3,707,505 United States Patent Office Patented Dec. 26, 1972 1. In respective control tests, no N-higher acyl amino acid 3,707,505 was added. ENZYME-CONTAINING DETERGENT COMPOSTON As is evident from Table 1, the stabilizing agents of the Itsutoshi Maeda, Yasuichi Nomura, Kohei Hashimoto, invention sharply reduce the loss of enzyme activity under Akira Shimizu, Koji Mitsugi, and Ryonosuke Yoshida, the test conditions. Kanagawa-ken, Japan, assignors to Ajinomoto Co., Inc., TABLE 1. Toyko, Japan Rate of residual No Drawing. Filed Dec. 28, 1970, Ser. No. 102,182 activity, percent Claims priority, application Japan, Dec. 30, 1969, Deter- Deters 45/1,752 O gent gent nt. C. C11d 3/04 Expt. ad base base U.S. C. 252-136 7 Claims No. N-higher acy amino acid A. B 1------None.------10 20 2------N-caproyl-L-valine.-- 28 25 8------N-capryl-L-valine----- 43 40 ABSTRACT OF THE DSCLOSURE 4. -- N-lauroylglycine.------36 38 5 5 ... N-lauroyl-D-alanine- 26 41 The enzymes present in an enzyme-bearing detergent 6 -- N-laturoylsarcosine- 64 39 cornposition are stabilized by small amounts of N-acyl 7 ... N-lauroyl-L-leucine. 28 31 8 N-lauroyl-L-threonine 32 29 amino acids whose acyl groups are derived from saturated 9------N-auroy-DL-methionine..... 3. 35 10------N-lauroyl-e-aminocaproic acid--- 32 30 or unsaturated fatty acids having 6 to 20 carbon atoms, 1l------N-auroyl-S-benzyl-L-cysteine. 32 28 and the enzymes are also stable in detergents in which the 20 12 N-myristoyl-L-waine.-- 30 39 13-- N-palmitoyl-L-valine. 55 3. N-acyl amino acids are the primary surfactant ingredients. 14-- -- N-palmitoylglycine.-- 30 39 15.-- -- N-palmitoyl-DL-alanine 35 40 a-sa 16-- N-palmitoylsarcosine- 34 45 17------N-palmitoyl-L-leucine.-- 35 32 18------N-palmitoyl-L-threonine------33 30 This invention relates to detergent compositions, and 19------N-palmitoyl-L-phenylalanine.--- 32 43 particularly to improved enzyme-bearing detergent com 25 20- -- N-stearoyl-L-valine----- 3. 35 2.-- -- N-undecylenoyl-L-valine 35 28 positions. 22.-- -- N-auroyl-L-aspartic acid- 26 40 Enzyme-bearing detergent compositions have recently 23- -- N-palmitoyl-L-aspartic aci 33 35 24- -- N-palmitoyl-L-asparagine.------37 45 found wide application. In addition to the enzyme, such 25------N-palmitoyl-D-homocysteic acid.------29 30 compositions may typically contain an anionic surfactant 26------N-hydrogenated tallowyl-glutamic acidi- 32 3. 30 27-- -- N-lauroyl-L-arginine.------36 35 as the primary active ingredient, such builders as sodium 28-- -- N-palmitoyl-L-arginine------48 55 tripolyphosphate, sodium silicate, Glauber's salt, and 29 -- N-hydrogenated tallowyl-L-arginine 38 58 30- - N-auroyl-L-arginine methyl ester. 50 36 addition agents such as carboxymethylcellulose, optical 31-- -- N-lauroyl-L-ornithine methyl este 25 38 brightening agents, dyes, perfumes, etc. 32. -- N,N-dimethyl-N-auroyl-L-ornithine.... 49 62 33------N-palmitoyl-L-lysine methyl ester----- 55 65 The known compositions suffer from the fact that 34------N-hydrogenated tallowyl-L-arginine----- 54 60 the anionic surfactants make the enzymes unstable. The 35 35------N-pairnitoyl-L-lysine methyl ester----- 54 55 alkaline builders inactivate the enzymes at temperatures 36------N-Stearoyl-L-serine------37 43 of 40 C. or more. The enzymes are even less stable in aqueous solutions having a pH of 10-11 as normally Detergent bases A and B were modified to contain caused by the other ingredients of the conventional com 40 0.4% protease, and batches of the modified bases were positions. further mixed with the stabilizing agents of the invention A practically useful detergent composition in which the listed in Table 2 in amounts of 0.8%. The powder com enzyme component is not significantly inactivated has positions so obtained were stored at 84% relative humid not been available heretofore, and it is an object of this ity and 37 C. in a desiccator over saturated aqueous am invention to provide an enzyme-bearing detergent com monium chloride solution for 7 and 14 days together position wherein the enzyme is relatively stable. 