Anti-fatigue composition containing lactobacillus

Abstract

To provide an anti-fatigue composition containing Lactobacillus.SOLUTION: The present disclosure relates to an anti-fatigue composition containing Lactobacillus. The composition includes at least one of Lactobacillus brevis GKEX, Lactobacillus plantarum GKK1 and Lactobacillus johnsonii GKJ2, is administered to a healthy subject for a continuous period of time to significantly improve fatigue-related biochemical indices and to prolong aerobic exercise time to exhaustion, and can be used as an active ingredient for preparation of various compositions for preventing fatigue and / or improving athletic ability.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to a probiotic composition, and in particular to an anti-fatigue composition containing Lactobacillus that effectively improves fatigue-related biochemical values ​​and extends the time it takes to become exhausted during aerobic exercise (hereinafter also referred to as "time to exhaustion"). [Background technology]

[0002] Modern people live busy lives and are under a lot of competitive pressure, which can lead to fatigue and overwork, which can be harmful to health. Fatigue is caused by excessive work, and as a result, physiological functions decline, leading to a decline in physical strength and athletic ability.

[0003] Fatigue can also be divided into mental fatigue and physical fatigue. Physical fatigue refers to a state in which the body cannot maintain normal function at a certain standard, resulting in a decline in physical strength and athletic ability. When physical fatigue continues, the human body remains fatigued. Excessive pressure and excessive work for a long time prevent the body from getting enough rest. If fatigue is not relieved even after rest, affects daily life, and activity is reduced by 50% or more, it may become chronic fatigue syndrome (CFS), and symptoms include muscle pain, generalized muscle weakness, generalized headache, sleep disorder, mental symptoms or neurological symptoms, which further affect work and daily life.

[0004] Probiotics are generally defined as bacteria that are beneficial to human health, can grow in the human intestinal tract, and are non-pathogenic. Usually, probiotics can achieve functions such as adjusting the constitution, changing some metabolic pathways in the human body, and improving the entire digestive tract and metabolic capacity. Currently, Lactobacillus occupies the majority of the most commonly used probiotic strains, and past studies have shown that in addition to the effects of improving obesity and strengthening immunity, certain strains of Lactobacillus have significant antioxidant activity, maintaining intestinal permeability, and inhibiting the growth of intestinal gas-producing bacteria. Summary of the Invention [Problem to be solved by the invention]

[0005] However, there is currently relatively little research into the anti-fatigue functions and athletic performance improvement of Lactobacillus, and there is an urgent need to develop an anti-fatigue composition containing Lactobacillus. [Means for solving the problem]

[0006] Accordingly, one aspect of the present invention provides a lactobacillus-containing anti-fatigue composition comprising at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2.

[0007] Another aspect of the present invention provides an anti-fatigue composition containing Lactobacillus, which comprises continuously administering to a healthy subject an oral composition containing an effective dose of Lactobacillus for a certain period of time, and which has the above-mentioned Lactobacillus strain as an active ingredient, thereby improving fatigue-related biochemical values ​​and extending the time until exhaustion occurs during aerobic exercise.

[0008] According to the above aspect of the present invention, there is provided a composition for anti-fatigue containing Lactobacillus. In one embodiment, the composition for anti-fatigue contains at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2. Lactobacillus brevis GKEX was deposited at the National Institute of Technology and Evaluation, Japan Patent Microorganism Deposit Center (NITE-IPOD, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on August 5, 2022, and has the deposit number BP-03696. Lactobacillus plantarum strain GKK1 was deposited at the NITE-IPOD on August 5, 2022, and has the deposit number BP-03702. Lactobacillus johnsonii strain GKJ2 was deposited at NITE-IPOD on November 6, 2020, with the deposit number NITE BP-03315.

[0009] In the above embodiment, the anti-fatigue composition may be an oral composition, such as a pharmaceutical composition or a food composition, etc. In the above embodiment, the anti-fatigue composition further comprises a pharmaceutical and food acceptable carrier, excipient, and / or additive.

