Impact of Soy- and Milk-Based Yogurts on Enamel Preservation: Erosion and Abrasion under Simulated Conditions

Impact of Soy- and Milk-Based Yogurts on Enamel Preservation: Erosion and Abrasion under Simulated Conditions | 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 Research Article Impact of Soy- and Milk-Based Yogurts on Enamel Preservation: Erosion and Abrasion under Simulated Conditions Rafaela Ricci Kim, Aline Silva Braga, Gabriela Pellizon Floret, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9348858/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Objectives This study evaluated the effect of soy-based and milk-based yogurts against enamel erosive-abrasive surface loss. Materials and Methods Ninety-eight bovine enamel specimens were randomly allocated to seven experimental groups (n = 14). Over seven days, specimens underwent six daily erosive cycles (0.05M citric acid, pH 2.3, 2 min), interspersed with remineralizing solution (pH 6.5). Post-acid preventive measures consisted of immersion in milk-based yogurt (pH 4.4), soy-based yogurt (pH 4.7), or tap water for 3 min twice daily. Under erosion-abrasion conditions, specimens underwent simulated toothbrushing using tin-containing fluoride toothpaste. Fluoride-free toothpaste served as abrasion control. Enamel surface loss was measured after 3 and 7 days using three-dimensional laser scanning microscopy. Calcium and phosphorus contents were quantified by optical emission spectrometry. Results At day 3, no significant differences were observed among yogurt- and water-treated groups subjected to abrasion with fluoride toothpaste. By day 7, abrasion with a tin-containing fluoride toothpaste combined with soy-based yogurt resulted in significantly lower enamel surface loss (7.9±3.3µm) compared with the fluoride toothpaste only (12.2 ± 2.7µm; p <0.05). Under erosion-only conditions, no significant differences were detected among preventive measures. Milk-based yogurt presented significantly higher calcium and phosphorus contents (Ca: 1434.1±190.3µg/g; P: 1033.8±128.1 µg/g) than soy-based yogurt (Ca: 168.4±15.5 µg/g; P: 623.2±47.7 µg/g). Conclusions Fermented soy-based yogurt may offer adjunctive protection in patients exposed to erosive and abrasive challenges. Clinical relevance : Soy-based yogurt significantly enhances anti-erosive efficacy of fluoride-stannous formulations, providing clinicians with an evidence-based dietary intervention to optimize preventive strategies against erosive enamel loss. Abrasion Enamel erosion Erosive tooth wear Fluorides Soy-based yogurt Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Erosive tooth wear (ETW) is defined as the progressive and irreversible loss of dental hard tissues, mainly enamel and dentin, caused by the chemical action of acids without bacterial involvement. This process may be exacerbated by associated mechanical factors, such as abrasion, commonly related to toothbrushing, or attrition [ 1 – 3 ]. ETW is a clinical and conceptual term that describes the cumulative outcome of chemical erosion, often modified by mechanical processes. In experimental studies, this condition is commonly quantified as enamel surface loss or wear, which refers to the measurable irreversible loss of mineralized enamel tissue over time, typically expressed in micrometer [ 4 ]. The acids affecting dental structures may have an extrinsic origin, derived from dietary sources (soft drinks, citrus fruits, vinegars, among others), or an intrinsic origin, resulting from conditions such as gastroesophageal reflux or frequent vomiting [ 5 , 6 ]. Epidemiological studies have reported a high prevalence of this condition, estimated to range from 30% to 50% in primary dentition and from 20% to 45% in permanent dentition [ 7 , 8 ]. The progression of ETW may significantly impair patients’ quality of life, being associated with dentin hypersensitivity, functional impairments such as chewing difficulties, and esthetic concerns, including tooth discoloration and shortening, which may negatively affect self-esteem and smile appearance [ 9 ]. Given its multifactorial nature, the clinical management of ETW requires careful assessment of the chemical, biological, and behavioral factors involved, as well as the implementation of evidence-based preventive strategies [ 10 – 12 ]. Among the natural protective factors against erosion, saliva plays a fundamental role by neutralizing and clearing acids, promoting the formation of the acquired pellicle, and providing calcium and phosphate ions necessary for remineralization processes [ 13 ]. Similarly, certain foods, particularly dairy products such as milk and cheese, have been associated with protective effects on enamel, mainly attributed to their mineral, protein and lipidic content, in especial to calcium interactions with the dental surface [ 14 – 16 ]. Previous studies have demonstrated that, although acidic foods increase dental erosion, the consumption of milk and milk-based yogurt may attenuate their effect [ 16 , 17 ]. Yogurt presents properties comparable to those of milk due to the presence of calcium, phosphate, and proteins, especially casein at high concentration. These components may contribute to enamel remineralization and to the formation of a protective layer on the tooth surface [ 16 – 19 ]. Evidence indicates that casein and its derivatives, such as casein phosphopeptides (CPPs), can adsorb onto enamel, forming a physical barrier against acids and promoting the retention of calcium and phosphate ions, thereby reducing demineralization [ 20 , 21 ]. Concurrently, there has been a markable increase in the consumption of plant-based foods (vegetarian and vegan style of life), driven by changes in dietary habits and growing environmental and health concerns [ 22 – 26 ]. In this context, plant-based yogurts have gained attention as alternatives to traditional milk-yogurt products. Recent studies have indicated that plant-based yogurts may exhibit a protective effect on dental enamel, even in the absence of milk-derived proteins such as casein [ 27 , 28 ]. This protective potential has been suggested to be related to the physicochemical characteristics of these products, including mineral content, buffering capacity, and the food matrix, rather than exclusively to the presence of casein-derived peptides. These findings highlight the need to further evaluate the role of plant-based yogurt formulations in their interaction with dental hard tissues, particularly considering their increasing consumption [ 27 – 29 ]. The available evidence remains limited, particularly regarding the mechanisms involved and the interaction of these products with other preventive strategies commonly adopted in clinical practice, such as the use of fluoridated toothpastes. To the best of our knowledge, no study has evaluated the protective effect of soy-based yogurt in an erosion–abrasion model or its interaction with fluoridated toothpastes. Moreover, many previous studies have employed experimental models based on continuous acidic exposure or prolonged contact times with foods or beverages, which do not adequately reflect the clinical dynamics of oral cavity [ 30 – 33 ]. Clinically, erosive challenges are typically short and repeated, interspersed with periods of salivary recovery and often associated with toothbrushing abrasion. Consequently, it remains unclear whether the protective effects attributed to foods, such as yogurt, persist under more clinically relevant conditions, especially when erosion is combined with abrasion. Therefore, the present study aimed to evaluate the protective effects of milk-based and soy-based yogurts on enamel surface loss under erosive conditions alone and when combined with abrasion, with particular emphasis on their interaction with the use of a tin-containing fluoride toothpaste following erosive challenges. Materials and Methods Experimental Design The experimental protocol was designed to simulate clinical preventive recommendations for erosive tooth wear, namely the consumption of yogurt immediately after acidic food or beverage intake, followed by toothbrushing with a tin-containing fluoride toothpaste. This in vitro model enabled the systematic evaluation of post-acid exposure preventive measures under controlled erosive–abrasive cycling conditions. The experimental design is illustrated in Fig. 1 a. Sample Size Calculation A sample size of 14 specimens per group was determined based on data from Esteves-Oliveira et al. [ 34 ] In that study, the difference in enamel surface loss between the control group (27.22±4.1µm) and the tin-containing fluoride group (3.31±2.0 µm) was 23.91µm, with a pooled standard deviation of 3.23 µm, resulting in a standardized effect size (Cohen’s d) of approximately 7.41. Assuming an alpha level of 0.05 and a target power of 0.80, this contrast would require fewer than one specimen per group. Therefore, the allocation of 14 specimens per group provides statistical power exceeding 99% for this benchmark effect. Tooth Sample Preparation A total of 98 enamel specimens were prepared from permanent bovine incisors and sectioned into dimensions of 4 mm⋅4 mm using two diamond discs mounted on a IsoMet® Low Speed Precision Cutter (Buehler®, Lake Bluff, USA).Throughout the study, specimens were stored in a 0.1% thymol solution, and only specimens without visible cracks or structural defects were included. Enamel surfaces were planarized and polished under continuous water irrigation using a Micro Grinder 400 polishing machine (Exakt®, Norderstedt, Germany).Each specimen was fixed with sticky wax at the center of an acrylic plate and initially polished from the dentin side, followed by the enamel side, to obtain planar and parallel surfaces. Final enamel polishing was performed sequentially using 800, 1200, 2400, and 4000 grit silicon carbide papers under water cooling, followed by a 2 min polishing step with a felt disc and diamond suspension.Enamel reduction by polishing was monitored using a micrometer standardized not to exceed 400µm [ 34 ]. The absence of dentin exposure was confirmed using a 3D laser scanning microscope (VK-3000, Keyence®, Osaka, Japan). Specimens were subsequently cleaned in distilled water using an ultrasonic bath and stored in 0.1% thymol at 4°C until use [ 34 ]. To ensure that enamel surface loss was consistently measured at the same location throughout the experiment, two reference marks were engraved on each specimen using a spherical bur (No.1011) (Hager & Meisinger®, Neuss, Germany), positioned on the left and right sides of the experimental area. Two control areas on each specimen were protected with removable adhesive tape during the erosive–abrasive cycling and uncovered only during the measurement procedures. Experimental Groups Specimens were allocated into seven experimental groups to isolate the effects of yogurt type (milk-based