Complex probiotic composition and its use for improving athletic performance in individuals with congenital poor aerobic fitness

Abstract

To provide a complex probiotic composition for improving exercise performance in individuals with congenitally low aerobic exercise capacity, and to provide uses thereof.SOLUTION: This complex probiotic composition comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24, and Clostridium butylicum strain GKB7, and effectively reduces serum lactate and serum urea nitrogen after aerobic exercise, reduces visceral fat, and / or improves glycogen concentration in the liver and muscles, by administering continuously for a certain period of time to subjects with congenitally low aerobic exercise capacity, and, furthermore, can be used as an active ingredient in the preparation of various compositions.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to a probiotic composition and its use, in particular to a complex probiotic composition and its use for improving exercise performance in individuals with congenital low aerobic fitness by lowering serum lactate and serum urea nitrogen after aerobic exercise, lowering visceral fat and / or improving liver and muscle glycogen concentration. [Background technology]

[0002] Factors such as cardiorespiratory fitness, muscular fitness, flexibility, age, sex, genetics, body shape, drugs, and environment all affect an individual's athletic performance. "Acquired" efforts such as training and dietary control can improve overall athletic ability. However, research has shown that even under the same conditions (e.g., sex, age, training content, etc.), athletic performance is still influenced by "innate constitution."

[0003] In a group of experimental mice of the same age and sex from the same mother, the exercise performance of the same batch of mice was found to be linearly distributed, not normally distributed. That is, some mice have innately strong athletic ability, while others have innately weak athletic ability. And the difference between the strongest and weakest athletic ability is very large. If we can improve or adjust the physical condition of those with innately weak aerobic ability, we can reduce the difference in the results of acquired exercise training between those with innately strong aerobic ability and those with strong aerobic ability.

[0004] A study has shown that individuals with congenital aerobic weakness not only have poorer exercise performance than their peers, but also have poorer physiological composition and metabolic parameters, such as a higher proportion of visceral fat (e.g., epididymal fat pads), higher blood glucose levels, and a lower ratio of red and white muscle. For example, selective breeding of mice with high or low willingness to exercise independently by humans has revealed that these two types of animals with congenital differences in exercise ability have significantly different health, aging, life expectancy, and susceptibility to risk factors for chronic diseases such as metabolic syndrome and cardiovascular complications.

[0005] Those with congenital low athletic ability are significantly more susceptible to physiological, psychological, behavioral and metabolic diseases than those with congenital high athletic ability (Thyfault & Morris, 2017). Therefore, if we can improve or adjust the congenital constitution, we can not only improve athletic performance, but also improve physiological conditions such as metabolism, and effectively prevent the occurrence of diseases.

[0006] Probiotics are generally defined as bacteria that are beneficial to human health, can grow in the intestinal tract of the human body, and are non-pathogenic. Probiotics can generally regulate the body constitution and change some metabolic pathways in the human body, improving the function of the entire gastrointestinal tract, metabolic capacity, etc.

[0007] In recent years, many studies have suggested that probiotics may improve exercise performance. One study found that Lactiplantibacillus plantarum was useful in improving the exercise endurance performance of normal mice and reducing the accumulation of lactic acid in serum after exercise. Another study showed that Ligilactobacillus salivarius reduced blood urea nitrogen and creatine kinase in serum after exercise in normal mice, improved glycogen in the liver and muscles, and shortened the physiological recovery time after exercise. Summary of the Invention [Problem to be solved by the invention]

[0008] However, current research is limited to evaluating whether probiotics enhance the athletic performance of general groups (i.e., groups with general aerobic fitness), and does not consider the influence of "innate factors" on athletic outcomes. Therefore, there is a need to develop a complex probiotic composition and its use to improve the athletic performance of people with innately low aerobic fitness. [Means for solving the problem]

[0009] Accordingly, one aspect of the present invention provides a composite probiotic composition for improving exercise performance in individuals with congenital poor aerobic fitness comprising Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7.

[0010] Next, another aspect of the present invention provides a use of a complex probiotic for preparing a composition for improving the exercise performance of a person with congenital low aerobic exercise capacity, comprising continuously administering to a subject an oral composition containing an effective dose of the complex probiotic for a certain period of time, the complex probiotic containing 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 individuals with congenital low aerobic fitness is provided. In one embodiment, the composite probiotic composition for improving the exercise performance of individuals with congenital low aerobic fitness comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7, where Lactobacillus rhamnosus strain GKLC1 was deposited at the Bioresource Conservation Research Center, Food Industry Development Research Institute (BCRC, No. 331 Food Road, Hsinchu, Taiwan) on February 12, 2018, and is designated as BCRC. 910828, Bifidobacterium lactis strain GKK24 was deposited at the BCRC on April 6, 2022 and has the accession number BCRC911121, and Clostridium butyricum strain GKB7 was deposited at the BCRC on April 1, 2022 and has the accession number BCRC 911120.

