Effect of low-sugar fermented milk with Lactobacillus paracasei 431 on Streptococcus mutans biofilm formation and enamel demineralization: in vitro study
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Abstract
Abstract
Objectives: This study investigates the effects of Lactobacillus paracasei 431 and sugar content in fermented milk on the growth and biofilm formation of S. mutans, as well as its potential to cause enamel demineralization.
Materials and methods: Two fermented milks were used: 1.) 6% sucrose, and 2.) 0.1% sucrose. S. mutans (ATCC25175) was cultured with fermented milk and filtered fermented milk to remove L. paracasei 431. Firstly, S. mutans growth was determined by using colony-forming units. Secondly, S. mutans biofilm formation was evaluated using a biofilm assay. Thirdly, S. mutans biofilms were grown on enamel slabs, then exposed twice per day to the fermented milks for five days, and surface hardness loss was measured. Biofilms on the enamel slabs were stained with live/dead dye and observed under a confocal microscope. The Kruskal-Wallis test was used to compare differences among groups, and post-hoc test contrasts for every pair of variables were used (Bonferroni Correction). A difference was considered statistically significant if it was p<0.05.
Results: The viable S. mutans count in fermented milk with 6% sucrose was lower than in filter-sterilized milk (p<0.05). Both fermented milks with 6% sucrose and 0.1% sucrose reduced S. mutans biofilm formation compared to filter fermented milk (p<0.05). Fermented milk with 0.1% sucrose showed less demineralization than 6% sucrose, though the difference was not significant (p>0.05).
Conclusions: Fermented milk with Lactobacillus paracasei 431 inhibited S. mutans growth and biofilm formation. No statistically significant difference was observed between fermented milk with 6% and 0.1% sucrose in the percentage of surface hardness loss.
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References
Morelli L, Capurso L. FAO/WHO guidelines on probiotics: 10 years later. J Clin Gastroenterol. 2012 Oct;46 Suppl:S1-2. doi: 10.1097/MCG.0b013e318269fdd5.
Luo SC, Wei SM, Luo XT, Yang QQ, Wong KH, Cheung PC, et al. How probiotics, prebiotics, synbiotics, and postbiotics prevent dental caries: an oral microbiota perspective. NPJ Biofilms Microbiomes. 2024 Feb;10(1):14. doi: 10.1038/s41522-024-00488-7.
Radaic A, Kapila YL. The oralome and its dysbiosis: New insights into oral microbiome-host interactions. Comput Struct Biotechnol J. 2021 Feb;19:1335-1360. doi: 10.1016/j.csbj.2021.02.010.
Coqueiro A, Bonvini A, Raizel R, Tirapegui J, Rogero MM. Probiotic supplementation in dental caries: Is it possible to replace conventional treatment?. Nutrire. 2018;43:1-9. doi: 10.1186/s41110-018-0064-3.
Lin X, Chen XI, Tu Y, Wang S, Chen H. Effect of probiotic lactobacilli on the growth of Streptococcus mutans and multispecies biofilms isolated from children with active caries. Med Sci Monit. 2017 Aug;23:4175-4181. doi: 10.12659/MSM.902237.
Rossoni RD, Velloso MDS, de Barros PP, de Alvarenga JA, Santos JDD, Santos Prado ACCD, et al. Inhibitory effect of probiotic Lactobacillus supernatants from the oral cavity on Streptococcus mutans biofilms. Microb Pathog. 2018 Oct; 123:361-367. doi: 10.1016/j.micpath.2018.07.032.
Cai JN, Jung JE, Lee MH, Choi HM, Jeon JG. Sucrose challenges to Streptococcus mutans biofilms and the curve fitting for the biofilm changes. FEMS Microbiol Ecol. 2018 Jul;94(7) doi: 10.1093/femsec/fiy091.
Wimolsantirungsri K, Asvanund Y, Mitrakul K, Srisatjaluk RL. Effect of bovine and plant based milks on Streptococcus mutans biofilm formation, biofilm pH Level and enamel demineralization in human primary teeth. Southeast Asian J Trop Med Public Health. 2023 Apr;54(2):71-86.
Srivoha N, Asvanund Y, Mitrakul K, Srisatjaluk R. Effects of alternative milk on Streptococcus Mutans biofilm formation and enamel demineralization in human primary teeth. European J Gen Dent. 2025 May;14(02):203-211. doi: 10.1055/s-0044-1792165.
Wu CY, He SJ, Mar K, Hsu CY, Hung SL. Inhibition of Streptococcus mutans by a commercial yogurt drink. J Dent Sci. 2019 Jun;14(2):198-205. doi: 10.1016/j.jds.2018.11.007.
