KR20210062660A - Probiotic strains for aging, muscle, bone, intestine, hyperlipidemia, skin and brain - Google Patents

Abstract

The first aspect of the present invention relates to the Lactobacillus fermentum strain deposited with the China Microbial Resource Bank under the accession number CGMCC 15536 and the entire gene sequence deposited with the gene bank under the accession number CP0333071. This strain is particularly beneficial for aging, bone and muscle health, intestinal balance, hyperlipidemia, skin, and brain health.

Description

Probiotic strains for aging, muscle, bone, intestine, hyperlipidemia, skin and brain

This application claims the benefit of the Malaysian patent application PI 2018703875 filed on October 19, 2018, and the contents of the Malaysian patent application are incorporated by reference in their entirety in this specification, and the Malaysian patent application is referred to as “Malaysia Priority Application. "It is said.

The present invention relates to the fields of medicine, microbiology and nutrition, and in particular to the probiotic strain Permentum DR9. These strains are particularly useful for slowing aging, treating and preventing muscle and bone degeneration, controlling intestinal dysfunction for imbalance of intestinal microbial population and metabolite concentration, and treating and preventing hyperlipidemia, interlipid accumulation, lipid metabolism, lipid disease and skin degeneration. It has the same health-beneficial biofunctionality.

The paper “ Lactobacillus strains alleviated aging symptoms and aging-induced metabolic disorders in aged rats ” was published online on December 28, 2018, after the priority date specified in the Malaysian priority application. The authors of the paper are the inventors of this application, and all rights are granted to the applicants of this application and the Malaysian priority application. That is, in other words, the thesis of the present applicants can be considered as a so-called "publication by the inventor" (that is, the subject of publication is directly or indirectly from the inventor, one or more co-inventors, or inventors and co-inventors. It must be attributed to the person who acquired it). The content of the paper was included in the Malaysian priority application (although the content was not disclosed on the priority filing date), and is also included in this application. Therefore, the effect of Lactobacillus fermentum DR9 strain on slowing aging and on the treatment and prevention of muscle and bone degeneration was not published on the filing date of the Malaysian priority application.

Thesis “ Lactobacillus sp. "Improved microbiota and metabolite profiles of aging rats " was published online on June 14, 2019, after the priority date specified in the Malaysia priority application. The authors of the paper are the same as the inventors of this patent application. The authors of are the inventors of the present application, and have granted all rights to the applicants of this application and the Malaysian priority application, in other words, the above papers of the applicants can be regarded as the so-called "publication by the inventor" (public subject matter Should be attributed to the inventor, one or more co-inventors, or to a person who directly or indirectly acquired the subject from the inventor and co-inventor) Although the content of the paper was not incorporated into the Malaysian priority application (therefore, the content of the priority application date) Therefore, the effect of Lactobacillus fermentum DR9 strain on intestinal regulation on imbalance of intestinal microbial population and metabolite concentration was not known at the filing date of the Malaysian priority application. (Not published).

The contents of the above-mentioned papers are incorporated herein by reference in their entirety.

Probiotics are defined as'live microorganisms that, when consumed in the right amount, give a healthy effect to the host (PAO/WHO, 2006). Lactobacillus bacteria (Lactobacillus) will demonstrate the health benefits from controlled environments to relieve intestinal and immune response (immune responses) regulation of metabolic disorders (metabolic disorders).

Aging is an age-dependent multifactorial process associated with physiological decline leading to an increase in age-specific mortality rate. The number of seniors aged 60 and over is expected to more than double by 2050 and more than triple by 2100, and will increase from 920 million globally in 2017 to 2.1 billion by 2050 and 3.1 billion by 2100. It is expected. Aging is inevitable, but current aging research aims to improve the quality of life as life expectancy increases.

Telomere length is often considered a biomarker of aging and age-related morbidity. Telomeres are a complex of nuclear proteins at the ends of eukaryotic chromosomes that decrease with each cell division. Telomere length shortens with cell division until a critical length is reached, causing cell senescence to be initiated. In addition to telomeres, the p53 gene is also a biomarker for aging. The P53 encodes a tumor suppression protein and serves as a major indicator for telomeric stress-induced senescence that induces aging. This affects cell cycle arrest, DNA repair, apoptosis and cellular senescence.

Aging is linked to oxidative stress. Progressive accumulation of oxidative damage to DNA, proteins and lipids is a contributing factor to the aging process. In addition, oxidative damage accelerates the aging process by reducing mitochondrial function by affecting the replication and transcription of mitochondrial DNA. Mitochondrial dysfunction is closely related to several age-related diseases such as Alzhe-imer's disease and Parkinson's disease. Oxidative stress can also cause metabolic disorders such as obesity, diabetes, and cardiovascular diseases.

Aging affects the musculoskeletal system. Muscle aging is sarcopenia, which is generally described as an involuntary loss of skeletal muscle mass and strength. In humans, muscle mass begins to decrease at age 40. Approximately 25% of adults over the age of 60 and 60% of the population by age 80 can experience muscle loss. Patients suffering from sarcopenia may experience low muscle strength and low muscle performance. They are also at risk of physical immobility, poor quality of life and death. Chronic inflammation, changes in hormone levels due to age (menopause), disrupted mitochondrial function, and muscle cell regeneration have been considered the root causes of sarcopenia.

The early onset of bone aging begins around the age of 30, when bone density begins to decrease in both men and women. In women who have experienced menopause, this condition is accelerated. An increased risk of falls and fractures with a decrease in bone mass and density is called osteoporosis. Worldwide, it is reported that one in three women over the age of 50 experience a fracture due to osteoporosis every year. There is currently no cure for sarcopenia, except for strength training, which is taken as a preventive measure. Therefore, there is a need to seek alternative therapy to promote healthy aging of the musculoskeletal system.

The gut is one of the largest lymphoid organs in our body with about 4 trillion bacteria. Because the gut microbiota regulates the physiology and survival of the host, the role of these bacteria in the aging process is indispensable. In humans, the gut microbiota is affected by dietary and health conditions, but remains relatively stable when it is established 3 years after birth. However, the gut microbiota is composition and diversity in older adults less rich in core bacterial families such as Bacteroidaceae, Ruminococcaceae, and Lachnospiraceae. ), and certain health-related species are more prevalent. It has been reported that changes in the gut microbiota are affected by gut barrier integrity, intestinal and various other old age-diseases. Gut-related disorders can be delayed and prevented through a healthy and balanced lifestyle. One of the approaches is to maintain a balanced gut microbiota, which is possible through diet intervention and or functional food supplementation. Improved gut integrity lowers oxidative stress and inflammatory markers. Fecal microbiome and metabolome analyses provide reliable and comprehensive information on fecal biomarkers, particularly for metabolic diseases.

Metabolic diseases such as hyperlipidemia increase the risk of cardiovascular diseases (CVD). Cardiovascular disease accounts for 31% of all deaths worldwide, according to the World Health Organization (WHO), and cardiovascular complications are considered a major factor in morbidity and mortality. The projected cost of treatment for cardiovascular disease is expected to be $104 billion by 2030. Elevated blood lipid levels and hyperlipidemia are well known as risk factors for cardiovascular disease. Hyperlipidemia can be broadly classified as an increase in isolated cholesterol, an increase in isolated triglycerides (TG), and an increase in both. Hyperlipidemia has also been shown to affect antioxidant status and lipoprotein levels in other organs, which in turn aggravate metabolic disturbances, cardiovascular disease and non-alcoholic fatty liver disease. fatty liver disease (NAFLD). Probiotics have shown an improved blood lipid profile in animal studies.

The skin separates the body from the outer environment and prevents excessive water loss and infection. In addition, skin is large and beautiful skin plays an important role in the cosmetic aspect, which can have a positive effect on social behavior. Due to aging, the skin displays the most pronounced signs of aging as it ages. Consumers invest a significant amount of their daily expenses in skin products. This has led cosmetics and pharmaceuticals companies to develop products that prevent or reverse skin aging.

The aging process affects the brain and emotions in a variety of ways, and the prevalence of anxiety is high in the aging population. This may be related to cognitive declines due to neuro-degenerative diseases such as Alzheimer's. In addition, the accumulation of cell-damaging reactive molecules produced during aging through cellular respiration often leads to oxidation of proteins and other cellular molecules, leading to brain degeneration. Proceed.

Biochemical processes and body metabolism in living organisms are regulated by adenosine monophosphate-activated protein kinase (AMPK). AMPK is an intracellular energy sensor that regulates the anabolic and catabolic pathways, and in particular acts as a major regulator of cellular energy homeostasis. In particular, when AMPK detects low levels of adenosine triphosphate (ATP), a signaling pathway is activated, where AMPK promotes the catabolic pathway to produce more ATP for various cellular metabolic activities. On the other hand, AMPK is the ATP consumption biosynthesis process (ATP), such as glucose production (gluconeo-genesis), lipid and protein synthesis, etc. through direct phosphorylation of several enzymes directly involved in this process. -It negatively regulates the consuming biosynthetic process, and regulates transcriptional control of metabolism by phosphorylating transcription factors, co-activators and co-repressors. Thus, AMPK is a biological switch that regulates ATP levels so that body functionality can be maintained at an optimal stage and controls body metabolisms.

When ingesting probiotics, many metabolites in the gut of the host are strengthened, enhancing metabolisms, including AMPK activation. Some of the metabolites are beneficial for health and are generally beneficial for metabolisms of the hosts. One of these metabolites is also known as 5-oxoproline or pyrocratamic acid. 5-oxoproline is produced through enzymatic hydrolysis in a variety of peptides. Thyrotropin-releasing hormone (TRH), known as protirelin, is a tripeptide consisting of 5-oxoproline, histidine, and proline, which is a medical treatment for spinocerebellar ataxia. It is used for 5-oxoproline has been shown to improve verbal memory functions in subjects affected by age-related memory decline. In addition, 5-oxoproline is found as an N-terminal modification in hormones and neuronal peptides, including peptides accumulated in Alzheimer's disease and familial dementia. Therefore, 5-oxoproline has the potential for anti-aging effects. Another important intestinal metabolite is ascorbic acid, a precursor for the biosynthesis of vitamin C. Vitamin C is a natural antioxidant that exists at the cutaneous level and exhibits specific antioxidant, anti-inflammatory, and photoprotective properties, and collagen synthesis. It is known as a biostimulator. Thus, ascorbic acid has the potential to improve metabolism and skin health.

Probiotics can also produce a variety of metabolites with high health potentials. One of these metabolites is succinic acid, which maintains homeostasis of the aging hypothalamus, has an anti-aging effect, and reverses menopausal symptoms without sex hormone replacement therapy. It recovers the gradual loss of functions related to cell aging and systemic aging, has antioxidant properties with the potential for the treatment of skin aging, and has the respiration and oxidation of muscles. It played an important role in oxidation). It has also been found that succinic acid is used to regulate the gut flora and is present as part of the composition in bones. Another metabolite is benzaldehyde, which is produced by some probiotic strains of phenylalanine. Benzaldehyde is used as a natural flavoring agent in products such as cheese and is commonly used in the food, beverage, pharmaceutical, perfume, soap and dye industries. Benzoaldehyde has been reported to have beneficial properties for health such as anti-tumors and antiviral. Benzoic acid is a fungistatic compound widely used as a food preservative and is spontaneous from many plant sources such as berries, fruits and pickled foods. Occurs as. Benzoic acid can be combined with glycine in the liver and excreted as hippuric acid, and is generally used to treat fungal skin diseases. Benzoic acid is also a common compound in pharmaceuticals such as topical antiseptics and inhalant decongestants. Among animals, benzoic acid is mainly present in omnivorous animals such as the intestines and muscles of the grouse (thunderbird). Since some lactobacilli can modify hippuric acid to produce benzoic acid in milk, benzoic acid is usually present in yoghurt. Benzoic acid has been reported to be helpful in increasing the retention of calcium and phosphorus, reducing femur weight, chloride, and increasing magnesium in bone.

Therefore, there is a need to provide solutions, especially probiotic bacteria, to improve the above-mentioned symptoms.

The problem to be solved in the present invention is to provide new compositions and useful treatments for the improvement of the above-mentioned conditions in a safe and effective manner.

The solution to the problem is based on the provision of a probiotic bacterial strain deposited under the accession number CGMCC 15536, also known as Lactobacillus fermentum DR9. The present invention has been found to be associated with useful biofunctionality, particularly in the improvement of various conditions and diseases described below.

Accordingly, the first aspect of the present invention relates to a Lactobacillus fermentum strain or a metabolite thereof, wherein the strain was deposited with the China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC 15536, and the whole genome sequence Is deposited in GenBank under accession number CP03371, and its metabolites include succinic acid, benzaldehyde, and benzoic acid.

