BRPI0904505A2 - caries removal composition, composition preparation process and caries removal process - Google Patents

Abstract

COMPOSITION FOR THE REMOVAL OF CARIES, PROCESS OF PREPARING THE COMPOSITION AND PROCESS OF REMOVAL OF CARIES The present invention has as its object a composition for the chemical-mechanical removal of caries, containing as an active removing agent an extract comprising bromelain. The present invention is also directed to a process for preparing such a composition and a process for removing cavities.

Description

Patent Invention Descriptive Report

Caries Removal Composition, Preparation Preparation Process ε Caries Removal Process

Field of the Invention

The present invention relates to a chemical-mechanical caries removal composition containing as main remover an extract comprising bromelain. The present invention is also directed to a process for preparing such composition and a caries removal process.

Background of the Invention

Contemporary dentistry advocates maximum conservation with minimal intervention to remove decayed tissue. It is minimally invasive, seeks patient comfort, avoids the opening of enamel-restricted lesions in which dental biofilm control is possible. In cavitated lesions only irreversibly demineralized infected tissue is removed, as healthy tissue is the best protection for the dentin-pulp complex (BUSATO et al, 2005; CORRÊA, 2005).

The high cost of dental treatment is also a challenge to modern dentistry, making it more affordable is an achievement to be achieved in this millennium. Based on these paradigms came the Atraumatic Restorative Treatment (ART), aiming at the manual (mechanical) removal of decayed tissue with curettes or chisels with cut and subsequent closure of the cavity with adhesive restorative material, such as glass ionomer cement (FRENCKEN, et (1996, FRENCKEN, et al, 1997).

The TRA was designed to be applied to refugee camps in underdeveloped countries where extraction would be the only treatment option. It is characterized by its low cost, can be performed in places without electricity, as it does not require the use of high and low speed motors. The absence of electricity and the idea that dental restoration always requires special electrical equipment are the main reasons for this situation. In contrast, ART enables cavity treatment to be performed on people living in resource-poor areas, without electricity or unable to maintain expensive dental equipment (FRENCKEN, 5 et al., 1997).

The drawback of ART is muscle fatigue caused by muscle tension during dentin curettage, often leading to ineffective removal, and decayed infected tissue may be left in the cavity. (AMERONGEN, 1996). Due to the mentioned aspects the use of techniques of

Chemical-mechanical removal of decayed tissue has been growing considerably. Some authors cite that such techniques combine the ability to remove only decayed, non-remineralizing carious dentin, while the second uninfected layer, which has remineralizing potential, remains - in the absence of discomfort in most cases (KUBOKI et al, 1977; ERICSON et al, 1999; MARAGAKIS et al, 2001). Although there are advantages over the conventional method because of the rapid treatment of caries removal, its cutting can produce unnecessary wear and tear making the dental remnant weakened (BANERJEE 20 et al, 2000a).

The chemical-mechanical removal method optimizes TRA1 and is an excellent option in areas with difficult access to dental treatment, being a reality in many areas of Brazil. The combination of the two techniques will bring better quality treatment and effectiveness in areas where traditional dentistry has no access. This being, unfortunately, the picture of oral health still present in various regions of our country, providing an improvement of dental treatment where only extraction is the only option of dental treatment (BUSSADORI, 2005).

Through the revised literature, two products on the market, 30 Carisolv® and Papacárie® have been shown to be effective in chemical and mechanical removal. Carisolv® has been around for over a decade, but its high cost by Brazilian standards makes it unviable for use in the public sector. Because Papacárie® is a newer product, it is still being researched, with a shelf life of about one year, with refrigeration (PAPACARIE, 2005). Carisolv® is composed of 5 two carboxymethylcellulose-based gels: a red gel containing 0.1 M amino acids (glutamic acid, leucine and lysine), NaCl1 NaOH1 erythrosine; and the second colorless gel containing sodium hypochlorite (0.5% NaCl), after mixing the gel chemically results in the formation of another product, chloramine. Chloramine is composed of the combination of sodium hypochlorite and ίο aminonitrogen, making the effect of hypochlorite less aggressive and longer (TONAMI, et al, 2003). The formulation of Papacárie® contains 3% papain and chloramine acting synergistically (PAPACARIE, 2005).

Caries is characterized by being an infectious, opportunistic, multifactorial disease, strongly influenced by dietary carbohydrates and the action of salivary components. It results in localized loss of milligrams of minerals from affected teeth caused by organic acids from the microbial fermentation of dietary carbohydrates (WEYNE; HARARI, 2002). Destructions, located in hard tissues (enamel, dentin and cementum), represent the symptoms of the disease (THYLSTRUP; 20 FEJERSKOV, 1988). Carious lesion is generally seen as an exclusively destructive process of dental structures.

