Composite probiotic composition for improving athletic performance of people with innate aerobic athletic ability and application of composite probiotic composition

Abstract

The invention relates to a composite probiotic composition for improving athletic performance of people with innate aerobic athletic ability and application of the composite probiotic composition. The composite probiotic composition comprises a lactobacillus rhamnosus strain GK L C1, a bifidobacterium lactis strain GK K 24 and a clostridium butyricum strain GK B 7, and can effectively reduce serum lactic acid and serum urine nitrogen after aerobic exercise, reduce visceral fat and / or increase the liver sugar content of the liver and muscles after being fed to a subject with poor innate aerobic exercise ability for a continuous period of time, so that the composite probiotic composition can be used for preventing and treating the liver and muscles. The compound is further used as an effective component and used for preparing various compositions.

Description

[Technical field]

[0001] The present invention relates to a probiotic composition and its use, and in particular to a composite probiotic composition and its use for improving the exercise performance of people with congenitally weak aerobic exercise capacity by reducing serum lactate and serum urea nitrogen after aerobic exercise, reducing visceral fat and / or increasing liver and muscle glycogen content. [Background technology]

[0002] Cardiorespiratory fitness, muscular fitness, flexibility, age, gender, genetics, body shape, drugs, environment and other factors will affect individual athletic performance. Although overall athletic performance can be improved through "acquired" efforts, such as training and diet control, research has found that under the same conditions (such as gender, age, training content, etc.), athletic performance is still affected by "innate constitution".

[0003] In a group of experimental mice of the same age and sex from the same mother, it was found that the exercise performance of the same batch of mice was not normally distributed, but linearly distributed; that is, some mice were born with strong exercise ability, some were born with weak exercise ability, and the difference between the strongest and the weakest was very large. If the physical fitness of those with weak innate aerobic exercise ability can be improved or adjusted, the difference in the results of acquired exercise training with those with strong innate aerobic exercise ability can be narrowed.

[0004] Studies have found that people with congenitally weak aerobic exercise capacity not only have poorer exercise performance than their peers, but also have poorer physiological composition and metabolic related values, such as a higher proportion of visceral fat (e.g., epididymal fat pad), higher blood sugar levels, and a lower ratio of red muscle to white muscle. For example, after selectively breeding mice with high or low willingness to run on a voluntary wheel, it was found that these two animals with different innate exercise abilities have completely different susceptibility to health, aging, life extension, and chronic disease risk factors, including metabolic syndrome and cardiovascular complications.

[0005] People with poor innate athletic ability are significantly more susceptible to physiological, psychological, behavioral and metabolic diseases than people with good innate athletic ability (Thyfault & Morris, 2017). Therefore, if the innate constitution can be improved or adjusted, in addition to improving athletic performance, it is also expected to improve physiological states such as metabolism and effectively prevent the occurrence of diseases.

[0006] Probiotics are generally defined as bacteria that are beneficial to human health, can reproduce in the human intestine and are non-pathogenic. Probiotics can usually regulate the body constitution, change the metabolic pathways of some parts of the human body, and improve the overall gastrointestinal tract, metabolic capacity and other functions.

[0007] In recent years, many studies have suggested that probiotics have the potential to improve athletic performance. One study found that Lactobacillus plantarum can help improve the athletic endurance performance of normal mice and reduce the accumulation of lactic acid in the serum after exercise. Another study found that Lactobacillus salivarius can reduce blood urea nitrogen and creatine kinase in the serum of normal mice after exercise, increase glycogen in the liver and muscles, and shorten the physiological recovery time after exercise.

[0008] However, current studies have only focused on the evaluation of probiotics to improve the exercise performance of the general population (i.e., the population with average aerobic exercise capacity), and have not considered the influence of "innate factors" on exercise outcomes. There is an urgent need to develop a composite probiotic composition and its use to improve the exercise performance of those with innate weak aerobic exercise capacity. [Summary of the invention]

[0009] Therefore, one aspect of the present invention is to provide a composite probiotic composition for improving the exercise performance of people with innate aerobic exercise capacity deficiency, which comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK 24 and Clostridium butyricum strain GKB7.

[0010] Secondly, another aspect of the present invention is to provide a composite probiotic for use as a composition for preparing a composition for improving the exercise performance of people with congenitally weak aerobic exercise capacity, comprising administering an oral composition containing an effective dose of the composite probiotic to a subject for a continuous period of time, and the composite probiotic contains the above-mentioned strain as an active ingredient.