45 with controls free from the N-higher acyl amino acids, It has now been found that the stability of the enzyme and the residual enzyme activity was determined in per in detergent compositions of the type described is en cent of the initial value. The improved shelf life of the hanced by the presence of an N-acyl amino acid, and its compositions according to this invention is evident from salts and esters, the N-acyl group being the radical of a Table 2. fatty acid having 6-20 carbon atoms. The stabilizing 50 agents of the invention are effective both in aqueous TABLE 2 solutions and in dry detergent powders. The stabilizing Rate of residual activity, agents of the invention will be referred to hereinafter as percent “N-higher acyl amino acids” for the sake of brevity. Detergent Detergent To demonstrate the effectiveness of the stabilizing 55 base A base B agents, two detergent bases were prepared. Base A con Expt. 7 4. 7 14 sisted of 25% sodium n-dodecylbenzene sulfonate, 30% No. N-higher acy amino acid days days days days sodium tripolyphosphate, 5% sodium metasilicate, 37% 87------None.------62 40 85 65 Glauber's salt, and 3% carboxymethylcellulose together 38------N-lauroyl-L-arginine methyl 84 64 94 82 60 ester hydrochloride. with 0.1% protease (60,000 units/g. as determined by 39------Sodium N-hydrogenated 100 O 98 95 the modified Anson method described hereinbelow). Base tallowyl-L-glutamate. B differed from Base A by substitution of sodium lauryl 40------N-palmitoyl-L-Waline.------94. 90 100 92 sulfate for the sodium n-dodecylbenzene sulfonate. Aqueous 2% solutions of the bases having a pH of 10 The N-higher acyl amino acids of the invention also were mixed with the N-higher acyl amino acids listed in 65 favorably affect the cleaning efficiency of detergent con Table 1 in a concentration of 0.2%, each mixture was positions containing enzymes. heated to 50° C. for 30 minutes, and the residual enzyme Samples of cotton fabric artificially soiled (Empa 116) activity was measured. The term "hydrogenated tallowyl' were soaked in a liquor ratio of 1:50 in aqueous 0.2% is used in this specification for brevity's sake for the acyl solutions of detergent base A without protease, with radicals of the fatty acids in hydrogenated beef tallow. 70 0.001% protease (60,000 u./g.) but without N-higher The term "cocoyl” will be used in an analogous manner. acyl amino acid and with 0.001% protease and varying 3,707,505 3. 4 amounts of N-palmitoyl valine (P-V) or sodium N-hydro was determined by the method of Yamada et al. J. Agric. genated tallowyl-L-glutamate (HGS). After 10 minutes Chem. Soc. Japan 36 (1962) 860), one unit being the agitation at 100 r.p.m. at 30° C., the washed fabric amount of enzyme liberating 1 u, equivalent of acid per samples were dried, and the rate of reflection L after minute. washing was compared with the rate of reflection L be TABLE 5 fore washing on a color-difference meter with Hunter Cleaning Standard (91.8). Cleaning efficiency CE (percent) was efficiency Detergent composition after- Rate of calculated according to the formula residual L-L Expt. Amt., 10 30 activity, O No. Ingredient g/dl. Illin. min. percent Detergent base A--- 0.2 The results obtained with the several detergent com 57------E.etergent base 0.0020.2 60 70 5 positions are listed in Table 3. rE,- - - -isulfate. ... 