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WO2019006077A1 - Low-agglomeration, enzyme-containing particles - Google Patents
Low-agglomeration, enzyme-containing particles Download PDF
Douglas A. Dale
Danisco Us Inc
2018-06-28 Application filed by Danisco Us Inc filed Critical Danisco Us Inc
C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
C11D17/00—Detergent materials characterised by their shape or physical properties
C11D17/0008—Detergent materials characterised by their shape or physical properties aqueous liquid non soap compositions
C11D17/0013—Liquid compositions with insoluble particles in suspension
C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
C11D3/16—Organic compounds
C11D3/38—Products with no well-defined composition, e.g. natural products
C11D3/386—Preparations containing enzymes, e.g. protease, amylase
C11D3/38672—Granulated or coated enzymes
C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
Described are compositions and methods relating 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.
LOW-AGGLOMERATION, ENZYME-CONTAINING PARTICLES
FILED OF THE INVENTION
[01] The present compositions and methods relate to enzyme-containing particles with reduced agglomeration for inclusion in cleaning and other low-water compositions. The particles demonstrate low agglomeration in storage, and release active enzyme upon dilution of the low-water compositions with water.
[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.
[05] Enzymes can be provided 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, without agglomerating, 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, particles capable of isolating and stabilizing enzymes in a liquid composition without agglomerating in manufacturing and/or storage are provided, comprising: (a) a core, including an active component, and/or a core having a first coated layer comprising an active component immediately deposited upon the core; and (b) an outer-most coated layer comprising a hydrophobic, water-insoluble, water-disintegrating- material having an amount of water solubility of less than about 1 mg/mL in water at 25°C; wherein the coated layer in (b) fully disintegrates 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, and wherein the particles exhibit reduced aggregation in the liquid composition compared to otherwise identical particles comprising a third coated layer comprising a water-soluble polymer having a solubility of greater than about 1 mg/mL in water at 25°C.
2. In some embodiments, the particles of paragraph 1 further comprise, between (a) and (b), at least one additional layer comprising a water-soluble polymer and an active ingredient.
3. In some embodiments, the particles of paragraph 1 further comprise, between (a) and (b) at least one additional layer comprising a water-soluble polymer lacking an active ingredient.
4. In some embodiments of the particles of paragraphs 1 or 2, the core lacks an active component.
5. In some embodiments of the particles of paragraphs 1 or 3, the core includes an active component.
6. In some embodiments of the particles of any of the preceding paragraphs, the outer-most 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 a liquid composition containing the particles is contacted with at least one additional volume of water at 25°C. 7. In some embodiments of the particles of any of the preceding paragraphs, the outer-most coating represents less than 8%, less than 7%, less than 6%, or even less than 5% of the overall weight of the particle.
8. In some embodiments of the particles of any of the preceding paragraphs, the outer-most consists essentially of, or consists of, a hydrophobic, water-insoluble, water- disintegrating-material having an amount of water solubility of less than about 1 mg/mL in water at 25°C.
9. In some embodiments of the particles of any of the preceding paragraphs, the core has a density defined by the equations:
wherein pc is the density of the core in in g/cm3, p/ is the mass density of the liquid composition in g/cm3, xc is the mass fraction of the core in the particle, Dc is the diameter of the core in μΜ, and ΌΡ is the diameter of the particle in μΜ.
10. In some embodiments, the particles of any of the preceding paragraphs 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.
1 1. In another aspect, a method for reducing the agglomeration of particles in manufacturing and/or storage is provided, comprising coating the particles in an outer-most layer comprising a hydrophobic, water-insoluble, water-disintegrating-material having an amount of water solubility of less than about 1 mg/mL in water at 25°C.
12. In some embodiments of the method of paragraph 1 1, the outer-most consists essentially of, or consists of, a hydrophobic, water-insoluble, water-disintegrating-material having an amount of water solubility of less than about 1 mg/mL in water at 25°C.
13. In some embodiments of the method of paragraph 1 1 or 12, the outer-most 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 a liquid composition containing the particles is contacted with at least one additional volume of water at 25°C.
14. In some embodiments of the method of any of paragraphs 11 -13, the outer-most coating represents less than 8%, less than 7%, less than 6%, or even less than 5% of the overall weight of the particle.
[07] These and other aspects and embodiments of the compositions and methods are described, below. DETAILED DESCRIPTION
I. Definitions and abbreviations
[08] 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.
[09] 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.
[10] 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/ml. 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. [11] As used herein, the term "water-insoluble material" refers to a material that is not soluble in water even when mixed, such as a material with a solubility of less than 1 mg/ml in water at 25°C.
[12] As used herein, the term "hydrophobic" refers to a material that is repelled from (or repels) water. That is, there is no attractive forces between the material and water.
