Lactobacillus composition for reducing serum urea nitrogen concentration and use thereof

Abstract

This invention relates to a lactobacillus composition that reduces serum urea nitrogen concentration after aerobic exercise. The lactobacillus composition comprises *Lactobacillus brevis* strain GKEX, *Lactobacillus plantarum* strain GKK1, and / or *Lactobacillus johnsonii* strain GKJ2. After being administered to healthy subjects for a period of time, it effectively improves fatigue-related biochemical values ​​and prolongs the time to exhaustion during aerobic exercise. Therefore, it can be used as an active ingredient in the preparation of various anti-fatigue and / or performance-enhancing compositions.

Description

Technical Field

[0001] The present invention relates to a probiotic composition and its use, and in particular to an anti-fatigue lactic acid bacteria composition and its use that effectively improves fatigue-related biochemical values ​​and prolongs the time to exhaustion during aerobic exercise. Prior Art

[0002] Modern life is busy and competitive, and these various pressures often leave us feeling tired or overworked, which can be detrimental to our health. Fatigue is caused by overwork and is accompanied by a decline in physiological function, leading to a decrease in physical strength and athletic ability.

[0003] Fatigue can be categorized as mental fatigue and physical fatigue. Physical fatigue refers to a condition in which the body's functions fail to maintain a certain standard, resulting in a decrease in physical strength and athletic ability. Persistent physical fatigue leaves the body in a state of constant exhaustion. Prolonged periods of excessive stress or overwork can prevent the body from adequately resting. When rest alone fails to alleviate fatigue, disrupting daily life and causing a reduction in activity by more than 50%, chronic fatigue syndrome (CFS) may develop. Symptoms may include muscle aches, generalized muscle weakness, widespread headaches, sleep disturbances, and psychiatric or neurological symptoms, which can affect work and daily life.

[0004] Probiotics are generally defined as bacteria beneficial to human health that can thrive in the human intestine without causing disease. Probiotics can regulate the body's constitution and alter certain metabolic pathways, ultimately improving overall gastrointestinal function and metabolic capacity. Lactobacillus currently makes up the vast majority of the most commonly used probiotic strains. Past research has shown that lactic acid bacteria, in addition to improving obesity and boosting immunity, exhibit significant antioxidant activity in certain strains, help maintain intestinal permeability, and inhibit the growth of gas-producing bacteria.

[0005] However, there is still relatively little research on the anti-fatigue function of lactic acid bacteria and their ability to enhance athletic performance. There is an urgent need to develop an anti-fatigue lactic acid bacteria composition and its use. Summary of the Invention

[0006] Therefore, one aspect of the present invention is to provide an anti-fatigue lactic acid bacteria composition comprising at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2.

[0007] Secondly, another aspect of the present invention provides a use of lactic acid bacteria as a preparation for an anti-fatigue lactic acid bacteria composition, comprising administering an effective dose of an oral lactic acid bacteria composition to healthy subjects for a continuous period of time, wherein the oral composition contains the above-mentioned lactic acid bacteria strain as an active ingredient, thereby improving fatigue-related biochemical values ​​and prolonging the time to exhaustion during aerobic exercise.

[0008] According to the above-mentioned aspects of the present invention, an anti-fatigue lactic acid bacteria composition is provided. In one embodiment, the anti-fatigue lactic acid bacteria composition comprises at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2. Lactobacillus brevis strain GKEX was deposited with the Bioresource Conservation and Research Center, Food Industry Development Research Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan) on January 21, 2022, with the deposit number BCRC 911099. Lactobacillus plantarum strain GKK1 was deposited with the BCRC on July 18, 2019, with the deposit number BCRC 910919. Lactobacillus johnsonii strain GKJ2 was deposited with BCRC on May 15, 2020, with the deposit number BCRC 910999.

[0009] In the above embodiment, the anti-fatigue lactic acid bacteria composition can be an orally administered composition, such as a pharmaceutical composition or a food composition. In the above example, the anti-fatigue lactic acid bacteria composition further includes a pharmaceutically and food-acceptable carrier, excipient, and / or additive.

[0010] In the above embodiment, the dosage form of the lactic acid bacteria composition may include, for example, powder, tablet, granule, microcapsule, ampoule or liquid spray.

