SANITIZING DISINFECTANT COMPOSITION

Description

SANITIZING DISINFECTANT COMPOSITION 1. State of the art In recent years, dental caries and biofilm formation have become among the most widespread infections and Improper management of this plant leads to the development of serious diseases and the creation of biofilm. Oral hygiene is an important measure for the prevention of oral diseases (dental caries and periodontitis) and prophylactic treatment for aspiration pneumonia, type 2 diabetes mellitus and cardiovascular diseases. Although tooth decay is a global health problem, it is preventable with appropriate interventions, especially in the early stages of life. However, some groups cannot easily perform regular oral hygiene care (tooth brushing or use of dental floss), including seriously ill patients, the elderly and those displaced by disasters. Furthermore, many individuals lack the knowledge and motivation to take proper oral hygiene care. Failure to maintain oral hygiene leads to the formation of microbial biofilms, called plaque. Dental, on the surface of the teeth. The formation of biofilm on the tooth surfaces by communities cariogenic bacteria is the initial step in the development of dental caries. Streptococcus mutans is a primary etiological agent of dental caries. The main virulence traits associated with S. mutans cariogenicity is due to the production of acid from fermentable carbohydrates in the diet, from acid tolerance and the formation of exopolysaccharides (EPS). Acid production promotes demineralization of tooth enamel and acid tolerance confers the survival in a low pH environment within dental plaques. EPS promotes formation of acidogenic biofilms on the tooth surface, which are acid-resistant bioaggregates mechanical toothbrushing. Although fluoride-based preparations protect the surfaces teeth from acid attacks, their effects are limited if not combined with control of the Dental plaque. The use of bactericidal compounds to eradicate cariogenic bacteria is controversial. because these compounds disturb healthy oral microflora and can lead to the growth of bacteria multidrug-resistant. Oral compositions currently used are known and in many different forms. They are often used for dental care or for the prophylaxis of dental diseases and appear in any physical form, e.g. liquid, solid, gaseous, aerosol, spray, nebulizer and combinations thereof suitable for create pastes, gels, toothpastes, mouthwashes, devices dedicated to their release and more. Just for example, known oral compositions are usually a mixture of alcohol and / or water and usually contain other ingredients. Alcohol, such as ethanol or propanediol, can be used for disinfection and / or preservation. Furthermore, many substances frequently used in oral compositions dissolve much better in a mixture of alcohol and water than in pure water, so Alcohol can also serve as a solubilizer. For example, known oral compositions are added antiseptic active ingredients such as chlorhexidine, some of which dissolve much better in alcohol-water mixtures than in pure water. In addition, some known oral compositions contain fluorides, which have the purpose of hardening the enamel. In terms of composition and mode of action, these known oral compositions are usually oriented primarily to a curative approach. Although a disinfectant effect is also expected and desired, that is, an overall reduction in the load of germs or bacteria, it plays only a subordinate role. However, numerous pathologies of the oral cavity, especially of an inflammatory nature, ranging from from simple gingivitis to periodontitis and peri-implantitis up to childhood and adult caries They also require a disinfection approach. In fact, periplantitis is the result of the action of pathogenic noxa in the anomalous presence of some bacterial species and their metabolic products (biofilms) in which a germicidal or disinfectant effect is desirable. Antibiotics are considered one of the most important weapons in the fight against these infections. bacterial. Since their introduction, they have significantly improved the quality of human life. in terms of health. However, these health benefits have been threatened in recent decades, because many commonly used antibiotics have become less effective against some diseases, no not only because many of them cause toxic reactions, but also because of the emergence of resistant bacteria to drugs. The development of resistance is a serious problem today, as bacterial resistance often does not limited to the specific antibiotic prescribed to the patient, but can generally be extended to other compounds of the same class. Bacterial resistance and its rapid increase are a source of great global public health concern and are emerging as one of the major challenges for human health. Treatment of bacterial infections with antibiotics is therefore useful, but their widespread use has led to an alarming resistance of microorganisms and, ultimately, to the recurrence of old infectious diseases. Therefore, an antibiotic-based approach to decimate the bacterial load in human oral cavity is