US3629123A

US3629123A - Stabilized amylase compositions

Info

Publication number: US3629123A
Application number: US883356A
Authority: US
Inventors: Anthony E O'reilly; Charles A Hong
Original assignee: Procter and Gamble Co
Current assignee: Procter and Gamble Co
Priority date: 1969-12-08
Filing date: 1969-12-08
Publication date: 1971-12-21
Anticipated expiration: 1988-12-21
Also published as: ES386245A1; SE367206B; CA941768A; NL7017809A; ZA708277B; JPS5120528B1; GB1306990A; FR2073004A5; AT313448B; IE34789L; DE2060072A1; IE34789B1; BE759964A

Abstract

A STABILIZED AMYLASE COMPLEX CONSISTING ESSENTIALLY OF AMYLASE AND HYDROLYSIS PRODUCTS OF CHEMICALLY-MODIFIED CROSS-LINKED PREGELATINIZED AMYLOPECTIN IS DESCRIBED, TOGETHER WITH A PROCESS FOR PREPARING SAID COMPLEX WHICH COMPRISES MIXING AMYLASE AND CROSS-LINKED PREGELATINIZED AMYLOPECTIN IN AN AQUEOUS SLURRY HAVING A TEMPERATURE IN THE RANGE OF 35*F. TO 110*F. AND A PH OF 4.5 TO 8.5, THE RATIO BY WEIGHT OF AMYLASE TO CROSS-LINKED PREGELATINIZED ANYLOPECTIN BEING IN THE RANGE OF 1:1 TO 1:200. A LAUNDRY DETERGENT COMPOSITION IS ALSO DECRIBED WHICH CONSISTS ESSENTIALLY OF FROM .001% TO 3% BY WEIGHT OF A STABILIZED AMYLASE COMPLEX MECHANICALLY MIXED WITH A GRANULAR DETERGENT COMPOSITION WHICH CONSISTS OF A DETERGENT AND A BUILDER. GRANULAR AMYLASE CONTAINING DETERGENT COMPOSITIONS ARE ALSO DESCRIBED WHICH CONTAIN A STABILIZED AMYLASE COMPLEX ATTACHED TO A WATER-SOLUBLE CARRIER GRANULE.

Description

United States Patent Oflice Int. Cl. Clld 7/42 US. Cl. 252-89 15 Claims ABSTRACT OF THE DISCLOSURE A stabilized amylase complex consisting essentially of amylase and hydrolysis products of chemically-modified cross-linked pregelatinized amylopectin is described, together with a process for preparing said complex which comprises mixing amylase and cross-linked pregelatinized amylopectin in an aqueous slurry having a temperature in the range of 35 F. to 110 F. and a pH of 4.5 to 8.5, the ratio :by weight of amylase to cross-linked pregelatinized amylopectin being in the range of 1:1 to 1:200.

A laundry detergent composition is also described which consists essentially of from 001% to 3% by weight of a stabilized amylase complex mechanically mixed with a granular detergent composition which consists of a detergent and a builder.

Granular amylase containing detergent compositions are also described which contain a stabilized amylase complex attached to a water-soluble carrier granule.

BACKGROUND This invention relates to a discovery for stabilizing enzymes and specifically for sustaining the enzymatic activity of amylase enzymes. Amylolytic enzymes such as a-amylase of Bacillus subtilis origin find many commercial applications based upon their suitability for the liquefaction of starch. Large quantities of amylase are consumed in the textile (desizing) and starch processing industries.

Within the last few years, enzyme-containing detergent compositions have been marketed and have met with considerable commercial success. These compositions containing amylases and proteases are effective in selectively degrading substrates such as starches and protein stains. It is well known, however, that enzymes are highly sensitive proteinaceous organic compounds which can be readily degraded and destroyed by environmental conditions such as those involved in processes for preparing detergent compositions, the many different types of ingredients which comprise a detergent composition, including 'perborate bleaching agents, as well as the dilferent storage and packaging conditions which are an important factor in marketing enzyme containing detergent compositions.

Virtually, all enzymes, including amylases and proteases are subject to the same inactivating conditions although perhaps to varying degrees. However, it is now known that proteases can be rendered substantially stable and proteolytic activity can be sustained for considerably longer periods of time by adding partially hydrolyzed and partially solubilized collagen to protease-containing detergent compositions. Until now, however, no over-all satisfactory method has been discovered for effectively stabilizing amylase enzymes or providing amylase-containing detergent compositions with sustained enzymatic activity.

SUMMARY OF THE INVENTION It has now been discovered that a stabilized amylase complex can be prepared having sustained enzymatic activity by mixing amylase and cross-linked pregelat- 3,629,123 Patented Dec. 21, 1971 inized amylopectin in an aqueous slurry having a temperature in the range of 35 F. to 110 F., and a pH of 4.5 to 8.5.

The amylase complex can be usefully employed in the form of the suspension formed by the reaction which occurs between amylase and cross-linked pregelatinized amylopectin. Alternatively, the amylase complex can be recovered as a solid from the suspension reaction mixture and used as such in any application where amylolytic enzymatic activity is needed.

There are several alternative embodiments for incorporating the amylase complex in detergent compositions. Most simply, the amylase complex in granular or powder form can be mechanically mixed with a granular detergent composition. By another embodiment, the amylase complex can be sprayed onto a suitable enzyme carrier granule (sometices called a carrier granule, granular carrier, or just a carrier) thereby becoming attached to the carrier granule. The carrier granule can then be added to a granular detergent composition. The carrier granule can be any material (porous or otherwise) to which the amylase can be attached or adsorbed and which would not interfere with the overall washing and laundering process or other intended purpose. According to preferred practice of this invention, however, the carrier granule is a granular detergent composition which can be either a granular detergency builder or a granular mixture of an organic synthetic detergent and a detergency builder.

A process for preparing the amylase complex per se and a process for preparing a granular amylase-containing laundry detergent composition are also described herein as preferred embodiments.

DETAILED DESCRIPTION OF INVENTION According to the present invention, it has now been discovered that a stabilized amylase composition can be prepared having sustained amylolytic activity by forminga unique complex between amylase and cross-linked pregelatinized amylopectin by mixing amylase, cross-linked pregelatinized amylopectin, and water. The result of practicing this invention results in the preparation of a stabilized amylase complex consisting essentially of amylase and hydrolysis products of chemically modified crosslinked pregelatinized amylopectin. The precise mechanism for the reaction which occurs leading to the stable formation is not known, nor is the structure of the complex known. Water in the mixture, however, is a critical factor. The water can be employed in any form, vapor, liquid, etc. Ordinarily the reaction between the amylase and the cross-linked pregelatinized amylopectin is performed in an aqueous solution by mixing amylase and cross-linked pregelatinized amylopectin in an aqueous slurry having a temperature in the range of 35 F. to 110 F., and a pH of 4.5 to 8.5 The proportions for mixing amylase to crosslinked pregelatinized amylopectin are important. A general range of proportions of 1:1 to 1:200 can be used; preferred is a range of 111.5 to 1:40. If proportions less than 1:1 are used, the insoluble complex will not be formed; likewise, proportions exceeding 1:200 do not result in the formation of the amylase complex. For optimum results, the temperature of the water should be between 50 F. and F. with a pH of 5.5 to 7.5, and most preferably about 6.8.

Temperatures below 35 F. may require special equipment such as cooling apparatus without any apparent advantage in yields of complex formation or stability of the resulting complex. Caution must be exercised in exceeding F because of damage to. the amylase. Acid conditions below pH 4.5 and alkaline conditions above pH 8.5 can adversely elfect the stability of the amylase as well as complex formation.

In practicing the complex preparation, the aqueous solution should be stirred while the reaction between the amylase and cross-linked pregelatinized amylopectin occurs. This can be performed by any suitable means such as any ordinary mixing device or specifically adapted apparatus.

