Storage-stable enzyme preparations, their production and use

Abstract

Described herein is a liquid enzyme preparation containing component (a): at least one salt according to general formula (I)
(R.sup.2).sub.3N.sup.+—(CH.sub.2).sub.nC(R.sup.3)(R.sup.4)—(O—X).sub.m—O—C(O)—R.sup.1A.sup.−  (I) wherein n is selected from 1 to 12, m is selected from zero to 50, R.sup.1 is selected from the group consisting of methyl, ethyl, —CH(OH)—CH(OH)—COOH, CH(OH)—CH.sub.3, (E)-CH═CHCOOH, (Z)—CH═CHCOOH, —C.sub.6H.sub.5, para-HO—C.sub.6H.sub.4—, o,p-dihydroxyphenyl, and 3,4,5-triydroxyphenyl, R.sup.2 are same or different and selected from C.sub.1-C.sub.10-alkyl, phenyl, R.sup.3 and R.sup.4 are same or different and selected from hydrogen and C.sub.1-C.sub.4-alkyl, X is C.sub.2-C.sub.4-alkylen, and A.sup.− is an inorganic or organic counteranion,
component (b): at least one enzyme selected from hydrolases (EC 3),
and
optionally component (c): at least one compound selected from enzyme stabilizers different from component (a), preservatives, and surfactants.

Claims

1. A liquid enzyme preparation containing component (a): at least one salt according to general formula (I)
(R.sup.2).sub.3N.sup.+—(CH.sub.2).sub.nC(R.sup.3)(R.sup.4)—(O—X).sub.m—O—C(O)—R.sup.1A.sup.−  (I) wherein n is selected from 1 to 12, m is zero, R.sup.1 is selected from the group consisting of methyl, ethyl, —CH(OH)—CH(OH)—COOH, CH(OH)—CH.sub.3, (E)-CH═CHCOOH, (Z)—CH═CHCOOH, —C.sub.6H.sub.5, para-HO—C.sub.6H.sub.4—, o,p-dihydroxyphenyl, and 3,4,5-triydroxyphenyl, or —O—C(O)—R.sup.1 together constitute a citrate, R.sup.2 are same or different and selected from C.sub.1-C.sub.10-alkyl and phenyl, R.sup.3 and R.sup.4 are same or different and selected from hydrogen and C.sub.1-C.sub.4-alkyl, X is C.sub.2-C.sub.4-alkylen, and A.sup.− is an inorganic or organic counteranion, component (b): at least one enzyme selected from hydrolases (EC 3), and optionally component (c): at least one compound selected from enzyme stabilizers different from component (a), preservatives, and surfactants.

2. The liquid enzyme preparation according to claim 1 wherein component (a) has a counterion selected from the group consisting of halide, sulphate, carbonate, tartrate, citrate, lactate, and methanesulfonate.

3. The liquid enzyme preparation according to claim 1 wherein R.sup.2 in compound according to general formula (I) are all methyl.

4. The liquid enzyme preparation according to claim 1 wherein said enzyme preparation contains component (a) in amounts in the range of 0.1 to 30% by weight relative to the total weight of the enzyme preparation.

5. The liquid enzyme preparation according to claim 1 wherein component (a) contains as impurity a compound (a′):
(R.sup.2).sub.3N.sup.+—(CH.sub.2).sub.nC(R.sup.3)(R.sup.4)—(O—X).sub.m—OHR.sup.1—COO—  (a′) wherein the variables R.sup.1, R.sup.2, X, n and m are the same as in the corresponding component (a).

6. The liquid enzyme preparation according to claim 1 wherein the enzyme preparation contains component (c), wherein component (c) comprises at least one enzyme stabilizer selected from the group consisting of boron-containing compounds and peptide aldehydes.

7. A method of making a detergent formulation, the method comprising mixing the liquid enzyme preparation of claim 1 in one or more steps with one or more detergent components.

8. The liquid enzyme preparation of claim 1, wherein the component (b) is selected from the group consisting of lipases (EC 3.1.1), endopeptidases (EC 3.4.21), triacylglycerol lipase (EC 3.1.1.3), and subtilisin type proteases (EC 3.4.21.62).

9. The liquid enzyme preparation of claim 1, wherein the surfactants are selected from the group consisting of non-ionic, amphoteric, and anionic surfactants.

