PRIORITY

[01] The present application claim priority to U.S. Provisional Application Serial No. 62/173,260, filed on June 9, 2015, which is hereby incorporated by reference in its entirety.

BACKGROUND

[01] The present compositions and methods relate to low density enzyme-containing particles for inclusion in cleaning and other low-water compositions. The particles remain in suspension without settling, and release active enzyme upon dilution of the low-water compositions with water.

BACKGROUND

[02] Enzymes are supplied in both liquid and solid forms for incorporation within products used in a variety of consumer and industrial applications, including laundry and dish cleaning, personal care, textile treatment, pulp and paper production, leather production, food and beverage processing, starch processing, decontamination, oil and gas drilling, production of biofuels, and production (or modification) of biopolymers and other chemicals.

[03] There is a broad need to compartmentalize enzymes or other actives in liquid formulas that contain such incompatible ingredients, so that they are stable during storage, but release quickly upon dilution in application. Many otherwise effective enzymes cannot be utilized because they are unstable in liquid formulations such as detergents.

[04] Aside from present a challenge in terms of stability, enzymes are immunogenic molecules and can present problems relating to exposure and sensitization. In some cases, the maximum amount of enzymes that can be added to a liquid cleaning formulation is determined by exposure risk, as opposed to performance or economics.

[OS] Enzymes can be provide in granular form in liquid detergent but granules invariably settle in liquid formulations such as detergents, resulting in non-uniform distribution of enzymes as well as the unappealing appearance of settled granules. Accordingly, there is a need for improved ways to compartmentalize enzymes in liquid formulations, such that they remain stable, retains catalytic potential until use in an application in which enzyme activity is desired, and remain uniformly suspended in a liquid for prolonged periods of time.

BRIEF SUMMARY OF THE INVENTION

[06] The 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.

1. In one aspect, a particle capable of isolating and stabilizing enzymes in a liquid composition is provided, comprising:

(a) a core having a density defined by the equations:

and

wherein p _c is the density of the core in in g/cm ³ , p _f is the mass density of the liquid composition in g/cm ³ , Xc is the mass fraction of the core in the particle, D _c is the diameter of the core in μΜ, and Dp is the diameter of the particle in μΜ;

(b) a first coated layer comprising enzyme and/or other active component, and optionally a density modifier having a density less than 1 g/cm ³ ; and

(c) a second coated layer comprising a water-soluble polymer having a solubility of greater than about 1 mg/mL in water at 25°C;

wherein the second coated layer fully dissolves within about 5 minutes when the liquid composition is diluted 1:1 with water at 25°, allowing the dissolution of the enzyme and/or active component into the diluted liquid composition.

2. In some embodiments, the particle of paragraph 1, has an overall true density of less than 1.6 mg/mL.

3. In some embodiments, the particle of paragraph 1, has an overall true density of less than 1.4 mg/mL.

4. In some embodiments, the particle of paragraph 1 , has an overall true density of less man 1.2 mg/mL.

5. In some embodiments of the particle of any of the proceeding paragraphs, the core is soluble or dispersible in water.

6. In some embodiments of the particle of any of the proceeding paragraphs, the core is non-salt containing. 7. In some embodiments of the particle of any of the proceeding paragraphs, the core comprises sugar, polymer, fat, wax, a salt of an inorganic or organic acid, clays, silica, alluminosilicate, or a combination, thereof.

8. In some embodiments, of the particle of any of the proceeding paragraphs, the core further comprises enzyme or other active ingredient.

9. In some embodiments, of the particle of any of proceeding paragraphs 1-8, the core does not comprise enzyme or other active ingredient.

10. In some embodiments of the particle of any of proceeding paragraphs 1-8, the core does not comprise enzyme.

11. In some embodiments of the particle of any of the proceeding paragraphs, the first coated layer further comprises stabilizer.

12. In some embodiments of the particle of any of the proceeding paragraphs, the first coated layer is a plurality of coated layers, optionally comprising the same or different enzyme and/or other active component.

13. In some embodiments of the particle of any of the proceeding paragraphs, the first coated layer further comprises polymer.

14. In some embodiments, of the particle of any of the proceeding paragraphs, the second coated layer comprises a water-soluble polymer having a solubility of greater than 10 mg/ml in water at 25°C.

15. In some embodiments of the particle of any of the proceeding paragraphs, the second coated layer comprises a polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polyethylene oxide, methylcellulose, hydroxyl propyl methylcellulose, polyvinyl pyrrolidone, polyacrylates, polymethacrylates, and copolymers and combinations, thereof.

[07] These and other aspects and embodiments of the compositions and methods are described, below.

BRIEF DESCRIPTION OF THE DRAWINGS

[08] Figure 1 is a graph showing the leakage of particles A-E in the detergent composition obtained from commercial Tide PODS® at 37°C over 35 days. Leakage was measured as the percentage of protease activity detected in the detergent composition based on the total amount of protease activity expected from the amount of protease coated onto the particle cores. [09] Figure 2 is a graph showing the stability of particles A-F in the detergent composition obtained from commercial Tide PODS® at 37°C over 40 days. Stability was measured as the percentage of residual protease activity in the particles based on the total amount of protease activity expected from the amount of protease coated onto the particle cores.

DETAILED DESCRIPTION

Introduction

[10] Described are low-density particles capable of isolating and stabilizing enzymes and other active components in low-water liquid compositions. Generally, the particles include a core, at least one enzyme and/or other active component-containing layer, and a water- soluble-polymer coating. An important feature of the low-density particles is that they have an overall particle density that is close to the density' of low-water liquid compositions, such as laundry detergents. Conventional particles have a significantly higher density. Higher density particles inevitably settle out of a suspension. In contrast, particles having a density similar to their surrounding liquid environment can remain suspended for prolonged periods of time.

Definitions and abbreviations

[11] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. [12] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations 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.

[13] As used herein, the term "water soluble polymer" refers to a polymer that is soluble in water in in an amount of at least 1 mg/g at 25°C. As used herein, an "aqueous medium" or "aqueous solution" is a solution and/or suspension in which the solvent is primarily 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). The aqueous medium may include 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, and the like. Exemplary aqueous media are laundry and dishwashing wash liquors. Materials such as textiles, fabrics, dishes, kitchenware, and other materials may also be present in or in contact with the aqueous medium

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

[15] As used herein, the term "substantially non-aqueous," with reference to a liquid laundry detergent composition, indicates that the detergent composition contains about 2- 5% water (w/w).

