Bifidobacterium longum subsp. longum OLP-01 Supplementation during Endurance Running Training Improves Exercise Performance in Middle- and Long-Distance Runners: A Double-Blind Controlled Trial

Bi dobacterium longum subsp. longumOlympic No. 1 (OLP-01) has been shown in previous animal experiments to improve exercise endurance performance, but this e ect has not been con rmed in humans, or more particularly, in athletes. Toward this end, the current study combined OLP-01 supplementation with regular exercise training in well-trained middle- and long-distance runners at the National Taiwan Sport University. The study was designed as a double-blind placebo-controlled experiment. Twenty-one subjects (14 males and seven females aged 20–30 years) were evenly distributed according to total distance (meters) traveled in 12 min to one of the following two groups: a placebo group (seven males and three females) and an OLP-01 (1.5 10 10 colony forming units (CFU)/day) group (seven males and four females). All the participants received placebo or OLP-01 supplements for ve consecutive weeks consisting of three weeks of regular training and two weeks

total distance (meters) traveled in 12 min to one of the following two groups: a placebo group (seven males and three females) and an OLP-01 (1.5 10 10 colony forming units (CFU)/day) group (seven males and four females). All the participants received placebo or OLP-01 supplements for ve consecutive weeks consisting of three weeks of regular training and two weeks of de-training. Before and after the experiment, the participants were tested for 12-min running/walking distance, and body composition, blood/serum, and fecal samples were analyzed. The results showed that OLP-01 signi cantly increased the change in the 12-min Cooper's test running distance and the abundance of gut microbiota. Although no signi cant change in body composition was found, OLP-01 caused no adverse reactions or harm to the participants' bodies. In summary, OLP-01 can be used as a sports nutrition supplement, especially for athletes, to improve exercise performance. Keywords:OLP-01;Bi dobacterium longum; probiotic; exercise performance; athletes Nutrients2020,12, 1972; doi:10.3390 /nu12071972 /journal/nutrients

Nutrients2020,12, 1972 2 of 14 1. Introduction Middle- and long-distance running is de ned as competitions of 800 m to 10,000 m and require both speed and endurance. From a performance optimization perspective, these events are very complex. For example, elite middle- and long-distance runners need to develop aerobic exercise systems similar to those of marathon runners, as well as some mechanical properties of elite sprinters, while possessing world-class anaerobic ability and superb tactical instincts to maintain the fastest possible speed throughout the race [1,2]. In addition to genetics, other factors, such as gender, age, physiological and psychological factors [3,4], training [5], metabolic variables [6], energy utilization [7], and nutritional supplements [8], have important impacts on exercise performance. Endurance can be de ned as the ability to maintain one's speed or power output for as long as possible [9]. However, endurance performance is related to fatigue, which is a complex physiological phenomenon. It can be divided into central fatigue and peripheral fatigue. The physical and psychological e ects during exercise depend on the type, intensity, duration, and energy expenditure of the exercise. They are used to describe the decline in physical function and the actual/perceived di culties associated with tasks or increased exercise [10]. To cope with long races and high-tension speeds, middle- and long- distance runners need more complex energy metabolism, which is characterized by aerobic metabolism, glycolysis, and adenosine triphosphate-creatine phosphate (ATP-cp), a combined energy metabolism of three energy supply systems [11]. As the distance increases, the type of metabolism gradually changes from a mixed metabolic process based on anaerobic metabolism to a mixed metabolic process based on aerobic metabolism [12]. These energy systems interact with tissues, including muscle types, bers, and mitochondria, to provide the large amounts of energy required for strenuous exercise. Furthermore, recently-described complex interrelationships between gut microbiota and systemic energy metabolism exert positive performance e ects in elite athletes [13]. Exercise training can cause changes in gut microbiota and increase their diversity and abundance [14]. Athletes have a higher short-chain fatty acid (SCFA) metabolism pathway [15], which involves digesting complex carbohydrates and fermentation in

