Use of lactic acid bacteria in preparation of composition for increasing muscle mass and reducing body fat

Abstract

The present invention relates to a lactic acid bacteria in preparation of an oral composition for increasing muscle mass, decreasing body fat and enhancing exercise performance. The lactic acid bacteria including Lactobacillus brevisGKEX can be administered to a healthy and non-athelic subject in a continuation period, for significantly increasing muscle mass and reducing body fat, as well as enhancing physical performance metrics such as muscle strength, maximal oxygen uptake (VO 2max), explosive force, and anaerobic power. Therefore, the lactic acid bacteria can be an active ingredient for use in the preparation of various compositions for increasing muscle mass, decreasing body fat and enhancing exercise performance.

Description

Technical Field

[0001] This invention relates to a lactobacillus composition and its use, particularly to the use of a short lactic acid bacteria strain GKEX in the preparation of compositions for muscle building, fat loss and improved athletic performance. Prior Technology

[0002] obesity

[0003] Obesity is a growing global health challenge, with rates rising sharply since the 1980s. Obesity is typically caused by energy intake exceeding energy expenditure, a phenomenon primarily attributed to changes in modern lifestyles, including the prevalence of high-calorie diets and declining levels of daily activity. Obesity is closely linked to a variety of health problems, such as cardiovascular disease, type 2 diabetes, certain cancers, and joint diseases. These diseases not only pose risks to individual health but also place a significant burden on global public health systems. Beyond individual health risks, obesity also has negative social and economic consequences, such as increased healthcare costs, reduced productivity, and increased burdens on social security systems.

[0004] Exercise and Muscle Building & Fat Loss

[0005] Besides weight control, exercise is also a key factor in building muscle and losing fat. Building muscle and losing fat is a fitness goal that refers to the process of simultaneously increasing muscle mass and reducing body fat, resulting in a more perfect physique. Weight training and adequate protein intake can increase muscle mass. Exercise stimulates muscle growth, and during the recovery process after training, muscles repair and strengthen. Increasing muscle mass not only enhances the body's metabolic rate, strength, and explosiveness, but also strengthens the support of bone structure, reducing the risk of osteoporosis.

[0006] Exercise increases energy expenditure, helps control weight, and reduces body fat. Regular aerobic exercise (such as running and swimming) and anaerobic exercise (such as weight training) can effectively promote fat metabolism and burning, improve endurance and stamina, and help reduce the risk of cardiovascular disease, type 2 diabetes, and other chronic diseases.

[0007] Building muscle and losing fat is a long-term process that requires consistent exercise and a healthy diet. This is the only way to consistently achieve your ideal physique and health results. Consistent exercise helps change your body composition, increasing muscle mass and reducing fat. Combining this with systematic training, gradually increasing intensity and difficulty, and incorporating different types of training such as aerobic exercise, strength training, flexibility training, and skill training can help improve athletic performance.

[0008] Currently, most sports nutrition supplements are antioxidants and protein supplements, which may put a burden on the liver and kidneys. Therefore, there is an urgent need to develop a lactic acid bacteria composition with muscle-building and fat-reducing functions and to improve athletic performance, as well as its uses.

[0009] Athletes improve athletic performance

[0010] Athletes need to increase muscle strength, explosiveness, and endurance through training to improve athletic performance. Athletes may need to train for several hours every day, which can lead to physical exhaustion. They often need to engage in high-intensity training, including strength training, aerobic training, and technical training. This training puts tremendous stress on muscles, bones, and joints and requires a high degree of endurance and perseverance.

[0011] Probiotics promote muscle gain, fat loss, and enhanced athletic performance

[0012] Probiotics can help maintain the balance of gut microbiota, inhibit the growth of harmful bacteria, and enhance the function of beneficial bacteria, thereby improving gut health. In addition, probiotics can alter the gut environment by producing organic acids, enzymes, and antibacterial substances, which can help improve intestinal problems such as indigestion, diarrhea, and constipation, and enhance the function of the immune system.

[0013] In recent years, an increasing number of studies have shown that probiotics have the potential to increase muscle mass and athletic performance as supplements. In six randomized controlled trials, probiotic supplements enhanced muscle mass and strength. In another study, consuming *Lactiplantibacillus plantarum* TWK10 for six weeks without exercise increased muscle mass, reduced body fat percentage, and decreased exercise fatigue index. Supplementing with probiotics alters the gut microbiota, increasing short-chain fatty acids in the gut, which can serve as an energy source, increasing ATP production and muscle fiber metabolic efficiency, thereby improving exercise endurance. Daily consumption of *L. brevis* FZU0713 for eight weeks effectively modulates liver genes (Acat2, Acox1, Hmgcr, Cd36, Srebp-1c, and Cyp7a1), aiding in fat metabolism.

[0014] Therefore, there is an urgent need to develop new benefits of other probiotics to effectively increase muscle mass, reduce fat, and improve athletic performance. Summary of the Invention

[0015] Therefore, one aspect of the present invention is to provide the use of Lactobacillus in the preparation of compositions for muscle gain, fat loss and improved athletic performance, wherein the Lactobacillus includes the Lactobacillus brevis strain GKEX.

[0016] Secondly, another aspect of the present invention is to provide the use of Lactobacillus in the preparation of a muscle-building composition, which involves administering a composition containing the short lactic acid bacteria strain GKEX to healthy, non-athlete subjects for a period of time to increase the subjects' muscle mass.