[0011] According to the above aspect of the present invention, a composite probiotic composition for improving the exercise performance of people with weak innate aerobic exercise capacity is provided. In one embodiment, the composite probiotic composition for improving the exercise performance of people with innate aerobic exercise capacity weakness comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7, wherein the Lactobacillus rhamnosus strain GKLC1 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee (CGMCC, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on January 12, 2018, with a deposit number of CGMCC 15202, and the Bifidobacterium lactis strain GKK24 was deposited in the National Institute of Technology for Product Evaluation and Research, Japan, on October 14, 2022, with a deposit number of NITE-IPOD, Room 2-5-8122, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan, with a deposit number of NITE BP-03766, and Clostridium butyricum strain GKB7 was deposited in NITE-IPOD on May 22, 2023, with the deposit number NITE BP-03889.

[0012] In the above embodiments, the composite probiotic composition can be, for example, an oral composition, such as a pharmaceutical composition or a food composition. In the above examples, the composite probiotic composition further comprises a carrier, excipient and / or additive acceptable to medicine and food.

[0013] In the above embodiments, the dosage form of the composite probiotic composition may include, for example, powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray or plug.

[0014] In the above embodiment, the aforementioned improvement in the exercise performance of people with congenitally weak aerobic exercise capacity includes reducing serum lactate and serum urea nitrogen after aerobic exercise, reducing visceral fat and / or increasing the glycogen content in the liver and muscles.

[0015] According to another aspect of the present invention, a composite probiotic is provided for use as a composition for preparing a composition for improving the exercise performance of people with innate aerobic exercise capacity weakness, comprising administering an oral composition containing an effective dose of the composite probiotic to a subject for at least 28 consecutive days, and the composite probiotic comprises Lactobacillus rhamnosus strain GKLC1 (CGMCC 15202), Bifidobacterium lactis strain GKK24 (NITE BP-03766) and Clostridium butyricum strain GKB7 (NITE BP-03889) as active ingredients.

[0016] In the above embodiment, the subject may be, for example, an adult, and the effective dosage of the compound probiotics for the subject may be, for example, 50 mg / 60 kg body weight / day to 300 mg / 60 kg body weight / day.

[0017] In the above embodiment, the subject may be, for example, a mouse, and the effective dosage of the compound probiotics to the subject may be, for example, 10.5 mg / kg / day to 63 mg / kg / day.

[0018] The composite probiotic composition for improving the exercise performance of people with congenitally weak aerobic exercise capacity and its use of the present invention, which comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7, can be administered to subjects with congenitally weak aerobic exercise capacity for a continuous period of time to effectively reduce serum lactic acid and serum urea nitrogen after aerobic exercise, reduce visceral fat and / or increase liver and muscle glycogen content, and then be used as an effective ingredient and prepared in various compositions.

[0019] It is to be understood that the foregoing general description and the following detailed description are merely exemplary, and are intended to provide further explanation of the invention as claimed.

[0020] Lactobacillus rhamnosus strain GKLC1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on January 12, 2018, with the deposit number CGMCC 15202, and the strain was confirmed to be alive on January 12, 2018.

[0021] The Bifidobacterium lactis strain GKK24 was deposited at the National Institute of Technology for Product Evaluation and Research, National Institute of Microorganisms and Technology (NITE-IPOD, Room 2-5-8122, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) on October 14, 2022, with the deposit number NITE BP-03766, and the strain was confirmed to be alive on December 7, 2022.

[0022] The Clostridium butyricum strain GKB7 was deposited at the National Institute of Technology for Product Evaluation and Research, National Institute of Microorganisms and Materials (NITE-IPOD, Room 2-5-8122, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) on May 22, 2023, with the deposit number NITE BP-03889. The strain was confirmed to be alive on May 24, 2023.

Brief Description of the Drawings

[0023] In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the attached drawings is as follows: [ Figure 1 ] is a bar graph showing the forelimb grip strength of mice in the control group and the experimental group according to one embodiment of the present invention. [ Figure 2 ] is a bar graph showing the weighted swimming exhaustion time of mice in the control group and the experimental group according to one embodiment of the present invention. [ Figure 3 ] is a bar graph showing the oral glucose tolerance test of mice in the control group and the experimental group according to one embodiment of the present invention. [ Figure 4 ] is a bar graph showing the serum lactate content of mice in the control group and the experimental group after exercise according to one embodiment of the present invention. [ Figure 5 ] is a bar graph showing the serum lactate levels of mice in the control group and the experimental group after exercise and rest according to one embodiment of the present invention. [ Figure 6 ] is a bar graph showing the serum urine nitrogen levels of mice in the control group and the experimental group after exercise and rest according to one embodiment of the present invention. [ Figure 7 ] is a bar graph showing the liver glycogen content of mice in the control group and the experimental group according to one embodiment of the present invention. [ Figure 8 ] is a bar graph showing the muscle glycogen content of mice in the control group and the experimental group according to one embodiment of the present invention. [ Fig. 9 ] is a bar graph showing the epididymal fat weight of mice in the control group and the experimental group according to one embodiment of the present invention. [Specific implementation method]

[0024] If the definition or use of a term in a reference is inconsistent or contrary to the definition of that term here, the definition of that term here shall apply rather than the definition of that term in the reference. Secondly, unless otherwise defined in the context, a singular term may include the plural and a plural term may include the singular.