[13] As used herein, the term hydrophilic-lipophilic balance (HLB) refers to the empirical expression of relationship of hydrophilic and hydrophobic groups of a surfactant.
[14] As used herein, the term "disintegrating material" refers to a material that is not soluble in water, but has the ability to break down from larger particles into smaller particles that are capable of being suspended in water when mixed.
[15] As used herein, the term "agglomeration" refers to the phenomena wherein individual particles come together to form groups or clusters of multiple particles. The association between the particles can either be a loose association or a tight association, including by covalent bonds formed between the particles.
[16] 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).
[17] 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).
[18] As used herein, a "non-aqueous" solution contains less than about 2% water (w/w).
[19] 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.
[20] 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).
[21] 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.
[22] 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.
[23] As used herein, "cold" water is water having a temperature between freezing and about 25°C.
[24] As used herein, "room temperature" is 25 °C.
[25] As used herein, "warm" water is water having a temperature between about 26°C and about 37°C.
[26] As used herein, "hot" water is water having a temperature between about 37°C and boiling.
[27] As used herein, a "low" pH is a pH of less than about 7.
[28] As used herein, a "high" pH is a pH of greater than about 7.
[29] As used herein, the term "contacting," means bringing into physical contact, such as by placing a unit-dose package in an aqueous solution.
[30] As used herein, a "solid" form of a chemical component refers to a powder, crystals, granules, aggregates, paste or wax thereof.
[31] As used herein, a "liquid" form of a chemical component refers to a liquid, gel, or slurry.
[32] As used herein, "true density" refers to the mass of a particle divided by its volume, excluding open pores and closed pores.
[33] 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.
[34] 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.
[35] As used herein, the term "UFC Solids" refers to ultrafiltrate concentrate from a fermenter/bioreactor, and is synonymous with enzyme concentrate solids.
[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 "hard surface cleaning composition" refers to detergent compositions for cleaning hard surfaces such as floors, walls, tile, bath and kitchen fixtures, and the like.
[43] 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. [44] 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).
[45] "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.
[46] 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.
[47] 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).
[48] The following abbreviations may be used in the description. Definitions are also provided as needed throughout the description.
°C degrees Centigrade
AU activity units
CaCh calcium chloride
Cm centimeter
cm3 cubic centimeters
D(0.5) median particle size where 50% of the particles are at or below the specified diameter
dH20 or DI deionized water
eq. equivalents
ETOH ethanol g or gm Grams (note, below)
H2O water
hr hour
M molar
melting temperature melting temperature
mg milligrams
min minute
mL and ml milliliters
mm millimeters
mM millimolar
MW molecular weight
N normal
Na2S04 sodium sulfate
NaOH sodium hydroxide
nm nanometer
PE polyethylene
PEG poly ethylenegly col
ppm parts per million
PVA poly(vinyl alcohol)
PVP poly(vinylpyrrolidone)
sec seconds
T1O2 titanium dioxide
U units
v/v volume/volume
w/v weight/volume
w/w weight/weight
wt% weight percent
g micrograms
μΐ. and μΐ microliters
μm micrometer
μΜ micromolar
II. Particles with hydrophobic or water-insoluble, water-disintegrating coatings
[49] It is often desirable to incorporate particles with active agents into low-water liquid detergents in order to provide cleaning or other benefits. Unfortunately, conventional particles having outer surfaces made from materials with water-soluble or hydrophilic properties have been shown to agglomerate during and/or following incorporation into the low water detergents, such as during manufacturing, mixing, handling, transportation and/or storage.
[50] The present materials and methods overcome this undesirable agglomeration phenomenon by using hydrophobic and/or water-insoluble materials on the outer surface of the particles to prevent agglomeration from occurring, which materials readily disintegrate when the low water detergent is diluted into a wash liquid.
[51] Generally, the particles include (i) a core, (ii) at least one enzyme and/or other active component-containing layer, (iii) one or more additional layers and (iv) an outer-most coating with hydrophobic and/or water-insoluble properties that will rapidly disintegrate when diluted 1 : 1 in water. These components are described in greater detail.
A. Hydrophobic or water-insoluble and water-disintegrating surface coatings
[52] A key feature of the present compositions and methods, is a particle having an outermost coating with hydrophobic and/or water-insoluble properties to prevent the particles from agglomerating during manufacture, handling, transportation and/or storage, but which readily disintegrates when diluted into a wash liquor.
[53] Exemplary materials that have the necessary hydrophobic and/or water-insoluble properties but which are readily disintegrating upon dilution include, but are not limited to, natural waxes, such as carnauba, beeswax, palmitic wax, candelilla wax, synthetic waxes such as paraffin wax and microcrystalline wax, low HLB surfactants such as those with values below HLB=6, hydrophobically modified polyvinyl alcohol, hydrophobically modified starch such as those modified with fatty acid side chains, hydrophobically modified cellulosic polymers.
