Abstract
biotics are posited to enhance exercise performance by influencing muscle protein synthesis, augmenting glycogen storage, and reducing inflammation. This double-blind study randomized 88 participants to receive a six-week intervention with either a placebo,Lactococcus lactis subsp.lactisLY-66,Lactobacillus plantarumPL-02, or a combination of both strains, combined with a structured exercise training program. We assessed changes in maximal oxygen consumption (VO 2max), exercise performance, and gut microbiota composition before and after the intervention. Further analyses were conducted to evaluate the impact of probiotics on exercise-induced muscle damage (EIMD), muscle integrity, and inflammatory markers
with either a placebo,Lactococcus lactis subsp.lactisLY-66,Lactobacillus plantarumPL-02, or a combination of both strains, combined with a structured exercise training program. We assessed changes in maximal oxygen consumption (VO 2max), exercise performance, and gut microbiota composition before and after the intervention. Further analyses were conducted to evaluate the impact of probiotics on exercise-induced muscle damage (EIMD), muscle integrity, and inflammatory markers in the blood, 24 and 48 h post-intervention. The results demonstrated that all probiotic groups exhibited significant enhancements in exercise performance and attenuation of muscle strength decline post-exercise exhaustion (p< 0.05). Notably, PL-02 intake significantly increased muscle mass, whereas LY-66 and the combination therapy significantly reduced body fat percentage (p< 0.05). Analysis of intestinal microbiota revealed an increase in beneficial bacteria, especially a significant rise inAkkermansia muciniphilafollowing supplementation with PL-02 and LY-66 (p< 0.05). Overall, the combination of exercise training and supplementation with PL-02, LY-66, and their combination improved muscle strength, explosiveness, and endurance performance, and had beneficial effects on body composition and gastrointestinal health, as evidenced by data obtained from non-athlete participants. Keywords:probiotics;Lactobacillus plantarumPL-02;Lactococcus lactissubsp.lactisLY-66; explosive strength; muscle health; exercise 1. Introduction Regular exercise has been extensively studied and scientifically validated for its nu- merous health benefits across a range of physical conditions, including cardiovascular disease, lung disease, metabolic syndrome, cancer, and sexual dysfunction [1]. Among the array of benefits conferred by exercise, it is particularly noteworthy that it can enhance maximal oxygen uptake, thereby fortifying cardiovascular robustness. Maximal oxygen uptake (VO2max), the maximum rate of oxygen consumption during intense and maximal exercise, serves as a crucial measure of cardiovascular health and aerobic endurance [2,3]. The ability to transform nutrients into adenosine triphosphate (ATP) via aerobic pathways Nutrients2024,16, 1921.
Nutrients2024,16, 1921 2 of 22 is directly linked to how effectively oxygen is utilized by the muscles, thereby enhancing athletic performance and supporting cardiovascular health, potentially reducing the risk of developing atherosclerosis [4]. Exercise plays a pivotal role in positively influencing the musculoskeletal system by enhancing muscular strength, endurance, and flexibility. These benefits not only help mitigate muscle loss but also serve as preventive measures against falls in the elderly [5]. Furthermore, regular physical activity significantly contributes to enhanced social well-being, improved quality of life [6], and increased longevity [7]. It is noteworthy that exercise can also be associated with elevated self-esteem and a reduced risk of anxiety in younger individuals [8,9]. Despite these advantages, many individuals experience post-exercise fatigue, which can hinder the maintenance of a regular exercise routine. This form of fatigue typically arises from exercise-induced muscle damage (EIMD). During the initial phases of muscle damage, mechanical trauma triggers an acute inflam- matory cascade, resulting in the release of