[0011] In the above embodiments, the anti-fatigue lactic acid bacteria composition has anti-fatigue ability and / or enhances exercise capacity, including prolonging the time to exhaustion in swimming, prolonging the time to exhaustion in running, reducing the serum lactate increase ratio and serum urea nitrogen concentration after aerobic exercise, and / or increasing the liver glycogen content.

[0012] According to another aspect of the present invention, a lactobacillus is used as a preparation for an anti-fatigue lactic acid bacteria composition, comprising administering an oral composition containing an effective dose of lactobacillus to healthy subjects for at least four consecutive weeks. In this embodiment, the lactobacillus is used as the active ingredient, wherein the lactobacillus comprises at least one of Lactobacillus breve strain GKEX (BCRC 911099), Lactobacillus plantarum strain GKK1 (BCRC 910919), and Lactobacillus johnsonii strain GKJ2 (BCRC 910999).

[0013] In the above embodiment, the healthy subject may be, for example, an adult, and the effective dose of the lactobacillus for the subject may be, for example, 50 mg / 60 kg body weight / day to 1900 mg / 60 kg body weight / day.

[0014] In the above embodiment, the healthy subject may be, for example, a mouse, and the effective dose of the lactobacillus for the subject may be, for example, 10.5 mg / kg body weight / day to 400 mg / kg body weight / day.

[0015] The anti-fatigue lactic acid bacteria composition and its uses described above, comprising at least one of the Lactobacillus breve strain GKEX, the Lactobacillus plantarum strain GKK1, and the Lactobacillus johnsonii strain GKJ2, can be administered to healthy subjects for a continuous period of time to effectively improve fatigue-related biochemical values ​​and prolong the time to exhaustion during aerobic exercise. The composition can then be used as an active ingredient in the preparation of various anti-fatigue and / or exercise performance-enhancing compositions.

[0016] It should be understood that the foregoing general description and the following detailed description are merely illustrative and are intended to provide further explanation of the invention as claimed. Simple diagram description

[0017] To make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described in detail as follows: FIG1 is a bar graph showing the weighted swimming exhaustion time of mice in the blank control group and the experimental group according to one embodiment of the present invention. FIG2 is a bar graph showing the running exhaustion time of mice in the blank control group and the experimental group according to one embodiment of the present invention. FIG3 is a bar graph showing the ratio of blood lactate elevation in mice of the blank control group and the experimental group after exercise according to one embodiment of the present invention. FIG4 is a bar graph showing the blood urea nitrogen concentrations of mice in the blank control group and the experimental group after exercise according to one embodiment of the present invention. FIG5 is a bar graph showing the liver glycogen content (mg / g liver tissue) of mice in the blank control group and the experimental group according to one embodiment of the present invention. Implementation Method

[0018] If a definition or use of a term in a reference is inconsistent or contrary to the definition of that term herein, the definition of that term herein shall apply and not the definition of that term in the reference. Furthermore, unless the context otherwise requires, singular terms may include plural terms and plural terms may include the singular.

[0019] As described above, the present invention provides an anti-fatigue lactic acid bacteria composition and its use, which, when administered to healthy subjects for a continuous period of time, can effectively reduce serum lactate after aerobic exercise, reduce serum urinary nitrogen after aerobic exercise, increase liver glycogen content, increase muscle glycogen content and / or reduce visceral fat.

[0020] In one embodiment, the term "Lactobacillus" herein may include strains of the same genus but different species, such as at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2. In one specific example, Lactobacillus brevis strain GKEX was deposited with the Bioresource Conservation and Research Center, Food Industry Development Research Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan) on January 21, 2022, with the deposit number BCRC 911099. Lactobacillus plantarum strain GKK1 was deposited with the BCRC on July 18, 2019, with the deposit number BCRC 910919. Lactobacillus johnsonii strain GKJ2 was deposited with the BCRC on May 15, 2020, with the registration number BCRC 910999. It should be noted that the present invention requires the use of specific strains of Lactobacillus for the resulting Lactobacillus composition to achieve the effects of improving fatigue resistance and / or enhancing exercise performance in healthy subjects. If the Lactobacillus composition is not selected from these specific strains, or if the strains are added or subtracted, or if some or all of the strains are replaced with other strains of the same species, the effects of improving fatigue resistance and / or enhancing exercise performance in healthy subjects cannot be expected.

