers are used in medical settings as well as in industrial products or processes but may also be used in the home, office or in public places. The use of chemical or natural sanitisers alone, due to the increasing resistance of microorganisms including bacteria, viruses and fungal pathogens, is required to be by more frequent application and in higher concentrations to remain effective. Alternatively the chemical disinfectants being used need to be replaced with new chemical disinfectants as resistance developes, in order for the product to be effective. However, many of these compounds and compositions are known to have toxic and/or corrosive content and are detrimental to human and animal health and to the natural environment, particularly at the high concentrations and frequency of use required for long-lasting effectiveness. Furthermore, certain species of bacteria can form spores or a biofilm to survive the effects of a conventional chemical or natural sanitiser.

The combination of an isolated probiotic Bacillus species together with a sanitiser has been found by the applicant to provide an effective solution for cleaning, degreasing and disinfecting surfaces with an advantage over the commonly available sanitisers on the market, in that less frequent applications of chemical sanitiser is required for effective use. The applicant has also surprisingly shown that the formulation of their product does not require the addition of a stabiliser, which has previously been required to be included in similar products, thereby saving on the cost and complexity of formulation and is still effective for use.

The sanitiser component of the product provides initial disinfection and sanitation by killing or inhibition of pathogenic and undesirable microorganisms upon application to the surface. Thereafter, the probiotic Bacillus bacteria continue to function, maintaining a healthy bacterial environment that limits the ingress of pathogenic or undesirable microorganisms through competition with undesirable/pathogenic bacteria by consuming the available food source.

The probiotic sanitiser of the invention can be effectively used to disinfect or sanitise any surface, including those in industrial, medical, pharmaceutical, agricultural, business, public or domestic spaces and including but not limited to: work surfaces, floors, walls, railings, handles, operating buttons, coveyers, processing and packaging equipment, and the like.

The invention is described by way of the following illustrated embodiments which are not to be construed as limiting in any way the scope of the invention.

EXAMPLE 1 :

Liquid probiotic sanitiser

Component

per 1,000 L

Bacillus sp. (about 6.00E+06 to about 1 ,00E+9 0,001 % - 30%

CFU) sanitising agent

cationic and/or non-ionic surfactants 5% - 15% water 40% - 80%

If desired, one or more additives including one or more colourants or fragrances may be added.

The diluted pH value is between 7 and 10.

Manufacturing Steps:

Step 1 : Add water into a mixing tank or vessel.

Step 2: Add each of the chemicals and Bacillus sp. and mix the product for 2 mins or until completely mixed.

Step 3: Decant into packaging for distribution, sale and use.

Directions for use:

Dilute probiotic Bacillus sanitiser product by adding to a bucket of water or spray bottle at 1 :10 parts water: probiotic Bacillus sanitiser and apply to the relevant surface with a mop, cloth or spray. Wipe down the surface or allow to dry naturally. It is also possible to dilute the probiotic Bacillus sanitiser in an alternative concentration as desired or specific to a particular application. EXAMPLE 2:

Dry probiotic Bacillus sanitiser

If desired, various additives may be included such as one or more colourants, or fragrances.

The diluted pH value is between 7 and 10.

Manufacturing Steps:

Step 1 : Add each of the dry chemicals and Bacillus sp. to a mechanical mixer in the quantities provided above.

Step 2: Turn on mixer and mix for 10 minutes or until thourghly mixed.

Step 3: Decant the finished dry probiotic Bacillus sanitiser into packaging for distribution, sale and use.

Directions for use:

• Mix 50g of dry probiotic Bacillus sanitiser into 10L of water in a container by stirring until dissolved.

• Store mixture in an air tight container for use the next day, or use the mixture within 3 hours of mixing.

• Allow minimum 30 seconds of contact with relevant surface for disinfecting or sanitising before wiping and or allow to air dry.

EXAMPLE 3:

Degreasing probiotic Bacillus sanitiser

If desired, one or more additives including colourants or fragrances may be added. The diluted pH value is between 9.5 and 10.5.

Manufacturing Steps:

Step 1 : Add the each of the dry chemicals and Bacillus sp. to a mechanical mixer in the quantities provided above.

Step 2: Turn on mixer and mix for 10 minutes or until thoroughly mixed.

Step 3: Decant the finished dry degreasing probiotic Bacillus sanitiser into packaging for distribution, sale and use.

