NZ211745A - Casein or phosvitin containing compositions - Google Patents

Description

4--5-6 J ..." 2-5 fc 81 116' fi6iK57./i6.. ftiw ijoo 3 0 0CTI987:- ooi. . 2 11745 Under tl;? provisions of Reflation 23 (I; tiie . ^ J Specification has been ante-dated, to 1. M Q.ik. 1915-1 j M __j Initials Patents Form No.5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION "CARIES INHIBITION" -I-,WE THE UNIVERSITY OF MELBOURNE, a body politic and corporate established under the Melbourne University Act of the State of Victoria, of Grattan Street, Parkville, Victoria, Australia, and THE VICTORIAN DAIRY INDUSTRY AUTHORITY a corporation constituted pursuant to the provisions of the Victorian Dairy Industry Authority Act 1977 of the State of Victoria, of Domville Avenue, Hawthorn, Victoria, Australia, hereby declare the invention, for yhich-I/we pray that a patent may be granted to -jne-/us, .and the method by which it is to be performed, to be particularly described in and by the following statement:- 211745 This invention relates to caries inhibition. The invention also relates to gingivitis inhibition.

The present invention provides an orally ingestible composition containing a caries and gingivitis inhibiting amount of a protein or a polypeptide or a salt thereof.

Preferably, the protein or polypeptide is a phospho-protein or a polyphosphopeptide.

Preferably, the protein or polypeptide is an acidic nhosphoprotein or polypeptide.

Preferably, the protein or polypeptide contains the amino acid sequence (X-Y-Z) where X and Z are a phosphoserine, phosphothreonine, phosphotyrosine , glutamate or asparate and Y is any amino acid.

Preferably, the protein or polypeptide contains a plurality of units each having the amino acid sequence (X-Y-Z) where X, Y and Z are as stated in claim 4.

Preferably, the protein or polypeptide contains a group of formula (X-Y-Z^ where X, Y and Z are as stated - and n is 1 or more.

Preferably, n' is 3 or more.

Preferably, X and Z are phosphoseri ne , Preferably, the protein or polypeptide is a polyphosphoserine.

Preferably, the phosphate groups of the polyserine are spaced at about 6.88 Anystrom Units spacing.

Preferably, the protein is a casein.

Preferably, the protein is a 1 pha-s-cas i n .

Alternatively, the protein is phosvitin.

Preferably, the prote in or polypeptide is in solution.

Preferably, the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of at least 45 nmol/min under the test conditions defined herein.

Preferably, the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of 211745 - at least 80 nmol/min under the test conditions defined herein.

Preferably, the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of at least 90 nmol/min under the test conditions defined herein.

Preferably, the protein or polypeptide is one exhibiting a reduction in calcium dissolution rate of at least 95 nmol/min under the test conditions defined herein.

Preferably, the protein or polypeptide is present as up to 10% by weight.

Preferably, the protein or polypeptide is present as up to 5% by weight. ' Preferably, the protein or polypeptide is present as up to 2% by weight.

Preferably, the composition additionally contains urea. 15 The composition of this invention may be in the form of a foodstuff, confectionery, dentifrice, tablet or comprise a Pharmacologically acceptable vehicle, solution of suspension for topical application to the teeth or a mouthwash. Other modes of administering the protein or polypeptide would be j acceptable if Pharmacologically acceptable. . I Of particular interest as compositions are chewing gum, breakfast foods, ice-cream and other frozen confectionery, confectionery, sweets and cakes as these are all known as ; caries problem foods.

Also of particular interest are dentifrices, mouth washes and preparations for topical application to teeth. a^- casein and other caseins are obtainable from milk, phosvitin is obtainable from egg yolks and other suitable phosphoproteins include those which are obtainable from cereals, 30 nuts and vegetables particularly in bran husks or sheaths.

In particular, rise, wheat, oat, barley and rye brans are a fruitful source of such phosphoproteins. In the case of rice a particular phosphoprotein, phytate, is of interest.

Polypeptides of interest include pplyphosphoserine, 35 polyglutamate acid and polyaspartate acid.