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

[17] As used herein, where a component is "provided in" a specified form (e.g. , nonaqueous, very low water, solid, and the like), this form refers to the final form as the component exists in the unit-dose package, not the form in which it may be added to another component that is then added to the unit-dose package.

[18] As used herein, the phrase "insufficient to substantially dissolve water-soluble packaging" means that a subject liquid does not dissolve more than 5% of a water-soluble material over a period of six months at room temperature (i.e., 25°C). [19] As used herein, the term "bounded" with reference to the contents of water-soluble packaging means the specified contents, whether liquid, solid, or a combination, thereof, are physically contained in a compartment, at least a portion of which is defined by water- soluble material. In some cases, the contents are fully bounded by water-soluble material, meaning that the entire compartment is defined by the water-soluble material, as in the case of a pouch made of water-soluble material. In some cases, the contents are only partially bounded by water-soluble material, meaning that only a portion of the compartment is defined by the water soluble material, and the remainder is defined by water-insoluble material, as in the case of a cup or dish covered by a lid made of water-soluble material.

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

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

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

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

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

[25] As used herein, a "low" pH is a pH of less than about 7.

[26] As used herein, a "high" pH is a pH of greater than about 7.

[27] As used herein, the term "contacting," means bringing into physical contact, such as by placing a unit-dose package in an aqueous solution.

[28] As used herein, a "solid" form of a chemical component refers to a powder, crystals, granules, aggregates, paste or wax thereof.

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

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

[31] As used herein, the terms "purified" and "isolated" refer to the removal of contaminants from a sample and/or to a material (e.g., a protein, nucleic acid, cell, etc.) that is removed from at least one component with which it is naturally associated. For example, these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system

[32] As used herein, the term "spray drying" refers to a method of producing a dry powder from a liquid or slurry by rapidly drying with a hot gas, as known in the art and discussed for example in US Patent 5,423,997 and WO2008/088751A2.

[33] As used herein "d50" refers to the size of the particles measured where 50% are above or below the mid-point within the population measured.

[34] As used herein, the term "UFC Solids" refers to ultrafiltrate concentrate from a fermentor/bioreactor, and is synonymous with enzyme concentrate solids.

[35] As used herein, the term "bleaching" refers to the treatment of a material (e.g. , fabric, laundry, pulp, etc.) or surface for a sufficient length of time and under appropriate pH and temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material. Examples of chemicals suitable for bleaching include but are not limited to

C10 ₂ , H2O2, peracids, N0 ₂ , etc.

[36] As used herein, "cleaning compositions" and "cleaning formulations" refer to compositions that may be used for the removal of undesired compounds from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes) etc. The term encompasses any materials/compounds selected for the particular type of cleaning composition desired. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.

[37] The terms further refer to any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e.g., laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).

[38] As used herein, the terms "detergent composition" and "detergent formulation" are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects. In some preferred embodiments, the term is used in reference to laundering fabrics and/or garments (e.g., "laundry detergents"). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., "dishwashing detergents").

[39] As used herein, the term "nonionic surfactant" refers to a surfactant molecule with a non-electrically charged polar group.

[40] As used herein, the term "anionic surfactant" refers to a surfactant molecule with a negatively charged polar group at the pH of the composition or the application of use. Salts used to complex or neutralize the surfactant, e.g., forming the monoethanolamine (MEA) salt of linear alkylbenzene sulfonate (LAS) are included I accounting herein for the mass or concentration of anionic surfactant.

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

[42] As used herein, the term "disinfecting" refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items. It is not intended that the present invention be limited to any particular surface, item, or contaminants) or microbes to be removed.

[43] As used herein the term "hard surface cleaning composition" refers to detergent compositions for cleaning hard surfaces such as floors, walls, tile, bath and kitchen fixtures, and the like.

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

[45] As used herein, "personal care products" means products used in the cleaning, bleaching and/or disinfecting of hair, skin, scalp, and teeth, including, but not limited to shampoos, body lotions, shower gels, topical moisturizers, toothpaste, and/or other topical cleansers. In some particularly preferred embodiments, these products are utilized on humans, while in other embodiments, these products find use with non-human animals (e.g., in veterinary applications).

[46] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single- stranded, double-stranded, triple-helical, etc.), which contain deoxy ribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or

ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides which encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-0-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin. The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. The terms "polynucleotide" and "nucleic acid" and "oligonucleotide" are used herein interchangeably. Polynucleotides of the invention may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), (0)NR ₂ ("amidate"), P(0)R, P(0)OR', CO or CH ₂ ("formacetal"), in which each R or R' is independently H or substituted or

unsubstituted alkyl (CI -20) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions.

[47] As used herein, "polypeptide" refers to any composition comprised of amino acids and recognized as a protein by those of skill in the art. The conventional one-letter or three- letter code for amino acid residues is used herein. The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[48] Related (and derivative) proteins encompass "variant" proteins. Variant proteins differ from a parent protein and/or from one another by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, variants differ by about 1 to about 10 amino acids.

[49] In some embodiments, related proteins, such as variant proteins, comprise any of at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity.

[50] As used herein, the term "contaminant" refers to any substance which by its contact or association with another substance, material, or item makes it undesirable, impure, and/or unfit for use.

[51] As used herein, the term "a contaminated item" or "item in need of

decontamination" refers to any item or thing in contact or associated with a contaminant and/or which needs to be decontaminated. It is not intended that the item be limited to any particular thing or type of item For example, in some embodiments, the item is a hard surface, while in other embodiments, the item is an article of clothing. In yet additional embodiments, the item is a textile. In yet further embodiments, the item is used in the medical and/or veterinary fields. In some preferred embodiments, the item is a surgical instrument. In further embodiments, the item is used in transportation (e.g., roads, runways, railways, trains, cars, planes, ships, etc.). In further embodiments, the term is used in reference to food and/or feedstuff ^' s, including but not limited to meat, meat by-products, fish, seafood, vegetables, fruits, dairy products, grains, baking products, silage, hays, forage, etc. Indeed, it is intended mat the term encompass any item that is suitable for decontamination using the methods and compositions provided herein.