[0025] As mentioned above, the present invention provides a composite probiotic composition for improving the exercise performance of people with congenitally weak aerobic exercise capacity and its use. After being administered to subjects for a continuous period of time, it can effectively reduce serum lactic acid after aerobic exercise, reduce serum urea nitrogen after aerobic exercise, increase liver glycogen content, increase muscle glycogen content and / or reduce visceral fat.

[0026] In one embodiment, the "complex probiotics" referred to herein may refer to probiotics of different species or genera, for example, Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7, wherein the Lactobacillus rhamnosus strain GKLC1 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee (CGMCC, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on January 12, 2018, with a deposit number of CGMCC15202, and the Bifidobacterium lactis strain GKK24 was deposited in the National Institute of Technology for Product Evaluation and Research, Japan (NITE-IPOD, Room 2-5-8122, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) on October 14, 2022, with a deposit number of NITE BP-03766, and Clostridium butyricum strain GKB7 was deposited in NITE-IPOD on May 22, 2023, with the deposit number NITE BP-03889. It should be noted that the present invention requires the use of a specific strain of composite probiotics, so that the resulting composite probiotic composition can play the role of improving the performance of people with congenital aerobic exercise capacity. If the composite probiotics are not selected from the above-mentioned specific strains, or the strains are increased or decreased, or part or all of them are replaced with other strains of the same species, it is not expected that the effect of improving the performance of people with congenital aerobic exercise capacity can be obtained.

[0027] The "innate aerobic exercise capacity weakness" referred to herein is based on the literature method published by Koch and Brittion (2001), using the swimming endurance time of mice as a screening index for high (high intrinsic, H) or low (low intrinsic, L) aerobic exercise capacity (aerobic exercise capacity, AEC). In one embodiment, a single mating selects male and female individuals in the bottom 20% of the aerobic exercise capacity ranking in the population for breeding of the next generation. Mating can be performed at least once, but the population that has been bred 5 times (inclusive) or more is more stable, and the population that has been bred 8 times (inclusive) or more is more stable. In a specific example, the population of the 8th generation of mating can be used as the innate aerobic exercise capacity weakness (L-AEC) for subsequent evaluation of exercise performance.

[0028] The evaluation items of "exercise performance" referred to herein may include, but are not limited to, serum lactate content and serum urinary nitrogen content after aerobic exercise, visceral fat ratio and / or liver and muscle glycogen content. In general, the exercise performance of L-AEC may include, but is not limited to, increasing serum lactate content and serum urinary nitrogen content after aerobic exercise, increasing visceral fat and / or reducing liver and muscle glycogen content. Therefore, in one embodiment, administering an oral composition containing an effective dose of a composite probiotic to a L-AEC subject for at least 28 consecutive days can significantly improve the exercise performance of L-AEC, including but not limited to reducing serum lactate after aerobic exercise, reducing serum urinary nitrogen after aerobic exercise, increasing liver glycogen content, increasing muscle glycogen content and / or reducing visceral fat. In other embodiments, the exercise performance of L-AEC improved by the above-mentioned composite probiotics can selectively include improving forelimb grip, improving aerobic exercise performance, delaying aerobic exercise exhaustion time (or prolonging weight-bearing swimming exhaustion time), increasing the ability to metabolize glucose (or increasing glucose tolerance), etc.

[0029] In one embodiment, the content of each strain of the composite probiotics is not particularly limited. In one example, the weight (mg) ratio or bacterial count (CFU) ratio of the Lactobacillus rhamnosus strain GKLC1 (CGMCC 15202), the Bifidobacterium lactis strain GKK24 (NITE BP-03766) and the Clostridium butyricum strain GKB7 (NITE BP-03889) can be, for example, 1:1:1, but in other examples, the weight (mg) ratio or bacterial count (CFU) ratio of the Lactobacillus rhamnosus strain GKLC1 (CGMCC 15202), the Bifidobacterium lactis strain GKK24 (NITE BP-03766) and the Clostridium butyricum strain GKB7 (NITE BP-03889) can also be a ratio other than 1:1:1, such as 1:1:(>1-4).

[0030] When used, the composite probiotic composition may be, for example, an oral composition, such as a pharmaceutical composition or a food composition. The usage of the aforementioned composite probiotics may include, but is not limited to, whole fermentation liquid, bacterial sludge (or bacterial precipitate, cellpellet), supernatant and lyophilized powder. In the above embodiments, the aforementioned whole fermentation liquid refers to the product including bacterial cells and culture liquid. The aforementioned bacterial sludge refers to the product after the supernatant is removed from the whole fermentation liquid. The aforementioned supernatant refers to the product after the bacterial sludge is removed from the whole fermentation liquid. The aforementioned lyophilized powder refers to the lyophilized powder prepared from the whole fermentation liquid, bacterial sludge and / or supernatant.

