This study will investigate the effect of postbiotics supplementation on gut microbiota (GM) composition, exercise-induced oxidative stress and performance following eccentrically biased exercise. The study will be a cross-over, randomized, double-blind, controlled study that will be conducted in two cycles. Participants will be randomly assigned into one of the two trials: i) Postbiotics supplementation for 4 weeks, ii) Placebo supplementation for 4 weeks. Participants will then perform a 45-min treadmill running at (-15% slope, \~70% VO2max) followed by a time-trial (0% slope, \~95% VO2max) until exhaustion. Before, as well as 24 h, 48 h and 72 h following exercise, assessment of GM composition and metabolites (short-chain fatty acids), exercise induced muscle damage (EIMD) (delayed onset of muscle soreness, creatine kinase), complete blood count (CBC), blood inflammatory status (tumor necrosis factor alpha, interleukin 6, C-reactive protein), gut inflammatory status (lipopolysacharides-binding protein, zonulin), blood redox status (total antioxidant capacity, catalase, protein carbonyls, reduced glutathione, oxidized glutathione, and performance (knee-extensors' and knee-flexors' isometric, concentric, eccentric torque, countermovement jump) will be performed. In addition, metabolism (lactic acid) will be assessed before and 4 min following exercise. Afterwards, the participants will receive the postbiotics supplement or the placebo for 4 weeks, and will repeat the exercise protocol and measurements of EIMD, CBC, blood inflammatory status, blood redox status and performance at the same time-points. At the second cycle, the participants will repeat the above procedures under the remaining condition. Between conditions, there will be a 14-day washout period. The results of the research will provide important information for coaches and physically active individuals, regarding the effectiveness of postbiotics to favorably change the gut microbiota composition and alleviate gut inflammation, exercise-induced inflammation and oxidative stress, and improve performance after intense exercise.
Acute, vigorous and/or unaccustomed eccentric exercise can induce muscle injury and inflammatory reactions, and oxidative stress, but also reduced muscle performance. For this reason, many professional as well as amateur athletes, often consume nutritional supplements such as antioxidants, anticipating to reduce inflammation and oxidative stress after intense exercise. The human gastrointestinal tract is inhabited by various microorganisms, called the gut microbiome (GM). GM, among other things, contributes to the normal functioning of the immune system, contributes to the production of short-chain fatty acids (SCFAs) and vitamin synthesis as well as the digestion and absorption of food, protects against enteropathogens and regulates inflammatory and redox responses. Recent evidence also suggests that GM may be involved in athletic performance. In contrast, disruption of GM composition (dysbiosis) is characterized by reduced diversity, reduced abundance of health-promoting bacteria, and increased abundance of gram-negative and other pathogenic bacteria and is associated with various metabolic diseases such as obesity, diabetes, and various forms of cancer, systemic inflammation, oxidative stress and reduced performance. Thus, the supplementation of several "biotics" has been emerged as a means to regulate the GM in favor of health-promoting bacteria. Postbiotics is defined as a "preparation of inanimate microorganisms and/or their components that confers a health benefit on the host". Evidence suggests that supplementation with postbiotics may regulate the GM, and consequently, strengthen the immune system, reduce intestinal permeability, improve antioxidant mechanisms, as well as accelerate recovery after exercise-induced inflammation, enhance adaptations to exercise, and improve performance. However, the scientific data regarding the possible beneficial effect of supplemental administration of postbiotics is limited. More research is needed, in order to determine the role of postbiotics supplementation on GM composition and function, exercise-induced inflammation and redox status, but also on performance after intense exercise. This study will investigate the potential of postbiotics supplementation to alter the GM composition and affect the recovery of exercise-induced oxidative stress and performance following intense, eccentrically biased acute exercise. The study will be cross-over, randomized, double-blind, controlled, and will be conducted in two cycles. The participants, will be primarily informed regarding the study procedures, as well as the benefits and possible risks, and will sign an informed consent form for participation in the study. Before the experimental procedure, they will be involved in a week of familiarization to the evaluation tests and the exercise protocol, at a low intensity. Participants will undergo baseline measurements: anthropometric characteristics (body height, body mass, body mass index) via a stadiometer-scale (Stadiometer 208; Seca, Birmingham, UK), body composition (amount of body fat, lean body mass, fat mass, bone density) via by dual emission X-ray absorptiometry (DXA, GE-Healthcare, Lunar DPX NT, Belgium), aerobic capacity (VO2max) via an automated online pulmonary gas analyzer (Vmax Encore 29, BEBJO296, Yorba Linda, CA, USA) during a graded exercise protocol on a treadmill (Stex 8025T, Korea), isokinetic strength (isometric, concentric and eccentric torque of the knee extensors and knee flexors) on an isokinetic dynamometer (Cybex, HUMAC NORM 360, Ronkonkoma, NY), and muscle power via the assessment of countermovement jump (CMJ) via an optical measurement system (Optojump next, Microgate, USA). In addition, the participants will record their diet via a 7-days recall before their participation in the first experimental condition, and dietary data will be analyzed with ScienceFit Diet 200A diet analysis program (Science Technologies, Athens, Greece), in order to estimate that they do not consume nutrients that may affect muscle injury, inflammation and oxidative stress (e.g. antioxidants, etc.). Participants will then be randomized in one of the two conditions: