## 3 Results
### Colonization of GMNL Lactobacillus Strains among Participants of VGA-1 or VGA-2 Group  
One hypothesis states that colonization on vaginal epithelium is one of the reason for the beneficial effects of probiotics consumption to BV [21]. To understand the colonization of GMNL _Lactobacillus_ strains among participants of the VGA-1 or VGA-2 group, we spread the vaginal discharges onto MRS agar plates (Figure 6A) and cultured the _Lactobacillus_ species, as described in the Materials and Methods section. Among the 37 enrolled participants, _Lactobacillus_ colonies were found in No. S017 and No. S025 of the VGA-1 group. GMNL-74 or GMNL-185 _Lactobacillus_ was confirmed by RAPD profiles, using arbitrary primers and PCR analysis (Figure 6B-E). From these data, the ameliorative effect of VGA-1 consumption in BV was suggested to be achieved mainly through a colonization independent mechanism. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**VGA-1, _n = 18_** & \multicolumn{2}{c}{**VGA-2, _n = 19_**} \\ \hline Patient & BV recurrence & Patient & BV recurrence \\ after-intervention & & after-intervention \\ BV history, _n = 8_ & 1 (12.5) & BV history, _n = 10_ & 2 (20) \\ \hline \hline \end{tabular} Values are given as numbers (percentages) of subjects after intervention.  
\end{table} 
Table 3: BV recurrence status of subjects with BV history after probiotic intervention. Figure 4: The effects of VGA-1/VGA-2 supplementation on the clinical characteristics in BV patients. The changes in (**A**,**B**) vaginal pH value; (**C**,**D**) Nugent score; and (**E**,**F**) clinical symptom scores of VGA-1 (\(n=18\)) and VGA-2 (\(n=19\)) patients during the follow-up period. Asterisks (*) represent statistical significance (* \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\)) between two groups from the [PERSON] matched-pairs signed rank test. Figure 5: BV recurrence rates of enrolled subjects before and after probiotic intervention. Stacked bar chart of BV recurrence occurred <3 month, 3–6 month, >6 month, respectively: (**A**) The VGA-1 group and (**B**) the VGA-2 group. The variables were analyzed using the chi-square test. Figure 6: RAPD profiles of _Lactobacillus_ isolated from vaginal swabs of BV patients who received VGA-1 probiotics. (**A**) Scheme of isolating _Lactobacillus_ strains from vaginal swabs. (**B–D**) The vaginal discharge was spread onto the MRS agar plates for isolating _Lactobacillus_ species. The genomic DNA extracted from the individual colonies grown on MRS agar plates were amplified with strain-specific primers (Lac P1-P4), respectively, followed by agarose gel electrophoresis. RAPD profiles of individual _Lactobacillus_ strains isolated from BV patient No. S017 received VGA-1 probiotics at week 4 (**B**,**C**) or No. S025, who received VGA-1 probiotics at week 4 (**D**,**E**). Parental _L. rhamnus_ GMNL-74 and _L. acidophilus_ GMNL-185 were used as the control strains for RAPD analysis. Consumption of VGA-1 or VGA-2 Reduces interleukin (IL)-6 Expression in the Vaginal Specimens of BV Patients  
Next, we examined the changes of pro-inflammatory cytokines, including IL-19, IL-6, and TNF-\(\alpha\), in the vagina of BV patients, after consumption of VGA-1 or VGA-2. A qRT-PCR method was used to quantify the mRNA expression of these three pro-inflammatory cytokines in vaginal discharges. A decreased IL-6 expression was observed at week 4 samples of VGA-1 and VGA-2 consumption group (Figure 7, \(p\) = 0.012 for VGA-1 and \(p\) = 0.007 for VGA-2). The difference of IL-18, IL-6, or TNF-\(\alpha\) expression at week 2 was not significant compared to baseline or week 4, irrespective of the VGA-1 or VGA-2 group. The inhibition of TNF-\(\alpha\) by GMNL _Lactobacillus_ strains was also observed in HeLa cells after 1 h incubation of _S. agalactiae_ (Figure S4). In addition, the four GMNL _Lactobacillus_ strains displayed an induction effect to the IL-10 production in human peripheral blood mononuclear cells (Figure S5).