[0031] The aforementioned examples applicable to pharmaceutical compositions may include, but are not limited to, medicines. Examples of composite probiotics applied to food compositions may include, but are not limited to, general foods, health foods, beverages, nutritional supplements, dairy products, or feeds, etc. In the aforementioned examples of oral compositions, the composite probiotic composition may selectively include carriers, excipients, and / or additives acceptable to medicine and food. In other examples, the dosage form of the composite probiotic composition may include, but are not limited to, powders, tablets, granules, suppositories, microcapsules, ampoules, liquid sprays, or plugs.

[0032] When the above-mentioned composite probiotics are used as a composition for preparing a composition for improving the exercise performance of people with weak innate aerobic exercise capacity, an oral composition containing an effective dose of composite probiotics can be administered to the subject for a continuous period of time. The aforementioned effective dose depends on the subject and is not particularly limited. For example, when the subject is an adult, the effective dose of the composite probiotics for adults can be, for example, 50 mg / 60 kg body weight / day to 300 mg / 60 kg body weight / day, but 75 mg / 60 kg body weight / day to 200 mg / 60 kg body weight / day is preferred, and about 100 mg / 60 kg body weight / day is more preferred. In another example, when the subject is a mouse, the effective dose of the composite probiotics for this subject can be, for example, 10.5 mg / kg / day to 63 mg / kg / day, but 15.75 mg / kg / day to 42 mg / kg / day is preferred, and about 21 mg / kg / day is more preferred. In other examples, the administration period may be, for example, continuous for at least 28 days, or may be longer or shorter.

[0033] It is understood that the following specific strains, specific formulations, specific dosages, specific detection methods, viewpoints, illustrations and embodiments are for illustration only and are not intended to be limiting conditions of the present invention. The main features of the present invention may be used in various embodiments without departing from the spirit and scope of the present invention. Therefore, those with ordinary knowledge in the technical field to which the present invention belongs can easily determine the necessary technical features of the present case without departing from the spirit and scope of the present invention, and make various changes and modifications to the present invention to adapt to different uses and conditions. Example 1 1.1 Source of strains

[0034] The bacterial strains used in this embodiment include a specific combination of probiotics, including Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24, and Clostridium butyricum strain GKB7. The Lactobacillus rhamnosus strain GKLC1 was deposited on January 12, 2018 at the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China), with a deposit number of CGMCC 15202, the Bifidobacterium lactis strain GKK24 was deposited on October 14, 2022 at the National Institute of Technology for Product Evaluation and Research, Japan (NITE-IPOD, Room 2-5-8122, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan), with a deposit number of NITE BP-03766, and the Clostridium butyricum strain GKB7 was deposited on May 22, 2023 at NITE-IPOD, with a deposit number of NITE BP-03889.

[0035] The strains GKLC1, GKK24 and GKB7 are all isolated from human bodies. For example, strain GKLC1 is isolated from breast milk. Strain GKK24 is isolated from infant feces, and strain GKB7 is isolated from feces. After purifying the DNA of strains GKK24 and GKB7, gene sequencing is performed to obtain the nucleic acid fragment sequences of the 16srRNA genes of strains GKK24 and GKB7, as shown in sequence identification numbers (SEQ ID NO): 1 and SEQ ID NO: 2, respectively. After sequence alignment, strain GKK24 was identified as Bifidobacterium lactis, and strain GKB7 was identified as Clostridium butyricum. The purification, gene sequencing and sequence alignment of the above DNA are performed using conventional methods and do not affect the subsequent evaluation of the improvement of innate aerobic exercise capacity of strain GKK24, so they are not repeated here. The strain GKK24 of Bifidobacterium lactis is Gram-positive, acid-fast negative, obligately anaerobic, non-motile, without spores, capsules and flagella. The strain GKB7 of Clostridium butyricum is Gram-positive, obligately anaerobic, motile, with spores and flagella.

[0036] In some examples, the above-mentioned Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7 are respectively inoculated on solid culture medium to activate the strains. In a preferred embodiment, the above-mentioned solid culture medium is commercially available MRS (deMan, Rogosa and Sharpe) agar or RCM (Reinforced Clostridial Medium) agar. After the colony is generated, a single colony is picked out and inoculated into a liquid culture medium for liquid culture. In a preferred embodiment, the strain culture temperature is 25°C to 40°C. In a preferred embodiment, the liquid culture time is 16 to 24 hours. In a preferred embodiment, the liquid culture medium is MRS liquid culture medium or RCM liquid culture medium. Fermentation culture is carried out after the liquid culture is completed. In a preferred embodiment, the formula of the fermentation medium is shown in Table 1 below.