10. A process for making an enzyme preparation, said process comprising the steps of mixing at least component (a): at least one salt that is a compound of general formula (I),
(R.sup.2).sub.3N.sup.+—(CH.sub.2).sub.nC(R.sup.3)(R.sup.4)—(O—X).sub.m—O—C(O)—R.sup.1A.sup.−  (I) wherein n is selected from 1 to 12, m is zero, R.sup.1 is selected from the group consisting of methyl, ethyl, —CH(OH)—CH(OH)—COOH, CH(OH)—CH.sub.3, (E)-CH═CHCOOH, (Z)—CH═CHCOOH, —C.sub.6H.sub.5, para-HO—C.sub.6H.sub.4—, o,p-dihydroxyphenyl, and 3,4,5-triydroxyphenyl, or —O—C(O)—R.sup.1 together constitute a citrate, R.sup.2 are same or different and selected from C.sub.1-C.sub.10-alkyl, phenyl, R.sup.3 and R.sup.4 are same or different and selected from hydrogen and C.sub.1-C.sub.4-alkyl, X is C.sub.2-C.sub.4-alkylen, and A.sup.− is a counteranion, inorganic or organic and component (b): at least one enzyme selected from hydrolases (EC 3).

11. The process of claim 10, wherein the component (b) is selected from the group consisting of lipases (EC 3.1.1), endopeptidases (EC 3.4.21), triacylglycerol lipase (EC 3.1.1.3), and subtilisin type proteases (EC 3.4.21.62).

12. A method of stabilizing at least one enzyme selected from hydrolases (EC 3) within a liquid enzyme preparation by the step of adding at least one salt of the general formula (I),
(R.sup.2).sub.3N.sup.+—(CH.sub.2).sub.nC(R.sup.3)(R.sup.4)—(O—X).sub.m—O—C(O)—R.sup.1A.sup.−  (I) wherein n is selected from 1 to 12, m is zero, R.sup.1 is selected from the group consisting of methyl, ethyl, —CH(OH)—CH(OH)—COOH, CH(OH)—CH.sub.3, (E)-CH═CHCOOH, (Z)—CH═CHCOOH, —C.sub.6H.sub.5, para-HO—C.sub.6H.sub.4—, o,p-dihydroxyphenyl, and 3,4,5-triydroxyphenyl, or —O—C(O)—R.sup.1 together constitute a citrate, R.sup.2 are same or different and selected from C.sub.1-C.sub.10-alkyl, phenyl, R.sup.3 and R.sup.4 are same or different and selected from hydrogen and C.sub.1-C.sub.4-alkyl, X is C.sub.2-C.sub.4-alkylen, and A.sup.− is a counteranion, inorganic or organic.

13. The method according to claim 12, wherein the enzyme is stabilized in the presence of at least one surfactant.

14. A method of using at least one salt of the general formula (I),
(R.sup.2).sub.3N.sup.+—(CH.sub.2).sub.nC(R.sup.3)(R.sup.4)—(O—X).sub.m—O—C(O)—R.sup.1A.sup.−  (I) wherein n is selected from 1 to 12, m is zero, R.sup.1 is selected from the group consisting of methyl, ethyl, —CH(OH)—CH(OH)—COOH, CH(OH)—CH.sub.3, (E)-CH═CHCOOH, (Z)—CH═CHCOOH, —C.sub.6H.sub.5, para-HO—C.sub.6H.sub.4—, o,p-dihydroxyphenyl, and 3,4,5-triydroxyphenyl, or —O—C(O)—R.sup.1 together constitute a citrate, R.sup.2 are same or different and selected from C.sub.1-C.sub.10-alkyl, phenyl, R.sup.3 and R.sup.4 are same or different and selected from hydrogen and C.sub.1-C.sub.4-alkyl, X is C.sub.2-C.sub.4-alkylen, and A.sup.− is a counteranion, inorganic or organic, the method comprising using the salt of the general formula (I) as an additive for at least one enzyme selected from hydrolases (EC 3), wherein said salt and said enzyme are solid and wherein stabilization of said enzyme occurs when said salt and said enzyme are contacted with at least one solvent.

