Various types of milk are given to children but may contribute to dental caries by promoting the growth of the cariogenic bacteria Streptococcus mutans. In this study, we aimed to determine level of S. mutans biofilm formation, enamel demineralization and pH of the biofilm in vitro using human primary incisor teeth exposed to unsweetened bovine milk and sweetened and unsweetened almond and soy milk products in order to compare their respective risk factors for contributing to caries formation. To determine the level of biofilm formation, we put 200 mu l of sterile pooled human saliva in each well of a 96-well polystyrene plate for 24 hours. We then removed the saliva and inoculated each well with 100 mu l of S. mutans at a concentration of 106 CFU/ml. The studied milks used for this study were: 1) bovine milk (Nongpho (R)), 2) unsweetened almond milk (Almond Breeze (R)), 3) almond milk with 2.7% sucrose (Almond Breeze (R)), 4) unsweetened soy milk (Lactasoy (R)) and 5) soy milk with 7% sucrose (Lactasoy (R)). Each milk product was diluted with sterile deionized water at a ratio of 1:2. We then added 100 mu l of each diluted tested milk sample to their respective wells. The study was performed in triplicate for each studied sample. We used Brain Heart Infusion (BHI) with 5% sucrose as a positive control and sterile deionized water as a negative control placing them in their respective wells. The plate was gently agitated and then incubated in 5% CO2 at 37 degrees C for 24 hours to allow a biofilm to form. The biofilm in each well was stained with 200 mu l of 0.05% crystal violet solution for 10 minutes and the optical density (OD) was read to determine the concentration of S. mutans biofilm formation. The OD (+/- standard deviation (SD)) values for the bovine milk, unsweetened almond milk, sweetened almond milk, unsweetened soy milk and sweetened soy milk were: 0.082 (+/- 0.001), 0.076 (+/- 0.026), 1.86 (+/- 0.152), 0.535 (+/- 0.217) and 1.6 (+/- 0.108), respectively. The sweetened plant-based milk samples (sweetened almond milk and sweetened soy milk) had significantly (p<0.05) greater biofilm formation than the unsweetened plant-based milk samples (unsweetened almond milk and unsweetened soy milk samples) and the bovine milk (p<0.05). There was no significant difference in biofilm formation between the bovine milk and the unsweetened plant-based milk samples so for the pH and demineralization parts of the study, we only compared the bovine milk with the sweetened plant-based milk samples. To assess for enamel demineralization, we cut 40, 4 x 4 mm enamel slab samples from 40 primary human incisor teeth and divided the cut slabs into 5 study groups by the type of solution the enamel slab was immersed in: Group 1: bovine milk (n = 10), Group 2: sweetened almond milk (n = 10), Group 3: sweetened soy milk (n = 10), Group 4 (positive control): 5% sucrose solution (n = 5), Group 5 (negative control) deionized water (n = 5). Each enamel slab was checked for surface hardness prior to and after being immersed in the respective studied solutions. The immersions occurred for 30 minutes at a time 4 hours apart 3 times a day for 5 consecutive days. The surface hardness readings before and after immersion were compared and the percentage of surface hardness loss (SHL) was calculated. The percents (+/- SD) of SHL in the bovine milk, sweetened almond milk, positive control and negative control were: 73 (+/- 6), 68 (+/- 11), 78 (+/- 5), 83 (+/- 8) and 73 (+/- 14), respectively. The percent SHL among the teeth samples exposed to sweetened almond milk was significantly (p<0.05) lower than the percent SHL among the teeth samples exposed to sweetened soy milk and the bovine milk (p<0.05). We measured the pH of biofilm formed on the enamel slab samples exposed to the studied milk samples for 6 days. The pH ranges during Days 1-6 of the biofilm on the bovine milk samples, sweetened almond milk samples and sweetened soy milk samples were: 6.3-6.7, 6.26.7 and 5.4-6.6, respectively. We compared the changes in pH between Day 1 and Day 6 for each of these three studied milk samples and found the sweetened soy milk had a significantly (p<0.05) greater pH-decrease than the other groups. In summary, there was significantly greater biofilm formation with the sweetened plant-based milks than the bovine milk and non-sweetened plant-based milks but no significant difference between the bovine milk and non-sweetened plant-based milks. There was a significantly greater percent SHL with the sweetened plant-based milks than the bovine milk and there was a significantly greater drop in the pH of the biofilm with the sweetened plant-based milks than the bovine milk and the drop was significantly greater with the sweetened soy-based milk than the sweetened almond-based milk. We conclude, sweetened plant-based milks increase the risk for primary incisor teeth caries formation greater than bovine milk but non-sweetened plant-based milks did not. Further in vivo studies are needed to determine if children who drink sweetened plant-based milks have more caries or not.