• Allow minimum 30 seconds of contact with relevant surface for disinfecting or sanitising before wiping and allow to air dry.

EXAMPLE 4:

Efficacy of reconstituted powder formulation in water of probiotic sanitiser with Bacillus so. (between about 6.00E+06 to about 1.00E+9 CFU) and sodium dichloro iso cvnurate (SDIC) >50PPM chemical sanitiser

1. Bacterial pathgens Efficacy of reconstituted powder formulation in water of probiotic sanitiser with Bacillus sp. (between about 6,00E+06 to about 1 ,00E+9 CFU), sodium dichloro iso cynurate (SDIC) >50PPM chemical sanitiser, and 10% surfactant plus soda ash or sodium chloride as a filler was tested against four pathogenic bacterial species by an accredited laboratory. The pass requirements in terms of the accredited test is that the product must demonstrate at least a 5 decimal log reduction in bacteria when diluted with hard water or undiluted.

1.1. Methods

The probiotic sanitiser was tested against four bacterial species: Enterococcus hirae ATCC 10541 , Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538 for a contact time of 5 minutes at 20 °C. An amount of 0.3 g/L bovine serum albumin as added as the interfering substance. Thereafter samples were tested at 37 °C for bacterial growth.

For the test to be valid, for each test organism:

• N is between 1.5x10⁸ and 5.0x10⁸ (8.17< log N < 8.70)

• No is between 1.5x10⁷ and 5.0x10⁷ (7.17< log N < 7.70)

• N_vo is between 30 and 160 (3.0x10¹ and 1.6x10²)

• A, B, C are equal to or greater than 0.5xN_vo

• Control of weighted mean counts: quotient is not lower than 5 and not higher than 15.

1.2. Results

Escherichia coli ATCC 10536 Test log reduction values

Pseudomonas aeruginosa ATCC 15442 Test log reduction values

Staphylococcus aureus ATCC 6538 Test log reduction values

Enterococcus hirae ATCC 10541 Test log reduction values

1.3. Conclusion

The probiotic sanitiser test product tested at either 60g / 10L or 90g / 10L complied with the pass criteria of SANS test 51276 (EN1276) standard, which requires at least a 99.999% kill (5 log reduction) in bacteria.

In addition, on culture of the probiotic sanitiser product after treatment, based on standard SANS 7932 the Bacillus strains remainng were found to survive, repopulate and continue to thrive having a Bacillus species (cfu/g) of 530,000.

2. Fungal pathogens

Efficacy of reconstituted powder formulation in water of probiotic sanitiser with Bacillus sp. (between about 6,00E+06 to about 1 ,00E+9 CFU), sodium dichloro iso cynurate (SDIC) >50PPM chemical sanitiser, and 10% surfactant plus soda ash or sodium chloride as a filler was tested against 2 pathogenic fungal strains by an accredited laboratory. The pass requirements in terms of the accredited test is that the product must demonstrate at least a 4 decimal log reduction in fungus when diluted with hard water or undiluted.

2. 1. Methods

The probiotic sanitiser was tested against two fungal strains: Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 for a contact time of 15 minutes at 20 °C. An amount of 0.3 g/L bovine serum albumin as added as the interfering substance. Thereafter samples were tested at 30 °C for bacterial growth.

For the test to be valid, for each test organism:

• N is between 1 .5x10⁷ and 5.0x10⁷ (7.17< log N < 7.70)

• No is between 1 .5x10⁶ and 5.0x10⁶ (6.17< log N < 6.70)

• Nvo is between 30 and 160 (3.0x10¹ and 1 .6x10²)

• A, B, C are equal to or greater than 0.5xN_vo

2.2. Results

Candida albicans ATCC 10231 Test log reduction values

Aspergillus brasiliensis ATCC 16404 Test log reduction values

2.3. Conclusion

The probiotic sanitiser test product tested at either 60g / 10L or 90g / 10L complied with the pass criteria of SANS test 53624:2011 (EN13624) standard, which requires at least a 99.99% kill (4 log reduction) in fungus.