The amount of water used for the complex reaction is important in terms of how much amylase and cross-linked pregelatinized amylopectin are reacted. The minimum amount of water should be used which affords good mixing and highest yields of complex formation. Satisfactory results are obtained when the proportion of water ranges from 1:4 to 200: 1, preferably 1: 1-1021, based on the total weight of amylase and cross-linked pregelatinized amylopectin; the reaction can be optimized readily for any given proportions of amylase to cross-linked pregelatinized amylopectin which are used. It is important that sufiicient water is used to dissolve the two reactants and form a solution. For this reason, if a proportion below 1:4 is used, the necessary solution is not achieved. Proportions in excess of 200:1 result in a solution which is too dilute to result in satisfactory formation of the amylase complex. Intimate contact of the reactants is necessary in the solution. The complex which is formed is insoluble in the reaction system and can be recovered.

The reaction between amylase and cross-linked pregelatinized amylopectin occurs regardless of which material is first added to the aqueous solution or if water is added to a granular mixture of amylase and cross-linked pregelatinized amylopectin. It has been observed, however, that overall best results including processing consideration are obtained by first preparing a slurry of water and amylase and then adding cross-linked pregelatinized amylopectin to said slurry. While no clear understanding of this preferred embodiment is known, it appears that this sequence provides the more favorable environment for complex formation. The result is that the amylase is protected more from conditions and deleterious ingredients which might otherwise tend to inactivate the amylase.

The amylase-cross-linked pregelatinized amylopectin complex can be prepared as a solid granular powder simply by drying off the water from the complex formation whether an aqueous slurry reaction medium is used for some alternative reaction technique involving amylase, cross-linked pregelatinized amylopectin and water. As noted above, amylase complex formation is aided by using a minimum but sufficient amount of water. Removal of water can be performed by any convenient method which does not involve conditions which could in anyway attack or destroy the amylase nor disrupt the complex structure. Conventional vacuum drying processes can be used satisfactorily as can spray drying processes or other suitable separation processes (filtration, centrifugation, etc). As regards these processes, however, temperature above ap proximately 120 F. should be avoided because such relatively high temperature can have deleterious effects upon amylase.

In addition, crystallization processes can also be used provided the amylase complex formation is not damaged.

The amylolytic enzymes, which form a stable complex with cross-linked pregelatinized amylopectin which complex provides amylolytic activity over a prolonged period of time are known materials and can be of fungal, plant, animal or bacterial origin. Suitable amylolytic enzymes include the a-amylases which are particularly well suited for breaking down starch molecules by attacking the a glycosidic linkages in starchy soils and stains. The resulting degraded short chains are then more easily removed from their environment with water or aqueous solutions of detergent. Examples of suitable amylolytic enzymes include the a-amylases of mold origin including those derived from Aspergillus oryzae, Aspergillus niger, Aspergi/IMS ll aceus, Aspergillus wenzii, Pelzcillium glau- 4 cum. The a-amylases derived from cereal grains, pancreatic sources and such bacteria as Bacillus subtilis, e.g., Bacillus subtilis var. amyloliquefaciens, Bacillus macerans, Bacillus mesenrericus and Bacillus thermophilus are also useful herein. These enzymes are active in the pH range of from about 4.5 to about 10 and at temperatures from about 30 F. to about 190 F. Optimum activity of these a-amylases is generally exhibited in the pH range of from about 5.5 to about 7.5.

Preferred amylolytic enzymes herein are the wamylases from the bacterial organism Bacillus subtilis var. amyloliqucfacicns. These amylases have high enzymatic activity and provide excellent starch-digestive properties which are especially useful in the laundering of textile materials containing soils and stains of a starchy nature and in the Washing of hard surfaces, e.g. dishes, pots and pans, having particles of starchy origin. These enzymes are also preferred for their ready availability and their degree of inherent resistance to detergent inactivation in detergent compositions.

The amylolytic enzymes useful herein can be employed in a pure state. The percentages and proportions used in this description are based on pure amylase as determined by activity analysis. Generally amylases are prepared and employed in the form of a powdered commercially available enzyme preparation wherein the amylolytic enzyme is present in an amount of from 2% to of the preparation. The remaining proportion, i.e., from 20% to 98%, comprises inert powdered materials. These powdered materials can comprise inorganic alkali metal salts such as sodium sulfate, sodium chloride, potassium silicate, sodium phosphate, inorganic alkaline earth metal salts such as calcium sulfate, magnesium sulfate, magnesium phosphate and the like; organic components such as non-enzymatic proteins, carbohydrates, organic clays, starches, lipids, color bodies, and the like.

The amount of amylolytic enzyme employed in this invention can vary within the prescribed ranges depending upon the activity of the enzyme or enzyme preparation, conditions of pH and the intended use of the composition. The stabilized amylase compositions of the invention should contain an amount of amylolytic enzyme sufficient to provide efficient starch-degrading properties for the intended purpose, e.g., to facilitate the removal of soils and stains from laundered textile materials or to aid the cleaning of soiled dishware.

Amylases vary in enzymatic activity depending upon their purity and pH in solution. Pure a-amylase, for example, has a specific activity of about 11,500,000 units per gram, while commercially available preparations of about 50,000 to about 2,000,000 amylase activity units per gram. The enzyme-containing detergent compositions of the present invention which contain about .001 to 3% by weight of a-amylase (calculated on the basis of pure aamylase) contain about 60 to 400,000 amylase activity units per gram of detergent composition.

Numerous methods have been described for the determination of tit-amylase activity. A modification of the saccharifying activity developed by P. Bernfeld: Adv. in Enzymology 12, 385 (1951) can be used in the determination of the activity of the a-amylases used in compositions of this invention. In this method, a sample of tit-amylase is permitted to catalyze the hydrolysis of the 1,4 a-glucosidic bonds of starch for 5 minutes at a pH of 6.0 and a temperature of 37 C. The reaction is stopped by the addition of an alkaline solution of 3,5-dinitrosalicylic acid and rochelle salt. The brown colour of the reduction product which is developed in the analytical sample is compared spectrophotometrically with that developed by standard solutions of maltose hydrate. One amylase activity unit is assigned for each 0.4 mg. of maltose hydrate produced during hydrolysis. In practice, it is found that the amount of maltose produced in the analytical procedure by a substance containing a given amount of rat-amylase, and therefore its measured activity in amylase activity units, can vary considerably as a result of slight variations in the test conditions or in the substances with which the a-amylase is associated. The activity of a particular sample can be measured consistently and reproducibly, and values such as those for the percentage amylase activity remaining after a storage test are reliable. The numerical values of amylase activity quoted in this specification, while they indicate the order of magnitude and are self-consistent, should not be taken to be exact in absolute terms.

Examples of commercial enzyme preparations suitable for use herein and the manufacturers thereof include: Diasmen tat-amylase (Daiwa Kasei KK, Tokyo, Japan); Rapidase rx-amylase THC250 (Rapidase, Seclin, France); Novo Bacterial a-amylase (Novo Industri, Copenhagen, Denmark); Wallerstein tat-amylase, Lot #545A (Wallerstein Company, Staten Island, N.Y.); Rhozyme-33 and Rhozyme H-39 (Rohm & Haas, Philadelphia, Pa.); the ot-amylase which is an integral part of Mileszyme (Miles Chemical Company, Elkhart, Ind.); the a-amylase which is an integral part of CRD-Protease (Monsanto DA-lO) derived from Bacillus subtilis (Monsanto Company, St. Louis, Mo.); tX-HIIlYlfiSG (Midwest Biochemical Company, Milwaukee, Wis.); Maxamyl (Koninklijke Nederlandsche Gist-En Spiritusfabreik, N.V., Delft, Netherlands). Mixtures of these materials can be employed in the compositions of the invention.

