A composition containing three strains of Lactobacillus, and its uses.

Abstract

This invention provides a mixture of three strains of Lactobacillus fermentum and Lactobacillus platarum, as well as its uses. [Solution] A composition comprising a mixture of Lactobacillus fermentum GB102, GB103 strains and Lactobacillus plantarum GB104 strain, and their lysates, cultures, or extracts of cultures as active ingredients, can be usefully used for the prevention or treatment of muscle-related diseases or obesity, such as by alleviating age-related muscle weakness symptoms or reducing age-related changes in the intestinal microbial environment.

Description

[Technical Field]

[0001] This invention relates to a composition containing three strains of Lactobacillus bacteria, and to its uses. [Background technology]

[0002] The microbiome refers to the microorganisms present in a particular environment and their entire bioinformation, encompassing the genetic information and derived products (genome, transcriptome, proteome, metabolome) of the microbiome (microbiota) within that environment. Therefore, the human microbiome refers to the microorganisms residing inside and outside the human body and their entire bioinformation.

[0003] The human body lives in symbiotic relationships with many microorganisms, and the intestines, in particular, provide an optimal environment for microorganisms to obtain nutrients and form systematic communities, making them home to the largest number of microorganisms. Intestinal microorganisms supply nutrients that cannot be produced by the host's enzymes alone and are deeply related to the host's metabolic and immune systems. At the same time, they have been reported to be associated with the development of various diseases, including irritable bowel syndrome, obesity, atopic dermatitis, depression, rheumatoid arthritis, autism spectrum disorder, and dementia.

[0004] In recent years, Western dietary habits and the indiscriminate use of antibiotics have led to imbalances in the gut microbiota, resulting in a deterioration of gut health. Research into gut microbiota and various diseases has highlighted the importance of gut microbiota, leading to increased interest in the subject.

[0005] Furthermore, with the extension of the average human lifespan, an aging society has emerged, and humanity is facing various challenges never experienced before. Socially and economically, it is expected that the increase in the elderly population and the decrease in the working-age population will lead to an increase in the dependency ratio per elderly person, and there is also a growing trend of concern for improving the quality of life of the elderly. Thus, as the social needs for a healthy and happy old age increase, research on the changes in pathological conditions associated with aging and the prevention of age-related diseases is being actively conducted.

[0006] In recent years, as the interest in lactic acid bacteria has increased, research results confirming the utility value of lactic acid bacteria for the prevention and treatment of age-related diseases have been reported. Korean Registered Patent No. 10-2049700 discloses the preventive or therapeutic activity of Lactobacillus reuteri ATG-F4 strain against muscle diseases, but it is an invention with each single strain as an active ingredient, and the fact is that no invention with a mixed strain as an active ingredient has been disclosed yet. Therefore, the inventors of the present invention attempted to discover strains for use as compositions applicable as foods and pharmaceuticals. Summary of the Invention Problems to be Solved by the Invention

[0007] One embodiment provides a composition comprising at least one, two, or three or more selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum GB104 strain, its lysate, and culture medium, deposited under deposit number KCTC 14107BP.

[0008] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The objective is to provide an anti-aging or antioxidant composition containing as an active ingredient a composition comprising at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0009] Another aspect is to provide a composition for preventing, improving, or treating aging-related diseases, comprising at least one, two, or more than two selected from the group consisting of any one selected from the group consisting of Lactobacillus fermentum GB102 strain belonging to the genus Lactobacillus, its lysate, and its culture solution, deposited under the accession number KCTC 14105BP; any one selected from the group consisting of Lactobacillus fermentum GB103 strain belonging to the genus Lactobacillus, its lysate, and its culture solution, deposited under the accession number KCTC 14106BP; and any one selected from the group consisting of Lactobacillus plantarum GB104 strain belonging to the genus Lactobacillus, its lysate, and its culture solution, deposited under the accession number KCTC 14107BP as an active ingredient.

[0010] Another aspect is to provide a method for suppressing aging, comprising the step of administering an effective amount of the composition to an individual who needs it.

[0011] Another aspect is to provide the use of the composition for the manufacture of an anti-aging composition.

[0012] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The objective is to provide a health functional food for the prevention or improvement of muscle-related diseases or obesity, which contains as an active ingredient a composition comprising at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0013] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The objective is to provide a pharmaceutical composition for the prevention or treatment of muscle-related diseases or obesity, comprising as an active ingredient a composition containing at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0014] Another embodiment provides a method for preventing or treating a muscle-related disease, comprising the step of administering an effective amount of the composition to an individual in need.

[0015] Another aspect is to provide the use of the composition for the manufacture of health functional foods for the prevention or improvement of muscle-related diseases.

[0016] Another embodiment provides a method for preventing or treating obesity, comprising the step of administering an effective amount of the composition to an individual in need.

[0017] Another aspect is to provide the use of the composition for the manufacture of health functional foods for the prevention or improvement of obesity.

[0018] Another aspect is to provide the use of the composition for the manufacture of pharmaceutical formulations for the prevention or treatment of muscle-related diseases.

[0019] Another aspect is to provide the use of the composition for the manufacture of pharmaceutical formulations for the prevention or treatment of obesity.

[0020] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The objective is to provide a feed composition containing as an active ingredient a composition comprising at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium. [Means for solving the problem]

[0021] One embodiment provides a composition comprising at least one, two, or three or more selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum GB104 strain, its lysate, and culture medium, deposited under deposit number KCTC 14107BP.

[0022] In one specific example, the bacterial strain may be a composition of live or dead bacteria.

[0023] In one specific example, the mixed bacterial strains contained in the composition are 10 3 ~10 16 It can be included in an amount of CFU / g.

[0024] In one specific example, the composition may be administered orally.

[0025] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The present invention provides an anti-aging composition, antioxidant composition, or composition for the prevention, improvement, or treatment of aging-related diseases, comprising as an active ingredient a composition containing at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0026] In one specific example, the anti-aging may be one or more selected from the group consisting of anti-aging of muscle cells, anti-aging of nerve cells, anti-aging of skin cells, and anti-aging of the intestinal microbial environment.

[0027] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The present invention provides a health functional food for the prevention or improvement of muscle-related diseases or obesity, comprising as an active ingredient a composition containing at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0028] In one specific example, the composition can reduce at least one of the following: joint stiffness, muscle loss, strength loss, velocity loss, balance loss, endurance loss, and agility loss.

[0029] In one specific example, the composition may be selected from the group consisting of those that improve motor performance, those that restore muscle coordination, mobility and walking ability, and those that increase muscle mass and grip strength.

[0030] In one specific example, the pharmaceutical composition for the prevention or treatment of muscle-related diseases may improve athletic performance, restore muscle coordination, mobility and walking ability, increase muscle mass and grip strength, or suppress muscle loss in the elderly.

[0031] In one specific example, the muscle-related disease may be one or more selected from the group consisting of sarcopenia, age-related sarcopenia, muscular atrophy, muscular dystrophy, disuse muscle atrophy, motor neuron disease, inflammatory muscle disease, neuromuscular junction disease, endocrine muscle disease, muscle degeneration, myotonia, amyotrophic axonal sclerosis, myasthenia gravis, myositis, muscle calcification, muscle ossification, muscle weakness-related diseases, and cachexia.

