Bio-based surfactant compositions for reducing multi-species biofilm formation in oral cavity

Abstract

The present invention relates to a composition comprising rhamnolipid and sodium lauroyl sarcosine. The composition can be used as an oral care composition to prevent or treat plaque and / or biofilm formation in the mouth and / or in the teeth.

Description

Technical Field

[0001] Dental plaque removal is one of the major challenges in human oral care. Dental plaque, or dental biofilm, is a highly organized microbial community that adheres to the solid surface of teeth. On the one hand, biofilms are a symbiotic component of the immune system; on the other hand, the uncontrolled activity of bacterial pathogens can lead to a variety of diseases, including dental caries, periodontitis, pulpitis, tonsillitis, and alveolar ulceration (Yaacob et al., 2014; Colombo et al., 2015; Valm, 2019).

[0002] The composition of dental plaque varies from person to person and depends on many factors; however, there are some typical characteristics that characterize dental plaque. Dental plaque is broadly divided into two categories: supragingival plaque and subgingival plaque. Supragingival plaque is found at or above the gingival margin, while subgingival plaque is found below the gingival margin and between the tooth and the gingival suture (Haffajee et al., 2008).

[0003] Dental biofilm formation occurs in several stages: 1) biofilm formation prior to planktonic attachment; 2) initial colonization, followed by proliferation of attached microorganisms; 3) secondary colonization / co-aggregation; and 4) biofilm maturation (Souza et al., 2016; Digel et al., 2020). Gram-positive cocci and bacilli dominate on the direct tooth surface, while Gram-negative bacilli, filamentous bacteria, and spirochetes dominate on the surface of the formed biofilm (Haffajee et al., 2008). In addition to bacteria, fungi are also involved in biofilm formation (Valm, 2019).

[0004] A mature biofilm is a multilayered structure consisting of a heterogeneous population of cells surrounded by an extracellular matrix. Nutrients and bacterial metabolic waste circulate through numerous channels within the matrix. The matrix is ​​composed of extracellular polysaccharides, proteins, and macromolecules such as nucleoproteins and lipids. The matrix protects bacteria from toxins, pH, osmotic pressure changes, ultraviolet radiation, and dehydration (Shah et al., 2013).

[0005] A shift in the balance towards pathogenic microbes is associated with a higher risk of oral diseases such as dental caries and periodontitis. These diseases are among the most common caused by alterations in the composition of the healthy oral microbiome and the introduction of pathogenic microbes from the environment (Tanner et al., 2018; Valm, 2019). Therefore, maintaining a healthy oral microbiome and timely removal of cariogenic biofilms are essential. Gingivitis is another disease that can be caused by dental plaque. In the absence of proper oral hygiene, gingivitis can progress to chronic periodontitis (Kurhan and Kantarci, 2018; Valm, 2019).

[0006] Given the above, timely removal of dental plaque is crucial, as uncontrolled plaque increases the risk of oral diseases. Several types of plaque control and prevention methods are currently available. These methods can be categorized into mechanical and chemical methods (Vyas et al., 2021). Healthcare professionals recommend annual professional cleaning at a dental clinic, specifically mechanical cleaning (Axelsson and Odont, 1981), which offers several types of professional treatments: mechanical cleaning, air polishing, ultrasonic cleaning, or laser cleaning. Between professional cleaning sessions, patients need to take preventative measures to prevent tartar formation and slow plaque buildup. Mechanical methods include the use of toothbrushes, dental floss, and gum protectors (Vyas et al., 2021). Of these methods, toothbrushes are the most commonly used (Mandal et al., 2017). For effective prevention, safe and effective chemical agents are needed as ingredients in preventative oral care products. Background Technology

[0007] Based on various methods, anti-plaque agents can be divided into three generations. First-generation agents, such as antibiotics, phenols, and other compounds, are compounds that reduce plaque levels by 20-50%. Second-generation methods are based on the use of chlorhexidine. Although it is highly effective (70-90%) in removing plaque, this method has many side effects, including: decreased taste, discomfort caused by a buttery taste, burning sensation, dry and tender mucosa, and epithelial desquamation. Third-generation agents block the adhesion of microorganisms to the tooth surface (Vyas et al., 2021).

[0008] Because many anti-plaque agents are ineffective or cause mucosal damage, there is an unmet need to develop new agents that are safe and highly effective in controlling plaque formation in oral soft tissues. This makes it very practical to incorporate compounds such as surfactants, especially those in the group of surfactants that are milder and more environmentally compliant, into oral care product formulations. The authors of this invention have found that compositions containing the biosurfactants rhamnolipid and sodium lauroyl sarcosinate are highly effective in preventing dental plaque formation.

[0009] Surfactants are chemically active compounds used in household chemicals, cosmetics, and oral care products. Surfactants are a key component of toothpaste, responsible for its foaming activity. They contribute to the even distribution of toothpaste in the mouth and aid in the cleaning process, particularly in removing debris, microbial biofilm, and plaque from the tooth surface. Another function of surfactants is to form micelles containing the flavor fillers in toothpaste (Lindenmüller et al., 2011; Aspinall et al., 2021).

