Isofloridoside: A Novel Inhibitor of Streptococcus mutans Biofilm Formation and Glucosyltransferase Activity

Isofloridoside: A Novel Inhibitor of Streptococcus mutans Biofilm Formation and Glucosyltransferase Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Isofloridoside: A Novel Inhibitor of Streptococcus mutans Biofilm Formation and Glucosyltransferase Activity Manami Kimijima, Naoki Narisawa, Yoichiro Hama, Tomoyo Nakagawa-Nakamura, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6568825/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Aug, 2025 Read the published version in BMC Research Notes → Version 1 posted 10 You are reading this latest preprint version Abstract Isofloridoside, a galactose-containing heteroside derived from marine red algae, has potential applications as a sweetener because it can activate the sweet taste receptor taste 1 receptor members 2 and 3. This study investigated the effects of isofloridoside on the growth and sucrose-dependent biofilm formation of the cariogenic bacterium Streptococcus mutans . The results showed that S. mutans did not grow when isofloridoside was used as the sole carbon source. Isofloridoside inhibited sucrose-dependent biofilm formation by S. mutans in a concentration-dependent manner, similar to galactose and glucose, but unlike melibiose, galactose-containing disaccharides. Biofilm inhibition induced by isofloridoside was associated with the inhibition of glucosyltransferase activity. Isofloridoside exhibited biofilm inhibition comparable to that of xylitol, an alternative sugar known to inhibit biofilm formation. The differential effects of isofloridoside and melibiose on biofilm formation may be due to structural differences that affect their interactions with S. mutans enzymes. These findings highlight the potential of galactose and its polysaccharides as regulators of S. mutans biofilm formation and suggest that isofloridoside is a promising alternative sweetener for caries prevention. Further research is required to elucidate the detailed mechanism of action, potential for resistance acquisition, taste, safety, and economic feasibility of isofloridoside as a caries-preventive agent. Isofloridoside Streptococcus mutans Biofilm formation Dental caries Glucosyltransferase (GTF) Alternative sweetener Galactose Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Dental caries is a major global health issue associated with oral microorganisms that form dental plaque biofilms on tooth surfaces. Streptococcus mutans induces dental caries and is crucial for plaque formation through acid production, acid tolerance, and the synthesis of extracellular water-insoluble glucans. S. mutans converts dietary sucrose into water-insoluble glucans via glucosyltransferases, enhancing plaque pathogenicity and bacterial adherence (for a review, see [1]). Glucosyltransferases (GTF) in S. mutans , encoded by gtfB and gtfC , specifically GTF-B (165.8 kDa) and GTF-C (153 kDa), respectively, catalyze water-insoluble glucan synthesis from sucrose, thereby promoting bacterial adherence and biofilm formation [2]. Thus, regulation of glucan synthesis in S. mutans effectively reduces the risk of dental caries. Xylitol, a sugar alcohol and natural sweetener with sweetness comparable to that of sucrose, was not metabolized by S. mutans . It demonstrates caries-preventive effects through its bactericidal properties and suppression of biofilm formation [3]. However, concerns remain regarding the health implications of prolonged xylitol consumption and potential emergence of bacteria-resistant strains [4]. Identifying effective alternative sweeteners may help reduce the incidence of dental cavities and offer a cost-effective approach for improving global oral health. Isofloridoside, featuring an α-1,1 bond between galactose and D- or L-glycerol, has been isolated from Lagenandra undulata and assists in the osmotic acclimation of marine algae [5]. It exhibits high antioxidant activity and activates the sweet taste receptor taste 1 receptor members 2 and 3 [6]. Although galactose and raffinose inhibit S. mutans GTF activity and biofilm formation [7,8], the effects of isofloridoside on the oral microbiome remain unclear. Based on its unique α-1,1 bond structure, this study experimentally investigated the potential of isofloridoside to inhibit S. mutans . MATERIALS AND METHODS Extraction and purification of isofloridoside Isofloridoside was extracted with pure water from dried sheets of nori ( Pyropia yezoensis ), the raw material of which was cultivated in the Ariake Sea, Saga, Japan. The extract was subjected to lyophilization, ion-exchange column chromatography, and ultrafiltration to obtain the purified crude product. Crystallization of the product in ethanol yielded purified isofloridoside crystals (> 95% yield). The structure and purity of isofloridoside were confirmed using gas chromatography-mass spectrometry, time-of-flight mass spectrometry, and 1 H- and 13 C-nuclear magnetic resonance analyses. Bacterial strains and culture conditions Bacterial cultures were maintained in brain-heart infusion broth (Becton Biosciences, San Jose, CA, USA). S. mutans UA159, NBRC13955, and nine clinical isolates [9] were grown at 37°C for 20 h in 5% CO 2 . Semi-defined minimal medium (SDM) [10] was used to culture cells for growth assays. Biofilm formation was assessed using tryptic soy broth without dextrose (TSB). Isofloridoside and commercially available D(+)-galactose, D(+)-glucose, glycerol, melibiose, xylitol, and sucrose (Fujifilm Wako Pure Chemicals, Osaka, Japan) were added to the medium. Growth assay Aliquots (10 mL) of overnight S. mutans cultures were centrifuged at 11,200 × g for 5 min, and the