Black Cumin Seed Oil Disrupts Structural Biomass and Attenuates Porphyrin-Associated Maturation in Mature Oral Microcosm Biofilms: An In Vitro Study

Background/Objective: Mature oral biofilms resist conventional antiseptics via dense extracellular polymeric substance (EPS) matrices. Consequently, the prolonged use of broad-spectrum agents like chlorhexidine (CHX) raises profound ecological concerns. Using a mature oral microcosm biofilm model, we evaluated the multifaceted efficacy of 0.5% black cumin seed oil (BCSO) in disrupting structural biomass, suppressing aciduric bacterial viability, and attenuating porphyrin-associated anaerobic biofilm maturation. Methods: Saliva-derived microcosm biofilms were cultivated on hydroxyapatite discs in a mucin-containing medium (0.5% sucrose) for 6 days. Mature biofilms were treated daily for 1 min for 6 days (n = 19/group) with 0.5% BCSO, 0.12% CHX (positive control), or 0.5% dimethyl sulfoxide (negative control). EPS and bacterial biovolumes were quantified using confocal microscopy. Viability was strictly evaluated as aciduric bacterial colony-forming units. Pathogenic potential was specifically assessed as porphyrin-associated maturation by the red-to-green fluorescence intensity ratio (RatioR/G) using quantitative light-induced fluorescence-digital technology. Results: Compared with the negative control, 0.5% BCSO significantly degraded the EPS matrix (p < 0.001) and bacterial biovolumes (p = 0.045), and reduced aciduric bacterial counts (p = 0.028). Importantly, 0.5% BCSO significantly reduced the RatioR/G (p = 0.015), compared with the negative control, indicating severely attenuated porphyrin-associated anaerobic maturation. In the CHX group, the reduction did not reach statistical significance compared with the negative control (p = 0.399). Conclusions: In vitro, 0.5% BCSO and 0.12% CHX effectively reduced structural biomass and aciduric bacterial viability. Furthermore, BCSO significantly reduced late-stage, porphyrin-associated anaerobic maturation compared with the negative control, whereas the reduction observed with CHX did not reach statistical significance. These findings highlight the potential of BCSO as a targeted natural antibiofilm adjunct, warranting rigorous in vivo and mechanistic validation before clinical translation.