Method for stabilizing both lipase and protease in liquid enzymatic laundry detergent

Abstract

This disclosure provides a method for stabilizing both lipase and protease in liquid enzymatic laundry detergent, comprising steps of: first, self-assembling conjugated linoleic acid with protease and lipase, respectively, to form vesicles encapsulated protease and vesicles encapsulated lipase at a low Ca.sup.2+ concentration; then, mixing the solution of vesicles encapsulated protease and the solution of vesicles encapsulated lipase, and then partially cross-linking conjugated linoleic acid molecules on the vesicles' surface. The obtained enzyme vesicles, after being concentrated, can be used directly in liquid laundry detergent. The lipase in the liquid laundry detergent will not be degraded by protease, and the enzymes in vesicles are able to resist against surfactant inhibition. Thus, the enzyme activities can be maintained in the liquid laundry detergent, and the enzymatic vesicles will be broken to release enzymes, when the liquid laundry detergent is used at a higher Ca.sup.2+ concentration such as in tap water.

Claims

1. A method for stabilizing both lipase and protease in a liquid enzymatic laundry detergent, comprising steps of: self-assembling, in a buffer solution at room temperature, containing conjugated linoleic acid and calcium salts; subsequently adding a protease solution and a lipase solution respectively into the buffer solution; followed by shaking with a shaker at 20-30 C. and 50 rpm until the encapsulation rates no longer increase; wherein the self-assembly step yields a solution of conjugated linoleic acid vesicles encapsulated protease and a solution of conjugated linoleic acid vesicles encapsulated lipase; and a step (b) of mixing the solution of conjugated linoleic acid vesicles encapsulated protease and the solution of conjugated linoleic acid vesicles encapsulated lipase to produce a mixture, and placing the mixed solution mixture in a reactor equipped with a magnetic stirrer and a stopper; adding [3-(3,4-dimethyl-9-oxo-9-hydro-thioxanthene-2-oxy)-2-hydroxypropyl] trimethylammonium chloride solution in dark; and then conducting nitrogen sweeping into the reactor for 20-25 minutes; sealing the reactor, stirring the solution and then radiating by an ultraviolet spot light to partially cross-link conjugated linoleic acid, and stop radiation when reaching a certain cross-linking degree; thus a solution of enzymatic vesicles was obtained in which the partially cross-linked conjugated linoleic acid serve as wall material and the enzymes serve as core material; and afterwards, lyophilizing to concentrate the solution of enzymatic vesicles to reach a water content of 10%-15%, and then adding the solution of enzymatic vesicles directly into a liquid laundry detergent to obtain liquid enzymatic laundry detergent in which both protease and lipase can maintain their enzyme activities in the liquid laundry detergent when the liquid laundry detergent is stored; and in tap water use, the vesicles rupture to release protease and lipase rapidly.

2. The method for stabilizing both protease and lipase in a liquid enzymatic laundry detergent according to claim 1, wherein the calcium salt is one or a mixture of two of calcium citrate, calcium citrate tetrahydrate, and tricalcium dicitrate.

3. The method for stabilizing both protease and lipase in a liquid enzymatic laundry detergent according to claim 1, wherein the concentration of the calcium salts is 0.1-0.30 mmol.Math.L.sup.1.

4. The method for stabilizing both protease and lipase in a liquid enzymatic laundry detergent according to claim 1, wherein with regard to the partial cross-linking of conjugated linoleic acid, stop radiating when reaching a certain cross-linking degree means stop cross-linking when the cross-linking degree reaches 35%-50%.

5. The method for stabilizing both protease and lipase in liquid enzymatic laundry detergent according to claim 1, wherein the liquid laundry detergent is a concentrated liquid laundry detergent with surfactants as main material and the surfactant concentration is between 20 wt % and 50 wt %.

6. The method for stabilizing both protease and lipase in liquid enzymatic laundry detergent according to claim 1, wherein the final concentration of conjugated linoleic acid is 200-600 mmol.Math.L.sup.1.