not desirable, especially for chronic or persistent disease models. Added to this is an unexpected event involving the chlorhexidine molecule, considered the golden standards for oral antibacterials. In fact, a study published in Antimicrobial Agents and Chemotherapy highlights that exposure at increasing concentrations of chlorhexidine, a widely used component in disinfectants and antiseptics for domestic but also hospital use, determines the appearance of strains of Klebsiella pneumoniae resistant even to colistin, considered the latest frontier in antibiotic treatment in multidrug resistance forms. A life full of successes for chlorhexidine, discovered by chance in Manchester in the laboratories of ICI, where new antimalarials were being researched. After a slow start spent in operating rooms as a disinfectant (Hibitane and Hibiscrub), in the late 1970s publications on its use in There were only a few dozen dentists. After that, the boom: mouthwash, gel, toothpaste; it seemed like a success. destined to never end, denying any space to aspiring competitors (who still remembers (Sanguinaria canadensis?). The eternal fight against oral pathogens seemed to be coming to an end. In reality, Harald Löe himself, one of the pioneers of the oral use of chlorhexidine, was the first to report the risk of resistance already in 1972. Now the risk is a certainty. Unlike antibiotics, where the attention is almost hysterical given the level of alert reached, the The problem of resistance to antiseptics is much less known (and feared), especially towards chlorhexidine while for others, such as triclosan, both in the USA and in Europe the authorities are already intervened by prohibiting its use in personal hygiene products (see box on this page). For a couple of years now, however, chlorhexidine has been under special surveillance, being suspected of inducing cross-resistance against colistin, a valuable antibiotic used against the most resistant bacteria. In the near future, it is likely that the dosage and indications for chlorhexidine will be restricted, similarly to antibiotics. It will perhaps disappear from toothpastes and soaps, while gels and mouthwashes will be used in a more targeted manner and for limited periods in patients in whom there is a demonstrated benefit. The same problem of bacterial resistance also arises for other oral antiseptics such as cetylpyridinium chloride, to which the appearance of resistant strains has been demonstrated in vitro proportional to the exposure. Shortly after the withdrawal of triclosan and 17 other compounds from personal hygiene products imposed by the FDA, an interesting article was published in the periodical of the American Society for Microbiology in which the authors exposed the heavy environmental alterations caused by the non-use motivated by antiseptics. According to the reported data, triclosan is the second most common chemical compound found in plants. urban sewage treatment plants, placing it ahead of pharmaceuticals and surfactants. In first place? Triclocarban, another antiseptic banned by the FDA. Considering that the solid residues of the purification are used in agriculture as fertilizers, some more scruples certainly cannot be be defined as alarmist and should make everyone think before purchasing a product for personal use. household advertised as “sanitizer”. The first known use of chlorhexidine in dentistry dates back to 1959, when it was used as an endodontic disinfectant; later, its ability to inhibit tooth decay was discovered animals and in 1970 the famous research by Harald Löe on dental students was published the toothbrush had been removed. In the group that, fortunately for them, used 0.2% chlorhexidine mouthwash as only form of oral hygiene, the teeth remained free of plaque, unlike the control group. In 1972 the same authors published the first reports of strains of S. sanguinis showing a reduced sensitivity after prolonged use of chlorhexidine mouthwash, but this data was not considered clinically important for many years. In 1980, it was discovered that resistance was a genetically transmitted characteristic. A few years ago some authors found in plaque dental strains with reduced sensitivity to chlorhexidine and resistant to many antibiotics, warning on the prolonged use of oral hygiene products containing this disinfectant. 2. Summary of the invention The invention is therefore based on the task of specifying an oral composition which, as far as intended use in the human mouth and oral cavity, has a germicidal effect with a high degree of tolerance for humans or human tissues. Furthermore, the environmental impact should be zero. As far as oral composition is concerned, this task is solved according to the invention as it is made up of a composition which presents as ingredients, in addition to various excipients aimed at achieving the desired composition variables, a sugar (D-Tagatose) and a trace element (Copper) of use consolidated as a food additive recognized and authorized worldwide thanks to a unique safety profile and effective biofilm activity. Furthermore, to date, there are no reports