FR2474051A1 - AQUEOUS COMPOSITIONS CONTAINING STABILIZED ENZYMES - Google Patents

Description

AQUEOUS COMPOSITIONS CONTAINING STABILIZED ENZYMES.

The present invention relates to a process for the long-term stabilization of an enzyme, in an aqueous solution, using an acid salt

  organic low molecular weight.

  It is well known that it may be advantageous to incorporate proteo-

  lytic or ct-amylolytic in cleaning compositions. These enzymes are interesting because of their ability to transform macromolecules, such as proteins and starches, into smaller molecules, so that they can be easily removed by washing with detergents and / or water. . More specifically, proteolytic enzymes are used to break down proteins and uLamylolytic enzymes are used to break down carbohydrates. Detergent compositions containing these enzymes have a wide field of application because they make it possible to remove protein and starchy soils such as stains from eggs, blood,

sauce, and the like.

  Detergent compositions are found on the market in the form of dry powder containing enzymes. However, there are problems inherent in these compositions. First, they should be stored away from moisture and heat so as to maintain its enzymatic activity.

dry powder compositions are not very suitable for certain applications

interesting cations such as cleaning stains, soaking detergent and stain removal which require direct application to the soiled surface. For these and other applications, it is desirable to have a liquid composition containing enzymes. In addition, it is advantageous to incorporate significant amounts of water into these compositions for reasons of economy and convenience of preparation. However, the addition of large amounts of water to an enzyme-containing composition raises particular problems due to the inherent instability of the enzymes.

  in the presence of water, which leads to a rapid decrease in their enzy-

matic, that is to say, their ability to break the macromolecules into smaller fragments. It is believed that this loss of enzyme activity could come from

of the hydrolyzing action of water on enzymes.

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A loss of enzymatic activity is also observed when the aqueous compositions containing enzymes are exposed to a temperature above 70C. In fact, if these compositions are subjected to temperatures of this

order for more than a few hours, we observe their complete deactivation.

Therefore, to have an enzyme-containing composition which can be used as indicated above, it is obvious that it does not

  It is not enough that the enzyme remains stable in water, that is to say that it conserves.

its enzymatic activity, but it must still be able to retain its activity even after prolonged storage at high temperature. Indeed, it is not uncommon to keep current products in reserves for a certain time at temperatures much higher than the temperature

  ambient, before delivering them to the consumer.

  Many attempts have already been made to stabilize enzymes in aqueous compositions, for example:

  - In US Patent No. 3,296,094 to Cayle, we use

uses partially hydrolyzed and solubilized collagen, and glycerol to stabilize an aqueous proteolytic enzyme composition. The amount of glycerol necessary to stabilize the composition is between 35 and

% of the total weight of the composition.

  - In McCarthy's U.S. Patent No. 3,557,002, we

  uses a monoalcohol and an alkoxylated alcohol to stabilize a protein enzyme

  lytic. Although the amount of alcohol used in this composition is less than that recommended by Cayle, the residual activity of the enzyme

this composition decreases after prolonged storage at a relatively low temperature.

tively high.

  - In US Patent No. 4,169,817 to Weber, either water-soluble salts such as Na2SO4-, K2SO4, NaCl or KCl and / or

glycerol, or alkylene glycols, to stabilize a proteolytic enzyme.

tick within compositions containing ionic (or ionizable) compounds and surfactants. Here, too, significant amounts of glycerol and / or other solids are required to achieve long-term stability of

the enzyme in these compositions.

  - In Innerfield's US Patent No. 3,682,842, an enzyme-ion binding agent such as. trichloroacetic acid or

  tungstic acid; a salt such as sodium chloride or amino sulfate

monium, an organic solvent such as ethanol and a surfactant so

  stabilize the mixture of proteolytic and amylolytic enzymes.

