KR100427638B1 - The compositons of enzyme complex which enhance enzyme stability and the cosmetic product containing the complex - Google Patents

Abstract

The present invention relates to a stabilized enzyme conjugate composition characterized in that it contains an enzyme adsorbed on calcium salt and a water-soluble chitosan and a cosmetic containing the same, wherein the enzyme conjugate composition of the present invention comprises calcium carbonate, calcium phosphate, calcium stearate or algae. One or two or more enzymes selected from proteolytic enzymes, starch degrading enzymes, lipolytic enzymes or lysozyme using one or two or more insoluble or insoluble calcium salt powders selected from calcium powders derived from egg shells of 0.0001 to 50 And is deacetylated chitosan or an oligomer thereof in an amount of 0.0001 to 50% by weight. The present invention also relates to a cosmetic containing the calcium salt chitosan enzyme complex in an amount of 0.001 to 70% by weight based on the total weight of the composition.

Description

Stabilized Enzyme Binder Composition and Cosmetics Containing the Same {THE COMPOSITONS OF ENZYME COMPLEX WHICH ENHANCE ENZYME STABILITY AND THE COSMETIC PRODUCT CONTAINING THE COMPLEX}

Currently, enzymes are applied in various industries such as medical industry, food industry, biotechnology, textile industry and cosmetics industry, and products containing various types of enzymes are commercialized. Among them, proteolytic enzymes are the most widely used enzymes in the world and are used for various purposes, and they are enzymes that hydrolyze peptide bonds, which are amino acid bonds. As a result, the skin color becomes brighter and fine skin is removed, which is an advantage of the cosmetics that directly apply proteolytic enzymes derived from plants and microorganisms.

However, these enzymes have the property of easily losing activity due to physical factors such as heat, light, pressure, chemical factors such as pH, complexing agents, various types of inhibitors according to the types of enzymes, and various metal ions. It is large but fairly unstable and its activity rapidly decreases over time. In the application of these enzymes to products, due to transport, distribution to customers and storage of stocks, it is usually time consuming to produce and use products containing enzymes, and they can also undergo wide temperature changes. In reality, the shelf life is short and there are many limitations in the application as it may be exposed to other conditions that exacerbate the decrease in activity.

Current methods for overcoming these drawbacks preserve enzymes in solutions such as saline and buffered saline, but these products do not provide sufficient stability when subjected to temperature stress or long term storage.

In addition, a method of dispersing the enzyme in the oil-soluble component by mixing with excipients in a moisture-free form or by mixing with a buffer or the like before use or minimizing contact of the enzyme with water is another method. come. However, such a method is cumbersome in use, and in many cases, due to such stabilization method, the function of the enzyme is not properly exerted.

Therefore, there is a need for a method of stabilizing the enzyme in an aqueous solution, such as adsorption (adsorption) to attach the enzyme to the surface of the unreacted material and entrapment (entrapment) in the material. Inclusive methods are used more than adsorption because they can immobilize various enzymes. However, the disadvantage is that the enzyme activity may be lost due to heat or chemical treatment during the immobilization process, so the physicochemical properties of the enzyme, such as maintaining the activity before and after treatment, should be considered.

Chitin is an important component of shellfish, shellfish and squid skeletal shells of crustaceans, and is a polysaccharide contained in the cell walls of insects, fungi, mushrooms and some bacteria. N-acetylglucosamine is a β-1,4 bond. Linked basic homopolymers.

Chitosan is obtained by deacetylating this chitin, and the IUPAC name is (1,4) 2-amino-2-deoxy-beta-D-glucosamine {(1,4) 2-amino-2-deoxy-beta -D-Glucosamine}. Chitosan is a basic polysaccharide with amino group like chitin, and was first used as a heavy metal adsorbent for wastewater treatment in 1970. Since then, it has been actively applied in various fields such as medical materials, food industry, biotechnology, textile industry and cosmetics industry. In particular, chitosan has been known to have excellent biocompatibility and biodegradability, no toxicity and genotoxicity to the human body, and has a very bright prospect as a new material and drug carrier for medical use due to less side effects such as immune response. In addition, it has thickening properties, excellent moisturizing properties, softness, and film formation properties, and thus has high functionality as a cosmetic raw material.