[52] As used herein, the term "decontamination" refers to the removal of substantially all or all contaminants from a contaminated item. In some preferred embodiments, decontamination encompasses disinfection, while in other embodiments, the term encompasses sterilization. However, it is not intended that the term be limited to these embodiments, as the term is intended to encompass the removal of inanimate contaminants, as well as microbial contamination, (e.g., bacterial, fungal, viral, prions, etc.). [53] As used herein, the term "disinfecting" refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items. It is not intended that the present invention be limited to any particular surface, item, or contaminants) or microbes to be removed.

[54] As used herein, the term "sterilizing" refers to the killing of all microbial organisms on a surface.

[55] As used herein, the term "sporicidal" refers to the killing of microbial spores, including but not limited to fungal and bacterial spores. The term encompasses compositions mat are effective in preventing germination of spores, as well as those compositions that render spores completely non-viable.

[56] As used herein, the terms "bactericidal," "fungicidal," and "viricidal" refer to compositions mat kill bacteria, fungi, and viruses, respectively. The term "microbiocidal" refers to compositions that inhibit the growth and/or replication of any microorganisms, including but not limited to bacteria, fungi, viruses, protozoa, rickettsia, etc.

[57] As used herein, the terms "bacteriostatic," "fungistatic," and "virostatic" refer to compositions that inhibit the growth and/or replication of bacteria, fungi, and viruses, respectively. The term "microbiostatic" refers to compositions that inhibit the growth and/or replication of any microorganisms, including but not limited to bacteria, fungi, viruses, protozoa, rickettsia, etc.

[58] The terms "recovered," "isolated," "purified," and "separated" as used herein refer to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated. For example, these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.

[59] "Water miscible" as used herein refers to a liquid forming a single thermodynamic liquid phase or isotropic phase upon mixing with water, at a specified ratio of water to the liquid.

[60] A "suspension" or "dispersion" as used herein refers to a two phase system wherein 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 spray dried particles as small as 1 micron in diameter or larger core-shell granules between 100 and 1,000 microns in diameter. [61] A "suspension aid" as used herein refers to a material added to a liquid composition to prevent or reduce sedimentation or floating of suspended particles. Suspension aids typically work by increasing either the viscosity or the yield stress of a carrier liquid. Fluids with a significant yield stress will flow only when stress is applied which is greater than the yield stress, and thus exhibit shear-thinning or thixotropic behavior. Effective suspension agents typically act by forming a reversible network of particles or fibers bridged by weak forces. Examples of suspending agents include, but are not limited to, xanthan gum and microfibrous cellulose, e.g., Cellulon (CP Kelco, San Diego, CA).

[62] The terms "immunogenicity," "immunogenenic," and related terms refers to the ability of an immunogen, e.g., an a-amylase polypeptide, to initiate or perpetuate an immune reaction in an animal, thereby causing the animal to develop sensitivity to the immunogen, resulting in the need to avoid or reduce further contact with the immunogen.

[63] The term "less immunogenic" means a given composition has a reduced potential to initiate or perpetuate and immune response in a population of animals.

[64] The phrase "humans having contact with the detergent composition" refers to any number of workers at a detergent manufacturing site or consumers who are exposed to a given detergent composition, including exposure to granules, liquids, and aerosols, such that they have a potential to develop an immune response to components of the composition.

[65] The following abbreviations may be used in the description. Definitions are also provided as needed throughout the description.

Low-density particles [66] Described are low-density particles capable of isolating and stabilizing enzymes and other active components in low-water liquid compositions. The particles include a core, at least one enzyme and/or other active component-containing layer, and a water-soluble- polymer coating. The low-density particles have an overall particle density close to the density of the low-water liquid composition in which they are intended to be suspended. This distinguishes then from conventional particles, which have a higher density', and tend to settle out of suspension.

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

Low-density cores

[68] The core of the particle is preferably made from one or more non-toxic and biodegradable materials. Preferably, the cores dissolve or disperse in water. As described, above, the cores may have a density similar to that of the low-water composition in which they are intended to be suspended liquid, such that they remain uniformly suspended in the carrier liquid without substantial settling. Most aqueous liquids have a density between 1.0 g/cm ³ and 1.3 g/cm ³ , depending on the dissolved solutes, and the density of the core should be within 0.5 g/cm ³ , 0.4 g/cm ³ , 0.3 g/cm ³ , 0.2 g/cm ³ , or even 0.1 g/cm ³ of the density of the liquid.

[69] The desired density of the cores depends on the relative size of the cores compared to the overall size of the particles. A larger core represents a larger portion of the overall particle, making its density more critical. A smaller core may represent only a small portion of the overall particle, making its density less critical. The desired density of the core can be selected based on Stoke' s law for calculating the settling velocity of a particle in a viscous medium:

[70] In the equation, above, v, is the particle's settling velocity (e.g. , m/s), which is vertically downwards if p and vertically upwards if g is gravitational

acceleration (m/s ² ), p _p is the mass density of the particle (e.g., kg/m ³ ), /? _/ is the mass density of the fluid (kg/m ³ ), μ is the dynamic viscosity (e.g., kg/m*s) of the water liquid in which the particle is suspended, and R is the particle radius (m). For convenience in view of the small size of the subject particles, other units may be used, for example, particle diameter and radius are preferably expressed in μηχ

[71] For a given liquid composition, the viscosity (μ) is held constant, so to maintain a constant settling viscosity the required density difference scales with the square of the particle radius or diameter and the other coefficients can be ignored since they cancel out of any ratio. An exemplary particle has a diameter of 250 um and a radius of 125 um. For this particle, the absolute value of the density difference between particle density (p„) and fluid density should be no more than 0.5 g/cm ³ , so any particle that is

larger or smaller than 250 μm. diameter is acceptable as long as the settling rate (v,) does not increase. With the liquid medium viscosity fixed, any particle will have the same v, when:

where Dp is the overall diameter of the particle. Such a particle will not settle (or rise) faster than v _s when for the maximum density the difference is given by:

