Deacidification of Cranberry Juice Reduces Its Antibacterial Properties against Oral Streptococci but Preserves Barrier Function and Attenuates the Inflammatory Response of Oral Epithelial Cells

Abstract

Cranberry (Vaccinium macrocarpon) may be a potent natural adjuvant for the prevention of oral diseases due to its anti-adherence, anti-cariogenic, and anti-inflammatory properties. However, the high titrable acidity of cranberry juice (CJ) has been reported to cause gastrointestinal discomfort, leading consumers to restrict their intake of this beverage. Electrodialysis with a bipolar membrane (EDBM) can reduce the organic acid content of CJ while retaining the flavonoids associated with potential health benefits. This study aimed to assess how the deacidification of CJ by EDBM impacts the antibacterial properties of the beverage against cariogenic (Streptococcus mutans, Streptococcus sobrinus) and commensal (Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius) streptococci, and how it affects oral epithelial barrier function and inflammatory response in an in vitro model. The removal of organic acids from CJ (deacidification rate ≥42%) reduced the bactericidal activity of the beverage against planktonic S. mutans and S. gordonii after a 15-min exposure, whereas only the viability of S. gordonii was significantly impacted by CJ deacidification rate when the bacteria were embedded in a biofilm. Moreover, conditioning saliva-coated hydroxyapatite with undiluted CJ samples significantly lowered the adherence of S. mutans, S. sobrinus, and S. oralis. With respect to epithelial barrier function, exposure to CJ deacidified at a rate of ≥19% maintained the integrity of a keratinocyte monolayer over the course of 24 h compared to raw CJ, as assessed by the determination of transepithelial electrical resistance (TER) and fluorescein isothiocyanate-conjugated dextran paracellular transport. These results can be in part attributed to the inability of the deacidified CJ to disrupt two tight junction proteins, zonula occludens-1 and occludin, following exposure, unlike raw CJ. Deacidification of CJ impacted the secretion of IL-6, but not of IL-8, by oral epithelial cells. In conclusion, deacidification of CJ appears to provide benefits with respect to the maintenance of oral health.

Keywords: antibacterial; cranberry juice; dental caries; electrodialysis; epithelial barrier; inflammation; oral streptococci; organic acids.

Figure 1

Effect of the deacidification rate (0, 19, 42, 60, and 79%) of cranberry juice on the viability of planktonic oral streptococci reported as log of the initial bacterial count (N₀) following 1-min and 15-min contacts. The effect of a ¼ dilution of raw CJ on bactericidal activity was also studied. (A) S. mutans, (B) S. sobrinus, (C) S. gordonii, (D) S. oralis, and (E) S. salivarius. Results are expressed as mean log (N/N₀) ± SD of triplicate assays from two independent experiments. Columns with different letters are significantly different from one another (Two-way ANOVA, Bonferroni test, p < 0.01). CHX: Chlorhexidine 0.12%. Ctrl: Dilution buffer.

Figure 2

Effect of the deacidification rate (0, 19, 42, 60, and 79%) of cranberry juice on the killing of biofilm-embedded (A) S. mutans, (B) S. sobrinus, (C) S. gordonii, (D) S. oralis, and (E) S. salivarius following 1- and 15-min exposures. A ¼ dilution of raw CJ was also included. Cell viability was assessed using FilmTracer LIVE/DEAD Biofilm Viability kit. A 100% value was attributed to the negative control (PBS). Results are expressed as means ± SD of triplicate assays from three independent experiments. Columns with different letters are significantly different from one another (Two-way ANOVA, Bonferroni test, p < 0.01). CHX: Chlorhexidine 0.12%. Ctrl: PBS.

Figure 3

Adherence of FITC-labelled (A) S. mutans, (B) S. sobrinus, (C) S. gordonii, (D) S. oralis, and (E) S. salivarius to saliva-coated HA preconditioned (5 min) with raw or deacidified (0, 19, 42, 60, and 79%) cranberry juice. A 100% value was attributed to the negative control. Results are expressed as means ± SD of triplicate assays from three independent experiments. Columns with different letters are significantly different from one another (ANOVA, Bonferroni test, p < 0.01). Ctrl: PBS.

Figure 4

Time-dependent relative TER measurements of a B11 keratinocyte monolayer exposed (5 min) to raw or deacidified (0, 19, 42, 60, and 79%) cranberry juice. The basal TER of each well was measured before the exposure to cranberry juice (t₀). The results are expressed as means ± SD of % of TER at t₀ (n = 3 from a representative experiment). Columns with different letters are significantly different from one another (Two-way ANOVA, Bonferroni test, p < 0.01). Ctrl: antibiotic-free K-SFM.

Figure 5

Time-dependent paracellular transport of FD-4 across a B11 keratinocyte monolayer exposed (5 min) to raw or deacidified (0, 19, 42, 60, and 79%) cranberry juice. FD-4 was added to the apical compartment, and the fluorescence of the basolateral compartment is reported. Results are expressed as means ± SD of triplicate assays from a representative experiment. Treatments with different letters are significantly different from one another (Two-way ANOVA, Bonferroni test, p < 0.01).

Figure 6

Immunofluorescence staining of (A) zonula occludens−1 (ZO-1) and (B) occludin in B11 oral keratinocytes 24 h after being challenged by raw or deacidified (0, 19, 42, 60, and 79%) cranberry juice for 5 min. Images from two independent experiments are presented.

Figure 7

Effect on the production of (A) IL-6 and (B) IL-8 of a 5-min exposure of GMSM-K oral epithelial cells to raw or deacidified (0, 19, 42, 60, and 79%) cranberry juice. A ¼ dilution of raw CJ was also included. Cells were incubated for 24 h post-exposure, following which the supernatants were collected and the secretion of cytokines was measured. Results are expressed as means ± SD of a triplicate assay. Columns with different letters are significantly different from one another (ANOVA, Bonferroni test, p < 0.01). Ctrl: DMEM+1% FBS.