Current status of whitening agents and enzymes in Dentistry

This study reviews the knowledge on the use of conventional dental whitening and the use of enzymes as a new approach in bleaching. A review of the literature was based on academic articles and on patents related to the use of enzymes in dental bleaching. Tooth whitening techniques used nowadays are well reported in the literature, and its mechanism of action consists of an oxidoreduction reaction with the release of free radicals. The great instability of radicals, when in contact with the tissues, promotes oxidation and reduction in the size of the pigment chains incorporated into them. These pigments are eventually broken down into smaller and smaller molecular chains and end up being diffused from the dental structure. In turn, the use of enzymes aimed at tooth whitening can be a less harmful alternative to the tooth because their specificity regarding the substrate makes them of great interest to perform specific reactions, reducing collateral effects. The use of proteolytic enzymes and oxidoreductases paired with the application of peroxides, can be a promising alternative for obtaining even better results in the dental bleaching process. Clinical Relevance: The combined application of several enzymes paired with the application of peroxides for tooth whitening can be a less harmful alternative for the teeth, reducing undesirable side effects and is therefore a promising alternative for dental bleaching. Clinical Relevance: The combined application of several enzymes together with the application of peroxides for tooth whitening can be a less harmful alternative to the tooth, reducing unwanted collateral effects and, thus, becoming a promising alternative for dental bleaching.


INTRODUCTION
The search for aesthetics and cosmetic care is highly valued, especially in dental offices. Teeth appearance has never been so important as today: a smile with pearly whites, well outlined and aligned, is commonly associated to a beauty standard in current society (Van der Geld et al., 2007). Chromatic alterations, whether of a single tooth or a group of teeth, cause discomfort related to the individual's aesthetics, negatively interfering in their social and professional life (Filho Menezes et al., 2006;Viegas et al., 2014).
The search for aesthetics and cosmetic care is highly valued, especially in dental offices. Teeth appearance has never been so important as today: a smile with pearly whites, well outlined and aligned, is commonly associated to a beauty standard in current society (Van der Geld et al., 2007). Chromatic alterations, whether of a single tooth or a group of teeth, cause discomfort related to the individual's aesthetics, negatively interfering in their social and professional life (Filho Menezes et al., 2006;Viegas et al., 2014).
Teeth are polychromatic structures arising from the overlapping of tissues with different characteristics (Joiner, 2004;Watts, Addy, 2001) and optical properties such as translucency, fluorescence, and opalescence (Priest, Lindke, 1999). Tooth color is associated with light reflection, dispersion, and absorption by the enamel (Vieira-Junior et al., 2018), dentin and pulp; however, the general chromatic appearance of the teeth is mainly related to the color of the dentin (Ten Bosch, Coops, 1995).
Dental color is determined by a combination of intrinsic chromatic characteristics and the presence of any extrinsic stain that may form on the tooth surface (Watts, Addy, 2001). Intrinsic staining is a result of problems and defects that occurred during odontogenesis due to some systemic disorders such as measles and scarlet fever, or due to dental trauma that may lead to internal bleeding, in addition to the darkening caused by the natural aging process (Alqahtani, 2014;Bernardon et al., 2010;Bizhang et al., 2009;Lo Giudice et al., 2016). Extrinsic color is related to the adsorption of chromogens, especially in the acquired pellicle of the enamel surface, which may later cause pigmentation (Alqahtani, 2014;Bernardon et al., 2010), Extrinsic chromatic changes may result from exaggerated or continuous exposure to some foods and beverages such as coffee, tea, soft drinks and dyes, in addition to smoking (Alqahtani, 2014;Bernardon et al., 2010;Bizhang et al., 2009;Lo Giudice et al., 2016).
Historically, dental bleaching techniques exist since ancient Egypt, where the population used vinegar to whiten teeth (Kihn, 2007;Lima et al., 2012). The Romans tried to use urine for the same purpose (Kihn, 2007). However, only in the late 19th century with the advent of the "organic chemistry revolution" these methods started to be further explored.
In the 19th century, the first reports on the use of peroxides arose, associated with an electric current to accelerate the reaction of oxygen release, thus promoting whitening in vital and non-vital teeth. In the 20th century, there are literature reports on the use of hydrogen peroxide together with other bleaching agents such as sodium perborate and carbamide peroxide associated with the application of electric current, heat source, and highintensity lamp, aimed at better results in tooth whitening (Fasanaro, 1992).
Dental bleaching is a non-invasive technique in which chemical agents are able to diffuse through the tooth surface, releasing free radicals that oxidize organic pigments of the teeth structure and, thus, cause whitening (Carey, 2014;Féliz-Matos, Hernández, Abreu, 2014;Ontiveros, Paravina, 2009;Torres et al., 2011;). Categorically, there are different approaches to the bleaching treatment (Joiner, 2006): I) At-home whitening, performed by the patient under supervision of a dental surgeon, using low-concentration bleaching agents in a high-frequency regime (Haywood, Heymann, 1989;Yey, Su, Lu, 2005); II) clinical bleaching, using highconcentration agents that are applied by a dental surgeon in a clinical environment; III) over-the-counter products (OTC), which have bleaching, abrasive or optical active principles added to the composition of toothpastes, rinses and strips, and are sold directly to the patient. Sometimes, OTC products do not have sufficient concentrations of bleaching agents for an in-depth tooth whitening, nor an ability to propagate to the dentin or potentiate the bleaching treatment performed by the dentist (Joiner, 2010;Vieira-Junior et al., 2019). In this category there are compounds capable of optically changing the perception of light or contributing to the removal of extrinsic pigments (Lima et al., 2008;Kugel et al., 2007), including those associated to the acquired pellicle of the enamel (Joiner, 2010).
Regardless of the technique and bleaching agent used, some adverse effects are reported, such as oral mucosa irritation, sensitivity, among others, which may be caused by both the whitening agents as well as by the device used during homemade treatment, transient sensitivity and changes in the physicochemical properties of hard dental tissues (Alqahtani, 2014;Kihn, 2007;Kugel et al., 2007;Vieira-Junior et al., 2006;Yey, Su, Lu, 2005).
The search for satisfactory whitening results with less adverse effects has been increasing, arising from a population highly driven by aesthetic standards and appearance (Kwon, Wertz, 2015). Thus, considering the growing demand for these cosmetic procedures and the need to minimize the adverse reactions associated, the search for alternative whitening methods, more conservative and effective, has attracted commercial attention (Kwon, Wertz, 2015). Given this context, biotechnology offers promising tools to meet such demand. This review describes whitening agents while focusing on the potential use of enzymes for this purpose, which provides advantages such as the reduction of adverse effects caused by conventional treatments.

