The present invention relates to a use for reducing and/or inactivating endotoxins.

Such endotoxins are part of the cell wall of gram-negative bacteria and cyanobacteria and can cause an airway irritation and/or an airway infection in humans, particularly in case of a high exposure to endotoxins.

Humans are predominantly exposed to endotoxins by inhalation.

A number of important sources of endotoxins are:

● Bacteria coming from the stool of people and/or mammals and/or manure, said bacteria being bound to dust particles which can spread via the air;

● Bacteria growing on plants and/or production crops and which when harvesting and/or storing and/or processing are released;

● Bacteria coming from the skin or fur of humans and/or animals;

● Bacteria in process water from factory processes, water purification, sludge processing and/or the like where recirculating process water is used.

Recirculating process water may result in the growth of bacteria, whereby endotoxins are thus released in the process water. The extent to which people are exposed to endotoxins can vary strongly.

For instance, a number of sectors have a higher risk of exposure to a high and/or hazardous amount of endotoxins, said sectors not being limited to the list below:

Agricultural/farming industry: o Poultry and livestock farming o Fruit and vegetable growing o Agricultural, forestry and/or manure processing; o Transport of animals; o Biodigesters; o Slurry pits;

● Processing and storing grains and/or corn and/or coffee beans and/or ground coffee and/or nuts and/or plants and/or vegetable, fruit and garden waste and/or herbs and/or animal materials and/or biological materials and/or flowers and/or the like;

● Other processing industries: o Waste processing; o Cotton processing; o Paper processing;

● Water purification and/or soil remediation and/or sewage works;

● Health care: o Physicians; o Dentists; o Nurse; o First Aid responders; o Disaster relief; • Rescue workers and firefighters;

• Construction: o Construction workers; o Remediation contractors; o Demolition workers;

● Pest control.

In addition, there is also a higher exposure to endotoxins in poorly ventilated spaces and/or offices and/or schools, in particular in places where a relatively large number of people and/or animals are present at the same time.

The existing "Heating Ventilation Air conditioning (HVAC) " installations can also cause a higher endotoxin content in the air.

The endotoxin content can be measured by means of a Limulus amebocyte lysate (LAL) test which is extremely sensitive and accurate.

In a recent recommendation the Health Council in the Netherlands proposed a limit of 90 endotoxin units/m3 for a working day in an enclosed space and a limit of 30 endotoxin units/m3 outdoors.

High exposure to endotoxins can result in an acute local disorder (typically lung and/or airway disorders), acute systemic disorders and chronic disorders, said disorders usually being pneumatic, disorders such as asthma, dyspnoea, COPD and the like. Various methods for reducing and/or neutralising endotoxins are already known.

Physical methods for neutralising and/or reducing exist, whereby typically ultraviolet light (UV light) is used.

Water or process water is radiated with UV light to inactivate endotoxins in the water. The inactivation of endotoxins is proportional to the dose of the UV light and the speed of the inactivation is approximately 0.55 EU/ml for every mJ/cm ² .

EU stands for "endotoxin Units here.

A disadvantage is tthhaatt the UV rays ccaann only reach to a certain depth such that the effectiveness is very limited.

It goes without saying that during radiation of the water with UV light, no people can be present in the space and/or near the UV light and/or the radiated water.

In addition, chemical methods exist for neutralising and/or reducing endotoxins.

On the one hand aluminium,hydroxide can be used.

The endotoxins hereby irreversibly adsorb to the surface of aluminium hydroxide. Adsorption occurs through electrostatic attraction and covalent bonds.

A disadvantage of this is that chemicals are needed for inactivating endotoxins, said chemicals possibly being harmful for the environment and/or requiring a separate post-treatment.

This naturally implies extra costs, which can be relatively high. Additionally , said chemical methods also require relatively high amounts of energy for reducing endotoxins, as well as for the post-treatment of the chemicals.

On the other hand endotoxins can be neutralised by photocatalysis. A photocatalyst is used for this, typically of the titanium dioxide semiconductor (TiO ₂ ).

Under the influence of radiation with UV light, electrons in the TiO2 can be excited, whereby electrons are elevated to a higher energy level. By transferring electrons said particles are transformed into radicals. These radicals can then cause the breakdown of organic components such as endotoxins .

This method also requires relatively high amounts of energy.

Such methods can lower the endotoxin content in water and/or the air slightly, albeit insufficiently.

The duration of action of the aforementioned methods is very limited. Thus, endotoxins can only be inactivated during the administration of the UV light or aluminium hydroxide. US 4832751A is also known which discloses a method for reducing endotoxins in cotton fibre or dust, whereby the cotton fibre or dust is detoxified,in a solution containing at least 95% ethanol and.hydrochloric acid; 95% ethanol and sodium hydroxide; and dimethylsulfoxide.

The useability of the aforementioned methods is limited and during their application no people and/or animals may or should be present.

US5413785A discloses a method for reducing the quantity of endotoxin circulating in the blood plasma ooff a living subject affected by an endotoxin-mediated clinical state.

