WO2020050553A1 - Souche probiotique pour une infection des voies respiratoires supérieures, le stress, l'anxiété, les troubles de la mémoire et cognitifs et le vieillissement - Google Patents

Souche probiotique pour une infection des voies respiratoires supérieures, le stress, l'anxiété, les troubles de la mémoire et cognitifs et le vieillissement Download PDF

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WO2020050553A1
WO2020050553A1 PCT/KR2019/011130 KR2019011130W WO2020050553A1 WO 2020050553 A1 WO2020050553 A1 WO 2020050553A1 KR 2019011130 W KR2019011130 W KR 2019011130W WO 2020050553 A1 WO2020050553 A1 WO 2020050553A1
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strain
metabolite
ageing
hfd
treatment
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Yong-Ha Park
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Clinical Nutrition Intl (M) Sdn Bhd
Lii Run Sdn Bhd
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Priority to KR1020217006268A priority Critical patent/KR20210042334A/ko
Priority to CN201980056940.3A priority patent/CN112930392B/zh
Publication of WO2020050553A1 publication Critical patent/WO2020050553A1/fr

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    • A—HUMAN NECESSITIES
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    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
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    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
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    • A61P3/00—Drugs for disorders of the metabolism
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Definitions

  • the present invention relates to the fields of medicine, microbiology and nutrition and, particularly, to the probiotic bacterial strain Lactobacillus plantarum DR7. It has several biofunctionalities beneficial for health, especially for the prevention and treatment of upper respiratory tract infection, stress, anxiety, memory and cognitive dysfunctions, and slowing down ageing signs.
  • Lactobacillus plantarum DR7 alleviated stress and anxiety while improving memory and cognition in stressed adults via the serotonin and dopamine pathways was published on March 18, 2019 (18.03.2019), thus, later to the priority date set forth by the MY priority application.
  • the content of this paper was incorporated in the MY priority application (so that content was not published at the date of filing of the priority application) and it is also included in the present application. Therefore, the effects of the Lactobacillus plantarum DR7 strain in the improvement of memory and cognition, and in the alleviation of stress and anxiety were not known (published) at the filing date of the MY priority application.
  • Lactobacillus plantarum DR7 improved upper respiratory tract infections via enhancing immune and inflammatory parameters was published on April 3, 2019 (03.04.2019), thus, later to the priority date set forth by the MY priority application.
  • the content of this paper was incorporated in the MY priority application (so that content was not published at the date of filing of the priority application) and it is also included in the present application. Therefore, the effects of Lactobacillus plantarum DR7 in improving upper respiratory tract infections and enhancing immune and inflammatory parameters were not known (published) at the filing date of the MY priority application.
  • 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.
  • URTI Upper respiratory tract infection
  • URTI is a general term encompassing any infection that affects the upper respiratory system, including the common cold, laryngitis, sinusitis, and pharyngitis. These infections represent one of the most common health concerns affecting global communities. In the United States, adults have 2 to 3 incidences of URTI each year and children up to 5 times annually; however, over 20% of adults experience an URTI every 4 weeks.
  • AD Alzheimer's disease
  • a ⁇ amyloid beta
  • Ageing is an age-dependent multifactorial process associated with physiological decline leading to an increase in age-specific mortality rate. While ageing is inevitable, current ageing researches aim to improve the quality of life amid increasing life expectancies.
  • Ageing processes affect the brain in many ways, ranging from cellular to functional levels which comprise declines in abilities affecting sensory, motor, memory and higher cognitive functions. Severe cognitive declines are associated with neurodegenerative diseases such as Alzheimer's disease and deterioration of memory. Meanwhile, ageing is also associated with metabolic diseases such as hyperlipidaemia that increases the risk of cardiovascular diseases (CVD). World Health Organization estimated that on a global level, CVD accounts for 31% of all deaths while cardiovascular complications are considered as the main factors of morbidity and mortality. Thus, natural and dietary strategies should be explored to benefit the ageing populations with hyperlipidaemia. Ageing also affect physical performance, in which we can measure in terms of endurance capacity. An organism may experience cellular and molecular changes during ageing, in which the age changes overall performance of an individual.
  • CVD cardiovascular diseases
  • the ability to perform muscular work deteriorates with age. This phenomenon is possibly due to a breakdown in the control mechanisms that is responsible for complex movement. Moreover, accumulation of cell-damageing reactive molecules produced via cellular respiration could also lead to ageing. These molecules can react and result in oxidation of proteins and other cellular molecules. Ageing is also a main factor in the onset of various brain degenerative disorders such as Alzheimer's disease.
  • 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 shortens with each cell division until it reaches a critical length and initiate cell senescence.
  • Ageism deteriorates physical and mental functioning and affects overall well being when the body is unable to function as well as compared to an early adulthood. For instance, hormone levels fall when the glands that produce specific hormones fail to function in optimum levels, and then affect organs' ability to carry out biochemical processes. In a more specific example, a woman loses her ability to reproduce when ovulation ceased and uterus and ovaries shrunk due to sharp fall in the levels of sex hormones during menopause. In addition, an aged population may suffer from hearing loss, vision difficulties, sleep disturbance, continence, and changes in body shape and size due to muscle loss and calcium loss. Deterioration in skin complexion, such as wrinkles, age spots and dryness, can also be arisen from factors like ageing and hormonal changes.
  • Biochemical processes and body metabolism in a living organism are highly affected by age factor.
  • the biochemical processes and metabolism can be regulated by adenosine monophosphate-activated protein kinase (AMPK).
  • 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 activities such as fatty acid oxidation and autophagy.
  • 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 and senescence.
  • the activation of AMPK via phosphorylation was found to play a role in reducing influenza viral infections via activation of macrophages, indicating the importance of immunomodulatory functions during viral attacks.
  • Hyperlipidemia can broadly be classified as isolated elevation of cholesterol, isolated elevated TG and elevations of both. Hyperlipidemia, which refers to increased levels of cholesterol and triglycerides in the blood, is among the known risk factors for the development of cardiovascular diseases. 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). Probiotics have been shown to improved blood lipid profile in animal studies.
  • Dysbiosis of the gut microbiota may occur due to diseases, diet and ageing. Both ageing and high-fat diet have been recognized as one of the main factors that alters the composition of gut microbiota.
  • the gut microbiota is a highly metabolically active community of microorganisms inhabiting all niches along the intestine and is now recognized as a critical regulator of its host homeostasis. It is hypothesized that modifying the gut microbial balance would induce a shift in the gut metabolic environment that can, in turn, affect our own metabolism. Recent research has led to increasing recognition of the association between gut microbiota and metabolites with host energy metabolism, development of metabolic diseases and immunity and bone health.
  • 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.
  • the solution is based on the provision of the probiotic bacterial strain Lactobacillus plantarum deposited under the accession number CGMCC 15535, also referred as Lactobacillus plantarum DR7.
  • 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 plantarum or a metabolite thereof, wherein the strain was deposited in the China General Microbiological Culture Collection Centre (CGMCC) under the accession number CGMCC 15535 and full genome sequence deposited in GenBank under accession number CP031318, and wherein the metabolite comprises 2-hydroxyisocapric acid and 3-phenyllactic acid.
  • CGMCC China General Microbiological Culture Collection Centre
  • An aspect of the invention relates to Lactobacillus plantarum DR7 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 plantarum DR7 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 plantarum DR7 or a metabolite thereof for use in the prevention and treatment of upper respiratory tract infection by exerting anti-inflammatory and immune modulatory protection.
  • a further aspect of the invention relates to the strain Lactobacillus plantarum DR7 or a metabolite thereof for use in the prevention and treatment of stress, anxiety, memory and cognitive dysfunctions by enhancing e.g. the serotonin and dopamine pathway.
  • Another aspect relates to the strain Lactobacillus plantarum DR7 or a metabolite thereof for use in the prevention and treatment of neurodegenerative diseases.
  • the invention relates to the strain Lactobacillus plantarum DR7 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 plantarum DR7 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 plantarum DR7 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 plantarum DR7 or a metabolite thereof for use as probiotic in gut modulation against imbalanced gut bacterial populations and metabolites concentrations.
  • Lactobacillus plantarum DR7 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 plantarum DR7, 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 2-hydroxyisocapric acid and 3-phenyllactic acid.
  • Lactobacillus plantarum DR7 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.
  • FRAP ferric reducing antioxidant potential
  • TAA thiobarbituric acid
  • FIG. 6. Behavioural assessment via (A) Morris water maze, (B) Open field test and (C) T-maze of rats after 12 weeks of treatment. Young (normal diet), Young HFD (young rats with high fat diet), Old (D-galactose induced ageing rats; 600mg/kg/day), Old HFD (D-galactose induced ageing rats with high fat diet), Old HFD DR7 (D-galactose induced ageing rats with high fat diet receiving L. plantarum DR7 log 10 CFU/day), Old HFD statin (D-galactose induced ageing rats with high fat diet receiving lovastatin 2mg/kg/day). Results are expressed as mean; error bars (SEM); n 6. Means of the same treatment group at different days are significantly different (P ⁇ 0.05).