METHODS AND MATERIAL
A narrative review of the literature was performed with the following inclusion criteria: original articles of quantitative and qualitative research, in English, to get information on the subject under scrutiny. The search was based on scientific articles using the keywords: Tooth Whitening, Tooth Bleaching Agent, Biotechnology, Enzymes, using as criterion articles published in the last 12 years. In addition, classical references pertaining to the history of dental bleaching use were included. The search for patents related to the use of enzymes in dental bleaching was carried out using the Software Orbit Intelligence of Questel with the following research words: Dental AND Bleaching AND Enzyme, Toothpaste AND Enzyme, Laccase AND Tooth, Catalase AND Tooth OR Dental, Peroxidase AND Tooth.

Conventional approaches use for dental bleaching
The most commonly used active ingredient in various dental bleaching techniques is hydrogen peroxide. Whitening agents can be made available in various forms, such as gels, stripes, tubes of toothpaste, among others (Joiner, 2010;Lima, Araújo, 2006), under the presentation of hydrogen peroxide, carbamide peroxide or sodium perborate.
The action mechanism suggested for dental bleaching consists of an oxidoreduction reaction with release of free radicals (Kwon, Wertz, 2015). The high instability of radicals when in contact with the intended tissues oxidizes and reduces the size of pigments chains incorporated to them (Kwon, Wertz, 2015). Such pigments are cleaved in increasingly smaller molecular chains and end up being spread from the tooth structure (Eimar et al., 2012;Kwon, Wertz, 2015;Marson et al., 2008) or, due to the reduced size of their chains, they indirectly promote the absorption of light, making the tooth apparently whiter (Sulieman et al., 2004). A description regarding conventional bleaching techniques will be presented with the aim of presenting a broad discussion and providing a critical comparison of biotechnological compounds.