Another aspect of the present invention is Lactobacillus fermentum for use as a therapeutic or pharmaceutical It relates to a strain or a metabolite thereof. Optionally, this aspect is Lactobacillus Fermentum. It can be expressed as a method of probiotic treatment by administering an effective amount of DR9 or its metabolite as needed. The term “probiotic” as used herein refers to a living microorganism that, when administered in an appropriate amount, provides a health benefit to the host.

Another aspect of the present invention is a strain Lactobacillus fermentum for use in slowing aging signs. DR9 or its metabolites.

Another aspect of the present invention is the strain Lactobacillus fermentum for use in the prevention and treatment of muscle and bone degeneration. DR9 or its metabolites.

In another aspect, the present invention is a strain Lactobacillus fermentum for use in slowing aging by improving energy metabolism and preventing telomere shortening against aging. DR9 or its metabolites.

Another aspect of the present invention is to prevent and treat hyperlipidaemia and hepatic lipid accumulation by preventing an increase in triglycerides level, inflammation, and accumulation of lipids. Strain Lactobacillus Fermentum for Use DR9 or its metabolites.

Another aspect of the present invention is a strain Lactobacillus fermentum for use in the prevention and treatment of cholesterol reduction and cardiovascular diseases. DR9 or its metabolites.

In another aspect, the present invention is a strain Lactobacillus fermentum as a probiotic for gut modulation for imbalance of intestinal bacterial population and metabolite concentration. It relates to the use of DR9 or its metabolites. Its gut metabolites consist of ascorbic acid and 5-oxoproline.

Another aspect of the present invention is the strain Lactobacillus fermentum for use in the prevention and treatment of skin degeneration. DR9 or its metabolites.

In another aspect, the present invention provides a strain Lactobacillus fermentum for use as a probiotic in the treatment and prevention of anxiety and Alzheimer's Disease. DR9 or its metabolites.

Lactobacillus Fermentum It should be noted that DR9 has all the advantages and medical applications described above, whereas in other cases the strain should be used for each medical application.

Another aspect of the present invention relates to a composition constituting the strain Lactobacillus fermentum DR9 and metabolites of the strain, wherein the strain is lyophilized and has an amount of ^{10 4} -10 ^{12 cfu / g in the composition, and metabolism} Products include succinic acid, benzaldehyde, and benzoic acid.

Lactobacillus fermentum mentioned above The medical use of DR9 and its metabolites may be selectively manifested by the use of strains or metabolites thereof to produce food supplements, pharmaceuticals, infant formulas, and edible products. In addition, it may appear as a method for the treatment and prevention of the above-mentioned symptoms, and includes administration to an experimental subject in need of an effective amount of the strain and its metabolite. The test subjects are mainly mammals, especially humans.

When referring to the strain used in the present specification, the term “effective amount” means that the strain in the composition is sufficient to treat symptoms in a positive direction within the scope of a valid medical judgment, while avoiding serious side effects. Represents a sufficiently low amount of colony forming units (cfu).

Terms used in the aspects and claims of the present invention are widely understood, and in this description, Lactobacillus fermentum The general meaning of the DR9 strain is briefly referred to as “DR9”.

For the purpose of easy understanding of the invention, specific embodiments are shown in the accompanying drawings through review when considering the following detailed description, the invention, and its configuration and operation, and their advantages can be immediately understood and recognized. have.
Figures 1a and 1b are the percentage of (A) adenosine monophosphate activated protein kinase (AMPK) activation and (B) AMPK activation in the presence of cell-free supernatant and AMPK inhibitors of various strains of lactic acid bacteria (LAB). It is shown in comparison.
2A and 2B are human bone osteosarcoma (U2OS), mouse muscle (C2C12), human neuroblastoma (SH-SY5Y), human ovary (OVCAR3), human keratin Comparison of AMPK phosphorylation in cell lines including human keratinocytes (HaCaT), human prostate (DU145), human colorectal (CaCo-2), etc. And these are Lactobacillus Fermentum DR9 was treated in a cell-free supernatant and non-fermented MRS medium (MRS) broth for 24 hours. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and compound C (compound C) were used as positive and negative controls, respectively.
Figures 3a, 3b and 3c . (A) Measurement of telomere length in blood. (B) Serum malondialdehyde (MDA) concentration in rats after 12 hours of administration. (C) Performance evaluation (distance, time, speed, work, power) of the treadmill exhaustion test at an incline for aging-induced rats. (D) Expression of p53 gene in bone (tibia) and muscles (soleus and gastrocnemius). The p53 gene expression was determined through real-time polymerase chain reaction (qPCR). Young rats: non-dose rats injected with 0.9% saline daily by subcutaneous injection (n=6); Aged rats receiving Lactobacillus fermentum DR9 (600 mg/kg of D-galactose daily, administered by subcutaneous injection, and daily 1×10 ¹⁰ CFU of DR9, n=6); Aged rats receiving metformin (600 mg/kg of D-galactose daily, administered by subcutaneous injection, and 300 mg/kg of metaformin daily, n=6); Aged rats receiving statins (ageing is induced via D-galactose, daily doses of 2 mg/kg lovastatin). Results are average; Error bars (standard error, SEM); Expressed as n=6. Statistical analysis was performed through independent t-tests.
Figures 4a, 4b, 4b and 4d. (A) Superoxide dismutase (SOD) and sulfation gene mRNA expression levels of gastrocnemius (left) and tibia (right) samples isolated from the right hind leg of mice after 12 weeks of administration . (B) mRNA expression of AMP-activated protein kinase (AMPKα2) subunit. (C) mRNA expression of genes IGF-1 and MyoD related to myogenesis from gastrocnemius muscle. (D) mRNA expression of TNF-alpha, IL-6, and TRAP related to osteoclastoge-nesis in tibia. (E) Levels of IFN-γ and tibial IL-1β in gastrocnemius muscle isolated from mice after 12 weeks of administration. Young rats: non-dose rats injected daily with 0.9% saline by subcutaneous injection (n=6); Aged rats by subcutaneous injection of 600 mg/kg of D-galactose daily, Aged-DR9: ^{aged rats receiving daily 1 x 10 10} CFU of Lactobacillus fermentum DR9, n=6. Aged-metformin: aged rats dosed with 300 mg/kg of metaformin daily, n=6. Results were expressed as mean±standard error (SEM). Statistical analysis was performed through independent t-tests.
Figures 5a and 5b. Hematoxylin and eosin (H&E) staining (magnification, x10) of gastrocnemius muscle sections of the left hind limb of rats after 12 weeks of dosing. (a) Young rats: Non-dose rats that received 0.9% saline daily by subcutaneous injection (n=6). (b) Rats aged by subcutaneous injection of 600mg/kg of D-galactose daily (n=6). (c) Aged-DR9: Aged-DR9: ^{aged rats receiving 1 x 10 10} CFU of Lactobacillus fermentum DR9 daily (n=6). (d) Aged-metformin: aged rats dosed with 300 mg/kg of metaformin daily (n=6). (e) Myonuclei count of the gastrocnemius muscle section in the left hind limb of rats after 12 weeks of dosing. Results were expressed as mean±standard error (SEM). Statistical analysis was performed through independent t-tests.
Fig. 6. Hematoxylin and eosin (H&E) staining (magnification, x4) of the proximal tibial section demineralized from the left hind limb of mice after 12 weeks of dosing. (a) Young rats: Non-dose rats that received 0.9% saline daily by subcutaneous injection (n=6). (b) Rats aged by subcutaneous injection of 600mg/kg of D-galactose daily (n=6). (c) Aged-DR9: Aged-DR9: ^{aged rats receiving daily 1 x 10 10} CFU of Lactobacillus fermentum DR9 (n=6). (d) Aged-metformin: aged rats receiving 300 mg/kg of metaformin daily (n=6). The black arrows indicate the bone physis & growth plate.
Figures 7a, 7b and 7c. (A) Distribution profile of the bacterial phylum, (B) Relative abundance of the Firmicutes/Bacteroidetes ratio, (C) Induction of aging for 12 weeks Blautia, Bacteriodetes, Lactobacillus genus, determined from rat feces. Young rats: non-dose rats receiving daily subcutaneous injection of 0.9% saline (n=6); Aged rats by subcutaneous injection of 600 mg/kg of D-galactose daily, Aged-DR9: ^{aged rats receiving daily 1 x 10 10} CFU of Lactobacillus fermentum DR9, n=6. Aged-metformin: aged rats receiving 300 mg/kg of metaformin daily. Results were expressed as mean±standard error (SEM). Statistical analysis was performed through independent t-tests.
8a and 8b. Sparse partial least square discriminant analysis (sPLS-DA) score plot: (A) short chain fatty acids, (B) soluble metabolites, (C) inducing aging for 12 weeks 5-oxoproline from rat feces afterwards. Metabolites were detected and analyzed using GC-MS. (D) Interferon-gamma (IFN-γ) and tumor necrosis detected in the distal colon of mice using multiplex ELISA and normalized to protein content measured by Bradford method. Cytokines levels of tumor necrosis factor alpha (TNF-α). Young rats: non-dose rats receiving daily subcutaneous injection of 0.9% saline (n=6); Aged rats (n=6) by subcutaneous injection of 600 mg/kg of D-galactose daily, Aged-DR9: ^{aged rats receiving daily 1 x 10 10} CFU of Lactobacillus Permentum DR9 (n=6) . Aged-metformin: aged rats receiving 300 mg/kg of metaformin daily (n=6).
9a and 9b. (A) Relative abundance of Blautia in fecal samples. (B) Fecal microbiota analysis of genus distribution of phylum Firmicutes after 12 weeks of dosing. Young rats: non-dose rats receiving daily subcutaneous injection of 0.9% saline (n=6); Aged rats (n=6) by subcutaneous injection of 600 mg/kg of D-galactose daily, Aged-DR9: ^{aged rats receiving daily 1 x 10 10} CFU of Lactobacillus Permentum DR9 (n=6) . Aged-statin: aged rats receiving 2 mg/kg of lovastatin daily (n=6).
10A and 10B. Analysis of short-chain fatty acid profiles in fecal samples of rats after 12 weeks of dosing. a) Heatmap normalized to a relative number between 2 and -2. Red indicates high concentration, blue indicates low concentration. (b) Absolute concentration of acetate, (c) Absolute concentration of propionate. Young-ND: normal diet; young-HFD: high-fat diet; aged-ND: aging is induced through D-galactose with a normal diet; aged-HFD: aging is induced through D-galactose along with a high fat diet; aged-HFD-Statin: aging is induced through D-galactose in conjunction with a high fat diet and administered daily at 2 mg/kg of lovastatin; aged-HFD-DR9: aging is induced via D-galactose with a high fat diet and ^{administered 10 10} CFU of Lactobacillus Fermentum DR9 daily. Results are average, error bars (standard error, SEM); Expressed as n=6. Statistical analysis was performed through independent t-tests.
11A and 11B. (A) mRNA gene expression of SCD1, (B) mRNA gene expression of IL-6, (C) mRNA gene expression of ABCG5 in liver of mice induced aging through 600 mg/kg of D-galactose daily for 12 weeks. (D) Serum biochemical parameters. ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet and 2 mg/kg daily lovastatin), HFD-DR9 (high fat diet and 10 ¹⁰ CFU lactobacillus permentum DR9 daily), aged metformin ( 300 mg/kg methformin daily); n=6. Statistical analysis was performed through independent t-tests.
12A and 12B. (A) Relative gene expression of AMPKα1, (B) intrahepatic cytokines (interleukin-4 (IL-4), interleukin-10 (IL-10)) levels, (C) daily for 12 weeks Hematoxylin and eosin staining (H&E staining) of the liver of rats whose aging was induced through 600mg/kg of D-galactose. ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet and 2 mg/kg of lovastatin daily), HFD-DR9 (high fat diet and 10 ¹⁰ CFU of lactobacillus permentum DR9 daily), (C ), (a) normal diet, (b) high fat diet, (c) high fat diet-statin, (d) high fat diet-DR9. Results are average, error bars (standard error, SEM); Expressed as n=6. Statistical analysis was performed through independent t-tests.
13A and 13B. (A) Skin elasticity, (B) cyclin-D1, (C) FAS, (D) mRNA gene expression in rat skin after 12 weeks of administration. Young rats: non-dose rats receiving daily subcutaneous injection of 0.9% saline (n=6); Aged rats (n=6) by subcutaneous injection of 600 mg/kg of D-galactose daily, Aged-DR9: ^{aged rats receiving daily 1 x 10 10} CFU of Lactobacillus Permentum DR9 (n=6) . Aged-metformin: aged rats receiving 300 mg/kg of metaformin daily (n=6). Results are average, error bars (standard error, SEM); Expressed as n=6. Statistical analysis was performed through independent t-tests.
14A and 14B. (A) Anxiety assessment through open field test. Young (control), Old (induction of aging through D-galactose), Old-statin (induction of aging through D-galactose and administration of 2mg/kg of lovastatin daily), Old-DR9 (induction of aging through D-galactose and daily) Lactobacillus fermentum DR9 dose of 10 log CFU. Results were expressed as mean, error bar (standard error, SEM); n=6. Statistical analysis was performed through independent t-tests. The eyes of Drosophila melanogaster showing formed hexagonal ommatidia and straight bristles were observed from GMR-OreR at (B) 250 times magnification and (C) 350 times magnification, while The rough eye phenotype with holes was observed at (D) 250 times magnification and (E) 350 times magnification from transgenic yellow Drosophila Aβ-42 (GMR-Aβ42). A well-formed eye shape and less damaged hexagonal eyes are from GMR-Aβ42.DR9 ingested 100 μL 1 x 10 11 CFU/mL of Lactobacillus fermentum strain DR9 (F) at a magnification of 250 times ((F) G) It was observed at 350 times magnification.
15A and 15B. (A) AMP-kinase activity of cell free supernatant (CFS), intracellular extracts (IE), cell wall debris of Lactobacillus fermentum DR9 using ADP-GLO Kinase Assay (AMP-kinase activation). Numerical values expressed the activity of AMPK in terms of ATP-ADP conversion as a percentage. n=6. (B) AMPK activity by lipid fraction extracted from cell-free supernatant of Lactobacillus fermentum DR9 against premature-senescence-induced U2OS cells. AICAR (2mM) and Dorsomorphin C (20 μM) were used as positive and negative controls, respectively. Error bars represent the standard error of the mean. n=3. Means vary considerably (p<0.05). ^ad statistical analysis was performed using one-way ANOVA. (C) Gas chromatography-mass spectrometry (GC-MS) color stratification of cell-free supernatant fractions produced from Lactobacillus fermentum DR9 (top) and unfermented MRS medium (bottom). Peak 1: butanedioic acid (succinic acid), Peak 2: benzaldehyde, Peak 3: unknown, Peak 4: benzoic acid.
Fig. 16. Gut concentrations of metabolites 5-oxoproline and ascorbic acid extracted from fecal samples of rats after 12 weeks of dosing. Young rats: non-dose rats receiving daily subcutaneous injection of 0.9% saline (n=6); Aged rats (n=6) by subcutaneous injection of 600 mg/kg of D-galactose daily, Aged-DR9: ^{aged rats receiving daily 1 x 10 10} CFU of Lactobacillus Permentum DR9 (n=6) . Aged-metformin: aged rats receiving 300 mg/kg of metaformin daily (n=6). Results were expressed as mean±standard error (SEM). Statistical analysis was performed through independent t-tests.