  • FIG. 7 Effects of a 12 week administration of Lactobacillus plantarum DR7 (gray bars) or placebo (white bars), on changes in scores as compared to week 0 for (A) PSS-10, (B) Stress, (C) Anxiety, (D) Depression, (E) Total scores DASS-42 in young adults (aged ⁇ 30 years old), normal adults (aged >30 years old) and all subjects.
  • FIG. 9 Relative hippocampal neurotransmitters gene expression of (A) Gamma-aminobutyric acid alpha subunit (GABA A2), (B) Tyrosine Hydroxylase (TH), (C) Dopamine beta-hydroxylase (DBH), (D) Indoleamine dioxygenase (IDO), and (E) Tryptophan Hydroxylase 1 (TPH1) after 12 weeks of treatment. (F) Hippocampal pathway of tryptophan metabolism to kynurenine via the actions of IDO or to serotonin via the actions of TPH.
  • GABA A2 Gamma-aminobutyric acid alpha subunit
  • TH Tyrosine Hydroxylase
  • DH Dopamine beta-hydroxylase
  • IDO Indoleamine dioxygenase
  • TPH1 Tryptophan Hydroxylase 1
  • FIG. 10 Effects of a 12-week administration of Lactobacillus plantarum DR7 on blood mRNA relative gene expressions (normalized using placebo as a control) for dopamine ⁇ -hydroxylase (DBH), tyrosine hydroxylase (TH), tryptophan hydroxylase-1 (TPH1), tryptophan hydroxylase-2 (TPH2), 5-hydroxytryptamine receptor-6 (5-HT6), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), glutamic acid decarboxylase (GAD65), gamma aminobutyric acid A-receptor ⁇ -5 (GABRA5), brain derived neurotrophic factor (BDNF) and cAMP response element binding (CREB) in (A) young adults (aged ⁇ 30 years old), (B) normal adults (aged >30 years old) and (C) all subjects. P-values indicated difference between treatment groups at individual time points. Results are expressed as mean, error
  • FIGS. 11A and 11B show microscopic images of (A) an eye of a GMR-OreR produced by crossing a wild-type Drosophila melanogaster (Oregon-R) and Glass multimer reporter-GAL4 (GMR-Gal4), at a magnification of 250 ⁇ , and (B) well-formed hexagonal ommatidia and straight bristles at a magnification of 350 ⁇ .
  • GMR-A ⁇ 42 transgenic Drosophila melanogaster A ⁇ 42
  • GMR-GAL4 Glass multimer reporter-GAL4
  • FIG. 12 Hippocampal cytokines levels of (A) Interleukin 1-beta (IL-1b), (B) Interferon-gamma (IFN- ⁇ ), and (C) Tumour necrosis factor-alpha (TNF- ⁇ ) in hippocampus of rats after 12 weeks of treatment.
  • Young normal diet
  • Young HFD young rats with high fat diet
  • Old D-galactose induced ageing rats; 600mg/kg/day
  • Old HFD D-galactose induced ageing rats with high fat diet
  • Old HFD DR7 D-galactose induced ageing rats with high fat diet receiving L.
  • FIG. 13 Relative hippocampal apoptosis gene expression of (A) Tumor protein (P53), (B) Tumor protein 19 (P19), (C) B-cell lymphoma-extra-large (BCL-XL), (D) BCL2 Associated X (BAX), and (E) Caspase 9 (CAS-9) fter 12 weeks of treatment.
  • A Tumor protein
  • B Tumor protein 19
  • C B-cell lymphoma-extra-large
  • BAX BCL2 Associated X
  • E Caspase 9
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-10 interleukin-1 ⁇
  • IL-10 interleukin
  • FIG. 15. (A) Graphical illustration of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation activity in Dulbecco's Modified Essential Medium (DMEM), lipid fraction of a blank de Mann, Rogosa, Sharpe (MRS) broth, lipid fraction of Lactobacillus plantarum strain DR7 cell-free supernatant, protein fraction of a blank MRS broth, protein fraction of L. plantarum strain DR7 cell-free supernatant, polysaccharide fraction of a blank MRS broth, and polysaccharide fraction of L. plantarum strain DR7 cell-free supernatant.
  • AMPK 5' adenosine monophosphate-activated protein kinase
  • FIG. B Graphical illustration of relative phosphorylation of AMPK using a blank de Mann, Rogosa, Sharpe (MRS) broth, a commercial AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), Lactobacillus plantarum strain DR7 cell-free supernatant, L. plantarum strain DR7 cell-free supernatant added with a commercial AMPK inhibitor, Compound C, Compound C only and, lovastatin only.
  • AICAR 5-aminoimidazole-4-carboxamide ribonucleotide
  • FIG. 16 shows a chromatogram of (a) a blank de Mann, Rogosa, Sharpe (MRS) broth and (b) Lactobacillus plantarum strain DR7 cell-free supernatant, where intensity peaks of 2-hydroxyisocaproic acid and 3-phenyllactic-acid are labelled as HICA and PLA, respectively.
  • FIG. 17 Telomere length (T/S ratio) as measured from the rat's blood collected after 12 weeks of treatment.
  • Y young control
  • O Aged control via D-galactose induced ageing
  • O+S D-galactose induced ageing, receiving lovastatin 2 mg/kg/day
  • FIGS. 19A; 19B and 19C (A) Assimilation of cholesterol by different strains of lactobacilli (1%, v/v inoculum) in de Mann, Rogosa, Sharpe (MRS) broth (containing 0.3% (w/v) ox-bile and 60 ⁇ g/mL cholesterol) upon fermentation for 24 h at 37°C. Accumulation of cholesterol in (B) intestinal cells (HT-29) and (C) hepatic cells (HepG2) upon incubation for 24 h at 37°C, in the presence of cell free supernatant (CFS) from different strains of lactobacilli (50% v/v).
  • MRS hepatic cells
  • Serum-free Dulbecco's modified Eagle's medium (DMEM) used contained 1 mM of cholesterol and 0.03% of ox-bile.
  • Blank MRS sterile de Mann, Rogosa, Sharpe (MRS) broth at a final concentration of 50% (v/v) in serum-free DMEM; 30242.
  • HMGCR HMG-CoA reductase
  • Non-treated HepG2 cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) without any treatment; blank MRS: HepG2 cultured in sterile de Mann, Rogosa, Sharpe (MRS) broth at a final concentration of 50% (v/v) in serum-free DMEM.
  • F mRNA gene expression of HMG-CoA reductase in hepatic cell (HepG2) in the presence of different treatments for 3 h at 37°C under 5% CO2.
  • AICAR AMPK activator
  • Compound C AMPK inhibitor
  • Non-treated HepG2 cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) without any treatment; blank MRS: HepG2 cultured in sterile de Mann, Rogosa, Sharpe (MRS) broth at a final concentration of 30% (v/v) in serum-free DMEM; CFS-DR7, HepG2 cultured in CFS from L. plantarum DR7 at a final concentration of 30% (v/v) in serum-free DMEM.
  • G Relative gene expression of AMPK in hepatic cell (HepG2) upon incubation with different treatments for 3 h at 37°C under 5% CO2.
  • AICAR AMPK activator
  • Compound C AMPK inhibitor
  • Non-treated HepG2 cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) without any treatment
  • blank MRS HepG2 cultured in sterile de Mann, Rogosa, Sharpe (MRS) broth at a final concentration of 30% (v/v) in serum-free DMEM
  • CFS-DR7 HepG2 cultured in CFS from L. plantarum DR7 at a final concentration of 30% (v/v) in serum-free DMEM.
  • TG Serum triglyceride
  • 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
  • FIG. 20 Relative gene expression of (A) SCD1, (B) IL-6, (C) ABCG5, and (D) ABCG8 in liver 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
  • FIG. 21 Relative gene expression of (A) SR-B1, (B) LDL-R, (C) ABCA1, (D) Apo A1 in liver 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
  • FIG. 22 Relative gene expression of (A) AMPK ⁇ 1 (B) AMPK ⁇ 2 in liver 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
  • FIG. 23 Liver anti-inflammatory cytokine IL-4 level in liver 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
  • FIG. 24 Hematoxylin and eosin (H&E) staining of liver sections of D-galactose induced ageing rats (600mg/kg/day) after 12 weeks of treatment.
  • H&E Hematoxylin and eosin 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-DR7 high fat diet, receiving L. plantarum DR7 (10 10 CFU/day).