of the emergence of strains resistant to D-Tagatose or Copper, unlike chlorhexidine. The invention is based on the idea that the desired germicidal effect of the oral composition can be achieved by specifically providing an alternative agent for the treatment of infectious diseases, avoiding the use of antibiotics and the associated risk of developing resistance. This latter approach, i.e. combined or synergistic therapy against resistant microorganisms, can lead to new ways of treating infectious diseases and will likely be a potential area for further investigations in the future. The mode of action of combination therapy differs significantly from that of the drugs themselves which act individually; therefore, selecting an appropriate combination is essential and important and requires understanding the possible interaction and synergistic effects. The advantages of this approach are: greater efficacy, reduction of side effects, greater stability or bioavailability of free active ingredients, achievement of an adequate therapeutic effect relatively low doses compared to a synthetic drug. Considering that the oral mucosa has its own absorption (see tablets sublingual anti-inflammatories, trinitrin and others), it is not advisable to introduce substances potentially toxic in the oral cavity as has happened up to now, favoring instead the use of food additives, practically harmless to humans and of which D-Tagatose and Copper are part. Quite surprisingly, it was found that D-Tagatose and Copper, preferably in its form colloidal, especially if appropriately combined with each other, not only enhance the effect of the individual elements, but also determine a notable stability of the product after packaging. In a particularly advantageous and independently inventive project, the use of the The above-described oral composition is intended to reduce the germ load in the mouth or pharynx human and / or for dental care or devices used in the oral cavity and surrounding anatomical tissues. The advantages obtained with the invention consist in particular in the fact that the envisaged combination of D-Tagatose and a trace element, in particular copper, giving up antibiotics and the formation of resistances that can be triggered, allows to obtain particularly well tolerated reductions and equally very effective of the germ or bacterial load in the oral cavity. Surprisingly, the composition shows synergistic effects, so as to allow a particularly effective reduction of the bacterial load and the related treatment of inflammation resulting from infections. 3. Summary For intended use in the human mouth and pharynx, an oral composition must have a significantly greater germicidal effect than known oral compositions, with a high degree of tolerance for humans or human tissues and with the exclusion of the possible formation of resistances. To this end, according to the invention, an oral composition is provided, which comprises as components a sugar and a trace element Preferably, as an antimicrobial agent, active on the biofilm, a combination of copper and D-Tagatose is provided 4. Summary of the invention The development of specific behavioral measures for cariogenic bacteria should reduce the dental plaque formation. Several studies have been conducted to determine whether the growth and S. mutans adhesion is inhibited by various natural products, such as green tea catechins, cranberry constituents, citrus lemon oil, and mushroom extract. However, these studies do not evaluated biofilm formation. Sugar alcohols (polyols) are alternative candidates. used for the prevention of tooth decay. Rare sugars are generally monosaccharides and their derivatives are rarely found in nature. Recently, rare sugars have been at the center of attention as sugar substitutes for health, thanks to their equivalent sweetness but at much lower calorie content than sucrose. These sugars are supposed to reduce calorie intake, thus reducing the risk of type 2 diabetes mellitus and obesity. The ketohexose D-tagatose has 92% of the sweetness but 38% of the calories of sucrose. D-tagatose It is not a preferential substrate for bacterial fermentation and D-tagatose has been reported It is not readily catabolized by many lactic acid bacteria or pathogens such as Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Bacillus cereus and Yersinia enterocolitica. D-Tagatose has been shown to suppress the growth of aerobic bacteria. attics in minced ham, thus extending the shelf life of these products by 7-10 days. These results indicate that foods containing D-tagatose can suppress bacteria oral cariogenic. In fact, it has recently been reported that D-tagatose inhibits the production of acid, Growth and water-insoluble glucan production of S. mutans GS-5 in the presence of sucrose. The inhibitory effects of D-tagatose on the growth and biofilm formation of S. mutans GS-5. Monitoring the growth of S. mutans over a 24-hour period revealed that D- tagatose prolonged the lag phase without interfering with final cell yield. This lag growth was also observed in the presence of 1% sucrose, although it was