Low-aggregation enzyme-containing particles

Abstract

Compositions and methods are described for low-density enzyme-containing particles for inclusion in cleaning and other low-moisture compositions. The particles remain in suspension without settling and release active enzyme upon dilution of the low-moisture composition with water. [Selection diagram] None

Description

[Technical field]

[0001] The present compositions and methods relate to enzyme-containing particles that exhibit reduced clumping for inclusion in cleaning and other low moisture compositions. The particles exhibit reduced clumping during storage and release active enzyme upon dilution of the low moisture composition with water. [Background technology]

[0002] Enzymes are supplied in both liquid and solid forms for incorporation into products used in a variety of consumer and industrial applications, including laundry and dishwashing, personal care, textile treatment, pulp and paper making, leather manufacturing, food and beverage processing, starch processing, pollution removal, oil and gas drilling, biofuel production, and the production (or modification) of biopolymers and other chemicals.

[0003] There is a widespread need to compartmentalize enzymes or other actives in liquid formulations containing such incompatible ingredients so that they are stable during storage, but are rapidly released upon dilution during application. Many other effective enzymes are unavailable due to their instability in liquid formulations such as detergents.

[0004] Aside from stability challenges, enzymes are immunogenic molecules and can cause problems related to exposure and sensitization. In some cases, the maximum amount of enzyme that can be added to a liquid cleaning formulation is dictated by exposure risk as opposed to performance or economics.

[0005] Although enzymes can be provided in granular form in liquid detergents, the granules always settle in liquid formulations such as detergents, resulting in uneven distribution of the enzyme as well as the unsightly appearance of settled granules.Therefore, there is a need for improved methods to compartmentalize enzymes in liquid formulations so that they remain stable, retain their catalytic potential, and remain uniformly suspended in the liquid for extended periods of time without agglomeration until use in an application where enzyme activity is desired. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides low density particles for isolating and stabilizing enzymes in aqueous compositions, and methods of use thereof. Aspects and embodiments of the invention are described in the following numbered paragraphs.

[0007] 1. In one aspect, there is provided a particle capable of isolating and stabilizing an enzyme in an aqueous composition without agglomeration during manufacture and / or storage, comprising: (a) a core comprising an active ingredient, and / or a core having a first coating layer comprising an active ingredient immediately deposited on the core; and (b) an outermost coating layer comprising a hydrophobic, water-insoluble moisture-disintegrating material having a solubility in water in an amount of less than about 1 mg / mL in water at 25° C., wherein the coating layer of (b) completely disintegrates within about 5 minutes upon 1:1 dilution of the liquid composition with water at 25° C., allowing for dissolution of the enzyme and / or active ingredient in the diluted liquid composition, and wherein the particle comprises a third coating layer comprising a water-soluble polymer having a solubility of more than about 1 mg / mL in water at 25° C., and exhibits reduced aggregation in the liquid composition compared to otherwise identical particles.

[0008] 2. In some embodiments, the particle according to paragraph 1 further comprises at least one additional layer between (a) and (b) comprising a water-soluble polymer and an active ingredient.

[0009] 3. In some embodiments, the particle according to paragraph 1 further comprises at least one additional layer between (a) and (b) comprising a water-soluble polymer that is devoid of an active ingredient.

[0010] 4. In some embodiments of the particle according to paragraph 1 or 2, the core is devoid of an active ingredient.

[0011] 5. In some embodiments of the particles described in paragraphs 1 or 3, the core comprises an active ingredient.

[0012] 6. In some embodiments of the particles described in any one of paragraphs 1-5 above, after a liquid composition containing the particles is contacted with at least one additional volume of water at 25° C., the outermost coating disintegrates within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 1 minute, within 30 seconds, or even within 15 seconds.

[0013] 7. In some embodiments of the particle described in any one of paragraphs 1-6 above, the outermost coating represents less than 8%, less than 7%, less than 6%, or even less than 5% of the total weight of the particle.

[0014] 8. In some embodiments of the particles described in any one of paragraphs 1-7 above, the outermost layer consists essentially of or consists of a hydrophobic, water-insoluble, moisture-disintegrating material having an aqueous solubility of less than about 1 mg / mL in water at 25° C.

[0015] 9. In some embodiments of the particle described in any one of paragraphs 1-8 above, the core has the formula: ρ c ≦(ρ f +31250 / D p 2 ) * x c / (D c / D p ) (1 / 3) and ρ c ≧(ρ f -31250 / D p 2 ) * x c / (D c / D p ) (1 / 3) [In the formula, ρ c is g / cm 3 is the density of the core at f is g / cm 3 is the mass density of the liquid composition at c is the mass fraction of the core in the particle, and D c is the diameter of the core in µm, D pwhere is the diameter of the particle in μM].

[0016] 10. In some embodiments, the particles of any one of paragraphs 1-9 above have an overall true density of less than 1.6 mg / mL, less than 1.4 mg / mL, or even less than 1.2 mg / mL.

[0017] 11. In another aspect, a method is provided for reducing agglomeration of particles during manufacture and / or storage, comprising coating the particles with an outermost layer comprising a hydrophobic, water-insoluble moisture-disintegrating material having an aqueous solubility of less than about 1 mg / mL in water at 25° C.

[0018] 12. In some embodiments of the method of paragraph 11, the outermost layer consists essentially of or consists of a hydrophobic, water-insoluble, moisture-disintegrating material having an aqueous solubility of less than about 1 mg / mL in water at 25° C.

[0019] 13. In some embodiments of the method according to paragraphs 11 or 12, after contacting the liquid composition containing the particles with at least one additional volume of water at 25° C., the outermost coating disintegrates within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 1 minute, within 30 seconds, or even within 15 seconds.

[0020] 14. In some embodiments of the method of any one of paragraphs 11-13, the outermost coating represents less than 8%, less than 7%, less than 6%, or even less than 5% of the total weight of the particle.

[0021] These and other aspects and embodiments of the present compositions and methods are described below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] I. Definitions and Abbreviations Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. In the practice of the present invention, methods and materials similar or equivalent to those described herein are utilized, but preferred methods and materials are described herein. Thus, the terms defined immediately below are more fully explained by reference to the entire specification. Also, as used herein, the singular forms "a", "an" and "the" include the plural forms unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively. It should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary depending on the context used by those skilled in the art.

[0023] It is intended that every numerical upper limit given throughout this specification include every lower numerical limit, as if such lower numerical limit were expressly written herein. Every numerical lower limit given throughout this specification will include every higher numerical upper limit, as if such higher numerical limit were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0024] As used herein, the term "water-soluble polymer" refers to a polymer that dissolves in water in an amount of at least 1 mg / mL. As used herein, an "aqueous medium" or "aqueous solution" is a solution and / or suspension in which the solvent is predominantly water (i.e., the solvent is at least 50% water, at least 60% water, at least 70% water, at least 80% water, or even at least 90% water). Aqueous media may contain any number of dissolved or suspended components, including, but not limited to, surfactants, salts, buffers, stabilizers, complexing agents, chelating agents, builders, metal ions, additional enzymes, and substrates. Exemplary aqueous media are laundry and dishwashing cleaning solutions. Materials such as textiles, fabrics, dishes, kitchen utensils, and other materials may also be present in or in contact with the aqueous medium.