[0012] In the above embodiment, the composite probiotic composition may be, for example, an oral composition, for example, a pharmaceutical composition or a food composition. In the above embodiment, the composite probiotic composition further comprises a pharmaceutical and food acceptable carrier, excipient and / or additive.

[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 suppository.

[0014] In the above examples, the improvement in exercise performance of the individual with innately low aerobic exercise capacity includes reducing serum lactate and serum urea nitrogen after aerobic exercise, reducing visceral fat, and / or improving liver and muscle glycogen concentrations.

[0015] According to another aspect of the present invention, there is provided the use of a probiotic complex for the preparation of a composition for improving exercise performance in individuals with congenital poor aerobic fitness, comprising administering to a subject an oral composition comprising an effective dose of a probiotic complex for at least 28 consecutive days, the probiotic complex comprising Lactobacillus rhamnosus strain GKLC1 (BCRC 910828), Bifidobacterium lactis strain GKK24 (BCRC 911121) and Clostridium butyricum strain GKB7 (BCRC 911120) as active ingredients.

[0016] In the above embodiment, the subject may be, for example, an adult, and the effective dose of the complex 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 dose of the composite probiotic for the subject may be, for example, 10.5 mg / 20 g body weight / day to 63 mg / 20 g body weight / day. (Effects of the Invention)

[0018] The above-mentioned composite probiotic composition for improving the exercise performance of subjects with congenital low 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 continuously administered to subjects with congenital low aerobic exercise capacity for a certain period of time to effectively reduce serum lactate and serum urea nitrogen after aerobic exercise, reduce visceral fat and / or improve glycogen concentration in liver and muscle, and can also be used as an active ingredient in the preparation of various compositions.

[0019] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide a further explanation of the invention for which protection is sought. [Brief description of the drawings]

[0020] To make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, reference is made to the accompanying drawings as follows. [Figure 1] 1 is a bar graph showing the forelimb grip strength of control and experimental mice according to one embodiment of the present invention. [Diagram 2] 1 is a bar graph showing the time to exhaustion in mice of a control group and an experimental group swimming under load according to an embodiment of the present invention. [Diagram 3] 1 is a bar graph showing oral glucose tolerance tests of control and experimental mice according to one embodiment of the present invention. [Figure 4] 1 is a bar graph showing serum lactate concentrations after exercise in control and experimental mice according to an embodiment of the present invention. [Diagram 5] 1 is a bar graph showing serum lactate concentrations after exercise and rest in control and experimental mice according to one embodiment of the present invention. [Figure 6] 1 is a bar graph showing serum urea nitrogen concentrations after exercise and rest in control and experimental mice according to an embodiment of the present invention. [Figure 7] 1 is a bar graph showing liver glycogen concentrations in control and experimental mice according to one embodiment of the present invention. [Figure 8] 1 is a bar graph showing muscle glycogen concentrations in control and experimental mice according to one embodiment of the present invention. [Figure 9] 1 is a bar graph showing epididymal fat weights of control and experimental mice according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] If a definition or usage of a term in a reference document contradicts or is contrary to a definition of such term herein, the definition of the term herein shall govern in place of the definition in the reference document. Secondly, unless the context clearly indicates otherwise, singular terms may include plural terms and plural terms may include the singular term.

[0022] As described above, the present invention provides a composite probiotic composition and use thereof for improving the exercise performance of individuals with congenital low aerobic exercise capacity, which, when continuously administered to a subject for a certain period of time, can effectively reduce serum lactate after aerobic exercise, reduce serum urea nitrogen after aerobic exercise, improve liver glycogen concentration, improve muscle glycogen concentration, and / or reduce visceral fat.

[0023] In one embodiment, the term "composite probiotic" referred to herein may refer to probiotics comprising different species or different genera, for example, Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24, and Clostridium butyricum strain GKB7, where Lactobacillus rhamnosus strain GKLC1 was deposited at the Bioresource Conservation Research Center, Food Industry Development Research Institute (BCRC, No. 331 Food Road, Hsinchu, Taiwan) on February 12, 2018, with deposit number BCRC 910828, and Bifidobacterium lactis strain GKK24 was deposited at the BCRC on April 6, 2022, with deposit number BCRC 910828. 911121, and Clostridium butyricum strain GKB7 was deposited at the BCRC on April 1, 2022, with the deposit number BCRC 911120. It should be noted that the present invention requires the selection of a composite probiotic of a specific strain, and this alone allows the resulting composite probiotic composition to have the effect of improving the exercise performance of those with congenital low aerobic exercise capacity. The composite probiotic is not selected from the above specific strains, or if the strains are increased or decreased, or part or all are replaced with other strains of the same species, the effect of improving the exercise performance of those with congenital low aerobic exercise capacity cannot be expected.

