The invention relates to the use of insoluble enzyme preparations, having reduced safety concerns compared to the free enzyme, in certain applications that involve direct contact of enzymes with humans either by handling, utilizing, or uptake of such enzymes or enzyme containing compositions, which is particularly useful in care compositions and food. Being proteins, free enzymes are likely to stimulate an immunological response in man and animals, including an allergic response, under certain conditions. This risk is diminished by the insoluble enzyme preparations of the invention.

The industrial use of enzymes, notably enzymes of microbial origin, has become increasingly common. Enzymes are important constituents of many compositions as well as useful processing aids in numerous industries (including but not limited to the starch industry, the dairy industry, the detergent industry and the food or baking industry, as well as the animal feed industry). Furthermore, enzymes have a potential as beneficial additives in personal and oral care as well as housekeeping compositions.

However, many addressable enzyme applications are associated with potential safety concerns, e.g. with regard to the exposure of workers to air-borne enzyme dust, and generally to the dustiness of the available enzyme containing compositions. Those safety concerns encompass potential adverse health issues of enzymes due to their immunogenicity and the potential induction of respiratory allergies, including asthma. Many granular compositions, for example, but not limited to, useful in the food and feed industries are spray dried. These compositions tend to be dusty in handling. But also consumers are exposed to the same risk if enzyme containing compositions are applied under conditions that might enable the formation of airborne aerosols in form of dust or liquid droplets containing either dried or solubilized enzyme molecules.

Airborne particles or liquid droplets may settle out under their own weight but also may remain suspended for some time, depending on their weight and size. The best definition for particle diameter is given by the particle aerodynamic diameter, which represents the diameter of a hypothetical sphere with a density of 1 g/cm ³ . This hypothetical sphere has the same terminal settling velocity in air compared to the particle in question independent from its true geometric shape, its size and density. The aerodynamic diameter indicates the ability of the particle to penetrate and deposit at different areas of the respiratory tract in humans. For example, particles with aerodynamic diameter bigger than 50 μηι have a terminal velocity below 7 cm/s and hence do not usually remain airborne very long. Particles with aerodynamic diameters below 100 μηι may become, but hardly remain airborne. However, particles with aerodynamic diameters below 1 μηι remain airborne as the settling caused by gravity is negligible and the movement in air is more important than the sedimentation. For liquid droplets it has to be considered that their persistence in air is not the same due to potential coalescence of droplets.

Particles remaining airborne are inhaled through the nose or the mouth. The particle aerodynamic diameter and the breathing rate determine the probability of inhalation. The inhaled particles might be exhaled or deposited at the respiratory tract. Bigger particles (> 30 μηι) are deposited in the region of the head.

During nasal breathing, particles are captured by filtration by the nasal hairs and retention is facilitated by the mucus.

During oral breathing filtration efficiency is not the same. Particles that are not deposited in the head region will enter the tracheobronchial region and may later be eliminated by mucociliary clearance or may enter the body by dissolution in case they are soluble. Only about 1 % of small particles with aerodynamic diameters of 10 μηι are able to reach the alveolar region, which can be considered as the upper limit for entering this region. Maximum deposition at the alveolar region is given for particles of about 2 μηι. The vast majority of particles larger than 2 μηι deposited in regions further up. Even particles below 2 μηι are deposited to a lower extent (only about 10-15% in case of 0.5 μηι) as they are predominantly exhaled and not deposited.

Therefore the particle fractions can be distinguished as follows:

- Inhalable particulate fraction: fraction of total airborne particles that enters the body through the nose or mouth in course of breathing. The aerodynamic diameter of the corresponding particles is below 100 μηι.

- Thoracic particulate fraction: sub inhalable particulate fraction that can penetrate into the tracheo-alveolar region of the lung. The aerodynamic diameter of the corresponding particles is below 30 μηι.

- Respirable particulate fraction (alveolar fraction): sub inhalable particulate fraction that can penetrate into the alveolar region of the lung (the gas-exchange region of the lungs) and is pertinent to the development of severe airborne related diseases. The aerodynamic diameter of the corresponding particles is below 10 μηι.

Clearance of particles from the respiratory tract can occur by mucociliary clearance (in the trachea and bronchi), the bronchiole movement (peristaltic movements of the bronchioles, coughing, and sneezing) and through phagocytosis (particles engulfed by macrophage cells, which are transported upwards and out of the respiratory system). Further teaching is disclosed in the guidelines of the International Standards Organisation, the American Conference of Governmental Industrial Hygienists, and the European Committee for Standardisation as well as in Nieboer et al. (2005).

When soluble allergens such as enzymes are inhaled as aerosols in the form of dust or liquid droplets and resorbed in the alveolar region of the lung, they may give rise to the formation of specific antibodies due to the contact with cells of the humoral immune system. This process is called sensitization and is a response of the immune system to the foreign protein. This is especially true for industrial enzymes which are applied in dusty form, thereby exposing workers and users handling the enzyme to the risk of developing respiratory disease symptoms. Enzymes need to be dissolved and diffusible to result in an allergenic reaction of the human. Remarkably, there is no clear scientific evidence that enzymes are a cause of sensitization by skin contact or ingestion (except for enzyme compositions containing proteolytic enzymes that may cause eye and skin irritation upon their action) as such enzymes are not getting in contact with cellular components of the humoral immune system. In the context of this application inhalant allergens are therefore understood as preparations containing dissolved enzyme potentially resulting in allergenic response.

The general mechanism behind an allergic response is divided in a sensitization phase and a symptomatic phase.

The sensitization phase involves a first exposure of an individual to a solubilized allergen, triggering a humoral immune response. This event activates specific T- and B- lymphocytes, and may lead to the production of allergen specific IgE antibodies. These IgE antibodies eventually facilitate allergen capturing and presentation to T-lymphocytes at the onset of the symptomatic phase.

The symptomatic phase is initiated by a second exposure to the same or a resembling antigen. The specific IgE antibodies bind to the specific IgE receptors on mast cells and basophils, among others, and capture at the same time the allergen. The polyclonal nature of this process results in bridging and clustering of the IgE receptors, and subsequently in the activation of mast cells and basophils. This activation triggers the release of various chemical mediators involved in the early as well as late phase reactions of the symptomatic phase of allergy.

Prevention of sensitization and allergy in susceptible individuals is therefore a research area of great importance.

Since the introduction of enzymes into certain industries, including the detergent industry, and in awareness of the inherent risks, a lot of measures have been taken to improve the formulation of enzyme containing compositions, e.g. by applying granulation and coating of the enzyme and thus to avoid the formation of enzyme containing dust.

In the detergent industry for example, many granulates are composed of a core particle upon which an enzyme containing layer is added. The core may also in itself contain enzyme. Additional layers may be added to confer desired properties, e.g. solubility rate (WO 90/09440). However, this strategy is not applicable to all industries and compositions, as well as it does not eliminate the safety concerns with regard to the consumer in case of formation of airborne aerosols during or after use of enzyme containing compositions, particularly after the solubilization of the enzyme.

Therefore, also various attempts to reduce the immunogenicity of polypeptides and proteins itself have been made. It was found that small changes in an epitope may affect the binding to an antibody. This may result in a reduced importance of such an epitope, maybe by converting it from a high affinity to a low affinity epitope, or may even result in epitope loss, i.e. that the epitope cannot sufficiently bind an antibody to elicit an immunogenic response. Methods to identify epitopes on proteins and alter these epitopes by protein engineering in order to modify the immunogenicity have been described recently (US 201 1 /0045572).

However, in today's state of heightened awareness of enzyme-related safety risks, there remains a continuing need for enzyme compositions that circumvent the adverse health effects of enzymes as mentioned and explained above.

Enzyme granules or enzymes with engineered epitopes which are currently available lack certain properties rendering them unsuitable for use in many care compositions. For example, granules are solubilized and the enzyme is released in order to be available for the desired biocatalytical reaction. However, the formation of airborne aerosols containing immunogenic enzymes cannot be excluded after the granule has disintegrated.

Epitope engineering cannot be applied to all enzymes with the same success, which is why additional measures are necessary to assure the consumer's safety.

Therefore, it is an object of the invention to provide enzyme compositions having advantages to the prior art composition, particularly low allergenic enzyme compositions.

Figure 1 schematically visualizes the differences between aerosols derived from a composition containing free enzyme and compositions containing the insoluble enzyme preparation according to the invention.

Figure 2 schematically visualizes surface functionalities accessible by entrapment of the single-use-composition in coatings and paints.

Figure 3: illustrates the results of activity measurement in the soluble fraction and insoluble fraction of toothpaste containing insoluble enzyme preparation according to the invention and commercially available toothpaste containing soluble enzyme as analyzed in Example 1 . Soluble and insoluble fractions were obtained after resuspension and centrifugation of toothpaste. The colorimetric assay indicates GOX-activity as brownish colour. S = soluble fraction; P = insoluble fraction.

Figure 4: illustrates activity contained in aerosols formed by an ultrasonic fog generator from toothpaste containing insoluble enzyme preparation according to the invention and commercially available toothpaste as analyzed in Example 1 , containing soluble enzyme in ultimate proximity (0 cm) to the ultrasonic fog generator, and in a distance of 30 cm.

This object is solved by the present invention, which relates to the use of an insoluble enzyme preparation comprising an enzyme which is immobilized on a solid carrier for providing enzyme activity to a single-use-composition by dispersing the insoluble enzyme preparation in the single-use-composition, wherein preferably, after the intended usage of the single-use-composition, the insoluble enzyme preparation is disposed of together with the single-use-composition, or remains within the single-use-composition. Thus, preferably, the insoluble enzyme preparation is not intended to be recycled or used repeatedly. Typically, the insoluble enzyme preparation is dispersed in the single-use-composition in which it evolves its enzymatic activity in the course of the intended usage of the single-use- composition, and with which it is subsequently disposed of, or in which it subsequently remains after the intended single-usage.

Further, the invention relates to the use of a single-use-composition which is provided with enzyme activity by an insoluble enzyme preparation comprising an enzyme which is immobilized on a solid carrier, wherein the insoluble enzyme preparation is dispersed in the single-use-composition, and wherein preferably, after the intended usage of the single-use- composition, the insoluble enzyme preparation is disposed of together with the single-use- composition, or remains within the single-use-composition. By definition, the single-use- composition is not intended to be recycled or used repeatedly. Preferably, the single-use- composition is provided with enzymatic activity by the insoluble enzyme preparation dispersed therein, and the insoluble enzyme preparation evolves its enzymatic activity in the course of the intended usage of the single-use-composition. The insoluble enzyme preparation is subsequently disposed of together with the single-use-composition, or subsequently remains therein after the intended single-usage.

Still further, the invention relates to a single-use-composition which is provided with enzyme activity by an insoluble enzyme preparation comprising an enzyme which is immobilized on a solid carrier, wherein the insoluble enzyme preparation is dispersed in the single-use- composition, and wherein preferably, after the intended usage of the single-use-composition, the insoluble enzyme preparation is disposed of together with the single-use-composition, or remains within the single-use-composition.