  - In US Patent No. 3,676,374 to Zaki et al., A mixture of alkane sulfonates or aolefin sulfonates is used with 247405 1 of the alkyl alkyleneoxy hydroxyl or sulfate derivatives to stabilize an enzyme proteolytic incorporated into a liquid detergent composition containing water. With these compositions, it is also possible to use various stabilizing agents such as CaCl2, MgCl, lactates and acetates of calcium and magnesium, soluble in water. U.S. Patent Application No. 023363 of 03/23/1979 in the name of the Applicant indicates that aqueous solutions of enzymes can be stabilized for long periods by adding an alkanolamine and an organic acid to the solution. It has now been found that the stability of the enzyme in an aqueous medium containing small amounts of alcohol can be preserved by adding an alkanolammonium salt,

  of ammonium or alkali metal of an organic acid of low molecular weight.

The present invention relates to aqueous compositions containing

stabilized enzymes capable of being used as cleansing agents

toyage, said enzymes having a long shelf life stability.

  The object of the present invention is also to provide this stability by the addition of small amounts of stabilizing or stabilizing agent.

relatively inexpensive.

According to the present invention, minimal amounts of an agent are sufficient

stabilizer of enzymes and an alcohol to obtain an excellent stabilization

  long-term reading of the compositions and which can be stored even at

  high ture. These compositions are particularly effective as

  cleaning preparations usable in a wide range of applications.

The compositions of the present invention contain the ingredients below (all the amounts below are by weight): a) from 0.01 to 15% of an enzyme stabilizing agent, consisting of an alkali metal salt, d ammonium or alkanol ammonium of an organic acid of low molecular weight such as formic, acetic, propionic, butyric and valeric acids, b) from about 0.006 to 5% of an enzyme such as proteases, Ut- amylases and their mixtures, c) from 1 to 25% approximately of an alcohol of formula ROH, where R is an alkyl radical containing 1 to 6 carbon atoms, and

from about 10 to 90% water.

  According to the present invention, it has been found that an alkali metal, ammonium or alkanol ammonium salt of certain low molecular mass organic acids is capable, in the presence of a low molecular weight monoalcohol, of stabilizing the enzymes. proteolytics or ctamylolytics and their mixtures, in an aqueous medium. In addition, it has been found that the enzyme, thus stabilized, retains its activity for long periods, from

around 12 to 18 months.

  The main constituents of the compositions of the present invention

are water, enzy mes, stabilizers and an alcohol.

  Water can constitute from 10 to 90% of the total weight of the composition of the present invention and, preferably, from 40 to 90%. Although it is not essential, it is preferable to use deionized water.

The enzymes stabilized according to the process of the invention are the pro-

  teases, α-amylases and mixtures thereof.

  Proteases, of bacterial or fungal origin, can be classified

into three different categories: acid, neutral and alkaline proteases.

All fall within the scope of the invention. Although not easy to enter

  Proteases can also be used in the above categories.

  of animal or vegetable origin without departing from the scope of the present invention.

  These enzymes are active in the pH range which ranges from approximately 3 to 11.

  They generally have an optimum activity between pH 6 and approximately 10.

  Proteases catalyze the hydrolysis of peptide bonds in proteins, polypeptides and the like. By breaking connections

  peptide, it forms free amino and acid groups on-of-

  short chain cules which can be easily removed by washing with water and / or detergent. All of the following categories of proteases can be used in the context of the present invention, but preferably

uses the one whose activity is optimum at pH between 6 and 9.

  As an example of a protease preferably used, mention may be made of the protease

serine.

The α-amylases exhibit optimum activity at acidic pHs. These enzymes catalyze the breakdown reactions of starch molecules into molecules to

  short chain which can then be easily removed by washing with water -

  and / or detergent. Α-amylases can be obtained from ani-

males, cereal seeds or bacteria or fungal sources.

  The enzyme component of the present invention can be added as a commercial enzyme preparation. In general, they are found on the market in the form of dry powder, solution or suspension and they usually contain from 2 to 80% of active enzyme in association with an inert support such as sodium sulfate or calcium, sodium chloride,

  glycerol, nonionic surfactants, or mixtures thereof, and which

  titrates the others 20 to 98% of the mixture. As examples of preparations of

  commercial protease likely to enter into the compositions of the pre-

sente invention include the following preparations: SavinaseO, Savinase 8, dC in suspension; EsperaseO Esperase 8, O in suspension and Alcalas sold by NovoIndustri A / S, Copenhagen, Denmark; and "High Alkaline Protease", Maxatase e 'and Alkaline Protease 201PO, sold by G.B. Fermentation Inc, Des

Plains Ill.