EXAMPLE 5:

Efficacy of liquid formulation probiotic sanitiser with Bacillus SD. ( between about 6,00E+06 to about 1,00E+9 CFU ) and quaternary ammonium compound

1% chemical sanitiser

1. Bacterial pathgens

Efficacy of liquid formulation in water of probiotic sanitiser with Bacillus sp. (between about 6,00E+06 to about 1,00E+9 CFU), quaternary ammonium compound 1% chemical sanitiser, and 10% surfactant was tested against three pathogenic bacterial species by an accredited laboratory. The pass requirements in terms of the accredited test is that the product must demonstrate at least a 5 decimal log reduction in bacteria when diluted with hard water or undiluted.

The probiotic sanitiser was tested against three bacterial species: Enterococcus hirae ATCC 10541, Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538 for a contact time of 5 minutes at 20 °C. An amount of 0.3 g/L bovine serum albumin as added as the interfering substance. Thereafter samples were tested at 37 °C for bacterial growth.

For the test to be valid, for each test organism:

• N is between 1.5x10⁸ and 5.0x10⁸ (8.17< log N < 8.70)

• No is between 1.5x10⁷ and 5.0x10⁷ (7.17< log N < 7.70)

• vo is between 30 and 160 (3.0x10¹ and 1.6x10²)

• A, B, C are equal to or greater than 0.5xN o • Control of weighted mean counts: quotient is not lower than 5 and not higher than 15.

1.1. Results

Pseudomonas aeruginosa ATCC 15442 Test log reduction values

Staphylococcus aureus ATCC 6538 Test log reduction values

Enterococcus hirae ATCC 10541 Test log reduction values

1.2. Conclusion

The probiotic sanitiser test product tested at a dilution of 10% v/v or undiluted complied with the pass criteria of SANS test 53727:2011 (EN13727:2003) standard, which requires at least a 99.999% kill (5 log reduction) in bacteria.

2. Fungal pathogens

Efficacy of of liquid formulation in water of probiotic sanitiser with Bacillus sp. (between about 6,00E+06 to about 1 ,00E+9 CFU), quaternary ammonium compound 1% chemical sanitiser, and 10% surfactant plus soda ash or sodium chloride as a filler was tested against 2 pathogenic fungal strains by an accredited laboratory. The pass requirements in terms of the accredited test is that the product must demonstrate at least a 4 decimal log reduction in fungus when diluted with hard water or undiluted.

2.1. Methods

For the test to be valid, for each test organism:

• N is between 1.5x10⁷ and 5.0x10⁷ (7.17< log N < 7.70)

• No is between 1.5x10⁶ and 5.0x10⁶ (6.17< log N < 6.70)

• Nvo is between 30 and 160 (3.0x10¹ and 1.6x10²)

• A, B, C are equal to or greater than 0.5xN_vo

2.2. Results

Candida albicans ATCC 10231 Test log reduction values

Aspergillus brasiliensis ATCC 16404 Test log reduction values

2.3. Conclusion

The probiotic sanitiser test product tested at tested at a dilution of 10% v/v or undiluted complied with the pass criteria of standard SANS test 53624:2011 (EN13624) standard, which requires at least a 99.99% kill (4 log reduction) in fungus.

Claims

1. A probiotic Bacillus sanitiser for killing or inhibiting the growth of microorganisms on a surface, comprising or consisting of: a. as active ingredients, at least one species of Bacillus, at least one sanitising agent, and at least one surfactant; and b. optionally water, and/or additives including one or more colourants, fragrances, effervescent compounds, fillers including soda ash (sodium bicarbonate) or sodium chloride, degreasing agents, and cleaning agents, where no stabiliser is included.

2. The probiotic Bacillus sanitiser according to claim 1 , wherein the Bacillus comprises any one or more isolated GRAS (generally classified as safe) Bacillus species, including B. subtilis, B. pumilus, B. megaterium, B. licheniformis, B. coagulans, B. mojavensis, or B. amyloliquefaciens.

3. The probiotic Bacillus sanitiser according to either of claims 1 or 2, wherein the sanitising agent comprises any one or more of the following chemical sanitiser(s): chlorine, sodium dichloroisocyanurate (SDIC), chlorine dioxide, hypochlorous acid, hydrogen peroxide, sodium hypochorite, quaternary ammonium, alcohol including ethanol or isopropyl alcohol, or any derivative of the above.

4. The probiotic Bacillus sanitiser according to either of claims 1 or 2, wherein the one or more chemical sanitiser(s) is SDIC and/or quaternary ammonium.