[0032] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The present invention provides a pharmaceutical composition for the prevention or treatment of muscle-related diseases or obesity, comprising as an active ingredient a composition containing at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0033] In one specific example, the composition can reduce at least one of the following: joint stiffness, muscle loss, strength loss, velocity loss, balance loss, endurance loss, and agility loss.

[0034] In one specific example, the composition may be selected from the group consisting of those that improve motor performance, those that restore muscle coordination, mobility and walking ability, and those that increase muscle mass and grip strength.

[0035] In one specific example, the muscle-related disease may be one or more selected from the group consisting of sarcopenia, age-related sarcopenia, muscular atrophy, muscular dystrophy, disuse muscle atrophy, motor neuron disease, inflammatory muscle disease, neuromuscular junction disease, endocrine muscle disease, muscle degeneration, myotonia, amyotrophic axonal sclerosis, myasthenia gravis, myositis, muscle calcification, muscle ossification, muscle weakness-related diseases, and cachexia. [Effects of the Invention]

[0036] A composition comprising a mixture of Lactobacillus fermentum GB102, GB103 strains and Lactobacillus plantarum GB104 strain, and their lysates, cultures, or extracts of cultures as active ingredients, can be usefully used for the prevention or treatment of muscle-related diseases or obesity, such as by alleviating age-related muscle weakness symptoms or reducing age-related changes in the intestinal microbiota. [Brief explanation of the drawing]

[0037] [Figure 1] This graph shows the change in the uniformity of the bacterial flora when a mixed bacterial strain and herbal medicine are administered together, using a specific example. [Figure 2] This graph shows the results of principal coordinate analysis (PCoA) of the intestinal flora in a specific example of combined administration of mixed bacterial strains and herbal medicine. [Figure 3]This graph shows the frequency differences between major strains within the Firmicutes phylum when combined with the administration of mixed bacterial strains and herbal medicines, using a specific example. [Figure 4] This graph shows the frequency differences between major lineages within the Bacteroidetes phylum when combined administration of mixed bacterial strains and herbal medicines, using a specific example. [Figure 5] This graph shows the change in the ratio of Firmicutes / Bacteroidetes phyla upon administration of a specific example of a mixed bacterial strain composition. [Best Mode for Carrying Out the Invention]

[0038] One embodiment provides a composition comprising at least one, two, or three or more selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum GB104 strain, its lysate, and culture medium, deposited under deposit number KCTC 14107BP.

[0039] In one specific example, Lactobacillus fermentum GB102 strain, Lactobacillus fermentum GB103 strain, and Lactobacillus plantarum GB104 strain may be live or dead. More specifically, the dead strain may be dead due to heat treatment.

[0040] In this specification, the genus Lactobacillus sp. refers to the former genus Lactobacillus (Lactobacillus sp.) prior to the name change, which included Lactiplantibacillus plantarum and Limosilactobacillus fermentum.

[0041] The aforementioned Lactobacillus has been renamed Limosilacto bacillus or Lactiplantibacillus, and the renamed strain names can be used interchangeably in this specification. For example, Lactobacillus fermentum has been renamed Limosilactobacillus fermentum, and Lactobacillus plantarum has been renamed Lactiplantibacillus plantarum.

[0042] In this specification, the term "culture" can be used interchangeably with "supernatant," "conditional culture medium," or "adjusted medium," and can mean the entire medium containing the Lactobacillus strain, its metabolites, excess nutrients, etc., obtained by culturing the Lactobacillus strain for a certain period of time in a medium capable of supplying nutrients so that the Lactobacillus strain can grow and survive in vitro. The culture medium may mean the culture medium obtained by removing the cells from the bacterial culture medium obtained by culturing the strain. The medium can be selected from known liquid or solid media, and may be, but is not limited to, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, or BL agar medium.

[0043] In this specification, the term “lysate” means a solution or suspension in an aqueous medium of broken microbial cells, such as Lactobacillus fermentum or Lactobacillus plantarum. Cell lysates include macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, and lipids, and / or micromolecules such as amino acids, sugars, and fatty acids, or fractions thereof. Furthermore, the lysate may include cell fragments that are smooth or granular in structure.

[0044] Various known methods can be used to achieve the cytolysis of the aforementioned microorganisms, and any method capable of achieving microbial cytolysis can be used. For example, cell release / destruction can be carried out by enzymes, chemically or physically. Non-limiting examples of enzymes and enzyme mixtures include proteases such as proteinase K, lipases or glycosidases; non-limiting examples of chemicals include ion-permeable carriers, detergents such as sodium dodecyl sulfate, acids or bases; and non-limiting examples of physical means include high pressure such as a French press, osmotic pressure, and temperature such as heat or cold. Furthermore, methods using appropriate combinations of enzymes other than proteolytic enzymes, acids, bases, etc., can also be used.

[0045] The culture medium may include the culture medium itself obtained by culturing the bacterial strain, its concentrate, or its freeze-dried form, or the culture supernatant obtained by removing the bacterial strain from the culture medium, its concentrate, or its freeze-dried form.

[0046] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The objective is to provide an anti-aging or antioxidant composition containing as an active ingredient a composition comprising at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0047] In this specification, the term "contains as an active ingredient" means that a Lactobacillus strain, endoplasmic reticulum derived from said strain, lysate of said strain, culture medium, or extract of said culture medium is added, and includes the addition of various components as auxiliary components for purposes such as drug delivery and stabilization, and is formulated into various forms.

[0048] In this specification, the term “therapeutic effective dose” means the amount of a mixed strain composition for the methods and uses of the present invention, or a pharmaceutical composition containing a mixed strain for the methods and uses of the present invention, that a researcher, physician, or other clinician seeks to derive from a patient’s biological or medical response or desired therapeutic effect. The therapeutic effective dose of a mixed strain composition may vary depending on factors such as the individual’s condition, age, sex, and weight. The therapeutic effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or harmful effect.

[0049] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The objective is to provide a composition for the prevention, improvement, or treatment of aging-related diseases, comprising as an active ingredient a composition containing at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0050] In one specific example, a mixed strain or composition of mixed strains of the Lactobacillus genus may possess antioxidant activity. Without being limited to a specific theory, the natural process of aging makes the body vulnerable to oxidative damage. Anti-aging effects are primarily related to antioxidant properties and free radical scavenging ability. Furthermore, without being limited to a specific theory, oxidative damage has been proposed as one of the main causes of skeletal muscle loss that occurs with aging. Identifying free radicals as accelerators of the aging process may mean that their suppression can limit the harmful deformations they inflict on organisms (especially skeletal muscle). That is, if molecules with antioxidant capacity can respond to oxidative damage, this can play a crucial role in preventing the onset of aging-related conditions, including the detoxification process. Oxidative damage is the basis of the pathophysiological mechanisms that cause sarcopenia (and other age-related diseases), and interventions to enhance endogenous antioxidant defenses (e.g., administration of antioxidants) can suppress aging. For example, resveratrol, a polyphenol compound found in red wine, has been reported to slow the aging of Caenorhabditis elegans by reducing mitochondrial respiration (Wood et al., 2004). Furthermore, oxidative stress has been shown to cause an imbalance between cells responsible for bone formation and resorption, increasing bone turnover. Therefore, since antioxidants are known to suppress and / or improve the effects of oxidative stress, a mixed strain of Lactobacillus exhibiting antioxidant activity may be useful for anti-aging or the prevention, improvement, or treatment of age-related diseases.

