DE2000820A1 - Oral care products with a neutral prothease content - Google Patents

Description

DR. BERG DIPL.-ING. STAPF

PATENTANWÄLTE β MUNICH 2. HILBLESTRASSE

• Pr. To Dtyl-Irifl. Head. I MQwchwi 8, Hllblwtrofl «20 ·

BuZticiMn

19th

January 9, 1970

Lawyer file no. 19 224

Monsanto Company .800 North Lindbergh Boulevard St. Louis, Miss. 63166 / USA

"Oral care products containing neutral

Protease "

The invention relates to oral care preparations containing enzymes and in particular oral care preparations with a content of neutral protease.

χ / χ 009930/1964

Many undesirable conditions are known which result from inadequate dental hygiene, such as dental caries, infections, periodontal diseases, etc. One of these conditions, which develops very quickly after one to two days of light brushing of the teeth, is the formation of a soft mass on the teeth Tooth surface, which tends to be grounded mentioned as a coating. The coating appears to occur more stages in IP, the first of which a deposit of Speichelschleirastoff s on the Zahnot ^ rfläche is, which is then coated v / ith un ". Later deeply imbued with bacteria, v / hich in Cav iiuncihöhle are present. These bacteria reproduce them continuously and, depending on the type of bacteria, they can excrete by-products that are harmful to teeth and gums.

The source of material for their port planting are any I.
A! Food particles that remain in the mouth. During the later stages of plaque formation, the deposit is also impregnated with significant amounts of hydroxyapatite, which gradually hardens to form stone. Most of the time, a dentist removes this stone by scraping it off.

Various attempts have been made to contain the Bela _; ~ - Education made, for example, the inclusion of

009830/1964

1-Aiz; .Λ mixtures in conventional oral care products. These products contained multi-enzyme components such as EB proteases, lipases and amylases to break down or break down the various components of the toppings, such as proteins, starches and fats. However, many of these mixtures are obtained from animals and plants, and in view of the large quantities required for dental products, they are not available in sufficient quantities, and furthermore, the cost of these mixtures is high because of the limited availability. Other mixtures, available from ükroorganisi.ien, have components, one of which in turn, another with a reduction in activity. did which can inactivate enzymes. Likewise, many mixtures of components with extreme ρ values for oinu have optimal activity, which makes their effectiveness i :. Area of the oral cavity is lowered.

Line oral care preparation, which contains only a single unzyn, which is effective in terms of delaying de: ul iuriis or removal of the layer, would, among other things, represent an advance in the state of the art.

In accordance with the present invention _; :; * ■ "* u>-d:" - r. That preparations containing a neutral protease

009830/1964 " ^k "

-H-

and contain a pharmacologically acceptable carrier for the oral cavity effective in retarding formation or in the removal of a covering. In these preparations there are no more than trace amounts of more alkaline Protease and amylase present. It is completely unexpected that a single, in an oral care preparation included enzyme just as or even more effective than a mixture of I contained in these preparations Enzymes would be. In addition, a single enzyme is not sensitive to attack by> other enzymes, as can be the case with enzyme mixtures. ·

A neutral protease, as used in this application,

refers to a metallo-enzyme which ^! y its optimal activity at a p _H ~ value of about 6 * · 'has to about 8 is inhibited by metal chelating agents, however, not affected by such inhibitors such as diisopropyl fluorophosphate (DIP), and hydrolyzed substrates such as Purylacryloylglycyl-L -leucinamide (PAGLA), but has no activity against esters such as p-nitrophenyl acetate or N-CBZ-glycine p-nitrophenyl ester. A metalloenzyme is one that contains a metal necessary for activity. The alkaline protease as used in the present

009830/1964 " ⁵ "

ORtQtNALlNSPECTBJ

Application used refers to an enzyme that has optimal activity at a p "value of about 8 to about 11 unfolded which is inhibited by DPP but not by metal chelating agents and which an activity towards esters such as N-CBZ-glycinp-nitrophenyl ester, but not to PAGLA.

The neutral protease useful in the present invention can be derived from animals, plants or microorganisms be won. It is preferred to use neutral proteases of microbial origin, because these in considerable Quantities can be produced in an economical manner. These microorganisms can either produce predominantly neutral protease or a mixture of neutral protease and other enzymes.