[0010] In the above embodiments, the dosage form of the anti-fatigue composition may include powder, tablet, granule, microcapsule, ampoule, or liquid spray.

[0011] In the above embodiment, the anti-fatigue composition has anti-fatigue and / or athletic performance improving functions, including extending the exhaustion time of swimming, extending the exhaustion time of running, reducing the serum lactate increase rate and serum urea nitrogen concentration after aerobic exercise, and / or increasing liver glycogen content.

[0012] According to another aspect of the present invention, there is provided a composition for anti-fatigue containing lactobacillus, comprising administering an oral composition containing an effective dose of lactobacillus to a healthy subject for at least 4 weeks. In this embodiment, lactobacillus is an active ingredient, and the lactobacillus includes at least one of Lactobacillus brevis strain GKEX (BP-03696), Lactobacillus plantarum strain GKK1 (BP-03702), and Lactobacillus johnsonii strain GKJ2 (NITE BP-03315).

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

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

[0015] The above-mentioned lactobacillus-containing anti-fatigue composition of the present invention comprises at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2, and when continuously administered to healthy subjects for a certain period of time, it can effectively improve fatigue-related biochemical values ​​and extend the exhaustion time of aerobic exercise, and can further be used as an active ingredient to manufacture various compositions for improving anti-fatigue and / or athletic performance.

[0016] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further understanding of the invention as claimed. In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, reference is made to the accompanying drawings in which: [Brief description of the drawings]

[0017] [Figure 1] 1 shows a bar graph of exhaustion time during weight-loaded swimming for mice in a blank control group and an experimental group according to one embodiment of the present invention. [Diagram 2] 1 shows a bar graph of running exhaustion time for mice in blank control and experimental groups according to one embodiment of the present invention. [Diagram 3] 1 shows a bar graph of the increase rate of blood lactate after exercise in mice of a blank control group and an experimental group according to an embodiment of the present invention. [Figure 4] 1 shows a bar graph of blood urea nitrogen concentration after exercise in mice from a blank control group and an experimental group according to an embodiment of the present invention. [Diagram 5] 1 shows a bar graph of liver glycogen content (mg / g liver tissue) of mice in blank control and experimental groups according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] If a definition or usage of a term in a reference is inconsistent with or contrary to a definition of that term in this specification, the definition in the reference shall not be used and the definition in this specification shall be used instead. Also, unless otherwise defined in the context, singular terms may include pluralities and plural terms may include the singular.

[0019] As described above, the present invention provides an anti-fatigue composition containing Lactobacillus, which, when continuously administered to healthy subjects for a certain period of time, can effectively reduce serum lactate after aerobic exercise, reduce serum urea nitrogen after aerobic exercise, increase liver glycogen content, increase muscle glycogen content, and / or reduce visceral fat.

[0020] In one embodiment, the term "lactobacillus" as used herein may refer to different strains of the same genus, such as at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2. In one embodiment, Lactobacillus brevis strain GKEX was deposited at the National Institute of Technology and Evaluation, Japan Patent Microorganisms Depositary (NITE-IPOD, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on August 5, 2022, and has the deposit number BP-03696. Lactobacillus plantarum strain GKK1 was deposited at NITE-IPOD on August 5, 2022, with the deposit number BP-03702. Lactobacillus johnsonii strain GKJ2 was deposited at NITE-IPOD on November 6, 2020, with the deposit number NITE BP-03315. In the present invention, it is necessary to select a specific strain of lactobacillus, and it is additionally explained that only the obtained composition for anti-fatigue containing lactobacillus can exert the effect of anti-fatigue and / or improving athletic ability on healthy subjects. If the lactobacillus is not selected from the above specific strains, or the strain is increased or decreased, or a part or all of it is replaced with another strain of the same species, it cannot be expected that the effect of anti-fatigue and / or improving athletic ability will be obtained on healthy subjects.