The appearance of carious lesions is the pathognomonic sign of the activity of this disease, which is mediated by the presence of plaque, which will only be considered cariogenic if it occurs in interactions of the factors under critical conditions over a certain period of time. Factors are classified as primary (essential) and secondary (modulatory) factors. The essential factors that influence are the microorganisms, the host and the substrate (diet). Secondary or modulating factors are saliva and fluorine. Thus, the carious process presents a dynamic where there are periods of remineralization and demineralization (MALTZ, 2000; WEYNE; HARARI, 2002; CURY1 2001; ZANIN, 2003). The objective of chemical-mechanical removal is to remove the outermost layer of caries (infected), leaving the demineralized portion, which can be remineralized and repaired (KUBOKI et al., 1977; ERICSON et al., 1998; MARAGAKIS, et al, 2001; PORTO et al, 2001; CORRÊA, 2005;

BARATA, 2007).

Research in the field of dentistry has been advancing, seeking alternative means that preserve the dentin structure as much as possible, such as chemical-mechanical removal. During clinical procedures, the use of drills often leads the practitioner to remove dentin structures that would be likely to undergo remineralization. With this, contemporary dentistry faces constant challenges in the search for products that selectively act on decayed tissue, promoting treatment with minimal, atraumatic and holistic intervention (Cederlund et al., 1999; FURE et al., 2000; BANERJEE, 2000a ; BANERJEE, 2000b; MARAGAKIS, 2001; 15 PORTO et al, 2001; CORRÊA, 2005; BARATA, 2007).

For the removal of decayed tissue, currently, the opening of lesions restricted to the enamel, in which the control of the dental biofilm is possible. In cavitated lesions, a more conservative posture is recommended, with less removal of dentin tissue (BUSATO et al, 2005; CORRÊA, 2005). 20 Considering the aforementioned aspects, this study tested the bromelain extractatophilized using the CMC as a vehicle, aiming to use a different active principle from the other products mentioned.

Bromelain is a proteolytic enzyme present in the pineapple (Ananas cosmosus L Merril) pulp, stalk, stem, bark, crown and leaves. Plant 25 belonging to the Bromeliaceael botanical family comprising approximately 46 genera and about 1,700 species of herbaceous, epiphyte and terrestrial plants.

Bromelain is a cysteine-like protease, an oligosaccharide acid protein composed of covalently linked molecules in the peptide chain, having a molecular weight of approximately 35,000 Daltons. It has a reactive sulfhydryl group which is essential for its catalytic activity. Its proteolytic mechanism of action is based on protein hydrolysis in oligopeptides and amino acids. Proteases cleave peptide bonds by nucleophilic attack. When its proteolytic fraction is purified and extracted, the result is potent proteolytic activity in vitro. Eight stem bromelain proteolytic subtractions were detected. The two largest were F4 and F5 and the most active proteinase was F9, which makes up 2% of total proteins. It is estimated that 50% of the proteins in F4 and F5 are glycosylated and F9 is unglycolized, which theoretically would justify its higher proteolytic potential. The optimal pH of F4 and F5 is between 4.0 and 4.5 and that of F9 is close to neutral. Complete bromelain extract shows activity from pH 4.5 to 9.8 (KELLY, 1996). In addition to bromelain, there are two cysteine-like proteases on the pineapple stem: ananine and cosmosine, but the largest protease consists of bromelain (ROWAN et al., 1990).

Currently, commercial bromelain is obtained from both the fruit and other parts of the pineapple is prepared by juice centrifugation, filtration and lyophilization. The process results in a yellow powder whose enzymatic activity can be determined on different substrates such as casein, gelatin or chromogenic tripeptides. In aqueous solution bromelain rapidly deteriorates by self-digestion (MAURER, 2001).

With the advancement of the field of enzymology, several techniques of enzyme extraction emerged. The techniques are based mainly on the precipitation power of albuminoid substances by organic salts and solvents such as ammonium sulfate, ethyl alcohol, methanol, isopropanol and acetone. Acetone is the most convenient precipitant in industrial preparation as it is recovered by distilling the supernatant resulting from centrifugation (OLIVEIRA, 2001).