[0024] Here, "individuals with low aerobic capacity" are those with high (H) or low (L) aerobic exercise capacity (AEC) as screened for by the swimming endurance time of mice according to the method published by Koch and Brittion (2001). In one embodiment, male and female individuals with the lowest 20% of aerobic capacity in the group are selected and mated once to breed the next generation. Mating may be performed at least once, but groups mated 5 times (inclusive) or more are stable, and groups mated 8 times (inclusive) or more are more stable. In one embodiment, the group mated for the 8th generation is later evaluated for exercise performance as individuals with low aerobic capacity (L-AEC).

[0025] The evaluation item referred to herein as "exercise performance" may include, but is not limited to, serum lactate concentration and serum urea nitrogen concentration after aerobic exercise, visceral fat percentage and / or liver and muscle glycogen concentration. In general, the exercise performance of L-AEC subjects may include, but is not limited to, improving serum lactate concentration and serum urea nitrogen concentration after aerobic exercise, improving visceral fat and / or reducing liver and muscle glycogen concentration. Thus, in one embodiment, administering an oral composition containing an effective dose of a complex probiotic to an L-AEC subject for at least 28 consecutive days can significantly improve the exercise performance of the L-AEC subject, including, but not limited to, reducing serum lactate after aerobic exercise, reducing serum urea nitrogen after aerobic exercise, improving liver glycogen concentration, improving muscle glycogen concentration and / or reducing visceral fat. In other embodiments, the improved exercise performance of L-AEC subjects by the above-mentioned complex probiotics may further selectively include improved forelimb grip strength, improved aerobic exercise performance, delayed aerobic exercise exhaustion time (also referred to as prolonged time to exhaustion in weight-bearing swimming), improved glucose metabolic capacity (also referred to as improved glucose tolerance), and the like.

[0026] In one embodiment, the concentration of each strain of the above-mentioned complex probiotics is not particularly limited.In one embodiment, the weight (mg) ratio or bacterial count (CFU) ratio of Lactobacillus rhamnosus strain GKLC1 (BCRC 910828), Bifidobacterium lactis strain GKK24 (BCRC 911121) and Clostridium butyricum strain GKB7 (BCRC 911120) may be, for example, 1:1:1, while in other embodiments, the weight (mg) ratio or bacterial count (CFU) ratio of Lactobacillus rhamnosus strain GKLC1 (BCRC 910828), Bifidobacterium lactis strain GKK24 (BCRC 911121) and Clostridium butyricum strain GKB7 (BCRC 911120) may be other than 1:1:1, for example, 1:1:(>1-4).

[0027] When applied, the composite probiotic composition may be an oral composition, such as a pharmaceutical composition or a food composition. The use forms of the composite probiotic may include, but are not limited to, the whole fermentation broth, sludge (also called cell pellet), supernatant, and freeze-dried powder. In the above examples, the whole fermentation broth refers to a product containing bacteria and culture broth. The sludge refers to a product obtained after removing the supernatant from the whole fermentation broth. The supernatant refers to a product obtained after removing the sludge from the whole fermentation broth. The freeze-dried powder refers to a freeze-dried powder produced from the whole fermentation broth, sludge, and / or supernatant.

[0028] The application of the complex probiotics to pharmaceutical compositions may include, but is not limited to, pharmaceuticals. The application of the complex probiotics to food compositions may include, but is not limited to, general foods, health foods, beverages, dietary supplements, dairy products, or feeds. In the oral composition, the complex probiotics composition may further include, but is not limited to, pharmaceutical and food acceptable carriers, excipients, and / or additives. In another example, the dosage form of the complex probiotics composition may include, but is not limited to, powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray, or suppository.

[0029] When the composite probiotic is used for preparing a composition for improving the exercise performance of a person with congenital low aerobic exercise ability, an oral composition containing an effective dose of the composite probiotic can be continuously administered to a subject for a certain period of time. The effective dose is determined by the subject and is not particularly limited. For example, when the subject is an adult, the effective dose of the composite probiotic to an adult may be, for example, 50 mg / 60 kg body weight / day to 300 mg / 60 kg body weight / day, but is preferably 75 mg / 60 kg body weight / day to 200 mg / 60 kg body weight / day, and is more preferably about 100 mg / 60 kg body weight / day. In another example, when the subject is a mouse, the effective dose of the composite probiotic to the subject may be, for example, 10.5 mg / kg / day to 63 mg / kg / day, but is preferably 15.75 mg / kg / day to 42 mg / kg / day, and is more preferably about 21 mg / kg / day. In other instances, the administration period may be, for example, at least 28 consecutive days, or may be longer or shorter.