  As examples of preparations of am-amylases which can be used here, mention may be made of Amalase THC sold by the company G.B. Fermentation Inc., Des Plaines,

Ill., And Termamyl 60, Termamyl 600 sold by Novo Industri A / S, Denmark.

  As an example of a commercial enzyme preparation containing a mixture of α-amylase and protease, mention may be made of Maxatase from GB Fermentation Inc. Preferably, "Savinase 8, dC" in suspension from Novo Industries is an enzyme preparation proteolytic-alkaline obtained from Bacillus Subtilis and containing approximately 6% by weight of enzyme and having an activity

of 8 kilo-units novo.

  The compositions of the present invention make it possible to stabilize from 0.006 to 5% approximately of active enzyme. Preferably, the proportion of enzyme is

between 0.006 and 2.5% by weight.

Stabilizers are alkali metal, ammonium or alkanol ammonium salts of low molecular weight organic acids such as

formic, acetic, propionic, butyric and valeric acids.

  These stabilizers can be used at about 0.01 to 15% by weight

total of the composition and preferably from 2 to 10% approximately and, more specifically

  cially, from 2 to 6% of the weight of the composition.

As mentioned above, ammonium alkanol such as monoethanol ammonium, diethanol ammonium and

triethanol ammonium.

  The fourth main constituent of the compositions of the invention is an alcohol which contributes to increasing the enzymatic stability and which, as a function of lowering viscosity and preventing the formation of films on the surface of the compositions of the present invention. These alcohols have the formula ROH, where R is an alkyl radical comprising there 6 carbon atoms, limits included, linear or branched. The amount of alcohol is between 1 and 25% of the total weight of

  the composition and, preferably, between 4 and 8%.

In addition to the main components listed above, the compositions of the present invention may contain other ingredients such as agents

  anionic or nonionic surfactants, organic solvents, solu-

bilisants and perfumes.

The incorporation of anionic or nonionic surfactants has the advantage of improving the stability of the enzymes in the compositions and, even more, of appreciably increasing their detergent properties. These anionic or nonionic surfactants can be incorporated at a rate of approximately 1 to 55%.

  and preferably 10 to 30% of the total weight of the composition.

As examples of nonionic surfactants, there may be mentioned:

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  (1) Ethoxylated fatty alcohols, of formula: RO- (CH2-CH20) nH, o: R is a radical containing 8 to 18 carbon atoms, and n is an integer between 1 and 500; such as, for example: (a) the product of the condensation of a mole of aliphatic alcohol containing 12 or 13 carbon atoms, linear or branched, with 6.5 moles, on average, of oxide of ethylene; (b) the condensation product of a mole of aliphatic alcohol containing 12 to 15 carbon atoms, linear or branched, with 9 moles of ethylene oxide, and

  (c) the condensation product of one mole of aliphatic alcohol, comprising

  both 12 to 15 carbon atoms, linear or branched, with 3 moles

ethylene oxide.

  Products (a), (b) and (c) can be found commercially under the trade names Neodol, Neodol 23-6.5, Neodol 25-9 and

  Neodol 25-3, respectively, sold by Company Shell Oil.

(2) Ethoxylated fatty acids, corresponding to the formula: O 'R-C-O- (CH2CH20) nH

o R and n have the same meaning as above in (1).

  (3) Ethoxylated alkyl phenols, of formula: R --- O (CH2CH20) nH, o: R is an alkyl radical containing 6 to 16 carbon atoms, and

n is an integer between 1 and 500.

As examples of anionic surfactants, there may be mentioned: (1) Soaps - corresponding to the formula: o Il R - C-OX, o: X is sodium, potassium or ammonium, and R is a fatty acid radical, saturated or unsaturated, linear or

branched, having 10 to 18 carbon atoms.

