Oral bacteria produce organic acids from sugars. If oral pH declines to approximately 5.5 due to organic acids produced, enamel demineralization of teeth begins. If organic acids produced from sugars by mutans streptococci are omitted or decreased and oral pH does not decline to 5.5, enamel demineralization is prevented by buffering action of saliva. Therefore, the degree of pH decline of the culture medium is measured during the incubation with oral bacteria (especially mutans streptococci) in the presence and absence of test substance with anticariogenic effects. Our procedure to measure pH decline is rapid and reliable. Cell suspension and phytochemical solution are added to 20 mM glucose solution in Stephan's buffer (pH 7.0) in a test tube (total volume, 1.0-2.0 mL), and incubated for 1060 min at 37°C under anaerobic conditions after mixing. During incubation, the pH of the reaction medium is periodically measured with a portable pH meter (Hashiguchi-Ishiguro et al, 2009). Another method uses an incubation period of 24-48 h. But oral pH declines to about 4 immediately after the ingestion of sugars as glucose and sucrose (Lingstrom et al, 2000). Actually, the pH of reaction medium resulted in an immediate decline within 30 min in our study. In practical eating, if we intake sugars from meals or snacks, the food is masticated and swallowed within a few minutes and the pH in oral cavity declines. Therefore, the rapid and reliable method is suitable for in vitro assay.
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