TWI847318B - Lactic acid bacterium and method for preventing or minimizing exercise-induced muscle damage - Google Patents

Abstract

Provided is a composition for use in preventing or minimizing exercise-induced muscle damage (EIMD) in a subject in need thereof. The composition includes Lactobacillus paracaseiPS23 and a carrier thereof. Also provided is a method of preventing or minimizing EIMD by administrating the composition.

Description

Lactic acid bacteria and methods for preventing or minimizing exercise-induced muscle damage

The present disclosure relates to compositions comprising probiotics, and in particular, to compositions and methods for preventing or minimizing exercise-induced muscle damage.

Exercise-induced muscle damage (EIMD) occurs after intense or prolonged exercise, especially when it involves eccentric muscle contraction ^[1] . It is characterized by delayed onset muscle soreness (DOMS), structural damage, changes in biochemical parameters, and acute local inflammation, such as increased creatine kinase (CK), lactate dehydrogenase (LDH), and myoglobin (Mb), as well as increased inflammatory and oxidative markers such as C-reactive protein (CRP) and malondialdehyde (MDA) ^[2-4] . Symptoms can persist for several days and worsen with continued exercise, leading to fatigue and a decrease in strength and performance. This makes it difficult to maintain the required intensity and can have a negative impact on the performance of athletes and amateurs. ^[5]

For professional athletes, EIMD will induce mild or moderate DOMS within 2 to 3 days after exercise training. However, for untrained people, EIMD will lead to severe DOMS and loss of muscle function, so a longer recovery period is required to fully repair the muscles ^[6] . Therefore, in addition to an appropriate training plan, different strategies must be adopted to delay or reduce muscle fatigue, improve training adaptability, and promote rapid recovery from high-intensity training or continuous high-intensity competition.

Current treatments for muscle repair or regeneration include massage, ultrasound, physical therapy, hyperbaric oxygen delivery, and medication. These treatments may provide some beneficial results, but are far from ideal in terms of, for example, effectiveness and efficiency.

Therefore, there is an urgent need to provide an effective method to prevent or reduce sports-related muscle damage and fatigue, and protect individuals from other adverse effects caused by exercise, thereby improving sports performance.

In the present disclosure, a lactic acid bacterium has been identified that can effectively protect an individual from exercising by, for example, reducing muscle damage, reducing inflammation, and aiding recovery from fatigue, thereby improving the individual's athletic performance.

In at least one embodiment of the present disclosure, the lactic acid bacteria is Lactobacillus paracasei PS23 (also referred to herein as Lactobacillus paracasei PS23 or PS23), which is deposited with DSMZ accession number 32322 or with the Bioresource Conservation and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) with BCRC accession number 910755.

The present disclosure provides a composition for preventing or minimizing EIMD in an individual in need thereof, wherein the composition comprises an effective amount of lactic acid bacteria and a carrier thereof. In some specific embodiments, the lactic acid bacteria is Lactobacillus paracasei PS23.

In at least one embodiment of the present disclosure, the lactic acid bacteria in the composition are in the form of a live strain, a heat-killed live strain, a culture, a lysate, an extract, and any combination thereof. In some embodiments, the lactic acid bacteria are a heat-killed live strain of PS23.

In at least one embodiment of the present disclosure, the composition is administered to the individual in an amount of at least 1×10 ⁶ colony-forming units (CFU) of lactic acid bacteria per day. For example, an effective amount of PS23 is at least 1 × 10 ⁶ CFU, at least 1 × 10 ⁷ CFU, at least 1 × 10 ⁸ CFU, at least 1 × 10 ⁹ CFU, at least 1 × 10 ¹⁰ CFU, or at least 1 × 10 ¹¹ CFU, including 5 × 10 ⁶ CFU, 5 × 10 ⁷ CFU, 5 × 10 ⁸ CFU, 2 × 10 ⁹ CFU, 3 × 10 ⁹ CFU, 4 × 10 ⁹ CFU, 5 × 10 ⁹ CFU, 6 × 10 ⁹ CFU, 7 × 10 ⁹ CFU, 8 × 10 ⁹ CFU, 9 × 10 ⁹ CFU, 2 × 10 ¹⁰ CFU, 3 × 10 ¹⁰ CFU, 4 × 10 ¹⁰ CFU, 5 × 10 ¹⁰ CFU, 6 × 10 ¹⁰ CFU, CFU, 7 × 10 ¹⁰ CFU, 8 × 10 ¹⁰ CFU, 9 × 10 ¹⁰ CFU, 2 × 10 ¹¹ CFU, 3 × 10 ¹¹ CFU, 4 × 10 ¹¹ CFU, 5 × 10 ¹¹ CFU, 6 × 10 ¹¹ CFU, 7 × 10 ¹¹ CFU, 8 × 10 ¹¹ CFU, and 9 × 10 ¹¹ CFU, but are not limited thereto.

In at least one embodiment of the present disclosure, preventing or minimizing EIMD includes at least one of increasing muscle strength, increasing muscle endurance, delaying or reducing muscle fatigue, accelerating muscle recovery from EIMD, and preventing or reducing DOMS.

In at least one embodiment of the present disclosure, the composition is used to further enhance physical fitness, improve athletic performance, reduce inflammation and/or reduce oxidative stress in an individual.

The present disclosure also provides a method for preventing or minimizing EIMD in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of PS23 and a carrier thereof.

In at least one embodiment of the present disclosure, the method further comprises enhancing physical fitness, improving athletic performance, reducing inflammation and/or reducing oxidative stress in the individual.

In at least one embodiment of the present disclosure, the composition modulates serum levels of at least one biomarker associated with muscle damage, oxidative stress, inflammation, or anabolic hormones.

In at least one embodiment of the present disclosure, the increase of at least one of creatine kinase (CK), myoglobin, thiobarbituric acid reactive substance (TBARS) and high-sensitivity C-reactive protein in the subject caused by EIMD is inhibited after administration of the composition.

In at least one embodiment of the present disclosure, the decrease in testosterone caused by EIMD in the subject is inhibited after administration of the composition.

