In Vitro, Ex Vivo, and In Vivo Evidence of Nitrate-Reducing Activity in Levilactobacillus brevis CD2: A Potential Tool for Oral and Systemic Health Applications

Growing evidence supports the use of nitrate-reducing bacterial strains as probiotics to enhance the benefits of nitrate metabolism for both oral and systemic health. This study aimed to test the nitrate reductase activity of <i>Levilactobacillus brevis</i> CD2 (DSM-27961/CNCM I-5566), a strain widely used as a starter culture in fermented foods and recognized for its multifaceted health-promoting probiotic properties. We also sought to determine whether the probiotic lysate enhances nitrate reduction ex vivo using six salivary samples from healthy subjects while evaluating its potential influence on pH and buffering capacity. Considering the established link between lactate metabolism and nitrite production, we assessed the salivary levels of D-lactate after a 3-hour incubation with or without <i>Lv. brevis</i>. The results indicate that <i>Lv. brevis</i> CD2 exhibits significant intrinsic and concentration-dependent nitrate reductase activity. Additionally, treatment with <i>Lv. brevis</i> for 3 h significantly increased nitrite generation across all saliva samples, with further enhancement observed after the addition of exogenous nitrates. <i>Lv. brevis</i> also significantly improved salivary pH and buffering capacity, particularly when combined with nitrate. Furthermore, the probiotic treatment resulted in reduced levels of salivary D-lactate. To further support and validate our in vitro and ex vivo findings, we evaluated the oral nitrate-reducing activity in saliva samples from healthy individuals treated for four weeks with <i>Lv. brevis</i> CD2 lozenges. Of note, the results indicated that the probiotic group showed a significant increase in oral nitrate-reducing capacity compared to baseline and placebo after four weeks of treatment. Overall, our study suggests that <i>Lv. brevis</i> CD2 acts as a nitrate-reducing probiotic, providing new insights into its health benefits and complementing findings from previous studies.