[0010] Despite their beneficial properties and important role in toothpaste compositions, many synthetic surfactants can cause irritation and pollute the environment upon entry (Akbari et al., 2018; Naughton et al., 2019; Aspinall et al., 2021). For example, large amounts of surfactant can cause mucosal irritation, while insufficient amounts exhibit significantly lower activity (Aspinall et al., 2021). Surfactants also enter the environment when used in the production of various raw materials in the pharmaceutical, food and feed, agricultural, and petroleum industries.

[0011] Safety concerns related to adverse environmental effects have prompted the development of standards and social pressure to transition to more sustainable production using more biodegradable agents and raw materials (Naughton et al., 2019). This has led to a reduction in the proportion of synthetic surfactants in toothpaste, down to their complete absence. However, surfactant-free toothpaste lacks the beneficial properties inherent in surfactant-based toothpaste. The preferred solution is to produce toothpaste containing biosurfactants or biosynthetic surfactant compositions to minimize harmful effects on both the oral mucosa and the environment.

[0012] Compared to synthetic surfactants, biosurfactants exhibit numerous advantages, such as low toxicity, bioavailability, biodegradability, high foaming activity, environmental friendliness, low cost due to the availability of natural raw materials, and a wide pH and salinity range in which they retain surfactant activity (Fracchia et al., 2015; Akbari et al., 2021). In addition to their use in oral care products, biosurfactants are also used in cosmetic products, pharmaceuticals, the food industry, agriculture, and textile dyeing. These multifunctional agents exhibit stabilizing, antimicrobial, moisturizing, emulsifying, and anti-adhesion activities (Banat et al., 2000; Fracchia et al., 2014).

[0013] Natural surfactants are produced from natural raw materials, some of which are produced using microorganisms. The production of biosurfactants with antibacterial, antifungal, and antiviral properties using bacteria has attracted considerable attention. Pseudomonas genus and Bacillus Bacteria of the genus *Cryptotympany* are the most promising, but given the pathogenicity of these genera, yeasts and non-pathogenic bacteria are preferred (Makkar and Cameotra, 2002; Naughton et al., 2019).

[0014] Surfactants can be further divided into chemically synthesized surfactants and surfactants obtained from natural raw materials, i.e., biosurfactants. The latter are classified based on molecular weight, source, and composition. High molecular weight biosurfactants include lipopolysaccharide-protein complexes, polysaccharide-protein-fatty acid complexes, or lipoprotein complexes, while glycolipids, lipopeptides, and phospholipids are low molecular weight biosurfactants. Low molecular weight biosurfactants are more efficient at reducing surface tension and interfacial tension, while high molecular weight biosurfactants are more potent emulsifiers (Banat et al., 2010).

[0015] Among the biosurfactants studied, glycolipids have attracted considerable attention due to their greatest therapeutic potential. In glycolipid molecules, carbohydrate fragments are linked to fatty acids of varying lengths. This group includes trehalolipids, cellobiolipids, mannosylerythritol lipolipids, rhamnolipids, and sophorolipids (Mnif and Ghribi, 2016; Marchant and Banat, 2012; Adu et al., 2020). Microbially produced glycolipids are exemplified by the following: Pseudomonas The rhamnolipid produced, by Candida The produced sophorolipids and mannosyl erythritol esters and the esters produced by Mycobacterium and Rhodococcus The trehalose produced (Santos et al., 2016; Vecino et al., 2017; Peyrat et al., 2019).

[0016] rhamnolipids are mainly composed of Pseudomonas aeruginosa and Burkholderia Other species in the genus synthesize rhamnolipides. Rhamnolipides are classified based on the number of deoxyrhamnose residues, which are linked to one or two fatty acid chains via O-glycosidic bonds. The length of the fatty acid chains varies from 8 to 14 carbon atoms, with 10-carbon rhamnolipides being the most common. One rhamnose yields a monorhamnolipide, and two rhamnose yields a dirhamnolipide (Abdel-Mawgoud et al., 2010; Elshikh et al., 2017).

[0017] Compared to other biosurfactants, rhamnolipids have the greatest expansion potential in the cosmetics and household chemicals markets (Müller et al., 2012). This can be attributed to the unique properties of rhamnolipids, namely their ability to reduce surface tension, low critical concentration for micelle formation, high detergency and foaming activity, antimicrobial properties, gradual substrate sorption, and excellent ability to form molecular complexes and liquid crystals. Furthermore, rhamnolipids retain their properties over a wide range of temperatures, pH levels, and salinities (Moussa et al., 2014; Sharma, 2016; Kumar and Das, 2018).

[0018] Some synthetic surfactants, though of artificial origin, are non-toxic and therefore can be used in biodegradable compositions. One such example is sodium lauroyl sarcosinate (SLS). This moderately biodegradable anionic surfactant is composed of fatty acids and the amino acid sarcosine (Tackie-Otoo et al., 2022). Studies of N-acylsarcosine derivatives have shown that SLS is not a potentially toxic chemical because its derivatives readily decompose into amino acids and fatty acids (Lanigan, 2001; Tripathy et al., 2018).

[0019] The literature reports the use of rhamnolipids and SLS as individual agents and as components of various compositions in the field of oral care products, particularly for inhibiting microbial growth.

