WO2020080736A1 - Probiotic strain for ageing, muscle and bone, gut, hyperlipidemia, skin and brain - Google Patents

Probiotic strain for ageing, muscle and bone, gut, hyperlipidemia, skin and brain Download PDF

Info

Publication number
WO2020080736A1
WO2020080736A1 PCT/KR2019/013279 KR2019013279W WO2020080736A1 WO 2020080736 A1 WO2020080736 A1 WO 2020080736A1 KR 2019013279 W KR2019013279 W KR 2019013279W WO 2020080736 A1 WO2020080736 A1 WO 2020080736A1
Authority
WO
WIPO (PCT)
Prior art keywords
strain
rats
metabolite
aged
treatment
Prior art date
Application number
PCT/KR2019/013279
Other languages
English (en)
French (fr)
Inventor
Yong-Ha Park
Original Assignee
Clinical Nutrition Intl (M) Sdn Bhd
Lii Run Sdn Bhd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Clinical Nutrition Intl (M) Sdn Bhd, Lii Run Sdn Bhd filed Critical Clinical Nutrition Intl (M) Sdn Bhd
Priority to KR1020217011441A priority Critical patent/KR20210062660A/ko
Priority to CN201980069020.5A priority patent/CN112955539A/zh
Publication of WO2020080736A1 publication Critical patent/WO2020080736A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes
    • A61K31/115—Formaldehyde
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/733—Fructosans, e.g. inulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/11—Pteridophyta or Filicophyta (ferns)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to the fields of medicine, microbiology and nutrition and, particularly, to the probiotic bacterial strain Lactobacillus fermentum DR9. It has several biofunctionalities beneficial for health, especially for slowing down ageing signs, prevention and treatment of muscle and bone degeneration, gut modulation against imbalanced gut bacterial populations and metabolites concentrations, prevention and treatment of hyperlipidaemia, hepatic lipid accumulation, lipid metabolisms and lipid diseases, and skin degeneration.
  • a probiotic is defined as 'live microorganisms that confer the health effects to the host when consumed in adequate amounts' (FAO/WHO, 2006). Lactobacillus exerts health benefits ranging from regulation of the gut environment, to alleviation of metabolic disorders and modulation of immune responses.
  • Ageing is an age-dependent multifactorial process associated with physiological decline leading to an increase in age-specific mortality rate.
  • the number of elderly aged 60 years and above is expected to increase by more than double by 2050 and to more than triple by 2100, rising from 962 million globally in 2017 to 2.1 billion in 2050 and 3.1 billion in 2100. While aging is inevitable, current aging researches aim to improve the quality of life amid increasing life expectancies.
  • Telomere length is often regarded as a biomarker of ageing and age-related morbidity. Telomeres are nucleoprotein complex at the ends of eukaryotic chromosomes which declines with each cell division. Telomere length shorten with each cell division until it reaches a critical length and initiate cell senescence.
  • the p53 gene is also a biomarker for aging. P53 encodes a tumor suppression protein, acts as a critical marker for telomeric stress-induced senescence. It affects cell cycle arrest, DNA repair, apoptosis, and cellular senescence.
  • Oxidative stress may lead to development of metabolic disorder such as obesity, diabetes, and cardiovascular diseases.
  • Muscle aging is usually referred as sarcopenia that described an involuntary loss of skeletal muscle mass and strength.
  • muscle mass starts to deplete from the age of 40.
  • Approximately 25% of adult above 60 years old will experience muscle loss, and 60% of the population when we reach 80 years old.
  • Patients suffering from sarcopenia may experience low muscle strength and poor muscle performance. They are also at adverse risk pf physical immobility, poor quality life and death.
  • Chronic inflammation, altered hormone level due to age (menopause), disrupted mitochondrial function and muscle cell regeneration were believed to be the root cause of sarcopenia.
  • osteoporosis The loss of bone mass and density with increased risk of fall and fracture is termed osteoporosis. It was reported that globally one in every three women above 50 experience osteoporotic fracture annually. There is currently no treatment available for sarcopenia except for strength training which acts as a preventive measure. Hence, there is a need to search for alternative therapy to promote healthy aging of the musculoskeletal system.
  • Gut is one of the largest lymphoid organs in our body harbouring approximately 4 trillion of bacteria. As gut microbiota control host physiology and survival, the roles of these bacteria in the aging process are indispensable. In human, gut microbiota are affected by dietary and health conditions but remain relatively stable after establishment as early as 3 years after birth. However gut microbiota experience changes in composition and diversity in the elderly with lesser abundance of core bacterial families such as Bacteroidaceae, Ruminococcaceae and Lachnospiraceae, while certain health-associated species also become more prevalent. Changes in gut microbiota is reportedly affected by altered gut barrier integrity, intestinal immune homeostasis and various other old age-diseases.
  • Gut-related disorders can be delayed/ prevented through a healthy, balanced lifestyle.
  • One of the approaches is through maintenance of balanced gut microbiota, be it via diet intervention and/or functional food supplementation.
  • Probiotics are beneficial for a healthy gut and are well-known functional food used to improve and restore dysbiosis and gastrointestinal disease, modulate the composition of gut microflora and subsequently gut barrier function.
  • An enhanced gut integrity will lead to lower oxidative stress and inflammatory markers.
  • Fecal microbiome and metabolome analyses may provide reliable and comprehensive information to uncover fecal biomarkers of diseases, especially metabolic diseases.
  • CVD cardiovascular diseases
  • CVD cardiovascular diseases
  • WHO World Health Organization
  • CVD cardiovascular diseases
  • the estimated cost of CVD will be $1,044 billion by 2030.
  • Elevated levels of blood lipid, or hyperlipidemia are well-documented risk factors for CVD.
  • Hyperlipidemia can broadly be classified as isolated elevation of cholesterol, isolated elevated TG and elevations of both. Hyperlipidemia has also been shown to affect the antioxidant status of different organs as well as their lipoprotein levels which in turn can intensify metabolic disturbances and increases the risk of cardiovascular diseases as well as non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • Skin separates the body from the outer environment and prevent the body from excessive water loss and infection.
  • skin also plays crucial role in cosmetic aspect where young and beautiful skin may positively influence social behavior. Due to aging, the skin gives the most apparent sign of aging when one becomes older. Consumers invest a considerable amount of daily expense for skin products. This has attracted cosmetics and pharmaceuticals company to develop products attempting to prevent or reverse skin aging.
  • Ageing processes affect the brain and emotions in many ways, where the prevalence of anxiety is high among the ageing population. This may be associated with cognitive declines due to neuro-degenerative diseases such as Alzheimer's disease. Moreover, accumulation of cell-damageing reactive molecules produced via cellular respiration during ageing often result in oxidation of proteins and other cellular molecules, leading to the onset of brain degeneration.
  • AMPK adenosine monophosphate-activated protein kinase
  • AMPK is an intracellular energy sensor that regulates anabolic and catabolic pathways, and particularly acts as a master regulator of cellular energy homeostasis.
  • a signaling pathway is activated when the AMPK detects low adenosine triphosphate (ATP) level, where AMPK promotes catabolic pathways to generate more ATP for various cellular metabolic activities.
  • ATP adenosine triphosphate
  • AMPK negatively regulates ATP- consuming biosynthetic process, such as gluconeogenesis, lipid and protein synthesis, via a direct phosphorylation of a number of enzymes that are directly involved in these processes, and a transcriptional control of metabolism by phosphorylating transcription factors, co-activators and co-repressors.
  • the AMPK regulates the ATP level such that the body functionality can be maintained at an optimum stage, and is a biological switch controlling bodily metabolisms.
  • 5-oxoproline or also known as pyroglutamic acid.
  • 5-oxoproline is produced by enzymatic hydrolysis in a variety of peptides.
  • Thyrotropin-releasing hormone (TRH) also known as protirelin, is a tripeptide that consists of 5-oxoproline, histidine, and proline, and it is used for medical treatment of spinocerebellar ataxia.
  • 5-oxoproline was shown in improving some verbal memory functions in subjects affected by age-related memory decline. 5-oxoproline is also found as an N-terminal modification in many neuronal peptides and hormones that also include the accumulating peptides in Alzheimer disease and familial dementia. Thus, 5-oxoproline has potentials in anti-aging effects.
  • Another important gut metabolite is ascorbic acid, which is a precursor for the biosynthesis of vitamin C. Vitamin C is a natural antioxidant present at cutaneous level, displaying certain antioxidant, anti-inflammatory, photoprotective properties, and is a known biostimulator of collagen synthesis. Thus, ascorbic acid has potentials in enhancing metabolism and skin health.
  • Probiotics could also produce a wide array of metabolites that have great health potentials.
  • One of such metabolites is succinic acid, which reportedly have an anti-aging effect for maintaining the homeostasis of the aging hypothalamus, reversed menopausal symptoms without sex hormone replacement therapy, restored gradual loss in functions associated with cellular senescence and systemic aging, has antioxidative properties, with potentials for treatment of aging skin, and played important roles in respiration/oxidation of muscles.
  • Succinic acid has also been reported to be used for modulation of gut flora, and found present as a part of composition in bones.
  • Another metabolite is benzaldehyde that is produced by some strains of probiotics from phenylalanine.
  • Benzaldehyde is used as a natural flavoring agent, important in products such as cheeses, and commonly used in the food, beverage, pharmaceutical, perfume, soap, and dyestuff industries. Benzaldehyde has been reported to have beneficial health properties such as anti-tumors and antiviral.
  • Benzoic acid is a fungistatic compound that is widely used as a food preservative, and naturally occurring in many plant sources such as berries, fruits & pickled foods. It can be conjugated to glycine in the liver and excreted as hippuric acid and commonly used for the treatment of fungal skin diseases. It is a common compound for medicinal products such as topical antiseptics and inhalant decongestants.