The present invention also provides a process of inhibiting dental caries and/or tooth erosion and/or gingivitis comprising applying to the teeth.with a carrier a cariostatic flRfi ' - 2 I 1> V • and/or antigingivitis agent selected from a protein or a polypeptide or a salt thereof.

The present invention also provides a first test procedure for selecting among the proteins or polypeptides that may be used those that are most effective.

TEST 1 The purpose of this test is to determine hydroxyapatite dissolution and in this respect since tooth enamal appears to be largely composed of hydroxyapatite it is believed that useful comparisons can be made.

Double distilled, deinonised water (greater than 10 Mn/cm) was used throughout. Analytical reagent grade chemicals were obtained from Ajax Chemicals, Australia. Hydroxyapatite-spheroidal was purchased from BDH, and phosvitin from Sigma Chemical Co., Missouri, U.S.A. The milk proteins were fractionated by the method of Zittle and Custer (1), and their purity assessed by polyacrylamide gel electrophoresis using a modification of the method of Groves and Kiddy (2).

Methods Hydroxyapatite Dissolution Rate Assay.

A chromatography column (pharmacia K9/15) containing 1 g of hydroxyapaptite beads was used for the demineralisation assay. Tris 5 mM, pH 8.3 containing 50 mM NaCl and 20 mg/1 neomycin was used as the column influent buffer at 20°C and at a rate of 1.000± 0.003 ml./min. A protein solution (10 mg. of protein in 10 ml. -of ;i nf 1 uen-t buffer) was applied to the column and 1 ml. fractions were collected before and after protein application and assayed for total calcium, phosphate and protein. From these values a rate of dissolution (nmol calcium or phosphate per min) for each 1 ml fraction was obta ined.

Calcium, Phosphate and Protein Assays Inorganic phosphate was measured by the method of Itaya and Ui (3) and protein by the method of Bradford (4). The determination of calcium was by atomic absorption spectrophotometry using 1% lanthanum chloride to prevent phosphate s uppress i on .

Results The proteins used for the study are listed in Table 1, they are all acidic proteins and included four phosphoproteins and three non-phosphorylated proteins from the whey fraction of bovine milk. The effect of the individual proteins on 21 1745 - 5 hyroxyapatite dissolution rate is shown in Table 2.

Table 1. Properties of various phosphorylated and non-phosphorylated acidic proteins.

Protein Molecula r We i ght Phosphoserine Isoelectric Carbohydrate Content Point Content Phosvitin 35,50(5 11(3 1.5 + a$- Casein 23,613 8 4.1 B-casein 24,02(3 5 4.5 K-casein 19,037 1 3.7 + a-1 acta 1bumin 14,174 - 5.1 3-1actaglobulin 18,362 - 5.3 Bovine serum 66,210 - 4.7 albumin a. Residues per mol protein Table 2. Effect of phosphorylated and non-phosphorylated proteins on hyroxyapaptite dissolution rate.

Protei n Phosvi ti n a - casein B-casei n k-casei n a-lactal bumin 6-1actoglobulin Bovine serum albumi n Reduction in- calc i urn di ssoluti on rate3 (nmol/mi n) 93.1± 5.4b 100.1+ 4.1b 94.8±11.7b 56.3+ 8.9 2. 7± 1.7 17.1± 1.7 31.6+ 4.5 Reduction in phosphate di ssolution rate (nmol/mi n) 63.8+ 9.4 63.5+ 3.3 64.0+19.3 33.7+ 6.8 2.9+ 0.6 12.5+ 1.2 .5+ 3.3 a. means+SD, n = 3 b. not significantly different P>0.5 Amount of protein * bound (rug) 1.87+0.62 5.58+0.03 7.45+0.37 4.17±0.26 0.48+0.17 1.80+0.71 2.09±0.05 056 - - 1745 In a trial of the above test the dissolution rate of hydroxyapatite as measured by the rate of calcium and phosphate released from the hydroxyapatite column was constant over a two hour period calcium 353.6+3.9 nmol/min, phosphate 225.4+6.8 nr?ol/min. The dissolution rates obtained using different hydroxapatite columns showed greater variation, calcium 354.2+23.8 nmol/min, phosphate 229.6 30.8 nmol.min, n = 11. This intercolumn variation in dissolution rate could be attributable to different column packing resulting in a different HA surface area exposed.

