FR2807445A1 - PROCESS FOR STABILIZING AN ENZYME - Google Patents

Abstract

The present invention relates to a process for stabilizing an enzyme, in particular with a view to a cosmetic application, which consists in mixing the enzyme with a vehicle containing an effective amount of a vegetable oil or wax chosen from: a) - triglyceride oils of which at least 5% by weight of fatty acids consist of palmitoleic acid; b) - triglyceride oils of which at least 10% by weight of fatty acids consist of hydroxylated fatty acids; c) - triglyceride oils of which at least 40% by weight of the fatty acids consist of mono- or diunsaturated C20 to C24 fatty acids, d) - oils or waxes containing at least 50% by weight of fatty acid monoesters having in total 36 to 46 atoms of carbon and derived from an unsaturated fatty acid and an unsaturated fatty alcohol.

Description

i The present invention relates to a method for stabilizing an enzyme,

  especially for cosmetic application.

  We know that enzymes have limited stability over time when

  one seeks to incorporate them in cosmetic compositions.

  Various methods have therefore already been proposed for stabilizing enzymes. One of these methods consists in conditioning the enzyme in a phase where the enzyme is stable and in mixing during use this phase with a second phase conditioned separately to form an emulsion. Such a method is described in FR-A-2 163 348 (Richand), WO 96/10 990 (Knoll), WO 97/27 841 (GIST BROCADES), and WO 97/26 861 (NORD-MARK

ARZNEIMITTEL).

  Another method, described in FR 2 737 115 (L'OREAL), consists in

  decrease the activity of water by using polyols.

  Another method, described in FR 2 732 593 (BIOEUROPE), consists in

  immobilizing the enzyme on particles such as silica particles.

  With the aid of stability tests, the inventors have now demonstrated that the enzymes can be stabilized by certain vegetable oils. A subject of the present invention is thus a method for stabilizing an enzyme, in particular with a view to cosmetic application, which consists in mixing the enzyme with a vehicle containing an effective amount of a vegetable oil or wax chosen from: a ) - triglyceride oils of which at least 5% by weight of fatty acids consist of palmitoleic acid; b) - triglyceride oils of which at least 10% by weight of the fatty acids consist of hydroxylated fatty acids; c) - triglyceride oils of which at least 40% by weight of fatty acids consist of C20 to C24 fatty acids mono- or di-unsaturated, d) - oils or waxes containing at least 50% by weight of monoesters d fatty acids having a total of 36 to 46 carbon atoms and derived from an acid

  unsaturated fat and an unsaturated fatty alcohol.

  The specific vegetable oil or wax is advantageously present in a proportion by weight relative to the enzyme of at least 4/1 and, preferably, of

/ 1 to 60/1.

  The specific vegetable oils or waxes which are used in the present invention belong to 4 specific classes: a- triglyceride oils of which at least 5% by weight of the acids

  Fats are made up of palmitoleic acid.

  Examples include the following oils: * macadamia (containing 20 to 25% palmitoleic acid), 10. oatmeal (containing 22 to 29% palmitoleic acid), * sea buckthorn (containing 20 to 30% d 'palmitoleic acid),

  Avocado (containing 5 to 10% palmitoleic acid).

  b- Triglyceride oils of which at least 10% by weight of acids

  Fats are made up of hydroxy fatty acids.

  The hydroxylated fatty acids are essentially ricinoleic acid (OH C18: 1), dimorphecolic acid (OH C18: 2) and lesquerolic acid (OH

C20: 1).

  Examples include the oils of the following species: Castor oil (Ricinus communis) 85 to 90% of OH C18: 1 Ricinus zanzibarinus 90% of OH C18: 1 Hiptage madablota 70% of OH C18: 1 Hiptage benghalensis 85% of OH C18 : 1 Dimorphoteca pluvialis 62% OH C18: 2 Osteopermum ecklonis 72% OH C18: 2 Lesquerella fendleri, and others

  species of the same genus 57% of OH C20: 1.

  c- Triglyceride oils of which at least 40% by weight of acids

  Fats are made up of mono- or di-unsaturated C20-C24 fatty acids.

  These fatty acids are essentially eicosanoic acid (C20: 1 A13), eicosenoic acid (C22: 1 A5), docosenoic acid or erucic acid (C22: 1 A13), docosanedienoic acid (C22: 2 A5 , A13), tetracosenoic acid

(C24: 1).

  As examples, there may be mentioned the oils of the following species: Limnanthe (Limnanthes alba) containing 62.5% of C20: 1 A13 L. douglasii containing 62.5% of C20: 1 A13 Brassica napus containing 45% of C22: 1 A13 Crambe abyssinica containing 55% C22: 1 A13 Eruca sativa containing 45% C22: 1 A13

  Lunaria annua containing 43% of C22: 1 A13.

  d- Oils or waxes containing at least 50% by weight of fatty acid monoesters having a total of 36 to 46 carbon atoms and derived

  an unsaturated fatty acid and an unsaturated fatty alcohol.