Examples include Bacillus, Aspergillus or Streptomyces microorganisms, such as various B. subtilis strains such as B. subtilis strain NRRL B-3411 (U.S. Department of Agriculture Collection, Peoria, 111.), B. subtilis strain NRRL 644, B. subtilis strain IAM 1523 (Japanese Culture Collection), all of which produce a mixture of proteases and amylaee. Other Organisms include B. therraoproteolyticus, Streptomyces

009830/1964

OR! O «NAt

griseus, Aspergillus oryzae, Streptomyces rectus, Streptomyces naraensis and B. subtilis var amylosacchariticus, all of which are either a mixture of protease and Generate amyase or just neutral protease. The Streptomyces griseus strain K-I produces predominantly neutral ones Protease.

A particularly good source of neutral protease is an enzyme mixture produced by the B. subtilie strain NRRL Β-3 ^ 11. A method for producing this organism and the enzymes therefrom is described in US Pat. No. 3,031,380. The enzymatically active material produced by this organism has generally been found to be composed of neutral protease, alkaline protease and amyase. There are usually from about 700,000 to about 1.5 million casein units of neutral protease activity per gram of pesticide isolated and about 350,000 to 500,000 casein units of alkaline protease activity per oram, as would be the case by the casein digestion method (described below) is determined. There are about 300,000 to about 500,000 units of amylase activity as determined by the Bernfeld method, which is described below . As pointed out in the cited patent, the relative ratios of protease to amyase are

009830/1964

vary depending on the exact growth conditions of the microorganisms, but it has been found that the neutral and alkaline protease and the anylase are produced in at least considerable quantities, almost regardless of changes in the culture medium and other conditions of growth of the microorganisms.

Various analytical methods are available for determining the enzyme activity, and it can e.g. Protease activity by well known protein digestion methods using protein substrates such as e.g. of casein, hemoglobin, bovine serum albumin or gelatin. According to such attempts a protease catalyzes the hydrolysis of a protein (e.g. casein) in a certain unit of time controlled conditions of temperature, p ^ value and Substrate concentration; the reaction is stopped by adding trichloroacid and the solution is filtered. The solubilized debris in the filtrate is determined by either measuring the absorbance in the ultraviolet range determined by or visible by reaction with folinphenol Reagent and the absorbance in the visible Area (venessen and enzyme activity in the form of ii. .'iouivalents expressed. This procedure is

009830/196 *

BATH ORIGINAL

more fully in Journal of General Physiology, 30, (19 ^ 7) 291 and in Methods of Enzymology, Vol. 2, New York: Academic Press 1955 »33 described.

In this application, in the case where the neutral protease activity is expressed in casein units, understood that such activity is determined at a p "value of 7 and in the case where the alkaline protease is expressed in casein units, it is to be understood that such activity was determined at a p n of 10.

Other methods of determining protease activity make use of low molecular weight substrates in spectrophotometric studies, e.g. substrate FAGLA is specific for neutral protease and it is used to determine the activity of "neutral" protease, as described in Biochemical Biophysical Research Communications, 32 , 326 (1968).

The amylase activity is generally indicated by the well-known Dinitrosalicylic acid method by Bernfeld, described in Methods of Enzymology, Academic Press, 1955, Vol. I, page 149. Catalyzed according to this test

- 9 009330/1984

COpV

2000 320

Amylase the hydrolysis of starch into reducing sugars at a given time and temperature. The reaction is stopped and the color is changed by adding dinitrosalicylic acid developed. The optical density of the solution is determined according to a standard curve prepared with known Amounts of maltose hydrate, estimated. In this application is in the cases where units of activity of amylase are to be understood as meaning that the Bernfeld technique is used to determine such activity became.

The neutral proteases, taken as a group, have a different type from the alkaline proteases, taken as a group Specificity. For example, alkaline proteases have esterase activity, apparently as a result of their mechanism of action and not as a result of their p "-Optimum3, whereas die3 for neutral proteases is not the case. Attempts to prove this fact 3 are more complete in Arch. Biochem. Biophy3. 123 »(1968) 572. Since the specificity of neutral and alkaline proteases are different, different values will be obtained if, for example, in the studies use hemoglobin or bovine serum albumin instead of casein. For example, have a neutral one. Protease obtained from B. thermoproteolyticua

009830/1984

- Io -

and an alkaline protease obtained from B. subtilis, Activities of 12,800,000 and 3,500,000 units / g, in order if casein is the substrate; but when all other things being equal, hemoglobin is used, the activities are 240,000 and 400,000 units / g, in turn, i.e. the activities of both are lower than hemoglobin. Accordingly, the The enzyme units are specified in different types of units and can therefore be used in the applications Enzymes based on the same weight, the same casein activity or the same hemoglobin unit, etc. are used will.