0.002 60 71 11 TABLE 3 odium laurylsulfate- 0.2 5 59------E.“Tide' (trade name). --... o. 8330.2 67 75 Expt. No.------41 42 43 44 45 46, 47 48 49 60------{E.------0.002 } 68 76 Disodium N-hydrogenated Protease------61------tallowy DL-glutamate. 0.2 67 75 52 P-V, percent------0.01 0.001 0.0002 ------Protease------0.002 HGS, percent------0. 0.02 0.005 0, 001 Sodium N-lauroyl-DL- 0.2 CE, percent---- 60 68 71 67 69 69 67 73 73 62------glutanate. 5. 68 48 Protease------0.002 20 Sodium N-lauroyl-L- 0.2 The amount of N-higher acyl amino acid which sta 63------arginine. 4. 65 30 bilizes the enzyme in a detergent composition may be 0.1 Protease. 2 to 20% based on the weight of the detergent base or 64------3SES .002 69 80 42 N-palmitoyl-L-phenyl- 0.2 20% to 1000% based on the enzyme. The effects of 65.------alanine------63 79 46 varying amounts of N-palmitoyl valine (P-V) and sodium Protease------0.002 n-hydrogenated tallowyl-L-glutamate (HGS) on the re SE;odium N-hydrogenate BS A) ted 8.. 1. sidual activity of 0.01% protease in an aqueous 2% solu 66------tallowyl-L-glutamate. 67 82 73 tion of detergent base A after 10 minutes at 50 C. is Protease------0.002 evident from Table 4. Detergent base A wherein 0.2 sulfate was replaced by TABLE 4 30 67------sodiumSEE EYN-hydrogenated bene 66 77 60 tallowyl-L-glutanate. Expt. No.------50 5. 52 53 54 55 56 Protease------0, 002 Sodium N-cocoyl-L-gluta- 0.1 PV, percent------0.1 0, 01 0.002 ------mate. HGS, percent------0, 1 0, 01 0.002 68------Sodium laurylsulfate...... -- 0.1 68 8. 74 Activity, percent 41 72 62 57 74 65 60 Glauber's Salt------0. 35 St.Protease------N-cocoyl-L-gluta- 0.0020.2 Detergent compositions of the invention may be 38. powders, granules, pastes, or liquids. The stabilizers of 69.------Glauber's Salt------0. 50 60 50 the invention may be used to advantage in heavy-duty Protease------0.002 synthetic detergent compositions containing alkaline builder or in light-duty synthetic detergent compositions 40 without alkaline builder whose predominant detergent agent is an anionic, cationic, or nonionic surfactant. ABLE 6 Cleaning The N-higher acyl amino acids themselves have excel efficiency lent surface activity and are stable in hard water. They Detergent composition after- Rate of residual may be used, therefore, as predominant active ingredients 45 Expt. Amt., 10 30 activity, in detergent compositions which may further contain No. Ingredient g/dl. min. min, percent enzymes with or without builders. In such compositions, Sodium lauryl sulfate-...---- 0.2 the enzymes are very stable. 70------Amylase------0.002 39 59 80 Sodium iaurylsulfate.----- 0. When N-higher acyl amino acids constitute the primary Disodium N-hydrogenated 0.1 surfactant in enzyme bearing compositions, other syn 50 71.------talowyl-D-glutamate. 38 60 100 Amylase------0.002 thetic surfactants and builders may also be present. Good Sodium n-dodecyl benzene 0.2 detergency and foaming ability are achieved even in hard 72------sulfonate. 39 57 90 Amylase------0, 002 water without the use of alkaline builders, such as sodium Sodium n-dodecylbenzene 0. tripolyphosphate and sodium silicate, and the solutions sulfonate. 73------Disodium N-hydrogenated 0. 39 61 100 prepared from such compositions may be neutral or only 55 tallowyl-D-glutamate. slightly alkaline so as to further enhance the stability of mylase------0.002 the enzyme. The results of comparison tests between various deter gent compositions respectively containing protease, amy TABLE 7 lase (10,000 u./g. from B. subtilis), and lipase (20,000 60 Cleaning u./g.) as shown in Tables 5, 6, and 7 together with con efficiency Detergent composition after- Rate of trols. Cleaning efficiency was determined in the manner residual described with reference to Table 3 after 10 minutes and Expt. Amt, 10 30 activity, 30 minutes laundering. The residual enzyme activity was No. Ingredient g/dl. min. min. percent determined at ten times the concentrations indicated in 65 Sodium laurylsulfate.