[0025] As used herein, the term "water-insoluble substance" refers to a substance that does not dissolve in water upon mixing, such as a substance that has a solubility of less than about 1 mg / mL in water at 25°C.

[0026] As used herein, the term "hydrophobic" refers to a substance that is repelled by (or that repels) water, i.e., there is no attraction between the substance and water.

[0027] As used herein, the term hydrophilic lipophilic balance (HLB) refers to an empirical expression of the relationship of the hydrophilic and hydrophobic groups of a surfactant.

[0028] As used herein, the term "disintegrant" refers to a material that does not dissolve in water but has the ability to break down from larger particles into smaller particles and can be suspended in water when mixed.

[0029] As used herein, the term "agglomeration" refers to the phenomenon in which individual particles come together to form a mass or cluster of multiple particles. The association between the particles can be either a loose association or a tight association, including through covalent bonds formed between the particles.

[0030] As used herein, the term "low moisture" in reference to liquid laundry detergent compositions indicates that the detergent composition contains about 5-20% (w / w) water.

[0031] As used herein, the term "substantially non-aqueous" in relation to liquid laundry detergent compositions indicates that the detergent composition contains about 2-5% (w / w) water.

[0032] As used herein, a "non-aqueous" solution contains less than about 2% (w / w) water.

[0033] As used herein, when an ingredient is "provided in" a particular form (e.g., non-aqueous, very low moisture, solid, etc.), this form refers to the final form in which the ingredient is present in the unit dosage package, not the form in which it is added to another ingredient that is then added to the unit dosage package.

[0034] As used herein, the phrase "insufficient to substantially dissolve the water-soluble packaging" means that the liquid in question does not dissolve more than 5% of the water-soluble material over a period of six months at room temperature (i.e., 25°C).

[0035] As used herein, the term "compartmentalized" in reference to the contents of water-soluble packaging means that the particular contents, whether liquid, solid, or a combination thereof, are physically contained in a compartment, at least a portion of which is defined by a water-soluble material. In some cases, the contents are completely bounded by the water-soluble material, i.e., the entire compartment is defined by the water-soluble material, as in the case of a pouch made from a water-soluble material. In some cases, the contents are only partially bounded by the water-soluble material, i.e., only a portion of the compartment is defined by the water-soluble material, and the remainder is defined by a water-insoluble material, as in the case of a cup or dish covered by a lid made from a water-soluble material.

[0036] As used herein, the terms "suspended" and "dispersed" refer to the distribution of one component within another, for example, the distribution of a solid form of an acyl substrate within a water-soluble substance.

[0037] As used herein, "cold" water is water having a temperature between the freezing point and about 25°C.

[0038] As used herein, "room temperature" is 25°C.

[0039] As used herein, "warm" water is water having a temperature between about 26°C and about 37°C.

[0040] As used herein, "hot" water is water having a temperature between about 37° C. and the boiling point.

[0041] As used herein, a "low" pH is a pH below about 7.

[0042] As used herein, a "high" pH is a pH above about 7.

[0043] As used herein, the term "contacting" means physically contacting, such as by placing a unit dose package in an aqueous solution.

[0044] As used herein, a "solid" form of a chemical component refers to a powder, crystal, granule, aggregate, paste, or wax thereof.

[0045] As used herein, a "liquid" form of a chemical component refers to a liquid, gel, or slurry.

[0046] As used herein, "true density" refers to the mass of a particle divided by its volume (excluding open and closed pores).

[0047] As used herein, the term "spray drying" refers to a process known in the art and discussed, for example, in U.S. Pat. No. 5,423,997 and WO 2008 / 088751 A2, of producing a dry powder from a liquid or slurry by rapid drying with hot gases.

[0048] As used herein, "d50" refers to the measured particle size where 50% are above or below the midpoint within the measured population.

[0049] As used herein, the term "UFC solids" refers to the ultrafiltration concentrate from the fermentor / bioreactor and is synonymous with enzyme concentrated solids.

[0050] As used herein, "cleaning composition" and "cleaning formulation" refer to compositions that can be used to remove unwanted compounds from items to be cleaned, such as fabrics, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwash, toothpaste), etc. The terms encompass any material / compound selected for the particular type of cleaning composition desired. The specific selection of materials for the cleaning composition is readily made by considering the surface, item, or fabric to be cleaned, and the form of the composition desired for the cleaning conditions during use.

[0051] The term further refers to any composition suitable for cleaning, bleaching, disinfecting, and / or stabilizing any object and / or surface. The term is intended to include, but is not limited to, detergent compositions (e.g., laundry detergents and detergents for fine fabrics; hard surface cleaning formulations such as for glass, wood, ceramic and metal countertops, and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and pre-soil spotters for fabrics and laundry, as well as dish detergents).

[0052] As used herein, the terms "detergent composition" and "detergent formulation" are used in the context of mixtures intended for use in a laundry medium for cleaning solid objects. In some preferred embodiments, the terms are used in reference to laundering fabrics and / or clothing (e.g., "laundry detergent"). In alternative embodiments, the terms refer to other detergents, such as those used to clean dishes, cutlery, and the like (e.g., dishwashing detergent).

[0053] As used herein, the term "nonionic surfactant" refers to a surfactant molecule that has a non-charged polar group.

[0054] As used herein, the term "anionic surfactant" refers to a surfactant molecule that has a negatively charged polar group at the pH of the composition or intended use. It includes salts that form salts used to complex or neutralize surfactants, such as monoethanolamine (MEA) salts of linear alkylbenzene sulfonates (LAS), and is used herein to describe the mass or concentration of anionic surfactants.

[0055] As used herein, the phrase "detergent stability" refers to the stability of a detergent composition. In some embodiments, the stability is evaluated during detergent use, while in other embodiments, the term refers to the stability of a detergent composition during storage.

[0056] As used herein, the term "hard surface cleaning composition" refers to a detergent composition for cleaning hard surfaces such as floors, walls, tiles, bathtubs and kitchen fixtures.

[0057] As used herein, "non-fabric cleaning compositions" includes hard surface cleaning compositions, dishwashing compositions, personal care cleaning compositions (e.g., oral cleaning compositions, denture cleaning compositions, personal cleaning compositions, etc.), and compositions suitable for use in the pulp and paper industry.

[0058] As used herein, "personal care product" refers to products used to clean, whiten, and / or disinfect hair, skin, scalp, and teeth, including, but not limited to, shampoos, body lotions, shower gels, topical moisturizers, toothpastes, and / or other topical cleansers. In some particularly preferred embodiments, these products are utilized by humans, while in other embodiments, these products are also utilized by non-human animals (e.g., veterinary uses).