Various techniques can be used to separate neutral protease from enzyme mixtures, such as ion exchange chromatography, described in Journal of Biological Chemistry, 239, 3706 (1964) and in Agr. Biol. Chem. 30, 651 (1966). Another method is disclosed in pending application no. 752,460, filed August 14, 1968 and assigned to the same assignee as the present application. The starting material for this technique is water-clear fermentation beer containing the enzyme mixture obtained by filtration or centrifugation, or an aqueous extract of the enzyme mixture obtained by redissolving

- 11 -

009130/1964

ORIGINAL INSPECTS)

the raw, solvent-precipitated enzyme mixture. The ' Amylase is produced by solvent precipitation in the presence a calcium salt or by ammonium sulfate fractionation followed by starch absorption in the presence of aqueous ethanol to remove the last traces of amylase. Pigment is made by adsorption using DEAE cellulose or other anion exchange resins removed. The two proteases remaining in solution are activated by selective adsorption fractionated using hydroxyapatite as an adsorber. The neutral protease is adsorbed and subsequently elutes, whereas the alkaline protease is not adsorbed.

Other methods of stabilizing neutral protease in the mixture can be made using specific Inhibitors such as DPP or a potato inhibitor to inactivate the alkaline protease and careful solvent fractionation to remove the amylase must be performed.

The neutral protease is used in such an amount that that the oral care formulations of the present invention from about 50 casein units of activity to about 800,000 casein activity units per gram of oral care preparations and preferably about 100 casein

009830/1964

Activity units contain up to about 400,000 casein activity units per gram of the oral care preparations. The oral care preparations provide in general use a range of from about 1,000 xasein units of neutral protease activity to about 40,000 casein units of neutral protease activity, and preferably from about 5,000 casein units of neutral protease activity to about 20,000 casein units neutral protease activity. For example, one drop of a mouthwash concentrate with about 200,000 casein units of neutral protease activity per gram of the concentrate is diluted with about 50 ecm to 100 ecm of water and used as a mouthwash gives about 10,000 units of neutral protease activity per unit -Application off. In another example, a toothpaste is about 8000 τοη casein units of neutral Protetar activity per gram of dentifrice in the use of brushes turn tähne about 8000 casein units of neutral protease activity per Einsel application from. In yet another example, 50 ecm of a mouthwash which has about 400 casein units of neutral protease activity per gram of mouthwash, when used, releases about 20,000 units of neutral protease activity per one application.

- 13 -009830/1984

COPY

In the oral preparations of the present invention, there are no more than trace amounts per gram of preparation of alkaline protease and amylase present. A trace amount is less than 50,000 casein units alkaline protease activity and 50,000 units of amylase activity per 1,000,000 casein units of neutral Defined protease activity. It is preferred to have less than 20,000 casein units of alkaline protease activity and 20,000 units of amylase activity per 1,000,000 casein units of neutral protease activity to have.

The application or use of the preparations of the invention takes place, of course, in a conventional manner Way by putting an effective amount of neutral protease preparation in contact with the oral cavity or with the for brings a special application of the interesting part of the same.

The products in which neutral protease can be used with beneficial effects include toothpastes, Tooth powder, mouthwashes, mouthwash concentrates, Gargle washes, chewing gum, liquid concentrates, etc. Examples of typical recipes are given below Table I given.

