The “Vitaminic-strategy” Against the Oral Bacteria S. Mutans and F. Nucleatum, Agents of Caries and Halitosis

The oral cavity is one of the most complex human body environments. Indeed, the continuous variation of this habitat conditions reects the high dynamism of the resident microbial community. Two key actors in the oral diseases are the bacteria Streptococcus mutans and Fusobacterium nucleatum, both implicated in the formation of oral biolms and consequently in the generation of common pathologies such as caries and various gingival and soft tissue inammation diseases. In addition, F. nucleatum is also implicated in the halitosis phenomenon, thanks to its demonstrated ability to produce as second metabolite the hydrogen sulphide (H 2 S), one of the volatile sulphur compounds (VSCs) that, with methyl mercaptan (CH 3 SH) and the dimethyl sulphide (CH 3 SCH 3 ) 24 , is produced by periodontopathic anaerobic bacteria and causes the awkward bad breath in halitosis patients.


Abstract Background
The oral cavity is one of the most complex human body environments. Indeed, the continuous variation of this habitat conditions re ects the high dynamism of the resident microbial community. Two key actors in the oral diseases are the bacteria Streptococcus mutans and Fusobacterium nucleatum, both implicated in the formation of oral bio lms and consequently in the generation of common pathologies such as caries and various gingival and soft tissue in ammation diseases. In addition, F. nucleatum is also implicated in the halitosis phenomenon, thanks to its demonstrated ability to produce as second metabolite the hydrogen sulphide (H 2 S), one of the volatile sulphur compounds (VSCs) that, with methyl mercaptan (CH 3 SH) and the dimethyl sulphide (CH 3 SCH 3 ) 24 , is produced by periodontopathic anaerobic bacteria and causes the awkward bad breath in halitosis patients.

Methods
In this study, the oral preparation Vea® Oris constituted only by vitamin E and capric/caprylic acid was evaluated as a potential treatment of caries and periodontal diseases; the effect of the product at different concentrations on the growth and the ability of both strains to form bio lm was investigated.
Regarding to F. nucleatum also the in uence of Vea® Oris on the production of H 2 S was evaluated.

Results
Our in vitro results suggested that the Vea® Oris treatment could considerably reduce the growth and bio lm formation of both S. mutans and F. nucleatum. For F. nucleatum an appreciable reduction of the H 2 S production can be also obtained.

Conclusions
Overall, this study highlighted the potential of Vea® Oris as a more "natural" adjuvant to prevent the bio lm and plaque formation and to reduce the smelly odour of halitosis.

Background
The oral cavity is one of the most complex environments in the human body due to the continuous and simultaneously variation of homeostasis conditions. Over 700 diverse bacterial species have been identi ed as potential/usual colonizers of the many niches of the mouth, i.e., the surface of teeth and all the soft tissues of the oral mucosa such as the gingiva, the cheek, the tongue, the palate and the lips (1)(2)(3). The bacterial composition of the oral microbiota is responsive to different host intrinsic and host extrinsic factors. The host intrinsic factors are those factors not modulable by the host belonging to individually genetic factors (e.g., ethnicity, gender, host immune response, medical conditions, etc.), characteristics of the oral cavity such as the temperature, the saliva composition (pH, buffer capacity, hormones, and secreted substances) and characteristics of the attachment surfaces (e.g., roughness of tooth surface). The extrinsic factors come from the external environment, habits and lifestyle, are modulable or relatively modulable by the host and regard the hygiene, the diet, the use of drugs, smoking, medications, sex, access to dental care etc. (4)(5)(6)(7). All these factors contribute together and along the host ageing to establish in the oral cavity the conditions suitable for the colonization and the growth of different pools of microorganisms. As a result of this complex interaction, the balance between bene cial and pathogen species is continuously altered, passing from a state of symbiosis and health to a dysbiotic state with the upcoming of oral pathologies such as caries, gingivitis and periodontitis (2,4).