[0017] Furthermore, another aspect of the present invention provides the use of Lactobacillus in the preparation of a composition for fat reduction, wherein the composition containing the short lactic acid bacteria strain GKEX is administered to healthy, non-athlete subjects for a period of time to reduce the subjects' body fat.

[0018] Furthermore, another aspect of the present invention is to provide the use of Lactobacillus in the preparation of a composition for improving athletic performance, wherein the composition containing the short lactic acid bacteria strain GKEX is administered to healthy, non-athlete subjects for a period of time to improve the subjects' athletic performance.

[0019] In addition, another aspect of the present invention provides the use of Lactobacillus in the preparation of a composition for muscle gain, fat loss and improved athletic performance, which is administered to healthy non-athlete subjects for a period of time with a composition containing a specific dose of the short lactic acid bacteria strain GKEX.

[0020] According to the above-described form of the present invention, a use of Lactobacillus for preparing components for muscle gain, fat loss and improved athletic performance is proposed, wherein the Lactobacillus may include Lactobacillus brevis strain GKEX, and Lactobacillus brevis strain GKEX is deposited on January 21, 2022 at the Bioresource Conservation and Research Center of the Food Industry Research and Development Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan) with the deposit number BCRC 911099.

[0021] In the above embodiments, the aforementioned composition may be, for example, an oral composition.

[0022] In the above embodiments, the aforementioned components may be, for example, pharmaceutical components or food components.

[0023] In the above embodiments, the aforementioned composition further includes pharmaceutically and food-acceptable carriers, excipients and / or additives.

[0024] In the above embodiments, the dosage form of the aforementioned components may be, for example, powder, tablets, granules, capsules, ampoules, or liquid sprays.

[0025] According to another aspect of the present invention, a use of Lactobacillus for preparing a muscle-building composition is proposed, wherein the Lactobacillus comprises a short lactic acid bacteria strain GKEX, and the composition is administered orally to healthy, non-athlete subjects for at least six consecutive weeks to increase the skeletal muscle mass of the subjects.

[0026] According to another aspect of the present invention, a use of Lactobacillus for preparing a composition for fat reduction is provided, wherein the Lactobacillus comprises a short lactic acid bacteria strain GKEX, and the composition is administered orally to healthy, non-athlete subjects for at least six consecutive weeks to reduce the subjects' body fat.

[0027] According to another aspect of the present invention, a use is provided for Lactobacillus in the preparation of a composition for improving athletic performance, wherein the Lactobacillus comprises a short lactic acid bacteria strain GKEX, and the composition is administered orally to healthy, non-athlete subjects for at least six consecutive weeks to improve at least one of the subjects’ muscle strength, maximum oxygen uptake, explosive power, and anaerobic power.

[0028] According to another aspect of the present invention, a use of Lactobacillus for preparing a composition for muscle gain, fat loss and improved athletic performance is proposed, wherein the Lactobacillus includes a short lactic acid bacteria strain GKEX, and the short lactic acid bacteria strain GKEX was deposited on January 21, 2022 at the Bioresource Conservation and Research Center of the Food Industry Research and Development Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan) with the registration number BCRC 911099. The composition is administered orally to healthy non-athlete subjects for at least six consecutive weeks, and the Lactobacillus is administered to the subjects at a dose of 1 × 10¹⁰ CFU / 60 kg body weight / day to 3 × 10¹⁰ CFU / 60 kg body weight / day.

[0029] In the above embodiments, before administering the aforementioned components, the subject may selectively perform resistance training one to five times per week.

[0030] The use of the above-mentioned lactobacillus of the present invention in the preparation of compositions for muscle gain, fat loss and improvement of athletic performance, wherein the composition contains the short lactic acid bacteria strain GKEX, and the composition is administered orally to healthy non-athlete subjects for at least six consecutive weeks to increase the subjects' skeletal muscle mass, reduce the subjects' body fat and improve the subjects' athletic performance. Therefore, this lactobacillus can be used as an active ingredient to prepare various compositions for muscle gain, fat loss and improvement of athletic performance.

[0031] It is understood that the foregoing general description and the following detailed description are merely illustrative examples intended to provide further explanation of the claimed invention. Simple Explanation of the Diagram

[0032] To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the detailed description of the accompanying drawings is as follows: Figures 1A and 1B are bar-line graphs showing the maximum grip strength of the left hand (Figure 1A) and right hand (Figure 1B) before and after administering a composition containing strain GKEX to healthy, non-athlete subjects according to an embodiment of the present invention. Figure 2 is a bar-line graph showing the maximum oxygen uptake (VO2max) before and after administering a composition containing strain GKEX to healthy, non-athlete subjects according to an embodiment of the present invention. Implementation

[0033] If a cited reference provides a definition or usage of a term that is inconsistent with or contrary to the definition of that term herein, the definition herein shall apply, and the definition in the cited reference shall not apply. Secondly, unless the context otherwise defines, singular terms may include plural forms, and plural terms may include singular forms.

[0034] As previously stated, the present invention provides the use of Lactobacillus in the preparation of compositions for muscle gain, fat loss and improved athletic performance, wherein the Lactobacillus contains Lactobacillus brevis strain GKEX, which, when administered to healthy non-athlete subjects for a period of time, can effectively increase muscle mass, reduce fat and improve athletic performance.