pellets were resuspended in 2 mL of sterile distilled water. An aliquot (75 µL) was inoculated into 2.5 mL of SDM with each carbon source. Bacterial growth was measured at a wavelength of 600 nm. The number of viable cells was determined by diluting the culture medium with phosphate-buffered saline (pH 7.2) after 48 h in SDM containing 1% (w/v) isofloridoside and applying it to brain-heart infusion agar. Colonies on the agar medium were counted after overnight incubation. The number of viable cells in SDM was evaluated as colony-forming units. Biofilm formation assay Biofilm formation was quantified by assessing the cell adherence to 96-well polystyrene microtiter plates (Sumitomo Bakelite, Tokyo, Japan). An aliquot of S. mutans overnight culture (2 µL) and 100 µL of 2× TSB with 0.5% (w/v) sucrose were added to each well, and sterile distilled water was added to a final volume of 200 µL. The plates were incubated for 24 h. After incubation, the plates were washed with distilled water, and the adherent cells were stained with 0.01% (w/v) crystal violet. The dye was solubilized in 33% (v/v) acetic acid, and the A 590 was determined using a microplate reader (Colona Electric, Ibaraki, Japan). The biofilm formation rate was expressed as a relative value, with the control (sterile distilled water) value set to 1. Measurement of GTF inhibitory activities GTF activity was assessed by measuring the quantity of insoluble glucan produced from crude purified GTF, using sucrose as the substrate. A crude enzyme solution containing GTF from S. mutans was prepared as described previously [11]. The solution (1.0 mg protein/mL in 1% (w/v) sucrose) was incubated at 37°C for 1 h. The solution was inactivated by incubation at 90°C for 15 min, and then centrifuged at 10,000 × g for 10 min at 4°C. The precipitate was washed with sterile water and ethanol and then dried. The insoluble fraction was treated with 0.5 M sodium hydroxide at 37°C for 1 h. After centrifugation, insoluble glucan content was determined as glucose equivalents using the phenol-sulfuric acid method. The GTF inhibitory activity was expressed relative to that of insoluble glucans in sterile distilled water. Statistical analysis The means of at least three independent experiments were calculated along with their standard deviations. Data collected for analysis between the two groups were subjected to a t-test. RESULTS Effect of isofloridoside on the growth of S. mutans To evaluate the effect of isofloridoside on S. mutans , a concentration range of 1–5% (w/v) was selected, which is consistent with the range used in similar evaluations of galactose [7]. S. mutans UA159 could not proliferate when isofloridoside was provided as the sole carbon source at concentrations up to 5% (w/v) for 48 h (Fig. 1 A). Melibiose induced growth after 24 h of incubation, with a longer lag time than galactose or glucose. Glycerol did not induce cell growth after 48 h of incubation. S. mutans UA159, NBRC13955, and the nine clinical isolates showed no significant changes in viable cell counts after 48 h of treatment with 1% (w/v) isofloridoside, except for strains C2-1 and C8-1 (Fig. 1 B). Effect of isofloridoside on biofilm formation by S. mutans We explored the effects of isofloridoside on biofilm formation, which is crucial for the development of dental caries. To study the effects of saccharides and sugar alcohols on sucrose-dependent biofilm formation by S. mutans , 0.25% (w/v) sucrose was added to the medium. Figure 2 shows the significant concentration-dependent inhibitory effect of 1–5% (w/v) isofloridoside, similar to that of galactose and glucose, but unlike that of melibiose. Glycerol predominantly suppressed biofilm formation at a concentration of 1% (w/v); however, its effect was limited. Isofloridoside exhibited effects comparable to those of xylitol, another sugar substitute previously investigated for its ability to inhibit biofilm formation. We assessed the effect of isofloridoside on GTF, which is involved in biofilm formation. Isofloridoside exhibited strong concentration-dependent GTF inhibition at 1–5% (w/v), similar to galactose (Fig. 3 ). Melibiose, glucose, and glycerol also inhibited GTF activity but were less effective than isofloridoside and galactose. DISCUSSION The inhibitory effects of isofloridoside on S. mutans biofilm formation and GTF activity suggest its potential as a novel caries-preventive agent. Isofluoridoside inhibited S. mutans biofilm formation, which was comparable to that of known alternatives, such as xylitol. In this study, we explored the mechanisms underlying this phenomenon. α-Galactosidase from S. mutans , encoded by agaL (previously SMU_877), is a tetramer with a molecular weight of approximately 80,000 Da [12]. It belongs to the GH36 family and specifically targets α-1,6 galactosides, such as melibiose and raffinose [13]. No bacterial GH36 α-galactosidases acted on α-1,1 galactoside substrates, suggesting that this enzyme does not process α-1,1 linkages. Given that isofloridoside did not affect the growth of S. mutans , its effect on biofilm formation should be evaluated to understand its broad impact on caries prevention. Our findings support previous studies on the role of galactose in biofilm inhibition, highlighting the differential effects of isofloridoside and the need for further research on galactose-containing compounds. Rue et al . [7] reported significant biofilm inhibition by S. mutans at D-galactose concentrations ranging from 2–200 mM. Similar to d-galactose, raffinose inhibits biofilm formation, GTF gene expression, and glucan production [8]. Molecular docking suggests that galactose interacts with the Gtf-C active site in S. mutans [8]. The