US 2008/152601 describes desensitizing dentifrice components containing an enzyme such as papain and a second dentifrice component containing a surfactant such as sodium lauryl sulfate. The present invention differs from that document in that it presents the bromelain enzyme as it is not a dental plaque removal composition but a caries removal composition.

EP 1952801 describes a composition characterized by being a dental prophylactic containing bromelain active components in an amount of 0.01-1 wt% and xylitol in an amount of 1.5-20 wt%. The present invention differs from that document in that bromelain is lyophilized and does not use other prophylactic components such as xylitol.

WO 06130040 describes a caries prophylaxis composition comprising, among other ingredients, bromelain, xylitol and magnesium chloride or magnesium glycerophosphate. The present invention differs from this document in that it has lyophilized bromelain in its composition for mechanical caries removal.

WO 03043517 describes an oral composition for prophylaxis and / or cure of caries from compositions containing bromelain extract, among other extracts. The present invention differs from that document in that it contains lyophilized bromelain.

The main problem with such prior art compositions is the loss of enzymatic activity over time. The above compositions have the purpose of caries prevention, the present invention aims at the removal of decayed tissue when the caries pathology is already installed.

The present invention differs from these documents by using a bromelain extract in lyophilized form. Applicant observed that by lyophilizing the enzyme, it did not lose its activity rapidly as compared to non-lyophilized bromelain. Accordingly the present invention provides a more stable oral composition than compositions present in the prior art.

From what is clear from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity in the state of the art.

In a first aspect the present invention provides oral compositions for the chemical-mechanical caries removal comprising a lyophilized proteolytic enzyme.

An object of the present invention is an oral formulation comprising:

(a) a lyophilized extract of a proteolytic enzyme; and

b) A suitable vehicle.

In a preferred embodiment, lyophilized bromelain extract is present in concentrations of 0.5% to 5% w / w.

A further object of the present invention is a process for preparing the composition comprising the steps of:

(a) extract proteolytic enzymes from at least one plant belonging to the genus Ananas;

b) Lyophilize the extracted proteolytic enzymes; and

c) Incorporate lyophilized enzymes in a suitable vehicle.

In a preferred embodiment, the lyophilized bromelain extract is added to a carrier gel composed of carboxymethylcellulose.

In a preferred embodiment, the composition is within a pH range of 4.5 to 9.8.

In a second aspect, the present invention provides a caries removal process comprising the steps of:

a) apply a composition comprising a proteolytic enzyme

freeze-dried to a decayed tooth; and

b) remove the decayed portion of the tooth.

These and other objects of the invention will be immediately appreciated by those of skill in the art and by companies having an interest in the segment, and will be described in sufficient detail for their reproduction in the following description.

The examples shown herein are intended solely to exemplify one of the numerous ways of carrying out the invention, however, without limiting the scope thereof.

Proteolytic Enzyme

For the purposes of this invention, a proteolytic enzyme is any enzyme capable of breaking protein and / or peptide peptide bonds. Examples of such enzymes are bromelain enzymes found in plants belonging to the Bromeliaceae family, which has more than 50 genera.

Among the more than 50 genera belonging to the Bromeliaceae family is preferably the genus Ananas.

Examples of plants belonging to the genus Ananas include, but are not limited to, Ananas ananassoides, Ananas arvensis, Ananas bracteatus, Ananas comosus, Ananas erectifolius, Ananas genesio-linesii, Ananas guaraniticus, Ananas macrodontes, Ananas microcephalus, Ananas mordus, Ananas nanus, Ananas pancheanus, Ananas parguazensis, Ananas pyramidalis, Ananas strictus, Ananas viridis, and combinations thereof. Preferably the plant used is Ananas comosus.

Extractions can be performed from any part of the plant, with extractions from bark being preferred. Preferably, the plant used is derived from agroindustrial remains.

Orai Composition

The oral composition according to the present invention comprises:

(a) a lyophilized extract of a proteolytic enzyme; and

b) A suitable vehicle.

The composition may be in the form of toothpaste, gel, liquids or any other suitable form. Preferably the composition is in gel form, wherein the gelling agent is selected from the group comprising agents compatible with oral formulations, such as cellulose and its derivatives such as carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, gums, as guar gum, caroba, karaya, carrageenan and mixtures thereof.

The composition may further comprise additional agents such as chelating, flavoring, surfactant, thickener, bleach, preservative, antioxidant and mixture thereof. Such additives, when present, are incorporated in amounts that do not substantially affect the desired properties of the formulation.

Preparation Process

The process of preparing the oral composition comprises the steps of.