[0051] In one specific example, administration of a composition of mixed Lactobacillus strains can improve muscle strength that has decreased due to aging. Specifically, in one embodiment of the present invention, an increase in muscle strength (grip strength) was observed in a group of aging mice administered the composition of mixed Lactobacillus strains.

[0052] In one specific example, administration of a composition of mixed Lactobacillus strains can mitigate age-related changes in the gut microbiota. In another specific example, administration of a composition of mixed Lactobacillus strains has been shown to make the gut microbiota of aging mice similar to that of young mice, and to help prevent or treat age-related diseases such as obesity caused by the gut microbiota of aging mice. A recent paper reported that the gut microbiota of aging mice may cause obesity, and that the proportion of Firmicutes / Bacteroidetes increases in aging mice (Binyamin et al., Genome Medicine, 2020). This suggests that administration of a composition of mixed lactic acid bacteria strains, in one specific example, may have a positive effect on preventing and treating diseases such as obesity in aging mice.

[0053] As used herein, the term “anti-aging” includes delaying or preventing the aging of cells or individuals, or converting senescent cells into younger cells.

[0054] In one specific example, the anti-aging may be one or more selected from the group consisting of anti-aging of skeletal muscle cells, anti-aging of nerve cells, anti-aging of skin cells, anti-aging of immune cells, and anti-aging of the intestinal microbial environment.

[0055] In this specification, age-related diseases may be muscular age-related diseases (e.g., sarcopenia) or obesity.

[0056] Aging alters everything from a single cell in the body to all tissues and organs. Aging not only increases wrinkles and causes hunchback, but also changes the body's composition. The proportions of water, muscle protein, fat, and skeletal minerals in the body change. Comparing a 25-year-old to a 70-year-old, water, muscle mass, and minerals all decrease, but fat more than doubles. Age-related changes in body fat are directly linked to lifestyle-related diseases. Not only does fat increase in proportion, but its distribution changes; subcutaneous fat decreases while visceral fat increases. Although the exact reasons are not yet fully understood, visceral fat secretes more harmful cytokines such as TNF-α and IL-6, which contribute to lifestyle-related diseases like diabetes and hypertension, and impairs many physiological functions.

[0057] Furthermore, muscle mass decreases after the age of 30, influenced by a decline in growth hormone and male hormones. Generally, it is known that muscle mass decreases by 1-2% annually after the age of 50, and muscle strength can decrease by as much as 1.5-3%.

[0058] In this specification, the term "prevention" may mean any action that suppresses or delays the onset of a disease in an individual by administering a pharmaceutical composition in one aspect.

[0059] In this specification, the term “treatment” may mean any action by which the administration of a pharmaceutical composition in one aspect improves or beneficially alters the symptoms of an individual’s medical condition.

[0060] As used in this invention, the term "muscle aging" refers to the gradual weakening of muscle density and function as mitochondria within muscle fibers lose their activity or decrease in number due to aging, and includes sarcopenia.

[0061] The term "muscle aging" as used in this invention encompasses the decline of muscles that occurs with aging, such as a decrease in muscle function (muscle strength, muscle endurance, muscle explosive power, etc.) and muscle atrophy. Muscle atrophy refers to a decrease in muscle mass due to a reduction or shrinkage of muscle cells. Due to muscle aging, after the age of 30, a phenomenon may occur in which muscle density and function gradually weaken, making one more susceptible to falls and fractures. The causes of muscle aging may include a decrease in growth hormone and male hormones, a decrease in the body's ability to synthesize proteins, and a decrease in the ability to absorb proteins or calories related to maintaining muscle density.

[0062] The term "sarcopenia" as used in this invention refers to a disease in which normal muscle mass, muscle strength, and muscle function decrease due to nutritional deficiencies, decreased physical activity, aging, etc. Muscle loss generally begins in the 30s, decreases by 30% in those over 60, and by around 80, muscle mass decreases by half.

[0063] Sarcopenia can lead to complications such as diabetes, hyperlipidemia, and obesity, and is associated with a decline in overall bodily function and weakening of bones. In particular, sarcopenia is often linked to spinal aging in older adults, and the probability of developing lumbar disc herniation is very high.

[0064] In this specification, the term "muscle aging-related diseases" refers to diseases caused by the aforementioned changes and abnormalities in the condition of muscles due to aging, but in particular, age-related muscle loss is called age-related sarcopenia.

[0065] In one specific example, the muscle-related disease may be, but is not limited to, sarcopenia, age-related sarcopenia, atony, muscular atrophy, muscular dystrophy, disuse muscle atrophy, motor neuron disease, inflammatory muscle disease, neuromuscular junction disease, endocrine muscle disease, muscle degeneration, myotonia, amyotrophic axonal sclerosis, myasthenia gravis, myositis, muscle calcification, muscle ossification, muscle weakness-related diseases, and cachexia.

[0066] The aforementioned muscle aging-related diseases may be accompanied by muscle inflammation due to trauma, and the composition of the present invention has the effect of reducing or improving muscle inflammation.

[0067] In this specification, the term “obesity” refers to a condition in which there is an excess of body fat. Clinically, obesity may also refer to a body mass index (BMI) of 25 or higher in Korea, or 30 or higher according to the World Health Organization (WHO). Generally, obesity means having a body weight higher than normal, but even if a person does not have a high body weight, they may still be obese if the percentage of body fat among the body's components is high. Obesity can occur in both adults and children. Obesity can cause not only weight gain but also obesity-related diseases such as overeating, overdrinking and bulimia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux, arteriosclerosis, hypercholesterolemia, hyperuricemia, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, or polycystic ovary syndrome. Therefore, the composition can be used not only for obesity but also for the prevention or treatment of the obesity-related diseases. Furthermore, the composition can be used by individuals who wish to lose weight even if they are not obese.

[0068] The obesity can be caused by various factors. For example, the causes can be high-fat diet, reduced physical activity, genetics, psychological factors, endocrine system abnormalities, metabolic abnormalities, social and environmental factors. In particular, the obesity can be induced by a high-fat diet.