Lin YT, Chou CC, Hsu CY. Effects of Lactobacillus casei shirota intake on caries risk in children. J Dent Sci. 2017 Jun;12(2):179-84. doi: 10.1016/j.jds.2016.09.005.
Lodi CS, Oliveira LV, Brighenti FL, Delbem AC, Martinhon CC. Effects of probiotic fermented milk on biofilms, oral microbiota, and enamel. Braz Oral Res. 2015; 29:S1806-83242015000100229. doi: 10.1590/1807-3107BOR-2015.vol29.0033.
Choomroy D, Thanathornwong B, Techatanawat B, Techatanawat S, Boonmongkolraksa P. The effect of probiotic fermented milk on salivary Streptococcus mutans count. KKU Res J (Graduate Studies). 2021;21(4):185-195.
Cai JN, Kim D. Biofilm ecology associated with dental caries: understanding of microbial interactions in oral communities leads to development of therapeutic strategies targeting cariogenic biofilms. Adv Appl Microbiol. 2023 Jan;122:27–75. doi:10.1016/bs.aambs.2023.02.001.
Dashper S, Saion B, Stacey M, Manton D, Cochrane N, Stanton D, et al. Acidogenic potential of soy and bovine milk beverages. J Dent. 2012 Sep;40(9):736-741. doi: 10.1016/j.jdent.2012.05.004.
Kim KH, Choi CH. The effects of fermented milk intake on the enamel surface. J Korean Soc Dent Hyg. 2021;21(5):507-515. doi: 10.13065/jksdh.20210048.
Lodi CS, Sassaki KT, Fraiz FC, Delbem AC, Martinhon CC. Evaluation of some properties of fermented milk beverages that affect the demineralization of dental enamel. Braz Oral Res. 2010 Jan-Mar;24(1):95-101. doi: 10.1590/S1806-83242010000100016.
Nadelman P, Frazão JV, Vieira TI, Balthazar CF, Andrade MM, Alexandria AK, et al. The performance of probiotic fermented sheep milk and ice cream sheep milk in inhibiting enamel mineral loss. Food Res Int. 2017 Jul;97:184-90. doi: 10.1016/j.foodres.2017.03.051.
Nadelman P, Monteiro A, Balthazar CF, Silva HL, Cruz AG, de Almeida Neves A, et al. Probiotic fermented sheep’s milk containing Lactobacillus casei 01: Effects on enamel mineral loss and Streptococcus counts in a dental biofilm model. J Funct Foods. 2019 Mar:54:241-248. doi: 10.1016/j.jff.2019.01.025.
Mountcastle SE, Vyas N, Villapun VM, Cox SC, Jabbari S, Sammons RL, et al. Biofilm viability checker: an open-source tool for automated biofilm viability analysis from confocal microscopy images. NPJ Biofilms Microbiomes. 2021 May;7(1):44. doi: 10.1038/s41522-021-00214-7.
Wasfi R, Abd El‐Rahman OA, Zafer MM, Ashour HM. Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries‐inducing Streptococcus mutans. J Cell Mol Med. 2018 Mar;22(3):1972-1983. doi: 10.1111/jcmm.13496.
Marttinen AM, Haukioja AL, Keskin M, Söderling EM. Effects of Lactobacillus reuteri PTA 5289 and L. paracasei DSMZ16671 on the adhesion and biofilm formation of Streptococcus mutans. Curr Microbiol. 2013 Aug;67(2):193-199. doi: 10.1007/s00284-013-0352-3.
Zhao Z, Wu J, Sun Z, Fan J, Liu F, Zhao W, et al. Postbiotics derived from L. paracasei ET-22 inhibit the formation of S. mutans biofilms and bioactive substances: an analysis. Molecules. 2023 Jan;28(3):1236. doi: 10.3390/molecules28031236.
de Alvarenga JA, de Barros PP, de Camargo Ribeiro F, Rossoni RD, Garcia MT, dos Santos Velloso M, et al. Probiotic effects of Lactobacillus paracasei 28.4 to inhibit Streptococcus mutans in a gellan-based formulation. Probiotics Antimicrob Proteins. 2021 Apr;13(2):506–517. doi: 10.1007/s12602-020-09712-0.
Teanpaisan R, Piwat S, Tianviwat S, Sophatha B, Kampoo T. Effect of long-term consumption of Lactobacillus paracasei SD1 on reducing mutans streptococci and caries risk: a randomized placebo-controlled trial. Dent J (Basel). 2015 Apr;3(2):43-54. doi: 10.3390/dj3020043.