The stabilized amylase complex of the present invention which has the unexpectedly prolonged amylolytic activity is the unexpected result of a reaction between amylase and cross-linked pregelatinized amylopectin. The use of cross-linked pregelatinized amylopectin is a critical aspect of the present invention. Ordinary starch is not its equivalent in the present invention. Nor, are slightly modified forms of simple natural starches such as those which have been merely oxidized by heating (partial pyrolysis) or merely conventionally hydrolyzed with acids or enzymes. Lintner water-soluble starch, for example, while sometimes capable of improving the stability of amylase in a detergent composition cannot on an overall basis duplicate the surprisingly improved stability results which are obtained by the present invention. Practice of this invention using cross-linked pregelatinized amylopectin gives reliable and consistently improved results.

Thus, it is necessary to employ cross-linked pregelatinized amylopectin in combination with amylase and Water. Cross-linked pregelatinized amylopectin, in order to find application in the present invention, must be prepared from amylopectin as a starting material. Amylopectin is the branched-chain component of starch, amylose is the linear component. Amylose cannot be used as a suitable starting material in practicing the present invention. To the extent that amylose is present in an amylopectin source, in the context of this invention it is regarded as an impurity. It is a preferred embodiment of this invention to use pure amylopectin as a starting material which must be then cross-linked and pregelatinized before being reacted with amylase and Water to form a stabilized amylase complex. Thus, this component can be separated and recovered from any natural starch sources which contain amylopectin. Suitable sources include corn starch, potato starch, wheat starch, tapioca starch, rice starch, sweet potato starch, and the like. The single most preferred source of amylopectin to be crosslinked and pregelatinized for use herein is waxy maize. Waxy maize is a genetically controlled hybrid of corn which is comprised essentially 100% of amylopectin. The present invention should be practiced only with a pure amylopectin source or a natural starch source containing more than 90% amylopectin; and preferably greater than 95% amylopectin.

The amylopectin must be chemically modified by a treatment with a cross-linking agent. In cross-linking the amylopectin, only a slight degree of chemical modification is required but it is essential. To accomplish this, the amylopectin is reacted with from .005% to 0.25% of a cross-linking agent and preferably from .01% to .15 of a cross-linking agent. The cross-linking agent is a polyfunctional material and can be an aliphatic dihalide, such as propylene dichloride, ethylene dibromide, glycerol dichlorohydrin and dichlorobutane, epoxy halogen compounds such as epichlorohydrin and epibromohydrin, polyfunctional reagents such as phosphorous oxychloride, cyanuric chloride, and alkali metal polymetaphosphate. These cross-linking agents in reacting with amylopectin serve to chemically modify and mildly inhibit the amylopectin to such an extent that they are distinguishably distinct entities for practicing this invention and forming the desired stabilized amylase complex. It is impossible to define with precision the chemical reaction product between the preceding cross-linking agents and amylopectin due to the very complicated nature of the reaction.

Following cross-linking, the chemically modified crosslinked amylopectin must still be pregelatinized before being reacted with amylase in the prescribed practice of this invention. Pregelatinization can be carried out in a conventional manner by heating the cross-linked amylopectin in the presence of excess water until the amylopectin granules have broken. Pregelatinization processes can be performed for varying time and under different conditions. Any of such processes, when applied to the cross linked amylopectin described above, can be usefully employed in practicing this invention. The cross-linked pregelatinized amylopectin is then dried on drum rollers, for example, or any other conventional drying apparatus. In this form it can be mixed with amylase and Water to prepare the stabilized amylase complex of this invention. Typical production processes for pregelatinizing starches is described in considerable detail in chapter XXIII of Whistler and Paschalls book titled Starch Chemistry and Technology, vol. II, Industrial Aspects published by Academic Press.

Cross-linked pregelatinized amylopectins useful for the present invention are commercially available. For example, American Maize-Products Company, Indianapolis, Ind. manufactures a suitable product marketed as Amaizo Instant 721A. In addition, National Starch and Chemical Corporation, New York, N.Y., manufactures Instant Clearjel (trademark).

While the stabilized amylase complex prepared according to the present invention can be used in numerous processes in which amylolytic enzymatic activity is desired, the complex has special advantages in a detergent composition. In such applications sustained enzyme activity is an especially difiicult objective to meet. This is especially true, as noted above, because of the relatively harsh conditions employed in manufacturing detergent compositions and also because of the many different types of ingredients which are ordinarily used in detergent compositions, e.g., highly alkaline materials and perbo rate bleaching agents. Moreover, storage stability (carton stability) is an important factor which is closely examined in considering amylase (enzyme)-containing detergent compositions. In each of these respects, the stabilized amylase complex of the present invention provides unexpected and unsurpassed results.

In the simplest detergent composition embodiment, a laundry composition is provided consisting essentially of from .001% to 3% by weight of a stabilized amylase complex of the present invention mechanically admixed with from 97% to 99.999% of a granular detergent composition consisting essentially of a water-soluble organic synthetic detergent and a water-soluble detergency builder salt wherein the ratio of said detergent to said builder is in the range of 4:1 to 1:20. Preferably the stabilized amylase complex should be from .001% to 2% by weight and the granular detergent composition should be 98% to 99.999%. This composition can also contain up to 50% by weight of a perborate bleaching agent; preferably from 0.5% to 40% by weight. The most frequently used bleaching agent and the one preferred herein is sodium perborate. It is an especially valuable aspect of the present invention that the amount of sodium perborate which can be used without inactivating the amylase enzyme far exceeds anything known heretofore.

In an ordinary mechanical mixture of the type just described, i.e., a stabilized amylase complex and a granular detergent composition, it is possible to encounter segregation problems. This means that if the amylase complex is in the form of a powder having more finely divided particles than the granular detergent composition, the finer complex particles may tend to segregate to the bottom of the carton or package. This can be avoided, for example, by such embodiments as detergents in tablet form or packets containing suflicient content for a single washing application.

The segregation or settling-out problem is also solved by the embodiment of this invention which provides for a granular amylase-containing composition consisting essentially of a water-soluble carrier granule having attached thereto a stabilized amylase complex prepared by mixing amylase, cross-linked pregelatinized amylopectin, and water.

In this embodiment, the water-soluble granular carrier can be any material to which the stabilized amylase complex can be attached or adsorbed by any suitable means. For instance, the carrier can comprise an inorganic salt with which the complex can be agglomerated. Such granular carriers should be selected from materials which would be compatible with the intended use of such granules. By way of example, starch, cellulose, other natural or synthetic polymeric matrices and other nonoxidizing materials.

In addition, it is possible to attach the stabilized amylase enzymes to a granular carrier by using a suitably water-soluble adhesive (resinous) material such as polyvinyl acetate, and polyvinylalcohol.

In terms of a preferred embodiment, a granular amylase-containing laundry detergent composition consists essentially of a water-soluble granular carrier having attached thereto a stabilized amylase complex prepared by mixing amylase and cross-linked pregelatinized amylopectin in an aqueous slurry having a temperature in the range of 35 F. to 110 F. and a pH of 4.5 to 8.5, the ratio by weight of amylase to cross-linked pregelatinized amylopectin being in the range of 1:1 to 1:200 wherein the water-soluble granular carrier consists essentially of a granular detergent composition consisting essentially of a water-soluble detergency builder or a mixture of a water-soluble organic detergent and a water-soluble detergency builder wherein the ratio of detergent to builder is in the range of 4:1 to 1:20, and preferably 3:1 to 1:10.

In the embodiment of this invention in which a granular amylase-containing laundry detergent composition of this invention is provided which consists essentially of a water-soluble granular carrier having attached thereto a stabilized amylase complex of the present invention, and wherein said granular carrier is a detergent composition consisting essentially of an organic detergent and a water-soluble detergency builder having a ratio of detergent to builder of 4:1 to 1:20, the said granular amylase-containing laundry detergent composition consists of the following:

80 to 90% of a mixture of a water-soluble organic detergent and a water-soluble detergency builder salt wherein the ratio of said detergent to said builder is in the range of 4:1 to 1:20;

.0001 to 2% amylase,

.0001 to 4% hydrolyzed products of chemically modified cross-linked pregelatinized amylopectin, and

1 to 20% water.

