WO2025238215A1 - Process for producing an alcohol-free disinfectant - Google Patents

Abstract

The invention relates to a process for producing an alcohol-free disinfectant comprising at least one polyaminosaccharide and at least one carboxylic acid, comprising the steps of: - mixing the polyaminosaccharide with the carboxylic acid to obtain a mixture of the polysaccharide and the carboxylic acid; - treating the mixture in a shear film mixer to obtain a homogeneous disinfectant.

Description

Method for producing an alcohol-free disinfectant

The present invention relates to the technical field of disinfectants.

In particular, the present invention relates to an alcohol-free disinfectant and a method for producing an alcohol-free disinfectant.

Disinfection is the process of rendering dead or living material incapable of causing infection. It is therefore an essential component of antiseptic practices. Disinfection is typically carried out using chemical agents or physical methods. Physical methods include, in particular, ionizing radiation, ultrasound, and ultraviolet radiation, which act directly on pathogenic agents, killing or inactivating them.

Chemical disinfectants are also called disinfectants and are generally used to disinfect surfaces of the skin, clothing, equipment or rooms, but also to disinfect drinking water, food, seeds or waste.

Disinfectants are substances or mixtures of substances that, when applied to objects or surfaces, render them incapable of causing infection. The effect of a disinfectant is preferably generally microbicidal, i.e., bactericidal, fungicidal, virucidal, and sporicidal; therefore, the application of disinfectants is also referred to as sterilization with chemical agents.

The requirements for disinfectants are wide-ranging: In particular, a broad spectrum of activity is required, combined with low toxicity, high skin compatibility, and good material compatibility. Short contact times and long-lasting effects are further important criteria.

Common disinfectants are often based on high concentrations of alcohols, such as ethanol or isopropanol, or they contain specific active ingredients, such as aldehydes, halogens, peroxo compounds and/or quaternary ammonium compounds. A problem with disinfecting surfaces, especially in public spaces or on skin, or with wound disinfection, is that recontamination with pathogens from the environment occurs quickly after disinfection. While disinfectants are often highly effective when applied to the surface, they are rapidly removed, for example through evaporation or wiping, so recontamination is to be expected.

For this reason, film-forming disinfectants were developed, which are generally applied in the form of solutions or dispersions. After the solvent or dispersion medium evaporates, they remain on the surface, providing a longer-lasting microbicidal effect. It is particularly important for film-forming disinfectants that they have low toxicity and are also compatible with skin and materials. In particular, they must not adversely affect the properties of the surfaces to which they are applied.

Furthermore, there is a strong demand among users for hand and surface disinfectants that are free of alcohols, aldehydes, halogens, and peroxide compounds. Alcohols, for example, can dry out the skin or damage surfaces. Other ingredients in conventional disinfectants, on the other hand, can cause allergic reactions and are therefore viewed critically by users.

Sustainability and the ecological footprint of disinfectants are also playing an increasingly important role for many users. Aqueous formulations with naturally occurring active ingredients are particularly advantageous in this regard.

At the same time, the effect of a disinfectant should occur as quickly as possible and last as long as possible.

Biopolymers, especially chitosan, which have film-forming properties, are increasingly being used for disinfection. Chitosan, also called polyglusam or poly-D-glucosamine, is a naturally occurring biopolymer derived from chitin, the second most abundant biopolymer in nature after cellulose. Chitosan is a polyaminosaccharide and consists of 2-glucosamine or 2-amino-2-deoxy-β-D- Chitosan consists of glucopyranose monomers linked by β-1,4-glycosidic bonds and linearly. Technically, chitosan can be produced from chitin by deacetylation. This deacetylation can be achieved either by using a strong base, such as sodium hydroxide, or enzymatically. The resulting chitosan differs from chitin in its variable degree of deacetylation. Chitosan can also be obtained from certain fungi.

The antimicrobial effect of chitosan has been known for some time. In addition, chitosan has coagulating and hemostatic properties, which is why chitosan preparations are used, among other things, in the medical treatment of slow-healing wounds.

Chitosan possesses a broad microbiological spectrum of activity – it is bactericidal, virucidal, and fungicidal – and is also hypoallergenic and harmless to humans and ecotoxicology. These properties make chitosan particularly attractive for use in disinfectants.

However, to achieve the rapid reduction in germ count required for disinfection purposes, the antimicrobial potential of chitosan alone is not sufficient.

Therefore, it is advantageous to add further components to the disinfectant which, in combination with chitosan, lead to a significantly higher and faster reduction in the number of germs.

Since chitosan can be converted into a water-soluble form using organic acids, organic acids that themselves exhibit antimicrobial activity are preferable as combination components with chitosan. The phenolic acid salicylic acid, which is also known for its antimicrobial effect, has proven particularly suitable in this regard. Consequently, there are numerous attempts to use a combination of chitosan and salicylic acid in the hygiene and cosmetics sectors.

In US 2008 / 0045491 Al, a disinfectant for surfaces and also skin is described, which contains a water-soluble alcohol as its main component in addition to the combination of salicylic acid and chitosan.

CN 104622710 A also describes a combination of chitosan and salicylic acid, where the weight ratio of salicylic acid to chitosan is 1:1 to 1:1.8. Here too, alcohols such as ethanol are listed as additional components. or glycerin is used. The product CN 104622710 A is classified as a cosmetic, since the disclosed combination mitigates or eliminates the skin-irritating effect of free salicylic acid.

WO 02/49604 Al describes the application of the combination of salicylic acid and chitosan in moist wipes for skin care. Here, the combination of salicylic acid and chitosan serves as a multifunctional active ingredient that, on the one hand, prevents the growth of germs in the moist wipe, thus acting as a preservative, and on the other hand, is beneficial to skin care.

US patent 2007/0048356 describes antimicrobial compositions for coating surfaces. Such an antimicrobial composition may, for example, contain a combination of polyhexanide (PHMB), salicylic acid, and chitosan.

EP 4 088 784 Al describes antimicrobial compositions with three active components that can be used for cosmetic applications as well as for disinfection purposes. These three-component combinations each consist of a polysaccharide composed of amino sugar monomers, a carboxylic acid, and a polyamine or guanidine. The polysaccharide can be, among other things, chitosan. The carboxylic acid can be, among other things, salicylic acid. The three active components act synergistically, so that certain properties, such as water solubility and disinfection efficacy, are significantly improved compared to the individual components.