vs. soy-based) and brushing abrasion (Table 1 ). The inclusion of a fluoride-free toothpaste group served to control for the well-established protective effect of tin-containing fluoride toothpastes against abrasion of eroded enamel surfaces. Table 1 Composition of yogurt products and toothpastes Product type Commercial name Composition Soy-based yogurt Rewe Vegan Soja Natur Water, soy (13.3%), modified starch, starch, sea salt, natural flavoring, yogurt cultures Milk-based yogurt Rewe Bio Yogurt Mild 3.8% fat Milk and yogurt cultures Toothpaste type Full composition Tin-containing fluoride toothpaste Elmex® Opti-namel (Colgate-Palmolive ® , Świdnica, Poland) Aqua, glycerin, sorbitol, hydrated silica, hydroxyethylcellulose, aroma, cocamidopropyl betaine, titanium dioxide, olaflur (AmF), sodium gluconate, stannous chloride, alumina, chitosan, sodium saccharin, sodium fluoride, potassium hydroxide, hydrochloric acid Fluoride-free toothpaste Nenedent® children's (Dentinox ® , Berlin, Germany) Aqua, hydrated silica, glycerin, xylitol, propylene glycol, xanthan gum, aroma, sodium lauroyl sarcosinate, disodium EDTA, sodium chloride, sodium hydroxide All experimental groups were subjected to the same erosive and remineralization protocol. The experimental variables were the presence or absence of brushing abrasion, the use of fluoride-containing or fluoride-free toothpaste (Table 1 ), and the type of post-acid treatment (milk-based yogurt, soy-based yogurt or water), as summarized in Table 2 . Table 2 Experimental groups and preventive conditions. Group Post-Acid Treatment Abrasion Toothpaste (brand) pH SY + Sn/F Soy-based Yes Elmex® Opti-namel 4.75 MY + Sn/F Milk-based Yes Elmex® Opti-namel 4.75 W + Sn/F Water Yes Elmex® Opti-namel 4.75 W + NF Water Yes Nenedent® Children’s 7.89 MY Milk-based No – – SY Soy-based No – – W Water No – – Erosive and Abrasive Cycling Custom-made racks with two rows of Falcon tubes allowed precise and simultaneous handling of specimen groups across all stages of the erosion–abrasion cycle (Fig. 1 a). Demineralizing and remineralizing solutions were placed in tubes containing 45 mL each, whereas treatment solutions (milk-based yogurt, soy-based yogurt, or tap water) were placed in tubes containing a volume of 25 mL. All solutions were maintained at room temperature (25°C) and were changed before each exposition. Six erosive cycles per day were performed for seven consecutive days, at 8:00 a.m., 10:00 a.m., 12:00 p.m., 2:00 p.m., 4:00 p.m., and 6:00 p.m. Each erosive cycle consisted of immersion in 0.05 M citric acid (pH 2.3, 25°C) for 2 min, followed by rinsing with deionized water for 5s. After each erosive cycle, specimens were stored in a remineralizing solution. At 8:00 a.m. and 6:00 p.m., immediately after the first and last erosive challenges of the day, the treatment groups (soy-based yogurt, milk-based yogurt or water, both with or without brushing) were immersed in their respective treatment solutions for 3 min and rinsed with deionized water. For brushing groups, immediately after the treatment, the procedure was performed for 15 s using an electric toothbrush (Braun Oral-B Professional Care 8500, Precision Clean brush head) operating at approximately 8.800 oscillations and 40.000 pulsations per minute, mounted on a custom-designed 3D-printed brushing device, ensuring a constant load of 1.5 N and a standardized brushing motion for all specimens [ 35 ]. Immediately after brushing, specimens were immersed for 105 s in a toothpaste slurry (1 part toothpaste to 3 parts deionized water), resulting in a total toothpaste contact time of 2 min. Specimens were then rinsed with deionized water and stored in the remineralizing solution. Between erosive challenges throughout the day, specimens were maintained for 2 h in a supersaturated remineralizing solution containing 4.08 mM H₃PO₄, 20.10 mM KCl, 11.90 mM Na₂CO₃, and 1.98 mM CaCl₂ (pH 6.5), as previously described by Gerrard and Winter [ 36 ]. The remaining four daily challenges (10:00 a.m., 12:00 p.m., 2:00 p.m., and 4:00 p.m.) consisted exclusively of immersion in the citric acid solution for 2 min, followed by rinsing and storage in the remineralizing solution. The three experimental groups without abrasion (soy-based yogurt, milk-based yogurt and water) were subjected exclusively to the erosive-cycling but did not receive brushing or toothpaste slurry application. These groups were therefore exposed exclusively to the erosive challenge, followed by the corresponding post‑acid treatments (twice a day) and remineralization phases (Fig. 1 b). After completion of the six daily erosive cycles, all specimens, regardless of experimental group, were stored overnight in the remineralizing solution, simulating an extended period without acidic challenges. Scanning of Enamel Surfaces Enamel surface profiles were evaluated after the third (day 3) and seventh (day 7) day of the erosive cycling protocol. After day 3, all specimens were rinsed with deionized water, and the adhesive tapes protecting the control areas were carefully removed. Each specimen presented a central experimental area exposed to the erosive challenges, laterally delimited by two control areas marked on the right and left sides. Surface topography was analyzed using a 3D laser scanning microscope (VK-X3000, Keyence®, Osaka, Japan). The analyzed area comprised the central experimental region and the two adjacent control areas, resulting in a standardized measurement area of 3500⋅530µm. Scans were performed using a 20⋅ objective lens, as previously described [ 37 ]. After scanning on day 3, the adhesive tapes were repositioned in their original locations, and specimens returned to the erosive cycling protocol until day 7. At the end of day 7, the same cleaning and scanning procedures were repeated. Analysis of Enamel Surface Loss Image analysis was performed using the VK-X3000 MultiFileAnalyzer software (version 3.3.1.85, Keyence®,Osaka, Japan). The software processes the full 3D height map of the scanned area, containing millions of height points. Surface leveling was performed by selecting two reference regions within the control areas, from which the software generated a leveled reference plane. A representative mean height profile was then computed by averaging the height values across the entire width of the scan. Enamel surface loss was quantified as the vertical distance between the leveled reference plane and the mean eroded surface, expressed in micrometers (µm). To ensure precision the used tool performed the same measurements using 25 parallel line scans spaced at 30µm intervals across the experimental and control areas (Fig. 1 c). Analysis of calcium and phosphorus content: Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and pH Milk-based and soy-based yogurt samples, as well as a blank control, were analyzed in six replicates per group (n=6). All samples were subjected to the same acid digestion procedure. Briefly, 1.00±0.01g of each yogurt sample was placed in a borosilicate glass tube and treated with 5 mL of 65% HNO 3 and 1 mL of 30% H 2 O 2 . A blank tube containing the digestion reagents, but no yogurt sample was prepared as a control [ 38 ]. The tubes were covered with borosilicate watch glasses and heated at 80°C for 24 h to ensure complete digestion. Digestion vessels were weighed before and after heating to assess potential mass loss during the digestion process. After digestion, the samples were diluted 1:10 (v/v) with 2% HNO 3 . Elemental analysis of calcium (Ca) and phosphorus (P) was performed using an inductively coupled plasma optical emission spectrometer Avio®220 Max (PerkinElmer®, Waltham, USA), following standard procedures. Yttrium (20µg/mL) was used as an internal standard [ 39 ]. Measured concentrations were corrected by subtracting the values obtained from the blank control group, followed by multiplication by the dilution factor and adjustment based on recovery of the internal standard. The pH of yogurts were quantified using a 691 pH meter (Metrohm®, Filderstadt, Germany) under continuous stirring to prevent ion accumulation and ensure stable readings; the electrode was immersed directly in the original retail container, and measurements were recorded after stabilization. Statistical Analysis Statistical analysis was performed using GraphPad Prism (GraphPad Software®, San Diego, USA). Enamel surface loss data were analyzed using two-way analysis of variance (ANOVA), with time and treatment as fixed factors, followed by Tukey’s post hoc test for multiple comparisons. Calcium and phosphorus concentrations were evaluated only for the soy-based and milk-based yoghurt groups. Group differences were analyzed using an unpaired t -test with Welch’s correction to account for potential heterogeneity of variances. All tests were two-tailed, and the significance level was set at α= 0.05. Results Erosion–Abrasion Cycling Enamel surface loss under erosive–abrasive conditions are shown in Fig. 2 , while representative images of the enamel specimens after cycling are presented in Fig. 3 . At day 3, the water+no fluoride (negative control) group exhibited the highest mean enamel surface loss, differing significantly from the milk-based yogurt + Sn/F, soy-based yogurt+Sn/F, and water + Sn/F ( p<0.05 ). No statistically significant differences were detected among the three Sn/F groups (milk-based yogurt+Sn/F, soy-based yogurt+Sn/F, and water+Sn/F) at this time point (Fig. 2 ). By day 7, enamel surface loss increased in all experimental groups (Fig. 2 ). The water + no fluoride group continued to show the greatest enamel loss values and differed significantly from all other groups ( p<0.05 ). The soy-based yogurt+Sn/F group showed the lowest enamel surface loss at day 7 and differed significantly from the milk-based yogurt+Sn/F, water+Sn/F, and water+no fluoride groups (Figs. 2 and 3 ). Analysis of the time factor revealed a statistically significant increase in enamel surface loss from 3 to 7 days within all treatment groups ( p<0.05 ), indicating a progressive increase in enamel loss with longer exposure time under erosive–abrasive conditions (Figs. 2 and 3 ). Erosion–Only Cycling Enamel surface loss under erosive conditions without abrasion is presented in Fig. 4 , and representative images of the enamel specimens after erosive cycling are shown in Fig. 5 . At both day 3 and day 7, comparison among treatments (soy-based yogurt, milk-based yogurt, and water) revealed no statistically significant differences in enamel surface loss, as indicated by identical uppercase letters ( p>0.05 , Fig. 4 ). After 7 days, enamel surface loss increased in all experimental groups compared to day 3, indicating a progressive enamel loss pattern with increasing exposure time (Figs. 4 and 5 ). Analysis of pH, Calcium, and Phosphate The pH, calcium, and phosphorus values of the yogurt specimens are shown