[0020] Elshikh et al. studied non-pathogenic... Burkholderia tsinensis ( Burholderia Thailandensis The isolated rhamnolipids exhibited antimicrobial activity against several microorganisms found in the oral cavity, including... Streptococcus mutans ( Streptococcus mutans ), Oral streptococci ( Streptococcus oralis ), Blood Chain Globule bacteria ( Streptococcus sanguinis ), Neisseria mucosa ( Neisseria mucosa )and Actinomyces nelli ( Actinomyces naeslundii These microorganisms, along with other bacterial species, contribute to the formation of biofilms on tooth surfaces, which can lead to tartar buildup and dental caries (Kolenbrander et al., 2010). The authors found that rhamnolipids (alone and in combination with other antimicrobial agents such as tetracycline, chlorhexidine, sodium lauryl sulfate, and ciprofloxacin) inhibit biofilm formation and also disrupt existing biofilms (in the absence of any other components). The researchers demonstrated that rhamnolipids possess a strong ability to penetrate bacterial cells (Elshikh et al., 2017).

[0021] Yamasaki and others also specifically targeted Actinobacillus contamination Y4 Streptococcus mutans UA159 and Blood streptococcus ATCC10556 confirmed that rhamnolipids inhibit the growth and formation of biofilms in the oral cavity (Yamasaki et al., 2020).

[0022] Mynenivenkatasatya et al. investigated the efficacy of toothpastes containing SLS, as well as stabilized chlorine dioxide and sodium fluoride, particularly their effects on microbial biofilm formation. The authors demonstrated that the toothpastes examined exhibited high antimicrobial activity compared to controls (Mynenivenkatasatya et al., 2020).

[0023] The authors of this invention have demonstrated a unique synergistic effect of rhamnolipids when used in combination with SLS. The composition exhibits high activity against microbial biofilm formation compared to the individual components, namely the biosurfactant and SLS. This unique activity may be associated with the unique properties of each component, which are enhanced by concomitant use. For example, SLS is a “green” synthetic surfactant and an inhibitor of hexokinase, the first enzyme in the metabolic pathways of glucose and several other sugars (Carbon et al., 1995; Fosdick, 1956; Bajani et al., 2018). Therefore, these sugars are metabolized by bacteria, particularly by... Streptococcus mutans The lower degree of metabolism provides additional protection against plaque formation and caries (Forssten et al., 2010). When used with rhamnolipin (which is also a highly effective surfactant and exhibits antimicrobial activity, particularly disrupting the growth of fungi, bacteria, and other microorganisms), the composition provides effective control of plaque (Abalos et al., 2001; Benincasa et al., 2004; Louith and Kanlayavattanakul, 2009). The use of rhamnolipin in cosmetics, particularly in oral care products, has been previously described. However, the combination described herein and its unique properties are substantially innovative.

[0024] Patent application US2019307657A1 (EVONIK DEGUSSA GMBH [DE]), published on October 10, 2019, discloses the use of an oral care composition comprising at least one biosurfactant and at least one fluoride ion source. Patented biosurfactants include rhamnolipids, sophorolipids, lipids containing glucose residues, trehalose lipids, and cellulose lipids. The composition may also contain other non-biological surfactants. The authors of this patent highlight the advantages of the composition, including low levels of insoluble fluoride complexes, reduced tooth staining and mouth odor, better taste in oral care products, better fluoride deposition / retention on surfaces, and reduced plaque. Importantly, while US 2019 / 0307657A1 claims in paragraph

[0018] that "another advantage of the invention is that the composition reduces plaque," it provides no evidence, let alone any examples or data to support that claim. This contrasts sharply with the present invention. The embodiments of this application clearly demonstrate, with conclusive data, the content defined by the independent claims in the appended claims set, namely, the successful treatment of plaque and / or biofilm, or the prevention of plaque and / or biofilm formation. It is important to understand that the compositions of the present invention comprise two basic core components, namely rhamnolipid and sodium lauroyl sarcosinate. These two components can only be combined It only provides a statistically significant synergistic effect of biomembrane inhibition when acting in concert; while individual components do not provide this effect when acting alone. In the case of its counterpart, no biofilm inhibitory effect is provided compared to the control, which can be seen from the present application. Figure 2 show The data clearly showsUS 2019 / 0307657A1 provides no data on biofilm inhibition, whether for rhamnolipin and sodium lauroyl sarcosinate alone or in combination. Furthermore, US 2019 / 0307657A1 does not teach or suggest the importance of combined administration of rhamnolipin and sodium lauroyl sarcosinate for achieving a synergistic inhibitory effect on biofilms. Therefore, any claim that can be readily derived from US 2019 / 0307657A1 in an obvious manner from the invention as defined in the claims necessarily relies on impermissible hindsight reconstruction. The same applies to another prior art, EP 3 875 1001 A1, which relates to a composition comprising rhamnolipin and sodium lauroyl sarcosinate, used as a pharmaceutical or cosmetic for the regeneration of skin, mucous membranes, and skin appendages, wound treatment, or as a cosmetic ingredient for the protection, maintenance, and natural balance of said mucous membranes and skin appendages, but has no relevance to the subject matter of this application as defined in the claims, namely, using a combination of rhamnolipin and sodium lauroyl sarcosinate to achieve biofilm inhibition. Patent application WO2019133313A1 (LOCUS IP CO LLC [US]), published July 4, 2010, discloses the use of a composition for improving and / or maintaining oral health. This composition contains one or more purified biosurfactants and / or their derivatives, and optionally may contain one or more carriers, additives, and / or adjuvants. The claimed biosurfactants include glycolipids, such as rhamnolipin, sophorolipid, trehalolipid, and mannosyl erythritol lipolipide. Although the applicant states that the composition is intended for use in products for teeth whitening, removal and / or prevention of plaque, biofilm or tartar, and reduction of unfavorable microbial communities in the oral cavity, none of the claimed uses are supported by technical evidence. Instead, the authors provide the following examples: 1) Utilizing Bacillus 1) Producing lipopeptides; 2) Utilizing bumblebee candidiasis 3) Fermentation produces biosurfactants; 4) Variants of toothpaste compositions.