[0021] The anti-fatigue composition containing Lactobacillus has anti-fatigue function and / or exercise performance improvement function.Generally, the evaluation items of "anti-fatigue function" here include, but are not limited to, the decrease in serum lactate rise rate and serum urea nitrogen concentration after aerobic exercise, and / or the increase in liver glycogen content, and the evaluation items of "exercise performance improvement function" include, but are not limited to, the extension of the exhaustion time of aerobic exercise.In one embodiment, the oral composition containing an effective dosage of Lactobacillus can be continuously administered to healthy subjects for at least 4 weeks to effectively improve fatigue-related biochemical values ​​(e.g., decrease serum lactate rise rate and serum urea nitrogen concentration after aerobic exercise, and / or increase liver glycogen content), and significantly extend the exhaustion time of aerobic exercise (e.g., extend the exhaustion time of swimming, extend the exhaustion time of running).

[0022] In one embodiment, the content of each of the above Lactobacillus strains is not particularly limited. In one example, the weight (mg) ratio or cell count (CFU) ratio of the Lactobacillus brevis strain GKEX (BP-03696), Lactobacillus plantarum strain GKK1 (BP-03702), and Lactobacillus johnsonii strain GKJ2 (NITE BP-03315) may be, for example, 1:1:1, but in another example, the weight (mg) ratio or cell count (CFU) ratio of the above Lactobacillus brevis strain GKEX (BP-03696), Lactobacillus plantarum strain GKK1 (BP-03702), and Lactobacillus johnsonii strain GKJ2 (NITE BP-03315) may be a ratio other than 1:1:1, for example, 1:1: (>1 to 10).

[0023] In application, the anti-fatigue composition containing Lactobacillus may be an oral composition, for example, a pharmaceutical composition or a food composition. The above-mentioned use forms of Lactobacillus include, but are not limited to, whole fermentation broth, sludge (also called cell pellet), supernatant, and freeze-dried powder. In the above examples, the above-mentioned whole fermentation broth refers to a product containing bacteria and culture broth. The above-mentioned sludge refers to a product after removing the supernatant from the whole fermentation broth. The above-mentioned supernatant refers to a product after removing the sludge from the whole fermentation broth. The above-mentioned freeze-dried powder is prepared with the whole fermentation broth, sludge, and / or supernatant, and still contains other inseparable components.

[0024] The above-mentioned pharmaceutical composition can be applied to, but is not limited to, medicines.The above-mentioned food composition can be applied to, but is not limited to, general food, health food, beverage, nutritional supplement, dairy product or feed.In the above-mentioned oral composition, the anti-fatigue composition containing Lactobacillus can further optionally contain pharmaceutical and food acceptable carriers, excipients and / or additives.In another example, the dosage form of the anti-fatigue composition containing Lactobacillus can include, but is not limited to, powder, tablet, granule, microcapsule, ampoule, or liquid spray.

[0025] When the above-mentioned lactobacillus is used as a composition for improving the exercise performance of a subject, an oral composition containing an effective dose of lactobacillus may be continuously administered to the subject for a certain period of time. In one embodiment, the subject is not particularly limited, and may be, for example, a healthy subject. In this embodiment, the above-mentioned effective dose is not particularly limited, and is determined by the subject. For example, when the subject is an adult, the effective dose of lactobacillus for an adult may be, for example, 50mg / 60kg body weight / day to 1900mg / 60kg body weight / day, preferably 100mg / 60kg body weight / day to 1800mg / 60kg body weight / day, and more preferably about 100mg / 60kg body weight / day to about 1000mg / 60kg body weight / day. In another example, when the subject is a mouse, the effective dosage of lactobacillus for the subject may be, for example, 10.5 mg / kg body weight / day to 400 mg / kg body weight / day, preferably 15.75 mg / kg body weight / day to 300 mg / kg body weight / day, more preferably about 21 mg / kg body weight / day to about 205 mg / kg body weight / day. In another example, the administration period may be, for example, at least 4 weeks, or may be longer or shorter.

