This invention concerns a composition for repelling mosquitoes, consisting of a combination of non-pathogenic bacteria.

In particular, this invention concerns a composition of non-pathogenic bacteria of the Bacillus genus which is applied to a support or applied by means of a dispenser, with the purpose of repelling mosquitoes. A support is primarily understood to mean any surface the composition can be applied to, either permanently or sporadically, with the purpose of repelling mosquitoes.

Flying mosquitoes are a significant annoyance in bedrooms mostly. Mosquito bites and the corresponding itching are highly irritating problems.

Mosquitoes belong to the order of the Diptera and specifically the suborder of the Nematocera.

They are mainly attracted by CO ₂ , a product of breathing.

On the other hand, the heat and humidity of a body and skin are an important source of attraction for mosquitoes

(Culicidae) .

The use of both repellents and insecticides, including those with a mosquitocidal effect, are generally known from the state of the art. In the first case, odorous agents tend to be used.

As repellents, both DEET (N,N-diethyl- 'm-toluamide) and

Picaridine are well known.

The use of natural plant oils as a mosquito repellent is well known.

Citronella is a good example.

In addition, lemon eucalyptus oil or PMD (P-menthan-3, 8- diol) , cinnamon oil, rosemary oil and peppermint oil are included in the state of the art. WO2002/002065 describes an insect repellent derived from Foeniculum Vulgare fruit, using (+) -fenchone and E-9- octadecenoic acid.

A disadvantage of the use of the aforesaid agents is that they need to be applied frequently and that the odour may ^¬ be annoying.

Furthermore, the use of the Bacillus thuringiensis bacterium,, a Gram-positive spore-forming bacterium, and specifically the subspecies israelensis, containing various plasmids with a mosquitocidal effect, is known. The subspecies sphaericus also contains protein toxins which are lethal to mosquito larvae (Toxicity of Bacillus sphaericus Crystal Toxin to Adult Mosquitoes, Stray ea., Applied and Environmental Microbiology, Sept. 1988, p.2320-2321) . An example of this is CrylAa.

GB2376887 also describes the use of B. thuringiensis (Bti) , together with insect-repellent oils, to control mosquitoes .

WO01/91559 describes the formation of insecticidal parasporal protein crystals of B. thuringiensis as an insect repellent.

This document does not describe the use of a combination of Bacillus species to deter mosquitoes. Moreover the B. thuringiensis is not encapsulated in contrast to

US5352597 that is used as a biocide and wherein said B. thuringiensis are enclosed by starch.

WO9628973 concerns a controlled release of a bioactive agent, a B. thuringiensis variant for the treatment of water organisms.

JP6199616 describes an insect deterring composition using B. subtilis without encapsulation.

These documents do not teach to deter mosquitoes of a living body by using a well-defined combination of spores of non-pathogenic Bacillus species provided in a breakable biopolymer or synthetic capsule and applied to a support, such as a living body, preferable by a spray can.

This invention is aimed at applying a mosquito repellent, consisting of a mixture of non-pathogenic bacteria of the Bacillus genus, to a support or the appropriate location by means of a dispenser. The non-pathogenic bacteria can be applied both as spores and as living bacteria .

Mosquitoes are repelled from the location where the mixture of non-pathogenic bacteria is applied to a support, such as textiles, or sprayed by means of a dispenser.

In addition, this support may include human skin or animal fur, in order to repel mosquitoes.

The first advantage of this invention is that a longer- lasting effect can be achieved if the mixture of nonpathogenic bacteria, occurring both as spores and as living bacteria, is applied to a support. In addition, the high reproductivity of the bacteria ensures a long- lasting effect.

Since the mixture of non-pathogenic bacteria is odourless and does not contain any harmful chemicals, it does not cause any additional annoying effects, nor irritation of the airways or skin, which is a second advantage.