stronger inhibitory effect of isofloridoside on GTF activity may be attributed to its structural affinity for the active site of the enzyme, warranting further investigation using molecular docking. Isofloridoside, which contains galactose at the non-reducing end, similar to melibiose, inhibited biofilm formation in the presence of sucrose, which was linked to the inhibition of GTF activity. Galactose and its polymers are promising S. mutans biofilm regulators. However, melibiose, which is composed of galactose and glucose, was less effective than isofloridoside in inhibiting biofilm formation (Fig. 2 ). Biofilm formation by S. mutans is affected by factors such as changes in gene expression and cell surface structural modifications [1]. Studies on the effects of galactose and galactose-containing sugars on biofilm formation should evaluate multiple aspects beyond the inhibition of GTF activity. This study advances our understanding of galactose and its polysaccharides in S. mutans , potentially leading to new caries-preventive applications. Future research should explore the structure-activity relationships of galactose-containing compounds to optimize biofilm inhibition and investigate their synergistic effects with oral care products. The findings of this study indicated that isofloridoside did not significantly reduce the number of viable bacteria in S. mutans , except for certain strains (Fig. 1 B). Consequently, the potential to develop bacterial resistance to isofloridoside after prolonged use was considered minimal. Although our findings suggest the potential of isofloridoside for caries prevention, assessing its taste, safety, and economic feasibility is essential for determining its viability as an alternative sweetener. Future studies should examine the sensory properties of isofloridoside, conduct long-term safety assessments, and analyze its cost-effectiveness to evaluate its potential as a caries-preventive sweetener. Limitation of the study 1. Limited bacterial analysis: The effects were examined only in S. mutans and not in other oral bacteria. 2. Incomplete melibiose analysis: The effects of melibiose on S. mutans biofilm formation have been comprehensively studied. 3. Long-term safety data: The safety of prolonged isofloridoside use has not yet been evaluated. 4. Limited mechanisms: Inhibitory effects were observed; however, the underlying molecular mechanisms were unclear. 5. In vitro focus: May not fully represent the oral environment. 6. Limited strains: Testing may not capture full clinical strain diversity. 7. Lack of comparative data: Limited comparison with other anti-caries agents. 8. Economic aspects: Cost-effectiveness and production potential were not evaluated. 9. Sensory properties: Taste and palatability were not assessed in the present study. 10. Resistance potential: Long-term studies on bacterial resistance have not yet been conducted. Abbreviations GTF: Glucosyltransferase S. mutans : Streptococcus mutans Declarations Ethics approval and consent to participate not applicable Consent for publication not applicable Availability of data and materials not applicable Competing interests The authors declare that they have no competing interests Funding This work was partially funded by the Grants-in-Aid for the Development of Scientific Research (No. 22K02143) from the Ministry of Education, Science, and Culture of Japan. Authors' contributions The authors declare that they have no conflicts of interest. The funders had no role in the study design, collection, analysis, interpretation of the data, writing of the manuscript, or decision to publish the results. Acknowledgements not applicable References Lemos JA, Palmer SR, Zeng L, Wen ZT, Kajfasz JK, Freires IA, et al. The biology of Streptococcus mutans . Microbiol Spectr. 2019;7. Ren Z, Chen L, Li J, Li Y. Inhibition of Streptococcus mutans polysaccharide synthesis by molecules targeting glycosyltransferase activity. J Oral Microbiol. 2016;8:31095. Gasmi Benahmed A, Gasmi A, Arshad M, Shanaida M, Lysiuk R, Peana M, et al. Health benefits of xylitol. Appl Microbiol Biotechnol. 2020;104:7225–37. Marttinen AM, Ruas-Madiedo P, Hidalgo-Cantabrana C, Saari MA, Ihalin RA, Söderling EM. Effects of xylitol on xylitol-sensitive versus xylitol-resistant Streptococcus mutans strains in a three-species in vitro biofilm. Curr Microbiol. 2012;65:237–43. Li Y-X, Li Y, Lee S-H, Qian Z-J, Kim S-K. Inhibitors of oxidation and matrix metalloproteinases, floridoside, and D-isofloridoside from marine red alga Laurencia undulata . J Agric Food Chem. 2010;58:578–86. Akishino M, Aoki Y, Baba H, Asakawa M, Hama Y, Mitsutake S. Red algae-derived isofloridoside activates the sweet taste receptor T1R2/T1R3. Food Biosci. 2022;50:102186. Ryu E-J, An S-J, Sim J, Sim J, Lee J, Choi B-K. Use of d-galactose to regulate biofilm growth of oral streptococci. Arch Oral Biol. 2020;111:104666. Ham S-Y, Kim H-S, Cha E, Lim T, Byun Y, Park H-D. Raffinose inhibits Streptococcus mutans biofilm formation by targeting glucosyltransferase. Microbiol Spectr. 2022;10:e0207621. Kimijima M, Narisawa N, Hori E, Mandokoro K, Ito T, Ota Y, et al. Nattokinase, a subtilisin-like alkaline-serine protease, reduces mutacin activity by inactivating the competence-stimulating peptide in Streptococcus mutans . Pathogens. 2024;13:286. Li Y-H, Tang N, Aspiras MB, Lau PCY, Lee JH, Ellen RP, et al. A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation. J Bacteriol. 2002;184:2699–708. Goto I, Saga S, Ichitani M, Kimijima M, Narisawa N. Investigation of components in roasted green tea that inhibit Streptococcus mutans biofilm formation. Foods. 