Therefore, when the chitosan having such characteristics is used for the stabilization of the enzyme, it is stable to the human body and has excellent affinity with the skin or hair, and additional functions such as film formation and moisture retention can be expected.

However, such chitosan is generally not soluble in water or alcohol, and is dissolved in an inorganic acid such as an aqueous solution of organic acid such as formic acid, lactic acid, ascorbic acid, acetic acid, and dilute hydrochloric acid.

However, many kinds of oil-soluble substances, proteins and enzymes often exhibit high stability in the neutral or alkaline region. Therefore, recent researchers have developed various derivatives by applying chemical formulas to increase the solubility in the neutral or alkaline region of chitosan, and developed various derivatives, or improved water solubility by lowering the molecular weight of chitosan using enzymes, etc. Oligomers were developed. However, until now, stabilizing enzymes have been used as stabilizing aids by dissolving existing chitosan in acidic solutions.

As a result of research on various stabilization methods attempted so far, it has been found that the composition of an enzyme containing a part of a divalent cation such as calcium ions contributes to the stability of the enzyme. However, there have been no examples in which poorly soluble or insoluble salts such as calcium carbonate, calcium phosphate, calcium sulfate and calcium peroxide are used as stabilizing cycle agents.

Object of the present invention is one or two or more poorly soluble or insoluble calcium salt powders and deacetylated chitosan selected from calcium carbonate, calcium phosphate, calcium stearate, calcium sulfate, calcium peroxide or calcium powder derived from egg shell of algae It is to provide an enzyme stabilizing composition stabilized proteolytic enzyme, starch degrading enzyme, or hydrogen peroxide degrading enzyme using an oligomer.

Still another object of the present invention is to provide a cosmetic composition containing the calcium salt chitosan enzyme complex.

Other objects and functions of the present invention will become apparent to those skilled in the art from the following detailed description of the invention.

The present inventors first tried various stabilization methods for the purpose of improving the stability of protease as part of the research for applying various kinds of enzymes directly to cosmetics, and poorly soluble or insoluble calcium powder derived from eggshell of calcium carbonate or algae After adsorbing these enzymes onto the membrane, the membrane was formed using a water-soluble chitosan oligomer, thereby solving the conventional problems and confirming the fact that the efficacy and effect as a raw material for cosmetics increased, thereby completing the present invention.

Furthermore, the present inventors found that when insoluble calcium salt is used as a stabilizing mechanism of the enzyme, it is possible to protect the enzyme by converting hydrogen ions, which adversely affect the stability of the enzyme, into a weak acid form and blocking a sharp drop in pH.

The present invention relates to a stabilized enzyme conjugate composition characterized by containing a calcium salt-enzyme-chitosan complex produced by mixing calcium salts, enzymes and water soluble chitosan, which are poorly soluble or insoluble in neutral or basic aqueous solutions.

In addition, the present invention is a calcium salt which is poorly soluble or insoluble in the neutral or basic aqueous solution, one or two or more from calcium powder derived from calcium carbonate, calcium phosphate, calcium stearate, calcium sulfate, calcium peroxide or algae egg shell Selected.

In addition, the present invention is stabilized enzyme conjugate composition characterized in that the enzyme is one or two or more selected from protease, starch degrading enzyme, lipolytic enzyme or lysozyme is 0.0001 to 50% by weight relative to the total composition It is about.

Furthermore, the present invention relates to a cosmetic composition, characterized in that it contains 0.001 to 70% by weight of the stabilized enzyme conjugate composition.

In addition, the present invention relates to a cosmetic composition, characterized in that the cosmetic composition is a skin cosmetic or hair cosmetic.

More specifically, one or two or more poorly soluble or insoluble calcium salt powders selected from calcium carbonate, calcium phosphate, calcium stearate, calcium sulfate, calcium peroxide or algae egg shells are used to decompose protease and starch. One or two or more enzymes selected from enzymes, lipolytic enzymes or lysozymes are collected in an amount of 0.0001 to 50% by weight, and contain deacetylated chitosan or an oligomer thereof in an amount of 0.0001 to 50% by weight. It relates to a stabilized enzyme conjugate composition and a cosmetic containing the same.

Insoluble calcium salts used for the purposes of the present invention can be used without limitation calcium salts showing poor solubility or insolubility at pH of neutral and alkali.

As the enzyme adsorbed to the calcium salt of the present invention, various proteolytic enzymes, lipolytic enzymes or lysozyme may be used, and may adsorb an amount of 0.0001 to 50% by weight.