in Table 3 . The milk-based yogurt presented a lower pH (4.4) than the soy-based yogurt (4.7). Calcium concentration was significantly higher in the milk-based yogurt (1434.1±190.3 µg/g) compared with the soy-based yogurt (168.4±15.5 µg/g; p <0.0001). Similarly, phosphorus concentration was higher in the milk-based yogurt (1033.8±128.1 µg/g) than in the soy-based yogurt (623.2±47.7 µg/g; p =0.0004). Table 3 pH and mineral composition (calcium and phosphorus) of the yogurts. Yogurt pH Calcium (µg/g) Phosphorus (µg/g) Soy-based 4.70 168.48 ± 15.46 623.18 ± 47.73 Milk-based 4.36 1434.10 ± 190.31 1033.80 ± 128.09 The mineral composition (calcium and phosphorus) of the milk-based and soy-based yogurts, determined by inductively coupled plasma optical emission spectrometry (ICP-OES). Data are presented as mean ± standard deviation (n = 6). Unpaired t test with Welch´s correlation, Calcium p < 0.0001 and Phosphorous p = 0.0004 . Discussion The results of the present study demonstrated that the combination of soy-based yogurt with a toothpaste containing fluoride and tin (1450 ppm F, 3500 ppm Sn, 0.5% chitosan), under erosion–abrasion conditions, resulted in the lowest enamel loss values. The same protective effect was not observed when the soy-based yogurt was applied only between erosive challenges, in the absence of fluoride or tin, indicating that components of this yogurt likely react with residual components of the fluoridated toothpaste, both present on the dental surface, thereby protecting the enamel against erosive loss. It is important to emphasize that, in the present study, the effect of both the soy-based yogurt and the milk-based yogurt was observed only when combined with a toothpaste containing fluoride and tin. The literature consistently demonstrates that fluoridated toothpastes, especially when associated with stannous compounds, significantly reduce enamel loss under in in vitro erosion–abrasion models [ 40 , 41 ]. The combination of fluoride and tin is considered an effective and safe agent, presenting superior benefits compared with other fluoridated compounds, in addition to positive effects on dental plaque, gingiva, and dentin hypersensitivity [ 41 – 44 ]. Fluoride and tin interact directly with hydroxyapatite, the main mineral component of dental enamel. Stannous chloride (SnCl₂), being soluble in aqueous media, releases Sn²⁺ ions, which exhibit high affinity for the phosphate groups of hydroxyapatites, forming a modified mineral layer enriched with tin and fluoride that offers mechanical protection. The formation of this layer reduces the solubility of calcium phosphate under acidic conditions and decreases calcium release during erosive challenges, thereby increasing resistance to enamel loss [ 45 ]. This is the first study to evaluate a soy-based yogurt regarding erosive enamel loss, and despite its protective effect against enamel loss when combined with a fluoridated toothpaste, its calcium and phosphate contents were significantly lower than those of the milk-based yogurt, which showed a comparatively lower protective effect in this study. Therefore, the mechanism of action of this soy-based yogurt cannot be explained by its calcium and phosphate content. The presence of starch and modified starch in the soy-based yogurt may have contributed to the increased viscosity of the surface layer, favoring longer contact time with enamel surface and more stable mineral layer enriched with tin and fluoride, a factor recognized as relevant for modulation of tooth erosion [ 32 , 45 ]. More viscous media tend to reduce clearance and the immediate removal of active substances from the dental surface, potentially delaying the diffusion and elimination of fluoride ions and tin species, whose efficacy depends on surface retention and interaction with the enamel–medium interface [ 33 , 39 ]. Thus, it is plausible that the starch-rich matrix of the soy-based yogurt enhanced the action of the toothpaste containing fluoride, tin, and chitosan, although this mechanism still requires direct experimental confirmation [ 42 , 45 ]. In the literature, the study by Dos Santos et al [ 46 ] subjected enamel specimens to a static continuous erosion model, with direct immersion in beverages for 120 min without remineralization phases. The soy-based beverages were commercially available products (Addes®, Unilever) with pH varying from 3.9 to 4.2, while the non–soy-based beverages comprised pH values from 2.9 to 3.4. Under these conditions, beverages containing soy extract promoted lower enamel surface loss and smaller increases in surface roughness compared with non-soy beverages, although all drinks exhibited erosive potential. This attenuating effect of soy should be interpreted with caution, as prolonged and continuous acidic exposure favors chemical interactions between beverage components and the enamel surface and does not reflect the clinical dynamics of short acidic challenges interspersed with periods of salivary recovery. Furthermore, they tested beverage and not yogurt, which are more acidic and less viscous. With respect to yogurt, past in vitro erosive protocols frequently involved prolonged and repeated acidic exposures with accumulative several hours of contact time either [ 29 , 47 ], conditions that are far from clinical reality. In these aggressive experimental models, the prior application of milk and yogurt and extended storage in artificial saliva may have artificially enhanced the apparent reduction in demineralization. In addition, the use of outcome measures such as dental weight loss [ 47 ] or even confocal microscopy [ 29 ] limits clinical interpretation. Similarly, sequential demineralization–remineralization models, in that eroded enamel is immersed in milk or artificial saliva for up to 3 hours a prolonged period of exposure even with the application of sensitive methods to detect surface and mechanical changes, does not reflect real oral exposure patterns, restricting direct clinical extrapolation neither [ 48 ]. Therefore, the past protective effects attributed to yogurt should be interpreted cautiously, especially in continuous demineralization models in which dairy components remain present during acidic challenge, potentially overestimating protection [ 23 ]. In contrast, pH-cycling models that simulate short and repeated acidic challenges with remineralization periods better approximate clinical conditions. Under these circumstances, a previous study showed that fluoridated milk had greater protective potential, whereas non-fluoridated dairy products demonstrated limited effects gainst erosive enamel loss [ 16 ] consistent with the findings of the present study. Overall, evidence suggests that many of the past experimental protocols overestimate the protective effects of milk and yogurt, due to prolonged chemical interaction with enamel. Models incorporating short, repeated erosive challenges, salivary recovery, and abrasion when applicable, consistently demonstrate more limited protective effects, reinforcing that experimental design has a decisive influence on the magnitude and direction of the reported outcomes [ 16 , 23 , 29 , 46 ].Therefore, the protective effect of milk and derivates may be of less relevance in case of ETW under clinical conditions, which needs to be further investigated. Finally, limitations inherent to the in vitro model should be considered, such as the absence of human saliva, acquired pellicle formation, and physiological remineralization mechanisms, which favor progression of erosive loss at a higher rate than would occur in vivo [ 32 ]. On the other hand, inclusion of the erosion-only condition allowed isolation of the effect of yogurts on enamel, eliminating the influence of mechanical factors and fluoridated toothpaste, thereby contributing to a better understanding of the mechanisms involved. Conclusion Within the limitations of this in vitro erosion–abrasion study, the combination of soy-based yogurt with a fluoride–stannous toothpaste significantly reduced enamel surface loss, whereas the milk-based yogurt did not. The enhanced protection does not appear to be related to the yogurt’s mineral content, but may be associated with matrix-related factors that favor interaction with residual fluoride–stannous deposits on enamel, although this mechanism requires confirmation. Further in situ and clinical studies are required to confirm the clinical significance of these findings. Overall, these findings emphasize that protection against erosive enamel loss under clinically relevant cycling conditions is primarily attributable to fluoride, particularly when combined with stannous compounds. Yogurt, soy or milk-based, alone provides limited benefit. Declarations Ethical approval We declare that the work was approved by the Animal Ethics Committee of Bauru School of Dentistry – University of São Paulo (FOB/USP, CEUA:007/2026). Acknowledgement The authors gratefully acknowledge the technical support provided by the Department of Medical Materials Science and Technology, Institute for Biomedical Engineering, University Hospital Tübingen. The authors also acknowledge the financial support provided by São Paulo Research Foundation (FAPESP 2024/05183-0, 2024/20716-5). Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding Sources This study was financed by the São Paulo Research Foundation (FAPESP), Brazil. Process Number 2024/05183-0, 2024/20716-5 (first author). FAPESP provided financial support for the acquisition of the materials required to conduct the study. Data Availability Statement All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding authors. Author contributions Rafaela Ricci Kim: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, validation, writing – original draft, and writing – review and editing; Aline Silva Braga : conceptualization, data curation, formal analysis, investigation, methodology, validation, writing – original draft, and writing – review and editing; Gabriela Pellizon Floret: methodology; Ariadne Roehler , Jacob Schultheiss , Ana Carolina Magalhães: conceptualization, supervision, formal analysis, validation, and writing – review and editing; and Marcella Esteves-Oliveira: conceptualization, supervision, formal analysis, validation, and writing – review and editing and All authors: final approval of the version to be published. Declaration of generative AI and AI-assisted technologies in the manuscript preparation process During the preparation of this work, the authors used ChatGPT (OpenAI) to assist with figure design and text revision. 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Arch Oral Biol 104:133–140. 10.1016/j.archoralbio.2019.05.021 Kensche A, Pohl C, Basche S, Dürasch A, Henle T, Hannig M, Hannig C (2025) Bovine milk and milk protein – promotor or inhibitor of bacterial biofilm formation at the tooth surface? BMC Oral Health 25:992. 