Best mode for carrying out the invention

The Lactobacillus fermentum DR9 strain was extracted from fresh milk from Penang, and Clinical Nutrition Intl (M) Sdn. It was obtained through the provision of Bhd. The strain was identified as Lactobacillus fermentum , and was deposited with the China Microbial Resource Bank (CGMCC) located at the Microbiological Research Institute of the Chinese Academy of Sciences, No. 1, Seobukcheon-ro, Beijing, China. The deposited strain was assigned the accession number CGMCC 15536, and was deposited on April 2, 2018. In addition, the same applicant, Clinical Nutrition Intl (M) Sdn. Bhd. and Lii Run Sdn. Deposited by Bhd. The deposited strain is viable and all functions related to the deposit are maintained.

Species were identified through 16S rRNA gene sequencing.

The information on the 16s rRNA is as follows and the nucleotide sequence is SEQ ID: NO:1.:

Contig 26 (1,1491)

Contig Length: 1491 bases

Average Length/Sequence: 942 bases

Total Sequence Length: 1884 bases

Top Strand: 1 nucleotide sequence

Bottom Strand: 1 nucleotide sequence

Total: 2 bases

The whole genome sequence was deposited with GenBank under accession number CP033371 (November 4, 2018). (Access to revision history: CP033371.1 November 4, 2018).

By using the deposited strain as a starting material, the skilled person mutates additional variants or mutations thereof that maintain or enhance the relevant features and advantages of the strains of the invention described in this document. Alternatively, it can be obtained through re-isolation techniques. Accordingly, the present invention relates to the modification of the strains found in this document. As used herein, the term “variant” or “mutant” refers to a naturally occurring or specially developed strain obtained from the deposited strain, and mainly refers to the function of the deposited strain by mutation. Keep. These functions can be evaluated by a person skilled in the art using the methods described in the Examples section.

For example, the 16S rRNA gene or full genome sequence of the “mutant” strain considered herein is the 16S rRNA sequence SEQ ID NO: 1 or the CP033371 genome sequence of the strain identified in this document. sequence) and about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% base Share sequence identity.

In one specific embodiment, mutants are obtained using recombinant DNA technology. In another embodiment, the mutation is obtained by random mutagenesis. Therefore, another aspect of the present invention relates to a method of obtaining a mutation of the DR9 strain, wherein the obtaining method includes using the deposited strain as a starting material or applying mutagenesis. And the obtained variant or mutant maintains or enhances the biological functions of the deposited strain.

In certain embodiments, the strain is fermented in an artificial medium and sent to post-treatment after fermentation to obtain bacterial cells, and the resulting bacterial cells are liquid medium or solid medium. form). In particular, the post-treatment is drying, freezing, freeze-drying, fluid bed-drying, spray-drying, and refrigerating in a liquid medium. ), and in more detail, freeze-drying was selected.

The strain was produced through bacterial culture (or fermentation) in an appropriate artificial medium and under appropriate conditions. The expression “artificial medium” for microorganisms refers to a medium containing natural substances and optionally a polymer polyvinyl alcohol that expresses some functions of serums. It means synthetic chemicals such as ). In general, an appropriate artificial medium refers to nutrient broths containing elements including carbon sources (eg glucose), nitrogen sources (eg amino acids and proteins), water, salts, etc. required for bacterial growth. Growth media are either in liquid form or often mixed with agar or other gelling agents to obtain a solid medium. The strain may be cultured alone to form a pure culture, or various types of bacteria may be cultured separately and then combined in a desired ratio and mixed and cultured with other microorganisms. After cultivation, it can be used as purified bacteria according to the final formulation, or alternatively, it can be used as a bacterial culture or a cell suspension after appropriate post-treatment. In this description, the term “biomass” refers to a bacterial strain culture obtained through culture (or fermentation as a synonym for culture).

The term “post-treatment” means any treatment carried out on a biomass for the purpose of obtaining storable bacterial cells in the context of the present invention. The purpose of post-treatment is to slow down the rate of cellular deleterious reactions by reducing the metabolic activity of cells in the biomass. As a result of work-up, bacterial cells contain both solid and liquid forms. In the case of solid form, the stored bacterial cells can be powder or granules. In any case, solid and liquid forms, including bacterial cells, are not naturally occurring because they do not exist in nature and are the result of an artificial post-treatment process. In certain embodiments, post-treatment processes may require the use of one or more post-treatment agents. In the context of the present invention, the expression “post-treatment agent” refers to the compound used to carry out the post-treatment process described herein. Among the post-treatment agents, dehydrating agents, bacteriostatic agents, cryoprotective agents, inert fillers (also known as cryoprotectants), carrier materials (also known as core substances), etc., can be used alone or in combination without restrictions. Includes that.

There are two approaches to reducing the metabolic activity of bacterial cells, both of which use post-treatment. The first approach is to reduce the rate of all chemical reactions, which can be achieved by lowering the temperature through refrigerating or freezing using refrigerators, mechanical freezers, and liquid nitrogen freezers. . Alternatively, a substance that inhibits the growth of bacterial cells, i.e., a bacteriostatic agent (abbreviation: Bastatic) may be added to reduce the rate of all chemical reactions.

A second approach using post-treatment is to remove water from the biomass through a sublimation of water process using a lyophilizer. Suitable techniques for removing water from biomass are drying, freeze-drying, spraydrying or fluid bed-drying. The post-treatment process to create a solid form may include drying, freezing, freeze-drying, fluid bed-drying, or spraydrying.

The removal of water from the lyophilized bacterial suspension via sublimation under reduced pressure is particularly freeze-drying in the post-treatment. This process consists of three steps. first? In the step, the product is pre-freezing to form a frozen structure, and twice? In the step, most of the water is removed through primary drying, and in the final step, the bound water is removed through secondary drying. Due to the objective and predicted variability of industrial processes for the manufacture and isolation of cultures of lyophilized bacterial cultures, a certain amount of inert fillers known as lyoprotec-tants are commonly used. Include as. Its role is to standardize the content of live probiotic bacteria in the product. The following inert fillers are used in commercially available lyophilized cultures: sucrose, saccharose, lactose, trehalose, glucose ( glucose, maltose, maltodextrin, corn starch, inulin and other pharmaceutically acceptable nonhygroscopic fillers. Optionally, other stabilizing agents such as ascorbic acid or freeze-protecting agents are also used to form a viscous paste used for freeze. Either in, the material thus obtained can be ground to any suitable size, including powder.

Medical Use of Lactobacillus Fermentum DR9

Lactobacillus Fermentum DR9 has the advantage of being particularly useful as a probiotic. Probiotic bacteria must meet several requirements related to low toxicity, viability, adhesion and beneficial efficacy. The characteristics of each bacterial strain are unique and cannot be extrapolated from other strains of the same species.

Through the experiment of the Example part, it has been reasonably demonstrated herein that Lactobacillus fermentum DR9 strain has the following efficacy and utilization beneficial to health:

1. Lactobacillus fermentum DR9 can delay the signs of aging by activating AMPK, preventing telomere shortening, reducing lipid peroxidation, and improving physical endurance (see Example 1). A total of 18 lactic acid bacteria (LAB) strains were evaluated for the activation of AMPK, an intracellular energy sensor that mediates life extension. Cell-free supernatant (CFS) of Lactobacillus fermentum DR9 showed higher AMPK activation and was further evaluated. Phosphorylation of AMPK by DR9 was evident in most of the cell lines studied compared to Compound-C, known as an AMPK-inhibitor (P<0.05). In a study using Sprague-Dawley mice in which premature aging was induced through D-galactose, mice dosed with 10 log CFU of DR9 daily for 12 weeks prevented telomere shortening compared to the untreated control group. DR9 increased the gene expression of AMPKα1 in the liver, resulting in activation compared to the untreated control. DR9 was shown to reduce oxidative stress by lowering blood lipid peroxidation compared to the untreated control group. In the treadmill endurance test, mice fed DR9 showed longer running time, higher speed, more work and power, and better physical endurance compared to the untreated control group. DR9 lowered the expression of the p53 gene, known as a senescent biomarker, in the gastrocnemius and tibia compared to the control group.

Accordingly, as described above, an aspect of the present invention relates to Lactobacillus fermentum DR9 and its metabolites for use to slow down the signs of aging through AMPK activation, telomere shortening prevention, reduction of lipid peroxidation, and improvement of body endurance. Signs of aging are, for example, that reduced energy metabolism causes impairment of mobility and movements, and oxidation of plasma leads to impaired immunity.

Lactobacillus fermentum DR9 prevents and treats muscle and bone degeneration (see Example 2). The anti-aging mechanism of DR9 was evaluated in the gastrocnemius and tibia of rats whose aging was induced through D-galactose. DR9 showed improved expression of SOD in gastrocnemius muscle and tibia and improved expression of AMPKα2 in gastrocnemius muscle when compared to untreated aged rats. DR9 increased the mRNA expression of IGF-1 compared to the control, whereas the expression of MyoD decreased. In addition, DR9 decreased the expression of IL-6, TNF-α, and TRAP in the tibia, and decreased the expression of IL-1β tibia and IFN-sγ in the gastrocnemius, compared with the aged mice. The gastrocnemius muscles of DR9-treated rats had a greater number of myocytes (nuclei of muscle cells) with a denser arrangement of myofibers compared to untreated aging rats. The tibia of the DR9-treated rats was a normal bone trabecular line with less bone marrow adiposity and a more ordered arrangement compared to the untreated aged rats with a thinner network and sparse arrangement. trabeculae).

Therefore, as described above, the aspect of the present invention is to improve sulfation and energy metabolism while reducing atropy, osteoclastogenesis, inflammation and depletion of myonuclei. It relates to Lactobacillus fermentum DR9 or a metabolite thereof for use for the purpose of preventing and treating degeneration of muscles and bones by eliciting better mobility and physical endurance.