  • FIG. 25 The relative abundance of three major phyla; A) Actinobacteria, B) Bacteroidetes. C) Firmicutes, and D) ratio of Firmicutes over Bacteroidetes of rats fecal after 12 weeks of treatment.
  • FIG. 26 Analysis of absolute concentration of fecal acetate.
  • FIG. 27 Analysis of top 70 water-soluble metabolites in rat's fecal samples after 12 weeks of treatment; heatmap normalized to a relative value between 2 and - 2. Red color indicates higher concentration, and blue color indicates lower concentration.
  • Young-ND normal diet; young- HFD: high-fat diet; aged-ND: D-galactose-induced ageing with normal diet; aged-HFD: D-galactose-induced ageing with high-fat diet; aged-HFD-statin: D-galactose-induced ageing with high-fat diet, receiving lovastatin 2 mg/kg/day; aged-HFD DR7: D-galactose induced ageing with high-fat diet, receiving L. plantarum DR7 (10 10 CFU/day).
  • FIG. 28 Sparse partial least squares discriminant analysiss (sPLS-DA) plot of water soluble metabolites in rat's fecal samples after 12 weeks of treatment.
  • Young-ND normal diet
  • Young-HFD high fat diet
  • Aged-ND D-galactose induced ageing with normal diet
  • Aged-HFD D-galactose induced ageing with high fat diet
  • Aged-HFD-statin D-galactose induced ageing with high fat diet, receiving lovastatin 2 mg/kg/day
  • Aged-HFD-DR7 D-galactose induced ageing with high fat diet, receiving L. plantarum DR7 (10 10 CFU/day).
  • D-galactose (600mg/kg/day) was injected via subcutaneous injection.
  • Normal diet (ND) standard chow diet
  • High fat diet (HFD) standard chow diet with 25 % fat.
  • Lactobacillus plantarum DR7 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 plantarum 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.
  • CGMCC General Microbiological Culture Collection Centre
  • the deposited strain received the depository number CGMCC 15535 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.
  • 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 CP031318 genome squence 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 DR7 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 plantarum DR7 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 DR7 strain has, among others, the following effects and applications beneficial for health:
  • Lactobacillus plantarum DR7 improves upper respiratory tract infections via enhancing immune and inflammatory parameters, as shown by a randomized, double-blind, placebo-controlled study (see EXAMPLE 1 hereinafter). The aims of this study were to investigate the effects of Lactobacillus plantarum DR7 against upper respiratory tract infections (URTI) and elucidate the possible mechanisms underlying immunomodulatory properties.
  • the DR7 strain (9 log cfu/d) was administered for 12 wk in a randomized, double-blind, and placebo-controlled human study involving 109 adults. The administration of DR7 reduced the duration of nasal symptoms and the frequency of URTI after 12 and 4 wk, respectively, compared with the placebo.
  • the DR7 treatment suppressed plasma pro-inflammatory cytokines (IFN- ⁇ , TNF- ⁇ ) in middle-aged adults (30 to 60 yr old), while enhancing anti-inflammatory cytokines (IL-4, IL-10) in young adults ( ⁇ 30 yr old), accompanied by reduced plasma peroxidation and oxidative stress levels com-pared with the placebo.
  • Young adults who received DR7 showed higher expression of plasma CD44 and CD117 by 4.50- and 2.22-fold, respectively, compared with the placebo.
  • middle-aged adults showed lower expression of plasma CD4 and CD8 by 11.26- and 1.80-fold, respectively, compared with the placebo, indicating less T-cell activation.
  • both young and middle-aged adults who received DR7 showed enhanced presence of nonresting and mature NK cells compared with those who received the placebo.
  • DR7 alleviates the symptoms of URTI by improving inflammatory parameters and enhancing immunomodulatory properties.
  • an aspect of the invention relates to Lactobacillus plantarum DR7 or a metabolite thereof for use in the prevention and treatment of upper respiratory tract infection, by exerting anti-inflammatory and immune modulatory protection.
  • Lactobacillus plantarum DR7 alleviates stress and anxiety in adults as is demonstrated by a randomised, double-blind, placebo-controlled study (see EXAMPLE 2 hereinafter).
  • One hundred and eleven stressed adults were recruited based on moderate stress levels using the PSS-10 questionnaire.
  • the consumption of DR7 (1 ⁇ 10 9 cfu/day) for 12 weeks reduced symptoms of stress, anxiety, and total psychological scores as early as 8 weeks among stressed adults compared to the placebo group as assessed by the DASS-42 questionnaire.
  • Plasma cortisol level was reduced among DR7 subjects as compared to the placebo, accompanied by reduced plasma pro-inflammatory cytokines, such as interferon- ⁇ and transforming growth factor- ⁇ and increased plasma anti-inflammatory cytokines, such as interleukin 10.
  • DR7 better improved cognitive and memory functions in normal adults (>30 years old), such as basic attention, emotional cognition, and associate learning, as compared to the placebo and young adults.
  • the administration of DR7 enhanced the serotonin pathway, as observed by lowered expressions of plasma dopamine ⁇ -hydroxylase (DBH), tyrosine hydroxylase (TH), indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase accompanied by increased expressions of tryptophan hydroxylase-2 and 5-hydroxytryptamine receptor-6, while stabilising the dopamine pathway as observed via stabilised expressions of TH and DBH over 12 weeks as compared to the placebo.
  • DBH plasma dopamine ⁇ -hydroxylase
  • TH tyrosine hydroxylase
  • TH indoleamine 2,3-dioxygenase
  • tryptophan 2,3-dioxygenase accompanied by increased expressions of tryptophan hydroxylase-2 and 5-hydroxytry
  • an aspect of the invention relates to Lactobacillus plantarum DR7 or a metabolite thereof for use in the prevention and treatment of stress, anxiety, memory and cognitive dysfunctions.
  • Lactobacillus plantarum DR7 improves brain health enhancing the neurotransmitter serotonin and dopamine pathways against Alzheimer's Disease, and hippocampal inflammation and apoptosis (see EXAMPLE 3 hereinafter).
  • a study conducted in rats demonstrated that the administration of DR7 reduced anxiety accompanied by enhanced memory during behavioural assessments in aged-HFD rats. Hippocampal concentrations of three pro-inflammatory cytokines were increased during ageing but reduced upon administration of both statin and DR7.
  • Lactobacillus plantarum DR7 strain improves the the pathophysiology of a Drosophila melanogaster Alzheimer's disease (AD) model and alleviated neurodegeneration in the eye (see EXAMPLE 3 hereinafter). It was also showed the potential AD-reversal effects of DR7 strain through feeding to Drosophila melanogaster AD model. The administration of probiotic strains was able to rescue the rough eye phenotype (REP) seen in AD-induced Drosophila , with a more prominent effect observed upon the administration of Lactobacillus plantarum DR7.
  • AD Drosophila melanogaster Alzheimer's disease
  • REP rough eye phenotype
  • an aspect of the invention relates to Lactobacillus plantarum DR7 or a metabolite thereof for use in the prevention and treatment of neurodegenerative diseases.
  • the neurodegenerative disease is Alzheimer's Disease, wherein Lactobacillus plantarum DR7 enhances the neurotransmitter serotonin and dopamine pathways against Alzheimer's Disease, and hippocampal inflammation and apoptosis.
  • Lactobacillus plantarum DR7 prevents telomere shortening and enhances energy metabolism against ageing (see EXAMPLE 5 hereinafter).
  • Male Sprague-Dawley rats were fed with high fat diet (54% kcal fat) and injected with D-galactose daily for 12 weeks to induce ageing.
  • the effects of DR7 strain on age-related impairment such as telomere length, plasma lipid peroxidation, hepatic AMPK expression, as well as muscle performance were evaluated.
  • Administration of statin, and Lactobacillus plantarum DR7 (LP-DR7) significantly reduced the shortening of telomere and increased the expression of AMPK subunit- ⁇ 2.
  • AMPK subunit- ⁇ 1 was elevated in rats administered with LP-DR7 as compared to the control while statin did not exert such an effect.
  • LP-DR7 LP-DR7
  • statin did not exert such an effect.
  • AMPK subunit- ⁇ 1 was elevated in rats administered with LP-DR7 as compared to the control while statin did not exert such an effect.
  • an aspect of the invention relates to Lactobacillus plantarum DR7 or a metabolite thereof for use in slowing down ageing signs by prevention of telomere shortening and/or enhancing energy metabolisms against ageing.
  • 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 plantarum DR7 reduces cholesterol via phosphorylation of AMPK that down-regulated the mRNA expression of HMG-CoA reductase, thus has effects in liver lipid and energy metabolisms against increased triglycerides level, inflammation and accumulation of lipids (see EXAMPLE 6 hereinafter).