Dental bleaching with Hydrogen Peroxide
Hydrogen peroxide (H 2 O 2 ) is a naturally colorless liquid, slightly more viscous than water, and has a molar mass of 34.01 g.mol -1 . Due to its low molecular weight, it has a high capacity of penetration in the enamel and dentin (Ubaldini et al., 2003). It is typically used at a 35-38% concentration in clinical applications or 1.5-7.5% in at-home applications using individual devices (Bernardon et al., 2010;Kihn, 2007;Sulieman et al., 2006).
The H 2 O 2 dissociates into free radicals of oxygen (·O·), peroxyl (HO·) and perhydroxyl (HOO·), which are highly unstable and reactive since they have unpaired electrons in the valence band (Bernardon et al., 2010;Kihn, 2007;Sulieman et al., 2006). These free radicals diffuse in enamel and dentin and react by attacking chemical bonds present in high-molecular-weight pigments, fragmenting them into smaller molecules without the original coloration (Bernardon et al., 2010;Kihn, 2007;Sulieman et al., 2006). The reaction of H 2 O 2 with pigments is presented in Figure 1.

Carbamide Peroxide
Carbamide peroxide is the most widely used bleaching agent in at-home whitening, at concentrations between 10 and 22% applied in previously-made devices, although it is also used in office at a 35% concentration (Kihn, 2007;Soares et al., 2013). Carbamide peroxide (CH 6 N 2 O 3 ), when in contact with the oral environment and saliva, dissociates into hydrogen peroxide (H 2 O 2 ) and urea ((NH 2 ) 2 CO). Urea, in turn, decomposes into carbon dioxide (CO 2 ) and ammonia (NH 3 ), acting as a stabilizer, responsible for prolonging the life of the bleaching agent and maintaining the pH of the gel close to neutral (Navarra et al., 2014).
The formation of ammonia causes pH to rise, thus increasing the efficiency of the dissociation reaction of the hydrogen peroxide and, consequently, the production of free radicals, responsible for the breakdown of pigmented molecules, as explained in the previous section (Bernardon et al., 2010;Kihn, 2007).

Undesirable effects of conventional bleaching
Effects of bleaching agents on dental tissues have not been completely understood, their intensity and range of cases vary according to different methods and study designs (Attin, 2009;Joiner, 2007;Zeczkowski et al., 2015). However, it is worth noting that the human saliva plays an essential role in promoting the remineralization or reduction of demineralization and deleterious effects on teeth subjected to bleaching treatment (Zeczkowski et al., 2015).
In addition to these, whitening-related tooth sensitivity is also a pretty common adverse effect (Goldberg, Grootveld, Lynch, 2010;He et al., 2012;Li, 2011;Pretty, Edgar, Higham, 2003), Such sensitivity possibly comes from the diffusion of peroxides through dental substrates (Li, 2011), as the hydrogen peroxide is able to reach the dental pulp and have a cytotoxic effect, even when this gel is used at low concentrations (Kwon, Wertz, 2015).

Use of enzymes as whitening agents
Industrial biotechnology is an essential tool for developing new drugs and cosmetic assets (Fox, 2005;Orladelli et al., 2012). After the discovery of antibiotics, the use of enzymes is considered to be the most promising tool for the pharmaceutical industry and may bring economic and environmental benefits. They have paramount importance in the production of new medicines and products, in addition to having been increasingly popular in the cosmetics industry-also called Enzymes-cosmetics (Fox, 2005).
Among these cosmetic products, enzymes are mainly used in personal care products, skin exfoliation and anti-aging creams aimed at protecting the skin against external agents, promoting biological peeling, fighting free radicals, among others (Fox, 2005;Orladelli et al., 2012).
The use of enzymes in the treatment of stained teeth is possible due to the fact extrinsic tooth pigmentation derives mainly from the incorporation of organic compounds containing chromogens (the part of the organic molecule which generates color) in the acquired pellicle (Joiner, 2010;Watts, Addy, 2001). These compounds with color present in dark foods and beverages such as coffee or wine are usually derived from polyphenols (Joiner, 2010;Watts, Addy, 2001). Two different strategies may be applied: the first by removing the pellicle using proteases, and the second using enzymes to chemically change the color compounds, with specific enzymes (Wiliams, Prencipe, Masters, 2004). One of the main classes of polyphenols that cause stains in teeth are tannins, which are soluble in water and present a molecular weight from 500 to 3000 kDa, being present in different plant parts, such as bark, wood, leaves, fruits, roots, and seeds (Govindarajan et al., 2016). In dentistry, wine is a relevant tannin source, responsible for intensifying teeth pigmentation (Côrtes et al., 2013).
One of its most decisive biological characteristics is its ability to establish a complex with different minerals and macromolecules, such as proteins, cellulose, starch and others, affecting the degradation and absorption of these nutrients (Govindarajan et al., 2016). In the case of teeth, such complexation can be associated with the appearance of stains resulting from the association between food tannins and tooth components.
The ability of tannins regarding the complex is due to its structure constituted by multiple hydroxyl groups in their phenolic rings, capable of binding to proteins and chelating metals. Due to this, they can easily bind to tooth proteins (Chavez-González et al., 2012). However, tannin compounds present in foods feature an intense color, ranging from yellow to brown, being one of the classes of compounds responsible for tooth pigmentation; hence, one of the compounds that must be removed during the whitening process (FAO, 2019;Hertel et al., 2017).
The use of enzymes in dental bleaching is a less aggressive alternative for the body, especially compared to conventional methods (Patil et al., 2015). Enzymes can effectively accelerate the reactions it catalyzes (up to more than 10 12 times in comparison with the spontaneous non-catalyzed reaction) in mild conditions of pH and temperature. In addition, they usually present high specificity, which reduces unwanted side effects (Whitaker, Voragen Wong, 2003).
The use of enzymes in dental bleaching was firstly suggested in the 1960s, with the potential application of fungal proteolytic enzymes (Harrisson et al., 1963). Since then, several patents were filed, claiming the use of several enzymes in the dental bleaching process. Nonetheless, few articles on the application of enzymes as dental bleaching agents can be found (Joiner, 2010). Following, the main enzymes discussed in the literature are described, showing the current state of the knowledge on the topic. The mechanism of action of enzymes in bleaching is shown in Figure 2.