The deacetylated chitosan oligomer of the present invention is chitosan having a deacetylation degree of 50 to 100%, and the molecular weight of 10,000 to 1,000,000 can be effectively used in the present invention.

The enzyme stabilizing composition of the present invention may be formulated into a face wash, soap, cream, ointment, and the like, but which formulation is used and manufactured is a matter that can be extremely easily determined by those skilled in the art to which the present invention pertains.

In addition, the enzyme stabilizing composition of the present invention can be conveniently added to basic cosmetics such as foam cleansing, lotion, essence, lotion.

In the case of preparing the face wash and the soap, an enzyme stabilizing composition may be contained in the face wash and the soap base. In addition, fragrances, pigments, fungicides, antioxidants, preservatives, moisturizers and the like can be used, and thickeners, inorganic salts, synthetic polymer materials and the like can be used for the purpose of improving the properties.

In preparing creams, enzyme-stabilizing compositions are contained in a cream base of general oil type (O / W), and fragrances, chelating agents, pigments, antioxidants, and preservatives are used. It can be used by adding synthetic or natural materials such as vitamins.

The face wash, soap, and cream may be easily prepared according to a conventional manufacturing method, respectively, and may be prepared in any form such as liquid, cream, paste and solid.

Hereinafter, the method for preparing the enzyme stabilizing composition of the present invention will be described.

The enzyme to be stabilized is dissolved in a buffer of pH 5.0 to 9.5 where the enzyme exhibits the best stability. At this time, the amount of enzyme is appropriately used depending on the nature and activity of the raw enzyme, there is no particular limitation. However, it is preferably 0.0001 to 50% by weight. In addition, buffers are not particularly limited so long as they do not inhibit enzyme stabilization, and suitable buffers include various salts known in the art such as saline solution, borate solution and other conventional buffers such as phosphate and citrate. It can be chosen from an aqueous solution. When the aqueous solution is a saline solution, it is usually preferred that 0.1-2% sodium chloride is contained in the saline solution.

Insoluble calcium powder is finely pulverized using a grinding machine to increase the surface area as much as possible, add 10 to 500% by weight to the prepared enzyme solution, uniformly disperse it, and leave it at low temperature so that the enzyme solution is sufficiently adsorbed onto the insoluble calcium powder. do. At this time, the time required for adsorption varies depending on the conditions, but is in the range of 0.5 to 72 hours.

The adsorbed calcium-enzyme complex is used after filtration or centrifugation to remove the unadsorbed enzyme, or it is dried using a low temperature dryer or a freezing dryer. In this case, the time required for drying varies depending on various conditions, and the calcium-enzyme complex may be uniformly dispersed in the chitosan solution. For example, when using a lyophilizer, the range of 1 to 120 hours is preferable.

1 to 600% by weight of the calcium-enzyme complex obtained as described above after dissolving chitosan or an oligomer thereof having a deacetylation degree of 50 to 100% in a buffer of pH 5.0 to 9.5 showing the best stability of the enzyme. After homogeneously dispersing, the mixture is allowed to stand at low temperature so that the chitosan or chitosan oligomer sufficiently forms a coating on the calcium-enzyme complex. At this time, the time required for film formation varies depending on the conditions, but is in the range of 1 to 72 hours.

The formed chitosan-calcium-enzyme complex is removed by filtration or centrifugation of the excess chitosan solution. If necessary, the chitosan-calcium-enzyme complex can be molded into an appropriate size and shape according to the purpose and purpose of use. To dry. At this time, the time required for drying varies according to various conditions. For example, when using a freeze dryer, it is in the range of 1 to 120 hours.

Hereinafter, the present invention will be described in more detail based on the following Examples and Experimental Examples. However, these Examples and Experimental Examples are only for better understanding of the present invention, and the scope of the present invention in any sense is not limited to these Examples.

Example 1

The enzyme solution is prepared by dissolving trypsin in pH 8.0 tris aminomethane-hydrochloric acid buffer solution to 1,000 units / ml, and calcium carbonate is uniformly washed twice with distilled water to remove impurities, dried, and then ground uniformly. Chitosan with 80% deacetylation is prepared by heating and dissolving 3 g in 100 ml of pH 8.0 tris aminomethane-hydrochloric acid buffer. After all the experiments and reagents were brought to 4 ° C., 5 g of ground calcium carbonate was added to 10 ml of trypsin solution, uniformly dispersed, and left at 4 ° C. for 18 hours to sufficiently adsorb the enzyme solution to the calcium powder.