It has been surprisingly found that the allergenic potential of enzymes can be substantially reduced or even completely suppressed by coupling enzymes to particulate solid carriers, such as particulate silica and particulate metal oxides wherein the majority of the particles has a particle size within the range of from 0.1 to 500 μηι, preferably from 10 μηι to 500 μηι, more preferably from 10 μηι to 200 μηι, also preferably from 10 μηι to 100 μηι and most preferably from 10 μηι to 50 μηι. Such insoluble enzyme preparations render the enzyme permanently insoluble and non-respirable and thereby reduce their allergenic potential whereas the functionality of the enzyme is retained. As enzymes of such insoluble enzyme preparations will not reach susceptible areas of the respiratory tract and will not be resorbed and contacted with cells of the humoral immune system, they are not considered an inhalant allergen. In such insoluble enzyme preparations, no further activation of an enzyme or detachment of an entrapped enzyme is required. Further, it has been surprisingly found that the insoluble enzyme preparations according to the invention show high blendability and good dispersion characteristics, thereby facilitating their use in applications which were previously difficult to address. The insoluble enzyme preparations according to the invention are dispersible or blendable with other matrices like, but not limited to, food (i.e., baking), powders, cleaners, ointments, toothpastes or polymers (e.g. bioactive surfaces).

It has been surprisingly found that the insoluble enzyme preparations according to the invention are showing a reduced immunogenicity if present as aerosol in the form of dust or liquid droplets compared to free dry enzyme or the freely solubilized enzyme, while the activity in industrial applications is not affected.

The insoluble enzyme preparations according to the invention do not need to be solubilized to make the enzyme available and functional. Further, the contact of the enzymes with the humoral immune system of humans is avoided. Thus, the immunogenicity and safety concerns are reduced, if not abolished, compared to the usage of the free enzyme. This is of special interest in certain applications that involve direct contact of the single-use- compositions with humans.

Compared to particles composed of the free enzyme itself, which can be absorbed by the body through dissolution, the preparation in accordance to this invention renders the enzyme insoluble and therefore prevents, e.g. upon breathing, a direct contact of the enzyme with the humoral immune system.

Figure 1 schematically visualizes the differences between aerosols derived from a composition containing the free enzyme and compositions containing the insoluble enzyme preparation according to the invention.

The invention comprises the use of insoluble enzyme preparations as additives dispersed in single-use-compositions, e.g. personal care compositions, home care compositions, food compositions, paint compositions or feed compositions, which exhibit functionality through the activity of the enzyme.

The insoluble enzyme preparations according to the invention are preferably to be used in industry, housekeeping and/or medicine, such as personal care compositions (for example shampoo; soap; skin, hand and face lotions; skin, hand and face cremes; hair dyes; toothpaste deodorant; sanitary pad), food (for example in the baking industry), detergents and pharmaceuticals.

For the purpose of specification, the "enzyme" can be any enzyme molecule.

For the purpose of specification, a "solid carrier" according to the invention is a solid in particulate form.

For the purpose of specification, a "insoluble enzyme preparation" according to the invention comprises an enzyme that is substantially insoluble in pure water under ambient conditions. In this regard, "substantially insoluble" preferably means a solubility of not more than 10mg/100g water, more preferably not more than 1 .0mg/100g water, still more preferably not more than 0.5mg/100g water, yet more preferably not more than 0.1 mg/100g water, even more preferably not more than 5C^g/100g water, most preferably not more than 1 C^g/100g water, and in particular not more than 1 .C^g/1 OOg water.

For the purpose of specification, the term„coupled to carrier" shall mean that the enzyme is bound to a solid carrier by covalent bonds or by ionic or hydrophobic adsorption in the way that a leaching or a release of the enzyme from the solid carrier is avoided or at least reduced under the conditions of the intended application, in which the insoluble enzyme preparation shall be used. The term coupling shall explicitly exclude the entrapment of an enzyme in a matrix as well as the coupling of an enzyme to a non-particulate polymer. Moreover, the intended application explicitly excludes detachment or re-mobilization procedures to release the soluble enzyme and thus to reveal the enzymatic activity.

For the purpose of specification, an "insoluble enzyme preparation" shall refer to any enzyme or mixture of enzymes in an active form which is coupled to a solid carrier.

For the purpose of specification, an "additive" is a compound, composition or preparation which is added to a production process or used in an application which after its effect or action is not removed or isolated from the process.

For the purpose of specification, a "heterogeneous" mixture contains at least two components with different states of aggregation (liquid, solid, gaseous). Preferred heterogeneous mixtures are liquid-solid or solid-gaseous. For the purpose of specification, a "homogeneous" mixture contains one or more components with the same state of aggregation (liquid, solid, gaseous). Preferred homogeneous mixtures are liquid or solid.

For the purpose of specification, a "constituent" may refer to any substance, preparation, extract, chemical, powder, solid matter, complex, solvent, solution, oil, gas or liquid for instance.

For the purpose of specification, a "matrix material" comprises one or more constituents. The matrix material may be a homogeneous mixture or a heterogeneous mixture. Preferably, the matrix material is a heterogeneous mixture, more preferably liquid-solid or solid-gaseous.

For the purpose of specification, a "single-use-composition" refers to a composition which after its primary use is either discarded, used up or rendered in a form unsuitable for subsequent reuse. Further, the single-use-composition is to be understood as a preferably heterogeneous mixture or dispersion which comprises at least two constituents.

The insoluble enzyme preparation is dispersed in the single-use-composition such that the single-use-composition comprises the insoluble enzyme preparation as a constituent. Further constituents of the single-use-composition are typically customized for the intended usage of the single-use-composition. For the purpose of the specification, the single-use-composition comprises or consists of the insoluble enzyme preparation and a matrix material including said further constituents of the single-use-composition. The matrix material as such, i.e. in the absence of the insoluble enzyme preparation, may be a homogeneous mixture or a heterogeneous mixture. Preferably, the matrix material is a heterogeneous mixture, more preferably liquid-solid and likewise preferably solid-solid. The single-use-composition which comprises or consists of the insoluble enzyme preparation and the matrix material is preferably a heterogeneous mixture, more preferably liquid-solid, solid-solid, solid-gaseous, or liquid-gaseous, and most preferably liquid-solid.

Preferably, the macroscopic local content of the insoluble enzyme preparation that is dispersed within the matrix material is substantially the same within the single-use- composition. Thus, although the overall single-use-composition is preferably a heterogeneous mixture, at a macroscopic level the distribution of the insoluble enzyme preparation within the matrix material, i.e. the further constituents of the single-use- composition, is preferably substantially uniform. The enhanced safety and the reduced immunogenicity of insoluble enzyme preparations according to the invention can be evaluated by measuring solubility, aerosol formation and formation of respiratory fractions, e.g. compared to the free enzyme.

For measurements, aerosols can be produced by using a nebulizer or particle aerosol generator generating airborne aerosols starting from a liquid or solid sample obtained from the single-use-composition containing the free enzyme (comparative) or the insoluble enzyme preparation according to the invention. Aerosols from single-use-compositions that are solidified upon preparation (e.g. like coatings and paints) and that contain the free enzyme (comparative) or the insoluble enzyme preparation according to the invention can be generated by grinding or crushing under defined conditions before using a nebulizer or particle aerosol generator.

The nebulizer or particle aerosol generator can be supplemented with an aerosol collector or a particle sizing system which is e.g. based on scattered light measurements, in a setup considered as suitable by an artisan skilled in the art.

The aerosol collected in the aerosol collector may be analyzed regarding the amount of insoluble or soluble enzyme respectively, e.g. by determination of soluble protein content and activity extracted from filters contained in the collector; and/or its immunogenicity by contacting with an enzyme-specific antibody and quantification in an ELISA assay.

Computational analysis of data obtained from the particle sizing system may be used to quantify the tendency of the enzyme containing single-use-composition to form aerosols; and/or the non-respirable fraction of the enzyme containing or carrying particulates or droplets which are contained in aerosols derived from the single-use-compositions.

In a preferred embodiment of the invention, the content of soluble or solubilized enzymes in aerosols evolving from single-use-compositions containing the insoluble enzyme preparation according to the invention is reduced by at least 40%, more preferably at least 50%, still more preferably at least 60%, yet more preferably at least 70%, most preferably at least 80% and in particular at least 90%, relative to the content of soluble or solubilized enzymes in aerosols evolving from single-use-compositions containing the same quantity of the same enzyme not immobilized on a solid carrier.

In a preferred embodiment of the invention, the number of particles or droplets in aerosols derived from single-use-compositions containing the insoluble enzyme preparation according to the invention is reduced by at least 20%, more preferably at least 40%, still more preferably at least 50%, yet more preferably at least 65%, most preferably at least 80% and in particular at least 90%, relative to the number of particles or droplets in aerosols derived from single-use-compositions containing the same quantity of the same enzyme not immobilized on a solid carrier.

In a preferred embodiment of the invention, the average diameter of particles or droplets in aerosols derived from single-use-compositions containing the insoluble enzyme preparation according to the invention is increased by at least 1 %, more preferably at least 5%, still more preferably at least 10%, yet more preferably at least 15%, most preferably at least 20% and in particular at least 30%, relative to the average diameter of particles or droplets in aerosols derived from single-use-compositions containing the same quantity of the same enzyme not immobilized on a solid carrier.

In a preferred embodiment of the invention, the aerosol formation from single-use- compositions containing the insoluble enzyme preparation according to the invention is reduced by at least 20%, more preferably at least 40%, still more preferably at least 50%, yet more preferably at least 65%, most preferably at least 80% and in particular at least 90%, relative to the aerosol formation from single-use-compositions containing the same quantity of the same enzyme not immobilized on a solid carrier.

In a preferred embodiment of the invention, the enzyme-caused immunogenicity of single- use-compositions containing the insoluble enzyme preparation according to the invention is reduced by at least 20%, more preferably at least 40%, still more preferably at least 60%, yet more preferably at least 80%, most preferably at least 90% and in particular at least 95%, relative to the enzyme-caused immunogenicity of single-use-compositions containing the same quantity of the same enzyme not immobilized on a solid carrier.

Preferably, the enzyme-caused immunogenicity of single-use-compositions containing the insoluble enzyme preparation according to the invention is reduced by at least 20% relative to the enzyme-caused immunogenicity of single-use-compositions containing the same quantity of the same enzyme not immobilized on a solid carrier. The allergenic potential of the insoluble enzyme preparation according to the invention and aerosols derived thereof can be evaluated in in vivo or in vitro model systems.

Suitable in vitro assays for immunogenicity are e.g. assays based on cytokine expression profiles or other proliferation or differentiation responses of epithelial and other cells including B-cells and T-cells. Further, animal models for testing allergenicity can be set up to test a limited number of said insoluble enzyme preparations that show desired characteristics in vitro. Useful animal models include the guinea pig intratracheal model (GPIT) (Ritz et al., Fund. Appl. Toxicol., 21 , pp. 31 -37, 1993), the mouse subcutaneous model (mouse-SC) (WO 98/30682, Novo Nordisk), the rat intratracheal model (rat-IT) (WO 96/17929, Novo Nordisk), and the mouse intranasal model (MINT) (Robinson et al., Fund. Appl. Toxicol., 34, pp. 15-24, 1996).

Preferably, the immunogenicity of the insoluble enzyme preparation is measured in animal tests, wherein the animal is exposed to the insoluble enzyme preparation and the immune response is measured.

Specifically, it is of interest to determine the allergenicity by repeatedly exposing animals to the insoluble enzyme preparation according to the invention by the intratracheal route and following the specific IgG and IgE titers. These IgG and IgE titers are then compared to those obtained by repeatedly exposing animals to the free enzyme under the same conditions. Alternatively, the mouse intranasal (MINT) test can be used to assess the allergenicity of aerosols derived of the insoluble enzyme preparation according to the invention in comparison to the free enzyme. Moreover, as stated previously in US 201 1/0045572, the performance in ELISA correlates closely to the immunogenic responses measured in animal tests.

