Introduction
Probiotics are live microorganisms with many benefits for their host [1]. Saccharomyces boulardii is a well-known probiotic used for the treatment of diarrhea [2]. S. boulardii could increase the secretion of IgA and other immune secretory components in the small intestine [3]. Thus, stimulation of the host’s mucosal immunity is a mechanism by which S. boulardii acts against intestinal pathogens [4-6].
Probiotics like S. boulardii will increase the nutritional value of food and protect food from degradation; however, to do this, they need to survive in food processing techniques and gastrointestinal tract [7]. Microencapsulation is a technique that protects the microorganisms from external influences [8]. Among products used for microencapsulation, sodium alginate is widely used in microencapsulation of probiotic bacteria in concentrations of (0.5-5%) [9]. This study was designed to evaluate the effect of S. boulardii microencapsulation on IgA secretion in a rat model.
Materials and Methods
Animal model
Male Wistar rats weighing 100-110g were obtained from the Department of Laboratory Animals, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. They were kept at 20°C under 12:12h light/dark cycle. Standard pellet food and water was provided for them ad libitum. The rats were divided into three groups, each containing 10 rats: MT group of rats receiving microencapsulated S. boulardii, the control (C) group receiving normal saline, and the T group receiving uncapsulated S. boulardii. Probiotics were gavaged to rats at the dosage of 2g/kg for 8 continuous weeks.
Probiotic strain
S. boulardii (ATCC 74068) was obtained from the Mycology Research Center. It was cultured on sabouraud dextrose broth (Merck, Germany) supplemented with Chloramphenicol (0.05%) (SC) and incubated at 28°C, 180 rpm for 48h. The cells were centrifuged at 800g for 10 min and washed three times with sterile phosphate-buffered saline (PBS) before use. The number of S. boulardii yeast cells were adjusted to 2Í1010CFU/ml with a hemocytometer before microencapsulation.
Microencapsulation
Extrusion technique was used for microencapsulation of S. boulardii as described previously [10]. In brief, the cells were suspended in (2%) sodium alginate solution. Then, this mixture was injected to 0.1M calcium chloride using a sterile insulin syringe. The formed droplets were isolated from suspension with centrifugation.
IgA measurement
After 8 weeks, the rats were euthanized, and their gastrointestinal tracts were isolated. Their intestinal mucosa was isolated, according to Lim et al. (1981) method with brief modifications [11]. In brief, intestinal content was washed out with cold PBS containing penicillin/streptomycin (100µg/mL) and centrifuged at 500g for 10 minutes. IgA was measured in duodenal and jejunal washings using Enzyme Linked Immunosorbent Assay ELISA assay method according to manufacturer’s instructions.
Statistical analysis
The obtained data were analyzed in SPSS V. 21. Analysis of Variance (ANOVA) was applied to compare statistical changes in different groups. Tamhane’s T2 post hoc test was used to compare data between groups. A P-value less than (0.05) was considered significant.
Results
Table 1 shows the results of IgA production by duodenal and jejunal epithelial cells. Probiotic S. boulardii increased IgA secretion from duodenum and jejunum in comparison with the control group, and this increase was significant in the MT group and in the duodenum of T group (P<0.05). Interestingly, IgA secretion was significantly higher in the intestines of rats treated with microencapsulated S. boulardii (P<0.05).

Discussion
S. boulardii is a probiotic yeast mainly used in the treatment of gastrointestinal disorders. Enhancing the host immune system could be an important defense against infectious diseases of intestines, causing diarrhea [12]. In our study, S. boulardii increased the secretion of IgA from intestinal mucosa, which agreed with other studies [12-15]. However, this increase was higher in the duodenum of the T group and jejunum of the MT group. Nevertheless, this difference was not significant (P>0.05). The higher secretion of IgA in the jejunum of microencapsulated S. boulardii-treated rats could be the results of microencapsulation, which protected the yeast cells in the first parts of the intestinal tract and provided enough yeasts in jejunum that could affect IgA release by jejunal mucosa. Since intestinal infections are mostly initiated from jejunum, microencapsulation of S. boulardii could protect against intestinal disease as well as treatment of infectious disorders. It can be concluded that microencapsulation enhances the release of IgA by intestinal cells, and this could be an essential mechanism in the protection and treatment of intestinal illnesses.
Ethical Considerations
Compliance with ethical guidelines
The ethical issue of the present study was approved by Research Council of AJA University of Medical Sciences (Code: 1395.121)
Funding
This study was supported by vice Chancellor for Research of AJA University of Medical Sciences.
Authors' contributions
Designing: Ali Reza Khosravi, Zohreh Farahnejad and Hassan Ghorbani Choboghlo; Laboratory experiments: Donya Nikaein, Hassan Ghorbani Choboghlo and Reza Rahmani; Manuscript writing, editing and revising: Donya Nikaein, Hassan Ghorbani Choboghlo and Reza Rahmani.
Conflicts of interest
Authors declared no conflict of interest
Acknowledgements
Authors would like to thank AJA University of Medical Sciences for their technical support and Mr Asad Balal for his excellent cooperation.