Regular and optimized physical activities can be considered not only prevention but also therapy for different chronic diseases and related complications [1
]. Exercise, particularly aerobics, also significantly improves physiological adaption by enhancing cardiovascular capacity and energy metabolites [2
]. The balance between intensive exercise-induced oxidative stress and the antioxidant system play an important role in athlete health and in the prevention of molecular and cellular damage [3
]. Studies elucidated that functional ingredients, including vitamin C, vitamin E, green tea extract, and quercetin, regulate oxidative stress and tissue damage in athletes [4
]. Excessive exogenous antioxidants may have detrimental effects on physiological adaption, and antioxidants from a varied and balanced diet may be the best approach for the maintenance of optimal antioxidant status [6
]. In addition, a study reported that exercise increases microflora diversity and the number of beneficial microbial species and commensal bacteria with health benefits [7
], and microbiota improve exercise performance through antioxidative enzymes, as revealed through gnotobiotic animal model validation [8
]. Microbiota also regulate oxidative stress and inflammatory responses, as well as modulate metabolism and energy expenditure during intense exercise [9
Probiotics were demonstrated to have functional effects on gastrointestinal (GI) conditions, including diarrhea, inflammatory bowel disease (IBD), and liver disease, through microbiota composition modulation [10
]. Metabolic conditions (obesity and diabetes) can also be managed by probiotic and synbiotic supplementation from fermented food sources [11
]. Fermented milk, containing Lactobacillus delbrueckii
subsp. bulgaricus and Streptococcus thermophilus
, improved the behavior of aged mice as well as their redox state and immune cell function [12
]. A mixture of probiotics also improved the recovery of intestinal multibarriers in DSS-induced colitis by rebuilding the structure and diversity of gut microbiota [13
]. Our previous clinical and animal studies reported that L. plantarum
TWK10, isolated from Taiwanese pickled cabbage, exerted beneficial effects on body composition, energy production, and physiological adaption [14
]. In triathletes, L. plantarum
PS128 ameliorated inflammation, oxidative stress, fatigue, and injury-related biochemical indexes induced by intensive training [16
]. Thus, probiotics exert various functional effects, leading to health benefits and health promotion.
OLP-01 isolated from the gut microbiota of a weightlifting gold medalist showed the beneficial effects of fatigue mitigation and increased energy production [17
]. The exercise training was well-known for its benefits toward physiological adaption, and the probiotics were also able to exert multiple bioactivities. In this study, we hypothesized that OLP-01 combined with exercise intervention would facilitate exercise physiological adaption and performance. The aim was to reveal the different effects of probiotics on exercise training and, in particular, inform athletes regarding nutritional strategies.
2. Materials and Methods
OLP-01 (OLP-01) was selected and isolated from an elite weightlifting Olympic gold medalist. The strain was also further identified by an independent third party, the Food Industry Research and Development Institute (Hsinchu, Taiwan). OLP-01 was cultivated and maintained by Glac Biotech Co., Ltd. (Tainan, Taiwan), and lyophilized powder was applied at a concentration of 1.07 × 1011
CFU/g, equivalent to about 1 × 109
CFU/day for humans. Before supplementation, all aliquots of lyophilized powder were stored in a −20 ℃ refrigerator and prepared in sterilized saline buffer for daily use. The dose of OLP-01 used in current animal study (i.e., 1.03 × 1010
CFU/kg) was based on that used in a previous study [17
]; OLP-01 was administered for 6 weeks via oral gavage. The sedentary and exercise treatment groups received the same volume of saline according to individual body weight.
2.2. Experimental Design
Institute of Cancer Research (ICR) strain mice (5 weeks old), purchased from BioLASCO (Yi-Lan, Taiwan) and reared under specific pathogen-free (SPF) conditions, were used in this study. During the experimental duration, all animals were provided sufficient chow diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) and sterilized water ad libitum
and maintained on a 12 h light/dark cycle at 23 ℃ ± 2 ℃ and 50%–60% humidity. A veterinarian monitored the disease status and behavior of the animals. After 1 week of acclimation, the animals were randomly allocated into four groups (sedentary, exercise, OLP-01, and exercise + OLP-01). The animals had similar body weights at the beginning of the experiment and the indicated groups received the treadmill exercise protocol or/and probiotic supplementation for 6 weeks (Figure 1
). After 6 weeks of intervention, physical fitness was evaluated by measuring forelimb grip strength and by conducting exhaustive swimming, and biochemical indexes were also determined in mice undergoing acute and prolonged exercise challenges. Blood, relevant tissues, and feces were also sampled to determine the complete blood count (CBC), body composition, glycogen, and metabolites, as well as for histology analysis. The Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University approved all animal experiments in this study, and the study conformed to the guidelines of protocol IACUC-10801 approved by the IACUC ethics committee.