[0069] The composition according to one specific example may contain a mixed strain of Lactobacillus genus in an amount of 0.001% to 80% by weight based on the total weight of the composition. Also, the dosage of the mixed strain of Lactobacillus genus can be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. The strain is contained in the composition at a therapeutically effective amount or a nutritionally effective concentration. For example, one strain, a mixture of two strains, or a mixture of three strains is 10 3 ~10 16 CFU / g, 10 3 ~10 15 CFU / g, 10 3 ~10 14 CFU / g, 10 3 ~10 13 CFU / g, 103~10 12 CFU / g, 10 4 ~10 16 CFU / g, 10 4 ~10 15 CFU / g, 10 4 ~10 14 CFU / g, 10 4 ~10 13 CFU / g, 10 4 ~10 12 CFU / g, 10 5 ~10 16 CFU / g, 10 5 ~10 15 CFU / g, 10 5 ~10 14 CFU / g, 10 5 ~10 13 [[ID=5�]]CFU / g, 10 5 ~10 12 CFU / g, 10 6 ~10 13 CFU / g, 10 6 ~10 12CFU / g, 10 7 ~10 13 CFU / g, 10 7 ~10 12 CFU / g, 10 8 ~10 13 CFU / g, or 10 8 ~10 12 It is included in the composition in a CFU / g content, or as a culture of an equivalent number of live or dead bacteria. Specifically, for adult patients, 1 × 10 3 ~1 × 10 16 Live or dead bacteria at CFU / g can be administered in one or several divided doses. However, the dosage can be formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity, and those skilled in the art can appropriately control the dosage considering these factors. The number of doses can be one or two or more, within the range of clinically acceptable side effects, and the administration site can be one or two or more. For non-human animals, the dosage can be the same as for humans per kg (body weight), or a converted amount based on the above dosage, for example, by the volume ratio of organs (such as the heart) of the target animal and humans (e.g., average value). Possible routes of administration may include oral, sublingual, parenteral (e.g., subcutaneous, intramuscular, intra-arterial, intraperitoneal, intradural, or intravenous), rectal, topical (including percutaneous), inhalation, and injection, or insertion of an implantable device or substance. One example of an animal targeted for treatment is a human or other target mammal, specifically including humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, and pigs. According to one embodiment, the composition contains dead dried bacterial strains and can be administered in doses of 1g to 10g, 0.5g to 1.5g, 2.5g to 3.5g, or 4.5g to 5.5g, once to three times a day.

[0070] A pharmaceutical composition according to one specific example may contain pharmaceutically acceptable carriers and / or additives. For example, a pharmaceutical composition may include sterile water, physiological saline, conventional buffers (such as phosphoric acid, citrate, and other organic acids), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonic agents, or preservatives. For topical administration, it may also include combinations with organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and their chemical derivatives. When a pharmaceutical composition according to one specific example is prepared in a dosage form suitable for injection, Lactobacillus cells may be dissolved or dispersed in a pharmaceutically acceptable carrier, or frozen as a solution in which they are dissolved or dispersed.

[0071] A pharmaceutical composition according to one specific example may appropriately contain suspensions, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, reducing agents, antioxidants, etc., as needed depending on the method of administration and dosage form. Pharmacovigilant carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature "Remington's Pharmaceutical Sciences, 19th ed., 1995". A pharmaceutical composition according to one specific example may be manufactured in unit dose form by formulation using pharmaceutically acceptable carriers and / or excipients in a manner readily accessible to a person with ordinary skill in the art to which the invention belongs, or by being contained in a multi-dose container. The dosage form may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablet, or capsule.

[0072] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” as used herein means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective dose can be determined depending on factors including the type and severity of the patient’s disease, the activity of the drug, sensitivity to the drug, administration time, route of administration and elimination rate, duration of treatment, drugs used concurrently, and other well-known factors in the medical field. The compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or concurrently with conventional therapeutic agents, and can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that yields the maximum effect with the minimum amount without side effects, which can be readily determined by those skilled in the art.

[0073] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, and culture medium, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The objective is to provide a health functional food for the prevention or improvement of muscle-related diseases or obesity, which contains as an active ingredient a composition comprising at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, and culture medium.

[0074] In one specific example, the health functional food may further include a food-grade acceptable carrier.

[0075] In this specification, the term "food-safe" means that the compound exhibits properties that are non-toxic to cells or humans exposed to it.

[0076] In this specification, the term “improvement” may mean any action that at least reduces the severity of a symptom, for example, any parameter related to the treatment state. In this case, the health functional food may be used for the prevention or improvement of cancer, before or after the onset of the disease, simultaneously with or separately from a therapeutic agent.

[0077] In the aforementioned functional health food, the active ingredient may be added directly to the food or used in combination with other foods or food ingredients, and can be used as appropriate according to the usual methods. The amount of active ingredient mixed can be appropriately determined according to its intended use (prevention or improvement). Generally, when manufacturing food or beverages, the functional health food can be added in an amount of approximately 15% by weight or less, more specifically, approximately 10% by weight or less, relative to the raw materials. However, in the case of long-term intake for the purpose of health and hygiene, or for the purpose of health regulation, the amount may be less than the aforementioned range.

[0078] The aforementioned health functional food may be formulated into one selected dosage form from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids, further comprising one or more carriers, diluents, excipients, and additives. Foods to which the compound in one embodiment can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

[0079] Specific examples of the carrier, excipient, diluent, and additive may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0080] In addition to containing the active ingredients, the aforementioned health functional food may contain other ingredients as essential components without particular limitation. For example, various flavorings or natural carbohydrates may be included as additional ingredients along with a regular beverage. Examples of the above-mentioned natural carbohydrates may be monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; and polysaccharides, such as common sugars like dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavorings other than those mentioned above, natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycylhidin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates can be appropriately determined by those skilled in the art.

[0081] In addition to the above, a functional health food according to one embodiment may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and enhancers (such as cheese and chocolate), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Such components may be used independently or in combination, and the proportions of such additives may be appropriately selected by those skilled in the art.

[0082] The aforementioned functional health food can be provided in combination with conventionally known functional health foods for the prevention or improvement of muscle-related diseases or metabolic diseases, or with other existing functional health foods, the aforementioned other functional health foods for the prevention or improvement of muscle-related diseases or metabolic diseases may be conventionally known functional health foods for the prevention or improvement of metabolic diseases, existing functional health foods, or newly developed functional health foods.

[0083] If the aforementioned health functional food contains other health functional foods that have preventive or ameliorative effects on muscle-related diseases or metabolic diseases, it is important that they be mixed in an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0084] The aforementioned food compositions for the prevention or improvement of muscle-related diseases and obesity include all forms such as functional foods, nutritional supplements, health foods, and food additives, and these types of food compositions can be manufactured in various forms according to common methods known in the industry.

[0085] The compositions herein may be considered food supplements. Food supplements, also known as dietary supplements or nutritional supplements, may be considered other certain pharmaceutical products. They are prepared for use as a supplement to a diet and are intended to provide nutrients or beneficial ingredients that cannot be obtained or are not obtained in sufficient quantities through a normal diet. Most food supplements are considered foods, but sometimes they are considered drugs, natural health products, or nutraceutical products. In the sense of the present invention, food supplements include nutraceutical products. Generally, food supplements are sold over the counter without a prescription. When food supplements take the form of pills or capsules, they contain the same excipients as those used in pharmaceuticals. However, food supplements may also take the form of food fortified with several nutrients (e.g., infant formula). Therefore, in certain embodiments, the composition of the present invention is a food supplement.

[0086] The compositions according to the present invention may be administered as is, or mixed with a suitable food liquid or solid, or may be freeze-dried in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, syrups, or unit doses. They may also be in the form of monodoses of the freeze-dried composition, which are mixed in a separate liquid container provided together before administration.

[0087] The compositions of the present invention may be included in a variety of edible foods and products, such as milk products, for infants. As used herein, the term “edible product” has a broad meaning and includes any form of product ingested by an animal (e.g., a product received by sensory organs). The term “food product” is understood as an edible product that provides nutritional support to the body. Foods of particular interest are food supplements and infant formulas. Preferred foods include carrier materials such as oatmeal porridge, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins, and glycosylated proteins. In certain embodiments, the bacterial cells of the present invention are homogenized with other components, such as cereals or powdered milk, to constitute infant formula.

