Download PDF
CC BY 4.0 · Eur J Dent
DOI: 10.1055/s-0045-1809027
Original Article

The Inhibition of Streptococcus mutans Biofilms following Exposure to Different Chocolate Ingredients

Hadi A. Almoabid   *
1   College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
,
Leen Saleh Almutairi    *
1   College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
,
Abdul Samad Khan 
2   Department of Restorative Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
,
Mohammed A. Aljaffary
1   College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
,
Rasha AlSheikh
2   Department of Restorative Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
,
Khalid S. Almulhim
2   Department of Restorative Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
,
Abdulrahman A. Balhaddad 
2   Department of Restorative Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
› Author Affiliations
› Further Information
Also available at
   Permissions and Reprints

Abstract

Objectives

This project aimed to investigate the anticariogenic effect of four chocolate ingredients (polyphenol, theobromine, cacao, and flavanol) against Streptococcus mutans biofilms grown in vitro.

Materials and Methods

Stored S. mutans (UA 159) was transferred to Brain Heart Infusion (BHI) broth and incubated in aerobic incubator for 24 hours at 37°C in 5% CO2. Following this, 190 µL of each ingredient concentration (0.78–200 mg/mL) and 10 µL of the culture were added to a 96-well plate and incubated for 24 hours at 37°C in 5% CO2. Then, biofilms were fixed, stained with crystal violet, and analyzed for formation using a spectrophotometer. Control groups included negative control with only S. mutans and sterility control with BHI media.

Statistical Analysis

One-way analysis of variance and Tukey tests analyzed the data.

Results

Flavonoid at the 6.25 to 25 mg/mL concentrations reduced the S. mutans biofilms (p < 0.001) by 5- to 33-fold. Meanwhile, 50 mg/mL concentrations and higher completely eradicated biofilm growth. Similarly, cocoa concentrations ranging between 12.5 and 200 mg/mL revealed massive antibiofilm action from a 22-fold reduction at 12.5 mg/mL to complete biofilm eradication at 200 mg/mL. Polyphenol was the only ingredient showing biofilm inhibition at all concentrations ranging from almost 10-fold reduction to complete biofilm eradication, which were all significant (p < 0.001) compared to the control. Regarding theobromine, 3.125 mg/mL of it significantly increased the growth of S. mutans biofilms. At the concentration of 6.25 mg/mL, theobromine significantly (p < 0.001) inhibited the S. mutans biofilms by 3.35-fold. While at the range of 25 to 200 mg/mL, theobromine resulted in a reduction between 11-fold and complete biofilm eradication.

Conclusion

The findings suggest that flavonoid, cacao, polyphenol, and theobromine may serve as effective adjuncts in preventing dental caries by inhibiting S. mutans biofilm formation.

Keywords

antibacterial - biofilms - caries - chocolate - prevention

Authors' Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.


Data Availability Statement

The data supporting this study's findings are available from the corresponding author upon reasonable request.


* Both authors contribute equally




Publication History

Article published online:
06 May 2025

© 2025. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)

Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India

 