[0037] Table 1. Fermentation medium formula 1.2 Preparation of lyophilized powder

[0038] After the strains GKLC1, GKK24 and GKB7 are fermented and grown, the fermentation liquid containing the bacteria and the culture liquid is collected and centrifuged at a rate of 1000 rpm to 15000 rpm to obtain the bacterial mud. The obtained bacterial mud is mixed with a protective agent (i.e., 6% to 50% by weight of skim milk powder) and then freeze-dried. The aforementioned freeze-drying system is pre-frozen in a gradient setting mode, sequentially stored at 20°C to 0°C for 1 to 4 hours, then stored at 0°C to -20°C for 4 to 8 hours, and then stored at -196°C to -30°C for more than 8 hours. In a preferred embodiment, the temperature and time of freeze-drying are stored at -40°C for 2 hours, then stored at -20°C for 2 hours, then stored at 0°C for 2 hours, and finally at 20°C for about 10 hours or more. Afterwards, the above-obtained freeze-dried powder is stored at low temperature. In another preferred embodiment, the temperature of low temperature storage is -30°C to 4°C. The stored freeze-dried powder can be used as the raw material of the composite probiotic composition to be administered to the test animals in the following animal experiments. 1.3 Experimental animals

[0039] The following examples use a total of 24 8-week-old ICR strain male mice with congenitally weak aerobic exercise capacity, each weighing about 35 grams, purchased from Lesco Biotechnology Co., Ltd. The mice were housed in standard breeding cages, 3 mice per cage, the average temperature of the mouse room was about 22±2°C, the average humidity was 65±5%, the light and dark were set for 12 hours, and drinking water and standard feed were fully supplied. All animal experimental procedures were reviewed and approved by the Experimental Animal Care and Use Committee (IACUC) of Taiwan Sport University, and obtained the approval number IACUC-11011. The experimental animals were grouped in an S-shaped arrangement according to their weight and divided into 1 control group and 3 experimental groups, with 6 mice in each group. 1.4 Breeding of mice with congenitally weak aerobic capacity

[0040] According to the method published by Koch and Brittion (2001), the swimming endurance time of mice was used as a screening index for high (high intrinsic, H) or low (low intrinsic, L) aerobic exercise capacity (aerobic exercise capacity, AEC). Male and female individuals ranked in the bottom 20% of the aerobic exercise capacity in the population were selected for single mating to reproduce the next generation. The mating was carried out 8 times in total, and the population of the 8th generation was the population with inherently weak aerobic exercise capacity (L-AEC). 1.5 Screening method for mice with congenitally weak aerobic exercise capacity

[0041] After the mice had adapted for a week, they were subjected to an exhaustion swimming endurance test. In the test, a lead sheet weighing 5% of the body weight of the individual mice was fixed at the base of the mouse's tail, and the mice were placed in a bucket with a water depth of 40 cm and a surface diameter of 45 cm. The water temperature was controlled at 28±1°C, and the mice were allowed to swim continuously until exhaustion, and the mice were immediately rescued to prevent drowning. This test defines swimming exhaustion as the mouse sinking upright or being unable to swim back to the surface for air within 7 seconds after sinking into the water. The exhaustion swimming endurance test was conducted twice, with an interval of 72 hours each time to allow the mice to recover their physical strength. The better result of the two individual swimming results was taken as a comparison with others. The time it takes a mouse to swim to exhaustion represents its aerobic endurance performance; the worse the aerobic endurance performance, the shorter the time it takes to swim to exhaustion. 1.6 Experimental design

[0042] The experimental animals were orally administered with sterile water (control group) or the probiotic composition of Example 1 (experimental group) daily for four consecutive weeks (i.e., 28 days). The effective dose of the composite probiotic administered to the mice in the experimental group was 21 mg / kg / day (equivalent to an effective dose of 100 mg / 60 kg body weight / day for adults). After four weeks of intake, the following analyses were performed: forelimb grip strength (unit: g), weighted swimming exhaustion time (unit: minutes), blood glucose level (mg / dL) in the oral glucose tolerance test, serum lactate level after exercise (unit: mmol / L), serum lactate level after exercise and after rest (unit: mmol / L), serum urea nitrogen level after exercise and after rest (unit: mg / dL), liver and muscle glycogen content (mg / g liver or mg / g muscle), epididymal fat weight (which can represent the proportion of visceral adipose tissue) analysis. 1.7 Statistics

[0043] The values ​​described herein are expressed as mean ± SD, and analyzed using one-way ANOVA and commercially available software, with p < 0.05 used to determine statistically significant differences. Asterisks ** represent p < 0.05, and asterisks *** represent p < 0.005. Example 2: Evaluation of the composite probiotics of Example 1 to improve the athletic performance of L-AEC subjects 2.1 Assessing forelimb grip strength