In at least one embodiment of the present disclosure, the composition can be a nutritional composition, a food or a pharmaceutical composition. In some embodiments, the composition is in a form suitable for oral administration. In some embodiments, the composition is in the form of a concentrate, a paste, a powder, a suspension, a granule, a tablet, a pill, a capsule, a drink or a dairy product.

In at least one embodiment of the present disclosure, the lactic acid bacteria is the only active ingredient in the composition for preventing or minimizing EIMD. In some embodiments, the lactic acid bacteria may be combined with an additional active ingredient to prevent or minimize EIMD, wherein the additional active ingredient may be an active ingredient for preventing or minimizing EIMD.

In at least one embodiment of the present disclosure, the composition can be administered to the individual 1 to 4 times a day or 1 to 4 times a week. In some embodiments, the frequency of administration of the composition to the individual is selected from the group consisting of once a week, twice a week, three times a week, four times a week, once every three days, twice every three days, once every two days, once a day, twice a day, three times a day, and four times a day.

In at least one embodiment of the present disclosure, the composition is administered to the subject for at least one week. For example, the composition is administered to the subject for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks.

The composition provided by the present disclosure can effectively reduce inflammation and oxidative stress, and improve athletic performance and anti-fatigue. Therefore, by administering the composition, the method disclosed herein not only provides the benefit of restoring muscle damage, but also enhances physical performance, including increasing muscle strength, muscle endurance and physical fitness.

The following embodiments are used to illustrate the present disclosure. Based on the disclosure of this specification, a person with ordinary knowledge in the art can easily think of other advantages and effects of the present disclosure. The present disclosure can also be implemented or applied as described in different embodiments. For different aspects and applications, the following embodiments can be modified or changed to implement the present disclosure without violating the scope of the present disclosure.

It should also be noted that, as used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. The term "or" can be used interchangeably with the term "and/or" unless the context clearly indicates otherwise.

As used herein, the term "comprising" or "including" is used to refer to compositions, methods and their respective components included in the present disclosure, which are still open to including unspecified elements or steps, whether necessary or not.

As used herein, the term "about" generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean value as considered by one of ordinary skill in the art. Unless expressly provided otherwise, all numerical ranges, amounts, values and percentages disclosed herein, such as those for material amounts, durations, temperatures, operating conditions, amount ratios, etc., should be understood to be modified by the term "about" in all cases.

The present disclosure provides a composition for preventing or minimizing EIMD in an individual in need thereof, wherein the composition comprises an effective amount of lactic acid bacteria and a carrier thereof. In some specific embodiments, the lactic acid bacteria is Lactobacillus paracasei PS23, which has been deposited at the Leibniz Institute DSMZ-German Depository of Microorganisms and Cell Cultures (Inhoffenstr. 7 B, D-38124 Braunschweig, Germany) under the Budapest Treaty and has been granted DSMZ accession number DSM 32322 by the international depository.

As used herein, the phrase "effective amount" means the amount of active ingredient (e.g., PS23) that imparts the desired effect (e.g., preventing or minimizing EIMD) to the treated individual. As known to those skilled in the art, the effective dose will vary depending on the route of administration, the formulation used, the possibility of co-use with other therapies, and the condition to be treated.

In at least one embodiment of the present disclosure, the effective amount of PS23 may be at least 1×10 ⁶ CFU, at least 1×10 ⁷ CFU, at least 1×10 ⁸ CFU, at least 1×10 ⁹ CFU, at least 1×10 ¹⁰ CFU, or at least 1×10 ¹¹ CFU. In some embodiments, an effective amount of PS23 can range from about 1 × 10 ⁶ CFU to about 1 × 10 ¹³ CFU, from about 1 × 10 ⁶ CFU to about 1 × 10 ¹² CFU, from about 1 × 10 ⁶ CFU to about 1 × 10 ¹¹ CFU, from about 1 × 10 ⁶ CFU to about 1 × 10 ¹⁰ CFU, from about 1 × 10 ⁷ CFU to about 1 × 10 ¹³ CFU, from about 1 × 10 ⁷ CFU to about 1 × 10 ¹² CFU, from about 1 × 10 ⁷ CFU to about 1 × 10 ¹¹ CFU, from about 1 × 10 ⁷ CFU to about 1 × 10 ¹⁰ CFU, from about 1 × 10 ⁸ CFU to about 1 × 10 13 CFU, from about 1 × 10 ⁸ CFU to about 1 × 10 ¹³ CFU, CFU to about 1 × 10 ¹² CFU, from about 1 × 10 ⁸ CFU to about 1 × 10 ¹¹ CFU, or from about 1 × 10 ⁸ CFU to about 1 × 10 ¹⁰ CFU.

As used herein, the term "prevent" or "prevention" means preventive or avoidance measures against a disease, disorder, or symptoms or conditions thereof, including but not limited to the administration or administration of one or more active agents to an individual who has not yet been diagnosed with the disease, disorder, or symptoms or conditions thereof, but who may be susceptible to or predisposed to the disease, disorder, or symptoms or conditions thereof. The purpose of preventive or avoidance measures is to avoid, prevent or delay the occurrence of the disease, disorder, or symptoms or conditions thereof.

As used herein, the term "subject" means a human or an animal. Examples of subjects include, but are not limited to, rodents, mice, monkeys, guinea pigs, dogs, cats, cows, sheep, pigs, horses, rabbits, and humans. In some embodiments, the subject is a mammal, such as a primate, such as a human.

As used herein, the term "administering" or "applying" means placing an active ingredient in an individual's body by a method or route that allows the active ingredient to be at least partially localized at a desired site to produce a desired effect. In some specific embodiments, the composition of the present disclosure is a nutritional composition, a food or a pharmaceutical composition, and may be in any form suitable for oral administration. For example, the composition may be in the form of a concentrate, a paste, a powder, a suspension, a granule, a tablet, a pill, a capsule, a drink or a dairy product.

In at least one embodiment of the present disclosure, the nutritional composition may also include a dietary supplement and/or a functional food. As used herein, "dietary supplement" means a product made from a compound commonly used in food, but in the form of a tablet, powder, capsule, drink, or any other form not commonly associated with food, and which has a beneficial effect on human health. As used herein, "functional food" is a food that also has a beneficial effect on human health. In some embodiments, dietary supplements and functional foods may have a protective or therapeutic effect against a disease, disease, or its symptoms or conditions.