In a preferred embodiment of the invention, the allergenic potential (measured as IgE binding capacity) of aerosols derived from single-use-compositions containing the insoluble enzyme preparation according to the invention is reduced by at least 20%, more preferably at least 40%, still more preferably at least 60%, yet more preferably at least 80%, most preferably at least 90% and in particular at least 95%, relative to the allergenic potential (measured as IgE binding capacity) of aerosols derived under the same conditions from single-use- compositions containing the same quantity of the same enzyme not immobilized on a solid carrier. Preferably, the allergenic potential (measured as IgE binding capacity) of aerosols derived from single-use-compositions containing the insoluble enzyme preparation according to the invention is reduced by at least 20% relative to the allergenic potential (measured as IgE binding capacity) of aerosols derived under the same conditions from single-use- compositions containing the same quantity of the same enzyme not immobilized on a solid carrier.

In a preferred embodiment of the invention, aerosols from single-use-compositions comprising the insoluble enzyme preparation according to the invention contain at least 50%, more preferably at least 40%, still more preferably at least 60%, yet more preferably at least 80%, most preferably at least 90% and in particular at least 95% of non-respirable enzyme containing particulates or droplets containing immobilized enzyme.

Preferably, aerosols from single-use-compositions comprising the insoluble enzyme preparation according to the invention contain at least 50% of non-respirable enzyme containing particulates or droplets containing immobilized enzyme.

In a preferred embodiment of the invention, the majority of enzyme containing particulates or enzyme containing droplets in aerosols from single-use-compositions comprising the insoluble enzyme preparation according to the invention is non-respirable.

The insoluble enzyme preparations of the invention may be provided in different aerodynamic diameters as necessitated by the targeted application.

The aerodynamic diameter of the insoluble enzyme preparation may be adjusted by the size of the solid carrier particles and/or by the nature of the solid carrier, e.g. its density.

The insoluble enzyme preparation according to the invention comprises an enzyme which is coupled to the solid carrier.

The enzyme may be any enzyme suitable for use in enzyme based processes or suitable for conferring enzyme functionality to single-use-compositions.

The enzyme functionality can be assayed by specific protocols which are comprehensively published in literature, such as protease assays (WO 99/3401 1 , Genencor International; J.E. Ness et al, Nature Biotechn., 17, pp. 893-896, 1999), oxidoreductase assays (Cherry et al., Nature Biotechn., 17, pp. 379-384, 1999), and assays for several other enzymes (WO 99/45143, Novo Nordisk). The activity of lipases can be determined as described in "Methods of Enzymatic Analysis", Third Edition, 1984, Verlag Chemie, Weinheim, vol. 4.

The enzyme in the context of the invention may be any enzyme or combination of different enzymes. Accordingly, when reference is made to "an enzyme", it will in general be understood to include one enzyme or a combination of enzymes. Thus, the insoluble enzyme preparation of the invention may comprise several different enzymes.

It is to be understood that enzyme variants (produced, for example, by recombinant techniques) are included within the meaning of the term "enzyme". Examples of such enzyme variants are disclosed, e.g. in EP 251446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525610 (Solvay) and WO 94/02618 (Gist-Brocades NV).

Preferably, the insoluble enzyme preparations according to the invention comprise at least one enzyme selected from the group consisting of enzymes which are usually used or can be used in personal care compositions, such as shampoo, soap bars, skin lotion, skin creme, hair dye, toothpaste, deodorant, sanitary pad; household articles; agro chemicals; personal care compositions, such as cleaning preparations e.g. for contact lenses, cosmetics, toiletries; oral and dermal pharmaceuticals; compositions used for treating textiles; compositions used for manufacturing food, e.g. baking, and feed etc.

Preferably, the enzyme contained in the single-use-composition according to the invention is selected from, but not limited to, the group consisting of glycosyl hydrolases, carbohydrases, tannase, glucanases, pentosanases, hemicellulases, transferases, oxidoreductases, lipoxygenases, catalases, laccases, lactoperoxidases, isomerases, protein disulfide isomerases, mutanases, dextranases, amylases, peroxidases, proteases, lipolytic enzymes, phytases, polysaccharide lyases, hydrolases, transglutaminases and glucoseisomerases.

Preferably, the enzyme contained in the insoluble enzyme preparation which is used according to the invention to provide enzyme activity to a single-use-composition, is selected from, but not limited to, the group consisting of glycosyl hydrolases, carbohydrases, tannase, glucanases, pentosanases, hemicellulases, transferases, oxidoreductases, lipoxygenases, catalases, laccases, lactoperoxidases, isomerases, protein disulfide isomerases, mutanases, dextranases, amylases, peroxidases, proteases, lipolytic enzymes, phytases, polysaccharide lyases, hydrolases, transglutaminases and glucoseisomerases. In a preferred embodiment of the invention, the enzyme contained in the insoluble enzyme preparation which is used according to the invention to provide enzyme activity to a single- use-composition, is a glucose oxidase, a lactoperoxidase, a mutanase, a dextranase, a lysozyme, laccase or a tannase or a combination thereof. According to this embodiment, the single-use-composition containing the insoluble enzyme preparation is preferably a dentifrice or toothpaste or a chewing gum.

In another preferred embodiment of the invention, the enzyme contained in the insoluble enzyme preparation which is used according to the invention to provide enzyme activity to a single-use-composition, is a glucose oxidase, a lactoperoxidase, or a laccase or a combination thereof. According to this embodiment, the single-use-composition containing the insoluble enzyme preparation is preferably a personal care composition or a food composition.

In still another preferred embodiment of the invention, the enzymes contained in the insoluble enzyme preparation which is used according to the invention to provide enzyme activity to a single-use-composition, is a glucose oxidase together with at least one proteolytic or mucolytic enzyme or combinations thereof. According to this embodiment, the single-use- composition containing the insoluble enzyme preparation is preferably a sanitary pad.

In yet another preferred embodiment of the invention, the enzyme contained in the insoluble enzyme preparation which is used according to the invention to provide enzyme activity to a single-use-composition, is a proteolytic and/or saccharolytic enzyme. According to this embodiment, the single-use-composition containing the insoluble enzyme preparation displays preferably anti-algae activity.

In a further preferred embodiment of the invention, the enzyme contained in the insoluble enzyme preparation which is used according to the invention to provide enzyme activity to a single-use-composition, is a β-1 ,3-glucanase, a β-1 ,6-glucanase or a chitinase or a combination thereof. According to this embodiment, the single-use-composition containing the insoluble enzyme preparation displays preferably anti-mold activity.

In still a further preferred embodiment of the invention, the enzyme contained in the insoluble enzyme preparation which is used according to the invention to provide enzyme activity to a single-use-composition, is a glucanase, a protease, or a nuclease or a combination thereof. According to this embodiment, the single-use-composition containing the insoluble enzyme preparation displays preferably anti-biofilm activity. In a preferred embodiment of the invention, the relative weight content of the insoluble enzyme preparation which is contained in the single-use-composition according to the invention amounts to at least 0.01 %, more preferably at least 0.5%, still more preferably at least 7.5%, yet more preferably at least 15%, most preferably at least 20% and in particular at least 45% with respect to the total weight of the single-use-composition.

In a preferred embodiment of the invention, the amount of enzyme which is contained in the insoluble enzyme preparation according to the invention is at least 0.5%, more preferably at least 2%, still more preferably at least 4%, yet more preferably at least 7%, most preferably at least 10% and in particular at least 15% relative to the total weight of the insoluble enzyme preparation.

The enzyme is coupled to a solid particulate carrier.

The solid carrier particles may assume the form of spheres, rods, tubes or a mixture thereof.

The particle size of the insoluble enzyme preparation is important for the applicability in the targeted industries, particularly in the care industries, food, and baking industries. Due to its size range, the insoluble enzyme preparation according to the invention will blend well with other ingredients in food compositions (such as dough mixes, etc.).

In a preferred embodiment of the invention, at least 50 wt.-%, more preferably at least 60 wt.- %, still more preferably at least 70 wt.-%, yet more preferably at least 80 wt.-%, most preferably at least 90 wt.-% and in particular at least 99.999 wt.-% of the particles making up the solid carrier of the insoluble enzyme preparation preferably have a diameter in the range of from 0.1 to 500 μηι, more preferably 0.2 to 100 μηι or 0.2 to 500 μηι, still more preferably 0.5 to 50 μηι or 0.5 to 500 μηι, most preferably 2 to 30 μηι or 10 to 500 μηι and in particular 10 to 30 μηι or 10 to 50 μηι.

In a preferred embodiment, the solid carrier of the insoluble enzyme preparation has an average diameter in the range of from 0.1 μηι to 500 μηι.

Preferably, the amount of particles of the solid carrier which are not respirable is at least 50 wt.-%, more preferably at least 60 wt.-%, still more preferably at least 70 wt.-%, yet more preferably at least 80 wt.-%, most preferably at least 90 wt.-% and in particular at least 95 wt.-%, relative to the total amount of particles of the solid carrier. In a preferred embodiment, at least 50 wt.-% of the solid carrier has a size such that it is not respirable.

In a particularly preferred embodiment, the solid carrier of the insoluble enzyme preparation which is contained in the single-use-composition has an average diameter in the range of from 0.1 μηι to 500 μηι; and/or at least 50 wt.-% of the solid carrier has a size such that it is not respirable.

The solid carrier may have a surface area of 5 to 1 ,000 m ² /g, preferably 20 to 1 ,000 m ² /g, more preferably 100 to 700 m ² /g and most preferably 100 to 300 m ² /g.

In a preferred embodiment, the solid carrier is a porous material.

When the solid carrier is a porous material, the pore size is preferably of from 5 nm to 50 μηι, more preferably 5 to 1 ,000 nm, most preferably 10 to 500 nm and in particular 100 to 300 nm.

In a preferred embodiment, the melting point of the solid carrier is at least 50°C, more preferably at least 100°C, still more preferably at least 500°C, yet more preferably at least 1 ,000°C, most preferably at least 1 ,500°C and in particular at least 1 ,650°C or 2,000°C.

The particles forming the solid carrier may comprise inorganic, organic or both inorganic and organic material.

The solid carrier may further have a hydrophilic or hydrophobic surface.

In a preferred embodiment, the solid carrier comprises an inorganic material with a substantially hydrophilic surface, which is essentially insoluble in hydrophilic or hydrophobic liquids or mixtures thereof.

The solid carriers may be based on silicas (e.g. Sipernat 2200 from Evonik, Germany), zeolites (e.g. Wessalith MS330 from Evonik, Germany), aluminas, diatomaceous earth, ceramics such as disclosed in Yoshihiko Hirose et al. (Proceedings from 3rd International Symposium on Biocatalysis and Biotransformations, La Grande Motte, France, 1997, p. 238) and kaolins (e.g. kaolins subjected to acid, hydrothermal and baking treatment as disclosed in U.S. Pat. No. 5,614,401 ). Polymeric carriers on basis of synthetic or natural origin to be used for entrapment of said enzymes or coupling in non-particulate form are explicitly excluded from carriers in the meaning of this invention.

In a preferred embodiment of the invention, the solid carrier comprises metal oxides such as alumina, more preferably gamma alumina, silica, zirconia, silica magnesia, silica-zirconia- alumina etc.

In another preferred embodiment of the invention, the solid carrier is a porous particulate material which is selected from the group consisting of silica, zeolite, alumina, ceramic and kaolin.