  • benzoic acid present primarily in omnivorous animals, such as in viscera and muscles of the ptarmigan ( Lagopus mutus ). Some Lactobacillus can transform hippuric acid to produce benzoic acid in milk, thus benzoic acid is commonly present in yoghurt. Benzoic acid has been reported to benefit femur weight, decreased chloride and increased magnesium in bones accompanied by increased retention of calcium and phosphorus.
  • the problem to be solved by the present invention is to provide new compositions and remedies useful in the amelioration of the above-mentioned conditions in a safe and effective manner.
  • Lactobacillus fermentum DR9 The solution is based on the provision of the probiotic bacterial strain Lactobacillus fermentum deposited under the accession number CGMCC 15536, also referred as Lactobacillus fermentum DR9.
  • the present inventors have found that this strain has relevant biofunctionalities useful in the amelioration of different conditions and diseases, specially the described below.
  • a first aspect of the invention relates to a strain of Lactobacillus fermentum or a metabolite thereof, wherein the strain was deposited in the China General Microbiological Culture Collection Centre (CGMCC) under the accession number CGMCC 15536 and full genome sequence deposited in GenBank under accession number CP033371, and wherein the metabolite comprises succinic acid, benzaldehyde and benzoic acid.
  • CGMCC China General Microbiological Culture Collection Centre
  • An aspect of the invention relates to Lactobacillus fermentum DR9 or a metabolite thereof for use in therapy or as a medicament.
  • This aspect can alternatively be formulated as a method for probiotic treatment, comprising administering in a need thereof an effective amount of Lactobacillus fermentum DR9 or a metabolite thereof.
  • probiotic refers to live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.
  • Another aspect of the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use in slowing down ageing signs.
  • a further aspect of the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use in the prevention and treatment of muscle and bone degeneration.
  • the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use in slowing down ageing signs by prevention of telomere shortening and/or enhancing energy metabolisms against ageing.
  • Another aspect of the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use in the prevention and treatment of hyperlipidaemia and hepatic lipid accumulation by preventing the increase of triglycerides level, inflammation and accumulation of lipids.
  • Another aspect of the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use in the prevention and treatment of cardiovascular diseases and in the reduction of cholesterol.
  • the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use as probiotic in gut modulation against imbalanced gut bacterial populations and metabolites concentrations.
  • the gut metabolites thereof comprise of ascorbic acid and 5-oxoproline.
  • Another aspect of the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use in the prevention and treatment of skin degeneration.
  • the invention relates to the strain Lactobacillus fermentum DR9 or a metabolite thereof for use as probiotic in treating and preventing anxiety and Alzheimer's Disease.
  • Lactobacillus fermentum DR9 possesses all the advantages and medical applications described above together, while in other cases, a strain must be used per each medical application.
  • compositions comprising the strain Lactobacillus fermentum DR9, wherein the strain is freeze-dried and is in amount between 10 4 and 10 12 cfu/g in the composition; and to a composition comprising a metabolite of the strain, wherein the metabolite comprises succinic acid, benzalaldehyde and benzoic acid.
  • Lactobacillus fermentum DR9 or a metabolite thereof can be alternatively formulated as the use of the strain or a metabolite thereof for the manufacture of a food supplement, a medicament, an infant formula, an edible product or a food product for the treatment and prevention of the above-mentioned indications. Further, this may be alternatively formulated as a method for the treatment and prevention of the above-mentioned indications, comprising administering to a subject in need thereof an effective amount of the strain of the invention or a metabolite thereof.
  • the subject is primarily a mammal, and more particularly a human.
  • the term "effective amount" when referred to the strain as used herein is the amount of colony forming units (cfu) for the strain in the composition that is high enough to significantly modify the condition to be treated in a positive way but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment.
  • FIGS. 1A and 1B show an illustration for comparing percentage of (A) adenosine monophosphate-activated protein kinase (AMPK) activation, and (B) AMPK activation in presence of AMPK inhibitor with cell-free supernatant from different strains of lactic acid bacteria (LAB).
  • AMPK adenosine monophosphate-activated protein kinase
  • FIGS. 2A and 2B show an illustration for comparing AMPK phosphorylation in cell lines, including human bone osteosarcoma (U2OS), mouse muscle (C2C12), human neuroblastoma (SH-SY5Y), human ovary (OVCAR3), human keratinocytes (HaCaT), human prostate (DU145), and human colorectal (CaCo-2), treated with cell-free supernatant from Lactobacillus fermentum DR9 and unfermented De Man, Rogosa and Sharpe (MRS) broth for 24 hours.
  • 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and compound C were used as positive and negative control, respectively.
  • FIGS. 3A, 3B and 3C (A) Measurement of telomere length in the blood.
  • B Serum malondialdehyde (MDA) concentrations of rats after 12 weeks of treatment.
  • C Treadmill exhaustion test based on uphill treadmill performance (distance, time running speed, work, and power) evaluated on senescence induced rats.
  • FIGS. 4A, 4B, 4C and 4D mRNA expression levels of antioxidative gene, superoxide dismutase (SOD) of the gastrocnemius (left) and tibia (right) samples isolated from the right hind leg of rats after 12 weeks treatment.
  • SOD superoxide dismutase
  • AMPK ⁇ 2 AMP-activated protein kinase subunits
  • IGF-1 and MyoD myogenesis-related genes from gastrocnemius muscle.
  • D Osteoclastogenesis-related mRNA genes expression of TNF-alpha, IL-6 and TRAP in tibia.
  • FIGS. 5A and 5B Hematoxylin and eosin (H&E) staining of the gastrocnemius muscle sections from the left hind leg of rat after 12 weeks treatment (magnification, x10).
  • FIG. 6 Hematoxylin and eosin (H&E) staining of the decalcified proximal tibia sections from the left hind leg of rat after 12 weeks treatment (magnification, x4).
  • FIGS. 7A, 7B and 7C (A) Distribution profile of bacterial phylum, (B) relative abundance of Firmicutes/ Bacteroidetes ratio and (C) relative abundance of Blautia, Bacteriodetes and Lactobacillus genus determined in rats fecal samples upon 12 weeks of senescence induction.
  • FIGS. 8A and 8B Sparse partial least square discriminant analysis (sPLS-DA) score plot based on short chain fatty acids (A), water-soluble metabolites (B) and (C) 5-oxoproline from fecal samples of rats upon 12 weeks senescence induction. The metabolites were detected and analyzed using GC-MS.
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumour necrosis factor alpha
  • FIGS. 9A and 9B (A) Relative abundance of Blautia in fecal samples and (B) fecal microbiota analysis of the genus distribution in phylum Firmicutes after 12 weeks of treatment.
  • FIGS. 10A and 10B Analysis of short-chain fatty acid profiles in rat's fecal samples after 12 weeks of treatment.
  • FIGS. 11A and 11B mRNA gene expression of (A) SCD1, (B) IL-6, and (C) ABCG5 in liver of D-galactose induced ageing rats (600mg/kg/day) after 12 weeks of treatment.
  • FIGS. 12A and 12B Relative gene expression of AMPK ⁇ 1 (A) and cytokines level (interleukin-4 and interleukin-10) (B) in liver, and (C) hematoxylin and eosin (H&E) staining of liver sections of D galactose induced ageing rats (600mg/kg/day) after 12 weeks of treatment.
  • ND normal diet
  • HFD high fat diet
  • HFD-statin high fat diet, receiving lovastatin 2 mg/kg/day
  • HFD-DR9 high fat diet, receiving L. fermentum DR9 (10 10 CFU/day).
  • FIGS. 13A and 13B Skin elasticity (A), and mRNA gene expression of cyclin-D1 (B), FAS (C), GPX and SOD (D) of skin from rats after 12 weeks of treatment.
  • GMR-OreR Eyes of Drosophila melanogaster showing well-formed hexagonal ommatidia and straight bristles are observed from GMR-OreR at a magnification of 250 ⁇ (B), and of 350 ⁇ (C), while a rough eye phenotype with presence of holes are observed from transgenic Drosophila melanogaster A ⁇ 42 (GMR-A ⁇ 42) at a magnification of 250 ⁇ (D) and of 350x (E).
  • a well-formed eye shape and hexagonal ommatidia with less damaged bristles are observed from GMR-A ⁇ 42.DR9 fed with Lactobacillus fermentum strain DR9 at a dose of 100 ⁇ L 1 x 10 11 CFU/mL at a magnification of 250 ⁇ (F) and of 350x (G).
  • C GC-MS chromatogram of CFS fraction produced by 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 from fecal samples of rats after 12-weeks treatment.
  • Lactobacillus fermentum DR9 was isolated from fresh cow's milk from Penang and was obtained via courtesy of Clinical Nutrition Intl (M) Sdn. Bhd., Malaysia. The strain was identified as Lactobacillus fermentum and was deposited at China General Microbiological Culture Collection Centre (CGMCC) with address Institute of Microbiology Chinese Academy of Sciences, No. 1 West Beichen Road Chaoyang District Beijing, China. The deposited strain received the depository number CGMCC 15536 and deposit date April 2, 2018 (02.04.2018). It was deposited by the same applicants Clinical Nutrition Intl (M) Sdn. Bhd. and Lii Run Sdn. Bhd. The deposited strain is viable and keeps all their features related to their deposit.
  • CGMCC General Microbiological Culture Collection Centre
  • 16S rRNA gene sequencing Information on 16S rRNA is as follows and sequence is SEQ ID NO: 1:
  • the skilled person in the art can routinely, by conventional mutagenesis or re-isolation techniques, obtain further variants or mutants thereof that retain or enhance the herein described relevant features and advantages of the strain of the invention.
  • the invention also relates to variants of the strain disclosed herein.
  • the term "variant" or “mutant” of a strain refers to any naturally-occurring or specifically developed strain obtained from the deposited strain, mainly by mutation, that maintains the functions of the deposited strain. Functions can be assessed by the skilled in the art by any of the methods described in the EXAMPLES section.
  • the 16S rRNA gene or the full genome sequence of a "variant" strain as contemplated herein may share about 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent sequence identity with the 16S rRNA sequence SEQ ID NO: 1 or the CP033371 genome sequence of the strain disclosed herein.