  • Cell free supernatant (CFS) from Lactobacillus plantarum DR7 showed a higher ability to assimilate cholesterol, reduction in cholesterol accumulation in both HepG2 and HT-29 cells, accompanied by reduced mRNA expression of HMG-CoA reductase (HMGCR) in HepG2, compared to other lactobacilli.
  • L. plantarum DR7 exerted its effect via the AMPK pathway, typically via the phosphorylation of AMPK instead of the AMPK mRNA expression in HepG2.
  • L. plantarum DR7 can exert cholesterol lowering properties along the AMPK pathway, specifically via phosphorylation of AMPK that led to reduced expression of HMGCR.
  • an aspect of the invention relates to Lactobacillus plantarum DR7 or a metabolite thereof for use in the prevention and treatment of cardiovascular diseases and in the reduction of cholesterol.
  • Lactobacillus plantarum DR7 alteres lipid metabolism and alleviated NAFLD in ageing rats (see EXAMPLE 6 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.
  • DR7 reduced serum triglycerides level after 12-weeks as compared to the HFD control.
  • an aspect of the invention relates to Lactobacillus plantarum DR7 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.
  • 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 plantarum DR7 has effects on gut modulation against imbalanced gut bacterial populations and metabolites concentrations (see EXAMPLE 7 hereinafter).
  • Male Sprague-Dawley rats were fed with high fat diet and injected with D-galactose for 12 weeks to induce ageing.
  • the effect of Lactobacillus plantarum DR7, administration on the fecal microbiota profile, short-chain fatty acids and water-soluble compounds were analyzed. It was found that the administration DR7 altered the gut microbiota diversity and composition, and metabolites profiles. Analysis of the fecal water-soluble metabolites revealed that administration of L. plantarum DR7 led to higher fecal content of compounds related to amino acids metabolism such as tryptophan, leucine, tyrosine, cysteine, methionine, valine and lysine.
  • an aspect of the invention relates to Lactobacillus plantarum DR7 or a metabolite thereof for as probiotic in gut modulation against imbalanced gut bacterial populations and metabolites concentrations; that means, to ameliorate dysbiosis.
  • the strain of the invention exerts its health benefits via, among other mechanisms, the production of metabolites 2-hydroxyisocapric acid (HICA) and 3-phenyllactic (PLA).
  • HICA 2-hydroxyisocapric acid
  • PLA 3-phenyllactic
  • Such metabolite provides the slowing down of ageing signs by reducing telomere shortening. Furthermore, the metabolite induces activation of the AMPK pathway via AMPK phosphorylation. As such, the metabolite indirectly regulates lipid metabolism that are generally associated with ageing factor.
  • the slowing down of ageing signs is achieved by providing an anti-inflammatory and immune modulatory protection against upper respiratory tract infection.
  • the metabolite is capable of providing the slowing down of ageing signs to a subject upon ingestion.
  • the use of the metabolite is especially suitable for subjects suffering from age-related diseases, such as cardiovascular diseases, high cholesterol level and progressive neuronal dysfunction. Particularly, lipid fraction of the metabolite is suitable for treating the abovementioned age-related diseases.
  • the crude metabolite can be ingested, or further processed to mix with at least one excipient to form a health food or beverage.
  • the processing can be carried out in a milder manner, such that to maintain the nutritional value of the metabolite.
  • the slowing down of ageing signs is achieved by using the metabolite comprising HICA and PLA to activate adenosine monophosphate-activated protein kinase (AMPK) pathway via AMPK phosphorylation.
  • AMPK is an intracellular energy sensor that regulates anabolic and catabolic pathway cellular energy level. It is also an upstream kinase for critical metabolic enzyme such as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR).
  • HMGCR 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase
  • AMPK phosphorylates a wide range of downstream targets, where adenosine triphosphate (ATP)-producing pathways are increased and ATP-utilizing pathways are decreased.
  • AMPK reduces expression of HMGCR gene that is responsible for cholesterol and isoprenoid production. This shows that the metabolite exhibits cholesterol-lowering properties and is useful for lowering cholesterol or lipid level in the subject upon ingestion.
  • the slowing down of ageing signs is also achieved by activating the AMPK phosphorylation to reduce influenza viral infections through macrophage activation.
  • the macrophages can be activated by cytokines, such as interferon-gamma, and bacterial endotoxins, such as lipopolysaccharide, where the activation changes the macrophages to kill invading bacteria or infected cells.
  • the activated macrophages eliminates the invading bacteria or infected cell by releasing toxic chemicals and proteins, such as proteases, neutrophil chemotactic factors, reactive oxygen species, cytokines, eicosanoids and growth factors. Therefore, the metabolite of the present invention can exert an immune-modulatory protection against upper respiratory tract infections in the subject upon ingestion.
  • the metabolite disclosed herein can be used for promoting lipid metabolism.
  • the metabolite reduces serum triglyceride level in the body by down-regulating stearoyl-CoA desaturase 1 ( SCD1 ), and up-regulating ATP-binding cassette sub-family G member 5 ( ABCG5 ) and ATP-binding cassette sub-family G member 8 ( ABCG8 ) in the liver.
  • SCD1 stearoyl-CoA desaturase 1
  • ABCG5 up-regulating ATP-binding cassette sub-family G member 5
  • ABCG8 ATP-binding cassette sub-family G member 8
  • the down-regulation of SCD1 reduces synthesis of mono-unsaturated fatty acids and results in reduction of plasma triglyceride. Accordingly, triglyceride production in the body can also be restricted by the lowering of the SCD1 activity.
  • the up-regulation of ABCG5 and ABCG8 maintains a sterol balance via a selectively driven biliary neutral sterol secretion.
  • use of the metabolite reduces the risk of fat-associated health condition that is commonly related to ageing factor.
  • the metabolite comprising HICA and PLA provides the effect to slow down signs of ageing by preventing telomere shortening.
  • an aged animal model subjected to a diet containing the metabolite and high fat content exhibits higher telomere/single-copy-gene ratio than another aged animal subjected solely to a high-fat diet.
  • reactive oxygen species generated from mitochondrial oxidation is one of the factors that shortens the telomere.
  • the metabolite comprising HICA and PLA may correlate to mitochondrial oxidation, and then decreases generation of reactive oxygen species.
  • use of the metabolite reduces accumulation of amyloid beta peptides in the brain.
  • the amyloid beta peptides are crucial amino acids that contribute to dementia, progressive neuronal dysfunction, synaptic function impairment and Alzheimer's disease. As such, use of the metabolite prevents amyloid plaques formation in the brain.
  • 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 2-hydroxyisocapric acid and 3-phenyllactic 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 plantarum DR7 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
  • Metabolite of DR7 in the fermentation medium was collected via separation of cell pellet from the spent broth. Spent broth was separated into lipid, protein and polysaccharide fractions. 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). Approximately 100 ⁇ L of the sample extract were mixed with 500 ⁇ L a solvent mixture comprising 2% H 2 SO 4 and methanol in a 2 ml Eppendorf tube and then agitated under a temperature of 80°C for 2 hours.
  • FAME fatty acid methyl ester
  • GC-MS gas chromatography-mass spectrometry
  • HepG2 human hepatoma cell line
  • HT-29 human colorectal cell line
  • DMEM Dulbecco's modified Eagle's medium
  • HepG2 and HT29 cells seeded in 12-well plates (1 ⁇ 10 5 cells per well) were incubated in serum free DMEM containing 1 mM cholesterol and 0.03% ox-bile for 24 h, at 37°C, with 5% CO2. The cells were then washed twice using cold phosphate buffered saline (PBS, pH 7.4).
  • PBS cold phosphate buffered saline
  • Lipids were extracted using 2 mL of hexane:isopropanol (3:2) for 30 min. The extracts were collected into a glass tube and evaporated under nitrogen gas. The amount of cholesterol uptaken and retained in the cells were extracted and quantified using Amplex Red Cholesterol Assay kit as according to manufacturer's instruction. Intracellular protein was extracted from the cells in the 12-well plate using 2 mL of NaOH (0.1 N) with continuous shaking at 4°C for 16 h. Protein concentration was measured using Bradford's method. The intracellular cholesterol accumulation was expressed as per amount of intracellular protein content.
  • RNAs were isolated using TRISURE reagent (Bioline, UK) and was converted to cDNA by reverse transcription using RevertAid RT Kit (Thermo Scientific, USA) with random hexamer primer as according to manufacturer's instruction. Briefly, 1 ⁇ g of total RNA was reverse transcribed and amplified by in cubating the reaction mixture at 2°C for 5 min, followed 4°C for 60 min. The reaction was terminated by incubating the mixture at 7°C for 5 min. The cDNA was diluted 10 times using nuclease-free water and used as template in qPCR or stored at -8°C until use.