The method employs a formulated preparation comprising at least one persistent, acid-resistant , and colonising Lactobacillus species in combination with a biocompatible carrier. The uusseerr then introduces the formulated preparation to the gastrointestinal tract of the subject on one or on multiple occasions.

JP2000072659A discloses a method for inactivating endotoxin with simple equipment without using any organic solvent and ttoo obtain aann endotoxin inactivator, whereby the addition of ,an endotoxin inactivator is characteristic for the method. The endotoxin inactivator contains either (a) quaternary ammonium hydroxide, (b) quaternary ammonium hydroxide and a base, or (c) a quaternary ammonium salt and a base. JPS5865223A discloses an invention containing a composition comprising one or more antibodies that,are specific for one or more endotoxins, more specifically those produced by gram negative bacteria, mixed with a carrier medium. The carrier medium can be animal or human serum, milk or a synthetic material. The invention also relates to a method for producing a composition comprising an antibody or a mixture of antibodies specifically for endotoxin (s), including the steps of inoculating a suitable animal with an antigen or a mixture of antigens that is able to produce the correct immunological response in the animal and removing a bodily fluid comprising the resulting antibody or mixture of antibodies.

The purpose of the present invention is to provide a solution to at least one of the aforementioned and other disadvantages .

To this end, the invention relates to a use of a composition containing spores, which can grow into aerobic spore-forming bacteria, and/or aerobic spore-forming bacteria, whereby a use of such composition, reduces and/or breaks down airborne endotoxins whereby the use contains the following steps: providing an atomiser and the composition in the atomiser;

- atomising the composition in the air by means of an atomiser. An advantage of this is that the use of the composition has a prolonging action, in other words, the action of the composition is not limited to the moment of administration.

Thus, endotoxins will still be inactivated and/or reduced after applying the composition until none of the aforementioned spores and bacteria are present anymore.

In addition the composition is also self-sustaining, such that the composition needs to be applied less often for reducing endotoxins.

Self-sustaining is understood to mean here that the spores can grow into spore-forming bacteria which in turn can form spores again, such that new bacteria are formed regularly that can inactivate and/or reduce endotoxins.

The aforementioned composition is not harmful for people and other mammals either such that the composition can be administered in the presence of people and/or animals.

The aerobic spore-forming bacteria in the composition can reduce and/or break down free endotoxins and/or endotoxins bound to fine dust and/or endotoxins on a surface and/or endotoxins in a fluid and/or the like and/or inactivate active endotoxins.

Preferably, one or more of the following Bacilli from the

Bacillus genus are chosen for the composition: Bacillus subtilis and/or Bacillus pumilus and/or Bacillus licheniformis and/or Bacillus velezensis and/or a combination of two or more of the aforementioned Bacillus.

The present invention relates to a use of a composition as described above for breaking down and/or inactivating endotoxins in the air, said use containing the following steps: providing an atomiser and the composition in the atomiser;

- atomising the composition in the air by means of an atomiser.

In addition, the invention also relates to other uses of the composition for breaking down and/or inactivating endotoxins in process water and/or another fluid to be treated and/or in a biological mass and/or on a surface and/or in a HVAC.

It is understood that the use of the composition offers at least the same advantages as the aforementioned composition .

With the intention of better showing the characteristics of the invention, a preferred embodiment of a composition according to the invention and a use of the composition is described hereinafter, by way of an example without any limiting nature with reference to the accompanying drawings wherein: figure 1 schematically shows a perspective view of a composition according to the invention; figure 2 shows an atomiser for applying the composition of figure 1; figure 3 schematically illustrates the use of the composition in a fluid; figure 44 schematically illustrates the use of the composition in or on a biological mass; figure 5 schematically illustrates the use of the composition on a surface; figure 6 schematically illustrates the use of the composition in an HVAC installation; figure 7 shows a chart which shows the reduction of the endotoxin content.

Figure 1 shows a composition 1 according to the invention for reducing and/or inactivating active endotoxins.

The composition 1 contains spores 3, which can grow into aerobic spore-forming bacteria, and/or aerobic spore- forming bacteria 4.

The aforementioned aerobic spore-forming bacteria 4 can break down and/or inactivate endotoxins ooff gram-negative bacteria and/or cyanobacteria.

The composition 1 can be a powder containing spores 3, which can grow into aerobic spore-forming bacteria, and/or aerobic spore-forming bacteria 4.

However, it is also possible that the composition contains a medium on which the spores 3 which can grow into aerobic spore-forming bacteria, and/or aerobic spore-forming bacteria 4, are applied.

Preferably, such medium contains water and ethanol, whereby the ethanol content amounts, most preferably, to less than

5% of the total medium.

The relatively low content of ethanol ensures that the spores will not unintentionally germinate and grow into aerobic spore-forming bacteria.

Only after the ethanol content in the composition 1 is low enough, either through evaporation of the ethanol in the air or through dilution of the composition 1 in a fluid, more specifically dilution of the ethanol content, will the spores 3 germinate under favourable circumstances and grow into aerobic spore-forming bacteria 4.

The aforementioned spores 3 and/or aerobic spore-forming bacteria 4 from the Bacillus genus are chosen.