EP 1 786 264 Bl describes antimicrobial compositions consisting of an antiseptic (e.g., a biguanide), an enhancer (e.g., a beta-hydroxy acid such as salicylic acid), a hydrophilic component (e.g., a polyhydric alcohol), a surfactant, and a hydrophobic carrier substance. Optionally, a bioadhesive polymer, which may be chitosan, can be added. In one embodiment, the composition is prepared as a water-in-oil emulsion using a high-shear mixer.

CN 107 318 970 A discloses antibacterial coatings for pomegranates containing, for example, chitosan, sodium alginate, lecithin, citric acid and natamycin, the ingredients being homogenized in a “high-shear dispersing emulsifier”.

CN 117 814 239 A discloses a disinfectant which which includes chitosan and an organic acid.

Document KR 2020 0126078 A describes the production of antimicrobial solutions containing chitosan, hyaluronate and mandelic acid.

Document WO 2013/066322 Al discloses antimicrobial compositions that include chitosan and citric acid.

Document DE 10 2014 114488 Al relates to a mold control agent that contains, among other things, chitosan and an acid (e.g. citric acid, lactic acid, acetic acid and/or tartaric acid).

The known examples of applications combining chitosan and salicylic acid in disinfection are primarily intended for long-term protection, or for preventative or preservative effects. However, they reach their limits when rapid disinfection is required.

Especially in hand and surface disinfection, it is essential to achieve the required reduction in germ count very quickly. Preferably, a reduction factor of at least 5 log units for Enterococcus hirae (DSM 3320), Staphylococcus aureus (DSM 799), Pseudomonas aeruginosa (DSM 939), Escherichia coli K12 (DSM 11250) and Proteus mirabilis (DSM 788) and a reduction factor of at least 4 log units for Candida albicans (DSM 1386) should be achieved within 30-60 seconds in the quantitative suspension test according to EN 13727, EN 13624 or VAH 9.

Formulations known from the prior art using chitosan and salicylic acid as active ingredients are unable to achieve this.

In contrast, the object of the present invention is to alleviate or overcome the disadvantages of the prior art described above. The invention preferably aims to provide a disinfectant that has a broad spectrum of activity and good skin and material compatibility, is harmless to humans and ecotoxicology, and yet achieves a very rapid and high reduction in the number of germs on the treated surface.

In particular, the task is to provide an alcohol-free disinfectant which achieves a reduction factor of at least 5 lg levels for Enterococcus hirae (DSM) in the quantitative suspension test according to EN 13727, EN 13624 or VAH 9. 3320), Staphylococcus aureus (DSM 799), Pseudomonas aeruginosa (DSM 939), Escherichia coli K12 (DSM 11250) and Proteus mirabilis (DSM 788) and a reduction factor of at least 4 log levels for Candida albicans (DSM 1386) within 60 seconds.

Therefore, the present invention relates to a process for the production of an alcohol-free disinfectant containing at least one polyaminosaccharide and at least one carboxylic acid, comprising the steps of:

- Mixing the polyaminosaccharide with the carboxylic acid, resulting in a mixture of the polysaccharide and the carboxylic acid;

- Treating the mixture in a shear film mixer to obtain a homogeneous disinfectant.

The disinfectant according to the invention provides an efficient and very skin-friendly agent which can ensure a reduction in the number of germs in a very short time, i.e. less than 60 seconds, when disinfecting hands and surfaces.

The disinfectant according to the invention has a broad spectrum of activity and is harmless to humans and ecotoxicology, yet it achieves a very rapid and sufficient reduction in germ count. This is also surprising because the disinfectant according to the invention is produced without the involvement or addition of alcohol, i.e., it is purely aqueous. Accordingly, the disinfectant according to the invention is free of alcohols, in particular free of ethanol and isopropanol, which are otherwise very common in disinfectants. "Free of alcohols" in the context of the present invention means, at a minimum, that no alcohols are added during production and that the disinfectant does not derive its disinfecting effect from alcohols. More precisely, the disinfectant according to the invention is absolutely free of disinfecting alcohols or organic solvents such as acetone or acetonitrile, in particular free of ethanol or isopropanol. The disinfectant according to the invention therefore contains at least less than 1 wt%, preferably less than 0.1 wt%, in particular less than 0.01 wt%, ethanol, methanol, isopropanol, acetone or acetonitrile.

Surprisingly, it could be shown according to the invention that the combination of the components polyaminosaccharides and carboxylic acids, which are not sufficiently disinfecting in themselves, such as For example, the disinfectant effect of chitosan and salicylic acid (according to EP 4 088 784 Al) can be significantly enhanced by mixing the components under strong shear forces in a shear film mixer. This special manufacturing process therefore achieves a significant increase in the broad-spectrum disinfectant activity, making it possible to use this aqueous combination solution as a hand and surface disinfectant without the need for skin-damaging and/or skin-irritating ingredients.

The increase in activity may be due, among other things, to the fact that only the use of a shear film mixer makes it possible to dissolve the components, especially salicylic acid, in a sufficiently high concentration or to mix them homogeneously.

Preferably, after treatment in the shear film mixer, the disinfectant contains essentially no suspended particles, in particular no suspended particles visible to the naked eye. Preferably, when the disinfectant is filtered through a filter with a pore size between 12 pm and 15 pm (for example, Carl Roth Rotilabo circular filter type 11A / 110 mm / 12-15 pm), no suspended particles visible to the naked eye remain on the filter.

A shear film mixer, such as a shear film reactor, has the special property of enabling almost instantaneous, perfect homogenization of different components. Furthermore, in the mixing zone, which can be extremely thin (e.g., 50 micrometers), the components are forced into extremely small, water-dissolved parcels by the very high shear forces generated, for example, by a very rapidly rotating cylinder on this reaction film. This fragmentation of molecular clusters by means of eddy dispersion can contribute to a significant increase in the reactivity of the individual components.

This is especially the case if the molecular clusters are successfully broken down into the sub-Kolmogoroff region, which is not possible with conventional stirred reactors.