[0059] As used herein, "water-miscible" refers to a liquid that forms a single thermodynamic liquid phase, or isotropic phase, when mixed with water, given the ratio of water to the liquid.

[0060] As used herein, a "suspension" or "dispersion" refers to a two-phase system in which a discontinuous solid phase is dispersed within a continuous liquid phase. The solid phase can consist of very fine particles or larger granules, and the particles or granules can have a wide variety of shapes, morphologies, and structures. For example, the solids can be small spray-dried particles 1 micron in diameter, or larger core-shell granules 100-1,000 microns in diameter.

[0061] As used herein, "suspending aid" refers to a substance added to a liquid composition to prevent or reduce settling or floating of suspended particles. Suspending aids typically act by increasing either the viscosity or the yield stress of the carrier liquid. Fluids with significant yield stress will only flow when a stress higher than the yield stress is applied, and therefore exhibit shear thinning or thixotropic behavior. Effective suspending agents typically act by forming a reversible network of particles or fibers suspended by weak forces. Examples of suspending agents include, but are not limited to, xanthan gum and microfibrous cellulose, such as CELLULON (CP Kelco, San Diego, CA).

[0062] The following abbreviations may be used herein: Definitions are also provided where necessary for explanation. ℃ Celsius AU Activity Unit CaCl 2 Calcium chloride Cm Centimeters cm 3 Cubic centimeters D(0.5) The median diameter at which 50% of the particles are less than or equal to the specified diameter D(0.9) The median diameter at which 90% of the particles are less than or equal to the specified diameter dH 2 O or DI deionized water eq. equivalent amount ETOH Ethanol g or gm grams (note, below) H 2 O water hr time M Molar concentration Melting temperature Melting temperature mg milligram min mL and ml milliliters mm millimeters mM millimolar concentration MW molecular weight N regulations Na 2 SO 4 Sodium sulfate NaOH Sodium hydroxide nm nanometer PE Polyethylene PEG Polyethylene glycol ppm parts per million PVA Poly(vinyl alcohol) PVP Poly(vinylpyrrolidone) sec seconds TiO 2 Titanium dioxide U Units v / v volume / volume w / v weight / volume w / w weight / weight (wt%) Weight percent μg microgram μL and μl microliter μm micrometer μM micromolar

[0063] II. Particles with a hydrophobic, water-insoluble, moisture-disintegrating coating It is often desirable to incorporate particles with active agents in low moisture liquid detergents to provide cleaning or other benefits. Unfortunately, conventional particles having an outer surface made from a material with water solubility or hydrophilic properties have been shown to agglomerate during and / or after incorporation into the low moisture detergent during manufacturing, mixing, handling, shipping and / or storage.

[0064] The present materials and methods overcome this undesirable phenomenon of clumping by using hydrophobic and / or water insoluble materials on the outer surface of the particles, preventing clumping from occurring and allowing these materials to easily break down when the low moisture detergent is diluted in the wash liquor.

[0065] Generally, the particles comprise (i) a core, (ii) at least one enzyme and / or other active ingredient-containing layer, (iii) one or more additional layers, and (iv) an outermost coating having hydrophobic and / or water-insoluble properties that rapidly disintegrates upon 1:1 dilution with water. These components are described in more detail below.

[0066] A. Hydrophobic or Water Insoluble and Moisture Disintegrating Surface Coatings An important feature of the present compositions and methods are particles that have an outermost coating with hydrophobic and / or water-insoluble properties that prevent the particles from agglomerating during manufacture, handling, transportation and / or storage, yet readily disintegrate when diluted in the cleaning liquid.

[0067] Exemplary materials that have the required hydrophobic and / or water-insoluble properties, but that disintegrate easily upon dilution, include, but are not limited to, natural waxes such as carnauba, beeswax, palmitic acid wax, candelilla wax, synthetic waxes such as paraffin wax and microcrystalline wax, low HLB surfactants having values ​​less than HLB=6, hydrophobically modified polyvinyl alcohols, hydrophobically modified starches such as those modified with fatty acid side chains, hydrophobically modified cellulosic polymers.

[0068] Ideally, the melting point of the outer coating material should be high enough to remain solid during processing and storage, and therefore the melting temperature should be greater than 40°C, greater than 45°C, greater than 50°C, greater than 55°C, or even greater than 60°C, depending on processing conditions and application.

[0069] After contacting the low water liquid composition containing the particles with at least one additional volume of water at 25°C, the coating should disintegrate within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 1 minute, within 30 seconds, or even within 15 seconds.

[0070] The outer coating composition only needs to be incorporated at a level sufficient to provide the desired surface properties to avoid agglomeration and allow rapid disintegration when diluted in the washing liquid. Therefore, the outer coating should be as thin as possible. In some embodiments, the weight percent (wt / wt%) of the outermost coating relative to the whole particle should be less than 8%, less than 7%, less than 6%, or even less than 5%.

[0071] B. Coatings containing enzymes and other active ingredients The core (described below) may be coated with and / or optionally contain one or more of a wide variety of enzymes or other actives. Although this description focuses on enzymes, it will be apparent that similar particles can be used to provide a myriad of other active ingredients in a low moisture composition.

[0072] Exemplary enzymes include acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-β-1,4-glucanases, endo-β-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, and oxidases. , oxidoreductase, pectate lyase, pectin acetyl esterase, pectinase, pentosanase, perhydrolase, peroxidase, peroxygenase, phenol oxidase, phosphatase, phospholipase, phytase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, metalloproteases, additional serine proteases, and combinations thereof.

[0073] Examples of suitable proteases include, but are not limited to, subtilisins from the genus Bacillus (e.g., subtilisin, lentus, amyloliquefaciens, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168), including variants such as those described in, for example, U.S. Reissue Patent No. 34,606, U.S. Patent Nos. 5,955,340, 5,700,676, 6,312,936, and 6,482,628, all of which are incorporated herein by reference. Additional proteases include trypsin (eg, of porcine or bovine origin) and the Fusarium proteases described in WO 89 / 06270. In some embodiments, the protease is MAXATASE®, MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX™, EXCELLASE™, and PURAFAST™ (Genencor); ALCALASE®, SAVINASE®, PRIMASE®, DURAZYM™, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, NEUTRASE®, RELASE®, and ESPERASE® (Novozymes); BLAP™ and BLAP™ variants (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf, Germany), and KAP (B. alkalophilus subtilisin; Kao Corporation, Tokyo, Japan).Additional proteases are disclosed in WO 95 / 23221, WO 92 / 21760, WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO 11 / 072099, WO 10 / 056640, WO 10 / 056653, WO 11 / 140364, WO 12 / 151534, and the like. Frets are described in U.S. Patent Application Publication No. 2008 / 0090747, as well as U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, Reissue U.S. Pat. No. 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628.