i) Postbiotics supplementation (50mg/day of Heatkilled Lactobacillus plantarum L-137, Immuno-LP20TM) for 4 weeks, or ii) placebo supplementation for 4 weeks. Randomization of the conditions will be done by a software generating random integers available on the internet (Random.org). Participants will then perform an exercise protocol comprised of 45 min downhill running (-15% slope, \~70-75% VO2max) on a treadmill followed by a time-trial (0% slope, \~95% VO2max) until exhaustion. Before the exercise protocol, as well as 24 h, 48 h and 72 h after exercise, exercise-induced muscle damage (EIMD) \[delayed onset of muscle soreness (DOMS) via palpation of the knee extensors and knee flexors on a scale of 1 to 10 (1 = no pain at all; 10 = extreme pain), and muscle performance (CMJ, isometric, concentric and eccentric torque of the knee extensors and knee flexors)\], will be assessed. At the same time points, feces samples will be collected for the analysis of GM composition and function, and GM metabolites, as well as blood samples for the assessment of complete blood count (CBC), blood inflammatory status (creatine kinase, tumor necrosis factor alpha, interleukin 6, C-reactive protein), gut inflammatory status (lipopolysacharides-binding protein, zonulin), and blood redox status \[reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG ratio, total antioxidant capacity, catalase, protein carbonyls, uric acid, bilirubin). Furthermore, metabolism (lactic acid) will be assessed before and 4 min following exercise by analyzing capillary blood with a portable lactate analyzer (Lactate Plus, Nova Biomedical, USA). Afterwards, the participants will receive the postbiotics supplement or the placebo for 4 weeks, and will repeat the exercise protocol and measurements of EIMD, CBC, blood inflammatory status, blood redox status and performance at the same time-points. At the second cycle, participants will repeat the exact same procedures for the remaining condition. Between cycles, a 14-days washout period will be applied. Additionally, the 7-day diet recall from the first cycle, will be given to the participants to follow the same diet before the experimental exercise protocol at the second cycle.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
SCREENING
Masking
QUADRUPLE
Enrollment
15
The participants will consume one capsule of postbiotics supplement per day
The participants will consume one capsule of placebo per day
Department of Physical Education and Sport Science, Uninersity of Thessaly
Trikala, Thessaly, Greece
Changes in gut microbiota composition
Gut microbiota composition will be assessed in feces
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in butyrate
Butyrate will be assessed in feces
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in propionate
Propionate will be assessed in feces
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in acetate
Acetate will be assessed in feces
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in complete blood count
Complete blood count will be assessed in whole blood
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in creatine kinase activity
Creatine kinase activity will be assessed in serum
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in TNF-α concentration
TNF-α concentration will be assessed in serum
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in IL-6 concentration
IL-6 concentration will be assessed in serum
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in high-sensitivity C-reactive protein concentration
C-reactive protein concentration will be assessed in serum
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in lipopolysacharides-binding protein concentration
Lipopolysacharides-binding protein concentration will be assessed in serum
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in zonulin concentration
Zonulin concentration will be assessed in serum and in feces
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in protein carbonyls concentration
Protein carbonyls concentration will be assessed in plasma
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in malondialdehyde concentration
Malondialdehyde concentration will be assessed in plasma
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in reduced glutathione concentration
Glutathione concentration will be assessed in red blood cells lycate
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in oxidized glutathione concentration
Oxidized glutathione concentration will be assessed in red blood cells lycate
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in GSH/GSSG ratio
GSH/GSSG ratio will be assessed in red blood cells lycate
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in catalase concentration
Catalase concentration will be assessed in red blood cells lycate
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in total antioxidant capacity
Total antioxidant capacity will be assessed in red blood cells lycate
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in uric acid concentration
Uric acid concentration will be assessed in serum
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in bilirubin concentration
Bilirubin concentration will be assessed in serum
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in blood lactate concentration
Bilirubin concentration will be assessed in whole blood
Time frame: At baseline (pre), 4 min post-exercise
Changes in delayed onset of muscle soreness in the knee flexors and extensors of both limbs
Muscle soreness of the KF and KE will be assessed via palpation of the muscle belly and the distal regions following 3 squats, and the subjective pain will be recorded on a 10-point scale (1 = no pain, 10 = extreme pain)
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in countermovement jump height
Countermovement jump height will be measured with an optical system
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
Changes in isokinetic strength of knee extensors and knee flexors
Isometric, concentric and eccentric peak torque of the knee extensors and knee flexors of both limbs will be assessed on an isokinetic dynamometer
Time frame: At baseline (pre), 24 hours post-, 48 hours post-, 72 hours post-exercise
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