Impact of endurance exercise and probiotic supplementation on the intestinal microbiota: a cross-over pilot study

Background The human microbiota has a broad range of functions contributing to metabolic processes and the activities of our immune system. Its influence on health, well-being and chronic diseases are discussed in various studies. The intestinal microbiota and the mucosal integrity are influenced by diet, environment and other lifestyle factors, including physical activity. There are correlations between cardiorespiratory fitness and important markers of intestinal health. However, data linking endurance exercise to microbiota composition are sparse. Many endurance athletes take probiotics to reduce gastrointestinal symptoms linked to exercise or immunosuppression, but the longitudinal data is insufficient. This randomised, controlled cross-over pilot study will examine the impact of specific endurance training and probiotic supplementation on the intestinal microbiota and mucosa in healthy, athletic students. Objective The aim of this pilot study is to elucidate the impact of physical activity on the intestinal microbiota and mucosa with regard to the effects of a probiotic supplementation. Methods In this pilot study, thirty non-specifically trained student athletes will participate in an intervention consisting of a two-week rest (baseline) period, a four-week exercise programme and a four-week probiotic intervention using SymbioLactComp®. The exercise programme consists of three 60-min running workouts per week at 70–85% of the peak heart rate (HRpeak). Primary endpoint of this pilot study is the feasibility and practicality of the intervention as well as a sample size estimation. Furthermore, anthropometric measurements and information on nutrition and lifestyle will be obtained. The peak oxygen uptake (VO2peak) and peak heart rate (HRpeak) (determined during a shuttle run test) as well as selected blood and saliva parameters (haemogram, cytokines) will be evaluated. Changes to the intestinal microbiota will be analysed by stool diagnostics (KyberKompaktPRO®, KyberPlus®). The potential changes may include microbiota composition, bacterial metabolites and mucosa- and immune markers. Conclusion Results will be used for the design of a main randomised controlled trial with a larger collective based on feasibility, validity and sample size estimation as well as the potential effects of endurance exercise on intestinal microbiota and mucosa. Evidence-based information of an exercise-altered microbiota could be of importance for the prevention and therapy of intestinal or immune disorders. Trial registration German Clinical Trials Register: DRKS00011108. Retrospectively registered on 28 November 2016. Electronic supplementary material The online version of this article (10.1186/s40814-019-0459-9) contains supplementary material, which is available to authorized users.