[0030] It should be understood that the following specific strains, specific formulations, specific dosages, specific detection forms, aspects, examples and examples are merely illustrative and do not limit the present invention. Without departing from the spirit and scope of the present invention, the technical characteristics required for the present application can be easily determined, and various changes and modifications can be made to the present invention, and it can be applied to different uses and conditions. Example 1 1.1 Bacterial species source

[0031] The strains used in this example comprise a specific combination of probiotics including Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7. Lactobacillus rhamnosus strain GKLC1 was deposited at the Bioresource Conservation Research Center, Food Industry Development Research Institute (BCRC, No. 331 Food Road, Hsinchu, Taiwan) on February 12, 2018, with deposit number BCRC 910828; Bifidobacterium lactis strain GKK24 was deposited at the BCRC on April 6, 2022, with deposit number BCRC 911121; and Clostridium butyricum strain GKB7 was deposited at the BCRC on April 1, 2022, with deposit number BCRC 911120.

[0032] The above strains GKLC1, GKK24 and GKB7 are all isolated from the human body. For example, the GKLC1 strain is isolated from breast milk. The GKK24 strain is isolated from infant feces, and the GKB7 strain is isolated from feces. After purifying the DNA of the GKK24 and GKB7 strains, the gene sequences are sequenced to obtain the nucleic acid fragment sequences of the 16s rRNA genes of the GKK24 and GKB7 strains, which are shown in sequence identification numbers (SEQ ID NO: 1 and SEQ ID NO: 2, respectively. After sequence alignment, the GKK24 strain is identified as Bifidobacterium lactis, and the GKB7 strain is identified as Clostridium butyricum. The above DNA purification, gene sequencing and sequence alignment are performed by conventional methods, and do not affect the subsequent evaluation of the GKK24 strain for improving innate aerobic fitness, so that the description is omitted. Bifidobacterium lactis strain GKK24 is Gram-stain positive, acid-fast stain negative, obligately anaerobic, nonmotile, nonspore-forming, and lacks capsules and flagella. Clostridium butyricum strain GKB7 is Gram-stain positive, obligately anaerobic, motile, and lacks spores and flagella.

[0033] In some examples, the Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24, and Clostridium butyricum strain GKB7 were inoculated into solid medium to activate the bacterial species. In a preferred embodiment, the solid medium is commercially available MRS (deMan, Rogosa and Sharpe) agar or RCM (Reinforced Clostridial Medium) agar. After colonies were formed, a single colony was taken and placed in a liquid medium to perform liquid culture. In a preferred embodiment, the culture temperature of the strain 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 medium is MRS liquid medium or RCM liquid medium. After the liquid culture was completed, fermentation culture was performed. In a preferred embodiment, the formulation of the fermentation medium is shown in Table 1.

[0034] [Table 1] 1.2 Preparation of lyophilized powder

[0035] After the growth of the strain GKLC1, strain GKK24 and strain GKB7 by fermentation culture was completed, the entire fermentation liquid including the cells and culture liquid was collected and centrifuged at a speed of 1000 rpm to 15000 rpm to obtain a bacterial sludge. The obtained bacterial sludge was mixed with a protective agent (i.e., 6% by weight to 50% by weight of skim milk powder) and then freeze-dried. The freeze-drying was performed by pre-freezing in a gradient setting mode, sequentially storing at 20°C to 0°C for 1 to 4 hours, then storing at 0°C to -20°C for 4 to 8 hours, and then storing at -196°C to -30°C for more than 8 hours. In a preferred embodiment, the freeze-drying temperature and time are storing at -40°C for 2 hours, then storing at -20°C for 2 hours, further storing at 0°C for 2 hours, and finally storing at 20°C for about 10 hours or more. Then, the obtained freeze-dried powder was stored at a low temperature. In another preferred embodiment, the temperature of low temperature storage is -30°C to 4°C. The preserved lyophilized powder may be used as the source of a multiple probiotic composition administered to experimental animals in the following animal studies. 1.3 Test animals

[0036] In the following examples, 24 male mice with congenital low aerobic capacity of ICR breed, 8 weeks old, each weighing about 35 grams, purchased from LASCO Biotechnology Company were selected. The mice were housed in standard breeding cages with 3 mice per cage, and the mouse room had an average temperature of about 22±2°C, an average humidity of 65±5%, 12 hours of daylight and 12 hours of darkness, and sufficient drinking water and standard feed were provided. All animal experimental processes were reviewed and approved by the National Sport University Laboratory Animal Care and Use Committee (IACUC), and approval number IACUC-11011 was obtained. The test animals were arranged in an S-shape according to body weight, and then divided into one control group and three experimental groups, with 6 mice in each group. 1.4 Breeding mice with congenital low aerobic capacity