The effect of phosvitin on the dissolution rate of hydroxyapotite is shown in Figure 1. The protein caused an initial increase in the dissolution rate of phosphate which then decreased and approached a new steady state level; 63.8 nmol.min less than the rate prior to phosvitin application. The protein caused an immediate and marked drop in the calcium dissolution rate which then increased and approached a steady-state level 93.1 nmol/min less than that prior to phosvitin application. The amount of protein that passed through the column was measured and from this the amount retained was calculated 1.87 mg. The dissolution rate returned to the original value after phosvitin was eluted from the column with 10 ml of eluent buffer containing 1.5 M phosphate, followed by buffer not containing phosphate.

The trace for a$-casein was very similar to that of phosvitin except that the immediate drop in calcium dissolution rate was not as marked Fig. 2). The difference in the steady-state rates of calcium and phosphate released before and after as- casein application we re ve ry similar to those in Figure 1 (calcium, 100.1 nmol/min, phosphate 63.5 nmol/min).

The results obtained for 6-casein (Fig. 3) were characteristic of all the other proteins tested, except for the final steady-state rates of calcium and phosphate released. All protens (with the exception of phosvitin and as-casin) caused an initial increase in the dissolution rate of both calcium and phosphate which then decreased as the proteins passed out of the column. The mean differences between the steady-state dissolution rates before and after 056 211745 protein application, together with the amount of protein bound, for all proteins tested is presented in Table 2 above. The results show that the four phosphoproteins gave a marked reduction in the steady-state dissolution rate of HA with 5 phos vi ti n , o^-casein and g-casein all giving the same reduction in calcium and phosphate dissolution.

The results show that all the acidic proteins caused a reduction in the steady-state dissolution rate of hydroxyapatite. However, the greatest reduction was given by the four phospho-10 proteins; phosvitin, d^casein, B-casein and to a lesser extent K-casei n .

From the above and from other data which suggests that adjacent phosphate groups of polyserine compounds have a spacing of about 6.88 Angstrom units when in beta-pleated 15 sheet configuration and that calcium atoms in a hydroxyapatite surface along the c-axis will also be spaced at about 6.88 Angstrom units, we speculate that a phosphate group-calcium atom bond materially reduces calcium dissolution rates. v References: Zittle, C.A., Custer, J.H.: Purification and some properties of as~casin, J. Dairy Sci 46: 1183-1189, 1963.

Groves, M.L., Kiddy, C.A.: Polymorphism of y-casein in cow's milk. Arch. Biochem. Biophys 126: 188-193,1968.

Itaya, K., Ui, M.: A new micromethod for the color-25 imetrie determination of inorganic phosphate. Clin, Chim, Acta 14: 361-366, 1966.

Bradford, MM.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254, 1976 30 TEST 2 For the purpose of in vivo testing, the following test procedure for determining the effect of casein and whey protein on caries incidence in the Sprague-Dawley rat.

Materials and Methods.

Forty-five, weanling, male Sprague-Dawley rats, 18 days old, bred from animals fed a fluoride free diet were used. The rats were marked for indentification and then randomly distributed with respect to diet. They were housed i 056 211745 in raised-bottom stainless-steel cages, one group of 15 per cage and fed a powdered cariogenic diet with either deinonised water (control), a 2% casein solution or a 2% whey protein solution ad libitum. The cariogenic diet was a modified MIT -5 200 diet shown in Table 3.

Table 3 - Composition of modified MIT - 200 cariogenic diet.

Component % wt Sucrose a 67 Wheyprotein 2(8 Concentrate Salt mixture b 3 Cottonseed oil 3 Cellulose a 6 Vitamin mixture a ' b 1 15 a. Calcium and phosphate not detectable, fluoride content of complete diet was less than 0.2 g per g dry weight, b. Vitamin and Salt mixture described in detail elsewhere.

The animals were weighed daily and the amounts of powdered diet and liquid consumed over a 24 h period by each 20 group was measured. After 42 days on the diet, the animals were killed by cervical dislocation and treated as described bel ow.