Nevertheless, the new metagenomic approaches represented a vital advantage to unravel the correlations between different types of stimuli in the oral cavity and the enrichment of speci c bacterial species, with particular interest for those species that increase their growth as cause/effect of oral diseases (3,8). Also, through sequencing methods, the relationship between bene cial and pathogenic species of the oral cavity has been extensively studied as well as the composition of the polymicrobial bio lm assemblies detected in the oral cavity on the tooth, on the gingiva or on the tongue surface, and in minor part, on other soft tissue (7). The bio lm structure constitutes a protect sub-environment in which various bacterial species interact to protect themselves from the possible surrounding perturbations and thus represents a survival strategy of cells, whose formation is often di cult to inhibit or to remove (9)(10)(11). In the oral cavity, the bio lm causes the onset of the most prevalent infectious diseases, such as the plaque and dental caries and in ammation phenomena regarding the gingiva, which cause gingivitis, periodontitis, etc. (7,12). The formation of oral bio lm is arranged on the following subsequent three stages of attachment, colonization and bio lm development (13). The involved species are identi ed as "early" and "late" colonizers. The early colonizers belong to the streptococci genera; among them, S. mutans is particularly enriched in supragingival plaque, in childhood caries and dentinal and root caries lesions. Indeed, thanks to its rapid metabolism and strong acid tolerance it is recognized as the most cariogenic bacteria of the oral cavity (6,12,13). The early colonizer species are involved in the rst two stages of bio lm formation. Since their ability to bind salivary proteins, they adhere to nascent hard or soft tissues, colonize the oral niches and provide a substratum for subsequent colonizers (6, 13,14).
Between the rst and late colonizers, the Gram-negative obligate anaerobe F. nucleatum is considered a key actor for the stage of maturation of the oral bio lm. Thanks to its coating adhesion molecules and polysaccharide receptors, it is able to co-aggregate and bind both with the streptococcal early colonizers and the late colonizers, mainly Gram-negative anaerobes belonging to the genera of Bacteroidetes and Spirochaetes (13)(14)(15). Apart from the bridge role in the bacterial network of bio lm assemblages, F. nucleatum plays an active role as a periodontal pathogen, as demonstrated by its enhanced prevalence within the deep periodontal pockets. Moreover, in these niches, F. nucleatum triggers the production of matrix metalloproteinases by the host contributing initially to the periodontal in ammation and the onset of irreversible periodontal diseases (15). This pathogen is also characterized by a signi cant haemolytic activity and the ability to produce hydrogen sulphide (H 2 S). In particular, the H 2 S, similarly to the methyl mercaptan (CH 3 SH) and the dimethyl sulphide (CH 3 SCH 3 ) 24 , is one of the volatile sulphur compounds (VSCs) produced by periodontopathic anaerobic bacteria which cause the typical malodour of the halitosis (16-20). Consistently, the uncomfortable phenomenon of halitosis is often associated with the increase of VSCs producing species, in primis, F. nucleatum but also Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia and Eubacterium (16,17,19,20). Actions aimed to reduce the presence of F. nucleatum in the oral cavity could represent an excellent double-sides strategy to reduce both the formation of the oral bio lm causative of plaque and other frequent oral diseases and the uneasiness associated with the halitosis. An even more e cient approach to massive prevent/reduce the development of oral pathologies could be represented by the simultaneous inhibition of S. mutans as the principal rst colonizer of the oral bio lm. However, at the current point of knowledge, lifestyles modulation together with the use of innovative cosmetics or medicals are the only strategies to maintain or restore a balanced microbiome and the health of the oral cavity (2,4).
Indeed, mouthwashes and many dentistry products with antimicrobial compounds such as chlorhexidine, triclosan, cetylpyridinium chloride and chlorine dioxide are often used to treat the bad breath and to inhibit/disrupt the oral bio lm (17,18). However, the use of such products is always more often associated with the increase of the bacterial resistance as well as can cause the simultaneous killing of non-pathogenic commensal species with the consequent onset of undesirable dysbiosis states. The use of natural products is a more appreciated strategy to prevent and contrast the oral pathologies, even more if those substances impact more selectively on the pathogen species of the buccal microbiota. Moreover, the major acceptance of a "natural therapy" could modify the applicability of the therapy to a wider range of patients for ages and medical conditions, not only as curative but also as preventing therapy, both in paediatric and advanced ages. Products such as propolis, cranberry, tea, Galla chinensis, grapes, coffee, and cacao containing polyphenols have already demonstrated their activity against the oral bio lm, as well as hinokitiol, green tea powder, and eucalyptus extract made against the oral malodour (17,18).