15. A liquid detergent formulation comprising component (a): at least one salt according to general formula (I)
(R.sup.2).sub.3N.sup.+—(CH.sub.2).sub.nC(R.sup.3)(R.sup.4)—(O—X).sub.m—O—C(O)—R.sup.1A.sup.−  (I) wherein n is selected from 1 to 12, m is zero, R.sup.1 is selected from the group consisting of methyl, ethyl, —CH(OH)—CH(OH)—COOH, CH(OH)—CH.sub.3, (E)-CH═CHCOOH, (Z)—CH═CHCOOH, —C.sub.6H.sub.5, para-HO—C.sub.6H.sub.4—, o,p-dihydroxyphenyl, and 3,4,5-triydroxyphenyl, or —O—C(O)—R.sup.1 together constitute a citrate, R.sup.2 are same or different and selected from C.sub.1-C.sub.10-alkyl, phenyl, R.sup.3 and R.sup.4 are same or different and selected from hydrogen and C.sub.1-C.sub.4-alkyl, X is C.sub.2-C.sub.4-alkylen, and A.sup.− is an inorganic or organic counteranion, component (b): at least one enzyme selected from hydrolases (EC 3), optionally component (c): at least one enzyme stabilizers different from component (a), and at least one detergent component.

16. A method for removing stains comprising fat, comprising the step of contacting the stain with a detergent formulation according to claim 15, wherein at least one enzyme comprised in component (b) of the detergent formulation comprises at least one lipase (EC 3.1.1).

17. The method according to claim 16, wherein the stain is to be removed from a textile at a temperature ≤40° C.

18. The method of claim 16, wherein the at least one enzyme comprised in component (b) of the detergent formulation comprises at least one triacylglycerol lipase (EC 3.1.1.3).

19. The detergent formulation of claim 15, wherein the component (b) is selected from the group consisting of lipases (EC 3.1.1), endopeptidases (EC 3.4.21), triacylglycerol lipase (EC 3.1.1.3), and subtilisin type proteases (EC 3.4.21.62).

20. The detergent formulation of claim 15, wherein the component (c) is selected from the group consisting of boron containing compounds, phenyl boronic acid (PBA) or its derivatives, and 4-formyl phenyl boronic acid (4-FPBA).

Description

EXAMPLES

(1) The invention will be further illustrated by working examples.

(2) General remarks: percentages are weight percent unless specifically noted otherwise.

(3) Acetylcholine (A.12) was purchased from Sigma Aldrich. The counterion was chloride.

(4) The precursor of (A.14) can be produced directly instead of use of HCl in the ethoxylation of trimethylamine or via reaction of choline hydrogencarbonate with methanesulfonic, see Constantinescu et al in Chem. Eng. Data, 2007, 521280-1285.

(5) I. Synthesis of Salts (Component (a))

(6) Based upon the amounts of water distilled off and by IR spectroscopy it could be shown that the esterification reactions were complete.

(7) 90% methanesulfonic acid refers to a mixture from 10% water and 90% methanesulfonic acid.

(8) I.1 Synthesis of Inventive Salt (A.1):

(9) An amount of 225 g (1.5 mole) tartaric acid was dissolved in 280 g of a 75% by weight aqueous solution of choline chloride (1.5 mole). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 15 g of 90% by weight aqueous methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 617 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 7.8 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.1) was obtained.

(10) I.2 Synthesis of Inventive Salt (A.2):

(11) An amount of 150 g tartaric acid (1.0 mole) was dissolved in 374 g of a 75% by weight aqueous solution of choline chloride (2.0 mole). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 15 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 607 g of a yellowish liquid were obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.7 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.2) was obtained.

(12) I.3 Synthesis of Inventive Salt (A.3):

(13) An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 374 g of a 75% by weight aqueous solution of choline chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation, the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 607 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 10.3 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.3) was obtained.

(14) I.4 Synthesis of Inventive Salt (A.4):

(15) An amount of 210 g citric acid monohydrate (1.0 mol) was dissolved in 561 g of a 75% by weight aqueous solution of choline chloride (3.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 270 g diethylene glycol. An amount of 868 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 9.6 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.4) was obtained.

(16) I.5 Synthesis of Inventive Salt (A.5): An amount of 210 g citric acid monohydrate (1.0 mol) were dissolved in 485 g of a 75% by weight aqueous solution of choline methanesulfonate (2.0 mol). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C.

(17) A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 663 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 12.5 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.5) was obtained.

(18) I.6 Synthesis of Inventive Salt (A.6):

(19) An amount of 98.1 g maleic anhydride (1.0 mol) were mixed with 363 g of choline methanesulfonate (2.0 moles) as dry substance. The mixture was heated in a rotary evaporator to 135° C. After one hour of mixing an amount of 12 g of methanesulfonic acid (pure) was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of mixing the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 653 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.6) was obtained.