Introduction
" 3 Background and rationale 6a The intestinal microbiota composition plays a significant role in metabolism and immunity [1,2]. Certain species and their associated metabolic products influence physiological homeostasis. Metabolic and immune functions include degradation of food, synthesis of vitamins and bioactive compounds/ hormones (e.g. serotonin), training of the immune system, pathogen defense and proliferation of intestinal cells [3,4]. There is no complete definition of an intact microbiota; however, the following are believed to contribute: an enriched bacterial diversity (alpha diversity), a balanced Firmicutes/ Bacteroidetes ratio, an intact mucus layer and an abundance of certain species (e.g. Akkermansia muciniphila and Faecalibacterium prausnitzii) [5][6][7][8][9]. An altered microbiota contributes to the pathogenesis of diseases such as metabolic or (auto)-immune diseases [10][11][12][13]. Recent studies have highlighted that environment and lifestyle factors affect microbiota composition and its metabolic capacity [14,15]. There is a growing body of evidence indicating that physical activity is linked to specific markers of intestinal health [16][17][18][19].
Several studies determined a higher species richness (α diversity) in elite athletes and individuals with higher cardiorespiratory fitness (VO2peak) or higher training frequency than those with a sedentary lifestyle or lower fitness level [16,17,20].
Furthermore, an increase in number of the two commensal species Akkermansia muciniphila and Faecalibacterium prausnitzii was observed in athletes and highly active individuals [17,18,20]. This was related to a higher concentration of the health-promoting bacterial metabolite butyrate [18,20].
Some professional athletes suffer from immunosuppression or gastrointestinal symptoms, including abdominal pain, diarrhea or leaky gut syndrome. This is because excessive exercise reduces intestinal blood flow due to increased circulation in the strained muscles and heart. This may lead to microbial imbalances and mucosa disruption [19,[21][22][23][24]. An increased permeability of the intestinal mucosa and epithelium (intestinal barrier) has been associated with bacterial/ pathogen translocation to extraintestinal organs manifesting as intestinal or systemic inflammation [25][26][27].
Many athletes use probiotics to enhance barrier integrity and to improve gastrointestinal symptoms. Probiotic consumption as supplement may induce immune processes, including the synthesis of cytokines and immunoglobulins, thereby contributing to host health [28][29][30]. Previous research on trained athletes revealed the beneficial effects of probiotic intake on the marker for mucosal integrity zonulin and several cytokines. Interferon-gamma, tumor necrosis factoralpha and interleukins were all improved [19,29,[31][32][33].
Regular moderate exercise is well-known for its anti-inflammatory efficacy, but diet-driven effects can obscure the intestinal impact of sports per se [17,18]. Data that determines causality between microbiota composition, mucosa permeability and moderate endurance training are sparse. Longitudinal studies are needed to determine how the intestinal microbiota and mucosa as well as immune markers and cytokines vary with exercise as compared to a probiotic supplementation. A combination of exercise and probiotics may be a new method for the prevention and therapy of intestinal or immune-related diseases [34][35][36].
[2]Balducci S, et al. Anti-inflammatory effect of exercise training in subjects with type 2 diabetes and the metabolic syndrome is dependent on exercise modalities and independent of weight loss. Nutrition, metabolism, and cardiovascular diseases : NMCD. 2010;20 (8)  Choice of comparators In this controlled cross-over trial we will compare the potential influence of regular exercise training and probiotic supplement SymbioLactComp ® Training intensity uses the baseline shuttle run results (heart rate peak). Intensity of exercises will increase from 70% to 85% of the heart rate peak to improve the subject's basic endurance. The three 60-minute trainings per week will consist of extensive and intensive continuous methods, interval training and stretching. The probiotic period involves supplementation with the probiotic SymbioLactComp ® (SymbioPharm GmbH, Herborn, Germany). SymbioLactComp ® contains different lactic acid bacteria (Lactobacillus paracasei, Lactobacillus acidophilus, Lactococcus lactis and Bifidobacterium animalis subsp. lactis) as well as 30 µg of biotin. The total bacteria count of one sachet (2g) is ≥1 x 10 9 KBE/g. It will be mixed with water and taken daily at breakfast.

Objectives 7 Research Hypothesis
We hypothesize that a training period with regular running exercises may alter microbiota composition in healthy adults.
Participants receiving the intervention might further show higher rates of selected markers of intestinal health, like Akkermansia muciniphila, Faecalibacterium prausnitzii, Bifidobacterium spp. and lower rates of intestinal immune markers; e.g. zonulin, alpha-1-antitrypsin or secretory immunoglobulin A (sIgA).

Aims and study objectives
See below at "outcomes".
Trial design 8 This randomised, controlled, cross-over trial is conducted at the German Sport University Cologne in cooperation with the MVZ Institute of Microecology GmbH.

Methods: Participants, interventions, and outcomes
Study setting 9 Data will be collected through bachelor endurance courses at the German Sport University Cologne, Cologne, Germany.

Eligibility criteria 10 Inclusion criteria
Age between 18 and 29 and a body mass index of ≤ 25 kg/m 2 .