7. The method for stabilizing both protease and lipase in liquid enzymatic laundry detergent according to claim 1, wherein the amount of the protease or the lipase added is not more than 600 mg protein per gram of conjugated linoleic acid.

8. The method for stabilizing both protease and lipase in liquid enzymatic laundry detergent according to claim 1, wherein pH of the buffer solution ranges from 7.0 to 10.0; the citric acid-citrate and the cation in the buffer solution is one of sodium ion, potassium ion and ammonium ion.

9. The method for stabilizing both protease and lipase in the liquid enzymatic laundry detergent according to claim 1, wherein solvents for dissolving [3-(3,4-dimethyl-9-oxo-9-hydro-thioxanthene-2-oxy)-2-hydroxypropyl] trimethylammonium chloride and conjugated linoleic acid are same; and the amount of [3-(3,4-dimethyl-9-oxo-9-hydro-thioxanthene-2-oxy)-2-hydroxypropyl] trimethylammonium chloride used is 0.05 wt %-0.10 wt % of that of conjugated linoleic acid.

Description

DETAILED DESCRIPTION

(1) The formula of the liquid laundry detergent used in Embodiments 1-3 is as follows (in percentage by dry base):

(2) AES (fatty alcohol polyoxyethylene ether sodium sulfate): 21.5%;

(3) AEO-7 (fatty alcohol polyoxyethylene ether-7): 2.5%;

(4) AEO-9 (fatty alcohol polyoxyethylene ether-9): 2.5%;

(5) Trisodium citrate: 3.5%;

(6) Polyether defoamer: 0.05%;

(7) Citric acid: 0.1%;

(8) NaCl: 0.5%;

(9) Fluorescent brightener: 0.14%;

(10) Kathon: 0.05%;

(11) Essence (lemon flavor): 0.1%; and

(12) Water to make up to volume,

(13) pH=7.5.

(14) The formula of the liquid laundry detergent used in Embodiments 4-6 is as follows (in percentage by dry weight):

(15) AES: 29%;

(16) coconut oil polyoxyethylene ether-9: 8%;

(17) AEO-9: 5.5%;

(18) AEO-7: 2.5%;

(19) Trisodium citrate: 3.5%;

(20) Polyether defoamer: 0.05%;

(21) Citric acid: 0.1%;

(22) NaCl: 0.5%;

(23) Fluorescent brightener: 0.14%;

(24) Kathon: 0.05%;

(25) Essence (lemon flavor): 0.1%; and

(26) Water to make up to volume,

(27) pH=7.4.

(28) These formulae are similar to those of most commercially available liquid laundry detergents. The formulae may be modified as needed. Modifications on the formulae have little influence on the performance of stabilizing both protease and lipase provided in the present disclosure. The protease and lipase used in the following embodiments are all commercially available enzymes, some of which have been concentrated in the laboratory. The method of the present disclosure is not limited to proteases and lipases to be mentioned in the following embodiments.

Embodiment 1

(29) A Method for Stabilizing Both Alkaline Protease 2709 and Pseudomonas aeruginosa Lipase in a Liquid Enzymatic Laundry Detergent

(30) Vesicles encapsulated alkaline protease 2709 (909.1 mg protein/g, produced by Xintai Sinobest Biotech Co., Ltd., China) and vesicles encapsulated Pseudomonas aeruginosa lipase (839.6 mg protein/g, provided by Renewable Energy Laboratory, School of biotechnology, Jiangnan University, P.R. China) were respectively prepared and used as follows:

(31) (1) 11.52 g of conjugated linoleic acid was dissolved in 200 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.6, 0.01 mol/L) containing 0.25 mmol/L calcium citrate at room temperature, and mixed well; the solution was then divided into two halves, 3.17 g of alkaline protease 2709 was added in one half and 3.43 g of Pseudomonas aeruginosa lipase was added in another half; and, each of the two solutions was shaken in dark for 4 hours (at a shaking speed of 50 rpm) by a shaker at 25 C., to obtain two solutions of conjugated linoleic acid vesicles encapsulated enzyme, wherein the protease encapsulation rate was 91.2% and the lipase encapsulation rate was 92.3%.