Excess enzyme solution that was not adsorbed from the adsorbed calcium-enzyme complex was removed by centrifugation and dried sufficiently using a freezing dryer.

10 ml of chitosan solution was added to 5 g of the prepared calcium-enzyme complex, which was then uniformly dispersed and left at 4 ° C. for 18 hours to form a sufficient film of chitosan in the enzyme solution.

The excess unreacted chitosan solution was removed from the calcium-enzyme-chitosan complex by centrifugation, dried sufficiently using a freezing dryer, and then ground uniformly to obtain 4.5 g of the calcium carbonate-enzyme-chitosan complex.

Example 2

The enzyme solution was prepared by dissolving subtilisin Carlsberg in pH 8.5 tris aminomethane-hydrochloric acid buffer to 1,000 units / ml, and 4.3 g of calcium carbonate-enzyme-chitosan complex was obtained by the same experimental method as in Example 1.

Example 3

Enzyme solution was prepared by dissolving trypsin in pH 8.0 tris aminomethane-hydrochloric acid buffer solution to 1,000 unit / ml, and washing egg-derived eggshell powder obtained from farmed (2) with distilled water to remove impurities and drying. Prepare by grinding uniformly.

4.1g of the eggshell powder-enzyme-chitosan complex was obtained by the same experimental method as in Example 1.

Example 4

The enzyme solution is prepared by dissolving trypsin in pH 8.0 tris aminomethane-hydrochloric acid buffer solution to 1,000 units / ml, and washing the calcium phosphate powder twice with distilled water to remove impurities, drying and grinding it uniformly. 4.0g of calcium phosphate-enzyme-chitosan complexes were obtained by the same experimental method as in Example 1.

Example 5

The enzyme solution was prepared by dissolving α-starch degrading enzyme (1,4-α-D-Glucan-Glucanohydrolase) in pH 6.5 potassium phosphate buffer to 500 units / ml, and using calcium carbonate- 4.5 g of enzyme-chitosan complex was obtained.

Example 6 and Comparative Example 6 : Preparation of stabilized enzyme composition-containing gel composition

The protease-containing gel composition was prepared by uniformly mixing the composition by stirring, vigorizing, degassing, and cooling the trypsin stabilizing composition obtained in Example 1 at a composition ratio as shown in Table 1 below.

Raw material name (% by weight) Example 6 Comparative Example 6 Ethylenediaminetetradiacetic acid sodium Quantity Quantity DL-pantenol 0.3 0.3 1,3-butylene glycol 5.0 5.0 Carboxyvinyl Polymer 0.2 0.2 Cetearyl Alcohol / Cetearyl Glucoside 2.0 2.0 Fiji-20 / Methylglucosesquistearate 1.0 1.0 Dimethicone 3.0 3.0 Isostearyl lactate 4.0 4.0 Cetanol 3.0 3.0 Triethanolamine 0.2 0.2 Trypsin stabilizing composition 0.1 - Trypsin buffer lysate - 0.1 antiseptic Quantity Quantity Spices Quantity Quantity Purified water Remaining amount Remaining amount

Example 7 and Comparative Example 7 : Preparation of Protease-Containing Cream Composition

The composition of the anti-acne-inducing cream composition was prepared by uniformly mixing the mixture of the trypsin stabilizing composition obtained in Example 1 and the cream base in a homomixer with a composition ratio as shown in Table 2 below by stirring, stirring, degassing, and cooling in a homomixer. It was.

Raw material name (% by weight) Example 7 Comparative Example 7 Ethylenediaminetetradiacetic acid sodium Quantity Quantity DL-pantenol 0.3 0.3 Betaine 4.0 4.0 Polysorbate 60 1.0 1.0 Arachidyl Alcohol / Behenyl Alcohol / Arachidyl Glucoside 1.5 1.5 Stearic acid 2.0 2.0 Pyromethicone 4.0 4.0 Isostearyl lactate 3.0 3.0 Triethanolamine 0.04 0.04 Polyacrylamide / c13-14isoparaffin / laures-7 0.5 0.5 Trypsin stabilizing composition 0.1 - Trypsin buffer lysate - 0.1 antiseptic Quantity Quantity Spices Quantity Quantity Purified water Remaining amount Remaining amount

Example 8 and Comparative Example 8 Preparation of Protease-Containing Latex Composition

A proteinase-containing emulsion composition was prepared by uniformly mixing the composition of the trypsin stabilizing composition and the emulsion base obtained in Example 1 with a composition ratio as shown in Table 3 by stirring, stirring, degassing, and cooling in a homomixer. It was.