[0021] The aforementioned lactic acid bacteria composition has anti-fatigue properties and / or enhances exercise performance. Generally speaking, assessment criteria for "anti-fatigue properties" herein may include, but are not limited to, reducing the serum lactate rise ratio and serum urea nitrogen concentration after aerobic exercise and / or increasing liver glycogen content, while assessment criteria for "enhancing exercise performance" may include, but are not limited to, prolonging the time to exhaustion during aerobic exercise. In one embodiment, administration of an oral composition containing an effective dose of lactobacilli to healthy subjects for at least four consecutive weeks effectively improves fatigue-related biochemical parameters (e.g., reducing the serum lactate rise ratio and serum urea nitrogen concentration after aerobic exercise and / or increasing liver glycogen content) and significantly prolongs the time to exhaustion during aerobic exercise (e.g., prolonging the time to exhaustion during swimming or running).

[0022] In one embodiment, the content of each of the aforementioned Lactobacillus strains is not particularly limited. In one example, the weight (mg) ratio or colony count (CFU) ratio of the Lactobacillus brevis strain GKEX (BCRC 911099), the Lactobacillus plantarum strain GKK1 (BCRC 910919), and the Lactobacillus johnsonii strain GKJ2 (BCRC 910999) can be, for example, 1:1:1. However, in other examples, the weight (mg) ratio or colony count (CFU) ratio of the Lactobacillus brevis strain GKEX (BCRC 911099), the Lactobacillus plantarum strain GKK1 (BCRC 910919), and the Lactobacillus johnsonii strain GKJ2 (BCRC 910999) can also be a ratio other than 1:1:1, such as 1:1:(>1-10).

[0023] When used, the Lactobacillus composition can be, for example, an oral composition, such as a pharmaceutical composition or a food composition. The aforementioned Lactobacillus may be used in forms including, but not limited to, whole fermentation liquid, bacterial sludge (or cell pellet), supernatant, and lyophilized powder. In the above embodiments, the whole fermentation liquid refers to a product comprising bacterial cells and culture medium. The bacterial sludge refers to the product obtained by removing the supernatant from the whole fermentation liquid. The supernatant refers to the product obtained by removing the bacterial sludge from the whole fermentation liquid. The lyophilized powder refers to a product obtained from the whole fermentation liquid, bacterial sludge, and / or supernatant, and may contain other inseparable components.

[0024] Examples of pharmaceutical compositions include, but are not limited to, pharmaceuticals. Examples of Lactobacillus compositions for food use include, but are not limited to, general foods, health foods, beverages, nutritional supplements, dairy products, or feed. In the aforementioned oral composition examples, the Lactobacillus composition may optionally include pharmaceutically and food-acceptable carriers, excipients, and / or additives. In other examples, the dosage form of the Lactobacillus composition may include, but is not limited to, powders, tablets, granules, microcapsules, ampoules, or liquid sprays.

[0025] When the aforementioned Lactobacillus is used as a composition for improving a subject's athletic performance, an oral composition containing an effective dose of Lactobacillus can be administered to the subject continuously for a period of time. In one embodiment, the subject is not particularly limited and can be, for example, a healthy subject. In this embodiment, the effective dose depends on the subject and is not particularly limited. For example, when the subject is an adult, the effective dose of Lactobacillus for an adult can be, for example, 50 mg / 60 kg body weight / day to 1900 mg / 60 kg body weight / day, preferably 100 mg / 60 kg body weight / day to 1800 mg / 60 kg body weight / day, and more preferably about 100 mg / 60 kg body weight / day to about 1000 mg / 60 kg body weight / day. In another example, when the subject is a mouse, the effective dose of Lactobacillus for the subject can be, for example, 10.5 mg / kg body weight / day to 400 mg / kg body weight / day, preferably 15.75 mg / kg body weight / day to 300 mg / kg body weight / day, and more preferably about 21 mg / kg body weight / day to about 205 mg / kg body weight / day. In other examples, the administration period can be, for example, continuous for at least four weeks, or can be longer or shorter.

