Honors College Thesis
 

Developing a Method For Quantitation of Organic Acid Exometabolites Released By Streptococcus mutans Biofilm Grown Under Oral Cavity-like Conditions

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https://ir.library.oregonstate.edu/concern/honors_college_theses/3j333b197

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  • Tooth decay in the form of dental caries and cavities is estimated to be among the most common health conditions globally, with treatment of these oral diseases resulting in a massive, world-wide economic burden. The cause of this demineralization of teeth is rooted in the action of complex microbial biofilms formed on tooth surfaces. The bacteria most closely associated with the formation of dental caries is Streptococcus mutans, which can process fermentable carbohydrates, such as sugars, and produce high amounts of lactic acid and other organic byproducts as exometabolites. These acids can decrease the pH of the surrounding environment and cause the chemical demineralization of the hydroxyapatite surface of the enamel, resulting in dental caries. As such, analytical processes that can accurately quantitate these exometabolites in complex biological solutions are useful to relate factors to possible tooth decay. Our previous work established experimental conditions to study the growth of S. mutans biofilms under oral cavity-like conditions on different resin surfaces. During the growth process, basal medium mucin (BMM) was administered at a continuous flow rate over the biofilm, emulating saliva in the oral cavity. Here, we describe a protocol for determining accurate levels of the exometbolite lactic acid in BMM using liquid chromatography-tandem mass spectrometry. We hypothesized that the sample preparation method developed for this study would be successful in detecting lactic acid intensity in BMM samples and producing a standard curve for future quantitation when compared to lactic acid detection and standard curve production in samples produced using clean solvent. The standard curve produced in this experiment using 13C3-lactic acid prepared in media samples allows for future experiments to quantitate the levels of lactic acid and other organic acids in media. The analytical method is broadly applicable for study of the release of organic acid exometabolites dependent on growth conditions and the presence of dietary carbohydrates. This study will contribute to our understanding of the chemical mechanisms linked to tooth decay.
  • Keywords: S. mutans, biofilm, dental caries, lactic acid, organic acid, exometabolites, basal medium mucin, LC-MS, MRM, 3-NPH derivatization, Solid-phase extraction, standard curve
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