10.1186/s12903-025-06432-1 Manton DJ, Cai F, Yuan Y, Walker GD, Cochrane NJ, Reynolds C et al (2010) Effect of casein phosphopeptide-amorphous calcium phosphate added to acidic beverages on enamel erosion in vitro. Aust Dent J 55(3):275–279. 10.1111/j.1834-7819.2010.01235.x Schestakow A, Echterhoff B, Hannig M (2024) Erosion protective properties of the enamel pellicle in-situ. J Dent 147:105103. 10.1016/j.jdent.2024.105103 Shkembi B, Huppertz T (2023) Impact of dairy products and plant-based alternatives on dental health: food matrix effects. Nutrients 15(6):1469. 10.3390/nu15061469 Ferrazzano GF, Cantile T, Quarto M, Ingenito A, Chianese L, Addeo F (2008) Protective effect of yogurt extract on dental enamel demineralization in vitro. Aust Dent J 53(4):314–319. 10.1111/j.1834-7819.2008.00072.x Alcorta A, Porta A, Tárrega A, Alvarez MD, Vaquero MP (2021) Foods for plant-based diets: challenges and innovations. Foods 10(2):293. 10.3390/foods10020293 Salehi G, Díaz E, Redondo R (2023) Forty-five years of research on vegetarianism and veganism: A systematic and comprehensive literature review of quantitative studies. Heliyon 9(5):e16091. 10.1016/j.heliyon.2023.e16091 Asif N, Anwar O, Arif S, Anwar Z, Iahtisham-Ul-Haq, Ercisli S et al (2026) The rise of plant-based milk alternatives: exploring nutritional, health, and sustainability impacts. Food Chem X 34:103528. 10.1016/j.fochx.2026.103528 Carey CN, Paquette M, Sahye-Pudaruth S, Dadvar A, Dinh D, Khodabandehlou K et al (2023) The environmental sustainability of plant-based dietary patterns: a scoping review. J Nutr 153(3):857–869. 10.1016/j.tjnut.2023.02.001 Ramsing R, Santo R, Kim BF, Altema-Johnson D, Wooden A, Chang KB et al (2023) Dairy and plant-based milks: implications for nutrition and planetary health. Curr Environ Health Rep 10:291–302. 10.1007/s40572-023-00400-z Turaga SS, Sukhabogi JR, Doshi D, Jummala S, Billa AL (2024) Comparing the effect of animal and plant-based yogurt extracts on enamel demineralization: an in vitro study. Minerva Dent Oral Sci 73(3):161–168. 10.23736/S2724-6329.23.04804-0 West NX, Hughes JA, Addy M (2000) Erosion of dentine and enamel in vitro by dietary acids: the effect of temperature, acid character, concentration and exposure time. J Oral Rehabil 27(10):875–880. 10.1046/j.1365-2842.2000.00583.x Wiegand A, Attin T (2014) Randomised in situ trial on the effect of milk and CPP-ACP on dental erosion. J Dent 42(9):1210–1215. 10.1016/j.jdent.2014.07.009 Hara AT, Zero DT (2014) Aetiology of dental erosion: patient-related factors. The potential of saliva in protecting against dental erosion. Monogr Oral Sci 25:197–205. 10.1159/000360372 Shellis RP, Featherstone JD, Lussi A (2014) Understanding the chemistry of dental erosion. Monogr Oral Sci 25:163–179. 10.1159/000359943 Esteves-Oliveira M, Witulski N, Hilgers RD, Apel C, Meyer-Lueckel H, Eduardo Cde P (2015) Combined Tin-Containing Fluoride Solution and CO 2 Laser Treatment Reduces Enamel Erosion in vitro. Caries Res 49(6):565–574. 10.1159/000439316 Esteves-Oliveira M, Pasaporti C, Heussen N, Eduardo CP, Lampert F, Apel C (2011) Prevention of toothbrushing abrasion of acid-softened enamel by CO(2) laser irradiation. J Dent 39(9):604–611. 10.1016/j.jdent.2011.06.007 Gerrard WA, Winter PJ (1986) Evaluation of toothpastes by their ability to assist rehardening of enamel in vitro. Caries Res 20(3):209–216. 10.1159/000260937 Esteves-Oliveira M, Wollgarten S, Liebegall S, Jansen P, Bilandzic M, Meyer-Lueckel H et al (2017) A New Laser-Processing Strategy for Improving Enamel Erosion Resistance. J Dent Res 96(10):1168–1175. 10.1177/0022034517718532 Lee J, Park YS, Lee HJ, Koo YE (2022) Microwave-assisted digestion method using diluted nitric acid and hydrogen peroxide for the determination of major and minor elements in milk samples by ICP-OES and ICP-MS. Food Chem 373(Pt B):131483. 10.1016/j.foodchem.2021.131483 Poitevin E, Nicolas M, Graveleau L, Richoz J, Andrey D, Monard F (2009 Sep-Oct) Improvement of AOAC Official Method 984.27 for the determination of nine nutritional elements in food products by Inductively coupled plasma-atomic emission spectroscopy after microwave digestion: single-laboratory validation and ring trial. J AOAC Int 92(5):1484–1518 Ganss C, Marten J, Hara AT, Schlueter N (2016) Toothpastes and enamel erosion/abrasion - Impact of active ingredients and the particulate fraction. J Dent 54:62–67. 10.1016/j.jdent.2016.09.005 Lucchese A, Bertacci A, Lo Giudice A, Polizzi E, Gherlone E, Manuelli M et al (2020) Stannous Fluoride Preventive Effect on Enamel Erosion: An In Vitro Study. J Clin Med 9(9):2755. 10.3390/jcm9092755 Fiorillo L, Cervino G, Herford AS, Laino L, Cicciù M (2020) Stannous Fluoride Effects on Enamel: A Systematic Review. Biomimetics (Basel) 5(3):41. 10.3390/biomimetics5030041 Nicholson JW (2025) Stannous Fluoride in Toothpastes: A Review of Its Clinical Effects and Likely Mechanisms of Action. J Funct Biomater 16(3):73. 10.3390/jfb16030073 Babcock FD, King JC, Jordan TH The reaction of stannous fluoride and hydroxyapatite. J Dent Res 1978 Sep-Oct ; 57(9–10): 933–938. 10.1177/00220345780570092301 Schestakow A, Echterhoff B, Hannig M (2024) Erosion protective properties of the enamel pellicle in-situ. J Dent 147:105103. 10.1016/j.jdent.2024.105103 Santos EJLD, Meira IA, Sousa ET, Amaechi BT, Sampaio FC, Oliveira AFB (2019) Erosive potential of soy-based beverages on dental enamel. Acta Odontol Scand 77(5):340–346. 10.1080/00016357.2019.1570330 Basu SN, Kumar P, Gawali RA, Urs AB (2025) Protective Efficacy of Yogurt, Milk, and Fluoridated Tooth Creme Against Acidic Beverages on Human Teeth: Stereomicroscopic and Ultrastructural Analyses. Microsc Res Tech 88(6):1835–1847. 10.1002/jemt.24819 Larnani S, Song Y, Kim S, Park YS (2025) Examining enamel-surface demineralization upon exposure to acidic solutions and the remineralization potential of milk and artificial saliva. Odontology 113(1):201–212. 10.1007/s10266-024-00960-y Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviews received at journal 20 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor assigned by journal 14 Apr, 2026 Submission checks completed at journal 13 Apr, 2026 First submitted to journal 07 Apr, 2026 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. We do this by developing innovative software and high quality services for the global research community. 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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-9348858","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626500968,"identity":"fb5722f5-4091-4782-8e0a-98fcb0c71337","order_by":0,"name":"Rafaela Ricci Kim","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Rafaela","middleName":"Ricci","lastName":"Kim","suffix":""},{"id":626500969,"identity":"025b4d7a-9051-4e98-bf7d-f24307b4c85c","order_by":1,"name":"Aline Silva Braga","email":"","orcid":"","institution":"University Hospital Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Aline","middleName":"Silva","lastName":"Braga","suffix":""},{"id":626500970,"identity":"246054bd-2fe2-42a3-9ecd-85893d53b726","order_by":2,"name":"Gabriela Pellizon Floret","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Pellizon","lastName":"Floret","suffix":""},{"id":626500971,"identity":"65589691-263c-4837-881b-f7d478eb38e8","order_by":3,"name":"Ariadne Roehler","email":"","orcid":"","institution":"University Hospital Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Ariadne","middleName":"","lastName":"Roehler","suffix":""},{"id":626500972,"identity":"499709fe-7583-472b-8544-3204973615a1","order_by":4,"name":"Jacob Schultheiss","email":"","orcid":"","institution":"University Hospital Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Schultheiss","suffix":""},{"id":626500973,"identity":"38556649-9b16-4748-96ce-1dd50b206ffd","order_by":5,"name":"Ana Carolina Magalhães","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApklEQVRIiWNgGAWjYHACxgcVDAfALGZitTAbnCFVC5sEaVr420+nVRyouMNgcP7sAebCPURokTiTu+3GgTPPGAxu5CUwz3hGjDU3eLfd/th2GKiFx4CZ5wAROuSBWgoOgrScP0OkFgOgFgawlgM5RGoxPJO7WQLoFx5JoF8OzyBGi9zxsxs/AENMju/82YOPC4jRAgM8IESKBpiuUTAKRsEoGAXYAACDVz2oT9bTPQAAAABJRU5ErkJggg==","orcid":"","institution":"University of São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Ana","middleName":"Carolina","lastName":"Magalhães","suffix":""},{"id":626500974,"identity":"05ea32a1-8aca-4da4-9972-210d7f39bb23","order_by":6,"name":"Marcella Esteves-Oliveira","email":"","orcid":"","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Marcella","middleName":"","lastName":"Esteves-Oliveira","suffix":""}],"badges":[],"createdAt":"2026-04-07 19:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9348858/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9348858/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107834903,"identity":"5be997cc-11ee-48f6-8e5c-a3eed1ff193c","added_by":"auto","created_at":"2026-04-26 15:49:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":553194,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design and analytical workflow.\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9348858/v1/f569f26efa204ce67b5567b1.png"},{"id":107870406,"identity":"ae3a844d-6028-437b-acee-5a92b1c2f9f5","added_by":"auto","created_at":"2026-04-27 07:39:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28982,"visible":true,"origin":"","legend":"\u003cp\u003eErosion plus abrasion. Mean enamel surface loss (µm) after 3 and 7 days. Data were analyzed using two-way ANOVA followed by Tukey’s post hoc test (factors: time, \u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e; treatment, p\u0026lt;0.0001; interaction, \u003cem\u003ep=0.0001\u003c/em\u003e; n=14). Different uppercase letters indicate statistically significant differences among treatment groups. Different lowercase letters indicate statistically significant differences between time points (3 and 7 days).\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9348858/v1/20cf0cfc1378ee3b40cac235.png"},{"id":107870852,"identity":"f83f3654-5390-4322-a525-cc50acfd7ac5","added_by":"auto","created_at":"2026-04-27 07:41:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":735280,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional surface profilometry images showing enamel erosive-abrasive surface loss after 3 and 7 days of exposure to different yogurts and fluoride conditions: soy-based+Sn/F, milk-based+Sn/F, water+Sn/F, and water+no fluoride. Color maps represent surface height variations (µm).