[0035] In one embodiment, the term "lactobacterium" as used herein includes the Lactobacillus brevis strain GKEX. In a specific example, the aforementioned Lactobacillus brevis strain GKEX was registered on January 21, 2022, at the Bioresource Conservation and Research Center, Food Industry Research and Development Institute (BCRC, 331 Food Road, Hsinchu, Taiwan), with registration number BCRC 911099. In other embodiments, the Lactobacillus brevis strain GKEX may be used in combination with other Lactobacillus strains to further improve the effects of muscle gain, fat loss, and improved athletic performance. It should be noted that the present invention requires the selection of specific strains of Lactobacillus so that the resulting composition can exert the effects of improving muscle gain, fat loss, and improved athletic performance in healthy, non-athlete subjects. If the Lactobacillus is not selected from the aforementioned specific strain, or is replaced with other strains of the same species, then after administration to healthy, non-athlete subjects, the effects of muscle gain, fat loss, and improved athletic performance cannot be expected.

[0036] In application, the aforementioned composition containing the short lactic acid bacteria strain GKEX can be, for example, an oral composition, such as a pharmaceutical composition or a food composition. The aforementioned forms of use of *Lactobacillus* can include, but are not limited to, whole fermentation liquid, cell pellet (or bacterial precipitate), supernatant, and lyophilized powder. In the above embodiments, the aforementioned whole fermentation liquid refers to a product comprising bacterial cells and culture medium. The aforementioned cell pellet refers to the product after removing the supernatant from the whole fermentation liquid. The aforementioned supernatant refers to the product after removing the cell pellet from the whole fermentation liquid. The aforementioned lyophilized powder refers to a product prepared from whole fermentation liquid, cell pellet, and / or supernatant, and may also contain other inseparable components.

[0037] Examples of pharmaceutical compositions described above may include, but are not limited to, pharmaceutical products. Examples of Lactobacillus used in food compositions may include, but are not limited to, general foods, health foods, beverages, nutritional supplements, dairy products, or animal feed. In the aforementioned oral compositions, Lactobacillus compositions may selectively include pharmaceutically and food-acceptable carriers, excipients, and / or additives. In other examples, the dosage forms of the aforementioned compositions containing the short lactic acid bacteria strain GKEX may include, but are not limited to, powders, tablets, granules, capsules, ampoules, or liquid sprays.

[0038] When the aforementioned lactobacillus is used to prepare components for muscle building, fat loss, and improved athletic performance, the components containing an effective dose of lactobacillus can be administered to healthy, non-athlete subjects for a continuous period of time. In this embodiment, the aforementioned effective dose depends on the subject and is not particularly limited. For example, according to the conventional conversion method for adult and mouse drug dosages, if the effective dose for adults is 1 mg / kg body weight, the effective dose for mice can be converted to 12.3 mg / kg body weight. In this embodiment, when the subject is an adult, the effective dose of lactobacillus for adults can be, for example, from 1 × 10¹⁰ CFU / 60 kg body weight / day to 3 × 10¹⁰ CFU / 60 kg body weight / day, but 1.5 × 10¹⁰ CFU / 60 kg body weight / day to 2.5 × 10¹⁰ CFU / 60 kg body weight / day is preferred, and about 2 × 10¹⁰ CFU / 60 kg body weight / day is even more preferred. In another example, when the subject is a mouse, the effective dose of Lactobacillus for this subject may be, for example, 1 × 10⁹ CFU / kg body weight / day to 3 × 10⁹ CFU / kg body weight / day, but 1.5 × 10⁹ CFU / kg body weight / day to 2.5 × 10⁹ CFU / kg body weight / day is preferred, and about 2 × 10⁹ CFU / kg body weight / day is even better. Furthermore, in other examples, other doses of the short lactic acid bacteria strain GKEX may be used, for example, less than 1 × 10¹⁰ CFU / 60 kg body weight / day, or greater than 3 × 10¹⁰ CFU / 60 kg body weight / day. In other examples, the aforementioned administration period may be, for example, continuous for at least six weeks, or longer or shorter.

[0039] The aforementioned lactobacilli can be used to prepare components for muscle gain, fat loss, and improved athletic performance. Generally, the assessment criteria for "muscle gain" as used herein may include, but are not limited to, body composition metrics such as weight, body mass index (BMI), muscle mass, muscle thickness, and fascia thickness. The assessment criteria for "fat loss" as used herein may include, but are not limited to, body fat percentage. The assessment criteria for "athletic performance" as used herein may include, but are not limited to, physical performance metrics such as muscle strength (e.g., left-hand grip strength, right-hand grip strength), maximum oxygen uptake, explosive power (e.g., reverse jump, isotonic strength), and anaerobic power.

[0040] In one embodiment, prior to administration of the aforementioned components, the subject may selectively perform resistance training one to five times per week. This resistance training may utilize familiar assessment methods, and the invention is not particularly limited thereto. However, in some instances, resistance training may be, for example, the Wingate Anaerobic Test (WAnT). For instance, this test assesses anaerobic capacity for maximum cycling power, whereby the subject exerts their maximum power output under a percentage load of their body weight over a short period (e.g., 6-30 seconds).

[0041] It is understood that the specific ingredients, formulations, dosages, detection methods, viewpoints, examples, and embodiments described below are for illustrative purposes only and are not intended to limit the scope of the invention. The main features of the invention can be used in various embodiments without departing from its spirit and scope. Therefore, those skilled in the art can easily identify the essential technical features of this invention and make various modifications and refinements to suit different uses and conditions without departing from its spirit and scope.

[0042] Example 1: Preparation of the composition

[0043] 1.1 Source of strains

[0044] The bacterial strains used in this embodiment are specific probiotic strains, including Lactobacillus brevis strain GKEX. Lactobacillus brevis strain GKEX was deposited on January 21, 2022, at the Bioresource Preservation and Research Center, Food Industry Research and Development Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan), with registration number BCRC 911099.