[0088] Other embodiments include any of the following selected from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, culture medium, and culture medium extract, deposited under deposit number KCTC 14105BP; Lactobacillus fermentum GB103 strain, its lysate, culture medium, and culture medium extract, deposited under deposit number KCTC 14106BP; and Lactobacillus plantarum, a species of Lactobacillus deposited under deposit number KCTC 14107BP. The present invention provides a feed composition for the prevention or improvement of muscle-related diseases, comprising as an active ingredient a composition containing at least one, two, or three or more selected from the group consisting of the plantarum GB104 strain, its lysate, culture medium, and extract of the culture medium.

[0089] The aforementioned feed composition for preventing or improving aging-related diseases can be manufactured by adding the mixed bacterial strain composition within an appropriate effective concentration range according to various feed manufacturing methods known in the industry, and can be used as a feed additive composition for the purpose of preventing or improving aging-related diseases.

[0090] The term "feed" can mean any natural or artificial prescribed diet, single-serving diet, or components of such a single-serving diet, that an animal eats, ingests, and digests, or that is suitable for such purposes. The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include plant-based feeds such as grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, gourds, or grain by-products; and animal-based feeds such as proteins, lipids, oils and fats, mineralogy, single-cell proteins, zooplankton, or food products. [Modes for carrying out the invention]

[0091] The following are preferred embodiments to aid in understanding the present invention. However, these embodiments are provided to facilitate understanding of the present invention and do not limit the scope of the invention. The embodiments can be modified in various ways, and the embodiments are not limited to those disclosed below and can be implemented in various forms.

[0092] Embodiment 1: Isolation and identification of Lactobacillus fermentum and Lactobacillus plantarum strains

[0093] 1.1 Isolation of bacterial strains

[0094] The Lactobacillus fermentum and Lactobacillus plantarum strains of this invention were isolated from vaginal samples of healthy women who visited a hospital for a health checkup. Specifically, vaginal samples were collected with a cotton swab, inoculated into Rogosa SL (MRS) agar plates, and cultured in an anaerobic chamber at 37°C for 48 hours. Once the bacterial colonies had grown, a single colony was subcultured on a fresh MRS agar plate for pure isolation. After pure isolation, the strains were cultured using MRS medium.

[0095] 1.2 Selection of bacterial strains that inhibit fat accumulation

[0096] To select bacterial strains with lipid accumulation inhibitory activity, we confirmed the ability to inhibit pancreatic lipase activity and the ability to suppress the differentiation of 3T3-L1 adipocytes into adipocytes.

[0097] Specifically, the ability to inhibit the activity of pancreatic lipolytic enzymes was confirmed by diluting the bacterial strain 1-1 to a concentration of 0.1 mg / ml, then placing it in a plate with 0.167 mM p-nitrophenylpalmitate (PNP; Sigma, USA) solution, 0.061 M Tris-HCl buffer (pH 8.5), and 0.3 mg / ml lipase solution, reacting at 25°C for 10 minutes, and finally measuring the absorbance at 405 nm.

[0098] Furthermore, the ability of 3T3-L1 adipocyte progenitor cells to suppress adipocyte differentiation was measured using Oil Red O (Sigma, USA), which specifically reacts with intracellularly generated fat globules. After adipocyte differentiation was complete, the culture medium was removed, the cells were washed twice with PBS, fixed with 10% formalin at 40°C for 1 hour, washed twice with 60% isopropanol, and stained with 0.5% Oil Red O solution at room temperature for 30 minutes. After staining, the staining solution was removed and the cells were washed twice with distilled water. Once the distilled water had completely dried, isopropyl alcohol was added, and the absorbance was measured at 520 nm.

[0099] Finally, the 3T3-L1 cell viability of the bacterial strains obtained in 1-1 above was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The 3T3-L1 cells were 16 × 10⁶ 4 The cells / well concentration was dispensed into a 96-well plate, incubated for 24 hours, and then the medium was removed. To this, 100 μl of fresh DMEM medium was added to each of the lactic acid bacteria samples (100, 1,000 μg / ml) diluted to different concentrations, and incubated for 24 hours. Subsequently, 20 μl of MTT (Sigma, USA) solution prepared at 5 mg / ml was added, and incubated at 37°C for 4 hours. After incubation, the supernatant was removed, 200 μl of dimethyl sulfoxide (DMSO) was added, and the absorbance was measured at 546 nm.

[0100] From the results described above, the lipid accumulation inhibitory effect of each strain was confirmed, and among the prepared strains, Lactobacillus fermentum GB102 (hereinafter referred to as "GB102"), Lactobacillus fermentum GB103 (hereinafter referred to as "GB103"), and Lactobacillus plantarum GB104 (hereinafter referred to as "GB104") strains with low lipid cell accumulation inhibitory effect and cytotoxicity were finally selected. In addition, the strain name of Lactobacillus fermentum was changed to Limosilactobacillus fermentum, and the strain name of Lactobacillus plantarum was changed to Lactiplantibacillus plantarum. In the following embodiments, the strain names changed from existing strains are described in a mutually interchangeable manner.

[0101] 1.3 Molecular biological identification of selected bacterial strains

[0102] To identify the final selected Lactobacillus fermentum GB102, GB103, and Lactobacillus plantarum GB104 strains, analysis was performed using 16S rRNA gene sequences. 16S rRNA gene sequences obtained by PCR using 27F and 1492R Primers targeting bacterial 16S rRNA genes were analyzed using Sanger sequencing. The 16S rRNA sequences of Lactobacillus fermentum GB102, GB103, and Lactobacillus plantarum GB104 are shown in sequence lists 1, 2, and 3, respectively.

[0103] Furthermore, a homology analysis was performed on these strains, and as shown in Table 1 below, strains GB102 and GB103 showed the highest sequence similarity to the standard strain (type strain) of Limosilactobacillus fermentum, followed by a high sequence similarity to Limosilactobacillus gorilla. GB104, as a 16S rRNA sequence, shows a sequence similarity of over 98% to 18 species of the Limosilactobacillus genus, including the standard strain of Lactiplantibacillus plantarum, making species indistinguishable based on 16S rRNA gene sequences. GB104 was confirmed to be a strain belonging to Lactiplantibacillus plantarum through a comparison of the average nucleotide identity (ANI) values ​​of the genome sequences after genome analysis. In conclusion, it was confirmed that the two strains mentioned above correspond to Limosilactobacillus fermentum, and one strain corresponds to Lactiplantibacillus plantarum.