[0037] Following the method published by Koch and Brittion (2001), mice were screened for high (H) or low (L) intrinsic aerobic exercise capacity (AEC) by swimming endurance time. Male and female individuals with the lowest 20% aerobic exercise capacity were selected from the group and mated once to breed the next generation. Both were mated 8 times, and the 8th generation group was designated as the group with innately low aerobic exercise capacity (L-AEC). 1.5 Screening method for mice with congenital low aerobic fitness

[0038] After the mice were adapted for one week, a debilitating swimming endurance test was conducted. In the test, a lead sheet weighing 5% of the mouse's body weight was fixed at the base of the mouse's tail and placed in a bucket with a depth of 40 cm and a surface diameter of 45 cm. The water temperature was controlled at 28±1°C, and the mouse was made to swim until it was debilitated, after which it was immediately rescued to avoid drowning. In this test, swimming debilitation was defined as the mouse sinking straight down or being unable to return to the surface and ventilate on its own within 7 seconds after sinking underwater. The debilitating swimming endurance test was conducted twice, with the mice allowed to recover their strength at an interval of 72 hours each time, and the better score from the two swimming results of the individual was used for comparison with other mice. The time that the mouse swam until it was debilitated was defined as its aerobic endurance performance. The worse the aerobic endurance performance, the shorter the time that it swam until it was debilitated. 1.6 Study Design

[0039] The test animals were orally administered sterile water (control group) or the probiotic composition of Example 1 (experimental group) every day for 4 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 (corresponding to an effective dose of 100 mg / 60 kg body weight / day for adults). After 4 weeks of intake, the following were analyzed: forelimb grip strength (unit: g), time to exhaustion in swimming under load (unit: min), blood glucose concentration (mg / dL) in oral glucose tolerance test, serum lactate concentration after exercise (unit: mmol / L), serum lactate concentration after exercise and rest (unit: mmol / L), serum urea nitrogen concentration after exercise and rest (unit: mg / dL), liver and muscle glycogen concentration (mg / g liver or mg / g muscle), and epididymal fat weight (which may represent the proportion of visceral fat tissue). 1.7 Data Statistics

[0040] The values ​​in the text are shown as mean ± SD and were analyzed by one-way ANOVA and commercial software. The difference was judged to be statistically significant at p<0.05. Asterisks ** and *** indicate p<0.05 and p<0.005, respectively. Example 2: Evaluation of the improvement of exercise performance in L-AEC subjects by the combined probiotics of Example 1 2.1 Evaluation of forelimb grip strength

[0041] In this example, a test for forelimb grip strength was performed 30 minutes after administration of the probiotic composition of Example 1 on day 29. First, the base of the mouse's tail was grasped and lowered vertically so that both forelimbs were gripping a crossbar (diameter 2 mm, length 7.5 cm) connected to an AIKOH electronic push-pull gauge (Model-RX-5, Aikoh Engineering, Nagoya, Japan), and then the mouse's tail was pulled slightly backward so that the forelimbs were separated from the crossbar. The maximum grip strength of the mouse's forelimbs during this process was recorded using the push-pull gauge, and used as an index for evaluating grip strength.

[0042] Reference is made to FIG. 1, which is a bar graph illustrating the forelimb grip strength (g) of mice in control and experimental groups according to one embodiment of the present invention.

[0043] As shown in FIG. 1, the front limb grip strength of the mice in the control group was about 105 g, the front limb grip strength of the mice in the group orally administered with the GKK24 strain was about 130 g, the front limb grip strength of the mice in the group orally administered with the GKLC1 strain was about 130 g, and the front limb grip strength of the mice in the group orally administered with the GKB7 strain was about 134 g. This demonstrates that the front limb grip strength of the L-AEC mice was significantly improved after the probiotic strain of the present invention was continuously administered to the L-AEC mice for 4 weeks. 2.2 Exercise endurance - assessment of exhaustion time in load swimming

[0044] After completing the forelimb grip strength test, the mice were subjected to a load swimming exhaustion test with a lead sheet of 5% of the mouse's body weight fixed to the base of the mouse's tail. One week before the load swimming exhaustion test, the animals were adapted to swimming in an environment with a diameter of 28 cm, a water depth of 25 cm, and a water temperature of 27 ± 1 ° C. A test was performed with one mouse swimming, and the mouse was placed in a bucket and the test animal was forced to swim. During the entire experiment, the mouse's limbs were constantly moved, and if the limbs floated to the surface and could not move, they were stirred with a stirring rod nearby. Exercise endurance (load swimming exhaustion) performance was indicated by the time recorded until the mouse's head was completely submerged in the water without rising for 8 seconds (after swimming).