[0026] It is understood that the following specific strains, specific formulations, specific dosages, specific measurement methods, aspects, examples, and examples are merely for illustrative purposes and are not intended to limit 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 skilled in the art of the present invention can easily determine the necessary technical features of the present invention and make various changes and modifications to the present invention in order to suit various applications and conditions without departing from the spirit and scope of the present invention. EXAMPLES

[0027] Example 1

[0028] 1.1 Origin of the bacterial species

[0029] The strains used in this example include a specific combination of probiotics, including Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2. Lactobacillus brevis GKEX was deposited at the National Institute of Technology and Evaluation, Japan Patent Microorganism Deposit Center (NITE-IPOD, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on August 5, 2022, with the deposit number BP-03696. Lactobacillus plantarum strain GKK1 was deposited at the NITE-IPOD on August 5, 2022, with the deposit number BP-03702. Lactobacillus johnsonii strain GKJ2 was deposited at NITE-IPOD on November 6, 2020, with the deposit number NITE BP-03315.

[0030] The above-mentioned strains GKEX, GKK1, and GKJ2 are all isolated from the human body or plants, for example, strain GKEX is isolated from the fermentation liquid of organic vegetables and fruits, strain GKK1 is isolated from sour pepper, and strain GKJ2 is isolated from breast milk.

[0031] In some examples, the Lactobacillus brevis strain GKEX, the Lactobacillus plantarum strain GKK1, and the Lactobacillus johnsonii strain GKJ2 are inoculated into a solid medium to activate the bacterial species. In a specific example, the solid medium is commercially available MRS (deMan, Rogosa and Sharpe) agar or RCM (Reinforced Clostridial Medium) agar. After colonies are generated, a single colony is selected and inoculated into a liquid medium to perform liquid culture. In one example, the culture temperature of the strain is 25°C to 40°C. In one example, the liquid culture time is 16 to 24 hours. In one example, the liquid medium is MRS liquid medium or RCM liquid medium. After the liquid culture is completed, fermentation culture is performed. In one example, the formulation of the fermentation medium is shown in Table 1 below.

[0032] [Table 1]

[0033] 1.2 Preparation of lyophilized powder

[0034] After the growth of the above Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2 is completed by fermentation culture, the whole fermentation liquid containing the bacteria and the culture liquid is collected and centrifuged at a speed of 1000 rpm to 150000 rpm to obtain a bacterial sludge. The obtained bacterial sludge is mixed with a protective agent (i.e., 6% by weight to 50% by weight of skim milk powder) and then freeze-dried. In the above-mentioned freeze-drying, preliminary freezing is performed in a gradient setting mode, and the mixture is successively stored at 20°C to 0°C for 1 to 4 hours, then at 0°C to -20°C for 4 to 8 hours, and then at -196°C to -30°C for 8 hours or more. In a preferred embodiment, the temperature and time of freeze-drying are stored at -40°C for 2 hours, then at -20°C for 2 hours, then at 0°C for 2 hours, and finally at 20°C for about 10 hours or more. The freeze-dried powder obtained above is then stored at low temperature. In another preferred embodiment, the temperature of low temperature storage is -30°C to 4°C. The freeze-dried powder to be stored contains other inseparable components and may be used as a raw material for the anti-fatigue composition containing Lactobacillus, which is administered to experimental animals in the following animal experiments.

