Methylglyoxal

Glycation of commercially available fatty acid and globulin-free HSA (rHSA) was conducted as previously described^{86 (link)} by incubating 40 mg mL⁻¹ of HSA in 1 × PBS with 10 mM of methyl glyoxal (Sigma-Aldrich CAS:78-98-8) at 37 °C for 48 h. The product of this incubation (Gly-HSA) was dialyzed against 1 × PBS to remove the excess of methyl glyoxal. GlyHSA was lyophilized and stored at −20 °C. Control non-glycated HSA was treated identically to GlyHSA (i.e., incubation at 37 °C for 48 h) except that no methyl glyoxal was present in the reaction buffer. The glycation stage of GlyHSA was analyzed by MALDI TOF-MS as described previously.^{81 (link)}

All cranberry extracts were provided by Ocean Spray Cranberries Inc. The cranberry extracts were labeled by the manufacturers as “SWP” (subcritical water extract of presscake), “SWF” (subcritical water extract of fruit), “SWPE” (subcritical water extract of presscake with tannase), “Type R” (resin extract) and “RE” (resin extract with tannase). The extracts vary based on their composition and methods of extraction (18 (link)). For this experiment, we purchased manuka honey (Manuka Health, New Zealand) containing 550+ ppm MGO. Methylglyoxal (Sigma, USA) was added to distilled water to prepare a 550 ppm solution of Methylglyoxal, equivalent to the label-indicated content in the manuka honey. Commercially available mouthwashes Colgate Total, Listerine, and ACT were also used. To prepare a mix of extracts, 750 μL of one extract was added to 750 μL of another extract in a sterile Eppendorf tube and mixed well. Distilled water was used as a negative control. Chlorhexidine (2% vol/vol chlorhexidine gluconate) was used as a positive control.
Brain Heart Infusion (BHI) agar plates were used to culture S. mutans (ATCC 25175) by incubating streak plates in a 5% carbon dioxide (CO₂) incubator at 37°C overnight. To standardize the bacterial suspension used in each assay, a 0.5 McFarland standard was prepared (19 (link)).