[0045] Reference is made to FIG. 2, which is a bar graph showing the time (in minutes) to exhaustion in swimming under load for control and experimental mice in accordance with one embodiment of the present invention.

[0046] As shown in FIG. 2, the mice in the control group had a swimming exhaustion time of about 0.65 minutes, the mice in the GKK24 oral administration group had a swimming exhaustion time of about 1.20 minutes, the mice in the GKLC1 oral administration group had a swimming exhaustion time of about 0.94 minutes, and the mice in the GKB7 oral administration group had a swimming exhaustion time of about 1.15 minutes, demonstrating that the exercise endurance of the L-AEC mice could be significantly improved after four consecutive weeks of administration of the probiotic strain of the present invention to the L-AEC mice. 2.3 Oral Glucose Tolerance Test (OGTT)

[0047] Oral glucose tolerance test was used to evaluate the glucose metabolism ability of the aerobic weak lineage mice after 4 weeks of administration of the strain of this patent. First, 25% glucose solution was placed and then administered to the mice that had been fasted for 12 hours at a dose of 2.5g / kg BW, and the blood glucose levels of the mice at each time point were analyzed by Roche blood glucose meter (AccuChek®, Germany) at 0, 15, 30, 60, and 120 minutes.

[0048] Reference is made to FIG. 3, which is a bar graph illustrating oral glucose tolerance tests of control and experimental mice according to one embodiment of the present invention.

[0049] As shown in FIG. 3, the blood glucose changes (mg / dL, vertical axis) of the mice after oral administration of glucose and then oral administration of the strains GKK24, GKLC1, and GKB7 were all lower than those of the control group, demonstrating that the probiotic strains of the present invention can definitely contribute to improving the glucose tolerance of L-AEC mice after four consecutive weeks of administration to the L-AEC mice. 2.4 Evaluation of serum lactate concentrations after exercise and rest

[0050] After each group of mice had swum without load for 10 min and rested for 20 min, their blood samples were collected and centrifuged at 1500 × g and 4 °C for 10 min, and serum lactate concentrations were analyzed using a fully automated biochemical analyzer (Hitachi 7060, Hitachi, Tokyo, Japan).

[0051] Reference is made to FIG. 4, which is a bar graph showing serum lactate concentrations (mmol / L) after exercise in control and experimental mice according to one embodiment of the present invention.

[0052] The serum lactate concentration of the mice after exercise is shown in Figure 4. The serum lactate concentration of the control mice was about 6.55mmol / L, the serum lactate concentration of the mice orally administered with strain GKK24 was about 5.15mmol / L, the serum lactate concentration of the mice orally administered with strain GKLC1 was about 4.81mmol / L, and the serum lactate concentration of the mice orally administered with strain GKB7 was about 5.04mmol / L, demonstrating that the probiotic strain of the present invention can significantly reduce the accumulation of serum lactate after exercise in all L-AEC mice after 4 weeks of continuous administration.

[0053] Reference is made to FIG. 5, which is a bar graph showing serum lactate concentrations (mmol / L) after exercise and after rest in control and experimental mice according to one embodiment of the present invention.

[0054] As shown in FIG. 5, after the mice exercised and rested, the serum lactate concentration of the mice in the control group was about 5.59 mmol / L, the serum lactate concentration of the mice in the oral administration group of GKK24 strain was about 4.10 mmol / L, the serum lactate concentration of the mice in the oral administration group of GKLC1 strain was about 3.60 mmol / L, and the serum lactate concentration of the mice in the oral administration group of GKB7 strain was about 3.87 mmol / L, demonstrating that after the probiotic strain of the present invention was continuously administered to L-AEC mice for 4 weeks, it could significantly improve the serum lactate removal effect during rest after exercise in all cases. 2.5 Evaluation of serum urea nitrogen concentration after exercise and rest

[0055] After each group of mice had 90 min of unloaded swimming and 60 min of rest, their blood samples were collected and centrifuged at 1500 × g and 4 °C for 10 min, and the serum urea nitrogen concentration was analyzed using a fully automated biochemical analyzer (Hitachi 7060, Hitachi, Tokyo, Japan).

[0056] Reference is made to FIG. 6, which is a bar graph showing serum urea nitrogen concentrations (mg / dL) after exercise and rest in control and experimental mice according to one embodiment of the present invention.