In still another preferred embodiment of the invention, the solid carrier is produced from orthosilicates while the enzyme is entrapped and active without any later detachment or re- mobilization procedure e.g. as disclosed by Buthe (P. Wang (ed.), Nanoscale Biocatalysis, Methods in Molecular Biology, pp. 223, Springer 201 1 ).

Particles derived from such sol-gel process can be grinded down to the desired size in order to provide a suitable solid carrier for the production of insoluble enzyme preparations according to the invention.

In a preferred embodiment of the invention, the solid carrier comprises a hydrophilic inorganic material and is coated with a compound having organic hydrophobic moieties, thereby rendering the surface of the solid carrier substantially hydrophobic.

In this regard, it is further referred to JP 09000257-A, which describes an acid treated kaolin carrier which is coated with N-phenyl-gamma-aminopropyltrimethoxysilane. Further carriers are described in JP 08126489-A, wherein a water insoluble carrier is coated with a polymer forming a disulphide linkage with enzymes.

In a preferred embodiment, the surface of the solid carrier, which is preferably an inorganic material with a substantially hydrophilic surface such as silica or a metal oxide, is functionalized.

According to this embodiment, the surface of the solid carrier is preferably functionalized with NH ₂ -groups. Preferably, the functionalization of the surface of the solid carrier promotes the coupling of the enzyme to the solid carrier.

The solid carrier may comprise an organic polymer resin.

The resin may be an adsorbent resin, preferably a polyacrylate, a polymethacrylate (e.g. polymethyl methacrylate), polystyrene cross-linked with divinylbenzene, polyurethane or polypropylene. The resin may also be an ion exchange resin, preferably an anion exchange resin, e.g. a weakly basic anion exchange resin. A preferred anion exchange resin is a phenolic type Duolite resin from Rohm & Haas. Such polymeric resins can be grinded down to the desired size in order to provide a suitable solid carrier for the production of insoluble enzyme preparations according to the invention.

In a preferred embodiment, the solid carrier is selected from the group consisting of polyacrylates, polymethacrylates, polystyrene cross-linked with divinylbenzene, polyurethanes, polypropylene and phenolic type anion exchange resins.

Further suitable solid carriers may be selected from the group consisting of glass, glass fibers, carbon, natural clays, waxes, salts and minerals.

In a particularly preferred embodiment, the solid carrier is selected from the group consisting of particulate metal oxides; silica of artificial origin; silica of natural origin, e.g. natural silicate minerals (i.e. zeolithes and phyllosilicates like Kaolinit); industrially produced fumed or precipitated silica; and sol gels.

In a most preferred embodiment of the invention, the solid carrier is an industrially fumed silica.

In another most preferred embodiment of the invention, the solid carrier is an industrially fumed silica with an average particle size of 0.01 μηι to 1 μηι.

Industrially fumed silica are composed of primary particles with an average particle size range of 0.01 μηι to 0.09 μηι, which forms stable aggregates and agglomerates of less than 1 μηι particle size. They are advantageous for the immobilization of enzymes in comparison to other carriers, as to their low market prizes, allowing for cost efficient industrial production processes. The inherent properties of fumed silica further enable easy and efficient binding of enzymes to the carrier, through easy access to functional residues of the carrier. The immobilization rate of enzyme thereby can be significantly increased, overall lowering enzyme loss in the process and production cost accordingly. Fumed silica are advantageous in numerous industries and applications sensitive for rheologic parameters of liquid and free- flowing parameters of (solid) powder-like preparations.

It has been surprisingly found, that through immobilization of any enzyme to fumed silica particles, a larger particle size with an average particle diameter ranging from 0.1 μηι to 500 μηι or from 1 μηι to 500 μηι, preferable from 10 μηι to 500 μηι and most preferably in from 10 μηι to 50 μηι can be obtained. The increase of particle size is explained through advanced and stable agglomeration and aggregation of the fumed silica particles in the course of the immobilization process. The size increase observed surprisingly allows for the use of fumed silica in applications sensitive for inhalant allergenic properties.

In a preferred embodiment of the invention, the insoluble enzyme preparation comprises an enzyme immobilized on a fumed silica particle, with an average diameter of 1 μηι to 500 μηι, preferable from 10 μηι to 500 μηι. also preferably from 10 μηι to 200 μηι, even more preferably from 10 μηι to 100 μηι and most preferably in from 10 μηι to 50 μηι after the immobilization of the enzyme.

In another preferred embodiment of the invention, the insoluble enzyme preparation comprises an enzyme covalently immobilized on a fumed silica particle, with an average diameter of 1 μηι to 500 μηι, preferable from 10 μηι to 500 μηι, also preferably from 10 μηι to 200 μηι, even more preferably from 10 μηι to 100 μηι and most preferably from 10 μηι to 50 μηι after the immobilization of the enzyme.

In a preferred embodiment of the invention, the insoluble enzyme preparation comprises an enzyme immobilized on a fumed silica particle, of which a fraction of at least 50%, preferably 70%, more preferably 90% and most preferably more than 95% of the particles have a diameter in the range of 1 μηι to 500 μηι, preferable from 10 μηι to 500 μηι. also preferably from 10 μηι to 200 μηι, even more preferably from 10 μηι to 100 μηι and most preferably in from 10 μηι to 50 μηι after the immobilization of the enzyme.

In another preferred embodiment of the invention, the insoluble enzyme preparation comprises an enzyme covalently immobilized on a fumed silica particle, with an average diameter of 10 μηι to 50 μηι after the immobilization of the enzyme. In a preferred embodiment, when the single-use-composition is an oral care composition, such as toothpaste or a chewing gum, the solid carrier can also be an abrasive polishing material, since industrially used silica have abrasive and/or polishing properties and are therefore used in toothpaste.

In another preferred embodiment, the solid carrier is not carbon nanotubes, titanium dioxide, grain flour or starch.

In a preferred embodiment of the invention, the relative weight content of the solid carrier contained in the insoluble enzyme preparation which is comprised in the single-use- composition according to the invention amounts to at least 0.01 %, more preferably at least 0.5%, still more preferably at least 7.5%, yet more preferably at least 15%, most preferably at least 20% and in particular at least 45% with respect to the total weight of the single-use- composition.

The production of the insoluble enzyme preparation comprises the steps of: a) binding the enzyme to a solid carrier particle by contacting a liquid enzyme composition (comprising one or more enzymes) with a particulate solid carrier; or b) using deliberately chosen functional groups on the surface of the solid carrier for coupling the enzyme either by covalent attachment or adsorption; and/or c) using a crosslinker and if necessary a binder to covalently attach the enzyme to the particulate solid carrier.

The enzyme is coupled to the solid carrier by adsorption and/or covalent binding.

The skilled artisan will readily recognize that the coupling methods will be chosen according to the carrier properties and the requirements of the targeted application.

In a preferred embodiment, when the single-use-composition is hydrophobic, the coupling of the enzyme to the solid carrier prevents the agglomeration of the otherwise free enzymes within the hydrophobic single-use-composition. Furthermore, according to this embodiment, the coupling of the enzyme to the solid carrier is mandatory for obtaining a homogeneous distribution of the enzyme within the hydrophobic single-use-composition.

If necessary, coupling of the enzyme to the solid carrier can be facilitated by usage of a binder. Polyfunctional amines may be used as binders. The function of the polyfunctional amine is to provide a network of amine-groups available for covalent cross-linking with the cross-linking agent and the enzyme's amine-groups in case that the solid carrier has not been modified in advance. The poly-functional amine acts as binder and will provide a mechanical strength to the insoluble enzyme preparation and improve the overall cross-linking of the enzyme and thereby minimize the leaching of enzyme from the solid carrier.

Further, crosslinkers may be used in the coupling of the enzyme to the solid carrier.

The cross-linking agent is a poly- or bis-functional reagent that reacts with the activated silica carrier and/or the polyfunctional amine and the enzyme to produce covalent cross-linking.

The cross-linking agent can also react intermolecularly in between the polyfunctional amines as well as in between the enzymes.

An example for a suitable bi-functional crosslinker is glutardialdehyde.

For the covalent binding of an enzyme, silica-based and metal oxide-based materials have proven beneficial as carriers, according to literature, and also have a low price.

For example U.S. Pat. No. 5,776,741 describes the coupling of enzymes on a particulate silica carrier for synthesis in organic media. As being described in US 2007/0087418, several silica-based materials can be used. These materials can be activated or modified to allow for the covalent binding of enzymes or its simple adsorption (e.g. U.S. Pat. No. 4,384,045). It is also possible to modify the enzyme by means of protein engineering to promote the binding between the enzyme and the silica by adding specific tags to the enzyme, e.g. "silaffins" as described in US 2012/0034338.

Many other concepts for covalent coupling of enzymes to a solid carrier are described in literature.

A widely used approach to enzyme coupling might be referred to as the covalent cross- linking process and is exemplified by U.S. Pat. No. 4,071 ,409 (Messing et al.). According to the teaching of this patent, a support medium is modified or coated to present functionalities which can then be linked by way of a cross-linking agent to free functional groups of the enzyme. U.S. Pat. No. 4,141 ,857 describes the preparation of an enzyme support which is prepared by reacting a porous carrier with a polyamine and thereafter with a reactant. Other coupling processes are described in WO 95/22606 (Pedersen et al.) and WO 99/33964 (Christensen et al.).

WO 95/22606 describes a process, wherein an enzyme containing liquid is brought in contact with porous silica carrier in an extruder or a granulation apparatus. WO 99/33964 discloses a coupling process wherein the coupling is prepared in a fluid bed apparatus.

CA 2277371 describes a process for coupling of an enzyme by incubating a siliceous support having surface hydroxyl groups with a first aqueous solution comprising a polyaldehyde and subsequently allowing a second aqueous solution comprising an enzyme to come into contact with the modified support and finally removing the support from the solution. EP 133531 describes a process for coupling an enzyme by (a) introducing into an aqueous medium containing an enzyme an aqueous solution of polyethyleneimine; and (b) adding glutaraldehyde and chitosan to the aqueous medium to form a cross-linked reaction product; and (c) removing the cross-linked product from the liquid medium. In U.S. Pat. No. 4,888,285 (Nishimura et al.) a silica gel is modified by reaction with an aminosilane derivative in an organic solvent. The resulting aminated support is then linked to the enzyme in the presence of glutaraldehyde, tannic acid and chitosan.

EP 0206687 discloses a coupling process comprising mixing a dispersion of enzyme with polyazetidine prepolymer and glutaraldehyde followed by dewatering.

The enzyme may also be entrapped within the solid carrier, e.g. when the solid carrier is an orthosilicate, as disclosed by Buthe (P. Wang (ed.), Nanoscale Biocatalysis, Methods in Molecular Biology, pp. 223, Springer 201 1 ).

In a preferred embodiment of the present invention, the enzyme is covalently linked to the solid carrier.

In another preferred embodiment of the present invention, the enzyme is linked to the solid carrier by adsorption.

In still another preferred embodiment of the present invention, the enzyme is coupled to the solid carrier by covalent bonds and adsorption. In a preferred embodiment of the invention, the enzyme is coupled to a solid carrier wherein the solid carrier is an organic polymer resin, more preferably diatomaceous earth, still more preferably an alumina, yet more preferably a zeolite, ceramic and kaolin and most preferably fumed and precipitated silica.

The coupling of enzymes to a solid carrier is not new by itself, however, the prior art is merely directed at the use of immobilized biocatalysts in order to enable the separation and potential reuse of the biocatalyst.

Accordingly, the prior art discloses the preparation of immobilized enzymes for use in enzymatic modification of e.g. an organic compound, such as in synthesis processes in organic or aqueous medium. Usually an immobilized enzyme composition used in said processes may be reused several times.