(20) I.7 Synthesis of Inventive Salt (A.7):

(21) An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 437 g of a 70% by weight aqueous solution of beta-methyl choline chloride (HO—CH(CH.sub.3)—CH.sub.2—N(CH.sub.3).sub.3 Cl, 2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 676 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 12.3 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.7) was obtained.

(22) I.8 Synthesis of Inventive Salt (A.8):

(23) An amount of 105 g citric acid monohydrate (0.5 moles) was dissolved in 327 g of a 70% by weight aqueous solution of beta-methyl choline chloride (1.5 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 13 g of 90% methanesulfonic acid by weight methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 170 g diethylene glycol. An amount of 471 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 21.7 g triethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.8) was obtained.

(24) I.9 Synthesis of Inventive Salt (A.9):

(25) An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 520 g of a 70% by weight aqueous solution of beta-n-propyl choline chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 250 g propylene glycol. An amount of 774 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 11.9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.9) was obtained.

(26) I.10 Synthesis of Inventive Salt (A.10):

(27) An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 520 g of a 70% aqueous solution of dimethylmonobutylcholine chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g propylene glycol. An amount of 788 g of a yellowish liquid. An aliquot of 200 g of the liquid so obtained was neutralized with 11.4 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.10) was obtained.

(28) I.11 Synthesis of Inventive Salt (A.11):

(29) An amount of 105 g citric acid monohydrate (0.5 moles) was dissolved in 397 g of a 60% by weight aqueous solution of dimethyl n-octylcholine chloride (1.0 mole). Water was removed within 90 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 9.5 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g propylene glycol. An amount of 470 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.11) was obtained.

(30) I.13 Synthesis of Inventive Salt (A.13):

(31) An amount of 85 g gallic acid (3,4,5-trihydroxybenzoic-acid, 0.5 moles) was dispersed in 121 g of a 75% by weight aqueous solution of choline methanesulfonate (0.5 moles). Water was removed within 90 minutes in a rotary evaporator (2-l-flask)-oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 8 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 100 g diethylene glycol. An amount of 271 g of a yellowish liquid was obtained. An aliquot of 100 g of the liquid so obtained was neutralized with 4.6 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.13) was obtained.

(32) Comparative Salts:

(33) C-(A.15): Choline chloride, 75% by weight aqueous solution, commercially available from BASF SE

(34) C-(A.16):

(35) An amount of 75 g (0.5 mol) tartaric acid was portion-wise dissolved (15 g units) in 206 g of an 80% by weight aqueous solution of choline bicarbonate (1.0 mol). The solution was stirred until the CO.sub.2 evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-l-flask)-oil bath temperature of 120° C., 10 mbar. A clear substance was obtained that was diluted with 150 g diethylene glycol. 390 g of a clear solution were obtained, C-(A.16). No ester formation could be detected.

(36) C-(A.17): An amount of 105 g (0.5 mol) citric acid monohydrate was portion-wise dissolved (20 g units) in 206 g of an 80% by weight aqueous solution of choline bicarbonate (1.0 mol). The solution was stirred until the CO.sub.2 evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-l-flask)-oil bath temperature of 120° C., 10 mbar. A clear substance was obtained that was diluted with 150 g diethylene glycol. 412 g of a clear solution were obtained, C-(A.17). No ester formation could be detected.

(37) C-(A.18): An amount of 105 g (0.5 mol) citric acid monohydrate was portion-wise dissolved (20 g units) in 309 g of an 80% by weight aqueous solution of choline bicarbonate (1.5 moles). The solution was stirred until the CO.sub.2 evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-l-flask)-oil bath temperature of 120° C., 10 mbar. A clear substance was obtained that was diluted with 150 g diethylene glycol. 497 g of a clear viscous solution were obtained, C-(A.18). No ester formation could be detected.

(38) C-(A.19): citric acid monohydrate

(39) C-(A.20): monosodium salt of citric acid

(40) C-(A.21): disodium salt of citric acid

(41) C-(A.22): trisodium salt of citric acid

(42) C-(A.19), C-(A.20), C-(A.21) and C-(A.22) are known builder compounds used in detergents formulations.

(43) II. Application Tests

(44) II.1 Liquid Formulations

(45) Low water liquid detergent formulations substitute water with glycols as like diethylene glycol or DPG and hence solubility is inevitable. Salts (A.1) to (A.11) and C-(A.16) to C-(A.18) are each soluble in diethylene glycol and/or dipropylene glycol and hence can be formulated without water.

