Industrial sweet orange waste as a potential antimicrobial agent for the management of dental caries

Dental caries is a bacterial disease, caused by the buildup of plaque biofilm on the teeth and soft surfaces in the mouth. It remains the most prevalent chronic disease globally in both children and adults. The development of dental caries affects the structure of cementum, enamel, and dentin. Among all the conventional caries treatments, chlorhexidine (CHX) is well recognised as the gold standard treatment. However, the use of this treatment causes many side effects such as hampering healthy oral cells and eradication of health-associated oral microorganisms. It would therefore be beneficial to identify alternatives to CHX as an anticariogenic agent. Polyphenols have been reported to have many clinically significant effects on human health and are potential alternatives to CHX as an antimicrobial treatment. The sweet orange juice industries generate enormous amounts of solid waste, primarily peels which are an economically viable resource of polyphenols. Therefore, this project aims to investigate the antibacterial and anticariogenic properties of extracts from industrial sweet orange waste (ISOWE), a rich source of flavonoids. Extraction of polyphenols was conducted as microwave-assisted procedure under different temperature (70-90 °C) and time conditions (5, 10, and 15 minutes) with subsequent characterization revealing the flavanones hesperidin and narirutin, the flavonols quercetin, and flavones sinensetin, nobiletin, and tangeretin as main flavonoid compounds. Antimicrobial properties of these extracts and lead flavonoid compounds were explored as minimum inhibitory (MIC), bactericidal concentrations (MBC) and bactericidal kinetics, against Streptococcus mutans and Lactobacillus casei. Among the identified flavonoids, flavones were found to be the most effective antimicrobial compounds. The most potent extract, based on the MIC findings, was applied in different concentrations (40, 80, and 120 mg/mL) and in combination with CHX (0.1, 0.2%) to determine anticariogenicity in a 7-day dual species cariogenic model. Indeed, dose-dependent efficacy of ISOWE could be established and whilst CHX was more effective compared to ISOWE, synergistic action of ISOWE (120 mg/mL) with 0.1% CHX could be established which was indeed superior to 0.2% CHX alone. In addition, the effects of ISOWE in combination with CHX were investigated, on viability and proliferation of TR146 cells, a buccal epithelial cell line. Results of cell culture studies confirm absence of cytotoxic effects of ISOWE and indicate reduced cytotoxicity of 0.1% CHX when applied in combination. In addition, a complex cariogenic model using oral samples from human volunteers was developed to explore the efficacy of ISOWE against a diverse group of oral microorganisms using bacterial viability as well as 16S rRNA sequencing approach. The findings from the complex cariogenic model exhibited that the antimicrobial action of CHX is indiscriminate towards the different bacterial taxa including both pathogenic and health-associated oral microorganisms. On the other hand, ISOWE was more effective against pathogenic species and the synergy of ISOWE with CHX was also maintained in this complex cariogenic model. In conclusion, results from this research project suggest that ISOWE could be a potential natural antimicrobial agent for managing dental caries. As ISOWE and CHX demonstrated synergistic effects, further research should be conducted into optimising the combinatory effects e.g. into lowering CHX concentrations to reduce the negative impacts of CHX without compromising the antimicrobial efficacy.