pro-inflammatory cytokines such as interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and IL-1β, alongside heightened levels of creatine kinase (CK) and myoglobin [10,11]. Beyond the acute inflammatory response, pro- longed endurance exercise precipitates oxidative and metabolic stress, further exacerbating inflammation [12,13]. These physiological alterations result in a temporary decrease in muscle strength and performance. To modulate the dysregulated inflammatory response while continually optimizing athletic performance and preserving health, athletes have increasingly turned to dietary supplements in recent years. Among these, the utiliza- tion of probiotics for health enhancement and performance augmentation has attracted growing interest. Scientists have previously proposed the concept of the “gut–muscle axis”, suggesting a connection between the gut microbiota and muscle cells. It is believed that the gut microbiota, composed of four main phyla including Firmicutes, Bacteroidetes,Proteobacteria, andActinobacteria, may play a role in producing beneficial nutrients such as short-chain fatty acids (SCFAs) that can impact muscle cells [14,15]. The human gut microbiota consists of an incredibly vast number of microorganisms, with over 10 14 cells, which is approximately ten times the number of bacterial cells compared to the total count of human cells. Furthermore, the gut
Bacteroidetes,Proteobacteria, andActinobacteria, may play a role in producing beneficial nutrients such as short-chain fatty acids (SCFAs) that can impact muscle cells [14,15]. The human gut microbiota consists of an incredibly vast number of microorganisms, with over 10 14 cells, which is approximately ten times the number of bacterial cells compared to the total count of human cells. Furthermore, the gut microbiota has a genome size that is 150 times larger than the human genome [16]. This highlights the significant presence and potential impact of the gut microbiota on the human body’s normal homeostasis. However, the specific mechanisms by which the gut microbiota regulates the metabolism of muscle cells are still not fully understood. Further research is needed to uncover the intricate workings of the gut–muscle axis and the specific interactions between the gut microbiota and muscle cells. A human study reported that the probiotic strainBacillus coagulansdemonstrated potential in reducing exercise-induced muscle damage [17]. Another study focused on a probiotic strain isolated from humans,Bifidobacterium longumsubsp.longumOLP-01, which was found to enhance endurance running distance in middle- and long-distance runners [18]. Furthermore, this probiotic strain was associated with increased muscle grip strength and a reduction in fatigue induced by exhaustive exercise [19]. Additionally, our previous animal study investigated two probiotic strains,Lactococcus lactissubsp.lactis LY-66 andLactobacillus plantarumPL-02, which were isolated from an Olympic elite athlete. These strains were found to significantly elevate grip strength and endurance in tested mice. The mechanisms underlying these effects were proposed to involve improved fatty acid metabolism and increased glycogen storage levels in the liver and muscles, leading to enhanced exercise performance in the mouse model [20]. However, whether these two probiotic strains can similarly promote exercise performance in humans remains uncertain. While these studies show promising findings regarding the potential benefits of specific probiotic strains on exercise-related outcomes, it is important to note that further research is needed to validate and understand the precise mechanisms involved. Additionally, individual variations, the specific strains and dosages of probiotics, and other factors should be considered.
potential benefits of specific probiotic strains on exercise-related outcomes, it is important to note that further research is needed to validate and understand the precise mechanisms involved. Additionally, individual variations, the specific strains and dosages of probiotics, and other factors should be considered.