[0035] 1.3 Experimental animals

[0036] In the following examples, 6-week-old male ICR mice with an average weight of about 30 grams purchased from Lesco Biotechnology Co., Ltd. are used. All animal experimental procedures were reviewed and approved by the National Sports University Laboratory Animal Care and Use Committee (IACUC). Mice are kept in standard cages, and the mouse room has an average temperature of about 22 ± 2°C and an average humidity of 65 ± 5%, with 12-hour regular light and dark, and daily temperature and humidity values ​​are recorded. Food and water are given ad libitum. After the experimental animals are pre-bred for 2 weeks to adapt to the environment, they are arranged in an S-shape according to their body weight and divided into a total of four groups, one blank control (vehicle) group and three experimental groups (i.e., GKEX group, GKK1 group, and GKJ2 group), with six mice in each group.

[0037] The mice were subjected to a four-week animal experiment, during which lactobacillus was administered daily by tube (hereafter referred to as "tube administration"). After four weeks (from day 1 to day 28 of the experiment) of continuous administration, each exercise capacity and fatigue-related biochemical value were analyzed in sequence. Body weight, food intake, and water intake were recorded during the experiment period.

[0038] 1.4 Experimental design

[0039] The experimental animals were orally administered sterile water (blank control group) or the Lactobacillus of Example 1 (experimental group) daily for 4 weeks (i.e. 28 days). For the effective dose of Lactobacillus in the experimental group mice, the dose of Lactobacillus brevis strain GKEX was 21 mg / kg body weight / day; the dose of Lactobacillus plantarum strain GKK1 was 21 mg / kg body weight / day; and the dose of Lactobacillus johnsonii strain GKJ2 was 205 mg / kg body weight / day (for adults, the dose of GKEX was 100 mg / 60 kg body weight / day, the dose of GKK1 was 100 mg / 60 kg body weight / day, and the dose of GKJ2 was 1000 mg / 60 kg body weight / day). After 4 weeks of intake, the time to exhaustion in weighted swimming (corresponding to exercise endurance, unit: minutes), the time to exhaustion on a treadmill (corresponding to exercise endurance, unit: minutes), and blood biochemical values ​​before and after exercise [serum lactate content (unit: mmol / L), serum urea nitrogen content (unit: mg / dL), liver and muscle glycogen content (mg / g liver or mg / g muscle)] will be analyzed.

[0040] 1.5 Data Statistics

[0041] The values ​​described herein are expressed as mean ± standard deviation (mean ± SD), and were analyzed using one-way ANOVA and commercially available software, with p<0.05 being considered as a statistically significant difference. Asterisk ** indicates p<0.05, and asterisk *** indicates p<0.005.

[0042] Example 2: Evaluation of the anti-fatigue effect of the lactobacillus of Example 1

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

[0044] One week before the experiment, the animals are adapted to swimming (conditions: diameter 28 cm, water depth 25 cm, water temperature 27 ± 1 ° C). After four weeks of experiment, on the 31st day of the experiment, weights (5% of the mouse's body weight, for example, a lead sheet of 5% of the mouse's body weight is fixed to the base of the mouse's tail) are used to perform a weight-loaded swimming exhaustion experiment on the mouse. One week before the weight-loaded swimming exhaustion experiment, the animals are adapted to swimming and placed in an environment with a diameter of 28 cm, a water depth of 25 cm, and a water temperature of 27 ± 1 ° C. After fasting for 12 hours before swimming, feeding and administering Lactobacillus for 30 minutes, the experiment is performed using a single swimming method, the mice are placed in a water tank, the experimental animals are forced to swim, and the water temperature is controlled at 27 ± 1 ° C during the experiment. In addition, during the entire experiment, the limbs of the mice are kept moving, and if the mice float in the water and cannot move their limbs, a stirring rod is used to stir them nearby. Exercise endurance (weight-loaded swimming to exhaustion) performance was demonstrated by recording the time it took for the mouse's head to be completely submerged in water and not to float to the surface for 8 seconds, and the results are shown in Figure 1 .

[0045] FIG. 1 shows a bar graph of exhaustion time during weight-loaded swimming for mice in a blank control group and an experimental group according to one embodiment of the present invention.