AlKanderi S, AlFreeh M, Bhardwaj RG, Karched M. Sugar substitute Stevia inhibits biofilm formation, exopolysaccharide production, and downregulates the expression of streptococcal genes involved in exopolysaccharide synthesis. Dent J (Basel). 2023 Nov;11(12):267. doi: 10.3390/dj11120267.
Escobar E, Piedrahita M, Gregory RL. Growth and viability of Streptococcus mutans in sucrose with different concentrations of Stevia rebaudiana Bertoni. Clin Oral Investig. 2020 Sep;24(9):3237-3242. doi: 10.1007/s00784-020-03197-5.
Assaf D, Steinberg D, Shemesh M. Lactose triggers biofilm formation by Streptococcus mutans. Int Dairy J. 2015 Mar; 42:51-57. doi: 10.1016/j.idairyj.2014.10.008.
Zhu J, Liu J, Li Z, Xi R, Li Y, Peng X, et al. The effects of nonnutritive sweeteners on the cariogenic potential of oral microbiome. Biomed Res Int. 2021 Jun; 2021:9967035. doi: 10.1155/2021/9967035.
Lussi A, Schlueter N, Rakhmatullina E, Ganss C. Dental erosion–an overview with emphasis on chemical and histopathological aspects. Caries Res. 2011;45 Suppl 1:2-12. doi: 10.1159/000325915.
Prabhakar A, Kurthukoti A, Gupta P. Cariogenicity and acidogenicity of human milk, plain and sweetened bovine milk: an in vitro study. J Clin Pediatr Dent. 2010 Apr;34(3):239-247. doi: 10.17796/jcpd.34.3.lk08l57045043444.
Zulkapli R, Daslam D, Safiai NF, Zainal M, Zain NM. In vitro evaluation of the erosive effect of probiotic drink on tooth enamel. Eur J Gen Dent. 2020 May; 9:73-78. doi: 10.4103/ejgd.ejgd_166_19.
Li A, Ma Y, Cui N, Zhang X, Zheng Q, Du P, et al. Research progress of milk and dairy products to prevent caries. J Funct Foods. 2023 Nov;110:105837. doi: 10.1016/j.jff.2023.105837.
Chulibert ME, Ferrer A, Koch KE, Rigalli A. Effect of fermented milk with kefir grains on the in vitro demineralization of bovine tooth enamel. Actual Osteol. 2019;15(3):205-213.
Shimizu A, Yamamoto T, Nakashima S, Nikaido T, Sugawara T, Momoi Y. Measurement of surface hardness of primary carious lesions in extracted human enamel-Measurement of Knoop hardness using Cariotester. Dent Mater J. 2015;34(2):252-256. doi: 10.4012/dmj.2014-282.
Young DA, Nový BB, Zeller GG, Hale R, Hart TC, Truelove EL, et al. The American Dental Association caries classification system for clinical practice: a report of the American Dental Association Council on Scientific Affairs. J Am Dent Assoc. 2015 Feb;146(2):79-86. doi: 10.1016/j.adaj.2014.11.018.
Zhang T, Geng S, Cheng T, Mao K, Chitrakar B, Gao J, et al. From the past to the future: Fermented milks and their health effects against human diseases. Food Front. 2023 Dec;4(4):1747-1777. doi: 10.1002/fft2.304.
Schwendicke F, Korte F, Dörfer CE, Kneist S, Fawzy El-Sayed K, Paris S. Inhibition of Streptococcus mutans growth and biofilm formation by probiotics in vitro. Caries Res. 2017;51(2):87-95. doi: 10.1159/000452960.
Giacaman RA, Muñoz-Sandoval C. Cariogenicity of different commercially available bovine milk types in a biofilm caries model. Pediatr Dent. 2014 Jan-Feb;36(1):1E-6E.
Zhang G, Lu M, Liu R, Tian Y, Vu V, Li Y, et al. Inhibition of Streptococcus mutans biofilm formation and virulence by Lactobacillus plantarum K41 isolated from traditional Sichuan pickles. Front Microbiol. 2020 Apr;11:774. doi: 10.3389/fmicb.2020.00774.
Guo M, Yang K, Zhou Z, Chen Y, Zhou Z, Chen P, et al. Inhibitory effects of Stevioside on Streptococcus mutans and Candida albicans dual-species biofilm. Front Microbiol. 2023 Apr;14:1128668. doi: 10.3389/fmicb.2023.1128668.
Xiao J, Hara AT, Kim D, Zero DT, Koo H, Hwang G. Biofilm three-dimensional architecture influences in situ pH distribution pattern on the human enamel surface. Int J Oral Sci. 2017 Jun;9(2):74-79. doi:10.1038/ijos.2017.8.