(2) Alkyl benzene sulfonates - corresponding to the formula: R SO3X, o: X is ammonium, triethanol ammonium, sodium or potassium, and

  R is an alkyl radical having 8 to 18 carbon atoms.

  (3) Hydroxy alkane sulfonates - corresponding to the formula: OH I R - CHCH2-CH2 S03X, o: X is as above in (2), and R is an alkyl radical containing 10 to 15 carbon atoms. (4) Sulfonated fatty acids - corresponding to the formula: CH3 (CH2) n-COOH, o: S03X X is as above in (2), and

n is an integer between 12 and 18.

  (5) Sulfonated nonionic surfactants - corresponding to the formula: R - O - (CH2CH20) nH, o:

S03X

X is as above in (2), n is an integer between 8 and 16, and

  R has the same meaning as above in (1).

  (6) The fatty alcohol sulfates - corresponding to the formula: CH3 (CH2) nCH20 S03X, o: X has the same meaning as above in (2), and

n is an integer between 8 and 16.

  (7) Sulfated nonionic surfactants- corresponding to the formula: RO - (CH2CH20) n-SO3X, o: X has the same meaning as above in (2), R is an alkyl radical having 12 to 18 carbon atoms, and

  n is an integer between 1 and 50.

  (8) Mono- and di-esters of sodium sulfosuccinates - of formula:

O O

  Il I R1-O-C-CH-CH2 - CO-R2 o: SO3Na Ri is sodium, hydrogen or an alkyl radical containing 1 to 12 carbon atoms;

  R2 is an alkyl radical having 1 to 12 carbon atoms.

  Preferably used nonionic surfactants of the type

ethoxylated fatty alcohol.

  The compositions of the present invention may also contain organic solvents, such as isoparaffinic mixtures for petroleum distillation. They can constitute up to 75% of the weight of the composition and,

  preferably from 10 to 40% approximately.

The compositions containing said organic solvents can thus contain solubilizing agents such as, for example, sodium salts

benzene sulfonic, toluene sulfonic or xylene sulfonic acids.

  These solubilizing agents can constitute up to 10% of the weight of the compound.

  sition and, preferably, from 3 to 6% approximately.

  In addition to the components listed above, brighteners can be incorporated

  optics, softeners, anti-static agents, anti-static agents

  redeposition and small amounts of perfumes and dyes.

  In general, the pH of these compositions is close to 7, however, depending on the nature of the enzyme used, the pH can be increased by the addition of NaOH

or lower it with acetic acid.

The components of the compositions of the present invention can be mixed in any order. However, it is better to first prepare a mixture of salt, alcohol and water and then add

  enzymes to prevent their degradation or deactivation.

  We can add optional components, such as surfactants,

at any moment.

The compositions of the present invention have multiple uses.

You can, for example, use it as a stain remover; they can be used in domestic laundry operations such as soaking and as

  additive during the washing cycle of an automatic washing machine.

  Other advantages and characteristics of the present invention appear

  will read the following description and examples given as

illustrative but not limiting.

EXAMPLE 1

The following compositions are prepared and stored at 370 in stoppered glass containers, for the times indicated in Table I, below. We consider that a week of storage at 370 corresponds to approximately

  2 to 3 months of storage at room temperature.

  Each of these compositions has a pH of the order of 7.

TABLE T

  Sample No. 1 2 3 4 5 6 7 85 95 10 Ingredients% weight% weight% weight% weight% weight% weight% weight J weight% weight% weight * Neodol 25- 91 15 15 12.5 12.5 10 7, 5 5 15 18.25 15 * Neodol 23-6.52 15 15 12.5 12.5 10 7.5 5 15 18.25 15 * Savinase 8.03 suspension 1 1 1 1 1 1 1 1 1 1 * Acetate sodium 4 0 4 6 4 4 4 0 0 2 * Sodium propionate 0 4 0 0 0 0 0 0 0 0 Ethanol 7 7 7 5 5 5 5 8.8 8.8 8.0 * Water 58 58 63 63 70 75 80 61.6 59.1 59.1 *% Initial activity 100 100 100 100 100 100 100 100 100 100 *% Activity after 2 weeks 4 97 96 97 97 95 93 92 95 *% Activity after 4 weeks 4 99 95 97 95 94 94 93 60 63 95 *% Activity after 6 weeks 4 91 88 91 91 88 87 84 51 51 90 *% Activity after 8 weeks 4 93 92 97 91 95 95 89 88