[0074] Suitable proteases include neutral metalloproteases, including those described in WO 07 / 044993 and WO 09 / 058661. Other exemplary metalloproteases include nprE, a recombinant form of the neutral metalloprotease expressed in Bacillus subtilis (see, e.g., WO 07 / 044993), and PMN, a purified neutral metalloprotease from Bacillus amyloliquefaciens.

[0075] Suitable lipases include, but are not limited to, Humicola lanuginosa lipase (see, e.g., EP 258068 and EP 305216), Rhizomucor miehei lipase (see, e.g., EP 238023), Candida lipases, such as C. antarctica lipase (e.g., C. antarctica lipase A or B; see, e.g., EP 214761), Pseudomonas lipases, such as P. alcaligenes lipase and P. pseudoalcaligenes lipase. doalcaligenes lipase (see, for example, EP 218272), P. cepacia lipase (see, for example, EP 331376), P. stutzeri lipase (see, for example, GB 1,372,034), P. fluorescens lipase, Bacillus lipase (see, for example, B. subtilis lipase (Dartois et al., (1993) Biochem. Biophys. Acta 1131:253-260), B. stearothermophilus lipase (see, for example, JP-A-64-744992), and B. pumilus lipase (see, for example, WO 91 / 16422).

[0076] Additional suitable lipases include Penicillium camembertii lipase (Yamaguchi et al. (1991) Gene 103:61-67), Geotricum candidum lipase (see Schimada et al. (1989) J. Biochem. 106:383-388), and various Rhizopus lipases, such as R. delemar lipase (Hass et al. (1991) Gene 109:117-113), R. niveus lipase (Kugimiya et al. (1992) Biosci. Biotech. Biochem. 56:716-719), and R. oryzae lipase. Additional lipases include cutinases from Pseudomonas mendocina (see WO 88 / 09367) and Fusarium solanipisi (see WO 90 / 09446). Various lipases are described in WO 11 / 111143, WO 10 / 065455, WO 11 / 084412, WO 10 / 107560, WO 11 / 084417, WO 11 / 084599, WO 11 / 150157, and WO 13 / 033318. In some embodiments, the protease is one or more of M1 LIPASE™, LUMA FAST™, and LIPOMAX™ (Genencor); LIPEX®, LIPOLASE®, and LIPOLASE® ULTRA (Novozymes); and LIPASE P™ "Amano" (Amano Pharmaceutical Co., Ltd., Japan).

[0077] Suitable amylases include, but are not limited to, amylases of bacterial or fungal origin, or even mammalian origin. Many suitable ones are described in WO 9510603, WO 9526397, WO 9623874, WO 9623873, WO 9741213, WO 9919467, WO 0060060, WO 0029560, WO 9923211, WO 9946399, WO 0060058, WO 0060059, WO 9942567 ... Brochure No. 0114532, Brochure No. 02092797, Brochure No. 0166712, Brochure No. 0188107, Brochure No. 0196537, Brochure No. 0210355, Brochure No. 9402597, Brochure No. 0231124, Brochure No. 9943793, Brochure No. 9943794, Brochure No. 2004113551, Brochure No. 2005001064, Brochure No. 2005003311, Brochure No. 01 Brochure No. 64852, Brochure No. 2006063594, Brochure No. 2006066594, Brochure No. 2006066596, Brochure No. 2006012899, Brochure No. 2008092919, Brochure No. 2008000825, Brochure No. 2005018336, Brochure No. 2005066338, Brochure No. 2009140504, Brochure No. 2005019443, Brochure No. 2010091221, Brochure No. 2010088447, Brochure No. 0134784, Brochure No. 2006012902, Brochure No. 2006031554, Brochure No. 2006136161, Brochure No. 2008101894, Brochure No. 2010059413, Brochure No. 2011098531, Brochure No. 2011080352, Brochure No. 2011080353, Brochure No. 2011080354, Brochure No. 2011082425,Brochure No. 2011082429, Brochure No. 2011076123, Brochure No. 2011087836, Brochure No. 2011076897, Brochure No. 94183314, Brochure No. 9535382, Brochure No. 9909183, Brochure No. 9826078, Brochure No. 9902702, Brochure No. 9743424, Brochure No. 9929876, Brochure No. 9100353, Brochure No. 960 Brochure No. 5295, Brochure No. 9630481, Brochure No. 9710342, Brochure No. 2008088493, Brochure No. 2009149419, Brochure No. 2009061381, Brochure No. 2009100102, Brochure No. 2010104675, Brochure No. 2010117511, Brochure No. 2010115021, Brochure No. 2013184577, Brochure No. 9418314, International Application Nos. PCT / US12 / 70334 and PCT / US13 / 74282, PCT / US13 / 74282, PCT / US13 / 74282, PCT / US12 / 70334, ... Amylases are described in the following specifications: PCT / CN2013 / 077294, PCT / CN2013 / 077134, PCT / CN2013 / 077137, PCT / CN2013 / 077142, PCT / CN2012 / 087135, PCT / US12 / 62209, PCT / CN2013 / 084808, PCT / CN2013 / 084809, and PCT / US14 / 23458. Commercially available amylases include DURAMYL (registered trademark), TERMAMYL (registered trademark), FUNGAMYL (registered trademark), STAINZYME (registered trademark), STAINZYME PLUS (registered trademark),These include, but are not limited to, STAINZYME ULTRA®, and BAN™ (Novozymes), as well as one or more of POWERASE™, RAPIDASE®, and MAXAMYL® P, PREFERENZ® S100, PREFERENZ® S110, and PREFERENZ® S1000 (Genencor).

[0078] Suitable cellulases include, but are not limited to, those with color treatment benefits (see, e.g., EP 0 495 257). Examples include Humicola insolens cellulase (see, U.S. Pat. No. 4,435,307), as well as commercially available cellulases such as CELLUZYME®, CAREZYME® (Novozymes), and KAC-500(B)™ (Kao Corporation), and Primafast® GOLD (DuPont). In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or mutant cellulase, with a portion of the N-terminus deleted (see, e.g., U.S. Pat. No. 5,874,276). Additional suitable cellulases include those described in WO2005054475, WO2005056787, U.S. Patent Nos. 7,449,318, and 7,833,773.

[0079] Suitable mannanases are described in U.S. Patent Nos. 6,566,114, 6,602,842, 5,476,775, 6,440,991, and U.S. Provisional Patent Application No. 61 / 739267, all of which are incorporated herein by reference. Commercially available ones include, but are not limited to, MANNASTAR®, PURABRITE™, and MANNAWAY®.

[0080] In some embodiments, peroxidases are used in combination with hydrogen peroxide or sources thereof (e.g., percarbonate, perborate, or persulfate) in the compositions of the present teachings. In some alternative embodiments, oxidases are used in combination with oxygen. Both types of enzymes are used for "solution bleaching" (i.e., preventing the transfer of textile dyes from dyed fabrics to other fabrics when the fabrics are washed together in a wash liquor), preferably with an enhancer (see, for example, WO 94 / 12621 and WO 95 / 01426). Suitable peroxidases / oxidases include, but are not limited to, peroxidases / oxidases of plant, bacterial, or fungal origin. Some embodiments include chemically modified or genetically engineered mutants.