abolished by the addition of D-fructose. S. mutans biofilm formation was significantly inhibited by growth in sucrose media supplemented with 1 and 4% D-tagatose compared to that in a culture containing only sucrose, while S. mutans formed granular biofilms in the presence of this rare sugar. The inhibitory effect of D-tagatose on S. mutans biofilm formation was significantly more evident than that of xylitol. Growth in sucrose-based media supplemented with D-tagatose significantly reduced the expression of glucosyltransferase, exo-β-fructosidase and of D-fructose-specific phosphotransferase, but not the expression of fructose-specific phosphotransferase compared to culture containing only sucrose. Cell-associated glucosyltransferase activity in S. mutans was inhibited by 4% D-tagatose. These results indicate that D-tagatose reduces the production of water-insoluble glucan from sucrose by inhibiting the activities of glucosyltransferase. It is It is commonly assumed that colloidal copper has antibacterial properties. Copper generates toxic hydroxyl radicals that damage the cell membranes of Gram-negative and Gram-negative bacteria positive, including Enterococcus faecalis, present in infected root canals. Furthermore, the use of some polymers to stabilize colloidal copper increase its release time and may also decrease the risk of bacterial recolonization and biofilm formation inside the canals, enhancing the antimicrobial properties of these compounds. Copper is an essential element for the metabolism of animal and plant cells. It is a trace element present in most organisms and there are more than 30 types of proteins that contain it. Copper was the first metal used by man more than 9,000 years ago. Today, world copper consumption is approximately 18 million tons per year. Already in 2200 BC it was used to sterilize wounds and drinking water. In the 19th century it was observed that the Copper workers were immune to cholera and its use became widespread in the 20th century. They were conducted Laboratory and clinical studies to demonstrate its effectiveness. In recent years, copper has been used as a building material for hospitals and medical centers, since its antimicrobial properties are have proven useful in fighting infections. Colloidal copper, thanks to its properties antimicrobial is important and has potentially promising applications in the fight against growing number of pathogens resistant to currently available antimicrobials. These Pathogens pose a continuing threat to human and animal health (Usman et al., 2013 & Kruk et al., 2015). The aim of our studies was to evaluate and identify the effectiveness of colloidal copper and of D-tagatose in combination creating a synergistic effect with the consequent reduction of the charge bacterial biofilm and improving dentin sensitivity due to gingival retraction. This solution could find its maximum expression through the use of a gel, but also in other forms such as chewing gum, sweets and mouthwashes allowing direct application from the sites anatomical structures involved in the etiopathogenesis of biocide formation on the tooth surface. applications can be the most varied, from the prevention of caries in the pediatric population and not only. Other fields of application, but not exclusive, can be for example the application on orthodontic and prosthetic dental equipment up to the prevention of the formation of biofilm on implant abutments, often the cause of failure of implant prosthetic therapy. The new composition presents, in addition to the immediate synergistic effect on the formation of biofilm bacterial, an unexpectedly prolonged action over time which makes it a fundamental and effective in maintaining its effectiveness even during the night hours against the action cariogenic biofilm. To date there are devices with a limited action over time, often of chemical-synthetic origin, but which they have never shown lasting and effective action. Furthermore, the presence of copper guarantees a marked action against antibiotic-resistant bacteria and Today there are no publications demonstrating the development of a copper-resistant action by bacteria belonging to the Streptococcus mutans category. Furthermore, for the first time a composition with active ingredients classified as food additives and therefore equipped with a safety profile and tolerability never achieved with products of synthetic chemical origin and their degradation products can be identified as impurities known that cannot be determined as unknown impurities. In order to corroborate the theoretical assumption, an in vitro validation study was undertaken. 