  • the mutants are obtained by using recombinant DNA technology. In another embodiment, the mutants are obtained by random mutagenesis.
  • another aspect of the invention relates to a method to obtain a mutant of a DR9 strain, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the obtained variant or mutant further retains or enhances the biological functions of the deposited strain.
  • the strain has been fermented in an artificial medium and submitted to a post-treatment after the fermentation, to obtain bacterial cells, and the resulting bacterial cells are in a liquid medium or in a solid form.
  • the post-treatment is selected from the group consisting of: drying, freezing, freeze-drying, fluid bed-drying, spray-drying and refrigerating in liquid medium, and more particularly, is freeze-drying.
  • the strain is produced by cultivating (or fermenting) the bacteria in a suitable artificial medium and under suitable conditions.
  • artificial medium for microorganisms is to be understood a medium containing natural substances, and optionally synthetic chemicals such as the polymer polyvinyl alcohol which can reproduce some of the functions of serums.
  • suitable artificial media are nutrient broths that contain the elements including a carbon source (e.g. glucose), a nitrogen source (e.g. amino acids and proteins), water and salts needed for bacterial growth. Growth media can be liquid form or often mixed with agar or other gelling agent to obtain a solid medium.
  • the strains can be cultivated alone to form a pure culture, or as a mixed culture together with other microorganisms, or by cultivating bacteria of different types separately and then combining them in the desired proportions. After cultivation, and depending on the final formulation, the strains may be used as purified bacteria, or alternatively, the bacterial culture or the cell suspension may be used, either as such or after an appropriate post-treatment.
  • the term "biomass” is understood the bacterial strain culture obtained after cultivation (or fermentation as a term synonymous to cultivation).
  • post-treatment is to be understood in the context of the present invention, any processing carried out on the biomass with the aim of obtaining storable bacterial cells.
  • the objective of the post-treatment is decreasing the metabolic activity of the cells in the biomass, and thus, slowing the rate of cellular deleterious reactions.
  • the bacterial cells can be in solid or liquid form.
  • the stored bacterial cells can be a powder or granules.
  • both the solid and liquid forms containing the bacterial cells are not present in the nature, hence, are not naturally-occurring, since they are the result of artificial post-treatment process(es).
  • the post-treatment processes may in particular embodiments require the use of one or more of so-called post-treatment agent.
  • post-treatment agent refers to a compound used to perform the herein described post-treatment processes.
  • post-treatment agents are to be included, without limitation, dehydrating agents, bacteriostatic agents, cryoprotective agents (cryoprotectants), inert fillers (also known as lyoprotectants), carrier material (also known as core material), etc., either used alone or in combination.
  • the first one is decreasing the rate of all chemical reactions, which can be done lowering the temperature by refrigerating or freezing using refrigerators, mechanical freezers, and liquid nitrogen freezers.
  • decreasing the rate of all chemical reactions can be achieved by adding substances that inhibit the growth of the bacterial cells, namely a bacteriostatic agent, abbreviated Bstatic.
  • the second approach to carry out the post-treatment is to remove water from the biomass, a process which can involve sublimation of water using a lyophilizer. Suitable techniques to remove water from the biomass are drying, freeze-drying, spray-drying or fluid bed-drying. Post-treatments that result in solid form may be drying, freezing, freeze-drying, fluid bed-drying, or spray-drying.
  • the post-treatment is particularly freeze-drying, which involves the removal of water from frozen bacterial suspensions by sublimation under reduced pressure. This process consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most water, and secondary drying to remove bound water. Due to objective and expected variability of industrial processes for manufacturing and isolation of lyophilized bacterial cultures, the latter commonly contain certain amount of inert filler also known as lyoprotectant. Its role is to standardize the content of live probiotic bacteria in the product.
  • sucrose, saccharose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and other pharmaceutically acceptable non-hygroscopic fillers are also used.
  • other stabilizing or freeze-protecting agents like ascorbic acid, are also used to form a viscous paste, which is submitted to freeze-drying.
  • the so-obtained material can be grinded to appropriate size, including to a powder.
  • Lactobacillus fermentum DR9 has the advantage of being particularly useful as a probiotic.
  • Probiotic bacteria must fulfil several requirements related to lack of toxicity, viability, adhesion and beneficial effects. The properties of each bacterial strain are unique and cannot be extrapolated to other strains of the same species.
  • Lactobacillus strain DR9 strain has, among others, the following effects and applications beneficial for health:
  • Lactobacillus fermentum DR9 slows down ageing signs via activation of AMPK, prevention of telomere shortening, lowering of lipid peroxidation and enhancing physical endurance (see EXAMPLE 1 hereinafter).
  • a total of 18 strains of lactic acid bacteria (LAB) were evaluated on the activation of adenosine monophosphate-activated protein kinase (AMPK), an intracellular energy sensor mediating lifespan extension.
  • AMPK adenosine monophosphate-activated protein kinase
  • CFS cell-free supernatant
  • DR9 The phosphorylation of AMPK by DR9 was evident in most cell lines studied, compared to Compound-C the known AMPK-inhibitor (P ⁇ 0.05).
  • D-gal D-galactose
  • DR9 increased the gene expression of AMPK ⁇ 1 in the liver indicating activation as compared to the untreated control.
  • DR9 lowered blood lipid peroxidation as compared to the untreated control indicating reduced oxidative stress.
  • DR9 lowered gene expression of p53, a known senescent biomarker, in gastrocnemius muscle and tibia bone compared to the control.
  • an aspect of the invention relates to Lactobacillus fermentum DR9 or a metabolite thereof for use in the slows down ageing signs via activation of AMPK, prevention of telomere shortening, lowering of lipid peroxidation and enhancing physical endurance.
  • Ageing signs are e.g. reduced energy metabolisms leading to impairment in mobility and movements, and oxidation of plasma leading to impairment of immunity.
  • Lactobacillus fermentum DR9 prevents and treats muscle and bone degeneration (see EXAMPLE 2 hereinafter).
  • the anti-aging mechanism of DR9 was evaluated on gastrocnemius muscle and tibia of D-galactose induced aging rats.
  • DR9 demonstrated improved expression of SOD in gastrocnemius muscle and tibia bone and expression of AMPK ⁇ 2 in gastrocnemius muscle compared to the untreated aged rats.
  • DR9 also increased the mRNA expression of IGF-1 while reducing the expression of MyoD, in contrast to the aged controls.
  • DR9 also reduced the expression of IL-6, TNF- ⁇ and TRAP in tibia, and IL-1 ⁇ tibia and IFN- ⁇ in gastrocnemius when compared to the aged rats.
  • Gastrocnemius muscles of DR9 rats had higher number of myonuclei (nucleus of muscle cells) accompanied by denser arrangement of myofibers as compared to untreated aged rats.
  • Tibia of DR9 rats also showed more neatly arranged and normal bone trabeculae accompanied by lesser marrow adiposity as compared to the untreated aged rats which were arranged sparsely with thinner network.
  • an aspect of the invention relates to Lactobacillus fermentum DR9 or a metabolite thereof for use in prevention and treatment of muscle and bone degeneration via enhancing antioxidation and energy metabolisms while reducing atropy, osteoclastogenesis, inflammation and depletion of myonuclei, leading to better mobility and physical endurance.
  • Lactobacillus fermentum DR9 has effects on gut modulation against imbalanced gut bacterial populations and metabolites concentrations (see EXAMPLE 3 hereinafter).
  • the ratio of Firmicutes/Bacteroidetes was significantly lowered, while treatment with DR9 increased the ratio at the phylum level.
  • Study on the genus level showed that DR9 treated rats promoted the proliferation of Lactobacillus and Blautia while decreased the population of Bacteroides as compared to the aged rats.
  • Fecal short-chain fatty acid (SCFA) and water soluble metabolite profiles of DR9 rats resembled more of that from young rats as compared to old untreated rats.
  • DR9 also reduced cytokines levels of interferon gamma (IFN- ⁇ ) and tumour necrosis factor alpha (TNF- ⁇ ) in rats distal colon as compared to the untreated old rats.
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumour necrosis factor alpha
  • an aspect of the invention relates to Lactobacillus fermentum DR9 or a metabolite thereof for as probiotic in gut modulation against imbalanced gut bacterial populations and metabolites concentrations; that means, to ameliorate dysbiosis.
  • Lactobacillus fermentum DR9 enhances lipid metabolisms and alleviates NAFLD (see EXAMPLE 4 hereinafter).
  • different strains of Lactobacillus were tested for alleviating hyperlipidemia and liver steatosis via activation of AMPK in aged rats.
  • Male Sprague- Dawley rats were fed with a high fat diet (HFD) and injected with D-galactose daily over 12 weeks to induce ageing.
  • DR9 exerted positive effects ranging from hepatic gene expressions to liver histology as compared to the control; down-regulation of hepatic lipid synthesis and ⁇ -oxidation gene SCD1, up-regulation of hepatic IL-6 and sterol excretion gene of ABCG5 as compared to the HFD-aged control.
  • Administration of DR9 reduced serum triglycerides, total-cholesterol and LDL-cholesterol levels after 12-weeks as compared to the HFD-aged control.
  • DR9 also up-regulated hepatic energy metabolisms gene AMPK ⁇ 1 and increased liver concentrations of anti-inflammatory cytokines IL-4 and IL-10 as compared to the HFD-aged control, leading to lesser degree of liver steatosis.
  • this study illustrates that DR9 strain led to improved lipid profiles via activation of energy and lipid metabolisms, suggesting it as a promising natural intervention for alleviation of cardiovascular and liver diseases.
  • an aspect of the invention relates to Lactobacillus fermentum DR9 or a metabolite thereof for use in the prevention and treatment of hyperlipidaemia and hepatic lipid accumulation, by preventing the increase of triglycerides, total- and LDL-cholesterol levels, inflammation and accumulation of lipids.