009330/1934

copy

Table I.

paste powder Fluid-
opportunities rubber component 5 ¹ » % 95 % Polishes 20 % ι; i 10; { Humidifier 2 % Thickening
medium or
binder 2 % 3 % 2 ^ · G % Interfaces
active substances 20 % 68 % water 0.52 0.12 12 Si Sweeteners 20 JS 56 % alcohol 0.52 0.9 # H % Flavorings 20 ^ Base rubber

Polishing agents include tricalcium phosphate, dicalcium phosphate, and calcium pyrophosphate. Moisturizers include glycerol, Sorbitol or propylene glycol. Illustrative thickeners and binders include sodium carboxymethyl cellulose, Gum tragacanth, carrageenan gum and vinyl polymers. Surfactants include quaternary ammonium compounds and dioctyl sodium sulfosuccinate. Sweeteners include glucose or artificial sweeteners such as saccharin. Flavorings include anisole, or spearmint oil

009630/1864

- 15 -

ORfGJNAL INSPECTED

Clove oil. The base rubber includes chicle or synthetic resins.

These dog care preparations can be prepared in the usual way, BB as described in Kirk and Othmer, Encyclopedia of Chemical Technology, Ί, New York: Interscience, (195 ¹ I) 928-931.

The effectiveness of the neutral protease can be determined by in vitro or in vivo methods. An in vitro test is more advantageous in that there are no test items and no appreciable amounts of material are required and it can be done in a much shorter period of time can. An effective test is by making a synthetic covering, depositing a film of this covering on a glass plate, adding an enzyme solution to the film and testing for film removal. on in this way, a large number of enzymes can be "tested for plaque removal".

In summary, it can thus be said that the present invention relates to oral care preparations, which contain neutral protease and a carrier, for example a mouthwash concentrate containing a neutral protease produced by B. subtilis.

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009830/1964., Λ - _f ν ORfQINAL INSPECTED

- 1 -ι -

The following three games are presented to illustrate and in no way limit this invention. All Parts mean percentages by weight, based on the total preparation, unless expressly stated otherwise ■ is said.

EXAMPLE 1

Neutral protease is produced by inoculating a culture of B. subtili3 HRRL No. 3 * 111 in a sterile slurry of cereal grains or other nutritional material (such as rice bran, maize flour, fish meal, wheat bran, enzose (about 50 to 802 dextrose and the rest higher saccharides, which are the dried mother liquors from the dextrose production by enzymatic hydrolysis remaining from corn starch), soluble distillate residues, corn steep liquor, etc.) containing protein, carbohydrates, minerals and growth factors, the container being stirred and aerated by bubbling sterile air through the inoculated slurry. The p _H value can be regulated or left to itself in order to achieve its own natural p “value. Aliquots of the beer are removed at various times for testing purposes and, after the enzyme production has apparently reached a maximum,

- 17 τ 009330/1934

the growth ended and then nine (S) liters of the fermentation beer clarified by centrifugation and then with 200 g DEAE cellulose (diethylaminoethyl cellulose acetate gives a p n value of 6.4) at a p ^ value of 6.4 for 30 minutes stirred long to remove any pigment. The cellulosic resin is removed by filtration and the filtrate is again treated with another 100 g amount of DEAE cellulose in the same manner. 80 g of calcium acetate are added to the 8 liter filtrate, the powert is adjusted to 7 »5 and 2,240 g of ammonium sulfate are added with stirring. After stirring at 5 ° C for 30 minutes, the precipitate is removed by filtration and a further 900 g amount of ammonium sulfate is added to the supernatant and the precipitate is collected by centrifugation and reapplied in about 1 liter of a 0.1% calcium acetate solution dissolved. 100 g of powdered wheat starch and 120 ml of ethanol are added, the solution is stirred for 20 minutes and filtered. 2 liters of cold (5 °) acetone is' added to the filtrate and the ¹ precipitate collected by centrifugation, re-dissolved in 500 ml of 0.1 sodium ^ CaI-ciumacetat and lyophilized.

250 mg of this enzyme-containing solid partially purified are dissolved in 25 ml of 0.02 sodium calcium acetate solution ^, the _pH value to 7 »2 is adjusted and the solution

- 18 009930/1964

a 1.2 χ 7.0cm column of hydroxyapatite applied, and washed with 0.1 # calcium acetate. After the enzyme solution on the column with a little calcium acetate solution has been washed, the column is eluted with 0.2 M phosphate buffer of p "7.0. The eluted protein material (checked by the extinction at 280 mm) is at 5 ° against an 0.15 calcium acetate solution overnight dialyzed, centrifuged and lyophilized.