See page 10.

rPo 4: - o in

1-2-3-4-5)

- NOTES FROM TABLE I, above:

  1) Nonionic surfactant consisting of an aliphatic alcohol comprising

both 12 to 15 carbon atoms ethoxylated per 9 moles, on average of ethylene oxide. 2) Nonionic surfactant consisting of an aliphatic alcohol comprising

  both 12 or 13 carbon atoms, ethoxylated with 6.5 moles of ethylene oxide

lene on average.

  31 Commercial proteolytic enzyme sold by the company "Novo Industries" containing 6% of active enzymes with an activity of 8.0 kilounits

protease Novo.

  4) The residual activity level is evaluated by the trinitrobenzene sulfonate method, using casein as a substrate. The experimental error

on activities is 5%.

) Samples Nos. 8 and 9 do not comply with this invention

tion and they appear here only for comparison.

  Examination of these results indicates that the enzyme is rapidly deactivated

as shown in samples Nos. 8 and 9, if an agent is not used -

  stabilizer according to the present invention. Furthermore, as the results relating to sample No. 10 show, 2% of one of the stabilizing agents of the present invention is sufficient to obtain excellent stabilization.

long term.

EXAMPLE 2

  A composition is prepared in accordance with the present invention, containing: 4% by weight of sodium acetate as an enzyme stabilizer, 1% by weight of Savinase 8.0 in suspension, commercial enzyme preparation, sold by the Company Novo Industries, 7% by weight of ethanol, 15% by weight of nonionic surfactant, obtained by the reaction of a mole of aliphatic alcohol containing 12 to 15 carbon atoms with 9 moles of oxide d ethylene, sold by Shell Oil Company, under the trade name "Neodol 25-9",% by weight of nonionic surfactant, obtained by the reaction of one mole of aliphatic alcohol containing 12 or 13 carbon atoms with 6.5 moles of ethylene oxide, 0.25% by weight of perfume, 0.01% by weight of dye, and

  57.74% by weight of demineralized water.

  The cleaning power of this solution is then evaluated as a pre-stain remover: this composition is applied to two fabrics, one stained with blood, milk and ink, the other stained with used engine oil. After treating the tissue

  using the composition of the invention, they are washed using a compound

  detergent commercial product, sold by Procter & Gamble, under the brand name "Extra Action Tide". After washing, the tissues are left to dry and analyzed using a Gardener differential colorimeter with triple stimulation in order to evaluate the percentage of soil removed. This percentage

  is given in column 1 of Table II, below.

For comparison, we washed with Extra Action Tide ", then analyzed

stained fabrics as before. The results of these comparative tests fi-

  gure column 2 of said following Table II:

TABLE II

* Results expressed in% of soil removal.

These results clearly show that the compositions of the invention are

effective pre-stain removers.

EXAMPLE 3

  The same composition is evaluated as that of Example 2 as an additive for laundering. A tissue soiled with blood, milk and ink and another stained with used engine oil are washed separately with approximately 1.2 g of "Extra Action Tide" detergent composition and approximately 0.75 g of Example 2. These two washes are carried out in a tergotometer containing approximately 1 liter of water. After having leached fabrics, they are dried and the rate of elimination of stains is evaluated using the same instrument as in Example 2. The results of these tests are recorded in column 1

of Table III, below. -

  For comparison, stained fabrics were washed separately in a tergotometer as above using "Extra Action Tide" alone and added with a

COLUMN I 2

Pre-stain remover ..... according to the invention none Stain: Blood, milk and ink ..... 65.0 * 43.2 Used engine oil ..... 28.4 17.1

- - 12

commercial wash additive containing BIZ brand enzymes and

assessed the rate of soil removal by the same method as above.