[54] Ideally, The melting point of the outer coting materials should be high enough to remain solid during processing and storage. Accordingly, the melting temperature should be above 40°C, above 45°C, above 50°C, above 55°C, or even above 60°C, depending on the process conditions and application.
[55] 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 after the low-water liquid composition containing the particles is contacted with at least one additional volume of water at 25°C.
[56] The outer coating composition need only to be incorporated at level sufficient to impart the desired surface properties to avoid agglomerations and to allow for rapid disintegration upon dilution in wash liquor. Accordingly, the outer coating should be as thin as possible. In some embodiments, the percent weight (wt/wt%) of the outer-most coating relative to the particle as a whole should be less than 8%, less than 7%, less than 6%, or even less than 5%. B. Coating containing enzymes and other actives
[57] The cores (to be described, infra) are coated with and/or may optionally contain one or more of a wide variety of enzymes or other actives. While the present description is focused on enzymes, it will be apparent that a myriad of other active components can be provided in a low-water composition using the same particles.
[58] Exemplary enzymes include acyl transferases, a-amylases, β-amylases, a- galactosidases, arabinosidases, aryl esterases, β-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-β-Ι, 4-glucanases, endo- beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, 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.
[59] 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 proteases 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.
[60] 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 .
[61] Suitable lipases include, but are not limited to Humicola lanuginosa lipase (see e.g. , EP 258 068, and EP 305 216), Rhizomucor miehei lipase (See e.g. , EP 238 023), 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 218 272), P. cepacia lipase (See e.g., EP 331 376), P. stutzeri lipase (See e.g. , GB 1,372,034), P. fluorescens lipase, Bacillus lipase {e.g. , B. subtilis lipase (Dartois et al. (1993) Biochem. Biophys. Acta 1131 :253-260); B. stear other mophilus lipase (see e.g., JP 64/744992); and B. pumilus lipase (see e.g., WO 91/16422)).
[62] 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 ?. 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).
[63] 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, W09623874, W09623873, W09741213, W09919467, WO0060060, WO0029560, W09923211, W09946399, WO0060058, WO0060059, W09942567, WO0114532, WO02092797, WO0166712, WO0188107, WO0196537, WO0210355, WO9402597, WO0231124, W09943793, W09943794, WO2004113551, WO2005001064,
WO2005003311, WO0164852, WO2006063594, WO2006066594, WO2006066596, WO2006012899, WO2008092919, WO2008000825, WO2005018336, WO2005066338, WO2009140504, WO2005019443, WO2010091221, WO2010088447, WO0134784, WO2006012902, WO2006031554, WO2006136161, WO2008101894, WO2010059413, WO2011098531, WO2011080352, WO2011080353, WO2011080354, WO2011082425, WO2011082429, WO2011076123, WO2011087836, WO2011076897, W094183314, W09535382, WO9909183, WO9826078, WO9902702, W09743424, W09929876, WO9100353, WO9605295, WO9630481, WO9710342, WO2008088493, WO2009149419, WO2009061381, WO2009100102, WO2010104675, WO2010117511, WO2010115021, WO2013184577, W09418314, WO2008112459, WO2013063460, WO10115028, WO2009061380, WO2009100102, WO2014099523, WO2015077126A1, WO2013184577, WO2014164777, PCT/US12/70334, PCT/US 13/74282, PCT/CN2013/077294,
PCT/CN2013/077134, PCT/CN2013/077137, PCT/CN2013/077142, PCT/CN2012/087135, PCT/US 12/62209, PCT/CN2013/084808, PCT/CN2013/084809, and PCT/US 14/23458. 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™,
RAPIDASE® and MAXAMYL® P, PREFERENZ® SI 00, PREFERENZ® SI 10, and PREFERENZ® SI 000 (Genencor).
[64] Suitable cellulases include but are not limited to those having color care benefits (see e.g., EP 0 495 257). 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.
[65] 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 MANNAWAY®.
[66] 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.
[67] Suitable perhydrolases include the enzyme from Mycobacterium smegmatis. 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 smegmatis
perhydrolase, or homolog, includes the S54V substitution.
[68] 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 et al, J. Mol. Biol, 330:593-606 (2003)).
[69] 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).
[70] 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.
[71] The cores may further be coated with and/or contain one or more additional components, such as bleach catalysts, 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.
C. Additional coatings
[72] Depending on the particular embodiments of the present particles and methods, there may be included at least one non-aqueous, water-soluble coating 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 than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, or even greater than 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.
[73] 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, 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, , and; and mixtures, thereof.
D. Cores
[74] In some embodiments, the core of the present particles, which feature an outer, hydrophobic or water-insoluble, water-disintegrating, outer-coating, is not critical to the present compositions and methods, and may be of a conventional nature. Commonly used material are salts and sugars and other relatively inexpensive, water soluble materials. The core may be inert, or may feature active ingredients. In other embodiments, the core may include some, or even all the active agents, such as enzymes, mentioned, above.
[75] In particular embodiments, the core is selected such that the particles have an overall particle density close to the density of the low-water liquid composition in which they are suspended or intended-to-be suspended. This distinguishes further distinguishes the present particles from conventional particles, which typically have a higher density, and tend to settle out of suspension.