Nutrients2024,16, 1921 3 of 22 The aim of this study was to investigate the effects of PL-02, LY-66, and their combined supplementation on exercise performance, resilience to post-exercise muscle strength loss, and gastrointestinal health. Initially, we measured participants’ body composition, blood biochemical markers, exercise performance, and maximal oxygen consumption (VO2max) both prior to and following the probiotic intervention. For the assessment of muscle strength loss following exercise-induced muscle damage (EIMD), we conducted countermovement jump (CMJ) and isometric mid-thigh pull (IMTP) tests. Additionally, to measure the extent of muscle damage and inflammation, we analyzed specific biomarkers in the blood. To examine changes in the gut microbiota, we employed next-generation sequencing (NGS) technology pre- and post-intervention, providing a comprehensive analysis of the microbiota’s response to probiotic supplementation. 2. Materials and Methods 2.1. Experimental Test Samples The probiotic strains ofLactococcus lactissubsp.lactis(LY-66) (BCRC 911055 = CGMCC 21838) andLactobacillus plantarum(PL-02) (BCRC 911012 = CGMCC 20485) were isolated from the gut of a weightlifting Olympic gold medalist and obtained from Glac Biotech Co., Ltd (Tainan, Taiwan). [20]. The genetic sequences of these probiotic strains have been determined through sequencing techniques. Subsequently, the strains were encapsulated to produce a uniformly standardized dry beverage in appearance. 2.2. Participants The Harvard calculator (http://hedwig.mgh.harvard.edu/sample_size/size.html, accessed on 12 February 2022) was used to determine the sample size, assuming a parallel design with a significance level of 0.05, a power of 0.9, and a minimal detectable difference (following our pilot study) of 0.36 for the difference. A total of 84 patients entered this study. To account for the possibility of subject dropout, we added one person to each group to ensure minimum sample size requirements. A total of 88 healthy non-athlete adults aged 20–40 were included in this study, among which 44 participants were males and 44 were females. The exclusion criteria were as follows: smokers, cardiovascular disease, high blood pressure, BMI > 27, metabolic disease, asthma or within 6 months, those who are unable to engage in sports due to physical or neuromuscular injuries, those who have taken anti- inflammatory and analgesic drugs or probiotic-related products in the past month, students
participants were males and 44 were females. The exclusion criteria were as follows: smokers, cardiovascular disease, high blood pressure, BMI > 27, metabolic disease, asthma or within 6 months, those who are unable to engage in sports due to physical or neuromuscular injuries, those who have taken anti- inflammatory and analgesic drugs or probiotic-related products in the past month, students or related stakeholders of the program investigator. The Institutional Review Board of Landseed International Hospital (Taoyuan, Taiwan; LSHIRB No. 21-042-A2) reviewed and approved the conduct of this clinical study. The trial was as registered at as NCT06092723 on 23 October 2023. Furthermore, this study was executed by following the principles of the Declaration of Helsinki. 2.3. Experimental Design and Content A total of 88 healthy recruited participants were randomly and double-blindly divided into 4 groups of 22 people in each group (half male and half female), including (A) placebo group (2 sachets/day in 250 mL of water), (B) LY-66 probiotic group (7.5×10 9 CFU/sachet, 2 sachets/day in 250 mL of water), (C) PL-02 probiotic group (7.5×10 9 CFU/sachet, 2 sachets/day in 250 mL of water), (D) PL-02+LY-66 combined bacteria probiotic group 1:1 (7.5×10 9 CFU/sachet, 2 sachets/day in 250 mL of water). All participants included in the study were first numbered according to the order of registration, and then, randomly as- signed to four groups using a computer-generated random sampling method. The numbers were then redefined for identification purposes. Additionally, the sample manufacturer randomly assigned the codes A, B, C, and D to the four samples: placebo, PL-02, LY-66, and PL-02+LY-66. These coded samples were then distributed to the researchers who assigned them to the corresponding groups of participants. After the test and data analysis were completed, the sample manufacturer performed unblinding to reveal which samples corresponded to each group code. The probiotics were stored in a 4 ◦ C environment and