  • the strain of the invention is used in the prevention and treatment of Non-alcoholic fatty liver disease (NAFLD).
  • NAFLD Non-alcoholic fatty liver disease
  • Lactobacillus fermentum DR9 prevents and treats skin degeneration, senescence and oxidative stress (see EXAMPLE 5 hereinafter).
  • Male Sprague-Dawley rats were aged via injection with D-galactose daily for 12 weeks. The skin elasticity after 12 weeks of treatment was lower in old group compared to young group.
  • Administration of DR9 prevented the loss of skin elasticity as compared to the non-treated old group.
  • Cyclin-D1 a senescence biomarker was higher in the skin of old group as compared to young group.
  • Rats administered with DR9 had a lower expression of skin cyclin-D1 as compared to non-treated old group.
  • DR9 also upregulated the expression of skin FAS, an apoptosis biomarker as compared to old rat to facilitate with skin regeneration cycle.
  • Old rats showed higher expression of GPX, a biomarker of oxidative stress and lower expression of SOD, an antioxidative gene, while administration of DR9 reduced and increased such expressions, respectively.
  • Lactobacillus fermentum DR9 prevents and treats anxiety and Alzheimer's Disease (see EXAMPLE 6 hereinafter). Anxiety assessment are performed using the open field test. The administration of DR9 reduced movement of rats to the outer zone as compared to the untreated aged rats. Alzheimer's Disease are assessed using transgenic fruit flies, Drosophila melanogaster with amyloid beta A ⁇ 42 and targeted expressions for malformation of eyes using GMRGal4. The administration of DR9 prevented malformation of eyes with more well-formed shape of ommatidia and less damaged bristles, indicating less severity of Alzheimer's Disease.
  • an aspect of the invention relates to Lactobacillus fermentum DR9 or a metabolite thereof for preventing and treating skin degeneration via improved skin elasticity and skin cells regeneration while reducing senescence and oxidative stress.
  • Lactobacillus fermentum DR9 exerts its health benefits via, among other mechanisms, the production of metabolites succinic acid, benzaldehyde and benzoic acid (see EXAMPLE 7 hereinafter).
  • Succinic acid has anti-oxidative properties useful for treating skin aging and restoring gradual but significant loss in functions associated with cellular senescence and systemic aging.
  • presence of benzoic acid in the metabolites can decrease chloride and increase magnesium in bones, and then increases bone weight upon administration.
  • administration of the metabolites comprising benzoic acid can also increase retention of calcium and phosphorus in the bone, and then slow down the aging sign in the bones.
  • benzaldehyde in the metabolites also provides an anti-tumour and anti-viral properties to the subject, such that the metabolites can be optionally used for treating tumour or cancer.
  • Such metabolites confer the slowing down of aging signs in bone and muscle in a subject upon ingestion.
  • the composition is especially suitable for subjects suffering from age-related diseases, such as sarcopenia, osteoporosis and metabolic disorders.
  • the crude metabolites can be ingested, or further processed to mix with at least one excipient to form a health food or a beverage composition.
  • the processing can be carried out in a milder manner, preferably under low temperature and atmospheric pressure, to maintain nutritional value of the metabolites.
  • ingestion of the metabolites induce production of ascorbic acid in the subject.
  • the subject is a mammal.
  • the production of ascorbic acid can be induced directly from gastrointestinal tract of the subject, or indirectly from a gut microbiome within the gastrointestinal tract. Presence of ascorbic acid at cutaneous level removes toxin in the subject, and provides anti-oxidant, anti-inflammatory and photo-protective properties to the subject.
  • the anti-oxidant enzymatic activities provided by ascorbic acid is closely related to adenosine monophosphate-activated protein kinase (AMPK), where the activation of AMPK is important for subsequent aging-related pathways, particularly in conferring slowing down aging signs in bone and muscle.
  • AMPK adenosine monophosphate-activated protein kinase
  • ingestion of the metabolites induce production of 5-oxoproline in the subject.
  • the subject is a mammal.
  • the production of 5-oxoproline can be induced in colon upon ingestion, particularly for a process of forming glutathione. Formation of glutathione maintains intracellular redox homeostasis in the body to act against reactive oxygen species.
  • ingestion of the composition also induces a series of enzymatic hydrolysis that leads to production of thyrotropin-releasing hormone (TRH) comprising 5-oxoproline in hypothalamus that can be useful for treating spinocerebellar ataxia.
  • TRH thyrotropin-releasing hormone
  • 5-oxoproline also plays important roles in intracellular transport of free amino acids during metabolic processes. Furthermore, 5-oxoproline may physiologically induces AMPK activation, and then improves metabolic health and slows down aging signs in bone and muscle. The production of 5-oxoproline induced by ingestion of the composition leads to slowing down aging signs in bone and muscle.
  • the metabolites confer the slowing down of aging signs in bone and muscle by activating an AMPK pathway via AMPK phosphorylation.
  • AMPK is an energy-sensing network regulator that can transcriptionally reprogram the cells' metabolic reaction towards extrinsic stress, and AMPK is acting as a switch for cellular energy homeostasis.
  • AMPK also acts as a mediator of calorie restriction effects, where the effects include improved insulin sensitivity, improved metabolic rate, slower deterioration in biological functions, and weight loss.
  • the composition comprising the metabolites of L. fermentum strain DR9 may exert mimetic calorie restriction effect without requiring actual reduction in calorie intake, therefore allowing the composition to be used for metabolic diseases related to aging factors.
  • the metabolites confer the slowing down of aging signs in bone and muscle by preventing telomere shortening.
  • the telomere shortening is prevented by activating AMPK pathway via AMPK phosphorylation.
  • a senescence-induced animal model subjected to a diet comprising the metabolites show improved telomere length and higher anti-aging potential as compared to an animal model subjected to a controlled treatment comprising metformin.
  • the animal model subjected to the metabolites show absence of side effects associated with administration of metformin, where the side effects include hypoglycaemia and stomach upset.
  • the metabolites confer the slowing down of aging signs in bone and muscle by modulating metabolic markers, and protecting the bone and the muscle from senescence in an AMPK-dependent manner, particularly markers that are responsible for loss of bone strength and reduction in muscle mass. More particularly, the metabolites reduce p53 gene expression in localized gastrocnemius muscle and tibia, where p53 gene encoded for tumour suppression protein is a critical marker for telomeric stress-induced senescence and a regulator for cell cycle arrest, DNA repair, apoptosis and cellular senescence. The reduced p53 gene expression is correlated with promotion of AMPK phosphorylation, such that mitochondrial related gene can be upregulated to improve motor function in an aged subject.
  • an aged animal model subjected to the composition comprising metabolites of L. fermentum strain DR9 shows improved exercise performance in an uphill exercise test.
  • the metabolites of the present invention confer slowing down of aging signs in bone and muscle by protecting metabolic system and excretory system due to modulation of AMPK.
  • the metabolites are capable of restoring metabolic functions by activating AMPK pathway to regulate dynamics of mitochondrial for overcoming oxidative stress, minimizing energy consumption and minimizing exhaustion of resources.
  • the metabolites can improve lipid, liver and renal profiles, that could potentially reduce risks of cardiovascular diseases, obesity, diabetes, chronic kidney disease, and non-alcoholic fatty liver in late adulthood.
  • An aspect of the invention refers to a composition comprising the strain of the invention, wherein the strain is freeze-dried and is in amount between 10 4 and 10 12 cfu/g in the composition.
  • compositions comprising a metabolite of the strain, wherein the metabolite comprises succinic acid, benzaldehyde and benzoic acid.
  • the effective amount of the bacterial cells will be determined by the skilled in the art and will vary with the particular goal to be achieved, the age and physical condition of the patient being treated, the severity of the underlying disorder, and the final formulation.
  • the strain of the invention When administered orally, is present in the composition in an amount giving an effective daily dose of from 10 7 to 10 12 cfu, according to the current legislation, preferably from 10 9 to 10 11 cfu.
  • the expression "colony forming unit" (“cfu”) is defined as number of bacterial cells as revealed by microbiological counts on agar plates.
  • the strain is preferably in a concentration ratio of 1:1.
  • the general use of the strain of the invention is in the form of viable cells. However, it can also be extended to non-viable cells such as killed cultures or cell lysates (obtained by e.g. exposure to altered pH, sonication, radiation, temperature or pressure, among other means of killing or lysing bacteria) or compositions containing beneficial factors produced by the strain of the invention, such as the metabolite thereof comprising 2-hydroxyisocapric acid and 3-phenyllactic acid.
  • non-viable cells such as killed cultures or cell lysates (obtained by e.g. exposure to altered pH, sonication, radiation, temperature or pressure, among other means of killing or lysing bacteria) or compositions containing beneficial factors produced by the strain of the invention, such as the metabolite thereof comprising 2-hydroxyisocapric acid and 3-phenyllactic acid.
  • the composition is in a form selected from the group consisting of a food supplement, a medicament, an infant formula, an edible product and a food product.
  • the composition is in the form of tablets, capsules or or pills for oral administration.
  • composition of the invention may be prepared in any suitable form which does not negatively affect to the viability of the bacterial cells forming the composition of the invention. Selection of the excipients and the most appropriate methods for formulation in view of the particular purpose of the composition is within the scope of ordinary persons skilled in the art of pharmaceutical and food technology.