[76] The low density of the particles may be 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.
1. Cores made from low-density materials
[77] The core of the particle may be 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/cm3 and 1.3 g/cm3, depending on the dissolved solutes, and the density of the core should be within 0.5 g/cm3, 0.4 g/cm3, 0.3 g/cm3, 0.2 g/cm3, or even 0.1 g/cm3 of the density of the liquid.
[78] 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:
l')] In the equation, above, vs is the particle's settling velocity (e.g. , m/s), which is vertically downwards if pp > pf md vertically upwards if pp < pf ), g is gravitational acceleration (m/s2), pp is the mass density of the particle (e.g. , kg/m3), pf is the mass density of the fluid (kg/m3), 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 μηι.
[80] 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 μιτι and a radius of 125 μιτι. For this particle, the absolute value of the density difference between particle density (pp) and fluid density (p7), i.e. , (ρ,,-ρ/ or ApP ) should be no more than 0.5 g/cm3, so any particle that is larger or smaller than 250 μιτι diameter is acceptable as long as the settling rate (vs) does not increase. With the liquid medium viscosity fixed, any particle will have the same vs when:
(|Δρρ | * Όρ2) = (0.5)*(250)2 where is the overall diameter of the particle. Such a particle will not settle (or rise) faster than Vs when for the maximum density the difference is given by:
|Δρρ | < (0.5)*(250)2 / D/ or |Δρρ/| <_ 31250/D/
[81] Expressed in another way:
P;> /+ 31250/D/, to avoid settling
P;> > P/- 31250/D/, to avoid floating
[82] Using the latter formula, the maximum density difference (|Δρ^) required as a function of overall particle diameters (D^) can be calculated, as shown in Table 1 :
Table 1. Maximum density differences for different overall particle diameters
[83] The above relationship can also be extended to define the constraints on the density of the core (pc) within the overall particle (p^). The density of the core can be related to the density of the overall particle according to the relationship: pdpP = (rtic/vc)/ (ϊΏρ/γρ) where rric and mp represent the mass of the core and mass of the overall particle, respectively, and v^ and vc represent the respective volumes of the overall particle and the core. Rearranging:
[84] Expressing the volumes in terms of diameters of the core (Dc) and particle (ΌΡ) and representing the mass fraction of the core as xc, we obtain:
or we can show the particle density in terms of core density:
[85] 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 pc-p or Apcf.
|pc *(Dc/Dp)(1/3 xc -p/| <. 18750/D/
[86] Therefore:
[87] 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), hydroxy ethylmethyl 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.
[88] 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.
[89] 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 pay load to core weight ratio but are more readily aerosolized. Particles smaller than 10 μιτι, and especially smaller than 5 μιτι, should be avoided for respiratory tract safety reasons. Particles smaller than about 40 μιη are not visible to the human eye. Larger particles, e.g., greater than about 100 μηι, 150 μηι, or even 200 μιτι, 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 μπι, 50-150 μηι, 100-150 μηι, 100-200 μηι, 150-250 μηι, 200-250 μηι, 200-300 μηι, 250-300 μηι, 300-350 μηι, 300-400 μηι, 350-500 μηι, 400-550 μηι, 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.
[90] 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 μη 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.
2. Cores with density modifiers
[91] The overall density of the particles can also 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 that 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.
[92] Exemplary density modifiers are materials having a density of less than 1 g/cm3, and include starch, cellulose fibers, diatomaceous earth, feather particles, zeolites (such as used for molecular sieving), flour, milled plant derived fragments such as com 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 aluminosihcates 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.
3. Properties of particles with low-density cores
[93] 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/cm3, less than 1.5 g/cm3, less than 1.4 g/cm3, less than 1.3 g/cm3, or even less than 1.2 g/cm3, for example, 1.0-1.6 g/cm3, 1.0-1.5 g/cm3, 1.0-1.4 g/cm3, 1.0-1.3 g/cm3, and 1.0-1.2 g/cm3, 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/cm3, less than ±0.4 g/cm3, less than ±0.3 g/cm3, less than ±0.2 g/cm3, even less than ±0.1 g/cm3 or even less than ±0.05 g/cm3. 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.
[94] 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 enzymes 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.
III. Preparation of particles
[95] 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).
IV. Compositions containing the liquid enzyme suspensions
[96] The present 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™.
[97] 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.
[98] 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.
[99] 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. V. Methods of use
[100] The present particles may be used in any application where enzymatic activity is desired from a low-water liquid composition 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.
[101] 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.
[102] The 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.
[103] The following examples are intended to illustrate, but not limit, the low-density particles.
Evaluation of Particle Agglomeration in Low-Water Detergent Compositions
