Strain-Specific Effects of Vaginal Probiotics in formulation of Functional Fermented Milk designed for Women's Well-Being

Fermented milks are widely recognized as excellent carriers for probiotic bacteria, and growing evidence highlight the potential of functional foods to influence human health. It is already well established that probiotic strains exhibit strain-specific behaviors. This characteristic is crucial not only for selecting strains to achieve desired health effects but also for optimizing food formulations, as each strain may positively or negatively impact the final product's quality. This study, conducted as part of the FUNPRO-L project, aims to develop a functional fermented milk using skim milk, the starter culture ST-440 (Sacco) (a mix of Streptococcus thermophilus strains producing exopolysaccharides), and probiotics of vaginal origin with potential benefits for women's well-being. Five probiotic strains - Lactobacillus crispatus BC1, BC4, BC5 and Lactobacillus gasseri BC9, BC12 - at different concentrations (8, and 7 log CFU/ml) were initially tested for their survival by plate counting samples upon fermentation and after 28 days under refrigerated conditions (5°C). Subsequent evaluations focused on the three most promising strains (L. crispatus BC1, BC4, and BC5) at 8 log CFU/ml. Parameters assessed during 28 days of storage at 5 °C included pH, microbial viability (on M17 and MRS agar at 45°C and 37°C for 48 hours, for S. thermophilus and L. crispatus, respectively), water-holding capacity (WHC, measured gravimetrically), rheological properties (using a Rheometer), amino acid profiles, volatile molecule profiles (via SME-GC/MS), and antimicrobial activity against enteric and urogenital pathogens (using overlay assays). Among the tested strains, BC1 demonstrated the highest survival rate (around 0.5 log reduction), followed by BC5 (less than 1 log), while BC4 exhibited a reduction of more than 1 log during the 28 days of storage (Fig. 1). From a technological perspective, after 28 days of storage, fermented milk with BC5 maintained a higher value of WHC (23.3 vs 16.7 %) and had a significant greater yield stress value (1.16 Pa) and consistency index (3.30 Pa x sn) with respect to the other samples and control (0.17 Pa and 0.16 Pa x sn, respectively, for control). Presence of probiotic significantly enhanced the release of free amino acids, with BC5 contributing the most on the post-fermentation and BC4 during storage. Strain-specific effects of the strains were observed in volatile molecule profiles, with an increase in acetic acid and other short and medium chain fatty acids already after fermentation. In the antimicrobial assay, BC1 and BC5 exhibited the strongest and most sustained activity against Escherichia coli H10407 (ETEC), Yersinia enterocolitica, and Salmonella Typhimurium up to 28 days (Tab. 2). These findings position BC1 and BC5 as the most promising strains for the development of functional fermented milk targeting women's well-being. This work highlights the need to balance three key aspects—probiotic functionality and viability, technological characteristics, and sensory properties—to achieve an optimal formulation. The results will be applied to industrial-scale production and subsequent clinical trials to validate the product's health benefits for women