  • composition according to the invention can be formulated in a form in which the bacterial cells are the only active agent or are mixed with one or more other active agents and/or are mixed with pharmaceutically acceptable excipients or adequate additives or ingredients in the case of a food product.
  • the composition additionally contains one or more further active agents.
  • the additional active agent or agents are other probiotic bacteria which are not antagonistic to the bacterial cells forming the composition of the invention.
  • the bacterial cells may be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a bacterial culture which has been post-treated, and alone or together with suitable carriers or ingredients. Prebiotics can also be added.
  • composition can be in the form of a pharmaceutical product.
  • pharmaceutical product is understood in its widely meaning in this description, including any composition that comprises an active ingredient - in this case, the bacterial cells- together with pharmaceutically acceptable excipients.
  • pharmaceutical product is not limited to refer to medicaments.
  • pharmaceutically acceptable as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts.
  • the pharmaceutical product can adopt different forms or names depending on the product approval route and also depending on the country.
  • a medicament is a particular pharmaceutical product.
  • a medical food is considered in this description as another particular pharmaceutical product.
  • the terms "medical food” or “food for special medical purposes” are used in some countries to refer to a food specially formulated and intended for the dietary management of a disease that has distinctive nutritional needs that cannot be met by normal diet alone. They are defined in regulations such as the Food and Drug Administration's 1988 Orphan Drug Act Amendments in the United States, and the Commission Directive 1999/21/EC in Europe. Medical foods are distinct from the broader category of food supplements and from traditional foods that bear a health claim. Thus, in a particular embodiment, the composition of the invention is a medical food.
  • probiotic bacterial compositions such as the one disclosed herein, are considered as food supplements.
  • a food supplement also known as dietary supplement or nutritional supplement is considered another particular pharmaceutical product. This is a preparation intended to supplement the diet and provide nutrients or beneficial ingredients that are not usually ingested in the normal diet or may not be consumed in sufficient quantities.
  • food supplements are considered as food products, but sometimes they are defined as drugs, natural health products, or nutraceutical products. In the sense of the present invention, food supplements also include nutraceuticals. Food supplements are usually sold "over the counter", i.e. without prescription. If the food supplement adopts the form of a pill or a capsule, it comprises excipients which are the same as the used in medicaments.
  • a food supplement however can also adopt the form of a food product which is fortified with some nutrients (e.g. an infant formula).
  • the composition of the invention is a food supplement.
  • composition according to the invention can be administered as such or mixed with a suitable edible liquid or solid, freeze-dried in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, syrups or usually in the form of a unit dose. It can also be in form of monodoses of freeze-dried composition presented together with a separate liquid container to be mixed before administration.
  • composition of the invention can also be included in a variety of food products or edible products, such as milk products in case of infants.
  • edible product is used herein in its broadest meaning, including any type of product, in any form of presentation, which can be ingested by an animal; i.e. a product that is organoleptically acceptable.
  • food product is understood as an edible product which also provides a nutritional support for the body. Particularly interesting food products are food supplements and infant formulas.
  • the food product preferably comprises a carrier material such as oat meal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins.
  • the bacterial cells of the invention are homogenized with other ingredients, such as cereals or powdered milk to constitute an infant formula.
  • Another aspect of the present invention relates to a solid composition
  • a solid composition comprising a cryoprotectant; a freeze-dried biomass comprising the strain of the invention; and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier particularly is chosen from an emulsion, a gel, a paste, granules, a powder, and a gum.
  • Additional aspects of the invention provide an oral care product, a pharmaceutical composition, and edible product, a dietary supplement and a cosmetic composition comprising an effective amount of the composition as defined in the previous aspect.
  • the oral care product is a chewing gum, a tooth paste, a mouth spray, a lozenge, or an oral dispersible tablet.
  • the pharmaceutical composition, the edible product or the dietary supplement is a lozenge or an oral dispersible tablet.
  • compositions of the invention are in EXAMPLE 8 below.
  • Lactobacillus fermentum DR9 was isolated from fresh cow's milk in Penang and obtained via courtesy of Clinical Nutrition Sdn. Bhd. (Kuala Lumpur, Malaysia). All stocks cultures were preserved in 20% glycerol (-20°C), activated in sterile de Mann, Rogosa, Sharper (MRS) broth (Hi-Media, Mumbai, India) for three successive times using 10% (v/v) inoculums and incubated at 37°C for 24 h prior to use. The cultures were centrifuged at 12,000 x g for 5 min at 4°C and the pellets were resuspended in PBS (pH 7.5) to a final concentration of 1 x 10 11 CFU/mL.
  • PBS pH 7.5
  • Human neuroblastoma cell line SH-SY5Y (ATCC), human colorectal cell line Caco-2 (ATCC), human keratinocytes cell line HaCaT, human prostate cell line DU145 (ATCC), human bone osteosarcoma cell line U2OS (ATCC) and mouse muscle cell C2C12 (ATCC) were cultured in Dulbecco's Modified Essential Medium (DMEM), while human ovary cell line OVCAR3 (ATCC) was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium in presence of 10 ⁇ g/mL insulin.
  • DMEM Dulbecco's Modified Essential Medium
  • RPMI Roswell Park Memorial Institute
  • All cells are supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 50 ⁇ g/mL streptomycin at 37°C and 5% CO 2 . All cell lines were seeded to a final concentration of 5 ⁇ 10 4 cells/mL per well onto 96-well plate before the cells were treated with cell-free supernatant at 25% by volume for 24 hours. 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was measured using In-cell enzyme-linked immunosorbent assay (ELISA) Colorimetric Detection Kit according to manufacturer's protocol.
  • AMPK adenosine monophosphate-activated protein kinase
  • the supernatant was collected and filter-sterilized (pore size, 0.22 ⁇ m). This fraction was known as intracellular extract (IE).
  • the washed cell pellet was collected as the fraction known as cell wall (CW).
  • Activation of AMPK by CFS, IE and CW was performed using the AMPK (A1/B1/G1) Kinase Enzyme System coupled with ADP-GLO assay kit as per manufacturer's protocol (Promega, USA).
  • CFS of DR9 was collected via separation of cell pellet from the spent broth.
  • Spent broth was separated into lipid fraction and evaluated for AMPK activation using human bone osteosarcoma cell line U2OS (ATCC).
  • U2OS was cultured in Dulbecco's Modified Essential Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/ml penicillin, 50 ⁇ g/ml streptomycin at 37°C and 5% CO 2 .
  • DMEM Dulbecco's Modified Essential Medium
  • FBS fetal bovine serum
  • 2mM L-glutamine 100 U/ml penicillin
  • 50 ⁇ g/ml streptomycin 50 ⁇ g/ml streptomycin at 37°C and 5% CO 2 .
  • Cell lines were induced for senescence using H 2 O 2.
  • Cell lines were serially passaged to reach 80% confluency, after which they were detached using trypsin-EDTA solution (0.1% trypsin in 0.02% ethylenediamine tetraacetic acid) and centrifuged at 200 ⁇ g for 5 mins. The cells were then resuspended in serum-free DMEM to a final concentration of 5 ⁇ 10 5 cells/ml. An aliquot of 100 ⁇ l cells was seeded onto 96-well plate and incubated for 24 hours at 37°C with 5% CO 2 . Upon attachment of the cells, medium was discarded and replaced with 100 ⁇ l of treatment medium to induce premature senescence.
  • trypsin-EDTA solution 0.1% trypsin in 0.02% ethylenediamine tetraacetic acid
  • Treatment medium was prepared with serum-free DMEM containing H 2 O 2 at 100 ⁇ M for 96 h.
  • the lipid fraction was incubated with the U2OS cells while they were exposed to 100 ⁇ M of H2O2 for 96 h.
  • the viability of the cells was analyzed with MTT assay.
  • AMPK phosphorylation of the cells was quantified via cell-based ELISA method using In-cell ELISA Colorimetric Detection Kit (Thermo Scientific, USA) as per manufacturer's instructions.
  • the lipid fraction was condensed, hydrolysed and methyl-esterified for a fatty acid methyl ester (FAME) analysis using a gas chromatography-mass spectrometry (GC-MS).
  • FAME fatty acid methyl ester
  • GC-MS gas chromatography-mass spectrometry
  • the hexane layer was pipetted into an autosampler vial for FAME quantification. Approximately 1 ⁇ l of the hexane layer was injected into an Agilent 5977A gas chromatograph system equipped with a 5977 MSD mass spectrometer (Agilent Technologies Australia Pty Ltd; GC/MS), for identifying FAME. A BPX-70 column was used for separation.
  • a standard chow diet (Altromin, Germany) was used as ND while a standard chow diet added with 25 % (w/w) animal fat (ghee, 99 % fat content) was used as HFD.
  • Treatments were mixed into 1 g of food pellet and fed daily. The rats were placed in individual cage during feeding of treatment to ensure complete ingestion. At the end of the 12 weeks treatment period, the rats were fasted for 12 h before sacrificed by inhalation of carbon dioxide. All tissues were excised immediately and rinsed in saline. The tissues were used for gene quantification via real time PCR.
  • T/S ratio for an experimental DNA sample is T (the number of nanograms of the Standard DNA that matches the experimental sample for copy number of the telomere template) divided by S (the number of nanograms of the standard DNA that matches the experimental sample for copy number of the albumin). Average T/S is expected to be proportional to the average telomere length per cell.
  • Treadmill exhaustion test was carried out according to Castro and Kuang (2017).
  • the rats were first acclimatized inside the non-moving running chamber for 5 min, with 0° inclination, followed by running for 5 min with speed of 10 m/min and 0° inclination.
  • On the 2nd and 3rd day of acclimatization the rats were allowed to run for 10 min at 10° uphill inclination.
  • a transient and light electric stimulation (0.4 mA) was applied on the metal grid at the behind the treadmill platform to stimulate the rats to run.