(a) extract proteolytic enzymes from at least one plant belonging to the genus Ananas;

b) Lyophilize the extracted proteolytic enzymes; and

c) Incorporate lyophilized enzymes in a suitable vehicle.

Incorporation of the lyophilized extract into the CMC gel is performed immediately after its application to the dental cavity. For extraction of the bromelain enzyme, any part of at least one plant belonging to the genus Pineapple can be used, preferably using frozen Ananas cosmosus bark in a 0.1 M phosphate buffer solution, pH 7.0, in a 1: 1 to 4 ratio. ° C.

The obtained solution is filtered and then centrifuged to obtain the crude extract, which is discarded. Ethanol is added to the crude extract to give a first precipitate. To the supernatant of this centrifugation is added ethanol again. After 24 hours, the second precipitate is stored at -10 ° C for later freeze drying.

Bromelain extract should go through the lyophilization process, which is ideal for preserving bromelain proteolytic activity. Being a protease of plant origin it undergoes self-digestion losing its proteolytic activity. Lyophilization avoids self-digestion due to decreased enzyme water content. The result is a white or yellow powder retaining the above characteristics. In the process the material is frozen and the surrounding pressure reduced, thus allowing the frozen water to sublime directly from the liquid phase to the gas. Caries Removal Process

The caries removal process of the present invention is a process comprising the steps of:

a) applying a composition comprising a lyophilized proteolytic enzyme to a decayed tooth; and

b) remove the decayed portion of the tooth through blunt dentine curettes.

Example 1 - Preparation of Lyophilized Bromelain Extract The method used was that described by Freiman and Sabaa-Srur (1996). On the first day, frozen shells were centrifuged and 0.1 M phosphate buffer pH 7.0 was sequentially incorporated at a ratio of 1: 1 at 4 ° C. The obtained solution was filtered and then centrifuged at 5,500 rpm for 20 minutes at 5 ° C to give the crude extract. To the crude extract was added 96% commercial ethyl alcohol at -10 ° C in a 1: 1 ratio, standing for 24 hours in a refrigerator at 5 ° C. On the 2nd day, it was centrifuged at 5,500 rpm at 5 ° C for 20 minutes to give the 1st. Precipitate which has been stored at -10 ° C. To the supernatant of this centrifugation, 96% ethyl alcohol was added in a 1: 1 ratio, remaining at rest for 24 hours in a refrigerator at 5 ° C. On the 3rd day it was centrifuged at 5,500 rpm at 5 ° C for 20 minutes to obtain the 2nd. Precipitate which was stored at -10 ° C for later freeze drying.

The lyophilization process was performed on a Terroni model 3000 apparatus. The lyophilized extract was stored in a sterile petri dish wrapped in film paper in a 0 ° C refrigerator. Example 2. Characterization Assays

Proteolytic activity is determined with soluble collagen as substrate by a process known in the art in an agar-collagen-milk culture medium. Collagenolytic activity is performed according to the diameter of the halo in millimeters formed around the well by measuring the reverse of the caliper petri dish, as shown in Table 1.

Table 1: Classification of collagenolytic activity of lyophilized bromelain extracts by halo diameter measurement.

<table> table see original document page 12 </column> </row> <table>

To evaluate proteolytic activity a quantitative method was applied, in which casein was dissolved at 1% in 0.1 M acetate buffer pH 6.0. To each 1 ml of bromelain solution (1, 2 and 3%) was added 2 ml of casein. Then the solutions were incubated at 35 ° C for 10 minutes, the reaction was stopped with 5 mL of TCA acid in each solution, then ice-bathed for 5 minutes, filtered on Whatman No. 41 paper and after Therefore, the reading was done in a spectrophotometer. The results are shown in Table 2.

Table 2: Results obtained from collagenolytic activity, enzymatic activity, protein content and specific activity of solutions of lyophilized bromelain extract.

<table> table see original document page 12 </column> </row> <table> Example 3. Carboxymethylcellulose-based gel (CMC) formulation For the carboxymethylcellulose-based gel formulation three gel concentrations were tested containing lyophilized bromelain extract1 present in concentrations of 1%, 2% and 3% w / w. The gel was prepared in a previously standardized concentration of 3% CMC, in a single batch of 500mg, according to the following formula:

CMC .................................. 3%

Methylparaben .................. 0.1%

Propylparaben 0.1%

Glycerin ........................ 5%

Distilled water .................. 100%

Example 4. Standardization of dental elements involved in histopathological analysis.

Fifteen permanent molars with previous clinical indication of extraction were chosen as disposal products (CEDERLUND et al., 1999. TONAMI et al., 2003).