[0045] First, the short lactic acid bacteria strain GKEX was inoculated onto MRS agar solid medium and cultured at 37°C (or room temperature, i.e., 25°C to 40°C) for 16 hours to 3 days (16 hours to 5 days) to activate the strain. The solid culture medium described above is commercially available MRS (De Man–Rogosa–Sharpe) agar medium, which contains 1.0% peptone, 1.0% beef extract, 0.4% yeast extract, 2.0% glucose, 0.5% sodium acetate trihydrate, 0.1% polysorbate 80 (also known as Tween 80), 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 0.02% manganese sulfate heptahydrate, 0.005% manganese sulfate tetrahydrate, 1.0% agar, and a balanced volume of pure water, with the pH adjusted to 6.2 at 25°C.

[0046] After colonies have formed, select a single colony and inoculate it into MRS liquid medium. Incubate at 25°C to 40°C for 16 to 24 hours. Known pH values, agitation speed, and aeration rates can be used for liquid culture, and will not be elaborated further here. After liquid culture, proceed with fermentation culture. The fermentation medium formulation is shown in Table 1 below.

[0047] Table 1 Element Ratio (by weight percentage) carbohydrate 1~10% Yeast extract 0.1~5% Protein 0.1~5% Trace elements 0.01~2% Cysteine 0.01~0.1% Tween-80 0.05~1% pure water Balance

[0048] Fermentation culture can use familiar temperature, pH value, rotation speed, and aeration rate, which will not be elaborated here.

[0049] 1.2 Preparation of lyophilized powder

[0050] After fermentation, the complete fermentation broth (containing bacteria and culture medium) of the short lactic acid bacteria strain GKEX is collected and centrifuged at 1000 rpm to 15000 rpm to obtain a pellet. The aforementioned complete fermentation broth can be centrifuged directly. Alternatively, 15% by weight of skim milk powder can be selectively added to the complete fermentation broth, mixed thoroughly, and allowed to stand for 1 to 4 hours to allow the bacteria to aggregate. Then, centrifuge at 1000 rpm to 15000 rpm. Aggregation of the bacteria further increases the recovery rate.

[0051] The resulting bacterial sludge after centrifugation is mixed with 6 to 50% by weight of skim milk powder as a preservative and then freeze-dried. In a preferred embodiment, the pre-freezing temperature is set in a gradient, sequentially storing at 0°C to -20°C for 1 to 4 hours, then at -15°C to -40°C for 4 to 8 hours, and finally frozen at -196°C to -40°C for more than 8 hours. In another preferred embodiment, the freeze-drying temperature and time are approximately -5°C for about 2 hours, then at -20°C for about 6 hours, and finally at -50°C for about 16 hours.

[0052] The lyophilized powder obtained after freeze-drying is then stored at a low temperature. In a preferred embodiment, the storage temperature is -30°C to 0°C. The stored lyophilized powder is used as the raw material for oral samples to be administered to humans in the following human studies.

[0053] 1.3 Human Trials

[0054] The following examples recruited 52 participants (20 men and 36 women) from the National Taiwan University of Sport, aged between 20 and 39 years, and none of them were athletes. All participants did not meet the following exclusion criteria: (1) BMI ≥ 27; (2) history of metabolic disease, asthma, cancer, cardiovascular disease, or hypertension; (3) limb and neuromuscular motor dysfunction within the past 6 months, indicating an inability to exercise; (4) use of anti-inflammatory, analgesic, acute or chronic medications, or other drugs within the past month; (5) previous hepatobiliary and gastrointestinal surgery (excluding hernia and polyp removal); (6) smoking or drinking alcohol; (7) consumption of probiotic powder, capsules, or tablets (including yogurt and other related foods) within the past two weeks; (8) food or lactic acid bacteria product allergy; (9) being a student or associate of the principal investigator of this human trial. All eligible participants provided written informed consent before the start of the experiment. The trial was conducted after receiving approval from the Institutional Review Board of Lien Hsin Hospital (Taoyuan, Taiwan) (LSHIRB No. 22-022-A2) and was registered on the Clinical Trials.gov website (clinicaltrials.gov), registration number NCT05909475. This human trial was conducted in accordance with the guidelines of the Declaration of Helsinki.

[0055] 1.4 Experimental Design

[0056] After all participants were recruited, they were assigned numbers according to their registration order and randomly divided into two groups for a double-blind trial (each group contained 10 male and 16 female participants): a placebo group (2 capsules daily, each capsule containing 0 CFU of GKEX) and a GKEX group (2 capsules daily, each capsule containing 1 × 10¹⁰ CFU of GKEX). There were no significant differences between the placebo and GKEX groups in age (placebo group: 22.2 ± 1.9 years; GKEX group: 22.6 ± 2.4 years) and height (placebo group: 166.2 ± 8.9 cm; GKEX group: 168.6 ± 8.7 cm). The trial lasted 6 weeks, during which all participants took two capsules daily and underwent resistance training three times a week. Before and after administration of the aforementioned components (or before and after intervention, or before and after use), all participants underwent dietary record tests, exercise performance tests, and blood samples were collected for analysis and measurement of body composition indicators. Of these tests, only body composition needs to be measured every two weeks. Furthermore, all participants must maintain their original lifestyle and habits.