3. Lactobacillus fermentum DR9 affects imbalance of gut bacterial populations and gut modulation of metabolite concentration (see Example 3). In the aging model, the ratio of Firmicutes/Bacteroidetes decreased considerably, while administration of DR9 increased this ratio at the phylum level. Studies on genus levels showed that DR9-treated mice promoted the proliferation of Lactobacillus and Blautia, while reducing the number of Bacteroides compared to aged mice. The profiles of short-chain fatty acid (SCFA) and water soluble metabolite in the feces of DR9-treated rats were more similar to those of young rats when compared to untreated aged rats. Analysis of water-soluble metabolites showed that induction of aging through D-galactose had a significant effect on amino acids metabolism, such as a decrease in 5-oxoproline levels, whereas the DR9 administration reduced this. Was prevented. In addition, DR9 is a cytokine of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in the distal colon of mice compared to untreated aged mice. Reduced the figure. In the HFD group, male Sprague-Dawley rats were fed a high-fat diet, and D-galactose was injected for 12 weeks to induce aging. Administration of DR9 increased the intestinal concentration of Blautia compared to the HFD-age control. As a result of analysis of water-soluble metabolites in feces, DR9 administration was compared with HFD-age control, tryptophan, leucine, tyrosine, cysteine, methionine, valine and lysine. The contents of compounds related to amino acid metabolism such as (lysine) were shown in the excrement, and the contents of SCFAs such as acetate and propionate were high.

Therefore, as described above, an aspect of the present invention relates to Lactobacillus fermentum DR9 and its metabolites as a probiotic in the intestinal regulation of an unbalanced intestinal bacterial population and metabolite concentration. In other words, it means improving dysbiosis.

4. Lactobacillus fermentum DR9 enhances lipid metabolism and alleviates NAFLD (see Example 4). In this study, it was tested whether various strains of Lactobacillus alleviate hyperlipidemia and liver steatosis through AMPK activation in aged rats. Male Sprague-Dawley rats were fed a high fat diet and D-galactose was injected daily for 12 weeks to induce aging. Administration groups are as follows. i) normal diet, ii) HFD (high fat diet + D-galactose), iii) HFD-statin (high fat diet + D-galactose + lovastatin (2 mg/kg/day)), iv) HFD- Lactobacillus fermentum DR9 (high fat diet + D-galactose + lactobacillus fermentum DR9 (10 log CFU/day)). DR9 showed a positive effect from hepatic gene expression to hepatic histology compared to the control; Compared to the HFD-aged control, hepatic lipid synthesis and downregulation of β-oxidation gene SCD1 (β-oxidation gene SCD1), upregulation of sterol excretion genes of liver IL-6 and ABCG5 . DR9 administration reduced serum triglycerides, total-cholesterol, and LDL-cholesterol levels after 12 weeks compared to the HFD-aged control. In addition, DR9 upregulates the hepatic energy metabolisms gene AMPKα1 compared to the HFD-Aged control, and the anti-inflammatory cytokines IL-4 and IL-10 (anti-inflammatory cytokines IL-4 and IL-10). ) Increased the intrahepatic concentration, which lowers the degree of liver steatosis. Taken together, this study showed that the DR9 strain improved lipid profiles through activation of energy and lipid metabolism, indicating that this is a promising natural intervention for the alleviation of cardiovascular and liver diseases. .

Thus, as described above, another aspect of the present invention is lactobacillus fur for use in the prevention and treatment of hyperlipidemia and lipid accumulation in the liver by preventing an increase in triglycerides, total cholesterol and LDL-cholesterol levels, inflammation, and lipid accumulation. Mentum DR9 and its metabolites. In a specific embodiment, the strain of the present invention is used for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD).

5. Lactobacillus fermentum DR9 prevents and treats skin degeneration, senescence and oxidative stress (see Example 5). Male Sprague-Dawley mice were aged through daily infusion of D-galactose for 12 weeks. After 12 weeks of dosing, skin elasticity was lowered in the old group compared to the young group. Administration of DR9 prevented a reduction in skin elasticity compared to the untreated aging group. Cyclin-D1, a senescence biomarker, was higher in the skin of the aging group compared to the silver group. Mice dosed with DR9 showed lower expression of cyclin-D1 in the skin compared to the aging group not dosed. In addition, DR9 administration upregulates the expression of FAS in the skin, an apoptosis biomarker, in order to promote the skin regeneration cycle compared to aging mice. Aged mice showed high expression of GPX, a biomarker of oxidative stress, and low expression of SOD, an antioxidant gene, whereas administration of DR9 decreased and increased these expressions, respectively.

6. Lactobacillus Fermentum DR9 prevents and treats anxiety and Alzheimer's disease (see Example 6). Anxiety assessment was performed through an open field test. Administration of DR9 reduced migration to the outer zone compared to non-dose aged rats. Alzheimer's disease uses transgenic fruit flies and yellow fruit flies with amyloid beta Aβ42 (Drosophila melanogaster) and GMRGal4 to identify targeted expressions for the malformation of eyes. Was evaluated using. Administration of DR9 prevented ocular malformation, with better ommatidia and less damaged bristles, indicating that Alzheimer's disease is less severe.

Therefore, as previously mentioned, aspects of the present invention are Lactobacillus fermentum DR9 and its metabolites used for the purpose of preventing and treating skin degeneration through improvement of skin elasticity and skin cell regeneration while reducing aging and oxidative stress. It is about.

7. Lactobacillus fermentum DR9 exerts health benefits through the production of metabolites such as succinic acid, benzaldehyde and benzoic acid, among other mechanisms (see Example 7).

Succinic acid has anti-oxidative properties useful in treating skin agin and recovering the gradual and significant loss of functions related to cellular senescence and systemic aging. On the other hand, the presence of benzoic acid in the metabolite reduces bone chloride and increases magnesium, thereby increasing bone weight. Moreover, dosing of metabolites including benzoic acid also increases the amount of calcium and phosphorus in the bone, which can slow the signs of bone aging. In addition, benzoaldehyde in the metabolite also provides anti-tumour and anti-viral properties to the subject so that the metabolite can be selectively used in the treatment of tumors or cancers.

When ingested, these metabolites slow down the symptoms of the subject's bones and muscles. The composition is particularly suitable for subjects suffering from age-related diseases such as sarcopenia, osteoporosis and metabolic disorders. Crude metabolites are ingestable or may be mixed with at least one excipient and treated as a health food or beverage. These treatments must be carried out in an appropriate manner under low temperature and atmospheric pressure to maintain the nutrient value of the metabolites.

In another specific embodiment of the present invention, ingestion of metabolites induces the production of ascorbic acid in the subject. Preferably the subject is a mammal. The production of ascorbic acid can be induced directly in the gastroin-testinal tract of a subject or indirectly in the intestinal microbiome in the gastrointestinal tract. The presence of ascorbic acid at the skin level removes toxins from the subject and provides anti-oxidant, anti-inflammatory and photo-protective properties to the subject. In particular, anti-oxidant enzymatic activities provided by ascorbic acid are closely related to AMPK (adenosine monophosphate-activated protein kinase), and activation of AMPK is a continuous aging-related pathway, especially signs of aging in bones and muscles. It is important in the delay of.

In another specific embodiment of the present invention, ingestion of metabolites induces production of 5-oxoproline in a subject. Preferably the subject is a mammal. The production of 5-oxoproline can be induced in the colon when ingested in the process of forming glutathione. Glutathione formation fights reactive oxygen species and maintains the redox homeostasis of the body's intracellular redox environment. In addition, ingestion of the composition also induces a series of hydrolytic enzymes to treat a thyrotropin-releasing hormone, including 5-oxoproline in the hypothalamus, which is useful in the treatment of spinocerebellar ataxia. TRH) leads to production. In addition to improving language memory with TRH, 5-oxoproline plays an important role in the intracellular transport of free amino acids in metabolic processes. Moreover, 5-oxoproline physiologically induces AMPK activation, which improves metabolic health and slows down signs of aging in bones and muscles. The production of 5-oxoproline is induced by ingestion of the composition, which slows down the signs of aging in bones and muscles.

In another specific embodiment of the present invention, metabolites slow down the signs of aging in bones and muscles by AMPK activation through humanization of AMPK. Particularly, AMPK is an energy-sensing network regulator capable of reprogramming cells' metabolic reactions to extrinsic stress transcriptionally, and AMPK is an energy-sensing network regulator. It acts as a switch for cellular energy homeostasis). AMPK also acts as a mediator of calorie restriction effects, including improved insulin sensitivity, improved metabolic rate, delayed decline in biological function and weight loss. In addition, the composition containing the metabolite of Lactobacillus fermentum DR9 can exert a mimetic calorie restriction effect without reducing actual calorie intake, which is why metabolic diseases related to aging factors. ) To be able to use.

According to another specific embodiment of the present invention, metabolites slow down signs of aging in bones and muscles by preventing telomere shortening. Telomere shortening is prevented by activation of AMPK through AMPK phosphorylation. In one embodiment, an animal group in which aging is induced by eating a diet containing metabolites has improved telomere length and high anti-aging potential compared to an animal group receiving a controlled dosing containing metformin. potential). In addition, there were no side effects associated with metformin administration in the animal group that consumed metabolites, and side effects include hypoglycaemia and gastrointestinal upset.

In another specific embodiment of the present invention, metabolites slow the signs of aging of bones and muscles by regulating metabolic markers and protecting bones and muscles from aging in an AMPK-dependent manner. In particular, metabolic indicators are responsible for the reduction of bone strength and muscle mass. More specifically, the metabolite decreases the expression of the p53 gene in the localized gastrocne-mius muscle and tibia, where the p53 gene encoded for the tumor suppression protein is It is a critical marker for telomere stress and is a regulator of cell cycle arrest, DNA repair, apoptosis and cellular senescence. Decreased p53 gene expression is correlated with promoting AMPK phosphorylation, and by upregulating mitochondrial related genes, motor functions of aged subjects can be improved. In one exemplary embodiment, a group of aged rats ingesting a composition containing a metabolite of Lactobacillus fermentum DR9 showed improved exercise performance in an uphill exercise test.

Moreover, the metabolites of the present invention delay the signs of aging of bones and muscles by protecting the metabolic system and the excretory system due to the modulation of AMPK. Metabolites can restore metabolic functions by activating the AMPK pathway by regulating the dynamics of mitochondrial to maximize oxidative stress, minimize energy consumption, and minimize exhaustion of resources. have. In addition, metabolites improve lipid, liver and renal profiles, potentially leading to cardiovascular diseases, obesity, and diabetes in late adulthood. , May reduce the risk of chronic kidney disease and non-alcoholic fatty liver.

Product forms

Aspect of the invention relates to a composition comprising a strain of the present invention, wherein the strain is lyophilized (freeze-dried) and the amount of 10 ⁴ ~ 10 ¹² cfu / g of the composition.

Another aspect of the present invention relates to a composition comprising a metabolite of a strain, and the metabolite is succinic acid, benzaldehyde, and benzoic acid.

The effective amount of bacterial cells is determined by a person skilled in the art, which depends on the specific goal to be achieved, the age and physical condition of the patient being treated, the severity of the underlying disease and the final formulation. When administered orally, the strain of the present invention is present in the composition in an amount that provides an effective daily dose of ^{10 7} to 10 ¹² cfu, preferably 10 ⁹ to 10 ^{11 cfu, according to the current method.} The expression "colonizing unit (cfu)" is defined as the number of bacterial cells identified by microbiological counts on agar plates. When the strain is used in the form of the composition of the present invention, the strain preferably has a concentration ratio of 1:1.

A common use of the strains of the present invention is in the form of viable cells. However, it is a non-viable cell such as killed cultures or cell lysates (e.g., obtained by exposure to altered pH, ultrasound, radiation, temperature or pressure, other methods of killing or lysing bacteria). (non-viable cells) or a composition containing beneficial factors, the composition comprising 2-hydroxyiso-capric acid and 3-phenyllactic acid acid) is produced by the strain of the present invention, such as its metabolites.

In certain embodiments of the present invention, the composition is in a form selected from the group consisting of food supplements, medicines, infant formulas, edible products and food products.

In certain embodiments, the composition is in the form of tablets, capsules, and pills for oral administration.

The composition of the present invention can be prepared in a suitable form that does not negatively affect the viability of the bacterial cells forming the composition. The choice of additives and the most suitable formulation is within the scope of those skilled in the pharmaceutical and food technology, taking into account the specific purpose of the composition.