(46) 50 g of C-(A.19), C-(A.20), C-(A.21) and C-(A.22) were each combined in a flask together with 100 g diethylene glycol and heated at 100° C. for 30 minutes under stirring. The heating source was removed and the resulting white suspensions were cooled to ambient temperature over a period of 10 hours. The resulting slurries were filtered (paper filter) and the filter cakes washed twice with 50 g isopropanol. The isolated compounds C-(A.19), C-(A.20), C-(A.21) and C-(A.22) were gravimetrically determined, showing that in the absence of water C-(A.19), C-(A.20), C-(A.21) and C-(A.22) cannot be used due to insufficient solubility. The following amounts were obtained as filter cakes: C-(A.19): 44.0 g; C-(A.20): 45.8 g; C-(A.21): 47.2 g; C-(A.22): 48.2 g.

(47) II.2 Enzyme Stability

(48) The storage stability of Lipase and Protease in water was assessed at 37° C.

(49) Base test formulations were manufactured by making base formulations I to VI by mixing the components according to Table 1.

(50) The respective salt (component (a)) or comparative compound was added, if applicable, to the respective base formulation in amounts as indicated in Table 1.

(51) Enzyme (component (b)) was added, to the respective base formulation in amounts as indicated in Table 1. The amount of enzyme as provided in Table 1 refers to active protein. Either lipase or protease was added, depending on which enzyme activity was measured.

(52) Lipolase® 100 L (CAS-No. 9001-62-1, EC-No. 232-619-9) was purchased from Sigma-Aldrich. Savinase® 16.0 L (CAS-No. 9014-01-1, EC-No. 232-752-2) was purchased from Sigma-Aldrich.

(53) Water was added to accomplish the balance to 100.

(54) TABLE-US-00001 TABLE 1 liquid formulations wt % in formulation Ingredients reference I. II. III. IV. V. VI. Base formulation: (B.1) 6 15 8 — 35 30 25 (B.2) — — 6 8 — — — (B.3) 7.5 6 4 — 8 — 22 (B.4) 2 2 — — 10 12 6 (B.5) 8 4 8 4 14 — (B.6) — — 2.5 — — 5 — Sorbitol — 3 — — 3 — — PEI-EO20 — 3 5 3 5 5 — Propyleneglycol 8 4 — 8 6 4 Glycerol (G) or (E) 2.5 — — (G) 6 — (G) 6 (G) 8 Ethanol (E) Ca-formiate — 1 — 1 2 2 — Additives: Savinase 16.0 L 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Lipolase 0.4 0.4 0.4 0.4 0.4 0.4 0.4 component (a)** — 2.5 2.5 2.5 4.0 4.0 4.0 balance Water to 100 (B.1): n-C.sub.18-alkyl-(OCH.sub.2CH.sub.2).sub.25—OH (B.2): C.sub.10-C.sub.18-alkylpolygycoside blend (B.3): Sodium C.sub.10-C.sub.12-alkyl benzenesulfonate (B.4): Sodium cumenesulfonate (B.5): Sodium laurethsulfate - n-C.sub.12H.sub.25—O—(CH.sub.2CH.sub.2O).sub.3—SO.sub.3Na (B.6): n-C.sub.12H.sub.25(CH.sub.3).sub.2N.fwdarw.O **for comparative tests without inventive compounds those were replaced by the same amount of diethylene glycol.
Lipase Activity:

(55) Lipolase activity at certain points in time as indicated in Table 2 was be determined by employing pNitrophenol-valerate (2.4 mM pNP-C5 in 100 mM Tris pH 8.0, 0.01% Triton X100) as a substrate. The absorption was measured at 20° C. every 30 seconds over 5 minutes at 405 nm. The slope (absorbance increase at 405 nm per minute) of the time dependent absorption-curve is directly proportional to the activity of the lipase.

(56) Table 2 displays lipase activity in liquid formulations measured after storage; 1-30 days at 37° C. The lipolytic activity values provided in Table 2 were calculated referring to the 100% value determined in the reference formulation at the time 0.

(57) The nomenclature of formulations is as follows: the Roman number before the full stop characterizes the base formulation, the Arabian number the type of salt (A.# inventive salt (component (a)); C-(A.#) comparative compound). Zero (“0”): no salt, but diethylene glycol.