Nutrients2024,16, 1921 4 of 22 administered in a daily dosage of two sachets per intake, dissolved in 250 milliliters of water to prepare the beverage. This formulation, presented as a probiotic drink, served not only as a means of rehydration post-exercise but also facilitated the intake of probiotics. The intervention period lasted 6 weeks. The placebo contained anhydrous glucose, sodium citrate, lemon flavor, citric acid, sodium chloride, potassium citrate, sodium ascorbate, acesulfame potassium, and vitamin B complex. Probiotic-related products, anti-oxidant, anti-inflammatory drugs, or nutritional supplements were not allowed during the test. Subjects were required to photograph their meals on the day before the exercise test. Each photograph included a scale to facilitate accurate determination of portion sizes and nutri- tional content by the nutritionist. Additionally, subjects were asked to avoid extra exercise training from 48 h before and after the exercise test (Figure). The muscle-training regimen consisted of Tabata workouts conducted twice a week. Each session included the following exercises: (1) squats, (2) push-ups, (3) lunges, (4) high knees, (5) spiderman planks, (6) leg raises, (7) plank jacks, and (8) crunches. Each exercise was performed for 20 s followed by a 10 s rest. The complete Tabata workout comprised one set of these eight exercises. All subjects were required to train Tabata twice a week for a total of six weeks, a total of 12 times. The participation rate of each group was 100%, and no one was absent.Nutrients 2024, 16, x FOR PEER REVIEW 4 of 22 and data analysis were completed, the sample manufacturer performed unblinding to re- veal which samples corresponded to each group code. The probiotics were stored in a 4 °C environment and administered in a daily dosage of two sachets per intake, dissolved in 250 milliliters of water to prepare the beverage. This formulation, presented as a probi- otic drink, served not only as a means of rehydration post-exercise but also facilitated the intake of probiotics. The intervention period lasted 6 weeks. The placebo contained anhy- drous glucose, sodium citrate, lemon flavor, citric acid, sodium chloride, potassium cit- rate, sodium ascorbate, acesulfame potassium, and
250 milliliters of water to prepare the beverage. This formulation, presented as a probi- otic drink, served not only as a means of rehydration post-exercise but also facilitated the intake of probiotics. The intervention period lasted 6 weeks. The placebo contained anhy- drous glucose, sodium citrate, lemon flavor, citric acid, sodium chloride, potassium cit- rate, sodium ascorbate, acesulfame potassium, and vitamin B complex. Probiotic-related products, anti-oxidant, anti-inflammatory drugs, or nutritional supplements were not al- lowed during the test. Subjects were required to photograph their meals on the day before the exercise test. Each photograph included a scale to facilitate accurate determination of portion sizes and nutritional content by the nutritionist. Additionally, subjects were asked to avoid extra exercise training from 48 h before and after the exercise test (Figure 1). The muscle-training regimen consisted of Tabata workouts conducted twice a week. Each ses- sion included the following exercises: (1) squats, (2) push-ups, (3) lunges, (4) high knees, (5) spiderman planks, (6) leg raises, (7) plank jacks, and (8) crunches. Each exercise was performed for 20 s followed by a 10 s rest. The complete Tabata workout comprised one set of these eight exercises. All subjects were required to train Tabata twice a week for a total of six weeks, a total of 12 times. The participation rate of each group was 100%, and no one was absent. The relevant details of the experiment are as follows: the blood biochemistry, exercise tests, feces collection, maximum oxygen uptake, and body composition were tested before and after the intervention. Blood samples were collected 24 and 48 h after the exercise- induced muscle exhaustion for analysis of the inflammation damage indicator. Body com- position was measured every two weeks. During the exercise test process, sport protectors were on hand to assist and guide subjects for avoiding sports injuries. Figure 1. Experimental procedure description. 2.4. Body Composition Analysis Body composition was assessed utilizing the InBody 570 device (InBody, Seoul, South Korea), assessing total body weight, body fat percentage, muscle mass distribution, BMI, and basal metabolic rate. All assessments were conducted after participants had fasted for
sport protectors were on hand to assist and guide subjects for avoiding sports injuries. Figure 1. Experimental procedure description. 2.4. Body Composition Analysis Body composition was assessed utilizing the InBody 570 device (InBody, Seoul, South Korea), assessing total body weight, body fat percentage, muscle mass distribution, BMI, and basal metabolic rate. All assessments were conducted after participants had fasted for eight hours, following the methodology of a prior study [18]. Figure 1.Experimental procedure description. The relevant details of the experiment are as follows: the blood biochemistry, exercise tests, feces collection, maximum oxygen uptake, and body composition were tested before and after the intervention. Blood samples were collected 24 and 48 h after the exercise- induced muscle exhaustion for analysis of the inflammation damage indicator. Body composition was measured every two weeks. During the exercise test process, sport protectors were on hand to assist and guide subjects for avoiding sports injuries. 2.4. Body Composition Analysis Body composition was assessed utilizing the InBody 570 device (InBody, Seoul, Repub- lic of Korea), assessing total body weight, body fat percentage, muscle mass distribution, BMI, and basal metabolic rate. All assessments were conducted after participants had fasted for eight hours, following the methodology of a prior study [18].