2023;12:2502. Aduse-Opoku J, Tao L, Ferretti JJ, Russell RRB. Biochemical and genetic analysis of Streptococcus mutans α-galactosidase. J Gen Microbiol. 1991;137:757–64. Cervera-Tison M, Tailford LE, Fuell C, Bruel L, Sulzenbacher G, Henrissat B, et al. Functional analysis of family GH36 α-galactosidases from Ruminococcus gnavus E1: insights into the metabolism of a plant oligosaccharide by a human gut symbiont. Appl Environ Microbiol. 2012;78:7720–32. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 06 Aug, 2025 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Revision requested 20 Jun, 2025 Reviews received at journal 03 Jun, 2025 Reviews received at journal 16 May, 2025 Reviewers agreed at journal 15 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers invited by journal 14 May, 2025 Editor invited by journal 02 May, 2025 Editor assigned by journal 01 May, 2025 Submission checks completed at journal 01 May, 2025 First submitted to journal 30 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6568825","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":457432166,"identity":"064b41c0-6376-4f42-9542-ffd8eedaf520","order_by":0,"name":"Manami Kimijima","email":"","orcid":"","institution":"Nihon University Graduate School of Bioresource Sciences, Bioresource Utilization Sciences","correspondingAuthor":false,"prefix":"","firstName":"Manami","middleName":"","lastName":"Kimijima","suffix":""},{"id":457432167,"identity":"e809c7a3-e0ac-46c9-b96d-ef93a535f915","order_by":1,"name":"Naoki Narisawa","email":"data:image/png;base64,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","orcid":"","institution":"Nihon University Graduate School of Bioresource Sciences, Bioresource Utilization Sciences","correspondingAuthor":true,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Narisawa","suffix":""},{"id":457432168,"identity":"d2fc53f8-11c4-4a71-9bb0-a66f4c31966c","order_by":2,"name":"Yoichiro Hama","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Yoichiro","middleName":"","lastName":"Hama","suffix":""},{"id":457432169,"identity":"1de41fc5-6401-4e6b-a4db-469bc902b6a8","order_by":3,"name":"Tomoyo Nakagawa-Nakamura","email":"","orcid":"","institution":"Tokyo University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Tomoyo","middleName":"","lastName":"Nakagawa-Nakamura","suffix":""},{"id":457432170,"identity":"b2a06ce8-db2b-4743-a1d6-a8beb6cfba46","order_by":4,"name":"Tatsuro Ito","email":"","orcid":"","institution":"Nihon University School of Dentistry at Matsudo","correspondingAuthor":false,"prefix":"","firstName":"Tatsuro","middleName":"","lastName":"Ito","suffix":""}],"badges":[],"createdAt":"2025-05-01 03:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6568825/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6568825/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-025-07408-8","type":"published","date":"2025-08-06T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83042883,"identity":"c8de732d-bc50-4dfa-a04d-56722e767351","added_by":"auto","created_at":"2025-05-19 11:04:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of isofloridoside and other carbohydrates on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. mutans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The growth curve of \u003cem\u003eS. mutans\u003c/em\u003e UA159 is depicted with squares, circles, triangles, and diamonds representing the control and 1% (w/v), 2.5% (w/v), and 5.0% (w/v) treatments, respectively. The incubation period was either 48 or 34 h. The experiments were conducted independently on three occasions, and data from a representative trial are presented.\u003c/p\u003e\n\u003cp\u003e(B) The effect of isofloridoside on \u003cem\u003eS. mutans\u003c/em\u003e UA159, NBRC13955, and nine clinical isolates was assessed by viable cell counts. These counts were expressed as log CFU/mL after 48 h of incubation. Black bars denote the control, while gray bars represent 1% (w/v) isofloridoside. The experiments were independently conducted three times, with error bars indicating standard deviations. Different letters indicate significant differences based on the standard t-test between the two groups (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Kimijimaetal.Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6568825/v1/231c6c5cd00c9c10ae112822.png"},{"id":83042884,"identity":"9f8d2fed-4838-48aa-9404-f04bd7b9f82d","added_by":"auto","created_at":"2025-05-19 11:04:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of isofloridoside and other carbohydrates on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. mutans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiofilm levels were quantified as relative values, with the control value standardized at 1. The light gray, dark gray, and black bars represent 1%, 2.5%, and 5% (w/v) addition of isofloridoside and the other carbohydrates, respectively. The experiments were independently conducted three times, with error bars indicating standard deviation. Different letters indicate significant differences based on the standard t-test between two groups (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Kimijimaetal.Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6568825/v1/1b2de9973f9745a0da186ee1.png"},{"id":83043732,"identity":"6bbe6c73-ef90-4ff9-8a34-d3b9a9ace1df","added_by":"auto","created_at":"2025-05-19 11:12:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of isofloridoside and other carbohydrates on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. mutans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e GTF activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGTF activity was quantified as a relative value, with the control value standardized to 1. The light gray, dark gray, and black bars represent 1%, 2.5%, and 5% (w/v) addition of isofloridoside and the other carbohydrates, respectively. The experiments were independently conducted three times, with error bars indicating standard deviation.