The Effect of Oral Probiotics (Streptococcus Salivarius k12) on the Salivary Level of Secretory Immunoglobulin A, Salivation Rate, and Oral Biofilm: A Pilot Randomized Clinical Trial

We aimed to assess the effect of oral probiotics containing the Streptococcus salivarius K12 strain on the salivary level of secretory immunoglobulin A, salivation rate, and oral biofilm. Thirty-one consenting patients meeting the inclusion criteria were recruited in this double-blind, placebo-controlled, two-arm, parallel-group study and randomly divided into probiotic (n = 15) and placebo (n = 16) groups. Unstimulated salivation rate, concentration of salivary secretory immunoglobulin A, Turesky index, and Papillary-Marginal-Attached index were assessed after 4 weeks of intervention and 2 weeks of washout. Thirty patients completed the entire study protocol. We found no increase in salivary secretory immunoglobulin A levels and salivary flow rates in the probiotic group compared with placebo. Baseline and outcome salivary secretory immunoglobulin A concentrations (mg/L) were 226 ± 130 and 200 ± 113 for the probiotic group and 205 ± 92 and 191 ± 97 for the placebo group, respectively. A significant decrease in plaque accumulation was observed in the probiotic group at 4 and 6 weeks. Within the limitations of the present study, it may be concluded that probiotic intake (Streptococcus salivarius K12) does not affect salivation rates and secretory immunoglobulin A salivary levels but exhibits a positive effect on plaque accumulation. Trial registration NCT05039320. Funding: none.


Introduction
The oral cavity harbors the second largest microbiome in the human body. This microbial community, hosting over 700 species [1], is based on the interactions of microorganisms with the host environment as well as on their interaction with each other [2]. A healthy oral cavity is characterized by a dynamic balance between commensal (i.e., non-infectious) and opportunistic (cariogenic) microorganisms. This equilibrium can be disturbed by a high-carbohydrate diet, poor oral hygiene, some medications, and systemic diseases [3]. Dental caries, the most common non-communicable disease worldwide [4], are primarily caused by an imbalance in the oral microbiome, i.e., a predominance of cariogenic microorganisms, including various types of streptococci and lactobacilli, actinomyces, bacteroides, and bifidobacteria [5]. In this regard, the replacement of the cariogenic microorganisms with nose and throat) inflammatory diseases [35], halitosis [36], candidiasis [37], and dental caries [38][39][40]. Among the S. salivarius-containing probiotics, the two most promising strains are K12 and M18 [31].
Despite a large number of studies, there is still no consensus on the ways in which probiotics affect dental health indicators. Moreover, there is a paucity of literature on the use of S. salivarius (particularly K12 strain) as a probiotic for dental purposes. The aim of our study is to assess the effect of oral probiotics containing Streptococcus salivarius K12 strain on the salivary level of secretory immunoglobulin A (sIgA), salivation rate, and oral biofilm.

Ethical Approval
This clinical study was approved by the Local Ethics Committee (Protocol no. 34-20 (9 December 2020)) and registered on clinicaltrials.gov registry NCT05039320). This research received no external funding. The trial was designed following the principles of the modified Helsinki's code for human clinical studies (2013) and the CONSORT 2010 guidelines for reporting randomized clinical trials.

Study Design
The study assessed the effect of oral probiotics containing Streptococcus salivarius K12 on oral biofilm, salivation rate, and secretory immunoglobulin A (sIgA) salivary level. A double-blind, randomized, two-arm parallel-group study was conducted from September 2021 to November 2021.
General visit descriptions and study schedule are presented in Table 1.

Sampling Criteria
The patients visiting the Dental Institute were invited to participate in the study. Thirty-one healthy adult volunteers aged 20-24 years were enrolled and assigned to interventions by one of the study authors (DS). Written informed consent was obtained from all patients for participation in the study and publication of the data for research and education purposes. The patients were recommended to brush their teeth using standardized technique (Bass) and pea-sized amount of toothpaste without any antibacterial or antiplaque components twice daily.

Randomization
Subjects who met all inclusion and none of the exclusion criteria were randomized to one of the following study groups: Group 1 received lozenges containing a probiotic (Streptococcus salivarius K12); Group 2 received placebo lozenges. The allocation concealment was performed using containers numbered by a "third party" (person who did not participate in the study). The lozenges in unlabeled bottles were placed in the containers. The probiotics and placebo lozenges were identical in taste, color, texture, and size, but the placebo lozenges did not contain active bacteria. Each volunteer on enrolment received a container of lozenges. Neither study subjects nor researchers were aware of the type of lozenges used.

Interventions
All participants took probiotics/placebos for 4 weeks, 1 lozenge per day ( Table 2). The intervention was followed by a two-week washout period, during which the prescribed lozenges were not taken. This period was introduced to assess the stability of the achieved results. Table 2. Arms' characteristics.