  • the mRNA levels of HMG-CoA reductase and AMPK were determined by real-time PCR analysis using the Agilent AriaMx Realtime PCR System (Agilent Technologies, USA). Twenty microliter of PCR reactions consisted of 10 ⁇ L of 2xSensiFAST SYBR mix (Bioline, UK), 0.8 ⁇ L each of 10 ⁇ M forward and reverse primers and 1 ⁇ L of cDNA. The primer sequences are shown in Table 1, using amplification conditions suggested by the manufacturer. The 18S rRNA gene was used as the housekeeping gene for normalization of data. The mRNA expression was expressed as percentage change relative to the control.
  • Table 1 Primer sequences used in the real-time PCR analysis.
  • AMPK The activity of AMPK was assessed using a cell-based ELISA (RayBio cell-based protein phosphorylation ELISA kit, RayBiotech, USA) according to the instructions of the manufacturer. Briefly, HepG2 cells (1x10 4 cells/well) were seeded in 96-well plates and incubated 24 h at 3°C and 5% CO2 in serum free DMEM. After washing with 200 ⁇ L PBS twice, 100 mL of fixative solution was added to each well and incubated for 20 min at room temperature with shaking. Then the plate was washed 3 times using 200 ⁇ L TBST and incubated with a 100 ⁇ L quenching buffer for 20 min at room temperature, followed by addition of blocking buffer for 1 h at 3°C.
  • a cell-based ELISA RayBio cell-based protein phosphorylation ELISA kit, RayBiotech, USA
  • the plate was washed 3 times using 200 ⁇ L TBST, and 50 ⁇ L of rabbit anti-phosphorylated AMPK- ⁇ 1/2 (Santa Cruz Biotechnology; Thr172, 1:1,000 dilution in the blocking solution) was added and incubated for 3 h at room temperature with shaking. After washing, 50 ⁇ L of HRP-conjugated mouse antirabbit IgG (1:10,000 dilution in the blocking solution; Santa Cruz Biotechnology, USA) was added and incubated for 1.5 h at room temperature. After washing 3 times, 50 ⁇ L of TMB substrate (3,3,5,5-tetramethylbenzidine) was added to each well and incubated for 30 min with shaking at room temperature in the dark. Finally, 50 ⁇ L stop solution (H 2 SO 4 , 2N) was added, and the optical density was read at 450 nm.
  • HRP-conjugated mouse antirabbit IgG (1:10,000 dilution in the blocking solution; Santa Cruz Biotechnology, USA
  • TMB substrate 3,3,5,
  • 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.
  • MWM was performed in a non-reflective interior pool with diameter of 180 cm and height of 51 cm.
  • the pool was filled with water (19-22°C), divided into four zones (arbitrarily designated as north, south, east, and west quadrants) and contained a circular platform (20 cm diameter) in the center of the north quadrant that was submerged 1.5 cm below the surface of water (D'Hooge and De Deyn, 2001).
  • Each quadrant was marked with different objects and rats were shown the objects before the start of each assessment. Rats were trained over four trials per day for one day (once per each north, south, east, and west quadrants), before actual assessments for four consecutive days. Each day of assessment comprised of the similar four trials. Rats were gently placed in the water facing the wall at different start location and were allowed to swim freely to search for the escape platform within 60s. Time taken by each animal to locate the escape platform was recorded.
  • OFT was performed according previous study with modifications using an opaque rectangular box (32 cm H ⁇ 38 cm W ⁇ 52 cm L) with marked grids (Sprott and Eleftheriou, 1974). 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).
  • the animals were subjected to a T-maze comprising three arms, mainly an open arm without walls and two enclosed walls having dimensions of 40 cm high, 50 cm long and 15 cm wide. Each of the arms is attached to firm metallic legs 70 cm elevated from ground. Animals were gently placed on edge of the open arm of the T-maze that acts as a starting point for the experiment. Particularly, the experiment was conducted in two phases, where a first training phase was conducted by placing food the closed arms and then the animals were given 60 seconds to roam freely to enter either one of the closed arm. Upon entering, a cardboard was used to obstruct the animals for 30 seconds, to prevent the animals from leaving the chosen arm. Next, the animals were released and placed on the starting point to perform a second testing phase. Similarly, the animals were given 60 seconds to choose and enter either one of the closed arm. The experiment was repeated ten times per day and the result was recorded that the number of correct entries of the animals.
  • RNAlater Sigma, Missouri, USA
  • tissue samples were snap frozen and stored at -80°C until further analysis for Multiplex ELISA and real time PCR.
  • TC total cholesterol
  • TG triglyceride
  • LDL Low-density lipoprotein
  • HDL High-density lipoprotein
  • liver function total protein, albumin, globulin, Albumin/Globulin ratio (AG ratio), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • total bilirubin renal function
  • renal function sodium, urea, chloride, potassium, creatinine, uric acid, calcium and phosphate
  • the samples were processed using automatic tissue processor (Thermo ScientificTM Shandon Excelsior,USA). The samples were then embedded in paraffin wax and sectioned using cryotome. Sectioned samples were then stained with hematoxylin and eosin; and observed under light microscope.
  • 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.
  • DR7 and placebo products were manufactured by GN Pharmaceuticals Sdn. Bhd. (Selangor, Malaysia). Products did not contain any porcine or bovine ingredients; they were manufactured under GMP and certified halal by JAKIM (Department of Islamic Development Malaysia).
  • the probiotic product contained 1 ⁇ 10 9 CFU/ sachet of DR7 and maltodextrin as excipient (95%), while the placebo contained only maltodextrin (100%).
  • Each dose was 2 g of a light yellow powder supplied in an aluminum sachet, and all products were identical in taste.
  • inclusion criteria required male and female participants to be aged 18 to 60 years old and willing to commit to the study for the duration of the experiment.
  • Exclusion criteria included type 1 diabetes, long-term medication due to severe illness, HIV/AIDS, and glucose-6-phosphate dehydrogenase deficiency. All procedures involving human subjects were approved by the JEPeM-USM Review Panel on Clinical Studies of Universiti Sains Malaysia. Written informed consent was obtained from all subjects prior to the study.
  • URTI upper respiratory tract infection
  • baseline basic demographic questionnaire
  • health condition questionnaire every 4 week to record occurrences of upper respiratory illnesses (duration of respiratory-related illness, number of symptoms, number of illness episodes). All questionnaires were validated and available in 3 languages; namely, English, Malay, and Chinese (Lau et al., 2018a,b).
  • Blood samples were collected at baseline (week 0) and end of the study (week 12) for analysis of stress hormone cortisol, interleukin-1 ⁇ ,-4, and -10, tumour necrosis factor (TNF)- ⁇ and interferon (IFN)- ⁇ concentrations via ELISA.
  • Genes related to serotonin and dopamine pathway such as dopamine ⁇ -hydroxylase (DBH), tyrosine hydroxylase (TH), indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenasetryptophan hydroxylase-2 and 5-hydroxytryptamine receptor-6 were analyzed via real time PCR.
  • Gene expression of whole blood was also performed using 18S rRNA gene as a housekeeping gene for apoptosis and inflammatory genes.
  • Plasma membrane and hemolysate fractions of red blood cells (RBC) samples were evaluated for peroxidation of lipids and antioxidant potentials. Peroxidation of lipids and antioxidant potentials were assessed in plasma, membrane and hemolysate of red blood cells (RBC) samples. The RBC hemolysate and membranes were obtained as previously described. Briefly, whole blood was centrifuged for 20 minutes at 3500 ⁇ g to separate plasma and RBC pellet. The RBC pellets were washed with isotonic Tris-HCl buffer to remove buffy coat, resuspended in isotonic Tris-HCl buffer and centrifuged at 8000 ⁇ g for 20 minutes at temperature of 4°C for several times until the membranes appeared colorless.
  • the last colorless pellet was then rinsed twice with Tris-HCl buffer and collected as haemoglobin-free RBC membrane (RBC ghosts). Hemolysate were collected throughout the stages and pooled. RBC samples were standardized based on 20 ⁇ L of RBC pellet, while 20 ⁇ L of undiluted plasma was used. Lipid peroxidation was assayed by measuring the formation of malondialdehyde (MDA), while antioxidant potentials were determined using the ferric-reducing/antioxidant power (FRAP) assay.
  • MDA malondialdehyde
  • FRAP ferric-reducing/antioxidant power
  • Lactobacillus plantarum DR7 (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 DR7 were fed to designated Drosophila lines within 2 hours of solidification.
  • Table 2 List of Drosophila lines and treatment groups
  • HMDS hexamethyldisilazane
  • SEM scanning electron microscopy
  • EXAMPLE 1 Effects of Lactobacillus plantarum DR7 in anti-inflammatory and immune modulatory protection against upper respiratory tract infections
  • the clinical study in humans was a double-blind, randomized and placebo-controlled design study, where randomization was performed upon checking the inclusion and exclusion criteria.