(32) (2) The above two solutions of enzymatic vesicles were mixed in a cylindrical glass reactor (height-diameter ratio of 2:1) equipped with a stopper and a magnetic stirrer, and then the QTX solution of 0.06 wt. % of conjugated linoleic acid was then added in dark; oxygen was discharged by nitrogen sweeping for 20 minutes into the reactor before sealing the reactor. The solution was stirred under radiation by an ultraviolet spot light for 20 minutes at 25 C. The radiation is stopped when the cross-linking degree reached 36.2%. In this way, a solution of partially cross-linked conjugated linoleic acid vesicles encapsulated enzymes was obtained.

(33) (3) The above vesicle solution was concentrated with lyophilization until the water content reached 10%. The obtained enzymatic vesicles were added in the liquid laundry detergent in a proportion of 4 wt %. After 8 weeks at 25 C., in the liquid laundry detergent, the retention rate of protease activity was 85% and the retention rate of lipase activity was 84%. After six months, in the liquid laundry detergent, the retention rate of protease activity was 55% and the retention rate of lipase activity was 59%. 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water (hardness of 98 ppm, calculated in form of calcium carbonate), and after 1 min, the release rate of protease activity was 99.8% and the release rate of lipase activity was 100.1% in the solution.

Comparison Example 1

(34) Alkaline Protease 2709 and Pseudomonas aeruginosa Lipase were Directly Added in the Liquid Laundry Detergent.

(35) 0.78 g of alkaline protease 2709 and 0.84 g of Pseudomonas aeruginosa lipase were added together in 100 g of the liquid laundry detergent, and mixed well. After 8 weeks at 25 C., in the liquid laundry detergent, the retention rate of protease activity was 38% and the retention rate of lipase activity was 9%. After six months, the retention rate of protease activity was 31% and the retention rate of lipase activity was 3%. 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water, at 25 C. and after 1 min, the release rate of protease activity was 99.2% and the release rate of lipase activity was 100.3% in the solution.

Comparison Example 2

(36) Alkaline Protease 2709 and Pseudomonas aeruginosa Lipase were Stabilized by Sodium Alginate and Calcium Chloride.

(37) 3.17 g of alkaline protease 2709 solution and 3.43 g of Pseudomonas aeruginosa lipase solution were added, respectively, into 100 mL of the 3% sodium alginate solution under stirring. The enzyme solutions were slowly added into the 3% calcium chloride solution dropwise using a sterile syringe to obtain gel microspheres, respectively. The solution of gel microspheres was placed overnight at 40 C. for further hardening. The hardened gel microspheres were then obtained by vacuum filtration. The hardened gel microspheres were washed three times with sterile saline solution to remove calcium carbonate on the microsphere surfaces and then naturally dried until the water content reached 50%.

(38) 10 g of the protease gel microspheres and 10 g of the lipase gel microspheres were added into 100 g of the liquid laundry detergent and mixed well. After 8 weeks at 25 C., the retention rate of protease activity was 40.8% and the retention rate of the lipase activity was 14.6%. After six months, the retention rate of protease activity was 28% and the retention rate of lipase activity was 7%. 1.2 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water, at 25 C. and after 1 min, the release rate of protease activity was 10.2% and the release rate of lipase activity was 16.1% in the solution.

Embodiment 2

(39) A method for Stabilizing Both Serratia marcescens Alkaline Protease and Pseudomonas aeruginosa Lipase in Liquid Enzymatic Laundry Detergent

(40) Vesicles encapsulated Serratia marcescens alkaline protease (930.2 mg protein/g, from Serratia marcescens) and vesicles encapsulated Pseudomonas aeruginosa lipase (839.6 mg protein/g, provided by Renewable Energy Laboratory, Scholl of Biotechnology, Jiangnan University, China) were respectively prepared and used as follows:

