Analysis of the Impact of Used Probiotic Strains and Diet on Body Composition, Cardiopulmonary Efficiency and the Incidence of Gastrointestinal Disorders in Runners

The aim of the study was to assess the diet and the effect of probiotic therapy on reducing the incidence of gastrointestinal disorders and selected parameters determining the inflammation of the body, body composition, cardiopulmonary efficiency and selected serum biochemical parameters.

The approved of the Bioethics Committee of the Medical University, no. RI-002/81/2017, was obtained for the study. 70 people meeting the inclusion criteria were included in the intervention and observational study. The research was carried out at the Department of Dietetics and Clinical Nutrition at the Faculty of Health Sciences of the Medical University of Bialystok, Fitness Club Maniac Gym in Bialystok, as well as at the Hematological Diagnostics Department and the Biochemical Diagnostics Department of the University Clinical Hospital in Bialystok.

The study took place in the spring (March-June 2018) and autumn (September-December 2018), i.e. during of the most intense period in terms of participation in runners' competitions. The study was randomized using a double-blind trial. 35 persons selected in the draw were G1 group and the remaining G2 group (n=35). The tests were completely anonymous, the competitors gave their informed written consent to carry them out.

Interventional study was conducted using a probiotic or placebo. The observation period lasted three months. The intervention factor was the SANPROBI BARRIER multi-strain probiotic (commonly available in pharmacies) or placebo (created on the model of a probiotic capsule, specially for the needs of research, by Sanprobi Sp. z o.o.). Competitors used probiotic/ placebo for three months at a dose of 2x2 capsules daily (2.5 x 10 9 CFU / g (1 capsule)). The probiotic used in the study contained bacterial strains, i.e. Bifidobacterium lactis W52, Lactobacillus brevis W63, Lactobacillus casei W56, Lactococcus lactis W19, Lactococcus lactis W58, Lactobacillus acidophilus W37, Bifidobacterium bifidum W23, Lactobacillus salivarius W24. Players and researchers were not informed about the type of capsule taken (probiotic or placebo). Competitors were asked to use product A or B after blood tests.

Each player had 3 visits during the preliminary stage (W1) and 3 visits of the final stage (W2).

Preliminary stage (W1)

1. The first visit took place in the Department of Dietetics and Clinical Nutrition at the Medical University of Bialystok. It included qualification for the study based on the criteria for inclusion in the study group, discussion of the purpose of the study and signing the written consent for the study by the participants, as well as information about the protection of their personal data and full anonymity. Each participant received an individual code (e.g. A01, A02, B01, B02 etc.), which was used to identify the subject in further stages. Athletes were asked not to use other probiotic strains than those used in the study. All competitors completed a survey regarding demographic data, information on dietary supplementation used, the incidence of gastrointestinal disorders, and 3-day nutrition diary.
2. The second visit took place after a week. Each player was invited to the Maniac Gym Fitness Club in Bialystok. Body composition analysis was performed using an InBody 770 analyzer (InBody, Korea). To determine the level of cardiopulmonary efficiency using the Bruce protocol test on a medical treadmill were performed. Then, competitors were issued a referral for laboratory tests. During this meeting, in accordance with the simple draw product A (group G1) was given to every second person, and product B (group G2) for the remaining persons.
3. For morphological tests (blood count with smear, lipid profile, fasting glucose, C-reactive protein and ionogram) at the third visit for each competitor was collected blood from the vein in the amount of 10 ml.

Final stage (W2) The final stage consisted of three visits (similar to the preliminary stage). The first visit was made after 3 months of taking product A or B by the competitors. 66 people took part in the final stage of the study (G1 = 34 people, G2 = 32 people). One person from group G1 (product A) did not report for the final visit without providing a specific reason. Three people from the G2 control group (product B) did not participate in the final stage of the study (W2) due to injury (2 people) and without providing a specific reason (1 person).