[0057] As shown in FIG. 6, the serum urea nitrogen concentration of the mice in the control group was about 39.10 mg / dL, the serum urea nitrogen concentration of the mice in the oral administration group of the GKK24 strain was about 30.15 mg / dL, the serum urea nitrogen concentration of the mice in the oral administration group of the GKLC1 strain was about 30.85 mg / dL, and the serum urea nitrogen concentration of the mice in the oral administration group of the GKB7 strain was about 30.70 mg / dL. It was demonstrated that after the probiotic strain of the present invention was continuously administered to the L-AEC mice for 4 weeks, the serum urea nitrogen concentration at rest after exercise could be significantly reduced in all cases. 2.6 Assessment of glycogen concentration in liver and muscle tissue

[0058] After the test was completed, the liver and muscle tissues of the mice were taken and analyzed for glycogen concentration. 100 mg of liver or muscle tissue was homogenized with 0.5 ml of 10% glacial perchloric acid, and then centrifuged at 15,000xg and 4°C for 15 minutes. 30 μl of the supernatant was taken and added to a 96-well plate, mixed with 200 μl of iodine-potassium iodide reagent, and iodine was allowed to bind to glycogen for 10 minutes to produce a brown color. The glycogen concentration in the tissue was detected by ELISA (Tecan Infinite M200, Tecan Austria, Salzburg, Austria) at an absorbance wavelength of 460 nm. Standard glycogen purchased from Sigma was used as a calibration curve to convert the glycogen concentration in the tissue (mg / g tissue).

[0059] 7 and 8 are bar graphs showing glycogen concentrations (mg / g liver) in mouse liver (FIG. 7) and muscle (FIG. 8) of the control and experimental groups, respectively, according to one embodiment of the present invention.

[0060] As shown in FIG. 7, the glycogen concentration in the liver of mice in the control group was about 7.36 mg / g liver, the glycogen concentration in the liver of mice in the oral administration group of the GKK24 strain was about 10.77 mg / g liver, the glycogen concentration in the liver of mice in the oral administration group of the GKLC1 strain was about 13.57 mg / g liver, and the glycogen concentration in the liver of mice in the oral administration group of the GKB7 strain was about 14.31 mg / g liver, demonstrating that after four consecutive weeks of administration of the probiotic strain of the present invention to L-AEC mice, the glycogen concentration in the liver of all mice was significantly improved.

[0061] As shown in FIG. 8, the glycogen concentration in the muscle of the mice in the control group was about 1.07 mg / g muscle, the glycogen concentration in the muscle of the mice in the oral administration group of the GKK24 strain was about 2.21 mg / g muscle, the glycogen concentration in the muscle of the mice in the oral administration group of the GKLC1 strain was about 2.24 mg / g muscle, and the glycogen concentration in the muscle of the mice in the oral administration group of the GKB7 strain was about 2.24 mg / g muscle, demonstrating that after four consecutive weeks of administration of the probiotic strain of the present invention to L-AEC mice, the glycogen concentration in the muscles could be significantly improved in all cases. 2.7 Assessment of epididymal fat weight

[0062] After completion of the study, the epididymis of the mice are removed and weighed, and the weight of epididymal adipose tissue is recorded and may represent the percentage of visceral adipose tissue.

[0063] Reference is made to FIG. 9, which is a bar graph showing epididymal fat weight (g) of control and experimental mice according to one embodiment of the present invention.

[0064] As shown in FIG. 9, the epididymal fat weight of the mice in the control group was about 0.42 g, the epididymal fat weight of the mice in the oral administration group of strain GKK24 was about 0.32 g, the epididymal fat weight of the mice in the oral administration group of strain GKLC1 was about 0.30 g, and the epididymal fat weight of the mice in the oral administration group of strain GKB7 was about 0.31 g, demonstrating that the proportion of visceral fat tissue could be significantly reduced in all of the L-AEC mice after four consecutive weeks of administration of the probiotic strain of the present invention.

[0065] In summary, the specific strain, specific formulation, specific dosage, specific detection form, or specific evaluation method in the present invention are merely examples to describe the composite probiotic composition and its use for improving the athletic performance of individuals with congenital low aerobic fitness.However, those skilled in the art can understand that, without departing from the spirit and scope of the present invention, other strains, other formulations, other dosages, other detection forms, or other evaluation methods can also be applied to the composite probiotic composition and its use for improving the athletic performance of individuals with congenital low aerobic fitness, by combining two or three of Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24, and Clostridium butyricum strain GKB7, and are not limited to the above.For example, the composite probiotic composition may be, for example, a pharmaceutical composition or a food composition, and may selectively contain pharmaceutical and food acceptable carriers, excipients, and / or additives, and may be prepared into dosage forms such as powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray, or suppository.

[0066] According to the above examples, the composite probiotic composition for improving the exercise performance of subjects with congenital low aerobic exercise capacity of the present invention has the following advantages: the composite probiotic composition comprises Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7, and after being continuously administered to subjects with congenital low aerobic exercise capacity for a certain period of time, it can effectively reduce serum lactate and serum urea nitrogen after aerobic exercise, reduce visceral fat and / or improve glycogen concentration in liver and muscle, and can be used as an active ingredient in the preparation of various compositions in the future.