With the shear film mixers used here, this is easily achieved because the shear forces acting on the disinfection solution can be selected by varying the distance between the rotor and stator and by choosing the rotational speed. that a maximally highly dispersed distribution of the components can be achieved.

Another advantage of this technology is that it achieves a dispersion with a very narrow Gaussian distribution of the molecular units, for example, of chitosan. This, in turn, leads to an exceptionally stable dispersion, in which the larger and less reactive molecular clusters/conglomerates do not regress over time.

A further advantage of this technology is that the highly efficient dispersion is nevertheless very gentle. Without being limited to a specific mechanism or theory, a complex bond preferably forms as an acid/base complex with hydrogen bonds between the components polyaminosaccharide and carboxylic acid, in particular phenolic acid, so that the reactivity and film-forming properties of the disinfectant according to the invention are retained.

Accordingly, the shear film mixer is selected from mixing reactors capable of generating such sub-colmogoroff dispersions. Preferably, the shear film mixer is selected from shear film reactors, jet mixers, rotor-stator mixers, ultrasonic mixers, high-pressure homogenizers, rotor-stator colloid mills, microfluidic systems, vibrating mixers, kneading and roller mixers, planetary mixers, colloid mills, grinding balls or beads, or pressure homogenizers, preferably shear film reactors, jet mixers, ultrasonic mixers, high-pressure homogenizers, or microfluidic systems, particularly shear film reactors. Such mixers are capable of exerting the shear forces required according to the invention on the mixture of polyaminosaccharide and carboxylic acid, particularly as a complex compound, to ensure the reactivity of these mixtures, especially by providing them as complexes. In this context, a specialist also knows the individual manufacturing parameters for obtaining such sub-Kolmogoroff Eddies, which leads to the aqueous disinfectant according to the invention with an increased antimicrobial efficacy.

A shear film reactor is a special type of reactor used in chemistry and biotechnology. It is also known as a shear rate reactor. This reactor utilizes the shear force or shear stress that arises when a fluid is subjected to shear forces. (Liquid or gas) flows past a solid surface. This shear stress can cause molecules within the fluid to interact, mix, or react with each other. A shear film reactor is used to accelerate reactions by increasing the diffusion rate through shear stress, thereby improving the interaction between the reactants. This can be particularly useful in reactions where one of the reactants is contained in a solid or in an inert phase (as in the case of the present invention), since the shear force helps to overcome these obstacles. A particularly preferred shear film reactor according to the invention is, for example, the tube-in-tube device from AtlantiChem.

However, there are numerous other devices and mixing technologies for producing homogeneous mixtures using shear forces:

In a jet mixer, high-speed jets of liquids or gases are used to collide, creating intense shear forces and mixing materials.

In rotor-stator mixer technology, the rotation of a rotor within a stationary stator is used to generate shear forces and mix materials. The combination of rotational and shear forces contributes to homogenization.

In an ultrasonic mixer, ultrasound can be used to generate shear forces and to disperse and mix materials in liquids. Intense shear forces can be generated by applying high-frequency sound waves.

The high-pressure homogenizer uses high pressure to force materials through narrow gaps, generating intense shear forces. It is commonly used for the production of emulsions and suspensions.

Rotor-stator colloid mills use a rotating rotor and a stationary stator to force materials through narrow gaps and generate shear forces. They are particularly useful for the comminution and dispersal of materials.

Microfluidic systems utilize tiny channels and microstructures to precisely manipulate and mix liquids. By controlling flow patterns, shear forces can be generated to homogenize materials.

Vibrating mixers use vibrations to mix materials. to mix and homogenize. The continuous movement generates shear forces that contribute to the mixing.

Kneading and rolling mixers are often used in the food and pharmaceutical industries. They utilize mechanical movements such as kneading and rolling to mix and homogenize materials.

Planetary mixers use a planetary gear system to rotate a central mixer around a fixed axis while simultaneously moving around another axis. This creates complex motion patterns that contribute to homogenization.

Colloid mills use shear force to press and grind materials through narrow gaps. This can be used to produce suspensions and emulsions.

When mixing using grinding balls or beads, materials are placed together with grinding balls or beads in a container and mixed by vibration or rotation. The balls generate shear forces that contribute to homogenization.

Pressure homogenizers use high pressures to force materials through narrow gaps and generate shear forces. They are particularly useful for producing finely dispersed suspensions.

According to a preferred embodiment, the polyaminosaccharide comprises monomer units of optionally substituted amino sugars, in particular glucosamines and/or galactosamines, and is preferably selected from the group consisting of chitosan, hyaluronic acid, keratin sulfate, heparin, chondroitin sulfate, derivatives thereof with a molecular weight of 20 kDa or more, or mixtures thereof, in particular chitosan, chitosan derivatives with a molecular weight of 40 kDa or more, hyaluronic acid or mixtures thereof.

Suitable chitosan derivatives which can be used within the scope of the present invention are in particular hydroxypropylchitosan, N-octyl-N-trimethylchitosan, N-octyl-O-glycolchitosan, N-[(2-hydroxy-3-trimethylammonium)-propyl] chitosan chloride, carboxymethylchitosan and mixtures thereof.

Chitosan, in particular, is known as a suitable component in disinfectants, but until now it has not possessed the rapid disinfecting effect (without added alcohol). It was already known that long-chain chitosans form a film, leading to a longer-lasting disinfection effect on skin and surfaces. Short-chain chitosans In contrast, they are more "mobile" and increase the kinetics of the antimicrobial effect. However, in the composition according to the invention, the polyaminosaccharide, in particular chitosan, in combination with the carboxylic acid, could be formulated into a highly active agent that, on its own, i.e., without the addition of biocidal alcohols, exhibits a sufficiently efficient and rapid disinfecting effect. This mechanistically retains the known mode of action of both low-molecular-weight (e.g., below 50 kDa) and high-molecular-weight chitosans (e.g., 100 to 200 kDa), but significantly enhances their effect.