[0026] It should be understood that the specific strains, formulations, dosages, detection methods, concepts, illustrations, and examples described below are provided for illustrative purposes only and are not intended to limit the present invention. The key features of this invention may be applied to various embodiments without departing from the spirit and scope of the present invention. Therefore, those skilled in the art will readily be able to identify the essential technical features of this invention and make various modifications and refinements to suit different applications and conditions without departing from the spirit and scope of the present invention.

[0027] Example 1

[0028] 1.1 Source of strains

[0029] The strains used in this example include a specific combination of probiotics: Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2. Lactobacillus brevis strain GKEX was deposited with the Bioresource Conservation and Research Center, Food Industry Development Research Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan) on January 21, 2022, with the accession number BCRC 911099. Lactobacillus plantarum strain GKK1 was deposited with the BCRC on July 18, 2019, with the accession number BCRC 910919. Lactobacillus johnsonii strain GKJ2 was deposited with the BCRC on May 15, 2020, with the accession number BCRC 910999.

[0030] The aforementioned strains GKEX, GKK1, and GKJ2 were all isolated from humans or plants. For example, strain GKEX was isolated from organic fruit and vegetable fermentation broth, strain GKK1 was isolated from sour chili peppers, and strain GKJ2 was isolated from breast milk.

[0031] In some examples, the Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2 are inoculated onto solid culture media to activate the strains. In one embodiment, the solid culture media is commercially available MRS (deMan, Rogosa, and Sharpe) agar or RCM (Reinforced Clostridial Medium) agar. After colonies are formed, a single colony is selected and inoculated into liquid culture media for liquid culture. In one embodiment, the strain culture temperature is between 25°C and 40°C. In one embodiment, the liquid culture duration is 16 to 24 hours. In one embodiment, the liquid culture media is MRS liquid culture media or RCM liquid culture media. After completion of the liquid culture, fermentation is performed. In one embodiment, the fermentation medium formula is shown in Table 1 below.

[0032] Table 1. Fermentation medium formula Element Content (weight percentage, wt.%) carbohydrate 1~10 yeast extract 0.1~5 Protein 0.1~5 trace elements 0.01~2 Cysteine 0.01~0.1 Tween-80 0.05~1

[0033] 1.2 Preparation of lyophilized powder

[0034] After fermentation and growth of the Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2, the fermentation broth, containing the cells and culture medium, was collected and centrifuged at 1000 to 15000 rpm to obtain a bacterial slurry. The resulting slurry was mixed with a preservative (i.e., 6% to 50% by weight of skim milk powder) and freeze-dried. The freeze-drying process was performed using a gradient setting, with pre-freezing performed sequentially at 20°C to 0°C for 1 to 4 hours, followed by storage at 0°C to -20°C for 4 to 8 hours, and finally at -196°C to -30°C for more than 8 hours. In a preferred embodiment, the freeze-drying temperature and time were: storage at -40°C for 2 hours, then at -20°C for 2 hours, then at 0°C for 2 hours, and finally at 20°C for approximately 10 hours or more. The resulting freeze-dried powder is then stored at a low temperature. In another preferred embodiment, the storage temperature is between -30°C and 4°C. The stored freeze-dried powder contains other inseparable components and can be used as a raw material for the lactobacillus composition to be administered to test animals in the following animal experiments.

[0035] 1.3 Experimental animals

[0036] The following examples used 6-week-old male ICR mice, weighing approximately 30 grams, purchased from Lesco Biotechnology Co., Ltd. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University. Mice were housed in standard cages with an average temperature of approximately 22±2°C and an average humidity of 65±5%, under a 12-hour light and dark cycle. Temperature and humidity were recorded daily. Food and water were provided ad libitum. After two weeks of pre-housing, the experimental animals were acclimated to the environment and then divided into four groups arranged in an S-shaped pattern according to body weight: one blank control group (vehicle) and three experimental groups (i.e., GKEX, GKK1, and GKJ2), with six mice in each group.