Proteolytic enzymes
Proteolytic enzymes were the first to be described as having potential to be applied in dental bleaching based on the fact the external stains are initially incorporated to the teeth surface in the form of a pellicle comprised by saliva proteins (Harrisson et al., 1963). Bleaching using proteolytic enzymes occurs due to the hydrolysis and removal of the biofilm formed on the teeth surface over time, in addition to the breakdown of bonds between proteins and the compounds responsible for the color (Joiner, 2010;Kalyana et al., 2011). Furthermore, the use of proteolytic enzymes has other advantages, such as its potential to prevent bacterial growth, ability to act in pH close to neutral, and biocompatibility of many (such as papain), which do not present cytotoxicity nor secondary effects (Patil et al., 2015). Harrisson et al. (1963) tested proteases, amylases, cellulases and lipases in toothpaste compositions, showing that those containing a mixture rich in proteolytic enzymes of fungal origin were effective in reducing external stains after six months of use. According to the authors, a vessel compatible with high concentrations of enzymes and that releases them in a slower rate, such as chewing gums and tablets, could lead to even better results (Harrisson et al., 1963).
In 2011, Kalyana et al., studied the efficacy of the removal of stains in vitro using a toothpaste containing vegetal proteolytic enzymes (bromelain and papain). To evaluate whitening effectiveness, the authors photographed the teeth under standardized lighting conditions and calculated their luminosity. The authors observed that, on average, the luminosity of teeth brushed with the toothpaste containing enzymes was significantly higher than in the control group, showing a reduction in teeth stains. In a similar study, Patil et al., 2015, compared commercial toothpastes with chemical abrasives (perlite and calcium carbonate) with toothpastes containing enzymes (bromelain and papain) and found no statistical differences between both products. Thus, the whitening effect of the product with enzymes was considered similar to that of those containing abrasives, preventing the occurrence of new stains.
Catalase (EC 1.11.1.6) Catalase is an essential enzyme for aerobic organisms and it catalyzes the decomposition of hydrogen peroxide into water and hydrogen. Additionally, catalase is able to oxidize other hydrogen-releasing compounds, such as methanol, ethanol, formic acid, and phenols, using hydrogen peroxide as electron acceptor (Whitaker, Voragen Wong, 2003). Thus, catalase is not used to generate a direct whitening effect: its function on the bleaching process is to degrade peroxides during or after treatment with bleaching agents (hydrogen or carbamide peroxide).
It is well known that these peroxides can be degraded by the catalase and peroxidase of the dental pulp. Because of this, some authors evaluated the potential use of exogenous catalase as a way to neutralize residual peroxide from this process. Rotstein (1993), for example, demonstrated the enzyme was able to eliminate the peroxide in the first rinse, which did not occur with conventional rinsing (only water). In another study, Rotstein, Wesselink, Bab, 1993 show the use of catalase could protect the oral mucosa of rats with induced injuries, observing that, when applied in the tongue before treatments with hydrogen peroxide, the enzyme protects the tissue against adverse reactions Based on such properties, a patent request (EP1224925) was filed for a tooth-whitening formulation containing hydrogen peroxide as the main active ingredient and catalase as one of the possible activating compounds. The patent claims that the use of the catalase enzyme quickly increases the release rate of O 2 and H 2 O and the number of free hydroxyl radicals, which causes a fast whitening action and reduces the time required for the peroxide to act (Banerjee, Friedman, 2002). Similarly, another patent request (BR 102014010685-5 A2) claims that a 5-mg quantity of the enzyme applied for 3 minutes would be enough to break down all the H 2 O 2 (35 to 37%), with the advantages of not requiring the use of LEDs, not causing pH changes, and reducing the time of dental whitening (Silva et al., 2014).
Laccase (EC 1.10.3.2) Laccase is an enzyme of the group of "blue oxidases", named due to the copper present in their active sites, which gives a blue color to these proteins. This enzyme catalyzes the oxidation of several compounds, mainly phenolic compounds, including monophenols, o-and p-diphenols, methoxyphenol, aminophenols, in addition to aryl amines and even some inorganic ions (potassium ferrocyanide) (Chauhan, Goradia, Saxena, 2017;Whitaker, Voragen Wong, 2003). They use molecular oxygen as electron acceptors, forming water as a reaction byproduct.