Nutrients2024,16, 1921 5 of 22 2.5. Exercise Program to Induce Muscle Fatigue and Soreness An exercise regimen involving 100 repetitive jumps was designed to induce muscle fatigue and soreness. Participants performed sets of 10 consecutive jumps, each to be completed within 4 s, followed by a 90 s rest period before the next set. This cycle was repeated until 100 jumps were completed. During each jump, the participant’s knees were required to be bent to a 90-degree angle to standardize the movement across all participants. 2.6. Countermovement Jump Assessment (CMJ) Participants performed the CMJ on a Kistler force-measuring platform (9260AA, Kistler GmbH, Winterthur, Switzerland). They began by standing with hands on hips, performed a squat to a 90-degree knee bend, and then, jumped with maximum effort. Each participant completed three trials, with the platform calibrated to their individual weight to ensure accuracy. Measured parameters comprised the rate of force development (RFD), relative peak force, and jump height, offering detailed insights into each participant’s lower body power and force generation capabilities during the jumps. 2.7. Isometric Mid-Thigh Pull (IMTP) The IMTP was conducted to assess maximal force generation using a custom-built IMTP rack (Kairos Strength, Murphy, NC, USA) and a force plate (Type 9260AA, Kistler, Winterthur, Switzerland), as per the protocol outlined in a prior study [21]. Participants were required to exert their maximum strength on the bar for 3–5 s, with tests repeated at 2 min intervals. The primary metrics recorded were peak force (the maximum strength exerted) and relative peak force (the maximum strength relative to body weight). 2.8. Wingate Anaerobic Test (WAnT) The Wingate Anaerobic Test was conducted using the 894E anaerobic power bicycle (Monark Exercise AB, Dalarnas Lan, Sweden). Participants initially ramped up their cycling speed to 120 rpm, at which point a resistance equivalent to 7.5% of the participant’s body weight was automatically applied to the wheel. This test required participants to sprint at full strength for 30 s. Metrics assessed included relative mean power (W/kg), relative peak power (W/kg), and the fatigue index, calculated as the percentage drop from peak power to the lowest power
120 rpm, at which point a resistance equivalent to 7.5% of the participant’s body weight was automatically applied to the wheel. This test required participants to sprint at full strength for 30 s. Metrics assessed included relative mean power (W/kg), relative peak power (W/kg), and the fatigue index, calculated as the percentage drop from peak power to the lowest power level sustained [22]. 2.9. Measuring VO2max VO2maxwas assessed using the Bruce maximal treadmill protocol, established in 1973 [23]. Participants wore a face mask connected to a gas analysis system and had electrocardiogram electrodes placed in appropriate locations. The test started at a velocity of 7.2 km/hr, progressively increasing by 1.8 km/hr every two minutes until exhaustion. Data collection commenced once the participant’s heart rate reached 170 beats per minute and continued until exhaustion, defined by meeting at least two of three criteria: maximal heart rate (220 minus age), a respiratory exchange ratio over 1.1, or a rating of perceived exertion (RPE) above 18. 2.10. Physiological Observation and Serum Biochemical Analysis Venous blood samples were collected from participants following a minimum fasting period of 8 h, both before and after the intervention. Biochemical markers including AST, ALT, BUN, creatinine, uric acid, total protein, cholesterol profiles, and glucose levels were quantified utilizing a Hitachi 717 analyzer (Hitachi, Tokyo, Japan). Creatine kinase (CK) levels and IL-6 concentrations were determined using a Beckman Coulter AU5800 autoanalyzer (Beckman Coulter Inc., Brea, CA, USA) and an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA), respectively.
Description
This study investigates the effects of probiotics on exercise performance and muscle health.