[0057] 1.5 Resistance Training

[0058] All participants underwent resistance training three times per week using pneumatic resistance training equipment. This equipment included abdominal / back 5310, abduction / adduction 3520-HI5, push / pull 3120, leg extension / flexion 3530, twisting rehabilitation 5340, and leg press 5540 (AB Hur Oy, Kokola, Finland). The selection of this equipment was designed to enhance the strength of specific muscle groups, including the upper limbs, back, abdomen, and legs. The use of pneumatic resistance training equipment provided participants with a standardized platform for targeted training, promoting comprehensive strength development in the upper limbs, back, abdomen, and legs. Before the training program began, each participant underwent a 3RM (maximum repetitions) assessment, followed by the calculation of 1RM (maximum repetitions). This calculation used coefficient formulas from previous literature to ensure the determination of appropriate resistance training intensity.

[0059] The training program lasted four weeks, with gradually increasing intensity. In the first week, participants performed a set of 15 repetitions at 60% of their 1RM. In the second week, the intensity increased to 70% of their 1RM, with a set of 12 repetitions. The following week, the intensity further increased to 75% of their 1RM, still with a set of 12 repetitions. Until the final week, participants maintained 75% of their 1RM, completing two sets of 10 repetitions. Researchers adjusted the weight and number of sets of resistance equipment based on each participant's individual 1RM, adhering to the training plan. Participants were required to complete the specified movements and sets in each training session, with multiple professionals present to supervise the integrity and safety of the movements.

[0060] In addition, prior to administering the aforementioned components, subjects may selectively perform resistance training one to five times per week, such as the Wingate Anaerobic Test (WANT). For example, this test assesses anaerobic capacity for maximum cycling power during cycling, where the subject exerts their maximum power output at a percentage of their body weight over a short period (e.g., 6-30 seconds).

[0061] 1.6 Body Composition

[0062] To assess body composition, this embodiment uses a multi-frequency bioelectrical impedance analysis (BIA), such as the InBody 570 (In-body, Seoul, South Korea). This device measures the subject's body composition at frequencies of 1, 5, 50, 260, 500, and 1000 kHz over 60 seconds. During the measurement, the subject stands on the electrodes, cleans their hands and feet, holds the induction handle with arms extended at approximately 30°, and remains silent and still throughout the measurement. Furthermore, the subject fasts for 8 hours prior to the test to ensure optimal conditions for body composition assessment. During the experiment, the subject's body composition was measured every two weeks.

[0063] Because BIA measures skeletal muscle, it may be affected by body water content. Therefore, a comprehensive and reliable dual-energy X-ray absorptiometry (DXA) was used to perform measurements before and after the intervention to obtain additional bone mass data. This embodiment used the Lunar iDXA system from GE Healthcare (Chicago, IL, USA). Subjects were asked to lie supine on the testing bed with their bodies aligned with the center line and their limbs within the detection range to measure the distribution of lean body tissue, fat mass, and bone mineral density. DXA technology uses two different energy X-rays to scan a designated body site, and a scintillation detector receives the X-rays that penetrate the detection site to calculate and analyze parameters such as muscle mass and body fat.

[0064] 1.7 Ultrasound measurement of muscle and fascia thickness

[0065] The muscle and fascia thickness of the biceps brachii and quadriceps femoris muscles were measured using an ultrasound scanner (H1300, BENQ, Taipei, Taiwan). Subjects lay supine with palms facing upwards. For the biceps brachii, the measurement was taken along the straight line from the acromion to the elbow crease, with the ultrasound probe positioned one-third of the way from the elbow crease. For the quadriceps femoris, the probe was positioned along the straight line from the anterior superior iliac spine (ASIS) to the patella. Muscle thickness was analyzed by adding the thicknesses at both ends of the muscle and dividing by two.

[0066] 1.8 Maximum oxygen uptake (VO2max)

[0067] The assessment of subjects' VO2 max and exercise performance was conducted using a treadmill (Pulsar, h / p / cosmos, Traunstein, Bavaria, Germany) and an automated respiratory analyzer (Vmax 29c, Sensor Medics, Yorba Linda, CA, USA). Following the manufacturer's instructions, the flow sensor, as well as the O2 and CO2 sensors, were calibrated before each test to ensure measurement accuracy. The O2 and CO2 sensors used an automatic calibration function. Heart rate (HR) monitoring was performed using a Polar heart rate device. Following the Bruce protocol, the treadmill was started at 7.2 km / h, gradually increasing by 1.8 km / h every 2 minutes until the subject reached fatigue. Maximum O2 consumption was determined when the respiratory exchange rate (VCO2 / VO2, i.e., the volume ratio of CO2 production to O2 consumption) exceeded 1.10 and maximum heart rate (maximum HR = 220 - age) was reached. When calculating VO2max, the average of the three highest peak values ​​was taken as the individual VO2max value.

[0068] 1.9 Grip Strength Test

[0069] Grip strength was measured in kilograms using a Takei digital grip strength meter (TKK 5401, Takei Scientific Instruments Co., Ltd., Kamo, Niigata, Japan). Before the formal test, subjects were instructed to apply minimal force to the grip strength meter to ensure they understood the operating procedure and grip distance. Subjects were randomly assigned to either their dominant or non-dominant hand as the starting point for the test. During the test, subjects were instructed to apply maximum force to the grip strength meter with one hand for at least 5 seconds. To reduce the effect of fatigue, subjects alternated between using both hands at 60-second intervals. This alternation method was repeated three times, and the maximum grip strength of the dominant and non-dominant hands was recorded separately.