The aerobic spore-forming bacteria 4 in the composition 1 can reduce and/or break down the free endotoxins and/or endotoxins bound to fine dust and/or endotoxins on a surface and/or endotoxins in a fluid and/or the like and/or inactivate active endotoxins.

The concentration of the aforementioned spores 3 and/or aerobic spore-forming bacteria 4 in the composition 1 amounts to between 10 ⁶ KVE/ml and 50 x 10 ⁷ KVE/ml. Preferably, one or more of the following Bacilli from the

Bacillus genus are chosen: Bacillus subtilis and/or

Bacillus pumilus and/or Bacillus licheniformis and/or Bacillus velezensis and/or a combination of two or more of the aforementioned Bacillus.

As shown in figure 7 the aforementioned Bacilli can reduce and/or inactivate endotoxins, whereby the known "limulus

Amebocyte Lysate" test (LAL-test) is used for this measurement .

This chart shows the optical density at a wavelength of 545 nanometer.

To obtain the results, the Bacilli were grafted,in a Luria.

Bertani (LB) broth and.incubated for 48 hours at 30 °C.

The concentration of the endotoxins is 25000 EU before adding and/or incubating Bacilli.

The chart in figure 7 shows the quantity of endotoxins in the LB broth 48 hours after adding the Bacilli.

Legend of figure 7:

A) Bacillus velezensis

B) Bacillus licheniformis

C) Bacillus pumilus

D)Bacillus subtilis

E) Polymyxin B (Positive control)

F)Negative control Each of the aforementioned conditions was tested in the aforementioned LB broth with the aforementioned concentration of endotoxins.

Polymyxin B was used as positive control with, an optical density of 20. It is already known that polymyxin B binds and/or inactivates free endotoxins.

The negative control in said test only contains LB broth and the aforementioned concentration of endotoxins with an optical density of 150.

The aforementioned LAL-test snows that the aforementioned

Bacilli decrease the endotoxin content.

The invention is intended to reduce and/or break down the endotoxins in an indoor area and/or indoor environment and/or the like .

The present invention relates to a use of the aforementioned composition 1 for breaking down and/or inactivating endotoxins in the airy said use containing the following steps:

- providing an atomiser 5 and the composition 1 in the atomiser 5; atomising the composition 1 in the air by means of an atomiser 5.

Atomiser 5 is understood to mean an aerosol and/or spray and/or diffuser and/or the like here. It is not excluded that ventilation systems and/or airconditioners and/or the like can serve as atomiser 5 for diffusing the composition 1 in the air.

In an alternative application the composition 1 can also be used for breaking down and/or inactivating endotoxins in process water and/or other fluid to be treated, as shown in figure 3, whereby the use contains the following steps:

- providing the composition 1 and dosing thereof in the process water and/or other fluids 6 to be treated, that may or may not contain endotoxins.

Thus the composition 1 can be dosed in the process water of a paper factory and/or a sludge processing company and/or the like.

In another alternative application, aass shown in figure 4, the composition 1 can also be used for breaking down and/or inactivating endotoxins in a biological mass 7, said use at least containing the following steps:

- providing an endotoxin-containing biological mass 7 in a biodigester 8 and/or a compost bin and/or a silage and/or the like; dosing the aforementioned composition 1 in the endotoxin-containing biological mass 7 in a biodigester 8 and/or a compost bin and/or a silage and/or the like.

Biological mass 7 is understood to mean a mass of human and/or animal and/or vegetable and/orother biological origin, such as sludge and/or manure and/or the like. In yet another alternative application, the composition 1 can be used for breaking down and/or inactivating endotoxins on a surface 9, the composition 1 is applied on the aforementioned surface 9 by means of grafts, as shown in figure 5.

In this application the endotoxin-content in the air can also be reduced and/or endotoxins in the air can be inactivated, for example in a stable with animals and/or a recycling company.

A surface 9 can be grafted in different ways.

For example, the composition 1 can be applied on a surface 9 using an atomiser 5 by atomising oorr spraying the composition 1 directly on the relevant surface, like in the example of figure 5.

Incidentally, by atomising the composition 1 in the air using the aforementioned atomiser 5, surfaces 9 can also be grafted on which the composition 1 settles.

In an alternative use of the composition 1 endotoxins in a building can be reduced and/or broken downr whereby the use contains the steps of applying the composition 1 in an HVAC installation 10 and diffusing the composition though the air, as shown in figure 6.

Such HVAC installation 10 can be provided with a tube system 11 through which the air containing the composition 1 is blown. Consequently, several spaces 12 in one and the same building 13 can be provided with the composition 1 to reduce and/or break down the endotoxins in the air and on the surfaces on which the composition 1 settles.

Such HVAC installation 10 can thus also serve as aerosol.

In addition, it is also possible to use the invention in other sectors where the exposure to endotoxins is relatively high as specified above in the aforementioned list .

The present invention is by no means limited to the embodiments described as an example and shown in the figures, however, a use of such composition can be realised according to different variants without departing from the scope of the invention.