  • Eligible subjects were randomized at 1:1 ratio to two arms of the study according to a computer generated, blocked randomization list with a block size of four, stratified according to age (18-29, 30-39, 40-49, 50-60 years old), assigned to the probiotic group (DR7) and placebo group (maltodextrin) with treatment codes. Randomization was performed by the study statistician, who had no contact with the participants. The allocation sequence was not available to any member of the research team until the completion of the study.
  • the sample size was calculated for a parallel group study design involving one prevention arm and one placebo arm, and was based on a power design analysis. A total of 124 subjects were needed for this study, comprising of 62 subjects in each group (DR7 and placebo) and an additional 10% dropout. This calculation was based on the need for a continuous response variable from independent control and experimental subjects, with a ratio of control to subject fixed at 1:1, probability (power) of 0.9 and Type-I error probability associated with this test of null hypothesis of 0.05. Previous data have shown that for an intervention using probiotics against reducing duration of illness raised from pulmonary diseases and symptoms, a standard deviation of 1.7 days within group was observed, accompanied by a reduction of 1 day between treatment and placebo groups.
  • DR7 marginally reduced the duration for nasal, pharyngeal and general flu symptoms compared to the placebo in young adults (FIG. 1A).
  • Plasma pro-inflammatory cytokines such as interleukin-1 ⁇ , TNF- ⁇ and IFN- ⁇ were significantly reduced (p ⁇ 0.05) in adults upon administration of DR7 compared to placebo over 12-weeks, while DR7 only reduced IFN- ⁇ in young adults as compared to the placebo (p ⁇ 0.05; FIG. 2A and B).
  • Plasma anti-inflammatory cytokines such as IL-4 and IL-10 were significantly increased in young adults upon administration of DR7 compared to placebo over 12-weeks (p ⁇ 0.05; FIG. 2A and B), while no such effect was observed in adults.
  • DR7 decreased plasma pro-inflammatory cytokines such as TNF- ⁇ and IFN- ⁇ while increasing plasma anti-inflammatory cytokines such as IL-4 and IL-10 compared to placebo (p ⁇ 0.05; FIG. 2C).
  • DR7 did not affect antioxidant potentials in plasma, RBC membrane or hemolysate of young adults as compared to placebo (FIG. 3A)
  • DR7 marginally increased the antioxidant potential of RBC membrane in adults p ⁇ 0.10, FIG. 3B
  • significantly increased such a parameter in all subjects over 12-weeks as compared to placebo p ⁇ 0.05, FIG. 3C
  • the administration of DR7 also significantly decreased concentration of malondialdehyde (MDA) as determined by TBA in plasma of young adults, adults and all subjects as compared to placebo which showed an increase over 12-weeks (p ⁇ 0.05; FIG. 3 D,E and F).
  • MDA malondialdehyde
  • a marginal decrease in levels of TBA was also observed in RBC membrane of young adults and all subjects, and RBC hemolysate of all subjects upon administration of DR7 as compared to placebo over 12-weeks (p ⁇ 0.10, FIG. 3 D and F).
  • DR7 upregulated the expressions of plasma genes for CD44 and CD117 in young adults over 12-weeks as compared to the placebo (p ⁇ 0.05; FIG. 4A). Meanwhile, the administration of DR7 downregulated the expressions of plasma genes for CD4 and CD8 in adults over 12-weeks as compared to the placebo (p ⁇ 0.05; FIG. 4B). In comparing all subjects, such differences were lost where insignificant differences were observed between subjects of DR7 and placebo (FIG. 4C). Higher expressions of plasma CD44 and CD117 in young adults of DR7 group and lower expressions of plasma CD4 and CD8 in adults of DR7 group, showed the presence of less activated T-cells as compared to the placebo.
  • DR7 downregulated the expressions of plasma genes for NKp46 and NKp30 in young adults, CD56, NKp46 and NKp30 in adults, and CD56, CD94, NKp46 and NKp30 in all subjects over 12-weeks as compared to the placebo (p ⁇ 0.05; FIG. 4 D,E and F). This showed an enhanced presence of non-resting and matured NK-cells as compared to the placebo.
  • T-cells and NK-cells gene expressions showed that DR7 enhanced mucosal epithelial integrity, leading to lower infiltration of antigens which were sufficiently cleared by first defence of innate immunity NK-cells and thus adaptive immunity T-cells were less activated.
  • FIG. 5 shows that there is a correlation between clinical symptoms and blood parameters among the subjects.
  • the aims of this study were to investigate the effects of Lactobacillus plantarum DR7 against upper respiratory tract infections (URTI) and elucidate the possible mechanisms underlying immunomodulatory properties.
  • the DR7 strain (9 log cfu/d) was administered for 12 wk in a randomized, double-blind, and placebo-controlled human study involving 109 adults.
  • the administration of DR7 reduced the du-ration of nasal symptoms and the frequency of URTI after 12 and 4 wk, respectively, compared with the placebo.
  • the DR7 treatment suppressed plasma proinflammatory cytokines (IFN- ⁇ , TNF- ⁇ ) in middle-aged adults (30 to 60 yr old), while enhancing anti-inflammatory cytokines (IL-4, IL-10) in young adults ( ⁇ 30 yr old), accompanied by reduced plasma peroxidation and oxidative stress levels com-pared with the placebo.
  • Young adults who received DR7 showed higher expression of plasma CD44 and CD117 by 4.50- and 2.22-fold, respectively, compared with the placebo.
  • middle-aged adults showed lower expression of plasma CD4 and CD8 by 11.26- and 1.80-fold, respectively, compared with the placebo, indicating less T-cell activation.
  • both young and middle-aged adults who received DR7 showed enhanced presence of nonresting and mature NK cells compared with those who received the placebo.
  • DR7 alleviates the symptoms of URTI by improving inflammatory parameters and enhancing immunomodulatory properties.
  • EXAMPLE 2 Effects of Lactobacillus plantarum DR7 in the prevention and treatment of stress, anxiety, memory and cognitive dysfunctions.
  • the old High Fat Diet with DR7 and statin groups showed a reduced latency time starting from day-2 onwards, as compared to the control old HFD group which only showed a reduction starting from day-4 (mean difference -28.66; 95% CI -45.89 to -11.48; P ⁇ 0.05).
  • Both young (mean difference -8.75; 95% CI -26.48 to 8.93) and old groups (mean difference -8.75; 95% CI -25.97 to 8.47) administered with a normal diet showed reduced latency time from day-3 onwards (P ⁇ 0.05) in FIG. 6A. This shows that DR7 rats had better memory and used lesser time to find the hidden platform.
  • FIG. 6C shows that the animals treated with L. plantarum strain DR7 showed increase in number of correct entries in the T-maze test, indicating that the animals have improved memory after the treatment.
  • DR7 DR7
  • Plasma cortisol level was reduced among DR7 subjects as compared to the placebo, accompanied by reduced plasma pro-inflammatory cytokines, such as interferon- ⁇ and transforming growth factor- ⁇ and increased plasma anti-inflammatory cytokines, such as interleukin 10.
  • DR7 better improved cognitive and memory functions in normal adults (>30 years old), such as basic attention, emotional cognition, and associate learning, as compared to the placebo and young adults.
  • DR7 enhanced the serotonin pathway, as observed by lowered expressions of plasma dopamine ⁇ -hydroxylase (DBH), tyrosine hydroxylase (TH), indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase accompanied by increased expressions of tryptophan hydroxylase-2 and 5-hydroxytryptamine receptor-6, while stabilising the dopamine pathway as observed via stabilised expressions of TH and DBH over 12 weeks as compared to the placebo.
  • DBH dopamine ⁇ -hydroxylase
  • TH tyrosine hydroxylase
  • TH indoleamine 2,3-dioxygenase
  • tryptophan 2,3-dioxygenase accompanied by increased expressions of tryptophan hydroxylase-2 and 5-hydroxytryptamine receptor-6
  • EXAMPLE 3 Effects of Lactobacillus plantarum DR7 in enhancing the neurotransmitter serotonin and dopamine pathways against Alzheimer's Disease, and hippocampal inflammation and apoptosis.
  • DR7 did not exert any effects along the neurotransmitter pathways of GABA (FIG. 9A), dopamine (FIG. 9B) or norepinephrine (FIG. 9C). But, rats on DR7 showed reduced expression of IDO and increased the expression of the TPH1 in aged HFD rats, while statin increased the expression of the TPH1 in aged HFD rats compared to the old HFD control (mean difference -1.56; 95% CI -2.88 to -0.24; P ⁇ 0.05; FIG. 9E,F). This showed that DR7 may exert an anxiety-alleviating effect in aged High Fat Diet rats via the serotonin pathway.