10 g of laundry detergent was added to a clear 15 ml test tube. Approximately 0.2 g of particles was added and mixed to form a well-dispersed suspension. The tubes were placed on 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. The degree of agglomeration was visually assessed after 7 days. An ideal result was that all particles remain as individual particles that are not associated with any other particle. Less ideal results include the observation of groupings of small numbers of particles. The least ideal results are the agglomeration of tens to hundreds of particles, or more, together.
N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (AAPF-pNA) assay to measure protease activity
[104] The following reagent solutions were used:
AAPF substrate stock: 160 mM (i.e. , 100 mg/mL) suc-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 CaCh), Activity buffer: 100 mM Tris (pH 8.5 or 8.6) with 0.005% v/v Tween-80 (may optionally include 10 mM CaCh), 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).
[105] 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 of assay solution was added to each well of a 96-well plate.
[106] The assay was performed by adding 10 μΐ 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 °C (set in kinetic mode, over 2 minutes). The subtilisin protease activity (AU = activity units) was calculated as mOD4i5/min x dilution factor, where mOD4io refers to the optical density of the reaction product multiplied times 1000 as measured at 410 nm.
Making and Testing Particles
[107] Various particles were made using standard fluid bed methods, as exemplified in US 6413749, which is incorporated by reference. The particle types, labeled A-C, are summarized in Table 2. The composition of the core (Core), a first coating layer containing the enzyme(s) with or without a binder and/or with or without a density modifier (SP1), second coating layer (SP2), third coating layer (SP3), as applicable, are indicated. 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 2
Table 2. Description of particles
[108] The particles were tested for agglomeration performance criteria in low water laundry detergents. Particle B remained as individual particles suspended in the detergent after the evaluation period, whereas particle A had significant agglomeration with tens to hundreds of particles agglomerated together and no longer suspended in the detergent. Particles C are expected to have intermediate properties.
[109] 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.
CLAIMS What is claimed is:
1. Particles capable of isolating and stabilizing enzymes in a liquid composition without agglomerating in manufacturing and/or storage, comprising:
(a) a core, including an active component, and/or a core having a first coated layer comprising an active component immediately deposited upon the core; and
(b) an outer-most coated layer comprising a hydrophobic, water-insoluble, water- disintegrating-material having an amount of water solubility of less than about 1 mg/mL in water at 25°C;
wherein the coated layer in (b) fully disintegrates 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, and
wherein the particles exhibit reduced aggregation in the liquid composition compared to otherwise identical particles comprising a third coated layer comprising a water-soluble polymer having a solubility of greater than about 1 mg/mL in water at 25°C.
2. The particles of claim 1 , comprising, between (a) and (b) at least one additional layer comprising a water-soluble polymer and an active ingredient.
3. The particles of claim 1 , comprising, between (a) and (b) at least one additional layer comprising a water-soluble polymer lacking an active ingredient.
4. The particles of claims 1 or 2, wherein the core lacks an active component.
5. The particles of claims 1 or 3, wherein the core includes an active component.
6. The particles of any of the preceding claims, wherein the outer-most 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 a liquid composition containing the particles is contacted with at least one additional volume of water at 25°C.
7. The particles of any of the preceding claims, wherein the outer-most coating represents less than 8%, less than 7%, less than 6%, or even less than 5% of the overall weight of the particle.
8. The particles of any of the preceding claims, wherein the outer-most consists essentially of, or consists of, a hydrophobic, water-insoluble, water-disintegrating-material having an amount of water solubility of less than about 1 mg/mL in water at 25°C.
9. The particles of any of the preceding claims, wherein the core has a density defined by the equations:
10. The particle of any of the preceding claims, having 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.
1 1. A method for reducing the agglomeration of particles in manufacturing and/or storage, comprising coating the particles in an outer-most layer comprising a hydrophobic, water-insoluble, water-disintegrating-material having an amount of water solubility of less than about 1 mg/mL in water at 25°C.
12. The method of claim 1 1, wherein the outer-most consists essentially of, or consists of, a hydrophobic, water-insoluble, water-disintegrating-material having an amount of water solubility of less than about 1 mg/mL in water at 25°C.
13. The method of claim 11 or 12, wherein the outer-most 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 a liquid composition containing the particles is contacted with at least one additional volume of water at 25°C.
14. The method of any of claims 1 1 -13, wherein the outer-most coating represents less than 8%, less than 7%, less than 6%, or even less than 5% of the overall weight of the particle.
PCT/US2018/039927 2017-06-30 2018-06-28 Low-agglomeration, enzyme-containing particles WO2019006077A1 (en)
PCT/US2018/039927 WO2019006077A1 (en) 2017-06-30 2018-06-28 Low-agglomeration, enzyme-containing particles
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Family is looking for a housekeeper of their apartment in Monaco, Monte Carlo. The family offers an accommodation and food.
The candidate will travel with the family in Switzerland as well, and will be stewardess on their yacht in the summer.
Experience in Yachting required. The position is on a permanent contract, flexibility required.