[0104] [Table 1] JPEG2026086916000002.jpg46170

[0105] Embodiment 2: Genomic and comparative genomic analysis of Lactobacillus fermentum strains

[0106] To investigate the genome-based species identification and characterization of the GB102 and GB103 strains, the genome sequences of the strains were fully analyzed using next-generation sequencing technology (NGS) and bioinformatics techniques, and the functions of the genes contained in the genomes were estimated. Furthermore, the specificity of the strains was confirmed through comparative analysis with fully analyzed genome sequences of the same species. The strains were cultured in MRS liquid medium at 37°C under anaerobic conditions for 4 hours, and genomic DNA was extracted from the cultures using the MG Genomic DNA purification kit (MGMED, Inc., Korea). To obtain long read data with an average length of 10kb or more, sequence analysis was performed using a PacBio RS II instrument. To generate high-precision short read data with a sequence length of less than 500bp to complement the long read data, which would otherwise have lower accuracy, sequence analysis was performed using a NovaSeq 6000 instrument. The long read data were assembled into high-quality GB102 and GB103 draft genomes using the HGAP2 pipeline of the SMRT Analysis server. SNPs and InDel errors that may be present in the assembled draft genome sequence were corrected using long read data and fragment sequence data. Coding sequences (CDS) were predicted from the completed genome sequence using the Prodigal program, and rRNA and tRNA were predicted using the RFAM tool. The predicted CDS were subjected to homology searches (applying the BLAST algorithm) based on the publicly available UniProt database, GenBank nr database, Subsystem database, PFAM database, and COG database to predict their function. Standard strain B1 28 of the Lactobacillus fermentum species, which is publicly available in GenBank, was used. T (=ATCC 14931 T ) and Lactobacillus gorillae standard strain KZ01T Lactobacillus gastricus standard strain DSM 16045 T The average nucleotide identity (ANI) was calculated using the Jspecies program based on the genome sequences of the GB102 and GB103 strains. Analysis revealed that both GB102 and GB103 strains exhibited ANI values ​​of over 95% compared to the Lactobacillus fermentum standard strain, confirming their belonging to the Lactobacillus fermentum species (Table 2). Furthermore, the fact that GB102 and GB103 strains do not 100% match the standard strain indicates that they are novel species that have not been previously isolated or reported. Additionally, the large genomic distances between GB102 and GB103 strains confirmed that they are distinct strains. The inventors named the GB102 and GB103 strains "Lactobacillus fermentum GB102" (deposit number: KCTC 14105BP) and "Lactobacillus fermentum GB103" (deposit number: KCTC 14106BP), respectively, and deposited them with the Korean Collection for Type Cultures (KCTC), located at the Korea Institute of Biotechnology, on January 14, 2020.

[0107] [Table 2]

[0108] Embodiment 3: Genome and comparative genome analysis of Lactobacillus plantarum strains

[0109] To investigate the genome-based species identification and characterization of the GB104 strain, the strain's genome sequence was fully analyzed using next-generation sequencing technology (NGS) and bioinformatics techniques, and the function of the genes contained in the genome was estimated. Furthermore, the specificity of the strain was confirmed through comparative analysis with a fully analyzed genome sequence of the same species. The strain was cultured in MRS liquid medium at 37°C under anaerobic conditions for 4 hours, and genomic DNA was extracted from the culture using the MG Genomic DNA purification kit (MGMED, Inc., Korea). Sequence analysis was performed using a PacBio RS II instrument to obtain long read data with an average length of 10kb or more, and sequence analysis was performed using a NovaSeq 6000 instrument to generate high-precision fragment sequence data with a sequence length of less than 500bp to compensate for the long read data, which would otherwise have lower accuracy. The long read data was assembled into a high-quality GB104 draft genome using the HGAP2 pipeline of the SMRT Analysis server. Potential SNPs and InDel errors in the assembled draft genome sequence were corrected using long read data and fragment sequence data. CDS were predicted from the completed genome sequence using the Prodigal program, and rRNA and tRNA were predicted using the RFAM tool. The predicted CDS were then subjected to homology searches (using the BLAST algorithm) based on the publicly available UniProt database, GenBank nr database, Subsystem database, PFAM database, and COG database to predict their function. The GB104 genome is summarized in Table 3.

[0110] [Table 3]

[0111] Genomic analysis revealed that this strain possessed the plantaricin gene cluster, a group of bacteriocin biosynthesis genes associated with antimicrobial activity, which is found in Lactobacillus plantarum. However, since it did not 100% match the standard and reference strains, it is considered a novel species that has not been isolated or reported to date.

[0112] The inventors named the GB104 strain "Lactobacillus plantarum GB104" (deposit number: KCTC 14107BP) and deposited it with the Korean Collection for Type Cultures (KCTC), located at the Korea Institute of Biotechnology, on January 14, 2020.

[0113] Experimental Example 1: Verification of the antioxidant effect of a mixed bacterial strain (GB102 + GB103 + GB104) consisting of Lactobacillus strains.

[0114] To confirm the antioxidant capacity of substances containing GB102, GB103, and GB104 strains as active ingredients, or substances containing the strains and auxiliary ingredients (vitamin B2 and red ginseng), DPPH free radical scavenging activity was evaluated.

[0115] Specifically, the DPPH solution was prepared as a 0.2 mM solution in 100% methanol. The GB102, GB103, GB104 strains and the Lacticaseibacillus rhamnosus GG (formerly Lactobacillus rhamnosus GG, positive control group) strain were inoculated into MRS liquid medium and cultured at 37°C every 16 hours. Afterward, the culture solutions were centrifuged (3600 rpm, 15 minutes) to obtain cultured lactic acid bacteria. These were then washed twice with 1×PBS, and a mixture of the three lactic acid bacteria (GB102, GB103, GB104) was prepared in 5×10⁶ units. 9Samples with CFU / ml concentrations were prepared. Similarly, a comparative strain of Lacticaseibacillus rhamnosus GG was prepared in 5 × 10⁻⁶ units. 9 The preparation was done at a CFU / ml concentration.

[0116] For the auxiliary ingredients, samples were prepared at a concentration of 625 ug / ml using vitamin B2 and the contents of red ginseng capsules, while L-ascorbic acid (positive control group) was prepared at a concentration of 12.5 ug / ml.

[0117] A combination of three lactic acid bacteria: 125ug (200ul) of auxiliary ingredients, GB102, GB103, and GB104 (1x10). 9 CFU (200µl), a mixture of three lactic acid bacteria: GB102, GB103, and GB104 (1x10) 9 Each of the CFU and adjuncts (125 ug (200 ul)) was mixed with 200 ul of 0.2 mM DPPH solution and reacted in a dark room at room temperature for 30 minutes. The mixture was then centrifuged (12700 rpm, 5 minutes) to obtain 200 ul of supernatant, which was placed in a 96-unit immunoflat plate. The absorbance at 515 nm was measured using a Spectramax iD3 (molecular device). L-ascorbic acid (2.5 ug (200 uL)) and Lacticaseibacillus rhamnosus GG (1 x 10⁹ CFU (200 uL)) were used as positive controls. The results are shown in Table 4.

[0118] The DPPH radical scavenging activity was calculated using the following method.

[0119] DPPH radical scavenging activity (%) = (ODcontrol-ODsample) / (ODcontrol)x100

[0120] ODcontrol: Absorbance of PBS, ODsample: Absorbance of sample

[0121] [Table 4]

[0122] As shown in Table 4, Lacticaseibacillus rhamnosus GG 1 × 10 9 We were able to confirm DPPH radical scavenging activity of approximately 26.2% for CFU and approximately 53% for 2.5 ug of L-ascorbic acid. In comparison, 125 ug of auxiliary material showed approximately 14%, and a mixture of three lactic acid bacteria (GB102, GB103, GB104) showed approximately 1x10⁻¹⁰ activity. 9 CFU is approximately 44.4%, and it contains a mixture of three lactic acid bacteria: GB102, GB103, and GB104 (1x10). 9 We were able to confirm an effect of approximately 56.2% with CFU + auxiliary ingredient 125ug. This confirmed that the mixture of three lactic acid bacteria, GB102, GB103, and GB104, has superior antioxidant effects compared to the positive control lactic acid bacterium, Lacticaseibacillus rhamnosus GG.