(41) (1) Conjugated linoleic acid and calcium citrate were dissolved and mixed well in 0.01 mol/L borax buffer (pH 9.18) at room temperature. The final concentration of conjugated linoleic acid was 300 mmol/L and the concentration of calcium salt was 0.15 mmol/L. 5.25 g of Serratia marcescens alkaline protease and 5.81 g of Pseudomonas aeruginosa lipase were added, respectively, in 100 mL of the solution. That is, the amount of enzymes added in the solution was 565 mg protein per grain of conjugated linoleic acid. Each of the two solutions was mixed well, shaken for 6 hours by a shaker (at a shaking speed of 45 rpm) at 22 C., and self-assembled to obtain two solutions of conjugated linoleic acid vesicles encapsulated protease and lipase respectively, wherein the protease encapsulation rate was 92.5% and the lipase encapsulation rate was 93.2%.

(42) (2) The above two self-assembled solutions of conjugated linoleic acid vesicles encapsulated protease and lipase, obtained in the step (1), were placed together in the reactor. QTX solution of 0.065 wt % of the mass of conjugated linoleic acid, was then added in dark; and then the reactor was sealed after 20 minutes of nitrogen sweeping. The solution in the reactor was stirred under radiation by an ultraviolet spot light to partially cross-link conjugated linoleic acid. The radiation is stopped when the cross-linking degree reached 46.6%. In this way, a solution of partially cross-linked conjugated linoleic acid vesicles encapsulated protease and lipase was obtained. The above solution was concentrated with lyophilization until the water content reached 12%, and the obtained enzymatic vesicles were added directly into the liquid laundry detergent in a proportion of 3 wt %. After 8 weeks at 25 C., in the liquid laundry detergent, the retention rate of protease activity was 91.56% and the retention rate of lipase activity was 92.7%. 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water (hardness of 110 ppm, calculated in form of calcium carbonate), and after 1 min, the release rate of protease activity was 97.8% and the release rate of lipase activity was 100.1% in the solution.

(43) Embodiment 3

(44) A Method for Stabilizing Both Neutral Protease 1398 and Conn Alkaline Lipase in Liquid Enzymatic Laundry Detergent

(45) Vesicles encapsulated neutral protease 1398 (905.7 mg protein/g, provided by Xintai Sinobest Biotech Co., Ltd., China) and vesicles encapsulated alkaline lipase (860.3 mg protein/g, provided by Shenzhen Earth Conn Biological Technology Co., Ltd., China) were respectively prepared and used as follows:

(46) (1) Conjugated linoleic acid and calcium citrate tetrahydrate were dissolved in 0.01 mol/L disodium hydrogen phosphate-citric acid buffer (pH 8) at room temperature. The final concentration of conjugated linoleic acid was 240 mmol/L and the concentration of calcium salts was 0.25 mmol/L. After they were mixed well, 4.04 g of neutral protease 1398 and 4.26 g of lipase were added, respectively, into 100 mL of the solution. That is, the amount of neutral protease and lipase added in each solution was 585 mg protein per gram of conjugated linoleic acid. Each of the two solutions was shaken for 5.5 hours by a shaker (at a shaking speed of 50 rpm) at 25 C., and self-assembled to obtain two solutions of conjugated linoleic acid vesicles encapsulated protease and lipase, wherein the protease encapsulation rate was 91.9% and the lipase encapsulation rate was 90.7%.

(47) (2) The above two self-assembled solutions of conjugated linoleic acid vesicles encapsulated protease and lipase, obtained in the step (1), were placed together in a reactor; the QTX solution of 0.075 wt. % of the mass of conjugated linoleic acid was then added in dark; and then the reactor was sealed after 23 minutes of nitrogen sweeping. The solution in the reactor was stirred under radiation by an ultraviolet spot light to partially cross-link conjugated linoleic acid. The radiation is stopped when the cross-linking degree reached 48.9%. Therefore, a solution of partially cross-linked conjugated linoleic acid vesicles encapsulated both protease and lipase was obtained. The above solution was concentrated with lyophilization until the water content reached 12%, and the obtained enzymatic vesicles were added directly into the liquid laundry detergent in a proportion of 3 wt %. After 8 weeks at 25 C., in the liquid laundry detergent, the retention rate of protease activity was 83.9% and the retention rate of lipase activity was 87.8%.