[0070] 1.10 Reverse Jump Test (CMJ)

[0071] The reverse jump test is used to assess lower limb maximum speed, strength, and explosive power. In this assessment, subjects are instructed to place their hands on their hips and their feet on a Kistler force measurement platform (9260AA, Kistler GmbH, Winterthur, Switzerland). They are then instructed to squat until their knees are bent at 90 degrees, and immediately jump with maximum force to reach the highest possible vertical height. Each subject performs the reverse jump test three times, and data is collected at designated points. To ensure accuracy, the instrument is calibrated for each individual's body weight. Measured parameters include rate of force development (RFD), relative peak strength (N / kg), and jump height. These indicators collectively provide insight into lower limb dynamics, reflecting the subject's ability to generate force during vertical jumps.

[0072] 1.11 Isotonic Muscle Strength Test (IMTP)

[0073] Isotonic strength testing (IMTP) is used as a diagnostic tool for strength-time. For this purpose, a custom-designed IMTP testing apparatus and two force plates (model 9287BA, Kistler Instruments AG, Winterthur, Switzerland) were used. All subjects stood with their feet equidistant, the bar placed between their thighs to ensure an upright posture, neutral spine, and knee and hip angles set at 140 degrees, familiarizing them with the IMTP testing method. Careful evaluation was conducted during the practice test to confirm the symmetry of the force trail and the stability of the weighing cycle. To reduce the risk of measurement errors associated with postural changes, specific measurements were repeated at 2-minute intervals for each test phase. Recorded parameters included relative peak strength (N / kg) and rate of strength development (RFD). These data provide a nuanced understanding of the subjects' isotonic muscle strength generation capacity and its potential variations under a rigorous exercise program.

[0074] 1.12 Wingate Anaerobic Test (WAnT)

[0075] After a standardized warm-up procedure, all participants underwent the classic Wingate Anaerobic Test (WAnT) using a dynamic bicycle power meter (Monark 894E, Varberg, Sweden), performing a 30-second all-out sprint. Seat height was adjusted to each participant's satisfaction, and toe clips were used to prevent slippage off the pedals. A 5-minute warm-up of approximately 50 watts was performed before the initial test. Following the warm-up, two 3-second preparatory exercises were performed with a load of 3% of the participant's body weight to familiarize them with the resistance. At the start of the test, the resistance was set on the friction band of the power meter. The external load was individually estimated at 5% of body weight. Recorded results included relative average power (W / kg), relative peak power (W / kg), and fatigue index (%). These data assessed the participants' anaerobic capacity, thereby evaluating their average and peak power output and resistance to fatigue during short periods of high-intensity exercise.

[0076] 1.13 Data Processing and Statistical Analysis

[0077] All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS Statistics 25 (IBM Co., Armonk, NY, USA). Paired Student's t-tests were applied to data with a parent group to assess differences before and after the intervention. Post-hoc comparisons were performed using the Bonferroni test. For data without a parent group, the Wilcoxon sign ordination test was used. Unpaired Student's t-tests were used for comparisons between groups, while the Mann-Whitney U test was used for data without a parent group. In all analyses, p < 0.05 was considered statistically significant. Figures with * indicate a statistically significant difference (p < 0.05) between the experimental group (GNEX) and the control group (placebo), and were plotted using Prism v6.0 software.

[0078] Example 2: Evaluation Method

[0079] 2.1 Supplementing GKEX's effects on changes in body composition

[0080] Please refer to Table 1A, which shows the Inbody body composition measurements of subjects every two weeks according to an embodiment of the present invention. Different superscript letters (a, b) indicate significant differences between groups at the same time point (p < 0.05); * indicates a significant effect after use compared to before use (p < 0.05). The results in Table 1A show that, under the same resistance training intervention, there were no significant differences in weight, BMI, muscle mass, and body fat percentage between the placebo group and the GKEX group before use and at weeks 2, 4, and 6 after use. However, within-group comparisons revealed that, compared to before use, the placebo group showed a significant increase in weight and body fat percentage at weeks 2 and 4 after use, and a significant increase in BMI at week 4 after use (p < 0.05). On the other hand, compared to before use, the GKEX group showed a significant increase in muscle mass and a significant decrease in body fat percentage at weeks 2, 4, and 6 after use. Furthermore, to further understand the changes, we calculated the increments at weeks 2, 4, and 6. The results showed that at weeks 2, 4, and 6 after use, the placebo group had significant increases in body weight, BMI, and body fat percentage compared to the GKEX group (p < 0.05); at the same time, the GKEX group had significant increases in muscle mass changes compared to the placebo group at weeks 2, 4, and 6 after use (p < 0.05).

[0081] Table 1A

[0082] Please refer to Table 1B, which shows the measurement results of subjects using DXA whole-body scans according to an embodiment of the present invention. There were no significant differences in lean body mass (LMB), fat body mass (FBM), and bone mass between the placebo group and the GKEX group. However, after 6 weeks of GKEX supplementation, compared with before the intervention, lean body mass (LBM) increased significantly (by 1.01 times, p = 0.0231), while fat body mass (FBM) decreased significantly (by 4.88%, p = 0.0002). Furthermore, regarding the change in total body fat, the GKEX group reduced fat by 1.1 ± 1.3 kg after 6 weeks, significantly higher than the placebo group (0.2 ± 5.5 kg). This demonstrates that the probiotic GKEX helps to alter body composition.