(58) TABLE-US-00002 TABLE 2 lipase activity in the course of time of storage at 37° C. Formulation identifier Base for- com- mulation pound T0 1 d 3 d 6 d 10 d 15 d 20 d 25 d 30 d I. 0 95 89 77 68 53 38 30 22 16 I. (A.1) 96 96 93 94 89 85 82 64 72 I. (A.3) 101 100 98 95 91 88 85 68 78 I. (A.4) 103 100 99 96 95 93 90 86 85 I. (A.6) 97 96 94 92 89 85 80 78 75 I. (A.7) 95 95 91 85 81 76 69 65 59 I. C-(A.15) 97 95 80 67 55 41 32 22 20 I. C-(A.16) 95 90 81 68 51 40 34 25 24 I. C-(A.17) 97 90 80 70 53 42 38 33 29 I. C-(A.18) 98 91 84 72 55 45 39 32 29 II. 0 94 92 81 73 57 41 32 25 20 II. (A.2) 95 94 92 90 88 84 80 75 68 II. (A.5) 102 100 97 95 93 89 86 72 79 II. (A.6) 103 100 99 96 94 92 90 86 88 II. (A.8) 96 94 90 85 80 80 76 72 68 II. (A.9) 97 93 90 87 83 81 77 75 76 II. (A.10) 96 96 92 89 83 84 79 76 73 II. (A.12) 100 98 96 95 88 86 80 77 76 II. C-(A.15) 96 95 82 65 56 40 33 26 22 II. C-(A.22) 95 87 79 67 55 40 33 24 18 III. 0 96 93 83 74 63 51 42 30 24 III. (A.4) 100 98 96 93 90 85 84 82 77 III. (A.6) 104 100 101 97 94 90 89 86 82 III. (A.9) 97 95 93 88 84 80 77 73 71 III. (A.10) 96 96 91 86 82 79 76 73 69 III. (A.11) 97 96 93 84 83 76 71 70 63 III. (A.12) 102 98 97 95 91 86 80 78 74 III. C-(A.20) 100 92 79 70 51 39 30 22 16 III. C-(A.21) 101 93 78 68 50 39 31 25 20 III. C-(A.22) 98 92 76 66 48 37 28 23 19 IV. 0 88 85 81 70 60 55 46 39 33 IV. (A.1) 98 96 93 92 87 84 82 79 71 IV. (A.3) 99 100 98 95 90 88 85 80 74 IV. (A.4) 101 100 97 93 90 89 86 81 73 IV. (A.6) 96 96 91 89 86 85 81 78 75 IV. (A.7) 97 95 90 85 81 78 73 70 64 IV. C-(A.15) 94 95 82 72 59 44 36 30 23 IV. C-(A.16) 95 90 81 67 55 41 34 28 25 IV. C-(A.17) 96 93 86 74 63 51 46 38 33 IV. C-(A.18) 95 91 84 72 58 49 46 39 35 V. 0 83 80 75 68 59 50 41 36 30 V. (A.2) 97 94 90 87 84 81 78 74 69 V. (A.4) 101 98 94 90 87 84 80 76 71 V. (A.5) 101 100 98 96 94 88 84 77 74 V. (A.7) 96 95 92 88 84 80 75 70 66 V. (A.11) 97 96 91 89 85 77 73 68 60 V. (A.13) 95 96 91 87 80 70 61 52 45 V. C-(A.15) 98 95 86 74 63 54 41 39 30 V. C-(A.16) 96 92 85 70 65 58 49 37 28 VI. 0 82 79 72 63 54 47 38 30 25 VI. (A.4) 102 99 96 91 86 82 80 75 65 VI. (A.5) 99 97 95 91 83 78 73 70 63 VI. (A.6) 96 90 87 84 81 74 70 67 61 VI. (A.11) 97 92 88 84 83 78 75 72 65 VI. C-(A.18) 96 90 83 75 58 50 48 37 32
Protease Activity:

(59) Savinase activity at certain points in time as indicated in Table 3 was be determined by employing Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which was determined by measuring OD.sub.405. Measurement were done at 20° C.

(60) Table 3 displays protease activity measured in liquid formulations after storage for 1 to 30 days at 37° C. The proteolytic activity values provided in Table 3 were calculated referring to the 100% value determined in the reference formulation at the time 0.

(61) The nomenclature of formulations is as follows: the Roman number before the full stop characterizes the base formulation, the Arabian number the type of salt (A.# inventive salt (component (a)); C-(A.#) comparative compound). Zero (“0”): no salt, but diethylene glycol.