As used herein, the term "carrier" means a compound, material, composition and/or dosage form suitable for contact with the tissues of an individual. An "acceptable" carrier is one that is compatible with the active ingredient (e.g., PS23) of the disclosed composition (and capable of stabilizing the active ingredient) and is not harmful to the individual to be treated. Examples of carriers include colloidal silicon oxide, cellulose, microcrystalline cellulose, sodium lauryl sulfate, polyethylene glycol (PEG), alkyl glycol, sebacic acid, dimethyl sulfoxide, alcohols, magnesium stearate, calcium stearate, gelatin, fat, glycerin, dietary fiber, alginate, pectin, carrageenan, amidated pectin, xanthan gum, kelp gum, karaya gum, rhamnose gum, welan gum, gum ghatti and gum arabic.

The composition provided by the present disclosure can regulate the serum level of at least one biomarker related to muscle damage, oxidative stress, inflammation or anabolic hormones in an individual, thereby effectively reducing inflammation and oxidative stress after exercise, and improving exercise performance and fighting fatigue.

As used herein, the term "fatigue" refers to skeletal muscle fatigue and/or weakness. Muscle fatigue may be due to strenuous or repetitive physical activity or exercise that has symptoms of fatigue or affects muscle fiber and/or muscle function. Muscle fatigue can also be defined as a failure of athletic performance.

In at least one embodiment of the present disclosure, biomarkers associated with muscle damage, oxidative stress, inflammation and/or anabolic hormones include, but are not limited to, creatine kinase, myoglobin, thiobarbituric acid reactive substances (TBARS), high-sensitivity C-reactive protein (hs-CRP) and testosterone.

This disclosure will be illustrated using a variety of embodiments. The following embodiments should not be considered as limiting the scope of this disclosure. Embodiments

Materials and methods

The materials and methods used in Examples 1 to 4 are described in detail below. Materials used in this disclosure but not specified herein are commercially available.

1. Preparation of probiotic preparations

Lactobacillus paracasei PS23 was isolated from healthy human feces and deposited under DSMZ accession number 32322. To prepare the nutritional supplement, PS23 was inoculated into the culture medium, cultured at 37°C for 18 hours, collected by centrifugation, embedded in a preservative and excipient, and freeze-dried to produce a live PS23 strain powder with a final concentration of 1 × 10 ¹¹ colony forming units (CFU) per gram. For the preparation of heat-killed live strain of Lactobacillus paracasei PS23, PS23 was cultured as described above, heat-treated at 80°C for 1 hour, centrifuged, and then embedded in a protective agent and a plasticizer, and freeze-dried to make PS23 heat-killed live strain powder, with a final concentration of 1 × 10 ¹¹ cells per gram. Subsequently, the live strain powder of Lactobacillus paracasei PS23 and the heat-killed live strain powder were made into capsules, wherein the L-PS23 treatment capsule contained 10 billion (1 × 10 ¹⁰ ) CFU of live strain PS23, and the HK-PS23 treatment capsule contained 10 billion cells of heat-killed live strain PS23.

The placebo capsules contained only microcrystalline cellulose (MCC) and were compatible in size, color, and flavor to the L-PS23 and HK-PS23 treatment capsules.

2. Participants

The research described here was conducted in accordance with the guidelines of the Declaration of Helsinki. The experiments were approved by the Human Trial Committee of Lian-Shin International Hospital (Taoyuan, Taiwan) (LSHIRB No. 22-003-A2). Individuals aged 20 to 40 years were recruited for this study, but those with any of the following conditions were excluded: (1) a history of cardiovascular disease, hypertension, metabolic disease, asthma, or cancer; (2) a body mass index (BMI) ≥ 27; (3) limb disability, neuromuscular movement disorder, etc. within 6 months and unable to exercise; (4) taking anti-inflammatory drugs, analgesics, acute and chronic disease drugs, etc. in the past month; (5) conflict of interest with the principal investigator; (6) smoking and drinking habits; (7) taking probiotic powder, capsules or oral tablets (including yogurt and other related foods) within two weeks; (8) allergic to food or lactic acid bacteria products; (9) undergoing liver, gallbladder, gastrointestinal surgery (except hernia and polypectomy).

A total of 114 individuals from the National Sport University were recruited for this study, including 78 males and 36 females. All individuals were randomly assigned to 3 groups (i.e., L-PS23 group, HK-PS23 group, and placebo group), with an equal ratio of males and females in each group (26 males and 12 females in each group). All participants signed a written informed consent before the start of the experiment. During the experiment, a small number of individuals in each group withdrew from the experiment due to changes in physiological conditions, such as being diagnosed with COVID-19 or temporarily unable to cooperate with the experimental arrangements.

3. Experimental design

The study described here used a double-blind design. The experimental procedure is shown in Figure 1. All subjects were asked to take two capsules per day for 6 consecutive weeks, with subjects in the L-PS23 group taking L-PS23 capsules (1 × 10 ¹⁰ CFU/capsule, 2 capsules/day), subjects in the HK-PS23 group taking HK-PS23 capsules (1 × 10 ¹⁰ cells/capsule, 2 capsules/day), and subjects in the placebo group taking placebo capsules (2 capsules/day).

Participants visited the laboratory five times. Body composition, basal blood biochemical parameters, and exercise performance were measured before and six weeks after placebo, L-PS23, or HK-PS23 supplementation, immediately prior to exercise-induced muscle damage (EIMD). Biomarkers of muscle damage, indicators of inflammation and oxidative stress, and anabolic hormones were measured 3, 24, and 48 hours after EIMD.

All participants were asked to maintain their usual diet throughout the study and were asked to record their dietary intake for analysis by a professional nutritionist. The basic information and physical parameters of the individuals before the intervention are shown in Table 1 below.