[0123] The natural process of aging makes the body vulnerable to oxidative damage. Furthermore, anti-aging effects are primarily related to antioxidant properties and free radical scavenging ability. Moreover, oxidative damage has been proposed as one of the main causes of skeletal muscle loss that occurs with aging. Identifying free radicals as accelerators of the aging process may mean that their suppression can limit the harmful deformation they inflict on organisms (especially skeletal muscle). That is, if molecules with antioxidant capacity can respond to oxidative damage, this can play a crucial role in preventing the onset of aging-related conditions, including the disintegration process. Oxidative damage is the basis of the pathophysiological mechanisms that cause sarcopenia (and other age-related diseases), and interventions to enhance endogenous antioxidant defenses (e.g., administration of antioxidants) can suppress aging. For example, resveratrol, a polyphenol compound found in red wine, has been reported to slow aging in Caenorhabditis elegans by reducing mitochondrial respiration (Wood et al., 2004). Furthermore, oxidative stress has been shown to cause an imbalance between cells responsible for bone formation and resorption, increasing bone turnover. Therefore, since antioxidants are known to suppress and / or improve the effects of oxidative stress, a mixed strain of Lactobacillus exhibiting antioxidant activity may be useful for anti-aging or the prevention, improvement, or treatment of age-related diseases.

[0124] Experimental Example 2: Confirmation of the individual antioxidant effects of Lactobacillus strains GB102, GB103, and GB104.

[0125] To confirm the antioxidant capacity of each strain, GB102, GB103, and GB104, DPPH free radical scavenging ability was evaluated.

[0126] Specifically, a 0.2 mM DPPH (Alfa Aesar) solution was prepared using DPPH reagent in 100% methanol. The GB102, GB103, GB104 strains and the Lacticaseibacillus rhamnosus GG (positive control group) strain were inoculated into MRS liquid medium and incubated at 37°C for 16 hours. Subsequently, the culture solutions were centrifuged (3600 rpm, 15 minutes) to obtain cultured lactic acid bacteria. These were then washed twice with 1×PBS, and a mixture of the three lactic acid bacteria (GB102, GB103, GB104) was prepared in 5×10⁶ units. 9 Samples with CFU / ml concentrations were prepared. Similarly, a comparative strain of Lacticaseibacillus rhamnosus GG was prepared in 5 × 10⁻⁶ units. 9 Prepared at CFU / ml concentration. 1 × 10⁶ of each of the three lactic acid bacteria: GB102, GB103, and GB104. 9 CFU (200µl), 1x10 of a mixture of three lactic acid bacteria 9 200 µl of CFU was mixed with 200 µl of 0.2 mM DPPH solution, and the mixture was reacted in a dark room at room temperature for 30 minutes. The mixture was then centrifuged (12700 rpm, 5 minutes) to obtain 200 µl of supernatant, which was placed in a 96-well immunoplate. The absorbance at 515 nm was measured using Spectramax iD3 (molecular devices). The DPPH radical scavenging activity was calculated using the same method as in Experimental Example 1.

[0127] [Table 5]

[0128] As shown in Table 5, DPPH radical scavenging activity was confirmed for GB102 (64.1%), GB103 (41.0%), GB104 (34.5%), Lacticaseibacillus rhamnosus GG (38.1%), and GB102+103+104 (approximately 53%). This confirmed that each of the GB102, GB103, and GB104 strains possesses antioxidant effects.

[0129] Experimental Example 3: Verification of the efficacy of combined administration of mixed bacterial strains and / or herbal medicines for muscle strength improvement.

[0130] To confirm the effect of co-administration of a mixed bacterial strain consisting of Lactobacillus strains and / or herbal medicines on improving muscle strength that has declined due to aging, changes in muscle strength in aged mice were observed when a mixed bacterial strain (hereinafter referred to as "#7 lactic acid bacteria" or "mixed bacterial strain") consisting of three types of lactic acid bacteria, GB102, GB103, and GB104, and a mixture of four types of herbal medicines, along with auxiliary ingredients further containing inulin and vitamin B2, were administered individually or in combination.

[0131] The adjuncts administered in combination with the mixed bacterial strain included herbal medicines, inulin, and vitamin B2. The herbal medicines used were Andong yam, Job's tears, red ginseng powder, and reishi mushroom. The total amount of herbal medicines (Andong yam, Job's tears, red ginseng powder, reishi mushroom), inulin, and vitamin B2 was 70.20 mg. Including 30.30 mg of cryopreservative, each mouse was administered a total dose of 100.5 mg of the adjuncts.

[0132] [Table 6]

[0133] Specifically, the experimental group consisted of 16-month-old C57BL / 6 mice divided into four groups. Experimental group 1 (G1) was orally administered a mixed bacterial strain (#7 lactic acid bacteria) consisting of Lactobacillus fermentum GB102, GB103 strains (deposit numbers KTCT 14105BP, KCTC 14106BP), and Lactobacillus plantarum GB104 strain (deposit number KCTC 14107BP). The mixed bacterial strain (#7 lactic acid bacteria) was administered at a dose of 5 × 10⁶ per mouse. 8The bacteria were administered in CFU doses, and to maintain the guaranteed bacterial count, they were administered together with Bifidobacterium animalis sub sp. Lactis strains and Lactobacillus acidophilus obtained from Mediogen Co., Ltd. Experimental group 2 (G2) was orally administered a mixture of Chinese herbal medicine consisting of Andong yam, Job's tears, red ginseng powder, and reishi mushroom, along with auxiliary ingredients containing inulin and vitamin B2. The auxiliary ingredients were administered at a dose of 100.5 mg per mouse, as shown in Table 6 above. Experimental group 3 (G3) was orally administered the mixed bacterial strain and auxiliary ingredients together. Mice fed a normal chow diet (NCD) were set as the control group. Each control group and experimental group used 15 mice. The experimental conditions for the above experimental groups and control groups are summarized in Table 7 below.

[0134] [Table 7]

[0135] After 13 weeks of administration, each experimental group was tested for muscle strength improvement using a Grip Strength device. The mice gripped the wire portion of the Grip Strength device with both forelegs, and the pulling force (Grip Strength) was measured. The results are shown in Table 8.

[0136] [Table 8]

[0137] As shown in Table 8, compared to the control group, experimental group 1 (G1), and experimental group 2 (G2), experimental group 3 (G3), which was administered with a mixed bacterial strain and auxiliary ingredients, showed a significantly greater tendency for increased grip strength.

[0138] Based on these results, we confirmed that the combined administration of a mixed strain of Lactobacillus (#7 lactic acid bacteria) and a mixture of herbal medicines improved muscle strength that had declined due to aging.

[0139] Experimental Example 4: Verification of the effectiveness of measuring myostatin in serum.