Caries evaluation Fissure caries was assessed using the method of 25 Lonig, Marthaler and Muhlemann (1958)(5). The mandible was removed from each rat and placed in formal saline. The jaws were then sectioned and stained by the method of Konig et al ( 1958)(5), as described by Green and Hartles ( 1966) (6) to provide series of 100 m thick longitudinal mesio-distal sections 30 of the molar teeth. Only the main fissures of the first and second molar teeth were assessed for caries.

Results Diet Consumption The relative consumption of solid and liquid diet by 35 the three groups of rats was tested by an analysis of variance (by diet). This showed that the quanitites of both solid and liquid consumed by each group were not significantly different 056 9 (P>0.75).

Caries analysis.

The caries data shown in Table 4 were analysed in an analysis of variance table by diet. Table 4 Caries Experience Data Animals Number of carious fissures a Control Group Casein Group Whey Protein Group 7.92+2.06 1.87 + 2 .50 4.73±3.85 a. Maximum number possible 10.

The animals drinking the 2% casein solution has 76.5% less carious fissures than the control anima Is (p<0.001), and the animals consuming the 2% whey protein solution had 40.3* less carious fissures than the control group (p<0.01). Tht correlation of caries incidence with the final weight of the rat was tested for the three groups. No correlation attained significance (p>0.1).

Conclusion Acidic proteins in the drinking water substantially reduced caries incidence of male Sprague-Dawley rats, however the phosphorylated protein (casein) caused a greater reduction than the non-phosphorylated whey proteins.

References : (5) Koin K.G., Marthaler T.M. and Muhlemann H.R. 1958: Methodik der Kurzfristig erzeugten Rattenkaries. Dr. Zahn-Mund-u. Kieferheilk. 29, 99-127. (6) Green R.M. and Hartles R.L. 1966: The effect of di ffe ri ng high ca rbohydrates diets on dental caries in the albino rat. Br. J. Nutr. 20, 317-323.

Similarly, the initial and final weights showed no correlation, nor were weight gain and caries incidence correlated, j 056 211745 TEST 3 This test was to determine the effect of protein on the absorption of the bacterium Streptococcus mutans to bydrixyapatite.

Materials and Methods Hydroxyapatite discs were prepared by pressing 150 mg of hydroxyapatite (Bio-Gel HTP, Biorad Laboratories) for 5 min under 5 tons of pressure in a KBr press. The discs were hydrated then incubated with either various protein solutions or imidazole buffer (0.05M pH 7.0, containing 0.025 M NaCl). The adherence 3 of H-labelled S.mutans PK1 cells was studied by incubating 3 9 the pretreated discs with H-thymidine labelled cells (10 cells/ml) suspended in the imidazole buffer. The protein solutions used were all 5 mg/ml in imidazole buffer. The proteins and polypeptides studied were as-casin, B-casein, K-casein, phosvitin, bovine serum albumin, histone III, histone VIII, a-1 acta1bumi n , 3-lactogl obul in, poly-1-lysine and poly-1-glutamate . The caseins were prepared by selective precipitation and the other proteins were purchased from Sigma Chemical Co., Missouri, U.S.A.

Results The effect of pretreating hydroxyapatite discs with various protein solutions on the adherence of S.mutans cells is shown in Table 5.

Table 5. Effect of protein on S. mutans adherence to hydroxyapati te.

Type Number of S.mutans cells (Radio labelled) abso rbed (CPM) 22,081 2,723 2,968 7,939 2 ,989 4,069 25,382 22,611 Proteins Con trol a - casein B -casein < -casein phos vi ti n BSA histone III hi stone VIII acidic phosphoprotein acidic phosphoprotein acidic phosphoprotein acidic phosphoprotein acidic protein basic protein basic protein 056 21174-5 Protei ns Type Number of S.mutans cells (Radio labelled) absorbed a-lactalbumin acidic protein 3-1 actogl obul in acidic protein poly-lysine basic polypeptide poly-glutamate acidic polypeptide (CPM) 1(3,643 6,443 45,053 5,019 Conclusion All the acidic proteins and polypeptides (especially the phosphoprote ins) caused a reduction in bacterial adherence to hydroxyapatite. However, the basic proteins and polypeptides either had no effect or enhanced bacterial adherence to hydroxyapati te . using the above tests Applicants have formulated various compositions in accordance with this invention as follows. In general, the compositions contain from 0.5-20% by weight of protein or polypeptide.