[72] Expressed in another way:

[73] Using the latter formula, the maximum density difference (|Ap _py |) required as a function of overall particle diameters (Dp) can be calculated, as shown in Table 1: Table 1. Maximum density differences for different overall particle diameters

[74] The above relationship can also be extended to define the constraints on the density of the core (p _c ) within the overall particle (p _p ). The density of the core can be related to the density of the overall particle according to the relationship:

where nv and n¾, represent the mass of the core and mass of the overall particle, respectively, and \ _p and v _c represent the respective volumes of the overall particle and the core. Rearranging:

[75] Expressing the volumes in terms of diameters of the core (D _c ) and particle (Dp) and representing the mass fraction of the core as Xc, we obtain:

or we can show the particle density in terms of core density: [76] Therefore, the maximum density difference between the core and the fluid can be given by substituting the above expression to get the maximum density difference between the core and the fluid p /

[77] Therefore:

[78] Where larger particles are used, core density is critical and low density materials are preferable. Where smaller particles are used, the core density is less critical 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-hydrolylzed polyvinyl alcohol (PHPVA), polyethylene glycol (PEG), polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMC), hydroxypropylmethylcellulose (HPMC), intermediately-hydrolyzed PVA (IHPVA), fully -hydrolyzed PVA (FHPVA), plasticized PVA, carboxymethyl cellulose (CMC), carboxymethyl dextran (CMD), diethylaminoethyl dextran (DEAED), ethylhydroxyethyl cellulose (EHEC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hy<_roxyethylmethyl cellose HEMC), hydroxypropyl dextran (HPD) methyl cellulose (MC), polypropylene glycol (PPG), polypropylene oxide (PPO), polyvinylsulfuric acid (PVSA) and alginates, having a molecular weight such that the polymer is solid at room temperature), and combinations, thereof. Higher density materials include salts, such as sodium sulfate.

[79] The core may include fillers, buffers, stabilizers, plasticizers, distintegrants, extenders, lubricants, dyes, pigments, fragrances and the like, but all such components contribute to the density of the core, and must be selected accordingly. The core may include pockets of trapped air or other gases, which lower the density of the core. The core may include enzymes or enzymes may be coated onto a core that either includes or does not include enzymes. [80] The nominal diameter and size distribution of the particles is not critical but can be tailored to suit manufacturing, performance, safety, and other requirements. Smaller particles having an enzyme/active coating generally have a higher payload to core weight ratio but are more readily aerosolized. Particles smaller than 10 μm., and especially smaller than S μm., should be avoided for respiratory tract safety reasons. Particles smaller than about 40 μm. are not visible to the human eye. Larger particles, e.g., greater than about 100 μm., 150 μm., or even 200 μm., are visible to the human eye and may be brightly colored such that they are prominently visible 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., and the like. In some cases, the size distribution range is narrow, such that the particles are uniform in size. In some cases, the size distribution is not critical.

[81] Preferably, the cores dissolve or disperse in water within 15 min, 10 min, 5 min, 3 min, 2 min, or even 1, min following the dilution of the low-water liquid composition with at least one volume of water. In the case of smaller cores, e.g., less than about 40 μm., which are not visible to the human eye, it is not critical that the cores dissolve during the cleaning application (e.g., laundry cycle) but they are preferably biodegradable such that they do not accumulate in the environment.

Density modifiers

[82] The overall density of the particles can be modified by the incorporation of density' modifiers. Density modifiers can be included in the core, itself, or provided in a coating layer. Density modifiers can be included in the core, itself, or provided in an

enzyme/active-layer or coating layer. An advantage of providing the density' modifier in an enzyme/active-layer or coating layer is mat a preselected core can be fine-tuned for use in a given low-water composition simply by varying the amount of density modifier in a subsequently-applied coating.

[83] Exemplary density modifiers are materials having a density of less than 1 g/cm ³ , and include starch, cellulose fibers, diatomaceous earth, feather particles, zeolites (such as used for molecular sieving), flour, milled plant derived fragments such as corn cobs, soy grit, com syrup solids, among other small-particle, highly-porous materials. Other acceptable density modifiers include perlite and fumed silica (particularly, fumed silica that has been treated so as to be hydrophobic). It has been found that perlite and starch are especially useful for making roughly spherical low-density granules having a diameter of less than 700 μΜ via a fluidized-bed spray coating process. Other possible density modifiers include fly ash, borosilicate glass hollow spheres, fused glass hollowspheres, ceramic hollowspheres, plastic hollowspheres, hollow fibers (e.g., DACRON® (DuPont)), low density forms of silicates (such as sodium aluminosilicates used as flow aids for powders), low density forms of silicon dioxide (such as those used as flow aids for powders), sawdust, and/or aerogel shards.

Enzymes and other actives

[84] The cores are coated with, or contain, one or more of a wide variety of enzymes or other actives. While the present description is focused on the isolation and stabilization of enzymes, it will be apparent that a myriad of other active components can be provided in a low-water composition using the same particles.

[85] Exemplary enzymes include acyl transferases, a-amylases, β-amylases, a- galactosidases, arabinosidases, aryl esterases, β-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-β-1, 4-glucanases, endo- beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluromdases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, peroxygenases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, β-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, metalloproteases, additional serine proteases, and combinations, thereof.

[86] Examples of suitable proteases include but are not limited to subtilisins, such as those derived from Bacillus (e.g., subtilisin, lentus, amyloliquefaciens, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168), including variants as described in, e.g., U.S. Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, all of which are incorporated herein by reference. Additional protease include trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89/06270. In some

embodiments the protease is one or more of 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 Corp., Tokyo, Japan). Additional proteases are described in W095/23221, 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, U.S. Pat. Publ. No.

2008/0090747, and U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, US RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628.

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

amyloliquefacients.

[88] 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 lipase, such as C. antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g., EP 214 761), Pseudomonas lipases such as P. alcaligenes lipase and P. pseudoalcaligenes lipase (See e.g., EP 218272), P. cepacia lipase (See e.g., EP 331 376), P. stutzeri lipase (See e.g., GB 1,372,034), P. fluoresceins lipase, Bacillus lipase (e.g., B. subtilis lipase (Dartois et al. (1993) Biochem. Biophys. Acta 1131:253-260); B. stearothermophilus lipase (see e.g., JP 64/744992); and B. pumilus lipase (see e.g., WO 91/16422)).