According to the invention, however, the insoluble enzyme preparations are used as an "additive", which is added to a single-use-composition, or added in the course of a production process or used in an application which after its effect or action is not removed or isolated with the aim to reuse it.

According to the invention, a single-use-composition is provided with enzyme activity by an insoluble enzyme preparation comprising an enzyme which is immobilized on a solid carrier, wherein the insoluble enzyme preparation is dispersed in the single-use-composition.

Preferably, the enzyme activity of the single-use-composition provides antimicrobial properties or decontaminating or cleaning activity, in particular an antimicrobial effect, a whitening effect, a bleaching effect, a decolorizing effect, an anti-odor effect, and/or a bioorganic matter degrading effect.

The insoluble enzyme preparation is homogeneously dispersed in the single-use- composition. In a preferred embodiment, the single-use-composition is used to provide a bioactive surface.

The insoluble enzyme preparation is preferably dispersed homogeneously in the single-use- composition.

Preferably, the single-use-composition is a heterogeneous mixture, more preferably a solid- liquid heterogeneous mixture. Preferably, the single-use-composition contains a liquid constituent, such as water or another solvent which is suitable to be used in combination with an enzyme and the targeted application.

Suitable solvents may be C Ci ₈ alcohols, such as ethyl alcohol, propyl alcohol, isopropyl alcohol, isostearyl alcohol and lauryl alcohol.

A solvent may be regarded as being suitable if its presence does not counteract the enzymatic activity. The person in the art knows how to determine whether a solvent is suitable or not.

In a preferred embodiment, the single-use-composition contains a liquid constituent, preferably water.

In a preferred embodiment,

the enzyme displays the enzyme activity in the course of the intended usage of the single-use-composition; and/or the enzyme activity causes an antimicrobial effect, a whitening effect, a bleaching effect, a decolorizing effect, an anti-odor effect, and/or a bioorganic matter degrading effect; and/or after its intended usage, the single-use-composition is disposed of together with the insoluble enzyme preparation contained therein; or after the intended usage of the single-use-composition, the insoluble enzyme preparation remains within the single-use-composition.

In a preferred embodiment, an aerosol is formed as a side effect in the course of the intended usage of the single-use-composition.

In a preferred embodiment, the insoluble enzyme preparation is used for reducing the allergenic potential of the single-use-composition compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier; or for reducing the enzyme caused immunogenicity of the single-use-composition compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier.

The insoluble enzyme preparations of the invention can be used to prepare low allergenic enzyme single-use-compositions.

In a preferred embodiment, the insoluble enzyme preparation is used for providing enzyme activity to a single-use-composition which is selected from the group consisting of oral care compositions, personal care compositions, food compositions, cosmetic compositions, home care compositions, household compositions, detergent compositions, feed compositions, paint compositions, coating compositions, and agriculture compositions.

In a preferred embodiment, the single-use-composition containing the insoluble enzyme preparation is an oral care composition, more preferably a chewing gum or toothpaste; an ointment; a shampoo; a deodorant; a dry provender for animal nutrition; a dough for a bakery composition; a hard surface cleaner; a household-cleaner; a silicone elastomer; a coating; a paint; or a varnish.

In a preferred embodiment, the single-use-composition is a toothpaste, a sealant, a coating, a paint or a varnish.

In another preferred embodiment, the single-use-composition is an oral care composition, such as a chewing gum or a toothpaste.

In still another preferred embodiment, the single-use-composition is a personal care composition, such as a sanitary pad, an ointment, a shampoo or a deodorant.

In yet another preferred embodiment, the single-use-composition is a dough for bakery compositions.

In a further preferred embodiment, the single-use-composition is a home care composition, such as a hard surface cleaner.

In still a further preferred embodiment, the single-use-composition is a silicone elastomer. In yet a further preferred embodiment, the single-use-composition is a coating, a paint or varnish.

According to the invention, the insoluble enzyme preparations can be used for providing enzyme activity to single-use compositions, such as washing compositions or cleaning compositions.

The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. washing is often referred to as its washing ability, washability, detergency, or wash performance.

Examples of proteins, polypeptides, and enzymes, respectively, which are commonly used in compositions for washing and cleaning, include enzymes exhibiting protease, lipolytic enzyme, tannase, oxidoreductase, carbohydrase, transferase, such as transglutaminase, phytase and/or antimicrobial polypeptides.

In some applications, cleaning refers to the removal of biofilms. The biofilm formed, for instance, in lens cases can be removed by the action of glucanases and/or other hydrolytic enzymes.

The insoluble enzyme preparations of the invention can be used to prepare low allergenic enzyme single-use-compositions, e.g. low allergenic washing and cleaning compositions, respectively, such as low allergenic hard surface cleaners or low allergenic detergent compositions.

According to the invention, the insoluble enzyme preparations can be used for providing enzyme activity to single-use compositions, such as personal care compositions.

A particularly useful application area for low allergenic enzyme single-use-compositions is in personal care compositions where the end-user is in close contact with the protein contained therein, and where certain problems with allergenicity have been encountered in experimental set-ups (Kelling et al., J. All. Clin. Imm., 1998, Vol. 101 , pp. 179-187 and Johnston et al., Hum. Exp. Toxicol., 1999, Vol.18, p. 527).

According to the invention, the single-use-compositions can advantageously be used as personal care compositions, such as hair care and hair treatment compositions. This includes single-use-compositions such as shampoo, balsam, hair conditioners, hair waving compositions, hair dyeing compositions, hair tonic, hair liquid, hair cream, shampoo, hair rinse, hair spray.

Further, according to the invention, the single-use-compositions can be used as skin care compositions and cosmetics, such as skin cream, skin milk, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, nourishing essence, skin lotion, milky lotion, calamine lotion, hand cream, powder soap, transparent soap, sun oil, sun screen, shaving foam, shaving cream, baby oil, lipstick, lip cream, creamy foundation, face powder, powder eye-shadow, powder, foundation, make-up base, essence powder, whitening powder.

Still further, according to the invention, the single-use-compositions can be used as contact lenses' hygiene compositions. Such compositions include cleansers and disinfectants for contact lenses.

Proteases for example are effective ingredients in skin cleaning compositions, where they remove the upper layer of dead keratinaseous skin cells and thereby make the skin look brighter and fresher. Proteases are also well-known active ingredients for cleaning of contact lenses. They hydrolyze the proteinaceous soil on the lens, thereby making the protein soil soluble. Removal of the protein soil is essential for the wearing comfort. Further, proteases are used in toiletries, bath and shower compositions, including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps. Another application for proteases besides other mucolytic and/or antimicrobial enzymes are sanitary pads and wound dressings, where the absorption of viscoelastic body fluids is facilitated due to the degradation of high-molecular compounds as well as microbes can be suppressed or inactivated by the action of the enzymes.

Lipolytic enzymes can be used in cosmetics as active ingredients in skin cleaning and skin care compositions, e.g. in bath and shower compositions such as creams and lotions, or for removal of excessive skin lipids in anti-acne compositions. Lipolytic enzymes can also be used in hair cleaning compositions (e.g. shampoos) for effective removal of sebum and other fatty material from the surface of hair. Lipolytic enzymes are also effective ingredients in compositions for cleaning of contact lenses, where they remove lipid deposits from the lens surface.

The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with a substrate (e.g. glucose) that ensures production of H ₂ 0 ₂ , which then will initiate the oxidation of e.g. SCN ^" or into antimicrobial reagents (SCNO ^" or \ ₂ ) by a peroxidase (usually lactoperoxidase).

The enzymatic complex described above is known in nature from e.g. milk and saliva. Antimicrobial systems comprising the combination of an oxidase and a peroxidase are known in the cleaning of contact lenses.

Another application of oxidoreductases is oxidative hair dyeing and/or discoloration using oxidases, peroxidases and laccases. Protein disulfide isomerase (PDI) is also an oxidoreductase. It can be utilized for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds).

Free radicals formed on the surface of the skin (and hair) known to be associated with the ageing process of the skin (spoilage of the hair). The free radicals activate chain reactions that lead to destruction of fatty membranes, collagen, and cells. The application of free radical scavengers such as Superoxide dismutase into cosmetics is well known (R. L. Goldemberg, DCI, Nov. 93, p. 48-52).

Personal care compositions include ointments.

Preferably, an ointment is a homogeneous, viscous, semi-solid preparation, most commonly a greasy, thick oil (80% oil, 20% water) with a high viscosity, that is intended for external application to the skin or mucous membranes. They are can be used as emollients or for the application of active ingredients to the skin for protective, therapeutic, or prophylactic purposes and in applications where a degree of occlusion is desired. Ointments are preferably used topically on a variety of body surfaces (skin and the mucous membranes, vagina, anus, and nose).

An ointment may or may not be medicated. The ointment may comprise an ointment base. The medicaments can be dispersed in the ointment base, and are distributed after the drug penetrates into the living cells of the skin. The vehicle of an ointment is known as the ointment base. The choice of a base depends upon the clinical indication for the ointment. The different types of ointment bases comprise:

• Hydrocarbon bases, e.g. mineral oil, hard paraffin, soft paraffin, microcrystalline wax and ceresine

• Absorption bases, e.g. wool fat, beeswax • Water soluble bases, e.g. polyalkylene glycols like macrogol 200, 300, 400

• Emulsifying bases, e.g. emulsifying wax or surfactants like cetrimide

• Vegetable oils, e.g. olive oil, coconut oil, sesame oil, almond oil, and peanut oil.

Ointments are preferably formulated using hydrophobic, hydrophilic, or water-emulsifying ointment bases to provide preparations that are immiscible, miscible, or emulsifiable with skin secretions. They can also be derived from hydrocarbon (fatty), absorption, water-removable, or water-soluble bases.

An ointment composition can be a viscous solid at temperatures which are at or around body temperature. Although the physical properties of the ointment are temperature-dependent, at or near body temperature, the ointment typically has a viscosity of at least 10,000, preferably at least 15,000, and more preferably about 19,000 mPa-s. When spread on the skin or a mucosal surface, or a wound, lesion or ulcer it preferably forms a stable, coherent film.

The mineral oil which may be contained in an ointment is preferably pharmaceutical grade. The mineral oil can be present in an amount which produces an emulsion having the desired physical properties. The amount of mineral oil and/or polymers like polyalkylene glycol which may be present in the ointment compositions of the invention may be adjusted to provide the desired final viscosity. Also other hydrophilic polymeric equivalents of polyalkylene polymers can be employed. These polymers may also contribute to stabilize the emulsion by preventing its separation upon storage.

The ointment compositions are optionally stabilized with the inclusion of a non-ionic surfactant, of which many are known in the art to be useful in producing stable aqueous emulsions in the cosmetic and pharmacy fields. See e.g., Schwartz et at., "Surface Active Agents and Detergents," Vol. II, pp. 120-143 (Interscience Pub. Inc., N.Y., 1958). A preferred class of non-ionic surfactants are polyoxyethylene (ethoxylates) surfactants in which a polyoxyethylene block polymer chain is terminated with a less soluble group, e.g., an alkylphenol ether group, an alcohol or mercaptan ether group, an amide group, or a carboxylic acid ester group. The non-ionic surfactant may be present in the ointment compositions of the invention in a range from 0% to about 3% w/w, preferably from about 0.05 to 1 .5% w/w, and more preferably at about 0.7% w/w.