\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9348858/v1/0d7a5a1ba8a3fe293d82c734.png"},{"id":107834905,"identity":"8ce2af1f-2d70-444d-8fba-3b33c4cf3f4a","added_by":"auto","created_at":"2026-04-26 15:49:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22709,"visible":true,"origin":"","legend":"\u003cp\u003eErosion Only. Mean enamel surface loss (µm) after 3 and 7 days under erosive conditions without abrasion. Data were analyzed using two-way ANOVA followed by followed by Tukey’s post hoc test (factors: time, \u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e; treatment, \u003cem\u003ep\u0026gt;0.05\u003c/em\u003e; interaction, \u003cem\u003ep\u0026gt;0.05\u003c/em\u003e; n=14). Different uppercase letters indicate statistically significant differences among treatment groups. Different lowercase letters indicate statistically significant differences between time points (3 and 7 days).\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9348858/v1/6ed2c342959cd84158f529fc.png"},{"id":107834906,"identity":"c38d47fb-151b-4b1c-b244-840df1f780e0","added_by":"auto","created_at":"2026-04-26 15:49:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":530826,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional surface profilometry images showing enamel erosive surface loss after 3 and 7 days of exposure to different yogurts: soy-based, milk-based and water. Color maps represent surface height variations (µm).\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9348858/v1/bc724af371095d0b83070d66.png"},{"id":108181079,"identity":"42fcc69b-ba07-455e-b366-128a7ebc1eee","added_by":"auto","created_at":"2026-04-30 08:57:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2767393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9348858/v1/d5b5ad10-8440-42ee-b78e-2d9e43b966cb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of Soy- and Milk-Based Yogurts on Enamel Preservation: Erosion and Abrasion under Simulated Conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eErosive tooth wear (ETW) is defined as the progressive and irreversible loss of dental hard tissues, mainly enamel and dentin, caused by the chemical action of acids without bacterial involvement. This process may be exacerbated by associated mechanical factors, such as abrasion, commonly related to toothbrushing, or attrition [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. ETW is a clinical and conceptual term that describes the cumulative outcome of chemical erosion, often modified by mechanical processes. In experimental studies, this condition is commonly quantified as enamel surface loss or wear, which refers to the measurable irreversible loss of mineralized enamel tissue over time, typically expressed in micrometer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe acids affecting dental structures may have an extrinsic origin, derived from dietary sources (soft drinks, citrus fruits, vinegars, among others), or an intrinsic origin, resulting from conditions such as gastroesophageal reflux or frequent vomiting [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Epidemiological studies have reported a high prevalence of this condition, estimated to range from 30% to 50% in primary dentition and from 20% to 45% in permanent dentition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe progression of ETW may significantly impair patients\u0026rsquo; quality of life, being associated with dentin hypersensitivity, functional impairments such as chewing difficulties, and esthetic concerns, including tooth discoloration and shortening, which may negatively affect self-esteem and smile appearance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Given its multifactorial nature, the clinical management of ETW requires careful assessment of the chemical, biological, and behavioral factors involved, as well as the implementation of evidence-based preventive strategies [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the natural protective factors against erosion, saliva plays a fundamental role by neutralizing and clearing acids, promoting the formation of the acquired pellicle, and providing calcium and phosphate ions necessary for remineralization processes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, certain foods, particularly dairy products such as milk and cheese, have been associated with protective effects on enamel, mainly attributed to their mineral, protein and lipidic content, in especial to calcium interactions with the dental surface [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previous studies have demonstrated that, although acidic foods increase dental erosion, the consumption of milk and milk-based yogurt may attenuate their effect [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eYogurt presents properties comparable to those of milk due to the presence of calcium, phosphate, and proteins, especially casein at high concentration. These components may contribute to enamel remineralization and to the formation of a protective layer on the tooth surface [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Evidence indicates that casein and its derivatives, such as casein phosphopeptides (CPPs), can adsorb onto enamel, forming a physical barrier against acids and promoting the retention of calcium and phosphate ions, thereby reducing demineralization [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConcurrently, there has been a markable increase in the consumption of plant-based foods (vegetarian and vegan style of life), driven by changes in dietary habits and growing environmental and health concerns [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this context, plant-based yogurts have gained attention as alternatives to traditional milk-yogurt products.\u003c/p\u003e \u003cp\u003eRecent studies have indicated that plant-based yogurts may exhibit a protective effect on dental enamel, even in the absence of milk-derived proteins such as casein [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This protective potential has been suggested to be related to the physicochemical characteristics of these products, including mineral content, buffering capacity, and the food matrix, rather than exclusively to the presence of casein-derived peptides. These findings highlight the need to further evaluate the role of plant-based yogurt formulations in their interaction with dental hard tissues, particularly considering their increasing consumption [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The available evidence remains limited, particularly regarding the mechanisms involved and the interaction of these products with other preventive strategies commonly adopted in clinical practice, such as the use of fluoridated toothpastes. To the best of our knowledge, no study has evaluated the protective effect of soy-based yogurt in an erosion\u0026ndash;abrasion model or its interaction with fluoridated toothpastes.\u003c/p\u003e \u003cp\u003eMoreover, many previous studies have employed experimental models based on continuous acidic exposure or prolonged contact times with foods or beverages, which do not adequately reflect the clinical dynamics of oral cavity [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Clinically, erosive challenges are typically short and repeated, interspersed with periods of salivary recovery and often associated with toothbrushing abrasion. Consequently, it remains unclear whether the protective effects attributed to foods, such as yogurt, persist under more clinically relevant conditions, especially when erosion is combined with abrasion.\u003c/p\u003e \u003cp\u003eTherefore, the present study aimed to evaluate the protective effects of milk-based and soy-based yogurts on enamel surface loss under erosive conditions alone and when combined with abrasion, with particular emphasis on their interaction with the use of a tin-containing fluoride toothpaste following erosive challenges.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design\u003c/h2\u003e \u003cp\u003eThe experimental protocol was designed to simulate clinical preventive recommendations for erosive tooth wear, namely the consumption of yogurt immediately after acidic food or beverage intake, followed by toothbrushing with a tin-containing fluoride toothpaste. This \u003cem\u003ein vitro\u003c/em\u003e model enabled the systematic evaluation of post-acid exposure preventive measures under controlled erosive\u0026ndash;abrasive cycling conditions. The experimental design is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample Size Calculation\u003c/h3\u003e\n\u003cp\u003eA sample size of 14 specimens per group was determined based on data from Esteves-Oliveira et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] In that study, the difference in enamel surface loss between the control group (27.22\u0026plusmn;4.1\u0026micro;m) and the tin-containing fluoride group (3.31\u0026plusmn;2.0 \u0026micro;m) was 23.91\u0026micro;m, with a pooled standard deviation of 3.23 \u0026micro;m, resulting in a standardized effect size (Cohen\u0026rsquo;s d) of approximately 7.41. Assuming an alpha level of 0.05 and a target power of 0.80, this contrast would require fewer than one specimen per group. Therefore, the allocation of 14 specimens per group provides statistical power exceeding 99% for this benchmark effect.\u003c/p\u003e\n\u003ch3\u003eTooth Sample Preparation\u003c/h3\u003e\n\u003cp\u003eA total of 98 enamel specimens were prepared from permanent bovine incisors and sectioned into dimensions of 4 mm\u0026sdot;4 mm using two diamond discs mounted on a IsoMet\u0026reg; Low Speed Precision Cutter (Buehler\u0026reg;, Lake Bluff, USA).Throughout the study, specimens were stored in a 0.1% thymol solution, and only specimens without visible cracks or structural defects were included.\u003c/p\u003e \u003cp\u003eEnamel surfaces were planarized and polished under continuous water irrigation using a Micro Grinder 400 polishing machine (Exakt\u0026reg;, Norderstedt, Germany).Each specimen was fixed with sticky wax at the center of an acrylic plate and initially polished from the dentin side, followed by the enamel side, to obtain planar and parallel surfaces. Final enamel polishing was performed sequentially using 800, 1200, 2400, and 4000 grit silicon carbide papers under water cooling, followed by a 2 min polishing step with a felt disc and diamond suspension.Enamel reduction by polishing was monitored using a micrometer standardized not to exceed 400\u0026micro;m [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe absence of dentin exposure was confirmed using a 3D laser scanning microscope (VK-3000, Keyence\u0026reg;, Osaka, Japan). Specimens were subsequently cleaned in distilled water using an ultrasonic bath and stored in 0.1% thymol at 4\u0026deg;C until use [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo ensure that enamel surface loss was consistently measured at the same location throughout the experiment, two reference marks were engraved on each specimen using a spherical bur (No.1011) (Hager \u0026amp; Meisinger\u0026reg;, Neuss, Germany), positioned on the left and right sides of the experimental area. Two control areas on each specimen were protected with removable adhesive tape during the erosive\u0026ndash;abrasive cycling and uncovered only during the measurement procedures.