Raw material name Example 8 Comparative Example 8 Ethylenediaminetetradiacetic acid sodium Quantity Quantity Sodium lauryl sulfate 10.0 10.0 Laura Kiddie 3.0 3.0 Isostearyl lactate 2.0 2.0 glycerin 10.0 10.0 Hydroxyethyl Cellulose 50.0 50.0 Trypsin stabilizing composition 0.1 - Trypsin buffer lysate - 0.1 antiseptic Quantity Quantity Spices Quantity Quantity Purified water Remaining amount Remaining amount

Example 9 and Comparative Example 9 : Preparation of Protease-containing Cleansing Gel Composition

The cleansing gel composition was prepared by uniformly mixing the composition of the trypsin stabilizing composition and the cleansing gel base obtained in Example 1 with a composition ratio as shown in Table 4 by stirring, stirring, degassing, and cooling in a homomixer.

Raw material name Example 9 Comparative Example 9 Ethylenediaminetetradiacetic acid sodium Quantity Quantity Sodium lauryl sulfate 10.0 10.0 Laura Kiddie 3.0 3.0 Isostearyl lactate 2.0 2.0 glycerin 10.0 10.0 Hydroxyethyl Cellulose 50.0 50.0 Trypsin stabilizing composition 0.1 - Trypsin buffer lysate - 0.1 antiseptic Quantity Quantity Spices Quantity Quantity Purified water Remaining amount Remaining amount

Comparative Example 1

Trypsin is dissolved in pH 8.0 tris aminomethane-hydrochloric acid buffer to prepare to 1,000 units / ml, sodium azide is added to 0.02%, dissolved and stored at 4 ° C.

Comparative Example 2

Subtilisin Carlsberg is dissolved in pH 8.5 tris aminomethane-hydrochloric acid buffer to prepare to 1,000 units / ml, sodium azide is added to 0.02%, dissolved and stored at 4 ° C.

Comparative Example 3

α-starchase is dissolved in pH 6.5 potassium phosphate buffer and prepared to 500 units / ml. Sodium azide is added to 0.02%, dissolved and stored at 4 ° C.

Comparative Example 4

The enzyme solution is prepared by dissolving trypsin in pH 8.0 tris aminomethane-hydrochloric acid buffer solution to 1,000 unit / ml, and calcium carbonate is uniformly washed twice with distilled water to remove impurities, dried and then ground uniformly. After all the experiments and reagents were brought to 4 ° C., 5 g of ground calcium carbonate was added to 10 ml of trypsin solution, uniformly dispersed, and left at 4 ° C. for 18 hours to sufficiently adsorb the enzyme solution to the calcium powder.

The adsorbed calcium-enzyme complex is removed by centrifugation of excess enzyme solution and sufficiently dried using a freezing dryer.

Comparative Example 5

Enzyme solution was prepared by dissolving trypsin in pH 8.0 tris aminomethane-hydrochloric acid buffer solution to 1,000 unit / ml, dissolving calcium chloride to 50mM, adding sodium azide to 0.02%, and dissolving it at 4 ℃. keep it.

Comparative Example 10

Enzyme solution was prepared by dissolving trypsin in pH 8.0 tris aminomethane-hydrochloric acid buffer solution to 2,000 unit / ml, and chitosan with 80% deacetylation was prepared by heating and dissolving 6 g in 100 ml of pH 8.0 tris aminomethane-hydrochloric acid buffer solution. do. After all experiments and reagents were brought to 4 ° C., 10 ml of chitosan solution was added to 10 ml of trypsin solution, and then uniformly dispersed. The mixture was left at 4 ° C. for 18 hours, and then dried sufficiently using a freezing dryer. Trituration yielded 4.5 g of enzyme-chitosan complex.