Table 1. Participant characteristics and baseline physical fitness tests Basic Information Parameter (unit) Placebo L-PS23 HK-PS23 Physical characteristics Age (years) 21.6 ± 2.0 ^a 21.4 ± 1.3 ^a 21.8 ± 2.5 ^a Height (cm) 172 ± 9 ^a 172 ± 9 ^a 171 ± 9 ^a Weight (kg) 69.4 ± 12.4 ^a 69.7 ± 11.0 ^a 66.4 ± 11.6 ^a BMI (kg/m ² ) 23.2 ± 2.8 ^a 23.5 ± 2.4 ^a 22.7 ± 2.6 ^a CMJ RFD (N/kg/sec) 8.11 ± 0.95 ^a 8.03 ± 1.03 ^a 7.96 ± 1.08 ^a Relative peak force (N/kg) 15.11 ± 2.12 ^a 15.23 ± 2.22 ^a 15.09 ± 2.55 ^a Jumping height (cm) 30.7 ± 6.0 ^a 31.6 ± 7.6 ^a 32.0 ± 6.5 ^a IMTP Relative peak force (N/kg) 15.57 ± 2.32 ^a 15.31 ± 3.06 ^a 15.46 ± 2.51 ^a Peak RFD (N/sec) 9244 ± 1777 ^a 9219 ± 1821 ^a 9102 ± 1520 ^a Wingate Relative average power (W/kg) 6.17 ± 0.91 ^a 6.53 ± 0.94 ^a 6.45 ± 0.97 ^a Relative peak power (W/kg) 9.61 ± 1.59 ^a 9.40 ± 1.76 ^a 9.38 ± 1.73 ^a Fatigue index (%) 48.6 ± 3.9 ^a 47.8 ± 4.2 ^a 48.1 ± 4.6 ^a The same superscript letter (a) indicates no significant difference among the groups, p ＞ 0.05. BMI: body mass index; CMJ: countermovement jump; IMTP: isometric mid-thigh pull-up; RFD: rate of force development; Wingate: Wingate anaerobic test.

4. Body composition measurement

All subjects were asked to fast for at least 8 h before the measurement. During the measurement with the In-Body 570 device (In-body, Seoul, South Korea), the subjects stood on the bottom electrode, held the sensing handles with both hands, did not speak or move, and kept their arms open at a 30-degree angle to the body. Body composition was measured using a bioelectrical impedance analyzer (BIA) based on the multi-frequency principle at frequencies of 1, 5, 50, 260, 500, and 1000 kHz within 60 seconds.

5. Emotionally Induced Muscle Damage (EIMD)

To induce EIMD, all subjects were asked to perform 100 maximum vertical jumps. This method consisted of jumping once every 4 seconds for 10 sets of 10 jumps. All subjects were asked to bend their knees 90 degrees during each squat, and the maximum jump height for each subject was marked as a goal. The researchers encouraged the subjects to try their best to reach the maximum height goal during each jump.

6. Countermovement jump (CMJ) assessment

The CMJ is used to measure maximal speed, strength, and explosiveness of the lower body. For this test, participants were asked to place their hands on their hips and stand with both feet on a Kistler force platform (9260AA, Kistler GmbH, Switzerland). Afterwards, participants were asked to squat until their knees were bent to 90 degrees and then immediately jump as high as possible with maximal force. Each participant performed 3 repetitions. CMJ data were obtained at designated points and the instrument was calibrated for each participant's body weight. Measurement parameters included rate of force development (RFD), relative peak force, and jump height, where RFD is defined as the ability to generate force in a given time during a rapid voluntary contraction, which is often used to assess explosive strength.

7. Isometric mid-thigh pull-up (IMTP) test

The IMTP test has been shown to be a valid and reliable test of maximal lower extremity strength that is highly correlated with athletic performance and can be used to measure changes in explosiveness and as a marker of fatigue. In the test, a custom-made IMTP test apparatus (IMTP Rack, Kairos Strength, USA) and a force plate (model 9260AA, Kistler, Switzerland) were used. All individuals stood with their feet equally spaced apart, a barbell placed between their thighs, with their trunks erect, spine neutral, and knees and hips at a 140-degree angle, and then pulled the barbell with maximal force for 3 to 5 seconds.

Prior to testing, individuals should be familiar with the IMTP test and the exercise process to ensure proper force development and a consistent weight frequency. Each test was repeated at 2-minute intervals to reduce measurement errors associated with changes in posture. Measurement parameters included relative peak force and peak force rate.

8. Wingate Anaerobic Test (WAnT)

Participants were asked to complete 30-second all-out sprints on an anaerobic powered bicycle (894E, Monark Exercise AB, Sweden). The seat height was adjusted individually for each participant, and toe clips were used to prevent the foot from sliding off the pedals. Measurements were performed after a warm-up. As the individual sprinted at 120 revolutions per minute (rpm), a weight set to 7.5% was automatically dropped onto the friction belt of the bicycle to create additional resistance (e.g., a 60-kg person would add 4.5 kg of resistance). The individual was asked to sprint at full speed for 30 seconds. At the end of the test, the individual's fatigue index was recorded using the rating of perceived exertion. Parameters measured included relative mean power, relative peak power, and fatigue index.

9. Biomarker assessment of muscle damage, inflammation, oxidative stress and anabolic steroids

Blood-related markers are one of the most effective indirect methods for assessing muscle damage and inflammation. For example, creatine kinase (CK) usually increases after exercise and is therefore often used as a biomarker for skeletal muscle tissue damage.

Venous blood was collected from subjects before EIMD (i.e., six weeks after sample introduction and before EIMD occurred) and at 3, 24, and 48 hours after EIMD for biomarker assessment, including biomarkers of muscle damage, inflammation, oxidative stress, and anabolic steroids.

Creatine kinase (CK) and high-sensitivity CRP (hs-CRP) levels were analyzed by an automatic analyzer (AU 5820, Beckman Coulter, Inc., California, USA). Myoglobin and testosterone levels were analyzed by an automatic analyzer (DXI 800, Beckman Coulter, Inc., California, USA). Thiobarbituric acid-reactive substances (TBARS) levels were measured using a TBARS assay kit (No. 10009055, Cayman Chemical Company, Ann Arbor, Michigan, USA).

10. Serum biochemical analysis

Venous blood was collected from the individual before and after six weeks of sample intervention to analyze indicators such as liver function, kidney function, and blood lipids, in order to assess the individual's metabolism and health status and determine whether they were affected by the intervention. All subjects were asked to fast for at least 8 h, and then the following serum levels were assessed using an automatic analyzer (Hitachi 717, Tokyo, Japan): aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), blood urea nitrogen (BUN), creatine (CREA), uric acid (UA), and glucose.