[0025] Patent application US2021169757A1 (COLGATE PALMOLIVE CO [US]) discloses a composition for maintaining viscosity in oral care products and a method of application thereof. The composition comprises: 1) a basic amino acid; 2) a zinc ion source; and 3) a surfactant system containing one or more alkyl glycosides, acylglutamate esters, glycolipids, or combinations thereof. According to the authors, the composition possesses properties including reducing acid-producing bacterial communities, reducing the formation of oral microbial biofilms, and further reducing or inhibiting plaque formation. Despite these claimed properties, the applicant has only demonstrated the retention of toothpaste viscosity, flavor, and foaming activity. Furthermore, for toothpastes containing rhamnolipids, only the first two properties have been demonstrated.

[0026] Patent US7985722B2 (AURORA ADVANCED BEAUTY LABS [US]), published on July 26, 2011, discloses the use of rhamnolipin-based compositions for cleaning, disinfecting, and deodorizing living areas and workspaces. The compositions of this invention comprise 0.01% to 99.9% (preferably 0.01% to 70%) rhamnolipin, with the remainder being a carrier. The authors report that such compositions also form biofilms that prevent the proliferation of bacteria and fungi. The patent text, along with numerous other optional uses, indicates the use of the composition as a toothpaste ingredient. However, in embodiments related to oral care products, the authors only describe the use of the composition as an antimicrobial and antifungal coating on toothbrushes, where the antimicrobial properties and surface tension of the rhamnolipin are maintained for one week. Nevertheless, its activity has not been verified because no experiments have experimentally confirmed the rhamnolipin properties claimed by other oral care products.

[0027] Lauroyl sarcosine (LS) has been patented as an ingredient in many compositions, but these compositions do not contain rhamnolipids. For example, U.S. Patent US2022023180A1 (COLGATE PALMOLIVE CO[US]), published January 27, 2022, discloses compositions containing lauroyl sarcosine and betaine for use in oral care products. The authors of this patent describe several potential compositions and their foaming activities; however, their efficacy against microbial biofilms has not been demonstrated.

[0028] Therefore, the authors developed an innovative composition containing rhamnolipin and LS. Compared to the individual components, this composition unexpectedly exhibited a high synergistic effect in reducing plaque weight and can be used as an ingredient in cosmetics for highly effective cleaning of teeth and the oral cavity. Summary of the Invention

[0029] The present invention is set forth in the appended claims. The present invention relates to a composition comprising two components (A) and (B): (A) rhamnolipid; and (B) sodium lauroyl sarcosinate.

[0030] The rhamnolipid can be derived from Burkholderia Produced by bacteria, preferably by Pseudomonas Produced, more preferably by Pseudomonas aeruginosa The rhamnolipid produced, or even more preferably, is identified by CAS number 4348-76-9.

[0031] The level of component (A) in the composition may be selected from the following weight % range: 0.004-5.000; 0.006-4.000; 0.008-3.000; 0.010-2.000; 0.011-1.900; 0.012-1.800; 0.013-1.700; 0.014-1.600; 0.015-1.500; 0.015-1.400; 0.015-1.300; 0.015-1.200; 0.015-1.100; or 0.015-1.000. For example, the level of component (A) in the composition is selected from the following weight %: 0.004; 0.005; 0.006; 0.007; 0.008; 0.009; 0.01; 0.015; 0.02; 0.025; 0.03; 0.035; 0.04; 0.045; 0.05; 0.055; 0.06; 0.065; 0.07; 0.075; 0.08; 0.085; 0.0 9; 0.095; 0.1; 0.15; 0.2; 0.25; 0.3; 0.35; 0.4; 0.45; 0.5; 0.55; 0.6; 0.65; 0.7; 0.75; 0.8; 0.85; 0.9; 0.95; 1; 1.05; 1.1; 1.15; 1.2; 1.25; 1.3; 1.35; 1.4; 1.45; 1.5; 1.55; 1.6; 1.65; 1. 7; 1.75; 1.8; 1.85; 1.9; 1.95; 2.00; 2.05; 2.1; 2.15; 2.2; 2.25; 2.3; 2.35; 2.4; 2.45; 2.5; 2.55; 2.6; 2.65; 2.7; 2.75; 2.8; 2.85; 2.9; 2.95; 3.00; 3.05; 3.1; 3.15; 3.2; 3.25; 3.3; 3. 5; 3.4; 3.45; 3.5; 3.55; 3.6; 3.65; 3.7; 3.75; 3.8; 3.85; 3.9; 3.95; 4.00; 4.05; 4.1; 4.15; 4.2; 4.25; 4.3; 4.35; 4.4; 4.45; 4.5; 4.55; 4.6; 4.65; 4.7; 4.75; 4.8; 4.85; 4.9; 4.95; or 5.00. Furthermore, the level of component (A) in the composition may be selected from the following weight % range: 0.06-0.20; 0.07-0.19; 0.08-0.18; 0.09-0.17; 0.1-0.16; 0.11-0.15; 0.12-0.14; or 0.13-0.14.For example, the level of component (A) in the composition is selected from the following weight %: 0.06; 0.07; 0.08; 0.09; 0.1; 0.11; 0.12; 0.13; 0.14; 0.15; 0.16; 0.17; 0.18; 0.19; or 0.2.