The composition according to the present invention is that the bacterial cells are the only active agents, mixed with one or more other active agents, or mixed with pharmaceutically acceptable excipients or suitable additives or ingredients in the case of food. It can be manufactured in a form that is. In certain embodiments of the present invention, the composition additionally comprises one or more additional active agents. Preferably, the additional active agent is another probiotic bacteria that is not antagonistic to the bacterial cells forming the composition of the present invention. Depending on the formulation, the bacterial cells are purified bacteria, as a bacterial culture or as part of a bacterial culture, as a post-treated bacterial culture, and alone or as suitable carriers and components ( ingredients). Prebiotics can also be added.

The composition may be in the form of a pharmaceutical product. The term “pharmaceutical product” is understood in this document in its broader sense, including in this case, a composition comprising an active ingredient, such as a pharmaceutically acceptable additive, with bacterial cells. As used herein, the term “pharmaceutically acceptable” refers to excessive toxicity, irritation, or allergic reactions in contact with the tissues of a subject (eg human) within the scope of normal medical judgment. response), or compounds, materials, compositions and dosage forms that are suitable for use in accordance with a reasonable benefit/risk ratio without other problems or complications. . Each carrier, excipient, etc. should be “acceptable” in the sense that it is compatible with the other ingredients of the formulation. Suitable carriers, additives, etc. can be found in standard pharmaceutical texts.

Pharmaceutical products may be adopted in different forms or names depending on the product approval route and country. For example, a medicament is a specific medicinal product. In this description, medical food is considered to be another specific medicinal product. In some countries, the term “medical food” or food for special medical purposes” is a unique nutrition that cannot be met by specially formulated foods or normal diets. Refers to food for dietary management of diseases with distinctive nutritional needs, in the United States, the Orphan Drug Act Amendments of 1988 by the US Food and Drug Administration, and In Europe, it is regulated by the same legislation as Commission Directive 1999/21/EC Medical foods are different from a wide range of food supplements and traditional foods that claim health. In certain embodiments, the composition of the present invention is a medical food.

Often, the probiotic bacterial compositions disclosed herein are considered food supplements. Food supplements, also known as dietary supplements or nutritional supplements, are considered another specific pharmaceutical product. It will be prepared for use as a supplement to the diet and is intended to provide nutrients or beneficial ingredients that may not be eaten in a normal diet or in sufficient quantities. Most food supplements are considered food, but sometimes drugs, natural health products or nutraceutical products. In the meaning of the present invention, food supplements include health functional foods. Food supplements are usually sold over the counter without prescription. When food supplements take the form of pills or capsules, they contain the same excipients used in the drug product. However, food supplements may also take a form of food that is fortified with some nutrients (eg infant formula). Thus, in certain embodiments, the composition of the present invention is a food supplement.

The composition according to the present invention may be administered as it is or mixed with an appropriate edible liquid or solid, or tablets, pills, capsules, lozenges, granules, powders. ), suspensions, sachets, syrups, or freeze-dried in the form of a unit dose. In addition, it may be in the form of monodoses of the freeze-dried composition mixed in a separate liquid container provided together before administration.

The composition of the present invention may be included in various edible foods and foods such as milk products for infants. The term “edible product” as used in this document includes, in a broad sense, any form of product that can be consumed by an animal (for example, a product is received by the sensory organs). Products that can be imported). The term “food product” is understood as an edible product that provides nutritional support to the body. Of particular interest are food supplements and infant formulas. The food is preferably oatmeal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins. It contains carrier materials such as glycosylated proteins. In certain embodiments, the bacterial cells of the invention are homogenized with other ingredients such as cereals or powdered milk to make up infant formula.

Another aspect of the present invention is a cryoprotectant; Freeze-dried biomass containing the strain of the present invention; It relates to a solid composition comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are especially selected from emulsions, gels, pastes, granules, powders and gums. A further aspect of the invention provides an oral care product, a pharmaceutical composition, an edible product, a dietary supplement, a cosmetic composition comprising an effective amount of the composition as defined in the previous aspect. In certain embodiments, the oral care product is a chewing gum, tooth paste, mouth spray, lozenge, oral dispersible tablet. In certain embodiments, the pharmaceutical composition, edible product or dietary supplement is a lozenge or oral disintegrating tablet.

Examples of compositions of the present invention are shown in Example 8 below.

The word “comprise” and variations thereof in the detailed description and claims of the invention are not intended to exclude other technical features, additions, elements, or steps. Additional objects, advantages and features of the present invention will become apparent to those skilled in the art upon reviewing the detailed description, and can be learned through the embodiment of the present invention. Moreover, the present invention includes all possible combinations of the specific and preferred embodiments described in this document. The following examples and drawings are provided herein for illustrative purposes, without intending to limit the invention.

Mode for the Invention

Example

Materials and methods

Bacterial strain and culture

Lactobacillus Fermentum DR9 was extracted from fresh cow's milk in Penang, and Clinical Nutrition Intl (M) Sdn. It was obtained with the help of Bhd. All stocks cultures were preserved in 20% glycerol (-20°C), and serially washed in sterile MRS medium (Hi-Media, Mumbai, India) using 10% (v/v) inoculums. It was activated twice and incubated at 37° C. for 24 hours before use. The cultures were centrifuged at 12,000 xg for 5 minutes at 4°C, and the pellets were resuspended in PBS with an acidity of 7.5 (pH 7.5) to ^{a final concentration of 1 x 10 11} CFU/mL. .

In vitro experiment

Cell culture and AMPK activation

Human neuroblastic cell line SH-SY5Y (ATCC), human colon rectal cell line Caco-2 (ATCC), human keratin cell line HaCaT, human prostate cell line DU145 (ATCC), human bone osteosarcoma cell line U2OS (ATCC) and mouse muscle cell C2C12 ( ATCC) was cultured in Dulbecco's Modified Essential Medium (DMEM), and the human ovarian cell line OVCAR4 (ATCC) was cultured in RPMI 1640 medium in the presence of 10 μg/mL insulin. All cells were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 50 μg/mL streptomycin in 5% carbon dioxide at 37°C. do. All cell lines were seeded at a final concentration of ^{5 Х10 4} cells/mL per well in 96-well plates before cells were treated with cell-free supernatant at 25% volume for 24 hours. 5'adenosine monophosphate activated protein kinase (AMPK) phosphorylation was measured using an In-cell enzyme-linked immunosorbent assay (ELISA) Colorimetric Detection Kit according to the manufacturer's protocol.

Identification of metabolites

Culture fractionation method for AMPK analysis

The overnight (18 hours) incubation of activated DR9 was normalized to an optical density (600 nm) of 1.000±0.05 before centrifugation at 1100Xg for 15 minutes. The supernatant was collected and filtered-sterilized (pore size, 0.22 μm). This fraction is known as cell free supernatant (CFS). Collect remaining cell pellets from standardized cultures, wash three times with phosphate buffered saline (PBS) (10 mM, pH 7.4), resuspend in PBS, and ice bath After sonication in (ice bath) for 30 minutes, it was centrifuged at 3,500 X g for 15 minutes at 4°C. The supernatant was collected and filtered-sterilized (pore size, 0.22 μm). This fraction is known as an intracellular extract (IE). The washed cell pellet was collected in a fraction known as cell wall (CW). Activation of AMPK by CFS, IE, and CW was performed using the ADP-GLO assay kit and the linked AMPK (A1/B1/G1) kinase kinase system according to the manufacturer's protocol (Promega, USA).

AMPK activation using human bone osteosarcoma cell line (U2OS)

The CFS of DR9 was collected by separating cell pellets of spent broth. The medium used was separated into a lipid fraction and evaluated for AMPK activation using the human bone osteosarcoma cell line U2OS (ATCC). U2OS was cultured in Dulbecco's modified essential medium (DMEM) (Gibco, USA), 10% fetal bovine serum, 2mM L-glutamine, 100 U/mL penicillin, 50 μg/mL streptomycin was supplemented in 5% carbon dioxide at 37°C. Cell lines used H ₂ O ₂ to induce senescence. Cell lines were subcultured to reach 80% confluency, after which they were separated using trypsin-EDTA solution (0.1% trypsin in 0.02% ethylenediamine tetraacetic acid) and centrifuged at 200 X g for 5 minutes. Separated. Cells were resuspended in serum-free DMEM at a final concentration of ^{5 Х10 5 cells/ml.} Aliquots of 100 μl cells were seeded in a 96-well plate and incubated at 37° C. for 24 hours with 5% carbon dioxide. Upon cell attachment, the medium was discarded and replaced with 100 μl of Treatment medium to induce premature senescence. The treatment medium was prepared for 96 hours at 100 μM in serum-free DMEM containing _{H 2} O _2. The lipid fraction was incubated with U2OS cells during exposure to _{100 μM of H 2} O _{2 for 96 hours.} At the end of the treatment, the viability of cells was analyzed by MTT assay. Cellular AMPK phosphorylation was quantified by cell-based ELISA method using the In-cell ELISA Colorimetric Detection Kit (Thermo Scientific, USA) according to the manufacturer's instructions.

Identification of lipid fraction using GC-MS

The lipid fraction was condensed and hydrolysed and methylated for fatty acid methyl ester (FAME) analysis using gas chromatography-mass spectrometry (GC-MS). About 100 μL of sample extract was mixed with a 500 μL solvent mixture containing _{2% H 2} SO _{4 and methanol in a 2 ml Eppendorf tube, followed by 80} The mixture was stirred for 2 hours at a temperature of °C. Then, 500 μL of sodium chloride (NaCl) having a volume percentage of 0.9% and 500 μL of hexane were added to the tube containing the sample extract. The hexane layer was pipetted into an autosampler vial for FAME quantification To identify FAME, 1 µl of the hexane layer was added to a mass of 5597 MSD. It was injected into an Agilent 5977A gas chromatography system equipped with an analyzer (Agilent Technologies Australia Pty Ltd; GC/MS), and a BPX-70 column was used for separation.

Animal testing

Dosing group

All animal experiments were conducted with the approval of the USM Animal Care and Use Committee (USM/Animal Ethics Approval/2016/(724)). Male Sprague Dawley rats at 8 weeks were assigned to each group based on two experimental models; 1) An aging model using a statin as a positive control, 2) An aging model using a statin as a positive control and ingesting a high-fat diet (HPD).

In model (1), mice were assigned to one of the following groups (N = 6) for 12 weeks:

(One) Young (control), (2) Aged (D-galactose induction of aging) (3) Aged-metformin (induction of aging through D-galactose intake and administration of metformin at 300 mg/kg/day) (4) Aged-DR9 (D -Induction of aging through galactose intake and Lactobacillus fermentum DR9 administered 10 log CPU/day in a freeze-dried form using 20% (v/v) sucrose as a cryoprotectant.

In model (2), mice were assigned to one of the following groups (N = 6) for 12 weeks:

(1) Young (normal diet), Young HPD (young rats fed a high-fat diet), Old (rats induced aging through 600mg/kg/day of D-galactose), Old HPD (D- with a high-fat diet) Rats inducing aging through galactose), Old HPD DR9 (rats inducing aging through D-galactose with a high fat diet while administering Lactobacillus fermentum DR9 of log 10 CPU/day), Old HPD statin (2mg/day), Old HPD statin (2mg/day) Rats in which aging was induced through D-galactose along with a high fat diet while administering kg/day of lovastatin). A standard diet (Altromin, Germany) was used as ND, and a standard diet containing 25% (w/w) animal fat was used as HPD. Dosing was mixed in 1 g of food pellets and supplied daily. Rats were moved to individual cages during dosing to ensure complete ingestion. After the 12-week dosing period, I fasted for 12 hours before being sacrificed by inhalation of carbon dioxide. All tissues were immediately excised and rinsed with saline. Tissues were used for gene quantification through real-time PCR.

Telomere length measurement

Two standard curves (telomeres and albumin) were generated using a reference DNA sample (standard DNA) with a final DNA concentration range of 0.073-17.65 ng/μL. The T/S ratio of the experimental DNA sample is equal to T (the number of nanograms of standard DNA that matches the standard experimental sample to the number of copies of the telomere template) and S (the experimental sample to the number of copies of albumin). Is the value divided by the number of nanograms of standard DNA). The average T/S ratio is expected to be proportional to the average telomere length per cell.