11. Statistical analysis

All data are presented as mean ± SD. Statistical analyses were performed by SAS 9.0 (SAS Inst., Cary, NC, USA). Multiple group comparisons were performed by one-way analysis of variance (ANOVA). Repeated measures ANOVA was used to compare the differences between placebo, L-PS23, and HK-PS23 at specific time points during recovery using IBM SPSS Statistics version 24.1 (IBM, Co., Armonk, NY, USA). Paired t -tests with Bonferroni adjustment were used to compare treatment differences from pre-EIMD at each time point. Statistical significance was set at p < 0.05.

Example 1: Effects of six weeks of L-PS23 or HK-PS23 intervention on dietary intake and body composition

Table 2 shows the daily dietary intake (carbohydrates, proteins, and fats) and total calories of the individuals before and after the six-week intervention. The results showed that there were no significant differences among the three groups, and there were no significant changes in the same group before and after the intervention.

Table 2. Dietary intake of individuals before and after six weeks of L-PS23 or HK-PS23 intervention Dietary intake Before intervention After intervention Placebo L-PS23 HK-PS23 Placebo L-PS23 HK-PS23 Carbohydrates (g/day) 214 ± 69 ^a 213 ± 37 ^a 208 ± 34 ^a 214 ± 70 ^a 215 ± 48 ^a 213 ± 36 ^a Protein (g/day) 152 ± 23 ^a 155 ± 20 ^a 147 ± 27 ^a 149 ± 23 ^a 155 ± 19 ^a 149 ± 23 ^a Fat (g/day) 92 ± 5 ^a 93 ± 6 ^a 90 ± 5 ^a 93 ± 5 ^a 93 ± 9 ^a 92 ± 6 ^a Total calories (kcal/day) 2287 ± 313 ^a 2304 ± 163 ^a 2232 ± 153 ^a 2289 ± 315 ^a 2319 ± 219 ^a 2274 ± 194 ^a The same superscript letter (a) indicates no significant difference among the groups, p ＞ 0.05.

In addition, in terms of body composition, the changes in body composition of individuals before and after the intervention are listed in Table 3. The results showed that there were no significant differences in body weight, body mass index (BMI), lean body mass (LBM) and fat body mass (FBM) between the groups before and after the six-week intervention.

However, compared with pre-intervention, weight ( p = 0.0084), BMI ( p = 0.0087) and FBM ( p = 0.0044) of the placebo group increased significantly after six weeks of intervention, while LBM ( p = 0.0072) decreased significantly. After six weeks of intervention, weight ( p = 0.0036) and BMI ( p = 0.0028) of the HK-PS23 group decreased compared with pre-intervention. In addition, weight and BMI of the HK-PS23 group decreased significantly compared with the placebo group and L-PS23 group ( p < 0.05), and FBM of the HK-PS23 group also decreased significantly compared with the placebo group ( p = 0.0109).

Table 3. Body composition of individuals before and after six weeks of L-PS23 or HK-PS23 intervention Parameters Before intervention After intervention Difference Placebo L-PS23 HK-PS23 Placebo L-PS23 HK-PS23 Placebo L-PS23 HK-PS23 BW (kg) 69.4±12.4 ^a 69.7±11.0 ^a 66.4±11.6 ^a 70.1±12.9 ^a,* 70.0±11.0 ^a 65.6±11.7 ^a,* 0.7±1.4 ^b 0.3±2.3 ^b -0.8±1.6 ^a BMI (kg/m ² ) 23.2±2.8 ^a 23.5±2.4 ^a 22.7±2.6 ^a 23.5±3.0 ^a,* 23.6± ^2.5a 22.4±2.7 ^a,* 0.2±0.5 ^b 0.1±0.8 ^b -0.3±0.5 ^a LBM (kg) 31.8±6.7 ^a 31.7±6.3 ^a 30.2±6.2 ^a 31.5±6.7 ^a,* 31.7±6.4 ^a 30.2±6.0 ^a -0.3±0.6 ^a 0.0± ^0.9a -0.1±0.6 ^a FBM (%) 20.0±7.0 ^a 20.1±7.9 ^a 19.3±6.5 ^a 20.8±6.7 ^a,* 20.2±8.5 ^a 19.1±6.0 ^a 0.8±1.5 ^b 0.1±1.4 ^ab -0.2±1.7 ^a Different superscript letters (a, b) indicate significant differences between groups at the same time point, p < 0.05. * indicates significant effects after intervention compared with before intervention, p < 0.05. Difference represents the value of "after intervention" minus "before intervention". BW: body weight; BMI: body mass index; LBM: net body weight; FBM: fat body mass.

Example 2: Effects of L-PS23 or HK-PS23 intervention on biochemical characteristics before and after six weeks

Before and after the intervention, blood was collected from all individuals for basic blood parameter analysis to understand the individual's health and physiological status before the intervention, so as to determine whether there were any adverse reactions or side effects after six weeks of PS23 intervention.

As shown in Table 4, the results showed that liver function indicators (AST, ALT), renal function indicators (BUN, CREA, UA), lipid metabolism indicators (TC, TG, HDL-C, LDL-C) and blood sugar were all within the normal range, and there was no significant difference between the three groups before and after the intervention. In addition, there was no significant change in the same group after the intervention compared with before the intervention. In addition, no adverse events related to the intervention occurred during the study period.