  • the Drosophila eye is an ideal tissue for developmental neurobiologists as it is a simple neuroectoderm composed of photoreceptors and accessory cells.
  • Each eye is made up of 800 hexagonal shaped units termed as ommatidium, arranged in a crystalline array akin to the honeycomb cells of a beehive.
  • the ommatidia are aligned in columns across the eye to give an overall concave and egg-like shape.
  • the mechano-sensory bristles protruding at alternate vertices of each ommatidium that points at specific angles provides a second sensory field.
  • Ectopically expressing human A ⁇ 42 in the compound eyes using the pan-retinal GMR-GAL4 driver resulted in a strong neurodegenerative phenotype characterised by the rough eye phenotype (REP).
  • REP rough eye phenotype
  • the GMR-A ⁇ 42.nf adult eyes were severely distorted with fused individual eye units laced with multiple holes that gave the overall eye a "glazed appearance" (FIG. 11C,D).
  • the inter-ommatidial bristles have decreased in number while those present on the eye were pointing at random angles.
  • the feeding of DR7 was able to reverse the distortion of GMR-A ⁇ 42's eye at varying degrees (FIG. 11EF), to almost identical to the Control.
  • the ommatidia have assumed their original hexagonal shape with neither fused individual units nor any observable holes. Additionally, the number of bristles has increased significantly and the angles the bristles are pointing at have been rectified to a degree.
  • the quantification of morphological defects in the Drosophila eye was done using the software Flynotyper that calculates the phenotypic score (P-value) of each eye. A higher P-value represents increased disorderliness or altered symmetry of the ommatidia arrangement, and thus increased severity of the eye phenotype.
  • P-value phenotypic score
  • DR7 reduced neuronal apoptosis in the hippocampus via the regulation of senescing and apoptotic genes (FIG. 13).
  • the BCL-XL, BAX and CAS9 genes govern the processes of apoptosis and DNA damages (FIG. 13G).
  • the old HFD group exhibited higher expressions of BCL-XL, BAX and CAS9 as compared to the old ND group, indicating a need to combat and control apoptosis as induced by High Fat Diet. Low expressions of these genes were observed upon administration of DR7 and statin, suggesting that DR7 and statin reduced such a need to upregulate these genes amid conditions of lower apoptosis and oxidative stress.
  • DR7 increased anti-inflammatory cytokines and decreased pro-inflammatory cytokines in young adults, while only decreased pro-inflammatory cytokines in normal adults over 12 weeks in FIG. 14. As stress often triggers inflammatory responses, DR7 may have decreased stress levels in association with decreased inflammatory parameters in young adults, while a lesser reduction of both stress and inflammatory parameters were observed in normal adults.
  • EXAMPLE 4 Lactobacillus plantarum DR7 exerts its health benefits via the production of metabolites HICA and PLA.
  • AMPK 5' adenosine monophosphate-activated protein kinase
  • DMEM Dulbecco's Modified Essential Medium
  • the plantarum strain DR7 metabolite shows the highest AMPK phosphorylation activity, suggesting that the lipid fraction is highly responsible for AMPK phosphorylation of the metabolite.
  • the lipid fraction was further subjected to purification, characterization and identification.
  • the crude cell-free supernatant DR7 shows a comparable phosphorylation activity to a commercial AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR).
  • AICAR 5-aminoimidazole-4-carboxamide ribonucleotide
  • the effectiveness of the L. plantarum strain DR7 metabolite was further confirmed by comparing phosphorylation activity of a commercial AMPK inhibitor, Compound C and the cell-free supernatant added with Compound C.
  • the crude cell-free supernatant DR7 increases AMPK phosphorylation activity to a level similar to that of AICAR, while addition of Compound C diminishes the activity.
  • the lipid fraction of L. plantarum strain DR7 metabolite as determined by GC-MS analysis showed different peak profiles as compared the blank unfermented MRS medium (FIG. 16).
  • Two significant intensity peaks on the chromatogram (b) show the presence of 2-hydroxyisocapric acid (HICA) and 3-phenyllactic acid (PLA).
  • HICA 2-hydroxyisocapric acid
  • PLA 3-phenyllactic acid
  • Leucine is the precursor for the formation of HICA, upon conversion from KICA, via the actions of enzyme HaDH.
  • the gene for HaDH is found present in the whole genome sequence of DR7 (protein ID AYA79606.1).
  • Phenylalanine is the precursor for the formation of PLA, upon conversion from PPA, via the actions of enzymes HPT, LDH and AAT.
  • HPT protein ID AYA81108.1
  • AAT protein ID AYA79202.1
  • LDH protein ID AYA79316.1
  • EXAMPLE 5 Effects of Lactobacillus plantarum DR7 in prevention of telomere shortening and enhancing energy metabolisms against ageing.
  • telomere length as measured from the rat's blood collected after 12 weeks of treatment was as shown in FIG. 17. Results showed that D-galactose induced ageing rats (aged rats) had a significantly shorter telomere length (P ⁇ 0.05) as compared to the young rats. Meanwhile, it was also observed that non-treated aged group had a significantly shorter telomere length (P ⁇ 0.05) as compared to the aged-statin and aged-DR7 groups.
  • Treadmill exhaustion test was conducted to assess the endurance capacity of the rats after 12 weeks of treatment. Running time, distance and highest speed achieved till fatigue were estimated as maximal endurance capacity. As shown in FIG. 18, aged group showed significantly lower (P ⁇ 0.05) endurance capacity (distance, running duration, speed, work and power) as compared to young group. Results showed that aged-DR7 group had a significantly higher (P ⁇ 0.05) endurance capacity in terms of work and power as compared to the aged rats group. Running duration and speed was significantly higher in aged-DR7 group, accompanied by higher (P ⁇ 0.05) running distance as compared to the aged group.
  • telomere length Male Sprague-Dawley rats were fed with high fat diet (54% kcal fat) and injected with D-galactose daily for 12 weeks to induce ageing.
  • DR7 strain The effects of DR7 strain on age-related impairment such as telomere length, plasma lipid peroxidation, hepatic AMPK expression, as well as muscle performance were evaluated.
  • Administration of statin and Lactobacillus plantarum DR7 (LP-DR7) significantly reduced the shortening of telomere and increased the expression of AMPK subunit- ⁇ 2.
  • AMPK subunit- ⁇ 1 was elevated in rats administered with LP-DR7 as compared to the control while statin did not exert such an effect.
  • DR7 decreased muscle performance via increasing running distance, duration, speed, work and power as compared to the control while statin did not exert such an effect.
  • statin did not exert such an effect.
  • the study illustrated the potentials of DR7 strain in alleviation of age-related impairment.
  • EXAMPLE 6 Effects of Lactobacillus plantarum DR7 in liver lipid and energy metabolisms against increased triglycerides level, inflammation and accumulation of lipids.
  • L. plantarum DR7 was one of the strains that exhibited a higher reduction of total cholesterol from the culture media containing cholesterol micelle and were thus selected for subsequent analyses.
  • CFS of DR7 also significantly reduced cholesterol accumulation in HT-29 cells ( p ⁇ 0.05) as compared to the control (FIG. 19B,C) and reduced ( p ⁇ 0.05) cholesterol accumulation in HepG2 cells (approximately 19.7%) as compared to the control.
  • CFS-DR7 plantarum DR7
  • HMGCR mRNA a rate-limiting enzyme responsible for the biosynthesis of cholesterol
  • concentration tested varied from 10% to 50% (v/v), as higher concentration was not possible due to cell apoptosis at concentration above 50% (data not shown).
  • Lovastatin (1 ⁇ M) was included as a positive control. Cholesterol accumulation in HepG2 cells was significantly reduced at all concentrations studied, with similar effects as compared to lovastatin (FIG.
  • CFS-DR7 regulated the expression of HMGCR via increased expression of AMPK.
  • CFS-DR7 did not affect the expression of AMPK mRNA (FIG. 19G), indicating that the regulation was beyond a transcriptional level.
  • phosphorylation of AMPK leads to activation of the enzyme, while dephosphorylation inactivates it.
  • Our current data showed that CFS-DR7 significantly increased ( p ⁇ 0.05) the phosphorylation of AMPK to a similar level as the well-established AMPK activator (AICAR; Fig. 18H), while the addition of AMPK inhibitor (Compound C) diminished such an effect.
  • CFS-DR7 also significantly increased ( p ⁇ 0.05) the phosphorylation of AMPK to a level higher than lovastatin (1 ⁇ M).
  • lovastatin 1 ⁇ M
  • SCD1 Stearoyl-CoA desaturase 1
  • MUFA mono-unsaturated fatty acids
  • SCD1 enzyme was significantly down-regulated (P ⁇ 0.05) in HFD-DR7 (FIG. 20A) when compared with HFD group.