[0081] Suitable perhydrolases include those from Mycobacterium smegmatis, the enzyme, its properties, its structure, and numerous variants and homologs thereof, are described in detail in International Publication Nos. WO 05 / 056782A and WO 08 / 063400A, and U.S. Patent Publication Nos. 2008145353 and 2007167344, which are incorporated herein by reference. In some embodiments, the Mycobacterium smegmatis perhydrolase or homolog comprises an S54V substitution.

[0082] Other suitable perhydrolases include members of the carbohydrate esterase family 7 (CE-7 family), described, for example, in WO 2007 / 070609 and U.S. Patent Application Publication Nos. 2008 / 0176299, 2008 / 176783, and 2009 / 0005590. Members of the CE-7 family include cephalosporin C deacetylases (CAHs; EC 3.1.1.41) and acetyl xylan esterases (AXEs; EC 3.11.72). Members of the CE-7 esterase family share a common characteristic conserved motif (Vincent et al., J. Mol. Biol., 330:593-606 (2003)).

[0083] Other suitable perhydrolase enzymes include those derived from Sinorhizobium meliloti, Mesorhizobium loti, Moraxella bovis, Agrobacterium tumefaciens, or Prosthecobacter dejongeii (WO2005056782), Pseudomonas mendocina (U.S. Pat. No. 5,389,536), or Pseudomonas putida (U.S. Pat. Nos. 5,030,240 and 5,108,457).

[0084] The enzymes may be crystallized, precipitated, spray dried, freeze dried, and / or compressed and may be provided in their dry form or in a resuspended liquid form. The enzymes may be provided as ultrafiltration concentrates. They may be purified to a preselected level.

[0085] The core may be further coated with and / or contain one or more additional ingredients such as bleach catalysts, stabilizing systems, chelating agents, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, fragrances, colorants, filler salts, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, shrinkage inhibitors, anti-wrinkle agents, bactericides, fungicides, color speckle, silvercare, rust and / or corrosion inhibitors, alkalinity sources, dissolution agents, carriers, processing aids, pigments, pH adjusters, surfactants, builders, dye transfer inhibitors, precipitation aids, catalytic materials, bleach activators, bleach accelerators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay soil removal / anti-redeposition agents, whitening agents, structure elasticizers, fabric softeners, hydrotropes, processing aids, and / or pigments. Suitable examples of such additives and use concentrations are found in U.S. Pat. Nos. 5,576,282, 6,306,812, 6,326,348, 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014, and 5,646,101, all of which are incorporated herein by reference. Representative detergent formulations useful in the present invention include those found in WO 2013063460, WO 2003010266, WO 2006002755, WO 2006088535, and US 20110263475, all of which are incorporated herein by reference. Such auxiliary materials can be included in the core, enzyme layer, or polymer coating, so long as they do not adversely affect the described desired properties of the particle.

[0086] C. Additional Coatings Depending on the particular embodiment of the particles and methods, at least one non-aqueous, water-soluble coating may be included that is applied to the core or coated core to protect the enzyme and / or other active ingredient layer from the water present in the low-water liquid composition in which the particles are intended to be suspended. The coating must be non-toxic and biodegradable. The solubility of the coating in water must be greater than 1 mg / mL, greater than 2 mg / mL, greater than 3 mg / mL, greater than 4 mg / mL, greater than 5 mg / mL, greater than 6 mg / mL, greater than 7 mg / mL, greater than 8 mg / mL, greater than 9 mg / mL, or even greater than 10 mg / mL at 25° C. The coating must dissolve within 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, or even 15 seconds when the low-water liquid composition in which it is suspended is diluted with at least one volume of water.

[0087] Exemplary materials are linear or branched polymers with a molecular weight such that the polymer (or a mixture of different polymers) is solid at room temperature. Specific exemplary materials include, but are not limited to, synthetic polymers such as polyvinyl alcohol (PVA), polyvinyl acetate, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyethylene oxide (PEO), polyacrylic acid, polymethacrylic acid, pyrrolidone carboxylic acid, polystyrene sulfonate, and polyelectrolytes; fatty acids such as stearic acid, oleic acid, myristic acid, and palmitic acid; gums such as acacia, guar, xanthan, agarose, karaya, tragacanth, and locust bean; cellulosics. and mixtures thereof.

[0088] D. Core In some embodiments, the core of the particles, characterized by an outer, hydrophobic or water-insoluble, water-disintegrating, outer coating, is not critical to the compositions and methods and may be of a conventional nature. Commonly used materials are salts and sugars and other relatively inexpensive water-soluble materials. The core may be inert or may feature an active ingredient. In other embodiments, the core may include some or even all of the active agents, such as the enzymes listed above.

[0089] In certain embodiments, the core is selected so that the particles have an overall particle density that approaches the density of the low water liquid composition in which they are suspended or intended to be suspended, further differentiating the present particles from prior art particles which are typically denser and tend to settle out of suspension.

[0090] The low density of the particles can be achieved by one of two approaches, or a combination of both. The first approach is to use a low density core. Various materials for making the low density core are described below, and some are exemplified herein. The second approach is to use a more conventional medium to high density core in combination with a density modifier to reduce the overall density of the particle. These approaches can be easily combined, such that both the selection of the core material and the use of the density modifier contribute to the overall low density of the particle. Alternatively, the density modifier can be used to fine-tune the overall density of the particle based on a preselected core particle, such as when tailoring standardized particles for use with different low moisture compositions having different densities.

[0091] 1. Core made from low density material The core of the particle may be made of one or more non-toxic and biodegradable materials. Preferably, the core dissolves or disperses in water. As described above, the core may have a density similar to that of the low water composition intended to suspend the liquid, so that it remains uniformly suspended in the carrier liquid without substantial settling. Most aqueous liquids have a density of less than 1.0 g / cm depending on the dissolved solute. 3 ~1.3g / cm 3 and the density of the core is 0.5g / cm of the density of the liquid. 3 Within 0.4g / cm 3 Within 0.3g / cm 3 Within 0.2g / cm 3 or even 0.1 g / cm 3 It must be within.

[0092] The desired density of the core depends on the relative size of the core compared to the overall size of the particle. Larger cores represent a larger portion of the whole particle and their density becomes more important. Smaller cores represent only a small portion of the whole particle and their density becomes less important. The desired density of the core can be calculated using Stokes' law for calculating the settling velocity of a particle in a viscous medium:

number

[0093] In the above formula, v s is the settling velocity of the particle (e.g., m / s) (this is called ρ p >ρ f In the case of ρ, the vertical direction is downward. p <ρ f is vertically upwards), and g is the acceleration due to gravity (m / s 2 ) and ρ p is the mass density of the particle (e.g. kg / m 3 ) and ρ f is the mass density of the fluid (kg / m 3 ), and μ is the dynamic viscosity of the water liquid in which the particles are suspended (e.g. kg / m *s) and R is the radius of the particle (m). For convenience, other units may be used due to the small size of the particles of interest, for example, the diameter and radius of the particles are preferably expressed in μm.

