This double-blind randomized clinical trial was conducted on 40 students of medicine and pharmacy, with the age range of 19–35 years, at Isfahan University of Medical Sciences. A checklist of subjects was prepared at the beginning and end of this study. The exclusion criteria were students with orthodontic appliances or 6-month orthodontic treatments, students who had taken antibiotics in the last month, students undergoing incomplete dental treatments, and students with immune system diseases. Further, students who needed dental treatment during the intervention, used <90% of the consumables, needed to take antibiotics during the intervention, got bacterial and viral diseases during the intervention, and changed the number of times they brushed their teeth and the type of toothpaste and mouthwash or used a new oral hygiene method were excluded after the study. As shown in Figure 1, 47 volunteers enrolled for this study and 7 of them were excluded due to the study exclusion criteria. Four were undergone dental treatment and 3 had orthodontic appliances. The participants’ flow diagram is represented in Figure 1.

The subjects were randomly divided into two groups of 20 people each. To ensure the participants in both groups were equal in number, the block randomization method was used. The size of all the blocks was equal, and there were 40 blocks, including 20 participants in the intervention group (probiotic consumers) and 20 participants in the control group (mouth freshener tablets) in this two-group trial.

The randomization tool used was the random allocation software (version 2.0), which is able to perform block randomization in addition to simple randomization. Allocation concealment, which is used to implement a random sequence on the participants, was also used so that the assigned group was not known before the allocation of the individual. Sequentially, numbered, sealed, opaque envelopes were used, in which each of the random sequences created is recorded on a card, and the cards are placed inside the envelopes, respectively. To maintain a random sequence, the outer surface of the envelopes is numbered in the same order. Finally, the envelopes are glued and placed in a box. At the time of the participants’ registration, based on the order, in which the eligible participants were included in the study, one of the envelopes was opened and the allocated group of that participant was revealed. In this study, the double-blind method was used. For this purpose, the participants did not know whether the product they were using contained salivary probiotics or xylitol fresheners and the products were not delivered to them in the company’s original packaging. Furthermore, the statistician who analyzed the data was blind to the participants’ information. The randomization and blinding process were done by the main author.

During the equalization of oral hygiene before and during the study, signal toothpaste with a fluoride dose of 1450 ppm, manufactured in Iran and licensed by the Food and Drug Organization under license number 1515/Z/38, was used. The control group received xylitol freshener tablet (Iceberg tablet, a product of Shiva company, manufactured in Iran under health license number 7009/Z/56), and the intervention group received a probiotic tablet containing S. salivarius M18 and K12 (Lactogam tablet, Zist Takhmir Company, Company). It should be noted that the amount of xylitol in the mouth freshener tablets is much lower than the therapeutic and effective dose of xylitol and has no effect on the number of S. mutans. Both products have a mint flavor.

This study has been registered in the Ethics Committee of Isfahan University of Medical Sciences, with the ethics code IR.MUI.RESEARCH.REC.1400.297. It has also received the scientific code IRCT20210811052147N1 from the Center for Clinical Trials of Iran. All the information of the participants in this study was kept confidential, and only the main authors were aware of it. Moreover, the participants were fully aware of the research conditions. The results of the study were provided to the participants after the intervention.

According to the instructions of the probiotic product, the intervention group sucked one probiotic tablet every night before going to bed and after brushing their teeth for 20 days. The control group sucked a mouth freshener every night after brushing their teeth before going to bed for 20 days. Further, both groups were required not to make any new changes in their oral hygiene habits and to continue the training process before the intervention. The preintervention process means that they should not change the number of times they brush their teeth, or if they did not use mouthwash or dental floss, they would not do this during this 20-day period and should follow the instructions given. Participants were informed of all these conditions, and these health habits were determined through a checklist before and after the intervention. The oral health level before and after the study was determined by the plaque index using the common Silness–Loe method based on the WHO Oral Health Surveys, 5^th Ed., 2013. The students were contacted by phone once every 5 days and their cooperation was ensured. Furthermore, a text message was sent to the participants every night to remind them of taking probiotic pills and freshener tablets.

At the beginning and end of the intervention, unstimulated saliva samples of both groups were collected in sterile containers and cultured. To collect unstimulated saliva, the participants were asked to sit and rest for at least 2 h after consuming food and water between 9 and 11 am and pour their saliva samples into sterile and dry polyethylene vials through spitting and without chewing. The samples were collected at the Dentistry Facility of Isfahan University of Medical Sciences.

The TYCSB medium, which is a selective medium for S. mutans, was used to culture the bacteria. Then, 0.1 mL of each sample’s saliva was placed on the culture medium and cultured by a sterile loop in a linear manner. The plates were placed in an incubator at 35°C–37°C for 48–72 h under 5% carbon dioxide and 95% nitrogen. After incubation, the suspicious colonies were removed, heat-stained, and evaluated in terms of the presence of Gram-positive cocci. Then, catalase test was performed to confirm Streptococcus, and if catalase was negative, the presence of S. mutans was confirmed by performing differential sugar tests, including sorbitol and mannitol. Since the TYCSB culture medium is a specific medium for the growth of S. mutans, only this bacterium was expected to grow on the medium. Then, colony counting was performed, and the number of bacterial colonies was calculated based on cfu/mL and compared in both groups before and after the intervention. The samples were collected by the main author.

The data were collected through Excel software and analyzed by SPSS software (version 24, IBM, USA) using descriptive statistics (central tendency and dispersion) and inferential statistics (paired t-test and independent t-test). Furthermore, the normality of the data was checked using the Kolmogorov–Smirnov test. Since 0.1 mL of each sample’s saliva was cultured, the number of bacteria at the end of counting was multiplied by 10 to report it based on the CFU/mm unit.

This study was carried out as a free project and then a doctoral thesis in dentistry funded by Isfahan University of Medical Sciences.

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.