The hydrolysis products of cross-linked pregelatinized amylopectin are formed during the reaction between amylase, cross-linked pregelatinized amplopectin, and water.

In this embodiment, and all other embodiments of detergent compositions of this invention, the organic detergent can be selected from water-soluble, anionic nonionic, zwitterionic, ampholytic synthetic detergents or mixtures of detergents from these classes. Examples of each of these classes of detergents are given in detail below.

ORGANIC DETERGENTS The organic detergent compounds which can be utilized as optional components in the compositions of this invention are soap and anionic, nonionic, ampholytic and zwitterionic synthetic detergents and mixtures thereof and are explified as follows:

(a) Water-soluble soap: Examples of suitable soaps for use in this invention are the sodium, potassium, ammonium and alkanol ammonium (e.g. triethanolammoniurn) salts of higher fatty acids containing from about 10 to about 22 carbon atoms. Particularly useful are the sodium and potassium salts of the mixture of fatty acids derived from coconut oil and tallow, i.e., sodium and potassium tallow and coconut soap.

(b) Anionic synthetic non-soap detergents, a preferred class, can be broadly described as the water-soluble salts, particularly the alkali metal salts, of organic sulfuric reaction products having in their molecular structure an alkyl radical containing from about 8 to about 22 carbon atoms and a radical selected from the group consisting of sulfonic acid and sulfuric acid ester radicals. (Included in the term alkyl is the alkyl portion of higher alcyl radicals.) Important examples of the synthetic detergents which form a part of the preferred compositions of the present invention are the sodium or potassium alkyl sulfates, especially those obtained by sulfating the higher alcohols (C -C carbon atoms) produced by reducing the glycerides of tallow or coconut oil; sodium or potassium alkyl benzene sulfonates, in which the alkyl group contains from about 9 to about 15 carbon atoms, including those of the types described in U.S. Letters Pat. Nos. 2,220,099 and 2,477,383 (the alkyl radical can be a straight or branched aliphatic chain); sodium alkyl glyceryl ether sulfonates, especially those ethers of the higher alcohols derived from tallow and coconut oil; sodium coconut oil fatty acid acid monoglyceride sulfates and sulfonates; sodium or potassium salts of sulfuric acid esters of the reaction product of one mole of a higher fatty alcohol (e.g., tallow or coconut oil alcohols) and about 1 to 6 moles of ethylene oxide, sodium or potassium salts of alkyl phenol ethylene oxide ether sulfate with about 1 to about 10 units of ethylene oxide per molecule and in which the alkyl radicals contain from 8 to about 12 carbon atoms; the reaction product of fatty acids esterified with isethionic acid and neutralized with sodium hydroxide where, for example, the fatty acids are derived from coconut oil, sodium or potassium salts of fatty acid amide of a methyl tauride in which fatty acids, for example, are derived from coconut oil; and others known in the art, a number specifically set forth in U.S. Letters Pats. Nos. 2,486,921, 2,486,922 and 2,396,278. Other important anionic detergents, sulfonated olefins, are described in the copending application of Phillip E. Pfiaumer and Adriaan Kessler, Ser. No. 561,397 filed June 29, 1966.

(c) Nonionic synthetic detergents: One class can be broadly defined as compounds produced by the condensation of alkylene oxide groups (hydrophilic in nature) which an organic hydrophobic compound, which may be aliphatic or alkyl aromatic in nature. The length of the hydrophilic or polyoxyalkylene radical which is condensed with any particular hydrophobic group can be readily adjusted to yield a water-soluble compound having the desired degree of balance between hydrophilic and hydrophobic elements. Another class has semi-polar characteristics. Preferred classes of nonionic synthetic detergents are as follows:

(1) A class of nonionic synthetic detergents under the trade name of Pluronic. These compounds are formed by condensing ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. The hydrophobic portion of the molecule which, of course, exhibits water insolubility, has a molecular weight of from about 1500 to 1800. The addition of polyoxyethylene radicals to this hydrophobic portion tends to increase the Water solubility of the molecule as a Whole and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product.

(2) The polyethylene oxide condensates of alkyl phenols, e.g., the condensation products of alkyl phenols having an alkyl group containing from about 6 to 12 carbon atoms in either a straight-chain or branched-chain configuration with ethylene oxide, the said ethylene oxide being present in amounts equal to to 25 moles of ethylene oxide per mole of alkyl phenol. The alkyl substituent in such compounds may be derived from polymerized propylene, diisobutylene, octene, or nonene, for example.

(3) Those nonionic synthetic detergents derived from the condensation of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylene diamine. For example, compounds containing from about 40% to about 80% polyoxyethylene by weight and having a molecular Weight of from about 5,000 to about 11,000 resulting from the reaction of ethylene oxide groups with a hydrophobic base constituted of the reaction product of ethylene diamine and excess propylene oxide; said base having a molecular weight of the order of 2,500 to 3,000 are satisfactory.

(4) The condensation product of aliphatic alcohols having from 8 to 22 carbon atoms, in either straight chain or branched chain configuration, with ethylene oxide, e.g., a coconut alcoholethylene oxide condensate having from 5 to 30 moles of ethylene oxide per mole of coconut alcohol, the coconut alcohol fraction having from to 14 carbon atoms.

(5) The ammonia, monoethanol and diethanol amides of fatty acids having an acyl moiety of from about 8 to about 18 carbon atoms. These acyl moieties are normally derived from naturally occurring glycerides, e.g., coconut oil, palm oil, soybean oil and tallow, but can be derived synthetically, e.g., by the oxidation of petroleum, or by hydrogenation of carbon monoxide by the Fischer-Tropsch process.

(6) Long chain tertiary amine oxides corresponding to the following general formula wherein R is an alkyl radical of from about 8 to about 24 carbon atoms, R and R are each methyl, ethyl or hydroxyethyl radicals, R is ethylene, and n equals from 0 to about 10. The arrow in the formula is a conventional representation of a semi-polar bond. Specific examples of amine oxide detergents include: dimethyldodecylamine oxide; cetyldirnethylamine oxide: bis-(Z-hydroxyethyl) dodecylamine oxide; bis(Z-hydroxyethyl)-3-dodecoxy-1- hydroxypropyl amine oxide.

(7) Long chain tertiary phosphine oxides corresponding to the following general formula RRR"P O wherein R is an alkyl, alkenyl or monohydroxyalkyl radical ranging from 10 to 24 carbon atoms in chain length and R and R" are each alkyl or monohydroxyalkyl groups containing from 1 to 3 carbon atoms. The arrow in the formula is a conventional representation of a semi-polar bond. Examples of suitable phosphine oxides are found in U.S. Pat. 3,304,263 of Feb. 14, 1967 and include: di-

10 methyldodecylphosphine oxide; diethyldodecylphosphine oxide; dimethyl-(Z-hydroxydodecyl) phosphine oxide.

(8) Long chain sulfoxides having the formula wherein R is an alkyl radical containing from about 10 to about 28 carbon atoms, from O to about 5 ether linkages and from 0 to about 2 hydroxyl substituents, at least one moiety of R being an alkyl radical containing 0 ether linkages and containing from about 10* to about 18 carbon atoms, and wherein R is an alkyl radical containing from 1 to 3 carbon atoms and from one to two hydroxyl groups. Specific examples of these sulfoxides are: dodecyl methyl sulfoxide; 3-hydroxy tridecyl methyl sulfoxide; 3-methoxy tridecyl methyl sulfoxide; 3-hydroxy-4-dodecoxybutyl methyl sulfoxide.

(d) Ampholytic synthetic detergents can be broadly described as derivatives of aliphatic secondary and tertiary amines, in which the aliphatic radical may be straight chain or branched and wherein one of the aliphatic substituents contains from about 8 to 18 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfo, sulfato, phosphato, or phosphono. Examples of compounds falling within this definition are sodium-3-dodecylaminopropionate and sodium-3-dodecylaminopropane sulfonate.