Preferably, the disinfectant contains chitosan as a polyaminosaccharide, which has a degree of deacetylation of at least 70%, preferably at least 80%, even more preferably at least 90%, and particularly at least 95%. Deacetylation (of chitin, e.g., from shellfish, insects, or fungi) is a well-known standard process that can be carried out in many different ways. Chitosan can be obtained industrially from chitin by deacetylation, e.g., by treating chitin with (hot) sodium hydroxide or by enzymatic degradation. Both processes are used today on an industrial scale, with the alkaline procedure being more prevalent in terms of volume.

The degree of resulting deacetylation can vary considerably: Deacetylation can be complete or partial, resulting in a distribution of strongly deacetylated areas alongside weakly deacetylated areas, or a homogeneous deacetylation distribution, which has significant effects on the molecular shape. Simultaneously, this chemical intervention can decrease the polymer chain length (depolymerization), thereby adjusting the molecular weight, improving solubility, and reducing viscosity.

Preferably, the disinfectant according to the invention contains chitosan as a polyaminosaccharide, which has been deacetylated in a shear film mixer, in particular in a shear film reactor.

According to a preferred embodiment, the disinfectant according to the invention contains a carboxylic acid selected from citric acid, glycolic acid, tartaric acid, mandelic acid, malic acid, lactic acid, a phenolic carboxylic acid, or mixtures thereof, preferably an aromatic hydroxycarboxylic acid. in particular salicylic acid, which has proven to be particularly suitable for the process according to the invention.

Preferably, the mixing of the polyaminosaccharide with the carboxylic acid is carried out using a higher weight of the polyaminosaccharide.

According to a preferred embodiment, an emulsifier is added to the polyaminosaccharide/carboxylic acid mixture before the mixture is treated in a shear film mixer, in particular in a shear film reactor.

There are basically two different procedures for production in a shear film mixer.

All components of the disinfectant according to the invention can be premixed or dissolved and then fed to the shear film mixer to obtain the sub-colmogoroff microdispersion according to the invention.

However, the two main components, the polyaminosaccharide and the organic acid, can also be pre-dispersed and simultaneously fed into this shear film mixer at two separate inputs.

In both cases, additional emulsifier systems are used to facilitate pre-dispersion or dispersion and to contribute to better solubility of the polyaminosaccharide-acid complex formed.

Typical emulsifying systems include nonionic or amphoteric surfactants, such as betaines, alkyl polyglucosides (APG), or fatty acid esters such as sorbitan fatty acid esters, polysorbates (TWEEN), or sorbitan stearate (SPAN). Furthermore, the addition of urea, one of the smallest representatives of the so-called hydrotropes, has proven beneficial as a solubilizer. Urea is known for its property of disintegrating water clusters.

According to a preferred embodiment, the polyaminosaccharide/carboxylic acid mixture is first mixed with water and dissolved or finely dispersed at elevated temperature while stirring.

It is part of a particularly preferred embodiment of the invention that two different types of chitosan are used together as a polyaminosaccharide, one high molecular weight and one low molecular weight. Subsequently, after the mixture or complex has formed, an emulsifier is added while stirring, before the mixture is treated in a shear film mixer, in particular in a shear film reactor. Preferably, the polyaminosaccharide is mixed with the carboxylic acid in a particle size of 0.05 to 1 mm, preferably of 0.1 to 0.5 mm, in particular of 0.125 to 0.250 mm.

According to a preferred embodiment of the present invention, the disinfectant according to the invention comprises a polyaminosaccharide with a molecular weight of 100 to 200 kDa and a polyaminosaccharide with a molecular weight of 20 to 80 kDa, preferably comprising one polyaminosaccharide with a molecular weight of 100 to 200 kDa and one polyaminosaccharide with a molecular weight of 20 to 50 kDa. The weight ratio of high to low molecular weight can vary over relatively large ranges, with the higher molecular weight fraction being advantageous. The weight ratio can be between 6:1 and 2:1, but preferably 3:1. Furthermore, it has been found that it is advantageous for each polyaminosaccharide to have a polydispersity of 1.7 to 2.5, more preferably 1.4 to 1.7, and in particular 1.1 to 1.4.

The polydispersity D is a measure of the width of a molar mass distribution; it is calculated from the ratio of weight mean to number mean. The larger D, the wider the molar mass distribution. The mean molar masses required to determine the polydispersity, or to determine the mean molar masses as disclosed within the scope of the present invention, can be determined by various methods, e.g. (concerning a number mean M_{_n} ) via colligative properties using cryoscopy, ebullioscopy, or NMR spectroscopy, as well as by sedimentation analysis to determine the centrifuge mean, viscometry (i.e., rheological behavior in solution), rheology, or light scattering. The width of the distribution (i.e., the polydispersity D) can also be directly inferred from the different values. Gel permeation chromatography (GPC) and mass spectrometry (MALDI-TOF) are preferably used for the direct determination of the molar mass distribution, whereby, if in doubt, the polydispersity D is determined by GPC according to the invention. GPC and centrifugation are also used for preparative polymer fractionation.

Preferably, the polyaminosaccharide/carboxylic acid mixture consists of chitosan and salicylic acid, and the emulsifier system consists of urea, betaine, and salicylic acid.

Betaines are zwitterionic compounds with their The most important and simplest representative is trimethylglycine, a natural metabolic product. Betaines in general, and trimethylglycine in particular, are characterized by their excellent skin compatibility. As with the chitosan-salicylic acid mixture, the advantage of betaine salicylate, which can also form due to the excess by weight, lies in the fact that, through combination with either an amine-functional polysaccharide or a betaine, the carboxylic acid is converted into a more water-soluble form, and the irritation potential of, for example, salicylic acid is significantly reduced.

Preferably, the polyaminosaccharide/carboxylic acid mixture and an emulsifier system are fed separately and simultaneously to the shear film mixer, in particular the shear film reactor.

Preferably, the shear film mixer, in particular the shear film reactor, has a mixing zone, i.e., gap width between rotor and stator, which is 200 pm or less, preferably 150 pm or less, more preferably 50 pm wide.

According to a further aspect, the present invention further relates to an alcohol-free disinfectant obtainable by the inventive method, preferably filled in a ready-to-use container or in a storage-stable, transportable bulk container, in particular in a ready-to-use container with an application device for the disinfectant.

The present invention is further explained by the following examples, without of course being limited thereto.