According to the invention, the insoluble enzyme preparations can be used for providing enzyme activity to single-use compositions, such as oral care compositions e.g. dentifrice, oral washes and chewing gum. Proteases are also used in oral care compositions, especially for cleaning of dentures, but also in dentifrices.

Oxidoreductases are being utilized commercially as anti-microbial system in oral care compositions (mouth rinse, dentifrice, chewing gum) where it also can be combined with an amyloglucosidase to produce glucose, the substrate for the oxidoreductase. These systems are also known in cosmetic compositions for preservation. Oxidoreductases like laccases can be used for teeth whitening, and the presence of tannase enzyme can be beneficial in facilitating the breakdown of extrinsic stain. Tannases are useful in the hydrolysis of tannins, known to discolor the tooth surface.

Plaque formed on the surface of teeth is composed mainly of polysaccharides. They stick to the surface of the teeth and the microorganisms. The polysaccharides are mainly a-1 ,6 bound glucose (dextran) and a-1 ,3 bound glucose (mutan). The application of different types of glucanases such as mutanase and dextranase helps hydrolyzing the sticky matrix of plaque, making it easier to remove by mechanical action.

Oral care compositions include dentifrices.

A typical dentifrice comprises a water-phase, containing a humectant therein. The humectant is preferably glycerin, sorbitol, xylitol, and/or propylene glycol; but other humectants and mixtures thereof may be used as well. The humectant concentration typically is in the range of 5 to 70% by weight of the oral composition. Water is present typically in amount of at least 10% by weight, and generally about 15 to 30% by weight of the oral composition and should be preferably deionized and free of organic impurities.

There are more ingredients that can be used to compose a dentifrice such as thickening agents, surfactants, a source of fluoride ions, a synthetic anionic polycarboxylate, a flavoring agent, antitartar and coloring agents. Thickeners used in dentifrices include natural and synthetic gum and colloids. Inorganic thickeners include amorphous silica compounds which function as thickening agents and include colloidal silica compounds. The thickening agent is present in the dentifrice composition in amounts of 0.1 to 10% by weight.

Surfactants used in the composition are preferably detersive which confer detersive and foaming properties. Some surfactants are not compatible with enzymes, such as sodium lauryl sulfate as anionic surfactants. As a result, it is important to use a surfactant or a combination of surfactants that are compatible with the enzymes present in the oral composition and provide the requisite foaming characteristics. Examples of enzyme compatible surfactants solely or in combination include non-anionic polyoxyethylene surfactants such as Polyoxamer 407, Steareth 30, Polysorbate 20, and amphoteric surfactants such as cocamidopropyl betaine, and cocamidopropyl betaine lauryl glucoside. The total surfactant concentration in the dentifrice composition typically ranges from 2 to 10% by weight.

Typically dentifrice compositions contain a source of fluoride ions or fluorine providing component, as anti-caries agent in amount sufficient to supply about 25 ppm to 5,000 ppm of fluoride ions and include inorganic fluoride salts. Anti-tartar agents such as pyrophosphate salts including dialkali or tetraalkali metal pyrophosphate salts, long chain polyphosphates, and/or cyclic phosphates are typically used as well in concentrations of 1 to 5% by weight.

Enzyme stabilizing agents to protect certain enzymes from inactivation by chelating metal impurities may be used. Synthetic anionic polycarboxylates may also be used in dentifrice compositions as an efficacy enhancing agent for any antibacterial, anti-tartar or other active agent. Preferred are 1 :4 to 4:1 copolymers of maleic anhydride or acid with another polymerizable ethylenically unsaturated monomer, preferably methylvinylether/maleic anhydride. Generally, the anionic polycarboxylate is present in concentrations from 0.05% to 4% by weight.

Toothpaste typically contains a flavoring agent, which include essentials oils as well as various flavoring aldehydes, esters, alcohols, and similar materials. Examples of the essential oils include oils of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, lime, grapefruit, and orange. Also useful are such chemicals as menthol, carvone, and anethole. Of those, the most commonly employed are the oils of peppermint and spearmint.

The flavoring agent is incorporated at a concentration of 0.1 to 5% by weight. Various other materials may be incorporated in the dentifrice composition, including desensitizers, such as potassium nitrate, whitening agents; preservatives; silicones; and chlorophyll compounds. These additives, when present, are incorporated in the dentifrice composition in amounts which do not substantially adversely affect the properties and characteristics desired.

Oral care compositions further include chewing gums. Reagrding the composition of chewing gum, generally the following categories of ingredients can be found:

• Elastomers: used to provide elasticity or bounce, and can be of natural (e.g. latex) or synthetic (e.g. styrene-butadiene rubber, butyl rubber, polyisobutylene)

• Resins: used to provide a cohesive texture (e.g. terpene resins, and/or polyvinyl acetate)

• Waxes: used as softening agents

• Fats: used as plasticizers (e.g. hydrogenated vegetable oils)

• Emulsifiers: used as hydrating agent (e.g. lecithin or glycerol monostearate)

• Fillers: used to impart a texture (e.g. calcium carbonate, kaolin, or talc)

• Antioxidants: used to extend shelf-life.

When a chewing gum is used as oral hygiene preparation for plaque disrupting, gingivitis control, tartar control and microbial control, it contains an active ingredient, which may include, among others, enzymes.

In contrast to toothpaste and other rinse preparations, for which teeth are only being treated for a relatively short time, chewing gums tremendously extend the contact period for the active and teeth with respective beneficial effects. Some prior art methods have been disclosed for the incorporation of active or insoluble ingredients into sugar containing gum bases. U.S. Pat. No. 3,075,884 teaches a method for obtaining the release of solid active ingredients from a gum base by dispersing the solid active ingredient throughout the corn syrup ingredient of the gum prior to the admixture of the corn syrup with the gum base. U.S. Pat. No. 3,01 1 ,919 teaches a method for incorporating active ingredients, including phosphates, into slab chewing gum by coating the active ingredients with wet sugar.

A number of chewing gum compositions have been disclosed in the prior art which are said to inhibit or reduce plaque in the oral cavity. For example, U.S. Pat. Nos. 4,148,872, 4,150,1 12, 4,156,715, 4,156,716, 4,157,385, 4,159,315, 4,160,054, 4,160,820, 4,161 ,517, and 4,170,632, all to A. Wagenknecht et at., disclose chewing gum compositions effective in inhibiting or reducing plaque in the oral cavity. These chewing gum compositions contain a chewing gum base and a surface active agent, and, in some instances, a zinc compound or a plaque inhibiting flavor. In addition, a calcium carbonate abrasive may be included in the aforementioned chewing gum compositions. See also U.S. Pat. Nos. 3,974,293; 3,984,574; 3,651 ,206; 4,568,537; 4,474,749 and 4,828,820. U.S. Pat. No. 4,029,760 discloses pharmaceutical chewing gums for the treatment of gingivitis containing at least one carrageenin. U.S. Pat. No. 4,400,372, to J. C. Muller et at., discloses a chewing gum composition containing a chewing gum base, at least one non-toxic source of an acid and calcined kaolin particles having a median diameter of 2 micrometers or less, wherein substantially all of the kaolin particles are less than 20 micrometers in diameter.

According to the present invention, allnatural or synthetic gum bases may be contained in the chewing gum.. Examples of suitable gum bases include chicle, gutta percha, jelutong, balata, namaquland rubber, almeidana gum, abba rubber, gutta siak, gutta cotie, gutta kay, gutta hangkang, gutta penang, and yellow gutta. Further examples of gum bases include rosins, such as comarone resin, pontianak resin, copal gum, kauri gum, dammar gum, sweet bay gum, spruce gum, and balsams. Moreover, suitable gum bases include crown gum, nisperio, rosidinha, pendare, perillo, niger gutta, and tuno. Additional chewing gum base materials include elastomers such as polyisobutylene, polyisosprene, isobutyleneisoprene copolymers and copolymers of butadiene and styrene, hydrogenated or partially hydrogenated vegetable oils such as soy bean, cotton seed, corn, peanut, and palm or animal fats such as tallow and lard. In addition paraffin, beeswax, petroleum wax, polyethylenes, and polyvinylacetates may be employed.

Further descriptions of suitable chewing gum bases are found in U.S. Pat. No. 2,366,589 issued to Borglin; U.S. Pat. No. 3,821 ,417, issued to Westall et a!.; U.S. Pat. No. 4,041 ,179 issued to Stubits et a\. U.S. Pat. No. 3,984,574 issued to Comolloand U.S. Pat. Nos. 1 ,807,704 and 2,076,1 12. Further descriptions of suitable chewing gum bases are found in U.S. Pat. No. 4,357,355, to E. Koch et a!., U.S. Pat. No. 4,387,108, to E. Koch et al., and U.S. Pat. No. 4,518,615, to S. R. Cherukuri et al.

The gum base referred to above covers the nonnutritive, masticatory substance in chewing gum, as defined in the Federal Food, Drug and Cosmetic Act. In the regulation covering chewing gum ingredients under the Food Additives Amendment (Federal Register, p. 4419, May 9, 1962), paragraph (a) sets forth the ingredients permitted in chewing gum base under the regulation, and paragraph (c) defines the term "chewing gum base" as non-nutritive masticatory substance comprised of one or more of the ingredients named and so defined in paragraph (a) of this section. Suitable representative chewing gum bases which can be employed with facility in formulating chewing gum compositions are those disclosed, for example, in U.S. Pat. No. 2,284,804 of F. T. De Angelis and U.S. Pat. No. 2,137,746 of R. L. Wilson, U.S. Pat. No. 2,383,145 of J. E. Moose, U.S. 2,288,100 of G. J. Manson and U.S. Pat. Nos. 2,366,589; 3,821 ,417; 4,041 ,179 and 3,984,574.

According to the invention, the insoluble enzyme preparations may be used for providing enzyme activity to feed compositions and food compositions, e.g. flour and dough for cakes and bread, cereals, foodstuff containing soy beans, dairy products, sweets, drinks or soups.

The gluten in wheat flour is the essential ingredient responsible for the ability of flour to be used in baked foodstuffs. Proteolytic enzymes are sometimes needed to modify the gluten phase of the dough, e.g. a hard wheat flour can be softened with a protease. This may ensure a uniform dough quality and bread texture, and improve flavour. The gluten proteins are degraded either moderately or more extensively to peptides, whereby close control is necessary in order to avoid excessive softening of the dough.

Addition of lipolytic enzyme results in improved dough properties and an improved breadmaking quality in terms of larger volume, improved crumb structure and whiter crumb colour. The observed effect can be explained by a mechanism where the lipolytic enzyme changes the interaction between gluten and some lipid fragment during dough mixing. This results in an improved gluten network.

Several oxidoreductases are used for baking like glucose oxidase, lipoxygenase, peroxidase, catalase and combinations hereof. The dough strength is measured as greater resistance to mechanical shock, better oven spring and larger loaf volume. Traditionally, bakers strengthen gluten by adding ascorbic acid and potassium bromate. Some oxidoreductases, in particular glucose oxidase preparations with catalase activity, can be used to replace bromate in dough systems by oxidation of free sulfydryl units in gluten proteins. Hereby disulphide linkages are formed resulting in stronger, more elastic doughs with greater resistance.