Table 4. Blood biochemical parameters of individuals before and after six weeks of L-PS23 and HK-PS23 intervention Parameters Before intervention After intervention Placebo L-PS23 HK-PS23 Placebo L-PS23 HK-PS23 AST (U/L) 21 ± 6 ^a 22 ± 7 ^a 21 ± 7 ^a 21 ± 6 ^a 22 ± 6 ^a 121 ± 6 ^a ALT (U/L) 20 ± 6 ^a 22 ± 7 ^a 20 ± 7 ^a 20 ± 4 ^a 22 ± 7 ^a 21 ± 7 ^a TC (mg/dL) 179 ± 16 ^a 173 ± 18 ^a 174 ± 22 ^a 174 ± 21 ^a 178 ± 15 ^a 172 ± 18 ^a TG (mg/dL) 81 ± 24 ^a 84 ± 24 ^a 81 ± 18 ^a 82 ± 22 ^a 85 ± 22 ^a 82 ± 17 ^a HDL-C (mg/dL) 57.5 ± 9.3 ^a 56.7 ± 8.5 ^a 57.5 ± 7.6 ^a 58.0 ± 9.5 ^a 56.8 ± 9.1 ^a 58.2 ± 8.3 ^a LDL-C (mg/dL) 96.5 ± 17.7 ^a 98.2 ± 15.2 ^a 97.1 ± 17.3 ^a 96.5 ± 18.0 ^a 97.2 ± 15.2 ^a 96.0 ± 16.5 ^a BUN (mg/dL) 13.8 ± 2.5 ^a 14.9 ± 2.7 ^a 14.7 ± 3.1 ^a 13.5 ± 2.9 ^a 14.5 ± 2.3 ^a 14.4 ± 2.5 ^a CREA (mg/dL) 1.10 ± 0.10 ^a 1.10 ± 0.11 ^a 1.09 ± 0.12 ^a 1.06 ± 0.12 ^a 1.10 ± 0.11 ^a 1.07 ± 0.10 ^a UA (mg/dL) 5.7 ± 0.9 ^a 5.6 ± 0.9 ^a 5.4 ± 1.0 ^a 5.5 ± 0.8 ^a 5.6 ± 1.0 ^a 5.3 ± 1.1 ^a Glucose (mg/dL) 86 ± 6 ^a 84 ± 6 ^a 84 ± 8 ^a 84 ± 6 ^a 84 ± 7 ^a 84 ± 7 ^a The same superscript letter (a) indicates no significant difference among the groups, p ＞ 0.05. AST: aspartate transaminase; ALT: alanine transaminase; TC: total cholesterol; TG: triglyceride; HDL-C: high-density lipoprotein-cholesterol; LDL-C: low-density lipoprotein-cholesterol; BUN: blood urea nitrogen; CREA: creatine; UA: uric acid.

Example 3: Effects of L-PS23 and HK-PS23 supplements on athletic performance

(1) Vertical jump height

The vertical jump has been used to assess athletes' speed, maximum strength, explosiveness, and anaerobic performance ^[7] , and as a fatigue indicator of muscle damage and strength loss ^[8] .

Vertical jump height was measured by countermovement jump (CMJ) assessment at 3, 24, and 48 hours after EIMD. The results showed that the rate of force development (RFD) and the percentage change of relative peak force in the three groups (i.e., placebo group, L-PS23 group, and HK-PS23 group) were significantly reduced at 3, 24, and 48 hours after EIMD compared with those before EIMD ( p ＜ 0.05) (Figure 2A and Figure 2B).

In addition, for the inter-group comparison, Figure 2A showed that at 24 and 48 hours after EIMD, the RFD loss in the L-PS23 or HK-PS23 supplemented groups was significantly less than that in the placebo group and recovered significantly faster ( p < 0.0001), and at 3 hours after EIMD, the RFD loss in the HK-PS23 group was significantly less than that in the placebo group ( p < 0.05). Furthermore, as shown in Figure 2B, at 3 and 24 hours after EIMD, the relative peak strength loss in the L-PS23 and HK-PS23 supplemented groups was significantly less than that in the placebo group ( p < 0.05), and at 48 hours after EIMD, the HK-PS23 group recovered significantly faster than the placebo group ( p = 0.0008).

As shown in Figure 2C, at 24 hours after EIMD, the loss of jump height in the L-PS23 and HK-PS23 supplemented groups was significantly less than that in the placebo group ( p < 0.05). In addition, at 48 hours after EIMD, the HK-PS23 group recovered significantly faster than the placebo group ( p = 0.0097), and there was no significant difference compared with before EIMD. In addition, these results showed that RFD, relative peak strength, and jump height had significant time effects ( p < 0.0001).

(2) Isometric muscle strength

In the isometric mid-thigh pull-up (IMTP) test performed 3, 24, and 48 hours after EIMD, the percentage changes in relative peak strength and peak force rate of the placebo, L-PS23, and HK-PS23 groups were significantly reduced compared with those before EIMD ( p < 0.0001) ( Figure 3A and Figure 3B ). In addition, compared with the placebo group, the L-PS23 group had significantly less relative peak strength loss at 24 and 48 hours after EIMD, and the HK-PS23 group had significantly less relative peak strength loss at 3, 24, and 48 hours after EIMD ( p < 0.05) ( Figure 3A ).

On the other hand, compared with the placebo group, the L-PS23 group and the HK-PS23 group had significantly less loss in peak force rate at 3 and 24 hours after EIMD, and the recovery at 48 hours after EIMD was significantly better than that of the placebo group ( p < 0.05) (Figure 3B). In addition, these results showed that there was a significant time effect on relative peak strength and peak force rate ( p < 0.0001).

(3) Anaerobic exercise performance

The Wingate anaerobic test was used to evaluate the effects of EIMD on lower limb muscle strength and anaerobic strength. The Wingate anaerobic test at 3, 24, and 48 hours after EIMD showed that the percentage changes of relative mean power and relative peak power were significantly lower, and the percentage changes of fatigue index were significantly higher in the placebo group, L-PS23 group, and HK-PS23 group compared with those before EIMD ( p < 0.0001) (Figure 4A to Figure 4C).

In addition, at 3, 24, and 48 hours after EIMD, the relative mean power loss of the L-PS23 and HK-PS23 groups was significantly less than that of the placebo group ( p < 0.05) (Figure 4A). As shown in Figure 4B, at 24 and 48 hours after EIMD, the relative peak power loss of the L-PS23 and HK-PS23 groups was significantly less than that of the placebo group ( p < 0.05). As for the fatigue index, Figure 4C showed that the increase in fatigue index of the HK-PS23 group 24 hours after EIMD was significantly less than that of the placebo group ( p < 0.05), and the increase in fatigue index of the L-PS23 and HK-PS23 groups 48 hours after EIMD was significantly less than that of the placebo group ( p < 0.05). Furthermore, these results showed significant time effects for relative mean power, relative peak power, and fatigue index ( p < 0.0001).