This enzyme features potential applications in the food, paper and cellulose, fabrics, synthetic chemistry, and cosmetics industry, in addition to use in the bioremediation and bio-degradation of pollutant phenolic compounds (Shraddha et al., 2011). Considering that, as mentioned before, dark stains on teeth can have a polyphenolic character, and keeping this oxidation capacity of phenolic compounds through laccase in mind, Colgate-Palmolive listed in 1997 a patent request (WO9706775) of formulations containing this enzyme as a tooth-whitening agent (Aaslyng et al., 1997). This patent also vindicates the use of other oxidoreductases in dental bleaching formulation, such as glucose oxidase, hexose oxidase, L-amino acid oxidase, xylitol oxidase, galactose oxidase, pyranose oxidase, alcohol oxidase, peroxidase and haloperoxidase (Aaslyng et al., 1997). Years later, the Kobayashi-Pharmaceutical and the Takasago Perfumery also requested a patent (JP4637494) involving the application of laccase in dental bleaching. However, in this case, only a cleaning solution was tested, in artificial teeth. Peroxidase (EC1.11.1) Peroxidases are a group of oxidoreductases that use the peroxide as an electron acceptor instead of oxygen (Whitaker, Voragen Wong, 2003). The main specific enzymes of this group are NADH peroxidase (EC 1.11.1.1), glutathione peroxidase (EC 1.11.1.9) and lactoperoxidase (EC 1.11.1.7), in addition to non-specific enzymes collectively referred to as peroxidase (Hamid, Rehman, 2009).
Peroxidases are capable of oxidizing a broad range of substrates, using hydrogen peroxide or other peroxides as the electron acceptor. Thus, these enzymes feature a large number of industrial and analytical applications, such as in preparations of enzyme-linked immunosorbent assay (ELISA), bioremediation, synthetic dye degradation and bleaching in the paper and cellulose industry (Shraddha et al, 2011).
A recent study described that the horseradish peroxidase activation of high concentration bleaching gel accelerates the degradation of hydrogen peroxide, which could decrease the adverse effects on odontoblastlike cells (Ortecho-Zuta et al., 2018).
Similar to laccases, the degradation properties of several color compounds allow peroxidase enzymes to have potential application as dental bleaching agents, with the advantage of being able to be used concomitantly with peroxides (reducing agent). Starting from such a premise, the company Kin Laboratories patented (EP1224925) the use of peroxidase in toothpaste for dental bleaching (Pons et al., 2001). The efficiency of toothpaste containing lactoperoxidase (5%) combines with carbamide peroxide (3%) was evaluated in 20 volunteers for 21 days (Pons et al., 2001;Forner et al., 2012). The treatment consisted of teeth brushing using the toothpaste for 2 minutes, three times per day. Teeth color before and after treatment was recorded using spectrophotometry and analyzing color variations in teeth using the CIE L*a*b* standard. The authors have concluded that the use of the product containing low amounts of carbamide peroxide, when activated by peroxidase, was effective in whitening teeth even with a short time of exposure. Thus, the use of peroxidase proves to be able to increase the efficiency of dental bleaching by carbamide peroxide (Forner et al., 2012). Llena et al., (2016) observed increased luminosity and reduced b* component compared to the control group in double-blind clinical trials with the same toothpaste involving 48 participants with teeth colored A3 or higher than the Vita classical guide. The color variation among groups was not significant (p<0.064) but, according to the authors, the toothpaste containing enzyme could effectively modify the teeth color, progressively starting from the third to the ninth week of treatment, stabilizing after this period.
In an in vitro study, the aesthetic effectivity and cytotoxicity of a toothpaste containing 10% hydrogen peroxide and activated by radish peroxidase were evaluated. The authors observed the treatments with 10% peroxide plus enzyme presented statistically similar results (p<0.05) to that of the positive control (gel with 35% hydrogen peroxide). In addition, the authors have observed that the treatment containing radish peroxidase did not present cytotoxic effects, being statistically similar to the negative control .
As explained before, many teeth stains are based on tannins. Thus, tannase has been considered an enzyme for dental bleaching. The use of tannase can be useful for removing tannin compounds linked to the teeth proteins; this strategy has already been suggested in patents of oral hygiene products from Colgate-Palmolive (WO2004019899 and WO03094879). These documents cite the potential use of purified tannase of Aspergillus niger and A. allianceus to ease the removal of stains in the extrinsic portion of the tooth (Szeles et al., 2003;Wiliams, Prencipe, Masters, 2004). However, the use of these enzymes is not declared in the requests of the respective patents, as only the use of formulations containing papain and glucoamylase are vindicated (Szeles et al., 2003;Wiliams, Prencipe, Masters, 2004).