\u003c/p\u003e\n\u003ch3\u003eExperimental Groups\u003c/h3\u003e\n\u003cp\u003eSpecimens were allocated into seven experimental groups to isolate the effects of yogurt type (milk-based vs. soy-based) and brushing abrasion (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The inclusion of a fluoride-free toothpaste group served to control for the well-established protective effect of tin-containing fluoride toothpastes against abrasion of eroded enamel surfaces.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of yogurt products and toothpastes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProduct type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComposition\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoy-based yogurt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRewe Vegan Soja Natur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWater, soy (13.3%), modified starch, starch, sea salt, natural flavoring, yogurt cultures\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk-based yogurt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRewe Bio Yogurt Mild 3.8% fat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMilk and yogurt cultures\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eToothpaste type\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eFull composition\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTin-containing fluoride toothpaste\u003c/p\u003e \u003cp\u003e\u003cb\u003eElmex\u0026reg; Opti-namel\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(Colgate-Palmolive\u003cb\u003e\u0026reg;\u003c/b\u003e, Świdnica, Poland)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAqua, glycerin, sorbitol, hydrated silica, hydroxyethylcellulose, aroma, cocamidopropyl betaine, titanium dioxide, olaflur (AmF), sodium gluconate, stannous chloride, alumina, chitosan, sodium saccharin, sodium fluoride, potassium hydroxide, hydrochloric acid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eFluoride-free toothpaste\u003c/p\u003e \u003cp\u003e\u003cb\u003eNenedent\u0026reg;\u003c/b\u003e \u003cb\u003echildren's\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(Dentinox\u003cb\u003e\u0026reg;\u003c/b\u003e, Berlin, Germany)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAqua, hydrated silica, glycerin, xylitol, propylene glycol, xanthan gum, aroma, sodium lauroyl sarcosinate, disodium EDTA, sodium chloride, sodium hydroxide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll experimental groups were subjected to the same erosive and remineralization protocol. The experimental variables were the presence or absence of brushing abrasion, the use of fluoride-containing or fluoride-free toothpaste (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the type of post-acid treatment (milk-based yogurt, soy-based yogurt or water), as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental groups and preventive conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePost-Acid Treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbrasion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eToothpaste (brand)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSY\u0026thinsp;+\u0026thinsp;Sn/F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSoy-based\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElmex\u0026reg; Opti-namel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMY\u0026thinsp;+\u0026thinsp;Sn/F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMilk-based\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElmex\u0026reg; Opti-namel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW\u0026thinsp;+\u0026thinsp;Sn/F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElmex\u0026reg; Opti-namel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW\u0026thinsp;+\u0026thinsp;NF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNenedent\u0026reg; Children\u0026rsquo;s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMilk-based\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSoy-based\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eErosive and Abrasive Cycling\u003c/h3\u003e\n\u003cp\u003eCustom-made racks with two rows of Falcon tubes allowed precise and simultaneous handling of specimen groups across all stages of the erosion\u0026ndash;abrasion cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Demineralizing and remineralizing solutions were placed in tubes containing 45 mL each, whereas treatment solutions (milk-based yogurt, soy-based yogurt, or tap water) were placed in tubes containing a volume of 25 mL. All solutions were maintained at room temperature (25\u0026deg;C) and were changed before each exposition.\u003c/p\u003e \u003cp\u003eSix erosive cycles per day were performed for seven consecutive days, at 8:00 a.m., 10:00 a.m., 12:00 p.m., 2:00 p.m., 4:00 p.m., and 6:00 p.m. Each erosive cycle consisted of immersion in 0.05 M citric acid (pH 2.3, 25\u0026deg;C) for 2 min, followed by rinsing with deionized water for 5s. After each erosive cycle, specimens were stored in a remineralizing solution.\u003c/p\u003e \u003cp\u003eAt 8:00 a.m. and 6:00 p.m., immediately after the first and last erosive challenges of the day, the treatment groups (soy-based yogurt, milk-based yogurt or water, both with or without brushing) were immersed in their respective treatment solutions for 3 min and rinsed with deionized water.\u003c/p\u003e \u003cp\u003eFor brushing groups, immediately after the treatment, the procedure was performed for 15 s using an electric toothbrush (Braun Oral-B Professional Care 8500, Precision Clean brush head) operating at approximately 8.800 oscillations and 40.000 pulsations per minute, mounted on a custom-designed 3D-printed brushing device, ensuring a constant load of 1.5 N and a standardized brushing motion for all specimens [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Immediately after brushing, specimens were immersed for 105 s in a toothpaste slurry (1 part toothpaste to 3 parts deionized water), resulting in a total toothpaste contact time of 2 min. Specimens were then rinsed with deionized water and stored in the remineralizing solution.\u003c/p\u003e \u003cp\u003eBetween erosive challenges throughout the day, specimens were maintained for 2 h in a supersaturated remineralizing solution containing 4.08 mM H₃PO₄, 20.10 mM KCl, 11.90 mM Na₂CO₃, and 1.98 mM CaCl₂ (pH 6.5), as previously described by Gerrard and Winter [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The remaining four daily challenges (10:00 a.m., 12:00 p.m., 2:00 p.m., and 4:00 p.m.) consisted exclusively of immersion in the citric acid solution for 2 min, followed by rinsing and storage in the remineralizing solution.\u003c/p\u003e \u003cp\u003eThe three experimental groups without abrasion (soy-based yogurt, milk-based yogurt and water) were subjected exclusively to the erosive-cycling but did not receive brushing or toothpaste slurry application. These groups were therefore exposed exclusively to the erosive challenge, followed by the corresponding post‑acid treatments (twice a day) and remineralization phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eAfter completion of the six daily erosive cycles, all specimens, regardless of experimental group, were stored overnight in the remineralizing solution, simulating an extended period without acidic challenges.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eScanning of Enamel Surfaces\u003c/h2\u003e \u003cp\u003eEnamel surface profiles were evaluated after the third (day 3) and seventh (day 7) day of the erosive cycling protocol. After day 3, all specimens were rinsed with deionized water, and the adhesive tapes protecting the control areas were carefully removed. Each specimen presented a central experimental area exposed to the erosive challenges, laterally delimited by two control areas marked on the right and left sides.\u003c/p\u003e \u003cp\u003eSurface topography was analyzed using a 3D laser scanning microscope (VK-X3000, Keyence\u0026reg;, Osaka, Japan). The analyzed area comprised the central experimental region and the two adjacent control areas, resulting in a standardized measurement area of 3500\u0026sdot;530\u0026micro;m. Scans were performed using a 20\u0026sdot; objective lens, as previously described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter scanning on day 3, the adhesive tapes were repositioned in their original locations, and specimens returned to the erosive cycling protocol until day 7. At the end of day 7, the same cleaning and scanning procedures were repeated.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis of Enamel Surface Loss\u003c/h3\u003e\n\u003cp\u003eImage analysis was performed using the VK-X3000 MultiFileAnalyzer software (version 3.3.1.85, Keyence\u0026reg;,Osaka, Japan). The software processes the full 3D height map of the scanned area, containing millions of height points. Surface leveling was performed by selecting two reference regions within the control areas, from which the software generated a leveled reference plane.\u003c/p\u003e \u003cp\u003eA representative mean height profile was then computed by averaging the height values across the entire width of the scan. Enamel surface loss was quantified as the vertical distance between the leveled reference plane and the mean eroded surface, expressed in micrometers (\u0026micro;m). To ensure precision the used tool performed the same measurements using 25 parallel line scans spaced at 30\u0026micro;m intervals across the experimental and control areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n\u003ch3\u003eAnalysis of calcium and phosphorus content: Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and pH\u003c/h3\u003e\n\u003cp\u003eMilk-based and soy-based yogurt samples, as well as a blank control, were analyzed in six replicates per group (n=6). All samples were subjected to the same acid digestion procedure. Briefly, 1.00\u0026plusmn;0.01g of each yogurt sample was placed in a borosilicate glass tube and treated with 5 mL of 65% HNO\u003csub\u003e3\u003c/sub\u003e and 1 mL of 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. A blank tube containing the digestion reagents, but no yogurt sample was prepared as a control [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe tubes were covered with borosilicate watch glasses and heated at 80\u0026deg;C for 24 h to ensure complete digestion. Digestion vessels were weighed before and after heating to assess potential mass loss during the digestion process. After digestion, the samples were diluted 1:10 (v/v) with 2% HNO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eElemental analysis of calcium (Ca) and phosphorus (P) was performed using an inductively coupled plasma optical emission spectrometer Avio\u0026reg;220 Max (PerkinElmer\u0026reg;, Waltham, USA), following standard procedures. Yttrium (20\u0026micro;g/mL) was used as an internal standard [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMeasured concentrations were corrected by subtracting the values obtained from the blank control group, followed by multiplication by the dilution factor and adjustment based on recovery of the internal standard.