Example 1. Production of the disinfectant according to the invention

The disinfectant according to the invention (internal abbreviation: “PS001”) can be produced, for example, according to the following recipe and procedure (the specified molar masses of chitosan are to be understood as average values):

Chitosan I (120 kDa) 0.45% by weight

Chitosan II (40 kDa) 0.15 wt.% Salicylic acid 0.60 wt.%

Trimethylglycine 15 wt.%

Urea 5 wt.%

Water 78.8 wt.% (remaining amount) Chitosan I, chitosan II and salicylic acid are mixed with water, stirred and heated to approximately 70°C.

Once a cloudy solution/dispersion has formed, it is allowed to cool to approximately 30°C while continuously stirring. The solution/dispersion is then continuously fed into the shear film reactor via a first pump through a first shear film reactor inlet. Simultaneously, the emulsifier system, consisting of trimethylglycine and urea, is continuously fed into the shear film reactor via a second pump through a second shear film reactor inlet. The gap of the shear film reactor (tube-in-tube) is set to 50 pm and the rotation speed to 2000 rpm. At the same time, the shear film reactor is cooled with water to ensure that the temperature within the shear film does not exceed 30°C. After processing in the shear film reactor, a homogeneous mixture free of suspended solids is obtained.

An alternative option is to incorporate salicylic acid in excess by weight during production in the shear film reactor or subsequently up to the solubility limit.

Example 2. Preparation of the reference samples

In the preparation of comparison samples 1-4, a conventional magnetic stirrer is used instead of a shear film reactor to stir and mix the components. Any suspended solids are removed by filtration. This additional filtration step is necessary because, without the use of a shear film reactor, not all components can be completely dissolved.

The comparison sample 1 was prepared according to the following recipe and procedure (the specified molar masses of chitosan are to be understood as average values):

Chitosan I (120 kDa) 0.45% by weight Chitosan II (40 kDa) 0.15% by weight Salicylic acid 0.60% by weight

Trimethylglycine 15 wt.%

Urea 5 wt.%

Water 78.8 wt.% (remaining amount)

Chitosan I, chitosan II and salicylic acid are dissolved in water The mixture is stirred and heated to approximately 70°C. Once a cloudy solution/dispersion has formed, it is allowed to cool to approximately 30°C while continuing to stir. The emulsifier system, consisting of trimethylglycine and urea, is then added while stirring continuously. At the same time, care must be taken to ensure that the temperature of the solution/dispersion does not exceed 30°C. Any suspended solids are removed in a filtration step.

The comparison sample 2 was prepared according to the following recipe and procedure (the specified molar masses of chitosan are to be understood as average values):

Chitosan I (120 kDa) 0.45 wt.% Chitosan II (40 kDa) 0.15 wt.% Salicylic acid 0.20 wt.% Trimethylglycine 15 wt.% Urea 5 wt.% Water 79.2 wt.% (residual amount)

Chitosan I, chitosan II, and salicylic acid are mixed with water, stirred, and heated to approximately 70°C. Once a cloudy solution/dispersion has formed, it is allowed to cool to approximately 30°C while continuing to stir. The emulsifier system, consisting of trimethylglycine and urea, is then added while stirring continuously. At the same time, care must be taken to ensure that the temperature of the solution/dispersion does not exceed 30°C. Any suspended solids are removed in a filtration step.

The comparative sample 3 was prepared according to the following recipe and procedure (the specified molar masses of chitosan are to be understood as average values):

Chitosan I (120 kDa) 0.45 wt. % Chitosan II (40 kDa) 0.15 wt. % Salicylic acid 0.20 wt. % Trimethylglycine 15 wt. % Urea 5 wt. % PAP 0.10 wt. % Water 79.1 wt.% (remaining amount)

Chitosan I, chitosan II, salicylic acid, and phthalimidoperoxycaproic acid (PAP) are mixed with water, stirred, and heated to approximately 70°C. Once a cloudy solution/dispersion has formed, it is allowed to cool to approximately 30°C while stirring continuously. The emulsifier system, consisting of trimethylglycine and urea, is then added while stirring continuously. At the same time, care must be taken to ensure that the temperature of the solution/dispersion does not exceed 30°C. Any suspended solids are removed in a filtration step.

The comparison sample 4 was prepared according to the following recipe and procedure (the specified molar masses of chitosan are to be understood as average values):

Chitosan I (120 kDa) 0.45% by weight

Chitosan II (40 kDa) 0.15 wt.% Salicylic acid 0.20 wt.%

Trimethylglycine 15 wt.%

Urea 5 wt.%

Lactic acid 0.50 wt.%

Water 78.7 wt.% (remaining amount)

Chitosan I, chitosan II, salicylic acid, and lactic acid are mixed with water, stirred, and heated to approximately 70°C. Once a cloudy solution/dispersion has formed, it is allowed to cool to approximately 30°C while continuing to stir. The emulsifier system, consisting of trimethylglycine and urea, is then added while stirring continuously. At the same time, care must be taken to ensure that the temperature of the solution/dispersion does not exceed 30°C. Any suspended solids are removed in a filtration step.

Example 3. Evaluation of basic antimicrobial properties (EN 1040 - Suspension test with S. aureus)

To investigate the antimicrobial properties, the disinfectant PS001 according to the invention was first applied directly as a liquid in a suspension test on tested on the basis of EN 1040 with the clinically representative model germ Staphylococcus aureus DSM 799 .

For this purpose, the disinfectant according to the invention was mixed directly with the bacteria (in a ratio of 8 parts PS 001 to one part water and one part bacterial suspension with a concentration of ^10⁸ CFU/ml; CFU = colony-forming unit) and plated after a contact time of 5 minutes. The disinfectant PS 001 according to the invention was then pipetted into the respective sample tubes using a positive displacement pipette.

In addition, the disinfectant according to the invention was also diluted 1:10 with sterile ultrapure water and measured analogously to the undiluted sample. The results are shown in Table 1. The disinfectant PS 001 according to the invention showed a significant reduction within the contact time (especially in the undiluted sample, but also in the 1:10 diluted sample). Table 1:

Example 4. Practical test setup (surface test according to EN 13697)

In a next step, the disinfectant PS 001 according to the invention was investigated in a more practical test setup, a quantitative surface test based on EN 13697. For this purpose, a test suspension of S. aureus DSM 799 (50 pl with a concentration of 10 ^Å 8 CFU/ml) was pipetted onto stainless steel plates and allowed to dry for a maximum of 60 min. Then, 100 pl of the undiluted sample was pipetted onto the dried bacteria, held in contact for 5 min, and the stainless steel plates were subsequently extracted. The reductions were calculated with respect to blanks that had been in contact with 100 pl of 0.9% NaCl solution instead of the sample for 5 min and are listed in Table 2.