  The results of these tests are shown in columns 2 and 3, respectively,

from the following Table III.

TABLE III

COLUMN 1 2 3

Additive ..... Composition according to none BIZ 10. the invention Stain: Blood, milk and ink ..... 44.2 * 20.4 8.6 Used engine oil ....., 17.8 13 , 4 13.9

  * Rate of elimination of thighs.

From the above results, it is clear that one can employ

the compositions of the present invention as a laundry additive.

  Of course, the present invention is in no way limited to the examples

  and modes of implementation mentioned above; she is likely to name-

  various variants accessible to those skilled in the art, depending on the applications

envisaged and without departing from the spirit of the invention.

Claims (19)

  1.- Aqueous composition containing one or more stabilized enzymes characterized in that it contains: a) from 0.01 to 15% approximately of a fatty acid salt of low molecular mass such as sodium salts, ammonium or alkanol ammonium of formic, acetic, propionic, butyric or valeric acids; b) from about 0.006 to 5% of one or more proteases, a-amylases or their mixtures; c) from 1 to 25% approximately of an alcohol of formula ROH, where R is an alkyl radical containing 1 to 6 carbon atoms; d) from about 10 to 90% of water.   2.- Composition according to claim 1, characterized in that it rein-   also closes from i to about 55% of an anionic, nonionic surfactant, or their mixtures.   3.- Composition according to Claim 1, characterized in that the alkanolammonium is monoethanolammonium, diethanolammonium or triethanolammonium. 4.- Composition according to claim 1 or 2, characterized in that the   pH of the composition is between 6 and 10 approximately. 5.- Composition according to claim 4, characterized in that water concentration is between 40 and 90% approximately.   6.- Composition according to claim 5, characterized in that enzyme concentration is between 0.006 and 2.5% approximately. 7.- Composition according to claim 6, characterized in that   alcohol percentage is between 4 and 8% approximately.   8.- Composition according to claim 7, characterized in that   surfactant concentration is between about 10 and 30%. 9.- Composition according to claim 8, characterized in that salt concentration is between 2 and 6% approximately. that the pros- that the pros- that the pros- that the pros- that the pros-   10.- Composition according to claim 9, characterized in that the pH is between 6 and 9 approximately.   11.- Composition according to claim 10, characterized in that the   zyme is a proteolytic enzyme obtained from Bacilius Subtilis. 12.- Composition according to claim 11, characterized in that the salt   is sodium acetate or propionate.   13.- Composition according to claim 12, characterized in that the al- cool is ethanol. 14.- Composition according to claim 13, characterized in that the percentage of surfactant is about 30% and in that the surfactant is a mixture containing about 33.3% by weight of surfactant   anionic active and 66.6% by weight of nonionic surfactant. 15.- Composition according to claim 13, characterized in that the surfactant is an ethoxylated fatty alcohol of formula: RO- (CH2-CH20) nH, o: R is an alkyl radical containing 8 to 18 carbon atoms, and n is an integer between 1 and 500. 16.- Composition according to claim 15, characterized in that the surfactant is a mixture of: a) the condensation product of a mole of aliphatic alcohol containing 12 or 13 carbon atoms, linear or branched, with 6 , 5 moles, on average, of ethylene oxide, and b) the condensation product of a mole of aliphatic alcohol, having 12 to 15 carbon atoms, linear or branched, with 9 moles of oxide ethylene. 17. Composition according to claim 16, characterized in that the mixture of surfactants contains approximately 50% of constituent a) and % of constituent b).   18.- Composition according to claim 17, characterized in that it also contains, expressed by weight: a) from 1 to 10% approximately of solubilizing agent, and b) from 1 to 75% approximately of an isoparaffinic mixture petroleum distillates   having an average molecular weight of 154.   19. A composition according to claim 18, characterized in that the solubilizing agent constitutes approximately 3 to 6% of the composition, in that the isoparaffinic mixture of petroleum distillates constitutes approximately 10 to   40% of the composition and in that the solubilizing agent is xylene sul- sodium fonate.