Inclusion Criteria: Permanent molars with deep decayed dentin compromising the occlusal face and may extend to the mesial, distal, buccal and lingual surfaces, with or without endodontic involvement (CEDERLUND et al., 1999). The criteria were confirmed by digital radiographic evaluation (RVG Throphi) through the parallel angulation technique.

Exclusion criteria: root remnants, deciduous teeth and other dental elements other than permanent molars. Dental elements with pulp polyps were also excluded.

Control Group: Each tooth was sectioned in half. The mesial parts of the lower molars and buccal upper molars received bromelain gel, with group A nomenclature. The distal parts of the lower molars and upper palatine molars received CMC gel without the enzyme, control group, with group nomenclature. B.

After extraction, the teeth were rinsed under running water to remove organic debris (eg, blood, saliva and periodontal ligament). They were then individually stored in disposable cups immersed in 0.9% sodium chloride solution in a refrigerator at -50 ° C (IMPARATO, 1999; BANCO DE DENTES, 2008). Products such as sodium hypochlorite and hydrogen peroxide were avoided. The teeth, since they are contaminated, were always manipulated by professionals with EPIS (personal protective equipment).

The fifteen molars involved in the project were divided into three groups of five (1, 2 and 3). Each group was placed in a 17 cm long and 8.5 cm wide alginate swap, only about 1/3 of the roots 15 were immersed in the plaster. The teeth were then sectioned in half with a KG diamond floppy disk in a handpiece with cooling. The lower molars were sectioned longitudinally in the buccolingual direction and the upper molars in the mesio-distal direction, following the root anatomy. The gels at concentrations 1, 2 and 3% and the control were placed in four distinct 3mL syringes correctly identified.

The 1, 2 and 3% bromelain lyophilized extract gel was applied to Grupol a, 2a and 3a, respectively, for 30-40 seconds and sequentially the carious tissue was removed with blunt curves, repeating the 25 procedure for three consecutive times. In Groups 1b, 2b and 3b the gel without bromelain extract was applied using the above method.

It was stipulated that each group would be numbered according to the concentration of the bromelain gel, for example;

Group 1a: mesial side of lower and buccal molars of upper 30 molars receiving 1% bromelain gel. Group 2a: mesial side of lower and buccal molars of upper molars receiving 2% bromelain gel.

Group 3a: mesial side of lower and buccal molars of the upper molars that received the 3% bromelain gel.

Group 1b: distal side of lower molars and palatal molars

the upper teeth, each corresponding to their respective group 1a dental element, received CMC gel without bromelain.

Group 2b: distal side of mandibular molars and upper molar palatines, each corresponding to its respective dental element from group 2a, received CMC gel without bromelain.

Group 3b: distal side of lower molars and upper molar palatines, each corresponding to its respective dental element of group 3a, received CMC gel without bromelain.

All groups underwent histopathological analysis on 15 light microscopy stained with hematoxylin and eosin to evaluate gel effectiveness (YIP et al., 1999).

Histopathological analysis of teeth

The 15 (fifteen) dental elements involved in the work were decalcified for 30 days in a solution of 20% sodium citrate and 30% formic acid, suspended in gauze, and exchanged for a new solution identical to each one. 24 hours (MORSE, 1945). Then the teeth were rinsed in running water for 24 hours, dehydrated, diaphanized and paraffin embedded. The dental elements were embedded in paraffin. The blocks were cut in a longitudinal microtome at 5 pm of 25 thickness and stained with hematoxylin and eosin (Appendix B). Five cuts were obtained on each side of the tooth (A and B), ie 10 cuts of each tooth and totaling 150 cuts.

Qualitative histopathological analysis was performed by comparing sides A (gel with enzyme) and B (control). The dentin remnants were statistically evaluated by the presence of lesion (P) and absence of lesion (LA). In the presence of lesion (P), basophilic bacterial infiltrate was observed, represented by granular basophilic areas and thick contiguous prolongations. In the absence of injury (A), healthy dental tissue was represented by regular eeosinophil odontoblastic extensions. Absence of injury (A) was also considered as areas with discrete basophilic lines in between.

Group 1 (G1)

Side A.- received 1% bromelain gel (CMC).

Control-side, bromelain-free CMC gel

Table 3: Results of the hitstological sections of sample # 1, tooth 16 (right upper first molar) of group 1 (G1).

<table> table see original document page 16 </column> </row> <table>

Table 4: Results of the histological sections of sample # 3, tooth 46 (right lower first molar) of group 1 (G1).