  • the belt speed was set at 10 m/min for 5 min and increased 2m/min for every 2 min up to 46 m/min.
  • RNAlater (Ambion, Austin, TX, USA) at -80°C.
  • Total RNA was extracted from homogenized samples using TRI Reagent® (Sigma-Aldrich, Saint Louis, MO, USA) and first-strand cDNA was synthesized using ReverTra Ace- ⁇ -® kit (Toyobo, Kita-ku, Osaka, Japan).
  • the mRNA expression levels were determined with Agilent AriaMx Realtime PCR System (Agilent Technologies, Santa Clara, CA, USA).
  • the PCR reaction consisted of SensiFAST SYBR® mix (Bioline, Cricklewood, London, UK), 20 ng of cDNA and 10 ⁇ M of primers.
  • Gapdh Glyceraldehyde-3-phosphate dehydrogenase
  • Frozen tissue samples were disrupted or pulverized under liquid nitrogen using a cold stainless steel mortar and pestle.
  • the powdered samples were suspended in RIPA lysis buffer (Merck, USA) supplemented with protease and phosphatase inhibitors cocktail (Promega, USA) and further homogenized on ice using ultrasonic homogenizers (Labsonic, Sartorius, Germany).
  • the samples were subjected to centrifugation at 10,000 ⁇ g for 10 min at 4°C, and the supernatants were collected as protein lysates, separated into aliquots and stored at -80°C until subsequent analysis.
  • the inflammatory cytokines were assayed using MILLIPLEX ® MAP Rat Cytokine/Chemokine magnetic bead panel kit (Merck, USA) according to the manufacturer's instruction with overnight incubation at 4°C (18 h) under constant shaking. Washing of the plate was aided with a hand-held magnetic block. Standards for TNF- ⁇ , IFN- ⁇ and IL-1 ⁇ were provided by the manufacturer, each was run in duplicate. Signals were measured using a calibrated Luminex ® xMAP platform (Luminex, USA) and data was normalized to the protein concentration of the samples using Bradford method according to the manufacturer's protocol (Sigma Aldrich, USA).
  • Tensile strength testing machine (Shimazu, Japan) was used to measure the skin elasticity. A 0.025 probe was used to press the skin sample until it breaks. A graph was generated and the elasticity strength was calculated from the area of graph.
  • TC total cholesterol
  • TG triglyceride
  • LDL Low-density lipoprotein
  • HDL High-density lipoprotein
  • TC total cholesterol
  • TG triglyceride
  • LDL Low-density lipoprotein
  • HDL High-density lipoprotein
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • renal function sodium, urea, chloride, potassium, creatinine, uric acid, calcium and phosphate
  • Detection of water-soluble metabolites was conducted according to Tsugawa et al. 2011, where samples were derivatized prior to gas chromatography-mass spectrophotometry (GCMS) analysis. Extraction of short-chain fatty acids (SCFAs) in fecal samples was performed as previously described by Nakajima et al. 2017. Analysis was performed using gas chromatography-tandem mass spectrometry platforms on a GCMS-TQ8030 Triple Quadrupole Mass Spectrometer (Shimadzu, Kyoto, Japan).
  • GCMS gas chromatography-mass spectrophotometry
  • V1-V2 (27F-338R) region of the 16S rRNA gene was amplified using the isolated bacterial DNAs. PCR products were prepared as previously described (Kato T et al., 2014). 16S rRNA sequencing was performed using 454 GS Junior according to the manufacturer's instructions. The resulting 16S rRNA reads were analyzed using the QIIME pipeline.
  • OFT was performed using an opaque rectangular box (32 cm H ⁇ 38 cm W ⁇ 52 cm L) with marked grids. Each animal was gently transferred from its cage into the box and allowed to freely explore for five mins for three consecutive days. The number of entries into the central and outer zones was recorded. The performance and trajectory of each rat was recorded by a camera (GoPro, California, U.S.A).
  • Normal feed was prepared by boiling 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% (w/v) nipagin and 0.7% (v/v) propionic acid with constant mixing, before being aseptically transferred into plastic vials to be cooled and solidified. All feed was prepared in the absence of nipagin and propionic acid. Lactobacillus fermentum DR9 (100 ⁇ L) were added at 1 x 10 11 CFU/mL to the cooled feed prior to solidification and left to solidify in a laminar flow hood.
  • Fresh feed with DR9 were fed to designated Drosophila lines within 2 hours of solidification.
  • 5-10 virgin Drosophila (Gal4 or UAS line) and 3-5 male Drosophila of the corresponding parent line were placed into plastic vials containing food. Wild type Control crosses were done using virgin females of Oregon-R line mated with males of UAS-A ⁇ 42 while all GMR-A ⁇ 42 crosses were done using virgin females of UAS-A ⁇ 42 mated with male GMR-GAL4. Control and transgenic GMRA ⁇ 42. nf Drosophila lines were reared in normal feed without DR9. DR9 treated group employed transgenic GMR-A ⁇ 42 Drosophila . Parent Drosophila was removed after 4-5 days after mating. F1 progenies were collected 10-days post mating from each group for subsequent analyses.
  • HMDS hexamethyldisilazane
  • EXAMPLE 1 Lactobacillus fermentum DR9 slows down ageing signs via activation of AMPK, prevention of telomere shortening, lowering of lipid peroxidation and enhancing physical endurance.
  • DR9 exhibited higher AMPK activation as compared to positive control, the result is as seen in Figure 1A.
  • DR9 shows high AMPK activation in presence of Dorsormophin, an AMPK inhibitor, where the result is as seen in Figure 1B.
  • the AMPK phosphorylation by the cell-free supernatant of DR9 was further tested on seven different cell lines as seen in Figure 2A and 2B.
  • the cell-free supernatant of DR9 has effectively promoting AMPK phosphorylation in U2OS cells, with AMPK activity higher than the untreated and positive control respectively.
  • the cell-free supernatant DR9 show insignificant AMPK phosphorylation when tested on SH-SY5Y, OVCAR3 and DU145 cells.
  • telomere length of aged rats was significantly shortened as compared to the young rats, and the result is as seen in Figure 3A (A).
  • Lactobacillus treatment on the senescent-induced rat showed an improvement in telomere length, with the highest from the groups receiving DR9, as compared to the aged rats.
  • Metformin also prevents the shortening of telomere length significantly as compared to the aged rats.
  • Serum lipid peroxidation was determined by measurement of malondialdehyde (MDA) concentration.
  • MDA malondialdehyde
  • the serum MDA level was significantly higher (P ⁇ 0.05) in aged group as compared to young group.
  • aged-DR9 group had a significantly lower (P ⁇ 0.05) concentration of MDA in the serum as seen in Figure 3A (B).
  • Running distance was not affected by all treatments but the running capacity, such as time, speed, work and power are greatly affected upon senescence induction.
  • the running time, speed, work and power were significantly lower in aged rats as compared to the young rats.
  • DR9 was able to ameliorate the adverse effects of aging on exercise capacity, with improved performance in running time, speed, work and power as compared to the aged rats.
  • AMPK adenosine monophosphate-activated protein kinase
  • CFS cell-free supernatant
  • DR9 Lactobacillus fermentum DR9
  • DR9 10 log CFU/rat/day
  • DR9 increased the gene expression of AMPK ⁇ 1 in the liver indicating activation as compared to the untreated control.
  • DR9 lowered blood lipid peroxidation as compared to the untreated control indicating reduced oxidative stress.
  • rats fed with DR9 showed longer running time, higher speed, higher work and power as the untreated control, indicating better physical endurance.
  • DR9 lowered gene expression of p53, a known senescent biomarker, in gastrocnemius muscle and tibia bone compared to the control.
  • p53 a known senescent biomarker
  • EXAMPLE 2 Lactobacillus fermentum DR9 prevents and treats muscle and bone degeneration.
  • SOD superoxide dismutase
  • AMPK ⁇ 2 plays a key role in the regulation of exercise capacity.
  • IGF-1 affects differentiation of myotubes in skeletal muscles, leading to impaired size and function of the muscles.
  • L. fermentum DR9 significantly lowered the MyoD level compared to the aged rats (P ⁇ 0.05) as seen in Figure 4B (C).
  • TNF- ⁇ and IL-1 ⁇ act as signal molecules in the osteoclast differentiation and TRAP is osteoclast marker.
  • Results showed that TNF- ⁇ expression was highly elevated in aged rats, with 4.4- fold higher than its young counterparts (P ⁇ 0.001).
  • IL-1 ⁇ interleukin 1-beta
  • IFN- ⁇ interferon gamma
  • the young rats demonstrated neatly arranged and normal bone trabeculae as seen in Figure 6a as compared to the aged rats which were arranged sparsely with thinner network as seen in Figure 6b.
  • Aged rats treated with L. fermentum DR9 as seen in Figure 6c showed a neat network of trabeculae bone, bulkier and plumper bone and lesser marrow adiposity as compared to the aged rats.
  • the density of physis (growth plate) was also higher in young rats as seen in Figure 6a as compared to the aged control as seen in Figure 6b.
  • Rats with L. fermentum DR9 as in Figure 6c showed significant improvements in the growth plate.
  • DR9 The anti-aging mechanism of DR9 was evaluated on gastrocnemius muscle and tibia of D-galactose induced aging rats.
  • DR9 demonstrated improved expression of SOD in gastrocnemius muscle and tibia bone and expression of AMPK ⁇ 2 in gastrocnemius muscle compared to the untreated aged rats.
  • DR9 also increased the mRNA expression of IGF-1 while reducing the expression of MyoD, in contrast to the aged controls.
  • DR9 also reduced the expression of IL-6, TNF- ⁇ and TRAP in tibia, and IL-1 ⁇ tibia and IFN- ⁇ in gastrocnemius when compared to the aged rats.
  • Gastrocnemius muscles of DR9 rats had higher number of myonuclei (nucleus of muscle cells) accompanied by denser arrangement of myofibers as compared to untreated aged rats.
  • Tibia of DR9 rats also showed more neatly arranged and normal bone trabeculae accompanied by lesser marrow adiposity as compared to the untreated aged rats which were arranged sparsely with thinner network.
  • the study illustrated the potentials of DR9 strain in alleviation of muscle and bone impairment.
  • Lactobacillus fermentum DR9 modulates gut against imbalanced gut bacterial populations and metabolites concentrations.
  • Lactobacillus fermentum DR9 was able to restore changes of phylum in aging rats to resemble the young rats. Phyla Firmicutes and Bacteroidetes comprising near to 90% of the gut microbiota in the gut. The F/B ratio was observed significantly reduced in the natural aging process of both human and animals. However, Lactobacillus fermentum DR9 is able to restore ratio Firmicutes/Bacteroidetes in aging rats similar to the young rats as seen in Figure 7B.
  • Organisms in the Bacteroides genus were documented as opportunistic pathogens and the most commonly identified species was B. fragilis which cause abscess formation.
  • L. fermentum DR9 group has higher abundance of bacteria in the genus of Blautia and Lactobacillus as seen in Figure 7C.
