**2.1 Collect blood samples**

Every once two weeks, One day after exercise (Running a distance of 200 meters) 8 ml of blood was taken from all subjects Blood samples were collected in heparin tubes and immediately transferred to the laboratory. To separate the blood serum, samples were centrifuged at 5000 rpm for 15 minutes.

*Evaluation of the Effect of Fruit Juice Containing Bacillus Coagulans Probiotic Supplement… DOI: http://dx.doi.org/10.5772/intechopen.98370*

## **2.2 Measurement of immunoglobulin A& M levels**

In the present study, the levels of immunoglobulins A and M were measured using ELISA kits (ab196263 andab214568). The test steps were performed according to the protocol recommended by the kit manufacturer (abcam Inc., USA) And light absorption of the samples was read by ELISA reader (Biotech microplate reader ELX800).

#### **2.3 Lymphocyte cell count**

Lymphocyte counts were performed by flow cytometry. Blood samples were collected in heparinized tubes and then centrifuged. The cell layer was washed with phosphate buffered saline (PBS) twice, then 10 μl of monoclonal antibody was added and the samples were incubated in the dark for 20 min. To remove excess antibodies, wash twice with buffer. to measure adsorption Optical samples of fluorescent isothiocyanate fluorescein fluorochrome (ftic) were used. Then the tubes containing the sample were placed in a flow cytometer and the results were reported based on percentage.

#### **2.4 Statistical analysis**

Analysis of variance (ANOVA) was used to determine the effects of probiotics. Sample t-test was used to identify differences within the group (Beginning and end of the experiment). Values less than 0.05 were considered significant. All statistically The analysis was performed using Graphpad Prism software (version 6).

## **3. Results**

All participants completed the test. Taking a probiotic supplement did not cause any side effects. During the first month of the experiment, no significant change in the level of immune factors was observed. With increasing probiotic supplementation time, the experimental group had a significant increase in the levels of immunoglobulin A, immunoglobulin M and lymphocytes compared to the control group, That was arrangement it IgA (258 vs. 233 ug/dL) (**Figure 1**), IgM (159 vs. 133 ug/dl)

#### **Figure 1.**

*Immunoglobulin A level in experimental and control groups during the study time (0–90 days). \* standard deviation, \*\* means that the difference in the results in each row of values is not significant, \*\*\* means that the difference in results in each row is noticeable, (P < 0.05) different.*

#### **Figure 2.**

*Immunoglobulin M level in experimental and control groups during the study time (0–90 days). \* standard deviation, \*\* means that the difference in the results in each row of values is not significant, \*\*\* means that the difference in results in each row is noticeable, (P < 0.05) different.*


*\*Standard deviation.*

*\*\*means that the difference in the results in each row of values is not significant.*

*\*\*\*means that the difference in results in each row is noticeable.*

*(P < 0.05) different.*

#### **Table 1.**

*Lymphocyte cell count in both experimental and control groups one day after exercise (two 200 m speed) in terms of%.*

(**Figure 2**) and lymphocytes (37.6 vs. 33.6 cell/ul) (**Table 1).** In addition, there was a significant reduction in the incidence of respiratory infections among the supplement group compared to the control group.

#### **4. Discussion**

Athletes due to their readiness for various competitions endure a lot of physical and psychological stress.These tensions can lead to decreased immune system function and the development of multiple infections. Probiotics are natural compounds with a variety of benefits to increase the quality of life of people by improving their health by improving the function of the immune system [20–22]. The results of this study showed that consumption of fruit juice containing the probiotic *Bacillus coagulans* has a positive effect on improving the health and quality of life of athletes by improving the function of the immune system and reducing the risk of respiratory infections. A 2007 study by Kakonen et al. found that runners who took probiotic supplements were less likely to develop respiratory infections than other athletes [23]. The results of a 2016 study by Ahanchian et al. Also showed the effect of taking probiotic supplements on the prevention of respiratory infections [24]. In this study, it was found that taking probiotic supplements over time shows a positive effect on health. The results of the Zhang et al. study also showed that

#### *Evaluation of the Effect of Fruit Juice Containing Bacillus Coagulans Probiotic Supplement… DOI: http://dx.doi.org/10.5772/intechopen.98370*

taking probiotic supplements Lactobacillus casei after 16 weeks has a significant effect on increasing the level of immunoglobulins A [25]. Based on this, it can be concluded that to evaluate the beneficial effects of probiotics, it is necessary to take these natural supplements regularly and intermittently.

Michalikova and colleagues examined the effect of taking helveticus Lafti L10 on humoral safety in athletes. The results of this study showed that taking this probiotic supplement for fourteen weeks significantly improves the immune status of athletes [26]. The effect of *L. fermentum* supplement on respiratory and immune system of runners was evaluated by Batatinha and colleagues. The results of this study showed that taking this supplement has a positive effect on athletes' health [27]. Consumption of probiotics restores or maintains the balance of intestinal microbiota, reduces oxidative stress, and improves cardiovascular function in athletes. Strengthening the control system and inhibition of free radicals was observed in athletes using supplements containing several probiotic strains (L. rhamnosus® and *L. paracasei*) [28, 29]. Decreased gastrointestinal discomfort and endotoxin content in athletes during multi-component probiotic supplements (L. acidophilus CUL-60, L. acidophilus CUL-21, B. bifidum CUL-20, B. animalis subsp. Lactis CUL-34) Has been [30]. The results of the study by Strasser et al. Showed that the use of multicomponent probiotic supplements including (B. bifidum, B. lactis, *E. faecium*, L. acidophilus W22, *L. brevis* and L. lactis) impaired tryptophan levels during intens exercise By increasing serotonin levels, exercise induced stress is reduced and athletes' mental state is improved [31].

Probiotics directly affect the function of endocrine immune cells, modulate the immune system and strengthen the immune system, and most importantly, have a positive effect on the immune system without causing a harmful inflammatory response. Therefore, it seems that probiotics are a natural and healthy method that can increase host resistance in the face of injury and stress [32]. The effect of taking the probiotic supplement L-fermentum was evaluated in another study in 2014. The results showed that the level of influenza-specific immunoglobulin increased significantly with the use of this supplement compared to the control group [33].

The effect of consuming a mixture of probiotics on increasing the number of immune cells, including lethal t cells, cd4 cells, lymphocytes and monocytes, has been proven in a study by Wu et al. [34]. Considering the importance of maintaining health and improving the performance of athletes during competitions, based on the results of this study and other studies conducted in this field, it was found that taking probiotic supplements will have many positive effects on the health of people, especially professional athletes.