  As an example, mention may be made of jojoba oil, the two main fatty acids of which are as follows:

C20: 1 70 to 75%

C22: 1 0 to 15%,

  and the two main alkanols are as follows

C20: 1 50%

C22: 1 40%.

  The enzymes which can be stabilized according to the present invention can be of different types (proteases, lipases, phospholipases, phosphatases), of any origin (biotechnological, bacterial, fungal,

  vegetable) and in any form (powder, liquid, immobilized, vectorized).

  The vehicle used in the present invention may also contain a mineral oil which may be present in a proportion by weight relative to

the enzyme at least 10/1.

  Examples of mineral oils include paraffin oils

and petrolatum oils.

  The vehicle used in the present invention may also contain a silicone, in particular oils of the polydimethylsiloxane type (such as dimethicones, dimethiconols), modified polydimethylsiloxane oils (such as dimethicone copolyols), or volatile silicones (such as

cyclomethicones).

  The vehicle used in the present invention may also contain an ester chosen from fatty alcohol esters (C4-C36), fatty acid esters (C4-C36), polyoxyethylenic esters of fatty acids and d fatty alcohols such

  as Steareth 21, Isoceteth 20, esters of polyalcohols such as myreth3-

  myristate, polypropylene glycol - 2 myristyl ether propionate, esters of

  sucroses such as glucose stearate, methyl glucose sesquistearate.

  The vehicle used in the present invention may further contain a gelling agent. As an example, mention may be made of hydroxypropylmethylcellulose and hydroxyethylcellulose, xanthan gums, alginates, carbomers (for example Carbopol 1382), Polyquaterniums such as Polyquaternium 32,

Polyquaternium 39.

  Stabilized enzyme compositions according to the present invention 1o may contain in particular: 0.1 to 5% by weight of enzyme, 2 to 50% by weight of specific vegetable oil, to 50% by weight of mineral oil, 1 20% by weight of silicone, 1 to 30% by weight of gelling agent,

1 to 30% of ester.

  In vitro test results will be given below showing the stabilizing effect of specific vegetable oils in compositions of the type

  cosmetic and the effect of complementary constituents.

  The proteolytic activity of a protease (Keratoline from the company Sederma), an enzyme of the subtilisin type, is measured by in vitro hydrolysis of casein. After hydrolysis, the remaining proteins are precipitated by the acid. The assay of the released peptides is carried out according to the modified Lowery method: in an alkaline medium, the Cu2 + ions of the Folin reagent form with the amide bonds of the peptides a complex colored in blue, which is measured at 660 nm. The intensity of the hydrolysis is

  proportional to the value of the optical density.

  The compositions tested were the following Enzyme (Keratoline) 0.5% Vegetable oil 30.0% Mineral oil (isohexadecane) 38.9% Gelling agent (galactomannan) 3.0% Silica 2.0% Silicone (cyclomethicone) 10.0% Antioxidant (tocopherol acetate) 0.1% Ester (alkyl benzoate (C12 C15)) 15.0% Fragrance 0.5% Results Measurements were taken after 15 days and 1 month to observe the evolution of enzyme activity over time. The following results are obtained: Oils according to macadamia Limnanthes jojoba castor the invention

DO (TO) 0.68 0.83 1.29 0.45

  DO (15 days) 0.50 0.77 0.97 0.42 DO (1 month) 0.46 0.54 0.70 0.39 Other palm grape sweet almond oils

DO (T10O) 0.03 0.18 0.30

  DO (15 days) 0.00 0.14 0.23 DO (1 month) 0.00 0.00 It therefore appears that specific vegetable oils according to the invention are perfectly suited to preserve the activity of an enzyme over time in a finished product. On the other hand, the other oils (palm, grape, sweet almond), either destroy the enzymes from the start, or do not allow to preserve

enzyme activity.

  Other tests have been carried out with additional constituents.

  The compositions tested were as follows: 1 2 3 4 Enzyme (keratoline) 0.5% 0.5% 0.5% 0.5% Macadamia oil 30.0% 0.00 0.00 0.00 Mineral oil ( isohexadecane) 38.9% 68.9% 38.9% 38.9% Gelling agent (galactomannan) 3.0% 3.0% 3.0% 3.0% Silica 2.0% 2.0% 2.0 % 2.0% Silicone (cyclomethicone) 10.0% 10.0% 40% 10.0% Antioxidant (tocoterol acetate) 0.1% 0.1% 0.1% 0.1% 6 Ester (Benzoate d '' alkyl (C12 C15) 15.0% 15.0% 15.0% 45% Perfume 0.5% 0.5% 0.5% 0.5% The results were as follows: Composition Reference DO (TO) DO (15 days)% / T0 1 macadamia 0.68 0.50 73.5

2 mineral oil 0.92 NA * -

  3 silicone oil 0.55 NA * 4 ester 0.32 0.12

  * NA: not accessible because phase separation.