Test Example 1 Protease Activity Confirmation Experiment

0.6 g of emulsion casein is dissolved in 100 ml of buffer in which the enzyme exhibits optimal activity.

The enzyme solution is prepared so that the enzyme activity is about 10-20 units / ml using a buffer. Take exactly 1 ml of the test solution, leave for 3 min at 37 ± 1 ° C, and add 5.0 ml of casein solution previously warmed to 37 ± 1 ° C and shake immediately.

The solution was left at 37 ° C for 10 minutes, and 5.0 ml of trichloroacetic acid solution was added thereto, followed by immediate mixing. The solution was left to stand again at 37 ° C for 30 minutes and then centrifuged (10min × 10,000g) to accurately take 1 ml of filtrate, 2.5 ml of 0.55 M sodium carbonate solution and diluted folin`s solution (3-fold dilution). ) After adding 0.5 ml and leaving at 30 ± 2 ° C. for 30 minutes, absorbance A ₁ was measured at 660 nm by absorbance measurement. Separately, 1 ml of enzyme solution was correctly added, 5.0 ml of trichloroacetic acid solution was added to shake. After mixing, 5.0 ml of casein solution was added and left at 30 ± 2 ° C. for 30 minutes, followed by color development in the same manner as described above, followed by measurement of absorbance A ₂ at 660 nm by the absorbance measurement method. After obtaining the corrected absorbance (A ₁ -A ₂ ), the proteolytic activity was measured by measuring the amounts of tyrosine and tryptophan contained in the hydrolyzed material. One unit of proteolytic activity was defined as the amount of enzyme that produced 1 μg of tyrosine or tryptophan for 1 minute.

Test Example 2 Experiment for Confirming Starch Enzyme Activity

1 g of soluble starch is dissolved in 100 ml of the buffer in which the enzyme exhibits optimal activity.

The enzyme solution is prepared so that the enzyme activity is about 10-20 units / ml using a buffer. Take 1 ml of enzyme solution correctly, leave it at 37 ± 1 ° C for 3 minutes, and keep 5.0 ml of soluble starch, 3 ml of McCllvaine buffer and 1 ml of 0.1% calcium chloride solution, which were previously warmed to 37 ± 1 ° C. Add shake immediately to mix.

After leaving this solution at 37 ° C. for 30 minutes, 0.2 ml of the reaction solution was added to 10 ml of iodine solution, and the solution was measured for absorbance A ₁ at 660 nm using water as a reference solution. taking the ㎖ exactly heated at 100 ℃ 30 minutes and by color development after the enzymatic reaction by the method of the enzyme solution was modified enzymes are ways to measure the absorbance a ₂ in 660㎚ by the liquid in the absorption spectrum. Separately, 1 ml of distilled water was correctly taken and developed after the enzymatic reaction in the same manner as described above. The solution was measured for absorbance A _st at 660 nm by the absorbance measurement method. One unit of degradation activity was defined as the amount of enzyme that digests 10 mg of soluble starch for 30 minutes.

Test Example 3 Changes of enzyme activity over time in the aqueous solution of stabilized enzyme composition

The stabilizing compositions obtained in Examples 1 and 4 and 10 were dispersed in 20 ml of pH 8.0 tris aminomethane-hydrochloric acid buffer, in which 1 g of sodium azide was added to 0.02%, in a sealed container. After transfer, the samples were stored in a 37 ° C. thermostat for 30 days, and the samples obtained in Comparative Examples 1 and 5 were stored in a closed container for 30 days in a 37 ° C. thermostat, and each sample was activated by the method of Experiment 1 according to the storage period. Was measured. The results are shown in Table 5.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 1 37 100 97 91 86 83 Comparative Example 1 37 100 8 6 2 0 Comparative Example 4 37 100 77 61 49 28 Comparative Example 5 37 100 83 56 45 21 Comparative Example 10 37 100 95 51 38 17

Table 5 shows a stabilizing composition prepared by adsorbing trypsin to calcium carbonate as an enzyme stabilizing base and forming a film with water-soluble chitosan, a composition without forming a chitosan film, a composition with trypsin dissolved in a buffer, and a water-soluble calcium salt with trypsin dissolved in a buffer. The changes of enzyme activity over time of the added composition showed that the enzymatic activity of the composition using insoluble calcium salt and chitosan as a stabilizer was preserved a significant amount of enzyme activity even after one month. In Comparative Example 1 in which the enzyme was dissolved, enzymatic activity decreased drastically with time. In addition, when the composition using only insoluble chitosan and water-soluble chitoate were added, the degree of enzymatic activity was slower than that of Comparative Example 1, but the enzymatic activity decreased much over time.