Outcomes
Primary outcome measures included concentration of secretory immunoglobulin A in saliva and unstimulated salivary flow rate. Secondary outcome measures included Turesky modification of the Quigley-Hein plaque index (TQHPI) and the papillary marginal attached index (PMA). The evaluations were performed at baseline and 4 and 6 weeks by a single operator (DS).
TQHPI, PMA, and DMFT (decayed, missing, and filled teeth index) were accessed as described elsewhere [41][42][43]. Salivary concentration of sIgA was determined with the help of ELISA and using the salivary secretory IgA indirect enzyme immunoassay kit (8668 IgA secretory ELISA-BEST kit, VectorBest, Novosibirsk, Russia), in accordance with the manufacturer's instructions.
For unstimulated salivary flow measurements, participants refrained from eating, drinking, smoking, or conducting oral hygiene procedures for a minimum of 90 min prior to salivary collection. To avoid diurnal variations in saliva output, all measurements were Nutrients 2022, 14, 1124 5 of 13 taken in the morning. Participants were comfortably seated and, after a few minutes of relaxation, were trained to avoid swallowing saliva and asked to lean forward and spit all the saliva they produced every 2 min through a glass funnel and into a graduated test tube. The volume of the saliva collected over the 10-min period was measured. The flow rate was determined according to the following formula: Salivation rate (ml/min) = saliva volume (mL)/saliva collection time (min).

Statistical Analysis
The sample size for this pilot study was defined based on the sample sizes of similar studies [44][45][46]. All analyses were performed using per-protocol population. We analyzed all subjects who did not substantially deviate from the protocol as to be determined on a persubject basis by the study's principal investigator (KB) immediately before database lock.
Data were presented as means and standard deviations with 95% confidence intervals, medians and 25 and 75 percentiles, and percentages depending on the type of variables. The normality and sphericity of distribution of continuous variables were assessed with Shapiro-Wilk and Levene's tests, respectively. If the assumptions of normality and sphericity were met, repeated measures mixed ANOVA was performed followed by the post hoc Tukey's test with adjustment for multiple comparisons. If the aforementioned assumptions were not met, the differences between the groups were assessed using the Mann-Witney U-test and the differences within the groups at different study timepoints were assessed with Friedman test with post hoc comparisons. The same non-parametric tests were used for the analyses of categorical and ordinal variables. Fisher's exact test was used to access the frequencies of categorical variables in the groups.

Results
The study sample included 31 volunteers (27 females and 3 males) aged 20-24 years (mean: 21.2 ± 0.8 years). They were divided into the placebo group (14 females and 2 males) and the probiotic group (14 females and 1 male) using a random sequence generator. There were no significant differences between the groups in age, gender distribution, DMFT, and the decay component of DMFT values (Table 3).  Figure 1 shows the patient flow diagram. Of the 31 individuals included in this study, 30 completed the entire study protocol. One participant from the probiotic group was lost  Figure 1 shows the patient flow diagram. Of the 31 individuals included in this study, 30 completed the entire study protocol. One participant from the probiotic group was lost to follow-up due to sickness not related to the intervention (COVID- 19). No adverse effects were registered.  The baseline, outcome, and washout values of salivary IgA, unstimulated salivary flow rate, and dental indices are presented in Table 4. We found no statistically significant differences in the salivary secretory immunoglobulin A (sIgA) levels and unstimulated salivary flow rates between the individuals who took probiotics and placebos. After a 4-week probiotic intervention and a 2-week washout period, the study participants had significantly lower TQHPI values than the controls. At baseline, seven participants (three in the probiotic group and four in the control group) had PMA index values greater than 0. The PMA values tended to decrease in the probiotic group at the outcome and washout timepoints, although these changes did not reach the level of statistical significance (p = 0.06081).  There were no statistically significant differences in the distribution of the study subjects across the levels of salivary sIgA and PMA in the probiotic and placebo groups at all study timepoints (Table 5). Table 5. Distribution of sIgA levels (low, normal, high) a and PMA (zero or greater than zero) in the study groups.

Probiotics (n = 14) Placebo (n = 16) Significance b
Level of sIgA Baseline, n (%)    Figure 2 shows the results of the correlation analysis of oral health indicators. We found a strong negative correlation between salivation rate and sIgA level (r = −0.62), p = 0.000239395). A moderate positive correlation was detected between the number of decayed teeth and TQHPI values (r = 0.57, p = 0.000968335). No significant correlation was observed between the number of the decay component of DMFT and sIgA level (r = 0.17, p = 0.379).