Exercise load test

The treadmill exhaustion test was performed with reference to the study of Castro and Kuang (2017). The rats were first acclimated for 5 minutes at a 0° slope in a stationary running chamber, and then ran for 5 minutes at a speed of 10 m/min on a 0° slope. On days 2 and 3 of acclimatization, the rats ran an uphill slope of 10° for 10 minutes. A temporary and light electrical stimulation (0.4 mA) was used on the metal electrode behind the treadmill platform to stimulate the rats to run quickly. On the fourth day of the experiment, the belt speed was set at a speed of 10 m/min for 5 minutes, and the speed was increased by 2 m/min every 2 minutes until reaching 46 m/min. The rats were allowed to run until their stamina was exhausted (not moving for 5 seconds even with electrical stimulation) or until they reached their maximal speed. The time, speed, and distance of the moment the rats were exhausted were recorded. Work and power were calculated using the following equation: Work (J) = weight (kg) x gravitational acceleration (9.81m/s2) x longitudinal velocity (m/sx slope) x time (s) . Power (W) = Day (J) / Hour (s).

Quantitative real-time PCR

Samples of mice were stored at -80°C in RNAlater (Ambion, Austin, TX, USA). Total RNA was extracted from homogenized samples using TRI Reagent® (Sigma-Aldrich, Saint Louis, MO, USA), and ReverTra Ace-α-®kit (Toyobo, Kita-ku, Osaka, Japan) Was synthesized using the first-strand cDNA (first-strand cDNA). The mRNA expression level was determined with the Agilent AriaMx Realtime PCR System (Agilent Technologies, Santa Clara, CA, USA). The PCR reaction consists of SensiFAST SYBR®mix (Bioline, Cricklewood, London, UK), 20 ng of cDNA and 10 μM of primers. Glyceraldehyde- 3-phosphate dehydrogenase (Gapdh) was used as an endogenous control.

Qualification of serum malondialdehyde

Whole blood was centrifuged (20 minutes, 3,500 g) to separate serum and RBC pellets. According to Chakraborty and Bhattacharyya (2001), lipid peroxidation was analyzed by measuring malondialdehyde (MDA) formation. Briefly, trichloracetic acid (Sigma-Aldrich) and 2-thiobarbituric acid were added to the sample, cooled (heated at 95° C. for 30 minutes before 30° C., and then centrifuged (2). Min, 12,000 g) The supernatant was read at 534 nm and the concentration of malondialdehyde was calculated using an extinction coefficient of 1.563 ^{×10 5 /(mol/L).cm.}

Multiple enzyme-linked immunosorbent assay (ELISA)

Frozen tissue samples were destroyed or crushed under liquid nitrogen using a cold stainless steel mortar and pestle. Powdered samples were suspended in RIPA lysis buffer (Merck, USA) supplemented with protease and phosphatase inhibitor cocktail ((Promega, USA), and ice using an ultrasonic homogenizer (Labsonic, Sartorius, Germany). The samples were further homogenized at 4°C for 10 minutes at 10,000 Хg, then the supernatant was collected as protein lysates, separated into aliquots, and then analyzed at -80°C ( MILLIPLEX ® MAP Rat Cytokine/Chemokine Magnetic Beat Panel Kid (Merck, USA) was incubated overnight at 4°C for 18 hours with continuous shaking according to the manufacturer's instructions. inflammatory cytokines were analyzed Washing of the plate was aided by a hand-held magnetic block Standards for TNF-α, IFN-γ, and IL-1β Signals were measured using a calibrated Luminex ®xMAP platform (Luminex, USA) and data were compiled by the manufacturer's protocol (Sigma Aldrich, USA) and the Bradford method ) To the protein concentration of the sample.

Histology

Samples were fixed in neutral-buffered formalin upon resection. Tibial samples were removed from calcification with continuous stirring for 72 hours in 5% formic acid, and the decalcifying solution was replaced every 24 hours. Thereafter, the tissues were trimmed, dehydrated and infiltrated into paraffin wax using an automated tissue processor (Thermo ScientificTM Shandon Excelsior, USA). Subsequently, the samples were fixed in paraffin wax and sectioned using a microtome. Sectioned samples (5 μm thick) were stained with hematoxylin for 15 minutes and eosin for 5 minutes and observed with a light microscope. In the case of the gastrocnemius, the image was captured by magnifying 10 times (10X magnification), and the number of nucleus of the gastrocnemius was quantified using ImageJ Cell Counter software. For the bone sample, the image was captured by magnifying it by 4 times.

Skin elasticity

A tensile strength tester (Shimazu, Japan) was used to measure skin elasticity. A 0.025 probe was used to apply pressure until the skin sample broke. A graph was created, and the elasticity strength was calculated from the area of graph,

Biochemical analysis

Serum lipid profile (total cholesterol (TC), triglyceride (TG), low density protein (LDL), high density protein (HDL)), liver function profile (total protein, albumin, globulin, somemin/globulin ratio (AG ratio), as Paric acid aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin), kidney function profile (sodium, urea, chloride, calum creatinine, uric acid, calcium, phosphate) Whole blood was analyzed.

Metabolite detection in fecal samples

The detection of water-soluble metabolites was performed on the basis of Tsugawa's 2011 study, where samples were derivatized prior to gas chromatograph mass spectrometry (GCMS) analysis. The extraction of short-chain fatty acids from fecal samples was based on a previously conducted Nakajima 2017 study. The analysis was performed using a gas chromatography tandem mass spectrometry platform of GCMS-TQ8030 Triple Quadrupole Mass Spectrometer (Shimadzu, Japan).

Fecal Microbiome Sequencing and Analysis

The Vl-V2 (27F-338R) region of the 16S rRNA gene was amplified using isolated bacterial DNAs. PCR products were prepared based on previous Kato T research in 2014. Sequencing of 16S rRNA (16S rRNA sequencing) was performed using 454 GS Junior according to the manufacturer's manual. The resulting 16S rRNA reads were analyzed using the QIIME pipeline.

Open Field Test (OFT)

The open field experiment (OFT) was performed using an opaque rectangular box (32 cm (H) X 38 cm (W) X 52 cm (L)) with marked grids. Each animal was gently transferred from cage to box and allowed to freely navigate in the box for 5 minutes each day for 3 consecutive days. The number of entries into the central and outer zones was recorded. The performance and trajectory of each rat were recorded using a camera (GoPro, California, USA).

Drosophila on Alzheimer's disease Drosophila melanogaster Research

Preparation of stock

All superparistokes used in this study were registered with Flybase (http://fybase.bio.indiana.edu) and were purchased from Bloomington's Drosophila Stock Center (Bloomington, USA): Oregon-R wild type (#5) , Glass Multiple Reporter GAL4 (#1104) (Moses and Rubin, 1991), UAS-A 42 (#33769). All targeted expression experiments were performed using GMRGal4. In the wild-type control, Oregon-R was crossed with GMR-GAL4 to generate GMR-OreR, whereas UAS-A 42 was crossed with GMR-GAL4 to express Aβ. GMR-Aβ was produced, the stock was maintained at 25° C., and the mating was maintained at 29° C. Normal feed 4% (w/v) corn starch, 5% (w/ v) Polenta, 10% (w/v) brown sugar, 0.7% (w/v) agar, 5% (w/v) heat-killed yeast, 3 Prepared by boiling and mixing% (w/v) nipagin, 0.7% (w/v) propionic acid, etc., and cooling and solidifying before being transferred aseptically to plastic vials. All foods were prepared in the absence of nipazin and propionic acid 100 μL of Lactobacillus Permentum DR9 was ^{added to the cooled feed to 1 x 10 11} CFU/mL before solidification, Allow to coagulate in a laminar flow hood Fresh feed containing DR9 was supplied to the designated Drosophila family within 2 hours of coagulation To prepare for mating, 5-10 birds of the same parent line Virgin Drosophila (Gal4 or UAS series) and 5-10 male Drosophila (male Drosophila) were placed with food in plastic vials. Wild-type control matings were made using virgin females of the Oregon-R line that crossed with UAS-Aβ males. The GMR-Aβ crossing was carried out using virgin female UAS-Aβ crossing with female GMR-Aβ Control and transgenic GMR-Aβ Drosophila were bred on normal feeds that did not contain DR9. One group used transgenic GMR-Aβ Drosophila. Parent Drosophila was removed 4-5 days after mating. Fl progenies were collected 10 days after mating in each group for subsequent analyses.

Scanning electron micrograph

Ten progenies of each group were overnight in McDowell-Trump fixative (Sigma-Aldrich) at 4℃ containing 0.1M phosphate buffer and 4% formaldehyde. Fixed. Samples were washed three times with phosphate buffer, and post-fixed in 1% (w/v) osmium tetroxide (Sigma-Aldrich) at 25° C. for 1 hour. Then, the samples were washed with distilled water and then dehydrated in 50%, 75%, 95% and 100% ethanol for 15 minutes each. Dehydrated specimens are immersed in hexamethyldisilazane (HDMS (Sigma)) for 10 minutes. Specimens were air dried overnight in a desiccator. Dried specimens were mounted, coated with gold, and observed through a scanning electron microscope (SU8010; Hitachi Ltd., Japan).

Results and conclusion

Example 1 : Lactobacillus fermentum DR9 slows the signs of aging by activating AMPK, preventing telomere shortening, reducing lipid peroxidation, and improving body endurance.

In vitro cell culture research results

DR9 showed higher AMPK activation compared to the positive control group, and the results are shown in FIG. 1A. DR9 shows high AMPK activation in the presence of an AMPK inhibitor, Dorsormophin, and the results are as shown in FIG. 1B. AMPK phosphorylation by the cell-free supernatant of DR9 was additionally tested in 7 different cell lines, as shown in Figs. 2A and 2B. The cell-free supernatant of DR9 effectively promoted AMPK activation in U2OS cells, and showed higher AMPK activation compared to the untreated positive control. The cell-free supernatant of DR9 showed insignificant AMPK activation when tested with SH-SY5Y, OVCAR3 and DU145 cells.

Animal test results

After induction of aging through D-galactose, the telomere length of the aged rats was considerably shortened compared to that of the young rats, and the results are as shown in FIG. 3A (A). Lactobacillus administration of aging-induced rats showed an improvement in terolmere length, and compared with aged rats, a high improvement was found in the DR9-treated group. Metformin significantly prevented telomere shortening compared to aged rats.

Lipids are susceptible to oxidation, and lipid peroxidation products are frequently reported as potential biomarkers for oxidative stress. Serum lipid peroxidation was measured by measuring malondialdehyde (MDA) concentration. Serum MDA levels were significantly higher in the aged group compared to the young group (P<0.05). Compared with the aged group, the aged-DR9 group had a significantly lower serum MDA concentration (P<0.05), as shown in FIG. 3A (B).

The results of the treadmill exhaustion test are as shown in FIG. 3B (C). Running distance was not affected by all treatments, but time, speed, work, and power were significantly affected by the induction of aging. Running time, speed, work and power were significantly lower in aged rats compared to younger rats. DR9 was able to ameliorate the adverse effects of aging on exercise performance and improved performance in running time, speed, work and power compared to aged rats.

As shown in FIG. 3C (D), p53 gene expression increased upon aging, whereas DR9 decreased p53 gene expression in muscles (gastric muscles) and bones (tibia), as shown in FIG. 3B (C). , It is associated with improved motor function in aged rats, which represents a potential protection against aging of bones and muscles.

conclusion:

A total of 18 lactic acid bacteria (LAB) were evaluated by activating AMPK, an intracellular energy sensor that regulates lifespan extension. The cell-free supernatant of Lactobacillus fermentum DR9 showed higher activation of AMPK and was further evaluated. Phosphorylation of AMPK by DR9 was evident in most of the cell lines studied when compared to Compound-C, known as an AMPK-inhibitor (P<0.05). Using D-galactose (D-gal)-induced premature senescent Sprague-Dawley rats, D-galactose-treated rats administered DR9 daily for 12 weeks at 10 log CFU per rat were treated with D-galactose (D-gal). Compared to the untreated control group, telomere shortening was prevented. DR9 increases gene expression of AMPKα1 in the liver, indicating activation compared to untreated controls. DR9 shows a decrease in oxidative stress by lowering lipid peroxidation in blood compared to the untreated control group. In exercise tolerance tests, mice treated with DR9 ran longer, showed faster speed, higher work and power, and showed better physical endurance compared to the untreated control group. DR9 lowered the gene expression of p53, which is known as a biomarker of aging, in the gastrocnemius and tibia compared to the control group. Using in vitro cell culture and in vivo aged rat models, this study demonstrated the potential of DR9 in alleviating age-related impairment.

Example 2: Lactobacillus Fermentum DR9 prevents and treats muscle and bone degeneration.

Animal test results

Antioxidative genes, superoxide dismutase (SOD), are associated with primary defense antioxidants to overcome oxidative stress. Aged mice significantly decreased SOD expression when compared to young mice. As shown in FIG. 4A (A), aged rats dosed with Lactobacillus fermentum DR9 showed an increase in SOD levels in the gastrocnemius muscle and tibia when compared with the aged rats.