The above results show that both L-PS23 and HK-PS23 can significantly prevent the decline in jump-related strength performance after EIMD, and have a better recovery effect, while also improving Wingate anaerobic power performance.

Example 4: Effects of L-PS23 and HK-PS23 supplements on biomarkers of muscle damage, inflammation, oxidative stress and anabolic hormones

The results of biomarker assessment are shown in Figures 5A to 5E and illustrate that all groups showed significant increases in CK, myoglobin, TBARS, and high-sensitivity C-reactive protein (hs-CRP) at 3, 24, and 48 hours after EIMD ( p < 0.05).

In addition, compared with the placebo group, the increase in CK activity at 24 and 48 hours after EIMD in the L-PS23 group and the HK-PS23 group was significantly reduced ( p < 0.05) (Figure 5A). The increase rate of myoglobin at 3 and 48 hours after EIMD in the L-PS23 group and the HK-PS23 group was significantly lower than that in the placebo group ( p < 0.05) (Figure 5B). As shown in Figure 5C, the increase rate of TBARS at 24 and 48 hours after EIMD in the L-PS23 group and the HK-PS23 group was significantly lower than that in the placebo group ( p < 0.05), and at 3 hours after EIMD, the increase rate of TBARS in the HK-PS23 group was also significantly lower than that in the placebo group and the L-PS23 group ( p < 0.05). The hs-CRP increase rates in the L-PS23 and HK-PS23 groups 24 and 48 hours after EIMD were significantly lower than those in the placebo group ( p < 0.05). At 48 hours after EIMD, the hs-CRP increase rate in the HK-PS23 group was also significantly lower than that in the L-PS23 group ( p < 0.0001) ( Figure 5D ).

Furthermore, as shown in Figure 5E, the percentage changes of testosterone at 3 and 24 hours after EIMD were significantly lower in the placebo, L-PS23, and HK-PS23 groups compared with before EIMD ( p < 0.0001). In addition, the percentage changes of testosterone at 48 hours after EIMD in the placebo group were still significantly lower than before EIMD, while the percentage changes of testosterone in the L-PS23 and HK-PS23 groups had recovered and were not significantly different from before EIMD. In addition, the HK-PS23 group had significantly less testosterone concentration loss at 3 and 24 hours after EIMD than the placebo group, and recovered significantly faster ( p < 0.05), and the loss at 24 hours after EIMD was also significantly less than that of the L-PS23 group ( p < 0.05).

These results suggest that supplementation with L-PS23 or HK-PS23 not only attenuates the elevation of CK or myoglobin after exercise, but also reduces oxidative stress and inflammation after EIMD. In addition, supplementation with L-PS23 or HK-PS23 significantly reduces testosterone loss and promotes faster recovery.

As shown above, supplementation with L-PS23 or HK-PS23 for six consecutive weeks significantly reduced muscle strength loss after EIMD and the levels of biomarkers of muscle damage, inflammation and oxidative stress in the blood, and was beneficial for accelerating recovery, indicating the protective, restorative and ameliorative benefits of supplementation with Lactobacillus paracasei PS23 after muscle damage.

Therefore, the probiotic composition provided by the present disclosure includes live strains or heat-killed live strains of Lactobacillus paracasei PS23, which can improve sports performance, such as reducing muscle strength loss, reducing the levels of biomarkers of muscle damage, inflammation and oxidative stress in serum, and accelerating muscle damage repair, strength and fatigue recovery. In addition, the probiotic composition provided by the present disclosure can prevent or minimize EIMD.

In addition, since the heat-killed probiotics (i.e., HK-PS23) also have excellent sports performance improvement effects, the probiotic composition containing HK-PS23 provided in the present disclosure is safer, has a longer shelf life, and is easier to transport and store.

It is obvious to a person skilled in the art that the basic concept can be implemented in many ways as the technology advances. Therefore, the specific embodiments are not limited to the above embodiments; on the contrary, they may vary within the scope of the patent application.

The specific embodiments described above can be used in any combination with each other. Several specific embodiments can be combined together to form another specific embodiment. The method disclosed herein may include at least one specific embodiment described above. It should be understood that the above benefits and advantages may relate to one specific embodiment or may relate to several specific embodiments. The specific embodiments are not limited to solving any or all of the above problems, nor are they limited to having any or all of the above benefits and advantages.

References

[1] Visconti, L.M.; Cotter, J.A.; Schick, E.E.; Daniels, N.; Viray, F.E.; Purcell, C.A.; Brotman, C.B.R.; Ruhman, K.E.; Escobar, K.A. Impact of varying doses of omega-3 supplementation on muscle damage and recovery after eccentric resistance exercise. Metabolism Open. 2021, 12:100133.

[2] Owens, D.J.; Twist, C.; Cobley, J.N.; Howatson, G.; Close, G.L. Exercise-induced muscle damage: What is it, what causes it and what are the nutritional solutions? European Journal of Sport Science. 2019, 19(1):71-85.

[3] Fatouros, I.G.; Jamurtas, A.Z. Insights into the molecular etiology of exercise-induced inflammation: opportunities for optimizing performance. Journal of Inflammation Research. 2016, 9:175-186.

[4] Salehi, M.; Mashhadi, N.S.; Esfahani, P.S.; Feizi, A.; Hadi, A.; Askari, G. The effects of curcumin supplementation on muscle damage, oxidative stress, and inflammatory markers in healthy females with moderate physical activity: a randomized, double-blind, placebo-controlled clinical trial. International Journal of Preventive Medicine. 2021, 12:94.

[5] Paulsen, G.; Mikkelsen, U.R.; Raastad, T.; Peake, J.M. Leucocytes, cytokines and satellite cells: what role do they play in muscle damage and regeneration following eccentric exercise? Exercise Immunology Review. 2012, 18:42-97.

[6] Peake, J.M.; Neubauer, O.; Della Gatta, P.A.; Nosaka, K. Muscle damage and inflammation during recovery from exercise. Journal of Applied Physiology (1985). 2017, 122(3):559-570.