Example 1. Flour: In a device for mixing dry substances, \% by weight of powdered sodium caseinate was blended with flour.

Example 2. Cereal: A breakfast cereal was sprayed with a solution of calcium caseinate in water. The cereal flakes were then dried to .produce a finished product containing 1% calcium caseinate.

Fxample 3. Bread: 2% by weight of calcium caseinate was added to the flour during the mixing of ingredients for the manufacture of bread.

Example 4. Cake mix: 1* by weight of calcium caseinate was added to the dry ingredients in the preparation of a cake mix. Example 5. Confectionery: In the preparation of confectionery 2% by weight of calcium caseinate was added to the final mixture. Example 5. Biscuit: In the preparation of-a biscuit mixture 5% by weight of calcium caseinate was added to the other dry ingredients during mixing.

Example 7. Beverage: A beverage was prepared in which 1% weight of calcium caseinate had been dissolved.

Having regard to the successful results obtained from 056 211745 Example 8. Tablet: A tablet was made containing 10% by weight of calcium caseinate together with excipients being flavouring matter and binding material.

In preparation of a typical dentifrice within the scope of this invention, the requisite salt and salts of the selected protein or polypeptide are incorporated into dentifrice compositions in any suitable manner depending on whether a powder, paste or liquid preparation is to be produced. For this purpose appropriate preparations of surface-active agents, binders, flavouring materials and other excipients required to achieve the required form of dentifrice are added.

The invention is further illustrated by the following examples: Example 9. Tooth paste: A toothpaste was prepared having the following composition:- Calcium caseinate .0% by wei ght Gum tragacanth 1 .0% " II Sacchari n 0 . 1% II Glycerin (B.P.) .0% 11 ' II Sodium lauryl sulphate 1 .0% " II Methyl parahydroxy benzoate 0 . 1% " II Flavouring and colouring 1 .0% " II Dibasic calcium phosphate .0% " II Water 36 .8% It Example 10. Toothpaste: A preparation as set out in example 9 was repeated but with the addition of 2% sodium fluoride in a sui table form.

Example 11. Toothpaste: A preparation as set out in example 9 was repeated but with the addition of 0.4% stannous fluoride in a sui table form Example 12. Toothpaste: A preparation as set out in example 9 was repeated but with the addition of 0.1% mono sodium fluoro-phosphate in a suitable form. 056 7.117 Example 13.

Example 14.

Tooth powder: The following preparation was made:-Calcium caseinate 5.0X by weight Soluble saccharin 0.IX " " Colour agent Trace " " Dibasic calcium phosphate 94. IX " " Tooth powder: A preparation as set out in example 13 was made but with the addition of IX mono sodium f1uorophosphate in a suitable form.

Example 15. Liquid dentifrice: A preparation was made consisting 10 o f:- Sodium alginate Calcium caseinate Sodium lauryl sulphate F1avouri ng 15 Colouring Water pH adjusted to 7.0 Example 16. Liquid dentifice: As for example 15 but with 0.5% sodium fluoride added.