<table> table see original document page 16 </column> </row> <table>

Table 5: Results of the histological sections of sample # 4, tooth 36 (left lower first molar) of group 1 (G1). <table> table see original document page 17 </column> </row> <table>

Group 2 (G2)

Side A- received 2% bromelain gel (CMC).

Control-side, bromelain-free CMC gel

Table 6: Results of the histological sections of sample No. 7, tooth 17 (right upper second molar) of group 2 (G2).

<table> table see original document page 17 </column> </row> <table>

Table 7: Results of the histological sections of sample # 9, tooth 38 (left lower third molar) of group 2 (G2).

<table> table see original document page 17 </column> </row> <table>

Claims (29)

ClaimsCaries Removal Compound, Compound Preparation Process ε Caries Removal Process 1. Caries removal composition comprising: (a) a lyophilized bromelain extract; and (b) a suitable vehicle. Composition according to Claim 1, characterized in that dasbromelin is derived from a plant belonging to the Bromeliaceae family. Composition according to Claim 2, characterized by the plant belonging to the genus Ananas. Composition according to Claim 3, characterized in that the plant belongs to the species Arianas cosmosus L Merril. Composition according to Claim 1, characterized in that bromelinaser is extracted from a part of the plant chosen from the flower, fruit, stem, leaves, bark and mixture thereof. Composition according to Claim 5, characterized in that bromelinaser is extracted from frozen shells of Ananas cosmosus L Merril. Composition according to Claim 1, characterized in that the bromelain extratolyophilisate is present in concentrations ranging from 0.1% to 10% w / w. Composition according to Claim 7, characterized in that the bromelain extratolyophilisate is present in concentrations ranging from 0.5% to -5% w / w. Composition according to Claim 1, characterized in that it comprises a pH range from 3 to 10. Composition according to Claim 9, characterized in that it comprises a pH range from 4.5 to 9.8. Composition according to Claim 1, characterized in that it may be in the form of toothpaste, gel or liquid. Composition according to Claim 11, characterized in that the composition is in gel form. Composition according to Claim 12, characterized in that the gelling agent is chosen from the group of cellulose and its derivatives as carboxymethylcellulose, methylcellulose, methylethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, gums such as guar gum, caroba, karaya, carrageenan or mixtures thereof. . Composition according to Claim 13, characterized in that the bromelain extract extract is added to a carrier compound of carboxymethylcellulose. Process for preparing the caries removal composition, comprising the steps of: a) Extracting proteolytic bromelain enzymes b) Lyophilizing the extracted bromelain proteolytic enzymes; and c) Incorporate the lyophilized enzymes in a suitable vehicle when applying the product to dental caries. Process for preparing the composition according to claim 10, characterized in that the plant to be extracted from the bromelain proteinaceous enzymes belongs to the Bromeliaceae family. Process for preparing the composition according to claim 11, characterized in that the plant belongs to the genus Ananas. Process for preparing the composition according to claim 12, characterized in that the plant is the species Ananas cosmosus. Method for preparing the composition according to claim 15, characterized in that the bromelain is extracted from a part of the plant which is chosen from flower, fruit, stem, leaves, bark and mixture thereof. Process for preparing the composition according to claim 15, characterized in that bromelain is extracted from the frozen shells of Ananascosmosus L MerriI. Process for preparing the composition according to claim 15, characterized in that it comprises a 0.01 to 0.2 M phosphate buffer solution. Process for preparing the composition according to claim 16, characterized in that it comprises a 0.1 M phosphate buffer solution. Composition preparation process according to Claim 17, characterized in that it comprises pH of the buffer solution in the range of 6.0 to 8.0. Composition preparation process according to Claim 18, characterized in that it comprises pH of the buffer solution in the range 7.0. Process for preparing the composition according to claim 15, characterized in that the proportion of frozen Ananas cosmosus shells is 1:10 of phosphate buffer solution. Process for preparing the composition according to claim 15, characterized in that the ratio of frozen Ananas cosmosus shells is 1: 1 phosphate buffer solution. Process for preparing the composition according to claim 15, characterized in that the temperature is in the range of 2 ° C to 6 ° C. Process for preparing the composition according to claim 15, characterized in that the temperature is 4 ° C. Caries removal process comprising the steps of: (a) applying a composition comprising a lyophilized proteolytic enzyme to a decayed tooth; and b) remove the decayed portion of the tooth, where proteolytic enzymes are incorporated into the composition at the time of application.