text:
Why Temperature Remains Constant during a Phase Change
Thanks to physics, we know that phase changes occur when materials change state, going from liquid to solid (as when water freezes), solid to liquid (as when rocks melt into lava), liquid to gas (as when you boil water for tea), and so on. When the material in question changes to a new state — liquid, solid, or gas (you can also factor in a fourth state: plasma, a superheated gas-like state) — some heat goes into or comes out of the process without changing the temperature.
You can even have solids that turn directly into gas. As dry ice (frozen carbon dioxide gas) gets warmer, it turns into carbon dioxide gas. This process is called sublimation.
Imagine you're calmly drinking your lemonade at an outdoor garden party. You grab some ice to cool your lemonade, and the mixture in your glass is now half ice, half lemonade (which you can assume has the same specific heat as water), with a temperature of exactly 0 degrees Celsius.
As you hold the glass and watch the action, the ice begins to melt — but the contents of the glass don't change temperature. Why? The heat (thermal energy) going into the glass from the outside air is melting the ice, not warming the mixture up. So does this make the equation for heat energy
useless? Not at all — it just means that the equation doesn't apply for a phase change.
If you graph the heat added to a system versus the system's temperature, the graph usually slopes upward; adding heat increases temperature. However, the graph levels out during phase changes, because on a molecular level, making a substance change state requires energy. After all the material has changed state, the temperature can rise again.
Phase changes of water.
Imagine that someone has taken a bag of ice and thoughtlessly put it on the stove. Before it hit the stove, the ice was at a temperature below freezing (–5 degrees Celsius), but being on the stove is about to change that. You can see the change taking place in graph form in the figure. (The specific heat of ice is around 2.1 x 103 J/kg-C)
that the temperature of the ice will increase linearly as you add more heat to it, as you see in the graph.
However, when the ice reaches 0 degrees Celsius, the ice is getting too warm to hold its solid state, and it begins to melt, undergoing a phase change. When you melt ice, breaking up the crystalline ice structure requires energy, and the energy needed to melt the ice is supplied as heat. That's why the graph in the figure levels off in the middle — the ice is melting. You need heat to make the ice change phase to water, so even though the stove adds heat, the temperature of the ice doesn't change as it melts.
As you watch the bag of ice on the stove, however, you note that all the ice eventually melts into water. Because the stove is still adding heat, the temperature begins to rise, which you see in the figure. The stove adds more and more heat to the water, and in time, the water starts to bubble. "Aha," you think. "Another phase change." And you're right: The water is boiling and becoming steam. The bag holding the ice seems pretty resilient, and it expands while the water turns to steam.
You measure the temperature of the water. Fascinating — although the water boils, turning into steam, the temperature doesn't change. Once again, you need to add heat to incite a phase change — this time from water to steam. You can see in the figure that as you add heat, the water boils, but the temperature of that water doesn't change.
What's going to happen next, as the bag swells to an enormous volume? You never get to find out, because the bag finally explodes.