of energy required for strenuous exercise. Furthermore, recently-described complex interrelationships between gut microbiota and systemic energy metabolism exert positive performance e ects in elite athletes [13]. Exercise training can cause changes in gut microbiota and increase their diversity and abundance [14]. Athletes have a higher short-chain fatty acid (SCFA) metabolism pathway [15], which involves digesting complex carbohydrates and fermentation in the colon. In addition, propionate and acetate are transported through the blood to various organs as a substrate for energy metabolism, particularly to hepatic cells for gluconeogenesis with propionate [16]. Butyric acid is transported to mitochondria and recombined under aerobic conditions to become acetyl-CoA, which enters the Krebs cycle, forms NADH, and then enters the electron transport chain, thereby generating ATP production and CO2[17]. Moreover, gut microbiota can also promote the metabolism and synthesis of secondary bile acid to directly modify mitochondrial biogenesis, in ammation, and intestinal barrier function, and in combination with SCFA can improve energy e ciency and anti-fatigue, thereby enhancing exercise performance [18,19]. In addition to exercise training, the proportion and time of dietary intake had a great in uence on athletes 0 energy expenditure and exercise performance [20]. Diet is also one of the important factors a ecting the gut microbiota and proportion [21]. Dietary or probiotic supplements are the most direct and e ective ways to increase the richness and diversity of gut microbiota. Probiotics are considered to be bene cial microorganisms for the host, since they bene t intestinal and physical health [22]. Di erent strains have di erent e ects, but exercise-related research on them is still in its infancy. Currently, most research on probiotics is focused on improving respiratory function [22] and reducing intestinal discomfort in athletes [23]. In the area of exercise performance, most studies have been based on animal models [24,25], and few studies have con rmed the bene cial e ects on the human body [26,27]. One six-week study found that supplementation withLactobacillus plantarumTWK10 can lead to signi cant improvements in endurance exercise performance and body composition in both male and female humans, and it also reduced fatigue indicators

exercise performance, most studies have been based on animal models [24,25], and few studies have con rmed the bene cial e ects on the human body [26,27]. One six-week study found that supplementation withLactobacillus plantarumTWK10 can lead to signi cant improvements in endurance exercise performance and body composition in both male and female humans, and it also reduced fatigue indicators [27]. Therefore, more experiments are needed to con rm the e cacy and mechanism of probiotics to improve sports performance. B. longumOLP-01 was isolated from a weightlifting gold medalist. In previous studies, it was found to signi cantly increase muscle strength and endurance exercise performance and to reduce the

Nutrients2020,12, 1972 3 of 14 fatigue index of untrained or trained mice [28,29]. Therefore, in the current study, we combined OLP-01 with three weeks of regular training and two weeks of de-training for middle- and long-distance runners at the National Taiwan Sports University to explore of the e ects of OLP-01 supplementation on endurance exercise performance and physiological adaptation. 2. Materials and Methods 2.1. Probiotic OLP-01, a human strain probiotic derived theBi dobacterium longum subsp. Longum, was isolated from an Olympic gold medalist in the women 0 s 48 kg weightlifting event. The OLP-01 in the current study was identi ed by an independent third party, the Food Industry Research and Development Institute (Hsinchu, Taiwan), and prepared and provided by Glac Biotech Co., Ltd. (Tainan, Taiwan) in the form of capsules containing the speci ed dose. Each OLP-01 capsule contained 5 10 9 colony forming units (CFU). The placebo capsules were indistinguishable in appearance from the OLP-01 capsules. The dosage was three capsules per day, one after each meal. 2.2. Participants Participants included healthy people aged 20 to 30 and well-trained middle- and long-distance runners, but excluded people with high blood pressure, asthma, or skeletal neuromuscular injuries in the upper or lower extremities. Participants were instructed to cooperate with the corresponding training courses and not to consume nutritional supplements, yogurt, Yakult, other probiotic-related products, or antibiotics during the experiment, and they abstained from alcohol consumption for 1 week before the exercise test. The study was approved and reviewed by the Landseed International Hospital Institutional Review Board (Taoyuan, Taiwan; LSHIRB No. 19-005-A2). After the experiment process and content were explained in detail, all volunteers provided written informed consent before participating. 2.3. Experimental Design Twenty-one (14 males and seven females aged 20–30 years) well-trained middle- and long-distance runners at the National Taiwan Sports University were recruited. We used a double-blind test in which the participants were evenly distributed according to total distance (meters) traveled in 12 min to one of two groups: a placebo group (seven males and three females) and an OLP-01 (1.5 10 10 CFU/day) group (seven males and four