(48) 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap, water (hardness of 105 ppm, calculated in form of calcium carbonate), and after 1 min, the release rate of protease activity was 100.3% in the solution.

Embodiment 4

(49) A Method for Stabilizing Both Alkaline Protease 2709 and Conn Alkaline Lipase in Liquid Enzymatic Laundry Detergent

(50) The alkaline protease 2709 and Conn alkaline lipase were encapsulated and used as follows:

(51) (1) Conjugated linoleic acid, a same amount of calcium citrate tetrahydrate and tricalcium dicitrate were dissolved in 0.01 mol/L borax buffer solution (pH 9.0) at room temperature, and mixed well. The final concentration of conjugated linoleic acid was 255 mmol/L and the concentration of calcium salts was 0.28 mmol/L. Then, 4.77 g of alkaline protease 2709 and 5.04 g of Conn alkaline lipase were added, respectively, into 100 mL of the solution. The amount of alkaline protease and lipase added in the solution each was 600 mg protein per gram of conjugated linoleic acid. Each of the two solutions was shaken individually for 8 hours by a shaker (at a shaking speed of 50 rpm) at 30 C., and self-assembled to obtain two solutions of conjugated linoleic acid vesicles encapsulated protease and lipase, wherein the encapsulation rate was 92.4% for the protease and 91.5% for the lipase.

(52) (2) The above two self-assembled solutions of conjugated linoleic acid vesicles encapsulated protease and lipase, obtained in the step (1), were placed together in a reactor; the QTX solution of 0.12% of the mass of conjugated linoleic acid was then added in dark, and then the reactor was sealed after 25 minutes of nitrogen sweeping. The solution in the reactor was stirred under radiation by an ultraviolet spot light to partially cross-link conjugated linoleic acid. The radiation is stopped when the cross-linking degree reached 46%. In this way, a solution of partially cross-linked conjugated linoleic acid vesicles encapsulated protease and lipase was obtained. The above solution was concentrated with lyophilization until the water content reached 13%, and the obtained mixture of vesicles encapsulated protease and vesicles encapsulated lipase was added directly in the liquid laundry detergent in a proportion of 2.9 wt %. After 8 weeks at 25 C., in the liquid laundry detergent, the retention rate of protease activity was 80.8% and the retention rate of lipase activity was 79.9%. 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water (hardness of 110 ppm, calculated in form of calcium carbonate), 1 min after, the release rate of protease activity was 100.1% and the release rate of lipase activity was 100.2% in the solution.

Comparison Example

(53) The two enzymes were directly added in the liquid laundry detergent.

(54) 0.8 g of each enzyme was added in the liquid laundry detergent. After 8 weeks at 25 C., the retention rate of protease activity was 46% and the retention rate of lipase activity was 6.7%. 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water, and after 1 min, the measured release rate of protease activity was 99.8% and the release rate of lipase activity was 99.1% in the solution.

Embodiment 5

(55) A Method for Stabilizing Both Neutral Protease 1398 and Pseudomonas aeruginosa Lipase in Liquid Enzymatic Laundry Detergent

(56) vesicles encapsulated neutral protease 1398 and vesicles encapsulated Pseudomonas aeruginosa lipase were respectively prepared and used as follows:

(57) (1) Conjugated linoleic acid and calcium chloride were added into 0.015 mol/L disodium hydrogen phosphate-citric acid buffer solution (pH 7.0) at room temperature, and mixed well. The final concentration of conjugated linoleic acid was 310 mmol/L and the concentration of calcium salts was 0.25 mmol/L. 4.25 g of neutral protease 1398 and 4.59 g of the lipase were added, respectively, into 100 mL of the above solution. That is, the amount of neutral protease and lipase added in the solution each was 570 mg protein per gram of conjugated linoleic acid. Each of the two solutions was mixed well, shaken for 8 hours by a shaker (at a shaking speed of 45 rpm) at 20 C., and self-assembled to obtain two solutions of conjugated linoleic acid vesicles encapsulated enzyme, wherein the encapsulation rate was 93.7% for the protease and was 90.8% for the lipase, respectively.