  • Blautia is known to degrade complex polysaccharides in the colon, thus plays a major role in nutrient assimilation and probiotics such as Lactobacillus were well documented to encourage a Blautia -enriched community in gut.
  • L. fermentum DR9 is able to restore gut SCFA metabolites in old rats that resemble young rats as seen in Figure 8A (A). Distinct clusters were observed, where young rats and aged rats treated with metformin were grouped closely; and aged rats treated with L. fermentum DR9 in one cluster as seen in Figure 8A (B). Old rats treated with L. fermentum DR9 have gut water soluble metabolites which resemble young rats.
  • glutathione is an antioxidant that removes reactive oxygen species due to oxidative stress.
  • L. fermentum DR9 alleviated pro-inflammatory cytokines TNF- ⁇ and IFN- ⁇ in the gut as seen in Figure 8B (D). Probiotics were well documented to exhibit immune-modulating properties both in-vitro and in-vivo.
  • Aged-HFD-DR9 group had a significantly higher abundance of Blautia which is a beneficial microorganism (P ⁇ 0.05) compared to aged-HFD group as seen in Figure 9A.
  • the genus Blautia in aged-HFD group was approximately 16% and significantly increased to approximately 30% upon treatment with L. fermentum DR9 as seen in Figure 9B.
  • Blautia is known as butyrate producer, and it has been reported that there is a positive correlation between Blautia and butyrate concentration in the fecal sample which can be seen in Figure 10A a.
  • High Fat Diet reduced the fecal acetate and propionate concentrations as seen in Figures 10B.
  • fecal acetate and propionate concentrations were significantly higher (P ⁇ 0.05) in aged-HFD with DR9 compared to aged-HFD group.
  • Propionate and acetate are able to alleviate gut diseases by reducing plasma and hepatic fatty acid content, lower cholesterol, and potentially improve insulin sensitivity.
  • DR9 also reduced cytokines levels of interferon gamma (IFN- ⁇ ) and tumour necrosis factor alpha (TNF- ⁇ ) in rats distal colon as compared to the untreated old rats.
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumour necrosis factor alpha
  • HFD high fat diet
  • male Sprague-Dawley rats were fed with a HFD and injected with D-galactose for 12 weeks to induce ageing.
  • the administration DR9 increased gut concentration of Blautia as compared to the HFD-age control.
  • Lactobacillus fermentum DR9 enhances lipid metabolisms and alleviates NAFLD.
  • SCD1 Stearoyl-CoA desaturase 1
  • ABCG5 is part of ABC transporters that function as heterodimers (ABCG5/G8) to increase sterol excretion from the liver.
  • High fat diet significantly reduced the mRNA expression of ABCG5 (P ⁇ 0.05) whereas expression of ABCG5 mRNA expression was significantly upregulated in HFD- DR9 (P ⁇ 0.05) as seen in Figure 11A (C).
  • Aged rats fed with DR9 showed reduced serum triglycerides, cholesterol and LDL-cholesterol levels, as compared to normal aged rats as seen in Figure 11B (D). DR9 did not exert any detrimental effects on other serum biochemical parameters studied.
  • AMPK is an energy sensor that plays an important role in sustaining cellular energy levels.
  • AMPK ⁇ 1 mRNA expression were significantly lower in HFD group as compared to ND group.
  • HFD-DR9 showed significantly higher expression of AMPK ⁇ 1 as compared to HFD group as seen in Figure 12A (A).
  • IL-4 and IL-10 are anti-inflammatory cytokines. Results showed that IL-4 and IL-10 were significantly increased in HFD group as compared to ND group. The administration of HFD-DR9 led to a lower (P ⁇ 0.05) IL-4 and IL-10 levels as seen in Figure 12A (B). These indicated that liver cells damages and inflammation were prevented by DR9, leading to a reduced need of IL-4 and IL-10.
  • HFD-DR9 Higher degree of lipid accumulation and less organized structure was observed in the HFD group when compared to the ND group.
  • HFD-DR9 group showed lesser lipid accumulation as compared to HFD group.
  • DR9 exerted positive effects ranging from hepatic gene expressions to liver histology as compared to the control; down-regulation of hepatic lipid synthesis and ⁇ -oxidation gene SCD1, up-regulation of hepatic IL-6 and sterol excretion gene of ABCG5 as compared to the HFD-aged control.
  • Administration of DR9 reduced serum triglycerides, total-cholesterol and LDL-cholesterol levels after 12-weeks as compared to the HFD-aged control.
  • DR9 also up-regulated hepatic energy metabolisms gene AMPK ⁇ 1 and increased liver concentrations of anti-inflammatory cytokines IL-4 and IL-10 as compared to the HFD-aged control, leading to lesser degree of liver steatosis.
  • this study illustrates that DR9 strain led to improved lipid profiles via activation of energy and lipid metabolisms, suggesting it as a promising natural intervention for alleviation of cardiovascular and liver diseases such as NAFLD.
  • Lactobacillus fermentum DR9 prevents and treats skin degeneration, senescence and oxidative stress.
  • Ageing process causes loss of skin elasticity.
  • the skin elasticity after 12 weeks of treatment was lower in old group compared to young group. It was observed that the old rats that were administered with LF-DR9 had a significantly higher skin elasticity as compared to non-treated old group as seen in Figure 13A (A).
  • Cyclin-D1 is one of senescence biomarkers.
  • the relative gene expression of cyclin D1 after 12 weeks of treatment was significantly higher in old group compared to young group. It was observed that the old rats that were administered with LF-DR9 had a significantly lower expression of cyclin-D1 as compared to non-treated old group as seen in Figure 13A (B).
  • Apoptosis is needed to remove inflammatory cells and granulation tissues, to promote the development of new skin cells during the regeneration cycle. Aging of skin is attributed to reduced capability of skin cells to regenerate.
  • DR9 upregulated the expression of FAS, an apoptosis biomarker in young rat skin as compared to old rat skin. The relative gene expression of FAS after 12 weeks was significantly lower in old group compared to young group, indicating reduced skin regeneration. It was observed that the old rats that were administered with LF-DR9 had a significantly higher expression of FAS as compared to non-treated old group as seen in Figure 13B (C).
  • the antioxidative gene, superoxide dismutase (SOD) is involved in the first line defense antioxidants to overcome oxidative stress while GPX is a biomarker of oxidative stress.
  • Old rats showed significant high of GPX expression when compared to the young rats.
  • Old rats treated with L. fermentum DR9 showed significantly lower GPX level compared to the non-treated old group as seen in Figure 13B (D).
  • Old rats showed low SOD expression when compared to the young rats.
  • Old rats treated with L. fermentum DR9 showed significantly higher SOD level compared to the non-treated old group as seen in Figure 13B (D), indicating reduced oxidative stress.
  • DR9 Male Sprague-Dawley rats were aged via injection with D-galactose daily for 12 weeks. The skin elasticity after 12 weeks of treatment was lower in old group compared to young group. Administration of DR9 prevented the loss of skin elasticity as compared to the non-treated old group. Cyclin-D1, a senescence biomarker was higher in the skin of old group as compared to young group. Rats administered with DR9 had a lower expression of skin cyclin-D1 as compared to non-treated old group. The administration of DR9 also upregulated the expression of skin FAS, an apoptosis biomarker as compared to old rat to facilitate with skin regeneration cycle.
  • Old rats showed higher expression of GPX, a biomarker of oxidative stress and lower expression of SOD, an antioxidative gene, while administration of DR9 reduced and increased such expressions, respectively.
  • GPX a biomarker of oxidative stress and lower expression of SOD
  • SOD an antioxidative gene
  • Lactobacillus fermentum DR9 prevents and treats anxiety and Alzheimer's Disease (AD).
  • the open field test is a validated assessment tool to evaluate anxiety in rodents. Anxiety is shown by prevalence of animals to stay in the outer corners and zone of the open field, rather than staying in the center of the open field.
  • the administration of DR9 reduced the number of entries of rats to the outer zone as compared to the untreated aged control (P ⁇ 0.05) as seen in Figure 14A (A).
  • the eyes of fruit flies, Drosophila melanogaster comprise of well-formed hexagonal ommatidia and straight bristles (hair-like structures) as seen in Figure 14A (B),(C).
  • Alzheimer's Disease mutants of Drosophila melanogaster develop a rough eye phenotype with presence of holes, malformation of ommatidia that losses hexagonal features, and broken bristle as seen in Figure 14B (D),(E).
  • the administration of DR9 prevented such a malformation, as shown by more well-formed shape of ommatidia and less damaged bristles as seen in Figure 14B (F), (G) indicating less severity of Alzheimer's Disease.
  • Lactobacillus fermentum DR9 exerts its health benefits via the production of metabolites succinic acid, benzaldehyde and benzoic acid.
  • Cell free supernatant of DR9 was fractionated into intracellular extracts (IE) and cell wall debris (CW).
  • IE intracellular extracts
  • CW cell wall debris
  • the lipid fraction of CFS was evaluated for AMPK-activation potentials using U2OS cell culture.
  • the lipid fraction activated AMPK in the U2OS cells, in a similar manner as AICAR, the known activator of AMPK, and better than Dorsomorphin C, which is a known inhibitor of AMPK as seen in Figure 15A (B).
  • the lipid fraction was subsequently characterized.
  • benzoic acid is reported to decrease chloride and increase magnesium in bones, and then increases bone weight upon administration followed by increasing retention of calcium and phosphorus in the bone.
  • benzaldehyde reportedly provided an anti-tumour and anti-viral properties to the host.
  • 5-oxoproline plays important roles in intracellular transport of free amino acids during metabolic processes, and has been found lowered in blood of human with type-2 diabetes.
  • AMPK is dysregulated in animals and humans with type-II diabetes, and that AMPK activation improves metabolic health and its activation has been used to treat type-II diabetes. This shows that DR9 enhanced production of 5-oxyproline in the gut, which induced AMPK activation physiologically.
  • Ascorbic acid is a commonly recognized antioxidant.
  • AMPK is one of the upstream proteins involved in antioxidant enzymes activities.
  • Lactobacillus fermentum DR9 exerts its health benefits via the production of metabolites namely succinic acid, benzaldehyde and benzoic acid.
  • DR9 and/or its metabolites induced production of 5-oxyproline and ascorbic acid the gut.
  • the study illustrated the potentials of DR9 strain and its metabolites in preventing and treating aging-induced bone, muscle & metabolic deterioration, and enhancing AMPK activation.
  • EXAMPLE 8 Compositions with Lactobacillus fermentum DR9