Further, since it was found that oral disorders and pathologies are also associated with the oxidative stress that alters the microbial balancing, the activity of antioxidant substances against the oral bacteria has been studied as well. Well known non-enzymatic antioxidants are able to neutralize the Reactive Oxygen Species (ROS) causative of many oral pathologies. They are fat-soluble vitamins (vitamin A, vitamin E-tocopherol and b-carotene), water-soluble vitamins (vitamin C and vitamin B complex), trace elements (zinc, magnesium), and bio avonoids (plant-derived) (21). The antioxidant compounds are generally assumed through a balanced diet with consumption of fruits and vegetables that contain considerable levels of those; interestingly, their presence is sensibly minor in individuals with oral diseases (4,21,22).
Regarding vitamin E, it is the major fat-soluble antioxidant in all cell membranes, exerts some antiin ammatory properties and enhances the humoral immune response. Its positive effect on the oral health has been already documented with improvements of all periodontal parameters, principally the decreasing of plaque index and bio lm formation, of probing depth, clinical attachment level, and bleeding on probing (21)(22)(23). Therefore, in this study, we in vitro tested the innovative product Vea® Oris against S. mutans and F. nucleatum, the two bacteria causatives of the most recurrent oral diseases. This product is composed of only two components, vitamin E as alpha-tocopherol and Caprylic/Capric Triglyceride obtained from coconut oil and glycerine. The effect of Vea® Oris at different concentrations was evaluated both on the growth and bio lm formation of S. mutans and F. nucleatum, and for F. nucleatum also on the production of the volatile compound H 2 S.

Methods
The strains tested in the present study were S. mutans DSM 20523 and F. nucleatum subsp. vincentii DSM 19507. The product tested was the Vea® Oris (by Hulka s.r.l.).
The strains were cultured in Columbia Broth at the temperature of 37°C for 48 hours. The Optical Density at 600 nm (600 nm O.D.) of grown cultures was measured and dilutions were prepared to obtain 4 ml inocula with a starting O.D. of 0,1. For each strain, aliquots of 150 ul were distributed into the wells of a 96 well plate both without and with Vea® Oris at concentrations of 5, 10 and 20% v/v (50, 100 and 187 ul/ml, respectively) ( Fig. 1, yellow and green wells). Also, experimental controls were produced with only Columbia Broth medium not added and added with the same concentrations of Vea® Oris (Fig. 1, grey wells). Each treatment was reproduced in triplicates and triplicates of 96 well plates were assessed. Liquid sterile para n was used to cover the surface of each inoculated well and to provide the suitable condition of oxygenation, also to standardize the growth conditions. The dye entrapped into the adherent cells was resolubilized, adding 160 ul of 33% glacial acetic acid. Finally, the Optical Density (O.D.) at 570 nm of each well was measured in triplicates using the Perkin-Elmer VICTOR X5 multilabel plate reader (24)(25)(26)(27). The O.D. value for each treatment was calculated as the average of measures.
The calculated values are to be considered as proportional to the amount of the staining resuspended, thus proportional to the number of cells xed at the well surface as a bio lm. Therefore, the bio lm formed by strains grown with 5, 10 and 20% v/v of Vea® Oris was compared to the bio lm formed without the product and also the effect of the increasing concentrations of Vea® Oris on the bio lm formation was evaluated. More precisely, the bio lm formed by the strains in the control conditions (without Vea® Oris) was considered as the 100% of bio lm formation; then, the reduction of bio lm due to the Vea® Oris treatments was estimated as the percentage difference between the bio lm formed without (100% of bio lm) and with 5, 10 and 20% of Vea® Oris (ANOVA, Tukey HSD with p < 0.05).