5. Experimentation 5.1. Experiment on the synergistic efficacy of colloidal copper and D-tagatose Experiment No. 1909 / 2023: Streptococcus mutans strain ATCC 25175 / experimental preparations of D-Tagatose, colloidal copper > 100nm. Combination of D-Tagatose and colloidal copper > 100nm Date: 14.09.2023-16.09.2023 Objective: Determination of the antimicrobial activity of D-Tagatose, copper preparations colloidal > 100nm. and combination of D-Tagatose and colloidal copper > 100nm. Materials: concentrated preparations of aqueous solutions: N. 6-8-1 - concentrated preparation of D-Tagatose, colloidal copper > 100nm, N. 6-8-2 - concentrated preparation of D-Tagatose; N. 6-8-3 - concentrated preparation of colloidal copper > 100nm; culture broth produced; mannitol - agar salt ; Test strain: 1. Streptococcus mutans strain ATCC 25175 Methods: A suspension of the test strains with a density of 0.5 McFarland was prepared using a Densi-La-Meter instrument. Further dilution of the preparations was done with nutrient broth 1:1. The determination of antimicrobial activity was carried out by incubation (exposure) to 37°C for 30 and 90 minutes for the original preparations and for 3 hours and 24 / 48 hours for the preparations diluted 1:1 with the macro method followed by inoculation on a dense culture medium. In the prepared solutions, thus as in the control tube, 106 microbial cells / ml of the suspensions were added strains under examination. Controls consisted of distilled water without antimicrobial substances with the strain cultured in test (for the original preparations) and from nutrient broth diluted 1:1 with distilled water without substances antimicrobial with the culture of the strain under examination (for diluted preparations). Results: Test strain control: Streptococcus mutans, ATCC 25175: inoculation in 30 minutes - 106 microbial cells / ml; inoculation in 90 minutes - 5 * 106 microbial cells / ml; inoculation in 3 hours - confluent growth of the test strain; within 24 hours, a marked increase in broth turbidity is observed. Table 1 - Determination of the antimicrobial activity of aqueous solution preparations in comparisons of the test strain Streptococcus mutans strain ATCC 25175 Colloidal copper > 100nm. The growth results of the strain under examination after incubation with the preparations Registration visual in a Inoculation on dense culture medium after nutrient medium liquid incubation incubation 24 / 30 min 90 min 3 hours 48 hours Preparation Concentration 10 CFU 10 CFU Colloidal copper > 99.99% 6-8-3 99.9999% of 99.9999% of clear / cloudy 100nm. inhibition inhibition inhibition Table 2 - Determination of the antimicrobial activity of aqueous solution preparations against of the test strain Streptococcus mutans strain ATCC 25175 D-Tagatose The growth results of the strain under examination after incubation with the preparations Registration visual in a Inoculation on dense culture medium after nutrient medium liquid incubation incubation 24 / 30 min 90 min 3 hours 48 hours Preparation Concentration 10 CFU 10 CFU 99.99% 6-8-2 D-Tagatose 99.9999% 99.9999% turbid / turbid inhibition inhibition inhibition Table 3 - Determination of the antimicrobial activity of aqueous solution preparations in comparisons of the test strain Streptococcus mutans strain ATCC 25175 Colloidal copper + D- Tagatose The growth results of the strain under examination after incubation with the preparations Registration visual in a Inoculation on dense culture medium after nutrient medium liquid incubation incubation 24 / 30 min 90 min 3 hours 48 hours Preparation Concentration no growth no growth no growth Colloidal copper + 6-8-3 100.00% 100.00% 100.00% clear / clear D-Tagatose inhibition inhibition inhibition 5.2. Conclusions In this design, all preparations demonstrated an antimicrobial effect, but to different degrees. They can be classified according to their effectiveness: No. 6-8-1 > No. 6-8-2 > No. 6-8-3. 6. Detailed description 6.1. Colloidal copper as an antibacterial. Copper has a bactericidal action, mainly linked to its ability to donate and accept electrons in a continuous process. It produces a hydroxyl radical that can participate in a series of reactions. adverse to cellular macromolecules, such as the oxidation of proteins and lipids. Hydrogen peroxide generated can lead to increased production of toxic hydroxyl radicals. It causes the displacement of iron from iron-sulfur clusters, can compete with zinc or other metal ions, important at the sites of protein binding; disrupts membranes, denatures DNA, and blocks cellular respiration. In turn, bacteria have developed copper tolerance mechanisms such as extracellular sequestration. of the ion, the impermeability of the external and internal bacterial membranes, the removal of proteins of copper (metallothionein) in the cytoplasm and periplasm and the efflux of copper from the cell (Grass et al., 2011). Copper alloys acting on abiotic surfaces have shown that, in addition to rapid death of antibiotic-resistant strains, causing the destruction of plasmid and genomic DNA, the which has an implication in preventing the spread of infections and gene transfer (Warnes et al., 2013 & Bagchi et al., 2013). Copper surfaces or their alloys are capable of eliminate 99.9% of pathogenic bacteria in a few hours, including resistant Staphylococcus aureus methicillin-resistant (MRSA), Escherichia coli, Pseudomonas aeruginosa, Listeria, or bacterial DNA lesions (Santo et al., 2011). The starting point for the clinical use of copper, specifically for disinfection of the root canal system, is reported in an