Flour has a varying content of amylases leading to differences in the baking quality. Addition of amylases can be necessary in order to standardize the flour. Amylases and pentosanases generally provide sugar for the yeast fermentation, improve the bread volume, retard retrogradation, and decrease the staling rate and stickiness that results from pentosan gums. Certain maltogenic amylases can be used for prolonging the shelf life of bread for two or more days without causing gumminess. Amylases selectively modify the gelatinized starch by cleaving from the non-reducing end of the starch molecules, producing low molecular weight sugars and dextrins. The starch is modified in such a way that retrogradation is less likely to occur. The produced low-molecular-weight sugars improve the baked goods and water retention capacity without creating the intermediate-length dextrins that result in gumminess in the finished product. The enzyme is inactivated during bread baking, so it can be considered as a processing aid that does not have to be declared on the label.

The bread volume can be improved by fungal a-amylases which further provide good and uniform structure of the bread crumb. Said a-amylases are endoenzymes that produce maltose, dextrins and glucose. Cereal and some bacterial α-amylases are inactivated at temperatures above the gelatinization temperature of starch, therefore when added to wheat dough it results in a low bread volume and a sticky bread interior. Fungamyl has the advantage of being thermolabile and is inactivated just below the gelatinization temperature. α-amylases are further used in the animal feed industry to be added to cereal-containing feed to improve the digestibility of starch.

Insoluble enzyme preparations containing a number of pentosanase and hemicellulase activities can improve the handling and stability of the dough, and improves the freshness, the crumb structure and the volume of the bread. By hydrolyzing the pentosans' fraction in flour, the flour will lose a great deal of its water-binding capacity, and the water will then be available for starch and gluten. The gluten becomes more pliable and extensible, and the starch gelatinizes more easily. Pentosanases can be used in combination with or as an alternative to emulsifiers.

Transglutaminases are being used for improvement of baking quality of flour e.g. by modifying wheat flour to be used in the preparation of cakes with improved properties, such as improved taste, dent, mouth-feel and a higher volume (see JP 1 -1 10147).

Phytases may advantageously be used in the manufacturing of food, such as breakfast cereal, cake, sweets, drinks, bread or soup etc., and animal feed. Bread with better quality can be prepared by baking divided pieces of a dough containing wheat flour etc. and phytase (see JP-0-3076529-A). Phytases may be used either for exploiting the phosphorus bound in the phytate/phytic acid present in vegetable protein sources or for exploiting the nutritionally important minerals bound in phytic acid complexes. Microbial phytase may be added to feedstuff of monogastric animals in order to avoid supplementing the feed with inorganic phosphorus (see US patent no. 3,297,548). Further phytases may be used in soy processing. Soybean meal may contain high levels of the anti-nutritional factor phytate which renders this protein source unsuitable for application in baby food and feed for fish, calves and other non-ruminants, since the phytate chelates essential minerals present therein (see EP 0 420 358). Proteases are also used for modifying milk protein. To coagulate casein in milk when producing cheese proteases such as rennet or chymosin may be used. The flavor development of blue roan cheese (e.g. Danablue), certain Italian type cheese, and other dairy products containing butter-fat, are dependent on the degradation of milk fat into free fatty acids. Lipolytic enzymes may be used for developing flavor in such products. In the brewery industry, proteases are used for brewing with un-malted cereals and for controlling the nitrogen content. In animal feed compositions, proteases are used so to speak to expand the animals digestion system.

Transglutaminases can be used for gelling of aqueous phases containing proteins. They may be used when producing spreads (DK patent application no. 1071/84 from Novo Nordisk A/S) or producing paste type food material e.g. used as fat substitution in foods as ice cream, toppings, frozen desserts, mayonnaises and low fat spreads (see WO 93/22930 from Novo Nordisk A S). Furthermore, transglutaminases may be used in the preparation of gels for yoghurt, mousses, cheese, puddings, orange juice, from milk and milk-like products, and binding of chopped meat product, improvement of taste and texture of food proteins (see WO 94/21 120 and WO 94/21 129 from Novo Nordisk A/S).

Certain bacteriolytic enzymes may be used e.g. to wash carcasses in the meat packing industry (see US patent no. 5,354,681 from Novo Industri A S).

According to the invention, the insoluble enzyme preparations can be used for providing enzyme activity to single-use-compositions, such as oral and dermal pharmaceuticals.

Different combinations of highly purified proteases (e.g. Trypsin and Chymotrypsin) are used in pharmaceuticals to be taken orally, and dermal pharmaceuticals for combating e.g. inflammations, edemata and injuries.

According to the invention, the insoluble enzyme preparations can be used for providing enzyme activity to single-use hard surface cleaning compositions.

Cleaning of hard surfaces e.g. in the food industry is often difficult, as equipment used for producing dairies, meat, sea food products, beverages etc. often has a complicated shape.

The use of surfactant compositions in the form of gels and foams comprising enzymes have shown to facilitate and improve hard surface cleaning. Enzymes, which advantageously may be added in such surfactant compositions, are in particular proteases, lipolytic enzymes, amylases and cellulases. Such hard surface cleaning compositions comprising enzymes may also advantageously be used in the transport sector, for instance for washing cars and for general vessel wash.

According to the invention, the insoluble enzyme preparations can be used for providing enzyme activity to single-use-compositions, such as coatings, paint and varnish.

Enzyme functionalized surfaces display interesting properties regarding self-decontamination and self-protection. The growth of algae and mold is undesired.

Algae cells are surrounded by a protein and polysaccharide rich cell wall, which is suspectible for proteolytic and saccharolytic enzymes (proven in various publications - e.g. anti-fouling in marine environments). Fungal cells and spores are surrounded by cell wall made of chitin, susceptible for hydrolysis by β-1 ,3-glucanase, β-1 ,6-glucanase and Chitinase (proven e.g. in crop protection).

In a preferred embodiment of the invention, embedding those enzymes in coatings, paint and varnish yields a protection against the settlement of algae and mold.

Also the growth of biofilms composed of a complex microbial flora embedded in a protein and polysaccharide containing matrix is undesired.

Therefore, in a preferred embodiment of the invention, surfaces can be protected against biofilm formation by using glucanases and/or other hydrolytic enzymes like alcalase and savinase that break down the adhesive polymers during the colonization of new surfaces produced by species responsible for fouling.

In yet another preferred embodiment, hydrolytic enzymes like, but not limited to, proteases, amylases, and lipases can be embedded in coatings, paints and varnish to enable the degradation of adhering stains made of organic matter to facilitate their removal.

Figure 2 schematically visualizes surface functionalities accessible by entrapment of the single-use-composition in coatings and paints.

Typically, (dispersion) paint is based on at least one polymer dispersion with pigments, fillers, thickeners, dispersants and additives in the following quantities: • 2-20% by weight of polymer dispersion (calculated as a solid compound)

• 2-35% by weight of pigments

• 5-60% by weight of fillers having a particle diameter of 0.1 -200 μηι

• 0.1 -3% by weight of thickeners

• 0.1 -2% by weight of dispersants, and

• a maximum of 5% by weight additives

and water to make up 100%, wherein the dispersion preferably has a viscosity of 2.0 to 5- 10 ² mPa-s.

It is preferred that the polymer dispersion be selected from polymers which are built up from specific monomers. Suitable monomers are for example carboxylic acid vinyl esters, especially vinyl acetate, vinyl propionate, furthermore N-vinylpyrrolidone and its derivatives, ethylenically unsaturated carboxylic acids, their esters, amides or anhydrides, and furthermore oc-olefins, especially ethylene and propylene as well as acrylonitrile.

Particularly preferred is the use of ethylenically unsaturated carboxylic acids, especially acrylic acid and methacrylic acid. The alcohol residue of the esters of the esters can comprise linear or branched alkyl chains, cycloaliphates or aromatics which can be chemically modified if necessary. The use of styrene and styrene derivatives is also particularly preferred.

Pigments are known from the prior art. Examples are titanium dioxide, iron oxide, chromium oxide, cobalt blue, phthalocyanine pigments, spinel pigments and nickel and chromium titanate. Organic pigments such as azoic pigments, quinacridone pigments and/or dioxazine pigments can also be used.

As fillers, silicates, carbonates, fluorite, sulphates and oxides can be considered. By particular preference the fillers are kaolin, mica, talcum and calcium carbonate, alone and in mixture. The fillers typically have a diameter of 0.1 to 200 μηι. The selection of the particle size of the fillers is also important for setting the viscosity. A further variant for controlling the viscosity of the paint consists in the surfaces of the filler being functionalized. It is furthermore important that a thickener is used, in particular all those polycarboxylate thickeners known in the prior art are possible as thickeners here. Examples of these are polycarboxylates, urethane thickeners, polysaccharides and cellulose ethers. A dispersion paint preferably also contain additives such as dispersants, stabilisers, anti-foaming agents, preservatives and/or hydrophobing agents. According to the invention, the insoluble enzyme preparations can be used for providing enzyme activity to single-use-compositions, such as sealants.

Enzyme functionalized surfaces expose interesting activities regarding self-decontamination and self-protection. The growth of algae and mold is undesired. Algae cells are surrounded by a protein and polysaccharide rich cell wall, which is suspectible for proteolytic and saccharolytic enzymes (proven in various publications - e.g. anti-fouling in marine environments). Fungal cells and spores are surrounded by cell wall made of chitin, susceptible for hydrolysis by β-1 ,3- Glucanase, β-1 ,6-Glucanase und Chitinase (proven e.g. in crop protection). In a preferred embodiment of the invention, embedding those enzymes in coatings, paint and varnish yields a protection against the settlement of algae and mold. Also the growth of biofilms composed of a complex microbial flora embedded in a protein and polysaccharide containing matrix is undesired. Therefore, in a preferred embodiment of the invention, surfaces can be protected against biofilm formation by using proteolytic enzymes like alcalase and savinase that break down the adhesive polymers during the colonization of new surfaces produced by species responsible for fouling and have proven beneficial. In yet another preferred embodiment, hydrolytic enzymes like, but not limited to, proteases, amylases, and lipases can be embedded in coatings, paints and varnish to enable the degradation of adhering stains made of organic matter to facilitate their removal.

Such sealants are a crosslinkable preparation, which generally includes a silane-curing preparation, in particular, a crosslinkable silicone composition or crosslinkable silyl group- containing prepolymers such as silan-modified polymers, silanized acrylates, silanized polyolefins, in particular silanized polyisobutene, or silanized polyurethanes or mixtures of these substances, wherein the silicone composition or preparation containing crosslinkable silyl group-containing prepolymers preferably comprises a sealing composition, in particular a joint sealing composition. The term "silanized" means in this connection that the compounds are those which crosslink by means of alkoxy, acetate, oxime, benzamide, or aminosilane groups. Insoluble enzyme preparations prepared according to the invention can be included in the sealant matrix as single-use-composition in order to reduce the tendency of mold formation on the sealant surface.

In a preferred embodiment of the invention, an insoluble enzyme preparation comprising an enzyme which is immobilized on a solid carrier is used to provide enzyme activity to a single- use-composition, wherein the enzyme displays the enzyme activity in the course of the intended usage of the single-use-composition; and/or the enzyme activity causes an antimicrobial effect, a whitening effect, a bleaching effect, a decolorizing effect, an anti-odor effect, and/or a bioorganic matter degrading effect; and/or the insoluble enzyme preparation is homogeneously dispersed in the single-use- composition; and/or the allergenic potential of the single-use-composition is reduced compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier; and/or the enzyme caused immunogenicity of the single-use-composition is reduced compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier; and/or

after its intended usage, the single-use-composition either is disposed of together with the insoluble enzyme preparation contained therein, or the insoluble enzyme preparation remains within the single-use-composition; and/or the single-use-composition is selected from the group consisting of oral care compositions, paint compositions and coating compositions; and/or the single-use-composition contains a liquid constituent; and/or the solid carrier has an average diameter of from 0.1 to 500 μηι; and/or at least 50 wt.-% of the solid carrier are not respirable; and/or the enzyme is covalently linked to the solid support; and/or the solid support is based on a metal oxide, preferably silica; and/or the single-use-composition contains a liquid constituent; and/or the enzyme is selected from the group consisting of glycosyl hydrolases, carbohydrases, tannase glucanases, pentosanases, hemicelluloses, transferases, oxidoreductases, lipoxygenases, catalases, laccases, lactoperoxidases, isomerases, protein disulfide isomerases, mutanases, dextranases, amylases, peroxidases, proteases, lipolytic enzymes, phytases, polysaccharide lyases, , hydrolases, transglutaminases and glucoseisomerases.