\u003c/p\u003e\n\u003cp\u003eDifferent letters indicate significant differences based on the standard t-test between two groups (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Kimijimaetal.Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6568825/v1/dce1ff1cbe38b4992505cbf2.png"},{"id":88814184,"identity":"c65b4c4f-a520-403b-ac3d-5934ae305141","added_by":"auto","created_at":"2025-08-11 16:08:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":660706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6568825/v1/b7c49e87-3bfd-4b81-afa3-1c93ddade36f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isofloridoside: A Novel Inhibitor of Streptococcus mutans Biofilm Formation and Glucosyltransferase Activity","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDental caries is a major global health issue associated with oral microorganisms that form dental plaque biofilms on tooth surfaces. \u003cem\u003eStreptococcus mutans\u003c/em\u003e induces dental caries and is crucial for plaque formation through acid production, acid tolerance, and the synthesis of extracellular water-insoluble glucans. \u003cem\u003eS. mutans\u003c/em\u003e converts dietary sucrose into water-insoluble glucans via glucosyltransferases, enhancing plaque pathogenicity and bacterial adherence (for a review, see [1]). Glucosyltransferases (GTF) in \u003cem\u003eS. mutans\u003c/em\u003e, encoded by \u003cem\u003egtfB\u003c/em\u003e and \u003cem\u003egtfC\u003c/em\u003e, specifically GTF-B (165.8 kDa) and GTF-C (153 kDa), respectively, catalyze water-insoluble glucan synthesis from sucrose, thereby promoting bacterial adherence and biofilm formation [2]. Thus, regulation of glucan synthesis in \u003cem\u003eS. mutans\u003c/em\u003e effectively reduces the risk of dental caries.\u003c/p\u003e \u003cp\u003eXylitol, a sugar alcohol and natural sweetener with sweetness comparable to that of sucrose, was not metabolized by \u003cem\u003eS. mutans\u003c/em\u003e. It demonstrates caries-preventive effects through its bactericidal properties and suppression of biofilm formation [3]. However, concerns remain regarding the health implications of prolonged xylitol consumption and potential emergence of bacteria-resistant strains [4]. Identifying effective alternative sweeteners may help reduce the incidence of dental cavities and offer a cost-effective approach for improving global oral health.\u003c/p\u003e \u003cp\u003eIsofloridoside, featuring an α-1,1 bond between galactose and D- or L-glycerol, has been isolated from \u003cem\u003eLagenandra undulata\u003c/em\u003e and assists in the osmotic acclimation of marine algae [5]. It exhibits high antioxidant activity and activates the sweet taste receptor taste 1 receptor members 2 and 3 [6]. Although galactose and raffinose inhibit \u003cem\u003eS. mutans\u003c/em\u003e GTF activity and biofilm formation [7,8], the effects of isofloridoside on the oral microbiome remain unclear. Based on its unique α-1,1 bond structure, this study experimentally investigated the potential of isofloridoside to inhibit \u003cem\u003eS. mutans\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExtraction and purification of isofloridoside\u003c/h2\u003e \u003cp\u003eIsofloridoside was extracted with pure water from dried sheets of nori (\u003cem\u003ePyropia yezoensis\u003c/em\u003e), the raw material of which was cultivated in the Ariake Sea, Saga, Japan. The extract was subjected to lyophilization, ion-exchange column chromatography, and ultrafiltration to obtain the purified crude product. Crystallization of the product in ethanol yielded purified isofloridoside crystals (\u0026gt;\u0026thinsp;95% yield). The structure and purity of isofloridoside were confirmed using gas chromatography-mass spectrometry, time-of-flight mass spectrometry, and \u003csup\u003e1\u003c/sup\u003eH- and \u003csup\u003e13\u003c/sup\u003eC-nuclear magnetic resonance analyses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial strains and culture conditions\u003c/h3\u003e\n\u003cp\u003eBacterial cultures were maintained in brain-heart infusion broth (Becton Biosciences, San Jose, CA, USA). \u003cem\u003eS. mutans\u003c/em\u003e UA159, NBRC13955, and nine clinical isolates [9] were grown at 37\u0026deg;C for 20 h in 5% CO\u003csub\u003e2\u003c/sub\u003e. Semi-defined minimal medium (SDM) [10] was used to culture cells for growth assays. Biofilm formation was assessed using tryptic soy broth without dextrose (TSB). Isofloridoside and commercially available D(+)-galactose, D(+)-glucose, glycerol, melibiose, xylitol, and sucrose (Fujifilm Wako Pure Chemicals, Osaka, Japan) were added to the medium.\u003c/p\u003e\n\u003ch3\u003eGrowth assay\u003c/h3\u003e\n\u003cp\u003eAliquots (10 mL) of overnight \u003cem\u003eS. mutans\u003c/em\u003e cultures were centrifuged at 11,200 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min, and the pellets were resuspended in 2 mL of sterile distilled water. An aliquot (75 \u0026micro;L) was inoculated into 2.5 mL of SDM with each carbon source. Bacterial growth was measured at a wavelength of 600 nm. The number of viable cells was determined by diluting the culture medium with phosphate-buffered saline (pH 7.2) after 48 h in SDM containing 1% (w/v) isofloridoside and applying it to brain-heart infusion agar. Colonies on the agar medium were counted after overnight incubation. The number of viable cells in SDM was evaluated as colony-forming units.