[0083] Table 1B

[0084] 2.2 Supplementing the effects of GKEX on muscle and fascia thickness

[0085] Please refer to Table 2, which shows the results of muscle and fascia thickness measurements in subjects according to an embodiment of the present invention. In Table 2, data are expressed as mean ± standard deviation (mean ± SD). Different superscript letters (a, b) indicate significant differences between groups at the same time point (p < 0.05); * indicates a significant effect after intervention compared to before intervention (p < 0.05). Delta is the value after intervention minus the value before intervention. After 6 weeks of GKEX supplementation, the thickness of the quadriceps femoris in the right and left legs was significantly increased compared to the placebo group (1.09-fold increase, p = 0.0124 and 1.09-fold increase, p = 0.0231, respectively). In intragroup comparisons, after 6 weeks of GKEX supplementation, compared with pre-intervention levels, there were significant increases in the right biceps brachii (1.08-fold increase, p < 0.0001), right quadriceps brachii (1.05-fold increase, p < 0.0001), left biceps brachii (1.08-fold increase, p < 0.0001), and left quadriceps brachii (1.03-fold increase, p = 0.0020). Furthermore, the changes in GKEX levels in the right biceps brachii (p < 0.0001), right quadriceps brachii (p < 0.0001), left biceps brachii (p < 0.0001), and left quadriceps brachii (p = 0.0037) groups were significantly higher than those in the placebo group. The time-effects (p < 0.05) and group-time interaction effects (p < 0.05) of the right biceps brachii, right quadriceps femoris, left biceps brachii, and left quadriceps femoris were all significant. However, no significant differences were found between groups in post-hoc tests.

[0086] Regarding fascia, there were no significant differences in the thickness of the right biceps brachii, right quadriceps femoris, and left biceps brachii before and after intervention between the placebo group and the GKEX group. The thickness of the left quadriceps femoris in the GKEX group was significantly greater than that in the placebo group before intervention and after 6 weeks of intervention. Furthermore, compared to pre-intervention, the GKEX group showed significant increases in the right biceps brachii (1.20-fold increase, p < 0.0001), right quadriceps femoris (1.12-fold increase, p = 0.0092), and left biceps brachii (1.09-fold increase, p = 0.0067). The changes in the GKEX group were significantly greater than those in the right biceps brachii only in the placebo group (p < 0.0001).

[0087] Table 2

[0088] 2.3 Supplementing the impact of GKEX on maximum grip strength

[0089] Please refer to Figures 1A and 1B, which are bar-line graphs 101 and 103 showing the maximum grip strength of the left hand (Figure 1A) and right hand (Figure 1B) before and after administration of a composition containing strain GKEX to healthy, non-athlete subjects according to an embodiment of the present invention. The results in Figures 1A and 1B show that there were no significant differences in maximum grip strength of the left and right hands between the placebo group and the GKEX group before and 6 weeks after the intervention. However, in intragroup comparisons, only the GKEX supplementation group showed a significant increase in maximum grip strength of both the right and left hands compared to before the intervention (an increase of 1.04-fold, p=0.0374, and 1.06-fold, p=0.0055, respectively).

[0090] 2.4 Effect of GKEX Supplementation on Maximum Oxygen Uptake (VO2max)

[0091] Figure 2 is a bar-line graph showing the maximal oxygen uptake (VO2max) before and after administration of a composition containing strain GKEX to healthy, non-athlete subjects according to an embodiment of the present invention. The results in Figure 2 show that before the intervention, there was no significant difference in VO2max between the placebo group and the GKEX group. After 6 weeks of intervention, the VO2max values ​​in the placebo group and the GKEX group were 42.9 ± 4.9 and 42.5 ± 4.8 (mL / min / kg), respectively, with no significant difference between the two groups. However, within-group comparisons, the VO2max in the GKEX group was significantly increased compared to before the intervention (an increase of 1.02-fold, p = 0.0109). VO2max exhibited a significant time effect (p < 0.05) and a group × time interaction effect (p < 0.05).

[0092] 2.5 Supplement to the impact of GKEX on maximum vertical jump height

[0093] Please refer to Table 3 for the results of the test motor performance (reverse jump test), where data are expressed as mean ± standard deviation (mean ± SD). Different superscript letters (a, b) indicate significant differences between groups at the same time point (p < 0.05); * indicates a significant effect after intervention compared to before intervention (p < 0.05). The results in Table 3 show that although there were no significant differences in relative peak strength, rate of strength development (RFD), and jump height between the placebo group and the GKEX group before and after intervention, after 6 weeks of GKEX supplementation, relative peak strength (increased by 1.02 times, p = 0.0005) and RFD (increased by 1.07 times, p < 0.0001) increased significantly.

[0094] Table 3

[0095] 2.6 Supplementing the effects of GKEX on isotonic muscle strength and athletic performance

[0096] Please refer to Table 3 for the results of the test motor performance (isotonic muscle strength test), where data are expressed as mean ± standard deviation (mean ± SD). Different superscript letters (a, b) indicate significant differences between groups at the same time point (p < 0.05); * indicates a significant effect after intervention compared to before intervention (p < 0.05). The results in Table 3 show that there were no significant differences in relative peak strength and rate of force development (RFD) between the placebo group and the GKEX group before and after intervention; however, after 6 weeks of GKEX supplementation, relative peak strength (increased by 1.05 times, p = 0.0032) and RFD (increased by 1.03 times, p = 0.0009) increased significantly.