  It is clear that replacing macademia oil with mineral oil, silicone oil or an ester does not allow

  stabilize the enzyme composition.

  Other concrete examples of compositions will be given below.

  stabilized according to the process of the invention.

  Example I: FACE milk A face milk formula was packaged in a bi-pack packaging, the two compartments mixing only at the time of use, so as to

form an emulsion on the skin.

  The composition of a first phase of anhydrous gel type is as follows: Enzyme (Keratoline) 0.5% Vegetable oil 30.0% Mineral oil (isohexadecane) 38.9% Gelling agent (galactomannan) 3.0% Silica 2.0 % Silicone (cyclomethicone) 10.0% Antioxidant (tocopherol acetate) 0.1% Ester (alkyl benzoate (C12 -C15) 15.0% The composition of the second phase (emulsion type) is as follows: Water demineralized 54, 450 Sequestrant (Na4 EDTA) 0.025 Softener (Allantoin) 0.200 Gelling agent (Xanthan gum) 4.075 Ester (Cocoate) 8,000 Preservatives (POBM) 1.650 Vegetable oil (macadamia) 4,000 Antioxidant (tocopherol acetate) 0, 200 Emulsion (Steareth. ..) 4,100 Silica 1,000 Demineralized Water 8,000 Moisturizer (Glycerin) 10,000 Sorbaine 0.600 Plant-Pro-Retinol Complex 0.200 Vitamins (Palmitate of Vit.A, Vit.E) 0.200 Fragrance 0.300 Ethanol 3,000 Example 2: Cake (Cast powder) Ester ( Cocoate) 20,400 Paraffin 65 5,000 Sunscreens 1,000 Triglyceride 8.0 00 Vegetable oil (macadamia) 9,500 Emulsifiers (Soy lecithin) 1,500 Antioxidant (tocopherol acetate) 0.100 Silicone 7,000 Enzyme (protease) 0.600 Moisturizer (glycerin) 6,000 Silica 5,000 Perfume 0.200 Fillers (TiO2) 27,500 Gelling agent (Bentone) 1,200 Nacre 7,000 Example 3: FACE and BODY emulsion Demineralized water 60,575 Humectant (Propylene glycol) 2,000 Moisturizer (Glycerin) 2,000 Sequestrant (Na 4 EDTA) 0.025 Gelling agents (Alginate) 0.100 Preservative (POBM) 0.200 Antioxidant (tocopherol acetate) 0.100 Vegetable oil (macadamia ) 12,000 Silicone (Cyclomethicone) 3,000 Trygliceride 2,000 Softeners (Bisabolol) 0.100 Thickener (Fatty Acid) 1,500 Emulsifiers (Fatty Alcohol) 4,000 Ester (Cocoate) 7,000 Enzymes (Proteases) 1,100 Perfume 0.300 Ethanol 3,000 Silica 1,000

Claims (7)

  1. Method for stabilizing an enzyme, in particular with a view to cosmetic application, which consists in mixing the enzyme with a vehicle containing an effective amount of a vegetable oil or wax chosen from a) - triglyceride oils, including at least 5% by weight of the fatty acids consist of palmitoleic acid; b) - triglyceride oils of which at least 10% by weight of the fatty acids consist of hydroxylated fatty acids; c) - triglyceride oils of which at least 40% by weight of io fatty acids consist of C20 to 024 fatty acids mono- or diunsaturated, d) - oils or waxes containing at least 50% by weight of monoesters of fatty acids having a total of 36 to 46 carbon atoms and derived from an acid   unsaturated fat and an unsaturated fatty alcohol.   2. The method of claim 1, wherein said vegetable oil   is present in a proportion by weight relative to the enzyme of at least 4/1.   3. The method of claim 1 or claim 2, wherein   the vehicle also contains mineral oil.   4. The method of claim 3, wherein the mineral oil is   present in a proportion by weight relative to the enzyme of at least 10/1.   5. Method according to any one of claims 1 to 4, in   which the vehicle further contains a silicone.   6. Method according to any one of claims 1 to 5, in   which vehicle also contains an ester chosen from fatty alcohol esters (C4-C36), fatty acid esters (C4-C36), polyoxyethylene esters   fatty acids and fatty alcohols, polyalcohol esters, sucrose esters.   7. The method of claim 6, wherein the vehicle contains further a gelling agent.