Exclusion criteria
Injuries, diseases (e.g. inflammatory bowel diseases), an antibiotic therapy just before or during the intervention and lasting gastrointestinal disturbances based on probiotic intake.
Interventions 11a Participants will receive a twelve-week intervention program including a two-week rest, a four-week endurance training period and a four-week probiotic period with SymbioLactComp ® .
Subjects will not perform endurance sports or take probiotics during the rest period, but will participate in the university courses during the program.
Training intensity uses baseline shuttle run results (HRpeak). The training goal is a 10km-run in 50 minutes (males) or 55 minutes (female). The intensity of exercises will increase from 70% to 85% of the HRpeak to improve the subject's basic endurance [39]. The three 60-minute trainings per week will consist of extensive and intensive continuous methods, interval training and stretching [39]. The exercises will be performed in groups and supervised by a sports scientist. Sensitivities will be checked using a Borg Scale; the heart rate will be measured by a pulse monitor and belt KBE/g. It will be mixed with water and taken daily at breakfast.
Venous blood samples (of approximately 25 mL) and saliva samples will be collected under medical supervision. Fecal samples and nutrition diary will be collected at everyones' home.
11b Criteria for discontinuing or modifying allocated interventions for a given trial participant are the following: Antibiotic therapy, injuries, pain, headache, other complaints that do not allow participation in the (sports) program. In addition, meteorism, diarrhea or other gastrointestinal complaints as side effects of taking probiotics, which also do not allow participation in the program. In case of appropriate symptoms, participants will take a break or cancel participation, if necessary after consultation with the study management and the attending physician.
11c Adherence Participants will keep a training diary as well as a food diary to improve adherence of the intervention program. Participants will get instructions of taking the probiotic supplement and collecting fecal samples as well as the shipping of the samples.
11d Relevant concomitant care and interventions during the trial: -Use of borg scale and pulse watch to determine the participants' physical load during the training. -Medical consultation during the probiotic period, if probiotic induced complaints will arise. -Medical supervision during the blood and saliva measurements Outcomes 12 Primary feasibility outcomes of this randomised control cross-over trial are to assess practicability of the intervention (physical activity and probiotic supplementation) and capability of study design as well as to provide reliable estimates for sample size calculation. Primary efficacy outcome is the potential influence of moderate endurance training on the intestinal microbiota (microbiota composition) and barrier function (selected mucosa and immune parameters) in healthy student athletes compared to a probiotic supplement (SymbioLactComp ® ). Secondary efficacy outcomes will be selected blood and saliva biomarkers (haemogram, selected cytokines). The study will have a half-year enrollment phase. Participants will be recruited through bachelor endurance courses (maximal participant rate around 320 students) within one semester. The planned period for the whole intervention is for a maximum of one year. Schematic of the study design:

Recruitment 15
Potential student athletes will be achieved via flyers and short talks in the bachelor endurance courses at the German University Cologne. Interested students will receive contact data from the study administration and will be invited to a personal meeting with the study director.

Methods: Assignment of interventions (for controlled trials)
Allocation: Sequence generation 16a Key elements of randomisation: method: random number table Type of randomisation: blocked randomisation Participants will be randomised in a 1:1 ratio and matched to group one (endurance-probiotics) or group two (probioticsendurance).
Allocation concealment mechanism 16b Implementati on 16c All patients who give consent for participation and who fulfil the inclusion criteria will be randomised. Randomisation will be requested by the study director.

Methods: Data collection, management, and analysis
Data collection methods 18a A standardised multistage 20 m shuttle run test will be performed at the outset of the study, measuring Heart rate peak and the VO2peak as an indicator for the participants' cardiorespiratory fitness. Participants will complete four detailed seven-day food diaries based on self-reported intake. A standardised protocol will be developed by a research dietitan supervisor. Furthermore, a personal history and lifestyle including physical activity, will be collected by a modified questionnaire (International Physical Activity Questionnaire at baseline. Venous blood samples will be collected using the BD Vacutainer ® Butterfly Safety Lok, EDTA tubes and SST TM II Advance serum tubes (Becton Dickinson, Heidelberg, Germany). The thawed serum samples will be used to measure the following cytokines: tumor necrosis factor-alpha, interleukin-6, interleukin-8, interleukin-1β by multiplex ELISA (Bio-Plex Pro Human Cytokine 8-plex ELISA Kit, Bio-Plex Pro Human Chemokine IL-1β Singleplex Set; Bio-Rad Laboratories GmbH, Munich, Germany).
Fecal analyses will be carried out at the Institute of Microoecology, Herborn, Germany. Bacteria will be enumerated using the KyberKompaktPro ® test, which combines the identification of viable bacteria by classical microbial analysis and quantitative polymerase chain reaction (qPCR). Human stool samples analysed for SCFA content will be freeze-dried and subsequently analysed using a gas chromatograph. Fecal calprotectin, eosinophil protein x (EPX), zonulin, beta-defensin 2 (β-defensin 2) and secretory immunoglobulin A (sIgA) c o n c e n t r a t i o n s w i l l b e m e a s u r e d b y a n E L I S A k i t (Immundiagnostik AG, Bensheim, Germany).
18b Retention and attendance rates will be assessed to calculate adherence. The retention rate will be analysed by the percentage of participants completing all sessions of the endurance training (twelve in total), shuttle run test (two in total), nutrition questionnaire (four in total), blood/saliva measurement (four in total) and stool diagnostics (four in total) present in each step of study. Attendance rate will be analysed by the mean of presences of the participants divided by the number of sessions offered during the four-week exercise period and the four-week probiotic period.