redpajama_set_name: RedPajamaCommonCrawl


We don't just see potential.
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Carol Liao (center) celebrates with her colleagues from The Boston Consulting Group.
EY's Kris Pederson accepting her Lifetime Achievement Award from Peggy Vaughan, her award presenter.
RSM's Beth Johnson with her award presenter, Steve Ems.


text:
Boston Red Sox |
Red Sox LHP Eduardo Rodriguez out for season…
Red Sox LHP Eduardo Rodriguez out for season due to heart condition
FORT MYERS, FL. – FEBRUARY 24: Eduardo Rodriguez #57 of the Boston Red Sox pitches during a spring training at JetBlue Park Complex on February 24, 2020 in Fort Myers, Florida. (Staff Photo By Matt Stone/MediaNews Group/Boston Herald)
By Steve Hewitt | stephen.hewitt@bostonherald.com | Boston Herald
PUBLISHED: August 1, 2020 at 5:08 p.m. | UPDATED: August 1, 2020 at 10:43 p.m.
Eduardo Rodriguez had seemed to be progressing toward a return in 2020, but that optimism met reality on Saturday.
Rodriguez, who had been working his way through complications from COVID-19, has been shut down for the season, chief baseball officer Chaim Bloom announced prior to Saturday night's game against the Yankees.
Rodriguez, who tested positive for COVID-19 last month and recovered, was discovered to have a case of myocarditis, which is inflammation of the heart muscle. After monitoring it since Rodriguez's initial shutdown last week, it was concluded that while the case is mild, it will not be resolved by the end of this season. The Red Sox expect him to make a full recovery and return to pitch in 2021.
"We are confident he's going to make a full recovery and that his long-term prognosis is excellent, but the fact of the matter is there just isn't enough time left this season to safely ramp him back up to pitching," Bloom said. "Obviously it's unfortunate, not something any of us are happy about, least of all Eduardo. But we need to make sure we're taking care of him. This is not something to mess around with so we're shutting him down."
Bloom cautioned that while Rodriguez's heart is functioning normally and the history of the ailment suggests long-term recovery, there isn't much data about it after recovery from COVID-19 since the virus is so new. Still, they are confident about his eventual return to full health.
The loss of Rodriguez, who was projected as the No. 1 starter after Chris Sale had Tommy John surgery, is another big blow to a Red Sox starting rotation that was expecting him back at some point this season and desperately needs a boost.
But on a bigger picture, the news was a reality check for the team with MLB's season in jeopardy right now due to the number of positive COVID-19 tests around baseball. Rodriguez, who will return home to Florida, told his teammates the news over Zoom earlier Saturday, and it hit home.
"Here is one of the best pitchers in the game last year and he's not able to perform on the field," Red Sox manager Ron Roenicke said. "And at a young age, young people aren't safe from this. It hits because it hits different people in different ways, it doesn't matter if you're young or old, you're more at risk when you're older but it hits the young pretty hard too at times. Eddie, just unfortunately, he's one of those guys that it hit hard and to lose an entire season is pretty rough on anybody. …
"You just feel bad for Eddie, with the year that he had last year and the year that he had in the offseason and coming into spring training hopefully following it with another great year. We hope this thing heals up and next year we have the same great pitcher."
Bloom said that regardless of Rodriguez's status, it didn't change any short-term plans he might have had to fortify a depleted rotation. But without Rodriguez, Bloom acknowledged that it "makes the mountain a little higher." He said they'll continue to scour the market and monitor his arms in Pawtucket, some of whom have performed well in intrasquad games.
As the future of the season continues to look uncertain, Bloom said he is still planning with the assumption that it will be a full season. He still believes the Red Sox can compete this year, but he's also balancing that with the future.
"We want to compete, we want to win right now, but consistent with everything I've said since the day that I got here, as much as we want to win right now, we need to be mindful of the future," Bloom said. "We can't do it at the expense of everything that comes after 2020, so I think recognizing that we're dealing with a lot more now with the virus and all the things that might impose on this season, we have to keep that same mindset. We certainly want to win this year, we need to make sure that we're setting ourselves up really well for the long term."
Martinez sits out
A night after he declared his swing is "awful" right now, J.D. Martinez was out of the lineup Saturday against the Yankees. After starting hot, going 5-for-9 in his first two games, the designated hitter is 2-for-23 since and he had a talk with Roenicke after Friday's loss.
"I listen to the players and sometimes when I ask them about how they're doing and they need a day, if it makes sense to do it I'm hoping by doing this that maybe we'll get the next two months that will be really good from him," Roenicke said. "They're hard decisions because we know how important all these games are." …
Jonathan Lucroy, who was designated for assignment last week, cleared waivers and is back with the organization. He was outrighted to the Red Sox' alternate training site in Pawtucket. … Austin Brice will start for the Red Sox in Sunday night's series finale against the Yankees.
Steve Hewitt | Sports reporter
More in Boston Red Sox
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Burn Ban lifted as of 1/28/19.
Development Permits are required in Wilson County before construction starts.
Court-appointed defense attorneys, please sign up for our new Indigent Defense system at https://attorney.cuc.org.


text:
Q: Puma: Unable to load application: NameError: uninitialized constant When I am trying to deploy an rails application to Heroku, I am getting this error:
Puma starting in single mode...
2017-02-27T15:08:03.908788+00:00 app[web.1]: * Version 3.7.0 (ruby 2.3.1-p112), codename: Snowy Sagebrush
2017-02-27T15:08:03.908789+00:00 app[web.1]: * Min threads: 5, max threads: 5
2017-02-27T15:08:03.908790+00:00 app[web.1]: * Environment: production
2017-02-27T15:08:05.942041+00:00 app[web.1]: ! Unable to load application: NameError: uninitialized constant Api::V1::AController
2017-02-27T15:08:05.942133+00:00 app[web.1]: bundler: failed to load command: puma (/app/vendor/bundle/ruby/2.3.0/bin/puma)
2017-02-27T15:08:05.942266+00:00 app[web.1]: NameError: uninitialized constant Api::V1::AController

Here is the link to the full log
If I try to run the following code on my dev environment, everything works just fine:
bundle exec puma -C config/puma.rb

I ran rails on my local machine using the -e production flag and it runs ok, no error appears. This happens only when I deploy to Heroku.
puma.rb
workers Integer(ENV['WEB_CONCURRENCY'] || 2)
threads_count = Integer(ENV['RAILS_MAX_THREADS'] || 5)
threads threads_count, threads_count

preload_app!

rackup      DefaultRackup
port        ENV['PORT']     || 3000
environment ENV['RACK_ENV'] || 'development'

on_worker_boot do
    ActiveRecord::Base.establish_connection
end

What I've tried:

*

*Created a new instance on Heroku just to be sure there was not a problem with the current instance

*Tested multiple versions of Puma

*Checked development.rb and production.rb to see what was different

What I am doing right now:

*

*Going through each commit to find what changed in the app and try to find the cause of the app

I believe I am overcomplicating the problem... Any thoughts on this?