\u003c/p\u003e \u003cp\u003eThe pH of yogurts were quantified using a 691 pH meter (Metrohm\u0026reg;, Filderstadt, Germany) under continuous stirring to prevent ion accumulation and ensure stable readings; the electrode was immersed directly in the original retail container, and measurements were recorded after stabilization.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism (GraphPad Software\u0026reg;, San Diego, USA). Enamel surface loss data were analyzed using two-way analysis of variance (ANOVA), with time and treatment as fixed factors, followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. Calcium and phosphorus concentrations were evaluated only for the soy-based and milk-based yoghurt groups. Group differences were analyzed using an unpaired \u003cem\u003et\u003c/em\u003e-test with Welch\u0026rsquo;s correction to account for potential heterogeneity of variances. All tests were two-tailed, and the significance level was set at α= 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eErosion\u0026ndash;Abrasion Cycling\u003c/h2\u003e \u003cp\u003eEnamel surface loss under erosive\u0026ndash;abrasive conditions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, while representative images of the enamel specimens after cycling are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt day 3, the water+no fluoride (negative control) group exhibited the highest mean enamel surface loss, differing significantly from the milk-based yogurt\u0026thinsp;+\u0026thinsp;Sn/F, soy-based yogurt+Sn/F, and water\u0026thinsp;+\u0026thinsp;Sn/F (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e). No statistically significant differences were detected among the three Sn/F groups (milk-based yogurt+Sn/F, soy-based yogurt+Sn/F, and water+Sn/F) at this time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBy day 7, enamel surface loss increased in all experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The water\u0026thinsp;+\u0026thinsp;no fluoride group continued to show the greatest enamel loss values and differed significantly from all other groups (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e). The soy-based yogurt+Sn/F group showed the lowest enamel surface loss at day 7 and differed significantly from the milk-based yogurt+Sn/F, water+Sn/F, and water+no fluoride groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnalysis of the time factor revealed a statistically significant increase in enamel surface loss from 3 to 7 days within all treatment groups (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e), indicating a progressive increase in enamel loss with longer exposure time under erosive\u0026ndash;abrasive conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eErosion\u0026ndash;Only Cycling\u003c/h2\u003e \u003cp\u003eEnamel surface loss under erosive conditions without abrasion is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, and representative images of the enamel specimens after erosive cycling are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt both day 3 and day 7, comparison among treatments (soy-based yogurt, milk-based yogurt, and water) revealed no statistically significant differences in enamel surface loss, as indicated by identical uppercase letters (\u003cem\u003ep\u0026gt;0.05\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). After 7 days, enamel surface loss increased in all experimental groups compared to day 3, indicating a progressive enamel loss pattern with increasing exposure time (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of pH, Calcium, and Phosphate\u003c/h2\u003e \u003cp\u003eThe pH, calcium, and phosphorus values of the yogurt specimens are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The milk-based yogurt presented a lower pH (4.4) than the soy-based yogurt (4.7). Calcium concentration was significantly higher in the milk-based yogurt (1434.1\u0026plusmn;190.3 \u0026micro;g/g) compared with the soy-based yogurt (168.4\u0026plusmn;15.5 \u0026micro;g/g; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001). Similarly, phosphorus concentration was higher in the milk-based yogurt (1033.8\u0026plusmn;128.1 \u0026micro;g/g) than in the soy-based yogurt (623.2\u0026plusmn;47.7 \u0026micro;g/g; \u003cem\u003ep\u003c/em\u003e=0.0004).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003epH and mineral composition (calcium and phosphorus) of the yogurts.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYogurt\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCalcium (\u0026micro;g/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ePhosphorus (\u0026micro;g/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoy-based\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e168.48\u0026thinsp;\u0026plusmn;\u0026thinsp;15.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e623.18\u0026thinsp;\u0026plusmn;\u0026thinsp;47.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk-based\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e1434.10\u0026thinsp;\u0026plusmn;\u0026thinsp;190.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1033.80\u0026thinsp;\u0026plusmn;\u0026thinsp;128.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eThe mineral composition (calcium and phosphorus) of the milk-based and soy-based yogurts, determined by inductively coupled plasma optical emission spectrometry (ICP-OES). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;6). Unpaired t test with Welch\u0026acute;s correlation, Calcium \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e and Phosphorous \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.0004\u003c/em\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of the present study demonstrated that the combination of soy-based yogurt with a toothpaste containing fluoride and tin (1450 ppm F, 3500 ppm Sn, 0.5% chitosan), under erosion\u0026ndash;abrasion conditions, resulted in the lowest enamel loss values. The same protective effect was not observed when the soy-based yogurt was applied only between erosive challenges, in the absence of fluoride or tin, indicating that components of this yogurt likely react with residual components of the fluoridated toothpaste, both present on the dental surface, thereby protecting the enamel against erosive loss.\u003c/p\u003e \u003cp\u003eIt is important to emphasize that, in the present study, the effect of both the soy-based yogurt and the milk-based yogurt was observed only when combined with a toothpaste containing fluoride and tin. The literature consistently demonstrates that fluoridated toothpastes, especially when associated with stannous compounds, significantly reduce enamel loss under in \u003cem\u003ein vitro\u003c/em\u003e erosion\u0026ndash;abrasion models [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe combination of fluoride and tin is considered an effective and safe agent, presenting superior benefits compared with other fluoridated compounds, in addition to positive effects on dental plaque, gingiva, and dentin hypersensitivity [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFluoride and tin interact directly with hydroxyapatite, the main mineral component of dental enamel. Stannous chloride (SnCl₂), being soluble in aqueous media, releases Sn\u0026sup2;⁺ ions, which exhibit high affinity for the phosphate groups of hydroxyapatites, forming a modified mineral layer enriched with tin and fluoride that offers mechanical protection. The formation of this layer reduces the solubility of calcium phosphate under acidic conditions and decreases calcium release during erosive challenges, thereby increasing resistance to enamel loss [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis is the first study to evaluate a soy-based yogurt regarding erosive enamel loss, and despite its protective effect against enamel loss when combined with a fluoridated toothpaste, its calcium and phosphate contents were significantly lower than those of the milk-based yogurt, which showed a comparatively lower protective effect in this study. Therefore, the mechanism of action of this soy-based yogurt cannot be explained by its calcium and phosphate content.\u003c/p\u003e \u003cp\u003eThe presence of starch and modified starch in the soy-based yogurt may have contributed to the increased viscosity of the surface layer, favoring longer contact time with enamel surface and more stable mineral layer enriched with tin and fluoride, a factor recognized as relevant for modulation of tooth erosion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. More viscous media tend to reduce clearance and the immediate removal of active substances from the dental surface, potentially delaying the diffusion and elimination of fluoride ions and tin species, whose efficacy depends on surface retention and interaction with the enamel\u0026ndash;medium interface [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Thus, it is plausible that the starch-rich matrix of the soy-based yogurt enhanced the action of the toothpaste containing fluoride, tin, and chitosan, although this mechanism still requires direct experimental confirmation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the literature, the study by Dos Santos et al [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] subjected enamel specimens to a static continuous erosion model, with direct immersion in beverages for 120 min without remineralization phases. The soy-based beverages were commercially available products (Addes\u0026reg;, Unilever) with pH varying from 3.9 to 4.2, while the non\u0026ndash;soy-based beverages comprised pH values from 2.9 to 3.4. Under these conditions, beverages containing soy extract promoted lower enamel surface loss and smaller increases in surface roughness compared with non-soy beverages, although all drinks exhibited erosive potential. This attenuating effect of soy should be interpreted with caution, as prolonged and continuous acidic exposure favors chemical interactions between beverage components and the enamel surface and does not reflect the clinical dynamics of short acidic challenges interspersed with periods of salivary recovery. Furthermore, they tested beverage and not yogurt, which are more acidic and less viscous.