The non-pathogenic bacteria of the Bacillus genus can be applied to a variety of supports, such as different textile fibres. This allows the mixture to be applied to both bedding and clothing or related textiles. The mixture can also be applied to textiles of sitting furniture or seats in transport means, such as car seats or corresponding seat covers. This is a third advantage.

The invention is detailed below. During laboratory tests in cube-shaped cages (1:50cm, h:50cm and b:50cm) with mice, containing 50-odd

mosquitoes, we discovered to our surprise that virtually no mosquitoes (96.2 % intrinsic defence) landed on a textile (10 by 10 cm at the bottom of the cage) , to which the mixture of non-pathogenic bacteria of the Bacillus genus is applied, in comparison with the cages containing a piece of textile without the mixture. The difference between the number of mosquito landings on a treated and an untreated piece of textile is called the intrinsic defence.

It was also found that the ambient defence, i.e. the difference between the number of landings on a target (= mice) representing a treated and untreated piece of textile, was 28.8 %. These tests were replicated three times.

Repeated tests on a human forearm in a closed area containing mosquitoes, with a textile-covered and a naked forearm, as well as with or without treatment, revealed that an average of 10.8 mosquito attacks occurred within a 5-hour timeframe. Where the naked forearm was treated with the emulsion of non-pathogenic bacteria, the number of attacks significantly decreased compared to an untreated forearm. Without treatment, 32 attacks were found, while with treatment the first mosquito attacks occurred only 3 hours later. In addition, it was found that the use of microcapsules, containing the nonpathogenic bacteria, significantly prolonged the

repelling properties. Similar tests revealed that the repelling effect lasted for 12 hours.

We conclude from this that the mixture of non-pathogenic bacteria of the Bacillus genus has a strong mosquito- repellent effect. In particular, our mixture consists of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilis and Bacillus megaterium.

It is primarily the interactive and combined effect of the various bacteria which achieves the mosquito- repellent effect.

The non-pathogenic bacteria of the Bacillus genus can be applied both as spores and as living bacteria, depending on the intended purpose. In the former case, a quick action (sporadic) is intended, while in the latter case a rather slow (permanent) , but mainly long-lasting, effect is achieved. Using a dispenser, the non-pathogenic bacteria are applied to the desired location or support. The non-pathogenic bacteria may be applied as an oil emulsion or gel.

In another possible embodiment the living bacteria or their spores are encapsulated in a capsule, consisting of a biopolymer or synthetic polymer. In this form, the nonpathogenic bacteria can be applied to a support and they will have a slower, but longer-lasting effect against mosquitoes.

When applying the mixture of non-pathogenic bacteria of the Bacillus genus, a dispenser such as an atomiser can be used, or they can be applied by means of impregnation or as a coating on the support in question. Textiles are understand to mean primarily mattresses, bedding, sheets, pillows, but also clothing and related items such as sleeping clothes and handkerchiefs. The textiles can be either woven or non-woven. They are then treated with the mixture in such a manner that it penetrates the fabric or the fibres of the textile.

Furthermore, the mixture can also be applied in textiles of sitting furniture, such as sofas in living rooms or seats in transport means, as well as corresponding seat covers .

Also, the mixture can be applied to human skin or animal fur to repel mosquitoes .

In another possible embodiment the mixture with nonpathogenic bacteria of the Bacillus genus is put in an automatic dispenser which atomises the mixture at the desired location. A dispenser is understood to mean primarily an atomiser dispensing the mixture in the air or on a textile at the desired location. Such automated devices are usually fitted with a pumping mechanism. This device may also include a timer to atomise the mixture at specific points in time.

Such dispensers are operated with electricity, batteries or a manual pumping mechanism.

If the non-pathogenic bacteria are encapsulated,

vibrations in the dispensers may cause the capsules with non-pathogenic bacteria to be broken by internal

frictional forces. These vibrations may be generated by ultrasonic waves .

This dispenser must be installed in such a manner that it is located near a potential target, such as a human body, a pet or a textile.