[7] Marián, V.; Katarína, L.; Dávid, O.; Matúš, K.; Simon, W. Improved maximum strength, vertical jump and sprint performance after 8 weeks of jump squat training with individualized loads. Journal of Sports Science and Medicine. 2016, 15(3):492-500.

[8] Hong, W.H.; Lo, S.F.; Wu, H.C.; Chiu, M.C. Effects of compression garment on muscular efficacy, proprioception, and recovery after exercise-induced muscle fatigue onset for people who exercise regularly. PLoS One. 2022, 17(2):e0264569.

The present disclosure may be more fully understood by reading the following description of specific embodiments and referring to the accompanying drawings.

Figure 1 shows the experimental process conducted in this disclosure. In the intervention period, "asterisks" represent exercise-induced muscle damage; "circles" represent pre-test/post-test (pre-EIMD), which includes body composition, dietary records, blood biochemistry tests, CMJ, IMTP and Wingate; "triangles" represent exercise performance tests (CMJ, IMTP, Wingate) and blood biomarker assessments of muscle damage, inflammation, oxidative stress and anabolic hormones.

Figures 2A to 2C show the effects of supplementation with L-PS23 and HK-PS23 for six weeks on the rate of force development (RFD) (Figure 2A), relative peak force (Figure 2B), and jump height (Figure 2C), which were measured by countermovement jump (CMJ) before EIMD and 3, 24, and 48 hours after EIMD. Data are presented as mean ± SD of 35 individuals in each group (n = 35). Different superscript letters (a, b) indicate significant differences between groups at the same time point ( p < 0.05), and * indicates significant differences between the same group at different time points and before EIMD ( p < 0.05). L-PS23: live strain of Lactobacillus paracasei PS23; HK-PS23: heat-killed live strain of Lactobacillus paracasei PS23.

Figures 3A and 3B show the effects of supplementation with L-PS23 and HK-PS23 for six weeks on changes in relative peak strength (Figure 3A) and peak rate of force development (peak RFD) (Figure 3B), which were measured by isometric mid-thigh pull (IMTP) test before EIMD and 3, 24, and 48 hours after EIMD. Data are presented as mean ± SD of 35 individuals (n = 35) in each group. Different superscript letters (a, b, c) indicate significant differences between groups at the same time point ( p < 0.05), and * indicates significant differences between the same group at different time points and before EIMD ( p < 0.05). L-PS23: live strain of Lactobacillus paracasei PS23; HK-PS23: heat-killed live strain of Lactobacillus paracasei PS23.

Figures 4A to 4C show the effects of supplementation with L-PS23 and HK-PS23 for six weeks on the changes in relative mean power (Figure 4A), relative peak power (Figure 4B), and fatigue index (Figure 4C), which were measured by Wingate test before EIMD and 3, 24, and 48 hours after EIMD. Data are presented as mean ± SD of 35 individuals (n = 35) in each group. Different superscript letters (a, b, c) indicate significant differences between the groups at the same time point ( p < 0.05), and * indicates significant differences between the same group at different time points and before EIMD ( p < 0.05). L-PS23: live strain of Lactobacillus paracasei PS23; HK-PS23: heat-killed live strain of Lactobacillus paracasei PS23.

Figures 5A to 5E show the effects of supplementation with L-PS23 and HK-PS23 for six weeks on the changes in serum levels of creatine kinase (Figure 5A), myoglobin (Figure 5B), thiobarbituric acid reactive substances (TBARS) (Figure 5C), high-sensitivity C-reactive protein (hs-CRP) (Figure 5D), and testosterone (Figure 5E), which were measured before EIMD and 3 hours, 24 hours, and 48 hours after EIMD. The data are presented as the mean ± SD of 35 individuals (n = 35) in each group. Different superscript letters (a, b, c) indicate significant differences between the groups at the same time point ( p < 0.05), and * indicates significant differences between the same group at different time points and before EIMD ( p < 0.05). L-PS23: live strain of Lactobacillus paracasei PS23; HK-PS23: heat-killed live strain of Lactobacillus paracasei PS23.

Lactobacillus paracasei PS23: Bioresource Collection and Research Center, Institute for Food Industry Development, November 24, 2016, deposit number BCRC 9107551; and German Deposit Center for Microorganisms and Cell Cultures, June 6, 2016, deposit number DSM 32322.

Claims (10)

A composition for preparing a drug for preventing or minimizing exercise-induced muscle damage in an individual, wherein the composition comprises an effective amount of lactic acid bacteria and a carrier thereof, and the lactic acid bacteria is Lactobacillus paracasei PS23, which is deposited with DSMZ registration number 32322 or BCRC registration number 910755, and wherein the composition accelerates muscle recovery within 3 to 48 hours of the occurrence of the exercise-induced muscle damage, thereby preventing or minimizing the exercise-induced muscle damage. The use as described in claim 1, wherein the prevention or minimization of muscle damage caused by exercise includes at least one of increasing muscle strength, increasing muscle endurance, delaying or reducing muscle fatigue, and preventing or alleviating delayed muscle soreness. The use as described in claim 1, wherein the composition further enhances physical fitness, improves athletic performance, reduces inflammation and/or reduces oxidative stress in the individual. The use as described in claim 1, wherein the composition regulates the serum level of at least one biomarker associated with muscle damage, oxidative stress, inflammation or anabolic hormones in the individual. The use as described in claim 4, wherein the increase of at least one of creatine kinase, myoglobin, thiobarbituric acid reactive substances and high-sensitivity C-reactive protein caused by exercise-induced muscle damage in the individual is inhibited. The use as described in claim 4, wherein the decrease in testosterone caused by exercise-induced muscle damage in the individual is suppressed. The use as described in claim 1, wherein the form of the lactic acid bacteria is selected from the group consisting of live strains, heat-killed live strains, cultures, lysate products, extracts, and any combination thereof. The use according to claim 1, wherein the composition is administered to the individual in an amount of at least 1×10 ⁶ colony forming units of lactic acid bacteria per day. The use as described in claim 1, wherein the lactic acid bacteria is the only active ingredient in the composition for preventing or minimizing exercise-induced muscle damage. The use as described in claim 1, wherein the composition is administered to the individual for at least one week.