--...-- 0.2 74------{E. ------0.833 41 59 50 the tables after 30 minutes heating to 50° C. (protease) Sodium laurylsulfate.------0.1 or 40 C. (amylase, lipase). Protease activity was deter 75------Disodium N-hydrogenated 0.1 42 6. 90 tallowyl-D-glutamate. mined by the modified Anson method in which casein is Lipase------0.002 used as the substrate, and 1 unit is the amount of protease Sodium n-dodecyl 0.2 76------benzene sulfonate. 39 58 46 which liberates 1 pig. equivalent of tyrosine per minute at 70 Lipase------0.002 37 C. Amylase activity was determined by the modified Sodium n-dodecyl 0.1 benzene sulfonate. Wohlgemuth method in which 1 amylase unit is the 77------Disodium N-hydrogenated 0.1 35 63 96 amount of enzyme which reduces the iodine color of 1 Lipase------tallowyl-D-glutamate. 0.002 ml. of 0.65% soluble starch solution to an absorbance of 0,300 at 660 mu in 10 minutes at 40° C. Lipase activity 3,707,505 5 6 The acyl groups of the N-higher acyl amino acids in The detergent compositions of the present invention the detergent compositions of the invention are those of may therefore be formulated as laundry detergents, for saturated or unsaturated fatty acids having 6 to 20 carbon general kitchen use, for use in automatic dish washers, as atoms, such as caproic, lauric, tridecanoic, decanoic, my shampoos, bar soaps, cleaning creams, tooth pastes, and ristic, pentadecanoic, palmitic, margaric, stearic, and for industrial use. oleic acid, also of the fatty acid mixtures obtained from The following examples illustrate the preparation of fats and oils, such as coconut oil, tallow oil, hydrogenated detergent compositions of the invention. tallow oil, olive oil, soybean oil, corn oil, and the like. The N-higher acyl amino acids may be used also in Example 1 the form of their salts with alkali metals (sodium, potas iO 100 litre of water was added to a mixture of 2.5 kg. of sium) or ammonium, with water soluble amines, particul sodium-n-dodecylbenzene sulfonate, 2.5 kg. of disodium larly alkanolamines (mono-, di-, or triethanolamine) and N-hydrogenated tallowyl-DL-glutamate, 6 kg. of sodium triethylamine, with basic amino acids if the acyl group is tripoly-phosphate, 1 kg. of sodium metasilicate, 7 kg. of attached to an acidic amino acid, or in the form of their Glauber's salt and 0.6 kg. of carboxymethylcellulose, and acid addition salts with mineral acids (hydrochloric or 5 the solution was spray-dried to obtain 18 kg. of white sulfuric), organic acids (acetic acid, lactic acid, acidic granular detergent. Laundry detergent was prepared by amino acids if the acyl group is attached to a basic uniformly mixing the above granular detergent with 360 amino acid). g. of granular protease. The amino acid moiety of the N-higher acyl amino Example 2 acid may be an acidic amino acid (such as glutamic acid, aspartic acid, homocysteic acid, cysteic acid, aminoadipic 20 Powder laundry detergent was prepared by adding 12 acid), a basic amino acid (such as lysine, arginine, orni g. of protease to a mixture of 250 g. of sodium N-hydro thine, oxylysine, diaminobutyric acid, histidine, isolysine), genated tallowyl-L-glutamate and 5 g. of N-lauroyl-L- or a neutral amino acid (such as glycine, alanine, valine, ornithine lauryl ester acetate. serine, proline, leucine, isoleucine, oxyproline, methionine, 25 phenylalanine, threonine, tryptophane, cystein, cystine, Example 3 tyrosine, norvaline, norleucine, aminobutyric acid, amino Laundry detergent was prepared by mixing 300 g. of isobutyric acid, diaminopimelic acid, diaminooxypimelic the commercial laundry detergent, "Tide' (trade name, acid, pantonine, taurine, aminovaleric acid, and amino produced by The Procter & Gamble Company) with 6 g. caproic acid). It may be optically active or racemic. 