Here too, the disinfectant PS 001 according to the invention shows a very high reduction after a 5-minute exposure time. Table 2:

Example 5. Evaluation of antimicrobial properties as a coating (Antiviral activity according to ISO 18184)

To investigate the antimicrobial properties of the disinfectant PS001 according to the invention in its cured state in the form of coatings, an investigation for antiviral activity was carried out using phi6 phages (Pseudomonas virus phi6) based on ISO 18184. Autoclaved cellulose filters (Whatman No. 1, diameter 45 mm) served as the sample carrier material; these were pre-impregnated with 400 g of PS001 each, dried overnight, and then tested in triplicate.

To determine antiviral activity, samples were inoculated with 0.2 ml of phage suspension at a concentration of ^10⁷ PFU/ml (PFU = Plaque Forming Unit). The test samples and half of the reference samples (untreated filter paper without an inoculated sample) were placed in sterile, sealed tubes and incubated at 25 ± 1 °C for 2 hours. The other half of the reference samples were extracted immediately, and the PFU count was determined using the double agar method on tryptone-sodium agar (TSA) after an incubation period of 24 hours at 25 ± 1 °C.

After a 2-hour incubation/contact time, the test samples and the remaining reference samples were also extracted, and the PFU count was again determined using the double agar method for TSA after an incubation period of 24 hours at 25 ± 1 °C. During the test, Pseudomonas sp. DSM 21482 was used as the host strain for the Pseudomonas phage phi6 (cystovirus phi6 Pseudomonas virus phi6).

The results are shown in Table 3. Here, too, a significant reduction of 99.90% (3 log steps) was measured; in this case, all phi6 phages on the coated samples were reduced (taking the detection limit into account). Table 3: Example 6. Evaluation of the antimicrobial properties of the samples as a coating (Antibacterial activity according to ISO 20743)

Analogous to the antiviral examination of the samples in the cured state in the form of coatings, an examination for antibacterial activity was carried out based on ISO 20743 with Staphylococcus aureus DSM 799 and Klebsiella pneumoniae DSM 789.

Autoclaved cellulose filters (Whatman No. 1, diameter 45mm) were again used as sample carrier material; these were also pre-soaked with 400 gl of the disinfectant PS 001 according to the invention, dried overnight and examined for the tests in six different determinations.

To determine antibacterial activity, samples were each inoculated with 0.2 ml of bacterial suspension (S. aureus and K. pneumoniae separately) at a concentration of ^10⁶ CFU/ml. Half of the test samples and half of the reference samples (untreated filter paper without an inoculated sample) were placed in sterile, sealed tubes and incubated at a temperature of 37 ± 1 °C for 24 hours. The other half of the test and reference samples were extracted immediately, and the CFU count was determined by pour plate assay on tryptone soya agar (TSA) after an incubation period of 24 hours at 37 ± 1 °C.

After 24 h incubation/contact time, the remaining test and reference samples were also extracted and the CFU count was again determined by plate casting method on TSA after an incubation time of 24 h at 37 ± 1 °C.

The antibacterial efficacy is calculated as the difference between the increase in bacterial concentration on the control sample and the increase in bacterial concentration on the antibacterial sample. The efficacy of the biocidal treatment can be quantified using the following formula; the results are shown in Table 4:

A = (1g Ct ~ 1g Co) - (1g Tt - 1g To) = F - G

A the value of the antibacterial effect;

F is the increase value on the control sample (null sample)

( F = 1g C _t -1g Co ) ;

G is the increase value on the antibacterial measurement sample ( G = lg Tt - 1g To ) ;

1g C _t the general logarithm of the arithmetic mean for the bacterial count, obtained from three control samples after incubation of 18 h to 24 h;

1g Co the general logarithm of the arithmetic mean for the bacterial count obtained from three control samples immediately after inoculation;

1g T _t the general logarithm of the arithmetic mean for the bacterial count, obtained from three antibacterial samples after incubation of 18 h to 24 h;

1g T _to the general logarithm of the arithmetic mean for the bacterial count obtained from three antibacterial samples immediately after inoculation.

Table 4:

This method also resulted in a significant reduction of more than 6 log levels; here, all bacteria on the coated samples were reduced (taking into account the detection limit).

Example 7. Evaluation of samples as a coating in comparison between biological usability and antimicrobial efficacy (investigation with fungi and bacteria based on ISO 846)

In addition to antibacterial and antiviral efficacy, the samples were also tested for their behavior towards molds. A classic method is the test according to ISO 846:2019 "Plastics - Evaluation of the effects of microorganisms," although this standard includes further subdivisions into other methods designed to examine different aspects. For example, Method A, "Mold Growth Test," describes the usability of the Method B involves testing the model fungal strains in the absence of a nutrient medium, while Method B aims to measure potential fungistatic efficacy through the use of a carbon-containing medium. In this study, Method B was adapted to also include testing in a medium without a carbon source. Method C, on the other hand, examines resistance to bacteria and can be seen as a complement to the antibacterial tests described above.

Autoclaved cellulose filters (Whatman No. 1, 45 mm diameter) served as the sample carrier material. These were pre-soaked with 400 µl of chitosan solutions each, dried overnight, and analyzed in triplicate for the respective tests. Untreated filter paper without a sample served as the control.

For the investigations regarding an inherent resistance to fungal infestation in the absence of organic matter and for measuring the potential fungistatic efficacy, a mixed suspension of fungal spores from the strains Penicillium funiculosum DSM 1944, Aspergillus niger DSM 1957, Paecilomyces varlotll DSM 1961, Chaetomium globosum DSM 1962 and Gliocladium virens DSM 1963 was prepared.