\u003c/p\u003e\n\u003ch3\u003eBiofilm formation assay\u003c/h3\u003e\n\u003cp\u003eBiofilm formation was quantified by assessing the cell adherence to 96-well polystyrene microtiter plates (Sumitomo Bakelite, Tokyo, Japan). An aliquot of \u003cem\u003eS. mutans\u003c/em\u003e overnight culture (2 \u0026micro;L) and 100 \u0026micro;L of 2\u0026times; TSB with 0.5% (w/v) sucrose were added to each well, and sterile distilled water was added to a final volume of 200 \u0026micro;L. The plates were incubated for 24 h. After incubation, the plates were washed with distilled water, and the adherent cells were stained with 0.01% (w/v) crystal violet. The dye was solubilized in 33% (v/v) acetic acid, and the A\u003csub\u003e590\u003c/sub\u003e was determined using a microplate reader (Colona Electric, Ibaraki, Japan). The biofilm formation rate was expressed as a relative value, with the control (sterile distilled water) value set to 1.\u003c/p\u003e\n\u003ch3\u003eMeasurement of GTF inhibitory activities\u003c/h3\u003e\n\u003cp\u003eGTF activity was assessed by measuring the quantity of insoluble glucan produced from crude purified GTF, using sucrose as the substrate. A crude enzyme solution containing GTF from \u003cem\u003eS. mutans\u003c/em\u003e was prepared as described previously [11]. The solution (1.0 mg protein/mL in 1% (w/v) sucrose) was incubated at 37\u0026deg;C for 1 h. The solution was inactivated by incubation at 90\u0026deg;C for 15 min, and then centrifuged at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. The precipitate was washed with sterile water and ethanol and then dried. The insoluble fraction was treated with 0.5 M sodium hydroxide at 37\u0026deg;C for 1 h. After centrifugation, insoluble glucan content was determined as glucose equivalents using the phenol-sulfuric acid method. The GTF inhibitory activity was expressed relative to that of insoluble glucans in sterile distilled water.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe means of at least three independent experiments were calculated along with their standard deviations. Data collected for analysis between the two groups were subjected to a t-test.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEffect of isofloridoside on the growth of\u003c/b\u003e \u003cb\u003eS. mutans\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the effect of isofloridoside on \u003cem\u003eS. mutans\u003c/em\u003e, a concentration range of 1\u0026ndash;5% (w/v) was selected, which is consistent with the range used in similar evaluations of galactose [7]. \u003cem\u003eS. mutans\u003c/em\u003e UA159 could not proliferate when isofloridoside was provided as the sole carbon source at concentrations up to 5% (w/v) for 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Melibiose induced growth after 24 h of incubation, with a longer lag time than galactose or glucose. Glycerol did not induce cell growth after 48 h of incubation. \u003cem\u003eS. mutans\u003c/em\u003e UA159, NBRC13955, and the nine clinical isolates showed no significant changes in viable cell counts after 48 h of treatment with 1% (w/v) isofloridoside, except for strains C2-1 and C8-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of isofloridoside on biofilm formation by\u003c/b\u003e \u003cb\u003eS. mutans\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe explored the effects of isofloridoside on biofilm formation, which is crucial for the development of dental caries. To study the effects of saccharides and sugar alcohols on sucrose-dependent biofilm formation by \u003cem\u003eS. mutans\u003c/em\u003e, 0.25% (w/v) sucrose was added to the medium. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the significant concentration-dependent inhibitory effect of 1\u0026ndash;5% (w/v) isofloridoside, similar to that of galactose and glucose, but unlike that of melibiose. Glycerol predominantly suppressed biofilm formation at a concentration of 1% (w/v); however, its effect was limited. Isofloridoside exhibited effects comparable to those of xylitol, another sugar substitute previously investigated for its ability to inhibit biofilm formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe assessed the effect of isofloridoside on GTF, which is involved in biofilm formation. Isofloridoside exhibited strong concentration-dependent GTF inhibition at 1\u0026ndash;5% (w/v), similar to galactose (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Melibiose, glucose, and glycerol also inhibited GTF activity but were less effective than isofloridoside and galactose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe inhibitory effects of isofloridoside on \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation and GTF activity suggest its potential as a novel caries-preventive agent. Isofluoridoside inhibited \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation, which was comparable to that of known alternatives, such as xylitol. In this study, we explored the mechanisms underlying this phenomenon. α-Galactosidase from \u003cem\u003eS. mutans\u003c/em\u003e, encoded by \u003cem\u003eagaL\u003c/em\u003e (previously SMU_877), is a tetramer with a molecular weight of approximately 80,000 Da [12]. It belongs to the GH36 family and specifically targets α-1,6 galactosides, such as melibiose and raffinose [13]. No bacterial GH36 α-galactosidases acted on α-1,1 galactoside substrates, suggesting that this enzyme does not process α-1,1 linkages.