DISCUSSION
Dental bleaching has become a major ally for dentistry, being able to reestablish teeth aesthetics by achieving good results while conservating dental structure (Joiner, 2006); however, they also feature limitations and risks. Given this context, the personal hygiene and cosmetics industry increasingly seeks to launch new whitening products that maintain healthy dental structure the most, preserving dental tissues.
Dental bleaching is currently much used in dentistry, is considered an effective treatment and is associated to an improvement in the quality of life (De Geus et al., 2016). Effective whitening treatments manage to break molecules of chromogens in dental depth, especially in the dentin and not being restricted to the surface. Some of the aforementioned agents could be incorporated into conventional gels to promote catalysis of bleaching reactions. Additionally, proteolytic or chromogen-specific enzymes could be associated with different vessels to potentiate the diffusion of such agents through the dental structure. Some of the compounds described may act in extrinsic pigmentation or in chromogen biofilms associated to the acquired pellicle of the enamel, which could reduce staining, especially in risk patients who are frequently exposed to pigments. In this category, these agents could be associated with oral-care products such as toothpaste and rinses, which provide frequent and daily exposure to the patient. Biotechnology agents discussed in this article are presented as promising alternatives. However, clinical trials addressing the effectivity and safety of these assets must be devised to confirm the findings described in this review.
In addition to the enzymes mentioned in this review, the study of other proteolytic enzymes and oxidoreductases in dental bleaching also presents considerable potential since the number of enzymes studied up to the moment is limited. Thus, we highlight the potential of new proteolytic enzymes, e.g. those obtained from microorganisms present in the dental microbiota, in dental bleaching. Furthermore, the use of oxidoreductases traditionally used in the treatments of biomass (lignin peroxidase and manganese peroxidase) are also interesting options to be tested. It is well known that such enzymes present greater redox potential than laccases and could degrade a broader range of polyphenols (Llena et al., 2016). Such enzymes are known for degrading pigments, a fact that has been even used as a way to quantify the activity of these enzymes (Hamid, Rehman, 2009). The use of new enzymes requires several studies, from in vitro trials to extensive clinical studies, to monitor possible secondary or adverse effects.

CONCLUSIONS
Although currently used conventional tooth whitening techniques such as hydrogen peroxide and carbamide peroxide together with their mechanism of action are well reported in the literature, some adverse reactions are reported, such as enamel demineralization, which can be caused both by concentration of the bleaching agent used, application time to obtain the desired color, increase in surface roughness, decrease in microhardness, bond strength of enamel and dentin, and a significant decrease in the concentration of calcium, phosphate and carbon in dental enamel. The use of biotechnology, together with new formulations, aims to promote teeth whitening, or the control of stains, and can provide fewer adverse reactions, which can render great interest in the industrial sector.