years) well-trained middle- and long-distance runners at the National Taiwan Sports University were recruited. We used a double-blind test in which the participants were evenly distributed according to total distance (meters) traveled in 12 min to one of two groups: a placebo group (seven males and three females) and an OLP-01 (1.5 10 10 CFU/day) group (seven males and four females). The experimental procedure is presented in Figure. All the participants received placebo or OLP-01 supplements for ve consecutive weeks, which included three weeks of regular training and two weeks of de-training. Before and after the experiment, the participants were tested for 12-min running/walking distance, and their body composition, blood/serum, and fecal samples were analyzed. During the experiment, all the subjects cooperated with the team for work and rest. The team dietitian speci ed the diet and provided the same meal to ensure the consistency of the diet. The basic demographics and characteristics of the subjects are listed in Table.

Nutrients2020,12, 1972 4 of 14Nutrients 2020, 12, x FOR PEER REVIEW 4 of 14 Figure 1. Experimental design. We used a double-blind test in which volunteers (21 subjects; 14 males and seven females) were assigned to two groups: a placebo group and an OLP-01 (1.5 × 1010 colony forming units (CFU)/day) group. They completed a five-week intervention consisting of three weeks of regular training and two weeks of de-training, and their physical fitness, physiological adaptations, and fecal samples were analyzed before and after the experimental intervention. Table 1. Basic information data of the subjects. Characteristic Placebo OLP-01 Age (y) 21.2 ± 0.4 21.6 ± 0.7 Height (cm) 168.7 ± 1.5 169.5 ± 2.3 Weight (kg) 57.1 ± 1.9 56.4 ± 1.1 BMI (kg/m 2 ) 20.0 ± 0.4 19.7 ± 0.3 Data are expressed as mean ± SEM. There were no significant differences in the basic information data between the two groups. 2.4. The 12-min Cooper Running/Walking Test The 12-min Cooper running/walking test was used as a preliminary and simple method to assess aerobic endurance and physical fitness [30]. A standard sports field of 400 m was marked every 10 m. The time was recorded from the start of running, and the distance traveled was recorded every 3 min (3rd, 6th, 9th, and 12th min). 2.5. Body Composition Body composition was measured according to the multi-frequency principle with the bioelectrical impedance analyzer (BIA) of the InBody 570 (In-body, Seoul, Korea), which provides frequency screenings of 1, 5, 50, 260, 500, and 1000 kHz within 60 s. After their palms and soles were cleaned, the subjects stood upright on the electrodes of the instrument, held the sensing handle in both hands with the arms away from the body at a 30° angle, and refrained from speaking or moving during the measurement period [26]. 2.6. Blood Routine and Serum Biochemical Analysis To understand the health status of the subjects and whether they were adversely affected by the OLP-01 supplemented training, we collected blood with an arm venous catheter for analysis at the beginning and end of the experiment. The physiological adaptations and