The samples were then placed upside down in Petri dishes, and 100 pl of the mixed spore suspension ( ^10⁶ spores/ml) was sprayed evenly onto the sample surface of the coated filter papers in triplicate for each batch. The following batches were tested: empty Petri dishes (Method A), Petri dishes filled with complete medium (Method B), and Petri dishes with agar without a carbon source (modified Method B). The sprayed samples were then incubated for 4 weeks at 29 °C and >90% relative humidity.

For sterility control, test samples were placed in separate Petri dishes; these were not inoculated with the fungal test strains, but were layered with 3 ml of a 70% aqueous ethanol solution before incubation and incubated in parallel with the inoculated samples for 4 weeks at 29 °C and >90% relative humidity.

The samples are visually assessed and evaluated according to the schemes listed in Table 5 below; the results are shown in Table 6: Table 5:

Table 6:

The low growth on the disinfectant PS001 according to the invention indicates an inhibition of the fungi, since the carrier material itself (cellulose filter paper) was completely covered with growth. The investigation for resistance to bacteria, as described in Method C, uses a similar experimental setup. For this, a suspension of Pseudomonas aeruginosa ATCC 13388 with a concentration of ^10⁶ cells/ml was prepared and mixed with mineral salt agar (agar without a carbon source) and poured into Petri dishes (final bacterial concentration: 5 x ^10⁴ cells per ml of agar). Subsequently, the samples were placed on the medium in triplicate and covered with additionally inoculated mineral salt agar.

For sterility control, the samples (covered with 70% aqueous ethanol solution) were also layered with uninoculated mineral salt zagar. All samples were incubated for 4 weeks at 29°C and >90% relative humidity.

Method C does not use a general assessment scheme; instead, any bacterial growth is documented. After 4 weeks of incubation, none of the samples showed growth of Pseudomonas aeruginosa.

Example 8. Quantitative suspension test according to EN 13727, EN 13624 or VAH 9

To verify the antimicrobial properties of the disinfectant PS 001 according to the invention and the comparison samples 1-4 (prepared according to Example 2), quantitative suspension tests were carried out according to EN 13727, EN 13624 and VAH 9, respectively. The determined reduction factors (RE) are listed in Table 7 and illustrated in Figure 1.

Table 7:

Example 9. Antimicrobial efficacy of another comparator product, which was produced without a step in the shear film mixer.

Another comparison product was manufactured as in Example 1, but without using a shear film reactor. Instead, a conventional stirrer was used to mix the components.

The test was performed in accordance with ISO 22196 (August 2011 edition): Measurement of antibacterial activity on plastic and other non-porous surfaces. The test organisms were Staphylococcus aureus ATCC 6538 P and Escherichia coli ATCC 8739.

The contamination of the test pieces (“Cont.”) was carried out by pipetting 0.4 ml of microbial suspension (2.5–10 x ^10⁵ CFU/ml) onto six test pieces (5 cm x 5 cm) and spreading it over an area of 4 cm x 4 cm using a spatula. The contaminated surfaces were dried in a safety cabinet without being covered. Three test pieces were used for the baseline determination immediately after drying. The remaining three test pieces were analyzed after a contact time of 1 h at 37 ± 2°C and 90–15% relative humidity.

The test bacteria were recovered by pouring 10 ml of SCDLP medium over them and rinsing four times by re-aspirating the same medium.

The bacterial count of the test specimens was determined by diluting the eluate and pouring 1.0 ml of counting agar over it. The test was carried out using a 3-fold determination.

This testing was carried out in two different series of experiments and yielded the following result:

Table 8

Test germ S. aureus E. coli S. aureus E. coli

ATCC 6538P ATCC 8739 ATCC 6538P ATCC 873

Germ suspension 5.88 5, 75 5, 45 5, 46

CFU/ml [1g]

Average of 4.02 1.46 3.26 5.46

CFU/ ^cm² [1g] of the negative control. Directly after contact.

Average of 4.02 1.50 3.22 2.67

CFU/ ^cm² [1g] of the negative control 1 h after control.

Average of 4.07 1.44 3.39 2.22

CFU/ ^cm² [1g] of the test pieces

1 hour after contact.

Value of anti-bacterial effect 0.00 0.06 0.00 0.46

CFU: colony-forming units R[lg] = (Ut-Uo) - (At-Uo) = U _t -A _t R<2 : weak antibacterial effect R>3 : strong antibacterial effect

Therefore, no relevant antibacterial effect could be demonstrated for the comparison product.

In a further series of experiments, the comparison product (produced as in Example 1, but without the mixture being produced using a shear film reactor; instead of the shear film reactor, a conventional stirrer was used to mix the components) was examined for its antiviral efficacy.

The testing was performed in accordance with ISO 21702 (2019 edition): Determination of the antiviral activity of plastics and other non-porous surfaces. The test organism was Bovine Coronavirus (strain "S379 Riems", RVB-0020, passage no. 3).

Neutralization was checked by pouring 10 ml of DMEM + 2% FBS medium over the sample and rinsing it four times by absorbing the same medium. Dilution to ^10⁻⁴ was achieved within 10 seconds using ice-cold DMEM + 2% FBS. The rinsing solution was DMEM + 2% FBS, the test temperature was 25 ± 1°C, the exposure time was 1 hour, and incubation took place at 36 ± 1°C for 7 to 14 days.

The titration procedure involved a viral titration on cells as a monolayer in 96-well microtiter plates. 0.5 ml of test product was diluted with 4.5 ml of ice-cold DMEM + 2% FBS to a dilution of ^0.10⁻⁸ . 100 pl of each dilution were pipetted into 8 wells of the microtiter plate.

Cytotoxicity and cytosensitivity were checked by pouring 10 ml of DMEM + 2% FBS medium over the sample and rinsing it four times by aspirating the same medium. 5 ml of the solution were then transferred to a new tube and 50 pl of virus suspension were added. The mixture was then incubated at 25°C for 30 min and subsequently diluted to ^10⁻⁴ within 10 s with ice-cold DMEM + 2% FBS.

This test was also carried out in two different series of experiments and yielded the following result:

Table 9

Bovine coronavirus test germ

Average of 3.23 2.47

TCIDso/ ^cm² [1g] of the negative control directly after contact. Mean of 2.64 1.30

TCIDso/cm ² [1g] of the negative control 1 h after contact.