2.3. Aerobic Exercise Training
Mice underwent aerobic exercise training on a motor-driven treadmill (model MK-680; Muromachi Kikai, Tokyo, Japan) for 6 weeks, with motivation maintained through an electric shock grid under veterinarian surveillance. Mice in the exercise training groups (exercise and exercise + OLP-01) were initially acclimated to running at a speed of 10 m/min for 2 days prior to the training protocol. The training protocol began at a speed of 12 m/min and 35 min/day in the first week, and the speed was increased by 2 m/min every week until the sixth week (22 m/min). The slope was also elevated from 0% to 5% in the third week and maintained at 10% from the fourth to sixth weeks. The training protocol was slightly modified from a previous study protocol, and moderate exercise intensity corresponded to 65%–70% of maximal oxygen uptake [18
2.4. Physical Activities
Physical activities included forelimb grip strength activities and exhaustive swimming, which were used to test anaerobic and aerobic capacities. Grip strength was assessed using a low-force testing system (Model-RX-5; Aikoh Engineering, Nagoya, Japan), with detailed procedures described previously [19
]. To determine exercise performance, swimming to exhaustion was conducted to assess endurance capacity based on survival instinct. The animals were loaded with a weight equivalent to 5% of individual body weight and forced to swim in a tank until exhaustion. The time from the start of the experiment to exhaustion was recorded as the endurance index.
2.5. Peripheral Fatigue-Associated Biochemical Variables
Exercise-induced peripheral fatigue can be reflected by related biochemical indexes when evaluating physiological adaption. The acute exercise protocol included 15 min of swimming without any weight load; blood was sampled through submandibular collection at the beginning, immediately after 15 min of swimming, and during the subsequent 20 min rest interval for analysis of the indexes, such as lactate and ammonia. The prolonged exercise protocol included swimming for 90 min; blood was collected immediately after 60 min of rest and analyzed for blood urea nitrogen (BUN), creatine kinase (CK), and lactate dehydrogenase (LDH) levels. The blood samples were assessed using an autoanalyzer (Hitachi 7060; Hitachi, Tokyo, Japan) and a CBC analyzer.
2.6. Short Chain Fatty Acid Analysis
Feces samples were obtained 1 day before euthanization. They were weighed, suspended in 1 mL of water with 0.5% phosphoric acid per 0.1 g of sample, and stored in −30 °C immediately after collection until homogenization. Feces were homogenized for 2 min and centrifuged for 10 min at 14.8 RPM. The supernatant was isolated for ethyl acetate (300 μL) extraction, and the organic phase was collected for Agilent 5977B GC-MS (Agilent Technologies; Palo Alto, CA, USA) analysis. The GC instrument was fitted with a Nukol™ Capillary GC Column (30 m × 0.25 mm id, 0.25 μm df) and helium was used as the gas carrier, injected at 1 mL/min. The column temperature was 90 °C initially and then increased to 150 °C at 15 °C/min, 170 °C at 5 °C/min, and finally to 250 °C at 20 °C/min; this temperature was maintained for 2 min (total time of 14 min). Solvent delay was 3.5 min. The detector was operated in the electron impact ionization mode (electron energy of 70 eV), with scanning conducted in the 30–250 m/z range. The temperatures of the ion source, quadrupole, and interface were 230 °C, 150 °C, and 280 °C, respectively. Short-chain fatty acids (SCFAs) were identified based on the retention time of standard compounds and with the assistance of the NIST 08 and Wiley7N libraries.
2.7. Clinical Biochemical Profiles
After 95% CO2 asphyxiation euthanization of mice, blood samples were immediately collected through cardiac puncture and the sera were separated through centrifugation at 1000× g for 15 min at 4 °C after complete clotting for analysis of clinical biochemical variables, including aspartate aminotransferase (AST), alanine transaminase (ALT), CK, glucose (GLU), blood urea nitrogen (BUN), creatinine (CREA), uric acid (UA), albumin (ALB), albumin (ALB), total cholesterol (TC), and total protein (TP), using an autoanalyzer (Hitachi 7060, Hitachi, Tokyo, Japan).