[89] Additional suitable lipases include Penicillium camembertii lipase (Y amaguchi et al. (1991) Gene 103:61-67), Geotricum candidum lipase (See, Schimadaet al. (1989) J.

Biochem. 106:383-388), and various Rhizopus lipases such as R delemar lipase (Hass et al. (1991) Gene 109: 117-113), aR niveus lipase (Kugimiya et al. (1992) Biosci. Biotech. Biochem. 56:716-719) and R. oryzae lipase. Additional lipases are the cutinase derived from Pseudomonas mendocina (See, WO 88/09367), and the cutinase derived from

Fusarium solani pisi (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 Ml LIPASE™, LUMA FAST™, and LIPOMAX™ (Genencor); LIPEX®, LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE P™ "Amano" (Amano Pharmaceutical Co. Ltd., Japan).

[90] Suitable amylases include, but are not limited to those of bacterial or fungal origin, or even mammalian origin. Numerous suitable are described in W09510603, W09526397,

Commercially available amylases include, but are not limited to one or more of

DURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYME ULTRA®, and BAN™ (Novozymes), as well as POWERASE™,

RAPID ASE® and MAXAMYL® P, PREFERENZ® S100, PREFERENZ® SI 10, and PREFERENZ® S1000 (Genencor).

[91] Suitable cellulases include but are not limited to those having color care benefits (see e.g., EP 0495257). Examples include Humicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307) and commercially available cellulases such as CELLUZYME®, CAREZYME® (Novozymes), and KAC-500(B)™ (Kao Corporation), and Primafast® GOLD (DuPont). In some embodiments, cellulases are incorporated as portions or fragments of mature wild-type or variant cellulases, wherein a portion of the N-terminus is deleted (See e.g., U.S. Pat. No. 5,874,276). Additional suitable cellulases include those found in WO2005054475, WO2005056787, U.S. Pat. No. 7,449,318, and U.S. Pat. No. 7,833,773.

[92] Suitable mannanases are described in U.S. Pat. Nos. 6,566,114, 6,602,842, 5, 476, and 775, 6,440,991, and U.S. Patent Application Number 61/739267, all of which are incorporated herein by reference). Commercially available include, but are not limited to MANNASTAR®, PURABRITE™, and MANN AWAY®.

[93] In some embodiments, peroxidases are used in combination with hydrogen peroxide or a source thereof (e.g., a 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., to prevent transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor), preferably together with an enhancing agent (See e.g., WO 94/12621 and WO 95/01426). Suitable peroxidases/oxidases include, but are not limited to those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments.

[94] Suitable perhydrolases include the enzyme from Mycobacterium srnegmatis. This enzyme, its enzymatic properties, its structure, and numerous variants and homologs, thereof, are described in detail in International Patent Application Publications WO 05/056782A and WO 08/063400A, and U.S. Patent Publications US2008145353 and US2007167344, which are incorporated by reference. In some embodiments, the Mycobacterium srnegmatis

peihydrolase, or homolog, includes the S54V substitution.

[95] Other suitable perhydrolases include members of the carbohydrate family esterase family 7 (CE-7 family) described in, e.g., WO2007/070609 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; E.C. 3.1.1.41) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72). Members of the CE-7 esterase family share a conserved signature motif (Vincent etal, J. Mol. Biol, 330:593-606 (2003)).

[96] Other suitable perhydrolase enzymes include those from Sinorhizobium meliloti, Mesorhizobium loti, Moraxella bovis, Agrobacterium tumefaciens, or Prosthecobacter dejongeii (WO2005056782), Pseudomonas mendocina (U.S. Patent No. 5,389,536), or Pseudomonas putida (U.S. Patent Nos. 5,030,240 and 5,108,457). [97] The enzymes may be crystalized, precipitated, spray dried, lyophilized, and/or compressed and provided in dry form, or resuspended liquid form, thereof. The enzymes may be provided as an ultrafiltration concentrate. They may be purified to a preselected level.

[98] As mentioned, above, in addition to the carrier liquid and enzymes, the present low density particles may further include one or more actives, such as bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments, and pH control agents, surfactants, builders, dye transfer inhibiting agents, deposition aids, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, structure elasticizing agents, fabric softeners, hydrotropes, processing aids and/or pigments. Suitable examples of such other adjuncts and levels of use are found in U.S. Patent 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 for the present invention include the detergent formulations found in WO2013063460, WO2003010266,

WO2006002755, WO2006088535, and US20110263475, all of which are hereby incorporated by reference. Such adjuvants can be included in the core, the enzyme layer, or the polymer coating, so long as they do not adversely affect the described desired properties of the particles.

Particle coatings

[99] At least one non-aqueous, water-soluble coating is applied to the core or coated core to protect the enzyme and/or other active component layer from water present in the low- water liquid compositions in which the particles are intended to be suspended. The coating should be non-toxic and biodegradable. The solubility of the coating in water should be greater man 1, greater man 2, greater than 3, greater man 4, greater than 5, greater than 6, greater man 7, greater than 8, greater than 9, or even greater man 10 mg/mL at 25°C. The coating should dissolve within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 1 minute, within 30 seconds, or even within 15 seconds when the low-water liquid composition in which they are suspended is diluted with at least one volume of water

[100] Exemplary materials are linear or branched polymers having a molecular weight such that the polymer (or mixture of different polymers) is/are solid at room temperature).. Specific exemplary materials include but are not limited to synthetic polymers, such as polyvinyl alcohol (PVA), polyvinyl acetate, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyethylene oxide (PEO), poly acrylic acid, poly methacrylic acid, pyrrolidone carboxylic acid, polystyrene sulfonates, 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; cellulosic materials, such as hydroxy propyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, carboxy methyl cellulose (CMC), methyl cellulose, and hydroxy ethyl cellulose; and other materials, such as cucurbuturil, polyethylimine, quaternary polyamine, carrageenan, pectins, chitosan, polysacharrides, poloxamers, polyanhydrides, polyhydroxyalkanoates, gluten, gelatin, sodium alginate, carrageenan, starch, dextrins, fatty alcohols, and natural waxes; and mixtures, thereof.