Since the genus Fusobacterium has been largely demonstrated as associated with the halitosis (16, 19,28,29), the strain F. nucleatum subsp. vincentii DSM 19507 was also investigated regarding its ability to form the malodourous product H2S under Vea® Oris treatments.
The F. nucleatum was cultivated in Columbia Broth at 37°C for 48 hours. After growth, the culture was centrifuged at 5000 rcf for 3 minutes to collect the cells at the bottom of the culturing tube. The bacterial pellet was resuspended into 40 ml of Sulphide Indole Motility (SIM) medium. The formulation of the SIM medium allows the detection of H 2 S produced by the strain, since it contains ferrous ammonium sulphate, which reacts with this volatile compound and forms ferrous sulphide, a black precipitate, that nally acts as the indicator of H 2 S presence (30,31). Aliquots of the F. nucleatum culture in SIM medium were treated with the test concentrations of Vea® Oris, i.e., 5, 10 and 20% v/v, respectively. In detail, 3 ml aliquots of the culture were distributed in 15 ml falcon tubes that contain 150 (5% v/v), 300 (10% v/v) e 600 (20% v/v) ul of Vea® Oris, respectively. Each Vea® Oris treatment was assessed in triplicates. Also, 3 ml triplicates of blank and control tubes were produced respectively with SIM medium alone and with F. nucleatum culture without Vea® Oris adding. Finally, 1 ml of liquid sterile para n was added at the top of the tubes to provide the anaerobic growth condition. The tubes were incubated at 37°C for 48 hours in static condition. After incubation, the formation of black precipitated was observed and spectrophotometrically estimated. The absorbance of all inocula was measured within the wide range 400-700 nm at 1 nm steps, with the Lambda 25 spectrophotometer (Perkin-Elmer). The nal values of absorbance for different treatments were calculated as the average of values measured for each set of triplicates. The absorbance spectrum of each treatment with Vea® Oris was compared with the control one (no Vea® Oris adding) (ANOVA, Tukey HSD with p < 0.05).

Results
Following are described the signi cant variations (ANOVA, Tukey HSD with p < 0.05) induced by Vea® Oris treatments on the growth and bio lm formation of S. mutans and F. nucleatum and only for F. nucleatum on the strain ability to produce H 2 S.
As showed in Fig. 2, when compared with the untreated control, the in vitro growth of S. mutans was only slightly increased by Vea® Oris at 5 or 10% (v/v), with percentage increments respectively of 17 and 14%. Surprising, Vea® Oris at a concentration of 20% did not exert a signi cant increment of the bacterial growth.
Conversely, the same Vea® Oris doses were all signi cantly active in reducing the bio lm formation capability of the cariogenic species S. mutans. A bio lm reduction of 26% was obtained with Vea® Oris at 5% (v/v) and of 32% with Vea® Oris at 10 and 20% (v/v).
Instead, the results obtained for F. nucleatum, showed in Fig. 3, revealed that Vea® Oris does not in uence the growth of the strain at any tested concentrations. On the contrary, regardless to applied concentration, it signi cantly reduces the bacterium ability to form bio lm. Indeed, interestingly the treatments with Vea® Oris at 5, 10 and 20 % (v/v) caused similar bio lm reductions of respectively 50, 52 e 40 %.
Moreover, regarding the effect of Vea® Oris on the ability of F. nucleatum to produce the "smelly" volatile compound H 2 S of halitosis, after the incubation time (see the methods section) the darkening of SIM medium was observed for all inocula, without or with Vea® Oris adding, as an expected consequence of H 2 S production by the tested strain. However, as showed in Fig. 4, differences of the medium darkening were detected between the untreated and Vea® Oris treated inocula (Fig. 4). With respect to the control, a progressive reduction of the dark precipitate was observed with the incremental concentrations of the Vea® Oris treatments.