  • References

  • 1 Bin-Jardan LI, Almadani DI, Almutairi LS. et al. Inorganic compounds as remineralizing fillers in dental restorative materials: narrative review. Int J Mol Sci 2023; 24 (09) 8295
    PubMedSearch in Google Scholar
  • 2 Bhadila GY, Baras BH, Balhaddad AA. et al. Recurrent caries models to assess dental restorations: a scoping review. J Dent 2023; 136: 104604
    PubMedSearch in Google Scholar
  • 3 Pitts NB, Zero DT, Marsh PD. et al. Dental caries. Nat Rev Dis Primers 2017; 3: 17030
    CrossrefPubMedSearch in Google Scholar
  • 4 AlQranei MS, Balhaddad AA, Melo MAS. The burden of root caries: updated perspectives and advances on management strategies. Gerodontology 2021; 38 (02) 136-153
    PubMedSearch in Google Scholar
  • 5 Balhaddad AA, Mokeem L, Melo MAS, Gregory RL. Antibacterial activities of methanol and aqueous extracts of Salvadora persica against Streptococcus mutans biofilms: an in vitro study. Dent J 2021; 9 (12) 143
    CrossrefPubMedSearch in Google Scholar
  • 6 Balhaddad AA, AlSheikh RN. Effect of eucalyptus oil on Streptococcus mutans and Enterococcus faecalis growth. BDJ Open 2023; 9 (01) 1-5
    PubMedSearch in Google Scholar
  • 7 Pancu DF, Scurtu A, Macasoi IG. et al. Antibiotics: conventional therapy and natural compounds with antibacterial activity-a pharmaco-toxicological screening. Antibiotics (Basel) 2021; 10 (04) 401
    PubMedSearch in Google Scholar
  • 8 Ye L, Zhang J, Xiao W, Liu S. Efficacy and mechanism of actions of natural antimicrobial drugs. Pharmacol Ther 2020; 216: 107671
    PubMedSearch in Google Scholar
  • 9 Poliński S, Kowalska S, Topka P, Szydłowska-Czerniak A. Physicochemical, antioxidant, microstructural properties and bioaccessibility of dark chocolate with plant extracts. Molecules 2021; 26 (18) 5523
    PubMedSearch in Google Scholar
  • 10 Dumbrava D, Popescu LA, Soica CM. et al. Nutritional, antioxidant, antimicrobial, and toxicological profile of two innovative types of vegan, sugar-free chocolate. Foods 2020; 9 (12) 1844
    PubMedSearch in Google Scholar
  • 11 Tan TYC, Lim XY, Yeo JHH, Lee SWH, Lai NM. The health effects of chocolate and cocoa: a systematic review. Nutrients 2021; 13 (09) 2909
    PubMedSearch in Google Scholar
  • 12 Venkatesh Babu NS, Vivek DK, Ambika G. Comparative evaluation of chlorhexidine mouthrinse versus cacao bean husk extract mouthrinse as antimicrobial agents in children. Eur Arch Paediatr Dent 2011; 12 (05) 245-249
    PubMedSearch in Google Scholar
  • 13 Bensid A, El Abed N, Houicher A, Regenstein JM, Özogul F. Antioxidant and antimicrobial preservatives: properties, mechanism of action and applications in food - a review. Crit Rev Food Sci Nutr 2022; 62 (11) 2985-3001
    PubMedSearch in Google Scholar
  • 14 Cherniienko A, Pawełczyk A, Zaprutko L. Antimicrobial and odour qualities of alkylpyrazines occurring in chocolate and cocoa products. Appl Sci (Basel) 2022; 12 (22) 11361
    Search in Google Scholar
  • 15 Balhaddad AA, Melo MAS, Gregory RL. Inhibition of nicotine-induced Streptococcus mutans biofilm formation by salts solutions intended for mouthrinses. Restor Dent Endod 2019; 44 (01) e4
    PubMedSearch in Google Scholar
  • 16 Fideles SOM, Ortiz AC, Reis CHB, Buchaim DV, Buchaim RL. Biological properties and antimicrobial potential of cocoa and its effects on systemic and oral health. Nutrients 2023; 15 (18) 3927
    PubMedSearch in Google Scholar
  • 17 Moynihan P. Sugars and dental caries: evidence for setting a recommended threshold for intake. Adv Nutr 2016; 7 (01) 149-156
    PubMedSearch in Google Scholar
  • 18 Zhao A, Sun J, Liu Y. Understanding bacterial biofilms: from definition to treatment strategies. Front Cell Infect Microbiol 2023; 13: 1137947
    PubMedSearch in Google Scholar
  • 19 Gondil VS, Subhadra B. Biofilms and their role on diseases. BMC Microbiol 2023; 23 (01) 203
    PubMedSearch in Google Scholar
  • 20 Ciofu O, Moser C, Jensen PØ, Høiby N. Tolerance and resistance of microbial biofilms. Nat Rev Microbiol 2022; 20 (10) 621-635
    PubMedSearch in Google Scholar
  • 21 Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358 (9276) 135-138
    CrossrefPubMedSearch in Google Scholar
  • 22 Chen H, Xie S, Gao J. et al. Flavonoid Baicalein suppresses oral biofilms and protects enamel hardness to combat dental caries. Int J Mol Sci 2022; 23 (18) 10593
    PubMedSearch in Google Scholar
  • 23 Demir S, Keskin G, Akal N, Zer Y. Antimicrobial effect of natural kinds of toothpaste on oral pathogenic bacteria. J Infect Dev Ctries 2021; 15 (10) 1436-1442
    PubMedSearch in Google Scholar