(e) Zwitterionic synthetic detergents can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, in which the aliphatic radical may be straight chain or branched, and wherein one of the aliphatic substituents contains from about 8 to 18 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfo, sulfato, phosphato, or phosphono. Examples of compounds falling within this definition are 3-(N,N-dimethyl-N-hexadecylammonio)propane-l-sulfonate and 3-(N,N-dimethyl-N hexadecylammonio)-2-hydroxy propane-l-sulfonate which are especially preferred for their excellent cool water detergency characteristics. See, for example, Snoddy et al., Canadian Pat. 708,148 issued Apr. 20 1965.

The detergency builder salt useful as a. carrier granule in this embodiment is a partially hydrated hydratable salt which has a pH in aqueous solution in the range of 4 to 12, and a vapor pressure not greater than about 13.15 mm. of Hg when measured at atmospheric pressure at 20 C., and which is hydrated not in excess of of its hydration capacity. Preferably, the hydratable salt should be not more than 50% hydrated based on its hydration capacity, should have a pH in aqueous solution in range of 4 to 11, and should be selected from the group consisting sodium tripolyphosphate, sodium pyrophosphate, sodium tetraborate, sodium acid pyrophosphate and mixtures thereof.

In order to facilitate a more complete understanding of the embodiment in which a partially hydrated hydratable salt is used as or in the enzyme carrier granule, the following description is provided:

HYDRATABLE SALTS Water-soluble hydratable salts employed as or in the granular carrier of the compositions of this invention have certain characteristics relative to pH and hydration characteristics. As used in the specification and claims the term hydratable salt defines one compound of the desired properties or a mixture of such compounds.

Both the granular carrier and the hydratable salt used thereas or therein should provide a pH in water solution in the range of from 4-12. Carrier granules and hydratable salt having pH values from 4-7 are provided when the particular powdered amylase chosen for attachment to the granular carrier prefers an acidic environment for optimum activity and stability. Neutral or slightly alkaline pHs of 7-8 are employed for analagous reasons. Granular carrier and hydratable salts providing pHs in the range of 4-8 generally function solely as a carrier for the powdered amylase. When the granular carrier, and the hydratable salt used therein, function as a detergent or a detergent component in addition to a carrier for the amylase, materials providing high pHs in the 4-12 range are used, preferably in the range of 611. Water-soluble hydratable builder salts, serving as or in a granular carrier as hereinafter described, either in granular form per se or as a portion of a multi-component detergent granule, provide pHs in the range of 4-11.

Preferably the hydratable salt and amylase are matched for pH, that is the inherent pHs of the amylase and salt are within a pH unit or two. In the composition of this invention, however, such pH matching is not essential since the moisture absorbing capacity of the partiallyhydrated hydratable salt keeps the moisture away from the enzyme/ carrier interface, thereby precluding any pH differential in the salt and enzyme from causing degradation of the amylase.

The hydratable salt employed as the granular carrier or in the granular carrier should hold its water of hydration tightly and should readily accept more water to fulfill the function required in this invention. Suitable hydratable salts are those having a vapor pressure not greater than about 13.15 mm. of Hg at 20 C. and atmospheric pressure. This vapor pressure corresponds to 75% relative humidity as measured over the hydratable salt at the same conditions. Hydratable salts with lower vapor pressures are preferred. Vapor pressure varies with the compound and with the amount of water of hydration already in the compound. An anhydrous salt has no vapor pressure. In the composition of this invention the hydratable salt Will always contain some water of hydration and will have some vapor pressure, depending on how much water was used to effect the attachment of the powdered enzyme to the granular carrier. In the process of this invention either anhydrous or partially-hydrated hydratable salts can be used as a starting material.

Another important characteristic of the hydratable salt is its capacity to take water of hydration. Preferably this capacity should be in the range of 0.1 to 1.3 lbs. of water per lb. of anhydrous hydratable salt. One pound of anhydrous sodium tripolyphosphate will hydrate about 0.3 pound of water.

Examples of suitable water-soluble hydratable salts for use as or in the granular carrier are as follows.

(I) Hydratable salts of an acid with a large, and a base with a small, dissociation constant providing a pH of about 4 to about 7.

(II) Hydratable salts of an acid and a base with approximately the same dissociation constant having a pH of approximately 7-8 Na SO Ammonium higher fatty acid soaps (C -C (NH SiO tetraammonium ethane hydroxy diphosphonate (III) Hydratable salts of an acid with a small, and a base with a large, dissociation constant (e.g. builder salts) having a pH of about 8-11 or 12 S S IO Na P- O trisodium methane or ethane hydroxy diphosphonate, trisodium methane or ethane diphosphonate, tetra sodium ethane triphosphonate, tetra sodium ethane hydroxytriphosphonate, tetra sodium propane tetra, penta or hexaphosphonate sodium higher fatty acid soaps (C C Na SiO N212HPO4 trisodium nitrilotriacetate di-, triand tetra sodium ethylenediarninetetraacetate The above list is only exemplary. Many other hydratable salts having the desired pH, vapor pressure and hydration characteristics can be employed. The cations of such hydratable salt can be alkali metal, such as sodium, lithium or potassium, ammonium, alkanolammonium (e.g. triethanolammonium) and alkaline earth metal such as calcium and barium. Mixtures of such salts can be used.

Many of these hydratable salts vary in hydration capacity, holding from 1 to 10 or 12 moles of Water.

The preferred hydratable salts are sodium builder salts such as sodium tripolyphosphate, sodium tetraborate, sodium pyrophosphate, sodium acid pyrophosphate and mixtures of these such as a mixture of sodium tripolyphosphate and sodium acid pyrophosphate in a weight proportion of about 1:1 to 5:1, preferably 3:1. These builder salts, in any of their partially hydrated forms have a vapor pressure of less than 13.15 mm. of Hg at 20 C. and one atmosphere. Sodium tripolyphosphate, sodium pyrophosphate and sodium tetraborate have pHs of about 9-10.

With the amylase attached to the granular carrier, the hydratable salt is preferably not more than hydrated (based on total hydration capability by weight) in order to provide capacity for additional hydration during any exposure of the product of free moisture. Desirably this figure is not more than about 50% of the total hydration capacity. Generally, about 33% hydration of the total hydration capacity of the hydratable salt is effected when the amylase is attached to the granular carrier.

GRANULAR CARRIER The hydratable salt can be used either per se as the granular carrier or can comprise a portion of a multicomponent granular carrier. The other components of the granular carrier, when the hydratable salt is only a portion thereof, can comprise diluent salts, organic detergents, detergency builder salts not having the necessary hydrate characteristics, and other detergent composition components of the usual type. The granular carrier of this invention ranges in particle size from about .075 mm. to about 3.33 mm. (about .003" to about Mi"). This size range corresponds to granules which pass through 6 mesh and remain on 200 mesh (Tyler standard screen). A preferred size range is 0.2 mm. to 2 mm. Laundry products, such as conventional spray-dried detergent granules are in these size ranges which are easy to measure and use without dustiness. The granular carrier preferably has a density in the range of from about 0.2 gram/cc. to about 0.8 gram/cc.