[0032] The level of component (B) in the composition may be selected from the following weight % ranges: 0.05-22.00; 0.10-11.00; or 0.20-5.50. For example, the level of component (B) in the composition may be selected from the following weight % ranges: 0.05; 0.06; 0.07; 0.08; 0.09; 0.1; 0.15; 0.2; 0.25; 0.3; 0.35; 0.4; 0.45; 0.5; 0.55; 0.6; 0.65; 0.7; 0.75; 0.8; 0.85; 0.9; 0.95; 1; 1.5; 2; 2.5 ; 3; 3.5; 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5; 9; 9.5; 10; 10.5; 11; 11.5; 12; 12.5; 13; 13.5; 14; 14.5; 15; 15.5; 16; 16.5; 17; 17.5; 18; 18.5; 19; 19.5; 20; 20.5; 21; 21.5; or 22. Furthermore, the level of component (B) in the composition may be selected from the following weight % range: 1.0-2.0; 1.1-1.9; 1.2-1.8; 1.3-1.7; 1.4-1.6; or 1.5-1.6. For example, the level of component (B) in the composition is selected from the following weight %: 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; or 2.0.

[0033] The weight ratio of sodium lauroyl sarcosinate and rhamnolipid in the combination can be as follows: from 0.07:2.0 to 0.004:0.2, respectively. In other words, within the range, the weight content can be as follows: 0.004; 0.005; 0.006; 0.007; 0.008; 0.009; 0.01; 0.015; 0.02; 0.025; 0.03; 0.035; 0.04; 0.045; 0.05; 0.055; 0.06; 0.065; 0.07 and 0.2; 0.25; 0.3; 0.35; 0.4; 0.45; 0.5; 0.55; 0.6; 0.65; 0.7; 0.75; 0.8; 0.85; 0.9; 0.95; 1; 1.5; 2.

[0034] The composition may be an oral care composition.

[0035] The oral care composition can be selected from mouthwash and toothpaste.

[0036] The composition may be an oral care composition, wherein the oral care composition is preferably a formulation selected from the following: film, aerosol, suspension, solution, tincture, cream, lotion, ointment, gel, powder or granules.

[0037] The composition may be used for non-medical and / or cosmetic purposes.

[0038] In another aspect, the present invention relates to the use of the compositions of the invention for the prevention or treatment of dental plaque and / or biofilm formation in the mouth and / or on the teeth.

[0039] The dental plaque and / or biofilm may originate from the oral flora in the mouth, wherein the oral flora may be a microbial-based oral flora, and more preferably, the microorganisms may be selected from... Streptococcus mutans , Oral streptococci , Blood Chain Globule bacteria , Neisseria mucosa and Actinomyces nelli .

[0040] In another aspect, the present invention relates to a pharmaceutical composition or medicine comprising the composition of the present invention.

[0041] In another aspect, the present invention relates to compositions of the invention intended for use as pharmaceutical compositions or medicines, wherein the medicine is preferably formulated for use in the oral cavity of a subject.

[0042] In another aspect, the medicine or the composition of the present invention can be used to treat a subject suffering from at least one of the following diseases or symptoms: gingivitis, stomatitis, halitosis, sensitive teeth, dental caries, or tartar. Example

[0043] Example 1. The inhibitory effects of aqueous solutions of sodium lauroyl sarcosinate, the biosurfactant rhamnolipin, and mixtures thereof at different concentrations on biofilm formation on a rough glass rod surface were tested. The test samples of the raw materials contained 35% sodium lauroyl sarcosinate. The test samples of the raw materials contained 40% rhamnolipin.

[0044] Sterile water was used as a negative control, and Corsodyl mouthwash containing 0.2% chlorhexidine was used as a positive control. The mass of the dried biofilm was weighed to determine the amount of plaque formed.

[0045] Dental plaque was induced to grow for 3 days by immersing a rough glass rod in fresh human saliva containing 0.1% sucrose. On days 2 and 3, biofilm growth was further stimulated by adding a nutrient broth containing tryptone soy broth (TSB), saliva, and sucrose. On day 1, the glass rod was treated once with the test component solution, and on days 2 and 3, it was treated twice. At the end of day 3, the biofilm was collected from the glass rod, and the plaque weight (g) was determined on a dry weight basis. A lighter weight indicated higher plaque resistance.

[0046] Experimental procedure: Secure the sterilized rod to the support frame inside the microbiology cabinet. From this moment on, the rod and support frame are used as a single unit.