[0044] In this example, the forelimb gripping strength test was conducted 30 minutes after feeding the probiotic composition containing Example 1 on the 29th day. First, the base of the mouse's tail was grasped and the mouse was lowered vertically, so that the two forelimbs grasped a horizontal bar (2 mm in diameter, 7.5 cm in length) connected to an AIKOH electronic push-pull force gauge (Model-RX-5, Aikoh Engineering, Nagoya, Japan), and then the mouse's tail was slightly pulled back to make the mouse's forelimbs leave the horizontal bar, and the maximum value of the mouse's forelimb gripping strength during the process was recorded using the push-pull force gauge as an evaluation index of gripping strength.

[0045] See also Figure 1 , which is a bar graph showing the forelimb grip strength (g) of mice in the control group and the experimental group according to one embodiment of the present invention.

[0046] like Figure 1As shown, the forelimb grasping strength of the mice in the control group was about 105g, the forelimb grasping strength of the mice in the oral strain GKK24 group was about 130g, the forelimb grasping strength of the mice in the oral strain GKLC1 group was about 130g, and the forelimb grasping strength of the mice in the oral strain GKB7 group was about 134g, which proved that the probiotic strain of the present invention can significantly improve the forelimb grasping strength of the L-AEC mice after being administered to the L-AEC mice for four consecutive weeks. 2.2 Assessment of exercise endurance - time to exhaustion in weighted swimming

[0047] After the forelimb grasping strength test is completed, a lead sheet of 5% of the mouse's body weight is fixed to the base of the mouse's tail, and the mouse is subjected to a weighted swimming exhaustion test. One week before the weighted swimming exhaustion test, the animals are allowed to adapt to swimming and are placed in an environment with a diameter of 28 cm, a water depth of 25 cm, and a water temperature of 27±1°C. The test is conducted in a single swimming manner. The mouse is placed in a water tank and forced to swim. The mouse's limbs are kept moving throughout the experiment. If the mouse floats on the water surface with its limbs motionless, a stirring rod is used to stir nearby. Exercise endurance (weighted swimming exhaustion) performance is shown by recording the time it takes for the mouse's head to be completely submerged in water for 8 seconds without surfacing.

[0048] See also Figure 2 , which is a bar graph showing the weight-loaded swimming exhaustion time (minutes) of mice in the control group and the experimental group according to one embodiment of the present invention.

[0049] like Figure 2 As shown, the weighted swimming exhaustion time of the control group mice was about 0.65 minutes, the weighted swimming exhaustion time of the oral strain GKK24 group mice was about 1.20 minutes, the weighted swimming exhaustion time of the oral strain GKLC1 group mice was about 0.94 minutes, and the weighted swimming exhaustion time of the oral strain GKB7 group mice was about 1.15 minutes, which proved that the probiotic strains of the present invention can significantly improve the exercise endurance of L-AEC mice after being administered to L-AEC mice for four consecutive weeks. 2.3 Oral Glucose Tolerance Test (OGTT)

[0050] The glucose metabolism capacity of aerobic weak-lineage mice after four weeks of ingestion of the patented strain was evaluated by an oral glucose tolerance test. A 25% glucose solution was prepared and administered to mice that had fasted for 12 hours at a dose of 2.5 g / kg BW. The glucose levels were measured at 0, 15, 30, 60, and 120 minutes using a Roche blood glucose meter ( Germany) to analyze the blood glucose levels of mice at different time points.

[0051] See also Figure 3, which is a bar graph showing the oral glucose tolerance test of mice in the control group and the experimental group according to one embodiment of the present invention.

[0052] like Figure 3 As shown, after oral administration of glucose, the blood glucose changes (mg / dL, vertical axis) of the mice orally administered with strain GKK 24, strain GKLC1, and strain GKB7 were all lower than those of the control group mice, proving that the probiotic strains of the present invention can indeed help improve the glucose tolerance of L-AEC mice after being administered to L-AEC mice for four consecutive weeks. 2.4 Evaluation of serum lactate levels after exercise and rest

[0053] Blood samples were collected from mice in each group after 10 minutes of unweighted swimming and 20 minutes of rest. The blood samples were centrifuged at 1500×g and 4°C for 10 minutes, and the serum lactate content was analyzed using an automatic biochemical analyzer (Hitachi 7060, Hitachi, Tokyo, Japan).

[0054] See also Figure 4 , which is a bar graph showing the serum lactate content (mmol / L) of mice in the control group and the experimental group after exercise according to one embodiment of the present invention.