The hydratable salts can comprise from 20% to of the granular carrier. Preferably the hydratable salt comprises from 40% to 90% of the granular carrier in order to provide ample hydration capacity for the carrier and to permit inclusion of other desired components in the granular carrier, such as an organic detergent. When an organic detergent is used with the hydratable salt, particularly a hydratable builder salt, the ratio of salt to detergent ranges from 1:4 to 20:1, generally from 1:1 to 9:1. The granular carrier can also contain minor materials: for example, corrosion inhibitors such as benzotriazole, antiredeposition agents such as sodium carboxymethyl cellulose, optical brighteners, bactericides, perfumes and dyes. The optional minor components are each commonly used in amounts up to 2 to 5% by weight of the granular carrier. I

The granular carrier with the amylase attached can be used per se as a pre-wash soaking agent or, when the carrier comprises a detergent component such as an hydratable builder salt, as a cleaning agent. The granular carrier with attached amylase can be admixed with other granular detergent materials of approximately the same particle size and density to form a multicomponent, heavyduty laundry detergent composition tailored to have a number of desirable characteristics. In such a case, the granular carrier with amylase preferably is in an amount ranging from about .001% to 20% by weight of such a multicomponent detergent composition and is uniformly distributed throughout the detergent composition, so that any random sample thereof has about the same formulation as any other sample. Use of less than about .001% carrier-l-en-zyme makes uniformity difficult; more than 20% results in a loss of the advantages of such blending, sometimes called master-batching. 'If desired, the carrier with enzyme granules can be dyed a bright color and admixed with a white or slightly colored granular detergent system to provide an overall composition having a distinctive speckled appearance, in accordance with the teachings of Canadian Pat. 577,479, Britt.

The enzyme carrier granule embodiment of this invention has the advantage of permitting efficient incorporation of enzymes in a multi-component detergent composition wherein the total proportion of granules to be treated with enzyme powder is relatively minor. The laundering advantages of enzymes are achieved with only a minor amount thereof in a detergent composition. Therefore, it is more efficient to blend a minor proportion of granular carrier having a moderate amount of attached enzyme with a major proportion of enzyme-free granules than to attach a very small amount of enzyme to each of the granules in a granular detergent product. Moreover, the granular carrier is designed to provide improved stability whereas the detergent composition may not provide conditions for optimum stability.

In a multi-component detergent composition comprising about .001% to 20% of the granular carrier with attached enzyme, the remaining 80% to 99.999% can be granular detergent materials of the conventional types. Generally such materials are mixtures of builders and organic detergents in weight ratios of 1:4 to 20:1. The builders can be of the hydratable type required for the granular carrier or can be of other types such as the polycarboxylate builder salts of US. Pat. 3,308,067, Diehl, issued Mar. 7, 1967.

The multi-component detergent composition in a preferred embodiment also contains up to 50% of a perborate bleaching agent; preferably from 0.5% to 40% and is preferably sodium perborate.

Whether the hydratable salt comprises all or only a portion of any particular granular carrier in the desired size range, it can be in any particulate physical form such as granular anhydrous sodium tripolyphosphate which is relatively dense, or a heat-dried or agglomerated hydratable salt-containing particle which has reduced density and expanded surface area formed as a result of a drying or agglomeration step. Conventional spray-dried synthetic detergent granules can be used since they comprise a significant portion of hydratable salt, particularly builder salt such as sodium tripolyphosphate.

A granular carrier which can be used advantageously comprises spray-dried detergent granules containing sodium tripolyphosphate and anionic organic synthetic detergent in a weight ratio ranging from 8:1 to 2: 1, containing less than 4% and preferably less than 2.5% moisture (water or hydration) and being free from the highly alkaline sodium silicate commonly used in spray-dried synthetic detergent granules.

A low density sodium tetraborate can also be used as a granular carrier. It has an average bulk density of about 0.4 to 0.6 gram per cc. and a moisture (water of hydration) content of less than 4%, preferably less than 2.5%. It is formed by heating sodium borate decahydrate as it passes downward through a chamber containing heated, upwardly-rising air in the neighborhood of 450 to 550 F.

The granular amylase-containing laundry detergent composition of this invention which consists essentially of a stabilized amylase complex attached to a watersoluble granular carrier which is a granular detergent composition can be prepared in any convenient manner.

The preferred method comprises the steps of mixing amylase and cross-linked pregelatinized amylopectin in an aqueous slurry having a temperature in the range of 35 to F. and a pH of 4.5 to 8.5 to form an aqueous suspension of a stabilized amylase suspension, agitating a water-soluble granular carrier which consists essentially of a partially hydrated hydratable detergency builder salt or a mixture of water-soluble organic detergent and a water-soluble partially hydrated hydratable builder wherein the ratio of said detergent to said builder is in the range of 4:1 to 1:20, subjecting said agitated carrier to a spray of said suspension of water and amylase complex whereby the amylase complex is attached to the granular carrier as a result of hydration of the partially hydrated hydratable salt portion of. said granular carrier.

The proportion of water to the partially hydrated hydratable builder salt is critical. The amount of water used should be the minimum amount necessary to effect a suitable spray and ensure effective attachment of the enzyme to the hydratable base granule. In no case should the amount of water sprayed on the base granule exceed the hydrating capacity of the hydratable salt portion of the enzyme carrier base granule. If too much water is used, free water may be present which adversely effects enzyme stability. In addition, free water presents caking problems not only in the processing but also when the product is packaged into cartons. Ordinarily when the hydratable salt is sodium tripolyphosphate the ratio of builder to water is in the range of about 4:1 to about 50:1, preferably 5:1 to 40:1.

Critical aspects in this process are the first step in which the stabilized amylase complex is formed, and attachment of the complex to the granular carrier by means of the hydration of the hydratable salt portion of the granular carrier, and the proportions of water and hydratable salt used in the process.

A unique and unexpected feature of the present invention and one of the ones which renders possible preparation of amylase-containing detergent compositions having sustained enzymatic activity even in the presence of a perborate bleaching agent is that the stabilized amylase complex is relatively insoluble at the temperature and pH conditions under which is made but is soluble under the more alkaline conditions provided by ordinary detergent compositions.

An alternative method of preparing the carrier granules is to prepare a slurry suspension of stabilized amylase complexas previously described and spraying this slurry suspension onto a bed of particles comprising a hydrate forming salt such as sodium tripolyphosphate or mixtures of sodium tripolyphosphate and sodium pyrophosphate, in a mixing device such as a pan agglomerator. Substantially anhydrous sodium tripolyphosphate is the preferred salt. The carrier granules so formed are given time so that the moisture in the slurry can be absorbed by hydration of the hydratable salt. These carrier granules are then blended with the remainder of a granular detergent composition. Preferably the free moisture content of the carrier granules is kept low, usually not over 10%.

While the present invention has been described in terms of stabilizing amylase, it is to be pointed out that other enzyme ingredients can also be used in practicing this invention without interfering with the primary objectives described herein. Thus, for example, proteases may also be added to the slurry in which the stable amylase complex is prepared together with any stabilizers therefor such as collagen. There may also be present colouring matter and dyes, substances which prevent subsequent dusting of the carrier granules, and the like.

Built detergent compositions of the present invention can contain, in addition to the hydratable builder salts described above, additional builder ingredients which are inorganic alkaline builder salts or organic alkaline sequestering builder salts. There is no need for these additional enzyme-free builders to have the hydration characteristics described above in conjunction with the preparation of amylase enzyme carrier granules.

The present invention is demonstrated by the following examples. These are merely illustrative. Obvious modifications can be carried out in light of the previous discussion and the appended claims. All percentages and proportions referred to in this description are by weight unless otherwise indicated.

In the following examples, the following two crosslinked pregelatinized amylopectin materials were used:

PREPARATION A A cross-linked pregelatinized amylopectin from a waxy maize source, cross-linked with 0.05% by weight of phosphorous oxychloride. This material had a hydration capacity of 16.36 as determined by the procedure described below under the heading Method for Estimating the Hydration Capacity of Cross-Linked Pregelatinized Amylopectin. It also had a gel strength of about 59 as measured by the Embedded Disc Method described below.