AMPKα2 plays an important role in regulating exercise capacity. After 12 weeks of administration of D-galactose, the expression of AMPKα2 in the gastrocnemius muscle of aged rats decreased by 72% compared to that of young rats (P=0.012). However, administration of Lactobacillus fermentum DR9 increased the expression of AMPKα2 2.7 times (P=0.029) higher than that of aged mice, respectively, as shown in FIG. 4A (B).

Low IGF-1 affects the differentiation of myotubes from skeletal muscles, impairing muscle size and function. Aged mice showed decreased expression of IGF-1 when compared to the young control group (P=0.015), whereas administration of Lactobacillus fermentum DR9 significantly increased IGF-1 levels (P=0.042) compared to the aged mice. Increased. The high expression of MyoD in the muscles of aged rats is due to cells trying to activate compensatory myogenesis to overcome the regression consequences of the high expression of MyoD in aged rats compared to the young control group (P=0.036). . Lactobacillus fermentum DR9 significantly reduced MyoD levels compared to aged mice (P<0.05), as shown in FIG. 4B (C).

The loss of bone mass and bone density during aging is because bone resorption (osteoblast formation) precedes bone formation (osteoblast formation). TNF-α and IL-1β act as signal molecules in osteoclast differentiation, and osteoclast-specific marker (TRAP) is an osteoclast indicator. The results showed that TNF-α expression was 4.4 times higher in aging mice than in young mice (P<0.001). Lactobacillus fermentum DR9 improved the expression of cytokines (P=0.005) when compared with aged mice. Lactobacillus fermentum DR9 was shown to significantly reduce the expression of IL-6 and TRAP when compared to the aged mice, as shown in Figure 4c (D).

Protein expression related to immune response, interleukin 1-beta (IL-1β), interferon gamma (IFN-γ), etc. were analyzed. Significant increase in IL-1β in tibia of aged rats was 90% higher than that of young rats (P<0.001). Lactobacillus fermentum DR9 lowered the level of IL-1β compared to the aged control group (P<0.05). Dosing of Lactobacillus fermentum DR9 was shown to reduce the level of IFN-γ in the gastrocnemius muscle of aging mice, as shown in FIG. 4D (E).

As shown in FIG. 5a b, it was observed that the amount of myocardium (nuclei of muscle cells) in the gastrocnemius of aging mice was depleted, and the arrangement of myonuclei was far apart from each other. As shown in Fig. 5ac, the aged rats dosed with Lactobacillus fermentum DR9 showed an increase in myocardium with a very dense arrangement in myofibers. The aging mice dosed with Lactobacillus fermentum DR9 showed a dramatic increase in myocardium with a very dense arrangement in myofibrils, and as shown in Fig.5b e, the amount of myocardium was three times higher than that of the elderly mice (P<0.001). Exerted. In the proximal tibia, as compared to the aging mice, which are rarely arranged in a thin network as shown in FIG. 6A, the young rats show a normal bone trabeculae arranged in an orderly manner, as shown in FIG. 6A. have. As shown in FIG. 6C, aging mice dosed with Lactobacillus fermentum DR9 showed less ordered network of bone trabecular lines, larger and more fluffy bones, and less marrow adiposity as compared to aging mice. The density of the growth plate was higher in young mice as shown in FIG. 6A compared to the aging control group as shown in FIG. 6B. As shown in FIG. 6C, mice dosed with Lactobacillus fermentum DR9 showed significant improvement of the growth plate.

conclusion:

The anti-aging mechanism of DR9 was evaluated in the gastrocnemius and tibia of rats induced by D-galactose. DR9 showed improved expression of SOD in gastrocnemius muscle and tibia and improved expression of AMPKα2 in gastrocnemius muscle compared to untreated aging mice. DR9 also decreased the expression of MyoD while increasing the mRNA expression of IGF-1, unlike the aging control. In addition, DR9 decreased the expression of IL-6, TNF-α and TRAP in the tibia compared to the aging control group, and also decreased the expression of IFN-γ in the tibia of IL-1β and gastrocnemius. The gastrocnemius muscles of DR9-treated rats had a higher number of myocytes (nuclei of muscle cells) with a denser array of muscle fibers than those of aging rats that did not. The tibia of DR9-treated rats showed a neatly arranged and normal bone trabeculae with less marrow adiposity compared to the undosed aging rats, which were rarely arranged in a thin network. Using an in vivo aged rat model, this study demonstrated the potential of the DR9 strain in alleviating muscle and bone damage.

Example 3 : Lactobacillus fermentum DR9 regulates the gut against an unbalanced gut bacterial population and metabolite concentration.

Animal study results using an aging model

At the phylum level, a total of 12 phylogenies were detected, and sequences not included in any phylogeny were marked as “unclassified”. As shown in Fig. 7a, the main phylums, Firmicutes, Bacteroidetes, and Actinobacteria, accounted for 93% of all confirmed phylums. Lactobacillus fermentum DR9 can restore changes of phylum in aged rats similar to that of young rats. The posterior bacillus and pseudobacilli occupied almost 90% of the rose organisms in the intestine. It was observed that the F/B ratio decreased rapidly during the natural aging process of both animals and humans. However, Lactobacillus fermentum DR9 can restore the ratio of posterior bacillus bacillus / bacillus bacillus in aged rats to a level similar to that of young rats, as shown in FIG. 7B.

Organisms within the bacterium have been documented as opportunistic pathogens, and the most commonly identified species is B. Fragilis, which causes abscess formation. Lactobacillus buffer momentum DR9 group has a more rich in bacteria, Blau thiazole (Blautia) and Lactobacillus (Lactobacillus), as illustrated in Fig. 7c. Blautia is known to break down complex polysaccharides in the colon, so it plays an important role in nutrient assimilation, and probiotics such as Lactobacillus are known to degrade complex polysaccharides in the colon. It is well documented as encouraging a rich gut group.

Short Chain Fatty Acid acts as an energy source of the host to regulate health, especially in obesity and immunity, and metabolic syndrome. Lactobacillus fermentum DR9 can restore the intestinal SCFA metabolites of aged mice to a level similar to that of young mice, as shown in FIG. 8A (A). As shown in Figure 8a (B), metformin (metformin)-administered aged rats and young rats were closely grouped, and Lactobacillus fermentum DR9-administered aged rats were classified into one cluster. Distinct clusters were discovered. Aged mice treated with Lactobacillus fermentum DR9 had gut water soluble metabolites similar to those of young mice.

As shown in Figure 8b (C), the aged rats receiving Lactobacillus fermentum DR9 significantly improved the amount of 5-oxoproline (P<0.05) in feces compared to the aging control group. 5-Oxoproline is involved in glutathione metabolism, and glutathione is an antioxidant that removes reactive oxygen species due to oxidative stress. Firmicutes phyla in aged rats treated with Lactobacillus fermentum DR9 had a positive alteration, whereas in aged rats, there was a probable correlation with changes in intestinal metabolites with low posterior bacillus levels. It showed a decrease in 5-oxoproline, highlighting the possible correlation.

Chronic inflammation is a common phenomenon of aging, but it was not observed in the colon of mice whose aging was induced through D-galactose. However, Lactobacillus fermentum DR9 alleviated pro-inflammatory cytokines TNF-α and IFN-γ in the intestine, as shown in FIG. 8B (D). Probiotics are well documented to exhibit immunomodulatory properties both in-vitro and in-vivo.

Animal experiment results using aging-HFD model

The Aged-HFD-DR9 group was much more abundant in Blautia, a beneficial microorganism (P<0.05), compared to the Aged-HFD group, as shown in FIG. 9A. The genus Blautia of the Aged-HFD group was approximately 16%, as shown in FIG. 9B, and increased significantly to approximately 30% after administration of Lactobacillus permentum DR9.

Blautia is known as a butyrate producer, and it has been reported that there is a positive correlation between Blautia and butyrate in a fecal sample, as shown in Fig. 10A. . The High Fat Diet reduced the concentration of acetate and propionate in feces, as shown in FIG. 10B. However, the Aged-HFD-DR9 group had significantly higher concentrations of acetate and propionate in feces compared to the Aged-HFD group (P<0.05). Propionate and acetate can reduce plasma and hepatic fatty acid content to alleviate gut diseases, lower cholesterol, and potentially improve insulin sensitivity. .

conclusion:

In the aging model, the ratio of Firmicutes/Bacteroidetes (ratio of Firmicutes/Bacteroidetes) was significantly lowered, and DR9 administration increased the ratio at the phylum level. Studies on genus levels showed that DR9-treated mice promoted lactobacillus and blutia, while reducing the number of bacteroids compared to aging mice. The profiles of short-chain fatty acids (SCFA) and soluble metabolites in the feces of DR9-treated rats were similar to that of young rats compared to non-dose aged rats. Analysis of soluble metabolites in feces showed that D-galactose-induced aging had a significant effect on amino acids metabolism, such as 5-oxoproline reduction, but DR9 administration prevented this reduction. . DR9 also reduced cytokine levels of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) compared to untreated aging mice. In the Aged-HFD model, male Sprague-Dawley mice were fed a high fat diet (HFD) and D-galactose was injected for 12 weeks to induce aging. Dosing of DR9 increased the intestinal concentration of Blautia compared to the HFD-age control. The analysis of water-soluble metabolites of excreta showed that DR9 administration compared with HFD-age control group, tryptophan, leucine, tyrosine, cysteine, methionine, valine and lysine. It has been shown to lead to a high content in the excretion of compounds related to amino acid metabolism such as (lysine), and lead to higher excretory SCFAs such as acetate and propionate. Using an in vivo animal model, this study demonstrates the potential of the DR9 strain in regulating the intestinal environment and health.

Example 4. Lactobacillus Fermentum DR9 enhances lipid metabolism and alleviates NAFLD.

Animal test results

Stearoyl-CoA desaturase 1 (SCD1) is a critical control point that regulates hepatic lipid synthesis and β-oxidation. The mRNA expression level of SCD1 in the liver was significantly down-regulated in the HFD-DR9 group as shown in Fig. 11A (A) when compared to the HFD group (P<0.05). The gene expression of IL-6 was significantly lower in the HFD group compared to the ND group. Dosing of Lactobacillus fermentum DR9 in HFD mice markedly upregulated the expression of IL-6, which is similar to that of mice on a normal diet, as shown in Fig. 11A (B). ABCG5 is part of ABC transporters and acts as a heterodimer (ABCG5/G8) that increases sterol excretion from the liver. High fat diet (HFD) significantly reduced the mRNA expression of ABCG5 (P<0.05), whereas, as shown in FIG. 11A (C), this expression was observed in the HFD-DR9 (P<0.05) group. It was significantly upregulated. As shown in FIG. 11B (D), the aged rats dosed with DR9 showed lower serum triglycerides, cholesterol, and LDL-cholesterol levels compared to normal aged rats. DR9 had no detrimental effects on the other biochemical parameters studied.

AMPK is an energy sensor and plays an important role in maintaining cellular energy levels. The mRNA expression of AMPKα1 was significantly decreased in the HFD group compared to the ND group. The HFD-DR9 group showed significantly higher mRNA expression of AMPKα1 compared to the HFD group, as shown in FIG. 12A (A).

During damage to liver cells, inflammation occurs in the liver, increasing the need for the action of anti-inflammatory cytokines. IL-4 and IL-10 are anti-inflammatory cytokines. As a result, IL-4 and IL-10 were significantly increased in the HFD group compared to the ND group. Administration of HFD-DR9 showed low IL-4 and IL-10 levels (P<0.05), as shown in FIG. 12A (B). This indicates that hepatocellular damage and inflammation are prevented by DR9, reducing the need for low IL-4 and IL-10.

Compared to the ND group, higher levels of lipid accumulation and less organized structure were observed in the HFD group. The HFD-DR9 group showed less lipid accumulation compared to the HFD group. As shown in FIG. 12B, it can be seen that there is a better structured structure in the HFD-DR9 group when compared with other administration groups.

conclusion:

DR9 showed positive effects from hepatic gene expressions to liver histology compared to the control group; Hepatic lipid synthesis and downregulation of β-oxidation gene SCD1, upregulation of hepatic IL-6 and sterol excretion genes of ABCG5 compared to HFD-aged controls. Administration of DR9 reduced serum triglycerides, total cholesterol and LDL-cholesterol levels after 12 weeks compared to the HFD-aged control. DR9 also lowered the degree of liver steatosis by upregulating the liver energy metabolism gene AMPKα1 and increasing liver concentrations of the anti-inflammatory cytokines IL-4 and IL-10 compared to the HFD-aged control. Taken together, this study is a promising natural intervention in alleviating cardiovascular diseases and liver diseases such as NAFLD by improving the lipid profiles of DR9 strain through activation of energy and lipid metabolism. Suggests that it is.