\u003c/p\u003e \u003cp\u003eGiven that isofloridoside did not affect the growth of \u003cem\u003eS. mutans\u003c/em\u003e, its effect on biofilm formation should be evaluated to understand its broad impact on caries prevention. Our findings support previous studies on the role of galactose in biofilm inhibition, highlighting the differential effects of isofloridoside and the need for further research on galactose-containing compounds. Rue \u003cem\u003eet al\u003c/em\u003e. [7] reported significant biofilm inhibition by \u003cem\u003eS. mutans\u003c/em\u003e at D-galactose concentrations ranging from 2\u0026ndash;200 mM. Similar to d-galactose, raffinose inhibits biofilm formation, GTF gene expression, and glucan production [8]. Molecular docking suggests that galactose interacts with the Gtf-C active site in \u003cem\u003eS. mutans\u003c/em\u003e [8]. The stronger inhibitory effect of isofloridoside on GTF activity may be attributed to its structural affinity for the active site of the enzyme, warranting further investigation using molecular docking.\u003c/p\u003e \u003cp\u003eIsofloridoside, which contains galactose at the non-reducing end, similar to melibiose, inhibited biofilm formation in the presence of sucrose, which was linked to the inhibition of GTF activity. Galactose and its polymers are promising \u003cem\u003eS. mutans\u003c/em\u003e biofilm regulators. However, melibiose, which is composed of galactose and glucose, was less effective than isofloridoside in inhibiting biofilm formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Biofilm formation by \u003cem\u003eS. mutans\u003c/em\u003e is affected by factors such as changes in gene expression and cell surface structural modifications [1]. Studies on the effects of galactose and galactose-containing sugars on biofilm formation should evaluate multiple aspects beyond the inhibition of GTF activity. This study advances our understanding of galactose and its polysaccharides in \u003cem\u003eS. mutans\u003c/em\u003e, potentially leading to new caries-preventive applications. Future research should explore the structure-activity relationships of galactose-containing compounds to optimize biofilm inhibition and investigate their synergistic effects with oral care products.\u003c/p\u003e \u003cp\u003eThe findings of this study indicated that isofloridoside did not significantly reduce the number of viable bacteria in \u003cem\u003eS. mutans\u003c/em\u003e, except for certain strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consequently, the potential to develop bacterial resistance to isofloridoside after prolonged use was considered minimal. Although our findings suggest the potential of isofloridoside for caries prevention, assessing its taste, safety, and economic feasibility is essential for determining its viability as an alternative sweetener. Future studies should examine the sensory properties of isofloridoside, conduct long-term safety assessments, and analyze its cost-effectiveness to evaluate its potential as a caries-preventive sweetener.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLimitation of the study\u003c/b\u003e \u003c/p\u003e \u003cp\u003e1. Limited bacterial analysis: The effects were examined only in \u003cem\u003eS. mutans\u003c/em\u003e and not in other oral bacteria. 2. Incomplete melibiose analysis: The effects of melibiose on \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation have been comprehensively studied. 3. Long-term safety data: The safety of prolonged isofloridoside use has not yet been evaluated. 4. Limited mechanisms: Inhibitory effects were observed; however, the underlying molecular mechanisms were unclear. 5. In vitro focus: May not fully represent the oral environment. 6. Limited strains: Testing may not capture full clinical strain diversity. 7. Lack of comparative data: Limited comparison with other anti-caries agents. 8. Economic aspects: Cost-effectiveness and production potential were not evaluated. 9. Sensory properties: Taste and palatability were not assessed in the present study. 10. Resistance potential: Long-term studies on bacterial resistance have not yet been conducted.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGTF: Glucosyltransferase\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. mutans\u003c/em\u003e: \u003cem\u003eStreptococcus mutans\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially funded by the Grants-in-Aid for the Development of Scientific Research (No. 22K02143) from the Ministry of Education, Science, and Culture of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest. The funders had no role in the study design, collection, analysis, interpretation of the data, writing of the manuscript, or decision to publish the results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLemos JA, Palmer SR, Zeng L, Wen ZT, Kajfasz JK, Freires IA, et al. The biology of \u003cem\u003eStreptococcus mutans\u003c/em\u003e. Microbiol Spectr. 2019;7.\u003c/li\u003e\n\u003cli\u003eRen Z, Chen L, Li J, Li Y. Inhibition of \u003cem\u003eStreptococcus mutans\u003c/em\u003e polysaccharide synthesis by molecules targeting glycosyltransferase activity. J Oral Microbiol. 2016;8:31095.\u003c/li\u003e\n\u003cli\u003eGasmi Benahmed A, Gasmi A, Arshad M, Shanaida M, Lysiuk R, Peana M, et al. Health benefits of xylitol. Appl Microbiol Biotechnol. 2020;104:7225\u0026ndash;37.\u003c/li\u003e\n\u003cli\u003eMarttinen AM, Ruas-Madiedo P, Hidalgo-Cantabrana C, Saari MA, Ihalin RA, S\u0026ouml;derling EM. Effects of xylitol on xylitol-sensitive versus xylitol-resistant \u003cem\u003eStreptococcus mutans\u003c/em\u003e strains in a three-species in vitro biofilm. Curr Microbiol. 2012;65:237\u0026ndash;43.\u003c/li\u003e\n\u003cli\u003eLi Y-X, Li Y, Lee S-H, Qian Z-J, Kim S-K. Inhibitors of oxidation and matrix metalloproteinases, floridoside, and D-isofloridoside from marine red alga \u003cem\u003eLaurencia undulata\u003c/em\u003e. J Agric Food Chem. 2010;58:578\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eAkishino M, Aoki Y, Baba H, Asakawa M, Hama Y, Mitsutake S. Red algae-derived isofloridoside activates the sweet taste receptor T1R2/T1R3. Food Biosci. 