A: Try to reproduce this problem on your local machine by setting all environmental variables to the same values as on Heroku. Or at least set all variables used by puma:
RAILS_ENV=production RACK_ENV=production WEB_CONCURRENCY=1 RAILS_MAX_THREADS=5 bundle exec puma -C config/puma.rb

Also I've noticed that you are using threads. Is it on purpose? I mean ruby ecosystem is not famous for thread safety. Try changing RAILS_MAX_THREADS to 1 and bump WEB_CONCURRENCY to higher number.

A: For me, the application was not using ActiveRecord, but there were still references to it. Because require "active_record/railtie" was commented out, it broke on production.

redpajama_set_name: RedPajamaStackExchange


text:
Q: Applying Green's Theorem I'm studying for a grad-school preliminary exam, and came across this problem which I am unable to solve.

Let $C$ be a closed curve in the plane $ax + by + cz = 0$ (where $a,b,c \in \mathbb{R}$ are not all zero $0$), enclosing a region with area $S$.  Evaluate
$$ I:= \oint\limits_C \left| \begin{array}{ccc}
dx & dy & dz \\
a  & b  & c  \\
x  & y  & z \end{array} \right|
$$
where the integral along $C$ is counterclockwise relative to the normal direction $(a,b,c)$ to the plane.

Expanding the determinant yields $$
I = \oint\limits_C (bz - cy)dx + (cx - az)dy + (ay - bx)dz. $$
If we call $\Omega$ the region bounded by $C$, an application of Green's Theorem (or Stokes Theorem, if you prefer) yields $$
I = 2\iint\limits_\Omega a dydz + bdzdx + c dxdy.
$$
From here, I'm not sure what to do; I suspect that it's a simple solution and that I've just forgotten my vector calculus.  Anyway, any help is appreciated.

EDIT: Substitute in $z = \frac{1}{c}\left(-ax - by \right)$ and then applying Green's Theorem doesn't bring me any closer.  Making such a substitution yields $$ I = \frac{a^2 + b^2 + c^2}{c} \iint_\Omega dxdy $$
If $c \neq 0$ as required to make such a substitution, it is not the case that $\iint dxdy = S$.

SECOND EDIT:  I've solved the problem, making use of the following version of Stokes' theorem: $$ \iint_\Omega (\nabla \times \mathbf{F} ) \cdot \mathbf{n} dS = \oint_{\partial \Omega} \mathbf{F} \cdot d\mathbf{r}. $$
The above can be written as \begin{align*} I &= \oint\limits_C (bz - cy,cx - az,ay - bx)\cdot d\mathbf{r} \\
&= \iint_\Omega (\nabla \times (bz - cy,cx - az,ay - bx)) \cdot \left(\frac{1}{\sqrt{a^2 + b^2 + c^2}}(a,b,c)\right) dS  \\
&=\iint_\Omega (2a,2b,2c) \cdot \left(\frac{1}{\sqrt{a^2 + b^2 + c^2}}(a,b,c)\right) dS  \\
&=  2 \sqrt{a^2 + b^2 + c^2} \iint_\Omega dS  \\
&= 2\sqrt{a^2 + b^2 + c^2} (\text{Area of } \Omega) \end{align*}

A: Use the plane equation as
$$ ax + by + cz = 0  \implies z = -\frac{a}{c}x -\frac{b}{c}y  \implies dz= -\frac{a}{c}dx - \frac{b}{c}dy $$
and substitute for $z$ and $dz$ in the integral
$$ \oint\limits_C (bz - cy)dx + (cx - az)dy + (ay - bx)dz. $$
Then Green's theorem is straightforward.


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to tell the difference between board versions. However, I'm looking for ways in software to distinguish which board I'm currently using. The OS may know what version it's running on, but I guess every distribution has it's own ways to remember the HW configuration. The OS may not even care if it's running on a CB2 20151211 TSD or on a CubieBoard2 20120909 DualCard.
However, the problem I'm trying to solve is much less complex: all I want to know (right now) is whether there is a battery backed up Real Time Clock available or not. I know the 20151211 has a RTC on board, but older versions of the CB2 have no RTC. I'd like know a way for the software to tell the difference.