[0094] For a given liquid composition, the viscosity (μ) is held constant, and the density difference required to maintain a constant settling viscosity is proportional to the square of the particle radius or diameter, and other factors can be ignored since they cancel out in any ratio. An exemplary particle has a diameter of 250 μm and a radius of 125 μm. For this particle, the particle density (ρ p ) and fluid density (ρ f ) and the absolute value of the density difference, i.e. (ρ p -ρ f or Δρ pf ) is 0.5g / cm 3 Therefore, the settling velocity (v s Any particle larger or smaller than 250μ in diameter is acceptable, as long as the viscosity of the liquid medium is not increased. (|Δρ pf | * D p 2 )=(0.5) * (250) 2 [In the formula, D p is the total diameter of the particle] v s Such particles have the following maximum density differences: |Δρ pf |<(0.5) * (250) 2 / D p 2 or |Δρ pf |≦31250 / D p 2 It does not sink (or rise) any faster than if it were given by

[0095] Alternatively: To avoid settling, ρ p ≦ρ f +31250 / Dp 2 , To avoid floating, ρ p ≧ρ f -31250 / D p 2 It is expressed as:

[0096] Using the latter formula, the total particle diameter (D p The maximum density difference (|Δρ pf |) can be calculated as shown in Table 1:

[0097] [Table 1]

[0098] Extending the above relationship, we can calculate the total particle size (ρ p ) the density of the core (ρ c ) constraints can also be defined. The density of the core is determined by the relationship: ρ c / ρ p =(m c / v c ) / (m p / v p ) [In the formula, m c and m p are the mass of the core and the mass of the whole particle, respectively, and v p and v c are the volumes of the whole particle and the core, respectively] This can be related to the total particle density by: ρ c =ρ c * m c / m p * (v p / v p )

[0099] Core diameter (D c ) and particle diameter (D p ) and the mass fraction of the core is x c Expressed as: ρ c =ρ p * x c / (D c / D p ) (1 / 3) or the particle density in terms of the core density: ρ p =ρ c * (D c / D p ) (1 / 3) / x c It can be shown that

[0100] Therefore, the maximum density difference between the core and the fluid, ρ c -ρ f or Δρ cf : |ρ p -ρ f |≦18750 / D p 2 |ρ c * (D c / D p ) (1 / 3) / x c -ρ f |≦18750 / D p 2 The maximum density difference between the core and the fluid can be obtained by substituting the above expression to obtain

[0101] therefore: To minimize settling, ρ c ≦(ρ f +31250 / D p 2 ) * x c / (D c / D p ) (1 / 3) To minimize settling, ρ c ≧(ρ f -31250 / D p 2 ) * x c / (Dc / D p ) (1 / 3)

[0102] When larger particles are used, the core density is important and low density materials are preferred. When smaller particles are used, the core density is less important and higher density materials such as salts can be used. Low density materials include sugars (e.g., sucrose and sorbitol), carbohydrates (e.g., starch and glycogen), saturated fatty acids (e.g., stearic acid, myristic acid, palmitic acid, and their derivatives), waxes (e.g., polyethylene wax), polymers (e.g., polyvinyl alcohol (PVA), partially hydrolyzed polyvinyl alcohol (PHPVA), polyethylene glycol (PEG), polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose (HPMC), intermediately hydrolyzed PVA (IHPVA), fully hydrolyzed polyvinyl alcohol (VPA), hydroxypropyl methylcellulose (HMP ... Examples of such materials include soluble PVA (FHPVA), plasticized PVA, carboxymethyl cellulose (CMC), carboxymethyl dextran (CMD), diethylaminoethyl dextran (DEAED), ethyl hydroxyethyl cellulose (EHEC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxyethyl methyl cellulose (HEMC), hydroxypropyl dextran (HPD), methyl cellulose (MC), polypropylene glycol (PPG), polypropylene oxide (PPO), polyvinyl sulfate (PVSA) and alginates, and combinations thereof. Higher density materials include salts such as sodium sulfate.

[0103] The core may include fillers, buffers, stabilizers, plasticizers, disintegrants, bulking agents, lubricants, dyes, pigments, fragrances, etc., all of which contribute to the density of the core and must be selected accordingly. The core may contain pockets of trapped air or other gases, which reduces the density of the core. The core may contain an enzyme, or the enzyme may be coated onto the core with or without the enzyme.

[0104] The nominal diameter and size distribution of the particles are not critical, but can be tailored to suit manufacturing, performance, safety, and other requirements. Smaller particles with enzyme / active coatings generally have a higher net weight to core weight ratio, but are more susceptible to aerosolization. Particles smaller than 10 μm, especially smaller than 5 μm, should be avoided for airway safety reasons. Particles smaller than about 40 μm are invisible to the human eye. Larger particles, for example, greater than about 100 μm, 150 μm, or even 200 μm, may be brightly colored to be visible to the human eye and to stand out in the enzyme suspension. Exemplary size ranges are 50-100 μm, 50-150 μm, 100-150 μm, 100-200 μm, 150-250 μm, 200-250 μm, 200-300 μm, 250-300 μm, 300-350 μm, 300-400 μm, 350-500 μm, 400-550 μm, etc. In some cases, the particle size distribution range is narrow so that the particles are uniform in size. In some cases, the particle size distribution is not critical.

[0105] Preferably, the cores dissolve or disperse in water within 15 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, or even 1 minute after diluting the low water liquid composition with at least one volume of water. For smaller cores, which are invisible to the human eye, e.g., less than about 40 μm, it is not important that the cores dissolve during the cleaning application (e.g., wash cycle), but it is preferred that they are biodegradable so that they do not accumulate in the environment.

[0106] 2. Core with density modifier The overall density of the particle can also be modified by incorporating a density modifier. The density modifier can be included in the core itself or can be provided in a coating layer. The density modifier can be included in the core itself or can be provided in the enzyme / active layer or coating layer. The advantage of providing a density modifier in the enzyme / active layer or coating layer is that a preselected core can be fine-tuned for use in a given low moisture composition by simply changing the amount of density modifier in the subsequently applied coating.

[0107] An exemplary density modifier is 1 g / cm 3 Materials having a density less than 700 μm include starch, cellulose fibers, diatomaceous earth, feather particles, zeolites (as used in molecular sieves), flours, ground plant-derived fragments such as corn cobs, soy grit, corn syrup solids, other small particles, and highly porous materials. Other acceptable density modifiers include perlite and fumed silica, especially fumed silica that has been treated to be hydrophobic. Perlite and starch have been found to be particularly useful for the production of approximately spherical, low-density granules having a diameter less than 700 μm by a fluidized bed spray coating process. Other possible density modifiers include fly ash, borosilicate glass hollow spheres, fused glass hollow spheres, ceramic hollow spheres, plastic hollow spheres, hollow fibers (e.g., DACRON® (DuPont)), low density forms of silicates (such as sodium aluminosilicates used as flow aids in powders), low density forms of silicon dioxide (such as those used as flow aids in powders), sawdust, and / or aerogel shards.