ex vivo study in which canals treated with sulfate copper pentahydrate showed a 6-logarithm reduction in colony-forming unit counts on the fourth day (Sánchez-Sanhueza et al., 2015) 6.2. Tagatose as an antibacterial Effects of D-tagatose on the growth of S. mutans GS-5. The effects of D-tagatose on the growth of S. mutans GS-5 were examined in BHI containing 1% sucrose. Sucrose increased the growth of S. mutans GS-5 compared to BHI alone and the pH of the sucrose culture dropped to less of 5.0 after 9 hours of incubation. Interestingly, D-tagatose delayed the transition of S. mutans growth to the logarithmic phase despite the presence of sucrose. Consequently, The decline in culture pH was also delayed by D-tagatose compared to that of D-tagatose alone. sucrose. This growth delay became more evident and entry into the stationary phase was delayed by 6 hours when the D-tagatose concentration was increased to 4%; however, they are not significant differences in final OD590 were observed after 24 hours of cultivation (data not shown). As demonstrated, the growth retardation of S. mutans GS-5 and the pH decline induced by D- tagatose were abolished when 1% D-fructose, but not D-glucose, was added to BHI containing 1% sucrose. These results indicate that the rare sugar Dtagatose inhibits the Sucrose catabolism in S. mutans GS-5. Effects of D-tagatose on biofilm formation in S. mutans GS-5 in vitro. Since sucrose metabolism is important for glucan production insoluble in water, necessary for biofilm formation, D-tagatose was predicted to inhibit S. mutans biofilm formation. The effects of D-glucose, xylitol were then evaluated. and D-tagatose on the biofilm formation of S. mutans GS-5 in vitro. The addition of 1% of Sucrose addition to growth media significantly increased biofilm formation by S. mutans GS-5 compared to unspiked media (P<0.05), which is consistent with many reports Previous studies. Supplementation with D-glucose and sucrose slightly reduced biofilm formation. of S. mutans GS-5 compared to non-supplemented media. biofilm formation of S. mutans compared to supplementation with sucrose alone (P2%). On the contrary, D-tagatose showed a clear Dose-dependent inhibition of S. mutans GS-5 biofilm formation To determine whether the effects on biofilm formation were caused by high osmolality, the biofilm formation by S. mutans GS-5 was compared in the presence of 1% sucrose and at 5%. The biofilm mass in the culture with 5% sucrose was significantly smaller compared to that of 1% sucrose (OD550 1.27±0.05 vs. OD550 1.09±0.07, respectively; P<0.01 (data not shown). However, the addition of 4% D-tagatose to the 1% sucrose culture reduced the biofilm mass to almost half that of the 5% sucrose culture (OD550 0.57±0.06 vs. OD550 1.09±0.07, P<0.01; data not shown). These results indicate that the effects of high osmolality were limited under the conditions used in this study. Microscopic examination Scanning electron microscopy of S. mutans GS-5 biofilms. S. mutans GS-5 was grown in 1 ml of BHI containing 1% sucrose with or without 1 or 4% xylitol or D-tagatose in 24-well plates with plastic disc inserts; the plates were incubated anaerobically at 37 ˚C for 72 hours and were biofilms formed on the plastic discs were compared. S. mutans GS-5 grew similarly in all tested media. However, a smaller amount of biofilm formed on the discs in the cultures. containing D-tagatose compared to those in other media (1% sucrose alone or 1% sucrose plus 1 or 4% xylitol). In particular, in cultures containing D-tagatose, multiple cellular aggregates of S. mutans GS-5, especially at the highest concentration, while in other cultures containing 1% sucrose formed homogeneous biofilms on the discs. The examination also SEM of the discs revealed less biofilm presence on the discs in the D-tagatose culture. quantification of S. mutans GS-5 biofilms on discs showed a significant reduction in presence of D-tagatose (P<0.05). The use of non-cariogenic sweeteners represents a method of prevention of dental caries and Sugar alcohols such as xylitol are widely used in chewing gum. Also the D-Tagatose is recognized as a tooth-friendly sweetener and is not fermented by bacteria cariogenic dental plaque. Consistent with previous studies, D-tagatose has been shown to be a non-fermentable sugar for dental plaque bacteria. S. mutans GS-5, and the results Gas chromatographic-mass spectrometric analysis revealed that 81.6% of the D-tagatose added to the culture media was retained, even after 48 h of S. mutans GS-5 (data not shown). Although the addition of 1% D-tagatose to the culture medium retarded the growth of S. mutans GS-5, the final growth yield was unchanged compared to sugar-free crops. However, the D- tagatose inhibited biofilm formation of S. mutans GS-5, indicating that the effect is caused by a mechanism other than growth inhibition. As demonstrated, D-tagatose inhibits cell-associated GTF activity, resulting in reduced release of D-fructose from sucrose. D-fructose (and sucrose) appears to be a potent inducer of gtfB expression. The addition of 1% sucrose to the culture medium induced the