Example 5 . Lactobacillus Fermentum DR9 prevents and treats skin degeneration, senescence and oxidative stress.

Animal test results

The aging process causes loss of skin elasticity. After 12 weeks of dosing, skin elasticity was lower in the aged group compared to the? It was observed that aging mice dosed with LF-DR9 had significantly higher skin elasticity compared to the aging group not dosed, as shown in FIG. 13A (A).

Cellular senescence has emerged as a basic process of physiological aging. Cyclin-D1 is one of the senescence biomarkers. After 12 weeks of dosing, the relative gene expression of cyclin D1 was significantly higher in the aged group compared to the? It was observed that aged mice dosed with LF-DR9 had significantly lower expression of cyclin D1 compared to the aging group not dosed as shown in FIG. 13A (B).

Apoptosis is required to eliminate inflammatory cells and granulation tissues and to promote the development of new cells during the regeneration cycle. The aging of the skin results from a decrease in the ability to regenerate skin cells. DR9 upregulates the expression of FAS, an apoptosis biomarker, in the skin of young mice compared to that of aged mice. After 12 weeks of dosing,? Indicates that the relative gene expression of FAS significantly decreased in the aging group compared to the group, thereby reducing skin regeneration. It was observed that aged mice dosed with LF-DR9 had significantly higher expression of FAS compared to the aging group not dosed, as shown in FIG. 13B (C).

The antioxidant gene, superoxide dismutase (SOD), is involved in first line defense antioxidants to overcome oxidative stress, while GPX is a biomarker of oxidative stress. Aged mice showed higher expression of GPX compared to young mice. Aged mice dosed with Lactobacillus fermentum DR9 showed significantly lower GPX expression compared to the aging group not dosed, as shown in FIG. 13B (D). Aged mice showed lower SOD expression compared to young mice. The aged rats dosed with Lactobacillus fermentum DR9 showed significantly higher SOD expression and reduced oxidative stress compared to the aging group not dosed, as shown in FIG. 13B (D).

conclusion:

Male Sprague-Dawley rats were aged through daily infusion of D-galactose for 12 weeks. After 12 weeks of dosing, skin elasticity was lower in the aged group than in the? Administration of DR9 prevented loss of skin elasticity compared to the undosed aging group. The aging biomarkers such as cyclin-D1 were higher in the skin of the aging group than in the silver group. The mice treated with DR9 showed lower expression of cyclin-D1 in the skin compared to the aging group without treatment. In addition, DR9 promoted the skin regeneration cycle by upregulating the expression of skin FAS, a biomarker of apoptosis, compared to the aged mice. The aged mice showed high expression of GPX, an oxidative stress biomarker, and low expression of SOD, an antioxidant gene, whereas administration of DR9 decreased and increased these expressions, respectively. Using an in vivo animal model, this study showed that the DR9 strain has the potential to enhance skin regeneration while inhibiting oxidative stress and aging.

Example 6. Lactobacillus Fermentum DR9 prevents and treats anxiety and Alzheimer's Disease (AD).

Animal test results

The open field test is a proven evaluation tool for evaluating anxiety in rodents. Anxiety is manifested by the prevalence of animals that do not stay in the center of the open field, but in the outer corners or areas of the open field. As shown in FIG. 14A (A), DR9 administration decreased the number of accesses to the outer area of the rats compared to the non-dosed aging control group (P<0.05).

Fruit fly experiment results

The eyes of the yellow fruit fly (Drosophila melanogaster) are composed of well-formed hexagonal ommatidia and straight bristles (hair-like structure), as shown in FIGS. 14A (B) and (C). However, as shown in FIGS. 14A (D) and (E), the Alzheimer's disease mutants of Drosophila have a presence of holes, a malformation of ommatidia, and a broken setae that have lost their hexagonal structure. bristle) with a rough eye phenotype. Dosing of DR9 prevented this deformity, as shown in the better-formed eye shape and less damaged bristles in Figs. 14B (F) and (G), indicating that Alzheimer's disease is less severe.

conclusion:

Aged male Sprague-Dawley rats showed high levels of anxiety, but decreased with DR9 administration. This showed the potential Alzheimer's reversal effect of the DR9 strain by supplying DR9 to the Drosophila Alzheimer's model. Administration of DR9 can treat the rough eye phenotype seen in Alzheimer-induced fruit flies.

Example 7 Lactobacillus fermentum DR9 exerts health benefits through the production of metabolites such as succinic acid, benzaldehyde and benzoic acid.

In vitro test results

The cell-free supernatant of DR9 was fractionated into intracellular extracts (IE) and cell wall debris (CW). Among all the fractions studied, the CFS fraction activated AMPK more than the IE and CW fractions, as shown in FIG. 15A (A). Therefore, the CFS fraction was then used for further analysis. The lipid fraction of CFS was evaluated for AMPK activation potential using U2OS cell culture. Lipid fraction activated AMPK in U2OS cells in a manner similar to AICAR known as AMPK activator, and Dorsomorphin C known as AMPK inhibitor (inhibitor of AMPK) as shown in FIG. 15A (B). ) Better than Thus, the lipid fraction was later characterized.

As shown in FIG. 15B (C), as a result of comparing the GC-MS chromatogram between the lipid fraction of DR9 and the lipid fraction of the empty and non-fermented MRS medium, four peaks were butanedioic acid (succinic acid; peak 1). , Benzoaldehyde (peak 2), unknown (peak 3), and benzoic acid (peak 4). This indicates that DR9 produced these metabolites and was released into the fermentation medium. Succinic acid has anti-oxidative properties useful in treating skin aging and restoring the gradual and significant loss of functions associated with cellular senescence and systemic aging. Have. On the other hand, benzoic acid decreases bone chloride and increases magnesium, thereby increasing bone weight and leading to increased retention of calcium and phosphorus. In addition, benzaldehyde is known to provide anti-tumour and anti-viral properties to the host.

Results from the animal study

When DR9 was administered for 12 weeks, as shown in FIG. 16, two fecal metabolites, 5-oxoproline, and ascorbic acid were identified by GC-MS in the fecal samples of rats. acid) significantly increased. These metabolites were induced to be produced in the gut of the host by the action of DR9. 5-oxoproline plays an important role in the intracellular transport of free amino acids in metabolic processes, and has been shown to be lowered in the blood of type-2 diabetes patients. AMPK is poorly regulated in animals and humans with type 2 diabetes, and AMPK activation promotes metabolic health, and its activation has been used to treat type 2 diabetes. This indicates that DR9 induces AMPK activation physiologically by enhancing the production of 5-oxoproline in the intestine. Ascorbic acid is a commonly known antioxidant. AMPK is one of the upstream proteins involved in antioxidant enzyme activity. In brain hippocampal neurons, for example, AMPK protects cells through inhibition of inflammation and inducing Nrf2 antioxidant pathways. This indicates that DR9 enhances the production of ascorbic acid in the intestine, which induces antioxidant effects through AMPK activation.

conclusion:

Overall, this study indicates that Lactobacillus fermentum DR9 exerts health benefits through the production of the metabolites succinic acid, benzoaldehyde and benzoic acid. In addition, the metabolites of DR9 and/or DR9 induce the production of 5-oxoproline and ascorbic acid in the intestine. Using in-vitro and in-vivo animal models, this study aims to prevent and treat aging-induced bone, muscle and metabolic deterioration, and to improve AMPK activation. The potential of the metabolite was shown.

Example 8. Composition of Lactobacillus Permentum DR9

ingredient function Amount(g) percent(%) Anti-aging: 2.0g powder sachet (2.0g mixed in 100ml water) Lactobacillus Fermentum DR9 Probiotic 0.02g 1.00% Superoxide dismutase (SOD) Active 0.01g 0.50% Tapioca starch
Rock melon juice powder additive
food 0.20g
1.77g 10.00%
88.50% Bone and muscle: 0.5g capsule Lactobacillus Fermentum DR9 Probiotic 0.02g 4.00% Hostale (horsetail) Active 0.25g 50.00% Tapioca starch additive 0.23g 46.00% Intestine: 2.0g powdered sachet (2.0g mixed in 100ml water) Lactobacillus Fermentum DR9 Probiotic 0.02g 1.00% Inulin Probiotic 0.78g 39.00% Tapioca starch
Apple juice powder additive
food 0.20g
1.00g 10.00%
50.00% Lipid, cholesterol, non-alcoholic fatty liver disease: 0.5g capsule Lactobacillus Fermentum DR9 Probiotic 0.02g 4.00% Oat Better Glucan Active 0.30g 60.00% Tapioca starch additive 0.18g 36.00% Skin: 2.0g powder sachet (mix 2.0g in 100ml water) Lactobacillus Fermentum DR9 Probiotic 0.02g 1.00% Green tea extract food 1.84g 92.00% Tapioca starch additive 0.08g 4.00% Pomegranate juice powder food 0.06g 3.00%

Note: The contents of previously mentioned papers describing the materials and methods used in this section are incorporated herein by reference in their entirety.

Sequencing Listing Free Text

Sequence list

16s rRNA of Lactobacillus Fermentum DR9

AACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGT GGGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTGGGACAGATGATTAGGG GAAGTCGAACAAGAG

China Microbial Resource Bank (CGMCC) CGMCC15536 20180402

Claims (21)

Lactobacillus fermentum strain, characterized in that it was deposited with the China Microbial Resources Bank under the accession number CGMCC 15536, and the entire genome sequence is deposited with the gene bank under the accession number CP033371. The Lactobacillus fermentum strain according to claim 1, wherein the strain is for use in delaying signs of aging. The Lactobacillus fermentum strain according to claim 1, wherein the strain is for use in the prevention and treatment of bone and muscle degeneration. The Lactobacillus fermentum strain according to claim 1, wherein the strain is for use as a probiotic in the intestinal regulation of the unbalanced intestinal microbial population and the concentration of metabolites. The Lactobacillus fermentum strain according to claim 1, wherein the strain is for use in the prevention and treatment of hyperlipidemia, liver lipid accumulation, lipid metabolism, and lipid disorders. The Lactobacillus fermentum strain according to claim 5, wherein the strain is for use in the prevention and treatment of non-alcoholic fatty liver disease (NAFLD). The Lactobacillus fermentum strain according to claim 1, wherein the strain is for use in the prevention and treatment of skin degeneration. The Lactobacillus fermentum strain according to claim 1, wherein the strain is for use in the prevention and treatment of anxiety and Alzheimer's disease. Succinic acid (succinic acid), benzoaldehyde (benzaldehyde) and benzoic acid (bensoic acid) a metabolite of Lactobacillus permentum strain, characterized in that it contains. The metabolite of Lactobacillus fermentum strain according to claim 9, wherein the metabolite is for use in delaying signs of aging. The metabolite of Lactobacillus fermentum strain according to claim 9, wherein the metabolite is used for prevention and treatment of bone and muscle degeneration. The metabolite of Lactobacillus permentum strain according to claim 9, wherein the metabolite is for use as a probiotic in the intestinal regulation of the unbalanced intestinal microbial population and the concentration of metabolites. The metabolite of Lactobacillus permentum strain according to claim 12, wherein the metabolite comprises ascorbic acid and 5-oxoproline. The metabolite of Lactobacillus permentum strain according to claim 9, wherein the metabolite is for use in the prevention and treatment of hyperlipidemia, liver lipid accumulation, lipid metabolism, and lipid disorders. 15. The metabolite of Lactobacillus fermentum strain according to claim 14, wherein the metabolite is for use in the prevention and treatment of non-alcoholic fatty liver disease (NAFLD). The metabolite of Lactobacillus fermentum strain according to claim 9, wherein the metabolite is for use in the prevention and treatment of skin degeneration. The metabolite of Lactobacillus fermentum strain according to claim 9, wherein the metabolite is for use in the prevention and treatment of anxiety and Alzheimer's disease. A composition comprising a Lactobacillus fermentum strain, characterized in that the strain is lyophilized and has ^{an amount of 10 4} to 10 ^{12 cfu/g in the composition.} A composition comprising a metabolite of a Lactobacillus permentum strain, characterized in that it contains succinic acid, benzaldehyde, and benzoic acid. The composition according to claim 18 or 19, wherein the composition is selected from the group consisting of food supplements, medicines, infant formulas, edible products and foods. 21. The composition of claim 20, wherein the composition is selected from the group consisting of tablets, capsules or pills.

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