[0140] Myostatin ELISA analysis was performed on serum samples obtained after necropsy from aged mice in a control group (PBS), a mixed bacterial strain, a secondary ingredient, and a mixed bacterial strain plus secondary ingredient.

[0141] Serum analysis was performed using the DGF-8 / Myostatin DuoSet (R&D systems) ELISA, and the detailed experimental procedures followed the protocol provided by the manufacturer. Briefly describing the general analytical method, the product's Capture antibody was incubated overnight at 4°C on a 96-well flat immunoplate (SPL) the day before, followed by three washings using 0.05% Tween 20 in PBS wash buffer the next day. Subsequently, the sample was blocked at room temperature for 1 hour with 1% BSA in PBS sample diluent, followed by three washings using wash buffer. The sample was diluted to the dilution ratio that falls within the myostatin standard range, and after sample loading of 100 μl each, it was incubated at room temperature for 2 hours, followed by three washings using wash buffer. The Detection antibody was incubated at room temperature for 1 hour, followed by three washings using wash buffer, and then streptavidin-HRP was incubated at room temperature for 20 minutes, followed by three washings using wash buffer. Finally, after adding the TMB-substrate solution and incubating at room temperature for 20 minutes, the reaction was stopped by adding a 2N H2SO4 solution, and the absorbance at 450 nm was measured using a SpectraMax iD3 (Molecular devices) instrument.

[0142] [Table 9]

[0143] As shown in Table 9, the results of myostatin analysis in serum obtained from aging mice showed that the control group (PBS) had a myostatin level of 4689.8±364.7 ng / ml, the mixed strain alone had a level of 4439.7±251.2 ng / ml, and the auxiliary ingredient alone had a level of 4363.2±200.6 ng / ml. The group that ingested both the mixed strain and the auxiliary ingredient measured 3721.8±173.2 ng / ml, and a statistically significant decrease in myostatin was observed in the group that ingested both the mixed strain and the auxiliary ingredient compared to the group that ingested each ingredient alone.

[0144] Experimental Example 5: Verification of changes in the gut microbiota, anti-aging, and anti-obesity activity in animal models by concomitant administration of a mixed strain of lactic acid bacteria and / or herbal medicine.

[0145] To analyze the changes in the gut microbiota, anti-aging, and anti-obesity activity induced by the combined administration of a mixed strain of lactic acid bacteria and / or herbal medicine in aging mice, a total of three groups were constructed. A control group consisted of 15 16-month-old C57BL / 6 aging mice orally administered phosphate-buffered saline (PBS) for 32 weeks, and 10 young C57BL / 6 mice (10 mice). The experimental group consisted of 15 16-month-old C57BL / 6 aging mice orally administered the mixed strain and auxiliary ingredients used in Experiment 2 together for 32 weeks. After the experiment, each mouse was necropsied, and the cecum was excised and frozen at -80°C. Genomic DNA was extracted from the frozen samples using FastDNA® SPIN Kit for soil (MP Bio). The V3 / V4 region of the 16S rRNA gene in the microbial community was amplified by PCR using 341F and 805R primers (Table 10, SEQ ID NOs. 4 and 5) fused with barcode sequences from each extracted DNA sample.

[0146] [Table 10]

[0147] The amplified products were purified using AMPure XP beads (Beckman). Subsequently, large-volume sequencing data was generated from the purified products using the MiSeq (Illumina) platform. Denoising and paired sequencing of the generated large-volume sequences were performed using the DADA2 program. OTU clustering and microbial diversity analysis of the assembled sequence data were performed using the publicly available Mothur pipeline (Schloss PD et al. 2009. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology 75:7537-7541). The results are shown in Figures 1-5.

[0148] As shown in Figure 1, alpha diversity analysis of the gut microbiota based on 16S rRNA gene sequences revealed that the uniformity of the gut microbiota (Shannon evenness index) was significantly lower in aged mice compared to young mice. In one specific case, aged mice treated with a mixed strain of lactic acid bacteria and a Chinese herbal medicine did not show a significant difference from the young mouse group; in fact, the uniformity of the gut microbiota was evaluated as significantly higher in aged mice than in young mice. This suggests that the combined administration of a mixed strain of lactic acid bacteria and a Chinese herbal medicine may have a positive impact on gut health by restoring or preventing the decline of the uniformity of the gut microbiota in aged animals.

[0149] As shown in Figure 2, principal coordinate analysis (PCoA) of the gut microbiota community structure based on weighted-UniFrac distance confirmed that the gut microbiota community of aging mice differed from that of young mice. In contrast, the gut microbiota community structure of aging mice administered a mixed strain of lactic acid bacteria and herbal medicine was found to be more similar to that of young mice than to that of general aging mice. This suggests that, as a specific example, the combined administration of a mixed strain of lactic acid bacteria and herbal medicine regulates the gut microbiota community structure of aging animals to be similar to that of young animals.

[0150] As shown in Figures 3 and 4, it was confirmed that the combined administration of a mixed bacterial strain and herbal medicine in one specific example caused a significant difference in the frequency of major lineages inhabiting the intestines of aging mice: Lachnospiraceae and Ruminococcus from the phylum Firmicutes, and Muribacula, Rikenellaceae, and Prevotellaceae from the phylum Bacteroidetes.

[0151] Next, as shown in Figure 5, this resulted in a significant difference in the Firmicutes / Bacteroidetes ratio, a known indicator of obesity in the gut microbiota, between aged and young mice. This means that, as a specific example, the combined administration of a lactic acid bacteria mixed strain and herbal medicine made the gut microbiota of aged mice similar to that of young mice, and may help prevent or treat age-related diseases such as obesity caused by the gut microbiota of aged mice. Furthermore, it was shown that when aged mice were administered the lactic acid bacteria mixed strain and herbal medicine together, the ratio tended to be lower than that observed in the aged mouse control group. Recent papers have reported that the gut microbiota of aged mice may cause obesity, and that the Firmicutes / Bacteroidetes ratio increases in aged mice (Binyamin et al., Genome Medicine, 2020). This suggests that the combined administration of a mixed strain of lactic acid bacteria and herbal medicine, as described in one specific example, may have a positive effect on the prevention and treatment of diseases such as obesity in aging mice.

[0152] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that it can be readily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the embodiments described above should be understood to be illustrative in all respects and not limiting. [Accession Number]

[0153] Depository name: Korea Institute of Biotechnology Accession number: KTCT14105BP Date of acceptance: 2020114 Depository name: Korea Institute of Biotechnology Accession number: KTCT14106BP Date of acceptance: 2020114 Depository name: Korea Institute of Biotechnology Accession number: KTCT14107BP Date of acceptance: 2020114

Claims

[Claim 1] Any of the following selected substances from the group consisting of Lactobacillus fermentum GB102 strain, its lysate, and culture medium, deposited under deposit number KCTC 14105BP: Any of the following selected from the group consisting of Lactobacillus fermentum GB103 strain belonging to the genus Lactobacillus, deposited under deposit number KCTC 14106BP, its lysate, and culture medium, and A composition comprising at least one, two, or three or more elements selected from the group consisting of Lactobacillus plantarum GB104 strain, belonging to the genus Lactobacillus, deposited under deposit number KCTC 14107BP, its lysate, and culture medium.

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