[0037] The mice underwent a four-week animal experiment, during which they were gavaged daily with Lactobacillus (hereinafter referred to as "gavage") for four consecutive weeks (equivalent to days 1 to 28 of the experiment). Various biochemical markers related to exercise performance and fatigue were analyzed sequentially. Body weight, food intake, and water intake were recorded throughout the experiment.

[0038] 1.4 Experimental design

[0039] The experimental animals were orally administered sterile water (blank control group) or the lactobacilli of Example 1 (experimental group) daily for four consecutive weeks (i.e., 28 days). The effective doses of lactobacilli administered to the experimental mice were as follows: 21 mg / kg body weight / day for the Lactobacillus brevis strain GKEX; 21 mg / kg body weight / day for the Lactobacillus plantarum strain GKK1; and 205 mg / kg body weight / day for the Lactobacillus johnsonii strain GKJ2 (equivalent to adult doses of 100 mg / 60 kg body weight / day for GKEX, 100 mg / 60 kg body weight / day for GKK1, and 1000 mg / 60 kg body weight / day for GKJ2, respectively). After four weeks of intake, the following analyses were performed: weighted swimming exhaustion time (equivalent to exercise endurance, unit: minutes), treadmill exhaustion time (equivalent to exercise endurance, unit: minutes), and various blood biochemical indices before and after exercise [including serum lactate content (unit: mmol / L), serum urea nitrogen content (unit: mg / dL), liver and muscle glycogen content (mg / g liver or mg / g muscle)].

[0040] 1.5 Statistics

[0041] Numerical values ​​presented herein are expressed as mean ± standard deviation (SD) and analyzed using one-way ANOVA using commercially available software. Statistical significance was determined at p < 0.05. Asterisks ** indicate p < 0.05, and asterisks *** indicate p < 0.005.

[0042] Example 2: Evaluation of the Anti-Fatigue Efficacy of Lactobacillus in Example 1

[0043] 2.1 Assessment of exercise endurance - time to exhaustion in weighted swimming

[0044] One week before the experiment, the animals were acclimated to swimming (conditions: 28 cm diameter, 25 cm depth, 27 ± 1°C). After four weeks of testing, on day 31, mice were subjected to a weighted swimming test to exhaustion using a weighted load (e.g., a lead sheet weighing 5% of the mouse's body weight attached to the base of the mouse's tail). One week before the weighted swimming test, the animals were acclimated to swimming in a 28 cm diameter, 25 cm depth, and 27 ± 1°C water environment. They were fasted for 12 hours before swimming and, 30 minutes after Lactobacillus administration, were tested using a single swimming technique. Mice were placed in a water tank and forced to swim. The water temperature was maintained at 27 ± 1°C throughout the test. Furthermore, the mice were kept in motion throughout the experiment. If the mice floated on the water surface without moving their limbs, they were stirred with a stirrer. Exercise endurance (weighted swimming to exhaustion) was measured by recording the time it took for the mouse's head to remain completely submerged in water for 8 seconds without surfacing. The results are shown in Figure 1.

[0045] Please refer to FIG1 , which is a bar graph showing the weighted swimming exhaustion time (in minutes) of mice in the blank control group and the experimental group according to one embodiment of the present invention.

[0046] As shown in Figure 1, the weighted swimming time to exhaustion in the blank control group (Vehicle) was 5.02±0.83 minutes, the weighted swimming time to exhaustion in the GKJOY group was 9.15±0.73 minutes, the weighted swimming time to exhaustion in the GKK1 group was 7.01±0.8 minutes, and the weighted swimming time to exhaustion in the GKJ2 group was 8.68±0.94 minutes. Statistical analysis showed that the swimming time to exhaustion in the GKJOY, GKK1, and GKJ2 groups was significantly increased compared to the blank control group (Vehicle) by 1.82 times (p=0.0059), 1.40 times (p=0.0006), and 1.73 times (p<0.0001), respectively. Therefore, the probiotic strains GKJOY, GKK1, and GKJ2 of the present invention were shown to significantly prolong swimming time to exhaustion when administered to mice for four consecutive weeks.