These ndings were con rmed through the spectrophotometric analysis. The absorbance spectra obtained for the untreated samples clearly differed from the spectra obtained for the samples treated with Vea® Oris (Fig. 5).
The range of maximal absorbance for all samples was comprised between the wavelengths of 400-420 nm; among this range, the curves of Vea® Oris treatments showed respect to the control, a progressive reducing of absorbance along the increments of Vea® Oris concentration (Fig. 6). This result suggested that the production of H 2 S by F. nucleatum was inversely in uenced by the adding of Vea® Oris in a concentration-dependent manner.
To get inside the quantitative differences in H 2 S producing in absence/presence of Vea® Oris, the wavelength of maximum absorbance for the control strain was identi ed as the wavelength at which the dark precipitate is detected. The absorbance value is to be intended as proportional to the dark precipitate produced and nally to the H 2 S produced by the strain. Therefore, at the identi ed wavelength, the absorbance measured for the control was compared with the ones measured under the Vea® Oris treatments.
In detail, the heatmap in Fig. 7A shows that the major absorbance values for the control were measured within the sub-range of the wavelength 400-409 nm with the maximum value registered at 407 nm. Comparing the absorbance measured for the treated samples within this range with the ones of the control, the progressive reduction of absorbance (H 2 S production) along the increments of Vea® Oris is clearly highlighted (Fig. 7B). Finally, comparing the absorbance measured for the treated samples with the control at the single wavelength of maximal absorbance i.e., 407 nm, a reduction of 6, 9 and 13% was calculated for the treatment with 5, 10 e 20% (v/v), respectively (Fig. 7C).

Discussion
In this study, the action of the oral preparation Vea® Oris was investigated against S. mutans and F. nucleatum, two oral bacteria that can be considered as the key actors of the most common oral diseases.
S. mutans is one of the most cariogenic species of the mouth environment (6,18,32); since its ability to colonize the oral surfaces, its rapid metabolism and the strong acid tolerance, it manages the rst stage of the bio lm formation as the principal early colonizer (12,15). F. nucleatum intervenes in the later stage of the oral bio lm maturation, when it acts as bridge-species between the early colonizers and the late colonizers, the latter ones needed to assemble the complex structure of a mature and resistant oral bio lm (6,13,15). Moreover, F. nucleatum is also implicated in the development of gingivitis and periodontal diseases as well as, thanks to its ability to produces the malodorous sulphur compound H 2 S, its enrichment in the oral cavity is associated to the development of halitosis (16,17,20,28).
The product Vea® Oris tested in this study is made by only two components from natural sources, the vitamin E (α-tocopherol acetate) and the caprylic/capric triglyceride. This formulation was tested at the concentration of 5, 10 and 20% (v/v) on cultures of S. mutans and F. nucleatum. The effect of the Vea® Oris treatments on the growth and the bio lm formation of both strains was evaluated. In adding for F. nucleatum the in uence on the H 2 S production was also estimated. Regarding to the effect of Vea® Oris on the in vitro growth of the two oral strains, the growth of F. nucleatum was not promoted by the product. A weak stimulation was obtained for S. mutans with the lower concentrations of 5% and 10% (v/v), whereas at the higher tested concentration of 20% (v/v), no signi cant variation was registered. More interestingly, regarding the bio lm formation, the results suggested that the combination of vitamin E with capric/caprylic acid clearly decrease the in vitro ability of both S. mutans and F. nucleatum to assemble the bio lm structure. Indeed, depending on Vea® Oris concentration, a percentage reduction of bio lm ranging from 26-32% for S. mutans and 40-52% for F. nucleatum was estimated.