[0046] As shown in FIG. 1, the exhaustion time of the blank control group (Vehicle) during weight-loaded swimming was 5.02±0.83 minutes, the exhaustion time of the GKEX group was 9.15±0.73 minutes, the exhaustion time of the GKK1 group was 7.01±0.8 minutes, and the exhaustion time of the GKJ2 group was 8.68±0.94 minutes. Here, according to the statistical analysis results, the exhaustion time of the GKEX, GKK1, and GKJ2 groups was significantly increased by 1.82 times (p=0.0059), 1.40 times (p=0.0006), and 1.73 times (p<0.0001), respectively, compared to the blank control group (Vehicle). Thus, it is shown that the probiotic strains GKEX, GKK1, and GKJ2 of the present invention can all significantly extend the time to exhaustion during swimming when administered continuously to mice for 4 weeks.

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

[0048] After four weeks of experiment, on the 33rd day, a running fatigue experiment was conducted, and the mice were fasted for 12 hours before running. During the test, the mice were placed on a track, and an electric shock area was set at the end of the track, and the experimental animals were forced to run. The conditions of the treadmill were based on the conventional method, with the starting speed of 10 meters / minute (m / min), the gradient of 5%, and the speed was increased by 2 m / min per minute after 5 minutes. If the mouse fell into the electric shock area several times or could not move forward in the electric shock area for more than 5 seconds, it was judged to be exhausted, and the time from the start of running to exhaustion was recorded, and the results are shown in Figure 2.

[0049] FIG. 2 shows a bar graph of running exhaustion time (min) for mice in the blank control group and the experimental group according to one embodiment of the present invention.

[0050] As shown in FIG. 2, the running exhaustion time of the blank control group (Vehicle) was 8.25±1.14 minutes, the running exhaustion time of the GKEX group was 15.32±1.02 minutes, the running exhaustion time of the GKK1 group was 11.63±0.97 minutes, and the running exhaustion time of the GKJ2 group was 15.04±2.36 minutes. Here, according to the statistical analysis results, the running exhaustion time of the GKEX group, GKK1 group, and GKJ2 group was significantly increased by 1.86 times (p<0.0001), 1.41 times (p=0.0001), and 1.82 times (p<0.0001), respectively, compared to the blank control group (Vehicle). Thus, it was shown that the probiotic strains GKEX, GKK1, and GKJ2 of the present invention could significantly extend the time to exhaustion during running when administered continuously to mice for 4 weeks.

[0051] 2.3 Evaluation of fatigue-related biochemical values ​​after exercise

[0052] To evaluate the effect of Lactobacillus on blood biochemical values ​​related to fatigue after exercise, the change in blood lactate concentration before and after exercise in mice was measured according to the "Method for evaluating the anti-fatigue function of health foods" described below, and the increase rate of blood lactate after exercise was calculated. The results are shown in Figure 3.

[0053] FIG. 3 shows a bar graph of the increase rate of blood lactate after exercise in mice of the blank control group and the experimental group according to one embodiment of the present invention.

[0054] As shown in Figure 3, the lactate increase ratios of the blank control group (Vehicle), GKEX group, GKK1 group, and GKJ2 group were 1.85 ± 0.21, 1.45 ± 0.16, 1.46 ± 0.10, and 1.43 ± 0.15, respectively, where statistical analysis results showed that the lactate increase ratios of the GKEX group, GKK1 group, and GKJ2 group were significantly lower than the blank control group (Vehicle) by 21.9% (p = 0.0006), 21.1% (p = 0.0009), and 22.9% (p = 0.0003), respectively. Therefore, it was shown that the probiotic strains GKEX, GKK1, and GKJ2 of the present invention can significantly reduce the lactate increase ratio when continuously administered to mice for 4 weeks.