[0047] 2.2 Assessment of exercise endurance - treadmill exhaustion time

[0048] After four weeks of testing, a running exercise exhaustion test was performed on day 33, after a 12-hour fast. Each mouse was assigned a track with an electric shock zone at the end. The animals were forced to run. Treadmill conditions followed conventional methods: an initial speed of 10 meters per minute (m / min) and a 5% incline. After 5 minutes, the speed increased by 2 m / min per minute until the mouse repeatedly landed in the shock zone or was unable to move forward in the shock zone for more than 5 seconds. This was considered exhaustion. The time from the start of running to exhaustion was recorded. The results are shown in Figure 2.

[0049] Please refer to FIG2 , which is a bar graph showing the running exhaustion time (in minutes) of mice in the blank control group and the experimental group according to one embodiment of the present invention.

[0050] As shown in Figure 2, the time to exhaustion in the blank control group (Vehicle) was 8.25 ± 1.14 minutes, the time to exhaustion in the GKJOY group was 15.32 ± 1.02 minutes, the time to exhaustion in the GKK1 group was 11.63 ± 0.97 minutes, and the time to exhaustion in the GKJ2 group was 15.04 ± 2.36 minutes. Statistical analysis showed that the time to exhaustion in the GKJOY, GKK1, and GKJ2 groups was significantly increased compared to the blank control group (Vehicle) by 1.86 times (p < 0.0001), 1.41 times (p = 0.0001), and 1.82 times (p < 0.0001), respectively. Therefore, the probiotic strains GKJOY, GKK1, and GKJ2 of the present invention were shown to significantly prolong the time to exhaustion in mice when administered for four consecutive weeks.

[0051] 2.3 Assessment of biochemical indices related to fatigue after exercise

[0052] To evaluate the effects of Lactobacillus on post-exercise and fatigue-related blood biochemical markers, the following "Method for Evaluating the Anti-Fatigue Function of Health Foods" was used to measure changes in blood lactate concentrations in mice before and after exercise, and the post-exercise blood lactate increase ratio was calculated. The results are shown in Figure 3.

[0053] Please refer to FIG3 , which is a bar graph showing the ratio of blood lactate increase in mice in the control group and the experimental group after exercise according to one embodiment of the present invention.

[0054] As shown in Figure 3, the lactate elevation ratios for the blank control (Vehicle), GKJOY, GKK1, and GKJ2 groups were 1.85±0.21, 1.45±0.16, 1.46±0.10, and 1.43±0.15, respectively. Statistical analysis showed that the lactate elevation ratios for the GKJOY, GKK1, and GKJ2 groups were significantly lower than those for the blank control (Vehicle) by 21.9% (p=0.0006), 21.1% (p=0.0009), and 22.9% (p=0.0003), respectively. Therefore, the probiotic strains GKJOY, GKK1, and GKJ2 of the present invention were shown to significantly reduce lactate elevation ratios when administered to mice for four consecutive weeks.

[0055] Secondly, to evaluate the effects of Lactobacillus on fatigue-related blood urea nitrogen (BUN) levels, 0.2 mL of blood was collected from the subjects 30 minutes after feeding with Lactobacillus on the test day (experimental day 35). After 90 minutes of unweighted swimming and 60 minutes of rest, blood was collected at three time points for BUN analysis. The results are shown in Figure 4.

[0056] Please refer to FIG4 , which is a bar graph showing the blood urea nitrogen concentrations of mice in the blank control group and the experimental group after exercise according to one embodiment of the present invention.

[0057] As shown in Figure 4 , the blood urea nitrogen concentrations in the blank control group (Vehicle), GKJOY group, GKK1 group, and GKJ2 group were 35.23±2.62 mg / dL, 28.98±1.42 mg / dL, 32.57±1.22 mg / dL, and 26.05±2.69 mg / dL, respectively. Statistical analysis showed that the blood urea nitrogen concentrations in the GKJOY group, GKK1 group, and GKJ2 group were significantly lower than those in the blank control group (Vehicle) by 17.7% (p<0.0001), 7.57% (p=0.0064), and 26.06% (p<0.0001), respectively. Therefore, the probiotic strains GKJOY, GKK1, and GKJ2 of the present invention were shown to significantly reduce blood urea nitrogen concentrations in mice when administered for four consecutive weeks.