Although no studies have been so far conducted with a similar mixture of vitamin E (alpha-tocopherol) and capric/caprylic acid, our results are consistent with the hypothesis of a synergistic antimicrobial/antibio lm action of the two components. Undoubtedly, the vitamin E as alpha-tocopherol acetate has already con rmed its antibio lm action against bacteria associated with Urinary tract infections (UTIs), mainly belonging to the genera Staphylococcus and Proteus (27,33). Regardless of the methods used for the bio lm estimation and depending on the strain tested, the application of alphatocopherol acetate induced a reduction of bio lm around 50% or more (27,33). Unfortunately, very few investigated is the potential action of tocopherols on the growth of the buccal bacteria and on their ability to form the oral bio lm. However, Smolarek et al. showed that toothpaste added with tocopherols produced an in vitro antimicrobial activity against S. mutans and E. faecalis (34); although in this study, no characterization as alpha, beta, gamma, and delta-tocopherol has been performed, the results clearly suggested the potential bene cial use of tocopherols for the maintenance of the oral health. Nevertheless, some important experimental ndings suggested that, among the others, the tocopherol acetate is the vitamin E ester more suitable and potentially more active for the treatment of the oral tissues. Indeed, the acetate tocopherol could be signi cantly delivered to the surface of the gingival tissues, it could be metabolized by the buccal and gingival epithelium to the free form of vitamin E, and nally, it is able to deeply penetrate to the metabolically active gingival tissue (35)(36)(37). At the same time, our hypothesis that the effect of Vea® Oris is the result of a synergistic action of the two components is consistent with some previous observations about the bactericidal effects of medium-chain fatty acids (MCFAs), whom the capric/caprylic acid of Vea® Oris belong to. Among the lacking literature describing the activity of MCFAs against oral bacteria, the study conducted by Huang et al. con rmed that the short, medium and long-chain fatty acids exhibit patterns of inhibition against a panel of oral bacteria comprising S. mutans, Streptococcus gordonii, Streptococcus sanguis, Candida albicans, Aggregatibacter actinomycetemcomitans, F. nucleatum, and Porphyromonas gingivalis (38). The inhibitory action of fatty acids appeared clearly species-speci c, since it is related to the pro le of fatty acids produced by each species itself; it could be thought as a "system of cross-balancing" of the oral microbiota ecology constituted by a pool of molecules active against competitors of the same environment. For instance, it was demonstrated that caprylic acid (25 ug/ml) completely inhibits the growth of F. nucleatum and reduces by 50% the growth of S. mutans. Interestingly, these results are consistent with the evidence that F. nucleatum produces butyric, isovaleric, and propionic acids but does not produce capric/caprylic acid to whom, therefore, it is sensitive. Similarly, since S. mutans is a major acidogenic and aciduric microorganism in the oral cavity, it showed greater resistance to the effects of the major part of the fatty acids, comprising the capric/caprylic acid (38).
Instead, a potential antibio lm action of capric/caprylic acid has not been highlighted. Rather, the 1monoglyceride of capric acid, the monocaprin, tested against S. aureus, C. albicans, and S. mutans showed inhibitory action against the strains in the planktonic phase, but no effect when the same strains were tested in the bio lm growing state (39).
Then, we are con dent to suppose that the highlighted effect of Vea® Oris on the growth and bio lm formation of S. mutans and F. nucleatum is the result of combined action of the two-component, more focused on the antimicrobial action for the caprylic/capric acid, whereas to the antibio lm effect for the alpha-tocopherol acetate. The synergistic nature of the interaction seems to be further con rmed by previous studies, at least regarding the antimicrobial activity of caprylic/capric acid. Indeed, medium-chain fatty acids such as caprylic and lauric acids demonstrated an enhanced bactericidal effect when used in association with essential oils (i.e. carvacrol, eugenol, b-resorcylic acid, trans-cinnamaldehyde, thymol, and vanillin) that originates by extraction from plant and seeds, similarly to the alpha-tocopherol (40,41). Consistently, we are con dent to suppose a similar relationship of synergism between caprylic/capric acid and the alpha-tocopherol acetate of Vea® Oris as well. The enhancing activity may be due to the same mechanism of synergism identi ed by Chapple et al. (2013) for ascorbate and αtocopherol. These two compounds, recycling each other, produce together increased antioxidant effect respect to the ascorbate alone (42).