In a preferred embodiment of the invention, the single-use-composition contains an insoluble enzyme preparation comprising an enzyme which is immobilized on a solid carrier; and/or is provided with enzyme activity by the insoluble enzyme preparation; and/or is provided with oxidative and/or hydrolytic and/or antimicrobial activity by the insoluble enzyme preparation; particularly with antimicrobial, whitening, bleaching, decolorizing, anti-odor, and/or bioorganic matter degrading activity; and/or contains a liquid constituent, preferably water; and/or forms an aerosol in the course of the intended usage; and/or is a toothpaste, a sealant, a paint or a varnish; and/or is disposed of after its intended usage together with the insoluble enzyme preparation contained therein; or remains together with the insoluble enzyme preparation contained therein after its intended usage; and/or displays a reduced allergenic potential compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier; and/or displays a reduced enzyme caused immunogenicity compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier.

In a preferred embodiment of the invention, the single-use-composition is used as a low allergenic composition which has reduced allergenic potential compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier; and/or has reduced enzyme caused immunogenicity compared to a composition containing the same quantity of the same enzyme not immobilized on a solid carrier; and/or is selected from the group consisting of oral care compositions, personal care compositions, food compositions, cosmetic compositions, home care compositions, household compositions, detergent compositions, feed compositions, paint compositions, coating compositions, and agriculture compositions.

The following examples, which are partly prophetic, further illustrate the invention but are not to be construed as limiting its scope.

Example 1 (toothpaste containing glucose oxidase): 10 g fumed silica (Aerosil® 200, Evonik Industries AG) were dried overnight under vacuum at 150°C. The dried material was boiled overnight under reflux in 300 ml_ toluol (dry) and 15 ml_ (3-aminopropyl)-triethoxysilane. The resulting material was washed with toluol and acetone to yield aminopropyl-functionalized silica. After drying, the functionalized silica was resuspended in 1 % aqueous glutaraldehyde solution and stirred at RT for 4h. The aminopropyl-glutaraldehyde-functionalized silica (APG-Si) was washed twice with 200 ml water and dried under vacuum. The amount of 2 g of APG-Si was incubated over night with a 10 ml_ solution of 100 kU glucose-oxidase from Aspergillus niger (GOX) in Na-acetate buffer (50 mM, pH 5.5) at 4°C. The immobilized enzyme (GOX-APG-Si) was washed three times with Na-acetat-buffer and - optionally - freeze-dried. The amount of 1 g of commercial toothpaste (Elmex® homeopathy compatible mint-free toothpaste, GABA GmbH) was mixed with varying amounts (5 to 200 mg) of immobilized GOX-APG-Si (either wet or freeze-dried) to yield an enzyme containing toothpaste.

In order to demonstrate the underlying principle of the invention the presence/absence of soluble vs. insoluble GOX activity contained in enzyme-containing toothpaste according to the invention and commercial enzyme containing toothpaste was analysed. The commercial enzyme-containing toothpastes used were biotene ^® (GlaxoSmithKline, USA) and enzycal ^® (Curapox International AG, CH). 50 mg of each toothpaste was suspended in 200 ml of Na- acetate buffer (50 mM, pH 5.5) and the soluble-fraction was separated from the insoluble- fraction by centrifugation (16,000xg, 5 min). The insoluble-fraction was washed two times using 1 ml Na-acetat buffer (50 mM, pH 5.5). The volume of 100 μΙ of the soluble-fraction and the entire amount of the washed insoluble-fraction were separately mixed with potassium iodide-assay solution (100 mM potassium iodide and 100 mM glucose in 50 mM Na-acetat buffer (pH 5.5)) to yield a final volume of 1 .5 ml each. Enzyme activity is detected by oxidation of glucose to glucono-lactone and the concomitant formation of H ₂ 0 ₂ , which oxidises potassium iodide to brown iodine. As expected the enzyme activity of commercial toothpastes is almost exclusively in the soluble-fraction, while the enzyme activity of the enzyme containing toothpaste according to the invention shows enzyme activity exclusively in the insoluble-fraction (Figure 3).

In order to demonstrate that the enzyme containing toothpaste according to the invention does not form an enzyme-containing aerosol, an ultrasonic fog generator was loaded with a diluted sample of each toothpaste. Samples were prepared by suspending 1 g of each toothpaste in 30 ml_ buffer (50 mM acetate-buffer, pH 5.5). The aerosol generated by the ultrasonic device was collected on test patches (paper) in ultimate proximity to the device and in a distance of 30 cm. Test patches were soaked in an assay-solution of 0.2 mg/ml o- dianisidine-HCI, 100 mM glucose and 80 U/ml of horse radish peroxidise (HRP). If GOX- activity is present, H ₂ 0 ₂ is produced from glucose oxidation and used by the HRP to oxidize o-dianisidine to a red-coloured state. The relative amount of the red-coloured state was analysed by using a standard image evaluation software. The results are summarized in Figure 4.

Example 2 (ointment containing protease):

10 g of the hydrophilic fumed silica Aerosil® 200 (Evonik Industries AG) functionalized with oxirane groups are suspended in 50 ml_ phosphate buffer (0.1 M, pH 6.0), contacted with an aqueous phosphate buffer solution containing the solubilized protease from Bacillus licheniformis with 1 .5 kU/mL and incubated under shaking at room temperature for 20 h. The enzyme loaded silica is separated from the mixture by centrifugation and washed with the phosphate buffer to remove excess amounts of the free enzyme. Activity of the enzyme silica preparation is measured using azocasein as substrate in colorimetric assay. The enzyme silica preparation is suspended at a concentration of 10% (w/w) in an ointment base composed of Vaseline. In parallel the free enzyme is added to the same ointment base to compare performance of both an ointment containing the enzyme silica preparation according to the invention and a ointment containing the free enzyme. In the course of performance testing, a sample of 100 mg of the ointment is emulsified in 1 .5 ml_ in order to separate the highly viscous hydrocarbon base and the solid enzyme silica preparation from each other and to use the remaining aqueous phase to generate an aerosol by using a nebulizer. The aerosol is collected for analysis of activity. Moreover, the content of solubilized enzyme in the aqueous phase of the said emulsion is analyzed.

Example 3 (flour containing alpha-amylase):

10 g of the hydrophilic precipitated silica Sipernat® 50S (Evonik Industries AG) is suspended in 50 ml_ phosphate buffer (0.1 M, pH 7.0) and contacted with the solubilized alpha-amylase from Bacillus licheniformis at a final concentration of 50 kU/mL and incubated under shaking at room temperature for 15 h. The enzyme loaded silica is separated from the mixture by centrifugation and washed with the phosphate buffer to remove excess amounts of the free enzyme and finally lyophilized. Activity of the enzyme silica preparation is measured using an amylase activity assay kit (Sigma). The dry enzyme silica preparation is admixed to flour at a concentration of 2.5% (w/w) and used in baking experiments to appraise the enzyme performance in comparison to a flour composition containing the free enzyme. Moreover, dusty aerosols are prepared from the formulated flour containing either the enzyme silica preparation or the free enzyme and analyzed regarding the particle size distribution and enzyme content. Moreover, 100 mg of the ointment is suspended in 1 .5 ml_ and the content of solubilized enzyme in the resulting aqueous phase is analyzed.

Example 4 (household cleaner containing lipase):

10 g of the hydrophilic fumed silica Aerosil® 90 (Evonik Industries AG) functionalized with NH ₂ groups are suspended in 50 ml_ phosphate buffer (0.1 M, pH 6.0) containing 12.5% glutardialdehyde and incubated under shaking at room temperature for 5 h. The activated silica is separated from the mixture by centrifugation and washed with the phosphate buffer to remove excess amounts of glutardialdehyde. The silica is re-suspended in an aqueous phosphate buffer solution containing the solubilized lipase from Candida rugosa with 2.5 kU/mL and incubated under shaking at room temperature for 20 h. The enzyme loaded silica is separated from the mixture by centrifugation and washed with the phosphate buffer to remove excess amounts of the free enzyme. Activity of the enzyme silica preparation is measured using tributyrin as substrate in a titrimetric assay. The enzyme silica preparation is suspended at a concentration of 10% (w/w) in a commercial household cleaner. In parallel the free enzyme is added to the same household cleaner to compare performance of both a household cleaner containing the enzyme silica preparation according to the invention and a household cleaner containing the free enzyme. In the course of performance testing, a sample of 100 mg of the household cleaner is diluted in 1 .5 ml_ in order to generate an aerosol by using a nebulizer and to collect the aerosol for analysis of activity. Moreover, the content of solubilized enzyme in the aqueous phase of the said dilution is analyzed.

Example 5 (silicone sealant containing Ivsinq enzymes):

10 g of the hydrophilic precipitated silica Sipernat® 50S (Evonik Industries AG) is suspended in 50 ml_ phosphate buffer (0.1 M, pH 7.0) and contacted with the solubilized mixture of lysing enzymes from Trichoderma harzianum at a final protein concentration of 100 mg/mL and incubated under shaking at room temperature for 15 h. The enzyme loaded silica is separated from the mixture by centrifugation and washed with the phosphate buffer to remove excess amounts of the free enzyme and finally lyophilized. Activity of the enzyme silica preparation is measured by detection of glucose released from a beta-glucan. The dry enzyme silica preparation is suspended to an off-the-shelf silicone sealant at a concentration of 10% (w/w) and applied as sealant. After hardening the sealant is cut into slices and enzyme distribution is appraised with a fluorescent microscope in comparison to a silicone preparation containing the free enzyme. Moreover, possibly present free enzyme is extracted from the sealant slices and quantified.

Example 6 (wall paint containing Ivsinq enzymes):

10 g of the hydrophilic precipitated silica Sipernat® 50S (Evonik Industries AG) is suspended in 50 ml_ phosphate buffer (0.1 M, pH 7.0) and contacted with the solubilized mixture of lysing enzymes from Trichoderma harzianum at a final protein concentration of 100 mg/mL and incubated under shaking at room temperature for 15 h. The enzyme loaded silica is separated from the mixture by centrifugation and washed with the phosphate buffer to remove excess amounts of the free enzyme and finally lyophilized. Activity of the enzyme silica preparation is measured by detection of glucose released from a beta-glucan. The dry enzyme silica preparation is suspended to an off-the-shelf wall-paint at a concentration of 15% (w/w) and painted onto a test-surface. After curing the enzyme distribution is appraised with a fluorescent microscope in comparison to a wall-paint containing the free enzyme. Moreover, dusty aerosols are prepared from the cured wall-paint by grinding with abrasive paper. The resulting particles are analyzed regarding the particle size distribution and enzyme content. Moreover, 100 mg of the ointment is suspended in 1 .5 ml_ and the content of solubilized enzyme in the resulting aqueous phase is analyzed.