The above procedure then yielded 17 g of a partially purified enzyme-containing pesticide. Ten 250 mg parts of this pesticide were used purified from hydroxyapatite in the manner indicated to yield about 570 mg of a neutral protease which is a Had activity of about 12.5 x 10 casein units per gram.

A mouthwash concentrate is prepared by adding a neutral protease to a mixture of 80 parts by weight Ji glycerol, and 20 wt -% produced alcohol, so that the activity / ccm about 8OOO casein units is the mixture.. This mixture is produced as a concentrate which has to be diluted when used and is used as a mouthwash. ·.

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00 9 830 / 1Θ64

ORIGINAL INSPECTED

The effect of the neutral protease on the plaque is assessed by estimating the plaque area on the subjects' teeth before, during and after using a mouthwash; containing the above mixture, determined. A test group of volunteers was selected and their teeth checked and the surface area by a numerical evaluation method once for 2 consecutive weeks Establishment of a standard pavement value for each estimated for the j balance of the experiment. First were the teeth Professionally cleaned to remove all soft debris. The test subjects then use it daily about 50 ecm of water over a period of 6 weeks containing 20 drops of the above mixture as a mouthwash. Your teeth are cleaned at weekly intervals Checked for the size of the coating.

The pavement area is estimated using an indexing board manufactured by the Amural Company. Everyone visible tooth is divided into 6 segments, 3 in the upper half and 3 in the lower half. For every segment a rating of 1 is given for a trace of deposit, a rating of 2 for a deposit which less than half of the segment covered and one

Rating of 3 for a deposit covering more than half. In this way an overall rating derived for each visible tooth after each check. '

009830/1964

- 2o ORIGINAL INSPECTH)

Another group of volunteers underwent the same procedure, but the mouthwash used by these test subjects contained a mixture of enzymes from about 8000 casein units of neutral protease, JJOOO casein units of alkaline protease and 4000 units of amylase. This enzyme mixture is used in the same way obtained from B. subtilis NRRL B-3 ^ 11, with except that the alkaline protease and the amylase not removed ". The plaque rating for each visible tooth is then determined.

A dog water concentrate containing neutral protease, was "prepared according to the above procedure and distributed to 8 panelists. These panelists used the preparation in the manner mentioned and their coating ratings are given in Table II. A Another mouthwash concentrate was prepared containing the enzyme mixture according to the above procedure and given to 8 test persons who met the above instructions for use followed. Their plaque rating is shown in Table III.

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009830/1964

Table II

Remaining portion of the
original covering
after 6 weeks Percentage
reduction
after 6 weeks Test subject
No. 62 38 1 37 63 2 35 65 3 38 62 4th 62 38 5 27 73 6th 33 67 7 ' 35 65 8th Table III Remaining portion of the
original covering
after 6 weeks Percentage
reduction
after 6 weeks Test subject
No. 53 47 1 38 62 2. 34 66 3 33 61 4th 42 58 5 30 .. 70 6th 40 60 7th 40 60 8th

009830/1964

- 22 -

From the above data it can be easily seen that the neutral protease significantly reduces the deposit.

Other suitable mouthwash concentrates were prepared by using the neutral protease of Example 1 in which

same level of activity by a neutral protease obtained from B. subtilis NRRL No. 9 ¹ II, a neutral protease from B. subtilis IAM No. 1523 (Japanese culture collection), a neutral protease produced by Streptomyces griseus, a neutral protease obtained from B. thermoproteolyticus and a neutral protease obtained from B. Äubtilis var amylosacchariticus replaced.

Example 2

In order to examine the effectiveness of various neutral protease enzymes, a synthetic coating was used manufactured. A group of volunteers who hadn't brushed their teeth for 2 days brushed themselves with one

clean dry toothbrush and collect the saliva and toothbrushes in a sterile sachet.

For transferring the saliva samples to sterile plate cultures of nutrient agar (Difco, 31 g / liter, sterilized and poured into sterile plastic petri dishes) an annealed inoculating loop was used.

- 23 -009830/1964

The inoculated plates were incubated for 48 hours at 37 ⁰ C and a plentiful, microbial growth obachtet be ^. Visually different colonies were removed, slides with Gram staining were produced and examined. Selected colonies were secondary cultured on sterile nutrient agar slants for maintenance in a culture collection.