Example 17. Mouthwash: The following preparation was made:- Sodium caseinate 2.0% by weight Sodium fluoride 0.5% " " Flavouring Trace " " Colouring Trace " " Water 97.5% " In the above, casein was used principally because of economics but in lieu phosvitin or other material might be used. 1.5X by weight 5.0% " 1.0% " Trace " Trace " 92.5% " I 211745 3 Casein which 1s disclosed above 1s difficult to us* as 4 a result of poor solubility 1n water even wh*n present as a salt. 6 It has ba*n now found that digests of casein are also 7 effective and tend to have mcr*as*d water solubility as 8 compared to the undigested casein. 9 The digestion can be effected chemically or proteolytlcally. 11 Preferred digesting agents are the proteases, trypsin, 12 pepsin, chymotrypsln and pronase. 13 The following example Illustrates the preparation of 14 tryptle digest of casein: EXAMPLE 1 16 An aqueous mixture containing 10 gm casein, 200 mgm 17 trypsin (trypslntpck obtained from Sigma Chemical Company. 18 Missouri, U.S.A.) and 1 litre of a 100 mm ammonium 19 bicarbonate buffered solution having a pH of 8.S3 was prepared. Kept at 37°C for 2 hours and then subjected to 21 freeze drying under vacuum to remove the water and ammonium 22 bicarbonate to produce s tryptle digest of casein as a dry 23 powder. 24 The so obtained tryptle digest of casein was readily soluble 1n the pure water and also 1n addle solutions such 26 as exist In carbonated beverages and fruit juice. 27 The test procedures reported 1n table 2 of the primary 28 disclosure were repeated 1n respect of the tryptle digest of 29 casein and showed a reduction 1n calcium dissolution rate of 6S0404,csspe.052,unlcldv, • '-y ■*] i J ") 1 109.? + /-3.2 nmol/min, and • reduction In phosphate 2 dissolution rate of 67.4 + /-5.6 nmol/min. these reductions 3 1n dissolution rate shows the active peptides ere not 4 destroyed by the enzymatic hydrolysis.

The test procedure reported 1n table 5 above were 6 repeated 1n respect of the tryptle digest of casein and gave 7 1.9 + / — 0-6 * 10 ' it the number of s. mutans cells 8 adsorbed. 9 The test procedure described 1n test 4 above was repeated In respect of the tryptle digest of casein and 11 showed that It would alto Inhibit "carles-like" changes 1n 12 enamel slabs In situ. 13 Tha following additional examples are given: 14 EXAMPLE II Examples 1, 5. 7. 8, 9 and 17 of the primary disclosure 16 were repeated using an equel weight of the tryptle digest of 17 casein In lieu of calcium caseinate. 18 EXAMPLE III 19 Carbonated beverage: 0.5X by weight of the tryptle digest of casein of Example I was added to a commercially 21 available carbonated beverage. 22 EXAMPLE IV 23 Fruit Juice: 0.5Z by weight of the tryptle digest of 24 casein of Example I was added to a commercially available fruit Juice.

The claims form part of the disclosure of this specification. Modifications and adaptations may be made to the above described without departing from the spirit and scope of this invention which includes every novel feature and combination of features disclosed herein. 850404, csspe. 052, unlddv.

Claims (11)

Q\W4S

1 SEil £k£I2 11

2 1. An orally ingaatibla composition In the form of a

3 foodstuff, confietlonary, mouth wash, tablat, loxenge or

4 capsule containing at laaat 51 by waight of a water aolubli by weight 5 salt of casein or at least 2%JoT a water aolubli aalt of S alpha-s casain or at loaat 2% by weight of a water soluble 7 aalt of phosvitin. » 8 2. An orally ingastible compoaition as claimed in claim 1, 9 containing at least 10X by weight of a water aolubla aalt of by weight 10 caaein or at least Sl/of a water soluble salt of alpha-s 11 casein. 12 3. An orally ingestibla composition as claimed in claim 1, 13 containing at least 2f by weight of a water soluble aalt of 14 alpha-s casain. 15 A, An orally ingastib1 a composition as claimed In any 18 preceding claim In the form of a foodstuff or confectionery.

5. An orally iigaatibli composition In the form of a foodstuff, confectionery, dentifrice, mouthwash,' tablet, lozenge or capsule containing a carles and gingivitis inhibiting amount of water soluble chemical or proteolytic digest of casein or a salt thereof.

B. A composition as claimed in Claim 5, wherein said digest ia a trypsin, pepsin, chymotrypsln or pronase digest of Casein or a salt thereof.

7. A composition as claimed in claim 5, wherein the digest of essoin is present as up to 2% by weight.

8. A eompositior as claimed in claim 5, in the form of a foodstuff.

9. A composition as claimed in claim S, in the form of 1 7 1 8 1 9 20 21 22 23 24 25 26 27 28 Vs, ■'/ - 17 - 21174S 1 confectionary. 2

10. A eompoiitioi-i Claimed in claim 5, in t h • form of » 3 bavaraga. 4

11. An orally ingaatibla compoaition aubatan11 a 11y as 5 haralnbafori doacribad with rafaranet to any ono of the 8 Examples. 7 a a