[0108] 3. Characteristics of particles with low density cores Low density particles are defined by the formula provided above. In some embodiments, the particles have a density of 1.6 g / cm 3 Less than 1.5g / cm 3 Less than 1.4 g / cm 3 Less than 1.3 g / cm 3 Less than, or even less than 1.2 g / cm3 Less than, for example, 1.0 to 1.6 g / cm 3 , 1.0~1.5g / cm 3 , 1.0~1.4g / cm 3 , 1.0~1.3g / cm 3 , and 1.0 to 1.2 g / cm 3 and the difference between the overall true density of the particles and the density of the low water liquid composition in which the particles are intended to be suspended is ±0.5 g / cm 3 Less than ±0.4g / cm 3 Less than ±0.3g / cm 3 Less than ±0.2g / cm 3 Less than or even ±0.1g / cm 3 Less than, or even ±0.05 g / cm 3 The density is less than 100%. This allows the particles to remain substantially suspended in the liquid composition without falling out of suspension, as is typical for conventional particles. The true density can be calculated as described in Example 3. As mentioned above, the particles can be large enough to be visible to the human eye, e.g., to complement the appearance of the low moisture composition in which they are intended to be dissolved, or small enough to be invisible to the human eye. If the particles are intended to be visible, they can include dyes and pigments.

[0109] When present in a liquid suspension, the enzyme is dissolved in the carrier liquid at less than 1 gram per liter for at least the first 30 days of storage at 25° C., with less than 20% of the enzyme dissolved within the carrier liquid phase. The enzyme is catalytically active upon dilution of the particles in suspension with at least one volume of water, and exhibits most of its original catalytic ability within minutes of dilution. In some embodiments, the enzyme exhibits at least about 50, 60, 70, 80, 90, 95%, or essentially all of its original catalytic ability in less than 1 minute, less than 2 minutes, less than 3 minutes, less than 4 minutes, or less than 5 minutes at a preselected temperature.

[0110] III. Preparation of particles The particles can be made by methods known to those skilled in the art of particle production, including, but not limited to, fluidized bed coating, granulation, spray drying, drum granulation, high shear agglomeration, or a combination of these techniques. Most preferably, the granules are made by a fluidized bed spray coating process (as exemplified below).

[0111] IV. Compositions Containing Liquid Enzyme Suspensions The particles may be included in low moisture compositions such as those used in cleaning, disinfecting, decontamination, textile processing, feed, and food. The compositions may contain 5-20% water by weight. In some embodiments, compositions containing enzyme suspensions contain either about 5-10%, 10-15%, or 15-20% water by weight (w / w). Exemplary liquid laundry detergent compositions in which particles can be suspended include, but are not limited to, PUREX® ULTRAPACKS (Henkel), FINISH® QUANTUM (Reckitt Benckiser), CLOROX™ 2 PACKS (Clorox), OXICLEAN MAX FORCE POWER PAKS (Church & Dwight), TIDE® STAIN RELEASE, CASCADE® ACTION PACS, TIDE® and ARIEL® PODS™, and GAIN FLINGS (Procter & Gamble), ALL™ MIGHTY PACS (Sun Products), KIRKLAND SIGNATURE™ ULTRACLEAN PACS™.

[0112] The enzyme of interest present in the low density particles is stable in the low moisture composition for at least 9 days at 37° C. and is catalytically active upon dilution of the low moisture composition in at least one volume of water, in some embodiments, the enzyme of interest is stable at low moisture for about 2 weeks, 1 month, 2 months, or 3 months or more at 25° C. and exhibits at least about 50, 60, 70, 80, 90, 95%, or essentially all of its initial catalytic potential upon dilution in water.

[0113] When the low moisture composition is a detergent composition, it may contain one or more surfactants, builders, bleaches, bleach precursors, bleach activators, enzyme stabilizers, complexing agents, chelating agents, foam control agents, corrosion inhibitors, antistatic agents, dyes, fragrances, bactericides, fungicides, and activators, as well as any other ingredients typically found in laundry, dishwashing (including automatic and hand dishwashing), and other cleaning compositions.

[0114] In some embodiments, the detergent composition does not contain boron or borates. In some embodiments, the detergent contains low (e.g., submM) concentrations of calcium. In some embodiments, the detergent composition contains low (e.g., submM) concentrations of Period IV metals, such as K, Ca, Mn, Fe, Co, Ni, Cu, Zn.

[0115] V. How to use The particles may be used in any application where enzyme activity is desired from a low water liquid composition that is intended to be pre-diluted with at least one volume of water during use, such that upon dilution, at least about 50, 60, 70, 80, 90, or 95% of the enzyme is soluble and catalytically active in the diluted composition.

[0116] In some embodiments, the application is cleaning, and activation is performed in a bucket or other container that contains the container to be washed. For laundry detergent compositions, activation is typically performed in a washing machine. For dishwashing detergent compositions, activation is typically performed in a dishwasher. For textile compositions, activation is typically performed in a suitable bath. For food, beverage, or feed, activation is performed when required to deliver active enzyme to the application site.

[0117] The particles are particularly useful as components of cleaning compositions, such as detergent compositions (e.g., laundry detergent compositions or dishwashing detergent compositions). Particularly preferred are liquid laundry detergent compositions. Such cleaning compositions typically include a cleaning adjunct, or preferably a combination of cleaning adjuncts. Typically, the cleaning adjunct is present in the composition in an amount of 0.001-99.9 wt%, more typically 0.01-80 wt% of the cleaning adjunct. Exemplary formulations containing suitable cleaning adjuncts in the form of unit dose laundry detergent compositions are provided below. Such unit dose formulations may include one, two, three, or more compartments. The components of each compartment may be different or identical, but the overall / total components of the unit dose formulation have the same composition.

[0118] The following examples are illustrative of low density particles, but are not limiting. EXAMPLES

[0119] Example 1 Evaluating Particle Aggregation in Low Moisture Detergent Formulations 10 g of laundry detergent was added to a clear 15 mL test tube. Approximately 0.2 g of particles were added and mixed to form a well-dispersed suspension. The tube was placed in an end-over-end mixer and rotated at low RPM at room temperature (i.e., 25° C.), representing nominal movement under manufacturing and storage conditions. After 7 days, the degree of agglomeration was visually assessed. The ideal result would be for all particles to remain as individual particles not associated with any other particles. Non-ideal results include the observation of clustering of a few particles. The least ideal result would be clumping of tens to hundreds, or more, of particles.