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Molecular Biology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Dermatology (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Polymers & Plastics (AREA)
  • Psychiatry (AREA)
PCT/KR2019/013279 2018-10-19 2019-10-10 Probiotic strain for ageing, muscle and bone, gut, hyperlipidemia, skin and brain WO2020080736A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1020217011441A KR20210062660A (ko) 2018-10-19 2019-10-10 노화, 근육, 뼈, 장, 고지혈증, 피부 및 뇌를 위한 프로바이오틱 균주
CN201980069020.5A CN112955539A (zh) 2018-10-19 2019-10-10 针对衰老、肌肉和骨骼、肠道、高脂血症、皮肤和大脑的益生菌菌株

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MYPI2018703875 2018-10-19
MYPI2018703875 2018-10-19

Publications (1)

Publication Number Publication Date
WO2020080736A1 true WO2020080736A1 (en) 2020-04-23

Family

ID=70282983

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/013279 WO2020080736A1 (en) 2018-10-19 2019-10-10 Probiotic strain for ageing, muscle and bone, gut, hyperlipidemia, skin and brain

Country Status (3)

Country Link
KR (1) KR20210062660A (zh)
CN (2) CN111067922A (zh)
WO (1) WO2020080736A1 (zh)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111718873A (zh) * 2020-06-19 2020-09-29 江南大学 一株具有缓解骨质疏松功效的发酵乳杆菌及其应用
WO2022045513A1 (ko) * 2020-08-25 2022-03-03 고려대학교 산학협력단 락토바실러스 퍼멘텀을 포함하는 갱년기 예방 또는 치료용 조성물
CN114591861A (zh) * 2022-03-22 2022-06-07 华南农业大学 一株发酵乳杆菌wc2020及其应用
CN114634899A (zh) * 2022-05-17 2022-06-17 山东锦鲤生物工程有限公司 发酵乳杆菌及其应用
NL2035174A (en) * 2022-11-23 2023-09-21 Chongqing Tianyou Dairy Co Ltd Lactobacillus fermentum and applications of lactobacillus fermentum in preparation for improving hyperlipidemia

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115181695B (zh) * 2022-06-27 2023-08-29 广东粤港澳大湾区国家纳米科技创新研究院 一种植物乳杆菌5b4m2及其应用
CN117821282A (zh) * 2023-07-14 2024-04-05 浙江大学 约氏乳杆菌ljsd001及其在延缓衰老中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110014166A1 (en) * 2009-07-17 2011-01-20 Korea Research Institute Of Bioscience And Biotechnology Probiotics Spore-forming Lactic Acid Bacteria SL153
WO2011068398A1 (en) * 2009-12-01 2011-06-09 Universiti Putra Malaysia Probiotic composition obtained from lactobacillus plantarum strains ul4, tl1, rs5, r111, rg14 or combinations thereof
WO2015013334A1 (en) * 2013-07-23 2015-01-29 Myriant Corporation Method of producing succinic acid and other chemicals using facilitated diffusion for sugar import
WO2016201380A1 (en) * 2015-06-10 2016-12-15 Synlogic, Inc. Bacteria engineered to treat disorders involving the catabolism of a branched chain amino acid
WO2018002248A1 (en) * 2016-06-30 2018-01-04 Yun NV Preservation of microorganisms

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150174218A1 (en) * 2006-04-28 2015-06-25 Naidu Lp ANGIOGENIN COMPLEXES (ANGex) WITH ANABOLIC HORMONES FOR IMPROVING BONE HEALTH

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110014166A1 (en) * 2009-07-17 2011-01-20 Korea Research Institute Of Bioscience And Biotechnology Probiotics Spore-forming Lactic Acid Bacteria SL153
WO2011068398A1 (en) * 2009-12-01 2011-06-09 Universiti Putra Malaysia Probiotic composition obtained from lactobacillus plantarum strains ul4, tl1, rs5, r111, rg14 or combinations thereof
WO2015013334A1 (en) * 2013-07-23 2015-01-29 Myriant Corporation Method of producing succinic acid and other chemicals using facilitated diffusion for sugar import
WO2016201380A1 (en) * 2015-06-10 2016-12-15 Synlogic, Inc. Bacteria engineered to treat disorders involving the catabolism of a branched chain amino acid
WO2018002248A1 (en) * 2016-06-30 2018-01-04 Yun NV Preservation of microorganisms

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111718873A (zh) * 2020-06-19 2020-09-29 江南大学 一株具有缓解骨质疏松功效的发酵乳杆菌及其应用
CN111718873B (zh) * 2020-06-19 2022-09-06 江南大学 一株具有缓解骨质疏松功效的发酵乳杆菌及其应用
WO2022045513A1 (ko) * 2020-08-25 2022-03-03 고려대학교 산학협력단 락토바실러스 퍼멘텀을 포함하는 갱년기 예방 또는 치료용 조성물
CN114591861A (zh) * 2022-03-22 2022-06-07 华南农业大学 一株发酵乳杆菌wc2020及其应用
CN114634899A (zh) * 2022-05-17 2022-06-17 山东锦鲤生物工程有限公司 发酵乳杆菌及其应用
CN114634899B (zh) * 2022-05-17 2022-08-16 山东锦鲤生物工程有限公司 发酵乳杆菌及其应用
NL2035174A (en) * 2022-11-23 2023-09-21 Chongqing Tianyou Dairy Co Ltd Lactobacillus fermentum and applications of lactobacillus fermentum in preparation for improving hyperlipidemia

Also Published As

Publication number Publication date
KR20210062660A (ko) 2021-05-31
CN112955539A (zh) 2021-06-11
CN111067922A (zh) 2020-04-28

Similar Documents

Publication Publication Date Title
WO2020080736A1 (en) Probiotic strain for ageing, muscle and bone, gut, hyperlipidemia, skin and brain
WO2020050553A1 (en) Probiotic strain for upper respiratory tract infection, stress, anxiety, memory and cognitive dysfunctions, and ageing
AU2020202144B2 (en) Novel lactobacillus having various functions, and use thereof
Li et al. Vitexin alleviates high-fat diet induced brain oxidative stress and inflammation via anti-oxidant, anti-inflammatory and gut microbiota modulating properties
WO2019151843A1 (ko) 락토바실러스 플란타럼 kbl396 균주 및 그 용도
Tang et al. A synbiotic consisting of Lactobacillus plantarum S58 and hull-less barley β-glucan ameliorates lipid accumulation in mice fed with a high-fat diet by activating AMPK signaling and modulating the gut microbiota
WO2019226003A1 (ko) 락토바실러스 가세리 kbl697 균주 및 그 용도
WO2011010770A1 (ko) 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물
WO2019226002A1 (ko) 락토바실러스 크리스파투스 kbl693 균주 및 그 용도
Yi et al. Effect of LTA isolated from bifidobacteria on D-galactose-induced aging
WO2017204374A1 (ko) 혈당 조절 효능을 갖는 신규 유산균 및 이의 용도
WO2011136573A2 (ko) 비만 치료 및 항산화 활성을 갖는 효모 가수분해물
WO2021066549A1 (ko) 락토바실러스 애시도필러스 kbl409 균주 및 그 용도
Kim et al. Sodium butyrate ameliorates neurotoxicity and exerts anti-inflammatory effects in high fat diet-fed mice
WO2020218781A1 (ko) 불멸화 줄기세포 유래 엑소좀 풍부 배양액 및 장미꽃봉오리 추출물을 유효성분으로 포함하는 기능성 조성물
WO2023055188A1 (ko) 신규 프로바이오틱스 및 이의 용도
Kovačević et al. Fructose induces visceral adipose tissue inflammation and insulin resistance even without development of obesity in adult female but not in male rats
Li et al. Oligosaccharides isolated from Rehmannia glutinosa protect LPS-induced intestinal inflammation and barrier injury in mice
Li et al. Role of polyphenols from Polygonum multiflorum Caulis in obesity-related disorders
Sivamaruthi et al. A comprehensive review on functional properties of fermented rice bran
Long et al. Gut microbiota in ischemic stroke: Where we stand and challenges ahead
WO2023058801A1 (ko) 락토바실러스 애시도필러스 kbl402 또는 kbl409 균주를 포함하는 장 질환 개선, 예방 또는 치료용 조성물
WO2014065640A2 (ko) 외톨개모자반 추출물 또는 이의 분획물을 유효성분으로 함유하는 염증성 질환 치료 또는 예방용 조성물
Zhang et al. Study of the in vitro properties of oligopeptides from whey protein isolate with high Fisher's ratio and their ability to prevent allergic response to β-lactoglobulin in vivo
Ewe et al. Fermented soymilk as a nutraceutical

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19873873

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 20217011441

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19873873

Country of ref document: EP

Kind code of ref document: A1