Particle properties

[101] The present low-density particles are defined by the formulae provided above. In some embodiments, the particles have an overall true density (i.e., the mass of a particle divided by its volume, excluding open pores and closed pores) of less than 1.6 g/cm ³ , less than 1.5 g/cm ³ , less than 1.4 g/cm ³ , less than 1.3 g/cm ³ , or even less than 1.2 g/cm ³ , for example, 1.0-1.6 g/cm ³ , 1.0-1.5 g/cm ³ , 1.0-1.4 g/cm ³ , 1.0-1.3 g/cm ³ , and 1.0-1.2 g/cm ³ , and the difference between the overall true density of the particles and the density the low-water liquid composition in which they are intended to be suspended is less than ±0.5 g/cm ³ , less than ±0.4 g/cm ³ , less man ±0.3 g/cm ³ , less than ±0.2 g/cm ³ , even less than ±0.1 g/cm ³ or even less than ±0.05 g/cm ³ . This allows the particles to remain substantially suspended in the liquid composition without falling out of suspension, as is typical of conventional particles. True density can be calculated as described in Example 3. As mentioned, above, the particles can be sufficiently large to be visible to the human eye, e.g., to compliment the appearance of the low-water composition in which they are intended to be dissolved, or can sufficiently small to be invisible to the human eye. Where the particles are intended to be visible, they can include dyes and pigments.

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

Preparation of particles

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

Compositions containing the liquid enzyme suspensions

[104] The low-density particles may be included in low-water compositions, such as those used for cleaning, disinfection, decontamination, textile processing, feed, and food. The compositions may 5-20% water by weight. In some embodiments, the composition containing an enzyme suspension contains any of about 5-10%, 10-15%, or 15-20% water by weight (w/w). Exemplary liquid laundry detergent composition in which the particles may 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® ACTIONPACS, TIDE® and ARIEL® PODS™ and GAIN FLINGS (Procter & Gamble), ALL™ MIGHTY PACS (Sun Products), KIRKLAND SIGNATURE™ ULTRACLEAN PACS™.

[105] Enzyme(s) of interest present in the low-density particles are stable in low- water compositions for at least 9 days at 37°C and are catalytically active upon dilution of the low water compositions with at least one volume of water. In some embodiments, an enzyme of interest is stable in the low water for about 2 weeks, 1 month, 2 months, or 3 months or longer 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. In some embodiments, an enzyme of interest is stable in the low water for about 2 weeks, 1 month, 2 months, or 3 months or longer at 37°C and exhibits at least about 50, 60, 70, 80, 90, 95% or essentially all of its initial catalytic potential upon dilution in water.

[106] Where the low water 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 regulators, corrosion inhibitors, anti- electrostatic agents, dyes, perfumes, bactericides, fungicides, and activators, and any other ingredients typically found in laundry, dishwashing (including automatic and hand dishwashing), and other cleaning compositions.

[107] In some embodiments, the detergent composition does not contain boron or borate. In some embodiments, the detergent contains a low (e.g., submillimolar) level of calcium. In some embodiments, the detergent composition contains low (e.g., submillimolar) levels of period IV metals, e.g., K, Ca, Mn, Fe, Co, Ni, Cu, Zn.

[108] An advantage of the present low-density particles is that they allow the use of greater amounts of enzymes in a given application without creating increased risk of sensitization as the result of immunoreactivity, while avoiding the particle settling that occurs with conventional particles. This is an important consideration for, e.g., workers in laundry detergent manufacturing facilities and consumers of laundry detergents. In some embodiments, the use of the particles in liquid enzyme suspensions allows the inclusion of 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more enzymes that would be acceptable in a comparable detergent composition that did not include the particles.

Methods of use

[109] The described low-density particles may be used in any application where enzymatic activity is desired from a low-water liquid compositions intended to be diluted prior with at least one volume of water in use. Upon dilution, at least about 50, 60, 70, 80, 90, or 95% of the enzyme is soluble and catalytically active in the diluted composition.

[110] In some embodiments, the application is cleaning and activation is performed in a bucket or other container, including a container to be cleaned. In the case of a laundry detergent composition, activation is typically performed in a washing machine. In the case of a dishwashing detergent composition, activation is typically performed in a dishwasher. In the case of a textile composition, activation is typically performed in a suitable bath. In the case of a food, beverage, or feed, activation is performed where needed to deliver active enzyme to the site of application.

[111] The low-density enzyme containing particles are particularly useful as components of a cleaning composition, such as a detergent composition, e.g., a laundry detergent composition or a dishwashing detergent composition. Especially preferred is a liquid laundry detergent composition. Such cleaning compositions typically comprise a cleaning adjunct, or preferably a combination of cleaning adjuncts. Typically the cleaning adjunct will be present in the composition in an amount from 0.001 to 99.9 wt%, more typically from 0.01 to 80 wt% cleaning adjunct. An exemplary formulation with suitable cleaning adjuncts in the form of a unit dose laundry detergent composition is provided, below. Such a unit dose formulations can comprise one, two three or more compartments. The components in each compartment may be different or the same, but the overall/total ingredients of the unit dose formulation have the same composition:

[112] The following examples are intended to illustrate, but not limit, the low-density particles.

EXAMPLES

Example 1

Enzyme activity assays

[113] The following assays may be used to measure enzyme activity.

A. 2-Aminobeiizoyl-L-alanylglycyl-L-leucyl-L-alanino-4-nitroben zylaniide (Abz- AGLA-Nba) assay to determine neutral metalloprotease activity

[114] Equipment! Temperature controlled microplate mixer (Eppendorf Thermomixer), temperature controlled microplate fluorescence reader (Molecular Devices SpectraMax M5, Gemini EM), and clear-bottom 300 μΐ, shallow 96-well microplates (Costar).