(62) TABLE-US-00003 TABLE 3 protease activity in the course of time of storage at 37° C. Formulation identifier Base for- com- mulation pound T0 1 d 3 d 6 d 10 d 15 d 20 d 25 d 30 d I. 0 98 98 86 67 49 38 30 23 8 I. (A.1) 96 94 91 75 59 48 42 36 29 I. (A.4) 100 97 95 76 60 50 45 36 32 I. (A.5) 99 96 94 73 58 49 44 38 33 I. (A.6) 96 93 87 75 59 50 45 40 29 I. C-(A.15) 98 92 85 64 47 39 31 22 8 I. C-(A.16) 98 91 84 66 49 38 30 23 10 I. C-(A.17) 98 90 81 70 49 38 30 23 12 III. 0 96 95 86 71 51 40 33 21 12 III. (A.6) 92 96 92 82 69 59 50 41 34 III. (A.8) 94 95 93 78 68 60 52 42 33 III. (A.12) 96 96 92 80 70 61 50 39 31 III. C-(A.17) 92 94 83 71 53 42 34 27 14 III. C-(A.18) 94 94 85 72 54 43 35 28 14 V. 0 83 98 86 70 49 38 30 23 13 V. (A.1) 84 93 88 80 62 54 46 38 30 V. (A.2) 87 90 90 83 66 58 50 44 36 V. (A.6) 88 91 89 84 70 60 52 43 35 V. C-(A.18) 83 90 83 64 50 40 33 25 16 VI. 0 87 93 86 70 49 38 30 23 11 VI. (A.4) 84 90 88 76 68 61 53 44 30 VI. (A.7) 85 90 84 74 66 59 50 40 31 VI. (A.9) 83 87 85 75 67 60 51 40 30 VI. (A.11) 86 91 87 77 69 58 49 42 32 VI. C-(A.15) 85 90 84 66 47 36 29 20 10
II.3 Textile Cleaning Tests

(63) The detergent performance of formulations in cleaning two types of test fabrics was carried out. Testing cloth samples comprised a complex soil containing proteinaceous and fatty components due to CFT process as well as test cloth samples contained a fatty/particulate type of soil.

(64) The test was performed as follows: a multi stain monitor containing 8 standardized soiled fabric patches, each of 2.5×2.5 cm size and stitched on two sides to a polyester carrier was washed together in a launder-O-meter with 2.5 g of cotton fabric and 5 g/L of the liquid test laundry detergent, Table 4.

(65) The conditions were as follows: Device: Launder-0-Meter from SDL Atlas, Rock Hill, USA. Washing liquor: 250 ml, washing time: 60 minutes, washing temperature: 30° C. Water hardness: 2.5 mmol/L; Ca:Mg:HCO.sub.3 4:1:8

(66) Fabric to liquor ratio 1:12 After the wash cycle, the multi stain monitors were rinsed in water, followed by drying at ambient temperature over a time period of 14 hours.

(67) The following pre-soiled test fabrics were used:

(68) CFT C-S-10: butter on cotton

(69) CFT C-S-62: lard, colored on cotton

(70) CFT C-S-68: chocolate ice-cream on cotton

(71) EMPA 112: cocoa on cotton

(72) EMPA 141/1: lipstick on cotton

(73) EMPA 125: monitor for tensid

(74) wfk20D: pigment and sebum-type fat on polyester/cotton mixed fabric

(75) CFT C-S-70: chocolate mousse

(76) wfk=wfk test fabrics GmbH, Krefeld

(77) EMPA=Swiss Federal Institute of Materials Testing

(78) CFT=Center for Test Material B.V.

(79) The total level of cleaning was evaluated using color measurements. Reflectance values of the stains on the monitors were measured using a sphere reflectance spectrometer (SF 500 type from Datacolor, USA, wavelength range 360-700 nm, optical geometry d/8°) with a UV cutoff filter at 460 nm. In this case, with the aid of the CIE-Lab color space classification, the bright-ness L*, the value a* on the red-green color axis and the b* value on the yellow-blue color axis, were measured before and after washing and averaged for the 8 stains of the monitor. The change of the color value (Δ E) value, defined and calculated automatically by the evaluation color tools on the following equation:
ΔE=Δ Delta a*2+Δ Delta b*2++Δ Delta L*2,

(80) Δ E is a measure of the achieved cleaning effect. All measurements were repeated six times to yield an average number. Note that higher Δ E values show better cleaning. A difference of 1 unit can be detected by a skilled person. A non-expert can detect 2 units easily. The results are shown in Table 5

(81) R.sub.w=washed soil reflectance

(82) R.sub.o=unsoiled reflectance

(83) The increase in detergency due to the builder was calculated as: A total of 6 replications of each cloth were run during this study; a statistical confidence level of 90-95% was calculated.

