Subjects

endurance trained men (triathletes, runners, cyclists) participated in this trial. Inclusion criteria: male, healthy, 30–45 years, non-smokers, trained (maximum oxygen uptake, VO 2max > 45 mL . kg-1 . min-1), no dietary or nutritional supplement use within four weeks prior to the first exercise test. Exclusion criteria: smokers, men who failed eligibility testing for exercise - as described by the Austrian and German standards in sports medicine [24], men who significantly changed training regimen during the study, chronic or excessive alcohol consumption, recent surgery or illness, body fat > 20%. Body fat content and distribution was estimated by a computerized optical device Lipometer (Möller Messtechnik, Graz, Austria), as described by Möller, et al. [25]. Besides inclusion and exclusion criteria, a standard blood chemistry panel was determined after an overnight fast and all subjects completed a medical history. Subjects characteristics are presented in Table 1.

Table 1 Baseline characteristics, performance data, clinical chemistry and nutrition data of 23 trained men 1 Full size table

Ethical aspects, recruitment and randomization

All subjects provided written informed consent prior to participating in this investigation. This study was conducted according to the guidelines of the Declaration of Helsinki for Research on Human Subjects 1989 and was approved by the Ethical Review Committee of the Medical University of Graz, Austria. The trial was registered under http://www.clinicaltrials.gov, identifier: NCT01474629.

The study focused trained men and was advertised in the largest sports magazine of Austria. After a telephone screening conducted by the research team, 29 men volunteered for eligibility testing. From those, 24 men were eligible and entered the study program.

Subjects were randomized into blocks of six and sequentially numbered. To guarantee a balanced VO 2max distribution between groups (probiotics versus placebo) we conducted stratification via VO 2max rank statistics. Randomization code was held by a third party (Union of Sport and Exercise Scientists Austria) and handed over for statistical analyses after collection of all data.

Study design and time schedule

This was a randomized, placebo controlled, double-blinded study. All eligibility testing (blood panel, eligibility for exercise, clinic check-up, medical history questionaire, one-on-one interview) was finalized at least four weeks prior to the first exercise test. At the morning of the first exercise test a standardized breakfast (3 hours prior to exercise) was provided. After the test, the investigator dispensed the randomized sachet supply according to the man’s VO 2max -ranking. After 14 weeks taking the powder from sachets as directed, they returned their remaining sachets and the same test procedure was repeated. All subjects were checked by the physician before each exercise test.

Dietary and lifestyle assessment

Subjects were instructed to maintain their habitual diet, lifestyle and training regimen during the fourteen weeks study and to duplicate their diet before each exercise testing/blood collection appointment as described below. Before the first triple step test, men completed a 7-day food record for nutrient intake assessment. Subjects subsequently received copies of their 7-day diet records and were instructed to replicate the diet prior to the second exercise tests. The breakfast before each exercise test was standardized for the entire cohort to limit nutrient variation due to self-selection on the morning scheduled for blood draws. The composition of this standardized breakfast 3 hours prior to the strenuous exercise tests is shown in Table 2. Diet records were analyzed for total calories, protein, carbohydrate, fat, cholesterol, fiber, water, alcohol, and several vitamins, minerals, and fatty acids using “opti diet” software 5.0 (GOEmbH, Linden, Germany).

Table 2 Composition of the standardized breakfast 3 hours prior to the strenuous triple step test ergometry Full size table

Treatment

The men randomized to probiotics (n = 11) received boxes with sachets containing multi-species probiotics (Ecologic®Performance, produced by Winclove b.v., Amsterdam, the Netherlands; the product is also branded as OMNi-BiOTiC®POWER). The probiotic supplement contained of a matrix and six probiotic strains: Bifidobacterium bifidum W23, Bifidobacterium lactis W51, Enterococcus faecium W54, Lactobacillus acidophilus W22, Lactobacillus brevis W63, and Lactococcus lactis W58. The matrix consisted of cornstarch, maltodextrin, vegetable protein, MgSO 4 , MnSO 4 and KCl. The placebo consisted of the matrix only. The minimum concentration was 2.5 × 109 colony forming units (CFU) per gram. Subjects were instructed to take 2 sachets a 2 g per day (4 g/day), equivalent to 1010 CFU/day, with 100–125 mL of plain water per sachet, one hour prior to meals and throughout 14 weeks. Those subjects randomized to placebo (n = 12) received identical boxes and sachets with the same instructions for intake.