[0058] 2.4 Assessment of liver glycogen content

[0059] To evaluate the effects of Lactobacillus on hepatic glycogenolysis, after the 90-minute swim test, all animals were allowed to rest for 2 days (experimental day 39) and sacrificed 30 minutes after the last feeding. Additionally, the mouse livers were harvested, washed with saline, wiped dry, and weighed. Tissue was excised from the same location and aliquoted and frozen at -80°C for subsequent analysis of glycogenolysis.

[0060] This example directly quantifies glycogen content using conventional chemical analysis methods. Briefly, first, the tissue sample to be tested was removed and added to 5 volumes (w / v) of tissue homogenate, and the different tissues were homogenized using a Bullet Blender (Next Advance, Cambridge, MA, USA). Next, the tissue homogenate was dispensed into microcentrifuge tubes and centrifuged at 4°C and 12,000×g for 15 minutes. The upper extract was removed and directly analyzed for glycogen content. The analysis method is based on Huang et al., Chicken essence improves exercise performance and ameliorates physical fatigue. Nutrients 6(7): 2681-2696 (2014), which is incorporated herein by reference. 30 μl of supernatant was added to a 96-well plate and mixed with 200 μl of iodine-potassium iodide reagent. Iodine was allowed to bind to glycogen for 10 minutes, resulting in a brown color. The absorbance at 460 nm was measured using an ELISA (Tecan Infinite M200, Tecan Austria, Salzburg, Austria). A calibration curve was constructed using a commercially available glycogen standard (Sigma) to calculate changes in hepatic glycogen storage in different groups of animals. The results are shown in Figure 5.

[0061] Please refer to FIG5 , which is a bar graph showing the liver glycogen content (mg / g liver tissue) of mice in the blank control group and the experimental group according to one embodiment of the present invention.

[0062] As shown in Figure 5, the liver glycogen levels in the blank control (Vehicle), GKJOY, GKK1, and GKJ2 groups were 15.75±2.81 mg / g, 21.82±1.19 mg / g, 25.98±3.00 mg / g, and 25.16±1.71 mg / g, respectively. Statistical analysis showed that the liver glycogen levels in the GKJOY, GKK1, and GKJ2 groups were significantly increased by 1.51-fold (p=0.0005), 1.65-fold (p<0.0001), and 1.60-fold (p<0.0001), respectively, compared to the blank control (Vehicle). Therefore, the probiotic strains GKJOY, GKK1, and GKJ2 of the present invention were shown to significantly increase liver glycogen levels in mice when administered for four consecutive weeks.

[0063] In summary, the present invention uses specific strains, specific formulations, specific dosages, specific detection methods, or specific evaluation methods merely to illustrate the anti-fatigue lactic acid bacteria composition and its uses. However, those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, the anti-fatigue lactic acid bacteria composition and its uses may also be formulated using two or three of the Lactobacillus brevis strain GKEX, the Lactobacillus plantarum strain GKK1, and the Lactobacillus johnsonii strain GKJ2, using other formulations, other dosages, other detection methods, or other evaluation methods, and are not limited to the aforementioned. For example, the lactobacillus composition may be a pharmaceutical composition or a food composition, optionally containing pharmaceutically and food-acceptable carriers, excipients, and / or additives, and may be formulated in dosage forms such as powders, tablets, granules, microcapsules, ampoules, and liquid sprays.

[0064] According to the above embodiments, the anti-fatigue lactic acid bacteria composition of the present invention has the advantage that this lactobacillus composition contains at least one of the Lactobacillus breve strain GKEX, the Lactobacillus plantarum strain GKK1, and the Lactobacillus johnsonii strain GKJ2. When administered to healthy subjects for a continuous period of time, it can improve fatigue-related biochemical values ​​and prolong the time to exhaustion during aerobic exercise. In the future, it can be used as an active ingredient in the preparation of various anti-fatigue and / or exercise performance-enhancing compositions.

[0065] Although the present invention has been disclosed above with reference to several specific embodiments, other embodiments are possible. Therefore, the spirit and scope of the present invention as claimed herewith should not be limited to the embodiments contained herein.