[0120] Example 2 N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (AAPF-pNA) assay for measuring protease activity The following reagent solutions were used: AAPF substrate stock: 160 mM (i.e., 100 mg / mL) suc-AAPF-pNA dissolved in dimethyl sulfoxide (DMSO), stability buffer: 100 mM MES (pH 5.5) containing 0.005% v / v Tween 80 (10 mM CaCl 2 (optionally containing 0.005% v / v Tween-80), Activity Buffer: 100 mM Tris (pH 8.5 or 8.6) (10 mM CaCl 2 (optionally containing AAPF-pNA), Assay solution (substrate stock diluted 1:100 in activity buffer): 1.6 mM AAPF-pNA in 100 mM Tris (pH 8.5 or 8.6).

[0121] Procedure: An enzyme standard curve was prepared by performing serial dilutions of purified subtilisin protease (0.5-10 ppm) in stability buffer. Test samples were prepared to achieve protease concentrations of 1-10 ppm in stability buffer. Assay solution was prepared by diluting substrate stock 1:100 in activity buffer. 200 μL of assay solution was added to each well of a 96-well plate.

[0122] The assay was performed by adding 10 μL of diluted protease enzyme solution to each well of the assay solution plate. The solution was mixed for 10 seconds and the change in absorbance was measured at 410 nm in a microplate reader at 25° C. (set in kinetic mode over 2 minutes). Subtilisin protease activity (AU=activity units) was expressed as mOD 415 Calculate as mOD / min × dilution factor, where mOD is the 410 means the optical density of the reaction product measured at 410 nm and multiplied by 1000.

[0123] Example 3 Particle preparation and testing A variety of particles were produced using standard fluidized bed techniques as exemplified in US Pat. No. 6,413,749, which is incorporated by reference. Particle types are labelled A-C and are summarised in Table 2. The composition of the core (core), the composition of the enzyme-containing first coating layer (SP1), with or without a binder and / or with or without a density modifier, the composition of the second coating layer (SP2) and, where applicable, the composition of the third coating layer (SP3) are given. All particles contained the indicated amount of mutant subtilisin protease (enz), allowing protein release and leakage to be measured using the standardised protease activity assay described in Example 2.

[0124] [Table 2]

[0125] The particles were tested for clumping performance criteria in low moisture laundry detergents. Particle B remained as individual particles suspended in the detergent after the evaluation period, while Particle A showed significant clumping where tens to hundreds of particles clumped together and were no longer suspended in the detergent. Particle C is expected to have intermediate properties.

[0126] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety into this specification for all purposes and to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A particle capable of isolating and stabilizing an enzyme in a liquid composition without agglomeration during manufacture and / or storage, comprising: (a) a core comprising an active ingredient, and / or a core having a first coating layer immediately deposited thereon comprising an active ingredient, the active ingredient being an enzyme-containing core; and (b) an outermost coating layer comprising a hydrophobic, water-insoluble, moisture-disintegrating material having a solubility in water of less than 1 mg / mL in water at 25° C., the moisture-disintegrating material comprising natural waxes and / or synthetic waxes; Including, the coating layer of (b) completely disintegrates within 5 minutes when the liquid composition is diluted 1:1 with water at 25° C., allowing the enzyme and / or active ingredient to dissolve in the diluted liquid composition; A particle comprising a third coating layer comprising a water-soluble polymer having a solubility of greater than 1 mg / mL in water at 25° C., and which exhibits reduced aggregation in the liquid composition compared to an otherwise identical particle.

2. 10. The particle of claim 1, comprising at least one additional layer between (a) and (b) comprising a water-soluble polymer and an active ingredient.

3. 10. The particle of claim 1, comprising at least one additional layer between (a) and (b) that comprises a water-soluble polymer that is devoid of an active ingredient.

4. 3. The particle of claim 1 or 2, wherein the core is devoid of an active ingredient.

5. A particle according to claim 1 or 3, wherein the core comprises an active ingredient.

6. 6. The particle of any one of claims 1 to 5, wherein the outermost coating disintegrates within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 1 minute, within 30 seconds, or even within 15 seconds after contacting a liquid composition containing the particle with at least one additional volume of water in a 1:1 ratio with the liquid composition at 25°C.

7. A particle according to any one of claims 1 to 6, wherein the outermost coating represents less than 8%, less than 7%, less than 6% or even less than 5% of the total weight of the particle.

8. 8. The particle of any one of claims 1 to 7, wherein the outermost layer consists essentially of or consists of a hydrophobic, water insoluble, moisture disintegrating material having an aqueous solubility of less than 1 mg / mL in water at 25°C.

9. The core has the formula: ρ ｃ ≤ (ρ ｆ + 31250 / D ｐ ２ ) ＊ x ｃ / (D ｃ / D ｐ ) （１／３） and r ｃ ≧(ρ ｆ -31250 / D ｐ ２ ) ＊ + ｃ / (D ｃ / D ｐ ) （１／３） [In the formula, ρ ｃ is g / cm ３ is the density of the core at ｆ is g / cm ３ is the mass density of the liquid composition at ｃ is the mass fraction of the core in the particle, D ｃ is the diameter of the core in μM, D ｐ 9. The particle according to claim 1, having a density defined as:

10. 10. The particles of any one of claims 1 to 9, having a total true density of less than 1.6 mg / mL, less than 1.4 mg / mL, or even less than 1.2 mg / mL.

11. 1. A method for producing particles capable of isolating and stabilizing an enzyme in a liquid composition without agglomeration during production and / or storage, comprising: (a) producing a core comprising an active ingredient and / or a core having a first coating layer comprising an active ingredient immediately deposited on said core, said active ingredient being a core comprising an enzyme; and (b) producing the particles by coating the core with an outermost layer comprising a hydrophobic, water insoluble, moisture disintegrating material having a solubility in water of less than 1 mg / mL in water at 25° C., the moisture disintegrating material comprising natural waxes and / or synthetic waxes; the coating layer of (b) completely disintegrates within 5 minutes when the liquid composition is diluted 1:1 with water at 25° C., allowing the enzyme and / or active ingredient to dissolve in the diluted liquid composition; The method of claim 1, wherein the particles comprise a third coating layer comprising a water-soluble polymer having a solubility of greater than 1 mg / mL in water at 25° C. and exhibit reduced aggregation in the liquid composition compared to otherwise identical particles.

12. 12. The method of claim 11, wherein the outermost layer consists essentially of or consists of a hydrophobic, water insoluble, moisture disintegrating material having an aqueous solubility of less than 1 mg / mL in water at 25°C.

13. 13. The method of claim 11 or 12, wherein after contacting a liquid composition containing the particles with at least one additional volume of water in a 1:1 ratio with the liquid composition at 25°C, the outermost coating disintegrates within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 1 minute, within 30 seconds, or even within 15 seconds.

14. A method according to any one of claims 11 to 13, wherein the outermost coating represents less than 8%, less than 7%, less than 6% or even less than 5% of the total weight of the particle.

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