Exercise tests

Each subject was instructed not to perform physical training 3 days prior to any exercise test.

For eligibility testing all subjects performed an incremental cycle ergometer exercise test (EC 3000, Custo med GmbH, Ottobrunn, Germany) at 80 rpm. After a three minute rest phase sitting inactive on the ergometer, work rate started at 60 W for three minutes and was increased 20 W every minute until voluntary exhaustion. This allowed subjects to reach exhaustion within 15–18 minutes. A standard electrocardiogram was recorded during the entire test, which was supervised by a physician. Respiratory gas exchange variables were measured throughout the incremental exercise tests using a breath-by-breath mode (Metalyzer 3B, Cortex Biophysik GmbH, Leipzig, Germany). During these tests, subjects breathed through a facemask. Oxygen uptake (VO 2 ), carbon dioxide output (VCO 2 ), minute ventilation (V E ), breathing rate (BR) and tidal volume (V T ) were continuously obtained. Heart rate (HR) was monitored throughout the tests using a commercially available heart rate monitor (Polar Vantage NV, Polar Electro Finland).

For the triple step test ergometry we used the same test protocol as described above but repeated the incremental exercise test for two times, so that three step tests until voluntary exhaustion were integrated in one single bout of exercise. After the first and second part of this triple test subjects performed for 15 minutes with 60 W at 80 rpm. After the third part subjects continued exercise for three minutes with 60 W and 80 rmp and stopped then. The whole test procedure lasted between 80 and 90 minutes, depending on duration of each step test/part.

Blood pressure was controlled after each 100 W and after the last step of each ergometry. Gas exchange variables were monitored continuously throughout the step tests as described above. During the 15 minutes intervals between the ergometry step tests the facemask was removed to consume 750 mL of plain water, in total over the whole test procedure.

Fourteen weeks later this procedure was repeated on the same cycle ergometer, with the same investigator, standardized room temperature (20°C) and humidity (60%).

Blood and feces collection

We conducted blood collections in supine position from a medial cubital vein at each triple ergometry test: before exercise (Pre) and within 10 min post exercise (Post). Venous blood was collected to determine carbonyl proteins (CP), malondialdehyde (MDA), total oxidation status of lipids (TOS), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). After centrifugation for 10 minutes plasma was removed and samples were frozen at −70°C until analysis.

For zonulin and α1-antitrypsin from feces the subjects collected samples at baseline and after 14 weeks with standardized stool tubules within 24 hours prior to bringing the sample in a cool bag to the laboratory. All samples were analyzed within 72 hours after dispensing. Throughout the 14 weeks treatment the subjects recorded a stool protocol to monitor stool appearance with help of the Bristol stool scale/chart [28].

Stool analyses

Zonulin and α1-antitrypsin were analyzed with commercially available ELISA kits (Immundiagnostik AG, Bensheim, Germany).

The zonulin analysis is based on a competition between the free antigen in the samples or standards and the antigen coated on the wells of the microplate. Standards, samples and the primary anti-zonulin antibody are transferred directly into the precoated microplate wells. The antigen in the samples competes with the antigen immobilized on the wells of the microplate for the binding sites of the specific anti-zonulin antibody. A peroxidase-conjugated antibody is used for detection, and tetramethylbenzidine as a peroxidase substrate. The enzymatic reaction is terminated by acidic stop solution. The quantification is based on the optical density at 450 nm. Data are expressed in ng/mL.