Reagents and Solutions: 4-Morpholineethanesulfonic acid (MES, Sigma, catalog # M-3671) buffer (52.6 mM MES/NaOH, 2.6 mM CaCl ₂ , 0.00526% (v/v) Tween-80, pH 6.5), 48 mM Abz-AGLA-Nba (Bachem catalog # H-6675) stock solution prepared in dimethylformamide (DMF) stored at room temperature shielded from light, assay solution (50 mM MES, 2.5 mM CaCl ₂ , 0.005% (v/v) Tween-80, 5% DMF, 2.4mM Abz-AGLA-Nba, pH 6.5), enzyme dilution buffer (50 mM MES, 2.5 mM CaCl ₂ , 0.005% (v/v) Tween-80, pH 6.5), substrate dilution buffer (50 mM MES, 2.5 mM CaCl ₂ , 0.005% (v/v) Tween-80, 5% DMF, pH 6.5). Enzyme stock solutions were diluted with enzyme dilution buffer to a concentration of approximately 1 ppm (1 ug/mL). B. amyloliquefaciens neutral metalloprotease expressed in

B. subtilis (NprE protease) was diluted to concentrations below 6 ppm (6 μg/mL).

[115] Procedure: Each enzyme dilution was assayed in triplicate.200 μί, assay solution was added in a 96-well microplate and shielded from light. The assay was started by transferring 10 μί of the working enzyme solution to the assay microplate. The solutions were mixed vigorously for 15 seconds. The assay plate was immediately transferred to the microplate reader and fluorescence intensity measurements were recorded at excitation of 350 run and emission of 415 nm to measure the proteolytic activity as the rate of appearance of the Abz-AG product. The reader was set to calculate the rate of RFU/sec (relative fluorescence units per second).

B. assay

to determine protease activity

[116] The following reagent solutions were used:

AAPF substrate stock: 160 mM (i.e., 100 mg/mL) sue- AAPF-pNA dissolved in

dimethylsulfoxide (DMSO), Stability buffer: 100 mM MES (pH 5.5) with 0.005% v/v Tween 80 (may optionally include 10 mM CaCl ₂ ), Activity buffer: 100 mM Tris (pH 8.5 or 8.6) with 0.005% v/v Tween-80 (may optionally include 10 mM CaCl ₂ ), Assay solution (substrate stock diluted 1:100 into activity buffe)r: 1.6 mM AAPF-pNA in 100 mlM Tris (pH 8.5 or 8.6).

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

[118] The assay was performed by adding 10 ul of diluted protease enzyme solution to each well of the assay solution plate. The solutions were mixed for 10 seconds, and the absorbance change was measured at 410 nm in a microplate reader at 25 T (set in kinetic mode, over 2 minutes). The subtilisin protease activity (AU = activity units) was calculated as mOD ₄ i ₅ /min x dilution factor, where mOD ₄ io refers to the optical density of the reaction product multiplied times 1000 as measured at 410 nm

C. Megazyme (Ceralpha) assay for alpha amylase activity determination

[119] This assay is a modification of the Megazyme alpha amylase assay procedure (Ceralpha method) (ICC Standard No. 303) (Megazyme International Ireland). Entire contents of one vial of the substrate, [non-reducing end-blocked p-nitrophenyl

maltoheptaoside (BPNPG7, 54.5 mg)] were dissolved in 10.0 mL of distilled water. 10 μΐ. of the substrate solution was added to wells of a 96-well plate and the plate warmed up to 40°C for 5 minutes. Ten microliters of enzyme samples (diluted in 50 mM sodium malate, 50 mM sodium chloride, 2 mM calcium chloride 0.005% sodium azide buffer pH 5.4) were added per well. A standard curve was assayed alongside each sample set. The plate contents were mixed at 900 rpm at 40°C for exactly 10 minutes. After incubation, 150 μί. of stopping buffer (20% (w/v) Trizma buffer ~pH 9) was added to each well. The solution was mixed at 900 rpm for 15 seconds and the end point absorbance was read at 400 nm

D. Para-nitrophenyl butyrate (pNB) assay to determine aryl esterase activity

[120] Aryl esterase activity was measured by hydrolysis of /Miitrophenylbutyrate (Sigma, N9876, 4-Nitrophenyl butyrate) dissolved in DMSO (Sigma #154938). The reaction mixture was prepared by adding 40 μΐ, of 100 mM pNB to 10 mL of assay buffer (0.1 M Tris-HCl pH 9.2). The background rate of hydrolysis was measured before the addition of enzyme at 405 nm The reaction was initiated by the addition of 10 μί, of diluted enzyme samples to 190 μΐ, of the reaction mixture and the change in absorbance at 410nm was measured at room temperature.

E. SDS-PAGE for protein analysis

[121] For quantitative protein determination, samples were analyzed by running the NuPAGE® Bis-Tris Electrophoresis System (Invitrogen, Carlsbad, CA). NuPAGE® Novex 4-12% Bis-Tris acrylamide gels (1 mm thickness, 8 cm x 8 cm, 15-well) were used with the NuPAGE® MES [2-(N-morpholino) ethane sulfonic acid] SDS Running Buffer pH 7.3-7.7. Samples were prepared according to the manufacturer's recommendation. Typical run conditions consisted of 200 V constant for 35 minutes. After electrophoresis, the gels were stained using the SimplyBlue™ Staining kit. Stained/destained gels were imaged using an Epson Perfection 3170 Photo scanner. Images were scanned using the Epson Scan version 2.65A in professional mode at 24-bit color and 300 dpi. Analysis of gel images were performed with ImageJ image processing toolkit. The relative intensity of each protein band was determined by measuring the integral area of each protein band, using this application. The integrated area consists of the area under the curve once the baseline on both sides is subtracted. Integrated areas for test samples and aliquots of known amount for each enzyme were determined, running all samples from same enzyme on one gel. The values obtained for the known protein samples were used to create a standard curve. A single parameter linear regression fit going through the origin (0) was generated and used to calculate the amount of enzyme (ppm) in each test sample, extrapolating the amount of protein corresponding to the signal (integrated area). Results are reported as ppm protein detected. Example 2

Method for determining particle size

A. Sieve shaker method

[122] In a first method, average particle size was determined using a RO-TAP® sieve shaker (W.S. Tyler, Mentor, Ohio, USA) and compatible 16, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, and 100 mesh sieves. 50 grams of particles was loaded into the shaker with the sieve stack installed in order of decreasing mesh size and shaker was run for five minutes. The weight of particles collected by each sieve was determined using an open pan balance, and the percentage of the total weight of particles is calculated. The relationship between the mesh size and the particle size cut-off is provided in Table 2.