\u003c/p\u003e \u003cp\u003eWith respect to yogurt, past \u003cem\u003ein vitro\u003c/em\u003e erosive protocols frequently involved prolonged and repeated acidic exposures with accumulative several hours of contact time either [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], conditions that are far from clinical reality. In these aggressive experimental models, the prior application of milk and yogurt and extended storage in artificial saliva may have artificially enhanced the apparent reduction in demineralization. In addition, the use of outcome measures such as dental weight loss [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] or even confocal microscopy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] limits clinical interpretation. Similarly, sequential demineralization\u0026ndash;remineralization models, in that eroded enamel is immersed in milk or artificial saliva for up to 3 hours a prolonged period of exposure even with the application of sensitive methods to detect surface and mechanical changes, does not reflect real oral exposure patterns, restricting direct clinical extrapolation neither [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the past protective effects attributed to yogurt should be interpreted cautiously, especially in continuous demineralization models in which dairy components remain present during acidic challenge, potentially overestimating protection [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In contrast, pH-cycling models that simulate short and repeated acidic challenges with remineralization periods better approximate clinical conditions. Under these circumstances, a previous study showed that fluoridated milk had greater protective potential, whereas non-fluoridated dairy products demonstrated limited effects gainst erosive enamel loss [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] consistent with the findings of the present study.\u003c/p\u003e \u003cp\u003eOverall, evidence suggests that many of the past experimental protocols overestimate the protective effects of milk and yogurt, due to prolonged chemical interaction with enamel. Models incorporating short, repeated erosive challenges, salivary recovery, and abrasion when applicable, consistently demonstrate more limited protective effects, reinforcing that experimental design has a decisive influence on the magnitude and direction of the reported outcomes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].Therefore, the protective effect of milk and derivates may be of less relevance in case of ETW under clinical conditions, which needs to be further investigated.\u003c/p\u003e \u003cp\u003eFinally, limitations inherent to the \u003cem\u003ein vitro\u003c/em\u003e model should be considered, such as the absence of human saliva, acquired pellicle formation, and physiological remineralization mechanisms, which favor progression of erosive loss at a higher rate than would occur \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. On the other hand, inclusion of the erosion-only condition allowed isolation of the effect of yogurts on enamel, eliminating the influence of mechanical factors and fluoridated toothpaste, thereby contributing to a better understanding of the mechanisms involved.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWithin the limitations of this \u003cem\u003ein vitro\u003c/em\u003e erosion\u0026ndash;abrasion study, the combination of soy-based yogurt with a fluoride\u0026ndash;stannous toothpaste significantly reduced enamel surface loss, whereas the milk-based yogurt did not. The enhanced protection does not appear to be related to the yogurt\u0026rsquo;s mineral content, but may be associated with matrix-related factors that favor interaction with residual fluoride\u0026ndash;stannous deposits on enamel, although this mechanism requires confirmation. Further in situ and clinical studies are required to confirm the clinical significance of these findings.\u003c/p\u003e \u003cp\u003eOverall, these findings emphasize that protection against erosive enamel loss under clinically relevant cycling conditions is primarily attributable to fluoride, particularly when combined with stannous compounds. Yogurt, soy or milk-based, alone provides limited benefit.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthical approval\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that the work was approved by the Animal Ethics Committee of Bauru School of Dentistry \u0026ndash; University of S\u0026atilde;o Paulo (FOB/USP, CEUA:007/2026).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgement\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the technical support provided by the Department of Medical Materials Science and Technology, Institute for Biomedical Engineering, University Hospital T\u0026uuml;bingen. The authors also acknowledge the financial support provided by S\u0026atilde;o Paulo Research Foundation (FAPESP 2024/05183-0, 2024/20716-5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConflict of interest\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding Sources\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed by the S\u0026atilde;o Paulo Research Foundation (FAPESP), Brazil. Process Number 2024/05183-0, 2024/20716-5 (first author). FAPESP provided financial support for the acquisition of the materials required to conduct the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData Availability Statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eRafaela Ricci Kim:\u003c/u\u003e conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, validation, writing \u0026ndash; original draft, and writing \u0026ndash; review and editing; \u003cu\u003eAline Silva Braga\u003c/u\u003e\u003cem\u003e:\u0026nbsp;\u003c/em\u003econceptualization, data curation, formal analysis, investigation, methodology, validation, writing \u0026ndash; original draft, and writing \u0026ndash; review and editing; \u003cu\u003eGabriela Pellizon Floret:\u003c/u\u003e methodology; \u003cu\u003eAriadne Roehler\u003c/u\u003e, \u003cu\u003eJacob Schultheiss\u003c/u\u003e, \u003cu\u003eAna Carolina Magalh\u0026atilde;es:\u0026nbsp;\u003c/u\u003econceptualization, supervision, formal analysis, validation, and writing \u0026ndash; review and editing; and \u003cem\u003e\u003cu\u003eMarcella Esteves-Oliveira:\u003c/u\u003e\u003c/em\u003e conceptualization, supervision, formal analysis, validation, and writing \u0026ndash; review and editing and All authors: final approval of the version to be published.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDeclaration of generative AI and AI-assisted technologies in the manuscript preparation process\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work, the authors used ChatGPT (OpenAI) to assist with figure design and text revision. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCarvalho TS, Lussi A (2014) Combined effect of a fluoride-, stannous- and chitosan-containing toothpaste and stannous-containing rinse on the prevention of initial enamel erosion-abrasion. 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Odontology 113(1):201\u0026ndash;212. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10266-024-00960-y\u003c/span\u003e\u003cspan address=\"10.1007/s10266-024-00960-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Abrasion, Enamel erosion, Erosive tooth wear, Fluorides, Soy-based yogurt","lastPublishedDoi":"10.21203/rs.3.rs-9348858/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9348858/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study evaluated the effect of soy-based and milk-based yogurts against enamel erosive-abrasive surface loss.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNinety-eight bovine enamel specimens were randomly allocated to seven experimental groups (n = 14). Over seven days, specimens underwent six daily erosive cycles (0.05M citric acid, pH 2.3, 2 min), interspersed with remineralizing solution (pH 6.5). Post-acid preventive measures consisted of immersion in milk-based yogurt (pH 4.4), soy-based yogurt (pH 4.7), or tap water for 3 min twice daily. Under erosion-abrasion conditions, specimens underwent simulated toothbrushing using tin-containing fluoride toothpaste. Fluoride-free toothpaste served as abrasion control. Enamel surface loss was measured after 3 and 7 days using three-dimensional laser scanning microscopy. Calcium and phosphorus contents were quantified by optical emission spectrometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt day 3, no significant differences were observed among yogurt- and water-treated groups subjected to abrasion with fluoride toothpaste. By day 7, abrasion with a tin-containing fluoride toothpaste combined with soy-based yogurt resulted in significantly lower enamel surface loss (7.9±3.3µm) compared with the fluoride toothpaste only (12.2 ± 2.7µm; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Under erosion-only conditions, no significant differences were detected among preventive measures. Milk-based yogurt presented significantly higher calcium and phosphorus contents (Ca: 1434.1±190.3µg/g; P: 1033.8±128.1 µg/g) than soy-based yogurt (Ca: 168.4±15.5 µg/g; P: 623.2±47.7 µg/g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFermented soy-based yogurt may offer adjunctive protection in patients exposed to erosive and abrasive challenges.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eClinical relevance\u003c/em\u003e: Soy-based yogurt significantly enhances anti-erosive efficacy of fluoride-stannous formulations, providing clinicians with an evidence-based dietary intervention to optimize preventive strategies against erosive enamel loss.\u003c/p\u003e","manuscriptTitle":"Impact of Soy- and Milk-Based Yogurts on Enamel Preservation: Erosion and Abrasion under Simulated Conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-26 15:49:12","doi":"10.21203/rs.3.rs-9348858/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T04:17:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T02:26:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-20T16:54:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156726735595729013134340313837260366690","date":"2026-04-20T12:47:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38020457616005545994043805635033872436","date":"2026-04-20T11:44:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T04:15:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T20:10:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-14T03:04:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Oral Investigations","date":"2026-04-07T19:14:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"200b665a-3c91-487d-a4f8-dd3589c3f24a","owner":[],"postedDate":"April 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T07:53:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-26 15:49:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9348858","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9348858","identity":"rs-9348858","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}