[0067] While the present invention has been disclosed above in terms of several specific embodiments, other embodiments may be realized, and therefore the spirit and scope of the present invention should not be limited to the description of the embodiments contained herein. [Explanation of symbols]

[0068] none [Deposit of biological materials] Domestic deposit information (please note in the order of depository organization, date, and number) The Lactobacillus rhamnosus strain GKLC1 was deposited at the Bioresource Conservation Research Center, Food Industry Development Institute, Republic of China Foundation, No. 331 Food Road, Hsinchu, Taiwan, with the date of deposit on February 12, 2018, the deposit number being BCRC 910828, and the viability of the strain was confirmed on February 26, 2018. The Bifidobacterium lactis strain GKK24 was deposited at the Bioresource Conservation Research Center, Food Industry Development Institute, Republic of China Foundation, No. 331 Food Road, Hsinchu, Taiwan, with the deposit date of April 6, 2022, the deposit number of which is BCRC 911121, and the survival of the strain was confirmed on April 14, 2022. Clostridium butyricum strain GKB7 was deposited at the Bioresource Conservation Research Center, Food Industry Development Institute, Republic of China Foundation, No. 331 Food Road, Hsinchu, Taiwan, with the date of deposit on April 1, 2022, the deposit number is BCRC 911120, and the survival of the strain was confirmed on April 15, 2022.

Claims

1. The Lactobacillus rhamnosus strain GKLC1, Bifidobacterium lactis strain GKK24 and Clostridium butyricum strain GKB7 are included, wherein the Lactobacillus rhamnosus strain GKLC1 was deposited at the Bioresource Conservation Research Center, Food Industry Development Research Center (BCRC, No. 331 Food Road, Hsinchu, Taiwan) on February 12, 2018 and has the deposit number BCRC 910828, and the Bifidobacterium lactis strain GKK24 was deposited at the BCRC on April 6, 2022 and has the deposit number BCRC 910828. 911121, and the Clostridium butyricum strain GKB7 was deposited at the BCRC on April 1, 2022, with the accession number BCRC 911120, The subject with innately low aerobic fitness is an 8th generation individual ranked in the bottom 20% in aerobic endurance fitness, The subject is a mouse, and the subject has been adapted to swimming training for one week.

2. The complex probiotic composition for improving exercise endurance in subjects with congenital low aerobic fitness as described in claim 1, wherein the complex probiotic composition is an oral composition.

3. The complex probiotic composition for improving exercise endurance in subjects with congenital low aerobic fitness as described in claim 2, wherein the complex probiotic composition is a pharmaceutical composition or a food composition.

4. The composite probiotic composition for improving exercise endurance in subjects with congenital low aerobic exercise capacity according to claim 2, further comprising a pharmaceutical and food acceptable carrier, excipient and / or additive.

5. The composite probiotic composition for improving exercise endurance in subjects with congenital low aerobic fitness as described in claim 1, wherein the dosage form of the composite probiotic composition includes powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray or suppository.

6. The composite probiotic composition for improving exercise endurance in a subject with congenitally low aerobic fitness as described in claim 1, wherein the improvement in exercise endurance in the subject with congenitally low aerobic fitness comprises reducing serum lactate concentration and serum urea nitrogen concentration after aerobic exercise, reducing visceral fat percentage, and / or improving glycogen concentration in the liver and muscle.

7. 1. Use of a probiotic complex for the preparation of a composition for improving exercise endurance in a subject with congenital low aerobic fitness, comprising administering to said subject for at least 28 consecutive days an oral composition comprising an effective dose of a probiotic complex, said probiotic complex comprising active ingredients Lactobacillus rhamnosus strain GKLC1 (BCRC 910828), Bifidobacterium lactis strain GKK24 (BCRC 911121) and Clostridium butyricum strain GKB7 (BCRC 911120), The subject with innately low aerobic fitness is an 8th generation individual ranked in the bottom 20% of aerobic fitness, The subject is a mouse, and the subject has been adapted to swimming training for one week.

8. The use of a complex probiotic for preparing a composition for improving exercise endurance in a subject with congenitally low aerobic fitness as described in claim 7, wherein the improvement in exercise endurance in a subject with congenitally low aerobic fitness comprises reducing serum lactate and serum urea nitrogen after aerobic exercise, reducing visceral fat, and / or improving liver and muscle glycogen concentrations.

9. Use of a complex probiotic for preparing a composition for improving exercise endurance in a subject with congenitally low aerobic exercise capacity as described in claim 7, wherein the effective dose of the complex probiotic administered to the subject is 10.5 mg / kg / day to 63 mg / kg / day.

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