  • L. plantarum DR7 may exerted TG lowering effect via downregulation of SCD1 gene expression.
  • Gene expression of IL-6 was significantly lower (P ⁇ 0.05) in HFD group as compared to ND group and administration of L. plantarum DR7 significantly upregulated the expression of IL-6 (P ⁇ 0.05; FIG. 20B).
  • ABCG5 and ABCG8 are half-size 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 and G8 (P ⁇ 0.05; FIGs. 20C,D) whereas expression of ABCG5 and ABCG8 mRNA expression were significantly upregulated upon administration of L. plantarum DR7 (P ⁇ 0.05).
  • SR-B1 and LDL-R expression were significantly lower in HFD group as compared to the ND group (P ⁇ 0.05; FIG. 21 A,B).
  • HFD-DR7 group exhibit a significantly higher expression of SR-B1 and LDL-R (P ⁇ 0.05).
  • HFD-statin and HFD-DR7 group have a significant higher (P ⁇ 0.05) expression of ABCA1 as compared to HFD group (FIG. 21C).
  • Both statin and HFD-DR7 groups increased Apo-A1 levels as compared to the HFD group (FIG. 21D).
  • HFD-DR7 group showed significantly higher expression of AMPK ⁇ 1 and MPK ⁇ 2 as compared to HFD group.
  • L. plantarum DR7 may able to activate AMPK via down-regulation of SCD1 gene expression.
  • FIG. 23 showed that IL-4 was significantly increased in HFD group as compared to ND group. As compared to HFD group; HFD-statin and HFD-DR7 had a lower (P ⁇ 0.05) IL-4 level.
  • HFD-statin and HFD-DR7 groups showed lesser lipid accumulation as compared to HFD group (FIG. 24 C,D).
  • HFD-DR7 showed a better-organized structure compared to the other treatment groups.
  • Cell free supernatant (CFS) from Lactobacillus plantarum DR7 showed a higher ability to assimilate cholesterol, reduction in cholesterol accumulation in both HepG2 and HT-29 cells, accompanied by reduced mRNA expression of HMG-CoA reductase (HMGCR) in HepG2.
  • HMGCR HMG-CoA reductase
  • the reduction of HMGCR expression was also diminished in the presence of an AMPK inhibitor, suggesting that L. plantarum DR7 exerted its effect via the AMPK pathway, typically via the phosphorylation of AMPK instead of the AMPK mRNA expression in HepG2.
  • L. plantarum DR7 can exert cholesterol lowering properties along the AMPK pathway, specifically via phosphorylation of AMPK that led to reduced expression of HMGCR.
  • DR7 was 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.
  • HFD high fat diet
  • Treatments included i) normal diet, ii) HFD, iii) HFD-statin (lovastatin 2 mg/kg/day), iv) HFD- Lactobacillus plantarum DR7 (10 log CFU/day).
  • HFD high fat diet
  • HFD-statin lovastatin 2 mg/kg/day
  • HFD- Lactobacillus plantarum DR7 (10 log CFU/day).
  • the administration of DR7 reduced serum triglycerides level after 12-weeks as compared to the HFD control.
  • EXAMPLE 7 Effects of Lactobacillus plantarum DR7 on gut modulation against imbalanced gut bacterial populations and metabolites concentrations.
  • Actinobacteria was reduced upon ageing, regardless of with or without HFD (P ⁇ 0.05; FIG. 25a).
  • Actinobacteria, with its primary genus of Bifidobacterium is often associated with a healthier gut microbiota profile.
  • DR7 improved such a property in gut of ageing rats.
  • Bacteroidetes was higher (P ⁇ 0.05) in aged-ND group as compared to young-ND group while there was no significant difference in Firmicutes when comparing between the treatment groups (FIG. 25 b,c).
  • D-galactose induction significantly reduced (P ⁇ 0.05) the Firmicutes over Bacteroidetes (F/B ratio) while administration of L. plantarum DR7 significantly increased the F/B ratio of aged rats fed with HFD to a level similar to young-ND group (FIG. 25d).
  • Acetate was also reportedly claimed for maintenance of the gut barrier function via reduction of the luminal pH, which inhibits pathogenic.
  • Metabolites derived from the gut microbiota can have a direct role in human health and disease.
  • administration of L. plantarum DR7 led to higher fecal content of essential amino acids such as tryptophan, leucine, tyrosine, cysteine, methionine, valine, and lysine (FIG. 27) which are crucial to host amino acid homeostasis and health.
  • FIG. 28 the administration of DR7 has caused a shift in gut microbiota distribution.
  • Lactobacillus plantarum DR7 administration of Lactobacillus plantarum DR7 led to higher fecal content of compounds related to amino acids metabolism such as tryptophan, leucine, tyrosine, cysteine, methionine, valine and lysine
  • EXAMPLE 8 Compositions with Lactobacillus plantarum DR7

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Abstract

Selon un premier aspect, l'invention concerne une souche de Lactobacillus plantarum, déposée au Centre Général Chinois de Culture Microbiologique (CGMCC) sous le numéro d'accession CGMCC 15535, et une séquence génomique complète, déposée dans GenBank sous le numéro d'accession CP031318. La souche est particulièrement avantageuse pour les infections des voies respiratoires supérieures, le stress, l'anxiété, les troubles de la mémoire et cognitifs, la maladie d'Alzheimer, l'hyperlipidémie, l'équilibre intestinal et le vieillissement.
PCT/KR2019/011130 2018-09-03 2019-08-30 Souche probiotique pour une infection des voies respiratoires supérieures, le stress, l'anxiété, les troubles de la mémoire et cognitifs et le vieillissement WO2020050553A1 (fr)

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CN201980056940.3A CN112930392B (zh) 2018-09-03 2019-08-30 用于上呼吸道感染、压力、焦虑、记忆和认知功能障碍和衰老的益生菌

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CN112204129A (zh) * 2018-02-02 2021-01-08 Ko生物技术有限公司 植物乳杆菌kbl396菌株及其用途
CN112915109A (zh) * 2021-01-21 2021-06-08 吉林省农业科学院 植物乳杆菌dp189在制备预防和/或治疗阿尔茨海默病药物中的应用
CN113337428A (zh) * 2021-06-03 2021-09-03 海南大学 一株植物乳杆菌hnu082及其用途
CN113337427A (zh) * 2021-06-03 2021-09-03 海南大学 一种植物乳杆菌hnu082、组合物及其应用
WO2022094178A1 (fr) * 2020-10-30 2022-05-05 The Regents Of The University Of California Compositions et méthodes de diagnostic et de traitement de patients ayant des antécédents d'adversité précoce
WO2022131192A1 (fr) * 2020-12-16 2022-06-23 株式会社明治 Composition pour atténuer l'inflammation d'un tissu cérébral
CN115089619A (zh) * 2022-07-18 2022-09-23 南昌大学 植物乳杆菌zdy2013在具有缓解非酒精性脂肪肝功能的制剂中的应用
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CN115838653A (zh) * 2022-07-11 2023-03-24 四川维创天益生物科技有限公司 一种改善不良情绪的植物乳杆菌gm11及其用途

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112204129A (zh) * 2018-02-02 2021-01-08 Ko生物技术有限公司 植物乳杆菌kbl396菌株及其用途
CN112204129B (zh) * 2018-02-02 2023-12-15 Ko生物技术有限公司 植物乳杆菌kbl396菌株及其用途
WO2022094178A1 (fr) * 2020-10-30 2022-05-05 The Regents Of The University Of California Compositions et méthodes de diagnostic et de traitement de patients ayant des antécédents d'adversité précoce
WO2022131192A1 (fr) * 2020-12-16 2022-06-23 株式会社明治 Composition pour atténuer l'inflammation d'un tissu cérébral
CN112915109A (zh) * 2021-01-21 2021-06-08 吉林省农业科学院 植物乳杆菌dp189在制备预防和/或治疗阿尔茨海默病药物中的应用
CN113337428A (zh) * 2021-06-03 2021-09-03 海南大学 一株植物乳杆菌hnu082及其用途
CN113337427A (zh) * 2021-06-03 2021-09-03 海南大学 一种植物乳杆菌hnu082、组合物及其应用
CN113337428B (zh) * 2021-06-03 2023-06-06 海南大学 一株植物乳杆菌hnu082及其用途
WO2023230513A1 (fr) * 2022-05-26 2023-11-30 Pouda Biotech Llc Lactobacillus plantarum pda6 ayant des effets neurologiques et compositions le comprenant
CN115089619A (zh) * 2022-07-18 2022-09-23 南昌大学 植物乳杆菌zdy2013在具有缓解非酒精性脂肪肝功能的制剂中的应用

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