The preparation of the cross-linked pregelatinized amylopectin is performed by essentially the following procedure. After cleaning, the kernels of genetically pure waxy maize are first soaked for 35 to 40 hours in water containing sulfur dioxide, and when the kernels contain 45 to 50% moisture the separation of protein, germ, and fiber is facilitated. Any solubles in the corn are removed during the steeping process. The wet kernels are next fed to a type of pinmill which cracks the hull and loosens the germ. The resulting slurry is gravity separated with the germ floating off the top. The germ is removed and the remaining slurry is passed through entoleter-type pinmills and the coarse fiber filtered from the fine particles and gluten. The relatively pure amylopectin starch so recovered is then given further washings and chemical treatment. This comprises a cross-linking treatment with phosphorous oxychloride. Only a few tenths of, one percent of the phosphorous oxychloride is sufiicient to give adequate cross-linking. After cross-linking, the crosslinked amylopectin is subjected to additional cooking at 190 F. in order to eifect partial gelatinization. The crosslinked pregelatinized amylopectin is separated from the water, drum dried and sifted.

PREPARATION B A cross-linked pregelatinized amylopectin from a waxy maize source, cross-linked with .05 by weight of epichlorohydrin. This material had a hydration capacity of 13.42 as measured by the procedure described below under the heading Method for Estimating the Hydration Capacity of Cross-Linked Pregelatinized Amylopectin. The material had a gel strength of approximately 107 as measured by the Embedded Disk Method described below.

The procedure for preparing Preparation B was essentially the same as that described above in conjunction with Preparation A with the difference that epichlorohydrin was the cross-linking agent in place of the phosphorous oxychloride.

16 METHOD FOR ESTIMATING THE HYDRATION CAPACITY OF CROSS-LINKED PREGELATI- NIZED AMYLOPECTIN Apparatus (1) Balance (10.005 g.)

(2) Centrifuge (3) ml. round bottom centrifuge tubes, without the pourout, and stoppers.

Reagents 1) Distilled water (pH 6-7) Procedure 1) Weight 2.0 g. sample (as is) into 100 ml. centrifuge tube tared with stopper.

(2) Add 40 ml. distilled water, stopper, and shake vigorously to thoroughly suspend the sample.

(3) Allow suspension to stand for 10 minutes. During this time, mix by inverting 3 times at the end of 5 minutes and at the 10 minute period.

(4) Remove stopper and centrifuge for 15 minutes at 1,000 times gravity. Allow the centrifuge to stop without breaking.

(5) Carefully decant the supernatant and invert tube to drain. (Suction may be preferred for loosely-packed sediments.)

(6) Restopper and weigh tube and contents.

Calculations Hydration capacity (Wt. of tube-l-sediment.) (wt. of tube) Sample wt. (dry basis) Reference (1) Yamazaki, W. T. An alkaline water retention capacity test for the evaluation of cookie baking potentialities of soft winter wheat flours. Cereal Chem. 30: 242-246 (1953).

EMBEDDED DISC METHOD EXAMPLE I .5 g. crystalline a-amylase from Bacillus subtilis and 1 g. of the cross-linked pregelatinized amylopectin of Preparation A described above, both water-soluble substances, are added to 5 ml. of water (phosphate-buffered pH7.2). The temperature of this solution is 60 F. The ratio of amylase to cross-linked pregelatinized amylopectin is 1:10. The mixture is stirred for 5 minutes. The resulting suspension is centrifuged and an insoluble amylase complex is separated from the supernatant liquid. Since the initial reactants are both water-soluble, the complex which separates is a reaction product between amylase and crosslinked pregelatinized amylopectin. During the reaction, the cross-linked pregelatinized amylopectin is hydrolyzed and the ultimate complex consists of amylase and hydrolysis products of chemically modified cross-linked pregelatinized amylopectin,

Both the supernatant and the complex are tested for amylolytic activity. The amylase complex contains 78% of the amylolytic activity and the liquid supernatant contains only 22%.

1 7 EXAMPLE u If in Example I, Preparation B is substituted for Preparation A, substantially equivalent results are achieved.

EXAMPLE III A stabilized amylase complex is prepared by mixing .0667 part of Rapidase THC 250 (an amylase obtained from a Bacillus sublilis amyloliquefaciens source containing 5% active enzyme and the balance inert salts), .1667 part of a chemically modified cross-linked pregelatinized amylopectin described above as Preparation A, and 1.0 part water. A slurry is formed to which is added .25 part of a protease enzyme, Alcalase (16% active proteolytic enzyme derived from Bacillus subtilis and balance inerts comprising sodium and calcium sulfate) and .1667 part of powdered collagen derived protein. The temperature of the slurry is 100 F. and had a pH of 6.8. The proportions of amylase to chemically modified cross-linked pregelatinized amylopectin is 1:20.

The slurry consisted of a suspension of amylase complex formed by reaction between the amylase and chemically modified cross-linked pregelatinized amylopectin.

EXAMPLE IV The slurry from Example III is sprayed onto 5 parts of granular anhydrous sodium tripolyphosphate to form enzyme carrier granules. The ratio of water to tripolyphosphate is 1:5. The amylase complex attaches to the granular tripolyphosphate by hydration of the anhydrous salt. This results in the formation of a granular amylasecontaining composition which is useful as a detergent composition or a soaking composition for soiled garments.

EXAMPLE V 7.00 parts of enzyme carrier granules of Example IV are mixed homogeneously with 10 parts of sodium perborate and 83 parts of a spray dried granular detergent composition having the following composition.

Component: Parts by wt. Sodium alkyl benzene sulfonate derived from tetrapropylene 23.2 Sodium tripolyphosphate 24.0 Sodium silicate (SiO -Na O ratio of 2:1) 5.4 Sodium sulfate 39.0 Sodium toluene sulfonate 2.4 Water 6.0

This detergent composition is packed in outer wax laminated protective cartons and stored at constant conditions of 80 F. and 80% relative humidity for more than two months. Amylolytic activity is determined by saccharifying analysis at intervals with the results shown in Table I.

Comparisons are made with a Control composition which is alike in all respects except the omission of chemically modified cross-linked pregelatinized amylopectin from the initial slurry in the complex forming step.

TABLE 1 Product of this invention embodying a stabilized amylase Control complex Analyzed Analyzed amylase amylase level Percent level Percent activity, amylase activity, amylase units/ activity units/ activity gram 1 remaining gram 1 remaining 1 Activity of initial Rapidase amylase was 500,000 activity units per gram.

The analysis used to determine amylase activity is based on the principle that zit-amylase catalyzes the hydrolysis of a starch substrate to reducing sugars. The amount of re ducing sugars formed are measured colormetrically by reaction with dinitrosalicylic acid. Amylase activity is calculated from the amount of reducing sugars formed.

It can be seen from the data in Table I that the amylase activity of the product of this invention is surprisingly greater than the Control product. After 14 days the Control product retains 65% amylase activity as compared to 90.5% amylase activity to the product of this invention about a 35% improvement due to the stabilized amylase complex of this invention. After 28 days, the improvement is about 33%; after 59 days (approximately two months) the improvement is about 228%; after 84 days, the improvement is about 160%. These are unexpected and valuable enzyme stability performance advantages which are made possible by the stabilized amylase complex of the present invention.

EXAMPLE VI A stabilized amylase complex is prepared by preparing a slurry by mixing the following ingredients:

To 3.0 parts of water are added (1) 0.739 part of an enzyme preparation comprising 6.86% of a-amylase having 755,000 amylase activity units per gram, 6.33% alkaline protease having 465,000 protease activity units per gram and the balance inert materials such as calcium sulfate and sodium sulfate; (2) 0.279 part of an enzyme preparation comprising 5.68% of ot-amylase having 625,- 000 amylase activity units per gram, 5.10% alkaline protease having 75,000 protease activity units per gram and the balance inert materials such as calcium sulfate and sodium sulfate; (3) 0.239 part of an enzyme preparation comprising 1.82% of u-arnylase having 200,000 amylase activity units per gram, 5.44% of an alkaline protease having 400,000 protease activity units per gram and the balance inert materials such as calcium sulfate and sodium sulfate; (4) 1.186 parts of an enzyme preparation comprising 16.33% alkaline protease having 1,200,000 protease activity units per gram and the balance inert materials such as sodium and calcium sulfate; (5) 0.557 parts of an enbyme preparation comprising 6.25% of an alkaline protease having 458,000 protease activity units per gram and the balance inert materials such as sodium and calcium sulfate. The solution has a pH of 5.5 and a temperature of 100 F. The enzyme preparations are all from a Bascillus subtilis origin and contain the ordinary inert materials.