[0066] none

[0067] Domestic storage information (please note the order of storage institution, date, and number) The Lactobacillus brevis strain GKEX was deposited at the Bioresource Conservation and Research Center, Food Industry Development Institute, Republic of China, 331 Food Road, Hsinchu, Taiwan, on January 21, 2022, with the deposit number BCRC 911099. The strain was confirmed to be viable on February 10, 2022. Lactobacillus plantarum strain GKK1 was deposited at the Bioresource Conservation and Research Center, Food Industry Development Institute, No. 331, Food Road, Hsinchu, Taiwan, on July 18, 2019, with the accession number BCRC 910919. The strain was confirmed to be viable on July 26, 2019. Lactobacillus johnsonii strain GKJ2 was deposited at the Bioresource Conservation and Research Center, Food Industry Development Institute, No. 331, Food Road, Hsinchu, Taiwan, on May 15, 2020, with the accession number BCRC 910999. The strain was confirmed to be viable on May 28, 2020. Overseas deposit information (please note the order of deposit country, institution, date, and number) none

Claims

1. A lactobacillus composition that reduces serum urea nitrogen concentration after aerobic exercise, comprising *Lactobacillus brevis* strain GKEX, *Lactobacillus plantarum* strain GKK1, and *Lactobacillus johnsonii* strain GKJ2. Specifically, *Lactobacillus brevis* strain GKEX was deposited at the Bioresource Conservation and Research Center, Food Industry Research and Development Institute (BCRC, 331 Food Road, Hsinchu, Taiwan) on January 21, 2022, with registration number BCRC 911099; *Lactobacillus plantarum* strain GKK1 was deposited at BCRC on July 18, 2019, with registration number BCRC 910919; and *Lactobacillus johnsonii* strain GKJ2 was deposited at BCRC on May 15, 2020, with registration number BCRC 910919. 910999, and the weight (mg) ratio or cell count (CFU) ratio of the short lactic acid bacteria strain GKEX, the plant lactobacillus strain GKK1 and the johnsonia strain GKJ2 is 1:1:(1~10).

2. The lactobacillus composition that reduces serum urea nitrogen concentration after aerobic exercise as described in claim 1, wherein the lactobacillus composition is an oral composition.

3. The lactobacillus composition that reduces serum urea nitrogen concentration after aerobic exercise as described in claim 2, wherein the lactobacillus composition is a pharmaceutical composition or a food composition.

4. The lactobacillus composition for reducing serum urea nitrogen concentration after aerobic exercise as described in claim 3 further includes a pharmaceutically and food-acceptable carrier, excipient and / or additive.

5. A lactobacillus composition for reducing serum urea nitrogen concentration after aerobic exercise as described in claim 1, wherein one dosage form of the lactobacillus composition includes powder, tablets, granules, microcapsules, ampoules or liquid spray.

6. Use of a lactobacillus for preparing an oral composition for reducing serum urea nitrogen concentration after aerobic exercise, comprising administering an oral composition containing an effective dose of lactobacillus to a healthy subject for at least four consecutive weeks, wherein the lactobacillus is an active ingredient, and the lactobacillus is composed of a short lactic acid bacteria strain GKEX (BCRC 911099), a plant lactobacillus strain GKK1 (BCRC 910919), and a johnsonii strain GKJ2 (BCRC 910999), and the weight (mg) ratio or cell count (CFU) ratio of the short lactic acid bacteria strain GKEX, the plant lactobacillus strain GKK1, and the johnsonii strain GKJ2 is 1:1:(1~10).

7. Use of the Lactobacillus as claimed in claim 6 for the preparation of an oral composition for reducing serum urea nitrogen concentration after aerobic exercise, wherein the healthy subject is an adult, and the effective dose of the Lactobacillus administered to the healthy subject is from 50 mg / 60 kg body weight / day to 1900 mg / 60 kg body weight / day.

8. Use of the Lactobacillus as claimed in claim 6 for the preparation of an oral composition that reduces serum urea nitrogen concentration after aerobic exercise, wherein the healthy subject is a mouse, and the effective dose of the Lactobacillus administered to the healthy subject is from 10.5 mg / kg body weight / day to 400 mg / kg body weight / day.

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