The assay for analyses of α1-antitrypsin utilizes the “sandwich” technique with two selected polyclonal antibodies that bind to the glycoprotein forming a “sandwich” of capture antibody - human α1-antitrypsin - peroxidase-conjugate. Tetramethylbenzidine is used as peroxidase substrate. Finally, an acidic stop solution is added to terminate the reaction. The colour changes from blue to yellow. The intensity of the yellow colour is directly proportional to the concentration of α1-antitrypsin. Samples are quantified by referring their optical density to a lot-dependant master calibration curve and the use of a calibrator that is run with each test. Data are expressed in mg/dL.

Analyses of blood parameters

CP was analyzed with a commercially available ELISA (Immundiagnostik AG, Bensheim, Germany) via reaction of protein with dinitrophenylhydrazine (DNPH). The non-protein constituents and unconjugated DNPH are separated by ultracentrifugation. The proteins are adsorbed to an ELISA plate and incubated with anti-DNPH antibody followed by antibody-linked horseradish peroxidase. Absorbances are related to a standard curve prepared with oxidized serum albumin. The carbonyl protein content is calculated from the estimated carbonyl concentration and the total protein content of the sample. For this reason, a parallel determination of the protein content is required. Data are expressed in pmol/mg.

MDA was determined according to a previously described HPLC method by Pilz et al. [29] after derivatization with 2,4-DNPH. This method determines the protein bound MDA. The HPLC separations were performed with an L-2200 autosampler, a L-2130 HTA pump and a L-2450 diode array detector (all: VWR Hitachi Vienna; Austria). Detector signals (absorbance at 310 nm) were recorded and program EZchrom Elite (VWR) was used for data requisition and analysis. Data are expressed in nmol/mL.

Analysis of TOS: This assay (Immundiagnostik AG, Bensheim, Germany) determines total lipid peroxides and is performed by the reaction of a peroxidase with the peroxides in the sample followed by the conversion of tetramethylbenzidine to a colored product. After addition of a stop solution the samples are measured at 450 nm in a microtiter plate reader. The quantification is performed by the delivered calibrator. Data are expressed in μmol/L H 2 O 2 .

TNF-α was analyzed with a commercially available ELISA (Immundiagnostik AG, Bensheim, Germany) allowed quantitative determination of Tumor Necrosis Factor-α by using monoclonal antibodies and a horseradish peroxidase labeled conjugate. The amount of the converted substrate by the peroxidase is directly proportional to the amount of bound TNF-α and can be determined photometrically. Data are expressed in pg/mL.

IL-6 was also measured with commercial available ELISA kits (Invitrogen, LifeTech Austria, Vienna, Austria) using monoclonal antibodies specific for human IL-6. Based on the binding of streptavidin-peroxidase to antibodies the intensity of a colored adduct is directly proportional to the concentration of the cytokine and can be determined photometrically. Data are expressed in pg/mL.

Blood chemistry panel

Standard blood chemistry values were determined after overnight fast using EDTA plasma from peripheral venous blood. Analyses were conducted with a routine clinical chemistry analyzer (Abbott Diagnostics, Vienna, Austria).

Statistical analyses and sample size calculation

Per protocol analyses were performed using SPSS for Windows software, version 19.0. Data are presented as mean ± SD. Data for pre - post comparisons were adjusted for plasma volume changes as described elsewhere (except for CP, as it is expressed on protein) [30]. Statistical significance was set at P < 0.05. The Shapiro-Wilk test was used to determine normal distribution. Baseline characteristics, performance data, nutrient and clinical chemistry data, were compared by unpaired Student’s t-test. Data obtained for CP, MDA, TOS, TNF-α, and IL-6, were analyzed using a univariate, three-factorial, repeated measures ANOVA. Factors: treatment (probiotic supplementation and placebo), exercise (pre and post exercise), session (triple step test ergometry 1 and triple step test ergometry 2). For zonulin and α1-antitrypsin we used a two-factorial ANOVA (treatment, time). Significant interactions and main effects were analyzed by using Bonferroni correction.

Sample size calculation was based on oxidation markers CP and MDA. We estimated between 7 and 9 subjects per group - depending on parameter, standard deviation and effect size - to reach a probability of error (alpha/2) of 5% and 80% power. Allowing for a drop-out rate of 30%, 12 subjects per group were recruited.