[0047] On day 1, a sterile glass rod was pretreated with 5 mL of the test component solution for 2 minutes. The glass rod was then removed, washed with 5 mL of sterile distilled water, and immersed in 5 mL of saliva containing 0.1% sucrose. The test tube was placed in an incubator rack and then in an incubator (t=37℃) and shaker (250 rpm) for approximately 18 hours.

[0048] On the mornings of days 2 and 3, the test component solution was applied to the test component solution, then the sample was placed in nutrient broth and returned to the incubator and shaker. After incubation in the broth for 6 hours, the test component solution was applied to the test component solution again and the sample was transferred to saliva containing 0.1% glucose, then returned to the incubator for 18 hours.

[0049] On day 4, the test tube containing the rod was removed from the incubator for determination of the dry weight of the biofilm. To do this, the rod was dried and then weighed on an analytical balance accurate to four decimal places. After rehydration, the biofilm was removed, and the rod was weighed again. The dry weight of the biofilm was calculated using the following formula: Biofilm weight (g) = Weight of rods covered with biofilm (g) - Weight of rods (g) Experimental results: Table 1 and Figure 1 The results of the anti-biofilm activity obtained for the tested components are presented in the paper, in which... Figure 2 The core of this invention is summarized as follows: the claimed composition comprising a combination of rhamnolipid and sodium lauroyl sarcosinate has a statistically significant synergistic inhibitory effect compared with the control and the respective components at control levels.

[0050] Table 1. Dry weight (g) of biofilm Components Average biofilm weight (g) Standard deviation Biosurfactant rhamnolipid 0.01% 0.0026 0.0006 Sodium lauroyl sarcosinate 0.2% 0.0022 0.0006 Sodium lauroyl sarcosinate 0.2% + biosurfactant rhamnolipid 0.01% 0.0014 0.0002 Sodium lauroyl sarcosinate 2% 0.0020 0.0004 distilled water 0.0027 0.0005 Corsodyl (0.2% chlorhexidine) 0.0005 0.0002 As is evident from Table 1, the combination of 0.2% sodium lauroyl sarcosinate and 0.01% rhamnolipin provides a significant reduction in plaque formation. The plaque weights were almost identical when the rods were treated with 0.2% and 2% sodium lauroyl sarcosinate aqueous solutions, indicating no significant dose-dependent effect: increasing the concentration of this component tenfold did not lead to better efficacy in reducing plaque formation. However, adding only 0.01% rhamnolipin solution to a 0.2% sodium lauroyl sarcosinate solution resulted in a significantly lower biofilm weight, thus confirming a clear synergistic effect of the components at the tested concentrations.