[0055] like Figure 4 As shown, the serum lactate content of mice after exercise. The serum lactate content of the control group mice was about 6.55mmol / L, the serum lactate content of the oral strain GKK24 group mice was about 5.15mmol / L, the serum lactate content of the oral strain GKLC1 group mice was about 4.81mmol / L, and the serum lactate content of the oral strain GKB7 group mice was about 5.04mmol / L, proving that the probiotic strain of the present invention can significantly reduce the accumulation of serum lactate after exercise in L-AEC mice after four consecutive weeks.

[0056] See also Figure 5 , which is a bar graph showing the serum lactate levels (mmol / L) of mice in the control group and the experimental group after exercise and rest according to one embodiment of the present invention.

[0057] like Figure 5 As shown, after exercise and rest, the serum lactate content of the mice in the control group was about 5.59 mmol / L, the serum lactate content of the mice in the oral strain GKK24 group was about 4.10 mmol / L, the serum lactate content of the mice in the oral strain GKLC1 group was about 3.60 mmol / L, and the serum lactate content of the mice in the oral strain GKB7 group was about 3.87 mmol / L, which proved that the probiotic strain of the present invention can significantly improve the clearance effect of serum lactate at rest after exercise after being administered to L-AEC mice for four consecutive weeks. 2.5 Evaluation of serum urinary nitrogen levels after exercise and rest

[0058] After each group of mice swam for 90 minutes without weight bearing and rested for 60 minutes, blood samples were collected and centrifuged at 1500×g and 4°C for 10 minutes. The serum urea nitrogen content was analyzed using an automatic biochemical analyzer (Hitachi 7060, Hitachi, Tokyo, Japan).

[0059] See also Figure 6 , which is a bar graph showing the serum urea nitrogen levels (mg / dL) of mice in the control group and the experimental group after exercise and after rest according to one embodiment of the present invention.

[0060] like Figure 6 As shown, the serum urea nitrogen content of the control group mice was about 39.10 mg / dL, the serum urea nitrogen content of the oral strain GKK24 group mice was about 30.15 mg / dL, the serum urea nitrogen content of the oral strain GKLC1 group mice was about 30.85 mg / dL, and the serum urea nitrogen content of the oral strain GKB7 group mice was about 30.70 mg / dL, which proved that the probiotic strain of the present invention can significantly reduce the serum urea nitrogen content at rest after exercise in L-AEC mice after being administered for four consecutive weeks. 2.6 Assessment of glycogen content in liver and muscle tissue

[0061] After the experiment, the liver and muscle tissues of the mice were collected for analysis of glycogen content. 100 mg of liver or muscle tissue was homogenized with 0.5 ml of 10% glacial perchloric acid, and then centrifuged at 15,000 x g and 4°C for 15 minutes. 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 to turn brown, and ELISA (Tecan Infinite M200, Tecan Austria, Salzburg, Austria) was used to detect the absorbance wavelength of 460 nm. The standard glycogen purchased from Sigma was used as a standard curve to convert the glycogen content (mg / g tissue) in the detected tissue.

[0062] See also Figure 7 and Figure 8 , which respectively show the mouse livers of the control group and the experimental group according to one embodiment of the present invention ( Figure 7 ) and muscles( Figure 8 ) Bar graph of glycogenotype (mg / g liver).

[0063] like Figure 7As shown, the glycogen content in the liver of the control group mice was about 7.36 mg / g liver, the glycogen content in the liver of the oral strain GKK24 group mice was about 10.77 mg / g liver, the glycogen content in the liver of the oral strain GKLC1 group mice was about 13.57 mg / g liver, and the glycogen content in the liver of the oral strain GKB7 group mice was about 14.31 mg / g liver, which proved that the probiotic strain of the present invention can significantly increase the glycogen content in the liver after being administered to L-AEC mice for four consecutive weeks.

[0064] like Figure 8 As shown, the glycogen content in the muscles of the control mice was about 1.07 mg / g muscle, the glycogen content in the muscles of the mice in the oral strain GKK24 group was about 2.21 mg / g muscle, the glycogen content in the muscles of the mice in the oral strain GKLC1 group was about 2.24 mg / g muscle, and the glycogen content in the muscles of the mice in the oral strain GKB7 group was about 2.24 mg / g muscle, which proved that the probiotic strains of the present invention can significantly increase the glycogen content in the muscles of L-AEC mice after being administered for four consecutive weeks. 2.7 Assessment of epididymal fat mass

[0065] After the experiment, the epididymis of the mice was taken out, weighed, and the weight of epididymal adipose tissue was recorded, which can represent the proportion of visceral adipose tissue.

[0066] See also Fig. 9 , which is a bar graph showing epididymal fat weight (g) of mice in the control group and the experimental group according to one embodiment of the present invention.