angle, and refrained from speaking or moving during the measurement period [26]. 2.6. Blood Routine and Serum Biochemical Analysis To understand the health status of the subjects and whether they were adversely affected by the OLP-01 supplemented training, we collected blood with an arm venous catheter for analysis at the beginning and end of the experiment. The physiological adaptations and clinical biochemistry of the blood serum were assessed with an autoanalyzer (Hitachi 7060, Tokyo, Japan) for lactate, ammonia, creatinine kinase (CK), glucose, aspartate transaminase (AST), alanine aminotransferase (ALT), albumin, total protein (TP), total cholesterol (TC), triglyceride (TG), high-density lipid (HDL), low- density lipid (LDL), blood urea nitrogen (BUN), creatinine, and uric acid (UA) levels. 2.7. Bacterial DNA Extraction and 16S rRNA Sequencing Figure 1. Experimental design. We used a double-blind test in which volunteers (21 subjects; 14 males and seven females) were assigned to two groups: a placebo group and an OLP-01 (1.5 1010 colony forming units (CFU)/day) group. They completed a ve-week intervention consisting of three weeks of regular training and two weeks of de-training, and their physical tness, physiological adaptations, and fecal samples were analyzed before and after the experimental intervention. Table 1.Basic information data of the subjects. Characteristic Placebo OLP-01 Age (y) 21.2 0.4 21.6 0.7 Height (cm) 168.7 1.5 169.5 2.3 Weight (kg) 57.1 1.9 56.4 1.1 BMI (kg/m 2 ) 20.0 0.4 19.7 0.3 Data are expressed as mean SEM. There were no signi cant di erences in the basic information data between the two groups. 2.4. The 12-min Cooper Running/Walking Test The 12-min Cooper running/walking test was used as a preliminary and simple method to assess aerobic endurance and physical tness [30]. A standard sports eld of 400 m was marked every 10 m. The time was recorded from the start of running, and the distance traveled was recorded every 3 min (3rd, 6th, 9th, and 12th min). 2.5. Body Composition Body composition was measured according to the multi-frequency principle with the bioelectrical impedance analyzer (BIA) of the InBody 570 (In-body, Seoul, Korea), which provides frequency screenings of 1, 5, 50, 260, 500, and

The time was recorded from the start of running, and the distance traveled was recorded every 3 min (3rd, 6th, 9th, and 12th min). 2.5. Body Composition Body composition was measured according to the multi-frequency principle with the bioelectrical impedance analyzer (BIA) of the InBody 570 (In-body, Seoul, Korea), which provides frequency screenings of 1, 5, 50, 260, 500, and 1000 kHz within 60 s. After their palms and soles were cleaned, the subjects stood upright on the electrodes of the instrument, held the sensing handle in both hands with the arms away from the body at a 30 angle, and refrained from speaking or moving during the measurement period [26]. 2.6. Blood Routine and Serum Biochemical Analysis To understand the health status of the subjects and whether they were adversely a ected by the OLP-01 supplemented training, we collected blood with an arm venous catheter for analysis at the beginning and end of the experiment. The physiological adaptations and clinical biochemistry of the blood serum were assessed with an autoanalyzer (Hitachi 7060, Tokyo, Japan) for lactate, ammonia, creatinine kinase (CK), glucose, aspartate transaminase (AST), alanine aminotransferase (ALT), albumin, total protein (TP), total cholesterol (TC), triglyceride (TG), high-density lipid (HDL), low-density lipid (LDL), blood urea nitrogen (BUN), creatinine, and uric acid (UA) levels.