(84) Test formulations were manufactured by making formulations VII to XIII by mixing the components according to Table 14.

(85) The respective salt (component (a)) or comparative compound was added, if applicable, to the respective base formulation in amounts provided in Table 4.

(86) Lipolase® 100 L was added, if applicable, to the respective base formulation in amounts provided in Table 4.

(87) Savinase® 16.0 L was added, if applicable, to the respective base formulation in amounts provided in Table 4.

(88) Water was added to accomplish the balance to 100.

(89) TABLE-US-00004 TABLE 4 liquid laundry formulations Wt-% in formularion Ingredients VII. VIII. IX. X. XI. XII. XIII. Base formulation: (B.1) 8 8 8 35 35 35 35 (B.2) 6 6 6 — — — — (B.3) 4 4 4 8 8 8 8 (B.4) — — — 10 10 10 10 (B.5) 4 4 4 4 4 4 4 (B.6) 2.5 2.5 2.5 — — — — Sorbitol — — — 2 2 2 2 PEI-EO20 5 5 5 5 5 5 5 Propyleneglycol 4 4 4 8 8 8 8 Glycerol — — — — — — — Ca-formiate — — — 2 2 2 2 Additives: Savinase 16.0 L — — — — — 0.5 0.5 Lipolase — — 0.4 — 0.4 0.4 0.4 component (a)** — 2.5 2.5 — 2.5 2.5 4 balance Water to 100 (B.1): n-C.sub.18-alkyl-(OCH.sub.2CH.sub.2).sub.25—OH (B.2): C.sub.10-C.sub.18-alkylpolygycoside blend (B.3): Sodium C.sub.10-C.sub.12-alkyl benzenesulfonate (B.4): Sodium cumenesulfonate (B.5): Sodium laurethsulfate - n-C.sub.12H.sub.25—O—(CH.sub.2CH.sub.2O).sub.3—SO.sub.3Na (B.6): n-C.sub.12H.sub.25(CH.sub.3).sub.2N.fwdarw.O **for comparative tests without inventive compounds those were replaced by the same amount of diethylene glycol.

(90) The increase in detergency due to salt (component (a)) was calculated as: a total of 6 examples of each cloth were run during this study; a statistical confidence level of >90% was calculated. Table 5 shows the sum of ΔE of the above mentioned multi-stain monitor. The launder-O-meter tests were executed with freshly prepared formulation (to) and with storing at 37° C. during a 2-month storage temperature. As an approximation one week at 37° C. is equivalent to 3% weeks at 20° C.

(91) TABLE-US-00005 TABLE 5 Results of launder-O-meter tests Formulation identifier ΔE ΔE ΔE ΔE ΔE Base for- com- ΔE 1 2 4 6 8 mulation pound T0 week weeks weeks weeks weeks VII. 0 158 157 159 158 158 156 VIII. C-(A.21) 161 160 159 158 160 159 VIII. (A.12) 157 160 157 158 158 157 VIII. (A.4) 162 163 161 163 161 161 VIII. (A.2) 160 159 161 158 160 158 IX. 0 183 180 174 170 166 161 IX. (A.2) 184 183 180 178 177 173 IX. (A.3) 183 184 181 179 179 175 IX. (A.4) 185 185 183 181 182 181 IX. (A.7) 181 179 180 178 179 177 X. 0 164 164 163 162 163 163 XI. 0 188 186 180 174 169 164 XI. (A.5) 191 189 188 188 184 185 XI. (A.10) 185 187 187 185 182 180 XI. (A.12) 185 186 186 187 185 185 XII. 0 190 186 181 178 172 164 XII. (A.12) 190 189 188 188 188 184 XII. (A.2) 191 189 186 186 184 182 XII. (A.5) 191 194 193 191 189 188 XIII. 0 194 190 184 177 171 165 XIII. C-(A.15) 190 189 185 175 168 163 XIII. (A.8) 191 191 189 189 186 186 XIII. (A.10) 192 188 189 188 185 183 XIII. (A.12) 190 191 190 188 187 188