Data management 19
All data will be entered electronically at the Institute of Movement and Neuroscience, Department of Movement and Health Promotion, German Sport University Cologne. Participant files will be stored in a secure and accessible place and will be kept in locked cabinets. Access to the study data will be restricted. Data protection will be guaranteed through a password system.

Statistical methods
20a This pilot study intends to examine the confidence interval estimation, feasibility and practicability of the presented approach (physical activity and probiotic supplementation) (Leon et al., , Arain et al. 2004). Our trial is mainly conducted in preparation for a future definitive randomised controlled trial to assess efficacy of the intervention/ study design (Eldridge et al., 2016). All data will be entered into the database management software and analysed by the SPSS Version 25.0 (SPSS Inc., Chicago, IL, USA). The Shapiro-Wilk test will be used for variable normal distribution and the usual descriptive statistics for background information and mean values with a 95 % confidence interval. Data will be used to determine the most appropriate outcome measure for the main study and in sample size estimation. Descriptive statistics and confidence intervals will be also carried out to examine the trends in the analysed parameters. Depending on the normality of the underlying data, parametric or non-parametric (e.g. unpaired t test/ Mann-Whitney-U test) tests will be carried out. The level of statistical significance is p < 0.05. 20c All randomised participants, regardless of protocol adherence will be included in the electronical dataset. Missing data will be marked by using the variable 999.

Methods: Monitoring
Data monitoring 21a A data monitoring committee (DMC) is not needed because of the short duration as well as the known minimal risk of the intervention program (endurance training and probiotic intake). 21b

Harms 22
The study will monitor for the following adverse effects concerning the endurance training and probiotic supplementation: Muscle injuries, intolerance towards the probiotic SymbioLact.
" 13 Auditing 23 The study director will regularly review the dataset to determine whether the data reported are complete and accurate.

Ethics and dissemination
Research ethics approval 24 The study was approved by the ethic committee of the German Sport University Cologne (No. 136/2016).

Protocol amendments 25
Any amendments will be transparently described in trial reports.

Consent or assent
26a Subjects who meet the inclusion criteria, will obtain detailed information about the trial and will give written informed consent to the study director before starting the study.
26b Confidentiality 27 Participants' data will not be released outside of the study without the written permission of the participant. Furthermore, all studyrelated data will be stored securely in locked file cabinets in a room with limited access. Laboratory data will be identified by a coded Identification number only to maintain participant confidentially.
Declaration of interests 28 The authors declared that they have no conflict of interests.
Access to data 29 The study director (Laura Schmitz) and the medical director (Christine Graf) as well as the representative study director (Nina Ferrari) will have access to the final dataset of this study.
Ancillary and post-trial care 30 The attending physician ensures that this study is conducted in accordance with the Declaration of Helsinki (additions to Tokyo, Venice and Hong Kong).
Dissemination policy 31a The time between the completion of data collection and evaluation and the release of the study results will be reduce to a minimum. The trial results will be released to the participants as well as published, regardless of the statistical significance.
31b Only individuals who meet the criteria for authorship will be listed as authors. There won't make use of professional writers or of ghost authorship.
Datum, Ort, Unterschrift Teilnehmer/in " 15 *It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013 Explanation & Elaboration for important clarification on the items. Amendments to the protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT Group under the Creative Commons "Attribution-NonCommercial-NoDerivs 3.0 Unported" license.
Biological specimens 33 Aliquots with blood and saliva as well as fecal samples will be bar coded with a unique storage identifier. The scientists who will carry out blood, saliva and fecal analyses will not have access to personal identifiers and will not be able to link the results of the tests to personal identifier information. No individual results will be presented in publications.