Large amounts of selected Qrganisms were inoculated by inoculating a platinum loop full of the organisms from the slant cultures in flasks with sterile nutrient broth (Difco, 9 g / liter, 100 ml in 500 ml baffled flasks) and incubating in a rotating shaker (New Brunswick Scientific Co.) at 37 ° for 3 days. The ^! Organisms were then centrifuged at 5 ° in stainless steel bottles (Sorvall, SS-3 centrifuge with GSA-

Head, 9600 rpm for 30 minutes.) And resuspended in the ^: Minimal volume of water for transferring to a glass container. The mixed slurry of oral microorganisms is stored in the refrigerator.

The saliva is collected in a beaker and mixed with an equal volume of the cooled bacterial suspension. Sucrose is added to make a final concentration of 1% .

009830/1964

One ml of the bacterial / saliva mixture is added to a 17.78 χ 17.78 cm (7 χ 7 inch) glass plate (with sandblasted roughened surface) and the mixture with a bent glass rod, used for spreading of bacteria on plates, smeared. The film is spread out until it is identified as a is assessed uniformly.

The plate is then 'incubated at 37 ° C overnight dried. Enzyme solutions are made in distilled water and add 1 drop of each enzyme solution carefully placed on the plate and the plate incubated again at 37 ° C for half an hour. The plates are then gently heated over a Bunsen burner to repeatedly dry the film. The films are through careful pouring of a 1 ^ aqueous solution of Stained crystal violet (1 g / 100 ml) on the plate. After dyeing for 5 minutes, the excess of the color is poured off and gently immersed the plate in a bath of cold tap water, gently swirling it, then removed and air dried for observation.

The above procedure was performed on the enzymes shown in Table IV with the results reported there.

009830/1964 " ²⁵ "

Table IV . The effect of enzymes on the removal of synthetic plaque

A drop of enzyme solution, containing the units indicated below, is dripped onto the covering plate.

Results are reported as - (no piled removal)

up to +++ (complete piled removal)

enzyme

activity
u / mg (substrate)

Dilution mg / ml

Result

O
O

to

B, subtilis NRRL B-3411, Neutral protease

B. subtilis NRRL B-3411, Alkaline protease

B. subtilis var amyloäacchariticus,

Neutral protease

B. thermoproteolyticus, neutral protease

B. subtilis NRRL B-3411,

Anylase

110 (albumin)
270 (albumin)

195 (albumin)
240 (albumin)
15,000 (strength)

0.5
0.5
1

ro

CD CD O OO

From the above data it can easily be seen that the neutral proteases remove plaque.

Example 3

A toothpaste is prepared containing about 7,000 casein units of neutral protease activity per gram Toothpaste. The neutral protease is produced by B. subtilis NRRL B-3 ^ 11 and is produced in the same manner as obtained in Example 1. In addition to the enzyme, the toothpaste contains the following ingredients:

Sorbital-water mixture 43 %

Tragacanth gum

Saccharin

Dioctyl sodium sulfosuccinate Dicalcium phosphate dihydrate

Total 100 %

Suitable toothpastes are prepared by using a neutral protease obtained from B. thermoproteolyticus and one obtained from B. subtilis NRRL for the above neutral protease at the same activity threshold.

1 .5 % 1 .6 % 1 , 1055 rest

009830/1964

ORIGINAL fNSPECTEO

Example * J

A chewing gum formulation is made containing about 2000 casein units of neutral protease per gram Chewing gum. The neutral protease is produced by B. subtilis B-3 ^ 11 is generated in the same way as in Example 1 received. In addition to the neutral protease, the chewing gum contains:

Parts

Chicle · 100 ·

Gelatine kO

Water 60

Flavorings 20

Sucrose rest

A total of 520

Other suitable chewing gums are produced by replacing the aforementioned protease at the same activity threshold with a neutral protease " B. subtilis IAM 1523 and a neutral protease produced by B. thermoproteolyticus.

Example 5 A tooth powder is prepared containing 7000 casein

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Units of neutral protease per gram of powder. the neutral protease is produced by B. subtilis NRRL Β-3Ί11 and is produced in the same manner as in Example 1 obtain. In addition to the neutral protease, the tooth powder contains:

Parts

Carboxymethyl cellulose 5.0 Dioctyl sodium sulfosuccinate 15 »0 Peppermint oil 4.5 Dicalcium phosphate dihydrate remainder

A total of 500

Another suitable tooth powder is produced by replacing the above neutral protease at the same activity threshold with a neutral protease B. subtilis var amylsacchariticus.