30 of granular protease and 10 g. of dipotassium N-lauroyl The N-methyl-N-acyl and N-ethyl-N-acyl derivatives of DL-aspartate. these acids also may be used, such as N-acyl deriva Example 4 tives of N-methyleucine, N-methylglycine, N-methylala To a mixture of 500 g. of sodium lauryl sulfate, 10 g. nine, dimethyl ornithine, and also N-acyl peptide deriva of sodium N-palmitoyl-L-valine, 1.4 kg. of Glauber's salt tives. 35 and 50 g. of carboxymethylcellulose, 2 g of optical whit When the N-higher acyl amino acids of the invention ening agent was added and thereafter 24 g. of powder are used in the form of esters, the esters may be the lower from protease to obtain neutral laundry detergent in alkyl esters (methyl, ethyl, propyl, butyl) or higher alkyl powder. esters (such as octyl, lauryl esters). Example 5 Alkaline or neutral protease, amylase, and lipase may 40 be used in the detergent compositions of the invention 50 g. of protease was mixed with a mixture of 3 kg. of singly or in mixtures of two or more. sodium N-cocoyl-L-glutamate and 7 kg. of Glauber's salt. If a synthetic surfactant is used as the predominant The obtained mixture was dissolved in water to make 60 detergent component, it may be an alkyl benzene sul litres and spray-dried to produce neutral laundry deter fonate, the sulfate of a higher alcohol or of an alkyl 45 gent for wool and synthetic fibers. ethoxy ether, an ox-olefin sulfonate, another alkylaryl sul Example 6 fonate, a polyoxyethylene alkylphenyl ether, and may be 50 g. of triethanolamine lauryl sulfate, 10 g. of di mixed with a builder, other auxiliary agents (optical ethanolamide of lauric acid, 10 g. of N-lauroyl-L-iso bleach, perfume, dye, CMC to prevent redeposition), the leucine monoethanolamine salt and a small amount of N-higher acyl amino acid, and the enzyme. 50 If the N-higher acyl amino acid is the sole or predomi perfume were dissolved in water. 0.3 g of protease and nant surfactant, it may be combined with a builder, aux 1 g. of lipase were added thereto to prepare 300 g. paste iliary agents, and the enzyme. The detergent composition shampoo composition. in either case may be in the form of a powder, a shaped The monoethanolamine salt was obtained by acylating a solid body, a paste, granules, a cream, an emulsion, or a 55 mixture of 60% L-leucine, 30% L-isoleucine, L-valine, solution, as may be required. L-alanine, L-threonine and L-methionine to the N-lauroyl The detergent compositions of the invention have a long derivatives, and by converting the same to the mono shelf life because of the stabilizing effect of the N-higher ethanolamine salts. acyl amino acids on the enzymes, and only little of the Example 7 enzyme activity is lost during use. Detergency is ade 60 Deodorant cleaning cream was obtained by adding 0.1 quate, because the N-higher acyl amino acids are equal g. of protease and a very small amount of perfume to a or superior in this respect to conventional detergents with mixture of 35 g. of mineral oil, 12 g. of spermaceti, 2 g. which they are fully compatible. of beeswax, 5 g. of N-lauroyl-L-arginine methyl ester DL When the N-higher acyl amino acids constitute the a-pyrrolidone-carboxylate, 2 g of glycerin and 40 g. of predominant active agent in a detergent composition, the 65 