Average of 2.72 1.30

TCIDso/ ^cm² [1g] of the test pieces 1 h after contact. Value of the anti-viral effect: 0.00 0.00 TCIDso: Tissue culture infectious dose

R[lg] = (Ut-Uo) - (At-Uo) = U _t -A _t

M _v > 3.0: strong antiviral effect

Comparison of the control for cell sensitivity with antiviral non-treated and treated textile showed a difference of <0.5 lg levels.

Therefore, no relevant antiviral effect could be demonstrated for the comparison product. ...

Claims

Claims: 1. Method for the production of an alcohol-free disinfectant, containing at least one polyaminosaccharide and at least one carboxylic acid, comprising the steps of: - Mixing the polyaminosaccharide with the carboxylic acid, resulting in a mixture of the polysaccharide and the carboxylic acid; - Treating the mixture in a shear film mixer, resulting in a homogeneous disinfectant. 2. Method according to claim 1, wherein the polyaminosaccharide comprises monomer units of optionally substituted amino sugars, in particular glucosamines and/or galactosamines, and is preferably selected from the group consisting of chitosan, hyaluronic acid, keratin sulfate, heparin, chondroitin sulfate with a molecular weight of 20 kDa or more, or mixtures thereof, in particular chitosan with a molecular weight of 40 kDa or more, hyaluronic acid or mixtures thereof. 3. Method according to claim 1 or 2, wherein the disinfectant contains chitosan as a polyaminosaccharide having a degree of deacetylation of at least 70%, preferably at least 80%, more preferably at least 90%, and in particular at least 95%. 4. Method according to any one of claims 1 to 3, wherein the disinfectant contains chitosan as a polyaminosaccharide which has been deacetylated in a shear film mixer, in particular in a shear film reactor. 5. Method according to any one of claims 1 to 4, wherein the disinfectant contains a carboxylic acid selected from citric acid, glycolic acid, tartaric acid, mandelic acid, malic acid, lactic acid, polycarboxylic acids, a phenolic carboxylic acid, or mixtures thereof, preferably an aromatic hydroxycarboxylic acid, in particular salicylic acid. 6. A method according to any one of claims 1 to 5, wherein the mixing of the polyaminosaccharide with the carboxylic acid with a An excess of polyaminosaccharide by weight is added. 7. Method according to any one of claims 1 to 6, wherein an emulsifier is added to the polyaminosaccharide/carboxylic acid mixture before the mixture is treated in a shear film mixer, in particular in a shear film reactor. 8. A method according to any one of claims 1 to 7, wherein an emulsifier is added to the polyaminosaccharide/carboxylic acid mixture before the mixture is treated in the shear film reactor, and wherein the emulsifier consists of the group of nonionic or amphoteric surfactants, such as betaines, alkyl polyglucosides (APG), or fatty acid esters such as sorbitan fatty acid esters, polysorbates (TWEEN) or sorbitan stearate (SPAN), as well as urea; and mixtures thereof. 9. A method according to any one of claims 1 to 8, wherein an emulsifier is added to the polyaminosaccharide/carboxylic acid mixture before the mixture is treated in a shear film mixer, in particular in a shear film reactor, and the emulsifier is mixed with a carboxylic acid before this addition, so that a mixture of the emulsifier and the carboxylic acid is obtained, which is then added to the polyaminosaccharide/carboxylic acid mixture; wherein preferably the carboxylic acid in the emulsifier/carboxylic acid mixture is the same carboxylic acid as in the polyaminosaccharide/carboxylic acid mixture. 10. Method according to any one of claims 1 to 9, wherein the polyaminosaccharide in a particle size of 0.05 to 1 mm, preferably of 0.1 to 0.5 mm, in particular of 0.125 to 0.250 mm, is mixed with the carboxylic acid. 11. A method according to any one of claims 1 to 10, wherein the disinfectant contains a polyaminosaccharide with a molecular weight of 100 to 200 kDa and a polyaminosaccharide with a molecular weight of 1 to 50 kDa, preferably containing a polyaminosaccharide with a molecular weight of 100 to 200 kDa and a polyaminosaccharide with a molecular weight of 10 to 50 kDa, and each polyaminosaccharide comprising at least 25% of the total amount of polyaminosaccharides in the Disinfectant, wherein in particular the polyaminosaccharide has a polydispersity of 1.7 to 2.5, preferably of 1.4 to 1.7, especially of 1.1 to 1.4. 12. Method according to any one of claims 1 to 11, wherein the polyaminosaccharide/carboxylic acid mixture consists of chitosan and salicylic acid and the emulsifier/carboxylic acid mixture consists of urea, betaine and salicylic acid. 13. Method according to any one of claims 1 to 12, wherein the polyaminosaccharide/carboxylic acid mixture and an emulsifier/carboxylic acid mixture are fed separately and simultaneously to the shear film mixer, in particular the shear film reactor. 14. Method according to any one of claims 1 to 13, wherein the shear film mixer, in particular the shear film reactor, has a mixing zone that is 200 pm or less, preferably 150 pm or less, more preferably 50 pm or less wide. 15. Method according to any one of claims 1 to 14, wherein the shear film mixer is selected from shear film reactor, jet stream mixer, rotor-stator mixer, ultrasonic mixer, high-pressure homogenizer, rotor-stator colloid mill, microfluidic systems, vibrating mixers, kneading and roller mixers, planetary mixers, colloid mills, grinding balls or beads or pressure homogenizer, preferably shear film reactor, jet stream mixer, ultrasonic mixer, high-pressure homogenizer or microfluidic systems, in particular shear film reactor. 16. Method according to any one of claims 1 to 15, wherein the disinfectant contains essentially no suspended particles after treatment in the shear film mixer. 17. Alcohol-free disinfectant obtainable by a method according to any one of claims 1 to 16, preferably filled in a ready-to-use container or in a shelf-stable, transportable bulk container, in particular in a ready-to-use container with an application device for the disinfectant. Alcohol-free disinfectant obtainable by a method according to any one of claims 1 to 16, preferably filled in a ready-to-use container or in a storage-stable, transportable bulk container, in particular in a ready-to-use container with an application device for the disinfectant. ...