In addition, Vea® Oris demonstrated an interesting inhibitory effect on the ability of F. nucleatum to produce H 2 S, responsible with other volatile compounds, of the smelly odour in halitosis. The in vitro decrement of H 2 S induced by 5, 10, and 20% (v/v) of Vea® Oris ranged between only 5-12%, and interestingly the variation occurred in a product concentration-dependent manner. Although further investigations are needed to go insight the H 2 S inhibitory action of both components, we are con dent to speculate that the observed effect is principally attributable to the vitamin E. Indeed, Ben Lagha et al.
(2017) demonstrated that the green tea extract and the principal tea catechin, the epigallocatechin-3gallate (EGCG), at a high concentration of 2000 ug/ml markedly reduced the H 2 S production by F. nucleatum (about 80% of reduction); a weaker but signi cant reduction as well was veri ed at concentrations ≤ 500 ug/ml for both substances and also for the thea avins (43). A similar inhibitory action was veri ed for the thymoquinone, the aromatic compound obtained as the most prominent constituent of N. sativa seeds essential oil (44). Used at the concentration of 100 ug/ml it signi cantly decreases H 2 S level by 40% in F. nucleatum, and at an even lower concentration of 6.25 µg/ml it reduced of 45% the H 2 S production in Porphyromonas gingivalis as well. These data lead us to suppose that the aromatic molecular structure of alpha-tocopherol as for the aforementioned natural compounds makes them able to compete with some intermediate of the H 2 S production pathway. Going deeply inside the molecular mechanism of inhibition, we could speculate that the inhibitory action acts at genetic level as it is for the cetylpyridinium chloride (CPC), a quaternary ammonium compound often used in oral hygiene products (45). Daily use of mouth rinses containing CPC was found to reduce VSCs and oral malodor through both a direct antimicrobial activity against the oral halitosis bacteria such as F. nucleatum and Phorphiromonas gingivalis, and through the capacity of the molecule to suppress the expression of the bacterial genes mgl and cdl that lead the VSC production (45). Nevertheless, deeper investigations are needed to con rm the same genetic inhibition mechanism for the tested product.

Conclusions
The present study highlighted the potential of using Vea® Oris both to counteract the formation of bio lm and plaque and as adjuvant in the halitosis treatment. Not to be overlooked is that, since its natural derivation its application in the oral cavity prospects important advantages respect to a chemical derived product rstly in term of safety and approval by patients. Add to this, the antioxidant and antiin ammatory properties of vitamin E could contribute to the maintainance of the health state of the oral mucosa, as found for other mucosal tissues (46-51). These encouraging results certainly set the stage for further in vitro and desirable also in vivo studies. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
All the authors rmily declare they avoided circumstances that might affect their judgement or impartiality when performing their jobs, thus they declare that there are no con icts of interest.

Funding
This research was partially supported by the donation assigned from "Hulka s.r.l." to Prof. Roberto Di Marco and Prof. Germano Guerra, at University of Molise. The funding sustained the matherials and analysis costs. The sponsor had no role in any other step of the manuscript editing.
Authors' contributions L.P. conduced the investigations, formal analysis, conceptualization and writing. I.M. and G.P.P. contributed to the investigations, the writing and reviewing. M.A.C., N.V. and A.P. aied in procurement of resources and reviewing. D.N. contributed in data validation, and study supervision. G.G. was responsible of funding acquisition and supervision. R.D.M. was responsible for the funding acquisition, planned the study and managed the conceptualization, validation and supervision. All authors read and approved the fnal manuscript.  In uence of Vea® Oris at 5, 10 and 20% (v/v) on the growth and bio lm formation for S. mutans (left).

Figure 7
Variation of H2S production shown as variation of the absorbance values. Absorbance variation depicted by grey colour gradient for F. nucleatum cultures along with increments of Vea® Oris with respect to untreated samples (0%, CTR). The variation (grey gradient) is shown between the range 400-420 nm as absolute values for each treatment (A) and between the range of 400-409 nm as relative values between treatments(B). Absorbance variation (colour gradient) and percentage reduction (numerical values) registered for each treatment with respect to the untreated samples (C). The absorbance variation is proportional to the variation of H2S production.