  • 24 Ferrazzano GF, Amato I, Ingenito A, De Natale A, Pollio A. Anti-cariogenic effects of polyphenols from plant stimulant beverages (cocoa, coffee, tea). Fitoterapia 2009; 80 (05) 255-262
    PubMedSearch in Google Scholar
  • 25 Yan L, Zhang S, Zhou X, Tian S. Anti-biofilm and bacteriostatic effects of three flavonoid compounds on Streptococcus mutans . Biofouling 2023; 39 (03) 245-256
    PubMedSearch in Google Scholar
  • 26 Percival RS, Devine DA, Duggal MS, Chartron S, Marsh PD. The effect of cocoa polyphenols on the growth, metabolism, and biofilm formation by Streptococcus mutans and Streptococcus sanguinis. Eur J Oral Sci 2006; 114 (04) 343-348
    PubMedSearch in Google Scholar
  • 27 Farkash Y, Feldman M, Ginsburg I, Steinberg D, Shalish M. Polyphenols inhibit Candida albicans and Streptococcus mutans biofilm formation. Dent J 2019; 7 (02) 42
    Search in Google Scholar
  • 28 Falsetta ML, Klein MI, Colonne PM. et al. Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect Immun 2014; 82 (05) 1968-1981
    PubMedSearch in Google Scholar
  • 29 Taguri T, Tanaka T, Kouno I. Antibacterial spectrum of plant polyphenols and extracts depending upon hydroxyphenyl structure. Biol Pharm Bull 2006; 29 (11) 2226-2235
    PubMedSearch in Google Scholar
  • 30 Bouarab-Chibane L, Forquet V, Lantéri P. et al. Antibacterial Properties of polyphenols: characterization and QSAR (Quantitative Structure-Activity Relationship) models. Front Microbiol 2019; 10: 829
    PubMedSearch in Google Scholar
  • 31 Chagas MDSS, Behrens MD, Moragas-Tellis CJ, Penedo GXM, Silva AR, Gonçalves-de-Albuquerque CF. Flavonols and flavones as potential anti-inflammatory, antioxidant, and antibacterial compounds. Oxid Med Cell Longev 2022; 2022: 9966750
    PubMedSearch in Google Scholar
  • 32 Castellanos JS, Betancourt DE, Díaz-Báez D, Baldión PA. Effect of flavonoids from grape seed and cranberry extracts on the microbiological activity of Streptococcus mutans: a systematic review of in vitro studies. BMC Oral Health 2024; 24 (01) 662
    PubMedSearch in Google Scholar
  • 33 Saha S, Do T, Maycock J, Wood S, Boesch C. Antibiofilm efficacies of flavonoid-rich sweet orange waste extract against dual-species biofilms. Pathogens 2023; 12 (05) 657
    PubMedSearch in Google Scholar
  • 34 Prabu GR, Gnanamani A, Sadulla S. Guaijaverin – a plant flavonoid as potential antiplaque agent against Streptococcus mutans. J Appl Microbiol 2006; 101 (02) 487-495
    PubMedSearch in Google Scholar
  • 35 Rudin L, Bornstein MM, Shyp V. Inhibition of biofilm formation and virulence factors of cariogenic oral pathogen Streptococcus mutans by natural flavonoid phloretin. J Oral Microbiol 2023; 15 (01) 2230711
    PubMedSearch in Google Scholar
  • 36 Haraguchi H, Tanimoto K, Tamura Y, Mizutani K, Kinoshita T. Mode of antibacterial action of retrochalcones from Glycyrrhiza inflata. Phytochemistry 1998; 48 (01) 125-129
    PubMedSearch in Google Scholar
  • 37 Lakshmi A, Vishnurekha C, Baghkomeh PN. Effect of theobromine in antimicrobial activity: an in vitro study. Dent Res J (Isfahan) 2019; 16 (02) 76-80
    PubMedSearch in Google Scholar
  • 38 Cevallos González FM, Dos Santos Araújo EM, Lorenzetti Simionato MR. et al. Effects of theobromine addition on chemical and mechanical properties of a conventional glass ionomer cement. Prog Biomater 2019; 8 (01) 23-29
    PubMedSearch in Google Scholar
  • 39 Amaechi BT, Porteous N, Ramalingam K. et al. Remineralization of artificial enamel lesions by theobromine. Caries Res 2013; 47 (05) 399-405
    PubMedSearch in Google Scholar
  • 40 Durhan MA, Ozsalih S, Gokkaya B, Kulan PY, Kargul B. Caries preventive effects of theobromine containing toothpaste on early childhood caries: preliminary results. Acta Stomatol Croat 2021; 55 (01) 18-27
    CrossrefPubMedSearch in Google Scholar
  • 41 Srikanth RK, Shashikiran ND, Subba Reddy VV. Chocolate mouth rinse: effect on plaque accumulation and mutans streptococci counts when used by children. J Indian Soc Pedod Prev Dent 2008; 26 (02) 67-70
    PubMedSearch in Google Scholar
  • 42 Kibriya S, Srinivasan I, Setty JV, Anu S, Khan BS. Characterization of cocoa bean husk extract particles and its comparison as a mouthrinse with different vehicles in children aged 7-12 years. Int J Clin Pediatr Dent 2023; 16 (01) 54-59
    PubMedSearch in Google Scholar
  • 43 Rajendiran M, Trivedi HM, Chen D, Gajendrareddy P, Chen L. Recent development of active ingredients in mouthwashes and toothpastes for periodontal diseases. Molecules 2021; 26 (07) 2001
    PubMedSearch in Google Scholar
  • 44 Cheng L, Li J, He L, Zhou X. Natural products and caries prevention. Caries Res 2015; 49 (Suppl. 01) 38-45
    CrossrefPubMedSearch in Google Scholar