Nutrients2020,12, 1972 5 of 14 2.7. Bacterial DNA Extraction and 16S rRNA Sequencing Before the start and the designated end point, fecal specimens were collected from all subjects, and the DNA/RNA Shield—reagent was used in a fecal collection tube (Zymo Research Corp, Irvine, CA, USA). Each collection tube (with a spoon attached to the lid) was pre- lled with DNA/RNA Shield— (9 mL). Bacterial DNA was extracted by the cetyltrimethylammonium bromide/sodium dodecyl sulfate (CTAB/SDS) method, and the obtained nucleic acids (DNA and RNA) were stored at 80 C for subsequent analysis. DNA purity was determined by the ratio of OD 260 to OD 280 in the range of 1.8–2.0. Speci c primers 341F (F, forward primer; 5 0 -CCTAYGGGRBGCASCAG-3 0 ) and 806R (R, reverse primer; 5 0 -GGACTACNNGGGTATCTAAT-3 0 ) were used to amplify the highly variable V3-V4 regions of the bacterial 16S rRNA gene by PCR Zone. A sample preparation kit (Illumina, San Diego, CA, USA) without TruSeq DNA PCR was used to construct a double-ended library (each sample insertion size was 450–470 bp). The ampli ed DNA sizing was checked by TapeStation (Agilent Technologies, Santa Clara, CA, USA). High-throughput sequencing was performed on the Illumina HiSeq2500 platform. The sequences thus generated were ltered to obtain valid reads. Total reads were merged, low-quality and chimera sequence sequences were removed, and the clustered operational taxonomic unit (OTU) had 97% similarity to the Greengenes database. All OTU sequences and diversity analysis were performed with the CLC Microbial Genomics Module (Qiagen, Hilden, Germany), basespace (Illumina, San Diego, CA, USA) and Graphpad prism 7 (Graphpad Software, San Diego, CA, USA). Ap-value of less than 0.05 was considered statistically signi cant. 2.8. Statistical Analysis All the data are expressed as mean SEM. Statistical analyses were performed in SAS 9.0 (SAS Inst., Cary, NC, USA). Multi-group comparisons were analyzed by one-way analysis of variance (ANOVA), and within-group di erences (before vs. after OLP-01 supplementation), by paired Student'st-test. Statistical signi cance was set atp<0.05. 3. Results 3.1. E ect of OLP-01 Supplementation on Distance in 12-min Cooper Running/Walking Test As shown in Figure group achieved

analyses were performed in SAS 9.0 (SAS Inst., Cary, NC, USA). Multi-group comparisons were analyzed by one-way analysis of variance (ANOVA), and within-group di erences (before vs. after OLP-01 supplementation), by paired Student'st-test. Statistical signi cance was set atp<0.05. 3. Results 3.1. E ect of OLP-01 Supplementation on Distance in 12-min Cooper Running/Walking Test As shown in Figure group achieved distances of 907 26 and 888 21 m, 1791 52 and 1768 39 m, 2629 74 and 2625 65 m , and 3475 101 and 3496 84 m at the 3rd, 6th, 9th, and 12th min, respectively. There were no signi cant di erences between them. After ve consecutive weeks of OLP-01 intervention combined with three weeks of regular training and two weeks of de-training, the placebo group and the OLP-01 supplementary group achieved distancesof 927 28 and 928 28 m, 1795 57 and1840 48 m , 2602 83 and 2740 74 m, and 3419 112 and 3602 86 m at the 3rd, 6th, 9th, and 12th min, respectively. There were no signi cant di erences between them. Despite the lack of signi cant di erences in running distance before or after supplementation between the two groups, we further analyzed the di erences between post-test and pre-test to compare improvements in running distance. As shown in FigureB, the placebo group and the OLP-01 supplementary group exhibited changes in running distance of 21 4 and 40 11 m, 4 9 and72 14 m , 27 21 and 116 17 m, and 56 29 and 105 16 m at the 3rd, 6th, 9th, and 12th min, respectively. Compared with the placebo group, the improvements in the OLP-01 group were signi cantly greater at the 6th min (p=0.0014), 9th min (p=0.0001) and 12th min (p=0.0001). In addition, relative to the pre-test, the placebo group only exhibited improvement in running distance in the post-test at the 3rd minute (p=0.0051), and there were no signi cant di erences at the other time points. In the OLP-01 group, running distances in the post-test were signi cantly greater at the 3rd (p=0.0051), 6th (p=0.0004), 9th (p<0.0001), and

The study investigates the effects of OLP-01 on endurance performance in athletes.