5. The probiotic Bacillus sanitiser according to any one of claims 1 to 4, wherein the sanitising agent is a natural sanitiser comprising any one or more of essential oils or compounds including enzymes and/or flavonoids from antiseptic plants, including eucalyptus, neem, mint, rosemary, tea tree, witch- hazel, citrus peel, or lactic acid.

6. The probiotic Bacillus sanitiser according to any one of claims 1 to 5, wherein the surfactant is any one or more cationic and/or one or more non-ionic surfactant(s).

7. The probiotic Bacillus sanitiser according to claim 6, wherein the cationic surfactant is dimethyl distearyl ammonium chloride or sodium dodecylbenzene sulfonate.

8. The probiotic Bacillus sanitiser according to claim 6, wherein the non-ionic surfactant comprises a polar group that cannot be ionized in an aqueous solution, a fatty acid chain forming a hydrophobic component and a hydrophilic component containing non ionizable atoms of oxygen, nitrogen or sulphur, including any one or more of derivatives of polyoxyethylene (POE) or polyoxypropylene (POP), sugar esters and alkanolamides.

9. The probiotic Bacillus sanitiser according to any one of claims 1 to 8, wherein the pH of the probiotic sanitiser is between about 4.5 to about 9.5, or from about 5 to 9, or about 3.5 to 10.5, or about 8 to 9, or about 7 to 10, or about 9.5 to 10.5.

10. The probiotic Bacillus sanitiser according to any one of claims 1 to 9, which is formulated as a liquid or a dry (solid) form.

11. The probiotic Bacillus sanitiser according to claim 10, wherein the liquid form is formulated as a stock solution for further dilution by a user or distributor, or is formulated ready to use.

12. The probiotic Bacillus sanitiser according to claim 10, wherein the solid form is a granular or powder formulation which can be mixed with water to the desired liquid concentration by a user or distributor.

13. The probiotic Bacillus sanitiser according to claim 10 or 12, wherein the solid form further comprises a filler including soda ash (sodium carbonate) or sodium chloride.

14. The probiotic Bacillus sanitiser according to any one of claims 1 to 13, comprises degreasing components including soda ash (sodium carbonate), sodium tripolyphosphate, tetrapotassium pyrophosphate, sodium metasilicate anhydrous and sodium dodecylbenzene sulfonate.

15. The probiotic Bacillus sanitiser according to any one of claims 1 to 14 formulated as a liquid product comprising or consisting of per 1,000 L:

Bacillus sp. at between about 6,00E+05 to at least 0,0001% - 30% about 1 ,00E+11 colony forming units (CFU) depending on the CFU of the

Bacillis sp. used sanitising agent 10ppm - 1500ppm cationic and/or non-ionic surfactant(s) 5% - 15% water 40% - 80%

16. The probiotic Bacillus sanitiser according to any one of claims 1 to 14 formulated as a dry product comprising or consisting of per 1 ,000 g:

Bacillis sp. used soda ash or sodium chloride (as filler) 60%-80% sanitising agent 10ppm - 1500ppm cationic and/or non-ionic surfactant(s) 5%-15%

17. The probiotic Bacillus sanitiser according to any one of claims 1 to 14 formulated as a chemical degreasing product comprising or consisting of per 1,000 g:

Bacillis sp. used sanitising agent 10ppm - 1500ppm

sodium tripolyphosphate (technical grade) 10%- 100% tetra potassium pyrophosphate (technical 5%-10% grade) sodium metasilicate anhydrous 5%-10% sodium dodecylbenzene sulfonate 5%-10% surfactant sodium chloride

0,05%-30% soda ash (sodium carbonate) 30%-70%

18. A process for manufacturing a probiotic Bacillus sanitiser comprising or consisting of blending: a. as active ingredients, at least one species of Bacillus, at least one sanitising agent, and at least one surfactant; and b. optionally water, and/or additives including one or more colourants, fragrances, effervescent compounds, fillers including soda ash (sodium bicarbonate) or sodium chloride, degreasing agents, and cleaning agents, where no stabiliser is added.

19. The process according to claim 18, wherein the at least one surfactant is one or more cationic and/or non-ionic surfactant(s).

20. The process according to either claim 18 or claim 19, wherein the Bacillus comprises any one or more isolated GRAS (generally classified as safe) Bacillus species, including B. subtilis, B. pumilus, B. megaterium, B. licheniformis, B. coagulans, B. mojavensis, or B. amyloliquefaciens.