expression of the Dfructose-specific PTS and gtfB, indicating that the glucan production and energy metabolism pathways utilizing Dfructose are closely coordinated in S. mutans. This result is consistent with as reported by Shemesh et al., who demonstrated that D-fructose induces higher levels of gtfB expression relative to D-glucose in the early exponential phase. Therefore, the suppression of gtfB D-tagatose expression may be partially caused by a decrease in the supply of D-fructose. Furthermore, the genes encoding the EII component for the D-specific PTS genes also Fructose (ptsfru and ptsfru / man) were downregulated in the presence of D-tagatose. Growth retardation of S. mutans GS-5 by D-tagatose may also be due to limited D-fructose intake resulting from GTF inhibition, since the growth retardation induced by D-tagatose was reversed with the integration of D-fructose. It is therefore hypothesized that the alterations in the availability of this monosaccharide are responsible for the prolongation of the growth retardation phase of S. mutans GS-5 by D-tagatose. In contrast, ftf expression levels were not altered by D-tagatose in the presence of 1% sucrose. FTF produces water-soluble inulin-type fructan in S. mutans. Since fructose is digested by FruA to Dfructose, it is believed that this polymer of Fructose serves as an energy depot for S. mutans. It is hypothesized that the downregulation of fruA by part of the D-tagatose limits the D-fructose supply for S. mutans. This alteration of the availability of monosaccharides could influence the expression of fruA, which is known to sensitive to the control of carbon catabolite repression through the regulatory protein CcpA central. As mentioned, D-tagatose appears to inhibit the GTFs of S. mutans GS-5 and inhibition of cell-associated GTFs B or C, which produce water-insoluble glucan from from sucrose, is considered a primary mechanism underlying biofilm inhibition and retardation of growth of S. mutans GS-5 by D-tagatose. Since Dtagatose is an epimer of D- fructose at the C-4 position, its structural similarity to D-fructose could interfere with the binding or catalysis of sucrose by GTFs. Furthermore, the S. mutans GS-5 biofilm formed in the presence of D-tagatose was granular, while the biofilm formed in the culture with sucrose alone or sucrose plus xylitol was uniform. This difference could be related to the glucan-fructan imbalance caused by D-tagatose. Bautista et al. reported that many Human pathogens are unable to utilize D-tagatose and have shown that the sugar is metabolized by a limited number of lactobacilli. Probiotic lactobacilli have been reported suppress the growth of cariogenic bacteria and prevent tooth decay. Based on the result of the This study, according to which S. mutans GS-5 does not preferentially ferment D-tagatose, is expected that this sugar prevents the colonization of S. mutans on tooth surfaces by promoting ability of probiotic oral lactobacilli to resist colonization. Xylitol is widely used for the prevention of dental caries, although its effects in clinical studies remain controversial. S. mutans transports xylitol through a fructose-specific PTS and have emerged xylitol-resistant S. mutans strains lacking this PTS activity. Furthermore, the presence of sugars fermentables, such as sucrose, attenuate the effects of xylitol. Therefore, treatments are necessary Alternative prophylactics for tooth decay. Xylitol is a sugar that is non-fermentable to S. mutans. and exerts a toxic effect by causing energy expenditure for the absorption and export of This non-cariogenic sugar alcohol. The mechanism by which xylitol suppresses S. mutans appears to be different from that of D-tagatose described here; therefore, one might expect a synergistic effect due to their combination. However, a synergistic effect was not evident in the inhibition of biofilm formation of S. mutans GS-5, which could be related to interference with xylitol absorption, since D-tagatose downregulates genes D-fructose-specific PTS, ptsfru and ptsfru / man. In conclusion, D-tagatose appears to inhibit growth of S. mutans GS-5 and biofilm formation by interfering with GTF activity. This effect may be useful in the prevention of dental caries. Based on the results obtained by the In this study, foods or preparations containing D-tagatose could be useful tools for improve oral hygiene. D-tagatose may be able to suppress intermittent growth of S. mutans among oral hygiene activities. Furthermore, S. mutans produces a granular biofilm in the presence of D-tagatose, which could facilitate the removal of biofilm by mechanical brushing compared to homogeneous biofilms. In light of the bibliographical data and experimental evidence we can support and confirm that synergistic effect of efficacy between a trace element such as copper and a rare sugar D-Tagatose both in terms of both durability and effectiveness on biofilm-producing pathogens, the golden standard of which is represented by Streptococcus mutans 7. Composition examples Mouthwash D. Tagatosio ……… ………………… ………...