[0055] Next, to evaluate the effect of Lactobacillus on fatigue-related blood urea nitrogen (BUN) content, on the day of the test (experimental day 35), the rats were fed Lactobacillus for 30 minutes, then made to swim in water without weight for 90 minutes, and after resting for 60 minutes, 0.2 mL of blood was collected. Blood was collected at a total of three time points and subjected to BUN analysis. The results are shown in Figure 4.

[0056] FIG. 4 shows a bar graph of blood urea nitrogen concentration after exercise in mice from the blank control group and the experimental group according to one embodiment of the present invention.

[0057] As shown in FIG. 4, the blood urea nitrogen concentrations of the blank control group (Vehicle), GKEX group, GKK1 group, and GKJ2 group were 35.23±2.62 mg / dL, 28.98±1.42 mg / dL, 32.57±1.22 mg / dL, and 26.05±2.69 mg / dL, respectively. Here, according to the results of statistical analysis, the blood urea nitrogen concentrations of the GKEX group, GKK1 group, and GKJ2 group were significantly lower by 17.7% (p<0.0001), 7.57% (p=0.0064), and 26.06% (p<0.0001), respectively, than the blank control group (Vehicle). Therefore, it was shown that the probiotic strains GKEX, GKK1, and GKJ2 of the present invention were all able to significantly reduce blood urea nitrogen concentrations when continuously administered to mice for 4 weeks.

[0058] 2.4 Assessment of liver glycogen content

[0059] To evaluate the effect of Lactobacillus on liver glycogen content, all animals were allowed to rest for 2 days after the 90-min swim test (experimental day 39), and all animals were sacrificed 30 min after the last feeding. Mouse livers were also harvested and washed with saline, then dried and weighed. For subsequent tissue glycogen content analysis, tissues were cut at the same tissue site, aliquoted, and stored frozen at -80°C.

[0060] In this embodiment, glycogen is directly quantified according to conventional chemical analysis. Briefly, first, the tissue sample to be measured is taken out, and 5 volumes (w / v) of tissue homogenization solution are added, and different tissues are homogenized with a tissue homogenizer Bullet Blender (Next Advance, Cambridge, MA, USA). Then, the tissue homogenization solution is dispensed into a microcentrifuge tube, centrifuged at 4°C and 12000×g for 15 minutes, and a portion of the upper extract is taken out to directly analyze the glycogen content. For the analysis method, please refer to Huang et al., Chicken essence improves exercise performance and ameliorates physical fatigue. Nutrients 6(7): 2681-2696 (2014), which is also cited as a reference in this specification. 30 μl of the supernatant was added to a 96-well plate and mixed with 200 μl of iodine-potassium iodide reagent to bind iodine and glycogen for 10 minutes, resulting in brown color, and then the absorbance value at 460 nm was measured by ELISA (Tecan Infinite M200, Tecan Austria, Salzburg, Austria). In addition, a standard curve was made with a commercially available glycogen (Glycogen, Sigma) standard product, and the changes in liver glycogen reserves in animals from different groups were calculated. The related results are shown in Figure 5.

[0061] FIG. 5 shows bar graphs of liver glycogen content (mg / g liver tissue) of mice in blank control and experimental groups, respectively, according to one embodiment of the present invention.

[0062] As shown in Figure 5, the liver glycogen contents of the blank control group (Vehicle), GKEX group, GKK1 group, and GKJ2 group were 15.75 ± 2.81 mg / g liver tissue, 21.82 ± 1.19 mg / g liver tissue, 25.98 ± 3.00 mg / g liver tissue, and 25.16 ± 1.71 mg / g liver tissue, respectively, and the statistical analysis results showed that the liver glycogen contents of the GKEX group, GKK1 group, and GKJ2 group were significantly increased by 1.51 times (p = 0.0005), 1.65 times (p < 0.0001), and 1.60 times (p < 0.0001) respectively compared to the blank control group (Vehicle). Therefore, the probiotic strains GKEX, GKK1, and GKJ2 of the present invention can all significantly increase liver glycogen content after continuous administration to mice for 4 weeks.