2.8. Body Composition, Histology, and Glycogen Analysis
The important visceral organs, including the heart, liver, kidney, spleen, muscle (gastrocnemius and soleus), and cecum, alongside perirenal fat (white adipocyte tissue), were accurately excised and weighed after sacrifice and dissection for determining body composition. Then, the organs were preserved in 10% formalin for further paraffin-embedded procedures. Indicated tissue sections (4 μm) were collected from paraffin blocks and immersed in xylene and alcohol, followed by hematoxylin for 3 min and counterstaining with eosin for 1 min. Parts of the muscle and liver samples were kept in liquid nitrogen for glycogen content analysis. The analysis protocol was modified from a previous study; detailed procedures were as described previously [20
2.9. Statistical Analysis
Data were represented as the mean ± SD. Statistically significant differences in physical activity, biochemistry, lactate, body weight, body composition, growth curve, and glycogen content between the groups were analyzed using one-way and two-way analysis of variance (ANOVA), followed by multiple comparisons with post-hoc Tukey’s test. Data were considered statistically significant when the probability of a type I error was less than 0.05.
In the current study, B. longum OLP-01 isolated from an elite weightlifting athlete was combined with regular aerobic treadmill exercise training for six weeks to evaluate physiological effects and performance. Results demonstrated that B. longum OLP-01 combined with exercise training (the exercise + OLP-01 group) synergistically increased endurance capacity compared with training and probiotic-alone treatments (exercise and OLP-01 groups, respectively). Moreover, B. longum OLP-01 also significantly improved exercise-associated peripheral fatigue indexes and ameliorated oxidative stress-related injury indexes, possibly through inflammation regulation. From the perspective of ergogenic aid and health promotion, these probiotics could be further investigated in regard to functional activities in sport science.
The physiological effects of exercise are contingent on exercise intensity, type, and duration as well as energy demands, oxidative stress, and cardiovascular and metabolic adaptations [2
]. During exercise, central and peripheral fatigue can occur, alongside alterations in endocrine function, immune function, systemic inflammation, and oxidative stress [22
]. Peripheral fatigue can be evaluated using biomarkers related to energy metabolites, such as lactate, ammonia, and BUN, with these indexes reflecting ATP-generation efficiency and the associated metabolism during exercise. Oxidative stress caused by exercise results in reactive oxygen species (ROS) production with inadequate electron transfer through the mitochondrial respiratory chain, which is related to increased oxygen consumption for the energy demands of muscular contraction [24
]. ROS production is caused by the oxidation of proteins and lipids and is accompanied by a marked decrease in antioxidant capacity and an increased risk of tissue injury [25
]. Therefore, antioxidant and injury indexes, including glutathione, glutathione peroxidase, catalase, total antioxidant capacity, LDH, CK, AST, and ALT, were applied in previous studies on ergogenic acids [26
]. In a previous study [17
], OLP-01 was found to improve peripheral fatigue-associated indexes (NH3
, BUN, and CK) after acute exercise challenges without training intervention; these indexes were also improved by OLP-01-only treatment in the present study, with OLP-01 exerting similar physiological bioactivities after at least five weeks of supplementation. This study demonstrated that OLP-01 combined with training (exercise + OLP-01 group) significantly improved these indexes induced by exercise challenge and training.
Exercise training modulated physiological adaption and improved exercise performance. In a previous study, four weeks of aerobic swimming training significantly increased endurance capacity but not the lactate and BUN indexes [28
], and training intensity (six weeks of training with load that was 2% of body weight) was a critical factor for lactate and ammonia metabolism adaption in another study [29
]. In this study, exercise training (exercise group) with the treadmill protocol with gradually increasing intensity not only elevated endurance performance but also increased the lactate and BUN metabolic indexes, thereby providing physiological benefits (Table 2
and Figure 5
B). Pyruvate oxidation was enhanced by the exercise training-induced increase in pyruvate dehydrogenase activity, thereby reducing lactate production and accumulation during exercise [30
]. In addition, exercise-induced ammonia elevation was further metabolized as BUN through the urea cycle and removed by the kidneys as urine, which are considered to be fatigue biomarkers of the energy and metabolism balance. Exercise training significantly reduced creatinine and BUN levels, providing benefits regarding renal activities [31
]; citrulline is also considered an ergogenic aid from the perspective of ammonia metabolism [32
]. In this study, exercise training improved the modulation of BUN and ammonia metabolism, and the exercise + OLP-01 group showed significantly higher physiological adaption than the exercise group (Figure 5
Oxidative stress generated from excessive free radical production leads to inflammation and tissue injury during intensive exercise. Exercise training elevates antioxidant capacity and ameliorates oxidative stress to maintain a redox balance [33
]. The optimized exercise training intensity induces the production of reactive oxygen and nitrogen species, thereby improving physiological adaption and exerting nonlinear/hormetic effects. Excessive exogenous antioxidant supplementation interferes with the ROS/RNS signaling pathway for favorable muscle adaptation [34
]. Exercise training, regardless of the intensity, volume, and type of exercise and the target population, was reported to maintain the redox state balance and to exert health-related benefits [36
]. Regarding the antioxidant supplementation strategy, natural antioxidant nutrients/ingredients from a balanced and diverse diet may synergistically optimize antioxidant effects in conditions with high oxidative stress, such as intensive training [37
]. Moreover, probiotics exhibit antioxidant capacity through multiple possible mechanisms, including chelation, the production of antioxidant metabolites, the regulation of host antioxidant signaling pathways, and the modulation of host microbiota [38
]. Therefore, OLP-01 probiotics, in combination with exercise, could be considered as a nutritional supplementation strategy to improve physiological adaption-associated oxidative stress and inflammation.