[0067] like Fig. 9 As shown, the epididymal fat weight of the control group mice was about 0.42g, the epididymal fat weight of the oral strain GKK24 group mice was about 0.32g, the epididymal fat weight of the oral strain GKLC1 group mice was about 0.30g, and the epididymal fat weight of the oral strain GKB7 group mice was about 0.31g, which proved that the probiotic strain of the present invention can significantly reduce the proportion of visceral adipose tissue in L-AEC mice after being administered for four consecutive weeks.

[0068] In summary, the present invention uses specific strains, specific formulas, specific dosages, specific detection methods, or specific evaluation methods only to illustrate the composite probiotic composition and its use for improving the exercise performance of people with weak innate aerobic exercise capacity. However, those with ordinary knowledge in the technical field to which the present invention belongs should understand that, without departing from the spirit and scope of the present invention, two or three of the Lactobacillus rhamnosus strain GKLC1, the Bifidobacterium lactis strain GKK24, and the Clostridium butyricum strain GKB7, other formulas, other dosages, other detection methods, or other evaluation methods can also be used to improve the exercise performance of people with weak innate aerobic exercise capacity. Composite probiotic compositions and their uses are not limited to the above. For example, the composite probiotic composition can be, for example, a pharmaceutical composition or a food composition, which can selectively contain carriers, excipients and / or additives acceptable to medicine and food, and can be made into dosage forms such as powders, tablets, granules, suppositories, microcapsules, ampoules, liquid sprays or plugs.

[0069] According to the above embodiments, the composite probiotic composition for improving the exercise performance of people with congenitally weak aerobic exercise capacity of the present invention has the advantage that the composite probiotic composition comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7. After being administered to subjects with congenitally weak aerobic exercise capacity for a continuous period of time, the composite probiotic composition can effectively reduce serum lactic acid and serum urea nitrogen after aerobic exercise, reduce visceral fat and / or increase the glycogen content of the liver and muscles. In the future, the composite probiotic composition can be used as an effective ingredient and used to prepare various compositions.

[0070] 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 defined by the appended claims should not be limited to the embodiments contained herein.

Claims

1. A composite probiotic composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity, characterized in that: It comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7, wherein the Lactobacillus rhamnosus strain GKLC1 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on January 12, 2018, with a deposit number of CGMCC 15202, the Bifidobacterium lactis strain GKK24 was deposited in the Special Microbiological Depository Center of Japan's Independent Administrative Agency for Product Evaluation Technology Foundation on October 14, 2022, with a deposit number of NITE BP-03766, and the Clostridium butyricum strain GKB7 was deposited in the Special Microbiological Depository Center of Japan's Independent Administrative Agency for Product Evaluation Technology Foundation on May 22, 2023, with a deposit number of NITE BP-03889.

2. The composite probiotic composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity as claimed in claim 1, characterized in that: The composite probiotic composition is an oral composition.

3. The composite probiotic composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity as claimed in claim 2, characterized in that: The composite probiotic composition is a pharmaceutical composition or a food composition.

4. The composite probiotic composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity as claimed in claim 2, characterized in that: It also includes pharmaceutically and food-acceptable carriers, excipients and / or additives.

5. The composite probiotic composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity as claimed in claim 1, characterized in that: The dosage form of the composite probiotic composition includes powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray or plug.

6. The composite probiotic composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity as claimed in claim 1, characterized in that: The improvement of the exercise performance of those with congenitally weak aerobic exercise capacity includes reducing the serum lactate level and serum urea nitrogen level after aerobic exercise, reducing the proportion of visceral fat and / or increasing the glycogen content of the liver and muscles.

7. A use of a composite probiotic as a composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity, characterized in that: The invention comprises administering an oral composition containing an effective dose of the composite probiotic to a subject with congenitally weak aerobic exercise capacity for at least 28 consecutive days, wherein the composite probiotic comprises Lactobacillus rhamnosus strain GKLC1 (CGMCC15202), Bifidobacterium lactis strain GKK24 (NITE BP-03766) and Clostridium butyricum strain GKB7 (NITE BP-03889) as active ingredients.

8. Use of the composite probiotics as claimed in claim 7 as a composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity, characterized in that: The improvement of the exercise performance of those with congenitally weak aerobic exercise capacity includes reducing serum lactate and serum urea nitrogen after aerobic exercise, reducing visceral fat and / or increasing the glycogen content in the liver and muscles.

9. Use of the composite probiotics as claimed in claim 7 as a composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity, characterized in that: The subject is an adult, and the effective dose of the compound probiotic administered to the subject is 50 mg / 60 kg body weight / day to 300 mg / 60 kg body weight / day.

10. Use of the composite probiotics as claimed in claim 7 as a composition for improving the athletic performance of people with congenitally weak aerobic exercise capacity, characterized in that: The subject is a mouse, and the effective dose of the composite probiotic administered to the subject is 10.5 mg / kg / day to 63 mg / kg / day.

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