Patient will be cross-over to the different group of probiotic/placebo ("A" or "B" as described on the visit 2) and will be advised to consume 2 capsules in the morning and 2 capsules in the evening. Stool collection for microbial analysis, short chain fatty acids, zonulin and calprotectin concentration. This incredibly advanced functional system created by the mucous membrane of enterocytes and microorganisms that live there is unfortunately extremely susceptible to damages. The largest devastation in the natural bacterial microflora is made by antibiotics, still very willingly prescribed by doctors. Importantly, they cause changes in the bacterial composition of the digestive system not only during their application, but they also have influence on the probiotic barrier for many years after antibiotic therapy.

Metformin, the first-line drug in the treatment of type 2 diabetes (T2DM), may cause dose dependent undesirable side-effects like diarrhea, abdominal pain, nausea or bloating which may affect up to 20 % of patients treated with this drug. In the case of antibiotic therapy, it is recommended to consume the supplement when you start taking the antibiotic and continue supplementation for 14 consecutive days after the end of antibiotic therapy. To the best of our knowledge, this will be the first intervention study assessing the efficacy of both probiotic supplementation and a gluten-free diet and comparing the effect of both interventions and their combination in MDD patients. The protocol and the outline were developed by a multidisciplinary researcher team. The methodology employed in the study benefited greatly from our diverse set of knowledge, experience and perspectives. Considering the highly rigoristic selection criteria, the participant group is relatively large. Rigorous procedures established for this trial will be followed by well-trained researchers to ensure that research will be conducted in accordance with standards of best practice. The measurements listed above will be introduced to reduce the potential bias related to confounding variables (e.g. smoking, physical activity, and diet).

Visit 7 - month 5 (4 weeks ± 3 days from the visit 6) Gastrointestinal symptoms questionnaire administration. Patient's metformin intolerance assessment will be questionnaire based (questionnaire adapted from Laura J. Mc Creight et al.). Visit 8 - month 6 (4 weeks ± 3 days from the visit 7) Gastrointestinal symptoms questionnaire administration.

Patients will fill out questionnaires regarding the gastrointestinal symptoms associated with metformin at a dose they did not tolerate and at a time of reduced daily dose of metformin they do tolerate (patients are accepted to have gastrointestinal symptoms which they accept). Visit 3 - month 1 (4 weeks ± 3 days from the visit 2) Gastrointestinal symptoms questionnaire administration.

It is also known that patients with T2DM, in general, show evidence of gut dysbiosis followed by alterations of an intestinal barrier leading to an increase in intestinal permeability and elevated inflammatory state. To monitor cardiac contractions, a Garmin sporttester model HRM-Tri 010-10997-09 (United States) was used. 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).

Jang LG, Choi G, Kim SW, Kim BY, Lee S, Park H. The combination of sport and sport-specific diet is associated with characteristics of gut microbiota: an observational study. J Int Soc Sports Nutr. 2019 May 3;16(1):21. doi: 10.1186/s12970-019-0290-y.The immune system has a powerful impact on brain function and activity [ 8, 9]. In 1991, Smith proposed the macrophage theory of depression, where immune and inflammatory imbalances are the main factors leading to the onset or maintenance of MDD [ 10]. The author suggested that the intestine is a potential immune activation gate. Bidirectional signaling via the immune, endocrine, neural, and metabolic pathways occurs between the gastrointestinal (GI) tract and brain [ 11]. In his earlier works, Smith termed this connection the “food-gut-allergy-behavior axis” [ 10]. The intestines, having one of the largest surface areas interacting with the external environment, are exposed to more antigens than any other part of the body [ 12]. One of the proposed causes leading to macrophage activation is an abnormal immune response to food compounds in mechanisms other than classic allergy (i.e. immunoglobulin-E (IgE)-mediated food allergy). The interplay between genetic and environmental factors may lead to the disruption of gut proteins such as tight junctions (TJs) and subsequently to the loss in epithelial wall integrity and both gut and blood-brain barrier (BBB) permeability [ 13]. Disruption of the intercellular TJs may be the initial phenomenon related to uncontrolled molecule translocation (i.e. toxins, antigens, and bacteria) into the blood, which promotes proinflammatory cytokine synthesis and pro/anti-inflammatory imbalance [ 14].

At certain visits patients will undergo the tests measuring the intestinal permeability (blood and stool zonulin immunoenzymatic tests). The first of these (W1) included qualification for the study, completing the questionnaire and 3-day nutrition diaries. Vitale K, Getzin A. Nutrition and Supplement Update for the Endurance Athlete: Review and Recommendations. Nutrients. 2019 Jun 7;11(6):1289. doi: 10.3390/nu11061289. suplement diety wspomaga utrzymanie prawidłowej mikroflory jelitowej podczas antybiotykoterapii [3]. The second visit was conducted at the Fitness Club Maniac Gym, where the cardiopulmonary fitness test and body composition analysis were carried out.

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de Roos NM, Giezenaar CG, Rovers JM, Witteman BJ, Smits MG, van Hemert S. The effects of the multispecies probiotic mixture Ecologic(R)Barrier on migraine: results of an open-label pilot study. Benef Microbes. 2015;6(5):641-6. doi: 10.3920/BM2015.0003. Epub 2015 Apr 22. Electroencephalography (EEG) is a non-invasive method enabling the assessment of electrophysiological neural activity. Subjects participating in the study will sit with closed eyes throughout the EEG recording. The assessment will be carried out during the daytime, between 10:00a.m. and 2:00p.m. to ensure that participants will remain awake. The EEG recordings, after the initial calibration of the equipment, will last about 15min so that after cleaning, we will have at least five minutes of pure electrophysiological recording available from each participant. To ensure study comparability and reproducibility of the study, a standard procedure will be conducted, with 19 electrodes placed according to the International 10–20 system [ 69, 70].