[0051] References 1. Abalos A. et al. Physicochemical and antimicrobial properties of new rhamnolipids produced by Pseudomonas aeruginosa AT10 from soybean oilrefinery wastes / / Langmuir. – 2001. – Т. 17. – №. 5. – С. 1367-1371. 2. Abdel-Mawgoud AM, Lépine F., Déziel E. Rhamnolipids: diversity of structures, microbial origins and roles / / Applied microbiology and biotechnology. – 2010. – Т. 86. – №. 5. – С. 1323-1336. 3. Adu SA et al. Microbial biosurfactants in cosmetic and personalskincare pharmaceutical formulations / / Pharmaceutics. – 2020. – Т. 12. – №.11. – С. 1099. 4. Akbari S. et al. Biosurfactants—a new frontier for social andenvironmental safety: a mini review / / Biotechnology Research and Innovation.– 2018. – Т. 2. – №. 1. – С. 81-90. 5. Aspinall S. R., Parker J. K., Khutoryanskiy V. V. Oral careproduct formulations, properties and challenges / / Colloids and Surfaces B:Biointerfaces. – 2021. – Т. 200. – С. 111567. 6. Axelsson P., Odont D. Concept and practice of plaque-control / / Pediatr Dent. – 1981. – Т.3. – №. Sp. Issue. – С. 101-113. 7. Bajani D., Gharai D., Dey J. A comparison of the self-assemblybehaviour of sodium N- lauroyl sarcosinate and sodium N-lauroyl glycinatesurfactants in aqueous and aqueo-organic media / / Journal of colloid andinterface science. – 2018. – Т. 529. – С. 314-324. 8. Banat I. M., Makkar R. S., Cameotra S. S. Potential commercialapplications of microbial surfactants / / Applied microbiology andbiotechnology. – 2000. – Т. 53. – №. 5. – С. 495-508. 9. Benincasa M. et al. Chemical structure, surface properties andbiological activities of the biosurfactant produced by Pseudomonas aeruginosaLBI from soapstock / / Antonie Van Leeuwenhoek. – 2004. – Т. 85. – №. 1. –С. 1-8. 10. Carbon J. A. et al. The inhibition of anaerobic glycolysis bysodium N-lauroyl sarcosinate / / Archives of Biochemistry and Biophysics. –1955. – Т. 55. – №. 2. – С. 356-364. 11. Elshikh M. et al. Rhamnolipids and lactonic sophorolipids:natural antimicrobial surfactants for oral hygiene / / Journal of appliedmicrobiology. – 2017. – Т. 123. – №. 5. – С. 1111- 1123. 12. Forssten S. D., Björklund M., Ouwehand A. C. Streptococcusmutans, caries and simulation models / / Nutrients. – 2010. – Т. 2. – №. 3. –С. 290-298. 13. Fosdick L. S. Clinical experiment on the use of sodium N-lauroylsarcosinate in the control of dental caries / / Science. – 1956. – Т. 123. –№. 3205. – С. 988-989. 14. Fracchia L. et al. Industrial applications of biosurfactants / / Biosurfactants: production and utilization—processes, technologies, andeconomics. – 2014. – С. 245-260. 15. Fracchia L. et al. Potential therapeutic applications ofmicrobial surface-active compounds / / AIMS Bioengineering. – 2015. – Т. 2. –№. 3. – С. 144-162. 16. Kolenbrander P. E. et al. Oral multispecies biofilm developmentand the key role of cell– cell distance / / Nature Reviews Microbiology. –2010. – Т. 8. – №. 7. – С. 471-480. 17. Kumar R., Das A. J. Future Prospects and Scenario of Rhamnolipids / / Rhamnolipid Biosurfactant. – Springer, Singapore, 2018. – С. 137-141. 18. Kumar R., Das A. J. Industrial Applications of Rhamnolipid: AnInnovative Green Technology for Industry / / Rhamnolipid Biosurfactant. –Springer, Singapore, 2018. – С. 65-77. 19. Lanigan R. S. Final report on the safety assessment of CocoylSarcosine, Lauroyl Sarcosine, Myristoyl Sarcosine, Oleoyl Sarcosine, StearoylSarcosine, Sodium Cocoyl Sarcosinate, Sodium Lauroyl Sarcosinate, SodiumMyristoyl Sarcosinate, Ammonium Cocoyl Sarcosinate, and Ammonium LauroylSarcosinate / / International journal of toxicology. – 2001. – Т. 20. – С. 1-14. 20. Lindenmüller I. H., Lambrecht J. T. Oral care / / Topicalapplications and the mucosa. – 2011. – Т. 40. – С. 107-115. 21. Lourith N., Kanlayavattanakul M. Natural surfactants used incosmetics: glycolipids / / International journal of cosmetic science. – 2009. –Т. 31. – №. 4. – С. 255-261. 22. Makkar R., Cameotra S. An update on the use of unconventionalsubstrates for biosurfactant production and their new applications / / Appliedmicrobiology and biotechnology. – 2002. – Т. 58. – №. 4. – С. 428-434. 23. Mandal A. et al. New dimensions in mechanical plaque control: Anoverview / / Indian Journal of Dental Sciences. – 2017. – Т. 9. – №. 2. – С.133. 24. Marchant R., Banat I. M. Biosurfactants: a sustainablereplacement for chemical surfactants / / Biotechnology letters. – 2012. – Т.34. – №. 9. – С. 1597-1605. 25. Mnif I., Ghribi D. Glycolipid biosurfactants: main properties andpotential applications in agriculture and food industry / / Journal of theScience of Food and Agriculture. – 2016. – Т. 96. –№. 13. – С. 4310-4320. 26. Moussa T. A. A., Mohamed M. S., Samak N. Production andcharacterization of di- rhamnolipid produced by Pseudomonas aeruginosa TMN / / Brazilian Journal of Chemical Engineering. – 2014. – Т. 31. – С. 867-880. 27. Müller M. M. et al. Rhamnolipids—next generation surfactants / / Journal of biotechnology. – 2012. – Т. 162. – №. 4. – С. 366-380. 28. Mynenivenkatasatya S. R. et al. Effectiveness of a noveldentifrice containing stabilized chlorine dioxide, sarkosyl, and sodiumfluoride / / Dentistry journal. – 2020. – Т. 8. – №. 4. – С. 122. 29. Naughton P. J. et al. Microbial biosurfactants: current trendsand applications in agricultural and biomedical industries / / Journal ofapplied microbiology. – 2019. – Т. 127. – №.– С. 12-28. 30. Peyrat L. A. et al. Terrestrial microorganisms: cell factories ofbioactive molecules with skin protecting applications / / Molecules. – 2019. –Т. 24. – №. 9. – С. 1836. 31. Santos D. K. F. et al. Biosurfactants: multifunctionalbiomolecules of the 21st century / / International journal of molecularsciences. – 2016. – Т. 17. – №. 3. – С. 401. 32. Sharma D. Biosurfactants in food. – Cham, Switzerland : SpringerInternational Publishing, 2016. 33. Tackie-Otoo B. N. et al. Experimental investigation of N-lauroylsarcosine and N-lauroyl- L-glutamic acid as green surfactants for enhancedoil recovery application / / Journal of Molecular Liquids. – 2022. – Т. 362. –С. 119738. 34. Tripathy D. B. et al. Synthesis, chemistry, physicochemicalproperties and industrial applications of amino acid surfactants: A review / / Comptes Rendus Chimie. – 2018. – Т. 21. – №.– С. 112-130. 35. Vecino X. et al. Biosurfactants in cosmetic formulations: trendsand challenges / / Critical reviews in biotechnology. – 2017. – Т. 37. – №.7. – С. 911-923. 36. Vyas T. et al. Chemical plaque control-A brief review / / Journalof Family Medicine and Primary Care. – 2021. – Т. 10. – №. 4. – С. 1562. 37. Yamasaki R. et al. Rhamnolipids and surfactin inhibit the growthor formation of oral bacterial biofilm / / BMC microbiology. – 2020. – Т. 20.– №. 1. – С. 1-11。

Claims

1. A composition comprising at least two components (A) and (B): (A) Rhamnose lipids; and (B) Sodium lauroyl sarcosinate, It is used to treat plaque and / or biofilm in the mouth and / or on teeth, or to prevent the formation of plaque and / or biofilm in the mouth and / or on teeth.