[0063] From the above, in the present invention, a specific strain, a specific formulation, a specific dosage, a specific measurement method, or a specific evaluation method is only used to exemplify the composition for anti-fatigue containing Lactobacillus. However, those skilled in the art should understand that both or three of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2 may be used together without departing from the spirit and scope of the present invention, and other formulations, other dosages, other measurement methods, or other evaluation methods may also be used in the composition for anti-fatigue containing Lactobacillus, and are not limited to the above. For example, the composition for anti-fatigue containing Lactobacillus may be, for example, a pharmaceutical composition or a food composition, and may selectively contain a carrier, excipient, and / or additive that is acceptable for pharmaceuticals and foods, and may be prepared into a dosage form such as a powder, tablet, granule, microcapsule, ampoule, liquid spray, etc.

[0064] According to the above examples, the advantage of the lactobacillus-containing anti-fatigue composition of the present invention is that the composition contains at least one of Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2, and when continuously administered to healthy subjects for a certain period of time, it can improve fatigue-related biochemical values ​​and extend the exhaustion time of aerobic exercise, and in the future, it may be used as an active ingredient to manufacture various compositions that improve anti-fatigue and / or athletic performance.

[0065] While the present invention has been disclosed above in terms of several specific embodiments, other embodiments are possible, and therefore, the spirit and scope of the invention as defined by the appended claims should not be limited to the embodiments contained herein.

Claims

1. The Lactobacillus brevis strain GKEX, Lactobacillus plantarum strain GKK1, and Lactobacillus johnsonii strain GKJ2 are included in the Lactobacillus brevis strain GKEX. The Lactobacillus brevis strain GKEX was deposited at the National Institute of Technology and Evaluation (NITE-IPOD) of Japan Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on August 5, 2022. a composition for increasing liver glycogen content after aerobic exercise, wherein the Lactobacillus plantarum strain GKK1 was deposited at NITE-IPOD on August 5, 2022 and has the deposit number NITEBP-03702, and the Lactobacillus johnsonii strain GKJ2 was deposited at NITE-IPOD on November 6, 2020 and has the deposit number NITE BP-03315.

2. The composition for increasing liver glycogen content after aerobic exercise according to claim 1 , wherein the composition is an oral composition.

3. The composition for increasing liver glycogen content after aerobic exercise according to claim 2 , wherein the composition is a pharmaceutical composition or a food composition.

4. The composition for increasing liver glycogen content after aerobic exercise according to claim 3, further comprising a medicament or food acceptable carrier, excipient and / or additive.

5. The composition for increasing liver glycogen content after aerobic exercise according to claim 1 , wherein the composition is in the form of a powder, tablet, granule, microcapsule, ampoule, or liquid spray.

6. 1. A pharmaceutical composition for increasing liver glycogen content after aerobic exercise, comprising as active ingredients an effective dosage of lactobacillus consisting of Lactobacillus brevis strain GKEX (NITE BP-03696), Lactobacillus plantarum strain GKK1 (NITE BP-03702), and Lactobacillus johnsonii strain GKJ2 (NITE BP-03315), wherein the pharmaceutical composition is orally administered to a healthy subject for four consecutive weeks, and the weight ratio of the strains GKEX, GKK1, and GKJ2 is 1:1:9.

8.

7. 7. The pharmaceutical composition for increasing liver glycogen content after aerobic exercise according to claim 6, wherein the healthy subject is an adult and the effective dosage of the Lactobacillus administered to the subject is 50 mg / 60 kg body weight / day to 1900 mg / 60 kg body weight / day.

8. The pharmaceutical composition for increasing liver glycogen content after aerobic exercise according to claim 6, wherein the healthy subject is a mouse and the effective dosage of the lactobacillus administered to the subject is 10.5 mg / kg body weight / day to 400 mg / kg body weight / day.

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