To this solution is added 1.5 parts of a chemically modified cross-linked pregelatinized amylopectin which is Preparation B described above. The ratio of tat-amylase to Preparation B is about 1:20.

As the reaction proceeds between the amylase and the chemically modified cross-linked pregelatinized amylopectin, an insoluble amylase complex is formed between the a-amylase and the hydrolysis products of the chemically modified cross-linked pregelatinized amylopectin. The amylase complex has sustained amylase activity.

EXAMPLE VII Enzyme carrier granules are prepared in the following manner. 7.5 parts of the slurry of Example VI containing approximately 1.0% tat-amylase and 4% alkaline protease were sprayed onto 92.5 parts of enzyme carrier granules having the following formula:

Percent Sodium linear alkyl benzene sulfonate 4.05 Tallow alkyl sulfate 4.95 Sodium tripolyphosphate 55.0 Sodium dihydrogen pyrophosphate 15.0 Sodium sulfate 16.5 Water 3.5

The sodium tripolyphosphate is a hydratable salt containing less than of its hydration capacity. The moisture from the slurry spray is adsorbed in the granular carrier and the amylase (as well as the protease) is attached to the granular carrier.

EXAMPLE VIII Three parts of the enzyme carrier granules of Example VII are uniformly mixed with 97 parts of spray dried detergent granules having the following composition.

Percent Sodium linear alkyl benzene sulfonate 12.9 Sodium tripolyphosphate 45.5 Sodium silicates (SiO /Na O ratio 1.6/1) 5.4 Sodium sulfate 15.6 Fatty acid soap 2.7 Sodium perborate 2.7 Miscellaneous (brighteners, CMC etc.) 2.4 Moisture 11.0

The sodium perborate is incorporated in the above composition by fusing powdered sodium perborate tetrahydrate onto the surface of the spray-dried detergent at the base of the spray drying tower.

The product is packaged in protective (pattern coated) cartons and stored at 90 F., 80% relative humidity. Amylase activity measured at various storage intervals is shown below:

For comparative purposes a Control product was prepared in exactly the same manner except that the chemically modified cross-linked pregelatinized amylopectin was omitted from the product.

Control Invention Amylase Amylase activity, Percent activity, Percent units/ activity units/ activity gram remaining gram remaining Days EXAMPLE IX The detergent composition prepared in Example VIII is also stored at 80 *F. and 80% relative humidity. Amylase activity results are as follows. The unique sustained amylase activity is again demonstrated.

Control Invention Amylase Amylase activity, Percent activity, Percent units/ activity units/ activity gram remaining gram remaining The foregoing description of the invention has been presented describing certain preferred embodiments. It is not intended that the invention should be so limited since variations and modifications thereof will be obvious to those skilled in the art, all of which are within the spirit and scope of this invention.

20 What is claimed is: 1. A laundry composition consisting essentially of from .001% to 3% by weight of a stabilized amylase complex consisting essentially of amylase and hydrolysis products of chemically-modified, cross-linked, pregelatinized amylopectin in a ratio of 1:1 to 1:200 mechanically admixed with from 97% to 99.999% of a granular detergent composition consisting essentially of a watersoluble organic detergent and a water-soluble detergency builder salt wherein the ratio of said detergent to said builder is in the range of 4:1 to 1:20.

2. A laundry composition of claim 1 wherein the stabilized amylase complex is present in an amount in the range of 001% to 2% by weight.

3. A composition of claim 1 wherein there is also present up to 50% by weight of sodium perborate.

4. A composition of claim 3 wherein the amount of said perborate bleaching compound is in the range of 0.5% to 40%.

5. A granular amylase-containing laundry detergent composition consisting essentially of a water-soluble granular carrier having attached thereto a stabilized amylase complex prepared by mixing amylase and cross-linked pregelatinized amylopectin in an aqueous slurry having a temperature in the range of 35 F. to 110 F. and a pH of 4.5 to 8.5, the ratio by weight of amylase to crosslinked pregelatinized amylopectin being in the range of 1:1 to 1:200, wherein the water-soluble granular carrier consists essentially of a granular deterent composition consisting essentially of a water-soluble detergency builder or a mixture of a water-soluble organic detergent and a water-soluble detergency builder wherein the ratio of detergent to said builder is in the range of 4:1 to 1:20.

6. A composition of claim 5 in which the organic synthetic detergent is selected from anionic, nonionic, zwitterionic, ampholytic detergents or mixtures thereof and the detergency builder is selected from inorganic or organic alkaline builder salts.

7. A composition of claim 5 in which the detergency builder salt is a partially hydrated hydratable salt which has a pH in aqueous solution in the range of 4 to 12, and a vapor pressure not greater than about 13.15 mm. of Hg when measured at atmospheric pressure at 10 C., and which is hydrated not in excess of 90% of its hydration capacity.

8. A laundry composition of claim 7 wherein the hydratable salt is a hydratable builder salt which has a pH in aqueous solution in the range of 8 to 11.

9. A laundry composition of claim 8 wherein the hydratable salt is not more than 50% hydrated, based on its hydration capacity and the hydratable builder salt is selected from the group consisting of sodium tripolyphosphate, sodium pyrophosphate, and sodium tetraborate.

10. A laundry composition consisting essentially of a mixture of from about 001% to about 20% by weight of the granular amylase-containing laundry composition of claim 5 and about to about 99.999% of enzymefree detergent granules which consist essentially of a watersoluble organic detergent and a water-soluble detergency builder salt, wherein the ratio by Weight of detergent to builder is in the range of 4:1 to 1:20.

11. 'A composition of claim 10 wherein there is also present up to 50% by weight of sodium perborate.

12. A composition of claim 11 wherein the amount of said perborate bleaching compound is in the range of 0.5 to 40%.

13. A laundry composition of claim 10 wherein said granular amylase-containing laundry composition consists essentially of 80% to 99% of a mixture of a water-soluble organic detergent and a water-soluble detergency builder salt wherein the ratio of said detergent to said builder is in the range of 4:l to 1:20,

.0001 to 2% amylase,

.0001 to 4% hydrolysis products of cross-linked pregelatinized amylopectin, and p 1 to 20% water.

14. A process for preparing the laundry composition of claim 5 comprising the steps of mixing amylase and cross-linked pregelatinized amylopectin in an aqueous slurry having a temperature in the range of 35 F. to 110 F. and a pH of 4.5 to 8.5, the ratio by weight of amylase to cross-linked pregelatinized amylopectin being in the range of 1:1 to 1:200, to form a suspension of Water and a stabilized amylase complex,

agitating a water-soluble granular carrier which consists essentially of a partially hydrated hydratable water-soluble detergency builder salt or a mixture of a water-soluble organic detergent and Watersoluble partially hydrated hydratable builder salt wherein the ratio of said detergent to said builder is in the range of 4:1 to 1:20,

subjecting said agitated carrier to a spray of said suspension of water and stabilized amylase complex whereby the amylase complex is attached to the granular carrier as a result of hydration of the parti- 22 ally hydrated hydratable salt portion of said granular carrier, the amount of water used being insuflicient to exceed the hydrating capacity of the hydratable builder salt. 15. A process of claim 14 in which the hydratable salt is sodium tripolyphosphate or a mixture of sodium tripolyphosphate and sodium acid pyrophosphate in a proportion by weight of tripolyphosphate to pyrophosphate in the range of 1:1 to 5:1, the ratio of said hydratable salt to water is in the range of 4:1 to :1.

References Cited FOREIGN PATENTS 8/1946 Denmark. 12/1944 Switzerland.

US. Cl. X.R.

63, 68; 2S2Digest 12