2. The composition for use according to claim 1, wherein the rhamnolipid is derived from bacteria of the genus Burkholderia, preferably from the genus Pseudomonas, more preferably from Pseudomonas aeruginosa, and even more preferably from rhamnolipid having CAS number 4348-76-9.

3. The composition for use according to any one of the preceding claims, wherein the composition comprises component (A), and the component (A) in the composition comprises, in the following weight percentage range: i) 0.004 – 5.000; 0.006 – 4.000; 0.008 – 3.000; 0.010 – 2.000; 0.011 – 1.900; 0.012 – 1.800; 0.013 – 1.700; 0.014 – 1.600; 0.015 – 1.500; 0.015 – 1.400; 0.015 – 1.300; 0.015 – 1.200; 0.015 – 1.100; 0.015 – 1.000; or ii) 0.06-0.20; 0.07-0.19; 0.08-0.18; 0.09-0.17; 0.1-0.16; 0.11-0.15; 0.12-0.14; 0.13-0.

14.

4. The composition for use according to any one of the preceding claims, wherein the composition comprises component (B), and the component (B) in the composition comprises, in the following weight percentage range: i) 0.05 – 22.00; 0.10 – 11.00; 0.20 – 5.50; or ii) 1.0-2.0; 1.1-1.9; 1.2-1.8; 1.3-1.7; 1.4-1.6; or 1.5-1.

6.

5. The composition for use according to claim 1 or claim 2, wherein the weight ratio of sodium lauroyl sarcosinate to rhamnolipid in the composition is from 0.07:2.0 to 0.004:0.

2.

6. The composition for use according to any one of the preceding claims, wherein the composition is an oral care composition, and wherein the oral care composition is preferably selected from mouthwash and toothpaste.

7. The composition for use according to claim 6, wherein the oral care composition is a formulation selected from: films, aerosols, suspensions, solutions, tinctures, creams, pastes, lotions, ointments, gels, powders, and granules.

8. The composition for use according to any one of the preceding claims, wherein the composition is a pharmaceutical product, and wherein the pharmaceutical product is preferably formulated for oral administration to a subject.

9. The composition for use according to claim 8, wherein the composition or pharmaceutical product is further used for... - Prevention or treatment of oral diseases, wherein the oral diseases are preferably selected from dental caries, periodontitis, gingivitis, tonsillitis, stomatitis, halitosis, tartar, or - To treat pulp infection of the dental pulp, root or nerve of a subject's tooth, wherein the tooth is preferably a living natural tooth.

10. The composition for use according to any one of the preceding claims, wherein the plaque and / or biofilm is derived from oral microorganisms in the mouth, wherein the oral microorganisms are preferably selected from... Streptococcus mutans , Oral streptococci , Blood Chain Globule bacteria , Neisseria mucosa and Actinomyces nelli .

11. The composition for use according to any one of the preceding claims, wherein the plaque and / or biofilm is on a solid surface of the tooth, preferably supragingival plaque or subgingival plaque.

12. A composition comprising at least two of the following components (A) and (B): (A) Rhamnose lipids; and (B) Sodium lauroyl sarcosinate, Use in treating plaque and / or biofilm on an intraoral appliance or prosthesis of a subject or in preventing the formation of plaque and / or biofilm on an intraoral appliance or prosthesis of a subject, said intraoral appliance or prosthesis including dentures, partial dentures, palatal fillings, dental implants, crowns, and bridges, said treatment preferably performed ex vivo or outside the subject.

13. The use according to claim 12, wherein the rhamnolipid is derived from bacteria of the genus Burkholderia, preferably from the genus Pseudomonas, more preferably from Pseudomonas aeruginosa, and even more preferably from rhamnolipids having CAS number 4348-76-9.

14. The use according to claim 12 or claim 13, wherein the composition comprises component (A), and the component (A) in the composition comprises, in the following weight percentage range: i) 0.004 – 5.000; 0.006 – 4.000; 0.008 – 3.000; 0.010 – 2.000; 0.011 – 1.900; 0.012 – 1.800; 0.013 – 1.700; 0.014 – 1.600; 0.015 – 1.500; 0.015 – 1.400; 0.015 – 1.300; 0.015 – 1.200; 0.015 – 1.100; 0.015 – 1.000; or ii) 0.06-0.20; 0.07-0.19; 0.08-0.18; 0.09-0.17; 0.1-0.16; 0.11-0.15; 0.12-0.14; 0.13-0.

14.

15. The use according to any one of claims 12-14, wherein the composition comprises component (B), and the component (B) in the composition comprises, in the following weight percentage range: i) 0.05 – 22.00; 0.10 – 11.00; 0.20 – 5.50; or ii) 1.0-2.0; 1.1-1.9; 1.2-1.8; 1.3-1.7; 1.4-1.6; or 1.5-1.6.

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