Test Example 4 Changes of enzyme activity over time in the aqueous solution of stabilized enzyme composition

1 g of the stabilizing composition obtained in Example 2 was dispersed in 20 ml of pH 8.5 tris aminomethane-hydrochloric acid buffer, in which sodium azide was added at 0.02%, and transferred to a closed container for 30 days in a 37 ° C thermostat. After storage, the sample obtained in Comparative Example 2 was transferred to an airtight container, and then stored in a 37 ° C. thermostat for 30 days, and each sample was measured for activity by the method of Experimental Example 1 according to the storage period. The results are shown in Table 6.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 2 37 100 95 88 86 81 Comparative Example 2 37 100 80 50 24 10

Table 6 shows the changes in enzyme activity over time of the stabilizing composition prepared by adsorbing subtilisin to calcium carbonate as an enzyme stabilizing base and forming a film with water-soluble chitosan and a composition in which an enzyme was dissolved in a buffer. The enzymatic activity of the composition using chitosan as a salt and an encapsulant was preserved a considerable amount of enzymatic activity even after one month. However, in Comparative Example 2 in which the enzyme was dissolved in a buffer, the enzymatic activity rapidly decreased over time.

Test Example 5 Changes of enzyme activity over time in the aqueous solution of stabilized enzyme composition

1 g of the stabilizing composition obtained in Example 5 was dispersed in 20 ml of pH 6.5 potassium phosphate buffer to which sodium azide was added at 0.02%, transferred to a sealed container, and then stored in a 37 ° C. thermostat for 30 days. Samples obtained in Comparative Example 3 were transferred to an airtight container and then stored in a 37 ° C. thermostat for 30 days, and each sample was measured for activity by the method of Experimental Example 2 according to the storage period. The results are shown in Table 7.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 5 37 100 90 84 80 74 Comparative Example 3 37 100 74 52 31 19

Table 7 shows the changes in the enzyme activity over time of the stabilizing composition prepared by adsorbing α-starch dehydrogenase to calcium carbonate and forming a film with water-soluble chitosan as an enzyme stabilizing base and a composition in which the enzyme was dissolved in a buffer. The enzymatic activity of the composition using the insoluble calcium salt and chitosan as the film-forming agent was preserved a considerable amount of the enzyme activity even after one month. However, in Comparative Example 3 in which the enzyme was dissolved in the buffer, the enzymatic activity rapidly decreased. .

Test Example 6 Changes of enzyme activity over time in aqueous solution by stabilized base of stabilized enzyme composition

The stabilizing compositions obtained in Examples 1 and 3 to 4 were dispersed in 20 ml of pH 8.0 tris aminomethane-hydrochloric acid buffer, in which 1 g of sodium azide was added to 0.02%, and then transferred to a closed container. After storage for 30 days in a 37 ℃ thermostat, the sample obtained in Comparative Example 1 was transferred to an airtight container and stored in a 37 ℃ thermostat for 30 days and each sample was measured by the method of Experimental Example 1 according to the storage period. The results are shown in Table 8.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 1 37 100 97 91 86 83 Example 3 37 100 92 86 80 72 Example 4 37 100 95 88 83 80 Comparative Example 1 37 100 8 6 2 0

Table 8 shows the stabilizing composition prepared by adsorbing trypsin to calcium carbonate as an enzyme stabilizing base and forming a film with water-soluble chitosan, a composition adsorbed on eggshell calcium powder, a composition using calcium phosphate, and a composition in which only trypsin was dissolved in a buffer. The enzyme activity was changed according to the present invention. In the case of Examples 1, 3 and 4 using insoluble calcium salts, the enzyme activity was decreased even after one month compared to that in which the trypsin-soluble composition showed a rapid decrease with time. Many were preserved. The degree of stabilization according to the type of insoluble calcium salt used as the stabilization base may vary according to various variables, but calcium carbonate was the best, followed by calcium phosphate and eggshell calcium.