• The first visit took place at the Department of Dietetics and Clinical Nutrition (Medical University of Bialystok) where competitors were completed the questionnaires to determine the changes in the incidence of gastrointestinal disorders.
• The second visit was conducted at the Fitness Club Maniac Gym, where the cardiopulmonary fitness test and body composition analysis were carried out.
• Third visit took place at the Hematological Diagnostics Department of the University Teaching Hospital in Bialystok, where from each participant the blood was collected (from the vein) in an amount of 10 ml for biochemical tests (as during the initial stage at visit 3). 3-day nutrition diary: Each participant described customary meals consumed for the next three days, including two working days and one day off. The food diary includes hours of meals, the weight of products and dishes, and the amount of fluids consumed during the day. The competitors were asked to use the standard, current diet, without any significant changes. The energy value of the daily nutritional plan, the content of the main nutrients, vitamins, minerals, cholesterol and dietary fiber were taken into account in the evaluation of the three-day nutrition diary. Then, the percentage of compliance with the norm for the energy supply of diet and nutrients was assessed by comparing the results to Polish norms for healthy, adult men and women, and based on scientific research on the discipline of long-distance running.

Body composition analysis All competitors underwent body composition analysis using the InBody model 770 analyzer (South Korea). The body composition analyzer, using the electrical bioimpedance principle, uses low current flow using 6 frequencies and subsequent measurement of the resistance of individual tissues to determine body composition. The following parameters were assessed: body weight (kg), body fat content in kilograms and percent, visceral fat content (cm2), skeletal muscle mass (kg), body water content (kg). Before the test, the participants were informed about the need to stay fasting 8-12 hours and a minimum of 24 hours after physical activity. They were asked to avoid drinking liquids 1 h before the test. None of the competitors had an implanted pacemaker, which is an absolute exclusion criterion in the study of body composition analysis by electrical bioimpedance.

Analysis of cardiopulmonary efficiency In order to determine the cardiopulmonary efficiency of competitors, the Fitmate MED device (Cosmed, Italy) was used, which comprehensively determined cardiopulmonary efficiency and a medical treadmill adapted to this type of HpCosmos analysis (Germany). The Fitmate MED device is equipped with a sensor that enables the measurement of inhaled and exhaled air as well as a mask containing disposable mouthpieces and antibacterial filters. To monitor cardiac contractions, a Garmin sporttester model HRM-Tri 010-10997-09 (United States) was used. In cardiopulmonary fitness tests, a walking treadmill test was used according to Bruce's protocol. The exercise test included effort progression to increase walking speed and incline of the treadmill. The study was divided into 5 stages (3 minutes each). In the first of them, the walk took place at a speed of 2.7 km/h and a 10% incline of the treadmill. In the second stage, the treadmill accelerated to 4 km/h, and the incline increased to 12%. The third stage is a speed of 5.5 km / h and a incline 14%. The fourth stage is the speed increase to 6.8 km / h and the treadmill inclination 16%, and the last stage 5 is the speed of 8 km / h and the inclination of the treadmill 18%. Participants were informed that the cardiorespiratory fitness test should take place for a minimum of 2 weeks after intense physical effort (marathon, ultramarathon). During the test, the maximum test procedure was used to obtain more test parameters, and the test condition was considered to be 90% HRmax by the test subject, or the test could not be continued by interrupting it at the test subject's request. None of the competitors tested completed the test alone, they all reached the 90% HR max limit.

Laboratory tests The tests were performed after venous blood collection (from a vein in the arm) in the amount of 10 ml. Twice (during the initial and final stages) the following measurements were taken: complete blood count with smear, fasting glucose, total cholesterol, HDL, LDL and triglycerides, sodium, iron, potassium, magnesium, serum calcium, determination C-reactive protein (CRP) concentrations. In order to determine pro-inflammatory cytokines, blood was collected on a clot, it was subjected to centrifugation, and after clot separation, the obtained serum was stored at -80 degrees Celsius (until the tests were performed). To determine the concentration of interleukin 6 (IL-6) and tumor necrosis factor (TNF-alpha), the ELISA method (enzyme-linked immunosorbent assay) was used, using R\&D System kits (Bio-Techne, United States) according to the manufacturer's instructions.