Athletes typically participating in highly intensive training and competition, such as those participating in marathons and triathlons, may exhibit immunosuppression and inflammation, eventually leading to illness, including upper respiration infection and GI problems [39
]. Moreover, strenuous and prolonged exercise may cause splanchnic ischemia and dysregulation of the intestinal tight junction barrier, thereby increasing permeability, which may lead to local and systemic inflammation [40
]. In human and animal studies, PLR and NLR were elucidated as appropriate and potential indexes for systemic inflammation [41
]. In this study, exercise training significantly elevated PLR as a systemic inflammation index, and OLP-01 supplementation ameliorated the effects of exercise training to maintain homeostasis (Table 4
). Supplementation with multiple probiotic strains could therefore provide benefits on intestinal permeability, the immune system, intestinal microbiota, and inflammation and could also mitigate respiratory tract infections and GI symptoms for ensuring the overall health of athletes [43
Glycogen is an important energy source for meeting the energy demands and stability of physical activities. The intensity and duration of exercise training significantly affects the glycogen content, and carbohydrate availability may be a critical strategy for glycogenesis, with effects on higher performance and adaption [44
]. Studies also showed that exercise training upregulated metabolic enzymes and transport for higher glycogen bioavailability and replenishment rate [45
]. Butyrate, an SCFA, maintains blood glucose homeostasis and promotes glycogen metabolism through the GPR43-AKT-GSK3 signaling pathway [46
]. In this study, the exercise + OLP-01 group exhibited significantly elevated SCFAs, particularly acetate, propionate, and butyrate, compared with the sedentary and OLP-01 groups (Table 5
). A related study also demonstrated that probiotics (L. acidophilus
) regulated glycogen synthesis-related genes (GSK-3β and Akt) and glycogen content in tissues [47
]. In addition, the effects of bacteria-derived molecules and metabolites on the host immune system were shown to include regulation of gene expression for indicated functional activities [48
]. Therefore, OLP-01 supplementation also possibly regulated glucose metabolism, oxidative stress, and inflammation through other mechanisms, an area which needs further validation. Therefore, SCFAs may not be the only regulators that modulate glycogen synthesis in regard to OLP-01 supplementation.
A previous study elucidated that feces acetate content levels were associated with plasma acetate levels and that acetate restored the endurance capacity of antibiotic-treated animals, possibly by serving as an important energy substrate during exercise [49
]. In elite athletes, the indicated microbiota upregulated genes in a major pathway metabolizing lactate to propionate, which had a higher relative abundance post-exercise, as revealed by shotgun metagenomic analysis [51
]. The microbiome elucidates important roles in regard to endurance exercise by producing SCFAs [50
]. In addition, SCFAs also exert protective effects on the intestinal barrier function by inhibiting NLRP3 inflammasomes and autophagy to ensure intestinal permeability and health [52
]. Therefore, OLP-01 supplementation improved lactate metabolism after acute exercise without increasing SCFAs production. These data regarding the OLP-01 group indicated that there were other mechanisms at play from the exercise and OLP-01 group.
Therefore, OLP-01 supplementation possibly exerts functional effects through the modulation of beneficial physiological adaptions and the production of related metabolites, particularly when combined with exercise training. Exercise combined with OLP-01 probiotics demonstrated functional benefits on physical activities through modulation of inflammation and physiological adaption. The microbiota represented possible regulators of functional activities, but this area should be further investigated. Athletes often experience intestinal discomfort because of high-intensity exercise, psychological stress, and off-site training. Thus, appropriate probiotics could be considered to be an alternative nutritional strategy for athletes to improve both their physiological adaption and their exercise performance.