[0097] 2.7 Supplementing the impact of GKEX on anaerobic exercise performance

[0098] Please refer to Table 3 for the results of the test exercise performance (anaerobic exercise), where data are expressed as mean ± standard deviation (mean ± SD). Different superscript letters (a, b) indicate significant differences between groups at the same time point (p < 0.05); * indicates a significant effect after intervention compared to before intervention (p < 0.05). Table 3 shows that there were no significant differences in relative mean power, relative peak power, and fatigue index between the placebo group and the GKEX group before and after intervention. However, after 6 weeks of GKEX supplementation, relative mean power increased significantly (by 1.03 times, p = 0.0112).

[0099] In summary, the lactobacillus of this invention is used to prepare components for muscle building, fat reduction, and improved athletic performance. When taken orally, it strengthens body composition, increases muscle mass and thickness, and significantly reduces body fat percentage. Furthermore, it effectively enhances physical performance such as muscle strength, maximum oxygen uptake, explosive power, and anaerobic power. It has the effects of muscle building, fat reduction, and improved athletic performance.

[0100] In summary, the specific Lactobacillus strains, culture media, processes, or evaluation methods used in this invention are merely illustrative of the use of the Lactobacillus of this invention in preparing compositions for muscle building, fat loss, and improved athletic performance. However, those skilled in the art will understand that other Lactobacillus strains, culture media, processes, or evaluation methods can also be used to illustrate the use of the Lactobacillus of this invention in preparing compositions for muscle building, fat loss, and improved athletic performance without departing from the spirit and scope of this invention, and are not limited to those described above. For example, Lactobacillus compositions may be pharmaceutical or food compositions, selectively containing pharmaceutically and food-acceptable carriers, excipients, and / or additives, and may be formulated into dosage forms such as powders, tablets, granules, capsules, ampoules, and liquid sprays.

[0101] According to the above embodiments, the short lactic acid bacteria strain GKEX of the present invention, when administered to healthy non-athlete subjects for a continuous period of time, can significantly increase muscle mass, reduce body fat, and improve physical performance such as muscle strength, maximum oxygen uptake, explosive power, and anaerobic power. Therefore, this lactobacillus can be used as an effective ingredient and can be used to prepare various components for muscle building, fat reduction, and improvement of athletic performance.

[0102] While the invention has been disclosed above with reference to several specific embodiments, other embodiments are also possible. Therefore, the spirit and scope of the appended claims should not be limited to the embodiments described herein.

[0103] 101, 103, 201: Bar-Line Chart

[0104] Domestic storage information (please note in order of storage institution, date, and number) The Lactobacillus brevis strain GKEX was deposited at the Biological Resources Preservation and Research Center of the Food Industry Development Research Institute, Republic of China Foundation, No. 331, Food Road, Hsinchu, Taiwan, on January 21, 2022, with the deposit number BCRC 911099, and its survival was confirmed on February 10, 2022. Overseas storage information (please note in the order of storage country, institution, date, and number) none

Claims

1. The use of a lactobacillus in the preparation of a composition for muscle gain and fat loss, wherein the lactobacillus comprises a strain of Lactobacillus brevis, GKEX, which was deposited on January 21, 2022, at the Bioresource Conservation and Research Center, Food Industry Research and Development Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan), with the registration number BCRC 911099, and the lactobacillus was administered orally to a subject for at least two consecutive weeks to increase the subject's skeletal muscle mass and reduce the subject's body fat.

2. Use of the lactobacillus as described in claim 1 for the preparation of a composition for muscle building and fat loss, wherein the composition is an oral composition.

3. Use of the lactobacillus as described in claim 1 for the preparation of a composition for muscle building and fat reduction, wherein the composition is a pharmaceutical composition or a food composition.

4. Use of the lactobacillus as described in claim 1 for the preparation of a composition for muscle building and fat reduction, wherein the composition further comprises a pharmaceutically and food-acceptable carrier, excipient and / or additive.

5. Use of the lactobacillus as described in claim 1 for the preparation of a composition for muscle building and fat loss, wherein one dosage form of the composition includes powder, tablets, granules, capsules, ampoules or liquid spray.

6. Use of a lactobacillus for preparing a muscle-building composition, wherein the lactobacillus comprises a short lactic acid bacteria strain GKEX with accession number BCRC 911099, and the composition is administered orally to a healthy, non-athlete subject for at least two consecutive weeks to increase the skeletal muscle mass of the subject.

7. Use of a lactobacillus for preparing a fat-reducing composition, wherein the lactobacillus comprises a short lactic acid bacteria strain GKEX with accession number BCRC 911099, and the composition is administered orally to a healthy, non-athlete subject for at least two consecutive weeks to reduce the subject's body fat.

8. The use of a lactobacillus for preparing a composition for muscle gain and fat loss, wherein the lactobacillus comprises a short lactic acid bacteria strain GKEX, which was deposited on January 21, 2022, at the Bioresource Conservation and Research Center, Food Industry Research and Development Institute (BCRC, No. 331, Food Road, Hsinchu, Taiwan) with the registration number BCRC 911099. The composition was administered orally to a healthy non-athlete subject for at least two consecutive weeks, and the lactobacillus was administered to the subject at a dose of 1 × 10¹⁰ CFU / 60 kg body weight / day to 3 × 10¹⁰ CFU / 60 kg body weight / day.

9. Use of the lactobacillus as described in claim 8 for the preparation of a composition for muscle gain and fat loss, wherein the subject performs resistance training one to five times per week prior to administration of the composition.

10. Use of the Lactobacillus as described in claim 8 for the preparation of a composition for muscle gain and fat loss, wherein the composition is administered orally to the subject for at least six consecutive weeks, and the subject's muscle strength, maximum oxygen uptake, explosive power, and anaerobic power increase.

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