Discussion
In the present study, we assessed the effect of oral probiotics containing Streptococcus salivarius K12 on secretory immunoglobulin A salivary levels, salivation rates, and oral

Discussion
In the present study, we assessed the effect of oral probiotics containing Streptococcus salivarius K12 on secretory immunoglobulin A salivary levels, salivation rates, and oral biofilm in healthy adults. We found no increase in salivary secretory immunoglobulin A (sIgA) levels and salivary flow rates in the probiotic group compared with the placebo group. However, we observed a significant decrease in plaque accumulation in the probiotic group after 2 and 4 weeks of probiotic intake. A decrease in the PMA index was observed in the probiotic group, although the differences did not reach the level of statistical significance. This was possibly due to a small number of patients with gingivitis.
Salivary IgA is an important protein that participates in the prevention of oral diseases. sIgA level determination is widely used in dental science, as it can be collected noninvasively and is considered an indicator of health and disease [47]. Probiotics demonstrated beneficial effect on host immune response [48], although studies of the effect of probiotics on sIgA levels have shown conflicting results. Some studies found increased levels of salivary sIgA in adults [44,47,[49][50][51], elderly patients [52], and children [47,48] after probiotic intake, while others were unable to confirm such findings [23,[53][54][55][56]. One study even demonstrated a decrease in salivary sIgA after Bifidobacterium-containing probiotic intake [57]. A meta-analysis by Ebrahimpour et al. demonstrated no significant effect of probiotic intake on salivary sIgA levels compared to placebo [58], which is in line with our results. In the present study, the analysis of variance showed that neither the time factor (baseline/outcome/washout) nor the group factor (probiotic/placebo) affected the salivary sIgA levels.
Salivary flow rate is another crucial parameter for the maintenance of oral and systemic health [59]. There is some evidence that probiotics may affect salivary flow rate [59,60]. However, other studies did not confirm this effect of probiotics [61][62][63][64]. Our results are in agreement with the latter studies: we found no increase in unstimulated salivary flow rate after the intake of probiotics compared with placebo.
Differences in the effects of probiotics on various health indicators may be explained by the age of participants. In the studies involving elderly people, antibody responses might be different from healthy adults [58]. Moreover, intra-and inter-individual variations in saliva volume and its contents are influenced by a variety of factors, such as cigarette smoking [47,54], chronic and acute stress [47,54,58], depression [47,54], and circadian variation [47,65].
Immune-modulatory effects of probiotics in general and for particular species may be strain-specific [54]. To the best of our knowledge, there were no published reports directly comparable to ours. The only report partially comparable to ours was that of Ferrer et al., who assessed the effect of topical application of Streptococcus-containing probiotics on plaque accumulation, saliva quality, and salivary flow [22]. They found a significant increase in salivary flow rate at day 15 in the probiotic group compared with the placebo group. Furthermore, in the probiotic group, there was a decrease in the amount of dental plaque and gingival inflammation, but no differences were observed in the placebo group [22]. A similar effect on plaque formation was reported by Burton at al., who demonstrated that the probiotic strain S. salivarius M18 administered twice daily caused a significant reduction in plaque formation in children [66]. In the present study, we observed a significant decrease in TQHPI in the probiotic group after 2 and 4 weeks of probiotic intake.
However, the plaque-reducing effect of probiotics may also be strain-specific and product-specific. According to a meta-analysis by Nadelman et al., dairy probiotics increased plaque accumulation, possibly due to an increase in the amount of carbohydrates [67].
It could be expected that a decrease in plaque index would result in a decrease in gingivitis (PMA score). A number of studies have demonstrated that probiotics significantly improved various gingival health indicators, i.e., decreased gingival indices and bleeding on probing [22,42,68,69]. In our study, a decrease in the PMA index was observed in the probiotic group, although the differences did not reach the level of statistical significance. This was possibly due to a small number of patients with gingivitis due to good or moderate levels of oral hygiene in the majority of patients in both groups. Similarly, Montero et al. reported insignificant changes in the mean gingival index in general, although it significantly reduced at the sites of severe inflammation [70].
Although plaque accumulation rate is a rapidly changing variable and caries development is a relatively slow process, we found a moderate positive correlation between TQHPI values and the number of decayed teeth. There was no significant correlation between the value of the decay component of DMFT and sIgA level, although some authors hypothesized that the level of salivary sIgA may serve as a predictor of caries resistance in a patient [27,[71][72][73].
According to the literature, protein concentrations in saliva may also be dependent on the changes in salivary flow rates [74,75], so they may be sIgA levels [76,77]. For example, an increase in salivary sIgA in people with xerostomia was reported in pregnant women [78] and students experiencing stress because of exams [74]. We found a strong negative correlation between salivation rates and sIgA levels (p < 0.001). Similarly, an inverse correlation between salivary flow rates and salivary sIgA concentrations has been reported in previous studies [79][80][81][82][83].
We readily acknowledge several limitations to our study. This was a pilot study; the relatively small number of participants was defined based on the sample sizes of similar studies [44][45][46]. Further research with a larger sample size is planned based on the data generated in the present study. A four-week intake of probiotics and two-week washout period are relatively short periods for the assessment of the effect of probiotics; however, similar timepoints were used in the previous studies [47,54,84]. Moreover, although probiotics may affect different salivary components [25,58,63,85], we assessed the influence of probiotics on a single salivary protein (sIgA), having hypothesized that this parameter would be the most sensitive one to probiotic intake.

Conclusions
Within the limitations of this pilot study, it can be concluded that probiotic intake (Streptococcus salivarius K12) does not affect salivation rates and secretory immunoglobulin A salivary levels in healthy adults. However, a decrease in plaque accumulation was observed.

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.