21. The process according to any one of claims 18 to 20, wherein the one or more sanitising agent(s) are any one or more chemical sanitiser(s) comprising: chlorine, sodium dichloroisocyanurate (SDIC), chlorine dioxide, hypochlorous acid, hydrogen peroxide, sodium hypochorite, quaternary ammonium, alcohol such as ethanol or isopropyl alcohol or any derivative thereof.

22. The process according to claim 21 , wherein the one or more chemical sanitiser(s) is SDIC and/or quaternary ammonium.

23. The process according to any one of claims 18 to 20, wherein the one or more sanitising agent(s) are any one or more natural sanitiser(s) comprising: essential oils or compounds including enzymes and/or flavonoids from antiseptic plants, including eucalyptus, neem, mint, rosemary, tea tree, witch- hazel, or citrus peel or lactic acid.

24. The process according to any one of claims 19 to 23, wherein the cationic surfactant is dimethyl distearyl ammonium chloride or sodium dodecylbenzene sulfonate.

25. The process according to any one of claims 19 to 23, wherein the non-ionic surfactant comprises a polar group that cannot be ionized in an aqueous solution, a fatty acid chain forming a hydrophobic component and a hydrophilic component containing non ionizable atoms of oxygen, nitrogen or sulphur, including any one or more of derivatives of polyoxyethylene (POE) or polyoxypropylene (POP), sugar esters and alkanolamides.

26. The process according to any one of claims 18 to 25, further comprising a step of adjusting the pH of the formulated probiotic Bacillus sanitiser to between about 4.5 to about 9.5, or from about 5 to 9, or about 3.5 to 10.5 or about 8 to 9, between about 7 to 10 or between about 9.5 to 10.5.

27. The process according to any one of claims 18 to 26, wherein the step of blending further comprises or consists of a step of providing the water to a mixing tank followed by addition of each of the active components and any of the optional additives as desired, and mixing until blended into a liquid formulation of the probiotic Bacillus sanitiser before packaging.

28. The process according to claim 27, wherein the active components and optional additives are in a dry form.

29. The process according to any one of claims 18 to 26, wherein the active components and optional additives are in dry form and the step of blending further comprises or consists of a step of adding the dry active components and optional additives to a mixer and mixing until blended into a dry formulation of the probiotic Bacillus sanitiser before packaging.

30. The process according to claim 29, further comprising a step of forming the dry formulation of probiotic Bacillus sanitiser into a tablet by stamping the dry formulation into a mould before packaging.

31. A method of killing or inhibition of the growth of one or more pathogenic or undesirable microorganisms on a surface by application of the probiotic Bacillus sanitiser according to any one of claims 1 to 17.

32. The method according to claim 31, wherein the microorganisms include bacteria, viruses, fungi, protozoa or algae.

33. The method according to claim 31, wherein the microorganisms are bacteria and/or fungi.

34. The method according to any one of claims 31 to 33, wherein the surface comprises any surface present in an industrial, medical, pharmaceutical, agricultural, business, public or domestic space and includes work surfaces, floors, walls, railings, handles, operating buttons, coveyers, or processing or packaging equipment.

35. The method according to any one of claims 31 to 34, wherein the method further comprises a step of dilution in water of a liquid formulation of the probiotic Bacillus sanitiser to a desired concentration prior to application.

36. The method according to any one of claims 31 to 34, further comprising a first step of dissolving a dry formulation of the probiotic Bacillus sanitiser in water to obtain a liquid formulation of a desired concentration prior to application.

37. The method according to claim 36, wherein the liquid formulation of the probiotic Bacillus sanitiser is used within 3 hours of mixing, or if stored in an air tight container, within 12 hours of mixing.

38. The method according to any one of claims 31 to 37, wherein the application comprises spraying a liquid formulation of the probiotic Bacillus sanitiser at a desired concentration onto the surface and wiping the surface, followed by allowing the surface to dry naturally.

39. The method according to any one of claims 31 to 37, wherein the application comprises providing a bucket containing a liquid formulation of the probiotic Bacillus sanitiser at the desired concentration, and application of the liquid formulation of the probiotic Bacillus sanitiser to the surface by means of a mop or cloth dipped into the bucket and then wiping or mopping the surface, followed by allowing the surface to dry naturally.