2022;50:102186.\u003c/li\u003e\n\u003cli\u003eRyu E-J, An S-J, Sim J, Sim J, Lee J, Choi B-K. Use of d-galactose to regulate biofilm growth of oral streptococci. Arch Oral Biol. 2020;111:104666.\u003c/li\u003e\n\u003cli\u003eHam S-Y, Kim H-S, Cha E, Lim T, Byun Y, Park H-D. Raffinose inhibits \u003cem\u003eStreptococcus mutans\u003c/em\u003e biofilm formation by targeting glucosyltransferase. Microbiol Spectr. 2022;10:e0207621.\u003c/li\u003e\n\u003cli\u003eKimijima M, Narisawa N, Hori E, Mandokoro K, Ito T, Ota Y, et al. Nattokinase, a subtilisin-like alkaline-serine protease, reduces mutacin activity by inactivating the competence-stimulating peptide in \u003cem\u003eStreptococcus mutans\u003c/em\u003e. Pathogens. 2024;13:286.\u003c/li\u003e\n\u003cli\u003eLi Y-H, Tang N, Aspiras MB, Lau PCY, Lee JH, Ellen RP, et al. A quorum-sensing signaling system essential for genetic competence in \u003cem\u003eStreptococcus mutans\u003c/em\u003e is involved in biofilm formation. J Bacteriol. 2002;184:2699\u0026ndash;708.\u003c/li\u003e\n\u003cli\u003eGoto I, Saga S, Ichitani M, Kimijima M, Narisawa N. Investigation of components in roasted green tea that inhibit \u003cem\u003eStreptococcus mutans\u003c/em\u003e biofilm formation. Foods. 2023;12:2502.\u003c/li\u003e\n\u003cli\u003eAduse-Opoku J, Tao L, Ferretti JJ, Russell RRB. Biochemical and genetic analysis of \u003cem\u003eStreptococcus mutans\u003c/em\u003e\u0026alpha;-galactosidase. J Gen Microbiol. 1991;137:757\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eCervera-Tison M, Tailford LE, Fuell C, Bruel L, Sulzenbacher G, Henrissat B, et al. Functional analysis of family GH36 \u0026alpha;-galactosidases from \u003cem\u003eRuminococcus gnavus\u003c/em\u003e E1: insights into the metabolism of a plant oligosaccharide by a human gut symbiont. Appl Environ Microbiol. 2012;78:7720\u0026ndash;32.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Isofloridoside, Streptococcus mutans, Biofilm formation, Dental caries, Glucosyltransferase (GTF), Alternative sweetener, Galactose","lastPublishedDoi":"10.21203/rs.3.rs-6568825/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6568825/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIsofloridoside, a galactose-containing heteroside derived from marine red algae, has potential applications as a sweetener because it can activate the sweet taste receptor taste 1 receptor members 2 and 3. This study investigated the effects of isofloridoside on the growth and sucrose-dependent biofilm formation of the cariogenic bacterium \u003cem\u003eStreptococcus mutans\u003c/em\u003e. The results showed that \u003cem\u003eS. mutans\u003c/em\u003e did not grow when isofloridoside was used as the sole carbon source. Isofloridoside inhibited sucrose-dependent biofilm formation by \u003cem\u003eS. mutans\u003c/em\u003e in a concentration-dependent manner, similar to galactose and glucose, but unlike melibiose, galactose-containing disaccharides. Biofilm inhibition induced by isofloridoside was associated with the inhibition of glucosyltransferase activity. Isofloridoside exhibited biofilm inhibition comparable to that of xylitol, an alternative sugar known to inhibit biofilm formation. The differential effects of isofloridoside and melibiose on biofilm formation may be due to structural differences that affect their interactions with \u003cem\u003eS. mutans\u003c/em\u003e enzymes. These findings highlight the potential of galactose and its polysaccharides as regulators of \u003cem\u003eS. mutans\u003c/em\u003e biofilm formation and suggest that isofloridoside is a promising alternative sweetener for caries prevention. Further research is required to elucidate the detailed mechanism of action, potential for resistance acquisition, taste, safety, and economic feasibility of isofloridoside as a caries-preventive agent.\u003c/p\u003e","manuscriptTitle":"Isofloridoside: A Novel Inhibitor of Streptococcus mutans Biofilm Formation and Glucosyltransferase Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 11:04:29","doi":"10.21203/rs.3.rs-6568825/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-20T08:06:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-03T18:26:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-16T06:25:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267701638318904640025599803471631456832","date":"2025-05-15T06:20:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133198286182064578072180406814471346494","date":"2025-05-15T02:30:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T01:47:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-02T09:38:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-01T22:40:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-01T22:37:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-05-01T03:18:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"453fec32-7b6b-4155-9690-3cd5ce5d655a","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T16:02:57+00:00","versionOfRecord":{"articleIdentity":"rs-6568825","link":"https://doi.org/10.1186/s13104-025-07408-8","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2025-08-06 15:57:48","publishedOnDateReadable":"August 6th, 2025"},"versionCreatedAt":"2025-05-19 11:04:29","video":"","vorDoi":"10.1186/s13104-025-07408-8","vorDoiUrl":"https://doi.org/10.1186/s13104-025-07408-8","workflowStages":[]},"version":"v1","identity":"rs-6568825","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6568825","identity":"rs-6568825","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}