Test Example 7 Changes in Enzyme Activity of Proteases Containing Cosmetics with Time

The protease-containing cosmetic composition obtained in Examples 6 to 9 and Comparative Examples 6 to 9 was transferred to a closed container and stored in a 37 ° C. thermostat for 30 days, and each sample was stored in pH 8.0 tris amino according to the storage period. The activity was measured by the method of Experimental Example 1 by dispersing in 10 ml of methane-hydrochloric acid buffer. The results are shown in Tables 9, 10, 11 and 12.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 6 37 100 95 88 82 78 Comparative Example 6 37 100 36 19 8 0

Table 9 shows changes in enzyme activity over time of gel compositions containing a stabilizing composition prepared by adsorbing trypsin to calcium carbonate as an enzyme stabilizing base and forming a film with water-soluble chitosan, and a gel composition containing trypsin dissolved in a buffer solution. The enzymatic activity of the composition using insoluble calcium salt as a stabilizing base and chitosan as a film-forming agent preserved a considerable amount of enzymatic activity even after one month. Sharply reduced.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 7 37 100 92 84 79 75 Comparative Example 7 37 100 30 15 4 0

Table 10 shows the changes in the enzyme activity of the cream composition containing the trypsin stabilizing composition and the cream composition containing the trypsin dissolved in the buffer over time, and the enzyme activity of the composition using insoluble calcium salt as a stabilizing base and chitosan as a film forming agent. Although silver retained a considerable amount of enzymatic activity even after one month, Comparative Example 7 in which the enzyme was dissolved in a buffer rapidly decreased enzymatic activity over time.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 8 37 100 89 81 72 68 Comparative Example 8 37 100 24 11 0 0

Table 11 shows the changes in enzyme activity over time of the emulsion composition containing the trypsin stabilizing composition and the trypsin containing the trypsin dissolved in the buffer, and the enzyme activity of the composition using insoluble calcium salt as a stabilizing base and chitosan as a film forming agent. Although silver retained a significant amount of enzymatic activity even after one month, Comparative Example 8 in which the enzyme was dissolved in the buffer solution rapidly decreased with time.

division Storage temperature (℃) Relative activity by retention period (%) Initial activity 1 week 2 weeks 3 weeks 4 Weeks Example 9 37 100 96 87 85 81 Comparative Example 9 37 100 57 34 22 12

Table 12 shows the changes in enzyme activity over time of the cleansing gel composition containing the trypsin stabilizing composition and the trypsin containing the trypsin dissolved in the buffer. Enzyme activity was preserved a considerable amount of enzyme activity even after one month, but in Comparative Example 9 in which the enzyme was dissolved in the buffer solution, the enzyme activity rapidly decreased over time.

Amount of digested starch (mg) = (A ₂ -A ₁ ) ÷ A _st × 50 × dilution factor

As described above, the stabilized enzyme conjugate composition characterized by containing the enzyme and water-soluble chitosan adsorbed to the calcium salt according to the present invention gives high stability to the enzyme in the aqueous solution state for long-term storage in the aqueous solution state of the enzyme It is possible to increase the possibility of application of various types of products. Therefore, the stabilized enzyme conjugate composition characterized by containing an enzyme adsorbed on calcium salt and a water-soluble chitosan and a cosmetic containing it can effectively exhibit the original effect of the enzyme.

Claims (6)

Finely pulverized calcium salts showing poor solubility or insolubility in an neutral or basic aqueous solution are added to the enzyme solution, mixed and adsorbed to prepare a calcium-enzyme complex, followed by drying and mixing the water-soluble chitosan with the dried calcium-enzyme complex Stabilized enzyme conjugate composition produced by forming a water-soluble chitosan coating The calcium salt exhibiting poorly soluble or insoluble in the neutral or basic aqueous solution is selected from the group consisting of calcium carbonate, calcium phosphate, calcium stearate, calcium sulfate, calcium peroxide or calcium powder derived from egg shell of algae. Stabilized enzyme conjugate composition, characterized in that more than The stabilized enzyme according to claim 1 or 2, wherein the enzyme is one or two or more selected from proteolytic enzymes, starch degrading enzymes, and lysozyme, and is 0.0001 to 50% by weight relative to the total enzyme binder composition. Binder composition Cosmetic composition, characterized in that it contains the stabilized enzyme conjugate composition of claim 1 or 2. A cosmetic composition comprising the stabilized enzyme conjugate composition of claim 3 delete