009830/1964 " ²⁹ "

Claims (1)

Claims 1. An oral care preparation containing a pharmacologically acceptable carrier for the oral cavity and a neutral protease, which is present in an amount ranging from about 50 casein units of activity to about 800 To ensure casein units of activity per gram of the mentioned preparation and "where the mentioned preparation no more than a trace amount of amylase. or alkaline protease. 2. Preparation according to claim 1, characterized in that that the neutral protease is present in an amount ranging from about 100 casein units of activity to about 400,000 casein units of activity per gram of the aforementioned Ensure preparation. 3 .. · Preparation according to claim 1, characterized in that j that the aforementioned neutral protease is through a microorganism is produced. 4. Preparation according to claim 3, characterized in that said microorganism is a Bacillus microorganism out, AspergillUe microorganisms and / or Streptomyces f I Is a microorganism. , 009930/1994 - 3o - ORIGINAL INSPECTS) 5. Preparation according to claim 4, characterized in that that the neutral protease by a microorganism of the group B. subtilis, Streptomyces griseus, B. thermoproteoliticus, Aspergillus oryzae, Streptomyces rectus and / or Streptomyces naraensis is produced. 6. Preparation according to claim 5 »characterized in that said neutral protease by a B; subtilis from the group B. subtilis NRRL B-3411, B. subtilis NRRL 644, B. subtilis IAM 1523 and / or B. subtilis var amylosacchariticue. 7. Preparation according to claim 6, characterized in that said neutral protease by B. subtilis NRRL B-3411 is generated. 8. Preparation according to claim 5 »characterized in that said neutral protease is present in an amount is to ensure about 100 casein units of activity to about 400,000 casein units of activity per gram of said preparation. 9. Preparation according to claim 6, characterized in that said neutral protease is present ih an amount to provide from about 100 casein units of activity to about J 009130/1964 - 31 -. ORIGINAL INSPECTED 10,000 casein units of activity per gram of those mentioned Ensure preparation. Io .. Preparation according to claim 9 »characterized in that that the aforementioned neutral protease is caused by B. subtilis NRRL is produced. 11. Preparation according to claim 1, characterized in that said carrier from the group of toothpastes, Tooth powders, mouthwashes, mouthwash concentrates, liquid concentrates and / or chewing gum is selected. 12. Composition according to claim 11, characterized, in that the said neutral protease is present in an amount to provide about 100 casein units of activity to about l | ensure Kaeein 00 000 units of activity per gram of said composition. 13 * Preparation according to claim 3 *, characterized in that the said carrier is selected from the group of toothpastes, toothpowder, mouthwashes, mouthwash concentrates, liquid concentrate and / or chewing gum. 1 * 1. Preparation according to claim 4, characterized in that said carrier is selected from the group of toothpastes, 009830/19) 54 - 32 - Tooth powders, dog washes, mouthwash concentrates, liquids Concentrates and / or chewing gum is selected. 15. Preparation according to claim 5, characterized in that said carrier from the group of toothpastes, Tooth powders, mouthwashes, mouthwash concentrates, liquid concentrates and / or chewing gum is selected. 16. Preparation according to claim 15, characterized in that that the neutral protease is caused by B. subtilis NRRL B-3 ^ 11 is produced. 17. Preparation according to claim 15, characterized in that said carrier is a toothpaste. 18. Preparation according to claim 15, characterized in that said carrier is a mouthwash concentrate. 19. A preparation according to claim 15, characterized in that the neutral protease is present in an amount to ensure about 100 casein units of activity to about ¹ 100,000 casein units of activity per gram of said preparation. 20. Preparation according to claim l6, characterized in that - 33 009830/1964 that said neutral protease is present in an amount ranging from about 100 casein units of activity to about To ensure 400,000 casein units of activity per gram of said preparation. 21. Preparation according to claim 17 _3, characterized in that said neutral protease is present in an amount to ensure about 100 casein units of activity to about 400,000 casein units of activity per gram of said preparation. 22. Preparation according to claim 18, characterized in that said neutral protease is present in an amount is around 100 casein units of activity to about 400 To ensure casein units of activity per gram of said preparation.