…… ……… gr 10 Colloidal copper …… …… ………………… ………….. ppm 40 Copper chlorophyll …… … …………….......................... …..... gr 1 Demineralized water … ………… …….…………qb to 100 ml Oral topical gel (w / w) bio-ecological composition. 90% DEMINERALISED WATER BIOMER 0.6% D-TAGATOSE 9.3% COLLOIDAL COPPER 0.1% Oral topical gel (w / w) D.Tagatosio ……… ……… ………… ………........ … …………… …….gr 10 Colloidal copper ………… ………… ……………… … ……………ppm 40 Copper chlorophyll …… … ………… …………….… ………………….gr 1 Carbopol 940........................................................................................ 2.5 g Propylene glycol......................................................................................... 10 g Demineralized water qba................................................................ 100 g 8. Conclusion For intended use in the human mouth and pharynx, an oral composition must have a greater germicidal effect than known oral compositions, with a high degree of tolerance for humans or human tissues and excluding the possible formation of resistance. The risk / benefit ratio shows a clear improvement over the state of the art, as expected. from the code of ethics and the guidelines regulating the matter. To this end, according to the invention, provided an oral composition, which comprises as components a sugar and a trace element Di preference, as an active composition on the biofilm, a combination of copper and D- is given Tagatose 8.1. In light of what has been masterfully expressed The cases in which it can be presumed that an inventive activity exists are those in which the invention realizes: - an unexpected or surprising effect; - a synergistic effect generated by the combination of known elements; - a practical solution to a problem that has remained unsolved for a long time, even if the solution proposal seems to be quite simple after the inventor has described it in the patent; - a solution in which a technical prejudice is overcome. From CINQUANTINI B., PRIMICERI MV, Intellectual property and patents – practical guide, Rome, 2015, p. 19. This is considered application worthy of a patent grant. Louis Rosary frank inderst 02.10.2023 16:56:43 G M T +01:00

Claims

1) Microbiologically active composition comprising at least one or more trace elements of groups 10, 11 and 12 according to the Mendeleev periodic table in elemental form and / or its salts and / or derivatives and a sugar 2) Microbiologically active composition as per the application, wherein such elements are preferably colloidal copper and D-Tagatose 3) Microbiologically active composition according to the previous statements, wherein said elements have a proportional concentration between them of between 0.001% and 99.999% for each single element in any physical form, for example liquid, solid, gaseous, aerosol, spray, nebulised and combinations thereof 4) Microbiologically active composition according to the previous statements, in which said elements have a proportional concentration between them of between 0.001% and 99.999% for each single element in any physical form, for example liquid, solid, gaseous, aerosol, spray, nebulized and combinations thereof, dissolved or incorporated in excipients suitable for the desired formulations: gels, mouthwashes, toothpastes, washes and any device, including medical devices, for use on mucous tissue and dermis. 5) Microbiologically active composition according to the previous indications, including: D.Tagatose..................................................................................10g Colloidal copper..................................................................40ppm Copper chlorophyll..................................................................1g Demineralized water..................................................................as much as 100ml 6) Microbiologically active composition according to the previous indications including DEMINERALIZED WATER 90% BIOMER..................................................0.6% D-TAGATOSE.......................................9.6% COLLOIDAL COPPER...........................0.1% 7) Microbiologically active composition according to the previous indications including: D.Tagatose..................................................................................10g Copper >100 nm.............................................................40ppm Copper chlorophyll..................................................................1g Carbopol 940...........................................................2.5g Propylene glycol...........................................10g Purified water qba........................100g 8) Microbiologically active composition according to the previous indications for use against any microbiological pathogens with activity on human mucous membranes and dermis. 9) Microbiologically active composition according to the previous indications for use against any microbiological pathogen for veterinary use. 10) Microbiologically active composition according to the previous indications for use against any microbiological pathogen with activity on medical devices and / or personal protective equipment (PPE) and / or surfaces and / or environments.