(58) (2) The above two self-assembled solutions of conjugated linoleic acid vesicles encapsulated protease and lipase, obtained in the step (1), were placed together in a reactor; the QTX solution of 0.10% of the mass of conjugated linoleic acid was then added in dark; and then the reactor was sealed after 20 minutes of nitrogen sweeping. The solution in the reactor was stirred under radiation by an ultraviolet spot light to partially cross-link conjugated linoleic acid. The radiation is stopped when the cross-linking degree reached 50%. In this way, a solution of partially cross-linked conjugated linoleic acid vesicles encapsulated protease and lipase was obtained. The above solution was concentrated with lyophilization until the water content reached 14%, and the obtained mixture of vesicles encapsulated protease and vesicles encapsulated lipase was added directly in the liquid laundry detergent in a proportion of 6 wt %. After 8 weeks at 25 C., the retention rate of protease activity was 85.2% and the retention rate of lipase activity was 82.1%. 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water (hardness of 98 ppm, calculated in form of calcium carbonate), and after 1 min, the measured release rate of protease activity was 94%.

Embodiment 6

(59) A Method for Stabilizing Both Alkaline Protease 2709 and Conn Alkaline Lipase in Liquid Enzymatic Laundry Detergent

(60) Vesicles encapsulated alkaline protease 2709 and vesicles encapsulated Conn alkaline lipase were respectively prepared and used as follows:

(61) (1) Conjugated linoleic acid and calcium chloride were added into 0.01 mol/L borax-sodium hydroxide buffer solution (pH 10.0) at room temperature, and mixed well. The final concentration of conjugated linoleic acid was 590 mmol/L and the concentration of calcium salts was 0.30 mmol/L. After they were mixed well, 10.37 g of alkaline protease 2709 and 10.96 g of Conn alkaline lipase were added, respectively, into 100 mL of the solution. That is, the amount of alkaline protease and lipase added in the solution each was 550 mg protein per gram of conjugated linoleic acid. Each of the two solutions was shaken respectively for 7 hours by a shaker (at a shaking speed of 50 rpm) at 22 C., and self-assembled to obtain two solutions of conjugated linoleic acid vesicles encapsulated enzymes, wherein the protease encapsulation rate was 91.9% and the lipase encapsulation rate was 90.7%.

(62) (2) The above two self-assembled solutions of conjugated linoleic acid vesicles encapsulated protease and lipase, obtained in the step (1), were together placed in a reactor; the QTX solution of 0.10% of the mass of conjugated linoleic acid was then added in dark; and then the reactor was sealed after 25 minutes of nitrogen sweeping. The solution in the reactor was stirred under radiation by an ultraviolet spot light to partially cross-link conjugated linoleic acid. The radiation is stopped when the cross-linking degree reached 35%. In this way, a solution of partially cross-linked conjugated linoleic acid vesicles encapsulated protease and lipase was obtained. The above solution was concentrated with lyophilization until the water content reached 13%. The obtained mixture of vesicles encapsulated protease and vesicles encapsulated lipase was added directly in the liquid laundry detergent in a proportion of 1.5 wt %. After 8 weeks at 25 C., the retention rate of protease activity was 82.3% and the retention rate of lipase activity was 85.1%. 1 g of the liquid enzymatic laundry detergent was dispersed into 1 L tap water (hardness of 98 ppm, calculated in form of calcium carbonate), and after 1 min, the release rate of protease activity was 99.2% and the release rate of lipase activity was 98.9% in the solution.

(63) Although the present invention has been disclosed above by preferred embodiments, the present invention is not limited thereto. Various changes and improvements may be made by those skilled in the art without departing from the spirit of the present invention. Therefore, the protection scope of the present invention shall be subject to that defined by the appended claims.