Water. composition has excellent stability and detergency in hard Example 8 water without need for alkaline builders which impair Detergent for automatic dish washers was obtained by enzyme activity. The solutions are neutral or weakly alka mixing 100 g. of ammonium N-tallowyl-D-glutamate, 1 line so that enzymes not suitable for alkaline solutions g. of protease, 3 g. of lipase and 5 g. of amylase. may be employed singly or in mixtures. The solutions 70 may be employed for degreasing wool, as hair shampoos, Example 9 and as detergents for kitchen use. The N-higher acyl Detergent for kitchen use was obtained by dissolving 5 amino acids tend to protect the skin and rarely cause ir g. of potassium N-lauroyl-DL-alaninate and 0.5 g. of ritation. They also have antibacterial or bacteriostatic protease in 200 g. of a commercial detergent for kitchen effects. 75 and Fats Co., Ltd.). 3,707,505 7 8 Example 10 proline, leucine, isoleucine, oxyproline, methionine, phen A liquid dish washing detergent was obtained by adding ylalanine, threonine, tryptophane, cystein, cystine, tyro 0.1 g. of protease and 1 g. of amylase to a mixture of 15 sine, norvaline, norleucine, aminobutyric acid, aminoiso g. of lauryl alcohol ether sulfate, 10 g. of alkylamide of butyric acid, diaminopimelic acid, diaminooxypimelic coconut fatty acid, 5 g. of ethyl alcohol and 2 g of N acid, pantonine, taurine, aminovaleric acid, aminocaproic myristoyl-L-arginine butyl ester lactate. acid, and sarcosine. What we claim is: 4. A composition as set forth in claim 1, wherein said 1. A detergent composition consisting essentially of an N-acyl amino acid is an N-acyl derivative of an alpha anionic synthetic surfactant, at least one enzyme selected amino, beta-amino, or omega-amino acid. from the group consisting of protease, lipase, and amy 10 5. A composition as set forth in claim 1, wherein said lase, and an N-acyl amino acid, the amount of said sur N-acyl amino acid is in the form of a sodium, potassium, factant being sufficient to make said enzyme unstable in or ammonium salt thereof or in the form of an alkyl the absence of said N-acyl amino acid, and the amount ester thereof, said alkyl having up to 12 carbon atoms. of said N-acyl amino acid being sufficient to enhance the 6. A composition as set forth in claim 1, the amount stability of said enzyme in the presence of said surfac 5 of said N-acyl amino acid being between 20% and 1000% tant, the acyl group in the N-acyl moiety of said N-acyl of the amount of said enzyme. amino acid being the acid radical of a fatty acid having 7. A composition as set forth in claim 2, said builder 6 to 20 carbon atoms, the nitrogen of said N-acyl moiety being alkaline. being bound to the carbon chain in the amino acid moiety References Cited of said N-acyl amino acid. 20 UNITED STATES PATENTS 2. A composition as set forth in claim 1, wherein the 3,574,120 4/1971 Siebert ------252-527 remainder of said composition essentially consists of at 3,519,379 7/1970 Blomeyer ------252- DIG 12 least one builder selected from the group consisting of 2,962,448 11/1960 Dvorkovitz ------252-527 sodium tripolyphosphate, sodium metasilicate, and Glau 2,880,178 3/1959 Ruff et al. 252-52.7 X ber's salt. 25 3,051,627 8/1962 Bradford et al. -- 252-DIG 12 3. A composition as set forth in claim 1, wherein said N-acyl amino acid is an N-acyl derivative of an amino MAYER WEINBLATT, Primary Examiner acid selected from the group consisting of glutamic acid, aspartic acid, homocysteic acid, cysteic acid, aminoadipic U.S. C. X.R. acid, lysine, arginine, ornithine, oxylysine, diaminobutyric 30 195-63, 68; 252-Dig. 12, 527, 546 acid, histidine, isolysine, glycine, alanine, valine, serine,