Table 2. Mesh size versus particle size cut-off

B. Light scattering method

[123] In a second method, particle size was measured by laser diffraction, using a Malvern Instruments Mastersizer 2000 and associated Scirocco 2000 sample handling unit. The intensity of light (which is scattered as a laser beam as it passes through a dispersed particulate sample) is measured. The scattering pattern created is used to calculate the distribution of particle sizes that created the pattern. Approximately 1 g dry

powder/granular sample is dispersed in a controlled manner by vibrating the sample along the Scirocco 200 sample tray, and forcing it into the measurement chamber, or optical bench, under a pressure of 1 Bar. Whilst traversing the optical bench, a laser beam illuminates the dispersed particles. A series of detectors measure the intensity of light scattered by the particles over a range of angles. This scattering data is analyzed to produce the particle size range of the dispersed particles. For these calculations the refractive index of the sample is required (a standard of 1.52 was used). Both background and sample measurements were taken over a 12 second period, with a total of 12000 measurements being taken per sample.

Example 3

Determining particle true density by hexane displacement

[124] The density of hexane is first determined experimentally to take into account any impact of temperature on the measurements. On an analytical balance, 5 ml of hexane is added to a 5 ml volumetric flask and the value recorded. The density of hexane in g/ml is determined by dividing the mass by the volume. The calculated density will be used to calculate the volume of hexane when determining the density of particles.

[125] Approximately 1 g of particles is added to a 5 ml volumetric flask and the mass of the sample is recorded. Hexane is then added until the meniscus is at 5 ml and the mass of added hexane is recorded. The volume of hexane can be calculated from the experimental density calculation. The density of the sample can be calculated by dividing the mass of the sample by the volume of hexane displaced by the sample:

(1) Volume of hexane added = mass of hexane added / calculated hexane density

(2) Density of sample = mass of sample / (final volume - volume of hexane)

[126] The following measurement and calculation are provided as an example:

Mass of hexane in 5 ml volumetric flask = 3.3595 g

Density of methane = 3.3595 g/5 ml = 0.6719 g/ml

Mass of sample in 5 ml volumetric flask = 1.2307 g

Mass of hexane added to reach 5 ml = 2.9149 g

Volume of hexane added = 2.9149 g/0.6719 g/ml = 4.3383 ml

Density of sample = 1.2307 g/(5 ml - 4.3383 ml) = 1.86 g/ml

Example 4

Evaluating particle settling in laundry detergents

10 g of laundry detergent is added to a clear 15 ml test tube. Approximately 0.2 g of particles is added and mixed to form a well-dispersed suspension. At various time points (e.g., about 1 day, 1 week, 2 weeks and 1 month), the top 20% of the sample (i.e., the top 2 ml of the suspension) is visually examined to determine if any particles are still present. If particles are still present, they are scored "+." If no particles are present, they are scored "- ." Particles are considered non-settling if they are present in the top 20% of the sample after 1 month. Particles are considered settling if the top 20% is free of particles before the end of the assay period. A visual qualitative examination is generally sufficient to make the determination of whether the particles are settling or non-settling. Where a quantitative measurement is desired, the top 20% of the sample is carefully removed by pipetting and transferred to a fresh 15 ml test tube. The relative amount of particles in the top 20% and bottom 80% of the sample are measured based on absorbance at 600 nm and comparing the amounts to a standard curve. If less than 10% of the particles remain in the top 20% of the sample after 1 month, the particles are considered settling.

Example 5

Making and Testing Particles

[127] Various fluid bed particles were made using standard methods as exemplified in US6413749, which is incorporated by reference. The particle types, labeled A-G, are described in Table 3. The composition of the core (Core), second coating layer (SP2), third coating layer (SP3), as applicable, are indicated. The overall density (in g/cm ³ ) of the particles is and indicated as well as the median particle diameter, as determined by light scattering, as described, above. D(0.5) is the particle size distribution where 50% of the particles are at or below the specified diameter. D(0.9) is the particle size distribution where 90% of the particles are at or below the specified diameter. All particles included the indicated amount of a variant subtilisin protease (Enz), which allowed protein release and leakage to be measure using a standardized protease activity assay as described in

Example 1.

Table 3. Description of particles

n/d: no data; enz: enzyme; PVA: poly vinyl alcohol; exp: Expancel

[128] The particles were tested for several performance criteria in laundry detergents, including (i) settling, (ii) leakage of enzyme, and (iii) release of enzyme. Several of the particles were observed to have excellent overall performance in low-water detergent formulations.

[129] Table 4 summarizes the settling properties of particles A-F (described in Table 3) in the detergent composition from commercial Tide PODS® (i.e., the semi-opaque white component of the multi-chamber unit-dose product). Particle G refers to a commercially available competitor's product that includes the protease SAVINASE® (Novozymes). The settling assay was performed and scored as described in Example 4.

Table 4. Results of settling assay

[130] Particles B-F remained in suspension in the detergent compositions for at least a month. Particles A and G, with normal-density cores, settled rapidly in all tested detergent compositions.

[131] The leakage particles of A-E in the detergent composition obtained from commercial Tide PODS® at 37°C over 35 days is shown in the graph in Figure 1. Leakage was measured as the percentage of protease activity detected in the detergent composition based on the total amount of protease activity expected from the amount of protease coated onto the particle cores. Particle A (with a normal-density core) demonstrated very low leakage. Particle B demonstrated the greatest amount of leakage among the low-density particles with Particles C-E showing an intermediate an intermediate amount of leakage. The stability of particles A-F in the detergent composition obtained from commercial Tide PODS® at 37°C over 40 days is shown in the graph in Figure 2. Stability is measured as the percentage of residual protease activity in the particles based on the total amount of protease activity expected from the amount of protease coated onto the particle cores. Particles A, B, D, and F demonstrated the greatest stability, with particles F and particularly C demonstrating somewhat lower stability.

[132] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.